4 Hydrophilic Interaction Liquid Chromatography › uploads › short-url › nHoRuwZsaxbuG30CZu… · liquid chromatography (HILIC) [1] turns out to be a valuable complement for

4Hydrophilic Interaction Liquid ChromatographyXinmiao Liang, Aijin Shen, and Zhimou Guo

4.1Introduction

High-performance liquid chromatography (HPLC) has been recognized as apowerful analytical technique in many fields including pharmaceutical, environ-mental, food industries, clinical diagnosis, bioanalysis, and so on. Althoughreversed phase liquid chromatography (RPLC) has widely been used because ofits high resolution and superior reproducibility, the separation of polar andhydrophilic compounds is challenging owing to the insufficient retention. Nor-mal phase liquid chromatography (NPLC) and ion-exchange chromatography(IEX) can be utilized for resolving polar analytes. However, NPLC suffers frompoor reproducibility and low solubility of hydrophilic analytes, whereas IEX ismerely applicable for ionic compounds. In contrast, hydrophilic interactionliquid chromatography (HILIC) [1] turns out to be a valuable complement forthe resolution of polar compounds and has received increasing popularity duringthe past two decades [2–12].The concept of HILIC was coined by Alpert in 1990 [1], although it was origi-

nally introduced in the 1970s [13,14] for the analysis of carbohydrates. The maincharacteristic of HILIC is the combination of polar stationary phase withaqueous-organic mobile phase (most frequently acetonitrile). Generally, a watercontent of higher than 2% is essential for the formation of water-enriched layersemi-immobilized on the surface of the stationary phase. The retention of polaranalytes decreases with increasing polar solvent (water) in the mobile phase.Comparing with NPLC, the solubility of hydrophilic analytes is greatly improved,and the high organic component together with volatile buffer is suitable forcombining with mass spectrometric (MS) analysis. Meanwhile, low viscosity ofthe mobile phase enables the efficient separation at lower pressure than RPLC.In addition, HILIC affords alternative separation selectivity to RPLC and IEX forpolar compounds, thus enhancing the analytes’ coverage.Publications involving HILIC have increased rapidly since 2005 (as displayed

in Figure 4.1), covering stationary phases and chromatographic method develop-ment, retention behavior and mechanism, separation efficiency and peak shape,

63

Analytical Separation Science, First Edition. Edited by Jared L. Anderson, Alain Berthod,Verónica Pino Estévez, and Apryll M. Stalcup. 2015 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2015 by Wiley-VCH Verlag GmbH & Co. KGaA.

and numerous applications in diverse fields. Among them, a number of remark-able reviews have been contributed by different authors. In 2006, Hemström andIgrum provided a comprehensive summary of HILIC [7]. In 2008, Tanaka andothers presented an excellent review regarding the separation efficiency inHILIC [9]. Heck and coworkers summarized the application of HILIC in proteo-mics, along with the analysis of protein post-translational modifications [15]. In2011, Guo and Gaiki reviewed the retention and selectivity of HILIC stationaryphases [16]. Based on previous publications and research works, in this chapter,we will provide a general description of the principles and research progresses ofHILIC, including the separation mechanism, stationary phases, and practicalapplications.

4.2Separation Mechanism in HILIC

The separation mechanism in HILIC is not so well understood as that of RPLC.It is difficult to predict the retention or selectivity by the functional groups onthe HILIC stationary phase. As early as 1990, Alpert suggested the partitioningof analytes between the water-enriched layer on the surface of the stationaryphase and the hydro-organic mobile phase as the potential mechanism [1]. Theexistence of a surface water-enriched layer has been studied and validatedthrough chromatographic [17], spectrometric [18], and molecular dynamics sim-ulation methods [19]. Nonetheless, numerous investigations [6,7,20–25] regard-ing the mechanism characterization or application in HILIC reflect thecomplexity of interaction mechanism. As illustrated in Figure 4.2, apart from

Figure 4.1 Number of publications indexed on Web of Science with terms “HILIC” or“hydrophilic interaction liquid chromatography” or “hydrophilic interaction chromatography.”

64 4 Hydrophilic Interaction Liquid Chromatography

Figure 4.2 Schematic diagram of the interaction mechanism in HILIC.

hydrophilic partitioning, surface adsorption (such as hydrogen bonding anddipole–dipole interaction), as well as electrostatic interaction, may affect theretention of analytes. Moreover, the interaction mechanism exhibits significantdifferences among different stationary phases.In order to understand the retention mechanism in HILIC, several retention

models have been employed to preliminarily determine the relative contributionof partitioning and surface adsorption [7]. The relationship established for char-acterizing the partitioning interaction is

log k ´ � log kw � Sϕ (4.1)

where kw is the retention factor for the weaker eluent (organic solvent in HILIC)only as the mobile phase, ϕ is the volume fraction (concentration) of water inthe mobile phase, and S is the slope of the linear regression model. On the otherhand, the basic equation for describing the surface adsorptive interaction is

log k ´ � log k B � As

nBlogNB (4.2)

4.2 Separation Mechanism in HILIC 65

where k B is the retention factor with pure B (H2O in HILIC) as an eluent, As

and nB are the cross-sectional areas occupied by the solute molecule on thesurface and the B molecules, respectively. NB is the mole fraction of water inthe mobile phase. The relationship between log k´ and linear or logarithmicalfunction of water content in the mobile phase is supposed to indicate thepredominant retention mechanism in HILIC. However, sometimes the linear-ity of either log-linear plots or log–log plots is not satisfactory, indicating theabsence of pure partitioning or adsorption mechanism. Thus, the third modelwas proposed for characterizing solute–solvent–stationary-phase interac-tions [26].

ln k ´ � a � b ln φ � cφ (4.3)

where a is a constant relating to the molecular volume of solutes, the interactionenergy between solutes with the stationary phase and the mobile phase; b is thecoefficient involving the direct interaction between analyte and stationary phase;and c relates to the interaction energy between solutes and solvents. The reten-tion model based on Equation 4.3 was found to be suitable for describing theretention factors [22,26], revealing the diversity of intermolecular interactions inHILIC. In addition, the existence of multiple-interaction mechanism has beendemonstrated using the retention model based on a linear solvation energy rela-tionship (LESR) approach [25,27].

log k ´ � c � eE � sS � aA � bB � vV � d�D� � d�D� (4.4)

The capital factors represent the particular interaction properties of the analytes(solute descriptors), whereas lower case letters are the system constants relatedto the complementary effect of the stationary phase. c is a constant independentof the analytes and is dominated by the phase ratio and specific column parame-ters, such as porosity, as well as other properties. E is the excess molar refractionand model polarizability contributions from n and π electrons. S represents thesolute dipolarity/polarizability. A and B refer to the overall hydrogen bond acid-ity (H donor) and basicity (H acceptor). V is the McGowan characteristic vol-ume. D� reflects the negative charge carried by anionic and zwitterionic species,and D+ reflects the positive charge carried by cationic and zwitterionic species.The differential contribution of partitioning, hydrogen bonding, and electrostaticattractive/repulsive interaction among different HILIC columns represents thepredominant mechanistic interaction.In general, the relative contribution of partitioning and surface adsorption

mechanisms in HILIC depends on the comprehensive effect of the type of sta-tionary phase, the composition of the mobile phase, and the structures of theanalytes (as illustrated by the “LC retention Troika” in Figure 4.3). The contribu-tion of multiple intermolecular interactions is considered to be favorable forregulating the separation selectivity in HILIC.


4.3Stationary Phases for HILIC

In contrast to RPLC, stationary phases in HILIC are of polar characteristics(Table 4.1). The most frequently utilized chromatographic support in HILIC issilica gel (SiO2), which can easily be modified and is of high mechanical strength.

4.3.1

Conventional NPLC Stationary Phases for HILIC

Stationary phases for conventional NPLC can be employed in HILIC, such asunderivatized silica and amino-/cyano-/diol-modified silica. Bare silica possessessuperior stability at low pH and is not subject to the bleeding of bonded phasesfrom the column. It is favorable to combine with MS for selective and quantita-tive analysis. Thus, bare silica has appeared in a wide range of applications, espe-cially in pharmaceutical analysis. However, basic compounds may irreversiblyabsorb onto silanol groups owing to the strongly electrostatic interaction.Besides, the long-term stability of underivatized silica under neutral or basicconditions is poor. To improve the stability, ethylene-bridged hybrid silica(BEH HILIC) was introduced. However, the hydrophilicity and selectivity ofeither conventional underivatized silica or BEH HILIC are limited. Amino-modi-fied silica was initially applied for the separation of carbohydrates before theintroduction of the term “HILIC.” In 1975, Linden and Palmer reported the

Figure 4.3 LC retention “Troika.” (Reproduced with permission from Ref. [25].)

4.3 Stationary Phases for HILIC 67

Table

4.1

Represen

tativ

estationa

ryph

ases

forHILIC.

Types

ofpolar

group

sRe

presentativeco

lumn

Bon

dedpha

seCom

pan

y/ref.

Silica

Atlantics

HILIC

/BEH

HILIC

Und

erivatized

silica

Waters

Amino

Luna

NH2/TSK

gelN

H2

NH

2

Silica

Pheno

menex/

Tosoh

Cyano

YMC-PackCyano

CN

Silica

YMC

Diol

Inertsildiol

O

OH

OH

Silica

GLScience

Luna

HILIC

O

OO

OH

OH

OH

Silica

Pheno

menex


Amide

TSK

gelamide-80

H

OH

2N

H

R4 R

3R

1

R2n

m

Sili

ca

Tosoh

BEH

amide

Si

OO Olin

ker

ONH

2

Silica

Waters

Urea-based

Si

N H

O OO

H

NH

2

O

Silica

[28]

USP

-HILIC

Si

N H

O OO

H

NH

O

Si

N H

O

HO

OH

N H

O

Silica

[29]

Saccharides

Sorbitol-based

OC

C

O OO

H

HO

OH

HO

OH

CH

CH

3n

Silica

[30] (con

tinu

ed)


Table

4.1

(Con

tinued)

Types

ofpolar

group

sRe

presentativeco

lumn

Bon

dedpha

seCom

pan

y/ref.

ClickGlucose

Si

N NN

OO

OH

OH

HO

OH

O OO

H

Silica

[31]

ClickMaltose

Si

N NN

OO

O

O

OH

HO

OH

OH

OH

OH

OH

O OO

H

Silica

[31,32]

Clickβ-CD

N NN

(OH

) 6H N

(OH

) 14

Si

O OO

H

Silica

[31]

Galactose-based

Si

O OO

H

O

OH

N NN

O

HO

HO

OHOH

Silica

[33]


Lactose-based

Si

O OO

H

O

OH

N NN

OO

OH

HO

O

HO

HO

OHOH

HO

O

Silica

[33]

ClickChitooligosaccharides

O

OH

HO

OH

NH

2

OO

OH OH

NH

2

OO

OH OH

N

OH

nN

NN

H

NH

O

Si

O

O

OH

Silica

[34]

Prop

ylcarbam

atecyclofructan

6(CF6

)N H

O

O

CF

6S

iO O

OH

Silica

[35]

Dicarbamox

yl-hexylCF6

stationary

phase

N HN H

OH N

O

O

CF

6S

iO O

OH

Silica

[35]

Sulfo

natedCF6

stationary

phase

N HO

O

CF

6S

iO O

OH

SO

3

Silica

[36] (con

tinu

ed)


Table

4.1

(Con

tinued)

Types

ofpolar

group

sRe

presentativeco

lumn

Bon

ded

pha

seCom

pan

y/ref.

Poly-

aspartam

ide

Poly(succinim

ide)

silica

[PolyG

lycoplex

A]

H N

O

O

N HO

N

O

N

n

O

O

Silica

PolyLC

Poly(2-hydrox

yethylaspartam

ide)

silica

[PolyH

ydroxyethylA

]

H N

O

O

N H

O

HN

OH

O

H N

O

N H

HN

O

OH

n

Silica

PolyLC

Poly(2-sulfo

ethylaspartamide)

silica

[PolySulph

oethylA]

H N

O

O

N H

O

HN

SO

3-

O

H N

O

N H

HN

O

SO

3-

n

Silica

PolyLC


poly(asparticacid)silica[PolyC

ATA]

H N

O

O

N H

O O

H N

O

N H On

HO

OH

Silica

PolyLC

Zwitterion

icgrou

psZIC

–HILIC

N

SO

3

Silica

Merck

ZIC

–cH

ILIC

OPO

OO

N

Silica

Merck

3-P,P-diphenylpho

spho

nium

-prop

ylsulfo

nate

Si

PO O

OH

SO

3

Silica

[37]

Clicklysine

Si

O OO

H

N HN H

O

N

NN

CO

O

NH

3

Silica

[38] (con

tinu

ed)


Table

4.1

(Con

tinued)

Types

ofpolar

group

sRe

presentativeco

lumn

Bon

dedpha

seCom

pan

y/ref.

ClickTE-C

ys

OO

O

Si

Si

Si

OO

OO

Si

OO

Sili

ca G

el

SS

S

- OO

C

NH

3+

- OO

C

NH

3+

- OO

C

NH

3+

[39,40

]

ClickTE–GSH

OO

Si

Si

OO

O

Sili

ca G

el

S

NH

OO

H

NH

2

O

HN

HO O

O

Si

OO[41]


separation and quantitation of saccharides on amino-modified material usingacetonitrile and water as eluents [13,14]. Because of their good retention andspecial selectivity, amino-based materials still play an important role in the reso-lution of carbohydrates. Meanwhile, the existence of amino on the stationaryphase can efficiently eliminate the splitting or broadening of peak shape due toα/β sugar anomerization. But, amino-modified silica suffers from the formationof Schiff base with reducing sugars, affecting the accuracy of quantificationand altering the chemical characteristics of the bonded phase. Besides, amino-modified silica is not stable over a long period. Cyano-based silica displays poorretention for hydrophilic analytes owing to its low polarity. Diol-based silica canprovide additional hydrogen bonding and possesses higher polarity than cyano-based silica. Nonetheless, the hydrophilicity of both materials is limited.

4.3.2

Stationary Phases Developed for HILIC

After the introduction of HILIC, many stationary phases (academically and com-mercially) with diverse polar functional groups have been developed for improv-ing the hydrophilicity and separation selectivity in HILIC. Based on the inherentcharacteristics of bonded functional groups, HILIC stationary phases can bedivided into several categories, including polyaspartamide-, amide-, saccharides-,and zwitterionic-based materials.

4.3.2.1 Polyaspartamide-Based Stationary PhasesPolyaspartamide-based stationary phases were the first chemically modifiedmaterials designed for HILIC by Alpert [1]. Poly(succinimide) was bonded ontoaminopropyl silica to obtain the poly(succinimide) silica. Then, subsequent alka-line hydrolysis or modification with taurine or ethanolamine was carried out toform the corresponding poly(aspartic acid), poly(2-sulfoethyl aspartamide), andpoly(2-hydroxyethyl aspartamide) stationary phases [1,42,43]. With the combina-tion of hydrophilicity and unique electrostatic interaction, poly(2-sulfoethylaspartamide) silica revealed excellent selectivity for the separation of peptidesand proteins [9,15]. But, polyaspartamide-based materials present limited long-time stability and low efficiency [9].

4.3.2.2 Amide-Based Stationary PhasesDifferent from amino-modified silica, amide-based stationary phase is not sus-ceptible to Schiff-base formation. Besides, the stability of amide-based silica isgreatly improved compared to amino-modified silica. Among the commerciallyavailable amide-bonded phases, TSK Amide-80 column [4,44,45] proves to be agood choice because of its high hydrophilicity and separation efficiency. In addi-tion, urea or bidentate-bonded urea-modified silica [28,29] has been explored forHILIC.


4.3.2.3 Saccharides-Based Stationary PhasesSaccharides, which are composed of abundant hydroxyl groups, are highlyhydrophilic. The polyhydroxyl groups are beneficial for the formation of water-enriched layer and the enhancement of secondary interaction with polaranalytes, such as hydrogen bonding. Thus, saccharides represent an interestingalternative as bonded phases for HILIC. In 2007, Liang and others designed andsynthesized several saccharides-based HILIC materials through alkyne–azideclick chemistry. Monosaccharide (Click Glucose), disaccharide (Click Maltose),and oligosaccharides (Click β-CD, Click Chitooligosaccharides) were bondedonto silica [31,32,34]. Click Maltose material exhibited good selectivity in theenrichment of glycopeptides [46]. The analysis and preparation of oligosacchar-ides and peptides were successfully realized on the Click Maltose column [47,48].In 2008, a sorbitol-based material was developed by graft polymerization fromthe surface of silica [30]. The sorbitol-based silica exhibited better hydrophilicitythan underivatized silica. Lactose- and galactose-modified silica were synthesizedand applied in the resolution of sugar anomers [33]. Several cyclofructan 6(CF6)-based stationary phases were also developed by Armstrong and others forthe separation of small polar analytes [35,36].

4.3.2.4 Zwitterionic Stationary PhasesZwitterionic material is characterized by the existence of both positively andnegatively charged groups on the bonded phase. The incorporation of highhydrophilicity and unique ion-exchange interaction greatly improve the separa-tion selectivity in HILIC. Consequently, the application of zwitterionic materialin HILIC has received increasing popularity, especially in proteomics andmetabonomics. The typical zwitterionic stationary phases include sulfobetaine(ZIC–HILIC)- and phosphocholine (ZIC–cHILIC)-modified silica, of which theoppositely charged groups are distributed perpendicular to the silica surface. In2011, Liang and others designed a novel type of zwitterionic stationary phasewith a uniform distribution of both positive and negative charges that are paral-lel to the surface of the silica gel [39,40]. The zwitterionic material was facilelysynthesized by the modification of vinyl silica with cysteine through thiol-eneclick chemistry (Click TE-Cys). Click TE-Cys displayed high separation effi-ciency and better hydrophilicity than many commercial HILIC stationary phases(Figure 4.4). In addition, glutathione was immobilized onto vinyl silica to obtaina hydrophilic interaction/cation-exchange (HILIC/CEX) mixed mode zwitterionicmaterial [41]. In 2011, Armstrong and others developed a zwitterionic stationaryphase based on 3-P,P-diphenylphosphonium-propylsulfonate [37]. The resultingmaterial presented a higher efficiency and greater retention than ZIC–HILICand a bare silica column. Besides, lysine-based zwitterionic material [38] wasalso prepared and applied in the separation of cephalosporins and carbapenems.With expanding application of HILIC, the development of chromatographic

columns with higher efficiency, better hydrophilicity and selectivity is of greatsignificance. One way of increasing the separation efficiency is to minimize theparticle size and inner diameter of the column (capillary column). Stationary


phases with particle size less than 3 μm (Kinetex HILIC core–shell column) oreven 1.7 μm (BEH HILIC) have been successfully developed. Hydrophilic mono-lithic columns represent a novel trend among HILIC column techniques becauseof their good permeability, low resistance to mass transfer, and easy preparationwithin capillaries [9,49–52]. On the other hand, silica has been replaced bymetal-oxide such as titania or zirconia as packing material to improve the pHstability and introduce alternative selectivity [11,12]. Nonetheless, the selectionof appropriate column for separating particular compounds becomes more andmore complicated with the increasing diversity of HILIC stationary phases.

4.4Application of HILIC

4.4.1

Application in the Pharmaceutical Field

As the classical chromatographic technique, RPLC is the predominant approachfor qualitative and/or quantitative analysis of pharmaceutical components. How-ever, RPLC cannot provide sufficient retention and resolution with regard topolar drugs and their metabolites. HILIC has been proven to be a valuable alter-native to biological and nonbiological pharmaceutical assays [53–58], such as thedetermination of active ingredients in pharmaceutical formulations, the inter-mediates or impurities involving in drug development, and the quantification ofdrug compounds and/or their metabolites. The application of HILIC for the

Uracil Uridine Cytosine Cytidine Orotic acid0

1

2

3

4

5

6

7

8

9

10k

Click maltose

Click CD

ZIC-HILIC

Atlantis HILIC silica

TSKgel Amide-80

Tigerkin Diol

Venusil HILIC

Click TE-Cys

Figure 4.4 Comparison of capacity factors on Click TE-Cys and seven different HILIC columns.Mobile phase, 15mM HCOONH4 in ACN/H2O (85 : 15), pH 3.28. (Reproduced with permissionfrom Ref. [39].)

4.4 Application of HILIC 77

analysis of polar pharmaceuticals in complex biological samples (e.g., plasma,serum, or urine) can greatly increase the retention factors and separation selec-tivity, which is conducive to minimize matrix interference and ion suppression.Meanwhile, the high organic content in the mobile phase shows better compati-bility with LC–MS interface, thus improving the detection sensitivity. On theother hand, the commonly utilized sample cleanup procedures, such as proteinprecipitation (PP), liquid–liquid extraction (LLE), or solid-phase extraction,always generate extracts of high organic content. The extracts can be directlyinjected into HILIC columns without evaporation and reconstitution steps. Baresilica (Atlantis HILIC, YMC silica, etc.) is the most widely used stationary phasein pharmaceutical applications [55,57]. In contrast, a limited number of pharma-ceutical analysis was performed using chemically modified stationary phases thatare subject to the bleeding of bonded ligands.

4.4.2

Application in the Separation of Carbohydrates

As a large group of organic polar compounds, carbohydrates possess diverse bio-logical functions and play a significant role in the biochemical system. The sepa-ration and identification of carbohydrates are gaining more and more attention.Derivatization of carbohydrates with hydrophobic chromophore is alwaysrequired before the separation by conventional RPLC owing to the insufficientretention. High-performance anion-exchange chromatography (HPAEC), incombination with pulsed amperometric detection (PAD), has widely been usedfor analyzing carbohydrates. However, the mobile phase used in HPAEC isincompatible with mass spectrometry (MS). In comparison, HILIC provides ade-quate retention for carbohydrates and has become an efficient technique for theseparation of complex carbohydrates, quantification, or structure identifica-tion [2,10,43,47,59]. The resolution of small carbohydrates, native glycan cleavedfrom native bovine fetuin through hydrazinolysis, and derivatized complex car-bohydrates was successfully realized on the PolyGLYCOPLEX column withcomparable selectivity to HPAEC and RPLC [43]. In 2009, Wuhrer et al. com-prehensively reviewed the application of HILIC–MS in structural glycomics atboth glycan and glycopeptides levels (Figure 4.5), indicating that HILIC com-bined with MS is an attractive and powerful approach for the analysis of glycansand glycoconjugates [10]. Furthermore, the resolving ability of HILIC for oligo-saccharides with a high degree of polymerization has been greatly improved withincreasing hydrophilicity and selectivity of HILIC stationary phase [41].

4.4.3

Application in Proteome, Glycoproteome, and Phosphoproteome

RPLC is an indispensible technique for separating and purifying peptide mix-tures in proteomics. However, the separation selectivity in the analysis of com-plex proteomic samples based on one-dimensional RPLC separation is limited.


Therefore, the incorporation of online/offline multidimensional separationapproaches has received increasing popularity and widespread application.Strong cation exchange chromatography (SCX), which displays different reten-tion mechanism from RPLC, is commonly utilized for peptide separation. Butpeptides with the same charge always elute in narrow windows and the mobilephase is incompatible with MS. In contrast, HILIC is an attractive choicebecause of its distinct selectivity and superior orthogonality to RPLC. What ismore, HILIC presents higher resolution capacity than SCX [60]. In 2007,Mohammed and others explored an alternative two-dimensional liquid chroma-tography (2D-LC), that is, ZIC–HILIC hyphenated offline with RPLC for analyz-ing the cellular nuclear lysate, revealing its great potential as multidimensionalprotein identification technology (MudPIT) in proteomics [61]. In 2011, 2D-LCsystems based on ZIC–HILIC or ZIC–cHILIC in combination with RPLC werefurther applied for peptide identification from 1.5 μg of Hela lysate digestion(Figure 4.6). Approximately 20 000 unique peptides corresponding to over 3500proteins were successfully identified, demonstrating that HILIC can represent apowerful foundation for a sensitive multidimensional strategy [62]. In addition,HILIC/CEX mixed mode chromatography has been proved to be an excellentcomplement to RPLC. The great potential of HILIC/CEX in the separation ofpeptides (e.g., α-helical peptides and modified or deletion products of syntheticpeptides) and modified histone proteins was demonstrated by Hodges and Lind-ner [63]. Furthermore, the HILIC technique plays a significant role in the enrich-ment and analysis of protein post-translational modifications (e.g., glycosylationand phosphorylation) [8,10,45,64–66] that have significant effects on protein

Figure 4.5 Scheme of HILIC–MS approaches in structural glycomics. (Reproduced with per-mission from Ref. [10].)

4.4 Application of HILIC 79

functions. In 2008, Heck and coworkers reviewed the versatility of HILIC both inthe enrichment of phosphorylated, N-terminally blocked, and glycosylated pep-tides and in the separation of differentially modified histones [15].

4.4.4

Application in Metabolomics/Metabonomics

Metabolomics/metabonomics, aiming at the unbiased analysis and quantificationof metabolites in a biological system, can provide a comprehensive signature ofthe physiological state of an organism, as well as insights into specific bio-chemical processes [67]. NMR spectrometry and MS are the major analyticaltechniques for metabolite profiling, and MS exhibits better sensitivity. However,MS-based metabolomics is susceptible to matrix effects owing to the complexityof metabolites in biological samples. Consequently, the coupling of chromato-graphic separation to MS for reducing the interference is of great importance.RPLC–MS-based metabolomics is the most common approach for biofluidsanalysis. Nonetheless, RPLC is not applicable to polar components that areabundant in predominantly aqueous biofluids. The introduction of HILIC in MSmetabolomics provides an alternative choice for resolving highly polar metabo-lites [67–73]. Besides, the MS detection selectivity is greatly improved because ofthe high organic content in the mobile phase. HILIC–MS-based metabolomics

Figure 4.6 (a) Schematic design of thenano-online HILIC–MS systems (1) and first-dimensional separation configurations (2).After fractionation, the eluent was collected as1-min fractions in a 96-well plate containing10% formic acid and subsequently analyzed

by nano-RP–LC–MS. (b) Schematic workflow ofdifferent settings employed with the aboveoffline configuration shown in part (a2).(Reproduced with permission from Ref. [61].Copyright 2011 American Chemical Society.)


was first applied in plant metabolite profiling in 2002 [4]. With the combina-tion of HILIC (TSKgel Amide-80 columns) and electrospray quadrupole ion-trap mass spectrometry, the identification and quantification of polar metab-olites of plant origin were successfully realized. Both novel and general com-ponents, including oligosaccharides, glycosides, amino sugars, and sugarnucleotides, were detected from Cucurbita maxima phloem exudates. HILICcoupled to MS has been widely utilized for the analysis of cellular metabo-lites [70] (bacterial or microbial samples) such as water-soluble cellularmetabolites extracted from Escherichia coli, sulfur endogenous metabolitesrelated to glutathione biosynthesis from Saccharomyces cerevisiae cells, andso on. In addition, the metabolite profiling of urine samples in mammaliansystems based on the HILIC–MS approach can provide complementaryinformation involving polar metabolites to RPLC–MS-based metabolomics,thus increasing the coverage of metabolome [71].

4.5Conclusions and Outlook

Since the introduction of HILIC in 1990, the versatility and capability of HILICin the analysis of polar and hydrophilic compounds have widely been accepted.Applications concerning small compounds (nucleosides, nucleotides, etc.), phar-maceuticals, natural products, peptide mixtures, metabolites, and so on weresuccessfully achieved. The basic principles and mechanisms of HILIC have beengradually understood. Numerous stationary phases with distinct selectivitieshave been developed, and particular materials, including TSK Amide-80, ZIC–HILIC, Click Maltose, and Click TE-Cys, exhibit excellent chromatographicproperties. Nonetheless, the increasing complexity of samples such as in proteo-mics and metabolomics presents further challenges with regard to the separationselectivity and efficiency. Besides, the high organic content in the mobile phase isa disadvantage for the dissolution of polar analytes. Therefore, the developmentof novel HILIC stationary phase with superior hydrophilicity and resolution abil-ity is significant. On the other hand, the elaborate illustration of retention mech-anism is crucial for predicting the retention behaviors in HILIC.

References

1 Alpert, A.J. (1990) Hydrophilic-interactionchromatography for the separation ofpeptides, nucleic-acids and other polarcompounds. J. Chromatogr., 499,177–196.

2 Churms, S.C. (1996) Recent progress incarbohydrate separation by high-performance liquid chromatography based

on hydrophilic interaction. J. Chromatogr.A, 720, 75–91.

3 Strege, M.A. (1998) Hydrophilicinteraction chromatography electrospraymass spectrometry analysis of polarcompounds for natural product drugdiscovery. Anal. Chem., 70,2439–2445.

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4 Hydrophilic Interaction Liquid Chromatography › uploads › short-url › nHoRuwZsaxbuG30CZu… · liquid chromatography (HILIC) [1] turns out to be a valuable complement for