Poly(4-vinylpyridine) based novel stationary phase investigated under supercritical fluid chromatography conditions

Journal of Chromatography A, 1572 (2018) 119–127

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Poly(4-vinylpyridine) based novel stationary phase investigated
under supercritical fluid chromatography conditions
Kanji Nagai ∗ , Tohru Shibata, Satoshi Shinkura, Atsushi Ohnishi
DAICEL Corporation, CPI Company, Life Science Development Center, Innovation Park, 1239, Shinzaike, Aboshi-ku, Himeji, Hyogo, 671-1283, Japan

a r t i c l e

i n f o

Article history:
Received 31 May 2018
Received in revised form 7 August 2018
Accepted 16 August 2018
Available online 23 August 2018
Keywords:
Supercritical fluid chromatography
Stationary phase
Ligand
Selector
Polymer
Poly(4-vinylpyridine)

a b s t r a c t
A novel poly(4-vinylpyridine) based stationary phase was investigated for its performance under supercritical fluid chromatography (SFC) mode. Due to its unique structure, this stationary phase has high

molecular planarity recognition ability for aromatic samples possessing the same number of aromatic
rings and ␲-electrons. Taking advantage of the planarity recognition ability observed, separations of
structurally similar polycyclic aromatic hydrocarbons and steroids were achieved. This novel stationary phase afforded good peak symmetry for both acidic and basic active pharmaceutical ingredients even
when excluding the use of additives such as acids, bases, and salts. These findings may be attributed to the
polymeric pyridyl groups covalently-attached on silica gel, which will effectively shield the undesirable
interaction between residual silanol groups on the surface and the analytes. Moreover, the properties of
pyridyl group on the selector can be reversibly tuned to cationic pyridinium form by eluting trifluoroacetic
acid containing modifier. Column robustness toward cycle durability testing was also confirmed.
© 2018 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license
( />
1. Introduction
Supercritical fluid chromatography (SFC) is increasing inuse
in the analytical and preparative separation field [1]. SFC uses
supercritical or subcritical mobile phases consisting of pressurized carbon dioxide (CO2 ), usually mixed with a miscible organic
solvent (e.g. an alcohol). This technology has major advantages
over more conventional liquid chromatography (HPLC) or gas chromatography (GC), because it has a low viscosity allowing high
diffusivities and limited pressure drop. Therefore, high flow rates
can be applied without losing efficiency [2–8]. In addition, the
“green” aspect is a significant motivation for SFC because CO2 is a
nontoxic recycled material and generates no waste disposal issues.
The high-throughput potential together with ecological advantages
contribute to making SFC attractive technology for a wide range of
applications, not only for chiral [9–15], but also in the achiral field
[16–30].
The retention and separation mechanisms in SFC are likely to
depend on a combination of both mobile phase and stationary
phase (SP)[5]. A variety of SPs are currently available for use in
SFC mode. Most of these phases have been developed in and transferred from the commercially available portfolios of HPLC SPs (e.g.

∗ Corresponding author.

E-mail address: kn (K. Nagai).

reverse phase, normal phase, and/or HILIC). In parallel, there are
some activities to develop novel SPs specifically designed for SFC
use [31]. One of the most recognized SP dedicated to achiral SFC
separation is 2-ethylpyridine (2-EP) bonded silica phase. This 2-EP
SP affords good peak shapes especially for basic compounds, without any additive in the mobile phase [32]. Other novel SPs for SFC
have been developed by academic and industry groups [33–38].
Most of the SPs used for achiral SFC separations are composed of low-molecular-weight selectors covalently bonded onto
a solid support, usually silica gel. Polymer type selectors would
be expected to interact with analytes by utilizing multiple and
cooperative mechanisms and in addition possess high durability.
However, only a very limited number of examples have been introduced that utilize polymer-based ligands for achiral SFC separation
[36].
Based on the experience of our research team in the polymeric
field, we recently developed a novel poly(butylene terephthalate)
based column, which exhibited unique molecular recognition ability together with high robustness in cycle durability tests [38]. We
considered that these features may be attributed to the associated macromolecular effect and as a result we decided to develop
various polymer-based SPs and to evaluate their performance.
For the design of the novel polymer stationary phase series, we
attempted to prepare several polymers based on the ethylpyridine moiety, mainly as the commercial phases containing such
synthon are considered as benchmarks for many researchers. In

/>0021-9673/© 2018 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license ( />

120

K. Nagai et al. / J. Chromatogr. A 1572 (2018) 119–127

2.2. Instrumentation and chromatographic conditions

Fig. 1. Structure of poly(4-vinylpyridine) or P4VP-based selector of the column
DCpak P4VP.

this perspective, moving from a monomeric to a polymeric selector, we were expecting that the molecular recognition ability may
be improved by multiple concerted interactions between the more
abundant polymeric pyridyl ligand interactions with the analyte
sample. Similar to the recently reported poly(butylene terephthalate) selector, these polymer type selectors were anticipated to
display a high durability as the polymer layer on silica gel should
effectively shield any undesirable chemical interaction.
The HPLC separation behavior of pyridine containing polymer SP
has been studied by Ihara [39–41]. This SP showed good selectivity
particularly for planar and disk-like aromatic molecules in reverse
and normal phase HPLC modes. The investigations of the polymeric
phase in HPLC mode was the objective of this work.
In an earlier study, we focused on novel vinylpyridine polymers
and related vinyl heteroaromatic polymers, and evaluated their
performance in SFC mode. We tested various poly(vinylpyridine
isomers), including poly(2-vinylpyridine), poly(3-vinylpyridine),
and poly(4-vinylpyridine), together with poly(vinylimidazole)
[42,43]. These SPs afforded distinctive molecular recognition abilities, particularly for structurally-similar isomeric samples. Among
them, poly(4-vinylpyridine) (P4VP) SP was found to provide better molecular shape recognition performance (Fig. 1). The present
article focuses on this P4VP column and describes its separation
behavior by using various samples. Based on these results, its characteristics and suitable chromatographic conditions are discussed.

2. Materials and methods
2.1. Chemicals
The modifier used in this study was Japanese Industrial Standard
special grade methanol (MeOH) obtained from Nacalai Tesque Inc.
(Kyoto, Japan). Carbon dioxide of industrial grade (over 99.5%) was

purchased from Tatsumi Industry Co., Ltd. (Hyogo, Japan). Ammonium formate was obtained from Wako Pure Chemical Industries
(Osaka, Japan).
o-Terphenyl, triphenylene, anthracene, phenanthrene, pyrene,
chrysene, perylene, theobromine, trans-cinnamic acid, 3phenylphenol, adenine, and diethylamine were purchased
from Tokyo Chemical Industry Co. (Tokyo, Japan). 1,3,5-Tritert-butylbenzene,
2-acetylanthracene,
9-acetylanthracene,
3-acetylphenanthrene, 9-acetylphenanthrene, paraxanthine, fenoprophen, ketoprofen, naproxen, alprenolol, propranolol, atenolol,
pindolol, and cyanocobalamin were purchased from Sigma-Aldrich
Corporation (St. Louis, MO, USA). Trifluoroacetic acid (TFA), naphthacene and dexamethasone were purchased from Nacalai Tesque
Inc. (Kyoto, Japan). 2-Propanol (IPA), prednisone, estrone, prednisolone, estradiol, estriol, caffeine, theophylline, nicotinamide,
and pyridoxine were purchased from Wako Pure Chemical Industries (Osaka, Japan). N-hexane (nHex) was purchased from Kanto
Chemical Co. (Tokyo, Japan)

DCpak P4VP column (initially launched as DCpak SFC-B),
sized 150 mm × 4.6 mm (i.d.), was supplied by DAICEL Corporation (Tokyo, Japan). This selector is composed of immobilized
P4VP on 5 ␮m silica particle (N.B. also available on 3 ␮m).
A Silica 2-ethylpyridine (2-EP) column of 5 ␮m particle, sized
150 mm × 4.6 mm (i.d.), was purchased from Waters Corporation
(Milford, MA, USA). The SFC instrument used in this study is NexeraUC supplied by Shimadzu Corporation (Kyoto, Japan) equipped
with a CO2 pump, a modifier pump, a vacuum degasser, a column
oven, a multiple wavelength UV detector, and automated back pressure regulator (ABPR). Lab Solutions software (V 5.89) was used for
system control and data acquisition. Chromatographic conditions,
such as modifier, column temperature, ABPR pressure, total flow
rate, detection wavelength, sample concentration, and injection
volume were described in each figure, respectively.
2.3. Data analysis
Relative retention factor (k) and separation factor (˛) were calculated with the equations below.
k= (V/V 0 )–1,
˛=k2 /k1

(i)
(ii)

where V is the elution volume of an analyte and V0 is the
column void volume. V0 was estimated by injecting 1,3,5-tri-tertbutylbenzene as a non-retained marker. k1 and k2 in Eq. (ii) are the
retention factors of the first and second eluted peaks, respectively.
3. Results and discussion
3.1. Planarity recognition of aromatics
Considering the structure features of the poly(4-vinylpyridine)
SP, it was expected to interact with planar aromatic samples as a
result of the multiple aromatic pyridyl units covalently attached
on silica gel. Non-planar o-terphenyl (1) and planar triphenylene
(2) with the same number of aromatic rings and ␲-electrons will
provide detailed perception of the planarity recognition ability,
because they have been considered as indicator for molecular planarity recognition in HPLC [44,45] and SFC [46]. Fig. 2A shows the
SFC chromatogram of 1 and 2 by using P4VP, when its performance
was compared with commercially available 2-EP SP under isocratic
conditions. The retention time of non-planar 1 was almost identical
for the new selector and 2-EP, while that of planar sample 2 significantly increased for P4VP. The separation factor for P4VP selector
between 1 and 2 (˛: k2 /k1 ) reached 30.6, whereas that obtained
by 2-EP is 4.4. This result indicates that ␲-electron rich planar
2 could strongly interact with vinylpyridine polymer selector via
␲–␲ interaction.
Taking advantage of this high planarity recognition ability, commercially available polycyclic aromatic hydrocarbons (PAHs) were
analyzed. Fig. 3 shows the SFC chromatogram of eight PAHs (2–9)
under gradient condition. Eight peaks were well separated on the
P4VP column. Of particular note is that anthracene (4) and phenanthrene (5) have the same molecular weight and similar molecular
size and polarity. Therefore, these two compounds cannot be distinguished by MS detector, which mean that the only method to
separate 4 and 5 must be by column separation. This new selector achieved a baseline resolution for 4 and 5. On the other hand,

when these compounds were analyzed by 2-EP SP under the same
condition, 4 and 5 co-eluted. The slight adjustment of gradient
conditions was necessary to separate 4 and 5 in isocratic mode,
immediately after eluting 4 and 5, a linear gradient program started.


K. Nagai et al. / J. Chromatogr. A 1572 (2018) 119–127

121

Fig. 2. SFC chromatograms of o-terphenyl and triphenylene on (A) DCpak P4VP and
(B) 2-EP SPs. Modifier, MeOH (isocratic condition, 3%); temperature, 40 ◦ C; ABPR,
15 MPa; total flow rate, 3 mL/min; UV detection, 254 nm; sample concentration,
0.3 mg/mL in nHex/IPA = 9/1; injection volume, 1 ␮L.

The longer aspect ratio of the PAH analyzed resulted in a shorter
retention time. Wise et al. proposed length-to-breadth (L/B) ratio
for describing two dimensional aspect ratio of PAH, and the smaller
L/B ratio indicates the disk-like molecule [47]. Indeed, L/B ratio of
naphthacene (7), chrysene (8), and triphenylene (2) which have the
same number of aromatic rings and ␲-electrons is 1.89, 1.72, and
1.12, respectively. The elution order of 7, 8, and 2 is 7 < 8 < 2, which
is the inverse of L/B ratio. These tendencies are almost identical for
similar stationary phase used in HPLC mode [41].
Similar to the separation of non-substituted PAH separation,
regioselective acetylated anthracene (10, 12) and phenanthrene
(11, 13) were also examined under isocratic conditions. Although
these samples have almost the same molecular size and polarity,
the P4VP selector can recognize the slight structural differences and
the four peaks were well resolved as shown in Fig. 4.

From these results, the new column shows excellent planarity
recognition and molecular shape recognition of various aromatic
isomers and PAHs. Combining P4VP column with supercritical fluid
extraction (SFE) and subsequent SFC technique would enable us to
analyze the residual PAH in soil and atmosphere etc [21].
3.2. Separation of samples with different structural features
Inspired by the interesting planarity and molecular shape recognition, other type of planar sample families e.g. steroids mixtures
(14–19) were analyzed under isocratic conditions. Fig. 5 shows the
SFC chromatogram of six structurally related steroids. In particular, prednisone (14), prednisolone (16), and dexamethasone (18)

Fig. 3. SFC chromatogram of eight polycyclic aromatic hydrocarbons on (A) DCpak
P4VP and (B) 2-EP SPs. Inset shows magnified chromatogram. Modifier, MeOH (gradient condition); temperature, 40 ◦ C; ABPR, 15 MPa; total flow rate, 3 mL/min; UV
detection, 254 nm; sample concentration, 0.1 mg/mL (except 9), 0.3 mg/mL (9) in
nHex/IPA = 9/1; injection volume, 1 ␮L. The gradient started with 3% of MeOH, after
3 min hold at 3% of MeOH, linear gradient ramped up to 38% of MeOH over 14 min,
followed by 1 min hold at 38% of MeOH, then returned to 3% of MeOH over 2 min,
followed by 1 min hold at 3% of MeOH.

have almost same skeleton with some slight differences in the
substituents, and therefore they are difficult to separate. When
investigating these samples by using 2-EP SP under the same conditions, they were co-eluting. We note that the conditions applied
were only to compare the selectivity in exactly identical conditions.
A better separation might be possible for 2-EP by using different
gradient condition. The direct separation of such steroid mixtures in
SFC mode would have significance in steroid profiling, as potential
biomarkers[48] or also in anti-doping control.
The analysis of caffeine (20) and its demethylated derivatives,
theophylline (21), theobromine (22) and paraxanthine (23) was
also investigated. Fig. 6 shows the SFC chromatogram of the mixture. Good peak resolution with symmetrical peaks was observed
for these polar analytes as well as for less-polar PAH derivatives and

steroids. The longer retention of 21–23 than 20 may be attributed
to the hydrogen bonding interactions between the demethylated
proton of the analytes and the proton acceptor behavior of P4VP
selector.


122

K. Nagai et al. / J. Chromatogr. A 1572 (2018) 119–127

Fig. 4. SFC chromatogram of acetylated anthracene and phenanthrene on DCpak
P4VP SP. Modifier, MeOH (isocratic condition, 3%); temperature, 40 ◦ C; ABPR,
15 MPa; total flow rate, 3 mL/min; UV detection, 254 nm; sample concentration,
0.15 mg/mL in nHex/IPA = 9/1; injection volume, 1 ␮L.

This novel SP contains the basic poly(4-vinylpyridine) moiety
and one may guess that acidic samples would be strongly retained
and/or would show tailing peaks on it. In order to confirm this
point, we tested propionic acid nonsteroidal anti-inflammatory
drugs (NSAIDs), fenoprofen (24), ketoprofen (25), and naproxen
(26). Fig. 7 shows the SFC chromatogram of three NSAIDs under isocratic conditions without any additives, which gave three resolved
peaks. Surprisingly, their peak shapes were relatively symmetric,
with peak symmetry factors (Ps) for 24 of1.11, 1.15 for 25, and
1.19 for 26. These results indicate that the pyridine polymer ligand must efficiently shield the undesirable interactions between
analytes and residual silanol groups on SP.

Fig. 5. SFC chromatogram of six steroids on (A) DCpak P4VP and (B) 2-EP SPs.
Modifier, MeOH (isocratic condition, 30%); temperature, 40 ◦ C; ABPR, 15 MPa; total
flow rate, 3 mL/min; UV detection, 225 nm; sample concentration, 0.33 mg/mL in
nHex/IPA = 1/1; injection volume, 2 ␮L.

3.3. Effect of additives
In the SFC field, it is common to use additives as a third component in the mobile phase, such as acids, bases and salts. They
improve the peak shapes and/or increase the solubility in the
mobile phase especially for polar analytes [5,31,49]. Basic additives
are often used for basic samples; acidic additives for acids, but other
combinations are also possible. The current trend for both acidic
and basic sample analysis is to use volatile salts, such as ammonium formate and ammonium acetate. These additives are often
used when separating APIs in SFC in analytical as well as preparative
applications, because many APIs bear polar and/or ionizable groups
which can easily interact with the residual silanol groups, and often
result in deficient peak shapes (leading, tailing, and asymmetric
peaks) [31]. However, if a SP could afford good peak symmetry
without any additive, it would be considered advantageous. The
absence of additives would be a positive feature for preparative
applications (no salts to be recuperated together with the product), but also for analytical UV detection (the high UV absorption of

Fig. 6. SFC chromatogram of caffeine, theophylline, theobromine, and paraxanthine
on DCpak P4VP SP. Modifier, MeOH (isocratic condition, 5%); temperature, 40 ◦ C;
ABPR, 15 MPa; total flow rate, 3 mL/min; UV detection, 225 nm; sample concentration, 0.2 mg/mL in MeOH/IPA = 1/1; injection volume, 2 ␮L.


K. Nagai et al. / J. Chromatogr. A 1572 (2018) 119–127

123

any additive. In general, for such basic samples, their peaks were
broadened without any additives. However, when an appropriate
additive (e.g. 20 mM of ammonium formate) was used, the peak
shapes were substantially improved (Fig. 8B).

As expected, the P4VP column can produce relatively symmetrical peaks even in the absence of any additive (Fig. 8C). As for 2-EP,
after the addition of salts, peaks were further sharpened (Fig. 8D).
The fact that the P4VP column could gain sharp peaks without
any additives for these basic APIs may be attributed to the polymeric ligand effect. The covalently bonded P4VP chains may spread
on the porous silica gel surface and will contribute to reduce the
undesirable interactions between the residual silanol groups and
the basic analytes.

3.4. Effect of conditioning with different additive
Fig. 7. SFC chromatogram of nonsteroidal anti-inflammatory drugs on DCpak P4VP
SP. Modifier, MeOH (isocratic condition, 10%); temperature, 40 ◦ C; ABPR, 15 MPa;
total flow rate, 3 mL/min; UV detection, 210 nm; sample concentration, 0.2 mg/mL
in IPA; injection volume, 2 ␮L.

ammonium formate or ammonium acetate especially in gradient
conditions sometimes leads to unstable baselines).
Accordingly, we analyzed four ␤-adrenergic blocking agents
(␤-blockers) (27–30) in the presence and absence of ammonium
formate, to compare the chromatographic performance of the two
SPs. Fig. 8A shows the SFC chromatogram of four ␤-blockers under
gradient conditions obtained by using the 2-EP column without

As the new selector consists of basic poly(4-vinylpyridine) moieties, it can be envisaged to form a cationic pyridinium form by
reaction with strong acids such as trifluoroacetic acid (TFA) and
be converted to a quaternized amphiphilic salt form by reaction
with the corresponding alkyl halide. Recently, Ihara and Takafuji
reported amphiphilic poly(N-alkylpyridinium salt) based HPLC SPs
through quaternization reactions. Their separation mode can be
easily tuned by changing the N-alkyl side chain length [50,51].
The protonated pyridinium salt effect was investigated for

the P4VP phase by passing through TFA-containing modifier. We
selected neutral (2, 13), acidic (31, 32) and basic samples (21, 34)
for this test under isocratic conditions.

Fig. 8. SFC chromatograms of ␤-blockers (A, C) in the absence or (B, D) presence of ammonium formate on (A, B) 2-EP and (C, D) DCpak P4VP SPs. Modifier, MeOH (gradient
condition); temperature, 40 ◦ C; ABPR, 15 MPa; total flow rate, 3 mL/min; UV detection, (A, C) 220 or (B, D) 280 nm; sample concentration, 0.1 mg/mL in MeOH; injection
volume, 2 ␮L. The gradient started with 10% of modifier, after 1 min hold at 10% of modifier, linear gradient ramped up to 35% of modifier over 10 min, followed by 2 min
hold at 35% of modifier, then returned to 10% of modifier over 2 min, followed by 1 min hold at 10% of modifier.


124

K. Nagai et al. / J. Chromatogr. A 1572 (2018) 119–127

Fig. 9. (A–E) SFC chromatograms and (F) retention factor dependent of neutral, acidic, and basic samples on DCpak P4VP SP by using various modifier under isocratic
condition (10%). Before each analysis, the testing modifier was eluted for more than 30 min. for equilibration. Each modifier was, (A) MeOH, (B) MeOH/TFA = 100/1, (C) MeOH,
(D) MeOH/DEA = 100/1, and (E) MeOH. For detail, please see text. Temperature, 40 ◦ C; ABPR, 15 MPa; total flow rate, 3 mL/min; UV detection, 254 nm; sample concentration,
0.13 mg/mL in nHex/IPA = 1/1; injection volume, 2 ␮L.

Fig. 9A shows the SFC chromatogram of the six sample mixture with MeOH as modifier. These samples were eluted as
relatively symmetric peaks. After passing a TFA-containing modifier (MeOH/TFA = 100/1, (v/v)) for more than 30 min., the same
sample mixture was injected. Fig. 9B shows the corresponding
chromatogram and Fig. 9F the retention factor (k) dependence on
modifier composition. Their elution order dramatically changed.
Acidic samples (31, 32) eluted faster than in the initial analysis,
whereas basic theophylline (21) hardly changed its retention time
and more basic adenine (34) eluted significantly later. Surprisingly,
neutral samples (2, 13) also eluted faster than in the original analysis. However, by using MeOH again as a modifier during 30 min.,
retention time of all analytes were likely to recover the original profile (Fig. 9C). We then used a diethylamine (DEA)-containing MeOH
(MeOH/DEA = 100/1, (v/v)) as a modifier, and the same experiment was conducted. However, the elution order hardly changed

(Fig. 9D). After passing MeOH as a modifier again, the elution almost
reverted to that of the first injection (Fig. 9E).
Based on these results, we proposed the following retention
mechanism. As discussed in the previous section, the pyridyl groups
on the polymer side chains effectively mask the silanol groups on
silica gel surface, which lead to a shielding of undesirable interactions between silanols and analytes. When only MeOH was used as
modifier, the acidic samples (e.g. 31), can interact with the SP via

acid-base interaction, while such an interaction between SP and
basic analytes (e.g. 34) should not be expected (Fig. 10A). For this
reason, the elution of 34 might be faster than the one of 31. When
a TFA-containing MeOH was used as a modifier, the pyridyl groups
on the side chain are protonated (Fig. 10B). Contrary to Fig. 10A, the
protonated SP and 34 can interact. We note that the pyridyl groups
in the SP are more prone to protonate than 34 because pyridine is
more basic than 34 based on the pKa values of the corresponding
conjugate acids [52,53]. Hence, the elution of basic 34 was slower
than acidic 31. The reason why the retention time of neutral samples decreased under acidic conditions is still unclear. We envisage
that the electron density of the pyridyl ring in the SP might be
decreased by protonation, or MeOH may solvate the protonated
side chains, which will interfere with the SP and neutral analyte
interaction. In such a case, it may affect the retention behavior of
neutral samples in acidic conditions. After gradually passing MeOH
as modifier again, the protonated SP side chains gradually deprotonated to be in their original state. Therefore, the initial retention
behavior gradually recovered. DEA seems to have less effect on the
retention of these samples.
As demonstrated here, the elution order of this selector can be
reversely tuned through acid mediated pyridinium salt formation.

K. Nagai et al. / J. Chromatogr. A 1572 (2018) 119–127

125

Fig. 10. Postulated retention mechanism of DCpak P4VP in neutral conditions (A) and acidic conditions (B).

Fig. 11. Cycle dependent SFC chromatograms of water soluble vitamins on (A) 2-EP and (B) DCpak P4VP SPs. Modifier; MeOH (isocratic condition, 25%); temperature, 40 ◦ C;
ABPR, 15 MPa; total flow rate, 3 mL/min; UV detection, 230 nm; sample concentration, 0.2 mg/mL in MeOH; injection volume,2 ␮L.

3.5. Cycle durability tests
As mentioned in the introduction, polymer-type selectors were
expected to show good durability as we estimate that undesirable attack may be interfered with the polymer ligand layer on
the SP surface. Recently, we confirmed the column robustness of
poly(butylene terephthalate) selector by a range of cycle durability
testing [38].
In order to investigate the durability of a new P4VP column,
three water soluble vitamins (WSVs), nicotinamide (vitamin B3 ),

pyridoxine (vitamin B6 ), and cyanocobalamin (vitamin B12 ) were
selected.
Fig. 11A shows the cycle-dependent SFC chromatograms of
three WSVs by using 2-EP column under isocratic conditions. For
the first injection, three peaks were well separated and a characteristic long retention was observed for vitamin B12 . However, as the
cycle passed, the retention time gradually decreased for vitamin B3
and vitamin B6 , and sharply decreased for vitamin B12 . Fig. 11A also
shows the chromatograms after 20, 41, 60, and 80 cycles. The retention time continuously decreased and that of vitamin B12 reduced
to less than half of the original time. Fairchild et al. proposed that


126

K. Nagai et al. / J. Chromatogr. A 1572 (2018) 119–127

Fig. 12. Cycle versus retention factor (k) of (A) vitamin B3 , (B) vitamin B6 , and (C) vitamin B12 by using DCpak P4VP and 2-EP columns. Experimental condition is same as
Fig. 11.

silyl ether formation by a condensation reaction between silanols
and MeOH used as a modifier is a major contribution to retention
variation over time in SFC mode [54].
We investigated the same cycle test for the P4VP case (Fig. 11B)
In contrast to 2-EP case, the retention time of these samples did
not change after 20, 40, 60, and 80 cycles. Fig. 12 shows cycle
versus their retention factors (k). We note that the systematic cycle
investigation here reported was run over 80 sequential cycles in
brand-new column, however the P4VP columns used in this study
to generate all experimental data reported were periodically tested
with the standard samples and confirmed the durability and the
stability over several months.
4. Conclusion
A novel P4VP based column was designed and its performance
was evaluated under SFC conditions. This SP showed unique molecular shape recognition for planar molecules such as structurally
related polycyclic aromatic hydrocarbons and steroid mixtures.
The new SP afforded symmetric peaks for active pharmaceutical
ingredient analysis even in the absence of any additives, e.g. acids,
bases, or salts, probably due to the effective shield of residual
silanols by the polymeric pyridine selector. The surface chemical
properties of the new column can be easily converted to cationic
pyridinium form by eluting TFA containing modifiers, which dramatically change elution order of acidic, basic, and even neutral
analytes. This significant elution order change can be recovered to
the original state by passing through DEA containing modifier.

Additionally, the column performance did not change as a result
of cycle durability testing of water soluble vitamins.
The present study together with that of another our polymeric
SP [38,43,55] revealed that our synthetic polymer based selector
would be a versatile tool in SFC analysis. Furthermore, it can be
extended in use into preparative fields. Further investigations of
other sample application in different chromatographic modes and
deep insight of this SP is now in progress.
Acknowledgments
The authors wish to thank Dr. Pilar Franco and Tong Zhang in
Chiral Technologies Europe S. A. S. for valuable discussions. The

authors also appreciate Dr. Joseph M. Barendt and Ms Lorraine
Evangelista in Chiral Technologies, Inc. and Dr. Brian Freer in Chiral
Technologies Europe S. A. S for English grammatical correction.
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.

References
[1] C.F. Poole (Ed.), Supercritical Fluid Chromatography (Handbooks in Separation
Science), Elsevier, 2017.
[2] T.A. Berger, Separation of polar solutes by packed column supercritical fluid
chromatography, J. Chromatogr. A 785 (1997) 3–33, />1016/S0021-9673(97)00849-2.
[3] L.T. Taylor, Supercritical fluid chromatography for the 21 st century, J.
Supercrit. Fluids 47 (2009) 566–573, />09.012.
[4] G. Guiochon, A. Tarafder, Fundamental challenges and opportunities for
preparative supercritical fluid chromatography, J. Chromatogr. A 1218 (2011)
1037–1114, />[5] E. Lesellier, C. West, The many faces of packed column supercritical fluid
chromatography - A critical review, J. Chromatogr. A 1382 (2015) 2–13846,
/>[6] V. Desfontaine, D. Guillarme, E. Francotte, L. Nováková, Supercritical fluid

chromatography in pharmaceutical analysis, J. Pharm. Biomed. Anal. 113
(2015) 56–71, />[7] S. Fekete, J.L. Veuthey, D. Guillarme, Comparison of the most recent
chromatographic approaches applied for fast and high resolution separations:
theory and practice, J. Chromatogr. A 1408 (2015) 1–14, />1016/j.chroma.2015.07.014.
[8] A. Tarafder, Metamorphosis of supercritical fluid chromatography to SFC: an
Overview, TrAC Trends Anal. Chem. 81 (2016) 3–10, />1016/j.trac.2016.01.002.
[9] G. Terfloth, Enantioseparations in super- and subcritical fluid
chromatography, J. Chromatogr. A 906 (2001) 301–307, />1016/S0021-9673(00)00952-3.
[10] G.B. Cox, Enantioselective supercritical fluid chromatography using Daicel’s
“platinum series” polysaccharide-based columns, LC-GC North-Am. Appl.
Noteb. (2007) 31.
[11] L. Miller, Preparative enantioseparations using supercritical fluid
chromatography, J. Chromatogr. A 1250 (2012) 250–255, />1016/j.chroma.2012.05.025.
[12] P. Franco, T. Zhang, Common screening approaches for efficient analytical
method development in LC and SFC on columns packed with immobilized
polysaccharide-derived chiral stationary phases, in: G.K.E. Scriba (Ed.), Chiral
Sep. Methods Protoc., 2nd ed., Humana Press, Totowa, NJ, 2013, pp. 113–126,
/>[13] J. Lee, J.T. Lee, W.L. Watts, J. Barendt, T.Q. Yan, Y. Huang, F. Riley, M. Hardink, J.
Bradow, P. Franco, On the method development of immobilized
polysaccharide chiral stationary phases in supercritical fluid chromatography
using an extended range of modifiers, J. Chromatogr. A 1374 (2014) 238–246,
/>

K. Nagai et al. / J. Chromatogr. A 1572 (2018) 119–127
[14] S. Khater, M.A. Lozac’h, I. Adam, E. Francotte, C. West, Comparison of liquid
and supercritical fluid chromatography mobile phases for enantioselective
separations on polysaccharide stationary phases, J. Chromatogr. A 1467
(2016) 463–472, />[15] K. Zawatzky, M. Biba, E.L. Regalado, C.J. Welch, MISER chiral supercritical fluid
chromatography for high throughput analysis of enantiopurity, J. Chromatogr.
A 1429 (2016) 374–379, />[16] D. Thiébaut, Separations of petroleum products involving supercritical fluid

chromatography, J. Chromatogr. A 1252 (2012) 177–188, />1016/j.chroma.2012.06.074.
[17] L.T. Taylor, Packed column supercritical fluid chromatography of hydrophilic
analytes via water-rich modifiers, J. Chromatogr. A 1250 (2012) 196–204,
/>˛
[18] K. Ty´skiewicz, A. Debczak,
R. Gieysztor, T. Szymczak, E. Rój, Determination of
fat- and water-soluble vitamins by supercritical fluid chromatography: a
review, J. Sep. Sci. 41 (2018) 336–350, />201700598.
[19] G.F. Pirrone, R.M. Mathew, A.A. Makarov, F. Bernardoni, A. Klapars, R.
Hartman, J. Limanto, E.L. Regalado, Supercritical fluid
chromatography-photodiode array detection-electrospray ionization mass
spectrometry as a framework for impurity fate mapping in the development
and manufacture of drug substances, J. Chromatogr. B 1080 (2018) 42–49,
/>[20] C. West, E. Lemasson, S. Bertin, P. Hennig, E. Lesellier, Interest of
achiral-achiral tandem columns for impurity profiling of synthetic drugs with
supercritical fluid chromatography, J. Chromatogr. A 1534 (2018) 161–169,
/>[21] A.P. Wicker, D.D. Carlton, K. Tanaka, M. Nishimura, V. Chen, T. Ogura, W.
Hedgepeth, K.A. Schug, On-line supercritical fluid extraction—supercritical
fluid chromatography-mass spectrometry of polycyclic aromatic
hydrocarbons in soil, J. Chromatogr. B 1086 (2018) 82–88, />10.1016/j.jchromb.2018.04.014.
[22] O. Petkovic, P. Guibal, P. Sassiat, J. Vial, D. Thiébaut, Active modulation in neat
carbon dioxide packed column comprehensive two-dimensional supercritical
fluid chromatography, J. Chromatogr. A 1536 (2018) 176–184, .
org/10.1016/j.chroma.2017.08.063.
[23] J.L. Bernal, M.T. Martín, L. Toribio, Supercritical fluid chromatography in food
analysis, J. Chromatogr. A 1313 (2013) 24–36, />chroma.2013.07.022.
[24] K. Taguchi, E. Fukusaki, T. Bamba, Simultaneous analysis for water- and
fat-soluble vitamins by a novel single chromatography technique unifying
supercritical fluid chromatography and liquid chromatography, J. Chromatogr.
A 1362 (2014) 270–277, />[25] T. Yamada, Y. Nagasawa, K. Taguchi, E. Fukusaki, T. Bamba, 13 - polar lipid

profiling by supercritical fluid Chromatography/Mass spectrometry method,
in: M.U. Ahmad, X. Xu (Eds.), Polar Lipids, Elsevier, 2015, pp. 439–462, http://
dx.doi.org/10.1016/B978-1-63067-044-3.50017-0.
[26] E.L. Regalado, C.J. Welch, Separation of achiral analytes using supercritical
fluid chromatography with chiral stationary phases, TrAC Trends Anal. Chem.
67 (2015) 74–81, />[27] C. West, E. Lemasson, S. Bertin, P. Hennig, E. Lesellier, An improved
classification of stationary phases for ultra-high performance supercritical
fluid chromatography, J. Chromatogr. A 1440 (2016) 212–228, .
org/10.1016/j.chroma.2016.02.052.
[28] D.C. Patel, M.F. Wahab, D.W. Armstrong, Z.S. Breitbach, Advances in
high-throughput and high-efficiency chiral liquid chromatographic
separations, J. Chromatogr. A 1467 (2016) 2–18, />chroma.2016.07.040.
[29] A. Grand-Guillaume Perrenoud, D. Guillarme, J. Boccard, J.L. Veuthey, D.
Barron, S. Moco, Ultra-high performance supercritical fluid chromatography
coupled with quadrupole-time-of-flight mass spectrometry as a performing
tool for bioactive analysis, J. Chromatogr. A 1450 (2016) 101–111, http://dx.
doi.org/10.1016/j.chroma.2016.04.053.
[30] H. Segawa, Y.T. Iwata, T. Yamamuro, K. Kuwayama, K. Tsujikawa, T. Kanamori,
H. Inoue, Differentiation of ring-substituted regioisomers of amphetamine
and methamphetamine by supercritical fluid chromatography, Drug Test.
Anal. 9 (2017) 389–398, />[31] E. Lemasson, S. Bertin, C. West, Use and practice of achiral and chiral
supercritical fluid chromatography in pharmaceutical analysis and
purification, J. Sep. Sci. 39 (2016) 212–233, />201501062.
[32] A. Grand-Guillaume Perrenoud, J. Boccard, J.L. Veuthey, D. Guillarme, Analysis
of basic compounds by supercritical fluid chromatography: Attempts to
improve peak shape and maintain mass spectrometry compatibility, J.
Chromatogr. A 1262 (2012) 205–213, />2012.08.091.
[33] F.M. Chou, W.T. Wang, G.T. Wei, Using subcritical/supercritical fluid
chromatography to separate acidic, basic, and neutral compounds over an
ionic liquid-functionalized stationary phase, J. Chromatogr. A 1216 (2009)

3594–3599, />[34] J. Smuts, E. Wanigasekara, D.W. Armstrong, Comparison of stationary phases
for packed column supercritical fluid chromatography based upon ionic liquid
motifs: A study of cation and anion effects, Anal. Bioanal. Chem. 400 (2011)
435–447, />[35] R. McClain, M.H. Hyun, Y. Li, C.J. Welch, Design, synthesis and evaluation of
stationary phases for improved achiral supercritical fluid chromatography

[36]

[37]

[38]

[39]

[40]

[41]

[42]
[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]
[54]

[55]

127

separations, J. Chromatogr. A 1302 (2013) 163–173, />1016/j.chroma.2013.06.038.
C.G.A. da Silva, C.H. Collins, E. Lesellier, C. West, Characterization of stationary
phases based on polysiloxanes thermally immobilized onto silica and
metalized silica using supercritical fluid chromatography with the solvation
parameter model, J. Chromatogr. A 1315 (2013) 176–187, />10.1016/j.chroma.2013.09.055.
M. Dunkle, C. West, A. Pereira, S. Van Der Plas, E. Lesellier, Synthesis of
stationary phases containing pyridine, phenol, aniline and morpholine via
click chemistry and their characterization and evaluation in supercritical fluid
chromatography, Sci. Chromatogr. 6 (2014) 85–103, />4322/sc.2014.023.
K. Nagai, T. Shibata, S. Shinkura, A. Ohnishi, Poly(butylene terephthalate)
based novel achiral stationary phase investigated under supercritical fluid
chromatography conditions, J. Chromatogr. A 1549 (2018) 85–92, http://dx.
doi.org/10.1016/j.chroma.2018.03.032.
H. Ihara, W. Dong, T. Mimaki, M. Nishihara, T. Sakurai, M. Takafuji, S. Nagaoka,

Poly(4-Vinylpyridine) as Novel Organic Phase for RP-HPLC. Unique Selectivity
for Polycyclic Aromatic Hydrocarbons, J. Liq. Chromatogr. Relat. Technol. 26
(2003) 2491–2503, />H. Ihara, M. Fukui, T. Mimaki, A. Shundo, W. Dong, M. Derakhshan, T. Sakurai,
M. Takafuji, S. Nagaoka, Poly(4-vinylpyridine) as a reagent with
silanol-masking effect for silica and its specific selectivity for PAHs and
dinitropyrenes in a reversed phase, Anal. Chim. Acta 548 (2005) 51–57, http://
dx.doi.org/10.1016/j.aca.2005.05.056.
U.G. Gautam, A. Shundo, M.P. Gautam, M. Takafuji, H. Ihara, High retentivity
and selectivity for polycyclic aromatic hydrocarbons with
poly(4-vinylpyridine)-grafted silica in normal-phase high-performance liquid
chromatography, J. Chromatogr. A 1189 (2008) 77–82, />1016/j.chroma.2007.12.018.
K. Nagai, S. Shinkura, Stationary Phase for Supercritical Fluid
Chromatography, WO2016/152996, 2016.
C. West, E. Lemasson, K. Nagai, T. Shibata, P. Franco, S. Bertin, P. Hennig, E.
Lesellier, Synthesis and characterization of novel polymer-based pyridine
stationary phases for supercritical fluid chromatography, Submitted. (n.d.).
K. Kimata, K. Iwaguchi, S. Onishi, K. Jinno, R. Eksteen, K. Hosoya, M. Araki, N.
Tanaka, Chromatographic characterization of silica C18 packing materials.
Correlation between a preparation method and retention behavior of
stationary phase, J. Chromatogr. Sci. 27 (1989) 721–728, />1093/chromsci/27.12.721.
M. Mifune, Y. Mori, M. Onoda, A. Iwado, N. Motohashi, J. Haginaka, Y. Saito,
Separation characteristics of aminopropyl silica gels modified with
copper-phthalocyanine as high performance liquid chromatography
stationary phase, Anal. Sci. 14 (1998) 1127–1131, />analsci.14.1127.
K. Jinno, H. Mae, Molecular planarity recognition of polycyclic aromatic
hydrocarbons in supercritical fluid chromatography, J. High Resolut.
Chromatogr. 13 (1990) 512–515, />S. Wise, W.J. Bonnett, F.R. Guenther, W.E. May, A relationship between
reversed-phase C18 liquid chromatographic retention and the shape of
polycyclic aromatic hydrocarbons, J. Chromatogr. Sci. 19 (1981) 457–465,
/>J. Teubel, B. Wüst, C.G. Schipke, O. Peters, M.K. Parr, Methods in endogenous

steroid profiling – A comparison of gas chromatography mass spectrometry
(GC–MS) with supercritical fluid chromatography tandem mass spectrometry
(SFC-MS/MS), J. Chromatogr. A 1554 (2018) 101–116, />1016/j.chroma.2018.04.035.
E. Lemasson, S. Bertin, P. Hennig, H. Boiteux, E. Lesellier, C. West, Development
of an achiral supercritical fluid chromatography method with ultraviolet
absorbance and mass spectrometric detection for impurity profiling of drug
candidates. Part I: optimization of mobile phase composition, J. Chromatogr.
A 1408 (2015) 217–226, />M. Shahruzzaman, M. Takafuji, H. Ihara, Porous silica particles grafted with an
amphiphilic side-chain polymer as a stationary phase in reversed-phase
high-performance liquid chromatography, J. Sep. Sci. 38 (2015) 2403–2413,
/>M. Shahruzzaman, M. Takafuji, H. Ihara, Tuning of separation mode using
pyridinium salt-branched ionic polymer-grafted silica as stationary phase in
HPLC, Chem. Lett. 45 (2016) 13–15, />H.C. Brown, D.H. McDANIEL, O. HÄFLIGER, Dissociation constants, in: E.A.
BRAUDE, F.C. NACHOD (Eds.), Determ. Org. Struct. by Phys. Methods,
Academic Press, New York, 1955, p. iii.
R.M.C. Dawson, D.C. Elliott, W.H. Elliott, K.M. Jones, Data for Biochemical
Research, Oxford University Press, New York, 1959.
J.N. Fairchild, D.W. Brousmiche, J.F. Hill, M.F. Morris, C.A. Boissel, K.D.
Wyndham, Chromatographic evidence of silyl ether formation (SEF) in
supercritical fluid chromatography, Anal. Chem. 87 (2015) 1735–1742, http://
dx.doi.org/10.1021/ac5035709.
M. Douˇsa, 1H-tetrazole-5-amine immobilized on substituted polymer
Gel/Silica as a new stationary phase for hydrophilic interaction
chromatography, Chromatographia 81 (2018) 349–357, />1007/s10337-017-3452-6.