VYTAUTAS MAGNUS UNIVERSITY
Vilma Ratautaitė
REGULATION OF MORPHOLOGY OF
CONTINUOUS POLYMERIC BEDS FOR CAPILLARY
CHROMATOGRAPHY
Summary of doctoral dissertation
Physical Sciences, Chemistry (03P)
Kaunas, 2009
The scientific work was carried out in 2004-2008 at Vytautas Magnus
University, Faculty of Natural Sciences.
Research supervisor:
Doc. dr. Olga Kornyšova (Vytautas Magnus University, Physical Sciences,
Chemistry– 03P)
Evaluation board of Chemical Trend:
Chairman:
Prof. habil. dr. Arūnas Ramanavičius (Vilnius University, Physical
Sciences, Chemistry – 03P)
Members:
Prof. dr. Vytautas Getautis (Kaunas University of Technology, Physical
Sciences, Chemistry – 03P)
Prof. habil. Dr. Pavelas Duchovskis (Lithuanian Institute of Horticulture,
Biomedical Sciences, Ecology and environmental– 03B)
Prof. dr. Vitalis Briedis (Kaunas Medical University, Biomedical Sciences,
Pharmacy – 09B)
Doc. dr. Remigijus Ivanauskas (Kaunas University of Technology, Physical
Sciences, Chemistry – 03P)
Official opponents:
Prof. habil. dr. Audrius Padarauskas (Vilnius University, Physical Sciences,
Chemistry – 03P)
Dr. Evaldas Naujalis (Semiconductor Physics Institute, Physical Sciences,
Chemistry – 03P)
Public defense of the Dissertation will take place at the meeting of
Evaluation board at 11 a.m. on December 18th 2009 in the Auditorium 801 of the
Faculty of Natural Sciences at Vytautas Magnus University.
Address: Vileikos 8, LT-44404 Kaunas, Lithuania. Phone: +370 37 327
902.
The sending-out date of the summary of the doctoral dissertation is on 18th
November, 2009.
The dissertation is available at the libraries of Vytautas Magnus University
and Institute of Chemistry.
VYTAUTO DIDŽIOJO UNIVERSITETAS
Vilma Ratautaitė
IŠTISINIŲ POLIMERINIŲ SORBENTŲ, SKIRTŲ
KAPILIARINEI CHROMATOGRAFIJAI,
MORFOLOGIJOS REGULIAVIMAS
Daktaro disertacijos santrauka
Fiziniai mokslai, chemija (03P)
Kaunas, 2009
Disertacija rengta 2004-2008 metais Vytauto Didžiojo universitete, Gamtos
mokslų fakultete.
Mokslinis vadovas:
Doc. dr. Olga Kornyšova (Vytauto Didžiojo universitetas, Fiziniai mokslai,
Chemija – 03P)
Disertacija ginama Vytauto Didžiojo universiteto Chemijos mokslo
krypties taryboje:
Pirmininkas:
Prof. habil. dr. Arūnas Ramanavičius (Vilniaus universitetas, fiziniai
mokslai, chemija – 03P)
Nariai:
Prof. dr. Vytautas Getautis (Kauno technologijos universitetas, fiziniai
mokslai, chemija – 03P)
Prof. habil. dr. Pavelas Duchovskis (Lietuvos
daržininkystės institutas, ekologija ir aplinkotyra – 03B)
sodininkystės
ir
Prof. dr. Vitalis Briedis (Kauno medicinos universitetas, biomedicinos
mokslai, farmacija – 09B)
Doc. dr. Remigijus Ivanauskas (Kauno technologijos universitetas, fiziniai
mokslai, chemija – 03P)
Oficialieji oponentai:
Prof. habil. dr. Audrius Padarauskas (Vilniaus universitetas, fiziniai
mokslai, chemija – 03P)
Dr. Evaldas Naujalis (Puslaidininkių fizikos institutas, fiziniai mokslai,
chemija – 03P)
Disertacija bus ginama viešame Chemijos mokslo krypties tarybos posėdyje
2009 m. gruodžio mėn. 18 d. 11 val. Vytauto Didžiojo universiteto, Gamtos
mokslų fakulteto 801 auditorijoje.
Adresas: Vileikos g. 8, LT-44404 Kaunas, Telefonas (8 37) 327 902.
Disertacijos santrauka išsiuntinėta 2009 m. lapkričio mėn. 18 d.
Disertaciją galima peržiūrėti Vytauto Didžiojo universiteto ir Chemijos
instituto bibliotekose.
1.
Introduction
Relevance of the research. Miniaturization of chemical analysis is an up-to-date
problem because of usage of low amounts of various chemicals including very
aggressive and toxic solvents, therefore it agrees with principles of green chemistry.
Miniaturization of chromatographic analysis includes reduction of solvent usage by
means of decreased column inner diameter from 4.6 mm commonly used in HPLC to
100 µm or less used for capillary chromatography. Mobile phase flow rates through the
capillary column and the sample amounts are in nanoliters range, therefore the capillary
chromatography is called nanochromatography. Continuous (monolithic) beds are
characterized as a single piece adsorbent occupying capillary volume and are called as
“the fourth generation” of stationary phases. Pioneering research work dealing with
polymeric (acrylamide based) continuous beds was published in 1989 by S. Hjertén
research group. Polymer based continuous beds are formed using hydrophobic
interaction-based phase separation mechanism. During development of continuous bed
a degassed polymerization mixture of monomers, crosslinkers, additives and initiators
are drawn into capillary and left for a while. Continuous bed morphology is formed
during the polymerization induced phase separation. However, the effect of
polymerization mixture composition
on continuous bed morphology wasn’t
systematically investigated.
The main aim of the work was to evaluate polymerization conditions effect on
the continuous bed morphology and chromatographic properties and to apply prepared
continuous bed capillary columns for analysis of real sample.
The objectives of the research work are the following:

To synthesize polyacrylamide based continuous beds and evaluate
chromatographic properties and morphology of prepared capillary columns
varying following conditions:
-
Polymerization initiation method.
-
Polymerization buffer concentration.
-
Polymerization medium pH.
-
Total monomer concentration.
5
-
Periodate labile crosslinker concentration in the polymerization mixture.

Investigate the effect of different functional monomers on reversed phase
continuous bed chromatographic properties.

To apply prepared continuous bed for the real sample analysis.
Scientific novelty of the work. Systematic investigation of continuous beds by
means of various methods was performed. It was investigated how polyacrylamide
based continuous bed morphology is affected by polymerization buffer concentration
and pH. This research work revealed how investigated parameters affected continuous
bed surface charge (Zeta potential) and morphology. The effect on continuous bed
morphology of periodate labile crosslinker concentration was investigated. All
morphology changes were systematically investigated using the inverse size exclusion
chromatography, microscopy and electrochromatography methods.
For the determination of denatonium benzoate (ethyl alcohol denaturant) the
capillary chromatography and continuous polyacrylamide bed were applied for the first
time. Analysis time was shortened and solvent and sample amount were reduced.
Approbation of the research results. The results of the research work were
presented in 2 papers in scientific journals and 11 International and National
Conferences.
Structure of the dissertation. The dissertation consists of introduction, three
chapters, conclusions, list of references (199 entries) and a list of original scientific
publications. The materials of the dissertation are presented in 117 pages, 46 figures and
14 tables.
Literature review. Literature review gives the view of relevant publications
related to the topic of the dissertation. The review of continuous bed classification,
nomenclature, synthesis and used formats are described. The phenomenon of
polymerization induced phase separation, morphology regulation, continuous beds
chemical nature and applications are described.
2.
Experimental
Methods. Investigation of synthesized continuous beds was performed by means
of capillary liquid chromatography (cLC), capillary electrochromatography (CEC),
6
inverse size exclusion chromatography (ISEC), and microscopy. Amphiphilic
polyacrylamide based continuous bed was applied for evaluation ethyl alcohol
denaturant denatonium benzoate by means of capillary LC. All parameters were
calculated from at least 3 replicated measurements and RSD was lower 3%.
3.
Results and discussion
In this work continuous polyacrylamide based beds were prepared for the
evaluation of various parameters affecting continuous bed morphology. Continuous bed
was prepared according to principle of pioneering research work published by S.
Hjertén 1989 [J. Chromatogr. 473 (1989) 273]. Polymerization mixture composition of
all evaluated continuous beds is given in Fig. 1. The continuous bed formation was
accomplished after polymerization mixture including initiator was drawn into modified
fused silica capillary and polymerized in situ. Polymerization duration and temperature
effect on continuous bed morphology was evaluated.
Fig. 1. Evaluated continuous bed morphology affecting parameters and polymerization mixture
composition.
* - IPA – N-isoprylacrylamide, MA – methacrylamide, PDA – piperazine diacrylamide, HDDMA –1,6-hexanediol
dimethacrylate, VSA – vinylsulfonic acid sodium salt, HMAM – N-Hydroxymethylacrylamide, DATD - N,N′-diallyl
tartardiamide.
7
Regulation of amphiphilic continuous polyacrylamide based bed
morphology
Effect of polymerization initiation technique and polymerization
duration to continuous bed morphology
For polymerization initiation two methods were used. First polymerization
initiation method was to initiate polymerization with ammonium persulfate (APS) and
N,N,N′,N′-tetramethylethylenediamine (TEMED) at 25°C. The second polymerization
initiation method was to initiate polymerization only with APS, at elevated temperature
(65°C).
Increase of hydrodynamic
permeability, %
95
65
y = 0,1306x + 79,47
2
R = 0,9913
55
90
45
85
35
80
25
75
15
0
20
40
60
80
Reduction of theoretical plate
height , ×(-1) %
Hydrodynamic permeability of continuous bed was increased when
polymerization initiation method with APS and TEMED at 25°C was changed to
initiation with APS at 65°C (Fig. 2). Additionally it was found that effect of
polymerization initiation technique is stronger when polymerization buffer
concentration is higher.
100
Polymerization buffer concentration, mM
Fig. 2. The effect of polymerization initiation technique on the hydrodynamic permeability and of
theoretical plate height. Polymerization is initiated with APS plus TEMED at 25°C and initiated
with APS at 65°C. □ – is increase of hydrodynamic permeability (%), ◊ - decrease of theoretical
plate height (%).
Increase of hydrodynamic permeability at 25°C and 65°C temperature is
correlating with polymerization buffer concentration. Correlation coefficient was
0.9913. A very different effect of polymerization initiation technique is explained
according two aspects: 1) phase separation is affected when temperature is increased; 2)
8
Decrease of pore surface to
volume ratio, %
effect of temperature is correlating with phosphate buffer concentration, consequently
after increase of temperature and concentration the hydrodynamic permeability is
growing at different rate. Comparing continuous beds polymerized in 5 mM
concentration buffer hydrodynamic permeability was increased by 80%. And in the
same manner hydrodynamic permeability increased 92.7% if polymerization was
performed in 100 mM concentration polymerization buffer.
85
80
75
70
65
0
20
40
60
80
100
Polymerization buffer concentration, mM
Fig. 3. Polymerization initiation method effect on continuous bed pore surface to volume ratio
decrease (%) when polymerization performed in different concentration buffers.
ISEC revealed that temperature effect on pore surface to volume ratio is rather
similar if the polymerization is performed in 5 or 25 mM concentration buffer (Fig. 3).
However, at higher polymerization buffer concentrations effect of polymerization
technique is growing less. It can be concluded that effect of polymerization initiation
technique on continuous bed morphology is synergetic with phosphate buffer
concentration. Continuous beds polymerized at higher temperatures are more
hydrodynamically permeable, but pore surface to volume ratio is decreased.
The effect of polymerization duration was estimated on hydrodynamic
permeability, when polymerization initiation was performed only with APS at 65°C
(Fig. 4).
9
Hydrodynamic permeability,
nl/min
20
18
16
14
0
0,5
1
1,5
2
2,5
3
3,5
Duration of polymerization, hours
Fig. 4. Polymerization duration effect on hydrodynamic permeability
According to data presented in Fig. 4 it is obvious that hydrodynamic
permeability after 0.7 hour wasn’t changing very much. This indicates that continuous
bed morphology at investigated polymerization conditions was stable and
polymerization was completed after 0.7 h. After 0.7 h. such capillary column can be
washed and used for analysis.
According to the protocol of the continuous bed preparation the polymerization
mixture is prepared as follows: all comonomers, additives (such as ammonium sulfate)
are dissolved in phosphate buffer, after that polymerization mixture is degassed.
Polymerization starts after initiator is added. At the same moment polymerization
mixture is drawn in to modified fused silica capillary. Since the polymerization is
started in the same moment as initiator is added, the phase separation process is started
just before polymerization mixture is drawn in situ The effect of possible orientation of
the continuous bed morphology was evaluated by measurement of the hydrodynamic
permeability both ends of the capillary column. RSD% of hydrodynamic permeability
measurements (n=6) was 0.7%, from both ends. This means, that continuous bed
morphology isn’t orientated or irregular. It can be concluded that, continuous bed
morphology is isotropic.
Investigation of buffer concentration effect on continuous bed
morphology
The investigation of the polymerization buffer ionic strength effect on the
polyacrylamide-based continuous bed morphology was carried out polymerizing the
10
beds in 100, 50, 25, and 5 mM concentration phosphate buffers, pH 7. Concentrations of
other polymerization mixture components were kept constant.
The hydrodynamic permeability of the capillary columns was evaluated. As
shown in Fig. 5, the hydrodynamic permeability is increased as a higher ionic strength
of the polymerization medium is used.
The reduction of buffer concentration from 100 to 50 mM results in 4.8-fold
decrease in hydrodynamic permeability. Further decrease in buffer concentration gives a
less pronounced permeability drop: 20-fold decrease in the buffer concentration results
in 13.7-fold drop of the hydrodynamic permeability. The pore network in the
continuous beds is commonly regulated adding a lyotropic salt to the polymerization
medium. The minimum permeability is therefore determined by ammonium sulfate,
which is added at constant concentration to all the polymerization mixtures investigated.
The buffer concentration effect confirmed the positive ionic strength influence on the
hydrodynamic permeability of amphiphilic polyacrylamide-based continuous beds and
is in agreement with the ionic strength effect for nonpolar monomer composition
continuous beds.
Hydrodynamic permeability
(nl/min)
200
160
120
80
40
100 mM
50 mM
25 mM
0
pH 3
pH 4 pH 6
pH 7
5 mM
pH
8.5
Buffer
concentration
pH
10.5
Polymerization medium pH
Fig. 5. Evaluation of polymerization buffer concentration and pH effects on the polyacrylamidebased bed hydrodynamic permeability at relative pressure 1 bar/cm
Evaluation of the influence of mobile phase linear velocity in a range from 0.2 to
3.0 cm/min on separation efficiency (H(u)) was performed using DMF as unretained
marker. The minimum theoretical plate height and the maximum efficiency were
obtained for the continuous bed synthesized in 5 mM phosphate buffer (data are not
11
ζ, mV
0,9
ε
30
0,8
0,7
25
0,6
20
0,5
0,4
15
0,3
10
Electrokinetic porosity ε
Zeta potential ζ·(-1), mV
represented). The continuous bed synthesized in 100 mM phosphate buffer was the least
efficient. However the difference among all beds investigated was not significant and
did not exceed 4.3%. Therefore it can be concluded that the polymerization medium
buffer concentration has no significant effect on the separation efficiency of the
continuous bed synthesized.
0,2
5 mM
25 mM
50 mM
100 mM
Phosphate buffer concentration
Fig. 6. Influence of phosphate buffer concentration in polymerization mixture on Zeta potential and
electrokinetic porosity of the continuous bed. Mobile phase: 50% methanol/ 0.1% TEA and acetic
acid (2.5:1 (v/v)) buffer, pH 5.6.
Included in the polymerization mixture composition, the VSA monomer with
sulfonic acid groups provides a surface charge and the continuous bed can be used for
CEC in a broad pH range. In a liquid medium the charged surface forms an electrical
double layer. Such a double layer is characterized by the Zeta potential (ζ). The
experiments showed (Fig. 6) that the increase in ionic strength of polymerization
mixture increases the Zeta potential of the beds. In order to assess this phenomenon the
electrokinetic porosity of the beds was evaluated. Electrokinetic porosity is a
characteristic of the stationary phase, which indicates the fraction of the mobile phase,
which is transporting current. At constant total monomer concentration and monomer
composition, the increase in polymerization buffer concentration results in higher
electrokinetic porosity. At a constant mobile to stationary phase volume ratio, the
transport of ions (current) depends on the connectivity of the pores and channels in the
continuous bed. Therefore electrokinetic porosity is slightly different from the perfusive
properties of the bed, reflected in the hydrodynamic permeability as a macroscopic
indicator.
12
The electrokinetic porosity therefore may decrease when a big fraction of
restricted connectivity pores is present in the continuous bed, despite the fact that
monomer concentration and porosity of the bed (sum of channel and pore volume) are
kept constant. This assumption is supported by very different hydrodynamic
permeabilities of the beds synthesized (Fig. 5).
Table 1. Effect of phosphate buffer concentration in polymerization medium on continuous beds
characteristics and pore structure parameters
Polymerization buffer concentration (mM)
Porosimetric characteristics
5
25
50
100
V channels, (nl, %)
240 (54.4%)
245 (55.4%)
257 (58.3%)
291
(65.8%)
V pore, (nl, %)
107 (24.3%)
102 (23.1%)
87 (19.6%)
56 (12.7%)
53%
52%
47%
37%
Pore surface to volume ratio (m /mL)
25.6
15.6
12.4
9.7
Average pore size DS (nm)
78.2
128.3
161.3
207.1
Pore size polydispersity (U)
1.93
1.4
1.21
1.07
Porosity of the polymeric skeletons (%)
2
Since the monomer concentration in the polymerization mixture was constant, the
total porosity (sum of channel and pore volume) of the continuous beds was the same.
The fraction of pore to channels volume in the beds was highly affected by the ionic
strength of the polymerization medium. As shown in Table 1, the increase in buffer
concentration increased the flow channels fraction, with a maximum of 65.8% of total
capillary volume at 100 mM phosphate buffer in the polymerization medium. At 5 mM
buffer concentration the channel volume decreases to 54.4%. The pore volume is
increased correspondingly. It was estimated, that the fraction of pores in the polymeric
skeletons of the continuous bed synthesized in 5 mM phosphate buffer is 53% and in
100 mM it is decreased to 37%.
Due to the methodological differences using electrokinetic porosity evaluation
and ISEC characterization of beds, the results obtained by these methods cannot be
directly compared; however, they can provide important additional information
regarding the morphology of the bed. The electrokinetic porosity characterizes
interconnected pores along the capillary, whereas porosity determined using ISEC
describes all pores independently of their interconnectivity.
Evaluation of morphology by means of ISEC (Table 1) showed that maximum
pore surface to volume ratio is obtained in 5 mM phosphate buffer. It is decreased when
13
the polymerization medium buffer concentration is increased. Smaller average size and
higher pore size polydispersity are obtained at low ionic strength. High pore size
polydispersity decreases the hydrodynamic permeability of the bed. At higher ionic
strength of the polymerization medium, the phase separation of the polymer formed
results in larger size of pores with less pore size polydispersity. Such beds are more
hydrodynamically permeable due to larger perfusive channels. Such beds also have a
lower surface area. Pore size distribution curves are depicted in Fig. 7.
6
macropores >50 nm
mesopores from 2 to 50 nm
micropores
<2 nm
5
5 mM
25 mM
50 mM
100 mM
ΔV/Δ(lg D S)
4
3
2
1
0
1
2
4
7
13
18
26
38
55
78
134 195 324 507 768
Pore size, nm
Fig. 7. Pore size distribution of beds, obtained using different concentration phosphate buffer (pH
7) as a polymerization medium: 5, 25, 50, and 100 mM.
All the curves demonstrate multimodal pore size distribution with several pore
size maxima. Generally, the increase in the ionic strength of the polymerization medium
shifts the curves to the right (an increase in the average pore size) and downwards (a
decrease in the porosity of the polymeric skeletons). This is a common result of saltingout of the polymer occurring during the phase separation of the polymerized continuous
bed.
14
A
Polymerization
buffer
concentration
5 mM
B
Polymerization
buffer
concentration
25 mM
C
Polymerization
buffer
concentration
50 mM
Polymerization
buffer
concentration
D
100 mM
Fig. 8. Microscopic images of continuous beds when polymerization buffer was: A) 5 mM; B) 25
mM; C) 50 mM; D) 100 mM. Magnification was 1800 and 10,000.
If polymerization buffer concentration was increased the morphology of the bed
was changed (Fig. 8). Comparison of the continuous beds images showed that no
15
significant effect of polymerization buffer concentration on microstructure or pores was
if concentration was increased from 5 to 25 mM. Meanwhile, continuous bed prepared
at 100 mM polymerization buffer concentration was characterized extremely wide pores
and large microstructure. Microscopy images approved phosphate buffer concentration
effect on the continuous bed morphology results.
Polymerization medium pH effect on the continuous bed morphology
In order to assess the continuous bed dependence on the polymerization medium,
pH beds were synthesized using 50 mM phosphate buffers of different pH: pH 3, 4, 6, 7,
ζ
Zeta potential ζ·(-1), mV
31
ε
0,75
0,7
29
0,65
27
0,6
25
0,55
23
0,5
21
0,45
19
Electrokinetic porosity ε
8.5, and 10.5. All synthesized beds were mechanically rigid and firmly attached to the
capillary inner wall, so that they were not pushed out from the capillary columns using
pressures of up to 150 bars and higher. As shown in Fig. 5, the bed synthesized at pH
10.5 has extremely high hydrodynamic permeability (143 nL/min at relative pressure of
1 bar/cm). Beds synthesized in neutral and acidic buffers demonstrate lower
hydrodynamic permeability (30–42 nL/min at relative pressure of 1 bar/ cm), which can
be due to smaller perfusive channels and less connectivity of the channels.
0,4
pH 3
pH 4
pH 6
pH 7
pH 8.5
pH 10.5
Polymerization medium pH
Fig. 9. Influence of polymerization medium pH on Zeta potential and electrokinetic porosity.
The Zeta potential ζ (mV) was measured in order to clarify the polymerization
medium pH effect on surface charge of the continuous beds synthesized. It was
estimated that the Zeta potential was substantially lower, when polymerization is carried
out at low pH (3 and 4) or high pH (10.5). Much higher Zeta potential was
demonstrated by the beds polymerized in neutral and close to neutral polymerization
media (Fig. 9). Although the electrokinetic porosity was low for the bed polymerized at
16
pH 3 (ca. 0.5), it was much higher and quite constant, when polymerization medium pH
was ranging from pH 4 to 10.5 (ca. 0.65–0.7). A plausible explanation for such
observation can be the competition between polymerization and hydrolysis of the
polyacrylamide at extreme pH values (3 or 10.5) and high temperatures (65°C). Surface
hydrolysis can cause a cleavage of some functional groups (including sulfonic groups).
Therefore Zeta potential for the beds formed at low and high pH is smaller.
ISEC porosimetry data showed (Table 2) that using polymerization media of pH
from 3 to 8.5, there is no significant change in the volume of channels (55.8–59.3% of
total column volume) or pores (18.5–21.9% of total column volume). The total porosity
of the polymeric skeletons was between 45–50%. Performing the polymerization at pH
10.5, the channel volume increased to 64% and the pore volume decreased to 14.1%.
The total porosity of the skeletons decreased remarkably (to 39%). Big fraction of high
connectivity channels and shrinkage of the skeletons provided not only substantially
higher hydrodynamic permeability of the bed, but also an increase in the electrokinetic
porosity, although Zeta potential of this bed was lower.
Table 2. Effect of polymerization medium pH on the continuous bed porosimetric characteristics.
Porosimetric
characteristics
Polymerization medium pH
pH 3
pH 4
pH 6
pH 7
pH 8.5
pH 10.5
254
(57.6%)
246
(55.8%)
262
(59.3%)
257
(58.3%)
260
(58.8%)
284
(64%)
92 (20.8%)
97 (21.9%)
83 (18.9%)
49%
50%
46%
47%
45%
39%
15
15.2
13.4
12.4
12.8
7.9
Average pore size
(DS) (nm)
133.7
131.2
148.8
161.3
156.5
253.9
Pore size
polydispersity (U)
1.34
1.41
1.24
1.21
1.19
1.02
Vchannels (nL, %)
Vpore (nL, %)
Porosity of polymeric
skeletons (%)
Pore surface to
volume ratio (m2/mL)
87 (19.6%) 82 (18.5%)
62
(14.1%)
Pore size distribution curves are presented in Fig. 10.
Pore structure parameters, such as pore surface to volume ratio, average pore size,
and pore size polydispersity were only slightly affected by pH, varying it in the range
from pH 3 to 8.5. A tendency of some decrease in the pore surface to volume ratio was
observed due to the slight decrease in the pore size polydispersity and the increase in the
average pore size. A narrower pore size distribution and ca. two times larger pores of
17
the continuous bed were obtained at pH 10.5. This resulted in a dramatic decrease in the
relative pore surface (pore surface to volume ratio is 7.9 m2/mL).
6
pH 3
pH 6
pH 8.5
ΔV/Δ(lg D S)
5
pH 4
pH 7
pH 10.5
4
3
2
1
0
1
2
4
7
13
18
26
38
55
78
134
195
324
507
768
Pore size (nm)
Fig. 10. Continuous bed pore size distribution dependence on the polymerization medium pH.
All the continuous beds synthesized in the media of pH 3–8.5 showed very similar
pore size distribution curves with some fraction of mesopores (in the range of 2– 50nm)
and considerable fraction of macropores (above 50 nm). Continuous bed polymerized at
pH 10.5 provided quite a different pore size distribution profile, with a dominating
macropore peak in the pore size distribution curve at ca. 500 nm and almost no
mesopores. Such dramatic morphology change can be attributed to the same
phenomenon, i. e. ongoing competition between polymerization and hydrolysis of the
polyacrylamide at extreme polymerization medium pH 10.5, which caused a surface
charge difference. This can cause not only a cleavage of some ionic groups on the
polymeric surface, but a formation of the higher fraction of the macropores and
perfusive channels. Highly acidic pH 3 did not cause such high morphology
transformation, although it affected the polymer surface charge as reflected in the Zeta
potential change. From this it can be concluded that alkaline polymerization medium
may be applied for polymerization or subsequent pretreatment of the bed at higher
temperatures (e. g. 65°C) in order to modify not only the surface charge of the
continuous bed by the cleavage of ionic groups, but also to regulate the morphology of
the bed during the polymerization.
18
Effect of total monomer concentration %T on continuous polymeric
bed morphology
Effect of total monomer concentration on bed morphology was investigated using
three polymerization compositions with varying total monomer concentration (%T): 21,
25, and 30% using 25 mM phosphate buffer, pH 7 as polymerization medium. The ratio
of co-monomers in all cases was kept constant.
The total monomer concentration effect on the hydrodynamic permeability is very
strong. When %T increases from 21 to 25%, the hydrodynamic permeability decreases
3.7 times, i. e. from 46.07 to 12.44 nL/min. Further increase in %T to 30% decreases the
hydrodynamic permeability to 3.09 nL/min at relative pressure of 1 bar/cm.
Table 3. Effect of total monomer concentration on the electrokinetic porosity and Zeta potential
Total monomer concentration (%)
Zeta potential ζ (mV)
Electrokinetic porosity ε
21%
- 23.46 ± 0.66
0.773 ± 0.014
25%
- 22.1 ± 0.26
0.662 ± 0.012
30%
- 21.25 ± 0.60
0.598 ± 0.011
Evaluation of the electroosmotic properties of the beds showed that the increase in
%T from 21 to 30% slightly decreased Zeta potential (Table 3). The effect on the
electrokinetic porosity is similar. Higher values of Zeta potential at lower total
monomer concentration can be caused by certain arrangements of the monomers and
oligomers occurring during the polymerization and phase separation processes. Ionic
and polar monomers tend to agglomerate at the interface of the separating aqueous
phase (polar medium) and therefore to increase the relative surface charge of the formed
solid polymer compared to that of the polymer formed from much higher monomer
concentration. Higher viscosity (”molecular friction”) in the high monomer
concentration medium renders the composition of the separating solid phase more
uniform and the surface charge (density of the ionic groups at the interface with
separated aqueous phase) relatively lower. On the other hand, higher initial viscosity of
the medium causes faster phase separation and higher polydispersity of the polymeric
bed morphology. ISEC porosimetry results confirm (Table 4) that the increase in total
monomer concentration provides a dramatic increase in pore size polydispersity (U)
from 1.23 to 5.77 and a decrease in the average pore size from 151.6 to 22.3 nm, which
results in a 6.8-fold increase in pore surface to volume ratio.
19
Table 4. Effect of total monomer concentration %T on the porosimetric characteristics of the beds
Porosimetric characteristics
21%
25%
30%
V channels (nl, %)
257 (58.3%)
210 (48%)
201 (46%)
V pore (nl, %)
87 (19.6%)
117 (26.7%)
108 (24.7%)
93 (21.3%)
111 (25.3%)
128 (29.3%)
Pore surface to volume ratio (m /ml)
12.4
19.1
89.6
Pore size average, DS (nm)
161.3
105.0
22.3
Pore size polydispersity (U)
1.21
1.62
5.77
V stationary phase, (nl, %)
2
The influence of total monomer concentration on pore size distribution of the
continuous beds is presented in Fig. 11.
6
21%
5
25%
ΔV/Δ(log DS)
4
30%
3
2
1
0
1
2
4
7
13
18
26
38
55
78
134
195
324
507
768
Pore size, nm
Fig. 11. Pore size distribution curves obtained for the continuous beds polymerized from different
total monomer concentration mixtures.
For the bed, with a total monomer concentration of 21%, the pore size distribution
curve is shifted to right (to larger pores) with the maxima located in the macropores
range. When the total monomer concentration increases, the pore size distribution curve
shifts to left, i. e. to the smaller pores. It can be seen that for the highest total monomer
concentration bed (30%) the pores are distributed in a wide range and the dominating
pore size is in the mesopores range (curve maximum is between 38 and 55 nm).
Summarized results show that an increase in the polymerization buffer
concentration increases the hydrodynamic permeability, electrokinetic porosity, and
Zeta potential of the amphiphilic continuous beds synthesized. Low buffer
concentration results in small pores; however, the range of pore sizes in the continuous
20
bed polymerized was wide. The bed with large, but more uniform pores is polymerized
in buffer of higher concentration.
The pH of the polymerization medium has smaller influence on morphology,
compared to the buffer concentration. This demonstrates the ruggedness of the
continuous bed polymerization procedure, when pH shift does not much affect the
porous structure and the performance of the product. The hydrodynamic permeability is
remarkably smaller for the beds synthesized in acidic and neutral buffers compared to
the beds synthesized in alkaline buffers. Therefore alkaline media at higher
temperatures (e. g. 65°C) can be used as a tool to modify surface charge properties as
well as bed morphology polymerizing the amphiphilic polyacrylamide-based
continuous beds. The most efficient continuous bed was synthesized using buffer of pH
7.
The increase in total monomer concentration results in the decrease in the
hydrodynamic permeability of the continuous beds. It was demonstrated that the
electrokinetic porosity and Zeta potential decrease when increasing the total monomer
concentration. Porosimetric evaluation of the beds revealed that the increase in the total
monomer concentration decreases channel volume and mean pore size and increases
pore size polydispersity and pore surface to volume ratio.
The morphology of the continuous polyacrylamide based stationary phases is
controlled more efficiently by selecting the buffer concentration and the total monomer
concentration in the polymerization mixture, than by changing the buffer pH.
Continuous
crosslinker
bed
morphology
regulation
with
periodate
labile
Flexibility of preparation of continuous bed polymerization mixture gives an
opportunity to use different chemistry crosslinkers. Usage of DATD demonstrated that
cleavage of crosslinker with periodate creates additional porosity of the polymeric
skeleton, which may increase the analyte-accessible surface area and accessibility to the
active sites in the polymeric nonparticulate skeleton. Five capillary columns with
different DATD/PDA crosslinker ratio (Table 5) and one blank capillary column
(without labile crosslinker) were prepared. The amount of DATD was in the range 20 –
60 % of total crosslinker (TC) amount. Usage of higher amount of periodate labile
crosslinker DATD (more than 60 % of TC) was not recommended, since higher fraction
can cause the collapse of the continuous bed after periodate treatment due to insufficient
mechanical stability.
21
Table 5. Composition of polymerization mixture. Table II
Composition*
DATD**, mg
PDA***, mg
60% DATD
24
16
50% DATD
20
20
40% DATD
16
24
30% DATD
12
28
20% DATD
8
32
0% DATD
40
*VSA - 10 l, HMAM - 40 l, (NH4)2SO4 - 7 mg, APS/TEMED - 5/5 l, buffer - 150l
**DATD - periodate treatment unstable crosslinker
***PDA - periodate stable crosslinker.
60
60
50
50
40
40
30
30
20
20
10
10
0
0
20
Pore size
Volume,%
Pore size, nm
The use of PDA together with DATD makes the cleavage process controllable,
i.e. PDA is periodate stabile crosslinker, only DATD is periodate labile crosslinker.
Controlled cleavage can be performed changing ratio of periodate labile (DATD) and
periodate stabile (PDA) crosslinkers in polymerization mixture.
Skeleton
Channels
Skeleton
porosity
30
40
50
60
Fraction of DATD in crosslinker, %
Fig. 12. Effect of DATD/PDA ratio in polymerization mixture on continuous bed morphology. Solid
line for untreated capillary, dotted line – periodate treated capillary.
The periodate treatment increased average pore diameter (Fig. 12). The pore
diameter of the pretreated bed increased with the increase of DATD amount in the
polymerization mixture. It increased from 5.6 nm for untreated blank to 62.3 nm for
periodate treated 60 % DATD. Porosimetric investigation has shown that the cleavage
of labile crosslinker dramatically changes pore size distribution in the skeleton
22
log Mr
decreasing the micropore fraction and extending the macropore fraction in the bed (Fig.
13).
7,5
0% DATD
6,5
20% DATD
5,5
30% DATD
40% DATD
4,5
50% DATD
3,5
60% DATD
2,5
1,5
180
230
280
330
380
Elution volume, nl
a)
5
20% DATD
60% DATD
dV/d(logDs)
4
3
2
1
0
1
2
4
7
13 18 26 38 78 134 195 324 507 768
Pore diameter, nm
b)
Fig. 13. Inverse size exclusions porosimetric evaluation of the beds polymerized at different ratio of
periodate labile and periodate stabile crosslinkers: a - size exclusion chromatography calibration
graphs (calibration graphs with shaded symbols – for periodate untreated, unshaded symbols –
periodate treated continuous bed); b - pore size distribution curves, presented as derivatives of the
cumulative curves of the calibration graphs. Solid line for untreated capillary, dotted line –
periodate treated capillary. Samples: 0.5mg/ml benzene and polystyrene standards.
The pore size polydispersity parameter U was extremely high and similar for
periodate untreated and periodate treated beds (U=7.04 for untreated bed and 7.35
periodate treated). The pore surface to volume ratio was lower for periodate treated bed
due to the bigger average pore size (Fig. 14). As depicted in Fig. 14 the beds differ in
the skeleton porosities, stationary phase and channel volumes (Vp, Vs and Vch
correspondingly). Not surprisingly, periodate treatment increased the porosity of the
skeletons (for capillary with 60% DATD) by more than 20%. The skeleton (non-
23
Alteration
particulate stationary phase) fraction in the column, however, decreased dramatically
(by 56%), which changed also stationary-to-mobile phase ratio (from
Vs/Vmob=Vs/(Vch+Vp)=0.357 for untreated to 0.140 for periodate treated bed). Partial
decomposition of the polymeric skeleton by cleavage of DATD crosslinker also
manifested in highly increased (up to 57.6%) hydrodynamic permeability of the bed.
30
25
20
15
10
5
0
Mean pore size, nm
Surface area, m²/ml
20
30
40
50
60
Fraction of DATD in crosslinker, %
Fig. 14. Effect of DATD/PDA ratio on continuous bed mean pore size and surface area.
Employing DATD crosslinker as comonomer provides a possibility to modify
porous structure of the bed. Since 1,2-diol structure of DATD may be readily converted
to aldehyde groups using periodate treatment, it can be used for attachment of different
ligands of interest i.e. aldehyde group reactive small molecules or macromolecules. It
can be used for enhancement of the accessibility of the active centers within the
polymeric skeletons owing to partial decomposition of the crosslinked polymer. This
approach could be advantage if applied for instance for in situ preparation of the
efficient molecularly imprinted materials in order to wash out the template more
efficiently preventing bleeding of it from the polymeric matrix and increase
accessibility of the imprinted sites.
Summarizing it can be concluded that if during radical polymerization the
reacting polymerization mixture components are taken at constant concentration, main
tool of morphology regulation is the ionic strength. Effect of ionic strength similar both
if it was regulated polymerization buffer concentration or lyotropic salt concentration. It
should be paid attention to ionic strength effect at elevated temperatures, because effect
of polymerization initiation temperature is growing up when polymerization buffer
concentration is growing. Polymerization duration effect on continuous bed
hydrodynamic permeability depends on the chemistries polymerization mixture
monomers and polymerization initiation method. The effect of polymerization medium
24
pH is significantly lower than that of the concentration; however alkaline medium can
be used for continuous bed morphology modification. It was found that porosity and
electrokinetic porosity decreased if total monomer concentration was increased.
Periodate labile crosslinker offer a possibility at additional morphology regulation.
Regulation of amphiphilic polyacrylamide based continuous bed
morphology by means of monomers
Regulation of hydrophobicity
In most cases the main aim of the column investigation is increasing of column
efficiency, but it solves just a little part of sample analysis problems. The increased
column efficiency is limited if sample matrix is complicated. The increase of column
loadability and selectivity is a key of new generation of columns. One of the ways to
increase column selectivity is regulation of polymerization mixture monomer
composition and the increase of hydrophobic monomer concentration.
Initial continuous bed was prepared from monomers MA, IPA, VSA and
crosslinker PDA, where ratio of IPA and MA was 0.13. Solubility lgS of IPA was -1.34,
MA solubility was -0.87, PDA solubility was -0.36 and solubility of VSA was -0.32.
Solubility clarifies that increase of IPA concentration is limited, especially if in
polymerization mixture is included buffer at elevated concentration. Total monomer
concentration T% of estimated continuous beds was 21.7% and crosslinker
concentration was C% 40.9% (Fig. 15).
Retention factor for benzylbenzoate was 0.14, when continuous bed with IPA and
MA ratio 0.13 for sample analysis was used. When IPA and MA ratio was changed to
7.75, retention factor increased to 1.34. Respectively retention factor was 3.08 if IPA
and MA ratio was increased to 34. Methylene group selectivity was increased from 1.27
to 1.30. Good linearity was obtained for plots of logarithm of retention factor
dependence on methylene group number in alkyl benzoates. Linearity shows separation
mechanism based on hydrophobic interaction.
More effective hydrophobicity regulation is with alkyl ammonium salt monomer.
Alkyl ammonium salt monomer was prepared in alkaline conditions from 3-chlor-2hydroxypropyldimethyldodecylammoniumchloride and allylamine and has surfactant
properties. Total monomer concentration T% of investigated continuous bed was 30.6%
and crosslinker concentration C% was 36.8%. Effect of polymerization conditions on
retention factor was investigated (Fig. 16).
25
0,3
C
log (k)
0,1
-0,1
B
-0,3
-0,5
A
-0,7
-0,9
1
1,5
2
2,5
3
3,5
4
4,5
5
5,5
6
N CH2
a)
30
25
C
mAU
20
15
B
10
5
A
0
0
10
20
b)
30
40
50
60
70
Time, min
Fig. 15. Effect of IPA and MA ratio in polymerization mixture on continuous bed chromatographic
properties. a) Dependence of the logarithm of the retention factor on the methylene group number
in alkyl benzoates; b) Separation of alkyl benzoates using continuous bed prepared at three
different IPA and MA ratios in polymerization mixture A) 0,13; B) 7,75; C) 34. Sample: DMF and 6
alkyl benzoates (methyl, ethyl, propyl, N-butyl, N-amyl and benzyl benzates). Mobile phase: 50 %
of methanol with 50% TEA and acetic acid (0.1%) buffer, pH 5.7 (TEA – glacial acetic acid (2.5 ÷1
v/v)). Detection: UV, 220 nm.
Results indicate that shortened polymerization duration from 24 to 2 hours at
25°C didn’t change the selectivity of continuous bed substantially (  CH 2 was 1.47 and
1.48). When polymerization temperature increased from 25°C to 65°C (polymerization
duration was 2 hours), slope of retention factor logarithm dependence for alkyl benzoate
methylene group number decreased. Selectivity decreased from 1.48 to 1.4 and ΔΔG
increased from 0.95 to 0.81 kJ/mol.
Results could be explained by properties of alkyl ammonium salt monomer.
Alkylammonium salt comonomer because of hydrophilic and hydrophobic groups acts
as surfactant and its CMC is approx. equal to 8 mM. Commonly effect of temperature
on surfactant CMC has “U” form curve and the minimum is at temperature 25°C, so
alteration of CMC in water based solutions is easily forecasted. At elevated
temperatures in surfactant micelle phase appear nonhomogenity and polydispersity
26
lg (k)
changes and micelle transforms into higher ionization form. Consequently hydrophilic
bonds of surfactant and water at elevated temperatures are interrupted and because of
that surfactant hydrophobicity is increased. For continuous bed preparation used alkyl
ammonium salt monomer with surfactant properties and at elevated temperatures
surfactant hydrophilic and hydrophobic properties have substantial effect on monomer
dispersion in polymerization mixture during polymerization induced phase separation. It
can be concluded that effect of temperature is complex because of many factors like
hydrophobic interaction, hydrogen bond and parallel polymerization induced phase
separation.
0,3
0,2
0,1
0
-0,1
-0,2
-0,3
-0,4
A
B
C
2
3
4
5
N CH2
a)
20
18
16
A
mAU
14
12
10
B
8
6
4
2
C
0
0
b)
20
40
60
Time, min
Fig. 16. Effect of polymerization duration and temperature on continuous bed chromatographic
properties. a) dependence of retention factor logarithm on methylene group number of alkyl
benzoate. b) separation of alkyl benzoate mixture using continuous beds prepared at different
conditions. Polymerization conditions: A) 24 hour 25°C; B) 2 hours 25°C; C) 2 hours 65°C. Sample
and detection as in Fig. 12. Mobile phase: 50% methanol in water.
Usually hydrophobicity is controlled by means of monomers and regulation of
hydrophobicity by means of crosslinker is less used. In this case were used two
crosslinkers PDA and HDDMA. Solubility in water of HDDMA is -2.54 (compare with
PDA solubility -0.36). HDDMA in water was solubilized using alkylmmonium salt
comonomer AQ342 and additionally Triton X 100. Hydrodynamic permeability was
27
increased with ammonium sulfate. Total monomer concentration T% was 25.03% and
crosslinker concentration C% 42.38%. Estimated PDA and HDDMA ratios were 5:1,
2:1 and 1:1 (Fig. 17 and Table 6).
1,4
0,9
lg k
A
B
0,4
C
-0,1
2
3
a)
A
20
B
10
C
5
mAU
30
4
N CH2
0
0
10
20
30
40
50
60
70
Time, min
b)
Fig. 17. Effect of crosslinkers ratio in polymerization mixture on chromatographic properties. a)
retention factor logarithm dependence on alkyl benzoate methylene group number. b) separation
alkyl benzoates using continuous bed with different PDA and HDDMA ratio in polymerization
mixture. A) 5:1; B) 2:1; C) 1:1.
Table 6. Evaluation of alkyl benzoates chromatographic properties by means of capillary LC.
Mobile phase: 50% (v/v) methanol in water.
 CH 2
PDA and HDDMA ratio
ΔΔG, kJ/mol
5:1
1.69
-1.27
2:1
1.77
-1.38
1:1
1.89
-1.54
Effect of hydrophobic crosslinker concentration on chromatographic properties
was similar to usual effect of hydrophobic monomer. When HDDMA concentration in
polymerization mixture was increased, analysis time was longer, methylene group
selectivity was increased and consequently ΔΔGCH2 decreased. Additionally was
noticed, that into polymerization mixture included cationic and nonionic surfactants
28
interacts and self organization is changed. Continuous bed is affected not only by
additive concentration, but also on the interaction among them.
Summarized results of continuous bed regulation showed that continuous bed
morphology is affected by all components in polymerization mixture both reacting and
non reacting during radical polymerization.
Evaluation of ethyl alcohol denaturant, denatonium benzoate, by
capillary liquid chromatography with polyacrylamide based continuous
bed
The reason why denatonium benzoate (bitrex) was selected for continuous bed
application as real sample was the fact that denatonium benzoate is one of the most
effective protective agents for household liquids containing ethyl alcohol or other toxic
solvents from accidental consumption.
Why continuous bed application to denatonium benzoate analysis is relevant?
Usually used methods for denatured alcohol analysis are HPLC, CE etc. But till now
capillary liquid chromatography and polymer based continuous bed wasn’t used for this
purpose. Main advantages of capillary liquid chromatography are rapidity and smaller
quantities consumed mobile phases, thus, method more friendly for environment.
Denatured alcohol sample matrix is very simple (peak of bitrex was separated in
all investigated mobile phase composition cases) because of that optimization of
chromatographic conditions is fast. Effect of various mobile phase compositions was
evaluated.
Table 7. Determination of bitrex concentration in domestic chemistry commodities, where bitrex
usual component. Mobile phase: 90% ACN and 10% of 5 mM TFA and 5 mM NaOH, pH 6.7.
Detection: UV, 220 nm. Capillary column: effective length 9.7 cm, inner diameter 50 µm. Injection
volume 8.7 nl,
Household chemical goods
Bitrex concentration, mg/l
Window cleanser
0.30
Blackhead clearing cleanser
2.16
Shoe spray
3.80
Winter car window cleanser
2.65
Analysis time of bitrex in ethyl alcohol formulations by means of capillary LC
using polymer based continuous bed was about four minutes (Table 7). For one sample
29
analysis was sufficient approx. 400 nL of mobile phase. Limit of detection was 0.17
mg/L and limit of determination was 0.29 mg/L. Linearity was found in range from 0.17
mg/L to 36.6 mg/L. Relative standard deviation of peak area of replicated injections in
samples with household chemical goods samples varied from 2.7% to 2.9%.
Conclusions

Estimation of chromatographic properties and morphology of prepared continuous
bed for capillary chromatography showed that :
- Continuous beds synthesized at 65°C temperature when polymerization is
initiated with APS and without TEMED are more hydrodynamically permeable
than if polymerization is initiated with APS and TEMED at 25°C temperature. If
polymerization is initiated at 65°C temperature the prepared continuous beds are
more efficient, however pore surface to volume ratio is lower.
- When polymerization buffer concentration is increased the hydrodynamic
permeability, electrokinetic porosity and Zeta potential are increased. If
polymerization is performed at low buffer concentration (5 mM or 25 mM), the
polymerized continuous beds have small pores, but pore size polydispersity is
higher than at high buffer concentration (100 mM) polymerized continuous beds.
- Polymerization buffer pH effect on morphology is significantly lower than buffer
concentration. But because of hydrolysis, alkaline buffers (pH 10.5) are suitable
for modification of morphology of the beds.
- When the total monomer concentration increases (from 21% to 30%),
hydrodynamic permeability, Zeta potential and electrokinetic porosity of the
continuous bed is decreased respectively from 23.46 mV to 21.25 and from 0.773
to 0.598. Likewise volume of channels and average pore size is decreased;
however is increased pore size polydispersity and pore surface to volume ratio
(more than 4.5 times and 6.5 times respectively).
- If periodate labile crosslinker (DATD) amount in polymerization mixture is
increased (from 0 to 60%) changes of the polymeric continuous bed morphology
are obtained: after periodate cleavage macropore volume is increased and
respectively micro- and mesopore volume is decreased. If DATD concentration is
increased form 20% to 60%, after periodate cleavage average pore size is
increased by 13.9 nm and 28 nm
30
 If IPA, alkyl ammonium salt comonomer and HDDMA were used as functional
monomers, and IPA/MA ratio was increased from 7.75 to 34 selectivity of the
continuous bed reversed phase chromatography in 50 % methanol mobile phase was
increased from 1.27 to 1.30. If continuous bed with alkyl ammonium salt comonomer
polymerization temperature was increased from 25°C to 65°C, selectivity was
decreased from 1.48 to 1.4 and if amount of HDDMA in polymerization mixture was
increased from 20% to 50%, selectivity was increased from 1.69 to 1.89.
 Continuous bed was first time used for ethyl alcohol denaturant denatonium benzoate
analysis by means of capillary chromatography. It is rapid method and one analysis
is performed within 4 min. During one analysis was used approximately 400 nl of
mobile phase, sample amount was 8.7 nl. The limit of detection of the method was
0.17 mg/L and limit of quantification 0.29 mg/L. Good linearity was obtained for the
concentration range 0.17 mg/L - 36.6 mg/L.
List of original publications
Scientific Paper in the Journal Listed by ISI list
V. Ratautaitė, A. Maruška, M. Erickson, O. Kornyšova. „Effect of polymerization
conditions on morphology and chromatographic characteristics of polyacrylamide-based beds
(monoliths) for capillary electrochromatography and capillary liquid chromatography“.
Journal of Separation Science, 2009, 32, 2582 – 2591.
Paper in Reviewed Lithuanian Journal
V. Ratautaitė, O. Kornyšova, A. Maruška. „Etilo alkoholio denatūranto, denatonio
benzoato, nustatymas kapiliarinės skysčių chromatografijos metodu, naudojant ištisinį
polimerinį sorbentą“. Food Chemistry and technology, 2008, 42 (1), 78-85.
Proceedings of International Conferences
V. Ratautaitė, A. Maruška, O. Kornyšova. „The evaluation of monolithic (continuous
bed) acrylamide based adsorbents for nanochromatography by inverse size exclusion
chromatographic porosimetry: Comparison of two evaluation methodologies“. International
young scientist conference The Vital Nature Sign Proceedings [Kaunas, Vytautas Manus
University Press, 2007], Kaunas 2007 m. gegužės 18 d., p. 81-84. (oral presentation)
31
Proceedings of National Conferences
V. Ratautaitė, A. Maruška, O. Kornyšova. „Ištisinių akrilamidinių sorbentų
morfologijos priklausomybė nuo polimerizacijos iniciavimo ir buferio koncentracijos“.
Konferencija „Kur gamta siejasi su mokslu“, 19 May, 2006. Kaunas, p. 72-74. (Poster
presentation).
Abstracts of International and National Conferences
1.
A. Maruška, O. Kornyšova, V. Ratautaitė, R. Jarmalavičienė, R. Šurna, M.
Erickson. „Concepts, Development, Evaluation and Applications of Continuous Beds
(Monoliths)
for
Chromatographic
Microseparations
and
Sample
Clean-up“.
7th
Chromatography Conference, Białystok, Poland (oral presentation by habil. dr. A. Maruška).
10-13 September, 2006
2.
A. Maruška, O. Kornyšova, V. Ratautaitė, R. Jarmalavičienė, R. Šurna.
„Continuous Beds (Monoliths) for Capillary Liquid Chromatography and Capillary
Electrochromatography: Optimization of Synthesis and Evaluation“. Proceedings of the XXI
International Symposium on Physico-Chemical Methods of Separation, ARS Separatoria,
Torun, Poland (oral presentation by habil. dr. A. Maruška). 02-05 July, 2006
3.
V. Ratautaitė, O. Kornyšova, A. Maruška. Influence of polymerization buffer
concentration and pH to different hydrophobicity monolithic (continuous) beds morphology.
4th NoSSS International Conference, 26-29 August, 2007, Kaunas (poster presentation by).
ISBN 978-9955-12-232-6.
4.
A. Maruška, O. Kornyšova, V. Ratautaitė, R. Jarmalavičienė, R. Milašienė.
Continuous bed (monolithic) stationary phases for capillary format separations: current
status and applications. 50 jubileuszowy zjazd Polskiego towarzystwa chemicznego &
stowarzyszenia inśynierow i technikow przemyslu chemicznego & 11TH EuCheMS – DCE
International conference on chemistry and the environment. 9-12 September 2007 Toruń,
Poland (oral presentation by habil. dr. A. Maruška).
5.
A. Maruška, O. Kornyšova, R. Jarmalavičienė, V. Ratautaitė. Current status and
applications of the continuous bed (monolithic) stationary phases for capillary format
separations. 4th NoSSS International Conference, 26-29 August, Kaunas (oral presentation by
habil. dr. A. Maruška). ISBN 978-9955-12-232-6.
6.
A. Maruška, O. Kornyšova, R. Jarmalavičienė, V. Ratautaitė. Achievements and
perspectives in the continuous bed (monolithic) stationary phases design and applications.
VIII Konferencija Chromatograficzna, Application of chromatographic techniques in
32
environmental and clinical analysis, 21-23 April, 2008, Łodź, Poland (oral presentation by
habil. dr. A. Maruška).
7.
A. Maruška, O. Kornyšova, R. Jarmalavičienė, V. Ratautaitė. Design and
applications of continuous bed (monolithic) stationary phases for capillary and microchip
format separations: realities and prospects. Analysdagarna, 16-18 June, 2008, Göteborg,
Sweden. (oral presentation by habil. dr. A. Maruška).
8.
V. Ratautaitė, A. Maruška, O. Kornyšova. „Ištisinių akrilamidinių sorbentų
morfologijos priklausomybė nuo polimerizacijos buferio koncentracijos ir pH“. Konferencija
„Chemija ir cheminė technologija“ skirta prof. J. Janickio 100-sioms gimimo metinėms, 18
May, 2006. Kaunas University of Technology, 2006. Kaunas (poster presentation).
9.
V. Ratautaitė, A. Maruška, O. Kornyšova. Influence of polymerization initiation
technique to continuous acrylamide based bed chromatographic parameters and synthesis
repeatability. Chemistry 2007, Vilnius University, 12 October, 2007. (poster presentation).
ISBN 978-9955-33-093-6.
33
Ištisinių polimerinių sorbentų, skirtų kapiliarinei
chromatografijai, morfologijos reguliavimas
Reziumė
Atlikta ištisinių polimerinių sorbentų sintezė keičiant polimerizacijos terpės
koncentraciją, pH, bendrą monomerų koncentraciją bei perjodatui neatsparaus
tinklintojo kiekį. Įvertinta sintezės sąlygų įtaka chromatografinėms savybėms ir
morfologijai. Susintetinti ištisiniai poliakrilamidiniai sorbentai įvertinti naudojant
kapiliarinę slėgio chromatografiją, kapiliarinę elektrochromatografiją, inversinę dydžio
išskyrimo chromatografiją ir mikroskopiją.
Ištirta skirtingos prigimties monomerų (N-izopropilakrilamido, alkilamonio
druskos komonomero ir 1,6-heksandioldimetakrilato) įtaka chromatografinėms
savybėms keičiant šių monomerų kiekį polimerizacijos mišinyje.
Paruoštas ištisinis sorbentas buvo pritaikytas realaus bandinio analizei. Ištisinis
polimerinis sorbentas ir kapiliarinė skysčių chromatografija buvo pirmą kartą
panaudotas etilo alkoholio denatūranto, denatonio benzoato, nustatymui. Nustatyta, kad
tai spartus metodas atliekamas vos per 4 minutes. Vienai analizei yra sunaudojama
mažiau nei 400 nl eliuento, o bandinio tūris sudaro – 8,7 nl.
Acknowledgments
Many thanks for doc. dr. Olga Kornyšova and habil. dr. Audrius Maruška for help
to for discover of very attractive world of chromatography and continuous (monolithic)
beds. I am grateful for valuable advises and help when I was stopping with doubts.
A lot of thanks for UAB ArmGate for help with optical microscopy of continuous
beds in fused silica capillaries.
Many thanks for AB Stumbras for the samples used for continuous beds
applications.
I would like to thank to prof. Klaus K. Unger and doc. dr. Sami Franssila for
invitations to intensive courses Separation Science and Technology for Proteins and
peptide at the University of Utrecht, The Netherlands (2004) and Polymer Micro-and
nanofabrication at Palmse, Estonia (2007).
I express my special thanks to all my family as well as to my friends and
colleagues. Without you it would be nothing...
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Curriculum Vitae
Personal details
Name, surname:
Birth date and place:
Education
2004-2008
2002-2004
1997-2001
1997
Most significant facts
31 August - 6
September, 2007
22-26 November 2004
1 February - 30 April
2003
Vilma Ratautaitė
19 May, 1979, Ukmergės distr., Vepriai
PhD student at the Department of Chemistry Vytautas Magnus
University, Kaunas, Lithuania
MSc in Chemical Analysis, Vytautas Magnus University, Kaunas,
Lithuania
BSc in environmental sciences and mathematics, Vytautas Magnus
University, Kaunas, Lithuania
Graduated from secondary school of Vepriai, Vepriai, Lithuania
Attended NordForsk PhD course Polymer micro- and
nanofabrication, at Palmse (Estonia).
Attended course Separation science and technology for proteins
and peptides at Utrecht University, The Netherlands.
Studies at the department of chemistry of the University of
Marburg, Germany by SOCRATES/ ERASMUS program
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Vilma Ratautaitė
REGULATION OF MORPHOLOGY OF CONTINUOUS
POLYMERIC BEDS FOR CAPILLARY
CHROMATOGRAPHY
Summary of Doctoral Dissertation
Išleido ir spausdino – Vytauto Didžiojo universiteto leidykla
(S. Daukanto g. 27, LT-44249 Kaunas)
Užsakymo Nr. 141. Tiražas 50 egz. 2009 11 17.
Nemokamai.
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