jan havliš : national centre for biomolecular research :: laboratory of functional genomics and proteomics jan havliš : national centre for biomolecular research :: laboratory of functional genomics and proteomics SEPARATION  METHODS  ASEPARATION  METHODS  A Creative Commons: Attribution‐Noncommercial‐Share alike 3.0 Unported LicenseCreative Commons: Attribution‐Noncommercial‐Share alike 3.0 Unported License C7021 C7023 C7021 C7023 preparative separationpreparative separation : extraction L‐L, S‐L : SFE, ASE, SPE, SPME, MASE, TLC, HSE : extraction L‐L, S‐L : SFE, ASE, SPE, SPME, MASE, TLC, HSE SEPARATION; separation methodsSEPARATION; separation methods basics in separationbasics in separation separation methods A – syllabusseparation methods A – syllabus : liquid chromatography :: NPLC, RPLC, LC‐on‐chip, HIC, HILIC, UPLC, AC, IEC, SFC, PLC : gas chromatography :: GC : liquid chromatography :: NPLC, RPLC, LC‐on‐chip, HIC, HILIC, UPLC, AC, IEC, SFC, PLC : gas chromatography :: GC analytical separationanalytical separation 22 analytical separation methodsanalytical separation methods extraction as a separation model example extraction as a separation model example  : what the separation in fact is? : principles that allow it : what the separation in fact is? : principles that allow it recommended readingrecommended reading 33 J. C. Giddings, Unified separation science, Wiley 1991 C. F. Poole, The essence of chromatography, Elsevier 2003 R. L. Grob et al., Modern practice of gas chromatography, Wiley 2004 G. Guiochon et al. (eds.), Fundamentals of preparative and non‐linear chromatography, Elsevier 2006 J. Cazes (ed.), Encyclopedia of Chromatography, CRC Press 2010 L. R. Snyder et al., Introduction to modern liquid chromatography, Wiley 2010 D. Corradini (ed.), Handbook of HPLC, CRC Press 2011 J. C. Giddings, Unified separation science, Wiley 1991 C. F. Poole, The essence of chromatography, Elsevier 2003 R. L. Grob et al., Modern practice of gas chromatography, Wiley 2004 G. Guiochon et al. (eds.), Fundamentals of preparative and non‐linear chromatography, Elsevier 2006 J. Cazes (ed.), Encyclopedia of Chromatography, CRC Press 2010 L. R. Snyder et al., Introduction to modern liquid chromatography, Wiley 2010 D. Corradini (ed.), Handbook of HPLC, CRC Press 2011 sensitivity : ability to exclude false negative results :: TP / (TP + FN) sensitivity : ability to exclude false negative results :: TP / (TP + FN) specificity : ability to exclude false positive results :: TN / (TN + FP) specificity : ability to exclude false positive results :: TN / (TN + FP) precision : repeatability day‐to‐day series precision : repeatability day‐to‐day series accuracy : matching „reality“; reference, normalisation accuracy : matching „reality“; reference, normalisation selectivity : ability to acquire correct positive results :: TP / (TP + FP) selectivity : ability to acquire correct positive results :: TP / (TP + FP) result relevanceresult relevance result variabilityresult variability imprecise inaccurate imprecise inaccurate precise inaccurate precise inaccurate imprecise accurate imprecise accurate precise accurate precise accurate 44 I.I.separationseparation (s e p a r a t i o n)(s e p a r a t i o n) (s) + (e p a r a t i o n)(s) + (e p a r a t i o n) (s e) + (p a) + (r a) + (t i) + (on)(s e) + (p a) + (r a) + (t i) + (on) segregating component(s) of a mixture of substance in space (and time)segregating component(s) of a mixture of substance in space (and time) original mixtureoriginal mixture isolation, purification, preparation isolation, purification, preparation fractionationfractionation complete separationcomplete separation separation ← lat. separatus = SE‐ away + PAR‐ prepareseparation ← lat. separatus = SE‐ away + PAR‐ prepare (s) + (e) + (p) + (a) + (r) + (a) + (t) + (i) + (o) + (n)(s) + (e) + (p) + (a) + (r) + (a) + (t) + (i) + (o) + (n) partial separation partial separation 55 it is necessary that there is at least one different physico‐chemical propertyit is necessary that there is at least one different physico‐chemical property separation of two completely miscible substancesseparation of two completely miscible substances a driving force is based on such property that then transports and redistributes the substancesa driving force is based on such property that then transports and redistributes the substances another parameters influencing separation : equilibrium – distribution, dissociation : system structure – macroscopic, microscopic, molecular : flow – hydrodynamics, hydrostatics : mechanical processes – passing through pores another parameters influencing separation : equilibrium – distribution, dissociation : system structure – macroscopic, microscopic, molecular : flow – hydrodynamics, hydrostatics : mechanical processes – passing through pores α – separation factorα – separation factor 𝐜 𝐀 𝐈 𝐜 𝐀 𝐈𝐈 ≫1 𝐜 𝐀 𝐈 𝐜 𝐀 𝐈𝐈 ≫1 𝐜 𝐁 𝐈 𝐜 𝐁 𝐈𝐈 ≪1 𝐜 𝐁 𝐈 𝐜 𝐁 𝐈𝐈 ≪1 𝛂 𝐀,𝐁 ≫1𝛂 𝐀,𝐁 ≫1𝛂 𝐀,𝐁 𝐜 𝐀 𝐈 𝐜 𝐀 𝐈𝐈⁄ 𝐜 𝐁 𝐈 𝐜 𝐁 𝐈𝐈 𝐜 𝐀 𝐈 𝐜 𝐁 𝐈⁄ 𝐜 𝐀 𝐈𝐈 𝐜 𝐁 𝐈𝐈𝛂 𝐀,𝐁 𝐜 𝐀 𝐈 𝐜 𝐀 𝐈𝐈⁄ 𝐜 𝐁 𝐈 𝐜 𝐁 𝐈𝐈 𝐜 𝐀 𝐈 𝐜 𝐁 𝐈⁄ 𝐜 𝐀 𝐈𝐈 𝐜 𝐁 𝐈𝐈 II IIII AA AA BBBB cAcA II cAcA IIII cBcB II cBcB IIII optimal α valueoptimal α value 66 separation limitationsseparation limitations : physical :: achievable conditions of separation ::: temperature, pressure... : physical :: achievable conditions of separation ::: temperature, pressure... (s e p a r a t i o n)        (s) + (e) + (p) + (a) + (r) + (a) + (t) + (i) + (o) + (n)(s e p a r a t i o n)        (s) + (e) + (p) + (a) + (r) + (a) + (t) + (i) + (o) + (n)→ separating (ΔV≠0; ΔS<0) → separating (ΔV≠0; ΔS<0) mixing (ΔV=0; ΔS>0) ← mixing (ΔV=0; ΔS>0) ← diluting (ΔV≠0; ΔS>0) → diluting (ΔV≠0; ΔS>0) → ∆𝐒 𝐧 · 𝐑 · 𝐥𝐧 𝐕𝐬𝐭𝐚𝐫𝐭 𝐕𝐞𝐧𝐝 ∆𝐒 𝐧 · 𝐑 · 𝐥𝐧 𝐕𝐬𝐭𝐚𝐫𝐭 𝐕𝐞𝐧𝐝 : chemical :: equilibrium (of forms) ::: A1 ↔ A2 :: thermodynamic aspects (1st & 2nd laws of thermodynamics) ::: spontaneous change: ΔS > 0 in isolated system : chemical :: equilibrium (of forms) ::: A1 ↔ A2 :: thermodynamic aspects (1st & 2nd laws of thermodynamics) ::: spontaneous change: ΔS > 0 in isolated system (s e p a r a t i o n)        ( s   e   p   a   r   a   t   i o   n )(s e p a r a t i o n)        ( s   e   p   a   r   a   t   i o   n ) ::: spontaneous change: ΔG < 0::: spontaneous change: ΔG < 0 spontaneous separation happens if : work is done : it is heated : it is diluted spontaneous separation happens if : work is done : it is heated : it is diluted ∆𝐆 ∆𝐇 𝐓 · ∆𝐒 ⇒ 𝐝𝐆 𝐕 · 𝐝𝐩 𝐒 · 𝐝𝐓∆𝐆 ∆𝐇 𝐓 · ∆𝐒 ⇒ 𝐝𝐆 𝐕 · 𝐝𝐩 𝐒 · 𝐝𝐓 aim in separation : maximise separation and minimise dilution :: duel of driving forces of separation and dispersion aim in separation : maximise separation and minimise dilution :: duel of driving forces of separation and dispersion differential transport : each substance somewhere/somehow else differential transport : each substance somewhere/somehow else 77 example 1example 1 calculate the entropy change that follows separation of four derivatives of triptamine (5‐methoxy‐α‐ methyltryptamine, 5‐methoxy‐diisopropyltryptamine, 5‐methoxy‐dimetlyltryptamine and α‐ methyltryptamine), if the molar amount of each is 0.1 mmol. after the separation, each derivative of triptamine occupies ¼ of original sample volume. assess, whether the separation is spontaneous or not. calculate the entropy change that follows separation of four derivatives of triptamine (5‐methoxy‐α‐ methyltryptamine, 5‐methoxy‐diisopropyltryptamine, 5‐methoxy‐dimetlyltryptamine and α‐ methyltryptamine), if the molar amount of each is 0.1 mmol. after the separation, each derivative of triptamine occupies ¼ of original sample volume. assess, whether the separation is spontaneous or not. ΔS = n ∙ R ∙ ln(Vstart/Vend) ΔStot = ΔS1 + ΔS2 + ΔS3 + ΔS4 ΔStot = – 46.1 mJ∙K‐1 ΔStot < 0  process is not spontaneous ΔS = n ∙ R ∙ ln(Vstart/Vend) ΔStot = ΔS1 + ΔS2 + ΔS3 + ΔS4 ΔStot = – 46.1 mJ∙K‐1 ΔStot < 0  process is not spontaneous ?????? 88 separation methodsseparation methods there is no universal and absolute separation method : fundamental (separation) limitations : detection limitations there is no universal and absolute separation method : fundamental (separation) limitations : detection limitations basic separation principlesbasic separation principles :: continuative :: often in large scale :: continuative :: often in large scale basic separation typesbasic separation types : analytical: analytical : intermolecular interactions : geometry of molecules : external field influence : intermolecular interactions : geometry of molecules : external field influence :: high efficiency :: mostly in small scale :: high efficiency :: mostly in small scale : preparative: preparative 99 : pre‐step for instrumental analysis : increases quality of analytical separation : isolation of substances : pre‐step for instrumental analysis : increases quality of analytical separation : isolation of substances :: enrichment (molar ratio of wanted component <0.1) :: pre‐concentration (<0.9) :: purification (>0.9) :: enrichment (molar ratio of wanted component <0.1) :: pre‐concentration (<0.9) :: purification (>0.9) preparative separationpreparative separation : laboratory (discrete and continual; small volumes) : piloting (discrete and continual; large volumes) : operating (continual; large volumes) : laboratory (discrete and continual; small volumes) : piloting (discrete and continual; large volumes) : operating (continual; large volumes) 1010 does not serve directly to analysedoes not serve directly to analyse methods : discrete – in separate/discrete steps :: extraction, crystallisation, zone refining : continuative – in unseparated steps :: counter‐current extraction : continual – in inseparable steps :: filtration, electrolysis, distillation, chromatography methods : discrete – in separate/discrete steps :: extraction, crystallisation, zone refining : continuative – in unseparated steps :: counter‐current extraction : continual – in inseparable steps :: filtration, electrolysis, distillation, chromatography serves to analyse mixturesserves to analyse mixtures : distributive separation  :: chromatography : distributive separation  :: chromatography analytical separationanalytical separation : instrumental analysis (of fully) separated components : identification of a component : characterisation of a component (structure, physico‐chemical properties) : instrumental analysis (of fully) separated components : identification of a component : characterisation of a component (structure, physico‐chemical properties) : separation in force field :: electromigration :: mass spectrometry :: flow field fractionation : separation in force field :: electromigration :: mass spectrometry :: flow field fractionation : separation based on molecule geometry: separation based on molecule geometry 1111 two basic methods of separationtwo basic methods of separation separation methods – overviewseparation methods – overview separation property volatility distillation, sublimation solubility precipitation, zone refining, fraction crystallisation distribution constant extraction, partition chromatography (LL, GL) dissociation constant ion‐exchange and affinity chromatography surface activity adsorption chromatography (LS, GS), foam separation molecular geometry molecular sieve size + charge + mass electrophoresis, flow field fractionation, mass spectrometry different phase distribution of components : equilibrium – entropy influence :: rate of mass transfer through inter‐phase isn't controlling : non‐equilibrium                :: rate of mass transfer through inter‐phase is controlling different phase distribution of components : equilibrium – entropy influence :: rate of mass transfer through inter‐phase isn't controlling : non‐equilibrium                :: rate of mass transfer through inter‐phase is controlling different rate of distribution of components : through semi‐permeable membrane : in a force field different rate of distribution of components : through semi‐permeable membrane : in a force field 1212 separation based on phase equilibria G – L G – S L – L L – S distillation sublimation extraction precipitation foam separation molecular sieve partition LC fraction crystallisation partition GC adsorption GC molecular exclusion LC molecular sieve zone refining adsorption LC ion‐exchange LC separation based on differences in motion rate through membrane in field ultrafiltration electromigration methods reversed osmosis flow fractionation dialysis, electrodialysis mass spectrometry, ion mobility ultracentrifugation thermodiffusion 1313 gas chrom.gas chrom. size exclusion chrom.size exclusion chrom. electromigration methodselectromigration methods mass spectrometrymass spectrometry flow field fractionationflow field fractionation 00 11 22 33 44 55 66 77 88 99 1010 1111 log MWlog MW ions | monomers | polymers | virus | cells | particlesions | monomers | polymers | virus | cells | particles ion chromatographyion chromatography high‐performance liquid chrom.high‐performance liquid chrom. 1414 molecular equilibriummolecular equilibrium equilibrium in a closed systemequilibrium in a closed system equilibrium in an open systemequilibrium in an open system system boundaries impermeable to masssystem boundaries impermeable to mass system boundaries permeable to masssystem boundaries permeable to mass spontaneous processes water‐ice 9 °C, 101.33 kPa spontaneous processes water‐ice 9 °C, 101.33 kPa equilibrium water‐ice 0 °C, 101.33 kPa equilibrium water‐ice 0 °C, 101.33 kPa μB – chemical potential : change of G with amount of entering substance B μB – chemical potential : change of G with amount of entering substance B ∆𝐆 ∆𝐇 𝐓 · ∆𝐒 ⇒ 𝐝𝐆 𝐕 · 𝐝𝐩 𝐒 · 𝐝𝐓∆𝐆 ∆𝐇 𝐓 · ∆𝐒 ⇒ 𝐝𝐆 𝐕 · 𝐝𝐩 𝐒 · 𝐝𝐓 ∆𝐆 𝟎∆𝐆 𝟎 ∆𝐆 𝟎∆𝐆 𝟎 in → dnB > 0 out → dnB < 0 in → dnB > 0 out → dnB < 0 𝐝𝐆 𝛛𝐆 𝛛𝐧 𝐁 𝐓,𝐩,𝐧 𝐀 · 𝐝𝐧 𝐁 𝛍 𝐁 · 𝐝𝐧 𝐁𝐝𝐆 𝛛𝐆 𝛛𝐧 𝐁 𝐓,𝐩,𝐧 𝐀 · 𝐝𝐧 𝐁 𝛍 𝐁 · 𝐝𝐧 𝐁 1515 AA BB generalisation for i of substances at constant T and pgeneralisation for i of substances at constant T and p system of two immiscible phases creates closed system : equilibrium sets of substance A between phases I and II  system of two immiscible phases creates closed system : equilibrium sets of substance A between phases I and II  of two open systemsof two open systems μA – standard chemical potential (in given phase)μA – standard chemical potential (in given phase) 00 chemical potential of substance A in phase X (μA ) depends on : internal thermodynamic affinity of the substance to given phase; ↑ affinity  ↓ μA : dilution of the substance (entropy of dilution influence); ~ R∙T∙ln aA chemical potential of substance A in phase X (μA ) depends on : internal thermodynamic affinity of the substance to given phase; ↑ affinity  ↓ μA : dilution of the substance (entropy of dilution influence); ~ R∙T∙ln aA XX 00 aA – activity of substance AaA – activity of substance A 𝐝𝐆 𝐕 · 𝐝𝐩 𝐒 · 𝐝𝐓 𝛍𝐢 · 𝐝𝐧𝐢𝐝𝐆 𝐕 · 𝐝𝐩 𝐒 · 𝐝𝐓 𝛍𝐢 · 𝐝𝐧𝐢 𝐝𝐆 𝛍𝐢 · 𝐝𝐧𝐢𝐝𝐆 𝛍𝐢 · 𝐝𝐧𝐢 𝛍 𝐀 𝛍 𝐀 𝟎 𝐑 · 𝐓 · 𝐥𝐧 𝐚 𝐀𝛍 𝐀 𝛍 𝐀 𝟎 𝐑 · 𝐓 · 𝐥𝐧 𝐚 𝐀 ⇒ 𝛍 𝐀 𝐈 𝛍 𝐀 𝐈𝐈 ⇒ 𝛍 𝐀 𝐈 𝛍 𝐀 𝐈𝐈 𝐝𝐆 𝟎𝐝𝐆 𝟎 𝐝𝐆 𝐝𝐆𝐈 𝐝𝐆𝐈𝐈 𝛍 𝐀 𝐈𝐈 𝛍 𝐀 𝐈 · 𝐝𝐧 𝐀 𝟎𝐝𝐆 𝐝𝐆𝐈 𝐝𝐆𝐈𝐈 𝛍 𝐀 𝐈𝐈 𝛍 𝐀 𝐈 · 𝐝𝐧 𝐀 𝟎 𝐝𝐆𝐈 𝛍 𝐀 𝐈 · 𝐝𝐧 𝐀𝐝𝐆𝐈 𝛍 𝐀 𝐈 · 𝐝𝐧 𝐀 𝐝𝐆𝐈𝐈 𝛍 𝐀 𝐈𝐈 · 𝐝𝐧 𝐀𝐝𝐆𝐈𝐈 𝛍 𝐀 𝐈𝐈 · 𝐝𝐧 𝐀 1616 AA II IIII resulting ratio of substance A concentration in equilibrium in phases I and IIresulting ratio of substance A concentration in equilibrium in phases I and II K – distribution coefficientK – distribution coefficient practically in separations, we work with so‐called diluted solutionspractically in separations, we work with so‐called diluted solutions γA – activity coefficient of substance AγA – activity coefficient of substance A γA → 1 and cA → 0γA → 1 and cA → 0 pA – partial pressure of substance A (gas chromatography)pA – partial pressure of substance A (gas chromatography) ΔμA controls distribution of substance between two phasesΔμA controls distribution of substance between two phases00 D – distribution ratioD – distribution ratio 𝛍 𝐀 𝛍 𝐀 𝟎 𝐑 · 𝐓 · 𝐥𝐧 𝛄 𝐀 · 𝐜 𝐀 ⇒ 𝛍 𝐀 𝟎 𝐑 · 𝐓 · 𝐥𝐧 𝐜 𝐀𝛍 𝐀 𝛍 𝐀 𝟎 𝐑 · 𝐓 · 𝐥𝐧 𝛄 𝐀 · 𝐜 𝐀 ⇒ 𝛍 𝐀 𝟎 𝐑 · 𝐓 · 𝐥𝐧 𝐜 𝐀 𝐚 𝐀 𝐈𝐈 𝐚 𝐀 𝐈 𝐝𝐆 𝟎 𝐞 ∆𝛍 𝐀 𝟎 𝐑·𝐓 𝐊 𝐚 𝐀 𝐈𝐈 𝐚 𝐀 𝐈 𝐝𝐆 𝟎 𝐞 ∆𝛍 𝐀 𝟎 𝐑·𝐓 𝐊 𝐜 𝐀 𝐈𝐈 𝐜 𝐀 𝐈 𝐝𝐆 𝟎 𝐞 ∆𝛍 𝐀 𝟎 𝐑·𝐓 𝐃 𝐜 𝐀 𝐈𝐈 𝐜 𝐀 𝐈 𝐝𝐆 𝟎 𝐞 ∆𝛍 𝐀 𝟎 𝐑·𝐓 𝐃 𝛍 𝐀 𝛍 𝐀 𝟎 𝐑 · 𝐓 · 𝐥𝐧 𝐩 𝐀𝛍 𝐀 𝛍 𝐀 𝟎 𝐑 · 𝐓 · 𝐥𝐧 𝐩 𝐀 ∆𝛍 𝐀 𝟎 𝛍 𝐀 𝟎,𝐈𝐈 𝛍 𝐀 𝟎,𝐈 ∆𝛍 𝐀 𝟎 𝛍 𝐀 𝟎,𝐈𝐈 𝛍 𝐀 𝟎,𝐈 γA → 1 and cA → 0γA → 1 and cA → 0 ∆𝐆 𝟎 𝐑 · 𝐓 · 𝐥𝐧 𝐊∆𝐆 𝟎 𝐑 · 𝐓 · 𝐥𝐧 𝐊 at constant pressureat constant pressure 1717 equilibrium in open system with external fieldequilibrium in open system with external field system boundaries are permeable for energy of the fieldsystem boundaries are permeable for energy of the field : influences potential energy, which is additive to Gibbs free energy: influences potential energy, which is additive to Gibbs free energy ΔμA changes continuously in spaceΔμA changes continuously in spaceextext ΔμA is changed abruptly on phase boundaryΔμA is changed abruptly on phase boundaryintint separation in external field is different from separation between phasesseparation in external field is different from separation between phases generalisation for i of substances at constant T and pgeneralisation for i of substances at constant T and p 𝐝𝐆 𝐕 · 𝐝𝐩 𝐒 · 𝐝𝐓 𝛍𝐢 𝐢𝐧𝐭 𝛍𝐢 𝐞𝐱𝐭 · 𝐝𝐧𝐢𝐝𝐆 𝐕 · 𝐝𝐩 𝐒 · 𝐝𝐓 𝛍𝐢 𝐢𝐧𝐭 𝛍𝐢 𝐞𝐱𝐭 · 𝐝𝐧𝐢 𝐝𝐆 𝛍𝐢 𝐢𝐧𝐭 𝛍𝐢 𝐞𝐱𝐭 · 𝐝𝐧𝐢𝐝𝐆 𝛍𝐢 𝐢𝐧𝐭 𝛍𝐢 𝐞𝐱𝐭 · 𝐝𝐧𝐢 𝐃 𝐜 𝐀 𝐈𝐈 𝐜 𝐀 𝐈 𝐝𝐆 𝟎 𝐞 ∆𝛍 𝐀 𝟎 ∆𝛍 𝐀 𝐞𝐱𝐭 𝐑·𝐓𝐃 𝐜 𝐀 𝐈𝐈 𝐜 𝐀 𝐈 𝐝𝐆 𝟎 𝐞 ∆𝛍 𝐀 𝟎 ∆𝛍 𝐀 𝐞𝐱𝐭 𝐑·𝐓 𝐝𝐆 𝐝𝐆𝐢𝐧𝐭 𝐝𝐆 𝐞𝐱𝐭 ∆𝛍 𝐀 𝐢𝐧𝐭 ∆𝛍 𝐀 𝐞𝐱𝐭 · 𝐝𝐧 𝐀 𝟎𝐝𝐆 𝐝𝐆𝐢𝐧𝐭 𝐝𝐆 𝐞𝐱𝐭 ∆𝛍 𝐀 𝐢𝐧𝐭 ∆𝛍 𝐀 𝐞𝐱𝐭 · 𝐝𝐧 𝐀 𝟎 ∆𝛍 𝐀 𝐞𝐱𝐭 𝛍 𝐀 𝐞𝐱𝐭,𝐈𝐈 𝛍 𝐀 𝐞𝐱𝐭,𝐈 ∆𝛍 𝐀 𝐞𝐱𝐭 𝛍 𝐀 𝐞𝐱𝐭,𝐈𝐈 𝛍 𝐀 𝐞𝐱𝐭,𝐈 ∆𝛍 𝐀 𝐢𝐧𝐭 ∆𝛍 𝐀 𝟎 ∆𝛍 𝐀 𝐢𝐧𝐭 ∆𝛍 𝐀 𝟎 1818 intermolecular interactionintermolecular interaction ΔGA – partial molar Gibbs energy : influence of molar content change of component on system properties ΔGA – partial molar Gibbs energy : influence of molar content change of component on system properties ΔSA – partial molar entropy : randomness of actual molecular surround of substance A molecules ΔSA – partial molar entropy : randomness of actual molecular surround of substance A molecules 00–– within distribution of substance A between two phaseswithin distribution of substance A between two phases 𝐝𝐆 𝛛𝐆 𝛛𝐧 𝐁 𝐓,𝐩,𝐧 𝐀 · 𝐝𝐧 𝐁 𝛍 𝐁 · 𝐝𝐧 𝐁𝐝𝐆 𝛛𝐆 𝛛𝐧 𝐁 𝐓,𝐩,𝐧 𝐀 · 𝐝𝐧 𝐁 𝛍 𝐁 · 𝐝𝐧 𝐁 ∆𝐆 𝐀 𝟎 𝛛𝐆 𝛛𝐧 𝐀 𝐓,𝐩,𝐧 ∆𝛍 𝐀 𝟎 ∆𝐆 𝐀 𝟎 𝛛𝐆 𝛛𝐧 𝐀 𝐓,𝐩,𝐧 ∆𝛍 𝐀 𝟎 ∆𝐇 𝐀 𝟎 ≫ 𝐓 · ∆𝐒 𝐀 𝟎 ∆𝐇 𝐀 𝟎 ≫ 𝐓 · ∆𝐒 𝐀 𝟎 ∆𝛍 𝐀 𝟎 ∆𝐆 𝐀 𝟎 ∆𝐇 𝐀 𝟎 𝐓 · ∆𝐒 𝐀 𝟎 ∆𝛍 𝐀 𝟎 ∆𝐆 𝐀 𝟎 ∆𝐇 𝐀 𝟎 𝐓 · ∆𝐒 𝐀 𝟎 00–– 1919 00–– ΔHA – partial molar enthalpy : intermolecular interactions of substance A & substance of phases I & II ΔHA – partial molar enthalpy : intermolecular interactions of substance A & substance of phases I & II 𝐜 𝐀 𝐈𝐈 𝐜 𝐀 𝐈 𝐝𝐆 𝟎 𝐞 ∆𝛍 𝐀 𝟎 𝐑·𝐓 𝐃 𝐜 𝐀 𝐈𝐈 𝐜 𝐀 𝐈 𝐝𝐆 𝟎 𝐞 ∆𝛍 𝐀 𝟎 𝐑·𝐓 𝐃∆𝐇 𝐀 𝟎 𝟎 ⇒ ∆𝛍 𝐀 𝟎 𝟎∆𝐇 𝐀 𝟎 𝟎 ⇒ ∆𝛍 𝐀 𝟎 𝟎 ∆𝐇 𝐀 𝟎 𝐇 𝐀 𝟎,𝐈 𝐇 𝐀 𝟎,𝐈𝐈 ∆𝐇 𝐀 𝟎 𝐇 𝐀 𝟎,𝐈 𝐇 𝐀 𝟎,𝐈𝐈 ⇒ 𝐃 𝟏⇒ 𝐃 𝟏 𝐇 𝐀 𝟎,𝐈 𝐇 𝐀 𝟎,𝐈𝐈 𝐇 𝐀 𝟎,𝐈 𝐇 𝐀 𝟎,𝐈𝐈 the weaker the interaction → the lower the enthalpythe weaker the interaction → the lower the enthalpy exponent would have positive valueexponent would have positive value → substance A would appear in higher amounts in phase II, because→ substance A would appear in higher amounts in phase II, because enthalpy influenceenthalpy influence description of intermolecular interactionsdescription of intermolecular interactions intramolecular forces : water dissociation H2O → 2 H + O; 837 kJ∙mol‐1 intramolecular forces : water dissociation H2O → 2 H + O; 837 kJ∙mol‐1 ↔↔ rr intermolecular forces = van der Waals forces = weak forcesintermolecular forces = van der Waals forces = weak forces 𝐄𝐢𝐧𝐭𝐞𝐫𝐦𝐨𝐥 𝐄 𝐚𝐭𝐭𝐫𝐚𝐜𝐭 𝐄 𝐫𝐞𝐩𝐮𝐥𝐬𝐞𝐄𝐢𝐧𝐭𝐞𝐫𝐦𝐨𝐥 𝐄 𝐚𝐭𝐭𝐫𝐚𝐜𝐭 𝐄 𝐫𝐞𝐩𝐮𝐥𝐬𝐞 𝐄 𝒇 𝐫 𝐱 𝐄 𝒇 𝐫 𝐱 2020 intermolecular forces : water evaporation H2O (l) → H2O (g); 41 kJ∙mol‐1 intermolecular forces : water evaporation H2O (l) → H2O (g); 41 kJ∙mol‐1 electrostatic interactions : ion‐ion; dipole‐dipole, ion‐dipole, dipole‐induced dipole, disperse forces (London) :: hydrogen bridges – special case of dipole‐dipole bond (take part in e.g. solvation) electrostatic interactions : ion‐ion; dipole‐dipole, ion‐dipole, dipole‐induced dipole, disperse forces (London) :: hydrogen bridges – special case of dipole‐dipole bond (take part in e.g. solvation) Lewis interactions (theory of hard and soft acids and bases) : co‐ordination covalent, sharing of electron pairs and free binding orbitals Lewis interactions (theory of hard and soft acids and bases) : co‐ordination covalent, sharing of electron pairs and free binding orbitals interaction types interaction types  dipole‐dipole (ED) : substances with permanent dipoles – polar :: e.g. water, alcohols, halogen‐hydrocarbons... :: intensity depends on temperature dipole‐dipole (ED) : substances with permanent dipoles – polar :: e.g. water, alcohols, halogen‐hydrocarbons... :: intensity depends on temperature CH3OHCH3OH CHCl3CHCl3 δ+δ+ δ–δ– δ+δ+ δ–δ– inductive (EI) : induced dipoles – solvation by permanent dipoles : weak interaction :: e.g. water, ammonium, alcohols... :: intensity depends not on temperature inductive (EI) : induced dipoles – solvation by permanent dipoles : weak interaction :: e.g. water, ammonium, alcohols... :: intensity depends not on temperature (CH3)2C=O(CH3)2C=O C6H14C6H14 δ+δ+ δ–δ– hydrogen bridges : special case of dipole‐dipole interaction : strong interaction : the most general example of Lewis interaction (soft) hydrogen bridges : special case of dipole‐dipole interaction : strong interaction : the most general example of Lewis interaction (soft) CH3OHCH3OH H2OH2O δ+δ+δ–δ– δ+δ+ δ–δ– ∆𝐇 𝐀 𝟎 𝐄 𝐱 𝐱 𝐢𝐧𝐭𝐞𝐫𝐚𝐜𝐭𝐢𝐨𝐧 ∆𝐇 𝐀 𝟎 𝐄 𝐱 𝐱 𝐢𝐧𝐭𝐞𝐫𝐚𝐜𝐭𝐢𝐨𝐧 2121 disperse (EL) : „momentarily“ induced dipoles – primary non‐polar :: London forces : weak interactions :: e.g. CCl4, benzene... disperse (EL) : „momentarily“ induced dipoles – primary non‐polar :: London forces : weak interactions :: e.g. CCl4, benzene... C6H14C6H14 C8H18C8H18 ionic (EAB) : coulombic interactions between charged groups : or between permanent dipoles and charged groups : Lewis interactions (hard) : strong interactions :: e.g. water, ammonium, alcohols... ionic (EAB) : coulombic interactions between charged groups : or between permanent dipoles and charged groups : Lewis interactions (hard) : strong interactions :: e.g. water, ammonium, alcohols... C6H14C6H14 Cl–Cl– H2OH2Oδ+δ+ δ–δ– Na+Na+ inductive ionic : interaction ion‐induced dipole : weak interactions :: e.g. I3  bond inductive ionic : interaction ion‐induced dipole : weak interactions :: e.g. I3  bond– 𝐄 𝐋 𝐄𝐈 𝐄 𝐃 𝐄 𝐀𝐁𝐄 𝐋 𝐄𝐈 𝐄 𝐃 𝐄 𝐀𝐁 2222 relative strength of interactionsrelative strength of interactions polarity descriptionpolarity description polar bond polar bond polarisability (α) : measure of easiness of molecular electron clouds deformation polarisability (α) : measure of easiness of molecular electron clouds deformation p – dipole moment, E – electric field intensityp – dipole moment, E – electric field intensity N – Avogadro constant, n – refraction indexN – Avogadro constant, n – refraction index polarity : electric charge distribution leading to molecule as electric dipole polarity : electric charge distribution leading to molecule as electric dipole boron trifluorideboron trifluoride 𝐩 𝛂 · 𝐄𝐩 𝛂 · 𝐄 𝛂 𝟑𝛑 · 𝐍 𝟒 · 𝐧 𝟐 𝟏 𝐧 𝟐 𝟐 𝛂 𝟑𝛑 · 𝐍 𝟒 · 𝐧 𝟐 𝟏 𝐧 𝟐 𝟐 2323 the most often used descriptors of polarity : octanol‐water partition coefficient (Do/w; P; logP; logPo/w) the most often used descriptors of polarity : octanol‐water partition coefficient (Do/w; P; logP; logPo/w) : not suitable for ionisable substances :: octanol‐water distribution coefficient (KD,o/w; D)  : not suitable for ionisable substances :: octanol‐water distribution coefficient (KD,o/w; D)  𝐃 𝐨 𝐰⁄ 𝐜 𝐀 𝐨𝐜𝐭𝐚𝐧𝐨𝐥 𝐜 𝐀 𝐰𝐚𝐭𝐞𝐫𝐃 𝐨 𝐰⁄ 𝐜 𝐀 𝐨𝐜𝐭𝐚𝐧𝐨𝐥 𝐜 𝐀 𝐰𝐚𝐭𝐞𝐫  (pH – pKa) > 1  (pH – pKa) > 1  (pKa – pH) > 1  (pKa – pH) > 1 𝐥𝐨𝐠 𝐊 𝐃,𝐨 𝐰⁄ ,𝐚𝐜𝐢𝐝 𝐥𝐨𝐠 𝐃 𝐥𝐨𝐠 𝟏 𝟏 𝟏𝟎 𝐩𝐇 𝐩𝐊 𝐚 𝐥𝐨𝐠 𝐊 𝐃,𝐨 𝐰⁄ ,𝐚𝐜𝐢𝐝 𝐥𝐨𝐠 𝐃 𝐥𝐨𝐠 𝟏 𝟏 𝟏𝟎 𝐩𝐇 𝐩𝐊 𝐚 𝐥𝐨𝐠 𝐊 𝐃,𝐨 𝐰⁄ ,𝐛𝐚𝐬𝐞 𝐥𝐨𝐠 𝐃 𝐥𝐨𝐠 𝟏 𝟏 𝟏𝟎 𝐩𝐊 𝐚 𝐩𝐇 𝐥𝐨𝐠 𝐊 𝐃,𝐨 𝐰⁄ ,𝐛𝐚𝐬𝐞 𝐥𝐨𝐠 𝐃 𝐥𝐨𝐠 𝟏 𝟏 𝟏𝟎 𝐩𝐊 𝐚 𝐩𝐇 𝐥𝐨𝐠 𝐊 𝐃,𝐨 𝐰⁄ ,𝐚𝐜𝐢𝐝 ≅ 𝐥𝐨𝐠 𝐃 𝐩𝐊 𝐚 𝐩𝐇𝐥𝐨𝐠 𝐊 𝐃,𝐨 𝐰⁄ ,𝐚𝐜𝐢𝐝 ≅ 𝐥𝐨𝐠 𝐃 𝐩𝐊 𝐚 𝐩𝐇 𝐥𝐨𝐠 𝐊 𝐃,𝐨 𝐰⁄ ,𝐛𝐚𝐬𝐞 ≅ 𝐥𝐨𝐠 𝐃 𝐩𝐊 𝐚 𝐩𝐇𝐥𝐨𝐠 𝐊 𝐃,𝐨 𝐰⁄ ,𝐛𝐚𝐬𝐞 ≅ 𝐥𝐨𝐠 𝐃 𝐩𝐊 𝐚 𝐩𝐇 𝐊 𝐃,𝐨 𝐰⁄ 𝐚 𝐀 𝐨𝐜𝐭𝐚𝐧𝐨𝐥 𝐚 𝐀 𝐢𝐨𝐧𝐢𝐬 𝐰𝐚𝐭𝐞𝐫 𝐚 𝐀 𝐧𝐞𝐮𝐭𝐫 𝐰𝐚𝐭𝐞𝐫𝐊 𝐃,𝐨 𝐰⁄ 𝐚 𝐀 𝐨𝐜𝐭𝐚𝐧𝐨𝐥 𝐚 𝐀 𝐢𝐨𝐧𝐢𝐬 𝐰𝐚𝐭𝐞𝐫 𝐚 𝐀 𝐧𝐞𝐮𝐭𝐫 𝐰𝐚𝐭𝐞𝐫 2424 relative solvent permittivity (ε) : measure of intermolecular interactions in liquids relative solvent permittivity (ε) : measure of intermolecular interactions in liquids D – electric induction, E – electric field intensity D – electric induction, E – electric field intensity  ε0 – permittivity of vacuumε0 – permittivity of vacuum solvents usedsolvents used other interaction descriptionsother interaction descriptions water miscible water immiscible ε ε water 78 nitrobenzene 35 methanol 32 amylalcohol 16 1‐propanol 21 ethylacetate 6 pyridine 12 methyl‐isobutylketon 13 dioxane 2 chloroform 5 benzene 2 hexane 2 𝛆 𝐃 𝐄 𝛆 𝐃 𝐄 𝐃 𝛆 𝟎 · 𝐄 𝛂𝐃 𝛆 𝟎 · 𝐄 𝛂 2525 Hildebrand solubility parameter (δ) : measure of intermolecular interactions in substance it‐self Hildebrand solubility parameter (δ) : measure of intermolecular interactions in substance it‐self Δ Evap / V  – cohesive energetic density of substance energy necessary to evaporate a substance relative to volume : i.e. measure of interaction between molecules Δ Evap / V  – cohesive energetic density of substance energy necessary to evaporate a substance relative to volume : i.e. measure of interaction between molecules substance mixingsubstance mixing VA – molar volume of pure dissolved substanceVA – molar volume of pure dissolved substance –– A – dissolved substance (solute) B – dissolving substance (solvent) A – dissolved substance (solute) B – dissolving substance (solvent) 𝛅 ∆𝐄 𝐯𝐚𝐩 𝐕 𝛅 ∆𝐄 𝐯𝐚𝐩 𝐕 ∆𝐆 𝐦𝐢𝐱 ∆𝐇 𝐦𝐢𝐱 𝐓 · ∆𝐒 𝐦𝐢𝐱∆𝐆 𝐦𝐢𝐱 ∆𝐇 𝐦𝐢𝐱 𝐓 · ∆𝐒 𝐦𝐢𝐱 ∆𝐇 𝐦𝐢𝐱 𝐕 𝐀 · 𝛅 𝐀 𝛅 𝐁 𝟐 ∆𝐇 𝐦𝐢𝐱 𝐕 𝐀 · 𝛅 𝐀 𝛅 𝐁 𝟐 2626 : sort substances by increasing the polarity: sort substances by increasing the polarityexample 2 : part 1 example 2 : part 1 2727 2.70 2.49 2.70 2.49 3.10 2.93 3.10 2.93 7.71 7.13 7.71 7.13 1.50 1.67 1.50 1.67 ??? – 0.56 ??? – 0.56 1.90 1.63 1.90 1.63 – 2.27 – 1.54 – 2.27 – 1.54 0.90 1.14 0.90 1.14 ??? – 1.10 ??? – 1.10 – 0.17 – 0.22 – 0.17 – 0.22 – 3.45 – 3.75 – 3.45 – 3.75 5.18 4.02 5.18 4.02 3.70 3.38 3.70 3.38 0.90 – 0.10  0.90 – 0.10  – 2.29 – 2.62 – 2.29 – 2.62 11 22 33 44 55 66 77 8‐98‐9 8‐98‐9 1515 1414 1313 1010 1111 1212 example 2 : part 2 example 2 : part 2 2828 ?????? entropy influenceentropy influence in most cases within distribution of substance A between two phasesin most cases within distribution of substance A between two phases i.e. how the molecule of dissolved substance „fits“ between molecules of solvent : measure of necessary re‐orientation and re‐positioning of molecules i.e. how the molecule of dissolved substance „fits“ between molecules of solvent : measure of necessary re‐orientation and re‐positioning of molecules ΔSA : randomness of actual molecular surround of substance A moleculesΔSA : randomness of actual molecular surround of substance A molecules00–– : in a case of high difference between polarity of substance A and solvent :: re‐arrangement of solvent molecules (semi‐rigid structure) : in a case of sieve effect :: separation on porous media : in a case of high difference between polarity of substance A and solvent :: re‐arrangement of solvent molecules (semi‐rigid structure) : in a case of sieve effect :: separation on porous media so when does entropy influence the separation?so when does entropy influence the separation? ∆𝛍 𝐀 𝟎 ∆𝐇 𝐀 𝟎 𝐓 · ∆𝐒 𝐀 𝟎 ∆𝛍 𝐀 𝟎 ∆𝐇 𝐀 𝟎 𝐓 · ∆𝐒 𝐀 𝟎 ∆𝐇 𝐀 𝟎 ≫ 𝐓 · ∆𝐒 𝐀 𝟎 ∆𝐇 𝐀 𝟎 ≫ 𝐓 · ∆𝐒 𝐀 𝟎 2929 molecular geometry and structuremolecular geometry and structure porosity influenceporosity influence s – channel wall surface relative to volume units – channel wall surface relative to volume unit L – average contour length : for sphere equal to dp L – average contour length : for sphere equal to dp –– 𝐜 𝐀 𝐚𝐯𝐚𝐢𝐥𝐚𝐛𝐥𝐞 𝐜 𝐀 𝐭𝐨𝐭𝐚𝐥 𝐝𝐆 𝟎 𝐃 𝐜 𝐀 𝐚𝐯𝐚𝐢𝐥𝐚𝐛𝐥𝐞 𝐜 𝐀 𝐭𝐨𝐭𝐚𝐥 𝐝𝐆 𝟎 𝐃 𝐝 𝐤 𝟒 𝐬⁄𝐝 𝐤 𝟒 𝐬⁄ 𝐃 𝐞 𝐬·𝐋̅ 𝟐𝐃 𝐞 𝐬·𝐋̅ 𝟐 𝐃 𝛑 · 𝟎. 𝟓𝐝 𝐤 𝟎. 𝟓𝐝 𝐩 · 𝐋 𝛑 · 𝟎. 𝟓𝐝 𝐤 · 𝐋 𝐝 𝐤 𝐝 𝐩 𝐝 𝐤 𝟐 𝟏 𝐝 𝐩 𝐝 𝐤 𝟐 𝟏 𝐬 · 𝟎. 𝟓𝐝 𝐩 𝟐 𝟐 𝐃 𝛑 · 𝟎. 𝟓𝐝 𝐤 𝟎. 𝟓𝐝 𝐩 · 𝐋 𝛑 · 𝟎. 𝟓𝐝 𝐤 · 𝐋 𝐝 𝐤 𝐝 𝐩 𝐝 𝐤 𝟐 𝟏 𝐝 𝐩 𝐝 𝐤 𝟐 𝟏 𝐬 · 𝟎. 𝟓𝐝 𝐩 𝟐 𝟐 3030 dkdk dpdp ½ dp½ dp LL dp – particle diameter dk – separation channel diameter L – separation channel length dp – particle diameter dk – separation channel diameter L – separation channel length extractionextraction II.II. method based on substance distribution between two immiscible phasesmethod based on substance distribution between two immiscible phases phases usedphases used 3131 : liquid‐solid (leaching / infusing) :: maceration – infusion into one portion of extractant at ambient temperature :: digestion – infusion into one portion of extractant at elevated temperature :: percolation – continuous infusion of flowing extractant : liquid‐solid (leaching / infusing) :: maceration – infusion into one portion of extractant at ambient temperature :: digestion – infusion into one portion of extractant at elevated temperature :: percolation – continuous infusion of flowing extractant : liquid‐liquid (most common) :: batch extraction – one portion of extractant :: perforation – continuous circulation of extractant through liquid raffinate :: continuous extraction – repeated extraction by extractant portion ::: partition chromatography – continuative variant : liquid‐liquid (most common) :: batch extraction – one portion of extractant :: perforation – continuous circulation of extractant through liquid raffinate :: continuous extraction – repeated extraction by extractant portion ::: partition chromatography – continuative variant : liquid‐gas : solid‐gas : solid‐supercritical fluid : liquid‐gas : solid‐gas : solid‐supercritical fluid extraction liquid‐liquidextraction liquid‐liquid equilibrium state of compound in a system of two immiscible liquids : two phases; phase I and phase II equilibrium state of compound in a system of two immiscible liquids : two phases; phase I and phase II aA – activity of substance A in phases I and/or II sometimes more polar phase (water, w) and phase less polar (organic, o) aA – activity of substance A in phases I and/or II sometimes more polar phase (water, w) and phase less polar (organic, o) 1) original state 2) after equilibration 1) original state 2) after equilibration extraction process : transport in phase I – inter‐phase transfer – transport in phase II extraction process : transport in phase I – inter‐phase transfer – transport in phase II AA II IIII AA AA 1)1) 2)2) aAaA II aAaA IIII diffusiondiffusion inter‐phaseinter‐phase 3232 raffinate : phase, which originally contained sample extractant : phase, into which we extract extract : extracted sample raffinate : phase, which originally contained sample extractant : phase, into which we extract extract : extracted sample distribution constant (KD) : defines relation of separated substance to both phases Nernst distribution law; Nernst distribution constant distribution constant (KD) : defines relation of separated substance to both phases Nernst distribution law; Nernst distribution constant distribution ratio (D) : conditional distribution constant; depends on side reactions substitution of activity by analytical concentrations; easier determination distribution ratio (D) : conditional distribution constant; depends on side reactions substitution of activity by analytical concentrations; easier determination 𝐊 𝐃 𝐚 𝐀 𝐈𝐈 𝐚 𝐀 𝐈𝐊 𝐃 𝐚 𝐀 𝐈𝐈 𝐚 𝐀 𝐈 𝐃 𝐜 𝐀 𝐈𝐈 𝐜 𝐀 𝐈𝐃 𝐜 𝐀 𝐈𝐈 𝐜 𝐀 𝐈 𝐊 𝐃 𝐚 𝐀 𝐈𝐈 𝐚 𝐀 𝐈 𝐥𝐢𝐦 𝐀 →𝟎 𝐜 𝐀 𝐈𝐈 𝐜 𝐀 𝐈𝐊 𝐃 𝐚 𝐀 𝐈𝐈 𝐚 𝐀 𝐈 𝐥𝐢𝐦 𝐀 →𝟎 𝐜 𝐀 𝐈𝐈 𝐜 𝐀 𝐈 3333 menstruum : extractant in L‐S extraction marc : what remained left after extraction menstruum : extractant in L‐S extraction marc : what remained left after extraction conditions : pH, masking agents, etc... conditions : pH, masking agents, etc... if the compound is present in same form in both phasesif the compound is present in same form in both phases γ – activity coefficients of compound in both phasesγ – activity coefficients of compound in both phases V II/I – phase volumes ratio V II and V IV II/I – phase volumes ratio V II and V I distribution constant expressed using Hildebrand solubility parameters distribution constant expressed using Hildebrand solubility parameters 𝐥𝐧 𝐊 𝐃 𝐕 𝐀 𝐑 · 𝐓 · 𝛅𝐈 𝛅𝐈𝐈 · 𝛅𝐈 𝛅𝐈𝐈 𝟐𝛅 𝐀𝐥𝐧 𝐊 𝐃 𝐕 𝐀 𝐑 · 𝐓 · 𝛅𝐈 𝛅𝐈𝐈 · 𝛅𝐈 𝛅𝐈𝐈 𝟐𝛅 𝐀 𝐃 𝐜 𝐀 𝐈𝐈 𝐜 𝐀 𝐈 𝐧 𝐀 𝐈𝐈 𝐕 𝐈𝐈⁄ 𝐧 𝐀 𝐈 𝐕 𝐈⁄ 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐈 · 𝐕 𝐈 𝐕 𝐈𝐈 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐈 · 𝟏 𝐕 𝐈𝐈 𝐈⁄ 𝐃 𝐜 𝐀 𝐈𝐈 𝐜 𝐀 𝐈 𝐧 𝐀 𝐈𝐈 𝐕 𝐈𝐈⁄ 𝐧 𝐀 𝐈 𝐕 𝐈⁄ 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐈 · 𝐕 𝐈 𝐕 𝐈𝐈 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐈 · 𝟏 𝐕 𝐈𝐈 𝐈⁄ 𝐊 𝐃 𝐃 · 𝛄𝐈𝐈 𝛄𝐈⁄ ⇒ 𝐃 𝐊 𝐃𝐊 𝐃 𝐃 · 𝛄𝐈𝐈 𝛄𝐈⁄ ⇒ 𝐃 𝐊 𝐃 3434  n – molar amount of analyte in phase, m – weight of analyte in phase, V – phase volumen – molar amount of analyte in phase, m – weight of analyte in phase, V – phase volume distribution (mass) coefficient (DM)distribution (mass) coefficient (DM) 𝐃 𝐜 𝐀 𝐈𝐈 𝐜 𝐀 𝐈𝐃 𝐜 𝐀 𝐈𝐈 𝐜 𝐀 𝐈 𝐃 𝐌 𝐦 𝐀 𝐈𝐈 𝐦 𝐀 𝐈 𝐃 · 𝐕 𝐈𝐈 𝐕 𝐈 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐃 𝐌 𝐦 𝐀 𝐈𝐈 𝐦 𝐀 𝐈 𝐃 · 𝐕 𝐈𝐈 𝐕 𝐈 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ yield of separation (RA) yield of compound A yield of separation (RA) yield of compound A 𝐑 𝐀 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐭𝐨𝐭 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐈 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐈⁄ 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐈 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝟏 𝐑 𝐀 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐭𝐨𝐭 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐈 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐈⁄ 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐈 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝟏 𝐄 𝐀 % 𝟏𝟎𝟎 · 𝐑 𝐀𝐄 𝐀 % 𝟏𝟎𝟎 · 𝐑 𝐀 relation between DM and extraction yieldrelation between DM and extraction yield search for a such DM, at which E would be max : if DM > 1 → E > 50 % : at DM > 10 the increase in E is minimal search for a such DM, at which E would be max : if DM > 1 → E > 50 % : at DM > 10 the increase in E is minimal relation between D and extraction yieldrelation between D and extraction yield 𝐄 𝟏𝟎𝟎 · 𝐃 𝐌 𝐃 𝐌 𝟏 𝐄 𝟏𝟎𝟎 · 𝐃 𝐌 𝐃 𝐌 𝟏 𝐄 𝟏𝟎𝟎 · 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝟏 𝐄 𝟏𝟎𝟎 · 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝟏 3535 0.010.01 0.10.1 11 1010 100100 00 5050 100100 EE DMDM : when considering D & E relation, we have to consider VII/I : if we change the D value, it would change the E too : when considering D & E relation, we have to consider VII/I : if we change the D value, it would change the E too choice of extraction systemchoice of extraction system extraction system is given by properties ofextraction system is given by properties of extractant : non‐polar  :: unpolarisable :: polarisable : polar : ionic  extractant : non‐polar  :: unpolarisable :: polarisable : polar : ionic  + salt : increasing salt concentration means decreasing dielectric constant of water : binds solvent in solvation sphere : Δ ion concentration leads to extraction equilibrium shift  + salt : increasing salt concentration means decreasing dielectric constant of water : binds solvent in solvation sphere : Δ ion concentration leads to extraction equilibrium shift  polar substance : extractant must be solvent more polar than sample solvent non‐polar substance : extractant must be solvent less polar than sample solvent polar substance : extractant must be solvent more polar than sample solvent non‐polar substance : extractant must be solvent less polar than sample solvent extracted substanceextracted substance numeric value of D might be changed by equilibria numeric value of D might be changed by equilibria paria paribusparia paribus similia similibus solvuntursimilia similibus solvuntur 3636 : extracted substance : and both participating phases : extracted substance : and both participating phases pseudomolecular systempseudomolecular system presence of complexation agentpresence of complexation agent association in organic phaseassociation in organic phase simple extractionsimple extraction 2 (HA) II → (HA)2  II2 (HA) II → (HA)2  II benzoic acid; water / benzenebenzoic acid; water / benzene βn = [I3 –] / ([I2] ∙ [I–])  D = [I2] II / ([I2] I + [I3 –] I) βn = [I3 –] / ([I2] ∙ [I–])  D = [I2] II / ([I2] I + [I3 –] I)  RCOO– + H+ ↔ RCOOHRCOO– + H+ ↔ RCOOHRCOOH;  phase I / IIRCOOH;  phase I / II I2 + I– ↔ I3 –I2 + I– ↔ I3 – I2; CCl4 / water + I–I2; CCl4 / water + I– KHA = [RCOOH] / ([RCOO–] ∙ [H+]) KD = [RCOOH] II / [RCOOH] I KHA = [RCOOH] / ([RCOO–] ∙ [H+]) KD = [RCOOH] II / [RCOOH] I → D = [RCOOH] II / ([RCOOH] I + [RCOO–] I) → D = [RCOOH] II / ([RCOOH] I + [RCOO–] I)  KdimKdim 𝐃 𝐊 𝐃 𝟏𝟎𝐃 𝐊 𝐃 𝟏𝟎 𝐃 𝐊 𝐃 · 𝟏 𝟐𝐊 𝐝𝐢𝐦 · 𝐇𝐀 𝐈𝐈 𝐃 𝐊 𝐃 · 𝟏 𝟐𝐊 𝐝𝐢𝐦 · 𝐇𝐀 𝐈𝐈 𝐥𝐨𝐠 𝐃 𝐥𝐨𝐠 𝐊 𝐃 𝐥𝐨𝐠 𝛃 𝐧 · 𝐈𝐥𝐨𝐠 𝐃 𝐥𝐨𝐠 𝐊 𝐃 𝐥𝐨𝐠 𝛃 𝐧 · 𝐈 𝐃 𝐊 𝐃 𝟏 𝟏 𝐊 𝐃 · 𝐇 𝐃 𝐊 𝐃 𝟏 𝟏 𝐊 𝐃 · 𝐇 3737 system with ionic associatessystem with ionic associates system with metal chelatessystem with metal chelates KD = f ([H+], chelatogenic agent, ε, salting‐out agent)KD = f ([H+], chelatogenic agent, ε, salting‐out agent) M(H2O)m n+ + L– ↔ MLn + m H2OM(H2O)m n+ + L– ↔ MLn + m H2O KD = f ([H+], chelatogenic agent)KD = f ([H+], chelatogenic agent) H+ + L– ↔ HLH+ + L– ↔ HL HL, MLnHL, MLn Mn+;  phase I / II + HLMn+;  phase I / II + HL : liquid ion‐exchangers :: ternary amines – tri(2‐ethylhexyl)amine; methyldioctylamine : onionic systems (oxonionic ions) : liquid ion‐exchangers :: ternary amines – tri(2‐ethylhexyl)amine; methyldioctylamine : onionic systems (oxonionic ions) 3838 process of extractionprocess of extraction phase transfer goes on on inter‐phase : maximal inter‐phase area : from each place in solution as close as possible to inter‐phase phase transfer goes on on inter‐phase : maximal inter‐phase area : from each place in solution as close as possible to inter‐phase solvents which are mutually immiscible, but always mutually soluble → change of volume in contrast to the volume before shaking → use of solvents pre‐saturated with the other phase solvents which are mutually immiscible, but always mutually soluble → change of volume in contrast to the volume before shaking → use of solvents pre‐saturated with the other phase : batch extraction :: single and multi‐step : continuous extraction :: for low D, continual flow of solvent in raffinate solution  : batch extraction :: single and multi‐step : continuous extraction :: for low D, continual flow of solvent in raffinate solution  we care not to create an emulsion : it slows phase separation we care not to create an emulsion : it slows phase separation shaking the mixture of both phases in closed containershaking the mixture of both phases in closed container 3939 example 3example 3 determine the influence of other types of equilibria than distribution (dimerisation in organic phase, dissociation in aqueous phase) on extraction of benzoic acid from water to benzene. KD = 10, Kdim = 0.9 mol‐1∙l, pKa = 4.18, content of benzoic acid in benzene after extraction is 0.66 M. determine the influence of other types of equilibria than distribution (dimerisation in organic phase, dissociation in aqueous phase) on extraction of benzoic acid from water to benzene. KD = 10, Kdim = 0.9 mol‐1∙l, pKa = 4.18, content of benzoic acid in benzene after extraction is 0.66 M. a) dimerisationa) dimerisation b) dissociationb) dissociation 𝐃 𝐝𝐢𝐦 𝐊 𝐃 · 𝟏 𝟐𝐊 𝐝𝐢𝐦 · 𝐇𝐀 𝐈𝐈 𝐃 𝐝𝐢𝐦 𝐊 𝐃 · 𝟏 𝟐𝐊 𝐝𝐢𝐦 · 𝐇𝐀 𝐈𝐈 𝐃 𝐝𝐢𝐦 𝟏𝟎 · 𝟏 𝟐 · 𝟎. 𝟗 · 𝟎. 𝟔𝟔 𝟐𝟐𝐃 𝐝𝐢𝐦 𝟏𝟎 · 𝟏 𝟐 · 𝟎. 𝟗 · 𝟎. 𝟔𝟔 𝟐𝟐 𝐃 𝐝𝐢𝐬 𝐊 𝐃 𝟏 𝟏 𝐊 𝐃 · 𝐇 𝐃 𝐝𝐢𝐬 𝐊 𝐃 𝟏 𝟏 𝐊 𝐃 · 𝐇 𝐃 𝐝𝐢𝐬 𝟏𝟎 𝟏 𝟏 𝟏𝟎 · 𝟏𝟎 𝟒.𝟏𝟖 𝟏𝟎𝐃 𝐝𝐢𝐬 𝟏𝟎 𝟏 𝟏 𝟏𝟎 · 𝟏𝟎 𝟒.𝟏𝟖 𝟏𝟎 ?????? 4040 Freundlich isothermFreundlich isotherm column separation techniques, extraction on solid phase principle: adsorption from liquid phase on a solid, then desorption column separation techniques, extraction on solid phase principle: adsorption from liquid phase on a solid, then desorption sorption description – adsorption isothermsorption description – adsorption isotherm kf – Freundlich adsorption constant n = 0.4 – 1.0 (ideal) kf – Freundlich adsorption constant n = 0.4 – 1.0 (ideal) X – adsorbed quantity (g, mol) m – weight of sorbent (g) c – equilibrium concentration (mol∙l‐1) a, n – constants X – adsorbed quantity (g, mol) m – weight of sorbent (g) c – equilibrium concentration (mol∙l‐1) a, n – constants empiric formulasempiric formulas extraction liquid‐solidextraction liquid‐solid II – stationary phase (s) I – mobile phase (l) II – stationary phase (s) I – mobile phase (l) 𝐗 𝐦 𝐚 · 𝐜 𝟏 𝐧 𝐗 𝐦 𝐚 · 𝐜 𝟏 𝐧 𝐊 𝐃 𝐚 𝐀 𝐈𝐈 𝐚 𝐀 𝐈 ⇒ 𝐜 𝐀 𝐈𝐈 𝐊 𝐃 · 𝐜 𝐀 𝐈 ⇒ 𝐧 𝐀 𝐈𝐈 𝒇 𝐜 𝐀 𝐈 𝐊 𝐃 𝐚 𝐀 𝐈𝐈 𝐚 𝐀 𝐈 ⇒ 𝐜 𝐀 𝐈𝐈 𝐊 𝐃 · 𝐜 𝐀 𝐈 ⇒ 𝐧 𝐀 𝐈𝐈 𝒇 𝐜 𝐀 𝐈 𝐧 𝐀 𝐈𝐈 𝐤 𝐟 · 𝐜 𝐀 𝐈 𝐧 𝐤 𝐟 · 𝐩 𝐀 𝐈 𝐧 𝐧 𝐀 𝐈𝐈 𝐤 𝐟 · 𝐜 𝐀 𝐈 𝐧 𝐤 𝐟 · 𝐩 𝐀 𝐈 𝐧 4141 cc X/mX/m X/m ~ c1X/m ~ c1 X/m ~ c1/nX/m ~ c1/n X/m ~ c0X/m ~ c0 Langmuir isothermLangmuir isotherm Brunauer‐Emmett‐Tellerova isothermBrunauer‐Emmett‐Tellerova isotherm k2 – Langmuir adsorption constant k1 – maximal number of binding sites k2 – Langmuir adsorption constant k1 – maximal number of binding sites X – adsorbed quantity (g, mol) m – weight of sorbent (g) c – equilibrium concentration (mol∙l‐1) a – max. adsorbed quantity b – constant depending on a sorbent kind & size, sorbed substance & solvent properties X – adsorbed quantity (g, mol) m – weight of sorbent (g) c – equilibrium concentration (mol∙l‐1) a – max. adsorbed quantity b – constant depending on a sorbent kind & size, sorbed substance & solvent properties 𝐧 𝐀 𝐈𝐈 𝐤 𝟏 · 𝐤 𝟐 · 𝐜 𝐀 𝐈 𝟏 𝐤 𝟐 · 𝐜 𝐀 𝐈𝐧 𝐀 𝐈𝐈 𝐤 𝟏 · 𝐤 𝟐 · 𝐜 𝐀 𝐈 𝟏 𝐤 𝟐 · 𝐜 𝐀 𝐈 𝐗 𝐦 𝐚 · 𝐜 𝐜 𝐛 𝐗 𝐦 𝐚 · 𝐜 𝐜 𝐛 𝐧 𝐀 𝐈𝐈 𝐧 𝐦 · 𝐂 · 𝐩 𝐫 𝟏 𝐩 𝐫 · 𝟏 𝐩 𝐫 · 𝐂 𝟏 𝐧 𝐀 𝐈𝐈 𝐧 𝐦 · 𝐂 · 𝐩 𝐫 𝟏 𝐩 𝐫 · 𝟏 𝐩 𝐫 · 𝐂 𝟏 𝐩 𝐫 𝐩 𝐀 𝐈 𝐩 𝐀 𝐈𝐈𝐩 𝐫 𝐩 𝐀 𝐈 𝐩 𝐀 𝐈𝐈 𝐂 𝐞 𝐐 𝐚𝐝𝐬 𝐐 𝐜𝐨𝐧𝐝 𝐑·𝐓𝐂 𝐞 𝐐 𝐚𝐝𝐬 𝐐 𝐜𝐨𝐧𝐝 𝐑·𝐓 pA – equilibrium vapour tension pA – adsorbate vapour tension  pr – relative vapour tension C – constant nm – volume of monomolecular layer of adsorbate Qads – adsorption heat Qkond – condensation heat pA – equilibrium vapour tension pA – adsorbate vapour tension  pr – relative vapour tension C – constant nm – volume of monomolecular layer of adsorbate Qads – adsorption heat Qkond – condensation heat IIII II 4242 cc X/mX/m linear isothermlinear isotherm cc X/mX/m Langmuir isothermLangmuir isotherm BET isotherm BET isotherm  basic types of isothermsbasic types of isotherms isotherms: a – linear, b – Langmuir, c – anti‐Langmuir, d – chemisorptionisotherms: a – linear, b – Langmuir, c – anti‐Langmuir, d – chemisorption aa bb cc ddnAnA IIII nA or cA or pAnA or cA or pA II IIII 4343 Langmuir isothermLangmuir isotherm anti‐Langmuir isothermanti‐Langmuir isotherm description of single component extractiondescription of single component extraction separation yield (RA) yield of compound A separation yield (RA) yield of compound A remnant of substance A after extraction remnant of substance A after extraction 𝐑 𝐀 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐭𝐨𝐭 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐈 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐈⁄ 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐈 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝟏 𝐑 𝐀 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐭𝐨𝐭 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐈 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐈⁄ 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐈 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝟏 𝐧 𝐀 𝐈 𝐧 𝐀 𝐭𝐨𝐭 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ ⇒𝐧 𝐀 𝐈 𝐧 𝐀 𝐭𝐨𝐭 · 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ ⇒𝐜 𝐀 𝐈 𝐜 𝐀 𝐭𝐨𝐭 · 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐧 𝐀 𝐈 𝐧 𝐀 𝐭𝐨𝐭 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ ⇒𝐧 𝐀 𝐈 𝐧 𝐀 𝐭𝐨𝐭 · 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ ⇒𝐜 𝐀 𝐈 𝐜 𝐀 𝐭𝐨𝐭 · 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 4444 repeated extractionrepeated extraction : batch extraction  :: discrete, incontinuative ::: small yield : batch extraction  :: discrete, incontinuative ::: small yield solution : continuous extraction :: repeated extraction and fusing of organic phase thereafter solution : continuous extraction :: repeated extraction and fusing of organic phase thereafter potentially solvable by phase ratio change (VII/I > 10) : too large disproportion of volumes is not suitable :: problems with manipulation & mutual solubility of phases potentially solvable by phase ratio change (VII/I > 10) : too large disproportion of volumes is not suitable :: problems with manipulation & mutual solubility of phases 1st step1st step 2nd step2nd step ith stepith step 𝐧 𝐀 𝟏 𝐈 𝐧 𝐀 𝐭𝐨𝐭 · 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐧 𝐀 𝟏 𝐈 𝐧 𝐀 𝐭𝐨𝐭 · 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐧 𝐀 𝟐 𝐈 𝐧 𝐀 𝟏 𝐈 · 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐧 𝐀 𝟐 𝐈 𝐧 𝐀 𝟏 𝐈 · 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐧 𝐀 𝐢 𝐈 𝐧 𝐀 𝐭𝐨𝐭 · 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐢 𝐧 𝐀 𝐢 𝐈 𝐧 𝐀 𝐭𝐨𝐭 · 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐢 4545 extraction remnant after ith step extraction remnant after ith step 𝐧 𝐀,𝐢 𝐈 𝐧 𝐀 𝐭𝐨𝐭 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐢 ⇒𝐧 𝐀 𝐢 𝐈 𝐧 𝐀 𝐭𝐨𝐭 · 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐢 ⇒𝐜 𝐀 𝐢 𝐈 𝐜 𝐀 𝐭𝐨𝐭 · 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐢 𝐧 𝐀,𝐢 𝐈 𝐧 𝐀 𝐭𝐨𝐭 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐢 ⇒𝐧 𝐀 𝐢 𝐈 𝐧 𝐀 𝐭𝐨𝐭 · 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐢 ⇒𝐜 𝐀 𝐢 𝐈 𝐜 𝐀 𝐭𝐨𝐭 · 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐢 extraction yield after ith step extraction yield after ith step 𝐜 𝐀 𝐢 𝐈 𝐜 𝐀 𝐭𝐨𝐭 · 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐢 𝐜 𝐀 𝐢 𝐈 𝐜 𝐀 𝐭𝐨𝐭 · 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐢 𝐄 𝐀 𝐢 𝟏 𝟏𝟎𝟎 𝐄 𝟏𝟎𝟎 𝐢 · 𝟏𝟎𝟎𝐄 𝐀 𝐢 𝟏 𝟏𝟎𝟎 𝐄 𝟏𝟎𝟎 𝐢 · 𝟏𝟎𝟎 𝐑 𝐀 𝐢 𝟏 𝟏 𝐑 𝐀 𝟏 𝐢 𝐑 𝐀 𝐢 𝟏 𝟏 𝐑 𝐀 𝟏 𝐢 cIcI VII/I = 10 D = 10 VII/I = 10 D = 10 repeated  extractionrepeated  extraction N = number of repetitionsN = number of repetitions description of multiple component extractiondescription of multiple component extraction distribution separation factor (α(A,B) ) : is measure of selectivity distribution separation factor (α(A,B) ) : is measure of selectivity αOPTIM = 106αOPTIM = 106 ideal separation state A  II, B  I real separation A + B  II, B + A  I ideal separation state A  II, B  I real separation A + B  II, B + A  I : mixture contains at least compounds A and B : component A passes to phase II : component B stays in phase I : mixture contains at least compounds A and B : component A passes to phase II : component B stays in phase I 𝛂 𝐀,𝐁 𝐜 𝐀 𝐈𝐈 𝐜 𝐁 𝐈𝐈⁄ 𝐜 𝐀 𝐈 𝐜 𝐁 𝐈𝛂 𝐀,𝐁 𝐜 𝐀 𝐈𝐈 𝐜 𝐁 𝐈𝐈⁄ 𝐜 𝐀 𝐈 𝐜 𝐁 𝐈 𝛂 𝐨𝐩𝐭𝐢𝐦 𝟗𝟗. 𝟗 𝟎. 𝟏𝟎 𝐈𝐈 𝟎. 𝟏𝟎 𝟗𝟗. 𝟗 𝐈𝛂 𝐨𝐩𝐭𝐢𝐦 𝟗𝟗. 𝟗 𝟎. 𝟏𝟎 𝐈𝐈 𝟎. 𝟏𝟎 𝟗𝟗. 𝟗 𝐈 4646 AA BB II IIII BB AA check, if the extraction is plausiblecheck, if the extraction is plausible at what extent will the required compound A be separated from compound B?at what extent will the required compound A be separated from compound B? 𝛂 𝐀,𝐁 𝐜 𝐀 𝐈𝐈 𝐜 𝐁 𝐈𝐈⁄ 𝐜 𝐀 𝐈 𝐜 𝐁 𝐈 𝐜 𝐀 𝐈𝐈 𝐜 𝐀 𝐈 · 𝐜 𝐁 𝐈 𝐜 𝐁 𝐈𝐈 𝐃 𝐀 𝐃 𝐁 𝛂 𝐀,𝐁 𝐜 𝐀 𝐈𝐈 𝐜 𝐁 𝐈𝐈⁄ 𝐜 𝐀 𝐈 𝐜 𝐁 𝐈 𝐜 𝐀 𝐈𝐈 𝐜 𝐀 𝐈 · 𝐜 𝐁 𝐈 𝐜 𝐁 𝐈𝐈 𝐃 𝐀 𝐃 𝐁 𝐕 𝐈𝐈 𝐈⁄ 𝐕𝐈𝐈 𝐕𝐈 𝟏 𝐃 𝐀 · 𝐃 𝐁 𝐕 𝐈𝐈 𝐈⁄ 𝐕𝐈𝐈 𝐕𝐈 𝟏 𝐃 𝐀 · 𝐃 𝐁 1st extraction: in organic phase A (90.9%) and B (9.1%) 2nd extraction: in organic phase A (99.2%) and B (17.4%) 3rd extraction: ... 1st extraction: in organic phase A (90.9%) and B (9.1%) 2nd extraction: in organic phase A (99.2%) and B (17.4%) 3rd extraction: ... ideal caseideal case real casereal case DB < 10‐3; V I = V II, α(A,B) = 106 1. A ≈ 99.9 %, B ≈ 0.1 % DB < 10‐3; V I = V II, α(A,B) = 106 1. A ≈ 99.9 %, B ≈ 0.1 % DA =10.0, DB = 0.1, α (A,B) =100 1. A ≈ 90.9 %, B ≈ 9.1 % DA =10.0, DB = 0.1, α (A,B) =100 1. A ≈ 90.9 %, B ≈ 9.1 % EXTRACTION COMPLEATNESS vs. HIGHER CONTAMINATION!EXTRACTION COMPLEATNESS vs. HIGHER CONTAMINATION! 𝐕 𝐈𝐈 𝐈⁄ 𝟏 𝐃 𝐀 · 𝐃 𝐁 𝐕 𝐈𝐈 𝐈⁄ 𝟏 𝐃 𝐀 · 𝐃 𝐁 𝛂 𝐀,𝐁 𝐃 𝐀 𝐃 𝐁 𝛂 𝐀,𝐁 𝐃 𝐀 𝐃 𝐁 4747 capacity factor (k) : relation between separation factor and phase volumes capacity factor (k) : relation between separation factor and phase volumes separation yield, enrichment factor (S(A,B)) : state in phase II: A (99.9%), B (0.1%)  SA/B= 103 separation yield, enrichment factor (S(A,B)) : state in phase II: A (99.9%), B (0.1%)  SA/B= 103 preconcentration of extract : out of phase I we extract analyte into phase II (organic extractant) : we evaporate part of the organic solvent  V II,vap < V I preconcentration of extract : out of phase I we extract analyte into phase II (organic extractant) : we evaporate part of the organic solvent  V II,vap < V I 𝛂 𝐀,𝐁 𝐤 𝐀 𝐤 𝐁 𝛂 𝐀,𝐁 𝐤 𝐀 𝐤 𝐁 𝐒 𝐀,𝐁 𝐑 𝐀 𝐑 𝐁 𝐒 𝐀,𝐁 𝐑 𝐀 𝐑 𝐁 𝐧 𝐀 𝐈𝐈 𝐧 𝐁 𝐈𝐈 𝐒 𝐀,𝐁 · 𝐧 𝐀 𝐭𝐨𝐭 𝐧 𝐁 𝐭𝐨𝐭 𝐧 𝐀 𝐈𝐈 𝐧 𝐁 𝐈𝐈 𝐒 𝐀,𝐁 · 𝐧 𝐀 𝐭𝐨𝐭 𝐧 𝐁 𝐭𝐨𝐭 𝐤 𝐀 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐈 𝐜 𝐀 𝐈𝐈 · 𝐕 𝐈𝐈 𝐜 𝐀 𝐈 · 𝐕 𝐈 𝐊 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐤 𝐀 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐈 𝐜 𝐀 𝐈𝐈 · 𝐕 𝐈𝐈 𝐜 𝐀 𝐈 · 𝐕 𝐈 𝐊 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ continuative extractorcontinuative extractor : continual L‐L extraction : raffinate lighter than extractant : continual L‐L extraction : raffinate lighter than extractant Soxhlet extractorSoxhlet extractor condensercondenser flow of liquid solvent flow of liquid solvent flow of gaseous solvent flow of gaseous solvent extractor body extractor body aqueous sample solution aqueous sample solution solv.solv. solut.solut. heating bedheating bed boiling flaskboiling flask condensercondenser sample (s) w/ solv. (l) sample (s) w/ solv. (l) solventsolvent heating bed heating bed : continual S‐L extraction : (practically) constant volume of solvent : continual S‐L extraction : (practically) constant volume of solvent 4848  example 4example 4 ctot = orignal concentration, ntot = original molar amount (in phase I)ctot = orignal concentration, ntot = original molar amount (in phase I) oror oror 10 ml of 0.1 M of solution of compound X is separated between organic and aqueous phase, whereas distribution coefficient D = 10. calculate, how much (in mmol) of the X will be transferred into organic phase and how much it will remain in aqueous, if we extract into a) 30 ml, b) 10 ml, c) 1 ml? 10 ml of 0.1 M of solution of compound X is separated between organic and aqueous phase, whereas distribution coefficient D = 10. calculate, how much (in mmol) of the X will be transferred into organic phase and how much it will remain in aqueous, if we extract into a) 30 ml, b) 10 ml, c) 1 ml? 𝐜 𝐭𝐨𝐭 𝐧𝐭𝐨𝐭 𝐕 𝐈 𝐜 𝐭𝐨𝐭 𝐧𝐭𝐨𝐭 𝐕 𝐈 𝐃 𝐜 𝐈𝐈 𝐜 𝐈 𝐃 𝐜 𝐈𝐈 𝐜 𝐈 𝐜 𝐈𝐈 𝐃 · 𝐜 𝐈 𝐜 𝐈𝐈 𝐃 · 𝐜 𝐈 𝐧𝐭𝐨𝐭 𝐜 𝐈𝐈 · 𝐕 𝐈𝐈 𝐜 𝐈 · 𝐕 𝐈 𝐧𝐭𝐨𝐭 𝐜 𝐈𝐈 · 𝐕 𝐈𝐈 𝐜 𝐈 · 𝐕 𝐈 𝐧𝐭𝐨𝐭 𝐃 · 𝐜 𝐈 · 𝐕 𝐈𝐈 𝐜 𝐈 · 𝐕 𝐈 𝐧𝐭𝐨𝐭 𝐃 · 𝐜 𝐈 · 𝐕 𝐈𝐈 𝐜 𝐈 · 𝐕 𝐈 𝐧𝐭𝐨𝐭 𝐜 𝐈 · 𝐃 · 𝐕 𝐈𝐈 𝐕 𝐈 𝐧𝐭𝐨𝐭 𝐜 𝐈 · 𝐃 · 𝐕 𝐈𝐈 𝐕 𝐈 𝐜 𝐈 𝐧𝐭𝐨𝐭 𝐃 · 𝐕 𝐈𝐈 𝐕 𝐈 𝐜 𝐈 𝐧𝐭𝐨𝐭 𝐃 · 𝐕 𝐈𝐈 𝐕 𝐈 𝐜 𝐈𝐈 𝐧𝐭𝐨𝐭 𝐕 𝐈𝐈 𝐕 𝐈 𝐃⁄ 𝐜 𝐈𝐈 𝐧𝐭𝐨𝐭 𝐕 𝐈𝐈 𝐕 𝐈 𝐃⁄ ?????? 4949 example 5example 5 stepstep ntot =ntot = sumsum EE stepstep EE continuation of previous example (D = 10, ctot = 0.1 M): we would like to extract as much as possible of compound X of 10 ml aqueous phase (I) into 30 ml of organic one (II). is it better to use a) one‐step extraction into 30 ml or b) repeat thrice extraction into 10 ml and fuse them then? continuation of previous example (D = 10, ctot = 0.1 M): we would like to extract as much as possible of compound X of 10 ml aqueous phase (I) into 30 ml of organic one (II). is it better to use a) one‐step extraction into 30 ml or b) repeat thrice extraction into 10 ml and fuse them then? 𝐧 𝐀 𝟏 𝐈 𝐧 𝐀 𝐭𝐨𝐭 · 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐧 𝐀 𝟏 𝐈 𝐧 𝐀 𝐭𝐨𝐭 · 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐧 𝐀 𝟐 𝐈 𝐧 𝐀 𝟏 𝐈 · 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐧 𝐀 𝟐 𝐈 𝐧 𝐀 𝟏 𝐈 · 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐧 𝐀 𝐢 𝐈 𝐧 𝐀 𝐭𝐨𝐭 · 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐢 𝐧 𝐀 𝐢 𝐈 𝐧 𝐀 𝐭𝐨𝐭 · 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐢 ?????? 5050 example 6example 6 we would like to extract into organic phase 90 % of compound X although the distribution ratio equals D = 1. how to do that? we would like to extract into organic phase 90 % of compound X although the distribution ratio equals D = 1. how to do that? if VII = VI, thenif VII = VI, then : extraction into larger volume: extraction into larger volume  we need VII = VI ∙ 9 (nine times the volume of original phase) we need VII = VI ∙ 9 (nine times the volume of original phase) : repeated extraction into same volume: repeated extraction into same volume = 3.3, i.e. at least 4 steps, i.e. three repetitions= 3.3, i.e. at least 4 steps, i.e. three repetitions 𝐑 𝐀 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝟏 𝐑 𝐀 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝟏 𝐑 𝐀 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐈 𝟏 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄⁄ 𝐑 𝐀 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐈 𝟏 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄⁄ 𝐢 𝐥𝐨𝐠 𝟏 𝐑 𝐀 𝐢 𝐥𝐨𝐠 𝟏 𝐑 𝐀 𝟏 𝐢 𝐥𝐨𝐠 𝟏 𝐑 𝐀 𝐢 𝐥𝐨𝐠 𝟏 𝐑 𝐀 𝟏 𝐜 𝐀 𝐢 𝐈 𝐜 𝐀 𝐭𝐨𝐭 · 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐢 𝐜 𝐀 𝐢 𝐈 𝐜 𝐀 𝐭𝐨𝐭 · 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐢 5151 = 0.5, i.e. E = 50 %= 0.5, i.e. E = 50 % therefore we must choosetherefore we must choose III.III. preanalytical sample preparationpreanalytical sample preparation sample preparation lies mostly in separation : transferring analytes from matrix into solvent useful for analysis sample preparation lies mostly in separation : transferring analytes from matrix into solvent useful for analysis analysis it‐self needs not to represent the biggest problem of all analytics : fundamental can be the sample preparation analysis it‐self needs not to represent the biggest problem of all analytics : fundamental can be the sample preparation : sample preparation : separation : identification, quantification : sample preparation : separation : identification, quantification 5252 chemical analysischemical analysis samplesample liquid : L‐L extraction, perforation, dialysis, ultrafiltration : microextraction on solid phase, L‐S extraction liquid : L‐L extraction, perforation, dialysis, ultrafiltration : microextraction on solid phase, L‐S extraction solid : homogenisation, dissolution, evaporation / lyophilisation : S‐L extraction, Soxhlet, forced‐flow leaching : supercritical fluid extraction, microwave assisted extraction, accelerated solvent extraction, sonification assisted extraction solid : homogenisation, dissolution, evaporation / lyophilisation : S‐L extraction, Soxhlet, forced‐flow leaching : supercritical fluid extraction, microwave assisted extraction, accelerated solvent extraction, sonification assisted extraction 5353 extraction L‐L for the substances A and B : DA > DB : DB has low value (< 1) multiple step process, discrete, but consequent, not continuative extraction L‐L for the substances A and B : DA > DB : DB has low value (< 1) multiple step process, discrete, but consequent, not continuative : phases are automatically mixed : mobile phase is transferred into next compartment :: realised by centrifugal force substances with high KD are moving faster in the compartment system : phases are automatically mixed : mobile phase is transferred into next compartment :: realised by centrifugal force substances with high KD are moving faster in the compartment system (counter‐flow separation)(counter‐flow separation) counter‐current extraction (CCE)counter‐current extraction (CCE) 5454 Graig‐Post's extractor(1949) Graig‐Post's extractor(1949) Ronor's column : side view Ronor's column : side view instrumentationinstrumentation teflon ring teflon ring glass plate glass plate input for upper chamber input for upper chamber inter‐phase between light & heavy phases inter‐phase between light & heavy phases eccentrically positioned hole in plate eccentrically positioned hole in plate collecting tube w/ over‐flow collecting tube w/ over‐flow transfer unit transfer unit swing axisswing axis extraction unit extraction unit in supercritical state has the gas : density of a liquid in supercritical state has the gas : density of a liquid supercritical fluid : critical values (Tk, pk) supercritical fluid : critical values (Tk, pk) supercritical fluid extraction (SFE) supercritical fluid extraction (SFE)  5555 gas in supercritical state : is not a liquid, but a fluid :: only similar to liquid gas in supercritical state : is not a liquid, but a fluid :: only similar to liquid supercritical region supercritical region gasgas liquidliquid solidsolid pressurepressure critical pointcritical point temperaturetemperature triple pointtriple point carbon dioxidecarbon dioxide liquidliquid gasgas supercritical fluid supercritical fluid → increasing pressure & temperature →→ increasing pressure & temperature → all these properties are controllable by setting up pressure & temperature of the supercritical state : out of one substance, fluids of many different properties all these properties are controllable by setting up pressure & temperature of the supercritical state : out of one substance, fluids of many different properties properties of supercritical fluidsproperties of supercritical fluids : viscosity :: lower than liquids, similar as gases : density :: depends on pressure : diffusivity :: higher than liquids : solvation abilities :: as liquids : viscosity :: lower than liquids, similar as gases : density :: depends on pressure : diffusivity :: higher than liquids : solvation abilities :: as liquids physical properties of supercritical fluidsphysical properties of supercritical fluids gas supercritical fluid liquid density x103 [g∙ml‐1] 0.2 – 2 470 1600 – 600 diffusion coeff. x 104 [cm2∙s‐1] 1 – 4 2 – 7 0.02 – 0.2 viscosity x 104 [Pa∙s] 1 – 3 3 – 10 20 – 300 5656 cr. temperature [°C] cr. pressure [MPa] el. force δ  CO2 31.3 7.38 10.7 SF6 45.5 3.77 n‐C5H12 196.6 3.39 7.2 CCl2F2 111.7 4.02 CClF3 28.8 3.97 7.8 N2O 36.5 7.34 10.6 CHF3 25.9 4.75 CHClF2 96.0 5.01 NH3 132.3 11.35 13.2 Xe 16.6 5.91 critical values for some substances in SFEcritical values for some substances in SFE CO2 is very non‐polar eluent, polarity (elution force) is thus possible to increase adding organic solvent : typically 5 – 10 % of methanol CO2 is very non‐polar eluent, polarity (elution force) is thus possible to increase adding organic solvent : typically 5 – 10 % of methanol * elution (extraction) force δ is increasing w/ density* elution (extraction) force δ is increasing w/ density 1 – extraction column 2 – phase separator 3 – thermostat 4 – additive pump 5 – liquid CO2 pump 6 – sample 7 – analyte collector 8 – CO2 container 1 – extraction column 2 – phase separator 3 – thermostat 4 – additive pump 5 – liquid CO2 pump 6 – sample 7 – analyte collector 8 – CO2 container scheme of SFEscheme of SFE 5757 88 55 33 11 77 66 44 22 microvalvemicrovalve SFE advantages to classic extraction L‐LSFE advantages to classic extraction L‐L general SFE disadvantagesgeneral SFE disadvantages matrix effects (negative matrix influence) : interactions with sample and extraction solvent matrix effects (negative matrix influence) : interactions with sample and extraction solvent complex instrumentation : high temperatures & pressures : work with gases :: restrictor ::: new technological solutions complex instrumentation : high temperatures & pressures : work with gases :: restrictor ::: new technological solutions : 10 – 100x faster mass transfer : direct extraction force changes by changing the density :: by pressure or temperature : significant reduction of the extractant volume : some extractants are gases in normal conditions :: easy vaporisation = pre‐concentration : 10 – 100x faster mass transfer : direct extraction force changes by changing the density :: by pressure or temperature : significant reduction of the extractant volume : some extractants are gases in normal conditions :: easy vaporisation = pre‐concentration some disadvantages of extraction methods : high time and solvent consumption (Soxhlet) : low efficiency (sonification, microwaves) : matrix effects (SFE) some disadvantages of extraction methods : high time and solvent consumption (Soxhlet) : low efficiency (sonification, microwaves) : matrix effects (SFE) accelerated solvent extraction (ASE) accelerated solvent extraction (ASE)  5858 (PSE, pressurised solvent extraction)(PSE, pressurised solvent extraction) extractant is liquid, sample solidextractant is liquid, sample solid high temperature influencehigh temperature influence high pressure influencehigh pressure influence : higher solubility : faster desorption kinetics (+10 °C  2x ) : decrease in solvent viscosity : effective solvent diffusion into matrix :: elimination of matrix effects : higher solubility : faster desorption kinetics (+10 °C  2x ) : decrease in solvent viscosity : effective solvent diffusion into matrix :: elimination of matrix effects : solvents are under these conditions liquid :: changed boiling point : fast filling of the extractions cells : solvents are under these conditions liquid :: changed boiling point : fast filling of the extractions cells liquids in sub‐critical state, but under high pressure and temperatures give many advantages of supercritical state eliminating matrix effects of SFE liquids in sub‐critical state, but under high pressure and temperatures give many advantages of supercritical state eliminating matrix effects of SFE sample – solid or semi‐solid : water content less than 10 % :: if it is more than 10%, add PEG sample – solid or semi‐solid : water content less than 10 % :: if it is more than 10%, add PEG process of ASEprocess of ASE rough comparison : 1 h on Soxhlet is equal to 1 min ASE rough comparison : 1 h on Soxhlet is equal to 1 min ASE 5959 : extraction at high pressure & temperature (200 C and 20 MPa) : extraction cell: 10 – 33 ml : solvent consumption: 12 – 45 ml; ca 110 % of sample volume : total time of extraction (with cell filling): max 15 min (fast) : connectible to HPLC : extraction at high pressure & temperature (200 C and 20 MPa) : extraction cell: 10 – 33 ml : solvent consumption: 12 – 45 ml; ca 110 % of sample volume : total time of extraction (with cell filling): max 15 min (fast) : connectible to HPLC scheme of ASEscheme of ASE extraction cell extraction cell collection vial collection vial ovenoven pumppump solventsolvent extraction using microwave pulses in magnetron  : local overheating :: similar to ASE extraction using microwave pulses in magnetron  : local overheating :: similar to ASE speed of heating depends on : conductivity : heat capacity : dielectric constant speed of heating depends on : conductivity : heat capacity : dielectric constant mostly rotational energy transfer : no metallic parts – melting mostly rotational energy transfer : no metallic parts – melting oriented dipoles of solvent polar molecules oriented dipoles of solvent polar molecules thermally induced disorientation thermally induced disorientation microwave assisted solvent extraction (MASE)microwave assisted solvent extraction (MASE) 6060 extractant is liquid, sample solidextractant is liquid, sample solid K – conversion factor [cal]>[J] Cp – heat capacity of solvent m – weight of matrix Pabs – absorbed energy t – time of microwave field appl. Ti – initial temperature x – thermal losses K – conversion factor [cal]>[J] Cp – heat capacity of solvent m – weight of matrix Pabs – absorbed energy t – time of microwave field appl. Ti – initial temperature x – thermal losses ε' – dielectric constant : describes polarisability of molecules in elmag field ε" – dielectric loss factor : describes efficiency conversion of absorbed microwave radiation into heat δ – dissipation factor ε' – dielectric constant : describes polarisability of molecules in elmag field ε" – dielectric loss factor : describes efficiency conversion of absorbed microwave radiation into heat δ – dissipation factor extraction temperature Tfextraction temperature Tf energy dissipation in systemenergy dissipation in system 𝐓𝐟 𝐓𝐢 𝐏𝐚𝐛𝐬 · 𝐭 𝐊 · 𝐂 𝐩 · 𝐦 𝐱𝐓𝐟 𝐓𝐢 𝐏𝐚𝐛𝐬 · 𝐭 𝐊 · 𝐂 𝐩 · 𝐦 𝐱 𝛅 𝛆 𝛆 𝛅 𝛆 𝛆 6161 physical properties of MASE solvents physical properties of MASE solvents solvent boiling point [°C] viscosity [mPa∙s, 25 °C] heating speed [K∙s‐1] acetone 56 0.30 2.20 ethylacetate 77 0.43 1.78 ethanol 78 0.69 1.20 methanol 65 0.54 2.11 water 100 0.89 1.01 hexane 69 0.30 0.05 solvent diel. constant ε' [F.m‐1] diel. loss factor ε'' [F.m‐1] dissipation factor δ x104 acetone 80.00 12.0 1500 ethylacetate 20.70 11.5 5555 ethanol 23.90 15.2 6400 methanol 7.00 1.6 2286 water 1.88 1.9 x10‐4 1 x10‐1 hexane 6.02 3.2 5316 physical properties of MASE solventsphysical properties of MASE solvents 6262 heating (10 min) / cooling (30 min) cycle : frequency 2450 MHz : slow, but effective heating (10 min) / cooling (30 min) cycle : frequency 2450 MHz : slow, but effective sample container sample container revolving holder revolving holder solvent absorbing microwave energy : closed container : heating above the boiling temperature :: accelerated analyte extraction :: high temperature and pressure ::: < 200 °C; ~1.2 MPa solvent absorbing microwave energy : closed container : heating above the boiling temperature :: accelerated analyte extraction :: high temperature and pressure ::: < 200 °C; ~1.2 MPa MASE procedureMASE procedure 6363 solvent not absorbing microwave energy : closed or open container : cold solvent, analyte is heated :: useful for thermolabile compounds : possibility to use liquid CO2 (does not absorb) :: substitution for SFE solvent not absorbing microwave energy : closed or open container : cold solvent, analyte is heated :: useful for thermolabile compounds : possibility to use liquid CO2 (does not absorb) :: substitution for SFE coolercooler microwave heating microwave heating samplesample primary solvent primary solvent collection solvent collection solvent extraction L‐L → extraction L‐Sextraction L‐L → extraction L‐S extractant is solid, sample liquidextractant is solid, sample liquid : extraction column should not release plastic softeners and/or adsorb analyte : frits hold macroscopic impurities (dust, fibres) : extraction column should not release plastic softeners and/or adsorb analyte : frits hold macroscopic impurities (dust, fibres) serological pure polypropylene body serological pure polypropylene body polyethylene frits 20 μm pores polyethylene frits 20 μm pores stationary phase 40 μm particles stationary phase 40 μm particles solid phase extraction (SPE)solid phase extraction (SPE) 6464 stationary phase (SP) : same types as for HPLC (non‐polar, polar etc.) : graining less homogeneous, particles bigger than LC     stationary phase (SP) : same types as for HPLC (non‐polar, polar etc.) : graining less homogeneous, particles bigger than LC     before sample introduction – important SP – sedimented, wet (soaked), without contamination and activated : rinse of column by eluent : then by sample solvent :: min 2 column volumes before sample introduction – important SP – sedimented, wet (soaked), without contamination and activated : rinse of column by eluent : then by sample solvent :: min 2 column volumes : sample is eventually diluted :: saturation (over‐saturation) of column – sorbent capacity : pH is adjusted eventually : sample flows continually, slow at 1~ 5 ml∙min‐1 : sample is eventually diluted :: saturation (over‐saturation) of column – sorbent capacity : pH is adjusted eventually : sample flows continually, slow at 1~ 5 ml∙min‐1 low elution force solvents to rinse the matrix (weak interactions)  : max 1 – 2 column volumes low elution force solvents to rinse the matrix (weak interactions)  : max 1 – 2 column volumes basics of SPEbasics of SPE conditioning, equilibrationconditioning, equilibration sample introductionsample introduction column rinsingcolumn rinsing 6565 not necessary, only if needed : separation of volatile matrix components : inert gas (e.g. N2) or vacuum :: dry sorbent is an aim : max 1 – 10 minutes (washing out analyte) not necessary, only if needed : separation of volatile matrix components : inert gas (e.g. N2) or vacuum :: dry sorbent is an aim : max 1 – 10 minutes (washing out analyte) strong eluent at moderate flow : elutes strongly bond substances : total volume max 1 – 5 column volumes : to increase extraction efficiency – repeated elution with smaller volumes :: effect same to repeated extraction strong eluent at moderate flow : elutes strongly bond substances : total volume max 1 – 5 column volumes : to increase extraction efficiency – repeated elution with smaller volumes :: effect same to repeated extraction column dryingcolumn drying sample elutionsample elution 6666 disc SPEdisc SPE column SPEcolumn SPE if F = const. and S is small → p is high : increase of S → decrease of p if F = const. and S is small → p is high : increase of S → decrease of p high column & low area → high pressure & low speed high column & low area → high pressure & low speed  low column & high area → low pressure & high speed low column & high area → low pressure & high speed  A) overpressure B) underpressure C) centrifugal force D) en bloc A) overpressure B) underpressure C) centrifugal force D) en bloc filterfilter glass fore‐filter glass fore‐filter SPE discSPE disc SPE arrangementSPE arrangement 𝐩 𝐅 𝐒⁄𝐩 𝐅 𝐒⁄ 6767 : lower consumption of org. solvents → lower price (environment protection) : higher selectivity, yield and efficiency; separation of analyte from matrix : possible automation connected to flow‐through system with valves : lower consumption of org. solvents → lower price (environment protection) : higher selectivity, yield and efficiency; separation of analyte from matrix : possible automation connected to flow‐through system with valves basically, SPE is less efficient column chromatography basically, SPE is less efficient column chromatography advantagesadvantages SPE vs extraction L‐LSPE vs extraction L‐L : no complex manipulation by repeated extraction : till 50 theoretical plates, i.e. fifty extraction steps : complete separation of analyte and matrix : no complex manipulation by repeated extraction : till 50 theoretical plates, i.e. fifty extraction steps : complete separation of analyte and matrix 6868 conditions of SPE method choiceconditions of SPE method choice method packing sample polarity matrix conditioning elution reversed phase C18, C8, C2, cyclohexyl non‐polar polar (aqueous) MeOH > water > sample solvent non‐polar normal phase silica, alumina, graphite, CN, NH2 polar non‐polar sample solvent polar anex SAX, WAX positively charged polar non‐polar pH = pKa – 1‐2 pH = pKa – 1‐2 proper counter‐ion catex SCX, WCX negatively charged polar non‐polar pH = pKa + 1‐2 pH = pKa + 1‐2 proper counter‐ion (MSPD, matrix solid phase dispersion)(MSPD, matrix solid phase dispersion) disperse solid phase extraction (DSPE)disperse solid phase extraction (DSPE) : rubbing sample with suitable sorbent (C18) :: sample‐sorbent ratio 1 : 4 :: sample structure disruption by mechanical and hydrophobic forces :: large inter‐phase :: matrix = new sorption phase (more complex equilibria)  : resulting mixture into column (as within SPE) :: eventual addition of other sorbent; e.g. Florisil ::: non‐polar components on C18, polar on ‐OH of silica : analytes elution by suitable solvent : rubbing sample with suitable sorbent (C18) :: sample‐sorbent ratio 1 : 4 :: sample structure disruption by mechanical and hydrophobic forces :: large inter‐phase :: matrix = new sorption phase (more complex equilibria)  : resulting mixture into column (as within SPE) :: eventual addition of other sorbent; e.g. Florisil ::: non‐polar components on C18, polar on ‐OH of silica : analytes elution by suitable solvent QuEChERS (quick, easy, cheap, effective, rugged and safe; catchers) : L‐L extraction using organic solvent and solution of salts : DSPE of resulting mixture QuEChERS (quick, easy, cheap, effective, rugged and safe; catchers) : L‐L extraction using organic solvent and solution of salts : DSPE of resulting mixture 6969 advantages : isolation and purification in one step – especially in food analysis :: saving instrumentation, time and solvents advantages : isolation and purification in one step – especially in food analysis :: saving instrumentation, time and solvents : combination of sample collection and concentration : easy, fast, sensitive : collection in defined time :: quantitative adsorption : fibre with adsorbed analyte :: desorbed in a proper volume of a solvent   :: into GC injector ::: compound thermally desorbed there : combination of sample collection and concentration : easy, fast, sensitive : collection in defined time :: quantitative adsorption : fibre with adsorbed analyte :: desorbed in a proper volume of a solvent   :: into GC injector ::: compound thermally desorbed there isolation of organic compounds from gaseous and liquid samplesisolation of organic compounds from gaseous and liquid samples C. L. Arthur. J. Pawliszyn. Anal. Chem., 62 (1990) 21C. L. Arthur. J. Pawliszyn. Anal. Chem., 62 (1990) 21 SPE „inside out“ : adsorption and extraction goes on on/off surface of fibre, not inside of SPE column :: concentration of analyte on a fibre sunken in matrix ::: polymer or silica covered with adsorbent SPE „inside out“ : adsorption and extraction goes on on/off surface of fibre, not inside of SPE column :: concentration of analyte on a fibre sunken in matrix ::: polymer or silica covered with adsorbent solid phase micro‐extraction (SPME)solid phase micro‐extraction (SPME) 7070 SPMESPME thermal desorption thermal desorption septumseptum sorption rate : 2 (G) – 30 (L) min sorption rate : 2 (G) – 30 (L) min sensitivity : high in connection with GC‐MS :: IT detector  sensitivity : high in connection with GC‐MS :: IT detector  polydimethylsiloxane (PDMS) polydimethylsiloxane/divinylbenzene (PDMS/DVB) polyacrylate (PA) carbowax/divinylbenzene (CW/DVB) polydimethylsiloxane / carboxene (PDMS/CAR) polydimethylsiloxane / divinylbenzene /  carboxene1006 (PDMS/DVB/CAR) carbowax (CW/TPR) polydimethylsiloxane (PDMS) polydimethylsiloxane/divinylbenzene (PDMS/DVB) polyacrylate (PA) carbowax/divinylbenzene (CW/DVB) polydimethylsiloxane / carboxene (PDMS/CAR) polydimethylsiloxane / divinylbenzene /  carboxene1006 (PDMS/DVB/CAR) carbowax (CW/TPR) coloured screw coloured screw septumseptum ferruleferrule coaxial needle coaxial needle needle w/ fibre needle w/ fibre SPME fibre covered by SP SPME fibre covered by SP SPME instrumentationSPME instrumentation materials of SPME fibrematerials of SPME fibre 7171 s o r p t i o n d e s c r i p t i o n s o r p t i o n d e s c r i p t i o n sillica fibresillica fibre liquid polymer (c1, κ) liquid polymer (c1, κ) aqueous solution (c2) aqueous solution (c2) vialvial 7272 headspace – unfilled space in almost full bottle, can or other container after its sealing (Oxford English dictionary) headspace – unfilled space in almost full bottle, can or other container after its sealing (Oxford English dictionary) : extraction method for GC : extraction of volatile substances from non‐volatile matrices : extraction method for GC : extraction of volatile substances from non‐volatile matrices G = gas phase (headspace) L = liquid phase of sample G = gas phase (headspace) L = liquid phase of sample in G sample collection – gas drawing analyte in G is a gas in dynamic equilibrium above its own solution in G sample collection – gas drawing analyte in G is a gas in dynamic equilibrium above its own solution volatile substances volatile substances sample, solvent and matrix modifiers sample, solvent and matrix modifiers GG LL headspace extraction (HSE) headspace extraction (HSE)  7373 : equilibrium of sample in vial : as much as possible of analyte should go to G : analyte is taken from g and analysed in GC : equilibrium of sample in vial : as much as possible of analyte should go to G : analyte is taken from g and analysed in GC distribution coefficientdistribution coefficient phase ratiophase ratio VG/VL equal for standard & sample : else has the calibration no sense VG/VL equal for standard & sample : else has the calibration no sense 𝐊 𝐂 𝐋 𝐂 𝐆 𝐊 𝐂 𝐋 𝐂 𝐆 𝛃 𝐕 𝐆 𝐕𝐋 𝛃 𝐕 𝐆 𝐕𝐋 𝐂 𝟎 · 𝐕𝐋 𝐂 𝐋 · 𝐕𝐋 𝐂 𝐆 · 𝐕 𝐆𝐂 𝟎 · 𝐕𝐋 𝐂 𝐋 · 𝐕𝐋 𝐂 𝐆 · 𝐕 𝐆 𝐂 𝐆 𝐂 𝟎 𝐊 𝐕 𝐆 𝐕𝐋⁄ 𝐂 𝐆 𝐂 𝟎 𝐊 𝐕 𝐆 𝐕𝐋⁄𝐊 𝐂 𝐋 𝐂 𝐆 𝐊 𝐂 𝐋 𝐂 𝐆 7474K a βK a β CGCG ideal dependence profile ideal dependence profile K for solvents used in systems G‐LK for solvents used in systems G‐L solvent K 40 °C K 50 °C cyclohexane 0.077 n‐hexane 0.14 0.015 tetrachlorethylene 1.48 1,1,1‐trichlormethane 1.65 o‐xylene 2.44 toluene 2.82 benzene 2.90 2.5 dichlormethane 5.65 n‐butylacetate 31.4 ethylacetate 62.4 methylethylketone 139.5 11 n‐butanol 647 isopropanol 825 ethanol 1355 1150 dioxane 1618 increasing volatilityincreasing volatility also to suppress possible interactions in GC with inner coating of capillary : alcohols, acids, amines also to suppress possible interactions in GC with inner coating of capillary : alcohols, acids, amines derivatisations : esterification, acylation, silanisation, alkylation derivatisations : esterification, acylation, silanisation, alkylation can be conducted in situ in HSE vial : but contamination and pressure changes can be conducted in situ in HSE vial : but contamination and pressure changes suppression of matrix effectssuppression of matrix effects 7575 stabilisation by means of salts : NH3Cl, (NH3)SO4, NaCl, Na2SO4, K2CO3 stabilisation by means of salts : NH3Cl, (NH3)SO4, NaCl, Na2SO4, K2CO3 modification of HSEmodification of HSE MHE (multiple headspace extraction) : dynamic gas extraction MHE (multiple headspace extraction) : dynamic gas extraction FET (full evaporation technique) : gas phase >> phase of analyte FET (full evaporation technique) : gas phase >> phase of analyte TVT (total vaporisation technique) : vaporisation at elevated temperature TVT (total vaporisation technique) : vaporisation at elevated temperature VPC (vapour phase calibration) : external gaseous standard VPC (vapour phase calibration) : external gaseous standard EPICS (equilibration partition in close system) : two connected containers, equal concentrations, different volumes EPICS (equilibration partition in close system) : two connected containers, equal concentrations, different volumes VPT (variable phase ratio or variable volume technique) : series of same concentrations in vials w/ different G/L ratios VPT (variable phase ratio or variable volume technique) : series of same concentrations in vials w/ different G/L ratios different analytes (separated substances) have different affinity to stationary phase → → different analytes have different distribution between MP and SP → → different analytes are differently retained (time spent on SP) differently retarded (total time spent in system) different analytes (separated substances) have different affinity to stationary phase → → different analytes have different distribution between MP and SP → → different analytes are differently retained (time spent on SP) differently retarded (total time spent in system) chromatographic separation goes on in chromatographic bed (column or slab) : with stationary (fixed) phase (SP) = sorbent  : and mobile (movable) phase (MP) = eluent chromatographic separation goes on in chromatographic bed (column or slab) : with stationary (fixed) phase (SP) = sorbent  : and mobile (movable) phase (MP) = eluent theoretical fundaments of chromatographytheoretical fundaments of chromatography chromatography = dynamic repeated extractionchromatography = dynamic repeated extraction chromatographychromatography IV.IV. 7676 differential migrationdifferential migration basic principlebasic principle stepstep analyteanalyte stepstepequilibriumequilibrium equilibriumequilibrium MP flowMP flow MPMP SPSP eluenteluentsamplesample sorbentsorbent analyteanalyte eluateeluate equilibrium on columnequilibrium on column aA + aM ↔ aA + aMaA + aM ↔ aA + aM MM SSMM SS KA/D decreases to ~1/2 with temperature increase of temperature by 20 C : used in GC/LC KA/D decreases to ~1/2 with temperature increase of temperature by 20 C : used in GC/LC in GC pA – partial pressure of compound A in GC pA – partial pressure of compound A aA – concentration of analyte on SP surface  aM – concentration of MP in eluate aA – concentration of analyte in MP aM – concentration of MP on SP surface aA – concentration of analyte on SP surface  aM – concentration of MP in eluate aA – concentration of analyte in MP aM – concentration of MP on SP surface SS MM SS MM 𝐊 𝐀 𝐚 𝐀 𝐒 · 𝐚 𝐌 𝐒 𝐚 𝐀 𝐌 · 𝐚 𝐌 𝐌𝐊 𝐀 𝐚 𝐀 𝐒 · 𝐚 𝐌 𝐒 𝐚 𝐀 𝐌 · 𝐚 𝐌 𝐌 𝐚 𝐀 𝐒 𝐊 𝐃 𝐑 · 𝐓 · 𝐩 𝐀 · 𝛄 𝐀𝐚 𝐀 𝐒 𝐊 𝐃 𝐑 · 𝐓 · 𝐩 𝐀 · 𝛄 𝐀𝐃 𝐧 𝐀 𝐒 𝐦 𝐀 𝐒 𝐧 𝐀 𝐌 𝐕 𝐀 𝐌𝐃 𝐧 𝐀 𝐒 𝐦 𝐀 𝐒 𝐧 𝐀 𝐌 𝐕 𝐀 𝐌 𝐊 𝐀 𝐞 ∆𝛍 𝐀 𝟎 𝐑·𝐓𝐊 𝐀 𝐞 ∆𝛍 𝐀 𝟎 𝐑·𝐓 7777 AA S1S1 S2S2 stationary phasestationary phase physico‐chemical description of chromatographic processesphysico‐chemical description of chromatographic processes ideal linear chromatographyideal linear chromatography : is based on an idea of counter‐current extraction : column consists of ordered sections of the same volume ( ~ theoretical plate) : MP flow is discontinual, stepwise, adding MP volume over whole one section at a time : equilibrium on inter‐phase is much faster than MP flow rate : lateral diffusion is negligible : adsorption is controlled by linear isotherm  : is based on an idea of counter‐current extraction : column consists of ordered sections of the same volume ( ~ theoretical plate) : MP flow is discontinual, stepwise, adding MP volume over whole one section at a time : equilibrium on inter‐phase is much faster than MP flow rate : lateral diffusion is negligible : adsorption is controlled by linear isotherm  de facto : only point 5 can be guaranteed : points 3 and 4 are contradictive :: movement in all directions is of the equal rate de facto : only point 5 can be guaranteed : points 3 and 4 are contradictive :: movement in all directions is of the equal rate 7878 II I teoretical plate teoretical plate MP flowMP flow equilibriumequilibrium longitudinal diffusionlongitudinal diffusion fA – an aliquot of compound A in given phasefA – an aliquot of compound A in given phase when V II/I = 1when V II/I = 1 𝐤 𝐀 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐤 𝐀 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐟 𝐀 𝐈 𝐜 𝐀 𝐈 𝐜 𝐀 𝐭𝐨𝐭 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐟 𝐀 𝐈 𝐜 𝐀 𝐈 𝐜 𝐀 𝐭𝐨𝐭 𝟏 𝟏 𝐃 · 𝐕 𝐈𝐈 𝐈⁄ 𝐟 𝐀 𝐈 𝟏 𝟏 𝐤 𝐀 𝐟 𝐀 𝐈 𝟏 𝟏 𝐤 𝐀 𝐤 𝐀 𝐃𝐤 𝐀 𝐃𝐟 𝐀 𝐈𝐈 𝐤 𝐀 𝟏 𝐤 𝐀 𝐟 𝐀 𝐈𝐈 𝐤 𝐀 𝟏 𝐤 𝐀 𝐤 𝐀 𝐟 𝐀 𝐈𝐈 𝐟 𝐀 𝐈𝐤 𝐀 𝐟 𝐀 𝐈𝐈 𝐟 𝐀 𝐈 𝐤 𝐀 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐈 𝐜 𝐀 𝐈𝐈 𝐜 𝐀 𝐈 · 𝐕 𝐈𝐈 𝐈⁄ 𝐤 𝐀 𝐧 𝐀 𝐈𝐈 𝐧 𝐀 𝐈 𝐜 𝐀 𝐈𝐈 𝐜 𝐀 𝐈 · 𝐕 𝐈𝐈 𝐈⁄ 𝐟 𝐀 𝐈𝐈 𝟏 𝐟 𝐀 𝐈 𝐟 𝐀 𝐈𝐈 𝟏 𝐟 𝐀 𝐈 7979 MP SP II I 11 22 33 44 = n= n = r= r11 22 33  AA II IIII AA functional model based on counter‐current extraction (stochastic model)functional model based on counter‐current extraction (stochastic model) equilibriumequilibrium transport (r)transport (r) 11 = (fI + fII)0= (fI + fII)0 = (fI + fII)1= (fI + fII)1 = (fI + fII)2= (fI + fII)2 = (fI + fII)3= (fI + fII)3 fII fI 0 1 fII fI (fI)2∙fII 2fI∙(fII)2 (fII)3 (fI)3 2(fI)2∙fII fI∙(fII)2 (fI)2∙fII 2fI∙(fII)2 (fII)3 (fI)3 2(fI)2∙fII fI∙(fII)2 (fI)3∙fII 3(fI)2∙(fII)2 3fI∙(fII)3 (fII)4 (fI)4 3(fI)3∙fII 3(fI)2∙(fII)2 f I∙(f II)3 fI∙fII (fII)2 (fI)2 fI∙fII fI∙fII (fII)2 (fI)2 fI∙fII sumsum (fI)1(fI)1 (fII)1(fII)1 (fI)2(fI)2 (fII)2(fII)22(fI)1∙(fII)12(fI)1∙(fII)1 (fI)3(fI)3 (fII)3(fII)33(fI)2∙(fII)13(fI)2∙(fII)1 3(fI)1∙(fII)23(fI)1∙(fII)2 fI = 1/3fI = 1/3 fII = 2/3fII = 2/3 𝐟 𝟏,𝟐 𝐈 𝐟 𝟏,𝟐 𝐈 · 𝐟 𝐈𝐟 𝟏,𝟐 𝐈 𝐟 𝟏,𝟐 𝐈 · 𝐟 𝐈 𝐟 𝟐,𝟐 𝐈 𝐟 𝟏,𝟏 𝐈𝐈 · 𝐟 𝐈 𝐟 𝟐,𝟐 𝐈 𝐟 𝟏,𝟏 𝐈𝐈 · 𝐟 𝐈 𝐟 𝟐,𝟐 𝐈 𝐟 𝟏,𝟏 𝐈𝐈 · 𝐟 𝐈𝐈 𝐟 𝟐,𝟐 𝐈 𝐟 𝟏,𝟏 𝐈𝐈 · 𝐟 𝐈𝐈 𝐟 𝟏,𝟐 𝐈𝐈 𝐟 𝟏,𝟐 𝐈 · 𝐟 𝐈𝐈𝐟 𝟏,𝟐 𝐈𝐈 𝐟 𝟏,𝟐 𝐈 · 𝐟 𝐈𝐈 after r‐steps (fI + fII)r = 1after r‐steps (fI + fII)r = 1 8080  for n > 20 (r∙fA∙fA > 3) : gaussian dependence for n > 20 (r∙fA∙fA > 3) : gaussian dependence IIII II position (nmax) of compartment with maximal content of A after r transportsposition (nmax) of compartment with maximal content of A after r transports number of compartments with compound A : i.e. peak width number of compartments with compound A : i.e. peak width 𝐧 𝐦𝐚𝐱 𝐫 · 𝐟 𝐀 𝐈𝐈 𝐫 · 𝐤 𝐀 𝟏 𝐤 𝐀 𝐧 𝐦𝐚𝐱 𝐫 · 𝐟 𝐀 𝐈𝐈 𝐫 · 𝐤 𝐀 𝟏 𝐤 𝐀 𝐫 · 𝐟 𝐀 𝐈𝐈 · 𝐟 𝐀 𝐈 𝛔 𝐫 · 𝐤 𝐀 𝟏 𝐤 𝐀 𝟐 𝐫 · 𝐟 𝐀 𝐈𝐈 · 𝐟 𝐀 𝐈 𝛔 𝐫 · 𝐤 𝐀 𝟏 𝐤 𝐀 𝟐 𝐟 𝐀 𝐟𝐢𝐧 𝐈𝐈 𝐧! 𝐫! · 𝐧 𝐫 ! · 𝐟 𝐀 𝐈𝐈 𝐫 · 𝐟 𝐀 𝐈 𝐧 𝐫 𝐟 𝐀 𝐟𝐢𝐧 𝐈𝐈 𝐧! 𝐫! · 𝐧 𝐫 ! · 𝐟 𝐀 𝐈𝐈 𝐫 · 𝐟 𝐀 𝐈 𝐧 𝐫 𝐟 𝐀 𝐟𝐢𝐧 𝐈𝐈 𝐧! 𝐫! · 𝐧 𝐫 ! · 𝐃 𝐫 𝐃 𝟏 𝐧 𝐟 𝐀 𝐟𝐢𝐧 𝐈𝐈 𝐧! 𝐫! · 𝐧 𝐫 ! · 𝐃 𝐫 𝐃 𝟏 𝐧 𝐟 𝐀 𝐟𝐢𝐧 𝐈𝐈 𝟏 𝟐𝛑 · 𝐫 · 𝐟 𝐀 𝐈𝐈 · 𝐟 𝐀 𝐈 · 𝐞 𝐧 𝐫·𝐟 𝐀 𝐈𝐈 𝟐𝐫·𝐟 𝐀 𝐈𝐈·𝐟 𝐀 𝐈 𝐟 𝐀 𝐟𝐢𝐧 𝐈𝐈 𝟏 𝟐𝛑 · 𝐫 · 𝐟 𝐀 𝐈𝐈 · 𝐟 𝐀 𝐈 · 𝐞 𝐧 𝐫·𝐟 𝐀 𝐈𝐈 𝟐𝐫·𝐟 𝐀 𝐈𝐈·𝐟 𝐀 𝐈 𝐟 𝐀 𝐈𝐈 𝐟 𝐀 𝐈 𝐫 𝟏𝐟 𝐀 𝐈𝐈 𝐟 𝐀 𝐈 𝐫 𝟏 we putwe put 𝐟 𝐀 𝐈 𝟏 𝟏 𝐤 𝐀 𝐟 𝐀 𝐈 𝟏 𝟏 𝐤 𝐀 𝐟 𝐀 𝐈𝐈 𝐤 𝐀 𝟏 𝐤 𝐀 𝐟 𝐀 𝐈𝐈 𝐤 𝐀 𝟏 𝐤 𝐀 8181 intointo solved using binomial theoremsolved using binomial theorem after r transports there will particular content of A in nth compartment ( )after r transports there will particular content of A in nth compartment ( )𝐟 𝐀 𝐟𝐢𝐧 𝐈𝐈 𝐟 𝐀 𝐟𝐢𝐧 𝐈𝐈 non‐ideal linear chromatographynon‐ideal linear chromatography : out of the original assumptions of ideal linear chromatography, only point 5 is valid : out of the linear part of isotherm → zone shape distortion : description of chromatographic separation (efficiency of separation process) : chromatographic plate theory :: Martin‐Synge model (1941) : statistic (rate) theory :: van Deemter‐Zuiderweg model (1956) : stochastic / kinetic (rate) theory :: Giddings‐Eyring‐McQuarrie model (1963, 1999 – Cavazzini et al.) : out of the original assumptions of ideal linear chromatography, only point 5 is valid : out of the linear part of isotherm → zone shape distortion : description of chromatographic separation (efficiency of separation process) : chromatographic plate theory :: Martin‐Synge model (1941) : statistic (rate) theory :: van Deemter‐Zuiderweg model (1956) : stochastic / kinetic (rate) theory :: Giddings‐Eyring‐McQuarrie model (1963, 1999 – Cavazzini et al.) therefore, separation is possible only because the distance between maxima increases faster with increasing number of separations rather than the peak width therefore, separation is possible only because the distance between maxima increases faster with increasing number of separations rather than the peak width 𝐧 𝐦𝐚𝐱 𝒇 𝐫𝐧 𝐦𝐚𝐱 𝒇 𝐫 𝛔 𝒇 𝐫𝛔 𝒇 𝐫 8282 number of transports (r)number of transports (r) concentrationconcentration nmax,Anmax,A nmax,Bnmax,B σAσA σBσB zone of A is moving through system : signal detection of A, measuring its intensity (Isign) :: dependence Isign = f (t) – chromatogram ::: i.e. elution curve, concentration profile of analyte in zone zone of A is moving through system : signal detection of A, measuring its intensity (Isign) :: dependence Isign = f (t) – chromatogram ::: i.e. elution curve, concentration profile of analyte in zone concentrationconcentration position; timeposition; time graphical illustration of separationgraphical illustration of separation 8383 function Isign = f (t) gives specific shape – gaussian peak shape : chromatographic separation zone : chromatographic peak function Isign = f (t) gives specific shape – gaussian peak shape : chromatographic separation zone : chromatographic peak chromatogram analysis chromatogram analysis  signal intensity (Isign,x) in point x as a function of height (Isign,x = h) at maximum xmaxsignal intensity (Isign,x) in point x as a function of height (Isign,x = h) at maximum xmaxmaxmax 𝐈 𝐬𝐢𝐠𝐧,𝐱 𝐈 𝐬𝐢𝐠𝐧,𝐱 𝐦𝐚𝐱 · 𝐞 𝐱 𝐦𝐚𝐱 𝐱 𝟐 𝟐𝛔 𝟐 𝐈 𝐬𝐢𝐠𝐧,𝐱 𝐈 𝐬𝐢𝐠𝐧,𝐱 𝐦𝐚𝐱 · 𝐞 𝐱 𝐦𝐚𝐱 𝐱 𝟐 𝟐𝛔 𝟐 peak width is given in temporal (or longevity) unitspeak width is given in temporal (or longevity) units could be neglected and rectangle may be usedcould be neglected and rectangle may be used [s, min] [mm, cm][s, min] [mm, cm] peak areapeak area peak width of compound Apeak width of compound A 𝐰 𝟒𝛔𝐰 𝟒𝛔 𝐰 𝟏 𝟐⁄ 𝟐. 𝟑𝟓𝟒𝛔𝐰 𝟏 𝟐⁄ 𝟐. 𝟑𝟓𝟒𝛔 𝐰𝐢 𝟐𝛔𝐰𝐢 𝟐𝛔 𝐀 𝟏. 𝟎𝟔𝟒 · 𝐡 · 𝐰 𝟏 𝟐⁄𝐀 𝟏. 𝟎𝟔𝟒 · 𝐡 · 𝐰 𝟏 𝟐⁄ 𝐀 𝟏 𝟐 𝐡 · 𝐰𝐀 𝟏 𝟐 𝐡 · 𝐰 2.354σ2.354σ 2σ2σ 4σ4σ 0.5σ0.5σ 0.607σ0.607σ hh inflection pointinflection point : peak width between tangents at inflection points : peak width in half of peak height  :: FWHM – full width at half maximum : peak width between inflex points  : peak width between tangents at inflection points : peak width in half of peak height  :: FWHM – full width at half maximum : peak width between inflex points  8484 deformation of gaussian peak shape reflects adsorptiondeformation of gaussian peak shape reflects adsorption aa bb cc dd retention timeretention time detector signal detector signal dependence between K and peak shapedependence between K and peak shape KK K decreasesK decreases K increaseK increase frontingfrontingtailingtailing bb cc a – linear, b – Langmuir, c – anti‐Langmuir, d – chemisorptiona – linear, b – Langmuir, c – anti‐Langmuir, d – chemisorption aAaA SS 𝐊 𝐚 𝐀 𝐒 𝐚 𝐀 𝐌 𝒄𝒐𝒏𝒔𝒕.𝐊 𝐚 𝐀 𝐒 𝐚 𝐀 𝐌 𝒄𝒐𝒏𝒔𝒕. aa asymmetry measureasymmetry measure tailing factor : asymmetry measure USP (United States Pharmacopeia) tailing factor : asymmetry measure USP (United States Pharmacopeia) deformation of peak we observe through its asymmetrydeformation of peak we observe through its asymmetry symmetry measuresymmetry measure 𝐀 𝐭 𝐟⁄𝐀 𝐭 𝐟⁄ 𝐒 𝟏 𝐀⁄𝐒 𝟏 𝐀⁄ 𝐓 𝐟 𝐭 𝟐𝐟⁄𝐓 𝐟 𝐭 𝟐𝐟⁄ 100 %100 % 5 %5 % peak height (h)peak height (h) ff tt peak width peak width A, T > 1A, T > 1 A, T < 1A, T < 1 A, T = 1A, T = 1 𝐀 𝟐𝐓 𝟏𝐀 𝟐𝐓 𝟏 mutual relation of A and Tmutual relation of A and T 8585sometimes f and t are measured at 10 % hsometimes f and t are measured at 10 % h non‐ideal peak shapenon‐ideal peak shape complex separation process : impossible to establish exact mathematical (analytical) model :: combination of Gauss function and exponential function (tn) ::: allows to express deformations of ideal Gauss curve complex separation process : impossible to establish exact mathematical (analytical) model :: combination of Gauss function and exponential function (tn) ::: allows to express deformations of ideal Gauss curve integration of model elution curve : statistical moments of separation zone (M'n & Mn) integration of model elution curve : statistical moments of separation zone (M'n & Mn) nth normal momentumnth normal momentum 𝐈 𝐬𝐢𝐠𝐧,𝐭 𝐀 𝛕 · 𝟐𝛑 · 𝛔𝐭 𝟐 · 𝐞 𝐭 𝐑 𝐦𝐚𝐱 𝐌 𝟏 𝐭 𝟐 𝟐𝛔 𝐭 𝟐 · 𝐞 𝐭 𝛕 𝐝𝐭′𝐈 𝐬𝐢𝐠𝐧,𝐭 𝐀 𝛕 · 𝟐𝛑 · 𝛔𝐭 𝟐 · 𝐞 𝐭 𝐑 𝐦𝐚𝐱 𝐌 𝟏 𝐭 𝟐 𝟐𝛔 𝐭 𝟐 · 𝐞 𝐭 𝛕 𝐝𝐭′ τ – exponential element :: summary asymmetry contribution t' – auxiliary variable of integration τ – exponential element :: summary asymmetry contribution t' – auxiliary variable of integration nth central momentumnth central momentum 𝐌′ 𝐧 𝐭 𝐧 𝟎 · 𝐈 𝐬𝐢𝐠𝐧,𝐭 𝐝𝐭 𝐈 𝐬𝐢𝐠𝐧,𝐭 𝐝𝐭𝟎 𝐌′ 𝐧 𝐭 𝐧 𝟎 · 𝐈 𝐬𝐢𝐠𝐧,𝐭 𝐝𝐭 𝐈 𝐬𝐢𝐠𝐧,𝐭 𝐝𝐭𝟎 𝐌 𝐧 𝐭 𝐌′ 𝟏 𝐧 𝟎 · 𝐈 𝐬𝐢𝐠𝐧,𝐭 𝐝𝐭 𝐈 𝐬𝐢𝐠𝐧,𝐭 𝐝𝐭𝟎 𝐌 𝐧 𝐭 𝐌′ 𝟏 𝐧 𝟎 · 𝐈 𝐬𝐢𝐠𝐧,𝐭 𝐝𝐭 𝐈 𝐬𝐢𝐠𝐧,𝐭 𝐝𝐭𝟎 n ≥ 1n ≥ 1 n ≥ 2n ≥ 2 8686 timetime responseresponse exponentially deformed peak exponentially deformed peak symmetrical undeformed peak symmetrical undeformed peak A = AA = A h ≠ hh ≠ h 𝐌 𝟎 𝐀𝐌 𝟎 𝐀 𝐌′ 𝟏 𝐭′ 𝐑𝐌′ 𝟏 𝐭′ 𝐑 𝐌 𝟐 𝛔 𝟐 𝐌 𝟐 𝛔 𝟐 𝐍 𝐌′ 𝟏 𝟐 𝐌 𝟐 𝐭 𝐑 𝟐 𝛔 𝟐 𝐍 𝐌′ 𝟏 𝟐 𝐌 𝟐 𝐭 𝐑 𝟐 𝛔 𝟐 𝐌 𝟑 𝟎𝐌 𝟑 𝟎 𝐒 𝐌 𝟑 𝐌 𝟐 𝟑 𝐒 𝐌 𝟑 𝐌 𝟐 𝟑 𝐌 𝟒 𝟎𝐌 𝟒 𝟎 𝐄 𝐌 𝟒 𝐌 𝟐 𝟐 𝟑𝐄 𝐌 𝟒 𝐌 𝟐 𝟐 𝟑 zero momentumzero momentum other important statistical momentsother important statistical moments number of theoretical platesnumber of theoretical plates 𝛕 𝛔 𝟎 𝛕 𝛔 𝟎 𝐞 𝐑 𝐌′ 𝟏 𝐭 𝐑 𝛕 · 𝟏 𝛔 𝟐𝛕 𝐞 𝐑 𝐌′ 𝟏 𝐭 𝐑 𝛕 · 𝟏 𝛔 𝟐𝛕 𝛕 𝛔 𝟏 𝛕 𝛔 𝟏 𝛕 𝛔 𝟒 𝛕 𝛔 𝟒 𝐞 𝛔 𝟏𝟎𝟎 · 𝐌 𝟐 𝛔 𝐞𝐱𝐩 𝟐 𝐌 𝟐 𝐞 𝛔 𝟏𝟎𝟎 · 𝐌 𝟐 𝛔 𝐞𝐱𝐩 𝟐 𝐌 𝟐 8787 𝛔 𝐞𝐱𝐩~ 𝐰 𝟐 , 𝐰 𝟏/𝟐 𝟐 𝛔 𝐞𝐱𝐩~ 𝐰 𝟐 , 𝐰 𝟏/𝟐 𝟐 τ = 0 – no peak deformation; w – 100 %; h – 100 %τ = 0 – no peak deformation; w – 100 %; h – 100 % only for an ideal Gaussian peak : peak symmetry S (skew) :: M3 > 0 – tailing only for an ideal Gaussian peak : peak symmetry S (skew) :: M3 > 0 – tailing only for an ideal gaussian peak : vertical peak deformation E (excess) :: M4 > 0 – sharper than a Gaussian profile only for an ideal gaussian peak : vertical peak deformation E (excess) :: M4 > 0 – sharper than a Gaussian profile w – 124 %; h – 78 %w – 124 %; h – 78 % w – 215 %; h – 40 %w – 215 %; h – 40 % eR – error of retention time estimationeR – error of retention time estimation eσ – error of peak width estimationeσ – error of peak width estimation thermodynamic aspects of separationthermodynamic aspects of separation kinetic aspects of separationkinetic aspects of separation sample moving through columnsample moving through column thermodynamic and kinetic aspects of separation mutually coincide → they influence resolution of peaks thermodynamic and kinetic aspects of separation mutually coincide → they influence resolution of peaks : analytes are separated (retention time) : zones of analytes get broader :: separation vs dilution : analytes are separated (retention time) : zones of analytes get broader :: separation vs dilution influences on broadening of zones A during separation (peak width)influences on broadening of zones A during separation (peak width) influences extent of interaction between SP & analyte : eventually SP & MP influences extent of interaction between SP & analyte : eventually SP & MP 8888 influence of kinetic aspects influence of kinetic aspects influence of thermodynamics aspects influence of thermodynamics aspects thermodynamic aspects of separationthermodynamic aspects of separation chromatographic system description stationary phase volume VS [ml/cm3] mobile phase volume VM [ml] mobile phase flow rate FM [ml∙min‐1] linear mobile phase flow rate u [cm∙min‐1] length of column L [cm] column cross‐section A [cm2] chromatographic system description stationary phase volume VS [ml/cm3] mobile phase volume VM [ml] mobile phase flow rate FM [ml∙min‐1] linear mobile phase flow rate u [cm∙min‐1] length of column L [cm] column cross‐section A [cm2] 𝐕 𝐌 𝐅 𝐌 · 𝐭 𝐦𝐕 𝐌 𝐅 𝐌 · 𝐭 𝐦 𝐮 𝐋 𝐭 𝐦 𝐮 𝐋 𝐭 𝐦 8989 retention timeretention time tmtm timetime signalsignal void timevoid time tRtR injectioninjection retention quantitiesretention quantities measurable characteristics of analyte retentionmeasurable characteristics of analyte retention i‐th analyte retention volume  VR,i [ml] i‐th analyte retention time tR,i [min] void column volume Vm [ml] void retention time tm [min] i‐th analyte retention volume  VR,i [ml] i‐th analyte retention time tR,i [min] void column volume Vm [ml] void retention time tm [min] 𝐕 𝐌 𝐅 𝐌 · 𝐭 𝐦 𝐕 𝐦𝐕 𝐌 𝐅 𝐌 · 𝐭 𝐦 𝐕 𝐦 𝐕 𝐑 𝐢 𝐅 𝐌 · 𝐭 𝐑 𝐢 𝐕 𝐑 𝐢 𝐅 𝐌 · 𝐭 𝐑 𝐢 retention time : total time of A spent in separation column retention time : total time of A spent in separation column retention volume : MP volume gone through column in retention time retention volume : MP volume gone through column in retention time 𝐮 𝐅 𝐌 𝐀 𝐮 𝐅 𝐌 𝐀 thermodynamics of chromatography – analyte distributionthermodynamics of chromatography – analyte distribution adjusted retention quantitiesadjusted retention quantities adjusted  retention time t'R,i [min] adjusted  retention volume V'R,i [ml] adjusted  retention time t'R,i [min] adjusted  retention volume V'R,i [ml] MP moves through column in a constant flow rate : all molecules of A spent in mobile phase the same (void) time : retention time includes void time + adjusted retention time :: A spends adjusted retention time on stationary phase MP moves through column in a constant flow rate : all molecules of A spent in mobile phase the same (void) time : retention time includes void time + adjusted retention time :: A spends adjusted retention time on stationary phase 𝐭 𝐑 𝐢 𝐭 𝐑 𝐢 𝐭 𝐦𝐭 𝐑 𝐢 𝐭 𝐑 𝐢 𝐭 𝐦 𝐕 𝐑 𝐢 𝐕 𝐑 𝐢 𝐕 𝐦𝐕 𝐑 𝐢 𝐕 𝐑 𝐢 𝐕 𝐦 𝐕 𝐑 𝐢 𝐅 𝐌 · 𝐭 𝐑 𝐢 𝐕 𝐑 𝐢 𝐅 𝐌 · 𝐭 𝐑 𝐢 9090 relations between retention values and distribution constantrelations between retention values and distribution constant 𝐕 𝐑 𝐀 𝐕 𝐦 𝐊 𝐃 𝐀 · 𝐕 𝐒 𝐕 𝐑 𝐀 𝐕 𝐦 𝐊 𝐃 𝐀 · 𝐕 𝐒 𝐕 𝐑 𝐀 𝐊 𝐃 𝐀 · 𝐕 𝐒 𝐕 𝐑 𝐀 𝐊 𝐃 𝐀 · 𝐕 𝐒 𝐭 𝐑 𝐀 𝐕 𝐑 𝐀 𝐅 𝐌 𝐕 𝐦 𝐊 𝐃 𝐀 · 𝐕 𝐒 𝐅 𝐌 𝐭 𝐑 𝐀 𝐕 𝐑 𝐀 𝐅 𝐌 𝐕 𝐦 𝐊 𝐃 𝐀 · 𝐕 𝐒 𝐅 𝐌 𝐭 𝐑 𝐀 𝐕 𝐑 𝐀 𝐅 𝐌 𝐊 𝐃 𝐀 · 𝐕 𝐒 𝐅 𝐌 𝐭 𝐑 𝐀 𝐕 𝐑 𝐀 𝐅 𝐌 𝐊 𝐃 𝐀 · 𝐕 𝐒 𝐅 𝐌 void column time : retention time of inert A, moving with a front of MP :: all compounds stay in a system for at least tm  ::: tR > tm; total time of A spent in mobile phase void column time : retention time of inert A, moving with a front of MP :: all compounds stay in a system for at least tm  ::: tR > tm; total time of A spent in mobile phase void column volume : eluent gone through the column in void time :: elution time of un‐retained (inert) substance ::: interstitial volume of column (VM) void column volume : eluent gone through the column in void time :: elution time of un‐retained (inert) substance ::: interstitial volume of column (VM) distribution constantdistribution constant retention (capacity) factor (capacity ratio)retention (capacity) factor (capacity ratio) serves also to compare separation : kA in given system of SP and MP = const. serves also to compare separation : kA in given system of SP and MP = const. KA and kA do characterise selectivity, i.e. retention / retardation of on columnKA and kA do characterise selectivity, i.e. retention / retardation of on column kA > 1 → adequate retention, better separation qualitykA > 1 → adequate retention, better separation quality separation factorseparation factor 𝐊 𝐀 𝐚 𝐀 𝐒 𝐚 𝐀 𝐌 ⇒ 𝐃 𝐧 𝐀 𝐒 𝐧 𝐀 𝐌 · 𝐕 𝐈 𝐕 𝐈𝐈 𝐊 𝐀 𝐚 𝐀 𝐒 𝐚 𝐀 𝐌 ⇒ 𝐃 𝐧 𝐀 𝐒 𝐧 𝐀 𝐌 · 𝐕 𝐈 𝐕 𝐈𝐈 𝐤 𝐀 𝐧 𝐀 𝐒 𝐧 𝐀 𝐌 𝐊 𝐀 · 𝐕 𝐒 𝐕 𝐌 𝐤 𝐀 𝐧 𝐀 𝐒 𝐧 𝐀 𝐌 𝐊 𝐀 · 𝐕 𝐒 𝐕 𝐌 𝛂 𝐀,𝐁 𝐤 𝐀 𝐤 𝐁 𝐭 𝐑 𝐀 𝐭 𝐑 𝐁 𝐊 𝐀 𝐊 𝐁 𝛂 𝐀,𝐁 𝐤 𝐀 𝐤 𝐁 𝐭 𝐑 𝐀 𝐭 𝐑 𝐁 𝐊 𝐀 𝐊 𝐁 𝐤 𝐀 𝐭 𝐑 𝐀 𝐭 𝐦 𝐭 𝐦 𝐭 𝐑 𝐀 𝐭 𝐦 𝐭 𝐑 𝐀 𝐭 𝐑 𝐀 𝐭 𝐑 𝐀 𝐤 𝐀 𝐭 𝐑 𝐀 𝐭 𝐦 𝐭 𝐦 𝐭 𝐑 𝐀 𝐭 𝐦 𝐭 𝐑 𝐀 𝐭 𝐑 𝐀 𝐭 𝐑 𝐀 𝐤 𝐀 𝐕 𝐑 𝐀 𝐕 𝐦 𝐕 𝐦 𝐕 𝐑 𝐀 𝐕 𝐦 𝐤 𝐀 𝐕 𝐑 𝐀 𝐕 𝐦 𝐕 𝐦 𝐕 𝐑 𝐀 𝐕 𝐦 𝐭 𝐑 𝐀 𝐭 𝐑 𝐀 ⇒ 𝐤 𝐀 𝐤 𝐀 𝟏 𝐭 𝐑 𝐀 𝐭 𝐑 𝐀 ⇒ 𝐤 𝐀 𝐤 𝐀 𝟏 𝐤 𝐀 𝐭 𝐑 𝐀 𝐭 𝐦 ⇒ 𝐭 𝐑 𝐀 𝐋 𝐮 · 𝟏 𝐤 𝐀𝐤 𝐀 𝐭 𝐑 𝐀 𝐭 𝐦 ⇒ 𝐭 𝐑 𝐀 𝐋 𝐮 · 𝟏 𝐤 𝐀 9191 efficiency of chromatographic columnefficiency of chromatographic column kinetic aspects of separationkinetic aspects of separation zones of A do broaden during analysis : consequence of non‐ideal linear chromatography zones of A do broaden during analysis : consequence of non‐ideal linear chromatography characterises zone broadening of substance Acharacterises zone broadening of substance A 00 22 44 66 88 ‐2‐2 00 22 44 66 detector signaldetector signal time [min]time [min] tR,AtR,A tMtM wAwA w1/2,Aw1/2,A analyte Aanalyte A inert substance inert substance number of theoretical plates of columnnumber of theoretical plates of column Martin‐Synge model : measure of column efficiency Martin‐Synge model : measure of column efficiency 9292 𝐍 𝐭 𝐑 𝟐 𝛔 𝟐 𝐭 𝐑 𝛔 𝟐 𝐍 𝐭 𝐑 𝟐 𝛔 𝟐 𝐭 𝐑 𝛔 𝟐 𝐰 𝟒𝛔 ⇒ 𝐍 𝐭 𝐑 𝐰 𝟒⁄ 𝟏𝟔 𝐭 𝐑 𝐀 𝐰 𝐀 𝟐 𝐰 𝟒𝛔 ⇒ 𝐍 𝐭 𝐑 𝐰 𝟒⁄ 𝟏𝟔 𝐭 𝐑 𝐀 𝐰 𝐀 𝟐 𝐰 𝟏 𝟐⁄ 𝟐. 𝟑𝟓𝟒𝛔 ⇒ 𝐍 𝟓. 𝟓𝟒𝟓 𝐭 𝐑 𝐀 𝐰 𝟏 𝟐⁄ 𝐀 𝟐 𝐰 𝟏 𝟐⁄ 𝟐. 𝟑𝟓𝟒𝛔 ⇒ 𝐍 𝟓. 𝟓𝟒𝟓 𝐭 𝐑 𝐀 𝐰 𝟏 𝟐⁄ 𝐀 𝟐 relation of theoretical plate number, column length & σ : σ – standard deviation of peak position relation of theoretical plate number, column length & σ : σ – standard deviation of peak position 𝐍 𝐋 𝛔 𝟐 𝐍 𝐋 𝛔 𝟐 height equivalent of the theoretical plateheight equivalent of the theoretical plate effective number of theoretical plates of columneffective number of theoretical plates of column σ2 – mean of squared deviation of peak positionσ2 – mean of squared deviation of peak position 𝐇 𝛔 𝟐 𝐋 𝐇 𝛔 𝟐 𝐋 𝐍 𝐍 · 𝐤 𝟏 𝐤 𝟐 𝐍 𝐍 · 𝐤 𝟏 𝐤 𝟐 𝐍 𝟏𝟔 · 𝐭 𝐑 𝐀 𝐰 𝐀 𝟐 𝟓. 𝟓𝟒𝟓 · 𝐭 𝐑 𝐀 𝐰 𝟏 𝟐⁄ 𝐀 𝟐 𝐍 𝟏𝟔 · 𝐭 𝐑 𝐀 𝐰 𝐀 𝟐 𝟓. 𝟓𝟒𝟓 · 𝐭 𝐑 𝐀 𝐰 𝟏 𝟐⁄ 𝐀 𝟐 𝐇 𝐋 𝐍 𝐋 𝟏𝟔 · 𝐰 𝐀 𝐭 𝐑 𝐀 𝟐 𝐋 𝟓. 𝟓𝟒𝟓 · 𝐰 𝟏 𝟐⁄ 𝐀 𝐭 𝐑 𝐀 𝟐 𝐇 𝐋 𝐍 𝐋 𝟏𝟔 · 𝐰 𝐀 𝐭 𝐑 𝐀 𝟐 𝐋 𝟓. 𝟓𝟒𝟓 · 𝐰 𝟏 𝟐⁄ 𝐀 𝐭 𝐑 𝐀 𝟐 9393 : measure of column efficiency for higher k values : mostly in GC : measure of column efficiency for higher k values : mostly in GC : comparison of column with different length: comparison of column with different length II I theoretical platetheoretical plate MP flowMP flow equilibriumequilibrium longitudinal diffusionlongitudinal diffusion 𝛔 𝐫 · 𝐤 𝐀 𝟏 𝐤 𝐀 𝟐 𝛔 𝐫 · 𝐤 𝐀 𝟏 𝐤 𝐀 𝟐 r – transport number (~H), k'A ~ distribution ratio r – transport number (~H), k'A ~ distribution ratio  (HEPT)(HEPT) mass transfer against the concentration gradient (ΔS>0)mass transfer against the concentration gradient (ΔS>0) molecular diffusionmolecular diffusion c/t – increase of analyte concentration on unit area at time unit, c/z – convective transport (on z axis) at rate u, Dm – diffusion coefficient, l – diffusing particles trajectory, tD – time needed to travel its distance c/t – increase of analyte concentration on unit area at time unit, c/z – convective transport (on z axis) at rate u, Dm – diffusion coefficient, l – diffusing particles trajectory, tD – time needed to travel its distance 2nd Fick law2nd Fick law solution of 2nd Fick lawsolution of 2nd Fick law Eistein‐Smoluchowski equationEistein‐Smoluchowski equation mass transfer in the process of chromatographic separationmass transfer in the process of chromatographic separation Einstein equationEinstein equation σ2 – diffusing compound zone widthσ2 – diffusing compound zone width 9494 𝛛𝐜 𝛛𝐭 𝐃 𝐦 · 𝛛 𝟐 𝐜 𝛛𝐳 𝟐 𝐮 · 𝛛𝐜 𝛛𝐳 𝛛𝐜 𝛛𝐭 𝐃 𝐦 · 𝛛 𝟐 𝐜 𝛛𝐳 𝟐 𝐮 · 𝛛𝐜 𝛛𝐳 𝐭 𝐃 𝐥 𝟐𝐃 𝐦 𝐭 𝐃 𝐥 𝟐𝐃 𝐦 𝛔 𝟐 𝟐𝐃 𝐦 · 𝐭𝛔 𝟐 𝟐𝐃 𝐦 · 𝐭𝐜 𝟏 𝟐 𝛑 · 𝐃 𝐦 · 𝐭 𝟏 𝟐⁄ · 𝐞 𝐱 𝟐 𝟒𝐃 𝐦·𝐭 𝐜 𝟏 𝟐 𝛑 · 𝐃 𝐦 · 𝐭 𝟏 𝟐⁄ · 𝐞 𝐱 𝟐 𝟒𝐃 𝐦·𝐭 convectionconvection Hagen‐Poisseuille equationHagen‐Poisseuille equation uz(r) – forward flow rate of liquid in column axis in distance r from this axis, a – column diameter, Δp – pressure difference between input and output of column, μ – dynamic viscosity of liquid, L – length of column uz(r) – forward flow rate of liquid in column axis in distance r from this axis, a – column diameter, Δp – pressure difference between input and output of column, μ – dynamic viscosity of liquid, L – length of column ρ – liquid density, u – linear flow rate, dk – channel diameter, η – viscosityρ – liquid density, u – linear flow rate, dk – channel diameter, η – viscosity Re – Reynolds number : marks convection type Re – Reynolds number : marks convection type : ideal (piston) convection – Re ~ ∞ :: unfeasible in praxis : ideal (piston) convection – Re ~ ∞ :: unfeasible in praxis convection in open channelconvection in open channel 𝐑 𝐞 𝛒 · 𝐮 · 𝐝 𝐤 𝛈 𝐑 𝐞 𝛒 · 𝐮 · 𝐝 𝐤 𝛈 𝐮 𝐳 𝐫 𝐚 𝟐 𝐫 𝟐 𝟒⁄ · ∆𝐩 𝛍 · 𝐋 𝐮 𝐳 𝐫 𝐚 𝟐 𝐫 𝟐 𝟒⁄ · ∆𝐩 𝛍 · 𝐋 9595 : turbulent convection – Re ≥ 2100 :: basically better than laminar : turbulent convection – Re ≥ 2100 :: basically better than laminar : laminar (parabolic) convection – Re < 2100 :: most often in praxis : laminar (parabolic) convection – Re < 2100 :: most often in praxis [pwah‐ZAY][pwah‐ZAY] convection between porous particlesconvection between porous particles three types of space : stationary phase – unreachable : stagnant mobile phase – in pores and around particle : moving mobile phase three types of space : stationary phase – unreachable : stagnant mobile phase – in pores and around particle : moving mobile phase ξep – inter‐particle porosity, Vep – moving MP volume, Vcol – column volumeξep – inter‐particle porosity, Vep – moving MP volume, Vcol – column volume ξip – intra‐particle porosity, Vip – pore volume, Vcol – column volumeξip – intra‐particle porosity, Vip – pore volume, Vcol – column volume ξip ~ 0.4ξip ~ 0.4 ξep ~ 0.4ξep ~ 0.4 ξtot – total porosity, Vm – void volume, Vcol – column volumeξtot – total porosity, Vm – void volume, Vcol – column volume ξtot ~ 0.8 : ca 80 % of column is SP ξtot ~ 0.8 : ca 80 % of column is SP 𝛏𝐢𝐩 𝐕𝐢𝐩 𝐕𝐜𝐨𝐥 𝛏𝐢𝐩 𝐕𝐢𝐩 𝐕𝐜𝐨𝐥 𝛏 𝐞𝐩 𝐕𝐞𝐩 𝐕𝐜𝐨𝐥 𝛏 𝐞𝐩 𝐕𝐞𝐩 𝐕𝐜𝐨𝐥 𝛏𝐭𝐨𝐭 𝐕 𝐦 𝐕𝐜𝐨𝐥 𝛏𝐭𝐨𝐭 𝐕 𝐦 𝐕𝐜𝐨𝐥 9696 : turbulent convection – Re ≥ 100 : turbulent + laminar convection – Re = 1  100 : laminar (parabolic) convection – Re < 1 : turbulent convection – Re ≥ 100 : turbulent + laminar convection – Re = 1  100 : laminar (parabolic) convection – Re < 1 Darcy equationDarcy equation Kozeny‐Carman equationKozeny‐Carman equation 𝐁 𝟎 𝐝 𝐩 𝟐 · 𝛏 𝐞𝐩 𝟑 𝟏𝟖𝟎 · 𝟏 𝛏 𝐞𝐩 𝟐 𝐁 𝟎 𝐝 𝐩 𝟐 · 𝛏 𝐞𝐩 𝟑 𝟏𝟖𝟎 · 𝟏 𝛏 𝐞𝐩 𝟐𝐮 ∆𝐩 · 𝐁 𝟎 𝛏𝐢𝐩 · 𝛈 · 𝐋 𝐮 ∆𝐩 · 𝐁 𝟎 𝛏𝐢𝐩 · 𝛈 · 𝐋 Δp – pressure difference between inlet and outlet of column, dp – particle diameter Δp – pressure difference between inlet and outlet of column, dp – particle diameter premises : laminar convection : ξep ≤ 0.5 premises : laminar convection : ξep ≤ 0.5 average flow rateaverage flow rate specific coefficient of permeabilityspecific coefficient of permeability reasons for zone broadeningreasons for zone broadening eddy (turbulent) diffusion : different molecules must run different distances longitudinal molecular diffusion : molecules run from a place of higher concentration to places w/ lower mass transfer resistance in stationary phase : different molecules diffuse different deeply into SP mass transfer resistance in mobile phase : flow rate profile of MP inside of the channel is parabolic eddy (turbulent) diffusion : different molecules must run different distances longitudinal molecular diffusion : molecules run from a place of higher concentration to places w/ lower mass transfer resistance in stationary phase : different molecules diffuse different deeply into SP mass transfer resistance in mobile phase : flow rate profile of MP inside of the channel is parabolic influence of MP flow rate on zone broadeninginfluence of MP flow rate on zone broadening height equivalent of theoretical plate (H) depends on linear MP flow rate (u)height equivalent of theoretical plate (H) depends on linear MP flow rate (u) individual contributionsindividual contributions 𝐇 𝒇 𝐮𝐇 𝒇 𝐮 𝐇 𝐇 𝐕 𝐇 𝐏 𝐇 𝐒 𝐇 𝐌𝐇 𝐇 𝐕 𝐇 𝐏 𝐇 𝐒 𝐇 𝐌 9797 ξip – intraparticle porosity, ξep – interparticle porosity, γ – MP resistance (labyrinth / tortuosity) factor, Dm – analyte diffusion coefficient in MP ξip – intraparticle porosity, ξep – interparticle porosity, γ – MP resistance (labyrinth / tortuosity) factor, Dm – analyte diffusion coefficient in MP open channelopen channel longitudinal molecular diffusionlongitudinal molecular diffusion column with particlescolumn with particles 𝐇 𝐏 𝛔 𝟐 𝐏 𝐋 𝐁 𝐮 𝟐𝐃 𝐦 𝐮 𝐇 𝐏 𝛔 𝟐 𝐏 𝐋 𝐁 𝐮 𝟐𝐃 𝐦 𝐮 𝐁 𝟐𝐃 𝐦𝐁 𝟐𝐃 𝐦 𝐁 𝟐𝛄 · 𝐃 𝐦𝐁 𝟐𝛄 · 𝐃 𝐦 𝐇 𝐏 𝛔 𝟐 𝐏 𝐋 𝐁 𝐮 𝟐𝐃 𝐦 𝐮 · 𝟏 𝛏𝐢𝐩 𝛏 𝐞𝐩 𝟐𝐃 𝐦 · 𝛄 𝐮 𝐇 𝐏 𝛔 𝟐 𝐏 𝐋 𝐁 𝐮 𝟐𝐃 𝐦 𝐮 · 𝟏 𝛏𝐢𝐩 𝛏 𝐞𝐩 𝟐𝐃 𝐦 · 𝛄 𝐮 Einstein‐Smoluchowski equationEinstein‐Smoluchowski equation eddy diffusion eddy diffusion  𝐇 𝐕 𝛔 𝟐 𝐕 𝐋 𝐀 𝟐𝛌 · 𝐝 𝐩 · 𝐋𝐇 𝐕 𝛔 𝟐 𝐕 𝐋 𝐀 𝟐𝛌 · 𝐝 𝐩 · 𝐋 λ – empirical SP homogeneity factor dp – average SP particle size λ – empirical SP homogeneity factor dp – average SP particle size 𝐃 𝐄 𝛌 · 𝐝 𝐩 · 𝐮𝐃 𝐄 𝛌 · 𝐝 𝐩 · 𝐮 DE – formal effective diffusion coefficientDE – formal effective diffusion coefficient 9898 not dependent on unot dependent on u𝐇 𝐕 𝛔 𝟐 𝐕 𝐋 𝐀 𝒄𝒐𝒏𝒔𝒕.𝐇 𝐕 𝛔 𝟐 𝐕 𝐋 𝐀 𝒄𝒐𝒏𝒔𝒕. 𝛔 𝟐 𝟐𝐃 𝐦 · 𝐭 𝟐𝐃 𝐦 · 𝐋 𝐮 𝛔 𝟐 𝟐𝐃 𝐦 · 𝐭 𝟐𝐃 𝐦 · 𝐋 𝐮 𝐇 𝐏 𝛔 𝟐 𝐏 𝐋 𝐁 𝐮 𝐇 𝐏 𝛔 𝟐 𝐏 𝐋 𝐁 𝐮 mass transfer resistance in MP mass transfer resistance in MP  dp – average particle size, λ – eddy diffusion coefficient (0.5  1.5), x – system constant (0  0.33; x = 0 for GC, x = 0.33 for LC) dp – average particle size, λ – eddy diffusion coefficient (0.5  1.5), x – system constant (0  0.33; x = 0 for GC, x = 0.33 for LC) mass transfer resistance in SP mass transfer resistance in SP  out of MP into SPout of MP into SP f (k) – function proportional to capacity factor, DMP – diffusion coefficient in MP, dp – SP particle diameter, Q – shape coefficient (1/30 for sphere) f (k) – function proportional to capacity factor, DMP – diffusion coefficient in MP, dp – SP particle diameter, Q – shape coefficient (1/30 for sphere) 𝐇 𝐕 𝐇 𝐌 𝐀 𝐂 𝐌 · 𝐮 𝟐𝛌 · 𝐝 𝐩 𝟏 𝐱 𝐃 𝐦 𝐱 · 𝐮 𝐱 𝐇 𝐕 𝐇 𝐌 𝐀 𝐂 𝐌 · 𝐮 𝟐𝛌 · 𝐝 𝐩 𝟏 𝐱 𝐃 𝐦 𝐱 · 𝐮 𝐱 𝐇 𝐌→𝐒 𝐂 𝐌→𝐒 · 𝐮 𝒇 𝐤 · 𝐝 𝐩 𝟐 𝐃 𝐌𝐏 · 𝐮𝐇 𝐌→𝐒 𝐂 𝐌→𝐒 · 𝐮 𝒇 𝐤 · 𝐝 𝐩 𝟐 𝐃 𝐌𝐏 · 𝐮 𝒇 𝐤 𝐐 · 𝛏 𝐞𝐩 · 𝐤 𝟐 · 𝟏 𝛏𝐢𝐩 𝛏 𝐞𝐩 𝛏𝐢𝐩 · 𝟏 𝐤 𝟐 𝒇 𝐤 𝐐 · 𝛏 𝐞𝐩 · 𝐤 𝟐 · 𝟏 𝛏𝐢𝐩 𝛏 𝐞𝐩 𝛏𝐢𝐩 · 𝟏 𝐤 𝟐 9999 𝐇 𝐌 𝐂 𝐌 · 𝐮𝐇 𝐌 𝐂 𝐌 · 𝐮 𝐇 𝐒 𝐂 𝐒 · 𝐮𝐇 𝐒 𝐂 𝐒 · 𝐮 𝐇 𝐒 𝐂 𝐌→𝐒 𝐂 𝐒→𝐌 · 𝐮𝐇 𝐒 𝐂 𝐌→𝐒 𝐂 𝐒→𝐌 · 𝐮 out of SP to MPout of SP to MP qSP – constant of SP active surface shape (2/3 for thin layer), DSP – diffusion coefficient in SP, k – capacity ratio, dSP – thickness of SP active surface qSP – constant of SP active surface shape (2/3 for thin layer), DSP – diffusion coefficient in SP, k – capacity ratio, dSP – thickness of SP active surface 𝐇 𝐒→𝐌 𝐂 𝐒→𝐌 · 𝐮 𝐪 𝐒𝐏 · 𝐤 𝟏 𝐤 𝟐 · 𝐝 𝐒𝐏 𝟐 𝐃 𝐒𝐏 · 𝐮𝐇 𝐒→𝐌 𝐂 𝐒→𝐌 · 𝐮 𝐪 𝐒𝐏 · 𝐤 𝟏 𝐤 𝟐 · 𝐝 𝐒𝐏 𝟐 𝐃 𝐒𝐏 · 𝐮 𝐇 𝟐 𝟏 𝛏𝐢𝐩 𝛏 𝐞𝐩 · 𝐃 𝐦 𝐮 𝟐𝛌 · 𝐝 𝐩 𝟏 𝐱 · 𝐮 𝐱 𝐃 𝐦 𝐱 𝐪 𝐒𝐅 · 𝐤 𝟏 𝐤 𝟐 · 𝐝 𝐒𝐅 𝟐 𝐃 𝐒𝐅 · 𝐮 𝒇 𝐤 · 𝐝 𝐩 𝟐 𝐃 𝐦 · 𝐮𝐇 𝟐 𝟏 𝛏𝐢𝐩 𝛏 𝐞𝐩 · 𝐃 𝐦 𝐮 𝟐𝛌 · 𝐝 𝐩 𝟏 𝐱 · 𝐮 𝐱 𝐃 𝐦 𝐱 𝐪 𝐒𝐅 · 𝐤 𝟏 𝐤 𝟐 · 𝐝 𝐒𝐅 𝟐 𝐃 𝐒𝐅 · 𝐮 𝒇 𝐤 · 𝐝 𝐩 𝟐 𝐃 𝐦 · 𝐮 𝐇 𝐇 𝐏 𝐇 𝐕 𝐇 𝐌 𝐇 𝐌→𝐒 𝐇 𝐒→𝐌𝐇 𝐇 𝐏 𝐇 𝐕 𝐇 𝐌 𝐇 𝐌→𝐒 𝐇 𝐒→𝐌 𝐒 𝐌𝐒 𝐌 100100 van Deemter equation (van Deemter, Zuiderweg, Klinkenberg)van Deemter equation (van Deemter, Zuiderweg, Klinkenberg) low flow rate (C∙u is small) : H depends on B/u high flow (B/u is small) : H is directly proportional to C∙u low flow rate (C∙u is small) : H depends on B/u high flow (B/u is small) : H is directly proportional to C∙u Knox equationKnox equation for GCfor GC for LCfor LC 𝐇 𝐀 𝐁 𝐮 𝐂 𝐒 𝐂 𝐌 · 𝐮 𝐀 𝐁 𝐮 𝐂 · 𝐮𝐇 𝐀 𝐁 𝐮 𝐂 𝐒 𝐂 𝐌 · 𝐮 𝐀 𝐁 𝐮 𝐂 · 𝐮 𝐡 𝐀 · 𝛎𝟑 𝐁 𝛎 𝐂 · 𝛎𝐡 𝐀 · 𝛎𝟑 𝐁 𝛎 𝐂 · 𝛎 𝛎 𝐮 · 𝐝 𝐩 𝐃 𝐦 𝛎 𝐮 · 𝐝 𝐩 𝐃 𝐦 𝐡 𝐇 𝐝 𝐩 𝐡 𝐇 𝐝 𝐩 reduced parameters : dimensionless reduced parameters : dimensionless Golay equationGolay equation 𝐇 𝐁 𝐮 𝐂 · 𝐮𝐇 𝐁 𝐮 𝐂 · 𝐮 A → 0 for open tubular columns (OTC)A → 0 for open tubular columns (OTC) Giddings equationGiddings equation u >> E → van Deemter, u << E → 1st  term = 0u >> E → van Deemter, u << E → 1st  term = 0 includes in term E diffusivity of MPincludes in term E diffusivity of MP 𝐇 𝐀 𝟏 𝐄 𝐮 𝐁 𝐮 𝐂 · 𝐮𝐇 𝐀 𝟏 𝐄 𝐮 𝐁 𝐮 𝐂 · 𝐮 Huber(‐Hulsman) equationHuber(‐Hulsman) equation u >> E → similar to van Deemter equationu >> E → similar to van Deemter equation term D – turbulent mixingterm D – turbulent mixing 𝐇 𝐀 𝟏 𝐄 𝐮 𝐁 𝐮 𝐂 · 𝐮 𝐃 · 𝐮𝐇 𝐀 𝟏 𝐄 𝐮 𝐁 𝐮 𝐂 · 𝐮 𝐃 · 𝐮 101101 van Deemter curvevan Deemter curve curve minimum ≈ optimal flow rate : given column shows the highest efficiency :: minimal zone broadening of analytes curve minimum ≈ optimal flow rate : given column shows the highest efficiency :: minimal zone broadening of analytes 102102 00 22 44 66 88 1010 0.00.0 0.10.1 0.20.2 0.30.3 u [cm∙min‐1]u [cm∙min‐1] H[mm]H[mm] HH HVHV HPHP HS + HMHS + HM 𝛛𝐇 𝛛𝐮 𝐁 𝐮 𝟐 𝐂 𝟎 𝛛𝐇 𝛛𝐮 𝐁 𝐮 𝟐 𝐂 𝟎 𝐇 𝐨𝐩𝐭 𝐀 𝟐 𝐁 · 𝐂𝐇 𝐨𝐩𝐭 𝐀 𝟐 𝐁 · 𝐂 𝐮 𝐨𝐩𝐭 𝐁 𝐂 𝐮 𝐨𝐩𝐭 𝐁 𝐂 resolutionresolution resolution characterises : measure of relative separation : measure of mutual overlap of two neighbouring peaks resolution characterises : measure of relative separation : measure of mutual overlap of two neighbouring peaks R(A,B) > 1.5 – complete separation, at R(A,B) = 1.5 the overlap is 0.1 % R(A,B) > 1.5 – complete separation, at R(A,B) = 1.5 the overlap is 0.1 %  time [min]time [min] signalsignal ΔtRΔtR w1/2 = 2.354σw1/2 = 2.354σ w = 4σw = 4σ wAwA wBwB European Pharmacopoeia (Ph.Eur.), EDQM, Council of EuropeEuropean Pharmacopoeia (Ph.Eur.), EDQM, Council of EuropeUS Pharmacopoeia (USP)US Pharmacopoeia (USP) R(A,B) = 0.6R(A,B) = 0.6 R(A,B) = 0.8R(A,B) = 0.8 R(A,B) = 1.0R(A,B) = 1.0 R(A,B) = 1.25R(A,B) = 1.25 overlap 2 %overlap 2 % A/B = 1/1A/B = 1/1 1/41/4 1/161/16 𝐑 𝐀,𝐁 𝟐 𝐭 𝐑 𝐁 𝐭 𝐑 𝐀 𝐰 𝐀 𝐰 𝐁 𝟐 · ∆𝐭 𝐑 𝐰 𝐀 𝐰 𝐁 𝐱 𝟐 𝐱 𝟏 𝟒𝛔 𝐑 𝐀,𝐁 𝟐 𝐭 𝐑 𝐁 𝐭 𝐑 𝐀 𝐰 𝐀 𝐰 𝐁 𝟐 · ∆𝐭 𝐑 𝐰 𝐀 𝐰 𝐁 𝐱 𝟐 𝐱 𝟏 𝟒𝛔 𝐑 𝐀,𝐁 𝟏. 𝟏𝟖 𝐭 𝐑 𝐁 𝐭 𝐑 𝐀 𝐰 𝟏 𝟐⁄ 𝐀 𝐰 𝟏 𝟐⁄ 𝐁 𝐑 𝐀,𝐁 𝟏. 𝟏𝟖 𝐭 𝐑 𝐁 𝐭 𝐑 𝐀 𝐰 𝟏 𝟐⁄ 𝐀 𝐰 𝟏 𝟐⁄ 𝐁 103103 resolution factorsresolution factors efficiency factor  : mobile phase flow rate : column length : grain size, temperature, viscosity efficiency factor  : mobile phase flow rate : column length : grain size, temperature, viscosity selectivity factor (very important)  : stationary phase change : mobile phase change selectivity factor (very important)  : stationary phase change : mobile phase change capacity factor (practically k ≈ 3 to 10) : amount of SP in column : change of SP or MP : temperature (in LC less important) capacity factor (practically k ≈ 3 to 10) : amount of SP in column : change of SP or MP : temperature (in LC less important) efficiency  selectivity capacityefficiency  selectivity capacity let us presume for two neighbouring peaks : NA ≈ NB : α > 1 let us presume for two neighbouring peaks : NA ≈ NB : α > 1 𝐑 𝐀,𝐁 𝐍 𝟒 · 𝛂 𝐀,𝐁 𝟏 𝛂 𝐀,𝐁 · 𝐤 𝐁 𝟏 𝐤 𝐁 𝐑 𝐀,𝐁 𝐍 𝟒 · 𝛂 𝐀,𝐁 𝟏 𝛂 𝐀,𝐁 · 𝐤 𝐁 𝟏 𝐤 𝐁 R(A,B)R(A,B) αα NN kk αα NN kk 0.00.0 0.50.5 1.01.0 1.51.5 2.02.0 2.52.5 3.03.0 1.001.00 1.051.05 1.101.10 1.151.15 1.201.20 1.251.25 00 50005000 1000010000 1500015000 2000020000 2500025000 00 55 1515 20201010 2525 104104 chromatographic separation : model initial state chromatographic separation : model initial state influence of capacity factor changeinfluence of capacity factor change influence of selectivity changeinfluence of selectivity change influence of change of theoretical plates numberinfluence of change of theoretical plates number↑N↑N ↑α↑α ↑k↑k tmtm tt tt tt tt 105105 peak capacitypeak capacityability to separate : number of well‐resolved (R > x) peaks (n) during separation (tR0 to tRmax) ability to separate : number of well‐resolved (R > x) peaks (n) during separation (tR0 to tRmax) w – base peak width, k – capacity factorw – base peak width, k – capacity factor  general viewgeneral view 𝐧 𝟏 𝟏 𝐰 · 𝐝𝐭 𝐭 𝐑 𝐦𝐚𝐱 𝐭 𝐑 𝟎 𝟏 𝟏 𝟒𝛔 · 𝐝𝐭 𝐭 𝐑 𝐦𝐚𝐱 𝐭 𝐑 𝟎 𝐧 𝟏 𝟏 𝐰 · 𝐝𝐭 𝐭 𝐑 𝐦𝐚𝐱 𝐭 𝐑 𝟎 𝟏 𝟏 𝟒𝛔 · 𝐝𝐭 𝐭 𝐑 𝐦𝐚𝐱 𝐭 𝐑 𝟎 𝛔 𝐭 𝐑 𝟎 𝐍 · 𝐤 𝟏𝛔 𝐭 𝐑 𝟎 𝐍 · 𝐤 𝟏 𝐧 𝟏 𝐍 𝟒 · 𝟏 𝐤 𝟏 · 𝐝𝐭 𝐭 𝐭 𝐑 𝐦𝐚𝐱 𝐭 𝐑 𝟎 𝐧 𝟏 𝐍 𝟒 · 𝟏 𝐤 𝟏 · 𝐝𝐭 𝐭 𝐭 𝐑 𝐦𝐚𝐱 𝐭 𝐑 𝟎 𝐧 𝟏 𝐍 𝟒 · 𝟏 𝐤 𝟏 · 𝐥𝐧 𝐭 𝐑 𝐦𝐚𝐱 𝐭 𝐑 𝟎 𝐧 𝟏 𝐍 𝟒 · 𝟏 𝐤 𝟏 · 𝐥𝐧 𝐭 𝐑 𝐦𝐚𝐱 𝐭 𝐑 𝟎 N is not constant for different analytes, thus in different tR: N = f (tR)N is not constant for different analytes, thus in different tR: N = f (tR) simplified viewsimplified view 𝐧 𝟏 𝐍 𝟒 · 𝐥𝐧 𝐭 𝐑 𝐦𝐚𝐱 𝐭 𝐑 𝟎 𝐧 𝟏 𝐍 𝟒 · 𝐥𝐧 𝐭 𝐑 𝐦𝐚𝐱 𝐭 𝐑 𝟎 𝟏 𝐤 𝟏 𝟏 𝟏 𝐤 𝟏 𝟏 the most general formulathe most general formulaformula with efficiencyformula with efficiency 𝐧 𝐭 𝐫 𝐦𝐚𝐱 𝐰𝐥𝐚𝐬𝐭 𝐰𝐟𝐢𝐫𝐬𝐭 · 𝐥𝐧 𝐰𝐥𝐚𝐬𝐭 𝐰𝐟𝐢𝐫𝐬𝐭 𝐧 𝐭 𝐫 𝐦𝐚𝐱 𝐰𝐥𝐚𝐬𝐭 𝐰𝐟𝐢𝐫𝐬𝐭 · 𝐥𝐧 𝐰𝐥𝐚𝐬𝐭 𝐰𝐟𝐢𝐫𝐬𝐭 𝐧 𝐭 𝐑 𝐦𝐚𝐱 ∑ 𝐰𝐢 𝐣 𝐢 𝟏 𝐣 𝐧 𝐭 𝐑 𝐦𝐚𝐱 ∑ 𝐰𝐢 𝐣 𝐢 𝟏 𝐣 106106 : column arrangement (in tube, in capillary) : planar arrangement (inter cellulose fibres of e.g. paper) chromatographic analysis is conducted mostly in diluted analyte solutions  linear region of sorption isotherm : column arrangement (in tube, in capillary) : planar arrangement (inter cellulose fibres of e.g. paper) chromatographic analysis is conducted mostly in diluted analyte solutions  linear region of sorption isotherm basic chromatography arrangementbasic chromatography arrangement : adsorption :: sorption and desorption of analyte in phase system G‐S or L‐S : partition :: distribution of analyte between two phases G‐L or L‐L : ion‐exchange :: exchange of ion and counter‐ion in system of phases L‐S : affinity :: specific binding of two molecules through weak interactions : adsorption :: sorption and desorption of analyte in phase system G‐S or L‐S : partition :: distribution of analyte between two phases G‐L or L‐L : ion‐exchange :: exchange of ion and counter‐ion in system of phases L‐S : affinity :: specific binding of two molecules through weak interactions basic principles of chromatographybasic principles of chromatography 107107 adsorption chromatography LSC, GSC adsorption chromatography LSC, GSC partition chromatography LLC, (GLC) partition chromatography LLC, (GLC) exchange chromatography EC exchange chromatography EC 𝐊 𝐃 𝐚 𝐀 𝐒 𝐚 𝐀 𝐌𝐊 𝐃 𝐚 𝐀 𝐒 𝐚 𝐀 𝐌 𝐊 𝐄 𝐑 · 𝐗 𝐘 𝐑 · 𝐘 𝐗 𝐊 𝐄 𝐑 · 𝐗 𝐘 𝐑 · 𝐘 𝐗 R – (R +) – anex, (catex) X +, Y + (X –, Y –) – ion and counter‐ion R – (R +) – anex, (catex) X +, Y + (X –, Y –) – ion and counter‐ion exchange distribution of ions X and counter‐ions Y between surface of (ion) exchanger R and mobile phase solution exchange distribution of ions X and counter‐ions Y between surface of (ion) exchanger R and mobile phase solution different adsorption of molecules A between  solid surface of SP & fluid mobile phase MP (L, G) different adsorption of molecules A between  solid surface of SP & fluid mobile phase MP (L, G) S – SP, M – MPS – SP, M – MP different distribution of analyte molecules (A) between two completely immiscible fluids  (LL, GL) different distribution of analyte molecules (A) between two completely immiscible fluids  (LL, GL) 108108 : frontal :: continual introduction of mixture with constant concentration use : measurement of adsorption isotherms : industrial preparative separation :: isolation of major mixture component : frontal :: continual introduction of mixture with constant concentration use : measurement of adsorption isotherms : industrial preparative separation :: isolation of major mixture component : displacement :: introduction of mixture, eluted just after change of MP use : pre‐concentration (SPE), preparative separation, affinity & ionex chromatography : displacement :: introduction of mixture, eluted just after change of MP use : pre‐concentration (SPE), preparative separation, affinity & ionex chromatography : elution :: single introduction of mixture into continuously flowing MP use : partition and adsorption liquid chromatography : elution :: single introduction of mixture into continuously flowing MP use : partition and adsorption liquid chromatography basic chromatographic techniquesbasic chromatographic techniques A+B+CA+B+C stationary phase stationary phase mobile phasemobile phase CC B+CB+C AA BB CC A+BA+B B+CB+C A+BA+B BB AA A+B+C A+B A A+B+C A+B A A+B+CA+B+C AA A+BA+B CC AA BB CC BB AA 109109 quantitative structure‐retention relationship (QSRR)quantitative structure‐retention relationship (QSRR) study and description of chromatographic separationstudy and description of chromatographic separation complex modelling of retention properties numerically (hard modelling) asks for alternative ways : semi‐approximation (semi‐hard) : approximation (soft modelling) method of retention properties modelling complex modelling of retention properties numerically (hard modelling) asks for alternative ways : semi‐approximation (semi‐hard) : approximation (soft modelling) method of retention properties modelling : models including structural descriptors : models including solvation effects :: reduced linear solvation energy relationship (LSER) : models including distribution factors :: correlation between retention analyte in RPLC system and its distribution coefficient ::: e.g. in system n‐octanol / water or hexadecane (l) / hexadecane (g) : models including structural descriptors : models including solvation effects :: reduced linear solvation energy relationship (LSER) : models including distribution factors :: correlation between retention analyte in RPLC system and its distribution coefficient ::: e.g. in system n‐octanol / water or hexadecane (l) / hexadecane (g) methods of relations modellingmethods of relations modelling : combination of multilinear regression and artificial neural networks (MLR‐ANN) : comparative analysis of molecular fields (CoMFA) : combination of multilinear regression and artificial neural networks (MLR‐ANN) : comparative analysis of molecular fields (CoMFA) approaches within separation relations modellingapproaches within separation relations modelling V.V. 110110 :: angular momentum :: total energy :: polarisability :: ionisation potential :: dipole moment :: subpolarity – ability of analyte to create polar interaction with SP :: stericity – geometry of molecule :: hydrophobicity :: HOMO/LUMO energies of molecular orbitals :: free Gibbs energy of adsorption :: angular momentum :: total energy :: polarisability :: ionisation potential :: dipole moment :: subpolarity – ability of analyte to create polar interaction with SP :: stericity – geometry of molecule :: hydrophobicity :: HOMO/LUMO energies of molecular orbitals :: free Gibbs energy of adsorption descriptors of separated analytedescriptors of separated analyte : allow to describe system and interaction at least semi‐empirically : are used within development of separation methods : allow to describe system and interaction at least semi‐empirically : are used within development of separation methods important factors in regard to prediction of separation optimaimportant factors in regard to prediction of separation optima retention : polarisability of analyte and subpolarity; LUMO energy retention : polarisability of analyte and subpolarity; LUMO energy selectivity : hydrophobicity selectivity : hydrophobicity 111111 geometry and interaction properties : semi‐empirical quantum‐chemistry models – AM1, MNDO and PM3 geometry and interaction properties : semi‐empirical quantum‐chemistry models – AM1, MNDO and PM3 free Gibbs energy of adsorptionfree Gibbs energy of adsorption C – molar concentration of molecules in given state ( = property) π – hydrophobicity index, σ – electric property index, Es – stericity index C – molar concentration of molecules in given state ( = property) π – hydrophobicity index, σ – electric property index, Es – stericity index calculations of some complex descriptorscalculations of some complex descriptors 𝐥𝐨𝐠 𝟏 𝐂⁄ 𝐚 · 𝛑 𝐛 · 𝛔 𝐜 · 𝐄𝐬 𝐝𝐥𝐨𝐠 𝟏 𝐂⁄ 𝐚 · 𝛑 𝐛 · 𝛔 𝐜 · 𝐄𝐬 𝐝 ∆𝐆 𝐚𝐝𝐬 𝐑 · 𝐓 · 𝐥𝐨𝐠 𝐤 𝚽⁄∆𝐆 𝐚𝐝𝐬 𝐑 · 𝐓 · 𝐥𝐨𝐠 𝐤 𝚽⁄ 𝚽 𝐕 𝐒 𝐕 𝐌⁄𝚽 𝐕 𝐒 𝐕 𝐌⁄ 𝛂 𝐤 𝐁 𝐤 𝐀⁄𝛂 𝐤 𝐁 𝐤 𝐀⁄ 𝛂 𝐞 𝟏 𝐑·𝐓⁄ ·∆ ∆𝐆 𝐚𝐝𝐬𝛂 𝐞 𝟏 𝐑·𝐓⁄ ·∆ ∆𝐆 𝐚𝐝𝐬 Δ(ΔGads) – difference of free Gibbs energy of adsorption of two separated analytesΔ(ΔGads) – difference of free Gibbs energy of adsorption of two separated analytes 112112 holistic descriptor (hydrophobicity, stericity and electric properties) : weighted holistic invariant molecular descriptors (WHIP) holistic descriptor (hydrophobicity, stericity and electric properties) : weighted holistic invariant molecular descriptors (WHIP) capacity and selectivity factorscapacity and selectivity factors semi‐empirical models usedsemi‐empirical models usedsolvation parameter model : Poole :: valid to LLC, GLC or chemically bound SP ::: interaction solvent‐solute (solvation) solvation parameter model : Poole :: valid to LLC, GLC or chemically bound SP ::: interaction solvent‐solute (solvation) 113113 system descriptors – c, m, r, s, a, b, l (dependent on SP, MP & temperature)system descriptors – c, m, r, s, a, b, l (dependent on SP, MP & temperature) descriptors of solute (Kamlet‐Taft parameters) : R2 – interaction between π‐systems of solvent and solute : π2 – dipole‐dipole interactions : Σα2 – acidity of hydrogen bridges (donor) : Σβ2 – basicity of hydrogen bridges (acceptor)  : log∙L16 – KD,g‐l of solute in hexadecane (London forces) : Vx – molecular volume (McGowan method) descriptors of solute (Kamlet‐Taft parameters) : R2 – interaction between π‐systems of solvent and solute : π2 – dipole‐dipole interactions : Σα2 – acidity of hydrogen bridges (donor) : Σβ2 – basicity of hydrogen bridges (acceptor)  : log∙L16 – KD,g‐l of solute in hexadecane (London forces) : Vx – molecular volume (McGowan method) HH HH HH GCGC LCLC𝐥𝐨𝐠 𝐤 𝐜 𝐦 · 𝐕𝐱 𝐫 · 𝐑 𝟐 𝐬 · 𝛑 𝟐 𝐇 𝐚 · 𝛂 𝟐 𝐇 𝐛 · 𝛃 𝟐 𝐇 𝐥𝐨𝐠 𝐤 𝐜 𝐦 · 𝐕𝐱 𝐫 · 𝐑 𝟐 𝐬 · 𝛑 𝟐 𝐇 𝐚 · 𝛂 𝟐 𝐇 𝐛 · 𝛃 𝟐 𝐇 𝐥𝐨𝐠 𝐤 𝐜 𝐫 · 𝐑 𝟐 𝐬 · 𝛑 𝟐 𝐇 𝐚 · 𝛂 𝟐 𝐇 𝐛 · 𝛃 𝟐 𝐇 𝐈 · 𝐥𝐨𝐠 𝐋𝟏𝟔 𝐥𝐨𝐠 𝐤 𝐜 𝐫 · 𝐑 𝟐 𝐬 · 𝛑 𝟐 𝐇 𝐚 · 𝛂 𝟐 𝐇 𝐛 · 𝛃 𝟐 𝐇 𝐈 · 𝐥𝐨𝐠 𝐋𝟏𝟔 solvatophobic model : Horváth solvatophobic model : Horváth phenomenological model : LePree and Cancino phenomenological model : LePree and Cancino partition and displacement model (cavity model) : Jaroniec; Dill : creating cavity in SP : transfer of analyte into SP : closing cavity in MP partition and displacement model (cavity model) : Jaroniec; Dill : creating cavity in SP : transfer of analyte into SP : closing cavity in MP lattice model : Martire & Boehm, Dill lattice model : Martire & Boehm, Dill step 1step 1 step 2step 2 step 3step 3 SPSP MPMPanalyteanalyte SPSP MPMP eluent molecule eluent molecule analyte molecule analyte molecule sorbent molecule sorbent molecule unit of a square grid unit of a square grid 114114 van't Hoff's plotsvan't Hoff's plots retention phenomena dependence on temperatureretention phenomena dependence on temperature ∆𝐆 𝟎 ∆𝐇 𝟎 𝐓 · ∆𝐒 𝟎 ∆𝐆 𝟎 ∆𝐇 𝟎 𝐓 · ∆𝐒 𝟎 ∆𝐆 𝟎 𝐑 · 𝐓 · 𝐥𝐧 𝐊∆𝐆 𝟎 𝐑 · 𝐓 · 𝐥𝐧 𝐊 𝐥𝐧 𝐊 ∆𝐇 𝟎 𝐑 · 𝐓 ∆𝐒 𝟎 𝐑 𝐥𝐧 𝐤 · 𝐕 𝐌 𝐕 𝐒 𝐥𝐧 𝐊 ∆𝐇 𝟎 𝐑 · 𝐓 ∆𝐒 𝟎 𝐑 𝐥𝐧 𝐤 · 𝐕 𝐌 𝐕 𝐒 𝐥𝐧 𝐤 ∆𝐇 𝟎 𝐑 · 𝐓 ∆𝐒 𝟎 𝐑 𝐥𝐧 𝐕 𝐌 𝐕 𝐒 ~ ~ ∆𝐇 𝟎 𝐑 · 𝐓 ∆𝐒 𝟎 𝐑 𝐥𝐧 𝐕 𝐒 𝐕 𝐌 𝐥𝐧 𝐤 ∆𝐇 𝟎 𝐑 · 𝐓 ∆𝐒 𝟎 𝐑 𝐥𝐧 𝐕 𝐌 𝐕 𝐒 ~ ~ ∆𝐇 𝟎 𝐑 · 𝐓 ∆𝐒 𝟎 𝐑 𝐥𝐧 𝐕 𝐒 𝐕 𝐌 : determining exo‐ / endo‐thermicity of a process :: slope value : linear vs. non‐linear curve :: phase transitions in stationary phase (qS > qL) : „breaks“ in line :: analyte pKa changes (‐0.03 K‐1) : determining exo‐ / endo‐thermicity of a process :: slope value : linear vs. non‐linear curve :: phase transitions in stationary phase (qS > qL) : „breaks“ in line :: analyte pKa changes (‐0.03 K‐1) 𝐚~ ∆𝐇 𝟎 𝐑 𝐚~ ∆𝐇 𝟎 𝐑 𝐛~ ∆𝐒 𝟎 𝐑 𝐛~ ∆𝐒 𝟎 𝐑 1/T [K‐1]1/T [K‐1] ln Kln K ln K = f (1/T)ln K = f (1/T) 𝐥𝐧 𝐤 𝟐 𝐥𝐧 𝐤 𝟏 𝐥𝐧 𝐤 𝟐 𝐤 𝟏 𝐥𝐧 𝛂 ∆∆𝐇 𝟎 𝐑 · 𝐓 ∆∆𝐒 𝟎 𝐑 𝐥𝐧 𝐤 𝟐 𝐥𝐧 𝐤 𝟏 𝐥𝐧 𝐤 𝟐 𝐤 𝟏 𝐥𝐧 𝛂 ∆∆𝐇 𝟎 𝐑 · 𝐓 ∆∆𝐒 𝟎 𝐑 ΔG0[kj∙mol‐1]ΔG0[kj∙mol‐1] T [K]T [K] 00 ΔG0 < 0 K > 1 ΔG0 < 0 K > 1 ΔG0 > 0 K < 1 ΔG0 > 0 K < 1 115115 example 7 : part 1 example 7 : part 1 45 min w = 9.8 min 45 min w = 9.8 min 60 min w = 11.3 min 60 min w = 11.3 min time [min]time [min] tm = 5.5 mintm = 5.5 min 11 22 injecting two‐componential mixture onto 150 mm column C18 (=strong non‐ polar SP), with MP methanol : water 7:3 (v/v), MP flow‐rate 0.5 ml∙min‐1, we obtained following chromatogram: injecting two‐componential mixture onto 150 mm column C18 (=strong non‐ polar SP), with MP methanol : water 7:3 (v/v), MP flow‐rate 0.5 ml∙min‐1, we obtained following chromatogram: : calculate resolution, capacity factor and H of component 2 : which component has higher affinity to SP and how long it stayed on it during separation? : how would the separation parameters change (tR, R(A,B), N, k, α), if we change MP for 100% methanol? : calculate resolution, capacity factor and H of component 2 : which component has higher affinity to SP and how long it stayed on it during separation? : how would the separation parameters change (tR, R(A,B), N, k, α), if we change MP for 100% methanol? 116116 example 7 : part 2 example 7 : part 2 : calculate resolution, capacity factor and H of component 2: calculate resolution, capacity factor and H of component 2 : which component has higher affinity to stationary phase and how  long it stayed on it during separation? : which component has higher affinity to stationary phase and how  long it stayed on it during separation? : how would the separation parameters change (tR, R(A,B), N, k, α), if  we change MP for 100% methanol? : how would the separation parameters change (tR, R(A,B), N, k, α), if  we change MP for 100% methanol? R(A,B) = 1.42R(A,B) = 1.42 H = 0.3 mm  H = 0.3 mm   k2 = 9.9  k2 = 9.9   : it is component 2 : t'R2 = 54.5 min : it is component 2 : t'R2 = 54.5 min : ↓tR, ↓R(A,B), ≈ N, ↓k, ↓α: ↓tR, ↓R(A,B), ≈ N, ↓k, ↓α 𝐤 𝐀 𝐭 𝐑 𝐀 𝐭 𝐦 𝐭 𝐦 𝐤 𝐀 𝐭 𝐑 𝐀 𝐭 𝐦 𝐭 𝐦 𝐇 𝐋 𝟏𝟔 · 𝐰 𝐀 𝐭 𝐑 𝐀 𝟐 𝐇 𝐋 𝟏𝟔 · 𝐰 𝐀 𝐭 𝐑 𝐀 𝟐 𝐑 𝐀,𝐁 𝟐 𝐭 𝐑 𝐁 𝐭 𝐑 𝐀 𝐰 𝐀 𝐰 𝐁 𝐑 𝐀,𝐁 𝟐 𝐭 𝐑 𝐁 𝐭 𝐑 𝐀 𝐰 𝐀 𝐰 𝐁 ?????? 117117 : mobile phase (MP, liquid or supercritical fluid) : stationary phase (SP, solid matter or thin layer of liquid on solid carrier) : mobile phase (MP, liquid or supercritical fluid) : stationary phase (SP, solid matter or thin layer of liquid on solid carrier) : extraction L‐L : extraction L‐S : extraction L‐L : extraction L‐S liquid chromatographyliquid chromatography 118118 contact area (max), where the sorption/desorption of analyte happens : liquid flows between particles (~ μm) of sorbent contact area (max), where the sorption/desorption of analyte happens : liquid flows between particles (~ μm) of sorbent LC historyLC history 18341834 Friedlieb Ferdinand RungeFriedlieb Ferdinand Runge F. F. Runge, Farbenchemie I (1834)F. F. Runge, Farbenchemie I (1834) : chemist : he discovered the method of capillary migration :: Chemische Produktionsfabrik Oranienburg : chemist : he discovered the method of capillary migration :: Chemische Produktionsfabrik Oranienburg he introduced new terms : chromatography – studies on colours in painting :: Greek τό χρώμα (colour) a γράφειν (to write) : chromatograph – colour preparation apparatus : chromatology – colour studies and analysis he introduced new terms : chromatography – studies on colours in painting :: Greek τό χρώμα (colour) a γράφειν (to write) : chromatograph – colour preparation apparatus : chromatology – colour studies and analysis George FieldGeorge Field18351835 : chemist : worked in dye chemistry : chemist : worked in dye chemistry D. T. Day, Proc. Am. Phil. Soc., 36 (1897) 112  W. C. Mendenhall, Science, 17 (1903) 1007 D. T. Day, Proc. Am. Phil. Soc., 36 (1897) 112  W. C. Mendenhall, Science, 17 (1903) 1007 : geologist & petrologist : he used column to study influence of geological layers on oil : geologist & petrologist : he used column to study influence of geological layers on oil 18611861 Christian Friedrich SchönbeinChristian Friedrich Schönbein : chemist : lecture on use of capillary migration (Haarröhrchenanziehung) :: arrangement with hanging paper strip ::: water moves quicker than colours, and with these, each differently : chemist : lecture on use of capillary migration (Haarröhrchenanziehung) :: arrangement with hanging paper strip ::: water moves quicker than colours, and with these, each differently David Talbot DayDavid Talbot Day18971897 19001900 Christoph Friedrich GoppelsröderChristoph Friedrich Goppelsröder : chemist : described capillary migration as adsorption analysis :: inspiration by Schönbein's lecture :: used for analysis of (plant) colourants : chemist : described capillary migration as adsorption analysis :: inspiration by Schönbein's lecture :: used for analysis of (plant) colourants 119119 Mikhail S. Tswet (rus. Михаил Семёнович Цвет, it. Michail S. Tswett) Mikhail S. Tswet (rus. Михаил Семёнович Цвет, it. Michail S. Tswett) M. Tswett, Trav. Soc. Nat. Varsovie, 6 (1903) 14 M. Tswett, Ber. Dtsch. Botan. Ges., 24 (1906) 316 M. Tswett, J. Chem. Educ., 44 (1967) 238 M. Tswett, Trav. Soc. Nat. Varsovie, 6 (1903) 14 M. Tswett, Ber. Dtsch. Botan. Ges., 24 (1906) 316 M. Tswett, J. Chem. Educ., 44 (1967) 238 : botanist : separation of chloroplast pigments of different plant extracts :: glass column filled with CaCO3 using organic solvents :: chromatographic adsorption analysis (on column) : botanist : separation of chloroplast pigments of different plant extracts :: glass column filled with CaCO3 using organic solvents :: chromatographic adsorption analysis (on column) 19031903 120120 19311931 M. Steiger and T. Reichstein, Helv. Chem. Acta, 31 (1938) 546 A. J. P. Martin and R. L. M. Synge, Biochem. J., 35 (1941) 1358 R. Consden, A. H. Gordon and A. J. P. Martin, Biochem. J., 38 (1944) 224 A. Tiselius, Science, 94 (1941) 145 M. Steiger and T. Reichstein, Helv. Chem. Acta, 31 (1938) 546 A. J. P. Martin and R. L. M. Synge, Biochem. J., 35 (1941) 1358 R. Consden, A. H. Gordon and A. J. P. Martin, Biochem. J., 38 (1944) 224 A. Tiselius, Science, 94 (1941) 145 Zechmeister and von Cholnoky – book Die chromatographische Adsorptionsmethode Zechmeister and von Cholnoky – book Die chromatographische Adsorptionsmethode 19361936 121121 Kuhn and Lederer : re‐discovery of LC first application – carotenoid separation Kuhn and Lederer : re‐discovery of LC first application – carotenoid separation contemporarycontemporary since 1965since 1965 1940‐19491940‐1949 : micro‐, nano‐column LC; capillary LC : monolithic columns, LC‐on‐chip : separation at very high pressures : micro‐, nano‐column LC; capillary LC : monolithic columns, LC‐on‐chip : separation at very high pressures Halasz, Horvath, Kirkland et al., Regnier et al. : high performance liquid chromatography (HPLC) Halasz, Horvath, Kirkland et al., Regnier et al. : high performance liquid chromatography (HPLC) Martin and Synge – separation rate is limited by diffusion rate : of dissolved analyte from liquid phase : separation of small molecules, namely amino acids (AA in wool) Martin and Synge – separation rate is limited by diffusion rate : of dissolved analyte from liquid phase : separation of small molecules, namely amino acids (AA in wool) separation column separation column detectordetector outputoutput loading device loading device pumppump MP container MP container liquid chromatography arrangementliquid chromatography arrangement 122122 only for column filling with SPonly for column filling with SP : undisturbed and stabile flow of liquid : 0.001 – 10 ml∙min‐1 : pressure up to 40 MPa (400 bar, 395 atm, 5800 psi) : resistance to MP (influence of e.g. salts) : pressure and flow control  : thermostating possibility : undisturbed and stabile flow of liquid : 0.001 – 10 ml∙min‐1 : pressure up to 40 MPa (400 bar, 395 atm, 5800 psi) : resistance to MP (influence of e.g. salts) : pressure and flow control  : thermostating possibility : high flow rate > 10 ml∙min‐1 (perfusion separation, monoliths, affinity separation) : conventional 0.2 – 10 ml∙min‐1 : low flow rate < 0.2 ml∙min‐1 (micro‐ and capillary columns) : high flow rate > 10 ml∙min‐1 (perfusion separation, monoliths, affinity separation) : conventional 0.2 – 10 ml∙min‐1 : low flow rate < 0.2 ml∙min‐1 (micro‐ and capillary columns) MP deliveryMP delivery basic pump types according to flow ratebasic pump types according to flow rate constant pressure pumpconstant pressure pump analytical use : single‐action piston (syringe) pumps : double‐action piston (reciprocating) pumps analytical use : single‐action piston (syringe) pumps : double‐action piston (reciprocating) pumps constant flow pumpconstant flow pump 123123 psi (pound per square inch) 1 psi = 6 894.75729 Pa psi (pound per square inch) 1 psi = 6 894.75729 Pa atmosphere 1 atm = 1.01325 bar atmosphere 1 atm = 1.01325 bar Torr, mmHg (Torricelli) 1 Torr = 133.3224 Pa Torr, mmHg (Torricelli) 1 Torr = 133.3224 Pa bar (βάρος) 1 bar = 100 kPa bar (βάρος) 1 bar = 100 kPa standard pressure 101325 Pa standard pressure 101325 Pa pressure pulsespressure pulsessmall reservoir : limited use in gradient : problems with MP degassing small reservoir : limited use in gradient : problems with MP degassing injectinject syphonsyphon timetime pressurepressure asks for pulse dampenerasks for pulse dampener 124124 syringe pumpsyringe pump easy and robust : high pressures easy and robust : high pressures –– ++ versatility (isocratic, gradient) : high scale of flow‐rates versatility (isocratic, gradient) : high scale of flow‐rates reciprocating pumpreciprocating pump –– ++ timetime volumevolume AA BB A+BA+B MP filteringMP filtering removal of macroscopic impuritiesremoval of macroscopic impurities metal filter at MP inletmetal filter at MP inlet filtration apparatus : vacuum : fine filters :: 0.45 μm for HPLC :: 0.20 μm for UPLC filtration apparatus : vacuum : fine filters :: 0.45 μm for HPLC :: 0.20 μm for UPLC plastic filter at MP inlet : He degassing plastic filter at MP inlet : He degassing 125125 MP degassingMP degassing removal of gases dissolved in MP : dangerous expansion of bubbles (caisson disease) removal of gases dissolved in MP : dangerous expansion of bubbles (caisson disease) : boiling :: ideal and unpractical ::: also as reflux (4 min, – 100 % of gas) :: boiling under low pressure : boiling :: ideal and unpractical ::: also as reflux (4 min, – 100 % of gas) :: boiling under low pressure vacuumvacuum MP flowMP flow waterwater gasgas HeHe into LCinto LC injectioninjection outout inin temperaturetemperature : ultrasound sonication :: 30 min, – 30 % of gas :: optimal forestep for vacuum filtration : ultrasound sonication :: 30 min, – 30 % of gas :: optimal forestep for vacuum filtration time [min]time [min] gas removed [%]gas removed [%] refluxreflux ultrasoundultrasound vacuumvacuum heliumhelium 126126 : inert gas bubbling (He) :: 10 min, – 80 % of gas : inert gas bubbling (He) :: 10 min, – 80 % of gas : vacuum filtration :: 10 min, – 60 % of gas :: in‐line membrane degassing : vacuum filtration :: 10 min, – 60 % of gas :: in‐line membrane degassing elution and elution forceelution and elution force interaction of A and SP, MPinteraction of A and SP, MP: parameters of polarity : parameters of selectivity influences so‐called eluotropic (LSC; increasing polarity) & mixotropic (LLC; decreasing polarity) order : parameters of polarity : parameters of selectivity influences so‐called eluotropic (LSC; increasing polarity) & mixotropic (LLC; decreasing polarity) order elution force of MP (e) elution force of MP (e)  NP: hexane + isopropanol RP: water + methanol/acetonitrile NP: hexane + isopropanol RP: water + methanol/acetonitrile KD – distribution constant of analyte, Va – adsorbed layer volume, mSP – weight of SP, Vm – void volume, α – adsorbent activity parameter, S0 – free energy of solute adsorption, A – adsorption cross‐section of solute, e – elution force KD – distribution constant of analyte, Va – adsorbed layer volume, mSP – weight of SP, Vm – void volume, α – adsorbent activity parameter, S0 – free energy of solute adsorption, A – adsorption cross‐section of solute, e – elution force change of retention factor by change of elution force (e1 → e2)change of retention factor by change of elution force (e1 → e2) eluent – MP liquid coming into column eluate – MP liquid coming out of column effluent – liquid flowing out (of column) eluent – MP liquid coming into column eluate – MP liquid coming out of column effluent – liquid flowing out (of column) 𝐥𝐨𝐠 𝐊 𝐃 𝐥𝐨𝐠 𝐕𝐚𝐝𝐬 · 𝐦 𝐒𝐏 𝐕 𝐦⁄ 𝛂 · 𝐒 𝟎 𝐀 · 𝐞𝐥𝐨𝐠 𝐊 𝐃 𝐥𝐨𝐠 𝐕𝐚𝐝𝐬 · 𝐦 𝐒𝐏 𝐕 𝐦⁄ 𝛂 · 𝐒 𝟎 𝐀 · 𝐞 𝐥𝐨𝐠 𝐤 𝟏 𝐤 𝟐⁄ 𝛂 · 𝐀 · 𝐞 𝟏 𝐞 𝟐𝐥𝐨𝐠 𝐤 𝟏 𝐤 𝟐⁄ 𝛂 · 𝐀 · 𝐞 𝟏 𝐞 𝟐 127127 implementation of elution forceimplementation of elution force : isocratic :: elution force remains constant during elution : gradient :: elution force is changing (increasing) during elution : isocratic :: elution force remains constant during elution : gradient :: elution force is changing (increasing) during elution eA 0 & eB 0 – elution forces of solvents A and B, xB – molar ratio of B, α – parameter of adsorbent activity, AB – adsorption profile of solute B eA 0 & eB 0 – elution forces of solvents A and B, xB – molar ratio of B, α – parameter of adsorbent activity, AB – adsorption profile of solute B elution forceelution force timetime elution forceelution force timetime elution forceelution force timetime elution forceelution force timetime linearlinear step‐likestep‐like exponentialexponential saw‐likesaw‐like x % B 100 – x % A x % B 100 – x % A 2‐way valve2‐way valve pumppump columncolumn loadload mixermixer controllercontroller pumpspumps columncolumn injectioninjection mixermixercontrollercontroller high‐pressure gradienthigh‐pressure gradientlow‐pressure gradientlow‐pressure gradient 𝐞 𝐀𝐁 𝐞 𝐀 𝟎 𝐥𝐨𝐠 𝐱 𝐁 · 𝟏𝟎𝛂 · 𝐀 𝐁 · 𝐞 𝐁 𝟎 𝐞 𝐀 𝟎 𝟏 𝐱 𝐁 𝛂 · 𝐀 𝐁⁄𝐞 𝐀𝐁 𝐞 𝐀 𝟎 𝐥𝐨𝐠 𝐱 𝐁 · 𝟏𝟎𝛂 · 𝐀 𝐁 · 𝐞 𝐁 𝟎 𝐞 𝐀 𝟎 𝟏 𝐱 𝐁 𝛂 · 𝐀 𝐁⁄ 128128 isocratic or gradient? isocratic or gradient? : expensive : re‐equilibration after each measurement : expensive : re‐equilibration after each measurement : shorter analyses : higher resolution : higher peak capacity : high content of organic phase at the end of gradient keeps column : shorter analyses : higher resolution : higher peak capacity : high content of organic phase at the end of gradient keeps column elution force e (φ)elution force e (φ) desorption degree desorption degree 0 %0 % 100 %100 % isocratic elutionisocratic elution elution force eelution force e desorption degreedesorption degree 0 %0 % 100 %100 % gradient extentgradient extent elution forceelution force gradient elutiongradient elution 129129 (six‐way) valve(six‐way) valve loading of A onto column without interrupting of MP flowloading of A onto column without interrupting of MP flow manual injectionmanual injection injection deviceinjection device 130130 from pumpfrom pump onto columnonto column from pumpfrom pump onto columnonto column looploop into wasteinto waste from microsyringefrom microsyringe wastewaste automatic injectionautomatic injection (autosampler)(autosampler) injection unitinjection unit 6‐way valve 6‐way valve stepper motorstepper motor injection piston injection piston wastewaste onto columnonto column out of pumpout of pump solution with six‐way valve (Agilent)solution with six‐way valve (Agilent) 131131 onto columnonto column solution with multiple valves – 3x three‐way and 1x two‐way (Waters)solution with multiple valves – 3x three‐way and 1x two‐way (Waters) out of pumpout of pump injectionpistoninjectionpiston stepper  motorstepper  motor steppermotorsteppermotor 5 % of flow5 % of flow 95 % of flow95 % of flowinjection loopinjection loop needle washneedle wash wastewaste 132132 sample should be injected in a suitable solution regarding MP : strong eluent causes zone broadening, weak analyte focusation in zone sample should be injected in a suitable solution regarding MP : strong eluent causes zone broadening, weak analyte focusation in zone caffein & salicylamide; column C8, 4.6 x 150 mm isocratic elution: 18:81:1 acetonitrile‐water‐acetic acid (v/v) caffein & salicylamide; column C8, 4.6 x 150 mm isocratic elution: 18:81:1 acetonitrile‐water‐acetic acid (v/v) 133133 time [min]time [min] 100 % acetonitrile100 % acetonitrile 4.414.41 8.668.66 4.734.73 9.039.03 4.754.75 4.724.72 9.059.05 100 % water100 % water 1.2 % acetic acid1.2 % acetic acid MPMP caffeincaffein 9.009.00 MeOHMeOHsamplesample methanol zonemethanol zone water zonewater zonewaterwater A moving according to MP elution forceA moving according to MP elution force A moving w/ methanolA moving w/ methanol A moving according to MP elution forceA moving according to MP elution force sharp zonesharp zone container w/ SP included into flow of MPcontainer w/ SP included into flow of MP capillarycapillary length: 50 – 2000 mm diameter: 0.075 – 1.000 mm length: 50 – 2000 mm diameter: 0.075 – 1.000 mm : stable SP :: in flow (terminal frits) :: undisturbed SP column :: robust cover : stable SP :: in flow (terminal frits) :: undisturbed SP column :: robust cover length: 10 – 250 mm diameter: 1.0 – 4.6 mm length: 10 – 250 mm diameter: 1.0 – 4.6 mm length: 250 – 5000 mm diameter: 4.6 – 1000.0 mm length: 250 – 5000 mm diameter: 4.6 – 1000.0 mm i.d. ~ 300 μm – micro‐column ~   75 μm – nano‐column i.d. ~ 300 μm – micro‐column ~   75 μm – nano‐column microfluidicsmicrofluidics separation columnseparation column 134134 tubular / column : preparative (necessary capacity) : analytical tubular / column : preparative (necessary capacity) : analytical chipchip precolumn : silica (free ‐OH) : guarding pH changes  precolumn : silica (free ‐OH) : guarding pH changes  SP guardingSP guarding connectionsconnections pressure resistant : unified coil of screw thread pressure resistant : unified coil of screw thread feruleferule capillarycapillary nutnut seal endingseal ending pumppump precolumnprecolumn injectioninjection in‐line filterin‐line filter guard columnguard column columncolumn 135135 precolumnsprecolumns guard column : SP same as column : preconcentration guard column : SP same as column : preconcentration in‐line filter : filters MP in‐line filter : filters MP correctcorrect leakyleaky void volumevoid volume correctcorrect cutcut wrongwrong capillariescapillaries conducting MP between parts of instrumentationconducting MP between parts of instrumentation column ovencolumn oven keeps stable temperature temperatures > 40 °C : influence quality of separation Peltier cooler keeps stable temperature temperatures > 40 °C : influence quality of separation Peltier cooler electron flowelectron flow hole flowhole flow semiconductor type N, BiTe semiconductor type N, BiTe released heat released heat absorbed heat absorbed heat flow of electrons and holes transports the heatflow of electrons and holes transports the heat semiconductor type P, BiTe semiconductor type P, BiTe void volume in capillary connectionsvoid volume in capillary connections 136136 stainless steel, PEEK (polyether ether ketone), teflon : minimal volume – additive to void volume : inertness and proper diameter (Aris‐Taylor equation)) stainless steel, PEEK (polyether ether ketone), teflon : minimal volume – additive to void volume : inertness and proper diameter (Aris‐Taylor equation)) interaction types (generally)interaction types (generally) 137137 electrostatic interactionelectrostatic interaction interaction dipole‐dipoleinteraction dipole‐dipole hydrogen bondhydrogen bond van der Waals forcesvan der Waals forces other carriers, polar and non‐polarother carriers, polar and non‐polar silicasilica overview of main SP carriersoverview of main SP carriers : tempering to 150 °C  water removal = activation of silica : labile over pH 8 : tempering to 150 °C  water removal = activation of silica : labile over pH 8 aluminaalumina : similar properties to silica : modifications – amount of water bound, crystal structure : activation by drying [Al(OH)3  AlO(OH)  Al2O3] : at high water content (15%) separation effects appear :: besides of proton‐donoring hydroxyls appear on the surface also proton‐accepting centres : similar properties to silica : modifications – amount of water bound, crystal structure : activation by drying [Al(OH)3  AlO(OH)  Al2O3] : at high water content (15%) separation effects appear :: besides of proton‐donoring hydroxyls appear on the surface also proton‐accepting centres polystyrene‐divinylbenzene, activated carbon, polyamide, fluorisil (MgSiO3), ZrO2, porous glass,  kaolinite, MgO, CaCO3, CaSO4, infusorial earth, cellulose, Ca3(PO4)2 [Ca5(OH)(PO4)3] polystyrene‐divinylbenzene, activated carbon, polyamide, fluorisil (MgSiO3), ZrO2, porous glass,  kaolinite, MgO, CaCO3, CaSO4, infusorial earth, cellulose, Ca3(PO4)2 [Ca5(OH)(PO4)3] normal silanolnormal silanol siloxane bridgesiloxane bridge active silanolactive silanol 138138 SiO2. (H2O)x, by hydratation, silicate acid is createdSiO2. (H2O)x, by hydratation, silicate acid is created : interaction with silanol groups : silica surface is mildly acidic :: it has proton‐donor properties : interaction with silanol groups : silica surface is mildly acidic :: it has proton‐donor properties Al2O3, resp. Al(OH)3Al2O3, resp. Al(OH)3 bridged vicinalbridged vicinalvicinal silanolvicinal silanol hydrated silanol hydrated silanol geminal silanol geminal silanol bridged geminal silanol bridged geminal silanol column fillingcolumn filling : solid – particles directly of SP material : bound – SP bound (physically or chemically) to carrier carrier material – reactive : solid – particles directly of SP material : bound – SP bound (physically or chemically) to carrier carrier material – reactive 139139 solid polar SP (LSC) : silica (silicon oxide) – polar, acid, basic compounds : fluorisil (magnesium silicate) – polar, strongly acidic, basic compounds : alumina (aluminium oxide) – polar, basic, acidic compounds : organic polymers  (styrene‐divinylbenzene) solid polar SP (LSC) : silica (silicon oxide) – polar, acid, basic compounds : fluorisil (magnesium silicate) – polar, strongly acidic, basic compounds : alumina (aluminium oxide) – polar, basic, acidic compounds : organic polymers  (styrene‐divinylbenzene) chemically bound polar SP (LSC) : cyanopropyl (–CN) : aminopropyl (–NH2) : N‐propylethylene diamine (PSA) chemically bound polar SP (LSC) : cyanopropyl (–CN) : aminopropyl (–NH2) : N‐propylethylene diamine (PSA) physically bound polar SP (LLC) : dimethyl sulphoxide : water : propane‐1,3‐diol : ethane‐1,2‐diamine physically bound polar SP (LLC) : dimethyl sulphoxide : water : propane‐1,3‐diol : ethane‐1,2‐diamine chemically bound non‐polar SP (LSC) : ethyl (C2) : octyl (C8) : octadecyl (C18) : cyclohexyl (CH) : phenyl (PH) chemically bound non‐polar SP (LSC) : ethyl (C2) : octyl (C8) : octadecyl (C18) : cyclohexyl (CH) : phenyl (PH) chemically bound non‐polar SP : reaction of silica with alkylsilanes (R ~ 1 – 18 C atoms) chemically bound non‐polar SP : reaction of silica with alkylsilanes (R ~ 1 – 18 C atoms) 140140 solid non‐polar SP : activated carbon – almost non‐polar, non‐polar compounds : organic polymers (polymethyl methacrylates) solid non‐polar SP : activated carbon – almost non‐polar, non‐polar compounds : organic polymers (polymethyl methacrylates) physically bound non‐polar SP (LLC) : ethane‐1,2‐diol : squalane physically bound non‐polar SP (LLC) : ethane‐1,2‐diol : squalane chemically bound ion‐exchange SP : anion‐exchangers (anex) :: R‐NH3 +, R‐CH2N+(CH3)3 : cation‐exchangers (catex) :: R‐COO–, R‐SO3 – chemically bound ion‐exchange SP : anion‐exchangers (anex) :: R‐NH3 +, R‐CH2N+(CH3)3 : cation‐exchangers (catex) :: R‐COO–, R‐SO3 – anexesanexes catexescatexes 141141 quasi‐anex : quaternary amines (TEA, TBAH) quasi‐catex : sulphonic acids (pentane sulphonic acid) quasi‐anex : quaternary amines (TEA, TBAH) quasi‐catex : sulphonic acids (pentane sulphonic acid) solid ion‐exchange SP : zeolites (aluminium silicates) : clays : organic polymers (polystyrenes, acrylates) solid ion‐exchange SP : zeolites (aluminium silicates) : clays : organic polymers (polystyrenes, acrylates) physically bound ion‐exchange SP : ion‐pairing : uses combination of chemically bound non‐polar SP and ion pairing agents :: surface active substances (surfactants) physically bound ion‐exchange SP : ion‐pairing : uses combination of chemically bound non‐polar SP and ion pairing agents :: surface active substances (surfactants) mixed mode chromatographymixed mode chromatography ion‐exchangerion‐exchanger reversed phasereversed phase catexcatex anexanex single‐ligandsingle‐ligand zwitter‐ionzwitter‐iontippedtippedembeddedembeddedmulti‐ligandmulti‐ligandmultiphasemultiphase silicasilicasilicasilicasilicasilicasilicasilica (MMC)(MMC) : 1986 F. Ringier; AEC‐HIC combination : 1998 A. Štrancar; CLC monoliths : 1999 J. R. Yates; SCX‐RPLC : 1986 F. Ringier; AEC‐HIC combination : 1998 A. Štrancar; CLC monoliths : 1999 J. R. Yates; SCX‐RPLC : higher selectivity : higher loading capacity : one MMC column in cyclic system for 2D‐LC : higher selectivity : higher loading capacity : one MMC column in cyclic system for 2D‐LC : the most used combinations :: IEC/HIC, IEC/RPLC, HILIC/RPLC, HILIC/IEC, SEC/IEC : the most used combinations :: IEC/HIC, IEC/RPLC, HILIC/RPLC, HILIC/IEC, SEC/IEC 142142 IECIEC RPLCRPLC MMC RPLC/IEC MMC RPLC/IEC shielded stationary phasesshielded stationary phases : shielding against negative SP influences : multi‐modal separation :: restricted access material SP (RAM) : shielding against negative SP influences : multi‐modal separation :: restricted access material SP (RAM) proteinprotein hydrophilic layer hydrophilic layer hydrophobic layer hydrophobic layer small org. molecule small org. molecule respective mobile phasesrespective mobile phases polar SP + non‐polar basic MP chromatography with normal phases (SP polar)  elution force increases in following order : pentane, benzene, chloroform, acetone, acetonitrile, ethanol, methanol, water polar SP + non‐polar basic MP chromatography with normal phases (SP polar)  elution force increases in following order : pentane, benzene, chloroform, acetone, acetonitrile, ethanol, methanol, water ion‐exchanging MP solutions of inorganic acids and bases with defined ionic strength and given pH ion‐exchanging MP solutions of inorganic acids and bases with defined ionic strength and given pH non‐polar SP + polar basic MP chromatography with reversed phases (SP non‐polar) elution force increases in following order : water, methanol, ethanol, acetonitrile, isopropanol, tetrahydrofuran non‐polar SP + polar basic MP chromatography with reversed phases (SP non‐polar) elution force increases in following order : water, methanol, ethanol, acetonitrile, isopropanol, tetrahydrofuran 143143 basic types of sorbentsbasic types of sorbentsparticle sorbentparticle sorbent shape: spherical (regular shape) size for different column types : analytical 1.5 – 8.0 μm : preparative  > 10 μm shape: spherical (regular shape) size for different column types : analytical 1.5 – 8.0 μm : preparative  > 10 μm : smooth surface :: difficult manufacturing, more useful kinetic properties : smooth surface :: difficult manufacturing, more useful kinetic properties 144144 : superficial differentiation (SPP, surface porous particles) :: half‐way between particle and monolithic sorbent ::: core‐shell, poroshell, halo core: 1.7 – 4.5 μm shell: 0.25 – 1.00 μm shell pores: ~ 30 nm : superficial differentiation (SPP, surface porous particles) :: half‐way between particle and monolithic sorbent ::: core‐shell, poroshell, halo core: 1.7 – 4.5 μm shell: 0.25 – 1.00 μm shell pores: ~ 30 nm : pores (FPP, fully porous particles) :: historically older, negative influence on kinetic aspects : pores (FPP, fully porous particles) :: historically older, negative influence on kinetic aspects : pore volume :: cm3∙g‐1 (< 1); relative pore volume : surface area :: m2∙g‐1 (50 – 500) : pore size :: nm (5 – 50); 1 Å = 0.1 nm ::: < 10 nm ~ < 3000 Da ::: 10 – 30 nm ~ 3 – 10 kDa ::: > 30 nm ~ > 10 000 Da : pore volume :: cm3∙g‐1 (< 1); relative pore volume : surface area :: m2∙g‐1 (50 – 500) : pore size :: nm (5 – 50); 1 Å = 0.1 nm ::: < 10 nm ~ < 3000 Da ::: 10 – 30 nm ~ 3 – 10 kDa ::: > 30 nm ~ > 10 000 Da monolithic sorbentmonolithic sorbent macropores: ~1500 nm; mezopores: ~ 10 nm, micropores < 2 nmmacropores: ~1500 nm; mezopores: ~ 10 nm, micropores < 2 nm high flow rate at low pressure; large effective area → fast separation: seconds or minutes high resolution and high capacity high flow rate at low pressure; large effective area → fast separation: seconds or minutes high resolution and high capacity disadvantage: difficult manufacturingdisadvantage: difficult manufacturing porosity of ML SP almost 85 %; vs. particle SP ø 5 μm with porosity max 60 %porosity of ML SP almost 85 %; vs. particle SP ø 5 μm with porosity max 60 % with porous layer with porous layer  only capillary columns (i.d. 70 μm; 2 μm layer) : low pressure (70 MPa) within long columns (50 cm) only capillary columns (i.d. 70 μm; 2 μm layer) : low pressure (70 MPa) within long columns (50 cm) 145145 (porous layer open tube, PLOT)(porous layer open tube, PLOT) (silica‐rod, ML)(silica‐rod, ML) design: disc, tube, filled capillarydesign: disc, tube, filled capillary materialmaterial ML‐SP based on silica tetramethoxysilane (TMOS) tetraethoxysilane (TEOS)         +      acetic acid          +          polyethylenglycol (PEG) ML‐SP based on silica tetramethoxysilane (TMOS) tetraethoxysilane (TEOS)         +      acetic acid          +          polyethylenglycol (PEG) ML‐SP based on organic materials styrene‐divinylbenzene (S‐DVB) isooctane methacrylates tetrahydrofuran vinyl‐derivatives (vinylpyrrolidone, vinyl acetate) decanol ML‐SP based on organic materials styrene‐divinylbenzene (S‐DVB) isooctane methacrylates tetrahydrofuran vinyl‐derivatives (vinylpyrrolidone, vinyl acetate) decanol monomer + polymerisation agent + porogenic compoundsmonomer + polymerisation agent + porogenic compounds outer stabilisation : PTFE (polytetrafluoroethylene), PEEK (poly(ether‐ether‐ketone))  outer stabilisation : PTFE (polytetrafluoroethylene), PEEK (poly(ether‐ether‐ketone))  silica based monoliths of the first & second generationsilica based monoliths of the first & second generation : mezopores 11‐12 nm (1G) > 14‐16 nm (2G) : macropores 1.8‐2.0 μm (1G) > 1.1‐1.2 μm (2G) : decreasing SP heterogeneity : mezopores 11‐12 nm (1G) > 14‐16 nm (2G) : macropores 1.8‐2.0 μm (1G) > 1.1‐1.2 μm (2G) : decreasing SP heterogeneity 146146 molecularly imprinted polymersmolecularly imprinted polymers (MIP)(MIP) new type of SP : suitable for chiral or affinity separation : similar preparation to ML new type of SP : suitable for chiral or affinity separation : similar preparation to ML : decreasing total porosity 3.5 ml∙g‐1 (1G) > 2.9 ml∙g‐1 (2G) : decreasing surface area 320 m2∙g‐1 (1G) > 250 m2∙g‐1 (2G) : increasing number of theoretical plates 50 000 m‐1 (1G) > 155 000 m‐1 (2G) : decreasing total porosity 3.5 ml∙g‐1 (1G) > 2.9 ml∙g‐1 (2G) : decreasing surface area 320 m2∙g‐1 (1G) > 250 m2∙g‐1 (2G) : increasing number of theoretical plates 50 000 m‐1 (1G) > 155 000 m‐1 (2G) [mm∙s‐1][mm∙s‐1] [μm][μm] 1G1G 2G2G uu HH 2G2G1G1G 2000:12000:1 5000:15000:1 147147 chipchip structures etched into silicon (Si) platestructures etched into silicon (Si) plate advantages: analysis speed, size, low sample consumption (< nl)advantages: analysis speed, size, low sample consumption (< nl) disadvantages: too small volumes (surface tension, electrostatic interactions)disadvantages: too small volumes (surface tension, electrostatic interactions) use: pre‐separation and sample preparation for MS : proteolysis, desalting : pre‐concentration and desalting for ESI‐MS use: pre‐separation and sample preparation for MS : proteolysis, desalting : pre‐concentration and desalting for ESI‐MS MP delivery: centrifugal force, electrostatic forceMP delivery: centrifugal force, electrostatic force 148148 (lab‐on‐chip, LC‐on‐chip)(lab‐on‐chip, LC‐on‐chip) allow to gain information on separated analytes → signal (signal intensity) : speciality – MP recycling; eluate without sample with properties of pure MP allow to gain information on separated analytes → signal (signal intensity) : speciality – MP recycling; eluate without sample with properties of pure MP range of detected analytical information : universal detector : non‐selective detector :: presence (absorbance at one wavelength) : selective detector :: identity (UV‐Vis spectrum, mass spectrum, redox potential) :: structure (mass spectrum, NMR spectrum) range of detected analytical information : universal detector : non‐selective detector :: presence (absorbance at one wavelength) : selective detector :: identity (UV‐Vis spectrum, mass spectrum, redox potential) :: structure (mass spectrum, NMR spectrum) wastewaste collectioncollection base linebase line delaydelay VI.VI.detectorsdetectors 149149 fast response : if slow (slower than MP flow) :: signal distortion, low sensitivity signal stability : unstable signal :: loss of (quantitative) information selectivity, sensitivity, linearity fast response : if slow (slower than MP flow) :: signal distortion, low sensitivity signal stability : unstable signal :: loss of (quantitative) information selectivity, sensitivity, linearity influencing the nature of analyte by detection : non‐destructive detector :: no chemical change of detected analyte : destructive detector :: detected analyte irreversibly changed influencing the nature of analyte by detection : non‐destructive detector :: no chemical change of detected analyte : destructive detector :: detected analyte irreversibly changed detector hyphenation : high quality of detection (UV‐Vis + MS) detector hyphenation : high quality of detection (UV‐Vis + MS) FM,1FM,1 FM,2 = 0.5x FM,1FM,2 = 0.5x FM,1 FM,1FM,1 FM,2 = 0.5x FM,1FM,2 = 0.5x FM,1 mass dependent detector (MDD) : dm/dt (component mass flow in effluent) dependent on intake of component in detector :: at FM change, also peak height changes, but area remains the same mass dependent detector (MDD) : dm/dt (component mass flow in effluent) dependent on intake of component in detector :: at FM change, also peak height changes, but area remains the same concentration dependent detector (CDD) : dm/dV (component mass concentration in effluent) independent on intake of component in detector :: at FM change, peak area height changes, while height remains the same concentration dependent detector (CDD) : dm/dV (component mass concentration in effluent) independent on intake of component in detector :: at FM change, peak area height changes, while height remains the same t [min]t [min] II t [min]t [min] II 150150 CDDCDD MDDMDD dynamic rangedynamic range linearity is given as slope value (k) in range 0.98 to 1.02 or cA = ± 5 %linearity is given as slope value (k) in range 0.98 to 1.02 or cA = ± 5 % concentration range, in which change in concentration causes signal intensity changeconcentration range, in which change in concentration causes signal intensity change linear dynamic rangelinear dynamic range 𝐥𝐨𝐠 𝐑 𝐤 · 𝐥𝐨𝐠 𝐜 𝐀𝐥𝐨𝐠 𝐑 𝐤 · 𝐥𝐨𝐠 𝐜 𝐀 detector responsedetector response S – detector sensitivityS – detector sensitivityR – detector response R – detector response  𝐑 𝒇 𝐜 𝐀𝐑 𝒇 𝐜 𝐀 m – mass of analytem – mass of analyte 𝐑 𝐂𝐂𝐃 𝐒 · 𝐜 𝐀𝐑 𝐂𝐂𝐃 𝐒 · 𝐜 𝐀 𝐑 𝐌𝐃𝐃 𝐒 · 𝛛𝐦 𝛛𝐭 𝐑 𝐌𝐃𝐃 𝐒 · 𝛛𝐦 𝛛𝐭 elution curve areaelution curve area Fm must be constant for quantitative use of elution curve areaFm must be constant for quantitative use of elution curve area Fm – flow rateFm – flow rate elution curve area is independent on Fmelution curve area is independent on Fm 𝐀 𝐌𝐃𝐃 𝐑 · 𝐝𝐭 𝐭 𝟐 𝐭 𝟏 𝐒 · 𝐝𝐦 𝐝𝐭 · 𝐝𝐭 𝐭 𝟐 𝐭 𝟏 𝐒 · 𝐝𝐦 𝐭 𝟐 𝐭 𝟏 𝐒 · 𝐦𝐀 𝐌𝐃𝐃 𝐑 · 𝐝𝐭 𝐭 𝟐 𝐭 𝟏 𝐒 · 𝐝𝐦 𝐝𝐭 · 𝐝𝐭 𝐭 𝟐 𝐭 𝟏 𝐒 · 𝐝𝐦 𝐭 𝟐 𝐭 𝟏 𝐒 · 𝐦 151151 𝐀 𝐂𝐃𝐃 𝐑 · 𝐝𝐭 𝐭 𝟐 𝐭 𝟏 𝐒 · 𝐜 𝐀 · 𝐝𝐭 𝐭 𝟐 𝐭 𝟏 𝐒 · 𝐜 𝐀 · 𝐝𝐭 𝐭 𝟐 𝐭 𝟏 𝐒 · 𝐜 𝐀 · ∆𝐭 𝐒 · 𝐧 𝐕 · ∆𝐭 𝐒 · 𝐧 𝐅 𝐦 𝐀 𝐂𝐃𝐃 𝐑 · 𝐝𝐭 𝐭 𝟐 𝐭 𝟏 𝐒 · 𝐜 𝐀 · 𝐝𝐭 𝐭 𝟐 𝐭 𝟏 𝐒 · 𝐜 𝐀 · 𝐝𝐭 𝐭 𝟐 𝐭 𝟏 𝐒 · 𝐜 𝐀 · ∆𝐭 𝐒 · 𝐧 𝐕 · ∆𝐭 𝐒 · 𝐧 𝐅 𝐦 extra‐column contributions to zone broadening in LCextra‐column contributions to zone broadening in LC 𝛔𝐭𝐨𝐭 𝟐 𝛔 𝐜𝐨𝐥 𝟐 𝛔𝐢𝐧𝐣 𝟐 𝛔 𝐜𝐨𝐧 𝟐 𝛔 𝐝𝐞𝐭 𝟐 𝛔𝐭𝐨𝐭 𝟐 𝛔 𝐜𝐨𝐥 𝟐 𝛔𝐢𝐧𝐣 𝟐 𝛔 𝐜𝐨𝐧 𝟐 𝛔 𝐝𝐞𝐭 𝟐 𝛔𝐢𝐧𝐣 𝟐 𝐕𝐢𝐧𝐣 𝟐 𝐗 𝟐 𝛔𝐢𝐧𝐣 𝟐 𝐕𝐢𝐧𝐣 𝟐 𝐗 𝟐 𝛔 𝐝𝐞𝐭 𝟐 𝐕𝐝𝐞𝐭 𝟐 𝐘 𝟐 𝛔 𝐝𝐞𝐭 𝟐 𝐕𝐝𝐞𝐭 𝟐 𝐘 𝟐 σtot 2 – total zone broadeningσtot 2 – total zone broadening σinj 2 – broadening given by injection volume; X ≈ 1‐12 : dependent on injector shape σdet 2 – broadening given by detector cell volume; Y ~ X σinj 2 – broadening given by injection volume; X ≈ 1‐12 : dependent on injector shape σdet 2 – broadening given by detector cell volume; Y ~ X σcon 2 – broadening given by length and diameter of capillaries : Aris‐Taylor equation σcon 2 – broadening given by length and diameter of capillaries : Aris‐Taylor equation dt – capillary diameter, L – capillary length Fm – flow rate, Dm – diffusion coefficient dt – capillary diameter, L – capillary length Fm – flow rate, Dm – diffusion coefficient rt – capillary diameter u – linear flow rate rt – capillary diameter u – linear flow rate 𝛔 𝐜𝐨𝐧 𝟐 𝛑 · 𝐝𝐭 𝟒 · 𝐋 · 𝐅 𝐦 𝟑𝟖𝟒 · 𝐃 𝐦 𝛔 𝐜𝐨𝐧 𝟐 𝛑 · 𝐝𝐭 𝟒 · 𝐋 · 𝐅 𝐦 𝟑𝟖𝟒 · 𝐃 𝐦 𝛔 𝐜𝐨𝐧 𝟐 𝐮 · 𝐫𝐭 𝟐 · 𝐋 𝟐𝟒𝐃 𝐦 𝛔 𝐜𝐨𝐧 𝟐 𝐮 · 𝐫𝐭 𝟐 · 𝐋 𝟐𝟒𝐃 𝐦 152152 time constantstime constants of detectorof detector of A/D converterof A/D converter samplingsampling (τdet)(τdet) (τA/D)(τA/D) (τSR)(τSR) 𝛔𝐭𝐨𝐭 𝟐 𝛔 𝐆𝐚𝐮𝐬𝐬 𝟐 𝛕 𝟐 𝛔𝐭𝐨𝐭 𝟐 𝛔 𝐆𝐚𝐮𝐬𝐬 𝟐 𝛕 𝟐 τ = 4.0 sτ = 4.0 s 𝛔 𝐀 𝐃⁄ 𝟐 𝛕 𝐀 𝐃⁄ 𝟏𝟐 𝛔 𝐀 𝐃⁄ 𝟐 𝛕 𝐀 𝐃⁄ 𝟏𝟐 τ = 0.5 sτ = 0.5 s high SRhigh SR low SRlow SR for tR ~ min and n = 10 000 is τ ≈ 0.6 s (column 3.9x150 mm and Fm = 1.5 ml∙min‐1) : detectors should generally have τ < 1s, at best around 0.1 s for tR ~ min and n = 10 000 is τ ≈ 0.6 s (column 3.9x150 mm and Fm = 1.5 ml∙min‐1) : detectors should generally have τ < 1s, at best around 0.1 s influences the peak depiction : SR – sampling rate influences the peak depiction : SR – sampling rate noisenoise driftdrift peak heightpeak heightother factorsother factors 153153 detector noisedetector noise noise driftnoise drift determination of extra‐column contributions to zone broadeningdetermination of extra‐column contributions to zone broadening 𝛔𝐭𝐨𝐭 𝟐 𝛔 𝐜𝐨𝐥 𝟐 𝛔 𝐞𝐱𝐭𝐫𝐚𝐜𝐨𝐥 𝟐 𝛔𝐭𝐨𝐭 𝟐 𝛔 𝐜𝐨𝐥 𝟐 𝛔 𝐞𝐱𝐭𝐫𝐚𝐜𝐨𝐥 𝟐 𝐍 𝐭 𝐑 𝛔 𝐜𝐨𝐥 𝟐 𝐍 𝐭 𝐑 𝛔 𝐜𝐨𝐥 𝟐 𝛔𝐭𝐨𝐭 𝟐 𝒇 𝐭 𝐑 𝟐 𝐭 𝐑 𝟐 𝐍 𝛔 𝐆𝐚𝐮𝐬𝐬 𝟐 𝛔𝐭𝐨𝐭 𝟐 𝒇 𝐭 𝐑 𝟐 𝐭 𝐑 𝟐 𝐍 𝛔 𝐆𝐚𝐮𝐬𝐬 𝟐 slope 1/Nslope 1/N σtotσtot σextracolσextracol 22 tRtR 22 22 diode array detector (DAD)diode array detector (DAD)absorption photometric detectorabsorption photometric detector absorbance : noise 10‐4 : dynamic range 105 : sensitivity 10‐9 M absorbance : noise 10‐4 : dynamic range 105 : sensitivity 10‐9 M PDA – photodiode arrayPDA – photodiode array light source light source reference cell reference cell recorderrecorder mirrormirror lenslens monochromatormonochromator semipermeable mirror semipermeable mirror amplifieramplifier photomultiplierphotomultiplier measurement cell measurement cell holographic gridholographic grid photodiode fieldphotodiode field MP outflowMP outflow flow‐through cellflow‐through cell opticsoptics lamplamp CDD signal: light absorbance (B‐L‐B law) CDD signal: light absorbance (B‐L‐B law) 154154 : base line drift at gradient elution : eluate transparency at measuring wavelengths : optic path length is important (detection Z‐cell) : base line drift at gradient elution : eluate transparency at measuring wavelengths : optic path length is important (detection Z‐cell) fluorescence : noise 10‐5 : dynamic range 104 : sensitivity 10‐11 M fluorescence : noise 10‐5 : dynamic range 104 : sensitivity 10‐11 M fluorometric (fluorescence) detectorfluorometric (fluorescence) detector light sourcelight source monochromatormonochromator lenselense measurement cell measurement cell photomultiplierphotomultiplier amplifieramplifier recorderrecorder CDD signal: emission of wavelength λ2 after excitation at λ1 : λ2 > λ1 CDD signal: emission of wavelength λ2 after excitation at λ1 : λ2 > λ1 𝚽 𝐞𝐦𝐢𝐬 𝐤 · 𝚽 𝐞𝐱𝐜𝐢𝐭 · 𝛆 𝛌 · 𝐜 · 𝐥𝚽 𝐞𝐦𝐢𝐬 𝐤 · 𝚽 𝐞𝐱𝐜𝐢𝐭 · 𝛆 𝛌 · 𝐜 · 𝐥 155155 : limited linearity Φ = f (c) for higher concentrations : detector shielding from excitation radiation : optical path length is also important : limited linearity Φ = f (c) for higher concentrations : detector shielding from excitation radiation : optical path length is also important refractometric detectorrefractometric detector measurement cell measurement cell reference cell reference cell photomultiplierphotomultiplier light sourcelight source amplifieramplifier mirrormirror recorderrecorder mirrormirror refraction index : noise 10‐7 : dynamic range 104 : sensitivity 10‐6 M refraction index : noise 10‐7 : dynamic range 104 : sensitivity 10‐6 M CDD signal: light refraction angle (universal detector) CDD signal: light refraction angle (universal detector) : low sensitivity :: depends on difference in sample & MP refraction : high temperature influence : improper for gradient elution : for substances w/o other detection properties : low sensitivity :: depends on difference in sample & MP refraction : high temperature influence : improper for gradient elution : for substances w/o other detection properties amperometry : noise 10‐4 : dynamic range 106 : sensitivity 10‐12 M amperometry : noise 10‐4 : dynamic range 106 : sensitivity 10‐12 M electrochemical detectorelectrochemical detector CDD (amperometry) MDD (coulometry) signal: current coming from redox substance passing the measuring cell CDD (amperometry) MDD (coulometry) signal: current coming from redox substance passing the measuring cell coulometry : noise 10‐5 : dynamic range 107 : sensitivity 10‐14 M coulometry : noise 10‐5 : dynamic range 107 : sensitivity 10‐14 M MP flowMP flow working electrode working electrode reference electrode reference electrode auxiliary electrode auxiliary electrode 156156 carbon Pt, Au, Cu, Hg carbon Pt, Au, Cu, Hg : high sensitivity and response rate (temperature); selective detector – sugars : electrode passivation and consequent cleaning; MP – conductive (so no NP‐HPLC) : destructive detector : high sensitivity and response rate (temperature); selective detector – sugars : electrode passivation and consequent cleaning; MP – conductive (so no NP‐HPLC) : destructive detector conductivity detectorconductivity detector CDD signal: current coming from charged substance passing measuring cell CDD signal: current coming from charged substance passing measuring cell : AC voltage (X polarisation) : lower sensitivity; unspecific detector : MP – non‐conducting (no buffers in MP) : AC voltage (X polarisation) : lower sensitivity; unspecific detector : MP – non‐conducting (no buffers in MP) MP flowMP flow electrodeelectrodeelectrodeelectrode stainless Pt, Au stainless Pt, Au actuator electrode actuator electrode pick‐up electrode pick‐up electrode MP flowMP flow conductivity : noise 10‐3 : dynamic range 106 : sensitivity 10‐9 M conductivity : noise 10‐3 : dynamic range 106 : sensitivity 10‐9 M contactless conductivity detection contactless conductivity detection measured signalmeasured signal transformed into conductivitytransformed into conductivity light‐scattering detectorlight‐scattering detector scattering : noise 10‐8 : dynamic range 104 : sensitive 10‐8 M scattering : noise 10‐8 : dynamic range 104 : sensitive 10‐8 M effluateeffluate pressure regulator pressure regulator inert gasinert gas nebulisernebuliser amplifieramplifier photodetectorphotodetector wastewaste laserlaser analyteanalyte thermostated tube thermostated tube MDD signal: light scattering MDD signal: light scattering 157157 ELSD – evaporative light scattering detectorELSD – evaporative light scattering detector : volatile MP additives :: e.g. ammonium acetate : high sensitivity; universal detector : also for gradient elution : volatile MP additives :: e.g. ammonium acetate : high sensitivity; universal detector : also for gradient elution charged particles flow : noise 10‐6 : dynamic range 105 : sensitivity 10‐9 M charged particles flow : noise 10‐6 : dynamic range 105 : sensitivity 10‐9 M CAD – corona charged aerosol detector CAD – corona charged aerosol detector  corona charged aerosol detector corona charged aerosol detector  MDD signal: cationic current MDD signal: cationic current : volatile MP additives :: e.g. ammonium acetate : high sensitivity : also for gradient elution : volatile MP additives :: e.g. ammonium acetate : high sensitivity : also for gradient elution electrometerelectrometer signalsignal collectorcollector ion trap for fast ions ion trap for fast ions secondary gas charged (+) by corona discharge secondary gas charged (+) by corona discharge analyte molecule charginganalyte molecule charging dryingtubedryingtube waste big drops waste big drops primary gas nebulising primary gas nebulising eluateeluate nebulisernebuliser mass spectrometrymass spectrometry RPLC and NPLC mixing effluate with matrix : discrete points or continuous trace : off‐line or in‐line (endless band) RPLC and NPLC mixing effluate with matrix : discrete points or continuous trace : off‐line or in‐line (endless band) MDD signal: charged particles flow MDD signal: charged particles flow 158158 : improper for quantitation : high sensitivity and selectivity : universal detector : structural information : improper for quantitation : high sensitivity and selectivity : universal detector : structural information ion count : noise 10‐8 : dynamic range 102 – 104 : sensitivity 10‐18 M ion count : noise 10‐8 : dynamic range 102 – 104 : sensitivity 10‐18 M : connecting LC and MS :: (soft) ionisation : connecting LC and MS :: (soft) ionisation matrix assisted laser desorption/ionisation (MALDI)matrix assisted laser desorption/ionisation (MALDI) RPLC and NPLC dopant: acetone, toluene, hexane RPLC and NPLC dopant: acetone, toluene, hexane RPLC, HILIC 1:1 MeOH + strong acid (formic) RPLC, HILIC 1:1 MeOH + strong acid (formic) atmospheric pressure photoionisation (APPI)atmospheric pressure photoionisation (APPI) electrospray ionisation (ESI) : multiply charged ions electrospray ionisation (ESI) : multiply charged ions polar organic MP (HILIC, RPLC) volatile component : ammonium trifluoroacetate polar organic MP (HILIC, RPLC) volatile component : ammonium trifluoroacetate atmospheric chemical photoionisation (APCI) : not so soft ionisation atmospheric chemical photoionisation (APCI) : not so soft ionisation nuclear magnetic resonancenuclear magnetic resonance NMR probeNMR probe measuring cellmeasuring cell RF coilRF coil outlet capillaryoutlet capillary inlet capillaryinlet capillary LC columnLC column LC pumpLC pump injectioninjection wastewaste magnetmagnet : deuterated solvents : polar organic MP (RPLC) :: HTLC, SWLC : deuterated solvents : polar organic MP (RPLC) :: HTLC, SWLC nuclear spin : noise 10‐2 : dynamic range 104 : sensitivity 10‐7 M nuclear spin : noise 10‐2 : dynamic range 104 : sensitivity 10‐7 M CDD signal: spin of nuclear particles in magnetic field CDD signal: spin of nuclear particles in magnetic field 159159 : structural information : very expensive (1 l D20 ~140 EUR, 1 l AcN ~15 EUR) : structural information : very expensive (1 l D20 ~140 EUR, 1 l AcN ~15 EUR) record of LC‐NMRrecord of LC‐NMR allows to isolate part of separated sample (fraction) : separation into groups – fractionation controlled valve preceding waste outlet : leading liquid into collection vials allows to isolate part of separated sample (fraction) : separation into groups – fractionation controlled valve preceding waste outlet : leading liquid into collection vials collection of fractions : in defined time periods :: mixed separation zones :: does not require detector in‐line : at defined change of signal intensity in time :: collection of „pure“ zone collection of fractions : in defined time periods :: mixed separation zones :: does not require detector in‐line : at defined change of signal intensity in time :: collection of „pure“ zone necessity to know the capillary volume between detection cell and outlet necessity to know the capillary volume between detection cell and outlet collection of fractionscollection of fractions 160160 chemical derivatisation of sample before entering detection systemchemical derivatisation of sample before entering detection system : increasing sensitivity or allowing detection at all : increasing resolution or allowing separation at all : supressing unwanted sorption of substance on column : increasing sensitivity or allowing detection at all : increasing resolution or allowing separation at all : supressing unwanted sorption of substance on column derivatisation : change of separated substance elution :: separation efficiency and analysis time derivatisation : change of separated substance elution :: separation efficiency and analysis time sample derivatisationsample derivatisation : uses often autosampler :: derivate must be chemical individual and should be stable enough :: reaction must go on quantitatively and selectively :: reaction need no go on at high rate :: reaction with side products, under mild reaction conditions (pH, temperature) ::: no pre‐separation :: good separation of main product from side ones; different detection properties : uses often autosampler :: derivate must be chemical individual and should be stable enough :: reaction must go on quantitatively and selectively :: reaction need no go on at high rate :: reaction with side products, under mild reaction conditions (pH, temperature) ::: no pre‐separation :: good separation of main product from side ones; different detection properties 161161 : one‐step derivatisation (w/ creation of derivatisation agent in situ) : two‐step derivatisation :: reaction need not result in definitive chemical individual :: reaction need not to be quantitative; good reproducibility necessary :: reaction must go on at high rate, even under extreme conditions (pH, temperature) :: reaction agent surplus → dilution of MP by agent :: reaction may not be selective, side products of reaction are of no harm :: sample is separated in unchanged form :: expensive; special instrumentation and reactors, but automatic : one‐step derivatisation (w/ creation of derivatisation agent in situ) : two‐step derivatisation :: reaction need not result in definitive chemical individual :: reaction need not to be quantitative; good reproducibility necessary :: reaction must go on at high rate, even under extreme conditions (pH, temperature) :: reaction agent surplus → dilution of MP by agent :: reaction may not be selective, side products of reaction are of no harm :: sample is separated in unchanged form :: expensive; special instrumentation and reactors, but automatic : more common in GC or electromigration : more common in GC or electromigration pre‐column derivatisationpre‐column derivatisation post‐column derivatisationpost‐column derivatisation on‐column derivatisationon‐column derivatisation MPMP SPSP mobile phase composition mobile phase composition  flow rate / pressure (ml∙min‐1 / MPa)flow rate / pressure (ml∙min‐1 / MPa) temperature (20 – 60 °C)temperature (20 – 60 °C) load (X μl)load (X μl) detectordetector column (length, inner diameter)column (length, inner diameter) stationary phase type stationary phase type  isocratic: buffer concentration, % content of organic, pH, eventually Iisocratic: buffer concentration, % content of organic, pH, eventually I gradient: gradient profile – A – water component; B – organic component : e.g. 20 % A – 80 % B; 5 min 5 % A – 95 % B, 5 min 100 % B gradient: gradient profile – A – water component; B – organic component : e.g. 20 % A – 80 % B; 5 min 5 % A – 95 % B, 5 min 100 % B trade mark, type, particle size (Nucleosil100, C‐18, 5 μm)trade mark, type, particle size (Nucleosil100, C‐18, 5 μm) length x inner diameter (250x4 mm)length x inner diameter (250x4 mm) basic characteristic according to typebasic characteristic according to type definition of chromatographic system in LCdefinition of chromatographic system in LC 162162 peak area ≈ compound amount (concentration) : occasionally peak height peak area ≈ compound amount (concentration) : occasionally peak height retention time ≈ retention factor, distribution constant : compound identification (standard method) retention time ≈ retention factor, distribution constant : compound identification (standard method) spectroscopic detectors : UV‐Vis spectra : MS spectra (ESI / APCI; Qq / IT / o‐TOF) : NMR spectra (1H, 13C) spectroscopic detectors : UV‐Vis spectra : MS spectra (ESI / APCI; Qq / IT / o‐TOF) : NMR spectra (1H, 13C) : correct method: correct method calibration curve : x = 1  calibration line calibration curve : x = 1  calibration line linearisationlinearisation c – at least three orders of concentrationc – at least three orders of concentration analytical information in chromatogramanalytical information in chromatogram qualitative informationqualitative information quantitative informationquantitative information calibration curve methodcalibration curve method 𝐀 𝐤 · 𝐜 𝐱 𝐀 𝐤 · 𝐜 𝐱 𝐥𝐨𝐠 𝐀 𝐱 · 𝐥𝐨𝐠 𝐜 𝐥𝐨𝐠 𝐤𝐥𝐨𝐠 𝐀 𝐱 · 𝐥𝐨𝐠 𝐜 𝐥𝐨𝐠 𝐤 163163 A [s∙A.U.]A [s∙A.U.] c [M]c [M] y = 0.992∙x + 0.856 R = 0.995 y = 0.992∙x + 0.856 R = 0.995 (spiking)(spiking) assumes linear calibration dependenceassumes linear calibration dependence A1 – analyte peak area, unknown concentration c1 A2 – analyte peak area of unknown concentration c1 after addition of standard of known concentration cS V1 – sample volume, VS – standard solution volume A1 – analyte peak area, unknown concentration c1 A2 – analyte peak area of unknown concentration c1 after addition of standard of known concentration cS V1 – sample volume, VS – standard solution volume checking the calibration dependence linearity : the second addition of standard checking the calibration dependence linearity : the second addition of standard uses presence of substance of constant/defined concentration in chromatogramuses presence of substance of constant/defined concentration in chromatogram absolute quantitationabsolute quantitation A [s*A.U.]A [s*A.U.] c1c1 c [M]c [M] A1A1 A2A2 standard addition methodstandard addition method internal standard methodinternal standard method 𝐜 𝟏 𝐕𝐒 𝐕𝟏 · 𝐜 𝐒 𝐀 𝟐 𝐀 𝟏 · 𝐕𝟏 𝐕𝐒 𝐕𝟏 𝟏 𝐜 𝟏 𝐕𝐒 𝐕𝟏 · 𝐜 𝐒 𝐀 𝟐 𝐀 𝟏 · 𝐕𝟏 𝐕𝐒 𝐕𝟏 𝟏 𝐜 𝐀 𝐀 𝐈𝐒⁄ · 𝐜𝐈𝐒𝐜 𝐀 𝐀 𝐈𝐒⁄ · 𝐜𝐈𝐒 ∆𝐜 𝐀 𝟐 𝐀 𝟏 𝐀 𝐈𝐒⁄∆𝐜 𝐀 𝟐 𝐀 𝟏 𝐀 𝐈𝐒⁄ 164164 dilution influence : signal may decrease after addition :: graphical solution is then not possible dilution influence : signal may decrease after addition :: graphical solution is then not possible relative quantitationrelative quantitation number of in parallel measured values statistical evaluation : ≥ 7 according to Student : 3 – 7 according to Dean‐Dixon data normality test (D'Agostino‐Pearson test) number of in parallel measured values statistical evaluation : ≥ 7 according to Student : 3 – 7 according to Dean‐Dixon data normality test (D'Agostino‐Pearson test) μ – real value σ – xi value distribution around μ P(x) – probability density μ – real value σ – xi value distribution around μ P(x) – probability density μ estimation average μ estimation average for n = 3 median for n = 3 median σ estimation standard deviation σ estimation standard deviation relative standard deviationrelative standard deviation σ estimation Dean‐Dixonσ estimation Dean‐Dixon R – span kn – Dean‐Dixon coefficient R – span kn – Dean‐Dixon coefficient wherewhere accuracy and precisionaccuracy and precision measurement of retention time or peak areameasurement of retention time or peak area𝐱 𝟏 𝐧 · 𝐱 𝐢 𝐧 𝐢 𝟏 𝐱 𝟏 𝐧 · 𝐱 𝐢 𝐧 𝐢 𝟏 𝐬 𝟏 𝐧 · 𝐱𝐢 𝐱 𝟐 𝐧 𝐢 𝟏 𝐬 𝟏 𝐧 · 𝐱𝐢 𝐱 𝟐 𝐧 𝐢 𝟏 𝐬 𝐫 𝐬 𝐱 · 𝟏𝟎𝟎𝐬 𝐫 𝐬 𝐱 · 𝟏𝟎𝟎 𝐬 𝐑 𝐤 𝐧 · 𝐑𝐬 𝐑 𝐤 𝐧 · 𝐑 𝐑 𝐱 𝐦𝐚𝐱 𝐱 𝐦𝐢𝐧𝐑 𝐱 𝐦𝐚𝐱 𝐱 𝐦𝐢𝐧 165165 𝐱 𝐱 𝟐𝐱 𝐱 𝟐 (SD)(SD) (RSD)(RSD) 𝐭 𝛂 𝐭 𝟏 · 𝐬 𝐀 𝟐 𝐧 𝐀 𝟏⁄ 𝐭 𝟐 · 𝐬 𝐁 𝟐 𝐧 𝐁 𝟏⁄ 𝐬 𝐀 𝟐 𝐧 𝐀 𝟏⁄ 𝐬 𝐁 𝟐 𝐧 𝐁 𝟏⁄ 𝐭 𝛂 𝐭 𝟏 · 𝐬 𝐀 𝟐 𝐧 𝐀 𝟏⁄ 𝐭 𝟐 · 𝐬 𝐁 𝟐 𝐧 𝐁 𝟏⁄ 𝐬 𝐀 𝟐 𝐧 𝐀 𝟏⁄ 𝐬 𝐁 𝟐 𝐧 𝐁 𝟏⁄ confidence intervalconfidence interval outlying results Grubbs, T‐test outlying results Grubbs, T‐test L according to Dean‐DixonL according to Dean‐Dixon Kn α – Dean‐Dixon coefficient for αKn α – Dean‐Dixon coefficient for α if Ti < Tα (tabul.) result is not outlying if Ti < Tα (tabul.) result is not outlying Q‐test acc. to Dean‐DixonQ‐test acc. to Dean‐Dixon if Qi < Qα (tabul.) result is not outlying if Qi < Qα (tabul.) result is not outlying result identity Student t‐test result identity Student t‐test Lord u‐testLord u‐test if u or U < uα or Uα (tabul.) results are identical if u or U < uα or Uα (tabul.) results are identical for nA = nB = nfor nA = nB = n for nA ≠ nBfor nA ≠ nB sA 2 ≥ sB 2 → FA = sA 2/sB 2 sB 2 > sA 2 → FB = sB 2/sA 2 sA 2 ≥ sB 2 → FA = sA 2/sB 2 sB 2 > sA 2 → FB = sB 2/sA 2 if FA or B < Fα (tabul.) difference is insignificant if FA or B < Fα (tabul.) difference is insignificant if FA or B > Fα (tabul.) difference is significant if FA or B > Fα (tabul.) difference is significant if t < tα (tabul.) results are identical if t < tα (tabul.) results are identical t1 is tα for ν1 = (nA‐1) t2 is tα for ν2 = (nB‐1) t1 is tα for ν1 = (nA‐1) t2 is tα for ν2 = (nB‐1) Moore U‐testMoore U‐test μ value lies in interval L with probability 1‐α (95 – 99%) μ value lies in interval L with probability 1‐α (95 – 99%) 𝐓𝐢 𝐱 𝐱 𝐢 𝐒 𝐧 𝐓𝐢 𝐱 𝐱 𝐢 𝐒 𝐧 𝐬 𝟏 𝐧 · 𝐱𝐢 𝐱 𝟐 𝐧 𝐢 𝟏 𝐬 𝟏 𝐧 · 𝐱𝐢 𝐱 𝟐 𝐧 𝐢 𝟏 𝐐𝐢 𝐱 𝐢 𝐱 𝐢 𝟏 𝐑 𝐐𝐢 𝐱 𝐢 𝐱 𝐢 𝟏 𝐑 𝐮 𝐱 𝐀 𝐱 𝐁 𝐑 𝐀 𝐑 𝐁 𝐮 𝐱 𝐀 𝐱 𝐁 𝐑 𝐀 𝐑 𝐁 𝐭 𝐱 𝐀 𝐱 𝐁 · 𝐧 𝟏 𝐬 𝐀 𝟐 𝐬 𝐁 𝟐 𝐭 𝐱 𝐀 𝐱 𝐁 · 𝐧 𝟏 𝐬 𝐀 𝟐 𝐬 𝐁 𝟐 𝐋 𝐱 𝐬 · 𝐭 𝛂 𝐧 𝐋 𝐱 𝐬 · 𝐭 𝛂 𝐧 𝐋 𝐱 𝐊 𝐧 𝛂 · 𝐑𝐋 𝐱 𝐊 𝐧 𝛂 · 𝐑 𝐔 𝐱 𝐀 𝐱 𝐁 𝐑 𝐀 𝐑 𝐁 𝐔 𝐱 𝐀 𝐱 𝐁 𝐑 𝐀 𝐑 𝐁 𝐭 𝐱 𝐀 𝐱 𝐁 𝐬 𝐀 𝟐 𝐧 𝐀 𝟏⁄ 𝐬 𝐁 𝟐 𝐧 𝐁 𝟏⁄ 𝐭 𝐱 𝐀 𝐱 𝐁 𝐬 𝐀 𝟐 𝐧 𝐀 𝟏⁄ 𝐬 𝐁 𝟐 𝐧 𝐁 𝟏⁄ i = 2 or ni = 2 or n 166166 analysis of variance (ANOVA)analysis of variance (ANOVA) comparing more data sets (>2) of retention times or areas : day‐to‐day repeatability (inter‐day repeatability) : reproducibility, inter‐laboratory tests comparing more data sets (>2) of retention times or areas : day‐to‐day repeatability (inter‐day repeatability) : reproducibility, inter‐laboratory tests null hypothesis H0 : average values of individual samples are same (μ0 = μ1 = μ2 … μk) null hypothesis H0 : average values of individual samples are same (μ0 = μ1 = μ2 … μk) : we need to know, if the influence of some factor (different day of measurement), which assumes different values, on our studied quantity (tR) is statistically relevant : we need to know, if the influence of some factor (different day of measurement), which assumes different values, on our studied quantity (tR) is statistically relevant test on data homoscedasticity (same σ value in frame of individual days) : if not suited → non‐parametric Kruskal‐Wallis ANOVA test on data homoscedasticity (same σ value in frame of individual days) : if not suited → non‐parametric Kruskal‐Wallis ANOVA in case of null hypothesis H0 invalidity additive tests : Bonferroni test (tests on identity of averages) : Leven test (tests on identity of variance) in case of null hypothesis H0 invalidity additive tests : Bonferroni test (tests on identity of averages) : Leven test (tests on identity of variance) programmes used – Statistica, M$ Excell, SPSS, R…programmes used – Statistica, M$ Excell, SPSS, R… repeatability and reproducibilityrepeatability and reproducibility 167167 linear regression : x value is not subjected to error linear regression : x value is not subjected to error Y = a∙X + bY = a∙X + b standard deviationsstandard deviations correlation coefficientcorrelation coefficient confidence intervalconfidence interval outlying results Grubbs, T‐test outlying results Grubbs, T‐test segment significancesegment significance for regression parametersfor regression parameters for given xfor given x for individual valuefor individual value analyte concentration determinationanalyte concentration determination 𝐚 ∑ 𝐱 · ∑ 𝐲 𝐧 · ∑ 𝐱 · 𝐲 ∑ 𝐱 𝟐 𝐧 · ∑ 𝐱 𝟐 𝐚 ∑ 𝐱 · ∑ 𝐲 𝐧 · ∑ 𝐱 · 𝐲 ∑ 𝐱 𝟐 𝐧 · ∑ 𝐱 𝟐 𝐛 𝟏 𝐧 · 𝐲 𝐚 · 𝐱𝐛 𝟏 𝐧 · 𝐲 𝐚 · 𝐱 𝐫𝐱𝐲 𝐧 · ∑ 𝐱 · 𝐲 ∑ 𝐱 · ∑ 𝐲 𝐧 · ∑ 𝐱 𝟐 ∑ 𝐱 𝟐 · 𝐧 · ∑ 𝐲 𝟐 ∑ 𝐲 𝟐 𝐫𝐱𝐲 𝐧 · ∑ 𝐱 · 𝐲 ∑ 𝐱 · ∑ 𝐲 𝐧 · ∑ 𝐱 𝟐 ∑ 𝐱 𝟐 · 𝐧 · ∑ 𝐲 𝟐 ∑ 𝐲 𝟐 𝐋 𝐛 𝐛 𝐬 𝐛 · 𝐭 𝛂𝐋 𝐛 𝐛 𝐬 𝐛 · 𝐭 𝛂 𝐋 𝐘 𝐭 𝛂 · 𝐬 𝐲𝐱 · 𝟏 𝐧 𝐗 𝐱 𝟐 ∑ 𝐱 𝐢 𝟐 ∑ 𝐱 𝐢 𝟐 𝐧⁄ 𝐋 𝐘 𝐭 𝛂 · 𝐬 𝐲𝐱 · 𝟏 𝐧 𝐗 𝐱 𝟐 ∑ 𝐱 𝐢 𝟐 ∑ 𝐱 𝐢 𝟐 𝐧⁄ 𝐭 𝐛 𝐬 𝐛 𝐭 𝐛 𝐬 𝐛 𝐓 𝐘𝐢 𝐲𝐢 𝐬 𝐲𝐱 · 𝐧 𝐧 𝟐 𝐓 𝐘𝐢 𝐲𝐢 𝐬 𝐲𝐱 · 𝐧 𝐧 𝟐 𝐋 𝐘 𝐭 𝛂 · 𝐬 𝐲𝐱 · 𝟏 𝐧 𝐗 𝐱 𝟐 ∑ 𝐱𝐢 𝐱 𝟐 𝐋 𝐘 𝐭 𝛂 · 𝐬 𝐲𝐱 · 𝟏 𝐧 𝐗 𝐱 𝟐 ∑ 𝐱𝐢 𝐱 𝟐 𝐋 𝐚 𝐛 𝐬 𝐚 · 𝐭 𝛂𝐋 𝐚 𝐛 𝐬 𝐚 · 𝐭 𝛂 𝐬 𝐲𝐱 ∑ 𝐲𝐢 𝐘𝐢 𝟐 𝐧 𝟐 𝐬 𝐲𝐱 ∑ 𝐲𝐢 𝐘𝐢 𝟐 𝐧 𝟐 𝐬 𝐚 𝐬 𝐲𝐱 ∑ 𝐱 𝐢 𝐱 𝟐 𝐬 𝐚 𝐬 𝐲𝐱 ∑ 𝐱 𝐢 𝐱 𝟐 𝐬 𝐛 𝐬 𝐲𝐱 · 𝟏 𝐧 𝐱 𝟐 ∑ 𝐱𝐢 𝐱 𝟐 𝐬 𝐛 𝐬 𝐲𝐱 · 𝟏 𝐧 𝐱 𝟐 ∑ 𝐱𝐢 𝐱 𝟐 tα for ν = (n‐2) insignificant t < tα tα for ν = (n‐2) insignificant t < tα 168168 limit of detectionlimit of detection from value s0 of background x0from value s0 of background x0 from confidence intervalfrom confidence interval from linear regression, b ≠ 0 from linear regression, b ≠ 0  optimally: by sequential dilution till disappearance of the observable signaloptimally: by sequential dilution till disappearance of the observable signal c – known, low concentration H – height of peak; noise has no area c – known, low concentration H – height of peak; noise has no area time [min]time [min] absorbanceabsorbance s0 – standard deviation of x0s0 – standard deviation of x0 –– from linear regression, b = 0 from linear regression, b = 0  in HPLCin HPLC 𝐋𝐎𝐃 𝐱 𝟎 𝐰 · 𝐬 𝟎𝐋𝐎𝐃 𝐱 𝟎 𝐰 · 𝐬 𝟎 𝐱 𝟎 𝐡𝐢 𝐧 𝐢 𝟏 𝐧 𝟎𝐱 𝟎 𝐡𝐢 𝐧 𝐢 𝟏 𝐧 𝟎 𝐋𝐎𝐃 𝟑 · 𝐜 𝐇 · 𝐬 𝟎𝐋𝐎𝐃 𝟑 · 𝐜 𝐇 · 𝐬 𝟎 𝐋𝐎𝐃 𝟑 · 𝐬 𝟎 𝐚 𝐋𝐎𝐃 𝟑 · 𝐬 𝟎 𝐚 𝐋𝐎𝐃 𝐛 𝟑 · 𝐬 𝐛 𝐚 𝐋𝐎𝐃 𝐛 𝟑 · 𝐬 𝐛 𝐚 𝐋𝐎𝐃 𝐭 𝛂 · 𝐬 𝐚 𝐭 𝛂 · 𝐬 𝐲𝐱 · 𝟏 𝐧 𝐱 𝟐 ∑ 𝐱 𝐢 𝐱 𝟐 𝐋𝐎𝐃 𝐭 𝛂 · 𝐬 𝐚 𝐭 𝛂 · 𝐬 𝐲𝐱 · 𝟏 𝐧 𝐱 𝟐 ∑ 𝐱 𝐢 𝐱 𝟐 169169 (LOD)(LOD) : the lowest relevant detectable amount: the lowest relevant detectable amount limit of quantificationlimit of quantification upper limit of detectionupper limit of detection :: higher content than 99.85 % is not possible to determine directly:: higher content than 99.85 % is not possible to determine directly 𝐔𝐋𝐎𝐃 𝟏𝟎𝟎 𝐰 · 𝐬 𝟎𝐔𝐋𝐎𝐃 𝟏𝟎𝟎 𝐰 · 𝐬 𝟎 𝐋𝐎𝐐 𝟏𝟎 · 𝐬 𝟎 𝐚 𝐋𝐎𝐐 𝟏𝟎 · 𝐬 𝟎 𝐚 𝐋𝐎𝐐 𝐛 𝟏𝟎 · 𝐬 𝐛 𝐚 𝐋𝐎𝐐 𝐛 𝟏𝟎 · 𝐬 𝐛 𝐚 170170 (ULOD)(ULOD) : the highest relevant detectable amount: the highest relevant detectable amount tR [min]tR [min] signal intensitysignal intensity (LOQ)(LOQ) : the lowest relevant determinable amount: the lowest relevant determinable amount consists of two steps : first measured concentration; calculate sx1 here we can end and calculate MDL but better to approve the result: : next concentration, different from first, but similar; calculate sx2 : conduct F‐test on identity of both sets if F < Fα calculate sX12 and MDL consists of two steps : first measured concentration; calculate sx1 here we can end and calculate MDL but better to approve the result: : next concentration, different from first, but similar; calculate sx2 : conduct F‐test on identity of both sets if F < Fα calculate sX12 and MDL the lowest determinable concentration on a level of significance α = 0.01 in a sample with given matrixthe lowest determinable concentration on a level of significance α = 0.01 in a sample with given matrix : uses a low concentration : includes of steps of sample preparation : includes matrix : uses a low concentration : includes of steps of sample preparation : includes matrix approach is burdened by presumption of same dispersion all over the concentration scaleapproach is burdened by presumption of same dispersion all over the concentration scale method detection limitmethod detection limit 𝐌𝐃𝐋 𝐭 𝐧,𝛂 · 𝐬 𝐱 𝟏 𝐌𝐃𝐋 𝐭 𝐧,𝛂 · 𝐬 𝐱 𝟏 𝐬 𝐱 𝟏𝟐 𝟐 𝐧 𝟏 · 𝐬 𝐱 𝟏 𝟐 𝐧 𝟐 𝐬 𝐱 𝟐 𝟐 𝐧 𝟏 𝐧 𝟐 𝐬 𝐱 𝟏𝟐 𝟐 𝐧 𝟏 · 𝐬 𝐱 𝟏 𝟐 𝐧 𝟐 𝐬 𝐱 𝟐 𝟐 𝐧 𝟏 𝐧 𝟐 𝐌𝐃𝐋 𝐭 𝐧 𝟏 𝐧 𝟐,𝛂 · 𝐬 𝐱 𝟏𝟐 𝐌𝐃𝐋 𝐭 𝐧 𝟏 𝐧 𝟐,𝛂 · 𝐬 𝐱 𝟏𝟐 where n is number of degrees of freedomwhere n is number of degrees of freedom 171171 (MDL) (MDL)  method according to Hubaux‐Vosmethod according to Hubaux‐Vos works with confidence interval of linear regression of calibration dependence : to construct the calibration curve using linear regression : to calculate MDL as a xMDL value of confidence interval for x = 0 or to determine it graphically works with confidence interval of linear regression of calibration dependence : to construct the calibration curve using linear regression : to calculate MDL as a xMDL value of confidence interval for x = 0 or to determine it graphically for b ≠ 0for b ≠ 0 method of a use of standard deviation estimation of segment bmethod of a use of standard deviation estimation of segment b RDL (reliable detection limit) : the highest estimation of detection limit RDL (reliable detection limit) : the highest estimation of detection limit 𝐌𝐃𝐋 𝐛 𝐭 𝛂 · 𝐬 𝐛𝐌𝐃𝐋 𝐛 𝐭 𝛂 · 𝐬 𝐛 𝐌𝐃𝐋 𝟏 𝐚 · 𝐭 𝐧,𝛂 · 𝐬 𝐲𝐱 · 𝟏 𝐧 𝐌𝐃𝐋 𝟏 𝐚 · 𝐭 𝐧,𝛂 · 𝐬 𝐲𝐱 · 𝟏 𝐧 172172 yy xxMDLMDL RDLRDL check and assurance of reproducible conditions for separationcheck and assurance of reproducible conditions for separation parameters of material : pore volume, pore size, surface area, carbon content, modified surface coverage, end‐capping separation parameters : resolution, selectivity, void volume, efficiency other properties : inertness, hydrophobicity, metal ions influence, longevity parameters of material : pore volume, pore size, surface area, carbon content, modified surface coverage, end‐capping separation parameters : resolution, selectivity, void volume, efficiency other properties : inertness, hydrophobicity, metal ions influence, longevity stationary phase is manufactured in series we check : physical state : chemical state : topology reproducibility of SP : manufacturer‐to‐manufacturer : batch‐to‐batch stationary phase is manufactured in series we check : physical state : chemical state : topology reproducibility of SP : manufacturer‐to‐manufacturer : batch‐to‐batch evaluation of separation efficiencyevaluation of separation efficiency 173173 testing method : method of performance measurement : standard of good behaviour, which is comparable : universal test of good behaviour testing method : method of performance measurement : standard of good behaviour, which is comparable : universal test of good behaviour what must the test fulfilled : simplicity : illustrativeness : instructiveness what must the test fulfilled : simplicity : illustrativeness : instructiveness test measurements in LCtest measurements in LC 174174 observable separation parameters (one analyte) : N – number of theoretical plates, tR – retention time, Δp – pressure change observable separation parameters (one analyte) : N – number of theoretical plates, tR – retention time, Δp – pressure change 𝛑 𝐍 𝟐 𝐭 𝐑 · ∆𝐩⁄ 𝐍 𝐭 𝐑⁄ · 𝐍 ∆𝐩⁄𝛑 𝐍 𝟐 𝐭 𝐑 · ∆𝐩⁄ 𝐍 𝐭 𝐑⁄ · 𝐍 ∆𝐩⁄ π  (103 – 105), [tR] = s, [Δp] = MPa increase of η or k' → decrease of π w/ constant N η – viscosity MP, k' – capacity factor π  (103 – 105), [tR] = s, [Δp] = MPa increase of η or k' → decrease of π w/ constant N η – viscosity MP, k' – capacity factor index of good behaviour πindex of good behaviour π h – reduced height of theoretical plate Φ – column resistance parameter h – reduced height of theoretical plate Φ – column resistance parameter increasing E → behaviour deterioration increasing E → behaviour deterioration separation impedance Eseparation impedance E 𝐄 𝛑 · 𝛈 · 𝟏 𝐤 𝟏 𝐄 𝛑 · 𝛈 · 𝟏 𝐤 𝟏 𝐄 𝐇 𝟐 𝐊⁄ 𝐡 𝟐 · 𝚽𝐄 𝐇 𝟐 𝐊⁄ 𝐡 𝟐 · 𝚽 𝐄 𝐭 𝐑 · ∆𝐩 · 𝐍 𝟐 · 𝛈 · 𝟏 𝐤𝐄 𝐭 𝐑 · ∆𝐩 · 𝐍 𝟐 · 𝛈 · 𝟏 𝐤 void volume determinationvoid volume determination non‐interacting substance : runs through the system along with front of MP non‐interacting substance : runs through the system along with front of MP normal phase – metaxylene reversed phase – thiourine, aceton ionex – uncharged or same charge substance normal phase – metaxylene reversed phase – thiourine, aceton ionex – uncharged or same charge substance column testingcolumn testing testing mixture: contains series of substances with increasing retentiontesting mixture: contains series of substances with increasing retention normal phase – metaxylene, nitrobenzene reversed phase – aceton/uracil, benzene, toluene, naphthalene catex – uracil, cytosine anex – uridine, uridine monophosphate normal phase – metaxylene, nitrobenzene reversed phase – aceton/uracil, benzene, toluene, naphthalene catex – uracil, cytosine anex – uridine, uridine monophosphate in dependence on time (at constant flow rate) we observe : retention times of components : number of theoretical plates : symmetry of peaks : selectivity factor in dependence on time (at constant flow rate) we observe : retention times of components : number of theoretical plates : symmetry of peaks : selectivity factor column damaged by pressure, non‐additive void volume column damaged by pressure, non‐additive void volume efficiencyefficiency 175175 so‐called system peakso‐called system peak state of free silanols/end‐cappingstate of free silanols/end‐capping testing mixture: pyridine, phenol, toluene MP: acetonitrile : water 1:1 testing mixture: pyridine, phenol, toluene MP: acetonitrile : water 1:1 positive outcome: pyridine (charged) is eluted before phenol negative outcome: pyridine is eluted after phenol or is broadening positive outcome: pyridine (charged) is eluted before phenol negative outcome: pyridine is eluted after phenol or is broadening sensitivity to methylene groupssensitivity to methylene groups testing mixture: butylbenzene, amylobenzene MP: acetonitrile : water 4:1 testing mixture: butylbenzene, amylobenzene MP: acetonitrile : water 4:1 positive outcome: high selectivity (α)positive outcome: high selectivity (α) influence of heavy metal ions (DERT, dihydroxynaphthalene efficiency ratio test) influence of heavy metal ions (DERT, dihydroxynaphthalene efficiency ratio test) testing mixture: 2,7‐dihydroxynaphthalene, 2,3‐dihydroxynaphthalene MP: acetonitrile : water 1:1 testing mixture: 2,7‐dihydroxynaphthalene, 2,3‐dihydroxynaphthalene MP: acetonitrile : water 1:1 positive outcome: 2,3‐dihydroxynaphthalene (chelator) is not broadening : asymmetry ratio of both peaks should be ~ 1.0 positive outcome: 2,3‐dihydroxynaphthalene (chelator) is not broadening : asymmetry ratio of both peaks should be ~ 1.0 other properties of SP reversed phase other properties of SP reversed phase inertnessinertness hydrophobicityhydrophobicity sensitivity to metal ionssensitivity to metal ions 176176 test on β‐blocker separationtest on β‐blocker separation testing mixture: pindol, metoprolol, propranolol MP: 25 mM P, pH = 9.75, AcN/MeOH 35:10:55 testing mixture: pindol, metoprolol, propranolol MP: 25 mM P, pH = 9.75, AcN/MeOH 35:10:55 we observe: selectivity (α), resolution and peak asymmetry : difficult separation; complex influence of column state we observe: selectivity (α), resolution and peak asymmetry : difficult separation; complex influence of column state : regular testing (before measurement day and after) and records about it : MP filtering and degassing : not to expose column to harming conditions :: not to overload column by high pressure :: extreme pH only for a short period of time (max hours) :: max one night with MP w/ content of organic phase lower than 60 % AcN (70 % MeOH) :: after measurement on border limits (pH, salts, temperature) wash thoroughly ::: 5 % organic phase and then keeping MP column revitalisation : turn column up‐side down, MP flow from the other side : 5 % AcN, 60 % AcN, 100 % IPA or THF, 60 % AcN : regular testing (before measurement day and after) and records about it : MP filtering and degassing : not to expose column to harming conditions :: not to overload column by high pressure :: extreme pH only for a short period of time (max hours) :: max one night with MP w/ content of organic phase lower than 60 % AcN (70 % MeOH) :: after measurement on border limits (pH, salts, temperature) wash thoroughly ::: 5 % organic phase and then keeping MP column revitalisation : turn column up‐side down, MP flow from the other side : 5 % AcN, 60 % AcN, 100 % IPA or THF, 60 % AcN principles of SP storageprinciples of SP storage longevitylongevity 177177 column care guidecolumn care guide counter‐current liquid chromatography (CCC) normal‐phase liquid chromatography (NP‐HPLC, NPLC) reversed‐phase liquid chromatography (RP‐HPLC, RPLC) : ion‐pairing (IP‐RPLC) : ultra‐performance (UPLC) : high‐temperature (HTLC) : ultra‐performance at elevated temperature (ET‐UPLC) hydrophilic interaction liquid chromatography (HILIC) hydrophobic interaction liquid chromatography (HIC) counter‐current liquid chromatography (CCC) normal‐phase liquid chromatography (NP‐HPLC, NPLC) reversed‐phase liquid chromatography (RP‐HPLC, RPLC) : ion‐pairing (IP‐RPLC) : ultra‐performance (UPLC) : high‐temperature (HTLC) : ultra‐performance at elevated temperature (ET‐UPLC) hydrophilic interaction liquid chromatography (HILIC) hydrophobic interaction liquid chromatography (HIC) basic modes of liquid chromatographybasic modes of liquid chromatography VII.VII. 178178 ion‐exchange liquid chromatography (IEC) : ion exclusion chromatography (IXC) : chromatofocusing (CF) affinity chromatography (AC) : with immobilised metal ion (IMAC) super‐critical fluid chromatography (SFC) perfusion chromatography (PLC) chiral chromatography (XLC) planar chromatography (TLC) ion‐exchange liquid chromatography (IEC) : ion exclusion chromatography (IXC) : chromatofocusing (CF) affinity chromatography (AC) : with immobilised metal ion (IMAC) super‐critical fluid chromatography (SFC) perfusion chromatography (PLC) chiral chromatography (XLC) planar chromatography (TLC) mode % of use RPLC 36 IEC 18 NPLC 10 SEC 10 HILIC 8 XLC 7 HIC 2 AC 2 method was discovered in 1949 : both phases are realised by immiscible liquids of different density : gravitational & centrifugal arrangement method was discovered in 1949 : both phases are realised by immiscible liquids of different density : gravitational & centrifugal arrangement centrifugal counter‐current chromatography (CCCC)centrifugal counter‐current chromatography (CCCC) mobile phasemobile phase stationary phasestationary phase pressurepressure counter‐current chromatography (CCC)counter‐current chromatography (CCC) 179179 : hydrodynamic equilibrium with variable acceleration field :: Archimedean screw principle – biaxial rotation : system pressure max 2.5 MPa : high separation efficiency for low KD & short elution times : low SP stability & worse repeatability :: SP retention depends on angular velocity & flow rate : hydrodynamic equilibrium with variable acceleration field :: Archimedean screw principle – biaxial rotation : system pressure max 2.5 MPa : high separation efficiency for low KD & short elution times : low SP stability & worse repeatability :: SP retention depends on angular velocity & flow rate high speed CCC (HSCCC) : the most frequent contemporary variant of CCC :: ω up to 280G; 200 g of material per day : three columns on planetary arrangement (J‐type) high speed CCC (HSCCC) : the most frequent contemporary variant of CCC :: ω up to 280G; 200 g of material per day : three columns on planetary arrangement (J‐type) gravitational CCCgravitational CCC SPSP MPMP 180180 centrifugal partition chromatography (CPC)centrifugal partition chromatography (CPC) : hydrostatic equilibrium with constant acceleration field :: achieved by flowing MP, not SF : centrifugal force at monoaxial rotation holds liquid SP : MP is pushed though it :: system pressures ca 4 GPa (900G) : low separation efficiency, but still usable :: high flow rates & high capacity (~ 30 kg per day) : very quick wear of expensive rotation seals : hydrostatic equilibrium with constant acceleration field :: achieved by flowing MP, not SF : centrifugal force at monoaxial rotation holds liquid SP : MP is pushed though it :: system pressures ca 4 GPa (900G) : low separation efficiency, but still usable :: high flow rates & high capacity (~ 30 kg per day) : very quick wear of expensive rotation seals SPSP MPMP FGFG descending mode (MP heavier)descending mode (MP heavier) ascending mode (MP lighter)ascending mode (MP lighter) stationary phase : silica : modified silica (amido‐, amino‐, cyano‐) : polymer SP (polymethyl methacrylates, divinylbenzene) stationary phase : silica : modified silica (amido‐, amino‐, cyano‐) : polymer SP (polymethyl methacrylates, divinylbenzene) increasing retention on NPincreasing retention on NP increasing retention on RPincreasing retention on RP mobile phase : organic solvents :: n‐hexane, heptane, chloroform, alcohols : additives modify selectivity :: ion‐pairing NPLC (IP‐NPLC) :: diethanolamine, trifluoracetic acid :: (monovalent) salts – LiCl, NaCl, AgNO3 mobile phase : organic solvents :: n‐hexane, heptane, chloroform, alcohols : additives modify selectivity :: ion‐pairing NPLC (IP‐NPLC) :: diethanolamine, trifluoracetic acid :: (monovalent) salts – LiCl, NaCl, AgNO3 normal‐phase high‐performance liquid chromatography (NP‐HPLC)normal‐phase high‐performance liquid chromatography (NP‐HPLC) 181181 aqueous normal phase LC (ANP) : special SP (hydride Si‐H, sometimes Si‐COOH or Si‐alkyl) :: works also as a RPLC : mobile phase contains also water :: > 60 % of organic phase aqueous normal phase LC (ANP) : special SP (hydride Si‐H, sometimes Si‐COOH or Si‐alkyl) :: works also as a RPLC : mobile phase contains also water :: > 60 % of organic phase stable surface  : controlled modification shields silica end‐capping of free silanols : organic amides, trimethylsiloxanes stable surface  : controlled modification shields silica end‐capping of free silanols : organic amides, trimethylsiloxanes : (C1, C2, C4,) C8, C12, C18, C30, phenyl, perfluorophenyl (PFP) :: + variations (polar group spacing) : (C1, C2, C4,) C8, C12, C18, C30, phenyl, perfluorophenyl (PFP) :: + variations (polar group spacing) secondary silanolisation : polar sample, non‐polar eluent secondary silanolisation : polar sample, non‐polar eluent 1950 – RPLC description1950 – RPLC description reversed‐phase high‐performance liquid chromatography (RP‐HPLC)reversed‐phase high‐performance liquid chromatography (RP‐HPLC) 182182 stationary phasestationary phase : we add modifier into MP :: chiral selector ::: chiral LC :: pH change ::: ion‐suppression RP‐HPLC : we add modifier into MP :: chiral selector ::: chiral LC :: pH change ::: ion‐suppression RP‐HPLC :: surfactant ::: micellar RP‐HPLC (ctens > CMC; micellar liquid chromatography MLC) ::: ion‐pairing RP‐HPLC (quasi‐ionex) :: surfactant ::: micellar RP‐HPLC (ctens > CMC; micellar liquid chromatography MLC) ::: ion‐pairing RP‐HPLC (quasi‐ionex) :: sizable ion ::: ion interaction RP‐HPLC :: sizable ion ::: ion interaction RP‐HPLC – +SP – +SP analyteanalyte analyteanalyte interacting surfactantinteracting surfactant modifiermodifier 183183 easy adaptation of SP on different LC type easy adaptation of SP on different LC type  : so‐called non‐interaction mode of RPLC :: non‐interaction conditions – very high elution force ::: size/molecular exclusion LC (separates sccording to size) :::: sieving effect : so‐called non‐interaction mode of RPLC :: non‐interaction conditions – very high elution force ::: size/molecular exclusion LC (separates sccording to size) :::: sieving effect mobile phasemobile phase water – in RP low elution force increasing portion of organic phase  decreasing retention time (decreasing surface tension) : organic phase must be 100% miscible with water : onto column always min 5% or max 95 % organic phase in water :: SP dewetting water – in RP low elution force increasing portion of organic phase  decreasing retention time (decreasing surface tension) : organic phase must be 100% miscible with water : onto column always min 5% or max 95 % organic phase in water :: SP dewetting solvent  k with  content in 10% water – dimethyl sulphate (DMS) 1.5 x ethylene glycol 1.5 x acetonitrile (AcN) 2.0 x methanol (MeOH) 2.0 x acetone 2.2 x dioxane 2.2 x ethanol (EtOH) 2.3 x tetrahydrofuran (THF) 2.8 x isopropanol 3.0 x 75 % MeOH, 25 % H2O75 % MeOH, 25 % H2O 60 % MeOH, 40 % H2O60 % MeOH, 40 % H2O 50 % MeOH, 50 % H2O50 % MeOH, 50 % H2O 184184 dewetted SPdewetted SP viscosityviscosity % water content% water content ethanolethanol methanolmethanol acetoneacetone acetonitrileacetonitrile MP composition influence on its viscosity  MP composition influence on its viscosity  content of organic component changes MP viscosity : different polarity of both components : increasing of pressure in system problem within gradient elution : increased temperature decreases viscosity content of organic component changes MP viscosity : different polarity of both components : increasing of pressure in system problem within gradient elution : increased temperature decreases viscosity solvent viscosity [mP∙s] at 20 °C hexane 0.29 acetone 0.32 acetonitrile 0.34 THF 0.46 methanol 0.54 DMF 0.80 ethanol 1.08 isopropanol 1.90 185185 00 55 25251010 1515 2020 time [min]time [min] MP preparation influence on separation efficiency MP preparation influence on separation efficiencyvolumetric flask (1 l) w/ 400 ml of water : filled with MeOH up to scale line : 635 ml of MeOH → 1.59 : 1.00 volumetric flask (1 l) w/ 400 ml of water : filled with MeOH up to scale line : 635 ml of MeOH → 1.59 : 1.00 mixing 400 ml of water and 600 ml of MeOH : 400 ml of water → 1.50 : 1.00 :: resulting volume 965 ml mixing 400 ml of water and 600 ml of MeOH : 400 ml of water → 1.50 : 1.00 :: resulting volume 965 ml high‐pressure mixing of MP : A – water 0.4 & B – MeOH 0.6 ml∙min‐1 : ~ 1.45 : 1.00 high‐pressure mixing of MP : A – water 0.4 & B – MeOH 0.6 ml∙min‐1 : ~ 1.45 : 1.00 volumetric flask (1 l) w/ 600 ml of MeOH : filled with water up to scale line : 435 ml of water → 1.38 : 1.00 volumetric flask (1 l) w/ 600 ml of MeOH : filled with water up to scale line : 435 ml of water → 1.38 : 1.00 186186 volumecontractionvolumecontraction MeOHMeOH waterwater %% pH influencepH influence aqueous MP constituent : buffer (reproducible pH) :: concentration 50 mM and lower (system salinisation) :: always check solubility in given MP (influence of org. phase) addition of neutral salt increases surface tension a that increases retention aqueous MP constituent : buffer (reproducible pH) :: concentration 50 mM and lower (system salinisation) :: always check solubility in given MP (influence of org. phase) addition of neutral salt increases surface tension a that increases retention it is necessary to adjust pH of aqueous constituent of MP : change in 0.1 pH has a strong influence on retention it is necessary to adjust pH of aqueous constituent of MP : change in 0.1 pH has a strong influence on retention influences ionisable analytes (organic acids and bases) and polarisable analytesinfluences ionisable analytes (organic acids and bases) and polarisable analytes 187187 in RP – suppress ionisation any time it is possiblein RP – suppress ionisation any time it is possible selectivityselectivity 30% AcN 70% water 30% AcN 70% water 45% MeOH 55% water 45% MeOH 55% water 300x4.6 mm C‐18, 1.5 ml∙min‐1, detection 254 nm, 10 mg load300x4.6 mm C‐18, 1.5 ml∙min‐1, detection 254 nm, 10 mg load choice of MP according to elution force and interaction type (Snyder's triangle)choice of MP according to elution force and interaction type (Snyder's triangle) 1. hydrocortison, succinate 2. methylparaben 3. hydrocortison 4. propylparaben 5. hydrocortison, actate 6. cortison, acetate 1. hydrocortison, succinate 2. methylparaben 3. hydrocortison 4. propylparaben 5. hydrocortison, actate 6. cortison, acetate [min][min] [min][min] 188188 Snyder's triangleSnyder's triangle for interactions on RPfor interactions on RP change in content and type of organic phase means change of retentionchange in content and type of organic phase means change of retention type of organic phase : favours different interactions type of organic phase : favours different interactions content of organic phase : amplifies given interaction content of organic phase : amplifies given interaction 189189 proton‐acceptorproton‐acceptor proton‐ donor proton‐ donor dipole‐ dipole dipole‐ dipole triethylaminetriethylamine ethersethers tetrahydrofurantetrahydrofuran acetic acidacetic acid waterwater chloroformchloroform dichlormethanedichlormethane dichloretandichloretan benzene & derivatives benzene & derivatives nitromethanenitromethane acetonitrilacetonitril alcoholsalcohols acetoneacetone dioxanedioxane estersesters DMSO, pyridineDMSO, pyridine acidity α acidity α basicity β basicity β polarity πpolarity π THFTHF MeOHMeOH AcNAcN solvent‐buffer mixture w/ similar retention solvent‐buffer mixture w/ similar retention binary mixturebinary mixture quaternaryquaternary 50:5050:50 30:7030:70 1:1:11:1:1 ternaryternary 40:6040:60 1:11:1 A – ideal separationA – ideal separation B – high elution forceB – high elution force C – low elution forceC – low elution force D – improper column or flow rateD – improper column or flow rate E – improper column or flow rateE – improper column or flow rate m – individual parameter of each analyte in system log k0 – logarithm of extrapolated k value for water as MP φ – voluminal ratio of organic phase m – individual parameter of each analyte in system log k0 – logarithm of extrapolated k value for water as MP φ – voluminal ratio of organic phase 190190 AA BB CC DD EE isocratic elution modeisocratic elution mode Snyder & Soczewiński (logarithmic) equationSnyder & Soczewiński (logarithmic) equation Soczewiński‐Wachtmeister (semi‐logarithmic) equationSoczewiński‐Wachtmeister (semi‐logarithmic) equation 𝟎𝟎 𝟎𝟎 gradient steepness bgradient steepness b retention time dependence on gradient steepness and profileretention time dependence on gradient steepness and profile tG – time length of gradient Δφ – change of voluminal ratio of organic phase tG – time length of gradient Δφ – change of voluminal ratio of organic phase td – hold‐up or dwell time of gradienttd – hold‐up or dwell time of gradient td >> 0  separation begins at k0 (isocratic) and bpract < btheortd >> 0  separation begins at k0 (isocratic) and bpract < btheor 𝐭 𝐑 𝐭 𝐦 𝐛⁄ · 𝐥𝐨𝐠 𝟐. 𝟑 · 𝐤 𝟎 · 𝐛 𝟏 𝐭 𝐦 𝐭 𝐝𝐭 𝐑 𝐭 𝐦 𝐛⁄ · 𝐥𝐨𝐠 𝟐. 𝟑 · 𝐤 𝟎 · 𝐛 𝟏 𝐭 𝐦 𝐭 𝐝 increasing the flow rate causes the gradient steepness to reduceincreasing the flow rate causes the gradient steepness to reduce the gradient steepness can be used to alter retention, but also selectivitythe gradient steepness can be used to alter retention, but also selectivity 𝟎 𝟎𝟎 𝟎 191191 gradient elution modegradient elution mode 𝐛 𝐭 𝐦 · ∆𝛗 · 𝐚 𝐭 𝐆 𝐕 𝐦 · ∆𝛗 · 𝐚 𝐭 𝐆⁄ · 𝐅 𝐦⁄ ∆𝛗 𝐭 𝐆⁄ · 𝐕 𝐦 · 𝐚 𝐅 𝐦⁄𝐛 𝐭 𝐦 · ∆𝛗 · 𝐚 𝐭 𝐆 𝐕 𝐦 · ∆𝛗 · 𝐚 𝐭 𝐆⁄ · 𝐅 𝐦⁄ ∆𝛗 𝐭 𝐆⁄ · 𝐕 𝐦 · 𝐚 𝐅 𝐦⁄ [min][min] td = 3 mintd = 3 min td = 1 mintd = 1 min 10‐40 % B za 15 min 1 ml∙min‐1 100x4.6 mm 10‐40 % B za 15 min 1 ml∙min‐1 100x4.6 mm resolution within gradient elutionresolution within gradient elution average capacity factor within gradient elutionaverage capacity factor within gradient elution capacity factor within gradient elutioncapacity factor within gradient elution under td = 0under td = 0 peak capacity within gradient elutionpeak capacity within gradient elution 𝐑 𝐀,𝐁 𝐍 𝟒 · 𝛂 𝐀,𝐁 𝟏 · 𝐤 𝟏 𝐤 𝐑 𝐀,𝐁 𝐍 𝟒 · 𝛂 𝐀,𝐁 𝟏 · 𝐤 𝟏 𝐤 𝐤 𝟏 𝟏. 𝟏𝟓 · 𝐛 𝟏 𝐤 𝟎⁄⁄𝐤 𝟏 𝟏. 𝟏𝟓 · 𝐛 𝟏 𝐤 𝟎⁄⁄ 𝐤 𝟏 𝐛⁄ · 𝐥𝐨𝐠 𝟐. 𝟑 · 𝐤 𝟎 · 𝐛 𝟏𝐤 𝟏 𝐛⁄ · 𝐥𝐨𝐠 𝟐. 𝟑 · 𝐤 𝟎 · 𝐛 𝟏 𝐧 𝟏 𝐭 𝐆 ∑ 𝐰𝐢 𝐣⁄𝐣 𝐢 𝟏 𝐧 𝟏 𝐭 𝐆 ∑ 𝐰𝐢 𝐣⁄𝐣 𝐢 𝟏 192192timetime %B%B startingstarting ending %Bending %B isocratic runisocratic run tGtG cleaningcleaning conditioningconditioning reequilibrationreequilibration 1938 – TLC (Izmailov and Shreiber) 1944 – paper chromatography (PC; Consden et al.) 1938 – TLC (Izmailov and Shreiber) 1944 – paper chromatography (PC; Consden et al.) arrangement : vertical (ascendant, descendant) : horizontal (annular TLC) arrangement : vertical (ascendant, descendant) : horizontal (annular TLC) retardation factor : analyte identification retardation factor : analyte identification TLC plateTLC plate sample on startsample on start rising solventrising solvent MP reservoirMP reservoir loaded sample is  separated  into components loaded sample is  separated  into components mixture componentsmixture components more polarmore polar less polarless polar non‐polar  solvent interacts not with SP non‐polar  solvent interacts not with SP polar solvent interacts with SP polar solvent interacts with SP cellulosecellulose thin‐layer chromatography (TLC)thin‐layer chromatography (TLC) 𝐑 𝐟 𝐝 𝐀 𝐝𝐟 𝐑 𝐟 𝐝 𝐀 𝐝𝐟 startstart d0d0 dAdA dfdf frontfront 193193 reached distance df – MP front dA – analyte d0 – edge‐to‐start : no separation :: but a MP wicks reached distance df – MP front dA – analyte d0 – edge‐to‐start : no separation :: but a MP wicks visualisationvisualisation : scratching off SP layer, elution : direct elution from SP : scratching off SP layer, elution : direct elution from SP : sulphuric a./heat: destructive :: burned spots : cerium staining: destructive :: dark‐blue spots (polar compounds) : iodine staining: semi‐destructive :: iodine adsorption, not stable : UV irradiation: non‐destructive :: base is green, dark spots : sulphuric a./heat: destructive :: burned spots : cerium staining: destructive :: dark‐blue spots (polar compounds) : iodine staining: semi‐destructive :: iodine adsorption, not stable : UV irradiation: non‐destructive :: base is green, dark spots stationary phases (SP) : silica (SiO2) – on carrier RP‐18, chiral RP‐18, NH3 –, CN– : alumina (Al2O3) – on carrier : cellulose – paper  : polyamide 6 (polycaprolactam)  stationary phases (SP) : silica (SiO2) – on carrier RP‐18, chiral RP‐18, NH3 –, CN– : alumina (Al2O3) – on carrier : cellulose – paper  : polyamide 6 (polycaprolactam)  imbuing : sinking of TLC plate into solution :: heating for staining fixation imbuing : sinking of TLC plate into solution :: heating for staining fixation : different agents for different analyte classes are used  :: aminoantipyrin/K3Fe(CN)6 (aryls), AgNO3/H2O2 (halogenhydrocarbons), ninhydrin (amines), FeCl3 (amides), dithizone (metal ion), anisaldehyde (sugars), SbCl3/SbCl5 (lipids)... : different agents for different analyte classes are used  :: aminoantipyrin/K3Fe(CN)6 (aryls), AgNO3/H2O2 (halogenhydrocarbons), ninhydrin (amines), FeCl3 (amides), dithizone (metal ion), anisaldehyde (sugars), SbCl3/SbCl5 (lipids)... 194194 analysis and preparationanalysis and preparation : horizontal arrangement : MP forced‐flow : MP over‐pressured : horizontal arrangement : MP forced‐flow : MP over‐pressured : mobile phase is in reservoirs :: blue in Figure : separation start by their opening : MP out of the rises on porous plates : mobile phase is in reservoirs :: blue in Figure : separation start by their opening : MP out of the rises on porous plates glassy coverglassy cover plateplate startstart startstart MP reservoir MP reservoir MP reservoir MP reservoir high performance thin‐layer chromatography (HPTLC)high performance thin‐layer chromatography (HPTLC) MPMP samplesample SPSP MP flowMP flow 195195 imaging softwareimaging softwaredensitometerdensitometervisualisatorvisualisatorchromatographchromatographsampling robotsampling robot 𝐤 𝟏 𝐑 𝐟 𝐑 𝐟 𝐝 𝐟 𝐝 𝐀 𝟏𝐤 𝟏 𝐑 𝐟 𝐑 𝐟 𝐝 𝐟 𝐝 𝐀 𝟏 𝐍 𝟏𝟔 · 𝐝 𝐀 · 𝐝 𝐟 𝐰 𝐀 𝟐 𝐍 𝟏𝟔 · 𝐝 𝐀 · 𝐝 𝐟 𝐰 𝐀 𝟐 wA – zone widthwA – zone width thinner layer of sorbent – 0.20 vs. 0.25 mm : small grain diameter – 7 vs. 12 – 20 μm and low polydispersity of grain :: lower longitudinal diffusion, 10x lower limit of detection :: better price/output ratio : higher resolution on shorter runs df :: 50 mm vs. 100 – 120 mm  faster analysis : better optical properties for densitometry thinner layer of sorbent – 0.20 vs. 0.25 mm : small grain diameter – 7 vs. 12 – 20 μm and low polydispersity of grain :: lower longitudinal diffusion, 10x lower limit of detection :: better price/output ratio : higher resolution on shorter runs df :: 50 mm vs. 100 – 120 mm  faster analysis : better optical properties for densitometry disadvantages : smaller sample input than TLC (1 / 10 till 1 / 15) : higher demands on sample quality – purity : technical background for data evaluation :: good densitometer & imaging software disadvantages : smaller sample input than TLC (1 / 10 till 1 / 15) : higher demands on sample quality – purity : technical background for data evaluation :: good densitometer & imaging software TLC plateTLC plate samplesample MP solventMP solvent 1st dimension1st dimension 2nd dimension,  90°2nd dimension,  90° HPTLC vs. TLCHPTLC vs. TLC 2D TLC2D TLC migration distance [mm]migration distance [mm] analysis time [min]analysis time [min] 196196 (HP)TLC separation description(HP)TLC separation descriptionχ – system constant tf – MP flow time to front χ – system constant tf – MP flow time to front dp – particle diameter Dm – diffusion coefficient dp – particle diameter Dm – diffusion coefficient height equivalent of theoretical plateheight equivalent of theoretical plate resolutionresolution 00 141422 66 1010 df [cm]df [cm] 0.00.0 0.50.5 1.01.0 RfRf R(A,B)R(A,B) 0.000.00 0.250.25 0.500.50 0.750.75 1.001.00 H [μm]H [μm] 1010 2020 3030 4040 5050 HPTLCHPTLC TLCTLC HPTLC MP over‐pressured HPTLC MP over‐pressured TLC MP over‐pressured TLC MP over‐pressured 𝐑 𝐀,𝐁 𝐍 𝟏 𝐤 · 𝛂 𝟏 · 𝐤 𝟏 𝐤 𝐑 𝐀,𝐁 𝐍 𝟏 𝐤 · 𝛂 𝟏 · 𝐤 𝟏 𝐤 𝐝 𝐟 𝛘 · 𝐭 𝐟𝐝 𝐟 𝛘 · 𝐭 𝐟 𝐇 𝐚 · 𝐝 𝐟 𝟐 𝟑⁄ 𝐝 𝟎 𝟐 𝟑⁄ 𝐝 𝐟 𝐝 𝟎 𝐛 · 𝐝 𝐟 𝐝 𝟎𝐇 𝐚 · 𝐝 𝐟 𝟐 𝟑⁄ 𝐝 𝟎 𝟐 𝟑⁄ 𝐝 𝐟 𝐝 𝟎 𝐛 · 𝐝 𝐟 𝐝 𝟎 𝐚 𝟑 𝟐 · 𝐀 · 𝐝 𝐩 · 𝐝 𝐩 𝟐𝐃 𝐦 𝟑 𝐚 𝟑 𝟐 · 𝐀 · 𝐝 𝐩 · 𝐝 𝐩 𝟐𝐃 𝐦 𝟑 𝐛 𝟐𝐃 𝐦 𝛘 · 𝐑 𝐟 𝐛 𝟐𝐃 𝐦 𝛘 · 𝐑 𝐟 197197 white lightwhite light TLC plateTLC plate optical density  of reflectionoptical density  of reflection TLC           plateTLC           plate lamplamp apertureaperture lenseslenses samplesample displaydisplay amplifieramplifier sensorsensor colour filtercolour filter IR filterIR filter top viewtop view samplesample halohalo plateplate lightlight samplesample shadowshadow in‐material diffusion in‐material diffusion reflectionsreflections D – optical density R – reflexivity % D – optical density R – reflexivity % reflexive densitometric detectionreflexive densitometric detection 𝐃 𝐥𝐨𝐠 𝟏𝟎𝟎 𝐑⁄𝐃 𝐥𝐨𝐠 𝟏𝟎𝟎 𝐑⁄ 198198 RfRf opticaldensitaopticaldensita 2001 – new subtype of RPLC2001 – new subtype of RPLC : SP with particle size 1.3 – 1.7 μm (sub‐two micron) :: pressure up to 0.2 GPa (10x higher than by HPLC) :: at same MP flow rate, analyses are 3x faster : SP with particle size 1.3 – 1.7 μm (sub‐two micron) :: pressure up to 0.2 GPa (10x higher than by HPLC) :: at same MP flow rate, analyses are 3x faster ultra‐high performance liquid chromatography (UPLC)ultra‐high performance liquid chromatography (UPLC) 199199 competition to monolithic columnscompetition to monolithic columns bridged ethylsiloxane/silica hybrid (BEH)bridged ethylsiloxane/silica hybrid (BEH) new SPnew SP BEH withstands very high pressures : high range of pH 1 – 12 BEH withstands very high pressures : high range of pH 1 – 12 tetraethoxysilane (TECS) tetraethoxysilane (TECS) bis(tetraethoxysilane)ether (BTEE) bis(tetraethoxysilane)ether (BTEE) polyethoxysilane (BPEOS) polyethoxysilane (BPEOS) Si(OH)4Si(OH)4 hydrolysishydrolysis shields against hydrolysisshields against hydrolysis keeps shape and function also after hydrolysis keeps shape and function also after hydrolysis classic SP based on silicaclassic SP based on silica SP based on BEHSP based on BEH stability at pH > 10 ~300 h : normal SP based on silica ~40 h stability at pH > 10 ~300 h : normal SP based on silica ~40 h u [mm∙s‐1]u [mm∙s‐1] H[μm]H[μm] 1.7 μm 2004 1.7 μm 2004 UPLCUPLC 3.5 μm 90ies 3.5 μm 90ies 5 μm 80ies 5 μm 80ies 10 μm 70ies 10 μm 70ies development of separation efficiencydevelopment of separation efficiency 200200 from HPLC to UPLCfrom HPLC to UPLC use of HTLC presumes existence of respective SP with very stable carrier : solution – zircon‐particles covered by polybutadiene, polystyrene or carbon :: SP Discovery :: thermal stability up to 250 °C use of HTLC presumes existence of respective SP with very stable carrier : solution – zircon‐particles covered by polybutadiene, polystyrene or carbon :: SP Discovery :: thermal stability up to 250 °C subcritical water : change of retention thermodynamic between low (15 – 55 °C) & high temperatures (125 – 200 °C) :: change of distribution constant K leads to change of capacity factor k ::: how to change distribution constant K? → increasing temperature :: substitution of mild polar MP based on acetonitrile‐water mixture by pure water subcritical water : change of retention thermodynamic between low (15 – 55 °C) & high temperatures (125 – 200 °C) :: change of distribution constant K leads to change of capacity factor k ::: how to change distribution constant K? → increasing temperature :: substitution of mild polar MP based on acetonitrile‐water mixture by pure water HTLC ideal for on‐line 1H‐NMR detection by means of D2O : so‐called green chromatography HTLC ideal for on‐line 1H‐NMR detection by means of D2O : so‐called green chromatography high temperature liquid chromatography (HTLC)high temperature liquid chromatography (HTLC) 201201 superheated (water liquid) chromatography (SWC, SWLC) superheated (water liquid) chromatography (SWC, SWLC) 2003 – combination of UPLC and HTLC2003 – combination of UPLC and HTLC UPLC reaches N ~ 200 000 m‐1 RPLC reaches N ~ 10 000 – 25 000 m‐1 CZE reaches N > 1 000 000 m‐1 UPLC reaches N ~ 200 000 m‐1 RPLC reaches N ~ 10 000 – 25 000 m‐1 CZE reaches N > 1 000 000 m‐1 elevated temperature ultra‐HPLC (ET‐UPLC) elevated temperature ultra‐HPLC (ET‐UPLC)  202202 decreasing MP viscosity after increase of temperature, high pressure is compensated : u (80 °C) = 2.6 ∙ u (25 °C), pressure = const. :: u – linear flow rate : SP ø1 μm, pressure 180 MPa, temperature 90 °C :: N ~ 420 000 m‐1 decreasing MP viscosity after increase of temperature, high pressure is compensated : u (80 °C) = 2.6 ∙ u (25 °C), pressure = const. :: u – linear flow rate : SP ø1 μm, pressure 180 MPa, temperature 90 °C :: N ~ 420 000 m‐1 u [cm∙s‐1]u [cm∙s‐1] H [μm]H [μm] chromatography on „reversed reversed phase“ for very polar analytes or substances with many interacting groups  chromatography on „reversed reversed phase“ for very polar analytes or substances with many interacting groups  primary interactions : distribution between organic and aqueous phases secondary interactions : in aqueous phase between aqueous phase and SP :: hydrophilic (electrostatic) :: ionic primary interactions : distribution between organic and aqueous phases secondary interactions : in aqueous phase between aqueous phase and SP :: hydrophilic (electrostatic) :: ionic : charged – strong electrostatic interactions (silica, aminopropyls) : neutral polar – no electrostatic interactions (diols, amides) : zwitter‐ions – weak electrostatic interactions (sulphoalkyl betain, phosphatidylethanolamine) : charged – strong electrostatic interactions (silica, aminopropyls) : neutral polar – no electrostatic interactions (diols, amides) : zwitter‐ions – weak electrostatic interactions (sulphoalkyl betain, phosphatidylethanolamine) hydrophilic interaction liquid chromatography (HILIC)hydrophilic interaction liquid chromatography (HILIC) 203203 presumed retention mechanismpresumed retention mechanism stationary phasesstationary phases particle of  SP carrier particle of  SP carrier waterwater organic MPorganic MP analyteanalyte SPSP monolithic and polymer SP : polyfunctional polymer :: polymethyl methacrylate monolithic and polymer SP : polyfunctional polymer :: polymethyl methacrylate 𝐥𝐨𝐠 𝐤 𝐥𝐨𝐠 𝐤 𝟎 𝐦 · 𝐥𝐨𝐠 𝛗 𝐩𝐨𝐥𝐥𝐨𝐠 𝐤 𝐥𝐨𝐠 𝐤 𝟎 𝐦 · 𝐥𝐨𝐠 𝛗 𝐩𝐨𝐥 : organic component – max 97% :: min 3% water on SP hydratation sublayer : salt content – ammonium salts :: for low pH formiate, for high pH hydroxide :: regulates pH and also ionic strength :: defines interaction types  : organic component – max 97% :: min 3% water on SP hydratation sublayer : salt content – ammonium salts :: for low pH formiate, for high pH hydroxide :: regulates pH and also ionic strength :: defines interaction types  : organic component < 50 % :: on SP hydrophobic sublayer appears : requires specific SP type :: mixed‐mode stationary phase : organic component < 50 % :: on SP hydrophobic sublayer appears : requires specific SP type :: mixed‐mode stationary phase RPLC modeRPLC mode 204204 mobile phasemobile phase [min][min]tRtR HILICHILIC RPLC modeRPLC mode uraciluracil anilineaniline toluenetoluene uraciluracil anilineaniline toluenetoluene advantages : orthogonal to RPLC (substitution to NPLC) : advantageous for connection to MS due to high organic content disadvantages : complex and not yet precisely known retention mechanism advantages : orthogonal to RPLC (substitution to NPLC) : advantageous for connection to MS due to high organic content disadvantages : complex and not yet precisely known retention mechanism ionic interactions in HILIC A – ion pair with SP B – ion pair with sample anion C – ion pair with sample cation ionic interactions in HILIC A – ion pair with SP B – ion pair with sample anion C – ion pair with sample cation dual separation modedual separation mode electrostatic repulsion hydrophilic liquid chromatography (ERLIC)electrostatic repulsion hydrophilic liquid chromatography (ERLIC) HILIC modeHILIC mode uses repulsion of the same charges of SP and analyte : increases chances of other polar groups to influence retention :: coulombic interactions have higher chemical potential than polar ones :: organic component content in MP > 70 % :: increasing influence of salts and molecule spatial orientation uses repulsion of the same charges of SP and analyte : increases chances of other polar groups to influence retention :: coulombic interactions have higher chemical potential than polar ones :: organic component content in MP > 70 % :: increasing influence of salts and molecule spatial orientation 205205 repulsionrepulsion repulsionrepulsion repulsionrepulsion attractionattraction attractionattraction technique for separation of macromolecules : it is not RP‐HPLC technique for separation of macromolecules : it is not RP‐HPLC uses stimulated interactions of hydrophobic surface parts of macromolecule with SP SP: carrier (agarose, dextran) modified by small non‐polar group uses stimulated interactions of hydrophobic surface parts of macromolecule with SP SP: carrier (agarose, dextran) modified by small non‐polar group 1 – phenyl (Phe), 2 – octyl (C8), 3 – butyl (C4)1 – phenyl (Phe), 2 – octyl (C8), 3 – butyl (C4) sample is loaded in solution with ammonium sulphate (chaotropic agent) : w/ increasing entropy of water increases strength of hydrophobic interactions : stabilisation of proteins sample is loaded in solution with ammonium sulphate (chaotropic agent) : w/ increasing entropy of water increases strength of hydrophobic interactions : stabilisation of proteins MP serving for elution (KSCN and KClO4) has kosmotropic effect : disrupts interactions, but does not denature sample : addition of alcohol decreases surface tension of water and thus strongly desorbs (cleaning) MP serving for elution (KSCN and KClO4) has kosmotropic effect : disrupts interactions, but does not denature sample : addition of alcohol decreases surface tension of water and thus strongly desorbs (cleaning) P – carrier S – analyte L – SP modification H – hydrophobic analyte region W – water molecules P – carrier S – analyte L – SP modification H – hydrophobic analyte region W – water molecules hydrophobic interaction liquid chromatography (HIC)hydrophobic interaction liquid chromatography (HIC) 206206 11 3322 csl – concentration of chaotropecsl – concentration of chaotrope𝐥𝐧 𝐤 𝐥𝐧 𝐤 𝟎 𝐚 · 𝐜 𝐬𝐥𝐥𝐧 𝐤 𝐥𝐧 𝐤 𝟎 𝐚 · 𝐜 𝐬𝐥 displacement LC ion exchange – ions retained on solid surface (insoluble) are exchanged for ions contained in around flowing solution by means of contact with carrier displacement LC ion exchange – ions retained on solid surface (insoluble) are exchanged for ions contained in around flowing solution by means of contact with carrier ion exchange chromatography (IEC)ion exchange chromatography (IEC) D. T. Day – clays and zeolites have ion exchanging properties 1935 – the first synthetic ionex 1950‐1959 – start of intense IEC development D. T. Day – clays and zeolites have ion exchanging properties 1935 – the first synthetic ionex 1950‐1959 – start of intense IEC development VIII.VIII. 207207 exchanging ion bound strongly than the eluting ion by higher concentration exchanging ion bound strongly than the eluting ion by higher concentration startstart adsorptionadsorption desorptiondesorption washingwashing regenerationregeneration starting buffer counter‐ions OH– starting buffer counter‐ions OH– separated ions Cl– & Br– separated ions Cl– & Br– eluent ions NO3 – eluent ions NO3 – slow diffusion in polymer particle is substituted w/ fast diffusion in thin layer of polymerslow diffusion in polymer particle is substituted w/ fast diffusion in thin layer of polymer sample: proteins, nucleic acids and other big charged moleculessample: proteins, nucleic acids and other big charged molecules stationary phasestationary phase dowex, amberlite, Bio‐Rex, chelex... dowex, amberlite, Bio‐Rex, chelex...microporous spheres, diameter ca 10 μm : polystyrene‐divinylbenzene sample: amino acids, peptides and saccharides microporous spheres, diameter ca 10 μm : polystyrene‐divinylbenzene sample: amino acids, peptides and saccharides silica, glass, with polymeric coverage; particles must be porous : hydrophilic polymer coating silica, glass, with polymeric coverage; particles must be porous : hydrophilic polymer coating 208208 polymer porouspolymer porous pellicular boundpellicular bound SP surface enlargementSP surface enlargement : particle (5 μm) w/ R‐SO3 – anchored particles (0.1 μm) w/ active ‐SO3 – & anchoring ‐R3N+ : particle (5 μm) w/ R‐SO3 – rich on caverns (quasi‐monolith) : particle (4.5 μm) of highly cross‐linked polymer w/ surface layer active groups  (core‐shell) : particle (5 μm) w/ R‐SO3 – anchored particles (0.1 μm) w/ active ‐SO3 – & anchoring ‐R3N+ : particle (5 μm) w/ R‐SO3 – rich on caverns (quasi‐monolith) : particle (4.5 μm) of highly cross‐linked polymer w/ surface layer active groups  (core‐shell) HPAEC, high‐performance anion‐exchange chromatography HPCEC, high‐performance cation‐exchange chromatography HPAEC, high‐performance anion‐exchange chromatography HPCEC, high‐performance cation‐exchange chromatography : small particles (4 – 6 μm) of cross‐linked polymer (PSDVB) :: CarboPac™ carrier + MicroBead™ membrane with R‐NH3 + or R‐SO3 – :: the higher the cross‐linking, the smaller analytes ::: very high load capacity : often combined with pulse amperometric detector (PAD) : small particles (4 – 6 μm) of cross‐linked polymer (PSDVB) :: CarboPac™ carrier + MicroBead™ membrane with R‐NH3 + or R‐SO3 – :: the higher the cross‐linking, the smaller analytes ::: very high load capacity : often combined with pulse amperometric detector (PAD) high‐performance ion‐exchange chromatography (HPIEC)high‐performance ion‐exchange chromatography (HPIEC) 209209 amount (mols) of potentially ionisable groups related to 1 gram of dry ionex amount (mols) of potentially ionisable groups related to 1 gram of dry ionex quantitative measure of ion‐exchanging ability (of counterions)quantitative measure of ion‐exchanging ability (of counterions) capacitycapacity pHpH weak anex (WAX)weak anex (WAX) strong anex (SAX)strong anex (SAX) weak catex (WCX)weak catex (WCX) strong catex (SCX)strong catex (SCX) ion‐exchanger capacityion‐exchanger capacity strong base ‐NH(CH3)3 + OH– weak base secondary or tertiary amines strong base ‐NH(CH3)3 + OH– weak base secondary or tertiary amines strong acid ‐SO3 – H+ weak acid ‐COO– H+ strong acid ‐SO3 – H+ weak acid ‐COO– H+ 210210 actual capacityactual capacity total capacitytotal capacity amount of groups eventually exchangeable under given experimental conditions : the amount is pH dependent amount of groups eventually exchangeable under given experimental conditions : the amount is pH dependent ionic interaction typesionic interaction types e.g. reaction of saccharide with boratee.g. reaction of saccharide with borate they change relative retention or fully properties of ions : cation could be thus separated on anex they change relative retention or fully properties of ions : cation could be thus separated on anex ionic complexes with neutral moleculesionic complexes with neutral molecules complexes with ligandscomplexes with ligands neutralisation reactionsneutralisation reactions ligand exchangeligand exchange HA + R+ OH– → R+ A– + H2O B + R– H+ → R– + BH+ HA + R+ OH– → R+ A– + H2O B + R– H+ → R– + BH+ RM – metal / ion‐exchange ion pair L – ligand, forming complex with metal M X – analyte‐ligand RM – metal / ion‐exchange ion pair L – ligand, forming complex with metal M X – analyte‐ligand 211211 saccharide + B(OH)4 – → [saccharide∙borate]–saccharide + B(OH)4 – → [saccharide∙borate]– Fe3+ + 4 Cl – → FeCl4 –Fe3+ + 4 Cl – → FeCl4 – ion‐exchangers in acidic or basic forms are base of IECion‐exchangers in acidic or basic forms are base of IEC RM∙L + X → RM∙X + LRM∙L + X → RM∙X + L used within catexes conditioned with Ni2+ or Cu2+ : separation of amino acids and other bases used within catexes conditioned with Ni2+ or Cu2+ : separation of amino acids and other bases : solubility (salts, buffers) : retention by elution force : separation : solubility (salts, buffers) : retention by elution force : separation mobile phasemobile phase typical MP : aqueous solutions of salts, buffered and modified water‐miscible organic solvents :: methanol, acetonitrile, etc. typical MP : aqueous solutions of salts, buffered and modified water‐miscible organic solvents :: methanol, acetonitrile, etc. x RN(CH3)3 +OH– (SP) + Ax‐ (MP) →(RN(CH3)3 +)xAx– (SP) + x OH– (MP)x RN(CH3)3 +OH– (SP) + Ax‐ (MP) →(RN(CH3)3 +)xAx– (SP) + x OH– (MP) x RSO3 –H+ (SP) + Mx+ (MP) → (RSO3 –)xMx+ (SP) + x H+ (MP)x RSO3 –H+ (SP) + Mx+ (MP) → (RSO3 –)xMx+ (SP) + x H+ (MP) cation exchangecation exchange anion exchangeanion exchange 212212 elution force and selectivity : concentration of ions in buffer and of other salts :: increasing ionic strength means increasing elution force : pH : type and concentration of organic solvents elution force and selectivity : concentration of ions in buffer and of other salts :: increasing ionic strength means increasing elution force : pH : type and concentration of organic solvents distribution equilibriumdistribution equilibrium [Ca2+]SP and [H+]MP molar concentration in SP : concentration has values 0 – max, when all binding sites are occupied with one compound [Ca2+]SP and [H+]MP molar concentration in SP : concentration has values 0 – max, when all binding sites are occupied with one compound Ca2+ (MP) + 2H+ (SP) → Ca2+ (SP) + 2H+ (MP)Ca2+ (MP) + 2H+ (SP) → Ca2+ (SP) + 2H+ (MP) 𝐊 𝐂𝐚 𝟐 𝐒𝐏 · 𝐇 𝐌𝐏 𝐂𝐚 𝟐 𝐌𝐏 · 𝐇 𝐒𝐏 𝐊 𝐂𝐚 𝟐 𝐒𝐏 · 𝐇 𝐌𝐏 𝐂𝐚 𝟐 𝐌𝐏 · 𝐇 𝐒𝐏 213213 retention on anex increases with increase of pH (pH : 1  14), conversely on catex retention decreases with increasing concentration of organic solvent : effect is higher for less polar solvents change in selectivity is achievable by adding different solvents : methanol, ethanol, acetonitrile and dioxane retention on anex increases with increase of pH (pH : 1  14), conversely on catex retention decreases with increasing concentration of organic solvent : effect is higher for less polar solvents change in selectivity is achievable by adding different solvents : methanol, ethanol, acetonitrile and dioxane retentionretention controlled by pH of MPcontrolled by pH of MP other factors influencing retentionother factors influencing retention temperature 15 – 60 °C : change of MP viscosity with higher temperature  better separation : separation at 50 – 60 °C are advantageous (if column allows) : biochemical separations (enzymes) at 4 °C temperature 15 – 60 °C : change of MP viscosity with higher temperature  better separation : separation at 50 – 60 °C are advantageous (if column allows) : biochemical separations (enzymes) at 4 °C small changes of temperature easily influence the selectivity : useful if no other method leads to results small changes of temperature easily influence the selectivity : useful if no other method leads to results elutionelution [Ca2+]SP << [H+]SP [Ca2+]MP << [H+]MP [Ca2+]SP << [H+]SP [Ca2+]MP << [H+]MP SP affinity to Ca2+ in regard to H+ : generally the higher K, the higher affinity SP affinity to Ca2+ in regard to H+ : generally the higher K, the higher affinity concentration of H+ is constant in both phasesconcentration of H+ is constant in both phases eluting ion in surplus in both phaseseluting ion in surplus in both phases elution of Ca2+ by H+ ionselution of Ca2+ by H+ ions ion affinity to SPion affinity to SP bigger ions have higher affinity than smaller, polyvalent ions have higher affinity than monovalentbigger ions have higher affinity than smaller, polyvalent ions have higher affinity than monovalent for a typical catex, K decreases with ion diameter in order : monovalent Ag+ > Cs+ > Rb+ > K+ > NH4 + > Na+ > H+ > Li+ : divalent Ba2+ > Pb2+ > Sr2+ > Ca2+ > Ni2+ > Cd2+ > Cu2+ > Co2+> Zn2+ > Mg2+ > UO2 2+ for anex in this order citrate > SO4 2– > oxalate > I– > NO3 – > CrO4 2– > Br– > SCN– > Cl– > formate > acetate > OH– > F– for a typical catex, K decreases with ion diameter in order : monovalent Ag+ > Cs+ > Rb+ > K+ > NH4 + > Na+ > H+ > Li+ : divalent Ba2+ > Pb2+ > Sr2+ > Ca2+ > Ni2+ > Cd2+ > Cu2+ > Co2+> Zn2+ > Mg2+ > UO2 2+ for anex in this order citrate > SO4 2– > oxalate > I– > NO3 – > CrO4 2– > Br– > SCN– > Cl– > formate > acetate > OH– > F– 𝐇 𝐒𝐏 𝟐 𝐇 𝐌𝐏 𝟐 𝒄𝒐𝒏𝒔𝒕. 𝐇 𝐒𝐏 𝟐 𝐇 𝐌𝐏 𝟐 𝒄𝒐𝒏𝒔𝒕. Hofmeister ion seriesHofmeister ion series 214214 𝐊 𝐂𝐚 𝟐 𝐒𝐅 · 𝐇 𝐌𝐅 𝐂𝐚 𝟐 𝐌𝐅 · 𝐇 𝐒𝐅 𝐂𝐚 𝟐 𝐒𝐅 𝐂𝐚 𝟐 𝐌𝐅 𝐊 𝐂𝐚 𝟐 𝐒𝐅 · 𝐇 𝐌𝐅 𝐂𝐚 𝟐 𝐌𝐅 · 𝐇 𝐒𝐅 𝐂𝐚 𝟐 𝐒𝐅 𝐂𝐚 𝟐 𝐌𝐅 215215 atomic and ionic radiiatomic and ionic radii Zipax‐SAX w/ 1% TBAOH, 0.01 M borate, pH 9.2, 24 °C Zipax‐SAX w/ 1% TBAOH, 0.01 M borate, pH 9.2, 24 °C  1. caffeine 2. theobromine 3. coumarin 4. sorbate, potassium 5. ascorbic acid 6. benzoate, sodium 7. vanillin 8. ethylvanillin 1. caffeine 2. theobromine 3. coumarin 4. sorbate, potassium 5. ascorbic acid 6. benzoate, sodium 7. vanillin 8. ethylvanillin 9. methylparaben 10. ethylparaben 11. saccharin 12. propylparabene 13. butylparabene 9. methylparaben 10. ethylparaben 11. saccharin 12. propylparabene 13. butylparabene sodium nitrate content [mol∙l‐1]sodium nitrate content [mol∙l‐1] relativeretentionrelativeretention influence of ionic strength on separationinfluence of ionic strength on separation 216216 Zipax‐SAX anex, 0.01 M NaNO3, pH 5.7, 37 °CZipax‐SAX anex, 0.01 M NaNO3, pH 5.7, 37 °C 00 1010 4. phenobarbital4. phenobarbital 3. benzoic acid internal standard 3. benzoic acid internal standard 2. theofyline2. theofyline 1. ephedrine1. ephedrineA254 A254  [min][min] two ion‐exchange columns and conductivity detector : MP conductivity suppression two ion‐exchange columns and conductivity detector : MP conductivity suppression HCO3HCO3 MPMP suppression column suppression column separationcolumnseparationcolumn injectioninjection adsorption OH– + A–  A– + OH– adsorption OH– + A–  A– + OH– elution A– + HCO3 –  HCO3 – + A– elution A– + HCO3 –  HCO3 – + A– conductivity detector H2O, H2CO3, A– conductivity detector H2O, H2CO3, A– A–A– H+ + OH–  H2OH+ + OH–  H2O H+ + HCO3  H2CO3H+ + HCO3  H2CO3 IEC conductivity suppressionIEC conductivity suppression –– –– suppression column (Dionex)suppression column (Dionex) suppression micromembranessuppression micromembranes eluenteluent regeneration solution regeneration solution regenerantregenerant sealseal ion‐exchange membraneion‐exchange membrane ion‐exchange membraneion‐exchange membrane channel for  IEC eluentchannel for  IEC eluent channel for regenerantchannel for regenerant channel for regenerantchannel for regenerant 217217 analyte separation based on difference in distribution ratio between two liquid phases : MP and liquid immobilised on SP : similar to size exclusion chromatography (SEC) :: Donnan exclusion : separation of classes of uncharged species (ions are excluded) :: weak acids and bases, hydrophilic substances (saccharides, alcohols) analyte separation based on difference in distribution ratio between two liquid phases : MP and liquid immobilised on SP : similar to size exclusion chromatography (SEC) :: Donnan exclusion : separation of classes of uncharged species (ions are excluded) :: weak acids and bases, hydrophilic substances (saccharides, alcohols) : MP – according to analyte :: water or strong acid (heptafluorobutyric acid); for weak acids :: addition of organic solvent hastens separation (up to 40% AcN) : MP – according to analyte :: water or strong acid (heptafluorobutyric acid); for weak acids :: addition of organic solvent hastens separation (up to 40% AcN) RCOOHRCOOH eluenteluent Donnan double‐layer Donnan double‐layer : SP – WCX are used, eventually WAX :: Donnan equilibrium : SP – WCX are used, eventually WAX :: Donnan equilibrium the lower polarity, the higher retention : homologous lines are separated with increasing acidity : diacids have lower retention : double bond or aromatic ring are causing higher retention the lower polarity, the higher retention : homologous lines are separated with increasing acidity : diacids have lower retention : double bond or aromatic ring are causing higher retention ion exclusion chromatography (IXC)ion exclusion chromatography (IXC) 218218 RCOO–RCOO– H+H+ separation of analyte based on difference in isoelectric point value (pI) : it uses buffer abilities of charged ionex groups :: column with anex is equilibrated by buffer of higher value than the highest pI :: onto column, sample is inserted, with the starting buffer ::: compounds at pH above pI are negative and chatcheed on the top of column ::: compounds at pH below pI turn positive, migrate & bind in zone pH > pI ::: compounds with the highest pI are eluted first ::: separation at Δ pI = 0.05 resolution separation of analyte based on difference in isoelectric point value (pI) : it uses buffer abilities of charged ionex groups :: column with anex is equilibrated by buffer of higher value than the highest pI :: onto column, sample is inserted, with the starting buffer ::: compounds at pH above pI are negative and chatcheed on the top of column ::: compounds at pH below pI turn positive, migrate & bind in zone pH > pI ::: compounds with the highest pI are eluted first ::: separation at Δ pI = 0.05 resolution variant of IEC – 1978 Sluytermanvariant of IEC – 1978 Sluyterman mobile phases : ampholytes :: mixture of oligopeptides w/ different but close pKa ::: titration curve is almost linear – pH gradient :::: large number of small step in range of these pKa values : Pharmalyte 8 – 10.5 / Polybuffer 96 / Polybuffer 74 mobile phases : ampholytes :: mixture of oligopeptides w/ different but close pKa ::: titration curve is almost linear – pH gradient :::: large number of small step in range of these pKa values : Pharmalyte 8 – 10.5 / Polybuffer 96 / Polybuffer 74 chromatofocusing (CF)chromatofocusing (CF) limit bufferlimit buffer start bufferstart buffer limit bufferlimit buffer start bufferstart buffer volume [ml]volume [ml] volume [ml]volume [ml] pHpH pHpH 99 66 99 66 elution directionelution direction pH gradientpH gradient 44 99 pI ~ 8pI ~ 8 219219 stationary phases : PBE 96 (Polybuffer exchange 96) based on Sepharose stationary phases : PBE 96 (Polybuffer exchange 96) based on Sepharose involved are weak interactions : Coulombic : disperse : van der Waals involved are weak interactions : Coulombic : disperse : van der Waals specific interaction of immobilised ligand with analytespecific interaction of immobilised ligand with analyte very strong and specific interaction – multiple interactionvery strong and specific interaction – multiple interaction in praxis natural biogenic systems are suitable in praxis natural biogenic systems are suitable  affinity chromatography (AC)affinity chromatography (AC) 1987 – P. Cuatrecasas, M. Wilchek, Wolf prize for discovery of AC1987 – P. Cuatrecasas, M. Wilchek, Wolf prize for discovery of AC quasi‐displacement chromatographyquasi‐displacement chromatography 220220 carriercarrier ligandligand specific interactionspecific interaction elutionelution KD = 10‐4 – 10‐8 MKD = 10‐4 – 10‐8 M protein ↔ protein : antibody, antigen protein ↔ peptide : tag protein ↔ low mass compound : substrate, inhibitor, coenzyme, hormone, synthetic analogues protein ↔ protein : antibody, antigen protein ↔ peptide : tag protein ↔ low mass compound : substrate, inhibitor, coenzyme, hormone, synthetic analogues nucleic acid ↔ nucleic acid : complementary chain nucleic acid ↔ protein : histone, polymerase, binding protein nucleic acid ↔ nucleic acid : complementary chain nucleic acid ↔ protein : histone, polymerase, binding protein : no interaction with analyte : high amount of reactive groups : mechanical stability : porous : homogenous : no interaction with analyte : high amount of reactive groups : mechanical stability : porous : homogenous low‐pressure LC : cellulose (not homogenous) : polystyrene (not homogenous, strong hydrophobic, less pores) : PVA (volume change on rehydration and ionic strength) : dextran (less pores) : agarose (melts, sensitive to chemical influence /Gua, urea/) low‐pressure LC : cellulose (not homogenous) : polystyrene (not homogenous, strong hydrophobic, less pores) : PVA (volume change on rehydration and ionic strength) : dextran (less pores) : agarose (melts, sensitive to chemical influence /Gua, urea/) high‐pressure LC, FPLC : spheron – methacrylate (hydrophobic) : silasorb – SiO (sensitive to above pH 8) high‐pressure LC, FPLC : spheron – methacrylate (hydrophobic) : silasorb – SiO (sensitive to above pH 8) solid carriersolid carrier stationary phase stationary phase  221221 : sufficiently long :: steric hindrances, interactions with spacer, spacer aggregation  : HMDA or more methylene bridges or hydrophilic spacers (polyGly etc.) : sufficiently long :: steric hindrances, interactions with spacer, spacer aggregation  : HMDA or more methylene bridges or hydrophilic spacers (polyGly etc.) : covalently bound to carrier : covalently bound to carrier  spacerspacer ligandligand conditioning : regeneration, cleaning, equilibration conditioning : regeneration, cleaning, equilibration sample loading : sorption sample loading : sorption elution  : elution force :: disruption of weak bond elution  : elution force :: disruption of weak bond elution agent elution agent time [min]time [min] signal [A.U.]signal [A.U.] AC separation procedureAC separation procedure 222222 change of analyte conformationchange of analyte conformation displacement of analytedisplacement of analyte : Δt, ΔpH, ΔI, Δε :: solutions of salts, acids, bases, organic solvent : specific agents :: allosteric effects : Δt, ΔpH, ΔI, Δε :: solutions of salts, acids, bases, organic solvent : specific agents :: allosteric effects : low mass (free) molecular ligand: low mass (free) molecular ligand time [h]time [h] turbidimetryturbidimetry IgGsIgGs albuminalbumintransferrintransferrin NaClNaCl cibacron blue 3GAcibacron blue 3GA analysis of ligand binding to analyteanalysis of ligand binding to analyte isolation of analyteisolation of analyte selective separation : very complex mixtures without many clean‐up steps selective separation : very complex mixtures without many clean‐up steps : K' value must be in a range 1x10‐6 – 5x10‐3 mol∙l‐1 :: bond to immobilised ligand is generally weaker than to free ligand : K' value must be in a range 1x10‐6 – 5x10‐3 mol∙l‐1 :: bond to immobilised ligand is generally weaker than to free ligand : VM – void column volume, VR – elution volume of studied analyte : V0 – el. volume of unretained analogue of analyte (Mr) : cL – concentration of bound ligand : VM – void column volume, VR – elution volume of studied analyte : V0 – el. volume of unretained analogue of analyte (Mr) : cL – concentration of bound ligand use of ACuse of AC 𝟏 𝐕 𝐑 𝐕𝟎⁄ 𝐊 𝐕𝟎 𝐕 𝐌⁄ · 𝐜 𝐋𝟏 𝐕 𝐑 𝐕𝟎⁄ 𝐊 𝐕𝟎 𝐕 𝐌⁄ · 𝐜 𝐋 223223 dissociation constants of complex analyte – anchored ligand (K')dissociation constants of complex analyte – anchored ligand (K') dissociation constant determination of complex biopolymer – free ligand (K)dissociation constant determination of complex biopolymer – free ligand (K) principle – competitive elution : free and anchored ligands competitively bind analyte principle – competitive elution : free and anchored ligands competitively bind analyte 𝟏 𝐕 𝐑 𝐕𝟎⁄ 𝐊 𝐕𝟎 𝐕 𝐌⁄ · 𝐜 𝐋 𝐊 · 𝐜 𝐋 𝐕𝟎 𝐕 𝐌 · 𝐜 𝐋 · 𝐊⁄𝟏 𝐕 𝐑 𝐕𝟎⁄ 𝐊 𝐕𝟎 𝐕 𝐌⁄ · 𝐜 𝐋 𝐊 · 𝐜 𝐋 𝐕𝟎 𝐕 𝐌 · 𝐜 𝐋 · 𝐊⁄ : cL' – concentration of competitive ligand : cL' – concentration of competitive ligand  real systems – more binding sites for ligand & different binding sites w/ different affinityreal systems – more binding sites for ligand & different binding sites w/ different affinity carrier + chelating ligand + metal ioncarrier + chelating ligand + metal ion IDAIDA metal ions : Ag+, Al3+, Ca2+, Co2+, Cr3+, Cu2+, Eu3+, Fe3+, Hg2+, La3+, Mn2+ Nd3+, Ni2+, Yb3+, Zn2+, Ga3+ ... metal ions : Ag+, Al3+, Ca2+, Co2+, Cr3+, Cu2+, Eu3+, Fe3+, Hg2+, La3+, Mn2+ Nd3+, Ni2+, Yb3+, Zn2+, Ga3+ ... chelating ligandschelating ligands IDA – iminodiacetae  TACN – 1,4,7‐triazocyclonan NTA – nitrilotriacetate TREN – tris‐(2‐aminoethyl)‐amine TED – tris‐(carboxymethyl)‐ethylenediamine   IDA – iminodiacetae  TACN – 1,4,7‐triazocyclonan NTA – nitrilotriacetate TREN – tris‐(2‐aminoethyl)‐amine TED – tris‐(carboxymethyl)‐ethylenediamine   bond chelating ligand – metal ion enough strong (not to be washed out during separation) : but the stronger the bond is, the weaker the interaction with macromolecule  bond chelating ligand – metal ion enough strong (not to be washed out during separation) : but the stronger the bond is, the weaker the interaction with macromolecule  immobilised metal ion affinity chromatography (IMAC)immobilised metal ion affinity chromatography (IMAC) 224224 carriercarrierNi2+‐TEDNi2+‐TED His6 tagHis6 tag proteinprotein SFC vs. HPLC : fast separation : no use of organic solvents : high number of H, great ratio S / N, high resolution SFC vs. HPLC : fast separation : no use of organic solvents : high number of H, great ratio S / N, high resolution SFC vs. GC : higher resolution : analysis of thermolabiles (lower temperatures) SFC vs. GC : higher resolution : analysis of thermolabiles (lower temperatures) supercritical fluid chromatography (SFC)supercritical fluid chromatography (SFC) 1958 – Lovelock, method proposal 1962 – Klesper, Corwin and Turner, procedure 1980 – first commercial apparatus 2011 – Waters introduced UPSFC/UPC2 :: connectible with MS  1958 – Lovelock, method proposal 1962 – Klesper, Corwin and Turner, procedure 1980 – first commercial apparatus 2011 – Waters introduced UPSFC/UPC2 :: connectible with MS  ultraperformance convergence chromatography™ (UPC2) ultraperformance convergence chromatography™ (UPC2) 225225 ∆ 𝟏. 𝟐𝟓 · 𝐩 𝐤 · 𝛒 𝐒𝐅 𝛒𝐥𝐢𝐪⁄∆ 𝟏. 𝟐𝟓 · 𝐩 𝐤 · 𝛒 𝐒𝐅 𝛒𝐥𝐢𝐪⁄ increase of pressure  increase of density  increase of solvation force : pressure gradient in SFC ~ gradient in HPLC and temperature gradient of GC increase of pressure  increase of density  increase of solvation force : pressure gradient in SFC ~ gradient in HPLC and temperature gradient of GC supercritical CO2 ~ heptane : SFC ~ NPLC : additives for moderating polarity :: MeOH, HAc supercritical CO2 ~ heptane : SFC ~ NPLC : additives for moderating polarity :: MeOH, HAc increase of solubility (Δ)  increase of density  decrease of retentionincrease of solubility (Δ)  increase of density  decrease of retention capillary GCcapillary GC SFCSFC HPLCHPLC NN αα van Deemter curves comparisonvan Deemter curves comparison comparison of SFC with HPLC & GCcomparison of SFC with HPLC & GC 226226 u [mm∙s‐1]u [mm∙s‐1] H [μm]H [μm] SFCSFC HPLCHPLC UPLCUPLC UPSFC (UPC2)UPSFC (UPC2) : solubility of additives: solubility of additives : influence of pressure: influence of pressure solubility in CO2solubility in CO2 Tr = 1.01 = 308.0/304.2 KTr = 1.01 = 308.0/304.2 K log(y)log(y) pr = patm/72.8pr = patm/72.8 naphtalenenaphtalene pressure [bar]pressure [bar] solubility  [mol. fraction]solubility  [mol. fraction] 3.5% methanol3.5% methanol 3.5% acetic acid3.5% acetic acid pure CO2pure CO2 salicylic acidsalicylic acid SFC procedureSFC procedure detectors used for LC and GCdetectors used for LC and GC pressure sensor pressure sensor thermostatthermostat wastewaste from detectorfrom detector dynamicdynamic regulatorregulator staticstatic regulatorregulator system valvesystem valve into wasteinto waste 7 MPa7 MPa 0.1 MPa0.1 MPa 10 – 40 MPa10 – 40 MPa MP heatingMP heating new type of restrictornew type of restrictor 227227 MP containerMP container container & pump for additives container & pump for additives condensercondenser MP pumpMP pump heaterheater columncolumn detectordetector loadload restrictors separators restrictors separators silicon oil (Lukoil) in CH2Cl2, injection 60 nl, linear pressure change 8 – 36 MPa (30 min) detector: FID, 350 °C. restrictor Integral, column diameter 320 mm, L=145 mm, SP 5 μm C18 silicon oil (Lukoil) in CH2Cl2, injection 60 nl, linear pressure change 8 – 36 MPa (30 min) detector: FID, 350 °C. restrictor Integral, column diameter 320 mm, L=145 mm, SP 5 μm C18 application of SFCapplication of SFC time [min]time [min] 228228 enhanced fluidity liquid chromatography (EFLC)enhanced fluidity liquid chromatography (EFLC) exploitation of low viscosity mobile phases : mixing liquid and supercritical fluid :: no phase separation should happen (L & G) : transitional mode between LC & SFC (UPC2) : applicable to RPLC, NPLC, HILIC and even SEC exploitation of low viscosity mobile phases : mixing liquid and supercritical fluid :: no phase separation should happen (L & G) : transitional mode between LC & SFC (UPC2) : applicable to RPLC, NPLC, HILIC and even SEC within RP‐EFLC at molar fraction of CO2 0.3 : decreasing analysis time :: ~ 2x decrease : increasing separation efficiency :: ~ 2.5x decrease of viscosity causes 2x increase in separation efficiency within RP‐EFLC at molar fraction of CO2 0.3 : decreasing analysis time :: ~ 2x decrease : increasing separation efficiency :: ~ 2.5x decrease of viscosity causes 2x increase in separation efficiency molar ratio of CO2 in MeOHmolar ratio of CO2 in MeOH viscosity [mPa∙s]viscosity [mPa∙s] subcritical fluid chromatography : intermediate between SFC and EFLC : keeping supercritical pressure, but subcritical temperature :: even closer to liquid than supercritical liquid (density, diffusion coefficient) :: no phase separation easily happens subcritical fluid chromatography : intermediate between SFC and EFLC : keeping supercritical pressure, but subcritical temperature :: even closer to liquid than supercritical liquid (density, diffusion coefficient) :: no phase separation easily happens 229229 newer method (since beginning of 90'ies) SP particles have large lateral pores (POROS media) analyte carried by convective flow of MP and enters the particle interior lateral pores are mutually connected by short diffusion pores : cross‐linked structure with large space inside for interaction of A and carrier newer method (since beginning of 90'ies) SP particles have large lateral pores (POROS media) analyte carried by convective flow of MP and enters the particle interior lateral pores are mutually connected by short diffusion pores : cross‐linked structure with large space inside for interaction of A and carrier perfusion chromatography (PLC)perfusion chromatography (PLC) 230230 SP materialSP material poly(styrene‐divinylbenzene) polymer particle size 10, 20, 50 μm lateral pores 0.6 – 0.8 μm diffusion pores 0.05 – 0.15 μm poly(styrene‐divinylbenzene) polymer particle size 10, 20, 50 μm lateral pores 0.6 – 0.8 μm diffusion pores 0.05 – 0.15 μm conventional separationconventional separation perfusion separationperfusion separation MP flow creates across the particle a pressure gradient & causes convective flow through lateral pores (perfusion) : molecules of analyte in contact not only with particle surface, but also with binding sites interior : convective transport is much higher than diffusion transport controlled by concentration gradient MP flow creates across the particle a pressure gradient & causes convective flow through lateral pores (perfusion) : molecules of analyte in contact not only with particle surface, but also with binding sites interior : convective transport is much higher than diffusion transport controlled by concentration gradient principleprinciple 231231 effect of perfusion happens only at certain flow‐rates >1000 cm.h‐1effect of perfusion happens only at certain flow‐rates >1000 cm.h‐1 modern HPLC and FPLC – small analytical columns POROS : 4.6 x 100 mm, volume 1.7 mL, flow‐rate 10 mL∙min‐1 modern HPLC and FPLC – small analytical columns POROS : 4.6 x 100 mm, volume 1.7 mL, flow‐rate 10 mL∙min‐1 systems for perfusion chromatography generate pressures up to 20 MPa and high flow‐ratessystems for perfusion chromatography generate pressures up to 20 MPa and high flow‐rates PLC vs. LC : high capacity independent on flow‐rate : high resolution independent on flow‐rate : fast separation :: 10 – 100x LC, in order of minutes PLC vs. LC : high capacity independent on flow‐rate : high resolution independent on flow‐rate : fast separation :: 10 – 100x LC, in order of minutes enhancing quality of separation by serial connection of different LC modes : increasing the peak capacity (i = number of dimension) enhancing quality of separation by serial connection of different LC modes : increasing the peak capacity (i = number of dimension) connection problemsconnection problems multidimensional chromatographymultidimensional chromatography (mD‐LC)(mD‐LC) IX.IX. : heart‐cutting :: in following dimensions, separation of certain fractions; LC‐CL : comprehensive :: in following dimensions, separation of the whole eluate; LC×LC : heart‐cutting :: in following dimensions, separation of certain fractions; LC‐CL : comprehensive :: in following dimensions, separation of the whole eluate; LC×LC : separation in space :: slab techniques (e.g. TLC) : separation in space :: slab techniques (e.g. TLC) : separation in time :: column techniques : separation in time :: column techniques 𝐧 𝐦𝐃 𝐧𝐢.𝐃𝐧 𝐦𝐃 𝐧𝐢.𝐃 𝐧 𝟐𝐃 𝐧 𝟏.𝐃 𝐦 · 𝐧 𝟐.𝐃𝐧 𝟐𝐃 𝐧 𝟏.𝐃 𝐦 · 𝐧 𝟐.𝐃 232232 : incompatibility of solvents between modes :: miscible solvents : zone broadening between columns because of valves, loops and detector :: second separation dimension must be focusing : need for much faster separation in 2nd dimension than in the 1st :: technical solution of eluate transfer from one dimension to other : incompatibility of solvents between modes :: miscible solvents : zone broadening between columns because of valves, loops and detector :: second separation dimension must be focusing : need for much faster separation in 2nd dimension than in the 1st :: technical solution of eluate transfer from one dimension to other A – miscible solvents B – immiscible solvents A – miscible solvents B – immiscible solvents continuous connection for Acontinuous connection for A continuous connection for Acontinuous connection for A discontinuous connection for Adiscontinuous connection for A interphase between dimensionsinterphase between dimensions 233233 1D: long micro‐column with low flow rates 2D: short or monolithic column with high flow rates interface: 10‐way valve; system of two loops transports eluate from 1D to 2D 1D: long micro‐column with low flow rates 2D: short or monolithic column with high flow rates interface: 10‐way valve; system of two loops transports eluate from 1D to 2D 1D: long micro‐column with low flow rates 2D: several short or monolithic columns connected in‐parallel interface: 6‐way valve; loopless transport of a fraction from 1D to free 2D 1D: long micro‐column with low flow rates 2D: several short or monolithic columns connected in‐parallel interface: 6‐way valve; loopless transport of a fraction from 1D to free 2D 1D: long micro‐column with low flow rates 2D: short or monolithic column with high flow rates interface: 6‐way valve; through loop a fraction transported from 1D to free 2D 1D: long micro‐column with low flow rates 2D: short or monolithic column with high flow rates interface: 6‐way valve; through loop a fraction transported from 1D to free 2D discontinuous connection (A + B)discontinuous connection (A + B) 234234 1D: any column 2D: any column interphase – 6‐way valve : through capture column (RAM SP type) fractions are moved from 1D to 2D : through fraction collector fractions are moved from 1D to 2D : through evaporation loop fractions are moved from 1D to 2D :: MP1 removed from sample and new solubilisation in MP2 1D: any column 2D: any column interphase – 6‐way valve : through capture column (RAM SP type) fractions are moved from 1D to 2D : through fraction collector fractions are moved from 1D to 2D : through evaporation loop fractions are moved from 1D to 2D :: MP1 removed from sample and new solubilisation in MP2 pump 1 NT gradient pump 1 NT gradient pump 2 (isocratic)pump 2 (isocratic) column 1 IEC column 1 IEC column 2 SEC column 2 SEC detectordetector wastewaste ww MP2MP2 MP1MP1 wastewaste loop 1loop 1 loop 2loop 2 V1V1 V2V2 injectioninjection combined dimensionscombined dimensions 1D : HILIC; 2D : RP 1D : IEC; 2D : SEC, RP 1D : SEC; 2D : IEC, RP 1D : AC; 2D : RP 1D : HILIC; 2D : RP 1D : IEC; 2D : SEC, RP 1D : SEC; 2D : IEC, RP 1D : AC; 2D : RP RPLC; time [s]RPLC; time [s] IEC; time [min]IEC; time [min] record from 2D LC (IEC‐RP)record from 2D LC (IEC‐RP) dimension complementaritydimension complementarity to choose modes of both following dimensions so, that the separation selectivity would be maximalto choose modes of both following dimensions so, that the separation selectivity would be maximal 𝐎 ∑ 𝐛𝐢𝐧 𝐧 𝐦𝐚𝐱 𝟎. 𝟔𝟑 · 𝐧 𝐦𝐚𝐱 𝐎 ∑ 𝐛𝐢𝐧 𝐧 𝐦𝐚𝐱 𝟎. 𝟔𝟑 · 𝐧 𝐦𝐚𝐱 10 % ~ 0 %O10 % ~ 0 %O 100 %  fully ordered system 100 %  fully ordered system 63 % ~ 100 %O63 % ~ 100 %O bin – a position with value in 2D histogram nmax – max peak capacity : sum of all bins in histogram bin – a position with value in 2D histogram nmax – max peak capacity : sum of all bins in histogram highhighlowlow complementarity (othogonality)measurecomplementarity (othogonality)measure αα γγ NpNp ββ α'α' Np – effective area; practical peak capacity β – so‐called spreading angle Np – effective area; practical peak capacity β – so‐called spreading angle N1.DN1.D N2.DN2.D 235235 (orthogonality, O)(orthogonality, O) idealideal 𝐍 𝐩 𝐍𝐭𝐞𝐨𝐫 𝐍 𝟐.𝐃 𝟐 · 𝐭𝐚𝐧 𝛂 𝐍 𝟏.𝐃 𝟐 · 𝐭𝐚𝐧 𝛄𝐍 𝐩 𝐍𝐭𝐞𝐨𝐫 𝐍 𝟐.𝐃 𝟐 · 𝐭𝐚𝐧 𝛂 𝐍 𝟏.𝐃 𝟐 · 𝐭𝐚𝐧 𝛄 3.003.001.801.801.201.200.500.500.000.00 2.402.40 00 11 22 33 hh ff gg xx yy t1,xt1,x t1,yt1,y t2,xt2,x t2,yt2,y 𝐑 𝐀,𝐁 𝟎. 𝟓 · 𝐥𝐧 𝟏 𝐟 𝐠⁄ 𝟐⁄𝐑 𝐀,𝐁 𝟎. 𝟓 · 𝐥𝐧 𝟏 𝐟 𝐠⁄ 𝟐⁄ resolution in 2D LCresolution in 2D LC 236236 enhancing quality of separation by connecting different separation techniquesenhancing quality of separation by connecting different separation techniques hyphenated techniqueshyphenated techniques limited compatibility of principles : requires special interphases limited compatibility of principles : requires special interphases injection  injection   LC columnLC column pumppump MPMP injection valve for CE injection valve for CE waste valve waste valve CE capillary CE capillary high voltage high voltage detectiondetection check‐incheck‐in CE capillaryCE capillary from LC column from LC column to waste to waste combined / hyphenated techniquescombined / hyphenated techniques 237237 1D : LC; 2D : CE 1D : CE; 2D : LC 1D : LC; 2D : MS 1D : LC; 2D : CE 1D : CE; 2D : LC 1D : LC; 2D : MS preparative chromatographypreparative chromatography isolation and purification by means of LCisolation and purification by means of LC in extent of μg up‐to kg – purification of enzymes up‐to industrial scale in extent of μg up‐to kg – purification of enzymes up‐to industrial scale  non‐linear part of adsorption isotherm non‐linear part of adsorption isotherm  according to substance type : increasing load [g] leads to decrease of k' :: asymmetric peaks, strong tailing ::: concentration overloading : increasing load [g] leads to increase of k' :: asymmetric peaks, strong fronting ::: volume overloading according to substance type : increasing load [g] leads to decrease of k' :: asymmetric peaks, strong tailing ::: concentration overloading : increasing load [g] leads to increase of k' :: asymmetric peaks, strong fronting ::: volume overloading separation optimisation: yield, purity, speed separation optimisation: yield, purity, speed  preparative separation preparative separation analytical separation analytical separation amount [g]amount [g] k'k' k'k' σvσv 238238 methods of preparative LCmethods of preparative LC optimisation: flow rate; amount of sampleoptimisation: flow rate; amount of sample FM – voluminal flow rate r – column diameter  FM – voluminal flow rate r – column diameter  separation parameters conversion separation parameters conversion  x – max volume loaded L – column length  x – max volume loaded L – column length  𝐅 𝐌 𝟏 𝐅 𝐌 𝟐 𝐫𝟏 𝐫𝟐 𝐅 𝐌 𝟏 𝐅 𝐌 𝟐 𝐫𝟏 𝐫𝟐 𝐱 𝟏 𝛑 · 𝐫𝟏 𝟐 𝐱 𝟐 𝛑 · 𝐫𝟐 𝟐 · 𝐋 𝟐 𝐋 𝟏 𝐱 𝟏 𝛑 · 𝐫𝟏 𝟐 𝐱 𝟐 𝛑 · 𝐫𝟐 𝟐 · 𝐋 𝟐 𝐋 𝟏 239239 enlargement of system dimensionsenlargement of system dimensions positives: still symmetric peaks negatives: size of column and solvent consumption positives: still symmetric peaks negatives: size of column and solvent consumption (scale‐up)(scale‐up) system overloadingsystem overloading analytical separation analytical separation volume overloading volume overloading concentration overloading concentration overloading VinjVinj VinjVinj VinjVinj volume overloadingvolume overloading : at bad sample solubility in MP : rectangular peak shape : linear adsorption isotherm : controlled by column diameter : small SP particle size needed : at bad sample solubility in MP : rectangular peak shape : linear adsorption isotherm : controlled by column diameter : small SP particle size needed concentration overloadingconcentration overloading : at good sample solubility in MP : triangular peak shape : non‐linear adsorption isotherm : controlled by selectivity : small influence of SP particle size : at good sample solubility in MP : triangular peak shape : non‐linear adsorption isotherm : controlled by selectivity : small influence of SP particle size SP used and system parameters SP used and system parameters  optimisation on analytical column – same SP as in preparative mode optimisation on analytical column – same SP as in preparative mode  column diameter [mm] for α < 1.2 [mg] for α > 1.5 [mg] 4.6 2 – 3 20 – 30 9.4 10 – 20 100 – 200 21.2 50 – 200 500 – 2000 30, 50 > 200 > 2000 it is important to provide appropriate capillary diameterit is important to provide appropriate capillary diameter σ2 – zone broadening, FM – voluminal flow rate, L – column length, Dm – diffusion coefficient, r – capillary diameter  σ2 – zone broadening, FM – voluminal flow rate, L – column length, Dm – diffusion coefficient, r – capillary diameter  Aris‐Taylor equationAris‐Taylor equation𝛔 𝟐 𝛑 · 𝐫 𝟒 · 𝐅 𝐌 · 𝐋 𝟐𝟒 · 𝐃 𝐦 𝛔 𝟐 𝛑 · 𝐫 𝟒 · 𝐅 𝐌 · 𝐋 𝟐𝟒 · 𝐃 𝐦 240240 : critical is an extent of coverage by active layer (mol∙m‐2) :: controlled by the particle diameter ::: 5 μm for poorly separated mixtures ::: 7 – 10 μm for well separated mixtures   : critical is an extent of coverage by active layer (mol∙m‐2) :: controlled by the particle diameter ::: 5 μm for poorly separated mixtures ::: 7 – 10 μm for well separated mixtures   stationary phasestationary phase fraction collection fraction collection  detection controlleddetection controlled UV‐VisUV‐Vis MSMS monoisotopic peak of analyte is used monoisotopic peak of analyte is used  1st peak derivation is used1st peak derivation is used good signal filtering : noise smoothing (Savitzky‐Golay) good signal filtering : noise smoothing (Savitzky‐Golay) sharp peaks : cause lower losses by peak identification : important to minimise post‐column broadening : not too long capillaries : fast connection of PC and fraction collector sharp peaks : cause lower losses by peak identification : important to minimise post‐column broadening : not too long capillaries : fast connection of PC and fraction collector increaseincrease decreasedecrease chromatogramchromatogram inflex point inflex point = 95.5 %= 95.5 % = 68.3 %= 68.3 % 4σ4σ 2σ2σ tR [min]tR [min] 1st derivation1st derivation 241241 mobile phasemobile phase : suitable spectroscopic properties : volatility, boiling point (substance isolation from fraction) : viscosity (too high pressure) : purity : solubility : price (acetonitrile > heptane > acetone > methanol) : suitable spectroscopic properties : volatility, boiling point (substance isolation from fraction) : viscosity (too high pressure) : purity : solubility : price (acetonitrile > heptane > acetone > methanol) buffer pH trifluoroacetate < 1.5 ammonium formate 3.0 – 5.0 pyridinium formate 3.0 – 5.0 ammonium acetate 3.8 – 5.8 ammonium carbonate 5.5 – 7.5; 9.3 – 11.3 ammonium 8.3 – 10.3 volatile buffersvolatile buffers gas chromatographygas chromatography : mobile phase (MP, gas) : stationary phase (SP, liquid, solid, thin layer of liquid on carrier) : mobile phase (MP, gas) : stationary phase (SP, liquid, solid, thin layer of liquid on carrier) : extraction G‐L : extraction G‐S : extraction G‐L : extraction G‐S 19411941 GC historyGC history 19521952 19801980 19631963 capillary columns in GC – distinctive separation improvementcapillary columns in GC – distinctive separation improvement GC‐MS – first hyphenated techniqueGC‐MS – first hyphenated technique James and Martin : practical introduction of GC : separation of volatile fatty acids James and Martin : practical introduction of GC : separation of volatile fatty acids Synge and Martin – theoretic principles of GC ...very refined separations of volatile substances should be possible in a column in which permanent gas is made to flow over gel impregnated with a non‐volatile solvent ... Synge and Martin – theoretic principles of GC ...very refined separations of volatile substances should be possible in a column in which permanent gas is made to flow over gel impregnated with a non‐volatile solvent ... X.X. 242242 low concentrations of A, non‐ideal solution kH – Henry's constant pA – partial pressure of A over mixture low concentrations of A, non‐ideal solution kH – Henry's constant pA – partial pressure of A over mixture xA – molar ratio of A in mixture pA – pressure of saturated vapours of A xA – molar ratio of A in mixture pA – pressure of saturated vapours of A Raoult's lawRaoult's law partial pressurepartial pressure molar ratiomolar ratio 00 principal differences between GC and LCprincipal differences between GC and LCgas is compressible (liquid not)gas is compressible (liquid not) Henry isothermHenry isotherm 𝐩 𝐀 𝐩 𝐀 𝟎 · 𝐱 𝐀𝐩 𝐀 𝐩 𝐀 𝟎 · 𝐱 𝐀 𝐜 𝐀 𝐒 𝐤 𝐇 · 𝐩 𝐀𝐜 𝐀 𝐒 𝐤 𝐇 · 𝐩 𝐀 243243 Langmuir isothermLangmuir isotherm 𝐜 𝐀 𝐒 𝐜 𝐀 𝐦𝐚𝐱 𝐒 · 𝐊 𝐃 · 𝐩 𝐀 𝟏 𝐊 𝐃 · 𝐩 𝐀 𝐜 𝐀 𝐒 𝐜 𝐀 𝐦𝐚𝐱 𝐒 · 𝐊 𝐃 · 𝐩 𝐀 𝟏 𝐊 𝐃 · 𝐩 𝐀 cmax – maximal bound concentration on SPcmax – maximal bound concentration on SPSS distribution constant is strongly dependent of vapour pressure and volatility of analytedistribution constant is strongly dependent of vapour pressure and volatility of analyte 00 22 44 66 88 1010 00 22 44 66 88 1010 KD = 1KD = 1 cA,max = 0.01 mol∙m‐2cA,max = 0.01 mol∙m‐2SS pApA cAcA SS linear flow rate of carrier gas (MP)linear flow rate of carrier gas (MP) L – column length p – gas pressure u – linear flow rate indices:  i – on inlet x – in point x of length o – on outlet L – column length p – gas pressure u – linear flow rate indices:  i – on inlet x – in point x of length o – on outlet pressure gradient profile on column pressure gradient profile on column value profile  of linear flow rate value profile  of linear flow rate 0.00.0 0.20.2 0.40.4 0.60.6 0.80.8 1.01.0 00 11 22 33 44 55 66 po = 0.1 MPapo = 0.1 MPa pi = 0.5 MPapi = 0.5 MPa pxpx x / Lx / L 0.00.0 0.20.2 0.40.4 0.60.6 0.80.8 1.01.0 0.00.0 0.20.2 0.40.4 0.60.6 0.80.8 1.01.0 popo = 5= 5 pipi po = 5 pi uouo uxux uo ux x / Lx / L LL xxpipi pxpx popo uiui uxux uouo 244244 keeping constant flow rate : solely by column (input pressure checked; regulation by metal membrane) : pneumotoric serial resistance (capillary + needle valve) : constant mass flow (feed back) keeping constant flow rate : solely by column (input pressure checked; regulation by metal membrane) : pneumotoric serial resistance (capillary + needle valve) : constant mass flow (feed back) average linear MP flow rateaverage linear MP flow rate B0 – specific permeability of column [m2] (pi‐po) – pressure gradient [Pa] η – dynamic viscosity [Pa∙s] ε – sorbent inner porosity L – column length [m] B0 – specific permeability of column [m2] (pi‐po) – pressure gradient [Pa] η – dynamic viscosity [Pa∙s] ε – sorbent inner porosity L – column length [m] 𝐮 𝐁 𝟎 · 𝐩𝐢 𝐩 𝐨 𝛈 · 𝛆 · 𝐋 𝐮 𝐁 𝟎 · 𝐩𝐢 𝐩 𝐨 𝛈 · 𝛆 · 𝐋 compressibility factorcompressibility factor 𝐣 𝟑 𝟐 · 𝐩𝐢 𝐩 𝐨 𝟐 𝟏 𝐩𝐢 𝐩 𝐨 𝟑 𝟏 𝐣 𝟑 𝟐 · 𝐩𝐢 𝐩 𝐨 𝟐 𝟏 𝐩𝐢 𝐩 𝐨 𝟑 𝟏 𝐮 𝐣 · 𝐮 𝐨𝐮 𝐣 · 𝐮 𝐨 j – compressibility factorj – compressibility factor net retention volumenet retention volume retention volume / time of i‐th analyteretention volume / time of i‐th analyte corrected retention volume / timecorrected retention volume / time specific retention volumespecific retention volume retention quantitiesretention quantities 𝐕 𝐦 𝐅 𝐌 · 𝐭 𝐦 𝐕 𝐌𝐕 𝐦 𝐅 𝐌 · 𝐭 𝐦 𝐕 𝐌 𝐕𝐩 𝟐𝟕𝟑. 𝟏𝟓 · 𝐕 𝐍 𝐒 · 𝐓𝐤 𝐕𝐩 𝟐𝟕𝟑. 𝟏𝟓 · 𝐕 𝐍 𝐒 · 𝐓𝐤 𝐕𝐡 𝟐𝟕𝟑. 𝟏𝟓 · 𝐕 𝐍 𝐰 𝐋 · 𝐓𝐤 𝐕𝐡 𝟐𝟕𝟑. 𝟏𝟓 · 𝐕 𝐍 𝐰 𝐋 · 𝐓𝐤 𝐕 𝐑 𝐢 𝐅 𝐌 · 𝐭 𝐑 𝐢 𝐕 𝐑 𝐢 𝐅 𝐌 · 𝐭 𝐑 𝐢 𝐭 𝐑 𝐢 𝐭 𝐑 𝐢 𝐭 𝐦𝐭 𝐑 𝐢 𝐭 𝐑 𝐢 𝐭 𝐦 𝐕 𝐑 𝐢 𝐅 𝐌 · 𝐭 𝐑 𝐢 𝐕 𝐑 𝐢 𝐅 𝐌 · 𝐭 𝐑 𝐢 𝐕 𝐑 𝐢 𝐕 𝐑 𝐢 𝐕 𝐦𝐕 𝐑 𝐢 𝐕 𝐑 𝐢 𝐕 𝐦 𝐕 𝐍 𝐅 𝐌 · 𝐭 𝐑 𝐢 · 𝐣 𝐕 𝐑 𝐢 · 𝐣𝐕 𝐍 𝐅 𝐌 · 𝐭 𝐑 𝐢 · 𝐣 𝐕 𝐑 𝐢 · 𝐣 Vh [ml∙g ‐1] or Vp [ml∙m‐2]Vh [ml∙g ‐1] or Vp [ml∙m‐2] net retention volume related to 1 g or 1 m2 SP and to 0 °C net retention volume related to 1 g or 1 m2 SP and to 0 °C VN [min]VN [min] corrected retention volume adjusted to carrier gas compressibilitycorrected retention volume adjusted to carrier gas compressibility 245245 V'R,i [ml], t'R,i [min]V'R,i [ml], t'R,i [min] void volume / time of columnvoid volume / time of column VR,i [ml], tR,i [min]VR,i [ml], tR,i [min] Vm [ml], tm [min]Vm [ml], tm [min] wL – amount of immobilised SP (L) S – SF area (S) wL – amount of immobilised SP (L) S – SF area (S) temperature influencetemperature influence Tinj – injection head temperature Tcol – column thermostat temperature Tdet – detector temperature Tinj – injection head temperature Tcol – column thermostat temperature Tdet – detector temperature Tcol greater than Tboil while Tinj greater or equal to Tcol while Tdet greater than Tcol Tcol greater than Tboil while Tinj greater or equal to Tcol while Tdet greater than Tcol : higher Tcol leads to faster analysis : higher Tcol demands higher MP pressure on column inlet :: keeping u through column : higher Tcol leads to faster analysis : higher Tcol demands higher MP pressure on column inlet :: keeping u through column isothermic analysis: Tcol = const. analysis with temperature gradient: Tcol2 – Tcol1 > 0 isothermic analysis: Tcol = const. analysis with temperature gradient: Tcol2 – Tcol1 > 0 246246 temperature gradient 30 – 180 °Ctemperature gradient 30 – 180 °C isothermic separation 145 °Cisothermic separation 145 °C isothermic separation 45 °Cisothermic separation 45 °C 11 22 33 44 55 88 77 66 44 55 8877 66 44 55 99 11 22 33 30302020101000 30302020101000 30302020101000 time [min]time [min] separation column separation column injection device injection device outputoutput MP container MP container detectordetector GC arrangementGC arrangement 247247 gas sources : pressure containers : compressor : electrolyser gas sources : pressure containers : compressor : electrolyser: 0.5 – 400 ml∙min‐1 :: HP‐GC 1200 ml∙min‐1 : pressure up to 400 kPa :: HP‐GC 1 MPa : pressure and flow control : thermostating : 0.5 – 400 ml∙min‐1 :: HP‐GC 1200 ml∙min‐1 : pressure up to 400 kPa :: HP‐GC 1 MPa : pressure and flow control : thermostating advanced flow control (AFC) advanced flow control (AFC) advanced pressure control (APC) advanced pressure control (APC) u [cm∙s‐1]u [cm∙s‐1] H [mm]H [mm] 1.01.0 0.50.5 0.00.0 00 2020 4040 6060 8080 nitrogennitrogen heliumhelium hydrogenhydrogen MP deliveryMP delivery 248248 carrier gascarrier gas N2 (nitrogen) + cheap, safe – low thermal conductivity N2 (nitrogen) + cheap, safe – low thermal conductivity H2 (hydrogen) + high thermal conductivity, low viscosity – high diffusivity, explosive H2 (hydrogen) + high thermal conductivity, low viscosity – high diffusivity, explosive He (helium) + combines advantages of N2 & H2 – expensive He (helium) + combines advantages of N2 & H2 – expensive Ar (argon) especially for ECDAr (argon) especially for ECD purity – pre‐set guard column with molecular sievepurity – pre‐set guard column with molecular sieve must be chemically inert – always necessary to remove humidity and O2must be chemically inert – always necessary to remove humidity and O2 loading of A onto column : more difficult than by LC loading of A onto column : more difficult than by LC tubular columns ~ 200 μg capillary columns max 20 μg, opt 1 μg tubular columns ~ 200 μg capillary columns max 20 μg, opt 1 μg specially within capillary columns, inject small volume and do it quickly : slowly and large volume (overload) means broad zones and resolution loss specially within capillary columns, inject small volume and do it quickly : slowly and large volume (overload) means broad zones and resolution loss injection deviceinjection device 249249 liner of injector : heat evaporation and sample vapours mixing with carrier gas liner of injector : heat evaporation and sample vapours mixing with carrier gas inlet of carrier gas inlet of carrier gas septumseptum septum valveseptum valve separator valve separator valve evaporation cellevaporation cell column inletcolumn inlet heated metal block heated metal block linerliner necessity to (quickly) transform liquid and solid samples into gaseous state : without changing the nature of sample : requires heated space on the beginning of the column :: sometimes gasification on‐column volatility increment : chemical derivatisation :: silylation (N,O‐bis(trimethylsilyl)acetamide), silanisation (dimethylchlorsilane), acetylation (acetanhydride) necessity to (quickly) transform liquid and solid samples into gaseous state : without changing the nature of sample : requires heated space on the beginning of the column :: sometimes gasification on‐column volatility increment : chemical derivatisation :: silylation (N,O‐bis(trimethylsilyl)acetamide), silanisation (dimethylchlorsilane), acetylation (acetanhydride) sample evaporationsample evaporation 1 ml∙min‐11 ml∙min‐1 1 ml∙min‐11 ml∙min‐1 0 ml∙min‐10 ml∙min‐1 0 ml∙min‐10 ml∙min‐1 column inlet column inlet starting temperature of gradient starting temperature of gradient septumseptum injection needle injection needle on‐column injectionon‐column injection : similar to splitless injection :: after certain time, the valve is open & rest of the sample is washed out : injects precise amount : no evaporation during injection, until in the temperature gradient on column :: selective evaporation of compounds with lower boiling temperatures : instrumentally demanding :: necessity to restrict the pressure losses within injection : overloads column with liquid (1 μl for 50 cm of column) :: peak broadening (solution similar applies as to splitless injection) : similar to splitless injection :: after certain time, the valve is open & rest of the sample is washed out : injects precise amount : no evaporation during injection, until in the temperature gradient on column :: selective evaporation of compounds with lower boiling temperatures : instrumentally demanding :: necessity to restrict the pressure losses within injection : overloads column with liquid (1 μl for 50 cm of column) :: peak broadening (solution similar applies as to splitless injection) 250250 splitless injectionsplitless injection : suitable for classical packed columns :: and diluted samples : after a time without splitting, the valve is open :: meanwhile the sample is loaded on column ::: 10 – 40 s, opt 20 s (splitless time) :: the rest of the sample is washed out : suitable for classical packed columns :: and diluted samples : after a time without splitting, the valve is open :: meanwhile the sample is loaded on column ::: 10 – 40 s, opt 20 s (splitless time) :: the rest of the sample is washed out 251251 carrier gas flow rate e.g. 49 ml∙min‐1 carrier gas flow rate e.g. 49 ml∙min‐1 injectioninjection restrictorrestrictor separation valve closed separation valve closed septum valve 2 ml∙min‐1 septum valve 2 ml∙min‐1 waste 46 ml∙min‐1 waste 46 ml∙min‐1 flow rate 1 ml∙min‐1 flow rate 1 ml∙min‐1 majority of gaseous sample goes onto column majority of gaseous sample goes onto column advantages : majority of the sample goes onto column :: suitable for trace analysis advantages : majority of the sample goes onto column :: suitable for trace analysis disadvantages : slow mass transfer onto column :: zone broadening ::: a need for re‐concentration disadvantages : slow mass transfer onto column :: zone broadening ::: a need for re‐concentration 252252 split injectionsplit injection S – degree of sample splitting FM – column flow rate FS – splitter flow rate S – degree of sample splitting FM – column flow rate FS – splitter flow rate today, the most used way of injectiontoday, the most used way of injection 𝐒 𝐅 𝐌 𝐅𝐒 · 𝐅 𝐌 𝐒 𝐅 𝐌 𝐅𝐒 · 𝐅 𝐌 advantages : injection of a small volume :: sharp zones & low column polution advantages : injection of a small volume :: sharp zones & low column polution disadvantages : unsuitable for trace analysis : depends on the splitter geometry disadvantages : unsuitable for trace analysis : depends on the splitter geometry carrier gas flow rate e.g. 49 ml∙min‐1 carrier gas flow rate e.g. 49 ml∙min‐1 injectioninjection restrictorrestrictor separation valve open separation valve open septum valve 2 ml∙min‐1 septum valve 2 ml∙min‐1 waste 46 ml∙min‐1 waste 46 ml∙min‐1 flow rate 1 ml∙min‐1 flow rate 1 ml∙min‐1 majority of gaseous sample goes in waste majority of gaseous sample goes in waste sample re‐concentration : prevents zone broadening within direct and splitless injection sample re‐concentration : prevents zone broadening within direct and splitless injection cold trappingcold trapping : first few cm of column has negative temperature gradient :: ~ 250 °C (injection) decreases in capture region to ca 20 °C bellow solvent Tboil : sample components with low Tboil condensate with solvent : from the created thin film, the solvent is slowly evaporating : and thus re‐concentrate the components with low Tboil : first few cm of column has negative temperature gradient :: ~ 250 °C (injection) decreases in capture region to ca 20 °C bellow solvent Tboil : sample components with low Tboil condensate with solvent : from the created thin film, the solvent is slowly evaporating : and thus re‐concentrate the components with low Tboil : first few cm of column has negative temperature gradient :: ~ 250 °C (injection) decreases to 40 °C in capture region ::: ca about 150 °C lower than the compound with the highest Tboil : mobility of components with high Tboil is thus zero : and thus their re‐concentration is achieved : first few cm of column has negative temperature gradient :: ~ 250 °C (injection) decreases to 40 °C in capture region ::: ca about 150 °C lower than the compound with the highest Tboil : mobility of components with high Tboil is thus zero : and thus their re‐concentration is achieved solvent effectsolvent effect 253253 hyphenation of SFE with GC (cold‐trapping) hyphenation of SFE with GC (cold‐trapping) separation of supercritical fluid from sample increases quality GC analysisseparation of supercritical fluid from sample increases quality GC analysis a) w/o utilisationa) w/o utilisation b) w/ utilisationb) w/ utilisation separation by means of cold‐trapping 1. Tcol in time (t = 0) ≤ 25 °C 2. df ≥ 2 μm SP separation by means of cold‐trapping 1. Tcol in time (t = 0) ≤ 25 °C 2. df ≥ 2 μm SP SFE‐GC interfaceSFE‐GC interface direct SFE‐GCdirect SFE‐GC extraction cellextraction cell flow restrictorflow restrictor septum‐injectionseptum‐injection separated analyteseparated analyte capillary GC columncapillary GC column splittersplitter 254254 gas injectiongas injection step 1 equilibration step 1 equilibration step 2 HSE syphoning step 2 HSE syphoning step 3 injection step 3 injection injection in system with pressure equilibriuminjection in system with pressure equilibrium step 1 equilibration step 1 equilibration step 2 pressurising step 2 pressurising step 3 syphoning & injection step 3 syphoning & injection fromfrom into/frominto/from pressure valvepressure valve step 1 pressurising step 1 pressurising step 2 HSE syphoning step 2 HSE syphoning step 3 injection step 3 injection inlet inlet  outlet (column) outlet (column) hyphenation HSE‐GC hyphenation HSE‐GC  255255 packed tubular : analytical : preparative packed tubular : analytical : preparative length: 0.5 – 50.0 m diameter: 0.3 – 1.0 mm length: 0.5 – 50.0 m diameter: 0.3 – 1.0 mm length: 0.5 – 10.0 m diameter: 1 – 6 mm length: 0.5 – 10.0 m diameter: 1 – 6 mm length: 10 – 100 m diameter: 0.1 – 0.5 mm length: 10 – 100 m diameter: 0.1 – 0.5 mm length: 2 – 6 m diameter: > 6 mm length: 2 – 6 m diameter: > 6 mm separation columnseparation column ≡ 0.10 – minibore < 0.25 – narrow bore ≡ 0.32 – wide bore ≡ 0.45 – high speed megabore ≡ 0.53 – megabore ≡ 0.10 – minibore < 0.25 – narrow bore ≡ 0.32 – wide bore ≡ 0.45 – high speed megabore ≡ 0.53 – megabore 256256 capillary : open : filled capillary : open : filled GC separation of calamus oil components A – 50 m capillary column B – 4 m tubular column GC separation of calamus oil components A – 50 m capillary column B – 4 m tubular column carriers fine, solid and inert material (spherical silica) : active centres (silanols and siloxanes) cause tailing of more polar components :: suppression – silylation : serves directly as SP (GSC) : or is covered by thin liquid phase film (GLC) adsorbents : unspecific (activated carbon) : specific (silica, alumina, molecular sieves etc.) carriers fine, solid and inert material (spherical silica) : active centres (silanols and siloxanes) cause tailing of more polar components :: suppression – silylation : serves directly as SP (GSC) : or is covered by thin liquid phase film (GLC) adsorbents : unspecific (activated carbon) : specific (silica, alumina, molecular sieves etc.) basic type of sorbentsbasic type of sorbents packed tubular columnspacked tubular columns 257257 cover : glass, steel, copper, polymers cover : glass, steel, copper, polymers solid SPsolid SP non‐polar : methylated polysiloxane, squalene, apolane C‐87 non‐polar : methylated polysiloxane, squalene, apolane C‐87 mildly polar : phenylated polysiloxane strongly polar : polysiloxane with CH2‐CH2‐CN, ‐CH=CH‐CN, Carbowax 20M (PEG‐based) mildly polar : phenylated polysiloxane strongly polar : polysiloxane with CH2‐CH2‐CN, ‐CH=CH‐CN, Carbowax 20M (PEG‐based) quartz : surface enlargement by etching : polyimide cover increases mechanical stability SP universal non‐polar silicon phases or immobilised Carbowax quartz : surface enlargement by etching : polyimide cover increases mechanical stability SP universal non‐polar silicon phases or immobilised Carbowax capillary columnscapillary columns 258258 i.d. 100 – 530 μmi.d. 100 – 530 μm film thickness 0.1 – 8 μm film thickness 0.1 – 8 μm i.d. 320 – 530 μmi.d. 320 – 530 μm film layer thickness 6 – 60 μm film layer thickness 6 – 60 μm layer thickness 5 – 50 μm layer thickness 5 – 50 μm i.d. 320 – 530 μmi.d. 320 – 530 μm thin wall with outer polyimide cover (GSC)thin wall with outer polyimide cover (GSC) fused silica open tubularfused silica open tubular (FSOT)(FSOT) liquid SP anchored directly on the capillary wall (GLC)liquid SP anchored directly on the capillary wall (GLC) wall‐coated open tubular columnswall‐coated open tubular columns (WCOT)(WCOT) support‐coated open tubular columnssupport‐coated open tubular columns carrier is on capillary wall, SP is on it (GLC)carrier is on capillary wall, SP is on it (GLC) (SCOT)(SCOT) porous‐layer open tubular columnsporous‐layer open tubular columns layer of solid active sorbent on an inner capillary wall (GSC)layer of solid active sorbent on an inner capillary wall (GSC) (PLOT)(PLOT) column thermostatcolumn thermostat importance of temperature of GCimportance of temperature of GC optimal loading temperatures : Tboil of component with highest value + 30 – 50 °C optimal loading temperatures : Tboil of component with highest value + 30 – 50 °C : evaporation of liquid or solid sample : kinetic aspects of separation : evaporation of liquid or solid sample : kinetic aspects of separation 259259 kept with precision of 0.1 °C : thermostat range (Tlab + 4 °C) – 450 °C kept with precision of 0.1 °C : thermostat range (Tlab + 4 °C) – 450 °C wide range of Tboil of separated components : requires temperature programme / column gradient :: temperature change during experiment :: temperature may be increased gradually or in steps wide range of Tboil of separated components : requires temperature programme / column gradient :: temperature change during experiment :: temperature may be increased gradually or in steps optimal column temperature around Tboil of analyte : column temperature greater or equal to Tboil , thus tR = 2 – 30 min : minimal temperature means better resolution, but higher tR optimal column temperature around Tboil of analyte : column temperature greater or equal to Tboil , thus tR = 2 – 30 min : minimal temperature means better resolution, but higher tR flame ionisation detectorflame ionisation detector detected compound is volatile, in gaseous statedetected compound is volatile, in gaseous state concentration dependent detector (CDD) : dilution with carrier gas decreases sensitivity mass dependent detector (MDD) : carrier gas interferes not, depends on introduction rate into detector concentration dependent detector (CDD) : dilution with carrier gas decreases sensitivity mass dependent detector (MDD) : carrier gas interferes not, depends on introduction rate into detector hydrogenhydrogen columncolumn airair +300 V+300 V collection  electrode collection  electrodeignition spiral ignition spiral FIDFID MDD signal: current created by pyrolysis of carbon sample MDD signal: current created by pyrolysis of carbon sample ion current : noise 10‐13 : dynamic range 107 : sensitivity 10‐9 M ion current : noise 10‐13 : dynamic range 107 : sensitivity 10‐9 M detectorsdetectors 260260 differential thermal conductivity : noise 10‐5 : dynamic range 106 : sensitivity 10‐8 M differential thermal conductivity : noise 10‐5 : dynamic range 106 : sensitivity 10‐8 M thermal conductivity detectorthermal conductivity detector amplifieramplifier samplesample referencereferencesamplesample referencereference sourcesource TCD catharometer TCD catharometer CDD signal: sample molecules change (decrease) thermal conductivity of carrier gas : carrier gas must have high thermal conductivity (He, H2...) : temperature dependent, universal CDD signal: sample molecules change (decrease) thermal conductivity of carrier gas : carrier gas must have high thermal conductivity (He, H2...) : temperature dependent, universal from columnfrom column wastewaste current measuring current measuring 63Ni63Ni electron capture detectorelectron capture detector ECDECD MDD signal: analyte molecules decrease current generated by β‐emitter : halides, nitrites, cyano‐compounds, peroxides, anhydrides, organometals MDD signal: analyte molecules decrease current generated by β‐emitter : halides, nitrites, cyano‐compounds, peroxides, anhydrides, organometals ion current : noise 10‐12 : dynamic range 105 : sensitivity 10‐13 M ion current : noise 10‐12 : dynamic range 105 : sensitivity 10‐13 M 261261 ion current : noise 10‐14 : dynamic range 106 : sensitivity 10‐12 M ion current : noise 10‐14 : dynamic range 106 : sensitivity 10‐12 M helium ionisation detectorhelium ionisation detector HIDHIDdischarge electrodes discharge electrodes 500 V500 V auxiliary gas auxiliary gas discharge chamber discharge chamber columncolumn wastewaste metermeter 150 V150 V ionisation chamber ionisation chamber collection tables collection tables MDD signal: auxiliary gas is ionised first (He, Ar), its ions then secondary ionise sample molecules MDD signal: auxiliary gas is ionised first (He, Ar), its ions then secondary ionise sample molecules 262262 chemoluminiscence detectorchemoluminiscence detector datadataphoto.photo. columncolumn irradiationirradiation pump outlet pump outlet inlet of fluorine inlet of fluorine chemiluminiscence : noise 10‐13 : dynamic range 104 : sensitivity 10‐11 M chemiluminiscence : noise 10‐13 : dynamic range 104 : sensitivity 10‐11 M CDD signal: reaction of F (strong oxidant) with analyte CDD signal: reaction of F (strong oxidant) with analyte hydrogenhydrogen airair heated metal block heated metal block thermostat wallthermostat wallcolumncolumn flameflame region of chemoluminiscence region of chemoluminiscence wastewaste thermal filterthermal filter interference filterinterference filter sourcesource into amplifier into amplifier photomultiplierphotomultiplier flame photometric detectorflame photometric detector FPDFPD chemiluminiscence : noise 10‐12 : dynamic range 107 : sensitivity 10‐10 M chemiluminiscence : noise 10‐12 : dynamic range 107 : sensitivity 10‐10 M MDD signal: chemoluminiscence : selective S (394 nm), P (526 nm) MDD signal: chemoluminiscence : selective S (394 nm), P (526 nm) 263263 MDD signal: Rb/Ce glass thermoionisation electron emission enhanced by N or P presence MDD signal: Rb/Ce glass thermoionisation electron emission enhanced by N or P presence nitrogen phosphorus detectornitrogen phosphorus detector hydrogenhydrogen airair carrier gas (nitrogen)carrier gas (nitrogen) flameflame rubidium or caesium glass rubidium or caesium glass anodeanode connectors  of heating connectors  of heating heating spiraleheating spirale NPD – thermoionisation detectorNPD – thermoionisation detector ion current : noise 10‐12 : dynamic range 106 : sensitivity 10‐10 M ion current : noise 10‐12 : dynamic range 106 : sensitivity 10‐10 M ion current : noise 10‐13 : dynamic range 107 : sensitivity 10‐11 M ion current : noise 10‐13 : dynamic range 107 : sensitivity 10‐11 M lightproof cover lightproof cover sourcesource UV‐transparent window UV‐transparent windowwaste or secondary detector waste or secondary detector heated ionisation cell heated ionisation cell columncolumn thermostat wall thermostat wall UV lampUV lamp electrodeselectrodes metermeter datadata diode array diode array gridgrid columncolumn microwave reactor microwave reactor microwave „ignition“ microwave „ignition“ coolercooler mirrormirror atomic emission atomic emission atomic emission detectoratomic emission detector AEDAED atomic emission radiation : noise 10‐14 : dynamic range 104 : sensitivity 10‐11 M atomic emission radiation : noise 10‐14 : dynamic range 104 : sensitivity 10‐11 M MDD signal: microwave induced plasma : selective according to chosen emission wavelength : very expensive MDD signal: microwave induced plasma : selective according to chosen emission wavelength : very expensive photoionisation detectorphotoionisation detector PIDPID MDD signal: UV‐irradiation ionisation MDD signal: UV‐irradiation ionisation 264264 absorption of infrared radiation : noise 10‐12 : dynamic range 105 : sensitivity 10‐10 M absorption of infrared radiation : noise 10‐12 : dynamic range 105 : sensitivity 10‐10 M ion‐count : noise 10‐14 : dynamic range 103 : sensitivity 10‐15 M ion‐count : noise 10‐14 : dynamic range 103 : sensitivity 10‐15 M infrared detector infrared detector  mass spectrometric detectormass spectrometric detector columncolumnwastewaste IR sourceIR source KBr windowKBr window gilt glass tube gilt glass tube to IR detectorto IR detector IRDIRD CDD signal: IR absorbance CDD signal: IR absorbance MDD signal: ion count universal MDD signal: ion count universal analysers : quadrupole (Q, Qq) : ion trap (IT) : magnetic sector : time‐of‐flight (TOF) analysers : quadrupole (Q, Qq) : ion trap (IT) : magnetic sector : time‐of‐flight (TOF) ionisation : electron ionisation (EI) : chemical ionisation (CI) ionisation : electron ionisation (EI) : chemical ionisation (CI) MSMS GC‐MS interface : gaseous state, splitter GC‐MS interface : gaseous state, splitter 265265 multi‐dimensional gas chromatographymulti‐dimensional gas chromatography (2D‐GC)(2D‐GC) peak capacity in 2D‐GCpeak capacity in 2D‐GC modulatormodulator injectioninjection fast detectorfast detector the first dimension 0.20 to 0.32 mm ID the first dimension 0.20 to 0.32 mm ID the second dimension 0.10 to 0.15 mm ID the second dimension 0.10 to 0.15 mm ID modulators : allow transfer between dimensions modulators : allow transfer between dimensions injection loop injection loop electrically heated tube electrically heated tube rotating heated tube rotating heated tube cryogenic tube cryogenic tube dual cryogenic jet dual cryogenic jet high‐volumehigh‐volume low‐volumelow‐volume 𝐧 𝟐𝐃 𝟒 𝛑 · 𝐧 𝟏.𝐃 · 𝐧 𝟐.𝐃𝐧 𝟐𝐃 𝟒 𝛑 · 𝐧 𝟏.𝐃 · 𝐧 𝟐.𝐃 266266 definition of chromatographic system in GCdefinition of chromatographic system in GC temperature gradient profile initial temperature and its period, temperature increase; inlet temperature (e.g. 130 °C 1 min, 130 – 250 °C at 5 ° C∙min‐1, 250 °C 5 min; 250 °C)  temperature gradient profile initial temperature and its period, temperature increase; inlet temperature (e.g. 130 °C 1 min, 130 – 250 °C at 5 ° C∙min‐1, 250 °C 5 min; 250 °C)  MPMP SPSP carrier gas type carrier gas type  flow / pressure  (ml∙min‐1 / kPa)flow / pressure  (ml∙min‐1 / kPa) injection (X μl) injection type (event. splitting rate) injection (X μl) injection type (event. splitting rate) detectordetector stationary phase typestationary phase type length, inner diameter, manufacturer, SP type, film thickness 25m x 0.32 ID J&W DB‐5 DF – 1.0 length, inner diameter, manufacturer, SP type, film thickness 25m x 0.32 ID J&W DB‐5 DF – 1.0 basic characteristics according to typebasic characteristics according to type 267267 analytical information in chromatogramanalytical information in chromatogram retention time : compound identification (standard method)  spectroscopic detectors : UV‐Vis spectra : MS spectra (ESI / APCI; Qq / IT / o‐TOF) : NMR spectra (1H, 13C) retention time : compound identification (standard method)  spectroscopic detectors : UV‐Vis spectra : MS spectra (ESI / APCI; Qq / IT / o‐TOF) : NMR spectra (1H, 13C) specific retention volume (Vp)specific retention volume (Vp) relative retention time (rA,B) : comparison with internal standard relative retention time (rA,B) : comparison with internal standard Kovats retention indices (rA,B) : linear dependence of retention time logarithm of homologues on carbon number Kovats retention indices (rA,B) : linear dependence of retention time logarithm of homologues on carbon number qualitative informationqualitative information retention time formulationretention time formulation 𝐕𝐩 𝟐𝟕𝟑. 𝟏𝟓 · 𝐅 𝐌 𝐒 · 𝐓𝐜𝐨𝐥 𝐕𝐩 𝟐𝟕𝟑. 𝟏𝟓 · 𝐅 𝐌 𝐒 · 𝐓𝐜𝐨𝐥 𝐫 𝐀,𝐁 𝐭 𝐑 𝐀 𝐭 𝐑 𝐁 𝐫 𝐀,𝐁 𝐭 𝐑 𝐀 𝐭 𝐑 𝐁 268268 quantitative informationquantitative information peak area ≈ amount (concentration) of compound : because of narrow peaks frequently only height peak area ≈ amount (concentration) of compound : because of narrow peaks frequently only height : all components are eluted :: solvent does not count : all they have same response factor : all components are eluted :: solvent does not count : all they have same response factor internal normalisation method internal normalisation method  external standard method internal standard method standard addition method external standard method internal standard method standard addition method 𝐜% 𝐀%,𝐣 𝟏𝟎𝟎 · 𝐀𝐣 𝐀 𝐭𝐨𝐭 𝐜% 𝐀%,𝐣 𝟏𝟎𝟎 · 𝐀𝐣 𝐀 𝐭𝐨𝐭 thermostabilitythermostability test measurements in GCtest measurements in GC column testingcolumn testing testing mixture for uncoated carrierstesting mixture for uncoated carriers n‐decane, 1‐aminoacetate, 3,5‐dimethylpyrimidine, n‐dodecane, 1‐aminodecane, 2,6‐dimethyl‐aniline,  N,N‐dicyclohexylamine, 1‐aminododecane and n‐heptadecane MP – H2, Tinitial = 40 °C, Tterminal = 180 °C n‐decane, 1‐aminoacetate, 3,5‐dimethylpyrimidine, n‐dodecane, 1‐aminodecane, 2,6‐dimethyl‐aniline,  N,N‐dicyclohexylamine, 1‐aminododecane and n‐heptadecane MP – H2, Tinitial = 40 °C, Tterminal = 180 °C in dependence on time we observe : normalised retention times of components : height of peaks : symmetry of peaks in dependence on time we observe : normalised retention times of components : height of peaks : symmetry of peaks efficiencyefficiency testing mixture for coated carriers (Grob test)testing mixture for coated carriers (Grob test) methyl decanoate, methyl undecanoate, methyl dodecanoate, n‐decane, n‐undecane, n‐dodecane,  1‐octanol, nonanal, 2,3‐butanediol, 2,6‐dimethylaniline, 2,6‐dimethyl‐phenol, dicyclohexylamine, 2‐ ethylhexanoic acid MP – H2 or He, Tinitial = 40 °C, Tterminal = 100 °C, resp. 175 °C methyl decanoate, methyl undecanoate, methyl dodecanoate, n‐decane, n‐undecane, n‐dodecane,  1‐octanol, nonanal, 2,3‐butanediol, 2,6‐dimethylaniline, 2,6‐dimethyl‐phenol, dicyclohexylamine, 2‐ ethylhexanoic acid MP – H2 or He, Tinitial = 40 °C, Tterminal = 100 °C, resp. 175 °C column bleeding : decomposition of polymer materials in system :: SP material, septum column bleeding : decomposition of polymer materials in system :: SP material, septumn‐C22 MP – He, Tinitial = 40 °C, Tterminal = 300 °C n‐C22 MP – He, Tinitial = 40 °C, Tterminal = 300 °C TcolTcol released material released material 269269 inverse chromatographyinverse chromatography inverse (gas/liquid) chromatographyinverse (gas/liquid) chromatography (IC)(IC) classical chromatographyclassical chromatography inverse chromatographyinverse chromatography sample – mixturesample – mixture separation on SPseparation on SP separated mixture components separated mixture components probe substance or probe mixture probe substance or probe mixture interaction with sample (pseudo‐SP) interaction with sample (pseudo‐SP) one peak describing interaction one peak describing interaction : combination of frontal & elution chromatography :: probe in column until equilibrium ::: probe & studied material :: load of pseudo‐MP creates vacancies :: regions without probe : resulting chromatogram :: inverse towards elution method record metody : combination of frontal & elution chromatography :: probe in column until equilibrium ::: probe & studied material :: load of pseudo‐MP creates vacancies :: regions without probe : resulting chromatogram :: inverse towards elution method record metody study of termodynamic properties of materials (pseudo‐SP) : granular or fibrous p‐SP : infinite dilution IC (IC‐ID) :: small probe amount, elimination of their mutual interaction :: for sruface properties, transition temperatures, solubility : finite concentration IC (IC‐FC) :: monolayer on p‐SP, sometimes even more :: for desorption isotherms & surface inhomogeneities study of termodynamic properties of materials (pseudo‐SP) : granular or fibrous p‐SP : infinite dilution IC (IC‐ID) :: small probe amount, elimination of their mutual interaction :: for sruface properties, transition temperatures, solubility : finite concentration IC (IC‐FC) :: monolayer on p‐SP, sometimes even more :: for desorption isotherms & surface inhomogeneities ∆𝐆 𝐚 𝟎 𝒇 𝛘 𝐓∆𝐆 𝐚 𝟎 𝒇 𝛘 𝐓 probe 2probe 2 tNtN inertinert [min][min] ΔGa – change of free adsorption energy χT – probe molecular descriptor ΔGa – change of free adsorption energy χT – probe molecular descriptor 00 χT ~ carbon numberχT ~ carbon number ΔGaΔGa 00 alkane seriesalkane series ΔGaΔGa CH2CH2 timetime 270270