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Journal of Chromatography A, 1184 (2008) 474–503

Review

Separation efficiencies in hydrophilic interaction chromatography

Tohru Ikegami ∗, Kouki Tomomatsu, Hirotaka Takubo, Kanta Horie 1, Nobuo TanakaDepartment of Biomolecular Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan

Available online 3 February 2008

bstract

Hydrophilic interaction chromatography (HILIC) is important for the separation of highly polar substances including biologically active com-ounds, such as pharmaceutical drugs, neurotransmitters, nucleosides, nucleotides, amino acids, peptides, proteins, oligosaccharides, carbohydrates,tc. In the HILIC mode separation, aqueous organic solvents are used as mobile phases on more polar stationary phases that consist of bare silica,nd silica phases modified with amino, amide, zwitterionic functional group, polyols including saccharides and other polar groups. This reviewiscusses the column efficiency of HILIC materials in relation to solute and stationary phase structures, as well as comparisons between particle-
acked and monolithic columns. In addition, a literature review consisting of 2006–2007 data is included, as a follow up to the excellent review byemstrom and Irgum. 2008 Elsevier B.V. All rights reserved.
eywords: HILIC; Separation efficiency; Bare silica; Amino-silica; Amide-silica; Poly(succinimide)-silica; Sulfoalkylbetaine-silica; Diol-silica; CD-silica; Polymeronolithic columns; Silica monolithic column

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4752. Types of stationary phases for hydrophilic interaction chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4753. Separation efficiencies of particle-packed hydrophilic interaction chromatography mode columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 476

3.1. Columns packed with bare silica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4763.2. Columns packed with amino-modified silica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4793.3. Columns packed with amide-modified silica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4833.4. Columns packed with poly(succinimide)-modified silica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 485

3.4.1. PolyHydroxyethyl A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4863.4.2. PolyGlycoplex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4873.4.3. PolyCAT A and poly(Sulfoethyl A) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487

3.5. Columns packed with sulfoalkylbetaine-modified silica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4883.6. Columns packed with cyano- and diol-modified silica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4903.7. Columns packed with cyclodextrin-modified silica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4913.8. Columns packed with other functionality-modified silica, and polymer particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493

4. Separation efficiencies of monolithic hydrophilic interaction chromatography mode columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4934.1. Polymer monolithic columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493
4.2. Silica monolith columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author. Tel.: +81 75 724 7801; fax: +81 75 724 7710.E-mail address: [email protected] (T. Ikegami).

1 Present address: Eisai Co. Ltd., Japan.

021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2008.01.075

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 496

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501

mailto:[email protected]

dx.doi.org/10.1016/j.chroma.2008.01.075

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T. Ikegami et al. / J. Chrom

. Introduction

Recently, literature and research on hydrophilic interactionhromatography (HILIC) have been increasing drastically, alongith various stationary phases developed for HILIC. HILIC is aind of normal-phase liquid chromatography (NPLC), and hasttracted the attention of researchers that study the separationf polar compounds in a wide variety of scientific fields. InILIC mode, a mixture of water and organic modifiers in most

ases acetonitrile (MeCN) is employed with a polar stationaryhase. Structural variations in HILIC type stationary phasesre wider than those found in reversed-phase applications. Inddition to classical bare silica and aminopropyl-bonded sil-ca, silica gels modified with many polar functionalities such asmide, diol, cyano, derivatives of poly(succinimide), sulfoalkyl-etaine, cyclodextrin are applicable to HILIC mode separation.olymer-based stationary phases such as sulfonated polymers,nd diol-containing polymers can also be used. HILIC can ben alternative to reversed-phase chromatographic separation forolar compounds using isocratic and gradient elutions. Botharticle-packed columns and monolithic columns have beensed in HILIC. HILIC is suitable for electrospray ionizationESI)-mass spectrometry (MS), because of the compatibilityf the aqueous organic mobile phase to ESI-MS, which is aery powerful tool to detect and identify a wide range of polarompounds.

There are many examples of HILIC applications for thenalysis of small polar molecules including biomarkers, nucle-sides, nucleotides, carbohydrates, amino acids, peptides androteins, that contribute to the pharmaceutical chemistry, agri-ultural and food chemistry, medicinal chemistry, proteomics,etabolomics, and glycomics. However, it seems that only lim-

ted attention has been paid to the separation efficiencies inILIC. This review provides a perspective of HILIC type sta-

ionary phases, and discusses their separation efficiencies andetention tendencies.

First, various stationary phases for HILIC are introduced, andeparation efficiencies of columns packed with particles, anduitable analytes for each stationary phase are discussed. Then,he same discussion is applied to monolithic columns to revealhe differences in these types of support for stationary phases.

. Types of stationary phases for hydrophilic interactionhromatography

Although the acronym HILIC was already first suggested bylpert in 1990 [1], the number of publications regarding HILICas increased substantially since 2003, as outlined in the well-onstructed review by Hemstrom and Irgum [2]. NPLC has beensed widely to separate various compounds from non-polar com-ounds to highly polar compounds after chromatography wasrst introduced as a method of separation science [3,4]. NPLConsists of polar stationary phases, mainly bare silica or alumina,
nd less polar mobile phases formed by single organic solventsr mixed organic solvents, and has been the chromatographicainstream for about 70 years [5]. The chemical modifica-
ion of silica gels using chemical reactions began in the 1960s

(tYH

. A 1184 (2008) 474–503 475

6]. Reactions between alkylsilyl chlorides and silanol groupsSi–OH) is the most common method, and was reported in 19707].

The first example of polar functional groups seems to beublished in 1967 by Horvath et al., where glass beads haveoated with polyethylene imine [8]. At the early stage of theevelopment of stationary phase, the preparation of packingaterials containing polar functional groups such as amino, sul-

onic acid, carboxylic acid, ketone, and nitrile, for LC wereeported by Halasz et al. in 1973 [9]. The authors employedhemical reactions between Si–Cl bonds in silica (prepared byhlorination of Si–OH bonds using thionyl chloride, SOCl2)nd various amines that possessed the above-mentioned func-ional groups. These products were useful as polar bondedhases, with intermediate polarities between those of reversed-hases and those of bare silica gels. In the same year, Scott andrushka reported the preparation of a “polar bonded” phase, inhich silica particles were modified first with l-trimethoxysilyl--(4-chloromethylphenyl)ethane, and second with oligomers oflycine [10]. A similar stationary phase was reported by the sameroup to show that the height equivalent of theoretical plate, Hf the column was 0.4–18 mm for a linear velocity at 3.7 mm/s11].

The first generation of HILIC mode separation started in975. Linden et al. separated carbohydrates by an amino-silicahase, Bondapak (Waters, Milford, MA, USA) in a mixture ofeCN and water (75:25) [12]. Generally, amino-silica phases

re prepared by reacting trialkoxysilanes, RSi(OR′)3, whereand R′ stand for aminoalkyl and alkyl groups, respectively,

n silica and they can form polymeric phases with a certainhickness of the stationary phase to incorporate water. Beforehe introduction of HILIC separation, ion-exchange resin wassed to separate carbohydrates. The formation of a water-nriched layer on the surface of highly polar stationary phasesas reported in 1967 [13]. The existence of a water-enriched

ayer on the polar stationary phases under aqueous conditions,nd a partitioning equilibrium of analytes between a mobilehase and the “wet” stationary phase, are a basic, and anccepted mechanism for HILIC [1,2]. Alpert suggested thisechanism referring to research by Orth and Engelhardt [14],

nd the partitioning mechanism has been recently substantiated2].

The next generation of stationary phases for HILIC usediol- and amide-silica. The diol silica column has been usedainly for the separation of proteins [15,16]. According to

he Tosoh, producer of TSKgel Amide-80, this amide-silicaolumn has been available since at least 1985 [17]. This par-icular phase is described as “consisting of non-ionic carbamoylroups that are chemically bonded to the silica gel”, but thishase is mainly called as an amide bonded silica. The afore-entioned phase was used for the multidimensional mapping of

ligosaccharides with an octadecyl silica (ODS) phase for two-imensional mapping, and with ODS and diethylaminoethyl
DEAE) phases for three-dimensional mapping [18–21]. Afterhe application of this phase to the separation of peptides byoshida [22], the amide-silica phase became widely used inILIC.

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76 T. Ikegami et al. / J. Chrom

Stationary phases reported by Alpert contained polymerictructures of poly(succinimide) derivatives [1]. These columnsre manufactured by PolyLC (Columbia, MD, USA), andue to differences in functional groups, different phases suchs PolyGlycoplex, PolySulfoethyl aspartamide, and PolyHy-roxyethyl aspartamide are available [23]. These columnsave been widely used to separate various highly polarompounds, and recently, columns packed with 3 �m par-icles have been introduced to provide better separation24].

Recently, a HILIC phase prepared by graft polymerization toncorporate 3-sulfopropyldimethylalkylammonium inner salts,.e. sulfoalkylbetaine functional groups onto silica and polymerarticles has released. Sometime, this phase is called as a zwitte-ionic phase. These types of columns are available from SeQuantUme, Sweden), as ZIC-HILIC columns [25]. Originally, thishase was prepared for cation-exchange chromatography, andarlier reports described the separations of inorganic salts androteins in fully aqueous mobile phases [26–28]. This phase isidely accepted by many researchers, and used often in more

ecent publications.There are several stationary phases for HILIC mode sepa-

ation in addition to the above-mentioned phases, and they allossess highly polar functionalities, such as cyano, hydroxy,iol, carbohydrates and so on, that attract water molecules fromhe mobile phase to form water-enriched layers. These lay-rs seem to be important and facilitate partitioning equilibriaetween stationary and mobile phases.

In almost all published articles on HILIC mode separations,he elution order of analytes differs from their separation byPLC, and analyte retention times are described. However,

nformation on column separation efficiencies is still limited.ne of the reasons could be the use of gradient elutions in their

tudies.We would like to discuss the separation efficiencies of

arious HILIC phases in this review. Simple applications cov-red by two HILIC reviews [2,29] are omitted, and onlyiterature published between 2006 and 2007 were added tohis review. Therefore, articles containing isocratic separa-ions were mainly surveyed; however, HILIC studies used inpplications including proteomics, metabolomics, glycomics,edicinal science, agricultural and food chemistry reportedithin 2006–2007 are briefly mentioned. Comparisons of sep-

ration efficiencies between particle-packed and monolithicolumns are discussed herein. Since the separation efficiencyf each peak was estimated manually from the peak widtht half height, considerable errors can be included in the cal-ulation of separation efficiencies, N, and the column deadime, t0 was calculated by an estimation of column poros-ty, ε = 0.65 if there is no peak of the t0 marker. Plateeights depend on a variety of factors of dispersion param-ters corresponding to each part of HPLC systems, also oninear velocity of the eluent. Though data employed herein

ight be not under control of extra-column effects (extra-olumn volumes at injection, detection, and tubing, and sampleatrix), the chromatograms can be regarded as not the worst

esults.

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r. A 1184 (2008) 474–503

. Separation efficiencies of particle-packed hydrophilicnteraction chromatography mode columns

Stationary phases for reversed-phase separation are modifiedith silanes that possess alkyl chains, between C4 and C30, andainly C18. Not only “pure” alkyl groups, but also partially

olar alkyl groups are employed to increase the retention ofolar compounds. This approach does not seem to be so effec-ive for the separation of highly polar compounds. Comparedo stationary phases for RPLC, columns for HILIC separationave a wider variety of functional groups and are described asollows. All polar functionalities including anionic and cationicoieties would be able to form the water-rich regions on the

urface of packing materials, and they are useful for HILIC sep-ration. However, due to the wide variety of functionalities oftationary phases and target analytes, i.e. polar compounds, thenowledge on the applicability seems to be still limited. In thishapter, the fundamental characterization of columns such aslate number, N, and theoretical plate height, H, of stationaryhases for HILIC is discussed with some emphasis on retentionactors.

.1. Columns packed with bare silica

Bare silica is a classical phase in NPLC, and that is stillidely used in recent studies of HILIC, and especially withILIC-MS systems. Naidong reviewed the separation–detectionf bioanalytical samples by HILIC-MS/MS [29]. The mobilehase in HILIC, mainly a mixture of MeCN and aqueousuffer, is suitable and compatible for electrospray ionizationource that are interfaced to MS systems. Bare silica columnsmployed for HILIC separations include, Betasil (Thermocientific, Waltham, MA, USA) [30], Hypersil (Thermo Sci-ntific) [31], Inertsil (GL Science, Tokyo, Japan) [32], KromasilEKA Chemicals, Goteborg, Sweden) [33], Supelcosil LC-SiSigma–Aldrich, St. Louis, MO, USA) [34], Alltima (Alltech,icholasville, KY, USA) [35], Spheri5 Silica (Alltech) [36], andupersphere Si (Trentec, Gerlingen, Germany) [37]. There areany publications on the separations using Atlantis (Waters)

38–46]. At the time of the review by Naidong, the particle sizeor all these columns was 5 �m [29], but a 4 �m particle-packedolumn [37], and 3 �m particle-packed columns were used inecent studies [38,41–46]. Recently, an Acquity column packedith 1.7 �m particles was released from Waters for HILICode ultrahigh-performance liquid chromatography (UPLC)

eparations [47,48]. This column was applied to a LC–MSystem as a complementary separation method for RPLC–MSnd a gas chromatography–time-of-flight-MS (GC–TOF-MS) toomprehensively analyze metabolomes in human urine for theiagnosis of kidney cancer [49]. MS systems and nuclear mag-etic resonance (NMR) spectroscopy can be useful detectors forILIC mode separation. For HILIC-NMR/MS systems, water

nd formic acid were changed to D2O and DCO2D, but CH3CN
as used as an organic modifier [37].Almost all the literature on HILIC using bare silica columns
mploys gradient elutions, and it is difficult to evaluate theireparation efficiencies. Monolithic silica columns, Chromolith-

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i (Merck, Darmstadt, Germany) were used in a few publications50] and the use of monolithic silica capillary columns in HILICas recently reported [51,52]. Monolithic silica columns areiscussed in Section 4.2.

Bare silica columns were used to separate small polarompounds, especially pharmaceuticals, and examples of sepa-ations of carbohydrates, peptides or proteins using HILIC areimited. HILIC-MS/MS systems were employed for the analy-is of atenolol [31], acyclovir [36], propofol and its glucuronideerivatives [43], gabapentin and metformin [41], glutathione andts derivatives [39,44], iminoctadine triacetate [38], zanamivir45], levofloxacin [42], carvedilol [46], omeprazole and itserivatives [30], and spectinomycin [35], while UV–Vis detec-
ors were also used for the analysis of epirubicin and itsnalogues [33], carnosine, anserine, balenine, creatine, and cre-tinine in meat [40], pseudoephedrine, diphenhydramine, andextromethorphan [34].
mds

ig. 1. Separation of polar compounds on: (A) YMC-Pack NH2; (B) TSKgel Am50 mm × 4.6 mm I.D., 5 �m; mobile phase: MeCN/water (85/15, v/v) containing 20 metection: λ = 228 nm. (A) Compounds: (1) salicylamide; (2) salicylic acid; (3) 4-aminB) Compounds: (1) uracil; (2) adenosine; (3) uridine; (4) cytosine; (5) cytidine; and

. A 1184 (2008) 474–503 477

The retentivity and the separation efficiency of a bare silica,tlantis HILIC Silica was compared with three HILIC columns,

n amino-silica, YMS-Pack NH2, an amide-silica, TSKgelmide-80, and a sulfoalkylbetaine-modified silica, ZIC-HILIC,sing five aromatic carboxylic acids and five nucleic basesnd nucleosides [53] as shown in Fig. 1. Each column was50 mm × 4.6 mm I.D., 5 �m. The column dead time, t0 for theow rate 1.5 mL/min, was estimated to be 1.8 min assuming aorosity of 0.65. For the analytes employed, k and H were com-ared for four different stationary phases as shown in Fig. 2. Thetlantis HILIC Silica phase showed the smallest retention for

he 10 test samples, and the highest separation efficiency around= 25 000 (H = 10 �m).
As shown by Olsen, three bare silica columns from different
anufacturers, Zorbax SIL, Nucleosil, and YMC silica providedifferent retentivities, efficiencies, and peak shapes for nucleo-ides [54]. As shown in Fig. 3, peak tailing was observed in

ide-80; (C) ZIC-HILIC; and (D) Atlantis HILIC silica columns. Column:M ammonium acetate; column temperature: 30 ◦C; flow rate: 1.5 mL/min; UV

osaliclyic acid; (4) acetylsalicylic acid; and (5) 3,4-dihydroxyphenylaceticacid.(6) guanosine [53].

478 T. Ikegami et al. / J. Chromatogr. A 1184 (2008) 474–503

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he case of Nucleosil and Zorbax SIL for pyrimidines, while noignificant tailing was found for purines.

This kind of difference is also known for RPLC columns55,56], and recently reviewed by Neue recently [57], but theres limited detailed research on HILIC columns.

Compounds 1–4 (Fig. 4) were not separated on the KromasilR100-5SIL phase (250 mm × 4.6 mm I.D., 5 �m) and were

luted as a non-retained peak in MeOH, but partially separatedn THF, and completely separated in MeCN as shown in Fig. 5A.he separation efficiency in MeCN was estimated as N = 10 000,= 25 �m. Among three different buffer solutions of pH 5.0,

.2, and 2.9, the most acidic conditions afforded the best sep-ration efficiencies of N = 10 000–12 000, H = 21–25 �m, while
= 6000–9000, H = 28–42 �m was obtained at pH 5.0 [33].Naidong compared the separation efficiency of a HILIC
olumn, Betasil Silica with those of two RPLC columns, Inert-il ODS2 C18 and Chromolith SpeedROD RP-18e by the

wubi

inued ).

ackpressure-flow rate plot and the Van Deemter plot as shownn Fig. 6 [29]. The Betasil column showed an intermediate effi-iency between the Inertsil and the Chromolith RP columns.he optimal flow rate for the Betasil column was 1–2 mL/min,

linear velocity was estimated as u = 1.6–3.2 mm/s) and the plateeight was around 20 �m for fluconazole (k = ca. 2).

Phenyl-�-d-glucuronide (1), and glucuroconjugated metabo-ites (propofol-glucuronide (2), 1-quinol-glucuronide (3), and-quinol-glucuronide (4)) in Fig. 7 were analyzed on an AtlantisILIC column (150 mm × 2.1 mm I.D., 3 �m) with an AtlantisILIC guard cartridge (10 mm × 2.1 mm I.D.) in a mixture ofeCN/water/100 mM NH4OAc buffer (pH 5) (87/1/12, v/v/v)

t 25 ◦C and at a flow rate of 200 �L/min [43]. The efficiency
as estimated as N = 3500–4500, H = 33–43 �m, and these val-es were apparently worse than expected separation efficiencyy RP columns packed with 3 �m particles. The existence ofsopropyl group in 2–4 (Fig. 7) led to a decrease in retention

T. Ikegami et al. / J. Chromatogr. A 1184 (2008) 474–503 479

F e-80,u

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ig. 2. The k–H plots for the separation of Fig. 1. Column: (�) TSKgel Amidsing data from eight chromatograms by Guo and Gaiki [53].

f these glucuronides, compared to phenyl-�-d-glucuronide (1)hich showed a typical retention tendency in HILIC.
The user of this type of column must keep in mind that bare
ilica phases sometimes undergo severe irreversible adsorptionf analytes on the columns, as reported by Li and Huang: epiru-

ig. 3. The separation of pyrimidines (A) and purines (B) on three bare silicaolumns. Column: YMC SIL, Nucleosil Silica, Zorbax SIL 250 mm × 4.6 mm.D., 5 �m; Mobile phase: 5 mM phosphoric acid in MeCN–water (75:25) foryrimidines, (70:30) for purines; detection: UV, λ = 275 nm [54].

3

sud[A(C[BPsc[a1

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(�) YMC-Pack NH2, (�) Atlantis HILIC silica, and (×) ZIC-HILIC, plotted

icin was absorbed on the column under neutral or weakly acidiconditions [33].

.2. Columns packed with amino-modified silica

Many column manufactures supply 3-aminopropyl bondedilica phases for NPLC and HILIC modes. For example, columnssed for HILIC are Bondapak AX (Waters) [12], � Bon-apak Carbohydrate (Waters) [58], Spherisorb NH2 (Waters)59], YMC-Pack NH2 (YMC, Kyoto, Japan) [53,59], Lunamino (Phenomenex, Torrance, CA, USA) [60], Hypersil APS2

Thermo Science) [61,62], Zorbax NH2 (Agilent, Santa Clara,A, USA) [63], apHera NH2 (ASTEC, Whippany, NJ, USA))

64], Alltima Amino (Alltech) [65], PALPAK Type N (Takaraio, Otsu, Japan) [66], and Micropellicular [67]. YMC Packolyamine II (YMC) contains polyamine moieties on silicaurfaces [59]. Separation targets of amino-silica phases arearbohydrates [12,58,62,67,68], amino acids [64], peptides59], carboxylic acids and nucleosides [49,50,60], tetracyclinentibiotics [61], oxytocin analogues [59], and 2-amino-2-ethyl-,3-propanediol (AEPD) and tromethamine [63].

By comparing the retentivities of the four columns in Fig. 1,53], a 250 mm amino column, YMC-Pack NH2 gives N inhe range of 5000–17 000 (i.e., H = 15–50 �m) for benzoic aciderivatives, nucleic bases, and nucleosides peaks with k values

ig. 4. Structures of epirubicin and its analogues. (1) epirubicin, R = OH for A;2) epidaunorubicin, R = H for A; (3) doxorubicin, R = OH for B; (4) daunoru-icin, R = H for B.


Fig. 5. Effect of organic modifier on the separation of epirubicin and its ana-logues. (A) Column: Kromasil KR100-5SIL (250 mm × 4.6 mm I.D., 5 �m);mobile phase: sodium formate buffer (20 mM, pH 2.9) modified with variousorganic solvents (10:90, v/v). (B) Comparison of the chromatographic sepa-rFd

balc

Fig. 6. Influence of column type on the optimal flow rate for column efficiency.Column: (�) Chromolith Speed ROD RP-18e, (�) Betasil silica, (�) InertsilODS-2-C18; All columns used were 50 mm × 4.6 mm I.D., 5 �m (except for theChromolith Speed ROD RP-18e); Analyte: fluconazole; mobile phase: A 0.1%fRs

lOtnMwe6tdar

dtp

ation of epirubicin and its analogues at different buffer pH as shown in theigure. Compounds: (1) epidaunorubicin; (2) daunorubicin; (3) epirubicin; (4)oxorubicin [33].

elow 4. The retention tendency of the amino column is char-cteristic; for aromatic carboxylic acids, retention factors, k arearger than in the other three columns, and the separation effi-iency for these compounds was around N = 5000, which was

pbpt

Fig. 7. Structures of gl

ormic acid, B 0.1% formic acid in MeCN; For the Inertsil and Chromolith SpeedOD RP-18e, 25% B (the injection solvent was 100% water); For the Betasililica, 85% B (injection solvent was 100% MeCN) [29].

ess efficient than the other three columns as shown in Fig. 2.n the other hand, the amino column shows a similar separa-

ion efficiency to the ZIC-HILIC column for nucleic acids anducleosides. By studying on the relationship between k and theeCN concentration in the mobile phase, the amino columnas found to have significantly larger retention for salicylic acid

ven in 65% MeCN, while the k value decreased drastically in5% MeCN on other columns as shown in Fig. 8. For cytosine,he same study showed that the amino column has a similar ten-ency to other columns. By the Van’t Hoff plot for acetylsalicyliccid and salicylic acid, only the amino column showed positiveetention enthalpy.

The retention time of salicylic acid on the amino phaseecreases when a mobile phase with a lower MeCN concen-ration is used, or an increased salt concentration in the mobilehase is employed. These findings suggest that the hydrophilic
artition interaction and the ion-exchange interaction existetween analytes and the amino stationary phase for analytesossessing acidic functionalities [53]. For the carboxylic acids,he amino-silica column could generate only low efficiencies
ucuronides [43].

T. Ikegami et al. / J. Chromatogr

Fig. 8. The effect of MeCN content on the retention (A), and the Van’t Hoffplots (B) of acetylsalicylic acid. Column: (�) TSKgel Amide-80, (�) YMC-Pack NH2, (�) Atlantis HILIC Silica, and (×) ZIC-HILIC; 250 mm × 4.6 mmIpa

obsp

igpltbapwlo

rStN

afliYtAtrt9swav[dpd

tsatwaWdwwactcc

((tawaprkSNwA

e(ic

.D., 5 �m; (A) mobile phase containing 5 mM ammonium acetate; column tem-erature: 30 ◦C. (B) Mobile phase: MeCN/water (90/10, v/v) containing 10 mMmmonium acetate [53].

f N = ca. 5000, H = 50 �m. In addition, salt concentration anduffer pH effects indicated that the amino-silica phase was quiteensitive to these conditions in the case of analytes that possessedKa values in the pH range of the mobile phase employed.

Although amino-silica phases are useful separation median NPLC and HILIC, the reactive nature of amino functionalroups can cause several severe problems from an analyticaloint of view. For example, the irreversible adsorption of ana-ytes on the phase is often discussed: with aldehyde compounds,he primary amino group in the amino-silica phase forms a Schiffase, which results in the adsorption of carbonyl compounds,nd simultaneously changes the functionality of the stationaryhase. Thus, it is easy to understand why amino-silica phasesere not chosen to separate carbohydrates in more recent pub-

ications, though nobody ever produced any evidence that thisccurs under HILIC conditions with reducing sugars.

Oyler et al., employed amino-silica columns to sepa-
ate cyclic peptides, atosiban diastereomers. HILIC on apherisorb NH2 (250 mm × 4.6 mm I.D., 5 �m) separated
en atosiban diastereomers with a good separation efficiency,= 7000–11 000, H = 23–35 �m except for one diatereomer

s[rs

. A 1184 (2008) 474–503 481

t a flow rate of 0.5 mL/min [59]. However, under a fasterow rate, 1.2 mL/min, resolution of these compounds turned

nto worse, apparently. Separation of the same peptides on aMC Polyamine II column resulted in worse separation of

he analytes compared to the Sperisorb NH2 column [59].mong amino-silica phases, the Zorbax NH2 column was

ested over a period of several weeks, to take eleven separateuns with 1800 column volumes of the mobile phase. Afterhat, theoretical plate numbers for each peak were greater than4% of their original values. Moreover, retention factors andelectivities were hardly affected [54]. A very slow equilibriumhen substituting a citrate buffer by an acetate buffer on the

mino-phase was also reported. It took several hundred columnolumes of mobile phase to stabilize the baseline monitoring61]. Each brand of amino-silica columns showed significantlyifferent retention factors and selectivities for certain com-ounds. Comparisons of separation characteristics for threeifferent amino-silica columns are shown in Fig. 9 [54].

Contrary to the findings of Olsen, the self-decomposition ofhe stationary phase by amino groups in aqueous eluents washown, [69,70] and peak shape deterioration using HILIC werelso demonstrated [71]. Due to the self-decomposition reaction,he use of amino-phase in HILIC-MS causes higher backgroundsith ion suppression for certain analytes, and these phenomena

re not conductive to the sensitive detection of analytes [71].hen analyzing ginsenoides by LC–MS, these analytes were

etected as [M+Na]+ or [M+138]+ adducts [72]. The latter cationas caused by the addition of [NH3(CH2)3Si(OH)3]+, whichas caused by the stationary phase. There are several reports on

mino-phases based on different silica supports. Zirconia parti-les immobilized with amino functionalities were used in HILICo separate carbohydrates under a gradient elution. Six analytesontaining N-acetyl-chitooligosaccharides and maltooligosac-harides were completely separated [73].

Guo and Huang analyzed 2-amino-2-ethyl-1,3-propanediolAEPD) and tromethamine using three amino-silica columns150 mm × 4.6 mm I.D., 5 �m). The separation efficiency ofhe system for these amines in aqueous MeOH was estimateds 2800 (H = 54 �m, k = 0.2) and 3600 (H = 42 �m, k = 0.3),hile those in aqueous MeCN was 3700 (H = 41 �m, k = 1.2)

nd 6100 (H = 25 �m, k = 2.5). The separation by amino-silicahases, Zorbax NH2, YMC-Pack NH2, and Nucleosil NH2esulted in significantly different chromatograms, although

values of the three chromatograms were almost the same.lightly leading peaks were obtained by YMC-Pack NH2 anducleosil NH2, while Zorbax NH2 gave good shaped peaksith the highest separation efficiency of N = 4600 and 7300 forEPD and tromethamine [63].Amino-silica phases prepared from 3 �m particles were

mployed for the separation of tetracycline antibioticsFigs. 10 and 11). For the most retained solute, oxytetracycline,n 85% MeCN, N = 3800 (H = 13 �m) was obtained on an amino-olumn, ThermoHypersil APS2 (50 mm × 4.6 mm I.D.) with a
ame efficiency for a short-time retention factor stability test61]. After 5 weeks of daily use, theoretical plate numbers weree-evaluated to show they were greater than 90% of the initialeparation efficiency without change in retention factors [61].


Fig. 9. The separation of 2-amino-2-ethyl-1,3-propanediol (AEPD) and tromethamine. (A) Column: Zorbax NH2, 150 mm × 4.6 mm I.D., 5 �m; Mobile phase: (A)M lumnI ) Nua

caa((

tc

eCN/water (80/20), (B) MeOH/water (80/20) and (C) MeOH/water (90/10); Conjection volume: 50 �l. (B) Columns: (A) Zorbax NH2, (B) YMC-Pack NH2, (Cre the same to (A) [63].

A HILIC-ESI-MS/MS using an amino-silica column, Aste-apHera NH2 (150 mm × 4.6 mm I.D., 5 �m) was employed to
nalyze methionine, threonine, and taurine with a 60% MeOHs the mobile phase. A separation efficiency of N = 1500–2600H = 58–100 �m) was generated at a flow rate of 0.6 mL/minu = 3 mm/s) with a split ratio of 1/6 [64]. In a capillary elec-
aEvd

temperature: 25 ◦C; flow rate: 1 mL/min; samples: (1) AEPD, (2) tromethamine;cleosil NH2 column; mobile phase: MeCN/water (80/20, v/v). Other conditions

rochromatography, CEC mode, a polymer monolithic aminoolumn (280 mm × 100 �m I.D.) was employed to separate bile
cids and their conjugates [74] and saccharides [68] using anSI-Ion trap (IT)-MS as a detector. The separation efficiency isery high compared to the above-mentioned separations, and isiscussed later, in Section 4.1.


FC

3

ttu[aTtH

Uohscalttetum

p

ig. 10. Structures of tetracyclines [61]. (1) Tetracycline, R1 = R2 = H, (2)hlorotetracycline, R1 = Cl, R2 = H, Oxytetracycline, R1 = H, R2 = OH.

.3. Columns packed with amide-modified silica

HILIC type columns packed with amide-bonded silica par-icles are one of the most popular separation media. Amonghese, TSKgel Amide-80 from Tosoh (Tokyo, Japan) is widelysed for the separation of monosaccharides [75], peptides22,76], 2-pyridylamino (PA)-sugar [77], ammonium [78], par-lytic shellfish poison (PSP)-toxin [79], and amino acids [80]. ASKgel Amide-80 (250 mm × 2.0 mm I.D., 5 �m) was applied

o a metabolomics study of pumpkin phloem excudates with aILIC-ESI MS system [81].A column, GlycoSep N from ProZyme (San Leandro, CA,

SA) was also employed to separate carbohydrates includingligosaccharide [82], 2-aminoacridone (AA) derivatized carbo-ydrates [83], and derivatized glycan [84]. Due to the chemicaltability, i.e. low reactivity of the amide moiety, this type ofolumns was less sensitive to the pH of the mobile phase,nd the irreversible absorption of analytes to the column wasimited [85]. The Amide-80 column was used for the separa-ion of peptides by Yoshida [22,85], and two-dimensional orhree-dimensional mapping of oligosaccharides [18–20], how-ver, descriptions in these reports were limited to the retentionimes of peptides or retention factors of oligosaccharides, and
nfortunately, the separation efficiency of the column was notentioned.Yanagida et al. developed a method to separate oligomeric
roanthocyanidins, polyphenolic catechins contained in an apple

Fig. 11. Separation of three tetracycline compounds by the HILIC mode. Col-umn: ThermoHypersil APS2 (50 mm × 4.6 mm I.D., 3 �m); mobile phase:MeCN–5 mM citrate buffer (pH 3.5) (85:15); flow rate: 1.1 mL/min, detection:UV, l = 270 nm; compounds: (1) unretained, (2) CTC, (3) TC, (4) OTC [61].

Fig. 12. Structures of flavan-3-ols and oligomeric proanthocyanidins [86].


Fig. 13. Relationship between log k of 15 standards of oligomeric proantho-cyanidins in HILIC and log Po/w values measured by high-speed counter-currentcs

uppdfFvogabt

ptttw1

Fof

Fig. 15. Chromatogram of the 15 standards of flavan-3-ols and oligomericpm1

HfladTdApt(as

sMD

hromatography. Inset regression formula was calculated from the linear least-quare fit of all data (n = 15) [86].

sing a HILIC-MS system. The logarithm of the octanol–waterartition coefficient, log P values for standard samples ofolyphenolic catechins (Fig. 12) were plotted against eachegree of polymerization Dp, and the logarithm of retentionactors, log k on a TSKgel Amide-80 column as shown inig. 13 [86]. It was apparent that compounds with larger Dpalues simultaneously resulted in the higher hydrophilic naturef polyphenols, and larger log k values. Numbers of hydroxylroups in analytes were plotted against log k on amide-silicas shown in Fig. 14 to show there was a linear relationshipetween the two parameters. Still there are few examples ofhe structure–retention relationship studies using HILIC.

The complete separation of tetramers and pentamers ofroanthocyanidins would have been difficult under the condi-ions in the above-mentioned isocratic separation, because ofhe significantly broadened peak of the tetramer and the pen-
amer. The separation efficiency of peaks 4a and 5a in Fig. 15ere estimated as to be N = 1000 for both, with k = 7 (4a) and7 (5a), respectively [86].
ig. 14. Relationship between log k of the 15 standards in HILIC and the numberf hydroxyl groups in their structures. Inset regression formula was calculatedrom the linear least-square fit of all data (n = 15) [86].

ulf

baprdv1aetcfowr

8

roanthocyanidins. Column: TSKgel Amide-80 (250 mm × 4.6 mm I.D., 5 �m);obile phase: MeCN–water (84:16); flow rate: 1 mL/min; injection volume:

0 �l. The numbers of standard compounds were shown in Fig. 12 [86].

Simultaneous analyses of mono- and oligosaccharides byPLC using a TSKgel Amide-80 column with a post-columnuorescence derivatization showed detection limits of d-glucosend maltohexaose as 1.78 and 2.59 pmol [87]. Karlsson et al.emonstrated the separation of native monosaccharides on aSKgel Amide-80 column, followed by ELS detection, withetection limits of 0.3–0.5 �g for each saccharide [88]. N-cetylneuraminic acid (Neu5Ac) and glucuronic acid (GlcA)eaks were completely separated from other analytes, andheir separation efficiency was estimated to be N = ca. 2000H = 125 �m) at 40 ◦C, and N = 3000 (H = 83 �m) for Neu5Ac,nd N = 2400 (H = 104 �m) for GlcA at 60 ◦C, respectively ashown in Fig. 16.

Wuhrer et al. prepared a capillary column packed with amideilica (100 mm × 75 �m I.D., 5 �m) to evaluate a nano-LC-ESI-

S system for the analysis of underivatized oligosaccharides.ue to the lower degree of sample dilution of the capillary col-mn, very high sensitivity of the saccharides at low-femtomoleevel was achieved [89]. A similar column was also employedor the analysis of glycosilated proteins [90].

The TSKgel application reported that an isocratic separationy a TSKgel Amide-80 (250 mm × 4.6 mm I.D., 5 �m) usingMeCN–water (75:25) mixture gave 80 000 theoretical plates

er meter for mannitol at room temperature, at the optimal flowate, between 0.15 and 0.3 mL/min using a reflective index (RI)etector [17]. By a rough estimation, a 25 cm column can pro-ide N = 20 000 with H = 12.5 �m. Under a practical flow rate of.0 mL/min (u = 1.5 mm/s), the H value reached around 20 �m,nd thus N = 12 000. The temperature effect on the separationfficiency of the Amide-80 column was also studied. At 80 ◦C,he separation efficiency against a flow rate for a non-reducingarbohydrate, mannitol showed small differences in the rangerom 0.2 to 2.0 mL/min (u = 0.30–3.0 mm/s), while in the casef a reducing carbohydrate, d-glucose, the separation efficiency
as significantly lost under practical separation conditions (flow
ate at 1.0 mL/min) as shown in Fig. 17A.Very recently, Tosoh released a new column, TSKgel Amide-

0 packed with 3 �m particles [91]. By comparing H–u curves


F s. CoM :18);N nic ac

bFe7H

(awIiMTatrelc

cgvst

erwdf

3s

i[alpsraps[

ig. 16. Separation of monosaccharides by a TSKgel Amide-80 columneCN–ammonium formate buffer (concentration is shown in the Figures) (82-acetyl-d-glucosamine, (Neu5Ac) N-acetylnueraminic acid, (GlcA) d-glucuro

etween 3 and 5 �m particle-packed columns as shown inig. 17B, the new column provides better separation efficienciesven under fast separation conditions. The minimum H reaches�m at around u = 2.7 cm/min, i.e. 0.45 mm/s. At u = 1.0 mm/s,was estimated at around 8 �m.A mannitol peak obtained using a TSKgel Amide-80 column

150 mm × 4.6 mm I.D., 3 �m) in MeCN–water (75:25) gavevalue of N = 18 000 within 7 min. The separation efficiencyas comparable to a conventional column (250 mm × 4.6 mm

.D., 5 �m), but the 5 �m particle packed column gave a sim-lar separation efficiency around 10 min as shown in Fig. 18.

annitol in pharmaceutical formulations was analyzed using aSKgel Amide-80 column (250 mm × 4.6 mm I.D., 5 �m), withn ELS detection [75]. By a chromatogram obtained in a mix-ure of MeCN and 0.1% CF3CO2H in water (75:25) with a flowate of 1.0 mL/min, the separation efficiency for mannitol wasstimated as N = 13 000 (k = 2.5). The plate height was calcu-ated as 19 �m, thus the separation efficiency of the system wasomparable to that exhibited in Fig. 16.

The retentivity of Amide-80 was compared with other HILIColumns, as shown in Fig. 1 [53]. The 250 mm Amide-80 column
ave N in the range of 15 000–20 000 (i.e., H = 12–16 �m) for kalues below 5. For peaks with k = 4–5, the separation efficiencylightly worsened when compared to less retained analytes. Onhe Amide-80 column, acids were less retained in 85% MeCN,
Prw

lumn: TSKgel Amide-80, (250 mm × 4.6 mm I.D., 5 �m); mobile phase:compounds: (Fuc) l-fucose, (Man) d-mannose, (Gal) d-galactose, (GlcNAc)id [88].

xcept for 3,4-dihydroxyphenylacetic acid, which was stronglyetained giving a split peak with significant tailing. Comparedith the other three columns, the amide column showed smallerecreases in separation efficiencies by the increasing of retentionactors as shown in Fig. 2.

.4. Columns packed with poly(succinimide)-modifiedilica

Alpert reported new types of silica-based stationary phasesn 1983 by further modifying aminopropyl silica phases92]. The treatment of an aminopropyl silica phase with

poly(succinimide) resulted in the formation of cova-ent bonds to anchor polymers. This stationary phase,oly(succinimide) silica can be a platform for several HILICtationary phases. Hydrolysis or aminolysis reactions of theemaining succinimide rings using hydroxide, 2-aminoethanol,nd 2-aminoethylsulfonic acid leads to the preparation ofoly(aspartic acid), poly(2-hydroxyethyl)aspartamide, poly(2-ulfoethyl)aspartamide bonded silica phases as shown in Fig. 191].

These phases are available from PolyLC as PolyCAT A,olyHydroxyethyl A, PolyGlycoplex, and PolySulfoethyl A,espectively. Although these stationary phases are applied in aide variety of fields, their separation efficiencies have not been


Fig. 17. The Van Deemter plots on TSKgel Amide-80 columns. (A) Col-umn: TSKgel Amide-80, (250 mm × 4.6 mm I.D., 5 �m); mobile phase:MeCN/water = 80/20; temperature: 80 ◦C; detection: RI; sample: A, d-glucose,B, mannitol [14]. (B) Columns: (�) TSKgel Amide-80, (150 mm × 4.6 mm I.D.,3 �m), (�) TSKgel Amide-80, (250 mm × 4.6 mm I.D., 5 �m); mobile phase:MeCN/water = 75/25; temperature: 40 ◦C; detection: RI; Flow rate: 1.0 mL/min;injection volume: 10 �L; sample: mannitol [91].

Fig. 18. Comparison of chromatograms for sugar alcohols on TSKgel Amide-80 3 �m and 5 �m columns. A: TSKgel Amide-80, (150 mm × 4.6 mm I.D.,3 �m; B: TSKgel Amide-80, (250 mm × 4.6 mm I.D., 5 �m); Chromatographicconditions are the same to Fig. 17B except for the column temperature was25 ◦C. Compounds: (1) ethylene glycol; (2) glycerin; (3) erythritol; (4) xylitol;(5) mannitol; (6) inositol [91].

F

dea

3

aoadwb

Tapwtoc

(tpaHm(

ig. 19. Preparation scheme of poly(succinimide)-based stationary phases.

iscussed in detail. In this chapter, examples of application usingach phase are introduced first, and then separation efficienciesre discussed.

.4.1. PolyHydroxyethyl ASeparations with phosphorylated and non-phosphorylated

mino acids, dipeptides, derivatized maltooligoglycosides, andligothymidylic acid were shown by Alpert [1]. Peptides [59]nd glycopeptides by HPLC [93,94] and by CEC [95], carbohy-rate derivatives [96], and ionic compounds by CEC mode [97]ere separated using this stationary phase. This phase can alsoe used for solid phase extraction media [98].

Tolstikov and Fiehn compared the separation ability ofSKgel Amide-80 and PolyHydroxyethyl A columns usingmixture of amino acids and carbohydrates (standard com-

ounds) in HILIC-MS mode [99]. As shown in Fig. 20, peakidths by the PolyHydroxyethyl A column are wider than

hose by the TSKgel Amide-80 column, although the elutionrder of standard compounds is quite different for the twoolumns.

Tolstikov et al. used a PolyHydroxyethyl A column150 mm × 0.6 mm I.D., 3 �m) to analyze plant leaf extracts. Inhe mass chromatogram, metabolites were eluted in the order ofolar lipids, flavonoids, glucosinolates, saccharides, and amino
cids [81]. For the separation of dipeptide standards by a Poly-ydroxyethyl A column (200 mm × 4.6 mm I.D., 3 �m) with aixture of MeCN–triethylammonium phosphate (TEAP) buffer
10 mM, pH 2.8) (8:2), and a flow rate of 2 mL/min, provided


Fig. 20. HILIC/MS base peak chromatograms of a mixture of standards. Columns: (A) PolyHydroxyethyl A, 150 mm × 1.0 mm I.D., 5 �m; (B) TSKgel Amide-80, 250 mm × 2.0 mm I.D., 5 �m; mobile phases: A, MeCN, B, 6.5 mM ammonium acetate (pH 5.5, adjusted by acetic acid); gradient: 15–35% B from 10 to6 yl-d-g1 nitol;2

spoa

(oHHcHa

A(rosac

3

cet

mbMftssc5r(h(

3

g[101]. The ring opening reaction of succinimde residues on sil-ica gel by 2-aminoethylsulfonic acid lead to a poly(2-sulfoethylaspartamide) phase, which possessed strong cation-exchangeproperties. The aforementioned phase was employed to sep-

Fig. 21. Chromatogram of small carbohydrates. Column: PolyGlycoplex,200 mm × 4.6 mm I.D., 5 �m; mobile phase: MeCN–10 mM TEAP (pH

0 min for (A); 15–55% B from 10 to 60 min for (B); compounds: (1) N-acet-deoxynojirimycin; (6) l-alanyl-l-alanine; (7) 1,4-dideoxy-1,4-imino-d-arabi-amino-2-deoxy-d-glucose; (12) maltoheptaose [99].

eparation efficiencies N = 2300–2900 (H = 69–87 �m) foreaks of k = 6–25 [1]. With higher concentrations or higher pHsf buffer solutions, separation efficiencies, retention factors,nd selectivities decreased resulting in poorer resolutions.

Oyler et al., employed a PolyHydroxyethyl A columns200 mm × 4.6 mm I.D., 5 �m) to separate atosiban diastere-mers with a fair separation efficiency, N = 5000–6000,= 33–37 �m except for one diatereomer (N = 1700,= 119 �m). Among ten diastereomers, 3 peptides were

o-eluted with other peptides, and resolution with the Poly-ydroxyethyl A columns was worse compared to that with an

mino-silica column [59].Under completely aqueous conditions, a PolyHydroxyethylcolumn (100 mm × 4.6 mm I.D., 3 �m) with a TEAP buffer

10 mM, pH 2.8), and a flow rate of 0.5 mL/min, provided sepa-ation efficiencies of N = 4500–5000 (H = 20–22 �m) for peaksf k = 5–7 [1]. The use of smaller particles results in highereparation efficiencies in this HILIC column. However, the sep-ration efficiency by this phase is lower than conventional RPLColumns packed with 3 �m or 5 �m particles.

.4.2. PolyGlycoplexThe separation of oligosaccharides using a PolyGlycoplex

olumn was reported by Alpert et al. [23]. The same column wasmployed to separate N-(p-nitrobenzyloxy)aminoalditol deriva-ized oligosaccharides [100].

The HILIC separation of small carbohydrates containingonosaccharides, N-acetylneuraminic acids, and sialyl sugars

y a PolyGlycoplex column (200 mm × 4.6 mm I.D., 5 �m) ineCN10 mM TEAP (pH 4.4) (80:20) provided N = ca. 3000

or peaks of k = 10, 23, and 26. Plate heights were calculatedo be 57–66 �m at a flow rate of 1.0 mL/min (u = 1.6 mm/s) ashown in Fig. 21 [23]. Fig. 22 is an example of the HILIC modeeparation of p-nitrobenzyloxy (PNB) derivatives of xyloglu-ans (XG) on a PolyGlycoplex column (200 mm × 9.4 mm I.D.,
�m) in 70% MeCN. For peaks of XG7, XG8, and XG9, sepa-
ation efficiencies were estimated as N = 9500 (k = 4.5), 10 000k = 6.3), and 9000 (k = 8.4), respectively from Fig. 22. Plateeights were calculated as 20–22 �m at flow rate of 1.0 mL/minu = 0.37 mm/s) [23].

4Caas

lucosamine; (2) sucrose; (3) d-(+)-raffinose; (4) l-methionine; (5) N-methyl-(8) uridine-5-diphosphoglucose; (9) stachyose; (10) glucosaminic acid; (11)

.4.3. PolyCAT A and poly(Sulfoethyl A)The polyCAT A column was used to separate histones under a

radient elutions, and separation efficiencies were not estimated

.4) buffer (80:20, v/v); flow rate: 1.0 mL/min; detection: UV, l = 275 nm;ompounds: (A) monosaccharides; (B) 2,3-didehydro-2,6-anhydro-N-cetylneuraminic acid; (C), N-acetylneuraminic acid; (D) N-glycolylneuraminiccid; (E) sialyl(2 → 3)lactose; (F) sialyl(2 → 6)-N-acetyllactosamine; (G)ialyl(2 → 6)lactose; (H) disialyllactose [23].

488 T. Ikegami et al. / J. Chromatog

Fig. 22. Chromatogram of PNB derivatized xyloglucans. The XG7, XG8 andXG9 contain 0, 1 and 2 galactose (Gal) residues, respectively. The two peaksllv

apcams

e

F2(

sN(ebco97Nttkwe

Mpdpucbap

abeled XG8 represent isomers with Gal in alternative positions. Column: PolyG-ycoplex, 200 mm × 9.4 mm I.D., 5 �m; mobile phase: MeCN–water (70:30,/v); flow rate: 1.0 mL/min [23].

rate peptides using HPLC [102] and CEC [95], and purifyeptides [103], basic compounds [104], and basic pharmaceuti-als [105]. In a fully aqueous mobile phase, the phase was used ascation-exchange media, as reported by Gilar et al. [106]. Chro-
atograms by Alpert provided limited examples of an isocratic
eparation of amino acids by the use of this phase [1].For peaks in Fig. 23 until 8 min (k < 6), the separation

fficiencies were calculated as N = ca. 4000, H = 50 �m. The

ig. 23. Chromatogram of amino acids. Column: PolySulfoethyl A,00 mm × 4.6 mm I.D.; mobile phase: MeCN–5 mM TEAP (pH 2.8) buffer80:20, v/v); flow rate: 2 mL/min [1].

3

tpapdacHaianbg(ZidctriChdae[

r. A 1184 (2008) 474–503

eparation efficiency at the last peak was estimated as k = 12.5,= 3000, H = 67 �m. This separation was done at 2.0 mL/min

u = 3.1 mm/s) flow rate, and judging from the above-mentionedxample with the PolyGlycoplex column, the flow rate may haveeen too fast to provide optimal separation efficiency. The sameolumn was employed to separate cyclopeptides using mixturesf MeCN-15 mM TEAP buffer (pH 2.8) in the following ratios;5:5, 90:10, 75:25, or 60:40 at a 1 mL/min flow rate. In 60 and5% MeCN, the separation efficiency was poor, but in 90%,= 9000 for a peak of k = 3 was generated. In 95% MeCN, reten-

ion factor became larger, but the separation efficiency decreasedo N = 5000 for a peak of k = 3.3, and N = 3000 for a peak of= 9.1. Therefore, this kind of phase should be used for analytesith small k values (from 2 to 5) to provide optimal separation

fficiencies under isocratic conditions [1].Column bleeding with poly(Sulfoethyl A) was reported by

ihailova et al. [107]. The authors found several interferingeaks with m/z ratio of 526, 568, 659, 712 and 714 in a two-imensional LC–MS/MS study. Peaks were regarded as theroducts from column bleeding of the poly(Sulfoethyl A) col-mn used as a strong cation-exchange media. When a newolumn was tested, levels of interfering peaks were negligi-le. The authors concluded that the lifetime of the column isround 1 month, before significant column bleeding becomesroblematic.

.5. Columns packed with sulfoalkylbetaine-modified silica

Irgum et al. introduced sulfoalkylbetaine zwitterionic func-ionality, given as CH2CH2N(CH3)2

+CH2CH2CH2SO3− to

olymer supports to prepare ion-exchange materials for the sep-ration of inorganic compounds [26,27] and proteins [28]. Thishase can separate anion and cation species simultaneouslyue to its zwitterionic structure. Recently, similar function-lity was immobilized on silica surfaces, and this type ofolumn, the ZIC-HILIC, was released from SeQuant. The ZIC-ILIC column is one of the most popular HILIC columns,

nd is widely used to analyze aminoglycosides [108] includ-ng neomycin [109], morphine and its derivatives [110], smallnd polar compounds [111], metabolomes [112,113], glucosi-olates [114], and PSP-toxins [115]. This phase can alsoe used for solid phase extraction or trapping media of N-lycosylated peptides [116] and GlycosylphosphatidylinositolGPI)-anchored peptides [117]. Schettgen et al. employed aIC HILIC column (100 mm × 4.6 mm I.D., 3.5 �m) in an

sotope-dilution HILIC-MS system, using [2H4]lysine (lysine-4) as an internal standard [118]. Under their experimentalonditions, retention times for lysine and lysine-d4 werehe same, and no deuterium effect was observed on theetention times. Phytosiderophores and their metal complexesn plants were analyzed by a ZIC HILIC-ESI-MS system.omplexes of mugineic acid (N-(3-(3-carboxypropylamino)-2-ydroxy-3-carboxypropyl)azetidine-2-carboxylic acid) and its
erivatives with Fe2+, Fe3+, Ni2+, Zn2+, and Cu2+ were sep-rated under a gradient elution, whereas metal complexesluted earlier than their corresponding metal free ligands119].


F[

g[pdcsTsml

wZah

wamppaw

fitNchtmbo[

trbfrVwHdc

Fig. 25. Chromatograms from the separation of three peptides mixture.Columns: (A) KS-polyMPC, 150 mm × 4.6 mm I.D., 5 �m, (B) Kromasil C18,1a(

Hdata by the column manufacturer, SeQuant. For the separation ofnucleic bases by a ZIC-HILIC column (150 mm × 2.1 mm I.D.,5 �m), separation efficiencies were estimated as 13 000 for ade-nine (k = 1.1), 10 000 for guanine (k = 1.7), 12 000 for cytosine

ig. 24. Synthetic scheme of the zwitterionic stationary phase KS-polyMPC123].

Takegawa et al. have reported the separation of N-lycopeptides [120], PA-N-glycans and PA-N-glycopeptides121] using HILIC-ESI-MS systems with the ZIC HILIChase. To optimize and evaluate ZIC-HILIC LC–MS or two-imensional LC, ZIC-HILIC-RP-LC–MS systems, Heck ando-workers selected a set of peptides from digests of bovineerum albumin, �- and �-casein, as a standard sample [122].hey found that peptides possessing at least two acidic residueshowed longer retention times at pH 8, while peptides containingore than two basic residues or lacking acidic residues showed

onger retention times at pH 3.The orthogonality of the ZIC HILIC phase against RP phase

as also studied. The authors concluded that the separation usingIC-HILIC at pH 6.8 was better than at pH 3 for a comprehensivenalysis of peptides, although the orthogonality with RP wasighest at pH 3.

A novel zwitterionic HILIC stationary phase immobilizedith phosphorylcholine type polymers was reported by Jiang et

l. [123]. A zwitterionic phase, KS-PolyMPC was prepared byulti-step chemical modifications as shown in Fig. 24. A column

acked with this phase showed different elution orders for threeeptides from a RP phase derived from the same silica supports shown in Fig. 25. Apparently, the separation efficiency wasorse in the HILIC mode (top) than the RP mode (bottom).The separation efficiency of the KS-PolyMPC was estimated

rom the peak width at a half height for the chromatogramn 60% MeCN-buffer in Fig. 26 as N = 6000 for three pep-ides with k < 1.8, N = 4000 for two peptides with k < 4.0, and

= 1500, and bradykinin with k = 7.5, respectively. Since theolumn dimension was 150 mm × 4.6 mm I.D., 5 �m, the plateeight was estimated as H = 25–100 �m. The mobile phase con-ained a smaller fraction of MeCN compared to the usual HILIC

ode, and the elution order was different from that predictedy the hydrophilicity. The authors supposed that the separationf peptides was mainly controlled by ion-exchange interactions123].

By comparing four HILIC type columns, on their retentivi-ies and the separation efficiencies, separation efficiencies in theange of N = 12 000–22 000, i.e. H = 11–21 �m were obtainedy using a ZIC-HILIC column (250 mm × 4.6 mm I.D., 5 �m)or carboxylic acids, nucleic bases, and nucleosides [53]. Theetention enthalpy value of salicylic acid estimated from thean’t Hoff plots indicated that the ZIC-HILIC phase had some-
hat specific interactions for the separation as shown in Fig. 8.owever, no ion-exchange effect was observed for benzoic aciderivatives on the ZIC-HILIC phase under the experimentalonditions.
FKI(

50 mm × 4.6 mm I.D., 5 �m; Mobile phases: (A) MeCN/10 mM ammoniumcetate buffer (pH 7) (60:40, v/v) (B) MeCN/10 mM ammonium acetate bufferpH 7) (5:95, v/v); flow rate: 1 mL/min; detection: UV, λ = 214 nm [123].

Further assessments of the separation efficiencies of the ZIC-ILIC column were carried out, according to the application

ig. 26. Chromatogram from the separation of six peptides mixture on theSpolyMPC zwitterionic column. Columns: KS-polyMPC, 150 mm × 4.6 mm

.D., 5 �m, mobile phases: MeCN/10 mM ammonium acetate buffer (pH 6)60:40, v/v); flow rate: 1 mL/min; detection: UV, λ = 214 nm [123].

4 atog

(oa5abifpw(GfVItsEL

3

svga(issovrtcfp

gas(Ht[NsHsIMsboli

4a1

1abt

bsTls[CafdH

twAbciTfbopposuw9MattF

gcrdsflwac


k = 3.2), respectively [25]. Thus the plate height was in the rangef H = 12–15 �m. The separation of dehydroascorbic acid andscorbic acid by a ZIC-HILIC column (150 mm × 4.6 mm I.D.,�m) resulted in N = 9000–10 000, H = ca. 15 �m as mentionedbove. However, the separation of morphine and its glucuronidesy a ZIC-HILIC column (50 mm × 4.6 mm I.D., 5 �m) resultedn poorer separation efficiencies of N = ca. 1500 and H = 33 �mor the two glucuronides (k = 2.8 and 4.1), and N = 3400 for mor-hine (k = 6.1). A significant band broadening was observedhen separating oligopeptides using a ZIC-HILIC column

100 mm × 4.6 mm I.D., 5 �m). For neurotensin (k = 0.7) andly-His-Lys (k = 5.5), N = 3000, H = 33 �m was obtained, but

or bradykinin (k = 2.3), N = 1800, H = 55 �m was obtained. Thean Deemter plot on a ZIC-HILIC column (150 mm × 4.6 mm

.D.) revealed that the optimal flow rate was relatively low, lesshan 0.5 mL/min (u = 0.83 mm/s). The ZIC-HILIC shows goodtability and low column bleeding among 19 columns tested byLSD, suggesting that the column is suitable for high-sensitivityC–MS analyses [25].

.6. Columns packed with cyano- and diol-modified silica

A diol bonded phase has been prepared by immobilizingilanol groups using glycidoxypropyltrimethoxysilane, to pro-ide a hydrophilic and neutral stationary phase bonding, for aood recovery of proteins [15]. According to a column char-cterization using a quantitative structure retention relationshipQSRR) [124,125], the overall polarity of the diol-bonded phases close to the bare silica phase, although the diol-bonded phasehowed slightly stronger retention for test compounds under aupercritical fruid chromatographic conditions with a mixturef CO2 and MeOH (90:10) as a mobile phase [126]. The log kalues on the diol column (a product of Princeton Chromatog-aphy, Cranbury, NJ, USA) for the test samples were similar tohose of a bare silica phase (Kromasil), while those of a cyanoolumn (a product of Princeton Chromatography) showed dif-erent patterns with smaller k values than silica and amino-silicahases.

Diol-bonded phases possess hydrophilic surfaces, and silanolroups can be partially blocked by a silylating agent tovoid the irreversible absorption of polar analytes onto thetationary phase. On a diol type column, LiChrosorb-DIOL250 mm × 4.6 mm I.D., 10 �m, Merck), provided N = 5300,

= 47 �m for sucrose (k = 2.3) and N = 3400, H = 74 �m for lac-ose (k = 3.4) in 75% MeCN as estimated from a chromatogram58]. In the same report, an amino-silica phase (LiChrosorb-H2, 250 mm × 4.6 mm I.D., 10 �m, Merck) also separated the

ame samples under the same mobile phase to give N = 3200,= 78 �m for galactose (k = 1.8), N = 5600, H = 45 �m for

ucrose (k = 2.8) and N = 4700, H = 53 �m for lactose (k = 4.1).n the diol phase, galactose and lactose peaks were broad in 85%

eCN, due to anomerization [58]. Non-reducing saccharides,uch as sucrose, had no such effect. This situation was improved
y adding ethyldiisopropylamine (EDIPA) to the mobile phaser by the separation at a higher temperature: the N value for aactose peak in 85% MeCN at 30 ◦C was 600 with k = 6.0, whilet changed into N = 1300, k = 3.4 in 85% MeCN–0.1% EDIPA at
ooRs

r. A 1184 (2008) 474–503

0 ◦C. The k value for sucrose at 40 ◦C was less than 2/3 of thatt 30 ◦C, but the N value of each sucrose peak was constant at600.

A mixed mode HILIC column, Acclaim Mixed Mode HILIC-was released from Dionex. The stationary phase contained

lkyl chains which possessed 1,2-diol functionality, and coulde used in RP and HILIC modes with wider application rangehan conventional diol columns [127].

The log k–log k plot between a cyano-silica phase and aare silica showed that a significant number of analytes pos-essed different selectivities between the two columns [126].he application of the cyano-silica to the HILIC mode is still

imited. Determination of piperadine in pharmaceutical drugubstances by a cyano-silica, Alltima Cyano (Grace Alltech)128]. The separation efficiency of the cyano silica, Alltimayano (250 mm × 4.6 mm I.D.) in MeCN–water-0.15% nitriccid (95:4.85:0.15) as a mobile phase, with an ELSD detectionor piperadine, piperadine citrate, and estropipate (pipera-ine salt of estrone sulfate) was estimated as N = 13 000, and= 19 �m [128].A cyano-silica column, TSKgel CN-80T did not retain pep-

ides in MeCN–water with a gradient elution, while the peptidesere strongly adsorbed onto an amino-silica phase, and TSKgelmino-80 provided sufficient recovery [129]. A chromatogramy Padarauskas et al. provided one of the limited example of ayano-silica in HILIC [130]. For the separation of a denaturantn alcohol formulations, a cyano-silica column (Separon SGX,ESSEK, Prague, Czech Republic) possessed larger retention

or Bitrex (denatonium benzoate), crystal violet, and methylenelue than bare silica and amino-silica columns. In a mixturef MeCN–5mM NaTFA (90:10, v/v), the cyano-silica columnrovided N = 17 000 for peaks around k = 4.5, and N = 9500 foreaks at k = 10.3, respectively. However, the final optimizationf the separation conditions seemed to be carried out using a bareilica column [130]. The separation efficiency of a diol type col-mn, Inertsil Diol (150 mm × 4.6 mm I.D., 5.1 �m, GL Science)as tested using urea, sucrose, and glycine. In a mobile phase of0% MeCN, the k value of glycine was around 25, while in 80%eCN, it decreased into 4. The effect of temperature on ln k

nd on the plate height, H for the three analytes was examinedo show that the lnk values decreased linearly as the tempera-ure rose, while the H value did not change linearly as shown inig. 27 [131].

At elevated temperatures, an extra-column effect becamereater than a lower temperature in terms of separation effi-iency loses for less retained solute, while the decrease of theesistance of mass transfer at higher temperatures resulted in theecrease of plate height for a long retained analyte, glycine. Forucrose, the column provided H = ca. 18 �m at a 0.35 mL/minow rate at 30 or 40 ◦C. A conventional HPLC column packedith 5 �m particles, was categorized as slow in terms of sep-

ration, and at 1.0 mL/min, the H increased to 30 �m. Theolumn effectively separated amino acids in HILIC, although the
ptimal flow rate was somewhat slow, at 0.3–0.5 mL/min. Therthogonal separation characteristics of the diol phase againstPLC were examined for pharmaceuticals and impurities (exact
tructures were not shown, but they contained –NH2, –OH,


F lumn:w 31].

–(accsmdsIaosbiotsH

as[

([f

3

ohtbis

abii�arabyak

wbmitTwuhouotaaLtTaT

ig. 27. Effect of column temperature (a) on ln k and (b) on plate height (H). Co/w); flow rate: 1.0 mL/min; detection: RI (♦) urea; (�) sucrose; (�) glycine [1

NHCONH2, or –OCONH– functionalities). A diol columnYMC-Pack, 250 mm × 4.6 mm I.D., 5 �m) gave N = 24 000 forless retained compound (k < 1) and N = 20 000 for a retained

ompound (k > 4). A direct comparison of the separation effi-iency of the diol phase and RPLC was difficult, since theeparation in RPLC mode was carried out with a linear gradientode [132]. A sensitive HILIC LC-ESI–MS–MS system was

eveloped to analyze choline and acetylcholine in microdialy-is samples. A Lichrospher DIOL 100 column (125 mm × 4 mm.D., 5 �m, Merck) was employed for a gradient elution. The sep-ration of both target analytes was good [133]. The importancef the sample solvent was discussed: when samples were dis-olved in 90% MeCN, all analytes were concentrated as narrowands, while those dissolved in water showed band broaden-ng. A similar separation was reported using a longer columnf the same packing material in gradient elutions using a mix-ure of HCOOH in H2O–MeCN, and a gradient elution thattarted with hexane, ethyl acetate, MeCN, and 0.1% HCOOH in2O [134].Diol-bonded silica releases its silylation reagent slowly under

cidic conditions, and the lifetime of the phase is extended if theimilarly modified silica column is connected as a pre-column135].

In a CEC mode, a polymer monolithic cyano column270 mm × 100 �m I.D.) was employed to separate saccharides68] using ESI-IT-MS as a detector. This issue is discussedurther in Section 4.1.

.7. Columns packed with cyclodextrin-modified silica

Cyclodextrins (CD) are cyclic oligosaccharides that consistf � 1–4 linkages of d-glucose units. CDs possess a relativelyydrophobic cavities due to their unique toroid structures, and
he formation of inclusion compounds within their cavities haseen studied in molecular recognition chemistry. Due to theirnteresting structure and molecular functions, CDs are widelytudied not only in analytical (chromatographic) chemistry, but
tTNfl

inertsil diol 150 mm × 4.6 mm I.D., 5 �m; mobile phase: MeCN–water (80:20,

lso in organic chemistry, pharmaceutical science, enzymology,iochemical sciences, food and cosmetics sciences. Molecularnteractions that form inclusion complexes can be used as driv-ng forces in the separation chemistry. The well-known CD are, �, and �-CD, in which, six, seven, and eight glucose unitsre linked together with 174, 262, and 427 A3 cavity volumes,espectively [136]. Among these, �-CD is the most studiednd widely used in the field of separation science. The CD-onded silica phase has been used for chiral separations for manyears [137–139], and CD-bonded monolithic silica columns ofconventional size [140] and a capillary size [141] are also

nown.A Cyclobond I 2000 (�-CD bonded phase) column (ASTEC)

as used to separate native oligosaccharides including ara-inosides, celloside, cyclodextrins, isomaltoside, maltosides,annosides, and xylosides that possessed degrees of polymer-

zation (Dp) from 1 to 8, using 70% MeCN as a mobile phase,o estimate chromatographic parameters, k, Rs, and N [142].hroughout the measurements, resolution, a Rs of around 2.5as obtained to separate oligomers differing by one saccharidenit. The relationship between k and Dp is interesting: generally,igher Dp yields larger k values for all saccharides, however,ligosaccharides with the same Dp with different carbohydratenits sometimes lead to quite different k values. One of the reasonf the selectivities will be understood by that the columns func-ion in the HILIC mode through interaction with the hydrophilicqueous layer on the exterior of the CD molecule. The selectivity,lpha, for one unit of saccharides did not seem to be constant.arger molecules gave slightly smaller alpha values, and this

endency was different from �(CH2) in the RPLC mode [55].he separation efficiency was in the range of N = 2300–7800,nd usually around 4000 for a 25 cm column, H = ca. 60 �m.hese values were quite low value for a modern HPLC system,

hough they could have been better at very slow linear velocities.o separate �-, �-, and �-CDs at a 1.0 mL/min of flow rate, thevalue was 3000, but it became N = 7000 at a 0.05 mL/min of

ow rate [142].

4 atogr. A 1184 (2008) 474–503

obcf(oV�vFl((ITtse

tl

saarlu

Npa“iu

cpaNsspp

Fig. 28. The Van Deemter plots for saccharides on CD bonded phases. (A) Col-umn: �-CD, Cyclobond III, 250 mm × 4.6 mm I.D.; mobile phase: MeCN–water


Separation efficiencies were estimated for the separationf oligosaccharides up to tetramers, and sugar alcoholsy Cyclobond III (�-CD bonded phase) and Cyclobond Iolumns (25 cm in length) as N = 4000–13 000 (H = 19–63 �m)or oligosaccharides (k = 2.5–9.5), and N = 5000–13 000H = 19–50 �m) for sugar alcohols, even at a 1.5–2.0 mL/minf flow rate of the aqueous MeCN mobile phase [143]. Thean Deemter plot for fructose, sucrose, and lactose on a 25 cm-CD column (Cyclobond I) showed that the optimal linearelocity for these three saccharides was different as shown inig. 28. Relatively fast separation, u = 2 mm/s was preferred for

ong retained analytes such as sucrose (k = 2.71), and lactosek = 3.84), while u = 0.4 mm/s was the optimum for fructosek = 1.25). The same study on a 25 cm �-CD column (CyclobondII) also showed the same tendency, but with better efficiencies.he CD-bonded columns are stable and reproducible compared

o amino-silica phases. After 3000 injections of standardamples, the old column provided comparable selectivities andfficiencies to the new column.

The Cyclobond I column was applied to a LC-ESI–MS sys-em to analyze oligosaccharides up to 11 glucose units. Theimits of detection were as low as 50 pg for test analytes [144].

The Cyclobond I column was employed for the HILIC modeeparation of polar test compounds as shown in Fig. 29, andmino acids such as aspartic acid, proline, leucine, methionine,nd norvaline. The cyclodextrin-bonded column showed greateretention than that of TSKgel Amide-80 phase for several ana-ytes [145]. For a compound A, the chiral separation was realizednder the HILIC conditions.

A �-CD-bonded phase, Nucleodex beta OH (Macherey-agel, Duren, Germany) was used for the separation ofhosphorylated carbohydrates [146]. Novel silica-based station-ry phases modified with �-CD, glucose and maltose using aclick chemistry” developed by Lei and Liang [147], were exam-ned to separate nucleosides, saccharides, and sugar alcoholsnder HILIC conditions [148].

Judging from a chromatogram obtained under an isocraticonditions, the Click Maltose column (150 mm × 4.6 mm I.D.)rovided N = 8000–10 000 (H = 15–19 �m) for uracil (k = 1.0),denosine (k = 2.6), guanosine (k = 5.3), in MeCN–10 mMH4OAc buffer (85:15), respectively. The Click �-CD column

eparated 11 carbohydrate derivatives under a gradient modeeparation. Further detailed characterization of these stationaryhases is required to examine their potentials as a new stationaryhases.

(78:22, v/v); compounds: (�) ribose, (�) sorbitol, (�) sucrose, (©) glu-cose. (B) Column: �-CD, Cyclobond I, 250 mm × 4.6 mm I.D.; mobile phase:MeCN–water (85:15, v/v); compounds: (�) fructose; (�) sucrose; (�) lactose[143].

Fig. 29. Structures of test compounds [145].

atogr

3s

tipswtwhfaNaghpps

fif2Lch[c(c(aoNNt

wamftHvckopgptap

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ctma


.8. Columns packed with other functionality-modifiedilica, and polymer particles

Various silica-based phases modified with different func-ionalities from above-mentioned groups, are discussedn this category. The temperature responsive nature ofoly(N-isopropylacrylamide), PNIPAAm was incorporated toilica-based phases by Kanazawa et al. The column packedith PNIPAAm modified silica particles showed RP charac-

eristics over the lower critical solution temperature (LCST),hile it exhibited a hydrophilic nature at 5 ◦C [149]. PNIPAAmydrogel-modified silica phase was also reported. Startingrom amino-silica, immobilization of a radical initiator, 4,4′-zobis(4-cyanovaleric acid), and on-silica polymerization of-isopropylacrylamide with N,N′-methylenebis(acrylamide) ascrosslinking agent resulted in the preparation of the hydro-

el on silica surface [150]. The aforementioned phase showed aydrophilic nature under 5 ◦C, although a fully aqueous mobilehase was employed in these studies. These phases have theotential to be used in HILIC, but their separation efficiencieseem poor at lower temperatures.

A chiral stationary phase, Chirobiotic T (ASTEC), modi-ed with a macrocyclic antibiotic, teicoplanin was employedor chiral separations under HILIC conditions [145]. With a5 cm column, N = 7000–17 000 (H = 15–36 �m) was achieved.iu et al. reported [151] the synthesis and chromatographicharacterization of a silica-based phase functionalized by per-ydroxy cucurbit[6]uril, a macrocyclic compound with a cavity152]. The cavity of the cucurbit[6]uril was similar to that of �-yclodextrin [153]. In a mixture of MeCN and phosphate buffer5 mM, pH 3.48) (80:20, v/v), a good separation of alkaloids wasarried out on a column packed with the cucurbit[6]uril-silica150 mm × 4.6 mm I.D., 5–7 �m). For less retained solutes suchs narceine and berberine, N = 2000–4000 (H = 38–75 �m) wasbtained, while, for nicotine, significant broadening of the peak,= 600 (H = 250 �m) was observed. Interestingly, for brucine,= 8000 was provided though it had larger retention than nico-

ine.In 2007, a silica-based column, the Cosmosil HILIC phase

as released from Nacalai Tesque (Kyoto, Japan). The station-ry phase contains a 1,2,4-triazol group (the immobilizationethod of the polar heterocyclic compound to the silica sur-

ace is not shown), and possesses unique retentivity dueo the basic nature of the triazol [154]. By a CosmosilILIC column (250 mm × 4.6 mm I.D., 5 �m), water solubleitamins, carboxylic acids, amino acids, and polar pharma-euticals were separated with high efficiency; for analytes of= 0.5–6.5, N = 12 500–22 000, H = 11–20 �m and a flow ratef 1.0 mL/min were obtained. Application data for 154 com-ounds are available. For the separation of phosphorylatedlucose, amino acids (histidine and O-phospho-l-serine) andeptides, the separation efficiency was worse (H = 30–45 �m)han with other examples. The column can also be used as

weak anion-exchange stationary phase by changing mobilehases.

A cellulose-based column was used to separate N-linkedligosaccharides from hydrazinolysates of glycoproteins using

maaw

. A 1184 (2008) 474–503 493

mixture of 1-butanol–ethanol–water (4:1:1, v/v) or a mixturef ethanol–water (1:1, v/v) [155].

Polymer-based stationary phases with amino groups are alsonown. A column, Phenomenex AshaipakNH250D was appliedor the separation of 5-fluorouracil in plasma and tissue usingHILIC-APCI-MS system [156]. These types of phases were

mployed for the separation of PA-oligosaccharides [157], oraurine and methionine in matrices rich in carbohydrates [64].ccording to a chromatogram of the separation of taurine,

hreonine, and methonine on an ASTEC apHeraNH2 under iso-ratic conditions with 60% MeOH, the separation efficiencyy a column (150 mm × 4.6 mm I.D., 5 �m) was estimated as= 1500–2600 (H = 58–100 �m).Porous, spherical polystyrene–divinylbenzene particles func-

ionalized with neutral hydrophilic groups, Rogel-P (notentioned in detail) were invented by Yang and Verzele. A

olumn packed with particles, Rogel-P, was evaluated in 40%eCN. The efficiency of 50 000 plates/meter was obtained for

henol [158]. Generally, columns packed with polymer-basedarticle for the HILIC mode generate poorer separation effi-iencies than those of silica-based columns, and the use ofolymer-based materials should be limited under separationssing strong acidic or basic conditions.

. Separation efficiencies of monolithic hydrophilicnteraction chromatography mode columns

Monolithic materials, one-piece structure consisting of skele-ons and through-pores have recently attracted the attentions ofhromatographers recently. First, organic polymer monolithicolumns [159,160], then silica monolithic columns [161,162]ere reported. Monolithic materials possess several features

s supports for chromatographic stationary phases such asigher mechanical stabilities in comparison to particle-packedolumns, large through-pores which results in higher perme-bilities or lower backpressure than particle-packed columns,mall-sized skeletons to allow fast mass transfer within the sta-ionary phase, high porosities in monolithic columns lead to lowhase ratios, i.e. lower sample capacities and smaller retentivi-ies than particle-packed columns. These findings were reviewedepeatedly over the years [163–185], and judging from theseeviews, organic polymer monolithic columns have been mainlysed in CEC and for the separation of biological moleculesbiopolymers), while monolithic silica columns have beenmployed for both of HPLC and CEC mainly of small molecules.

.1. Polymer monolithic columns

Investigations on the use of organic polymer monolithicolumns is still limited, and there are only a few reports onhe subject even in 2007. Xie et al., prepared porous hydrophilic

onoliths using acrylamide and N,N-methylenebis(acrylamide)s starting materials, however, they did not report on their chro-
atographic characteristics [186]. Viklund and Irgum preparedpolymer monolith column containing zwitterionic function-
lity of sulfoalkylbetaine moiety in 2000 [187]. The columnas obtained by a photo-induced copolymerization of trimethy-

4 atogr. A 1184 (2008) 474–503

lmittm(ifa

lapH

Fu8n((v2d2


olpropane trimethacrylate (TRIM) and N,N-dimethyl-N-ethacryloxyethyl-N-(3-sulfopropyl)ammonium betaine (SPE)

n a glass column (150 mm × 2.7 mm I.D.). This column hashe potential for HILIC separation, but has been supplied forhe cation-exchange mode separation of proteins. The SPEonomer was co-polymerized with ethylene dimethacrylate

EDMA) in a glass tube (150 mm × 2.7 mm I.D.) by photo-nduced polymerization [28]. The column was evaluated in aully aqueous mobile phase for the separation of inorganic saltsnd proteins.

A similar methacrylate-based monolithic column in capil-
ary was reported by Smith et al., to provide N = 15 000 m−1
t a linear velocity of 2 mm/s [188]. The column was pre-ared from SPE and EDMA in a 100 �m I.D. capillary. UnderILIC conditions, no evidence of swelling or shrinking of the

ig. 30. Chromatograms obtained by a poly(SPE-co-EDMA) monolithic col-mn. Column: 285 mm × 100 �m I.D.; Detection: UV, λ = 214 nm; flow rate:00 nL/min; injection: 100 nL. (A) Mobile phase: MeCN–water (50 mM ammo-ium formate pH 3.0) (95:5, v/v); compounds: (1) toluene; (2) methacrylamide;3) acrylamide; (4) thymine; (5) uracil, (6) adenine, (7) thiourea, (8) cytosine.B) Mobile phase: MeCN–water (50 mM ammonium formate pH 3.0) (75:25,/v); Compounds: (B) benzoic acid, (3-HB) 3-hydroxybenzoic acid, (2-HB)-hydroxybenzoic acid, (3,4-DHB) 3,4-dihydroxybenzoic acid, (3,5-DHB) 3,5-ihydroxybenzoic acid, (3,4,5-THB) 3,4,5-trihydroxybenzoic acid, (2,6-DHB),6-dihydroxybenzoic acid [188].

Fig. 31. Chromatograms on a TEPIC–BACM monolithic column. Column:TEPIC–BACM, 36.4 cm × 100 �m I.D.; mobile phase: MeCN–water (90:10,v/v); detection: UV, λ = 210 nm (off column, 9 cm × 50 �m I.D.); pressure:5pc

cuipo(w

p((ecwMs(cMmo

FGRTRX

5 kg/cm2; flow rate: 0.5 mL/min (split method); temperature: 30 ◦C; com-ounds: adenine, thymine, uracil, cytosine, adenosine, guanosine, uridine, andytidine [189].

olumn bed was observed. When the estimation of the col-mn efficiency as above was adopted to two chromatogramsn this report, it was found that bases and neutral com-ounds were separated with N = 1900–4500 (H = 66–158 �m)n a column (300 mm × 100 �m I.D.), while N = 800–1300H = 230–375 �m) was obtained for benzoic acid derivativesith the same column as shown in Fig. 30.Polymer-based monolithic columns in capillaries were pre-

ared by reacting between tris(2,3-epoxypropyl) isocyanurateTEPIC) and 4-[(4-aminocyclohexyl)methyl]cyclohexylamineBACM) or trans-1,2-cyclohexanediamine (CHD) in the pres-nce of poly(ethylene glycol). This type of monolithicolumn apparently had a different morphology comparedith previously reported polymer monolith [189]. In 90%eCN, a TEPIC-BACM column (364 mm × 100 �m I.D.)

eparated nucleosides with efficiencies of N = 3200–27 000H = 13–113 �m) as shown in Fig. 31. Column efficien-
ies and permeabilities were quite high, and for benzene ineCN/20 mM sodium phosphate buffer pH 7.0 60/40 (v/v), opti-um plate height reached 5 �m within the linear velocity range
f 1–2 mm/s.

ig. 32. Structures of the bile acids. CA: R = OH, R1 = H, R2 = OH, X = OH;CA: R = OH, R1 = H, R2 = OH, X = NHCH2CO2H; GDCA: R = H, R1 = H,

2 = OH, X = NHCH2CO2H; GLCA: R = H, R1 = H, R2 = H, X = NHCH2CO2H;CA: R = OH, R1 = H, R2 = OH, X = NHCH2CH2SO3H; TDCA: R = H,

1 = H, R2 = OH, X = NHCH2CH2SO3H; TLCA: R = H, R1 = H, R2 = H,= NHCH2CH2SO3H [74].


Fig. 33. Normal-phase CEC/negative-ion ESI-MS analysis of the bile acidsin Fig. 32. Column: polymer monolith, 30 cm × 100 �m I.D.; mobile phase:MeCN–water–240 mM ammonium formate buffer (pH 3) (60:35:5, v/v/v); fieldstrength, 400 V/cm; injection, 6 kV, 5 s, compounds: a mixture of standards con-taining free bile acids, together with glycine and taurine conjugates (TLCA:0.2 mg/mL, TDCA: 0.1 mg/mL, TCA: 0.05 mg/mL, GLCA: 0.5 mg/mL, GDCA:0.2 mg/mL, GCA: 0.2 mg/mL, and CA: 0.1 mg/mL) [74].

Fig. 34. Electrochromatogram of a mixture of mono- and disaccharides. Col-umn: polymer monolithic cyano column, 27 cm × 100 �m I.D.; mobile phase:Msm

ebc

Fig. 36. Relationship between log tR and DP of linear maltooligomers. Exper-ipv

m[AcatuF

fpamMsta

Fa1

eCN–water–240 mM ammonium formate buffer (pH 3) (76:23:1, v/v/v); fieldtrength, 500 V/cm; injection, 4 kV, 10 s; compounds: each sugar (0.8 mg/mL);ass range: 100–600 Da [68].

A long column (150.5 cm) produced very high separationfficiencies of N = 140 000–210 000 (H = 7–11 �m) for alkyl-enzenes, but no such result has been reported for polarompounds [189].

ba

m

ig. 35. Electrochromatographic separation of anomeric maltooligosaccharides frommino column, 28 cm × 100 �m I.D.; mobile phase: MeCN–water–240 mM ammonikV, 10 s; compounds: dextrin sample, 4 mg/mL; Mass range: 150–1300 Da [68].

mental conditions were the same as shown in Fig. 37, except that the mobilehase was MeCN–water–240 mM ammonium formate buffer (pH 3) (60:39:1,/v/v) [68].

Que and Novotny reported on the preparation of poly-er monolithic columns containing amino functionalities,

2-(acryloyloxy)ethyl]trimethylammonium methyl sulfate (2-ETMA) and 3-amino-1-propyl vinyl ether (APVE) in a

apillary. The column (300 mm × 100 �m I.D.) was applied ton analysis of bile acids (Fig. 32) by a CEC-ESI-MS systemo generate very high separation efficiencies, 610 000 plates/msing 60% MeCN as a mobile phase as shown inig. 33 [74].

Que and Novotny prepared a cyano-phase polymer monolithor CEC mode also using 2-cyanoethyl acrylate as one com-onent of the column. This column and the above-mentionedmino column were employed in the analysis of neutralono-and oligosaccharides, and sugar alcohols in a CEC-ESI-S system [68]. The mono- and oligosaccharides were well

eparated on the cyano phase as shown in Fig. 34, in a mix-ure of MeCN–water–NH4OCHO buffer (76:23:1, v/v/v). At

higher concentration of MeCN, MeCN–water–NH4OCHO
uffer (95:4:1, v/v/v), anomers of fucose, N-acetylglucosamine,nd glucose were separated completely.
Sugar alcohols, fucitol, ribitol, xylitol, N-acetylglucosa-initol, glucitol, and mannitol were also separated using the

Dextrin 20 (degree of polymerization, DP) 1–9). Column: polymer monolithicum formate buffer (pH 3) (70:29:1, v/v/v); field strength: 500 V/cm; injection:

4 atogr. A 1184 (2008) 474–503

cvrMFatlt

cuctem

4

elmiaHsMcwflecomr

iAc1

Fig. 38. The Van Deemter plots by the monolithic silica columns with orwithout the hydrothermal treatment. Symbols: not tailored, (�) in CEC and(�) in nano-LC; 1 h tailored, (�) in CEC and (�) in-LC; column: SMC-2: not tailored, 46.6 cm × 75 �m I.D.; mobile phase: MeCN–water (95:5,v/v); Solute: dyphylline (k = 0.75); detection: UV, λ = 214 nm; 1 h tailored:L = 14 cm × 75 �m I.D. (effective length: 8.5 cm); mobile phase: MeCN–Tris0λ

eC

ltp

ifafgm(vta

F8t


yano column in MeCN–water–NH4OCHO buffer (95:4:1,/v/v). Using the amino column [74], maltooligosaccha-ides (degree of polymerization 1–9) were separated in

eCN–water–NH4OCHO buffer (70:29:1, v/v/v) as shown inig. 35. The separation efficiency of the column was calculateds 200 000 plates/m, H = 5 �m. Oligosaccharides were eluted inhe order of their molecular size as shown in Fig. 36, whereog tR was plotted against the degree of polymerization of mal-ooligosaccharides.

Even though they are used in CEC to separate polarompounds, probably due to their high backpressure dropnder HPLC conditions, applications with polymer monolithicolumns to HILIC are still limited. Without a few excep-ional columns, it is difficult to generate higher separationfficiencies than the particle-packed HILIC columns in HPLCode.

.2. Silica monolith columns

In spite of extensive researches on monolithic silica columns,xample of their use in the HILIC mode is still surprisinglyimited. Merck released a monolithic bare silica column, Chro-

olith Performance Si, (100 mm × 4.6 mm I.D.) [190], but theres only one example of its use in a HILIC separation. Packnd Risley evaluated the above-mentioned Chromolith Si in theILIC mode for inorganic ions such as lithium, sodium, potas-

ium, chloride, and drugs like naproxen and warfarin in 80%eCN with ELS detection [50]. For these substances, the effi-

iency was estimated as N = 1000–4800, for rapid separationith a flow rate of 2.0 mL/min. In 60% MeCN with a 1.0 mLow rate, the retentions of ionic species was smaller, and thefficiencies were better N = 5000, than those in 80% MeCN. Theolumn efficiency, however, is much lower compared to that ofctadecylsilylated monolithic silica column, Chromolith Perfor-ance C18, which generates H = 8 �m at around a 2 mm/s flow

ate.The HILIC mode separation using monoithic silica columns

n capillaries were recently reported by Demesmay et al. [51,52].lkaloids were analyzed in CEC by a bare monolithic silica

olumn (85 mm (effective length) × 75 �m I.D.) and in MeCN-mM Tris buffer (pH 7.5) (95:5, v/v) with high separation

wAsw

ig. 37. Separation of xanthines in CEC on unmodified monolithic capillary. Colum.5 cm); mobile phase: MeCN–Tris 1 mM (pH 7.5) (95:05, v/v); field strength: V = 30heophylline; (4) 1,7-dimethylxanthine; (5) b-hydroxyethyltheophylline; (6) dyphylli

.1 mM (pH 7.5) (98:2, v/v); solute: dyphlline (k = 0.3), detection: UV,= 214 nm [52].

fficiencies, N = 9300–17 500 as shown in Fig. 37 [51]. In theEC mode, the plate height reached to 5 �m (Fig. 38).

Since Puy et al. used a small sized (85 mm (effectiveength) × 75 �m I.D.) monolithic silica capillary column, theotal separation efficiency of the HPLC system was below 20 000lates.

Another approach to prepare HILIC type monolithic sil-ca columns utilizes polymerization of vinyl monomers havingunctional groups, such as amides, carboxylic acids, sulfoniccids, amines, and ammonium salts, to immobilize variousunctionalities onto silica surfaces modified with methacryloylroups. In this procedure, monolithic silica capillary columnsodified with 3-(methacryloxypropyl)trialkoxysilanes, or 3-

methacrylamidopropyl)trialkoxysilanes can be used as “uni-ersal platforms”. Into the column, a solution containinghe above-mentioned monomer, and a radical initiator suchs 2,2′-azobisisobutyronitrile or ammonium peroxydisulfate,ere charged, and polymerization reaction was carried out.
fter washing out polymers that did not bind to the column
urfaces, polymer-coated monolithic silica capillary columnsere obtained [191–197]. Starting from the modification of

n: W-SMC-2 silica monolithic column, 39 cm × 75 �m I.D. (effective length:kV; detection: UV, λ = 214 nm; Compounds: (1) naphthalene; (2) caffeine; (3)

ne [51].


F

s3m(3(p2pHepF

af

FaPvu([

Fig. 41. The Van Deemter plot with a column backpressure plot for thea poly(acrylamide)-coated monolithic silica column, 200T-PAAm. Column:200T-PAAm, 285 mm × 200 �m I.D.; mobile phase: MeCN–13 mM ammo-nium acetate buffer (pH 4.7) (80:20, v/v); temperature: 30 ◦C; detection:UtM

ccngmppc

cushown in Fig. 40 [191]. After the silica support was improvedto increase the phase ratio [198], the PAAm type column couldgenerate H = 7 �m for an optimal flow rate of 1 mm/s, as shownin Fig. 41 [194].

ig. 39. Synthetic scheme of the polymer-coated monolithic silica columns.

ilica using 3-methacryloxypropyltrimethoxysilane (MOP) or-methacrylamidopropyltriethoxysilane (MAS), various vinylonomers such as acrylamide (AAm) [191,194], acrylc acid

AA) [192,193], octadecyl methacrylate (OM) [196,197],-diethylamino-2-hydroxypropyl methacrylate (DAHMA), 2-triethylammonium)ethyl methacrylate chloride (DMAEA-Q),-styrenesulfonic acid sodium salt (pSSA), and 2-acrylamido--methylpropanesulfonic acid (AMPS) [195] were used. Theseolymer-coated monolithic columns were characterized asILIC phases for AA and AAm, RP phases for OM, cation-

xchange phases for pSSA and AMPS, and anion-exchangehases for DAHMA and DMAEA-Q, respectively as shown inig. 39.

This method for the stationary phase preparation has severaldvantages over the preparation of particle-packed columns asollows: (1) the MOP or MAS modified monolithic silica column

ig. 40. Chromatograms of nucleic bases and nucleosides separations onpoly(acrylamide)-coated monolithic silica column, PAMS-(100). Column:

AMS-(100)-1 (38 cm × 100 �m I.D.); mobile phase: MeCN–water (90:10,/v); temperature: ambient; detection: UV, λ = 260 nm, Linear velocity:= 1.2 mm/s at �P = 2.1 MPa; compounds: (1) thymine; (2) uracil; (3) adenine;

4) uridine; (5) adenosine; (6) cytosine; (7) guanine; (8) cytidine; (9) guanosine191].

FoAgT3Rm

V, λ = 210 nm; solute: guanosine (k = 3.8); sample volume: 1.0 �l injec-ion (split), column: ZIC-HILIC, 150 mm × 4.6 mm I.D., 5 �m; mobile phase:

eCN–buffer (80:20, v/v); temperature: 25 ◦C; solute: cytosine (k = 1.3) [194].

an be used as universal platforms, and various stationary phasesan be prepared by changing only the monomers; (2) there is noeed to prepare silanes possessing highly reactive functionalroups; (3) a good permeability and column efficiency can beaintained after the polymerization; (4) it is free from column

acking; (5) the amount of bonded phase can be controlled by theolymerization conditions; (6) the phase ratio can be increasedompared to the silica modification using chloroorganosilanes.

In earlier stages of this research, a monolithic silica columnoated with polyacrylamide (PAAm) generated H = 16 �m forridine in 90% MeCN (the bare silica showed H = 10 �m) as

ig. 42. Kinetic plot of log(t0/N2) vs. log N with the assumed maximum pressuref 20 MPa for the (�) MS-200T-PAAm, (�) the ZIC-HILIC, (�) the TSKgelmide-80, 5 �m, and (�) the TSKgel Amide-80, 3 �m columns. Chromato-raphic conditions for TSKgel Amide-80 columns are as follows. Column:SKgel Amide-80, 250 mm × 4.6 mm I.D., 5 �m and 150 mm × 4.6 mm I.D.,�m; mobile phase: MeCN–water (75:25, v/v); temperature: 40 ◦C; detection:I; Solute: mannitol (k = 2.5); sample volume: 10 �l injection. For other chro-atographic conditions, see Fig. 43 [194].

4 atogr. A 1184 (2008) 474–503

aPu5ewwwsFta

PWabeme

wbt[

Fig. 43. Plot of separation impedance against the linear velocity of themobile phase. Chromatographic conditions: column: poly(acrylic acid)-coatedmonolithic silica column, MS-200T-PAA(30), 200 mm × 200 �m I.D. (�), inMeCN–water(0.2% formic acid) (90:10, v/v) temperature: 25 ◦C; solute: adeno-s1p

at

FM(


By kinetic analyses in which log(t0/N2) values were plottedgainst log N values under an limiting of pressure of 20 MPa, theAAm modified column was compared with a ZIC-HILIC col-mn, and two TSKgel Amide-80 columns packed with 3 �m and�m particles to find that the monolithic type column could gen-rate the same separation efficiency as particle-packed columns,ith three time faster separation times than the columns packedith 5 �m particles, as shown in Fig. 42 [194]. This columnas operated with a fast linear velocity, u = 7.6 mm/s without

howing a severe loss in the separation efficiency as shown inig. 43. The above-mentioned column provides an example of

he separation of oligosaccharides in HILIC and detection usingLC-ESI–MS [194].

A monolithic silica column coated with poly(acrylic acid)AA, also generated H = 9–10 �m at a flow rate of 1.0 mm/s.hen the separation impedance E was compared to that ofparticle-packed column, the difference in column supports

ecame clear. The particle-packed column lost its columnfficiency at linear velocities higher than 1 mm/s, while theonolithic column showed no significant loss in its separation

fficiency until u = 3 mm/s as shown in Fig. 44 [194].Using the column in a LC-ESI–TOF MS system, 9 peptides

ere separated within 4 min. This type of stationary phase coulde used for a weak cation-exchange phase and a HILIC phaseo separate proteins, peptides, nucleosides, and oligosaccharides192]. The above-mentioned cation-and anion-exchange station-

bppr

ig. 44. LC–ESI–MS base peak chromatograms of a mixture of mono- di- and teCN–13 mM ammonium acetate buffer (80:20, v/v); linear velocities: (A) 3.9 mm/s

1) ribose; (2) sedoheptulose; (3) glucose; (4) sucrose; (5) maltose; (6) trehalose; (7)

ine (k = 3.8); injection volume: 2 �l split injection. Column: ZIC-HILIC (�),50 mm × 4.6 mm I.D., 5 �m; mobile phase: MeCN–buffer (80:20, v/v); Tem-erature: 25 ◦C; solute: cytosine (k = 1.3) [193].

ry phases [195] can also be used as HILIC phases, becausehey will easily form water-rich phases for partitioning equilibria
etween mobile and stationary phases. Other monolithic silicahases functionalized by such ion-exchange groups also have theotential to be used in the HILIC mode [184]. HILIC mode sepa-ations using the above-mentioned monolithic columns modified
risaccharides. Column: 200T-PAAm, 267 mm × 200 �m I.D.; mobile phase:, and (B) 7.6 mm/s; Detection: 2.2 kV Negative ESI-Ion Trap MS; Compounds:raffinose [194].

atogr

wa

5

o

.ms

TC

C

A

BA

P

AP

N

O

R

C

A

BA

PA

P

N

O

R

C

A

B

AP

AP

N

O

R


ith polar polymers will provide more efficient and faster sep-ration than particle-packed columns.

. Conclusion

The characteristics of stationary phases for HILIC in termsf the separation efficiencies are summarized in Table 1

sa(a

able 1omparison of the separation efficiencies for polar compounds by several types of H

olumn Bare silica

cids 10 �m, 4 aromatic carboxylic acid, 85%MeCN–20 mM AB, UV [53]

ases –mides, ureas 10 �m, salicylamide, 85% MeCN–20 m

[53]olyols 21–25 �m, epirubicines, 90% MeCN, 2

(pH 2.9), UV [33]mino acids –eptides –

ucleic bases and nucleosides 10–12 �m 6 compounds, 85% MeCN–2UV [53]

ligosaccharides and carbohydrates 33–43 �m by a 3 �m column, gluclonidMeCN–100 mM AB, [43]

emarks Irreversible adsorption

olumn Amide-silica

cids 14 �m, 4 aromatic carboxylic acid, 85%MeCN–20 mM AB, UV [53]

ases –mides, ureas 15 �m, salicylamide, 85% MeCN–20 m

AB, UV [53]olyols 250 �m, polyphenols, 84% MeCN, UVmino acids –

eptides Many examples in gradient elutions

ucleic bases and nucleosides 13–16 �m 6 compounds, 85%MeCN–20 mM AB, UV [53]

ligosaccharides and carbohydrates 7 �m by a 3 �m column, 13 �m by 5 �mcolumn, mannitol, 75% MeCN [91]

emarks Good recovery of analytes

olumn Sulfoalkylbetaine

cids 12–21 �m, 4 aromatic carboxylic acid, 85%MeCN–5 mM AB, UV [53]

ases 15 �m, morphine, 70% MeCN–20 mM AB, UV [25]

mides, ureas 13 �m, salicylamide, 85% MeCN–20 mM AB, UV [53]olyols 26–35 �m, 3 flavonoids, 75% MeCN–30 mM AB, UV

[25]mino acids –eptides 33 �m, neurotensin, 56 �m, bradykinin, 70%

MeCN–50 mM AB, UV [25]ucleic bases andnucleosides

17–21 �m 6 compounds, 85% MeCN–20 mM AB, UV[53] 36–84 �m for 6 nucleotides, 70% MeCN–100 mMAB, UV [25]

ligosaccharidesandcarbohydrates

33–38 �m, morphine glucuronides, 70% MeCN–20 mMAB, UV [25]

emarks Low column bleeding, Relatively slow optimal flowrates

. A 1184 (2008) 474–503 499

Due to the lack of separation examples in the isocraticode in many cases, the table is not fully completed. HILIC

eparation targets were roughly separated into eight groups,
uch as (1) acids, (2) bases, (3) polar small compounds likemides or ureas, (4) Polyols (5) amino acids, (6) peptides,7) nucleic bases and nucleosides, and (8) oligosaccharidesnd carbohydrates. Judging from the table, several sugges-
ILIC columns

Amino-silica

44–60 �m, 4 aromatic carboxylic acid, 85%MeCN–20 mM AB, UV [53]20–35 �m, 2 aminoalcohols, 80% MeCN, RI [63]

M AB, UV 50 �m, salicylamide, 85% MeCN–20 mM AB, UV [53]

0 mM FB 13 �m, 3 �m column, tetracyclines, 85% MeCN, 1 mMCB (pH 5), UV [61]58–100 �m, 3 amino acids, 60% MeOH, ESI-MS [64]22–95 �m, 10 �m column, disaccharides, 92%MeCN–2.5 mM AB [59]

0 mM AB, 14–20 �m 6 compounds, 85% MeCN–20 mM AB, UV[53]

es, 87% 43–180 �m by a 10 �m column, disaccharides, 85%MeCN [58]Irreversible adsorption, Slow column equilibration,column bleeding

Poly(succinimide)

–

–M –

[86] –50–67 �m, amino acids, 80% MeCN–5 mM TB (pH 2.8), UV[1]33–37 �m, atosiban, 89% MeCN–10 mM TB, UV [59],69–87 �m, dipeptides, 80% MeCN–10 mM TB, UV [1]–

57–66 �m, sialyl sugars, 80% MeCN–10 mM TB, 20–22 �m,xyloglucans, 70% MeCN–10 mM TB, PAD [23]Column bleeding

Diol/cyano

–

19 �m, piperadine and its salts, Cyano, 95% MeCN, ELSD [128]17 �m, methylene blue, Cyano, 90% MeCN–5 mM TFA, UV [130]28–30 �m, urea, Diol, 80% MeCN, RI [58]–

24–27 �m, glycine, Diol, 80% MeCN, RI [58]–

–

18–30 �m, sucrose, Diol, 80% MeCN, RI [58] 47 �m, sucrose, 74 �m,lactose, by a 10 �m Diol column, 75% MeCN, RI [58]

Column bleeding under acidic conditions


Table 1 (Continued )

Column Cyclodextrin Triazol

Acids – 11 �m, oxalic acid, 50% MeCN–10 mM PB (pH 7), UV17 �m, benzoic acid, 50% MeCN–10 mM AB, UV

Bases – 33 �m, benzamidine, 90% MeCN–10 mM AB, UVAmides, ureas – 17 �m, urea, 90% MeCN, UV 20 �m, cyanuric acid, 50%

MeCN–10 mM AB, UVPolyols 13–24 �m, norvaline, Chirobiotic T column,

50–80% MeCN–6.5 mM AB (pH 5.5) [145]11 �m, leucine, 13 �m, valine, 14 �m, isoleucine, 70%MeCN–10 mM AB, UV, 30 �m, histidine, 70%MeCN–10 mM CB, UV

Amino acids – 14 �m, 1,2,6-hexanetriol, 90% MeCN, ELSDPeptides – 35 �m, oxytocin, 70% MeCN–10 mM AB, UVNucleic bases and nucleosides 15–19 �m, nucleosides, maltose column, 85%

MeCN–10 mM AB, UV, [148]14 �m, uridine, 90% MeCN–10 mM AB, UV

Oligosaccharides and carbohydrates 19–63 �m, oligosaccharides, 78% MeCN, RI [143] 30 �m, glucose, 60%MeCN–20 mM PB (pH 7), RI 18 �m,glucose-1-phospate, 30 �m, glucose-6-phospate,60%MeCN–20 mM PB (pH 7), RI

Remarks All data are available in [154]

Column Triazol

Acids 18 �m, l-tartaric acid, 50% MeCN–10 mM PB(pH7), UV31 �m, pivalic acid, 50% MeCN–10 mM AB, UV

Bases 36 �m, p-phenylenediamine, 95% MeCN–10 mM AB, UVAmides, ureas 28 �m, nicotinamide, 95% MeCN–10 mM AB, UV

18 �m, allantoin, 90% MeCN–10 mM AB, UVPolyols 19 �m, phosphocreatine, 50% MeCN–10 mM PB (pH 7), UV

45 �m, O-phospho-l-serine, 50% MeCN–20 mM PB (pH 7), UVAmino acids 13 �m, meso-erythritol, 90% MeCN, ELSDPeptides 33 �m, angiotensin II, 50% MeCN–10 mM AB, UVNucleic bases and nucleosides 21 �m, cytidine, 80% MeCN–10 mM AB, UVOligosaccharides and carbohydrates 20 �m, sinigrin, 50% MeCN–10 mM AB, UVRemarks All data are available in [154]

S d. ABs

tf

atfplntsfsatatsmotreip

rossslbclHac

pscicptt

eparation efficiencies, analytes, mobile phases, and detection were summarizeodium phosphate buffer; TB: triethylammonium phosphate buffer.

ions for the selection of HILIC columns are given, asollows.

(1) Classical HILIC columns such as bare-silica columns andmino-silica columns are limited by the choice of analytes, dueo the strong adsorption phenomena of compounds with specificunctionalities. (2) The polymer-coated silica columns based onoly(succinimide) have retentivities for a wide range of ana-ytes, but separation efficiencies of these types of columns areot very high. (3) Amide-silica columns also show good reten-ivities for a wide range of analytes, and the use of the amideilica column packed with 3 �m particles may be the choiceor the efficient separation of highly polar compounds. (4) Theulfoalkylbetaine bonded silica column can be applied to the sep-ration of a wide range of analytes, but the optimal flow rate ofhe column is relatively lower than that of amide-silica columns,nd not suitable for fast separations. The mixed mode separa-ion of the HILIC and the ion-exchange LC will decrease theeparation efficiency. (5) Columns packed with carbohydrate-odified silica particles were used mainly for the separation of

ligosaccharides with a fair to good separation efficiencies, andhey may make an interesting research subject for chiral sepa-
ation using HILIC. (6) Cyano- and diol-silica columns can bemployed in HILIC, but the basic research for the scope and lim-tation of target analytes is required. (7) Newly released columnacked with triazol-bonded silica particles shows good sepa-
Htcc

: ammonium acetate buffer; CB: citrate buffer; FB: sodium formate buffer; PB:

ation efficiencies for a wide range of analytes. (8) Examplesf polymer-based monolithic columns are still limited, and theireparation efficiencies are not so high in the HILIC mode, thoughome polymer monoliths in capillaries can generate very higheparation efficiencies in the CEC mode. (9) Only limited mono-ithic silica columns for HILIC are commercially available, andare silica monolith columns show worse separation efficienciesompared with the RP mode. Monolithic silica column in capil-aries can be modified with various polar functionalities to haveILIC type monolithic silica columns. These columns gener-

te better kinetic performance compared to the particle-packedolumns.

Many researchers tend to use newly released smaller particle-acked columns, in many cases 3 �m particles except for bareilica columns packed with 1.7 �m particles. However, the basicharacteristic of the separation efficiency of these columns aregnored. It is difficult to know differences in separation effi-iencies between columns packed with 5 �m particles and thoseacked with 3 �m particles. There are no common test sampleso evaluate separation efficiencies of HILIC columns. In addi-ion, a survey of good t0 markers is also required. Although the
ILIC mode separation is said to be a good alternative to RPLC,
he knowledge of the basic chemistry of HILIC is limited whenompared to the RPLC. Generally lower performance of HILIColumns than RPLC columns is an interesting subject to study.

atogr

Itum

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fS1

R


nvestigations to elucidate the scope and limitation of each sta-ionary phase for HILIC should be carried out to more efficientlytilize this attractive separation mode in liquid chromatographyore efficiently.

cknowledgements

We acknowledge the financial support from a Grant-in-Aidor Scientific Research funded by the Ministry of Education,ports, Culture, Science and Technology, Nos. 17350036 and9550088.

eferences

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