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Monoliths in separation science
1st part
Dana Moravcová
Gustav Vigeland Sculpture Park,
Oslo, Norway
A single block or piece of stone of
considerable size.
Monolith???
A continuous stationary phase cast as a
homogeneous column in a single piece.
Monolithic chip
(A, B) – unmodified
(C, D) modified wall of chip. (D. A. Mair, Lab Chip 9 (2009) 877–88.)
Monolithic capillary column.
Monolithic pipette tip.
 An alternative to particle packed columns
 1967 – poly(ethylenglycol methacrylate) column – separation of proteins –
gel filtration, low permeability and separation efficiency (M. Kubín).
 1989 – compressed hydrophilic polyacrylamide gels – ion-exchange
chromatography of proteins (S. Hjertén).
Why monoliths?
SDS-gel
History
Wet state Dry state
Particle packed column, 5 µm particles.  Monolith = a rigid material with appropriate chemical, physical, and
mechanical properties (stability in a wide pH range, permanent porosity).
 Characteristic well-organized and highly porous structure
 Variable surface area, pore texture, surface chemistry
 Polymer-, inorganic-, and hybrid-monoliths
Rigid monoliths
 The 1990s – macroporous rigid monolithic materials based on
methacrylate and polystyrene-divinylbenzene copolymers suitable for
separation of proteins (F. Švec, J. M. J. Fréchet); silicagel-based
monolithic materials suitable for separation of small molecules
(K. Nakanishi, N. Soga, N. Tanaka).
Nowadays
 Acrylamide-,
methacrylate-,
and polystyrenebased
monoliths
 Alkoxysilanes -
tetramethoxysilane,
tetraethoxysilane
 Alkyltrialkoxysilanes or
polysilsesquioxanes as 1,2-
bis(trimethoxysilyl)ethane
Monolithic stationary phases
 The desired monolithic stationary phases can be prepared utilizing onestep
or multiple-modification preparation procedure.
 One-step preparation procedure – methacrylate monolithic capillary
columns
– butylmethacrylate BMA + ethylenedimethacrylate EDMA
 Multiple-modification preparation procedure – silicagel monolithic
capillary columns
– C18-stationary phases
– Sulfobetaine stationary phase
– Phosphonium ionic liquid stationary phase
– Liposome stationary phases
 Monolith – porous material
 Macropores > 50 nm, flow-through pores
 Mesopores 2-50 nm, surface area
 Micropores < 2 nm
 Material engineering
 Pore volume - mercury intrusion porosimetry
 Specific surface area - gas adsorption (BET)
 Infrared spectroscopy – presence of functional groups
 Elemental analysis
 Electron microscopy (SEM)
 Chromatography
 Permeability, porosity
 Separation efficiency
 Separation selectivity
 Inverse size-exclusion chromatography (ISEC)
Characterization of monolithic materials
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 Permeability
 Kozeny-Carman equation
F – mobile phase flow rate, η – viscosity of mobile phase, Δp – pressure drop, L – column length,
r – column radius, 0 – interstitial porosity, dp – „equivalent permeability particle diameter“.
The interstitial (through pore) and inner (mesopore) porosities calculated for polystyrene standard
with Mr = 2 700 000 in 100% THF.
 Porosity
 Column total porosity determined with uracil as a non-retained marker
compound
 Interstitial (through pore) porosity
 Inner (mesopore porosity) 0
0
 

 T
C
TO
i
V
VV
CV
V0
0 
C
TO
T
V
V

2F
F L
K
p r


 

  
0 3
0
180
(1 ) F
p
K
d 

  01000 ( , 0.4)p Fd K ODS   
Characterization of monolithic materials
 Separation efficiency
- the number of theoretical plates (n)
 N – the number of theoretical
plates per meter
 H - height equivalent to theoretical
plate
l – column length
 Retention factor k
2
,2/1
,
545.5









j
jR
w
t
n
l
N
H 
M
MjR
t
tt
k


,
Separation efficiency, retention factor
t R,j
t M
w 1/2,j
analyte j
Non-retained
compound
Detectorresponse
Characterization of monolithic materials
Column 1 2 3
Porogen 60 60 60
Monomer 40 40 40
BMA 44.5 44.5 44.5
EDMA 54.5 54.5 54.5
PrOH 60 62 64
BuOH 30 28 26
Water 10 10 10
% wt.
Monolithic methacrylate-based capillary columns
D. Moravcová et al., J. Sep. Sci. 2004, 27, 789–800.
 Monomers
– butylmethacrylate BMA
– ethylenedimethacrylate EDMA
 Pore forming solvents
– 1,4-butanediol BUT
– 1-propanol PROP
– water
 Initiator
– azobisisobutyronitrile AIBN
 Thermal polymerization
– 60°C, 24 hours
– 0.32 mm I.D. silanized
capillaries
Modification of inner wall of fused silica capillary
Reaction between silanol groups on the silica capillary surface and 3-(trimetoxysilyl)propyl
methacrylate.
20 hrs
Silanization
 Permeability
 Porosity
A - Inertsil ODS-2, 5 mm, 150 x 0.32 mm, Metachem, Torrance, USA
B - Biospher C18E, 5 mm, 141 x 0.32 mm, Labio Praha, Czech Republic
C - Chromolith CapRod RP-18e, 150 x 0.1 mm, Merck, Darmstad, Germany
Column 1 2 3 A B C
KF [cm2] 7.79E-10 2.38E-10 3.52E-11 2.25E-10 1.47E-10 8.66E-10
dperm [m] 7.6 3.8 1.9 7.2 5.6 5.1
Column 1 2 3 A B C
εT 0.710 0.680 0.650 0.590 0.650 0.847
εo 0.490 0.470 0.410 0.310 0.290 0.680
εi 0.220 0.210 0.240 0.280 0.360 0.167
Porosity and permeability of prepared columns
1) 2) 3)
Running conditions: 70% ACN, 30% water, UV detection 254 nm,
Fc = 2 µl/min, columns 0.32 mm I.D., l1,l2 = 240 mm, l3 = 140 mm.
5 m 5 m 5 m
Sample:
0 – uracil (unretained
compound)
1 – benzylalcohol
2 – benzaldehyde
3 – benzene
4 – toluene
5 – ethylbenzene
6 – propylbenzene
7 – butylbenzene
8 - amylbenzene
Column 1 2 3
kTOL 1,19 1,13 1,30
NTOL 4270 21860 30380
kAB 3,03 2,81 3,47
NAB 2090 9680 22110
Separation of alkylbenzenes
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 The methacrylete-based monolithic columns showed comparable
chromatographic performance as packed octadecylsilica capillary
columns.
 The results illustrate the importance of selection of appropriate
composition of the porogen solvent mixture.
 Column with 64% w/w of propanol in the porogen part showed better
chromatographic performance than the columns prepared using lower
propanol concentrations.
Conclusion
 Silicagel-based monolith
 Tetramethoxysilane
 Polyethylene oxide (Mr 10 000)
 Acetic acid, water
 Hydrolysis
≡Si-OR + H2O → ≡Si-OH + ROH
 Condensation of alcohole
≡Si-OH + RO-Si≡ → ≡Si-O-Si≡ + ROH
 Condensation of water
≡Si-OH + HO-Si≡ → ≡Si-O-Si≡ + H2O
Five steps of preparation – polymerization,
washing, drying, calcination, and
modification to appropriate stationary
phase.
Monolithic silica-based capillary columns
Silicagel-based monolith
Monolithic silica-based capillary columns
Isocratic separation of alkylbenzenes
(benzene -hexylbenzene, n = 0-6) in 80%
ACN/20 % water. Test mixture: 50 µL of
each in 20 mL of 80% v/v acetonitrile,
injection 60 nL loop, splitter, columns
150 x 0.1mm, Fc = 500 nl/min,
UV detection 220 nm.
 ODS column - chemical modification
C18-stationary phases (RPLC)
 ODM column - „grafting“ – two step
modification procedure
- silanization of
monolith by 3-trimethoxysilylpropyl
methacrylate
- radical polymerization
of 3-trimethoxysilylpropyl
methacrylate and octadecyl
methacrylate
Silica monolith modified by
octadecyldimethyl-N,N-diethylaminosilane
Analyte
Analyte
Analyte
Analyte
CH2-N-CH2-CH2-CH2-SO3
CH3
CH3
Electrostatic
Interaction
Electrostatic
Interaction Hydrophilic
Partitioning
H2O
H2O
H2O
H2O
H2O
H2O
H2O
H2O
H2O
ACN
ACN
ACN
ACN
 Alpert (A. J. Alpert, J. Chromatogr. A 499 (1990) 177.)
 Mobile phase: organic solvent (40-97% ACN) in water or volatile
buffer.
 Stationary phase: silica, amino-, diol-, polyhydroxyethyl-,
aspartamide-, cyclodextrin-, and zwitterion-based packings.
 Sample: highly hydrophilic polar analytes – small molecules –
drugs, peptides, carbohydrates, nucleosides, nucleotides.
Monomer:
[2-(methacryloyloxy)ethyl]-
dimethyl-(3-sulfopropyl)ammonium
hydroxide (MEDSA).
The retention processes in HILIC illustrated by hydrophilic
partitioning, and electrostatic interactions with either
positive or negative charges.
Hydrophilic Interaction Chromatography (HILIC)
 Monolithic silica
 Tetramethoxysilane, PEG 10 000, urea, 0.01M
acetic acid.
 1st modification
 3-Trimethoxysilylpropyl methacrylate,
ethanol, acetic acid, water.
 2nd modification
 15 mg/ml of [2-(methacryloyloxy)ethyl]-
dimethyl-(3-sulfopropyl)-ammonium
hydroxide (MEDSA) in methanol (30% v/v) and
xylenes (70% v/v).
 thermal polymerization
 80°C, 3 hours.
Preparation of monolithic capillary columns
The prepared monolithic column
(SEM).
Compound H [µm] N [tp/m] k u [mm/s]
Toluene 5.7 175 500 --- 1.5
Uracil 6.4 156 000 0.29 0.7
Cytosine 5.5 182 000 1.01 1.0
Van-Deemter dependency of
monolithic capillary column
(150 mm × 0.1 mm).
Mobile phase: 90% (v/v) ACN/
10% 5mM ammonium acetate
pH=6; UV detection 210 nm.
 – toluene
 – uracil
 – cytosine
u – mean linear velocity of
mobile phase
Separation efficiency of sulfobetaine monolithic capillary column.
Separation efficiency
D. Moravcová et al., J. Chromatogr. A 1270 (2012) 178– 185.
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Comparison of isocratic elution on
bare silica monolithic (A) and
sulfoalkylbetaine monolithic (B)
capillary columns.
Mobile phase: 95% (v/v) ACN/ 50 mM
ammonium formate, pH = 4.5, flow
rate 0.5 µl/min; Detection: UV 210
nm.
Isocratic separation
Sample:
toluene (t0 marker)
(1) Thymine
(2) Uracil
(3) 2-Deoxyuridine
(4) 5-Methyluridine
(5) Adenosine
(6) Uridine
(7) Cytosine
(8) 2-Deoxycytidine
(9) Cytidine
(10) 2-Deoxyadenosine
(11) Adenine
Uracil Thymine
Sample:
toluene (t0 marker), (1) Thymine, (2) Uracil,
(3) 2-Deoxyuridine, (4) 5-Methyluridine,
(5) Adenosine, (6) Uridine, (7) Cytosine,
(8) 2-Deoxycytidine,(9) Cytidine, () Guanosine.
Comparison of synthesized capillary column with
commercial ZIC®-HILIC column
Rs 1.26
Rs 0.81
ZIC®-HILIC
column
Capillary
column
Permeability 3.16 x 10-14m2 1.68 x 10-14m2
Porosity 0.74 0.79
HILIC separation.
Mobile phase: 90% ACN/10% 25 mM ammonium
formate (v/v) , pH = 4.5. UV detection 210 nm.
(A) synthesized monolithic capillary column
(150 mm x 0.1 mm), Fc = 0.5 μl/min*;
(B) ZIC®-HILIC column (150 mm × 2.1 mm, 5μm),
Fc = 200 μl/min*;
*mean linear velocity of the mobile phase is (A)
1 mm/s and (B) 1.2 mm/s.
Permeability and porosity of tested columns.
 The simple two-step modification of silica-based monolithic capillary
columns provides stable sulfoalkylbetaine stationary phase suitable for
separation of polar analytes.
 The high separation efficiency of original silica monolithic columns is
preserved even after modification by MEDSA.
 The synthesized column shows a long-term stability under the separation
conditions when the relative standard deviations for the retention times
of tested solutes were lower than 2% under the isocratic conditions and
lower than 3.5% under the gradient conditions.
 The ability of synthesized columns to separate modified nucleobases and
nucleosides such as thymine and uracil or 5-methyluridine and uridine
extends the application range of these columns to the field of proteomics
where separation of similar compounds with different levels of
methylation is required.
Conclusion
 Silicagel-based monolith modified by trioctyl(4-vinylbenzyl)phosphonium
chloride via 3-trimethoxysilylpropyl methacrylate
Phosphonium-based ionic liquid as stationary phase in
HPLC
Trioctyl(4-vinylbenzyl)phosphonium chloride
RP separation of alkylbenzenes on ILand
phenyl-columns.
Mobile phase: 60%ACN/40% ammonium
chloride (2mM in mobile phase); flow
rate 500 nL/min; UV detection at 210
nm. Sample: t0 – uracil, (0) benzene,
(1) toluene, (2) ethylbenzene,
(3) propylbenzene, (4) butylbenzene,
(5) pentylbenzene, (6) hexylbenzene,
(*) impurities.
IL-COLUMN
PHENYL-COLUMN
IL- COLUMN
SIO2-COLUMN
Sample:
toluene (t0 marker)
(1) Xanthine
(2) Thymine
(3) Uracil
(4) 2-deoxyuridine
(5) Cytosine
(6) 2-deoxycytidine
(7) Adenosine
(8) 5-methyluridine
(9) Uridine
(10) Cytidine
(11) Adenine
(12) Hypoxanthine
(13) Guanosine
SULFOBETAINE-COLUMN
PHENYL-COLUMN
HILIC separation of nucleobases and nucleosides.
Mobile phases: (A,B,C) 90% ACN, 10% ammonium acetate (5mM in mobile phase), pH = 4.5; (D) 90% ACN,
10% ammonium formate (2.5mM in mobile phase), pH = 4.5.
Flow rate 500 nL/min, UV detection at 210 nm.
Phosphonium-based ionic liquid as stationary phase in
HPLC
 The synthesized IL-columns possess distinct separation selectivity
compared to bare monolithic silica and phenyl-type as well as
zwitterionic stationary phase.
 The high separation efficiency of original silica monolithic columns is
preserved even after modification by phosphonium-based ionic liquid.
 These columns show mixed interactions and are suitable for multimodal
chromatography.
Conclusion
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 Silica-based monolith in capillary format (0.1 mm × 100 mm) was used
as a support for immobilization of liposomes. This new type of
biomimicking monolithic stationary phase was evaluated by capillary
LC and cryo-scanning electron microscopy (cryo-SEM).
 Acidic hydrolysis of mixture containing tetramethoxysilane,
polyethylene glycol, and urea.
4th
step
 Modification to aminopropylsilica monolithic capillary
column by (3-aminopropyl)trimethoxy-silane.
 Formation of imidoaldehyde groups by reaction of
amino groups with glutaraldehyde.
 Covalent bonding of preformed
liposomes prepared by probe
sonication; types: POPC, POPC/PS
(80/20 mol%).
3rd
step
2nd
step
1st
step
Figure 1. Liposome structure formed by phospholipids.
Liposomes as stationary phase in HPLC Preparation of liposomes
© Avanti Polar Lipids, Inc.
 Liposomes – wide variety of
lipids can be involved in the
preparation  different
biomembrane models
 Preparation of liposomes – the
lipid residues are hydrated in
phosphate buffer 
multilamellar vesicles (MLV)
 Extrusion or sonication leading
to formation of unilamellar
vesicles
– < 100 nm SUV
– > 100 nm LUV
Cryo-SEM images of the prepared
stationary phases.
(A) Silica-based monolith modified by
POPC liposomes;
(B) Detail of silica monolith modified by
POPC liposomes;
(C) Detail of aminopropylsilica monolith.
The structure of used phospholipids.
2-oleoyl-1-palmitoyl-sn-glycero-3-phosphatidyl
choline (POPC)
1,2-diacyl-sn-glycero-3-phospho-L-serine (PS)
Liposomes as stationary phase in HPLC
Separation of sulfa drugs on monolithic capillary columns.
(A) bare silica monolithic capillary column
(B) aminopropylsilica monolithic capillary column
(C) POPC-modified monolithic capillary column.
Sample:
arrow – methanol (t0 marker); 1 – sulfanilic acid; 2 –
sulfacetamide sodium; 3 – sulfafurazole; 4 – sulfanilamide; 5 –
sulfathiazole; 6 - sulfadimidine.
Running conditions:
Mobile phase 20 mM sodium phosphate, pH 7.4; columns (100
mm × 0.1 mm); flow rate 500 nL/min; UV-detection: 220 nm.
Separation of uric acid and its derivatives on liposomemodified
monolithic capillary columns.
(A) 80/20 mol% POPC/PS column
(B) POPC column.
Sample:
arrow – methanol (t0 marker); 1 – uric acid; 2 – xanthine; 3 –
etofylline; 4 - caffeine.
Running conditions:
Mobile phase 20 mM sodium phosphate, pH 7.4; columns (100
mm × 0.1 mm); flow rate 250 nL/min; UV-detection: 220 nm.
Liposomes as stationary phase in HPLC
 The cryo-SEM images confirmed that individual lipid vesicles persist in
their fully hydrated form as spherical vesicles even after bonding to the
monolithic silica back bone.
 The drug retention on the liposome-modified columns is caused by their
interactions with the immobilized liposomes, where electrostatic
interactions play a crucial role.
 The composition of the liposome mixture used for column preparation
significantly affects the retention of solute.
Conclusion