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(U)HPLC for Hyphenation
Prepared by
Gerard Rozing, Karlsruhe, Germany
Delivered by
Jan Preisler, Masaryk University, Brno, Czech Republic
6. 12. 2013 Page 1Methods of Chemical Research
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About the Author
Undergraduate and graduate studies at University of Amsterdam, 1964-1976.
Majors in Organic Chemistry and Chemical Engineering
Post-doctoral research at State University of Ghent, Belgium, 1977 and postdoctoral
training Analytical Chemistry, University of Amsterdam, 1978-1979
R&D Chemist, group & project Leader, R&D section manager, HPLC column
and HPLC system development at Hewlett-Packard, Waldbronn, Germany,
1979-’99
Since 2000, Agilent Technologies University Relations and External Scientific
Collaborations Manager
Since 2006 Agilent Research Fellow
Retired September 1, 2012. Since then, working on freelance basis. Visit my
website at http://www.rozing.com e-mail: gerard@rozing.com
6. 12. 2013Methods of Chemical Research 2
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Acknowledgements
Monika Dittmann, Stephan Buckenmaier, Udo Huber, Christian Scholz,
Konstantin Choikhet all at Agilent Technologies, Waldbronn, Germany
Paul Goodley, Chris Miller, Agilent Technologies, Santa Clara, USA
Oliver Schmitz, University of Duisburg-Essen, Germany
Achille Cappiello, Univ. Urbino, Italy
Remco van Soest, Eksigent part of AB Sciex, Dublin, USA
6. 12. 2013Methods of Chemical Research 3
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Hyphenation in Separation Science
Coupling of a separation method with a spectroscopic detection
method resulting in 3 (or more) dimensions of information
Detection Technique is coupled “on-line” with the separation
system
Detection Technique is coupled with the Separation System
through an interface in case of incompatibility between the
phases or systems
6. 12. 2013Methods of Chemical Research 4
Separation System
(w/w.o. detector)
HPLC
GC
CE
Detection Technique
UV-VIS
NMR
IR
Mass Spectrometry
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Hyphenation in Separation Science
Is not Multidimensional Separation Techniques
In this case the “x” is used
examples LCxLC coupling, LCxGC coupling etc.
6. 12. 2013Methods of Chemical Research 5
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Focus on LC-MS
Important aspects of LC-MS
Detector response type
Ionization mechanisms and interfaces
HPLC separation factors influencing ESI and APCI process and mass
detection
HPLC Column Technology, Special Techniques and New Developments
6. 12. 2013Methods of Chemical Research 6
12/5/2013
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Concentration Sensitive Detector in HPLC
Response proportional to concentration (e.g. UV detection)
Response independent of flow rate (infusion experiment!)
Chromatographic peak height does not change with flow rate (e.g in FIA or
neglecting any dispersion)
Chromatographic peak area is given by:
in case flow rate is constant (VR,i = F.tR,i ) and inversely proportional with flow
rate (peak width decreases in time domain)
In almost all cases, non-destructive
6. 12. 2013Methods of Chemical Research Slide 7
celliii LcAbs    ,, Lambert Beer’s law
   dttcFdvvcA iii  
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Mass Flow Sensitive Detector in HPLC
Response is proportional to mass/time (or cps)
Response increases with flow rate (infusion experiment!)
Chromatographic peak height increases with flow rate (e.g. in FIA
neglecting any dispersion) and the peak width decreases
Chromatographic peak area is given by:
and is independent of flow rate (peak width decreases in volume
domain)
In most cases a destructive detection method (FID, MS, ELSD,
ICP/MS
6. 12. 2013Methods of Chemical Research 8
t
m
aR i


 .
 dt
t
m
A i
i
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Important Aspects of LC-MS
Detector response
Ionization mechanisms and interfaces
6. 12. 2013Methods of Chemical Research 9
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Ionization Techniques in LC-MS
Soft, Atmospheric Pressure (in principle no fragmentation)
Electrospray Ionization- ESI
Chemical Ionization - APCI
Photo Ionization – APPI
Laser Ionization - APLI
Surface Ionization (MALDI, DART)
Soft, Vacuum Ionization
Matrix assisted laser desorption – MALDI
Hard, Vacuum Ionization (with fragmentation)
Particle Beam
Direct Electron Impact
Supersonic Molecular Beam
6. 12. 2013Methods of Chemical Research 10
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Pneumatically Assisted Electrospray Ionization
Charged solvent droplets formed
about 2 m in diameter
Electrical fields on the
cylinder, end plate and
capillary charge the droplets
6. 12. 2013Methods of Chemical Research 11
Courtesy of Agilent Technologies Kundenschulung
Initially Called Ion Spray
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Pneumatically Assisted Electrospray Ionization
Rayleigh Limit
+ +
+
+
+
+
+
++
+
-
-
-
-
-
+
+
+
+
+
+
+
++
+
--
-
-
-
+
+
+
+
++ --
+
+
+
+
++ --
+
+ Analyte Ions
Solvent Ion Cluster
from column
+ +
+
+
+
+
+
+
+
+
+
+
+ ++
+
+
TaylorCone
Nebulizing Gas
+ +
+
++++
+
+
++++
+
+ ++
+
+ +
+
+
+
+
+
+ +
+++
+ +
+
+
+ +
+
+
+
+
+
+
+ ++ +
+
+ +
+
+
+
+
+
+
+
+
+
+
++
+
+
+
+
+
+ ++
+
Solvent
evaporation
6. 12. 2013Methods of Chemical Research 12
Courtesy of Agilent Technologies Kundenschulung
An detailed explanation of the electrospray ionization process can be found at:
http://www.mcponline.org/content/early/2011/05/19/mcp.R111.009407/suppl/DC1
Polar/charged analytes
Ion Emission Model
Charge Residue Model
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Pneumatically Assisted Electrospray Ionization
Courtesy of Agilent Technologies Kundenschulung
Nebulizing Gas
From HPLC
Spray needle
MS Inlet
6. 12. 2013Methods of Chemical Research Slide 13
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Atmospheric Pressure Chemical Ionization
Gasphase
Analytes Evaporate!!
+
Analyte ions
+
+
+
+
+
+ +
++
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Proton Transfer
+ +
+
+
Neutral/Low Polarity
Analytes
in Aerosol
Solvent ionization by
charged nitrogen molecules
Courtesy of Agilent Technologies Kundenschulung
6. 12. 2013Methods of Chemical Research Slide 14
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APCI Detailed Mechanism- Gas Phase Ionization
Vcap
Corona
current
Nebulizer
Pressure
Fragmentor
Drying gas
Temperature
and Flow
Heater
Courtesy of Agilent Technologies Kundenschulung
6. 12. 2013Methods of Chemical Research Slide 15
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APCI-Interface
APCI corona
needle
Courtesy of Agilent Technologies Kundenschulung
6. 12. 2013Methods of Chemical Research Slide 16
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Atmospheric Pressure Photo Ionization
6. 12. 2013Methods of Chemical Research Slide 17
Courtesyof Oliver Schmitz, Univ. Duisburg/Essen,Germany
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Atmospheric Pressure Photo Ionization
Gasphase
Or a dopant (acetone) added is photoionized
which acts as reagent gas
Analytes evaporate!!
Analytes in
aerosol
Analyte ions+
+
+
+
+
+ +
++
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Photons ionize
analyte
+
+ +
+
+
+
+
+
+
+
h
h
Courtesy of Agilent Technologies Kundenschulung
Krypton 10.0 eV, 10.6 eV
6. 12. 2013Methods of Chemical Research Slide 18
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APPI-Interface
Lamp Source instead of
Discharge Needle Courtesy of Agilent Technologies Kundenschulung
6. 12. 2013Methods of Chemical Research 19
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Atmospheric Pressure Laser Ionization
6. 12. 2013Methods of Chemical Research Slide 20
Courtesyof Oliver Schmitz, Univ. Duisburg/Essen,Germany
Heater
Alternative for High MW substances:
Combine ESI with APLI!!
dual photon absorption
M + m h  M*
M* + n h  M+.
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Atmospheric Pressure Laser Ionization
6. 12. 2013Methods of Chemical Research Slide 21
Courtesyof Oliver Schmitz, Univ. Duisburg/Essen,Germany
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Summary
6. 12. 2013Methods of Chemical Research Slide 22
Courtesyof Oliver Schmitz, Univ. Duisburg/Essen,Germany
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Quoted from Marja-Liisa Riekkola, Helsinki, Finland*
6. 12. 2013Methods of Chemical Research Slide 24
“Many important advances in column materials and technology have contributed to improve the
resolution of analytes in liquid chromatography. As is well known, liquid chromatographic
separations critically depend on column type, choice of stationary phase, and type and composition
of the eluent employed as mobile phase. The selectivity of separations can be enhanced by adjusting
the stationary or mobile phase. The best separations are achieved through careful optimization of
conditions.
Liquid chromatography–mass spectrometry (LC–MS) has become increasingly popular in recent years.
Although three atmospheric pressure ionization (API) techniques (electrospray ionization,
atmospheric pressure chemical ionization and atmospheric pressure photoionization) are available to
facilitate the coupling of LC to MS, the MS detection is not always compatible with the solvents and
additives required in the preceding LC separation. Compromises must be accepted between the best
LC separation conditions, especially eluent composition, and the best ionization conditions if highest
selectivity and sensitivity are to be achieved.”
*J. Chromatography, 1216, 684 (2009)
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HPLC Separation Factors Influencing ESI and APCI
Process and Mass Detection
Interface Parameters (voltage(s), gases used)
Eluent Solvent Properties
Flow rate
Composition, volatility, viscosity, conductivity
Mobile phase additives, pH
Ion Suppression/Matrix effects
Practice of LC-MS
Use of inorganic buffers
Common background ions & contaminants
6. 12. 2013Methods of Chemical Research Slide 25
R. King et al., J. Am. Soc. Mass Spectrom., 2000, 11, 942–950
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Eluent Solvent Properties
6. 12. 2013Methods of Chemical Research 26
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Electrospray Ionization – Influence of Flow Rate
Pneumatically assisted electrospray
6. 12. 2013Methods of Chemical Research 27
Picture taken from G. Hopfgartner et al., J. Chrom. A, 647, 51 (1993)
Initially electrospray was not pneumatically
assisted – no direct countercurrent drying
gas, no nebulizer gas
Only working with very low LC flow rates.
Bruins and Henion introduce pneumatically
assisted electrospray (ion spray) *
*A.P. Bruins , Th. R. Covey , J. D. Henion, Anal. Chem., 1987, 59 (22), pp 2642–2646
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Influence of Flow Rate on Response in LC-MS*
Pneumatically Assisted ESI (Ion Spray)
6. 12. 2013Methods of Chemical Research Slide 28
F. Banks Jr., J. Chrom. A, 743, 99, 1996
Solv.: MeOH/Water 50/50, 0.1% AcOH
Source: Analytica of Branford
MS: SQ HP89A, 100-1000 m/z p.s.
This ESI works as an
Concentration
Sensitivity Detector
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Influence of Flow Rate on Response in LC-MS*
Pneumatically Assisted ESI (Ion Spray)
6. 12. 2013Methods of Chemical Research Slide 29
*F. Banks Jr., J. Chrom. A, 743, 99, 1996
Injection of equal amounts (50 pmol)
of methionine enkephaline on
columns with different i.d.
Signal height increase is 163x short
of 339x by column diameter ratio^2
Attributed to poor packing of the
microbore column
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Influence of Flow Rate on Response in LC-MS*
Pneumatically Assisted ESI vs. APCI
6. 12. 2013Methods of Chemical Research Slide 30
*W. Engewald et al., Journal of Chromatography A, 937 (2001) 65–72
ESI
APCI
Not all analyte ions are captured with the
same efficiency
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Nano-electrospray Ionization
Developed by Matthias Mann & Matthias Wilm*
Smaller droplets  generation of more ions
No orthogonal design – sprayer is 1 – 2 mm from MS
entrance
Higher sampling rate of ions into MS
6. 12. 2013 Page 31
*M. Wilm & M. Mann, International Journal of Mass Spectrometry and Ion Processes, 136, 167 (1994)
Result: dramatically higher sensitivity than standard ESI
Methods of Chemical Research
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Influence of Flow Rate on Response in NanoESI
6. 12. 2013Methods of Chemical Research Slide 32
R.D. Smith et al., Anal. Chem. 74, 4235 (2002)
Emitter tip orifice diameter
proportionally reduced
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Preliminary Conclusions – Flow Dependence ESI
The pneumatically assisted ESI interface (IF) tolerates maximally 1 mL/min
Pneumatically assisted ESI IF behaves largely like a concentration sensitive
detector
Reduction of column i.d. demands very low extra column dispersion and well
packed columns in order to exploit sensitivity gain with concentration
sensitive detection
Nano-ESI response increases dramatically at very low flow rates (<20 nL/min)
At low flow rate nor mass flow sensitive or concentration sensitive detector
because more ions reach the MS inlet
Ion suppression is much reduced (vide infra)
6. 12. 2013Methods of Chemical Research Slide 33
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Influence of Gradient Elution on Response in LC-MS*
Nano-electrospray Ionization
6. 12. 2013Methods of Chemical Research 34
Column 75 µm i.d.
Solvent 0.2% AcOH 0.1%
TFA in water vs. 0.1% TFA
in ACN, 0-100%
R.D. Smith et al, J. Amer. Soc. Mass Spectrometry, 20, 682 (2009)
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Influence of Gradient Elution on Response in LC-MS*
Nano-electrospray Ionization
6. 12. 2013Methods of Chemical Research Slide 35
C = cone jet
P = pulsed cone jet
M = multi jet
D = dripping
S = spindle
G. Valaskovic, J Am. Soc. Mass Spectrom., 2004, 15, 1201–1215
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HPLC Separation Factors Influencing ESI and APCI
Process and Mass Detection
Interface Parameters (voltage(s), gases used)
Eluent Solvent Properties
Flow rate
Composition, volatility, viscosity, conductivity
Mobile phase additives, pH
Ion Suppression/Matrix effects
Practice of LC-MS
Use of inorganic buffers
Common background ions & contaminants
6. 12. 2013Methods of Chemical Research Slide 36
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Frequently Used Mobile Phase Additives in ESI/MS
Positive Mode Negative Mode
Ammonium Acetate Ammonium Acetate
Ammonium Formate Ammonium Formate
Acetic Acid (pH 3-4) Ammonia/Ammonium Hydroxide (pH>7)
Formic Acid (pH 2-3) Triethylamine (pH >7)
Trifluoro-acetic Acid (pH 1-2) N-Methylmorpholin
• As additive or by post-column addition in case the solvent pH for optimal
separation differs from the pH for optimal ionization.
• Additives will cause an high background signal (TFA (m/z 113) in negative mode,
TEA (m/z 102) in positive mode), increase conductivity of the solvent and may
cause ion suppression
6. 12. 2013Methods of Chemical Research 37
Courtesy of Agilent Technologies Kundenschulung
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Solution Chemistry is Important
Positive Mode
Negative Mode
R1 R1
| |
:N - R2 + HA +HN - R2 + A|
|
R3 R3
Base Acid Analyte Cation
O O
|| ||
R-C-OH + :B R-C-O- + H:B+
Acid Base Analyte Anion
ESI mandates the formation of analyte ions in the eluent solution
6. 12. 2013Methods of Chemical Research 38
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Influence of Additive Concentration on Response
Pneumatically Assisted ESI
APCI
[Ammonium Acetate], mM
0
2
4
6
8
10
12
14
16
18
20
0 30 90 150 210 255 300
AverageArea,x100000
ESI
[Ammonium Acetate], mM
0
2
4
6
8
10
12
0 10 20 30 90 150 210 255 300
AverageArea,x1000000
6. 12. 2013Methods of Chemical Research 39
Courtesy of Agilent Technologies Kundenschulung
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Influence of Additive Concentration on Response
Pneumatically Assisted ESI
6. 12. 2013Methods of Chemical Research 40
Taken from: HPLC Analysis of Biomolecules, Technical Guide
Thermo Electron Corporation
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TFA Containing Solvents for Tryptic Peptides LC-MS
Ideally suited for RP LC since trypsine cleaves at lysine
or arginine leaving a basic peptide. With TFA is ion-pair
separation on RP column possible.
TFA neutralizes “hot” sites on the silica surface
TFA forms a strong ion pair with basic peptides
But spray instability due to high conductivity and high
surface tension of the solution has been reported
Strong signal reduction observed
6. 12. 2013Methods of Chemical Research 41
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FIA with 1% AcOH/0.25% TFA*
Pneumatically Assisted ESI
0.2% TFA
1.0% HOAc
min
Abundance
10.00 12.00 14.00 16.002.00 4.00 6.00 8.00
50000
100000
150000
200000
250000
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00
50000
100000
150000
200000
250000
min
Abundance
6. 12. 2013Methods of Chemical Research Slide 42
*A. Apffel et al., J. Chrom., 712 177 (1995)
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Remedy in Practice
Post-column addition of the “TFA-fix”
No compromise on chromatography
Additional hardware required (cost, reliability, mixing efficiency)
New stationary phases that have low silanophilic interactions allowing good
peptide separations without compromising chromatograph by using formic
acid etc.
Dionex Acclaim Pepmap
Waters CSH130 C18
Thermo BioBasic columns
Agilent AdvanceBio Peptide Mapping columns
6. 12. 2013Methods of Chemical Research 43
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Pneumatically Assisted API
ESI APCI
6. 12. 2013Methods of Chemical Research 44
In principle concentration sensitive
In many cases more sensitive
Wide flow rate range  nanobore –
normal bore columns
Solvent composition (gradient) OK
Mobile phase additives compromise
response
Tolerates low concentration of inorganic
buffers
High matrix effect
In principle mass flow sensitive
More selective, non-polar substances
No advantage at low flow rates
Organic solvent may have a large
influence on response
Chlorinated solvents will assist
ionization
Tolerate up to 100 mmol inorganic
buffers
Low matrix effect
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HPLC Separation Factors Influencing ESI and APCI
Process and Mass Detection
Interface Parameters (voltage(s), gases used)
Eluent Solvent Properties
Flow rate
Composition, volatility, viscosity, conductivity
Mobile phase additives, pH
Ion Suppression/Matrix effects in ESI/MS
Practice of LC-MS
Use of inorganic buffers
Common background ions & contaminants
6. 12. 2013Methods of Chemical Research Slide 45
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What is Matrix Effect/Ion Suppression in LC-ESI/MS*
Ionization efficiency of ESI depends
Solvent properties – mostly constant but for gradient elution
Source parameters
Compounds co-eluting with analyte
Standards are clean solutions
Different response for the same analyte concentration
in sample solution than in standard solution
Matrix effect depends on analyte concentration
6. 12. 2013Methods of Chemical Research 46
*Annelie Kruve, Univ. of Tartu, Estonia
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Example of Matrix Effect in LC-ESI/MS*
6. 12. 2013Methods of Chemical Research 47
*Annelie Kruve, Univ. of Tartu, Estonia
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Matrix Effect - Causes
Competition for available charges
(Keep in mind that a very low fraction from the analytes actually make it into the MS)
Interfering substances may cause increase of viscosity
and surface tension therewith hampering the
formation of droplet
Formation of solid particles including the analyte
As with TFA ion pair formation renders the analyte
neutral.
6. 12. 2013Methods of Chemical Research Slide 48
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Remedies for the Matrix Effect
Assess the scope of the effect by the post-column
addition method*
If possible prepare standard in sample matrix (e.g.
serum) and run it through the sample prep procedure
Address in the whole procedures the probable
mechanism of the matrix effect
Smaller droplets  nanoelectrospray!
Use another ionization method e.g APCI or Direct
Election Impact LC-MS interface (vide infra)
6. 12. 2013Methods of Chemical Research 49
*Matuszewski et al., Anal. Chem. 2003, 75, 3019-3030)
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HPLC Separation Factors Influencing ESI and APCI
Process and Mass Detection
Interface Parameters (voltage(s), gases used)
Eluent Solvent Properties
Flow rate
Composition, volatility, viscosity, conductivity
Mobile phase additives, pH
Ion Suppression/Matrix effects
Practice of LC-MS
Use of inorganic buffers
Common background ions & contaminants
6. 12. 2013Methods of Chemical Research Slide 50
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Using Non-volatile Buffers in the Mobile Phase
LC Conditions:
Mobile phase: 8% methanol in one of the following:
A: water
B: 0.2% acetic acid in water
C: 50 mM ammonium phosphate, pH 7
D: 50 mM sodium phosphate, pH 7
Flow rate: ESI - 0.3 ml/min; APCI - 0.7 ml/min
Injection: 1 µl of a mixture containing 10 ng/µl each of lincomycin, caffeine and
sulfachloropyradizine
Column: Zorbax Eclipse XDB C8 2.1 mm x 50 mm @ 30 °C
MS Conditions:
SIM ions:
Positive ion mode: 195, 285 and 407 amu
Negative ion mode: 193, 283 and 405 amu
Fragmentor: Ramped 70 V for 193/195; 50 V for 283/285; 80 V for 405/407
Vcap: ESI - 4000 V; APCI - 3000 V
Drying gas: ESI - 350°C, 10 l/min; APCI - 350 °C, 5 l/min
Nebulizer: ESI - 25 psig; APCI - 60 psig
6. 12. 2013Methods of Chemical Research 51
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Using Non-volatile Buffers in the Mobile Phase
Influence on Response
0.E+00
1.E+06
2.E+06
3.E+06
4.E+06
5.E+06
6.E+06
7.E+06
Area
B C D
Positive Ion Mode
Lincomycin Sulfachlorapyr. Caffeine
Mobile Phase Conditions:
(B) 0.2% acetic acid;
(C) 50 ammonium phosphate;
(D) 50 mM sodium phosphate
6. 12. 2013Methods of Chemical Research 52
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API-Spray Chamber after using a 25 mM Phosphate Buffer
6. 12. 2013Methods of Chemical Research 53
No comment needed
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HPLC Separation Factors Influencing ESI and APCI
Process and Mass Detection
Interface Parameters (voltage(s), gases used)
Eluent Solvent Properties
Flow rate
Composition, volatility, viscosity, conductivity
Mobile phase additives, pH
Ion Suppression/Matrix effects
Practice of LC-MS
Use of inorganic buffers
Common background ions & contaminants
6. 12. 2013Methods of Chemical Research Slide 54
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Common Contaminant & Background Ions
6. 12. 2013Methods of Chemical Research Slide 55
m/z Ion Compound
101 [M+Na]+ DMSO
102 [M+H]+ Triethylamine
104/106 [M+Cu]+ Acetonitrile
105 [2M+Na]+ Acetonitrile
120 [M+Na+CH3CN]+ DMSO
122 [M+H]+ Tris
123 [M+H]+ Dimethylaminopyridine
130 [M+H]+ Diisopropylethylamine
144 [M+H]+ Tripropylamine
145/147 [2M+Cu]+ Acetonitrile
146 [3M+Na]+ Acetonitrile
150 [M+H]+ Phenyldiethylamine
153 [M+H]+ 1,8-diazabicyclo[5.4.0]undec-7-ene
157 [2M+H]+ DMSO
159 [M+Na]+ Sodium trifluoroacetate
179 [2M+Na]+ DMSO
186 [M+H]+ Tributylamine
225 [M+H]+ Dicyclohexylurea
239/241 [(M.HCl)2-Cl]+ Triethylamine
242 M+ Tetrabutylammonium
243 M+ Trityl
257 [3M+H]+ DMSO
267 [M+H]+ Tributylphosphate
273 M+ Monomethoxytrityl
279 [M+H]+ Dibutylphthalate
301 [M+Na]+ Dibutylphthalate
317 [M+K]+ Dibutylphthalate
336 [M+H]+ Tributyl
371 [M+H]+ Polysiloxane,
391 [M+H]+ Diisooctyl phthalate
413 [M+Na]+ Diisooctyl phthalate
429 [M+K]+ Diisooctyl phthalate
445 [M+H]+ Polysiloxane
462 [M+NH4]+ Polysiloxane
449 [2M+H]+ Dicyclohexyl urea
798 [2M+NH4]+ Diisooctyl phthalate
803 [2M+Na]+ Diisooctyl phthalate
74 m/z units apart polydimethylcyclosiloxane
National Mass Spectrometry Facility UK
www.nmssc.ac.uk/documents/ESI_contam_and_bg_ions.pdf
Other sources
New Objective Inc.
http://www.newobjective.com/downloads/technotes/PV-3.pdf
Waters
https://www.waters.com/webassets/cms/.../docs/bkgrnd_ion_mstr_list.pdf
Alberta University
www.chem.ualberta.ca/~massspec/es_ions.pdf ‎‎
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Background Ions in LC-MS
Copper/Acetonitrile Adducts
6. 12. 2013Methods of Chemical Research Slide 56
Courtesy Daniel Thielsch, Agilent Technologies
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Avoid/Eliminate Contamination
Utmost cleanliness of lab articles, solvents etc.
Unlike UV-VIS, remember a MS “sees” everything!
Run solvent only – no HPLC column
Step gradient – monitor and identify background ions
Locate source of contamination
Replace parts, modules or clean system (see next page)
Run with HPLC column
Step gradient – monitor and identify background ions
Inject a blank sample
Use a sample divert valve to avoid sample salts and early
eluting sample components enter the MS
6. 12. 2013Methods of Chemical Research Slide 57
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Clean-up your HPLC System
Flush with water (no column, bypass UV-detection cell, outlet to waste) e.g. at 3
mL/min for 15-20 minutes to remove salts
Flush with i-propanol as above or at low flow rate overnight. Do blank sample injections
with i-propanol to clean injection path
Flush with organics cleaning solution as above
(e.g. from Agilent (50:25:15:10 acetonitrile/isopropanol/cyclohexane/dichloromethane)
Do blank sample injections with cleaning solution
Change back to isopropanol and flush. Do blank injections with i-propanol to clean
injection path
Flush with 100% methanol HPLC grade
Install column and flush with 100% methanol at elevated temperature
Switch to mobile phase. In case of gradient analysis do a reverse gradient.
After pumping down MS connect LC
As an alternative, one may use a solution of a few % formic acid in acetonitrile
Formal passivation with strong acid only after checking manufacturer literature
6. 12. 2013Methods of Chemical Research 58
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Focus on LC-MS
Important aspects of LC-MS
Factors Influencing ESI Process and Mass Detection
HPLC Column Technology, Special Techniques and New
Developments
Is separation prior to MS needed?
HPLC instrumental factors
What column diameter to use
HPLC Chip column technologies for LC-MS
Direct EI LC-MS
6. 12. 2013Methods of Chemical Research 59
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HPLC Column Technology, Special Techniques and
New Developments
Is separation prior to MS needed?
HPLC instrumental factors
What column diameter to use
HPLC Chip column technologies for LC-MS
Direct EI LC-MS
6. 12. 2013Methods of Chemical Research 60
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“Chromatographic separation is not
required when using MS. Extract
individual m/z values, do SIM or choose
precursor ions for MS/MS.”
Is separation prior to MS needed?
6. 12. 2013Methods of Chemical Research 61
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Why is Separation Needed?
MS will not/barely differentiate isomeric substances
(same MW but different structure of stereoisomers)
MS will not/barely differentiate isobaric substances
(same molecular formula but different molecules)
May mitigate matrix effect
6. 12. 2013Methods of Chemical Research Slide 62
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No Separation before MS
63
Diphenhydramine
M+H 256.17
Ketoprophen
M+H 255.10
0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00
Time, min
0.0
6.0e4
1.2e5
1.8e5
2.4e5
3.0e5
3.6e5
4.2e5
4.8e5
Intensity,cps
5:95 Water/Methanol, 0.2 % Acetic Acid
6. 12. 2013Methods of Chemical Research
Courtesy Udo Huber, Agilent Technologies Waldbronn
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Separation before MS
64
0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40
Time, min
0.0
6.0e4
1.2e5
1.8e5
2.4e5
3.0e5
3.6e5
4.2e5
4.8e5
Intensity,cps
Diphenhydramine
M+H 256.17
Ketoprophen
M+H 255.10
30:70 Water/Methanol, 0.2 % Acetic Acid
6. 12. 2013Methods of Chemical Research
Courtesy Udo Huber, Agilent Technologies Waldbronn
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HPLC Column Technology, Special Techniques and
New Developments
Is separation prior to MS needed?
HPLC instrumental factors
What column diameter to use
HPLC Chip column technologies for LC-MS
Direct EI LC-MS
6. 12. 2013Methods of Chemical Research 65
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HPLC Instrumental Factors – System Dwell Volume*
Volume from the point of mobile phase mixing to the
column head
Delays the arrival of eluent composition change
(gradient)
66
Delay Volume
*J.W. Dolan LCGC 2006, Vol 24, 458-466
6. 12. 2013Methods of Chemical Research
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67
Influence of Dwell Volume
Delay volume 700 μL
min0.5 1 1.5 2 2.5 3 3.5 4 4.5
mAU
0
100
200
300
400
Delay volume 120 μL
min0.5 1 1.5 2 2.5 3 3.5 4 4.5
mAU
0
100
200
300
400
Column: ZORBAX SB-C18 2.1 x 50 mm, 1.8 μm
Flow rate: 0.42 mL/min 3.7 min
2.6 min
6. 12. 2013Methods of Chemical Research
Courtesy Udo Huber, Agilent Technologies Waldbronn
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HPLC Instrumental Factors : Extra Column Dispersion
68
• “Dispersion is the sample bandspreading or dilution which occurs in
connecting tubing, sample valves, flow cells and in column end-fittings.”
• Connection capillaries (I.D. Length)
Dispersion
Peakheight: Reduced sensitivity
Peakwidth: Resolution loss
Aris-Taylor Gleichung
m
v
D
LFd



96
4
2 

6. 12. 2013Methods of Chemical Research
Courtesy Udo Huber, Agilent Technologies Waldbronn
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HPLC Instrumental Factors : Extra Column Dispersion
6. 12. 2013Methods of Chemical Research 69
min0.5 1 1.5 2 2.5
mAU
0
100
200
300
400
min0.5 1 1.5 2 2.5
mAU
0
50
100
150
200
250
300
Low extra column dispersion
Peak width 0.018 min
Peak width 0.019 min
Resolution 1.902
High extra column dispersion
Peak width 0.038 min
Peak width 0.037 min
Resolution 0.961
Courtesy Udo Huber, Agilent Technologies Waldbronn
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70
Influence of Poor Connections
min0 0.1 0.2 0.3 0.4
mAU
0
40
80
120
160
200
240
280
min0 0.1 0.2 0.3 0.4
mAU
0
20
40
60
80
100
120
140
6. 12. 2013Methods of Chemical Research
Courtesy Udo Huber, Agilent Technologies Waldbronn
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Recommendations for Sample Preparation
Positive ion ESI Negative ion ESI
6. 12. 2013Methods of Chemical Research 71
Dissolve samples in acid
Basic sites (N and O) bind to
proton to give the molecule a
positive charge
Other cat ions (Na+, or K+) may
also be used to form positive ions
Anions (Cl-) may be removed from
a molecule to form a positive ions
Dissolve samples in base
Acidic sites (acids) give up a
proton to form a negative ion.
• ESI works best when the samples are
free of salt
• Samples that contain salt can be
desalted in many ways (divert valve)
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HPLC Column Technology, Special Techniques and
New Developments
Is separation prior to MS needed?
HPLC instrumental factors
What column diameter to use
HPLC Chip column technologies for LC-MS
Direct EI LC-MS
6. 12. 2013Methods of Chemical Research 72
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What Column Diameter to Use?
6. 12. 2013Methods of Chemical Research Slide 73
colv
inji
colv
iinj
i
ii
Vcm
c
,
.0,
,
,
max,
22  



For a chromatographic separation
2max,
c
i
i
d
m
c 
When the column diameter is reduced:
For a concentration sensitive detector, response increases with the square root of
the diameter ratio in case the same amount of analyte is injected.
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Influence of Column Diameter – UV response
6. 12. 2013Methods of Chemical Research Slide 74
Mobile phase: Water/CAN, 0.1% FA
gradients from 5 – 90 %B in 15’
Inj. vol. 0.5 µL
Peak height increase
Courtesy Stephan Buckenmaier,
Agilent Technologies, Waldbronn
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Influence of Column Diameter – UV response
6. 12. 2013Methods of Chemical Research Slide 75
Chromatographic peak width increases
with decrease of diameter.
Increase in peak height below calculated
value
UV is concentration dependent
Courtesy Stephan Buckenmaier,
Agilent Technologies, Waldbronn
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Jetstream IF, Ion Funnel
6. 12. 2013Methods of Chemical Research Slide 76
1. Nebulizer w/ 50 µm needle
2. Nebulizing gas (35 psi)
3. Sampling capillary (Vcap 4 kV)
4. Drying gas (200°C, 13 L/min )
5. Sheath gas (400°C, 12 L/min)
6. Nozzle voltage (1500 V)
7. Thermal gradient focussing region
 Higher sampling from ion spray
Courtesy Stephan Buckenmaier,
Agilent Technologies, Waldbronn
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Influence of Column Diameter – MS response
with Jetstream IF and Ion Funnel
6. 12. 2013Methods of Chemical Research Slide 77
Advances in ionization and sampling
efficiency peak abundances are
maintained, independent of
column-ID, flow rates, and sample
concentration.
Courtesy Stephan Buckenmaier,
Agilent Technologies, Waldbronn
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Columns for NanoESI/MS
6. 12. 2013Methods of Chemical Research Slide 78
Nano-electrospray MS mandates flow rates 100 – 1000 nL/min.
For (U)HPLC to work properly the solvent has to move
with a velocity of 1-10 mm/s
u
F
dc
..
.4


Column I.D. must be between 0.05 and 0.15 mm
Sensitivity of NanoESI/MS increases dramatically at flow rates <50 nL/min
Proteomics research
 Ultra small samples
 Ultra high sensitivity mandated
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Example – NanoESI Interface Agilent
6. 12. 2013Methods of Chemical Research Slide 79
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Typical Set-up of Nanoflow HPLC Electro-Spray Ionization MS System
Sensitivity
75µm ID analytical column
Challenging to Set-up & Maintain
Multiple Parts
Possible leaks, misalignments
Robustness & Ease-of-use
Clogging of spray needle
After part replacement system can take hours
to stabilize
Chromatographic Fidelity
Rel. large extra column volume leads
to band broadening
Limited to peptide separation
Spray tip
Nano column
Tubings
Enrichment
Fittings
Valve
6. 12. 2013Methods of Chemical Research Slide 80
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Agilent HPLC-Chip Technology
Height 50 µm
Width 75 µm
Length 43 mm
Particle size 5 µm
6. 12. 2013Methods of Chemical Research Slide 81
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Stator
Rotor
Waste
Autosampler
Sixport valve
Trap Column
Nanospray tip
Column 43x0.075x0.05 mm
Nanoflow HPLC pump
HPLC-Chip MS
Essential Components for Nanoflow HPLC are
chip-integrated
 No extra column volume
 Chromatographic performance
is conserved
 Sensitivity for ESI LC-MS easily obtained!
 Avoids leaks and misalignments
6. 12. 2013Methods of Chemical Research Slide 82
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Analytical Pump
Waste
Analytical Column
Loading Pump
EC
P1 P2
P3
P4P5
P6
Spray Tip
RP-TiO2-RP
P4P1
TiO2
RP RP
(c)
HPLC-Chip - Phosphopeptide Enrichment Chip Design
6. 12. 2013Methods of Chemical Research Slide 83
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Thanks for your attention
PDF Copy can be found at http://www.rozing.com
(registration required)
6. 12. 2013Methods of Chemical Research 84