Accepted Manuscript
Title: Highly efficient monolithic silica capillary columnsmodified with poly(acrylic acid) for hydrophilic interactionchromatography
Authors: Kanta Horie, Tohru Ikegami, Ken Hosoya, NabilSaad, Oliver Fiehn, Nobuo Tanaka
PII: S0021-9673(07)01211-3DOI: doi:10.1016/j.chroma.2007.07.012Reference: CHROMA 347873
To appear in: Journal of Chromatography A
Received date: 23-4-2007Revised date: 5-7-2007Accepted date: 9-7-2007
Please cite this article as: K. Horie, T. Ikegami, K. Hosoya, N. Saad, O. Fiehn, N.Tanaka, Highly efficient monolithic silica capillary columns modified with poly(acrylicacid) for hydrophilic interaction chromatography, Journal of Chromatography A (2007),doi:10.1016/j.chroma.2007.07.012
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Highly efficient monolithic silica capillary columns modified with poly(acrylic 1
acid) for hydrophilic interaction chromatography 2
3
Kanta Horie,1 Tohru Ikegami,1* Ken Hosoya,1** Nabil Saad,2 Oliver Fiehn,2 Nobuo 4
Tanaka1 5
6
7 1 Department of Biomolecular Engineering, Kyoto Institute of Technology, Matsugasaki, 8
Sakyo-ku, Kyoto 606-8585, Japan 9 2 University of California, Davis, Genome Center, Davis, CA 95616-8816, USA 10
11
12
*Corresponding author: 13
Tohru Ikegami 14
Department of Biomolecular Engineering, Kyoto Institute of Technology 15
Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan 16
E-mail: [email protected] 17
Phone 81-75-724-7801, FAX 81-75-724-7710 18
19
**Present address 20
Graduate School of Environmental Studies, Tohoku University, Aramaki, Aoba-ku, 21
Sendai, 980-8579, Japan 22
23
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Abstract 23
Monolithic silica capillary columns for hydrophilic interaction liquid 24
chromatography (HILIC) were prepared by on-column polymerization of acrylic acid on 25
monolithic silica in a fused silica capillary modified with anchor groups. The products 26
maintained the high permeability (K = 5×10–14 m2), and provided a plate height (H) of 27
less than 10 µm at optimum linear velocity (u) and H below 20 µm at u = 6 mm/s for 28
polar solutes including nucleosides and carbohydrates. The HILIC mode monolithic 29
silica capillary column was able to produce 10 000 theoretical plates (N) with column 30
dead time (t0) of 20 s at a pressure drop of 20 MPa or lower. The total performance 31
was much higher than conventional particle-packed HILIC columns currently available. 32
The gradient separations of peptides by a capillary LC-electrospray mass spectrometry 33
system resulted in very different retention selectivity between reversed-phase mode 34
separations and the HILIC mode separations with a peak capacity of ca. 100 in a 10 min 35
gradient time in either mode. The high performance observed with the monolithic 36
silica capillary column modified with poly(acrylic acid) suggest that the HILIC mode 37
can be an alternative to the reversed-phase mode for a wide range of compounds, 38
especially for those of high polarity in isocratic as well as gradient elution. 39
40
1. Introduction 41
Monolithic silica capillary columns have been attracting considerable attention as a 42
high-performance separation medium because of higher permeability at a similar 43
column efficiency and higher mechanical stability than particle-packed columns [1–4]. 44
But most of these on the market are reversed-phase materials represented by octadecyl 45
modification, C18 [3,4]. The development of the materials for other separation modes 46
was necessary for the separation of highly polar compounds that showed limited extent 47
of retention in the reversed-phase mode. For this purpose, HILIC (hydrophilic 48
interaction liquid chromatography) mode separation has been studied recently. This 49
separation mode utilizes a polar stationary phase and a less polar mobile phase, which is 50
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commonly a mixture of an organic solvent and water, thus it belongs to the 51
normal-phase mode [5–16]. This chromatographic separation mode has an advantage 52
associated with the combination of HPLC with electrospray ionization mass 53
spectrometry (LC-ESI-MS) for the separation and detection of highly polar compounds 54
[17–23]. The mobile phases in the HILIC mode most frequently employed, a mixture 55
of acetonitrile and aqueous buffer, are compatible with the ESI-MS system. In contrast, 56
the conventional normal-phase LC using mixtures of polar organic solvents as mobile 57
phases is not suitable for the LC–ESI-MS application because of the low ionization 58
efficiency and low solubility of polar compounds in the solvent system. 59
There have been some reports on monolithic silica columns modified by an 60
on-column radical polymerization of acrylamide [24] and acrylic acid [25]. This 61
method of modification for a monolithic silica column was found to be useful for the 62
preparation of a polar stationary phase because the reagents for the bonding reactions 63
are compatible with various solvents and polar functionalities. The products 64
maintained the high permeability and separation efficiency of monolithic columns even 65
after the modification using polymerization. Monolithic silica capillary columns 66
modified with poly(acrylic acid) (PAA) as a weak cation-exchange (WCX) mode 67
separation medium could be used for HILIC mode separation because of the high 68
polarity of carboxylic acid functionality. These monolithic silica capillary columns 69
were shown to be useful as HILIC and cation-exchange mode columns [25]. There 70
have been other reports on the monolithic silica columns modified by polar functional 71
groups such as amino- [26], tertiary amine and quaternary ammonium- [27], sulfonate- 72
[27, 28], and phosphate groups [29]. However, the column efficiency and the kinetic 73
performance of the HILIC columns have not been reported in detail, while the studies 74
on the retention selectivity and the suitable mobile phase systems were described. 75
It is of much interest to evaluate the kinetic performance of the HILIC-mode 76
columns to demonstrate whether it is possible to achieve column efficiencies as high as 77
with columns in the reversed-phase mode. Here we report the chromatographic 78
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performance of a poly(acrylic acid)-coated monolithic silica column in 79
acetonitrile-water mixtures for isocratic separations of nucleosides and carbohydrates, 80
and in a gradient elution of peptides. 81
82
2. Experimental 83
2.1. Materials 84
HPLC- or LC/MS-grade acetonitrile (Wako, Osaka, Japan) and formic acid (Wako) 85
were used. Water was purified with a Milli-Q A10 system (Millipore, Billerica, MA, 86
USA). For the preparation of the stationary phases, 3-aminopropyltriethoxysilane 87
(Chisso, Tokyo, Japan), methacryloyl chloride, acrylic acid, and ammonium persulfate 88
(Wako) were used without further purification. 89
N-(3-Triethoxysilylpropyl)methacrylamide was prepared by adding methacryloyl 90
chloride (15.6 ml, 161 mmol) in dry toluene (15 ml) dropwise under stirring to a 91
mixture of 3-aminopropyltriethoxysilane (25 ml, 107 mmol) and dry triethylamine (22.5 92
ml, 161 mmol) in dry toluene (50 ml) in 0.5 h at 0 °C. The mixture was filtered 93
through a PTFE membrane filter (0.45 µm) to remove a salt formed, and toluene was 94
distilled in vacuo to give the desired product as a brown viscous liquid (28.5 g, 98%) 95
96
2.2. Preparation of monolithic silica columns with PAA stationary phase 97
Monolithic silica columns were prepared from tetramethoxysilane (TMOS) by 98
recently reported methods in 200 µm I.D. fused silica capillaries with minor 99
modifications [30]. (Throughout this report, 200T stands for a 200 µm I.D. capillary 100
column prepared from TMOS.) Then these columns were modified with a spacer, 101
N-(3-triethoxysilylpropyl)methacrylamide [24, 25]. A 1: 1: 1 mixture of the silane, 102
pyridine, and toluene was passed through the column (30 cm) for 24 h at 80 °C with 103
nitrogen (1 MPa) followed by a wash with toluene, and methanol for 24 h each by using 104
a HPLC pump (3 MPa). This spacer-bonding step was repeated twice to obtain 105
monolithic silica capillary columns modified with the methacrylamide functionality. 106
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Then a monomer solution, acrylic acid (10, 30, 50 µl) and ammonium persulfate (5 mg) 107
in water (1.0 ml) was charged into the column, and allowed to react at 60 °C for 2 h. 108
After the reaction, the column was washed with water quickly, using a HPLC pump at 109
20 MPa for 3 h. The washing process was repeated twice to obtain poly(acrylic acid) 110
modified monolithic silica capillary columns (MS-200T-PAA columns). All 111
chromatographic evaluations and applications were performed by using a column 112
prepared from the feed mixture containing 30 µl acrylic acid, unless otherwise noted. 113
114
2.3. Instrument and chromatographic measurements 115
The HPLC system consisted of Shimadzu LC-20AD prominence pumps and 116
MU701 detector (GL Science, Tokyo, Japan) with a capillary flow cell (GL Science, 117
cell volume 18 nl). Samples were injected using a 50 nl internal-loop sample injector 118
(VICI, C4-0004-.05, Schenkon, Switzerland) for evaluating the effect of monomer 119
concentration and the separation of peptides. The other injections for evaluating 120
chromatographic performance were performed by Model 7725 injector (Rheodyne, Park 121
Court, CA, USA) with split-flow and split-injection mode [2]. Throughout these 122
experiments, capillary columns were evaluated at 25°C. EZChrom Elite-2.61 data 123
processor (GL Science) was used for processing the chromatographic data. 124
125
2.4. NanoESI-MS analysis 126
All analyses were performed on a quadropole time-of flight (Q-TOF) micro mass 127
spectrometer (Micromass, Beverly, MA, USA) equipped with a nanoelectrospray probe. 128
The ESI chip consists of PicoTip EMITTER, SilicaTip, FS360-50-30-D (New 129
Objectives, Woburn, MA, USA), directly connected to the monolithic column using a 130
metal union. The typical operating conditions for the Q-TOF micro mass spectrometer 131
as follows: spray voltage, 3 kV; sample cone voltage, 40 V; source temperature, 20 °C. 132
The collision gas pressure was activated at 10 V. The Q-TOF instrument was operated 133
in the TOF-MS positive ion mode with the fullscan mass spectra. Data for each 134
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sample were acquired in the mass range between m/z 150 and 2000. Each spectrum 135
was integrated with a scan duration of 0.52 s and Interscan delay of 0.10 s. All data 136
were processed using MassLynx software version 4.0 from Waters. 137
138
2.5. Samples 139
Nucleosides (uridine, adenosine, guanosine) and sodium dodecylsulfate (Wako) 140
were dissolved in mobile phase at 10 µg/ml to obtain the nucleoside samples. 141
Carbohydrate samples (sucrose, trehalose, and raffinose) were purchased from Nacalai 142
Tesque (Kyoto, Japan), while 2-pyridylamino (PA-) derivatives of carbohydrates 143
(PA-arabinose, PA-glucose, PA-maltose, PA-maltotriose, PA-maltotetraose, 144
PA-maltopentaose, PA-maltohexaose, PA-maltoheptaose) were prepared as previously 145
described [24], and dissolved in water at 1 mg/ml. Peptides samples from 146
Sigma-Aldrich (St. Louis, MO, USA) included γ-Glu-His (γ-EH), 147
Asp-Ser-Asp-Pro-Arg (DSDPR), Val-Gly-Ser-Glu (VGSE), and the peptide standard 148
sample P2693 consisting of bradykinin fragment1-5, [Arg8]-vasopressin, bradykinin, 149
luteinizing hormone releasing hormone (LHRH), oxytosin, Metionine-enkephalin 150
(Met-enkephalin), bombesin, Substance P, Leucine-enkephalin (Leu-enkephalin). 151
They were dissolved in acetonitrile/water = 80/20 (0.2% formic acid) at 2.5 µg/ml for 152
HILIC mode and water (0.2% formic acid) at 2.5 µg/ml for reversed-phase mode. 153
Phosphorylase B digest sample was purchased from Waters and dissolved in water 154
(0.2% formic acid) at 1 nmol/ml concentration. 155
156
3. Results and Discussion 157
3.1. Preparation of poly(acrylic acid) bonded stationary phase on monolithic silica 158
The effect of a monomer concentration on retention property of the resulting 159
stationary phase was examined with three acrylic acid concentrations in the feed 160
mixtures. Although a higher acrylic acid concentration in the feed resulted in the 161
stationary phase showing the greater retention for polar compounds, which is usually 162
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preferable, a monomer concentration higher than 50 µl in 1 ml water resulted in a 163
plugged column after the polymerization reaction. At 50 µl of acrylic acid in 1 ml 164
water, the monomer is supposed to occupy about 5% of the mobile-phase volume in a 165
column, resulting in a high total porosity starting from a monolithic silica column 166
having about 89% total porosity. 167
Fig. 1 shows the separation of pyridylamino derivatives of glucose oligomers in 168
75% acetonitrile/25% water in the presence of 0.2% formic acid on MS-200T-PAA 169
prepared by using 10-50 µl acrylic acid in the feed, while keeping the amount of other 170
substances constant. Fig. 2 shows the separation of two disaccharides and a 171
trisaccharide at u = 1.9–2.7 mm/s in 60-80% acetonitrile on MS-200T-PAA(30). The 172
monolithic silica capillary columns modified with a radical polymerization of acrylic 173
acid provided highly efficient separations of the carbohydrate derivatives. For the 174
separation, MS-200T-PAA(30) was employed rather than MS-200T-PAA(50), because it 175
has enough selectivity of carbohydrate derivatives with shorter retention time, that led 176
to rapid separation of these compounds. The modification conditions for 177
MS-200T-PAA(50) seems to be the upper limit of the monomer concentration. The 178
results reflect the advantage of the use of monolithic silica support having structures 179
that can provide high chromatographic efficiency as a starting material. 180
Another advantage of the modification by an on-column polymerization on the 181
monolithic silica is that the amount of the polymer (the stationary phase) can be 182
controlled by changing the concentration of monomers in the reaction mixtures [31]. 183
The change of the k values for the separation in Fig. 1 is listed in Table 1. The 184
monolithic silica column with anchor groups only showed almost no retention for these 185
solutes, while in the case of polymer-coated column, the greater feed concentration of 186
acrylic acid resulted in the larger k values. The column prepared with the highest 187
concentration of acrylic acid in the feed, MS-200T-PAA(50), gave retention factor, k = 188
10.7, for maltopentaose (peak number 6 in Fig. 1), which is greater than that on 189
MS-200T-PAA(10) and MS-200T-PAA(30) by 8.1 and 2.4 times, respectively. 190
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Similar results were obtained in the preparation reversed-phase monolithic silica 191
capillary columns modified by an on-column radical polymerization of a hydrophobic 192
monomer, octadecyl methacrylate [31]. The poly(octadecyl methacrylate)-coated 193
monolithic silica columns showed greater k values for alkylbenzenes than a C18 194
monolithic silica column modified with a silylating agent, N, 195
N-diethylaminodimethyloctadecylsilane, by a factor of up to 2.5 as previously reported 196
[2]. The increase in the amount of the stationary phase by polymerization 197
modification will also be able to increase the sample loading capacity of monolithic 198
silica columns that possess higher porosities and smaller phase ratios than common 199
particle-packed columns. 200
Fig. 2 shows the effect of acetonitrile concentrations in the mobile phases on the 201
separation of saccharides using the poly(acrylic acid) coated monolithic silica columns. 202
Apparently, the retention of the saccharides decreased drastically with the decrease in 203
acetonitrile concentration. In this measurement, sodium dodecylsulfate was employed 204
to show the column dead time, to. In 80% acetonitrile (Fig. 3a), the k values for 205
sucrose, trehalose, and raffinose were about 5, 9, and 20, respectively, while in 60% 206
acetonitrile (Fig. 2c), the k values were below 1.1. In earlier studies on the 207
chromatographic separation of saccharides, aminopropyl silica gel columns were 208
employed using a mixture of acetonitrile and water [32, 33]. The k values for sucrose 209
and raffinose on the MS-200T-PAA(30) were comparable to or slightly smaller than 210
those on aminopropyl silica gel in 75-80% acetonitrile [32]. In the case of a 211
reversed-phase mode, monolithic silica C18 columns gave smaller k values compared to 212
the conventional particle-packed columns by a factor of 2-6, due to the lower phase 213
ratio, i.e. high porosity (over 80-95%) of the monolithic columns. The present 214
modification method of the monolithic silica columns by the on-column radical 215
polymerization can improve the retentivity of the columns by selecting the 216
polymerization conditions, as also demonstrated for the reversed-phase mode materials 217
[31]. 218
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219
3.2. Column efficiency of MS-200T-PAA(30) 220
Chromatograms of a separation of three nucleosides, uridine, guanosine, and 221
adenosine by the MS-200T-PAA(30) under two linear velocities, u, at 1.1 and 13.1 222
mm/s, are shown in Fig. 3. The column afforded column efficiency of about N = 223
20000 at u = 1.06 mm/s, and 6300 at u = 13.1 mm/s with back pressure up to 21.5 MPa, 224
for adenosine (peak No. 3). The solutes were eluted with good peak shape to show the 225
usefulness of the poly(acrylic acid) modified monolithic silica column for such a rapid 226
separation of polar compounds. The fast separation of polar compounds by the 227
monolithic silica column should be attractive in the field of life science including 228
proteomics, metabolomics, and glycomics. 229
The performance of MS-200T-PAA at different linear velocity, was evaluated by 230
the Van Deemter plot for adenosine (k = 3.8) in 90% acetonitrile at 25°C, as shown in 231
Fig. 4. In order to reduce the extra-column effect, a split injection was employed. At 232
the optimum linear velocity, the theoretical plate height, H was less than 10 µm, and the 233
H value was kept below 20 µm even at u = 6 mm/s, showing that the column was 234
suitable for fast separations. The column efficiency was compared with that of a 235
well-known commercially available HILIC column packed with 5 µm particles, 236
ZIC-HILIC (150 mm×4.6 mm I.D.). (The data for the column obtained for cytosine 237
(k = 1.3) in 80% acetonitrile at 25°C was taken from the handling manual of SeQuant 238
[34].) The particle-packed column provided the plate height, H, of 12-13 µm under the 239
optimum flow conditions, u = 0.5-1.0 mm/s), and the H value of 40 µm or more at u = 6 240
mm/s. Another commercially available column for HILIC, TSK Amide-80 showed 241
similar performance at optimum linear velocity [35]. 242
Compared to a particle packed column for reversed-phase mode, the 243
MS-200T-PAA column provided the lower separation efficiency at high speed. In the 244
present report, monolithic silica having an increased phase ratio [30] was employed as a 245
support. The silica support had smaller domain-size (a combined size of a skeleton 246
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and a through-pore), that lead to higher separation efficiency than the materials reported 247
before. A bare silica column, the MS-200T column used in this study provided H = 7 248
µm for a t0 peak of uracil in 80% methanol. The modification method does not require 249
a packing process to fabricate a column after the chemical modification of the silica 250
surface. This is a clear advantage of the modification by a radical polymerization in a 251
monolithic silica column resulting in the high separation efficiency after the chemical 252
modification. The high efficiency will be preserved, unless the stationary phase 253
hinders the solute mass transfer. 254
The permeability, K, was evaluated according to equation 1 [36], where ∆P, F, η, L, 255
r, u, and ε stand for the column back pressure, the flow rate of the mobile phase, the 256
viscosity of the mobile phase, the column length, the column radius, the linear velocity 257
of the mobile phase, and the porosity of the column, respectively. 258
259
K = FηL/πr2∆P = uηLε/∆P (1) 260
261
Permeability of MS-200T-PAA was calculated to be 5.08×10–14 m2 with the porosity of 262
0.87, while the permeability of the ZIC-HILIC column was calculated as 1.80×10-14 m2 263
with the porosity of 0.56. The monolithic silica column with increased phase ratio 264
provided much smaller permeability than monolithic silica-C18 columns reported earlier 265
[2] and slightly smaller than a polymer-coated monolithic silica column previously 266
reported for reversed-phase mode [24]. However, it still exhibited higher permeability 267
than common columns packed with 5 µm particles. 268
Separation impedance, E, allows comparison of total performance of the columns. 269
This parameter was suggested by Knox [37], and given in equation 2, where t0, ∆P, η, N, 270
H, and K stand for the elution time of an unretained solute, the column back pressure, 271
the viscosity of the mobile phase, the theoretical plate number, the theoretical height, 272
and the permeability, respectively. 273
274
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E = t0∆P/N2η = (∆P /N)×(t0/N)×(1/η) = H2/K (2) 275
276
In Fig. 5, the plot of E value against u was shown for the MS-200T-PAA (for adenosine, 277
k = 3.8) and the particle-packed column (for cytosine, k = 1.3) [34]. The minimum E 278
value observed with the MS-200T-PAA was smaller than that for the particle-packed 279
column by a factor of about 3.5, and the difference between the two columns was much 280
larger at higher linear velocity of the mobile phase. 281
Desmet and co-workers suggested the use of kinetic plots, where to/N2 was plotted 282
against a theoretical plate number, N, to discuss the most important parameters, a 283
maximal achievable theoretical plate number in certain analysis times, and a minimal 284
analysis time to achieve certain theoretical plate numbers, to operate a HPLC system at 285
the optimum performance [38]. The kinetic plot is useful to know the limit of the 286
column efficiency under a certain backpressure. In Fig. 6, the kinetic plots for the 287
MS-200T-PAA and the particle-packed columns were shown for the limiting pressure of 288
20 MPa, which is commonly used in conventional HPLC separations. Fig. 6 clearly 289
indicates that MS-200T-PAA can achieve separations faster than the particle-packed 290
column, and that the monolithic silica column will show the optimum performance at 291
around N = 300000. MS-200T-PAA can produce N = 20000 with t0 = 63 s, faster than 292
the particle-packed column by a factor of 2.5. The results also suggest the possibility 293
of very high column efficiency in HILIC mode, N = 150000 with t0 of about 1000 s, by 294
using a long monolithic capillary column. . 295
296
3.3. Separation of peptides with MS-200T-PAA column 297
The MS-200T-PAA was examined in a gradient separation of peptides, an 298
important subject of proteomics. Fig. 7a and b show the separations of peptides with a 299
variety of molecular weight and pI values studied by using the MS-200T-PAA column 300
and a MS-200T-C18 with an ESI-TOF-MS system as a detector. Gradient elution was 301
employed, a linear gradient of 5-50% acetonitrile-0.2% formic acid in 10 min for the 302
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MS-200T-C18 column, and a linear gradient of 90-10% acetonitrile-0.2% formic acid in 303
10 min for the MS-200T-PAA column. When the poly(acrylic acid)-modified column 304
is employed, the optimization of pH in the mobile phase is important, since not only the 305
chemical state of sample but also the chemical state of the stationary phase can be 306
altered by pH of the mobile phase, resulting in a significant difference in the retention of 307
the solutes. Under neutral conditions, the ionized form of carboxylic acid of the 308
stationary phase dominates, leading to the ion-exchange interactions between the 309
stationary phase and the solutes. Basic peptides such as bradykinin and Substance P 310
could not be eluted by increasing a water concentration in the mobile phase at neutral 311
pH. However, they could be eluted under the acidic conditions (0.2% formic acid), 312
where most carboxylic acid moieties are supposed to be in the neutral form. 313
As shown in Fig. 7a and b, the elution orders on the two stationary phases, C18 and 314
poly(acrylic acid), are significantly different. The peptides, containing hydrophobic 315
residues such as Leu-enkephalin and Met-enkephalin, eluted in the last part of the 316
chromatogram on the C18, while they eluted in the early part from the poly(acrylic acid) 317
phase. Basic peptides such as bradykinin and Substance P were well retained on the 318
MS-200T-PAA column. For these basic peptides, the acid-base interaction due to the 319
acidic nature of the PAA stationary phase seems to make dominant contribution even 320
under the acidic conditions in the HILIC mode. Another important feature of the 321
MS-200T-PAA is that the HILIC mode separation can provide large retention factors for 322
small peptides, which are eluted without much retention from the C18 column. For 323
example, highly polar peptides such as γ-Glu-His (γ-EH), Asp-Ser-Asp-Pro-Arg 324
(DSDPR), and Val-Gly-Ser-Glu (VGSE) eluted near the to peak in the case of C18 325
column, while they eluted in the middle range of gradient in the case of the 326
MS-200T-PAA column. The conventional proteome analysis using reversed-phase 327
HPLC as a separation medium could ignore these hydrophilic components, but the use 328
of the HILIC system for such analysis may provide a good alternative to the 329
reversed-phase separations. 330
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Fig. 7c show a rapid separation of these peptides (a peptide standard mixture from 331
Sigma, P2693 without γ-EH, DSDPR, and VGSE that were included in the sample for 332
Fig. 7a and b) at a back pressure of 21 MPa, with a linear gradient of acetonitrile 333
concentration 90%-10% in 3 min, followed by a hold at 10% acetonitrile until 3.5 min. 334
Even with the rapid gradient within 3 min, an excellent separation was obtained. The 335
gradient run can be repeated within 5 min including the column equilibration time. In 336
the 3 min gradient separation, the average peak width was about 3 seconds. Fast 337
gradient elution of polypeptides is desirable for the second dimension separation of the 338
two-dimensional (2D) HPLC system [39]. 339
A similar comparison of the MS-200T-PAA and the MS-200T-C18 columns for the 340
separation of phosphorylase B tryptic digest is shown in Fig. 8a and b. The peak 341
capacity, calculated as gradient time divided by peak width of a certain peak, was about 342
100 for 10 min for each separation mode. This result suggests that the HILIC mode 343
gradient separation can produce a similar peak capacity of the reversed-phase mode, for 344
the separation of peptides. The highly orthogonal selectivities and the high column 345
efficiencies of the two separation modes at high speed are attractive features for the 346
application in two-dimensional HPLC [40]. Such an application, however, will need 347
the optimization of injection conditions including the compatibility of the two mobile 348
phases. Although the mobile phases consist of acetonitrile and water, the mobile phase 349
strength shows opposite tendency with respect to the water content between the two 350
modes of liquid chromatography. 351
352
4. Conclusion 353
The MS-200T-PAA column, modified by an on-column polymerization of acrylic acid 354
on the silica surface modified with (3-methacrylamidopropyl)silyl groups as an anchor 355
group, showed much higher separation efficiency in comparison with a commercially 356
available HILIC-type particle-packed column. Based on the high permeability and 357
small-sized skeletons, the monolithic silica column was able to provide high efficiency 358
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even in very fast elutions higher than u = 10 mm/s. The retention factors on the 359
monolithic column for oligosaccharides were similar to or slightly smaller than those 360
provided by the aminopropyl silica columns, showing good retentivity in spite of its 361
much higher porosity than the particle-packed columns. The retentivity could be 362
controlled by the concentration of the monomer in the feed for the modification of silica 363
support. The MS-200T-PAA column was shown to be suitable for several types of 364
polar compounds in both the isocratic and the gradient modes, although the pH of the 365
mobile phase must be controlled to be acidic for the separation of peptides. 366
367
Acknowledgements 368
This work was supported in part by Grant-in-Aid for Scientific Research funded by 369
the Ministry of Education, Sports, Culture, Science and Technology, No. 14340234 and 370
17350036. K. Horie thanks Kyoto Institute of Technology for a scholarship of the 371
Engineer Training and Research Innovation Program. The supports by GL Sciences and 372
Merck KGaA, Darmstadt, are also gratefully acknowledged. 373
374
References 375
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435
Figure Captions 436
Figure 1, The separation of 2-pyridylamino (PA-) derivatives of glucose oligomers 437
in 75% acetonitrile/25% water in the presence of 0.2% formic acid on 438
MS-200T-PAA columns prepared by using 10-50 µL acrylic acid in the feed (Table 439
1). Columns: (a) MS-200T-silica, 200 mm×200 µm I.D., (b) MS-200T-PAA(10), 200 440
µm i.d.×200 mm, (c) MS-200T-PAA(30), 200 mm×200 µm I.D., and (d) 441
MS-200T-PAA(50), 200 mm×200 µm I.D. Mobile phase: acetonitrile/water (0.2% 442
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formic acid) = 75/25. Temperature: 25°C. Detection: UV 254 nm. Pressure drop: 443
3.8 MPa. Solute: 1: PA-arabinose, 2: PA-glucose, 3: PA-maltose, 4: PA-maltotriose, 5: 444
PA-maltotetraose, 6: PA-maltopentaose, 7: PA-maltohexaose, 8: PA-maltoheptaose, 445
Injection volume: 2 µl split injection. 446
447
Figure 2, The separation of sucrose, trehalose, and raffinose, u = 1.9–2.7 mm/s, 448
60-80% acetonitrile/water (13 mM ammonium acetate buffer, pH = 5.4) on 449
MS-200T-PAA. Column: MS-200T-PAA(30), 174 mm×200 µm I.D. 450
Temperature: 30°C. Detection: ESI-TOF-MS (2.8 kV, Negative). Solute: 1: sodium 451
dodecylsulfate, 2: sucrose, 3: trehalose, 4: raffinose (100 µg/ml for each solute). 452
Injection volume: 50 nl. Mobile phase: (a) acetonitrile/13 mM ammonium acetate 453
buffer (pH = 5.4) = 80/20, (b) acetonitrile/13 mM ammonium acetate buffer (pH = 5.4) 454
= 70/30, and (c) acetonitrile/13 mM ammonium acetate buffer (pH = 5.4) = 60/40. 455
456
Figure 3, Chromatograms obtained for nucleosides (uridine, guanosine and 457
adenosine). Column: MS-200T-PAA(30), 200 mm×200 µm I.D., Mobile phase: 458
acetonitrile/water (0.2% formic acid) = 90/10. Temperature: 25°C. Detection: UV 254 459
nm. Solutes: 1: uridine, 2: guanosine, 3: adenosine. Injection volume: 2 µl split 460
injection. (a) Linear velocity 1.06 mm/s, Pressure drop: 1.7 MPa. (b) Linear velocity 461
13.1 mm/s, Pressure drop: 21.5 MPa. 462
463
Figure 4, The Van Deemter plots obtained for HILIC mode elution. 464
MS-200T-PAA(30), 200 mm×200 µm I.D., (◆), in 90% acetonitrile/(0.2% formic 465
acid), at 25°C. Solute: adenosine (k = 3.8). Injection volume: 2 µl split injection. 466
ZIC-HILIC column, 150 mm×4.6 mm I.D., 5 µm particles, (△), in 80% 467
acetonitrile/buffer, at 25°C. Solute: cytosine (k = 1.3). 468
469
Figure 5, Plot of separation impedance against the linear velocity of the mobile 470
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phase. (◆) MS-200T-PAA(30), and (△) ZIC-HILIC column. For other 471
chromatographic conditions, see Figure 4. 472
473
Figure 6, Log (t0/N2) values plotted against log N for the two HILIC columns. 474
Columns were evaluated with the assumed maximum pressure of 20 MPa for (◆) 475
MS-200T-PAA and (△) the ZIC-HILIC column. For other chromatographic 476
conditions, see Figure 4. 477
478
Figure 7, LC–ESI-TOF-MS total ion chromatograms of peptides. (a) Column: 479
MS-200T-C18, 224 mm×200 µm I.D., Mobile phase: 5–50% acetonitrile (0.2% formic 480
acid) in 10 min linear gradient, (b) Column: MS-200T-PAA, 190 mm×200 µm I.D., 481
Mobile phase: 90–10% acetonitrile (0.2% formic acid) in 10 min linear gradient, (c) 482
Column: MS-200T-PAA, 190 mm×200 µm I.D., Mobile phase; 90–10-10% acetonitrile 483
(0.2% formic acid) in 3 min linear gradient followed by 0.5 min hold, Pressure drop; 20 484
MPa. Detection: ESI-TOF-MS (3 kV, Negative). Solutes: 1: γ-EH, 2: DSDPR, 3: 485
VGSE, 4: bradykinin fragment1-5, 5: [Arg8]-vasopressin, 6: bradykinin, 7: LHRH, 8: 486
oxytosin, 9: Met-enkephalin, 10: bombesin, 11: Substance P, 12: Leu-enkephalin for (a) 487
and (b), samples 4-12 (9 peptides) for (c). Sample concentration; 2.5 µg/ml, Injection 488
volume; 50 nl. 489
490
Figure 8, ESI-TOF-MS base peak chromatogram of phosphorylase B tryptic digest. 491
Detection: ESI-TOF-MS (3 kV negative). Solute: phosphorylase B tryptic digest. 492
Sample concentration: 1 nmol/ml. Injection volume: 50 nl. (a) Column: 493
MS-200T-C18, 238 mm×200 µm I.D.. Mobile phase: 5–60% acetonitrile (0.2% 494
formic acid) in 10 min linear gradient, (b) Column: MS-200T-PAA, 190 mm×200 µm 495
I.D.. Mobile phase: 90–10% acetonitrile (0.2% formic acid) in 10 min linear gradient. 496
497
498
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Table 1 Feed compositions for MS-200T-PAA columns and the retention factors
obtained for pyridylamino derivatives of maltopentaose on each stationary phase in 75%
acetonitrile/25% water in the presence of 0.2% formic acid.
Feed composition PA-Maltopentaose peak
ColumnAcrylic acid(l)
(NH4)2S2O7
(mg)H2O (ml) Retention factor (k)
MS-200T-MASa - - - 0.09
MS-200T-PAA(10) 10 5 1 1.32
MS-200T-PAA(30) 30 5 1 4.41
MS-200T-PAA(50) 50 5 1 10.7
a Monolithic silica modified with 3-methacrylamidopropylsilyl groups.
Tables
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