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

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

Acce

pted

Man

uscr

ipt

1

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: ikegami@kit.ac.jp 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

Page 1 of 27

Acce

pted

Man

uscr

ipt

2

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

Page 2 of 27

Acce

pted

Man

uscr

ipt

3

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

Page 3 of 27

Acce

pted

Man

uscr

ipt

4

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

Page 4 of 27

Acce

pted

Man

uscr

ipt

5

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

Page 5 of 27

Acce

pted

Man

uscr

ipt

6

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

Page 6 of 27

Acce

pted

Man

uscr

ipt

7

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

Page 7 of 27

Acce

pted

Man

uscr

ipt

8

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

Page 8 of 27

Acce

pted

Man

uscr

ipt

9

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

Page 9 of 27

Acce

pted

Man

uscr

ipt

10

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

Page 10 of 27

Acce

pted

Man

uscr

ipt

11

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

Page 11 of 27

Acce

pted

Man

uscr

ipt

12

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

Page 12 of 27

Acce

pted

Man

uscr

ipt

13

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

Page 13 of 27

Acce

pted

Man

uscr

ipt

14

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

[1] N. Tanaka, H. Kobayashi, K. Nakanishi, H. Minakuchi, N. Ishizuka, Anal. 376

Chem. 73 (2001) 420A. 377

[2] M. Motokawa, H. Kobayashi, N. Ishizuka, H. Minakuchi, K. Nakanishi, H. Jinnai, K. 378

Hosoya, T. Ikegami, N. Tanaka, J. Chromatogr. A 961 (2002) 53. 379

[3] K. Cabrera, J. Sep. Sci., 27 (2004) 843. 380

[4] G. Guiochon, J. Chromatogr. A (2007) in press. 381

[5] A.J. Alpert, J. Chromatogr. 499 (1990) 177. 382

[6] A.J. Alpert, M. Shukla, A.K. Shukla, L.R. Zieske, S.W. Yuen, M.A.J. Ferguson, A. 383

Mehlert, M. Pauly, R. Orlando, J. Chromatogr. A 676 (1994) 191. 384

[7] T. Yoshida, Anal. Chem. 69 (1997) 3038. 385

[8] T. Yoshida, J. Chromatogr. A 808 (1998) 105. 386

Page 14 of 27

Acce

pted

Man

uscr

ipt

15

[9] T. Yoshida, J. Biochem. Biophys. Methods 60 (2004) 265. 387

[10] X. Wang, W. Li, H.T. Rasmussen, J. Chromatogr. A 1083 (2005) 58. 388

[11] B.A. Olsen, J. Chromatogr. A 913 (2001) 113. 389

[12] S.C. Churms, J. Chromatogr. A 720 (1996) 75. 390

[13] R.S. Hodges, Y. Chen, E. Kopecky, C.T. Mant, J. Chromatogr. A 1053 (2004) 161. 391

[14] Y. Guo, S. Gaiki, J. Chromatogr. A 1074 (2005) 71. 392

[15] A.R. Oyler, B.L. Armstrong, J.Y. Cha, M.X. Zhou, Q. Yang, R.I. Robinson, R. 393

Dunphy, D.J. Burinsky J. Chromatogr. A 724 (1996) 378–383 394

[16] B.W. Pack, D.S. Risley J. Chromatogr. A 1073 (2005) 269. 395

[17] V.V. Tolstikov, O. Fiehn, Anal. Biochem. 301 (2002) 298. 396

[18] H.S. Cerny, M. Affolter, C. Cerny, Anal. Chem. 75 (2003) 2349. 397

[19] Y. Takegawa, K. Deguchi, S. Ito, S. Yoshioka, A. Sano, K. Yoshinari, K. 398

Kobayashi, S. Nishimura, Anal. Chem. 76 (2004) 7294. 399

[20] M.A. Strege, Anal. Chem. 70 (1998) 2439. 400

[21] Q. Song, W. Naidong, J. Chromatogr. B 830 (2006) 135. 401

[22] Y.S. Hsieh, J.W. Chen, Rapid Commun. Mass Spectrom. 19 (2005) 3031. 402

[23] R. Oertel, V. Neumeister, W. Kirch, J. Chromatogr. A 1058 (2004) 197. 403

[24] T. Ikegami, H. Fujita, K. Horie, K. Hosoya, N. Tanaka, Anal. Bioanal. Chem. 386 404

(2006) 578. 405

[25] T. Ikegami, K. Horie, J. Jaafar, K. Hosoya, N. Tanaka, J. Biochem. Biophys. 406

Methods 70 (2007) 31. 407

[26] D. Lubda, K. Cabrera, K. Nakanishi, W. Lindner, Anal. Bioanal. Chem. 377 (2003) 408

892. 409

[27] T. Ikegami, J. Ichimaru, W. Kajiwara, N. Nagasawa, K. Hosoya, N. Tanaka, Anal. 410

Sci. 23 (2007) 109. 411

[28] C.H. Xie, J.W. Hu, H. Xiao, X.Y. Su, J. Dong, R.J. Tian, Z.K. He, H.F. Zou, J. Sep. 412

Sci. 28 (2005) 751. 413

[29] B. Preinerstorfer, D. Lubda, W. Lindner, M. Lämmerhofer, J. Chromatogr. A 1106 414

Page 15 of 27

Acce

pted

Man

uscr

ipt

16

(2006) 94. 415

[30] T. Hara, H. Kobayashi, T. Ikegami, K. Nakanishi, N. Tanaka, Anal. Chem. 78 416

(2006) 7632. 417

[31] O. Núñez, T. Ikegami, W. Kajiwara, K. Miyamoto, K. Horie, N. Tanaka, J. 418

Chromatogr. A (2007) in press: available on-line 419

[32] J.C. Linden, C.L. Lawhead, J. Chromatogr. 105 (1975) 125. 420

[33] V. Kahle, K. Tesarík, J. Chromatogr. 191 (1980) 121. 421

[34] SeQuant, A Practical Guide to HILIC p 11, 422

http://www.sequant.com/sn/p_details.php?id=7. 423

[35] Tosoh, Separation Report, No. 055 424

http://www2.tosoh.co.jp/hlc/techinfo.nsf/SR01/160A562AFFC947FE49256A190014FC425

EA/$FILE/sr055.PDF 426

[36] U. D. Neue, HPLC Columns, Theory, Technology and Practice, Wiley-VCH, New 427

York, 1997. 428

[37] P.A. Bristow, J.H. Knox, Chromatographia 1977. 10. 279 429

[38] G. Desmet, D. Clicq, P. Gzil, Anal. Chem. 77 (2005) 4058. 430

[39] H. Kimura, T. Tanigawa, H. Morisaka, T. Ikegami, K. Hosoya, N. Ishizuka, H. 431

Minakuchi, K. Nakanishi, M. Ueda, K. Cabrera, N. Tanaka, Nobuo, J. Sep. Sci. 27 432

(2004) 897. 433

[40] M. Gilar, P. Olivova, A. Daly, J.C. Gebler, Anal. Chem. 77 (2005) 6426. 434

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

Page 16 of 27

Acce

pted

Man

uscr

ipt

17

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

Page 17 of 27

Acce

pted

Man

uscr

ipt

18

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

Page 18 of 27

Acce

pted

Man

uscr

ipt

Figure 1

Page 19 of 27

Acce

pted

Man

uscr

ipt

Figure 2

Page 20 of 27

Acce

pted

Man

uscr

ipt

Figure 3

Page 21 of 27

Acce

pted

Man

uscr

ipt

Figure 4

Page 22 of 27

Acce

pted

Man

uscr

ipt

Figure 5

Page 23 of 27

Acce

pted

Man

uscr

ipt

Figure 6

Page 24 of 27

Acce

pted

Man

uscr

ipt

Figure 7

Page 25 of 27

Acce

pted

Man

uscr

ipt

Figure 8

Page 26 of 27

Acce

pted

Man

uscr

ipt

1

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

Page 27 of 27