polar functional groups for the hilic method

225

11 Polar Functional Groups for HILIC Method

Zhigang Hao

11.1 INTRODUCTION

Using polar stationary phases such as bare silica paired with aqueous-organic mobile phases like water-acetonitrile (H2O-ACN) to separate hydrophilic ingredients can be traced back to the 1970s.1,2 The name hydrophilic interaction chromatography (HILIC) was coined by Alpert in 1990.3 Dr. Weng initiated the first HILIC review on bioanalytical application with underivatized silica column and mass spectrometer (MS) detection in 2003.4 Bare silica has no secondary modification on its surface, and it is still the most popular stationary phase in HILIC applications due to column stabil-ity and low back-pressure. Different types of silica materials (types A, B, and C) have been developed for method selectivity. The rapid growth of HILIC chromatography began around 2003 and since then hundreds of applications with many new station-ary phases have been successfully applied to polar analyte separation. Hemstrom and Irgum constructed review of the HILIC field in 2006 and attempted to ascertain the extent to which partition or adsorption accounted for the separation mechanism.5

In 2008, Prof. Laemmerhofer edited a special HILIC issue in Journal of Separation Science. Prof. Jandera reviewed HILIC stationary phases and differentiated the selec-tivity between HILIC and reverse phase modes.6 It was postulated that the mobile phase similarity in both HILIC and reverse phase systems provided an ideal com-bination for two-dimensional (2D) chromatographic separation. Polar analytes are separated in HILIC mode, usually in the first dimension, whereas less hydrophilic ones in reverse phase mode. Univariate and multivariate approaches in HILIC method development were summarized by Prof. Vander Heydens group.7 Parameters including

CONTENTS

11.1 Introduction ..................................................................................................22511.2 Ionic Functional Groups in HILIC Separation ............................................. 227

11.2.1 Effect of Stationary Phase Composition ...........................................22811.2.2 Effect of Buffer Concentration (Ionic Strength) ............................... 23311.2.3 Effect of Column Temperature ......................................................... 239

11.3 Nonionic Polar Functional Groups in HILIC Separation .............................24411.3.1 Effect of Mobile Phase Composition ................................................24411.3.2 Effect of Column Temperature ......................................................... 252

Acknowledgments ..................................................................................................256References ..............................................................................................................256

226 Hydrophilic Interaction Liquid Chromatography and Advanced Applications

stationary phase, column temperature, mobile phase composition, and flow rate for the detection of biological and nonbiological samples were discussed in this review. Our group discussed the importance of column temperature and mobile phase for HILIC selectivity.8 Drs. Nguyen and Schug highlighted the advantages of HILIC separation coupled with ESI-MS detection.9 The HILIC-MS method application in biological assays was reviewed by Dr. Hsieh.10 Several new HILIC studies were also included in this special issue. These articles gave readers an updated and representative overview of what can be accomplished using HILIC mode, the typical application fields, and the benefits for polar analytes as compared to traditional reverse phase chromatography.

The need to identify novel analytes such as those found in biological fluid metabolites and synthetic mixtures leads to analytical method development. With research advances in genomics, proteomics, and metabolomics, more and more small polar components have appeared in the analytical laboratory. These compounds are usually not retained well enough in a reverse phase high performance liquid chromatography (HPLC) sys-tem to satisfy the recommendation from the Center for Drug Evaluation and Research (CDER), which suggests a minimum capacity factor (k) value of >2.0 to ensure ade-quate separation of the analytes from un-retained matrix components.11 However, these analytes are retained more efficiently in HILIC mode, and the use of a high ratio of organic solvent such as ACN in the mobile phase is compatible with MS detection.

For reverse phase chromatography, retention is considered to be (ideally) controlled by partition since most functional groups such as silanols are covered/shielded by carbon chain materials (C8 or C18) in the stationary phase. Compared to reverse phase HPLC, columns for HILIC have a much wider variety of functional groups such as silanol, amine, amide, diol, cyano, poly-succinimide, sulfoalkylbetaine, and zirconia. All polar functionalities including anionic and cationic moieties on the HILIC packing material surface can absorb some water (0.5%1.0%) to form a stagnant water-enriched layer between the mobile and stationary phases, especially when the water ratio is low (usu-ally less than 40%) in the mobile phase. This layer is immobilized and can be considered as a portion of the stationary phase. The transition between adsorption and partition mechanisms is probably continuous as the water content in the mobile phase gradually increases.6,12 The term HILIC refers to practical application possibilities rather than to a special retention mechanism. Of course, this is the simplest way to consider the HILIC separation compared to reverse phase chromatography. The more hydrophilic an analyte, the more it associates with the stagnant water-rich layer and the later it elutes out. The functional moieties in the structure that convey this property to highly polar compounds are either charged groups or groups capable of entering strong dipolar or hydrogen bonds. An empirical formula can be used to describe analyte polarity:

Polarity

number of polar groupnumber of carbon

=

( )( )

(11.1)

where the polar group can be ionic, protic, CN, C=O, and CONH. The analyte can become very hydrophilic (the polarity is high) when a large number of polar groups relative to the number of carbons are present in a structure. A significant character-istic of polar analytes is that they contain multiple polar groups. If only partition dominates the HILIC separation, we can use the empirical formula (11.1) to predict

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Polar Functional Groups for HILIC Method 227

the elution order, which will be reversed compared to that in reverse phase chro-matography. However, the HILIC stationary phase is different from reverse phase materials in that polar functional groups bound on a HILIC column are not usually highly covered or shielded. They can be exposed directly to the mobile phase and even to the polar functional groups on analyte structures inside the stagnant liquid layer. The variety of polar functional groups bound on a HILIC phase exhibit more significant, strong, and different interactions with the hydrophilic functional groups on analyte structures. Some authors consider these secondary interactions undesir-able,13 whereas others utilized them for better method selectivity.14,15 The structural diversity of HILIC surface chemistries provides analysts with a lot of opportunity for better separation of polar analytes in terms of column selection and the manipulation of the experimental conditions. So far, less attention has been paid to electrostatic (or called as ionic), hydrogen-bond, and hydrophobic interactions for HILIC retention capability. More importantly, these interactions specifically correspond to the differ-ent functional group moieties in both analyte and HILIC phase structures. An elec-trostatic interaction (attraction or repulsion) is produced primarily by either cations or anions from both analytes and stationary phases. Experimentally, mobile phase pH, buffer species, concentration, and column temperature can impact upon this type of interaction. The hydrogen bond usually can be generated by protic groups, and its interaction strength can be affected by varying the mobile phase composition (either protic or aprotic organic solvents). The hydrophobic interaction comes mainly from nonpolar moieties in an analyte structure, and it becomes relatively strong only when a high ratio of water content (>40%) is present in the mobile phase. It is not a typical HILIC separation condition and will not be discussed in this chapter.

When polar analytes from different projects are sent to an analytical laboratory, columns are selected based on analyte structures. When an ionic group (either posi-tive or negative) is present in an analyte, logically the anionic or cationic stationary phase under a designed experimental condition should be selected to enhance or reduce the analyte retention due to electrostatic attraction or repulsion. If multiple protic groups are presented in an analyte such as a carbohydrate, a protic station-ary phase such as an amide column might be used for retention on the column due to strong hydrogen-bond interaction. Different polar functional groups from both analytes and stationary phases can exhibit varying levels of interaction strength in a HILIC separation. The most polar analytes usually contain more than one polar functional group and each group can contribute to some degree. The individual inter-action strength can be manipulated with different experimental conditions such as column selection, pH, buffer concentration, mobile phase composition, and column temperature. This chapter focuses on specific interactions between polar functional groups in both analytes and the stationary phase. For better understanding, ionic and nonionic groups will be reviewed and discussed in some detail separately.

11.2 IONICFUNCTIONALGROUPSINHILICSEPARATION

An ionic compound is a chemical compound in which ions are held together by the strong electrostatic force between oppositely charged groups. Small ionic compounds tend to dissolve in polar solvents like water and form strong electrostatic bonds. Usually,

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the positive-to-positive or negative-to-negative ions exhibit an electrostatic repulsive interaction. In a HILIC system, the ionic functional groups, like other polar groups, prefer to stay inside the stagnant water-rich layer and can interact with ions either inside the stagnant layer (usually from buffer) or bound on the stationary phase by either elec-trostatic attraction or repulsion. The partition retaining power of an analyte can be deter-mined by the empirical formula (Equation 11.1), as described earlier. The more polar groups an analyte possesses, the stronger its retaining power. The retention strength from ionic interaction can be determined by several factors. The first factor is the presence of ionic functional group(s) (either cationic or anionic) in the analyte structures or bound on the stationary phase under a specific pH condition. Certainly, the silanol, amine, and zwitterion chromatography (ZIC) stationary phases should be considered first in the HILIC separation of ionic analytes. The secondary factor is inclusion of buffer salts in the separation system. Different buffer species and their concentration could affect the ionic interaction between analytes and the stationary phase. The third factor is column temperature when a significant analyte transferring enthalpy is involved between the dynamic mobile phase and the stagnant water-rich layer. These three factors for ionic analyte separation will be reviewed and discussed in the following sections. The organic solvent compositions of the mobile phase can impact on ionic group retention in a HILIC system. However, this is more relevant to the hydrogen-bonding retention mechanism and will be reviewed in the section of nonionic functional groups in HILIC separation.

11.2.1 EffEctofStationaryPhaSEcomPoSition

The role and classification of the stationary phase in HILIC separation efficiencies have been reviewed by Prof. Tanakas group.16 Numerous ionic analytes, including acids, bases, amino acids, peptides, nuclei bases, and nucleosides, have been separated on bare silica, amino-silica, amide-silica, poly(succinimide), sulfoalkylbetaine, diol, cyano, cyclodextrin, and triazol columns. The application of the cyano-silica column to the HILIC mode is still limited probably because the cyano group does not have sufficient association with water molecules to generate an effective water-rich layer for partitioning. The residual silanol groups under the cyano-group perhaps are unable to stimulate accumulation of water due to the surface shielding by organic ligands.1618

In general, different HILIC stationary phases exhibit enough variability to retain a wide spectrum of analytes. For example, bare silica columns have been successfully applied for the separation of many nitrogen-containing analytes such as atenolol,19 carve-dilol,20 levofloxacin,21 doxazosin,22 donepezil,23 and glycyl-sarcosine.24 These nitrogen-containing analytes usually provide a positive charge under an acidic condition. However, electrostatic interaction between basic analytes and the different bare silica materials has not been discussed intensively enough to make an informed decision on column selec-tion. Type-A silica materials usually have a lower average pKa value and should provide a stronger electrostatic attraction to the positive charged group on polar analytes under most pH conditions. When this attraction is the secondary retaining power in the HPLC separation, tailing phenomenon can be observed. To eliminate interaction with the strong acidic silanol groups on the silica surface, newer silica materials, so-called types B and C, were developed. Silanol groups on type-B and type-C silica surfaces usually have a higher average pKa value. However, when the primary partition in HILIC separation

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cannot provide enough retention capability, the electrostatic attraction from type-A sil-ica materials can provide a very powerful retention for basic analytes compared with type-Bandtype-C silica materials. Of course, type-A silica may provide a stronger elec-trostatic repulsion to acidic functional groups, resulting in very weak analyte retention.

A comparison of nicotinic acid separation on different columns is a good example of electrostatic interaction in HILIC chromatography. Different types of stationary phases, Thermo Hypersil silica column and Phenomenex Luna NH2, were used to separate nicotinic acid. Nicotinic acid retention on the amino phase (Luna NH2) column shown in Figure 11.1 is much stronger than its retention on the Hypersil silica column shown in Figure 11.2, even though a much stronger mobile phase containing 30% water was used with the amino phase column. The different retentions from Figures 11.1 and 11.2 can be explained by electrostatic interaction of functional groups between analytes and the stationary phase. In Figure 11.2, the carboxyl group (RCOOH) on analytes was deprot-onated and the amine group (RNH2) on column was protonated under the experimental pH condition.15 The negative ion (RCOO) can be electrostatically attracted by the posi-tive ion (NH3+). Nicotinic acid and picolinic acid can be retained longer, even though a high ratio of water (30%) was presented in the mobile phase. However, Hypersil silica column in Figure 11.2 is a more acidic silica type-A material. The silanol group (Si-OH) bound on this material surface can be deprotonated into a negative ion (Si-O) under the pH condition employed. The negative silanol ions give a repulsive interaction to the negative carboxyl group (RCOO) on analytes and result in very short retention time even though a low ratio of water (2%) was presented in the mobile phase.

The repulsive interaction between silanol and carboxyl groups is further elucidated by the data in Table 11.1.14 A Hypersil silica column was used. The chemical structures of glycine (G), diglycine (DG), triglycine (TG), N-[1-deoxy-d-glycose-1-yl]-glycine (GG), N-[1-deoxy-d-glycose-1-yl]-diglycine (GDG), and N-[1-deoxy-d-glycose-1-yl]-triglycine (GTG) are shown in Figure 11.3. The equilibrium reactions for ionic func-tional groups in both analytes and stationary phase are shown in the following formula:

Si Si-OH RCOO RCOOH RNH RNH2 3 +

0.275

0.220

0.165

0.110

0.055

0.0000.00 2.00

AU

4.00 6.00min

Nico

tinic acid-6

.824

Picolin

ic acid-7

.840

8.00 10.00 12.00

FIGURE11.1 Representative chromatogram of nicotinic and picolinic acids at 70:30 ACN/buffer with a Phenomenex Luna NH2 column. The buffer was made by equal volume of 200 mM ammonium acetate and 100 mM glacial acetic acid in water. (Adapted from Christopherson, M.J. etal., J. Liq. Chromatogr. Relat. Technol., 29, 2545, 2006. With permission.)

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All equilibrium reactions will move to the right-hand side when mobile phase acidity increases. Amine NH(12) groups in the analyte structures should be fully protonated to positive ammonium NH(23)+ groups based on the pH values of different formic acid concentrations in the mobile phase (from 0.1% to 0.7%) and the pKa values of G, DG, TG, GG, GDG, and GTG listed in Table 11.1, even though the real pH values in aqueous organic mobile phase are slightly different from those in water media. Because their pKa values are 3.5, the neutral form of carboxyl groups in the DG, TG, GDG, and GTG structures will be dominant under mobile phase pH conditions from 2.20 to 2.68. The ionic form of silanol groups on the silica surface will decrease when the formic acid concentration increases because these groups usually have a broad range of pKa values. As expected, DG, TG, and their Amadori compounds, GDG and GTG, are retained less when formic acid content is increased because less ionic interaction between Si-O and NH(23)+ results in a weaker retention. It was interesting to note that retention times of G and GG were completely different from the other four analytes. They were retained longer when the formic acid con-tent was increased from 0.1% to 0.7% in the mobile phase. The observation can be explained by the equilibrium reactions described earlier. The pKa of silanol groups

0.2 0.6 1.0 1.4 1.8 2.2 2.6 3.0Time, min

ISTD

NiNH2

NiUAc

NiAc

1.5e5

1.5e5

Intensity, cps

1.5e5

1.5e5

3.4 3.8 4.2 4.6

FIGURE11.2 HILIC-APCI-MS/MS chromatograms for nicotinamide (NiNH2), nicotinic acid (NiAc), 6-methylnicotinic acid (ISTD), and nicotinuric acid (NiUAc), using high organic mobile phase containing 2% (solid line) or 5% (dotted line) water with a Thermo Hypersil silica column. (Adapted from Hsieh, Y. and Chen, J., Rapid Commun. Mass Spectrom., 19, 3031, 2005. With permission.)

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TABLE11.1RetentionTimesofSixAnalytes,G,DG,TG,GG,GDG,andGTGundertheHPLCConditionofHypersilSilicaColumn,1001mm,ParticleSize:3mwithMobilePhase2%Waterand98%MethanolandtheVariableFormicAcidContentsinBothSolventsfrom0.1%to0.7%andColumnTemperatureat30C

0.1%FA(pH:2.68)a

0.2%FA(pH:2.50)

0.3%FA(pH:2.41)

0.4%FA(pH:2.34)

0.5%FA(pH:2.28)

0.6%FA(pH:2.25)

0.7%FA(pH:2.20)

G 3.92 4.74 5.15 5.46 6.69 6.90 6.80

(pKa: 2.34, 9.6)

DG 10.62 10.52 10.31 9.90 8.87 8.67 8.26

(pKa: 3.5,8.0)

TG 13.62 13.11 12.49 11.87 10.43 10.12 9.61

(pKa: 3.5,8.1)

GG 3.05 3.15 3.35 3.35 4.38 4.69 4.79

(pKa: 3.5,8.5)b

GDG 8.20 7.59 7.48 7.28 6.87 6.56 6.35

(pKa: 3.5,8.0)b

GTG 10.28 9.15 8.63 8.22 7.29 7.09 6.99

(pKa: 3.5,8.0)b

Source: Adapted from Hao, Z. etal., J. Chromatogr. A, 1147, 165, 2007. With permission.a The pH values in this row were measured in water media and the real pH values in aqueous organic

mobile phase could be slight different in water media.b The pKa data for GG, GDG, and GTG were not measured and the data were just assumed to be similar

to G, DG, and TG, respectively.

O

O

ONH2HO

DG

TG

HN

O

GTGO

HOHN

HN

O O OH

OH OHOH

HN

O

GDG OHO

HN

HN

O

OH

OH

OHOH

O

GG

O

HOHN

OHOH

OH

OH

O

OHO

HN

O

HN NH2

HONH2

G

FIGURE11.3 Structures of G, DG, TG, GG, GDG, and GTG.

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is the average value of all silanols present on the silica surface. When the pH falls below 2.1, the ionization of silanols is suppressed with the exception of the most acidic ones.25 The silanol groups on type-A silica provide a lower average pKa value. A significant amount of the ionic form, Si-O, is presented on the stationary phase surface under the pH conditions listed in Table 11.1. At a pH of 2.68, the ionic form of carboxyl group, COO, is dominant in the G and GG structures due to their pKa values of 2.34 and 2.5, respectively. The electrostatic repulsion between COO and Si-O results in less retention. When formic acid content is increased, both carboxyl acid and silanol ionizations were suppressed. The decreased repulsive force provided a longer retention time. Indeed, the retention times were increased from 3.92 and 3.05 to 6.80 and 4.79 min for G and GG, respectively, when the formic acid concen-tration was changed from 0.1% to 0.7% (Table 11.1). A similar situation was found in sodium cromoglicate separation on an Atlantis HILIC-Si column. Its retention time became longer when the mobile phase pH increased from 4.2 to 5.8.26 Usually, a bare silica column cannot provide a very strong retention capability to acidic groups in polar analytes under an acidic condition without buffer applications.27,28

The repulsive interaction is not only present in the negative-to-negative groups but also in the positive-to-positive groups between analytes and the stationary phase. Dr. Liu etal. found that only a Zorbax-NH2 column caused four hydrazines to be eluted out before the void volume compared to three other columns (YMC-Pack Diol-120-NP, Amide-80, and ZIC-HILIC; see Figure 11.4).29 The Zorbax-NH2 col-umn also showed a weak retention to basic 4-(aminomethyl)pyridine and related compounds when compared with six other columns.18

11

1

1/2/3/4

3 4

4

1

23

4 (d)

(c)

(b)

(a)

T0

T0

T0

T0

22/3

0.750.50.25

00.250.50.75

11.25

0 5 10 15 20min

mV

25 30 35 40

FIGURE11.4 The separation of hydrazines on different columns. Parameters for the studies included 30C column temperature, 0.4 mL/min flow rate with splitter, CLND detector system set at 10, 50C combustion furnace, 50 mL/min argon, 280 mL/min oxygen, 75 mL/min makeup (argon), 30 mL/min ozone, 5C cooler, gain x1, and 750 V on PMT. The analyte concentrations were about 3070 g/mL in water/ethanol (20/80, v/v). Injection volume was 10 L. Mobile phase was formic acid/water/ethanol (0.5/20/80, v/v/v). 0.1% acetonitrile (v/v) in ethanol was used as a void volume marker. 1: 1,2-dimethylhydrazine, 2: 1,1-dimethylhydrazine,3: methylhydrazine, and 4: hydrazine. (a) Zorbax NH2, (b) Diol, (c) Amide-80, and (d) ZIC HILIC. (Adapted from Liu, M. etal., J. Chromatogr. A, 1216, 2362, 2009. With permission.)

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The secondary retaining force can be an undesired interference in HPLC separa-tion causing tailing or broad peak issues.13 But it can also be a very important reten-tion mechanism if we understand which column is able to provide such a retaining force for our analytes of interest. Switching from bare silica to an amino-phase for acidic or even basic functional groups should introduce a significant separation capa-bility. More recently, Alpert has coined another chromatographic acronym, ERLIC, for electrostatic repulsion hydrophilic interaction chromatography.30 The HILIC mobile phase (a high ratio of organic solvents) is combined with an ion-exchange column (a charged stationary phase) in this methodology. The separation strategy in ERLIC superimposes a second mode of chromatography that selectively reduces the retention of analytes that are usually the most strongly retained. Actually, many HILIC columns have this ion-exchange capability. For example, negative residual silanol groups are present in all silica-based HILIC columns,31 which not only pro-vide an extra retention capability to the basic or positive-charged functional groups but can also generate an electrostatic repulsion to acidic or negative-charged func-tional groups. The negative residual silanol groups are more significant in type-A silica material. For the amine column, the positive charge on the outer layer of the stationary phase can give a strong electrostatic attraction to acidic analytes, even though the residual silanols are present in the inner layer of the stationary phase. In contrast, the ZIC-HILIC stationary phase has an inner positive quaternary ammo-nium ion and an outer negative sulfonate ion separated by a three-methylene group, which has been reported for the separation of basic analytes such as hydrazines,29 mildronates,17 and acetylcholine.32 In addition, some degree of electrostatic interac-tion was found between ionizable analytes and the residual silanol groups under non-ionic (neutral) polar stationary phase surfaces such as diol,33 sorbitol methacrylate,34 and amide-silica.35 These initial studies are promising, but more studies are required to better understand the role of electrostatic interaction in HILIC separation.

11.2.2 EffEctofBuffErconcEntration(ionicStrEngth)

The buffer is a very important component when considering ionic interaction. The buf-fer can be used to control mobile phase pH, and pH can modify the ionization status not only for analytes but for stationary phases as well. Optimizing buffer concentra-tion can also generate the ion strength necessary to mediate the electrostatic inter-action between analytes and stationary phase.36 In addition, buffer composition can affect analyte retention or transition enthalpy between mobile and stationary phases. Retention enthalpy is an important parameter if column temperature needs to be con-sidered for improved analyte separation, which will be discussed in next section.

In general, at a low buffer concentration or a buffer-free condition, ionic analytes are effectively retained on stationary phases containing counter ionic groups. As the buffer concentration increases, a high level of organic composition in the mobile phase could force the buffer ions inside the stagnant water-rich liquid layer. Higher buffer ion concentration would drive more solvated salt ions into this liquid layer and result in an increase in volume or hydrophilicity of the liquid layer. The strength of the electrostatic interaction between analytes and the stationary phase would be weakened with an increase of the water-rich layer volume, thus resulting in a weak

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electrostatic interaction. If the electrostatic interaction is with counter ions, the elec-trostatic attraction would be weakened, resulting in a longer retention times. If the chargecharge interaction is with co-ions, the electrostatic repulsion would also be weakened, resulting in shorter retention times. Most experimental data, especially with ammonium acetate or formate buffer, support this rationale.29,3739 For example, room temperature ionic liquid (RTIL) imidazolium cations were not eluted (a very strong capacity factor) on diol stationary phase with a salt-free mobile phase due to their strong electrostatic interactions with the negative residual silanols under the diol stationary phase surface.40 A decrease in capacity factor was observed when the ammonium acetate concentration was raised from 5 to 20 mmol/L (shown in Figure 11.5). The negative residual silanols on a diol phase column was also reported for a uric acid separation.41 The negative silanol groups exert electrostatic repulsion on the negatively charged acids under the experimental conditions. Increasing the concen-tration of salt in the mobile phase would reduce this electrostatic repulsion leading to stronger retention.

The retention of anionic nicotinic and picolinic acids on an amine-silica column were reduced by increasing the buffer concentration, as shown in Figure 11.6.15 In another example, the retention time of acidic analytes (aspirin and salicylic acid) increased by about 20%40% on amide-silica, HILIC silica, and ZIC-HILIC col-umns but decreased sharply on the amine-silica column when the ammonium acetate concentration was increased from 5 to 20 mM (shown in Table 11.2).36 The buffer concentration can contribute to both electrostatic repulsion and attraction between analytes and stationary phases. The ionized residual silanol groups on amide-silica and silica columns can repulse the ionized carboxyl groups on analytesunderpH6.9.

1.80

1.60

1.40

1.20

1.00

0.80

0.60

0.40

0.20

0.000 5

543

10Ammonium acetate concentration (mM)

ln k

15 20 25

FIGURE11.5 Plots of ln k vs. ammonium acetate concentration in the mobile phase for three RTILs: (3) 1-butyl-2-methyl-3-methyl imidazolium bis-(trifluoromethylsulfonyl)-imide, (4) 1-hexyl-3-methyl imidazolium chloride, and (5) 1-methyl-3-octyl imidazolium chloride. Stationary phase: Uptisphere OH; mobile phase: ACN/H2O with 10 mM ammonium acetate at 0.2 mL/min. (Adapted from Rouzo, G.L. etal., J. Chromatogr. A, 1164, 139, 2007. With permission.)

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Anincrease in buffer ions would eliminate such repulsive interaction, resulting in a longer retention time. The ZIC-HILIC stationary phase has an inner positive qua-ternary ammonium ion and an outer negative sulfonate ion separated by a three-methylene group.29 The ascorbic acid (vitamin C) retention time on this column became longer when the ammonium acetate concentration increased from 10 to100 mM.42

6.0

5.0

4.0

3.0

2.050 90 130

Ammonium hydroxide concentration (mM)170 210

k 7.0

FIGURE11.6 Effect of buffer concentration on the retention of nicotinic acid () and picolinic acid (). (Adapted from Christopherson, M.J. etal., J. Liq. Chromatogr. Relat. Technol., 29, 2545, 2006. With permission.)

TABLE11.2RetentionTimeoftheModelCompoundsatDifferentAmmoniumAcetateConcentrationsintheMobilePhasea

Column Concentration(mM) SalicylicAcid Aspirin Cytosine

TSK-Gel Amide-80 5 2.07 3.06 6.84

10 2.39 3.65 7.19

20 2.61 4.14 8.01

YMC-Pack NH2 5 7.59 20.21 6.03

10 4.72 11.50 6.10

20 3.56 7.17 6.45

HILIC Silica 5 1.78 2.94 5.51

10 2.06 3.51 5.78

20 2.49 4.21 6.62

ZIC-HILIC 5 2.16 2.78 5.52

10 2.44 3.22 5.59

20 2.64 3.55 5.98

Source: Adapted from Guo, Y. and Gaiki, S., J. Chromatogr. A, 1074, 71, 2005. With permission from Elsevier.

a Mobile phase: acetonitrile/ammonium acetate solution (85/15, v/v). Column tempera-ture: 30C. Flow rate: 1.5 mL/min.D

ownl

oade

d by

[Was

hingto

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. Lou

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of M

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t 11:0

0 11 N

ovem

ber 2

015


Theelectrostatic repulsion between the analyte carboxyl group and sulfonate group on the outer layer of the stationary phase seems to be predominant. Analyte size may block or reduce the electrostatic attraction between analyte carboxyl ions and qua-ternary ammonium ions on the inner layer of the stationary phase. The ZIC-HILIC column has been reported for the separation of inorganic anions by ion chromatog-raphy.43 The retention behavior of four hydrazine analogues in different ammonium formate concentrations is shown in Figure 11.7. Since the HCl salts of hydrazine and 1,2-dimethylhydrazine were used and RI is a universal detector, the chloride peak was also observed. The retention of positively charged hydrazines decreased with increasing ionic strength, whereas the retention behavior of negatively charged chlo-ride was just the opposite (Figure 11.7a).

In conventional ion-exchange chromatography (IEC) with water media, the rela-tionship of the retention factor k of an analyte and the buffer concentration [C] is as described below44,45

log log constantk C = +s [ ]

where s is a constant slope, which is dependent on the overall charge of the ana-lytes and counter-ions. For a singly charged analyte and univalent counterion, the slope should be 1. Ammonium formate concentrations in the range of 530 mM were used for drawing the plots of log k vs. log [C] for hydrazines on a ZIC-HILIC column. The linear relationships observed for all hydrazines and for chloride (coef-ficient of determination r2 0.97) are shown in Figure 11.7b. All hydrazines in this

10,000

8,000

6,000

Cl

ClClCl

Cl

Cl

Cl/3

1

11

11

1

1

1

2

22

2

2

2

2

2/Cl

3

33

3

3

3

3

4

44

4

4

4

4

4

5 mM

10 mM20 mM

30 mM

40 mM

50 mM

70 mM

90 mM

4,000

2,000

5 10 15 20 25min(a)

nRIU

30 35 40

FIGURE11.7 (a) The effect of ionic strength on the retention of hydrazines on a ZIC-HILIC column. Column temperature was set at 30C. Isocratic runs with a mobile phase of 590 mM ammonium formate buffer pH 3.0/ethyl alcohol (20/80, v/v) (the buffer concentrations refer to the concentration before mixing with organic solvent). Flow rate was 0.4 mL/min with RI detection. The analyte concentrations were about 0.81.2 mg/mL in water/ethyl alcohol (20/80, v/v). The injection volume was 1 L. 0.5% toluene (v/v) in ethyl alcohol was used as a void volume marker. Chloride is from the HCl salts of hydrazine and 1,2-dimethylhydrazine. 1: 1,2-dimethylhydrazine, 2: 1,1-dimethylhydrazine, 3: methylhydrazine, and 4: hydrazine.

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study have negative slopes while chloride has a positive slope, which are indicative of the net electrostatic attraction and repulsion with the stationary phase, respectively. The absolute slope values in Figure 11.7b (0.3 for hydrazines and 0.1 for chloride) indicated other interactions, and mobile phase composition could also contribute to analyte retention. Positive intercepts were observed for all components, which provide evidence for the existence of additional retention mechanisms at infinite buffer concentration. Cumulatively, data in Figure 11.7 indicate that both ionic inter-action and hydrophilic interaction were involved in the separation of hydrazine ana-logues and their counterions under an aqueous organic mobile phase condition.

An exceptional example was found with the positive analytes (metformin hydrochlo-ride [MFH], cyanoguanidine [CGD], and melamine [MLN]) separated on an Atlantis HILIC-Si column.46 Ammonium acetate and ammonium formate buffers proved to

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

0.10.6 0.8 1.0

Log [Ammonium formate]

Log k

1.2 1.4

R2= 1.000

R2= 1.000R2= 1.000

R2= 1.000R2= 0.964

(b)

6

5

4

3

2

1

00 0.05 0.1 0.15 0.2 0.25 0.3 0.35

1/[Ammonium formate]

k

R2= 0.973

R2= 0.975

R2= 0.976

R2= 0.978

R2= 0.946

(c)

FIGURE11.7(continued) (b) Plot of logarithm of retention factor (k) against logarithm of buf-fer concentration (530 mM). (c) Plot of k against the inversed buffer concentration (530 mM). Hydrazine (), methylhydrazine (), 1,1-dimethylhydrazine (), 1,2-dimethylhydrazine (), and chloride (). (Adapted from Liu, M. etal., J. Chromatogr. A, 1216, 2362, 2009. With permission.)

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be unsuitable for UV detection at 218 nm. The retention of metformin increased when the concentration of sodium phosphate increased from 10 to 40 mM (Figure 11.8). It is not entirely clear whether sodium phosphate buffer has an impact upon ionic analyte separation in HILIC since the use of this buffer is limited with HILIC method.

Determining how buffer type modifies electrostatic interaction between analytes and stationary phases can be complicated. When ammonium acetate was replaced with ammonium formate in Drs. Guo and Gaiki study described previously,36 no significant change was found for the neutral analyte, cytosine. Little retention change was observed on amide-silica, HILIC silica, and ZIC-HILIC columns for acidic ana-lytes (aspirin and salicylic acid). A significant retention increase was obtained on an amine-silica column for acidic analytes, which might be related to formic acid size since these smaller sized ions might penetrate through the outer amine layer to suppress silanol ionization in the inner layer of stationary phase. Surprisingly, when ammonium acetate was replaced with ammonium bicarbonate, the latter caused drastic decreases in retention on all four HILIC columns and destroyed the separa-tion of the acids.

The study of buffer species is limited because some buffers that are typically used in reverse phase HPLC may not be suitable for HILIC due to their poor solu-bility in mobile phase containing a high level of ACN.36 In addition, the use of buf-fers is relatively limited when HILIC separation is coupled with mass spectrometry detection. Most nonvolatile buffers produce a very strong ionic suppression on MS detection signals. Therefore, the most common buffer solutions used in HILIC-MS

010 20 30

Phosphate buffer (mM)40

2

4

6

Retention factor (k)

8

10

12

14

CGDMLNMFH

FIGURE11.8 Effect of sodium buffer strength on the retention of MFH, CGD, and MLN on a 5 m, 250 4.6 Atlantis HILIC-Si column. Acetonitrile/buffer (84/16, v/v) with a pH of 3. Flow rate: 2.0 mL/min. (Adapted from Ali, M.S. etal., Chromatographia, 67, 517, 2008. With permission from Elsevier.)

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methods are ammonium acetate or formate. Practical HILIC applications coupled with MS detection only use organic acid to control the mobile phase pH because most HILIC stationary phases are stable under an acidic condition.

11.2.3 EffEctofcolumntEmPEraturE

Column temperature has long been recognized as an important parameter in HPLC sep-aration, especially after column and mobile phase selections have been made. Column temperature has a significant impact on analyte diffusivity, mobile phase viscosity, and analyte transferring enthalpy between mobile and stationary phases. In general, tem-perature elevation increases the diffusion coefficient and decreases the mobile phase viscosity, resulting in narrower peaks. But its drawback is shorter retention times. The total resolution could not be improved much under such a scenario. Only if increased column temperature would simultaneously retain various analytes longer on the column, then retention differences between individual analytes with the narrowed peaks would be enlarged and overall resolution could be improved. To achieve such a scenario, a positive transferring enthalpy (H value), or the negative slope in the vant Hoff plot, needs to be present. Small ionic analytes combined with a counterionic stationary phase in HILIC has provided such an opportunity. When ionic functional groups are present in small polar analytes, column temperature should therefore be considered in HILIC separations.

For example, when anionic aspirin (acetylsalicylic acid) was separated on four different HILIC columns, YMC-pack NH2, TSK-Gel Amide-80, Atlantis HILIC sil-ica (type-B), and SeQuant ZIC, only the positive amino stationary phase (YMC-pack NH2) obtained negative slopes or positive H values (Figure 11.9).36 The positive retention enthalpy indicated an endothermic process of transferring analytes from the mobile phase to the stationary phase. Guo etal. further investigated six organic acids, acetylsalicylic acid, salicylic acid, gentisic acid, hippuric acid, salicyluric acid, and -hydroxyhippuric acid, on the five columns (Figure 11.10b), and found

00.0028 0.0029 0.003 0.0031 0.0032

1/T (K)

ln k

0.0033 0.0034 0.0035

0.5

1

1.5

2

2.5

3

FIGURE11.9 The vant Hoff plots for aspirin on () TSK-Gel Amide-80, () YMC-Pack NH2, () HILIC Silica, and () ZIC HILIC columns. Mobile phase: ACN/water (90:10, v/v) containing 10 mM ammonium acetate. (Adapted from Guo, Y. and Gaiki, S., J. Chromatogr. A, 1074, 71, 2005. With permission.)

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25

20

15

10

5

25

20

15

10

5

Retention tim

e (min)

Retention tim

e (min)

4030

20

10 65(a)

(b)

Salt concentration (mM) ACN

content

(%)

70

65 70 75ACN content (%) Salt concentration (mM)

80 85 40 30 20 10

7580

85

16

14

12

10

6

8

16

14

12

8

10

6

Retention tim

e (min)

Retention tim

e (min)

4040

5060

3030

2020

10 10

Column temperature (C) Salt con

centratio

n (mM)

10 20Salt concentration (mM) Column temperature (C)

30 40 405060 30 20 10

FIGURE11.10 3D response surfaces for the amino phase (YMC-pack NH2 column) gener-ated by DOE software. (a) Influence of ACN and ammonium acetate concentration on acidic analyte retention. (b) Influence of ammonium acetate concentration and column temperature on acidic analyte retention. (Adapted from Guo, Y. etal., Chromatographia, 66, 223, 2007. With permission.)

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the amino phase had very different response surfaces. The retention time increased when the column temperature was elevated from 10C to 60C. Similarly, when pic-olinic and nicotinic acids were separated using the positive Phenomenex Luna NH2 stationary phase shown in Figure 11.1, negative slopes from both anionic analytes were obtained in the vant Hoff plots (shown in Figure 11.11).

In contrast, when cationic analytes are separated with an anionic stationary phase, the analytes which transfer enthalpy might also exhibit a better separation. The cat-ionic analytes, G, DG, TG, GG, GDG, and GTG (their structures are in Figure 11.3), were separated by four silica type-A columns (Figure 11.12). Their retention times increased when the column temperature was elevated no matter if methanol or ACN was used in the mobile phase.

When these six analytes were separated on bare silica type-B and type-C col-umns, much weaker retentions were observed for all six analytes (Figure 11.13). More importantly, the positive slopes or negative transferring enthalpies were obtained in vant Hoff plots when a column temperature program from 5C to 80C was applied for analyte separation. A possible rationale is higher average pKa values attributed to silanol groups on type-B and type-C silica material surfaces.

However, negative slopes were reported for the separation of the basic analogue epiru-bincin on a Kromasil KR100-5SIL bare silica column (Figure 11.14).47 More importantly, the column plate number (N) was increased from 32 to 55 K plates/m by elevating the column temperature from 25C to 40C (about 70% improvement in column efficiency).

For comparison, we also used this type of column to test the impact of column temperature on the separation of six analytes in our laboratory. In contrast, slopes for all six analytes were positive in the vant Hoff plots (Figure 11.15). Buffer solu-tion was not used in our experiments. It is not clear whether the different outcomes are the result of a difference in analyte characteristics or the sodium formate buffer used in epirubicin analogue separation. More studies with Kromasil KR100-5SIL are needed for a more definitive conclusion.

1.2

1

0.8

0.60.003 0.0031 0.0032

1/T0.0033 0.00350.0034

y=887.79x+4.0107

y=919.26x+3.9214

ln k

1.4

r2= 0.9922

r2= 0.9739

FIGURE 11.11 The vant Hoff curves for nicotinic acid () and picolinic acid () on a Phenomenex Luna NH2 column using 70:30 (acetonitrile:buffer). (Adapted from Christo-pherson, M.J. etal., J. Liq. Chromatogr. Relat. Technol., 29, 2545, 2006. With permission.)

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40.00

35.00

30.00

25.00

20.00

15.00

10.00

5.00

0.000.00270 0.00280 0.00290 0.00300 0.00310 0.00320

1/T (K1)(a)

Log k

0.00330

GTG

R2= 0.984R2= 0.992R2= 0.981R2= 0.995

R2= 0.993

R2= 0.980

GDGGGTGDGG

0.00340 0.003600.00350

18.00

16.00

14.00

12.00

10.00

8.00

6.00

4.00

2.00

0.000.00270 0.00280 0.00290 0.00300 0.00310 0.00320

1/T (K1)(b)

Log k

0.00330

GTGR2= 0.981

R2= 0.985

R2= 0.987

R2= 0.994

R2= 0.979

R2= 0.892

GDGGGTGDGG

0.00340 0.003600.00350

FIGURE11.12 The vant Hoff plots for G, DG, TG, GG, GDG, and GTG under HILIC condition of Hypersil silica column, 100 1.0 mm, particle size of 3 m with a flow rate of 100 L/min, the column temperature varying from 5C to 80C, mobile phase for top: water/ACN (25:75, v/v) containing 0.4% formic acid and mobile phase for bottom: water/MeOH (2:98, v/v) containing 0.4% formic acid. (Adapted from Hao, Z. etal., J. Sep. Sci., 31, 1449, 2008. With permission from Wiley-VCH Verlag Gmbh & Co. KGaA.)

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0.00

5.00

10.00

15.00

20.00

25.00

0.00270 0.00280 0.00290 0.00300 0.00310 0.00320 0.00330 0.00340 0.00350 0.003601/T (K1)

Log k

GTGGDGGGTGDGG

FIGURE 11.13 The vant Hoff plots for six analytes, G, DG, TG, GG, GDG, and GTG under HILIC condition of Atlantis silica column, 50 2.1 mm, particle size of 5 m with a flow rate of 100 L/min, column temperature varying from 5C to 80C, mobile phase of water/ACN (10:90, v/v) containing 0.4% formic acid. (Adapted Hao, Z. etal., J. Sep. Sci., 31, 1449, 2008. With permission from Wiley-VCH Verlag Gmbh & Co. KGaA.)

2.2

2.1

2.0

1.9

1.8

1.7

1.63.22 3.24 3.283.26

103/T (K1)3.32 3.343.30 3.36

ln k

2.3 EpidaunorubicinDaunorubicinEpirubicinDoxorubicin

FIGURE 11.14 Effect of temperature upon retention with acetonitrile as modifier. Conditions: Kromasil KR100-5SIL (5 m); mobile phase: 90% (v/v) ACN in sodium for-mate buffer (30 mM, pH 2.9) and UV detection at 254 nm for epirubicin and its analogues; flow rate: 1.0 mL/min; injection volume: 20 L. (Adapted from Dong, L. and Huang, J., Chromatographia, 65, 519, 2007. With permission.)

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11.3 NONIONICPOLARFUNCTIONALGROUPSINHILICSEPARATION

The nonionic polar functional group has a permanent dipole moment but lacks a complete electric charge. The permanent dipole occurs when two atoms in a functional group have substantially different electronegativity, one atom attracts an electron more than the other becoming more negative, while the other atom becomes more positive. The major and strongest interaction between nonionic polar functional groups is hydrogen bonding. The hydrogen bond can also provide powerful retaining power in HILIC, even though it is weaker than ionic interac-tion. Hydrogen-bond energy is within 530 kJ/mol while ionic bond energy is usually larger than 100 kJ/mol. Mobile phase composition and column temperature can dramatically impact upon hydrogen bonding between analytes and the station-ary phase.

11.3.1 EffEctofmoBilEPhaSEcomPoSition

In HILIC, the secondary hydrogen-bond retention mechanism can be distinguished from the primary partition retention mechanism. Hydrogen-bond strength can be

0.00270 0.00280 0.00290 0.00300 0.00310 0.003201/T (K1)

0.00330 0.00340 0.003600.00350

24.00

22.00

20.00

18.00

16.00

14.00

12.00

Log k

GTGGDGGGTGDGG

FIGURE11.15 The vant Hoff plots for six analytes, G, DG, TG, GG, GDG, and GTG, under HILIC condition of Kromasil KR100-5SIL silica column, 100 2.0 mm, particle size of 5 m with a flow rate of 100 L/min, the column temperature varying from 5C to 80C, mobile phase of water/acetonitrile (10:90, v/v) containing 0.4% formic acid. (Adapted Hao, Z. etal., J. Sep. Sci., 31, 1449, 2008. With permission from Wiley-VCH Verlag Gmbh & Co. KGaA.)

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explained by the neutral polar functional groups, including protic or aprotic. The polar protic groups can be both donors and acceptors of hydrogen bonds whereas aprotic solvents can be only hydrogen-bond acceptors. Partition is usually deter-mined by the entire analyte polarity or hydrophilicity, represented by the log P value, which is the logarithm of the octanolwater partition coefficient. Log P values are unavailable and an extra independent experiment is needed to determine them. For practical HILIC method development, the hydrogen-bond retention mechanism is more suitable to predict the retention capability of neutral functional groups within analytes and stationary phase. A separation study of neutral oligomeric proanthocy-anidins (structures in Figure 11.16) on an amide-silica column (TSK-Gel Amide-80) is a good example showing the contributions of hydrogen bonding and partitioning in a HILIC separation.48 The correlation between the logarithm of retention fac-tors(logk) and the number of hydroxyl groups in Figure 11.17 is even better than between logk and log P values in Figure 11.18 (r2 = 0.9501 vs. 0.7949, respectively).48

When hydrogen bonding is strongly involved in HILIC retention, switch-ing organic components in the mobile phase can be a very important strategy to improve analyte separation. These organic components can be subdivided into polar protic and aprotic solvents. Methanol, ethanol, isopropanol, and acetic acid are rep-resentative polar protic solvents. The typical polar aprotic solvents are ACN and tetrahydrofuran (THF). Because of their strong ability to hydrogen bond, polar protic solvents canmore effectively compete for polar active sites on the HILIC phase sur-face, perturbing the formation of water layers by replacing water molecules, thus

O

OH

R1HO

OHOH

OH

O

O

OH

R1HO

OH

OH

R2

R1

1a H : (+)-catechin

1b OH : (+)-gallocatechin

R1 R2

1c H H : ()-epicatechin1d OH H : ()-epigallocatechin1e H galloyl : ()-epicatechin gallate1f OH galloyl : ()-epigallocatechin gallate

EC

EC EC EC EC EC

EC EC EC EC

EC

EC EC ECEC C

EC EC

CC

3a

ECGC

3b

3c

4a

5a

2a

2b

2c

2d

(4 8)

(4 8)

(4 6)

2

3

46

8

2

3

46

8

EC EC

EC EC

C

GC

EC

FIGURE11.16 Chemical structures of monomeric flavan-3-ols and oligomeric proanthocy-anidins. (Adapted from Yanagida, A. etal., J. Chromatogr. A, 1143, 153, 2007. With permission.)

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producing a more hydrophobic stationary phase.49 As a consequence, analytes with strong hydrogen-bond capability are poorly retained.

Li and Huang compared the ability of various organic modifiers to retain epiru-bicin analogues using a Kromasil KR100-5SIL column (Figure 11.19).50 Methanol caused all four analytes to be eluted with no retention. Isopropanol has a longer alkyl chain and less hydrophilic character. It competes less strongly for the active

30

25

20

15

10

5

00.6 0.4 0.2 0.20 0.4 0.6 0.8 1.21 1.4

Log k

Num

ber o

f OH gr

oups

1a1c

2a

3b3c

4a

5a

1b1d

1e 1f 2c2b

3a

2d

y=12.341x+9.2541r2= 0.9501

FIGURE11.17 Relationship between log k of 15 standards in amide HILIC column (TSK-Gel Amide-80) and the number of hydroxyl groups (OH) in their structures. Inset regression formula was calculated from the linear least-square fit of all data (n = 15). (Adapted from Yanagida, A. etal., J. Chromatogr. A, 1143, 153, 2007. With permission.)

1

0

1

2

30.6 0.4 0.2 0.20 0.4 0.6 0.8 1.21 1.4

Log k

Log P

o/w

1a

1c

2a3b

3c

4a5a

1b1d

1e

1f

2c 2b 3a

2d

y=1.8177x0.4767r2= 0.7949

FIGURE 11.18 Relationship between log k of 15 standards in amide HILIC column (TSK-Gel Amide-80) and log P values measured by HSCCC. Inset regression formula was calculated from the linear least-square fit of all data (n = 15). (Adapted from Yanagida, A. etal., J.Chromatogr. A, 1143, 153, 2007. With permission.)

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sites and left more time for analytes to be retained. In contrast to methanol and isopropanol, the aprotic solvents ACN and THF provided more effective retention of the analytes on the column. The analytes were retained more strongly with ACN than THF because the latter is a better hydrogen-bond acceptor. Interestingly, the elution order of epirubicin and daunorubicin with ACN is different from THF and isopropanol (Figures 11.19 and 11.20). The major differences between these two structures are an extra hydroxyl group by the keto group in epirubicin and an inner hydrogen bond between the hydroxyl group and amine group in daunorubicin. Epirubicin was retained longer in the mobile phase containing ACN, where stron-ger hydrogen bonding occurs between analyte and the stationary phase. When ACN was replaced by THF or isopropanol, the retention contribution from such hydrogen bonding became weaker and ion-exchange interactions between the analyte and the stationary phase became stronger. Daunorubicin, with a higher pKa due to such inner hydrogen bonding, was retained longer. The four hydrazine analogues mentioned earlier were successfully separated by an aqueous ethanol mobile phase on a ZIC-HILIC column because ACN cannot be used with a chemi-luminescent nitrogen detector (CLND).36

A systematic comparison of selectivity (log values) between G vs. GG, DG vs. GDG, and TG vs. GTG was investigated on a Hypersil silica column with

140

120

100

80

60

40

20

0

0 5 10 15Time (min)

Acetonitrile

THF1

1

2

2

4

4

3

3

Isopropanol

Absorbance (m

AU)

Methanol

2+41+3

1+3+2+4

20 25 30

FIGURE11.19 Effect of organic modifier on separation of epirubicin and its analogues. Conditions: Kromasil KR100-5SIL (5 m); mobile phase: sodium formate buffer (20 mM, pH 2.9) modified with various organic solvents (10:90, v/v). Peaks: (1) epidaunorubicin, (2) daunorubicin, (3) epirubicin, (4) doxorubicin. (Adapted from Li, R.P. and Huang, J.X., J.Chromatogr. A, 1041, 163, 2004. With permission.)

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different organic solvents used in the mobile phase (Figure 11.21).14 When the methanol content was low and log < 0, G, DG, and TG eluted before GG, GDG, and GTG, respectively. When the methanol content was increased and log = 0, no separation occurred for each of the individual pairs. When the methanol con-tent was further increased and log > 0, G, DG, and TG eluted after GG, GDG,

O

O

OO

CH3

OH

HN

H

OOH

OHOH

OH CH3 O

O

OO

CH3

O H

HN

(2) (3)

H

OOH

OHOH

OH CH2OH

FIGURE11.20 Chemical structures of daunorubicin (2) and epirubicin (3).

0.100

0.080

0.060

0.040

0.0200 5 10

G-GGDG-GDGTG-GTG

15Log

20

%MeOH relative to ACN in mobile phase

25 30 35 40 450.000

0.020

0.040

0.060

0.080

0.100

FIGURE11.21 The plots of log (selectivity) vs. %methanol relative to acetonitrile in the mobile phase for three pairs of compounds, G vs. GG, DG vs. GDG, and TG vs. GTG, under HILIC condition on a Hypersil silica column, 100 1 mm, particle size of 3 m, 0.1% formic acid in all mobile phase solvents used, and column temperature at 30C. Total organic content (acetonitrile + methanol) in the mobile phase remained at 75%. (Adapted from Hao, Z. etal., J. Chromatogr. A, 1147, 165, 2007. With permission.)

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and GTG, respectively. The active silanol groups on the stationary phase surface can effectively be competed with either the hydroxyl groups from methanol or the neutral polar hydroxyl groups from Amadori compounds. The more active sides are occupied by methanol, the less are left for Amadori compounds, thus result-ing in a reversed elution order. A similar phenomenon was found in an analysis of morphine and its metabolite, morphine-3-glucuronide, and their elution order was switched when ACN was replaced by methanol in the mobile phase on the Inertsil silica and ZIC-HILIC columns.39,51

Switching a protic organic solvent with aprotic can improve separation selectivity not only for similar but also for completely different structures. For example, 50% ACN-H2O containing 0.1% formic acid as mobile phase was used to separate choline and arginine on a Hypersil bare silica column (Figure 11.22a). If the aprotic organic solvent ACN was replaced by the protic organic solvent methanol, the elution order in Figure 11.22a was switched to those shown in Figure 11.22b.8 The rationale behind this switching is hydrogen-bond competition. Abundant protic functional groups like

100RT: 0.0029.99

5.365.43

5.56

5.70

5.84

6.016.156.450.13

0.93Rela

tive a

bund

ance

2.10 3.40 4.40

Arginine

Choline

7.11 7.65 8.9911.19 13.65 15.47 16.8416.96 17.10

21.4518.8513.7013.2611.5810.288.946.925.243.391.290.13

0 2 4 6 8 10 12Time (min)(a)

14 16 18 20 22

4.52

17.56 20.89 22

1.29 3.39 5.225.43

6.90 8.1310.2812.3313.26 16.5816.72

16.96 17.1017.68

18.2619.1621.4

80

60

40

20

0100

80

60

40

20

0100

80

60

40

20

0

FIGURE11.22 HILIC-MS/MS chromatograms of choline and arginine under HILIC con-dition on a Hypersil silica column, 100 1 mm, particle size of 3 m. (a) The mobile phase is consistent of 50% water and 50% ACN and 0.4% formic acid in both solvents.

(continued)

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NH(12) and OH in the arginine structure can form hydrogen bonds with silanol groups on the silica surface or hydroxyl groups from the immobilized water-rich layer on the silica surface, contributing to arginine retention on the stationary phase. However, this hydrogen bond could be eliminated by a protic solvent such as metha-nol, thus resulting in a decrease in retention of arginine. The choline polarity may be higher or lower than arginine but its retention was less affected by ACN replacement with methanol compared to arginine.

Methanol was also preferred for the separation of taurine and methionine in a beverage matrix relative to ACN. Many carbohydrates such as glucose, fructose, and saccharose present in the beverage solution could be strongly retained on a HILIC column through a hydrogen-bonding retention mechanism if an ACNwater mobile phase was used, whereas with a methanolwater mobile phase they are not retained (Figure 11.23).52

Methanol has not always been successfully used to replace ACN for selecti-vity improvement. In general, ACN is often selected over methanol since it has many advantages including nearly ideal spectroscopic qualities, low viscosity, and

100RT: 0.0029.99

11.42

11.6611.9011.9412.08

12.2512.3912.5212.6913.0013.86

11.29

1.14

1.19 1.332.25 4.89 6.03 7.98

9.80

10.1110.17

10.6210.8911.4812.78 15.38 17.1018.47 20.05 22.4

Relat

ive ab

unda

nce

Arginine

Choline

0 2 4 6 8 10 12Time (min)(b)

14 16 18 20 22

1.76

1.761.14

3.75

3.75

4.60

4.60

6.32

6.32

7.00

7.00 8.34 9.30

11.66

11.90

13.3114.2015.57 17.2520.7521

9.399.69 9.80

14.6816.1618.21 20.75 21

80

60

40

20

0100

80

60

40

20

0100

80

60

40

20

0

FIGURE 11.22 (continued) (b) The mobile phase is consistent of 30% water and 70% methanol and 0.4% formic acid in both solvents. (Adapted from Hao, Z. etal., J. Sep. Sci., 31, 1449, 2008. With permission from Wiley-VCH Verlag Gmbh & Co. KGaA.)

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unique chromatographic properties. Acetonitrile is basically a polar solvent and it is miscible with water in all proportions. More importantly, ACN does not asso-ciate strongly with water and ACNwater mixtures remain binary in character. This simplifies the interactive theory and allows a more simple prediction of retention based on the ACN concentration in the partition retention mechanism. Methanol, on the other hand, not only interacts with analytes and the stationary phase but also forms a strong association with water so that at a high concentra-tion of water, the mobile phase behaves as a binary mixture of water and watermethanol associate. At high concentrations of methanol, the converse applies, that is, the mobile phase consists of a mixture of methanol and watermethanol associate. Between these extremes the mobile phase consists of a complex ternary mixture of methanol, watermethanol associate, and water. A review by Scott showed that there was some association between water with ACN and waterTHF, but not nearly to the same extent as water with methanol.53 At the point of maxi-mum association in water/methanol mixtures, the solvent contained nearly 60% of the watermethanol associate. In contrast, the maximum amount of waterTHF associate formed was only 17%, and that for waterACN as little as 8%. That is why methanol was considered to be too strong an elution solvent, leading to poor retention,54 and ACN usually provided sharper peaks than methanol.55

0 5 10Time (min)

15

a

bTau

Met

FIGURE 11.23 HILIC-ELSD analysis of a beverage diluted to 1/10 with mobile phase. (a) Beverage containing Met and Tau, (b) amino acid-free beverage. Column: apHera NH2 (150 mm 4.6 mm I.D., 5 m). Column temperature is at 37C. Mobile phase: MeOH/H2O (60:40, v/v) under isocratic elution mode; flow rate 0.6 mL/min; injection volume: 10 L. (Adapted from de Person, M. etal., J. Chromatogr. A, 1081, 174, 2005. With permission.)

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Thehydrogen-bonding interaction between methanol and analytes may introduce extra resonance structures and cause broad or tailing peaks.56,57

It is worthy to note that hydrogen bonding can become unstable at elevated tem-peratures.58 The broad and tailing peaks with a methanol mobile phase could be improved if the column is running at an elevated temperature, which will be dis-cussed in the next section. The effects of mobile phase composition and temperature are often complementary and a simultaneous optimization of these parameters could become a useful approach to control analyte retention and improve selectivity and peak shape.59 One should be aware, however, that polar analytes are poorly soluble in mobile phases with high concentrations of ACN.37

11.3.2 EffEctofcolumntEmPEraturE

Column temperature can change not only the analyte transferring enthalpy from mobile phase to stationary phase but can also have an effect on analyte structure. For example, when crystalline glucose, which is a single compound, is dissolved in an aqueous solution, tautomerization occurs. Ultimately an equilibrium mixture of at least five compounds is formed: the -pyranose, the -pyranose, the -furanose, the -furanose, and the aldehyde form.60 The transformation rate between different isomers is temperature dependent. The isomers - and -glucopyranoses have been separated at lower temperature on Ca2+-form Aminex HPX-87C column.61 Partially resolved double peaks were also observed for l-fucose on a carbamoyl-silica HILIC column (TSK-Gel Amide-80).35 Elevated temperature can preclude the existence of specific isomers of carbohydrate analytes. When isomers of the same analyte cannot be distinguished at higher temperatures, broad or split peaks will be narrowed down into a single peak.35,62

Amadori compounds can also exist as tautomers due to the carbohydrate por-tion of their structures. The different -, -, and acyclic anomers of Amadori compounds in Figure 11.24 have been confirmed by nuclear magnetic resonance.63

OHOH

OHO

NH COOH

OH

OH

OHO

NH COOH

(a) (b)

(c)

HOHN COOH

OH

OH

O

FIGURE 11.24 Chemical structures of open-chain and cyclic forms of N-(1-deoxy-d-xylose-1-yl)-glycine: (a), -anomer; (b), -anomer; (c), acyclic form. (Adapted from Davidek, T. etal., Anal. Chem., 77, 140, 2005. With permission.)

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Within the six structures of G, DG, TG, GG, GDG, and GTG seen in Figure 11.3, only three Amadori compounds contain a carbohydrate (glucose) portion. At a low temperature of 5C, broad/split peaks were observed when these compounds were separated on bare silica columns and became narrower single peaks at an elevated column temperature (Figure 11.25).14

Elevated column temperatures can narrow down the peak widths of analytes con-taining nonionic polar functional groups, especially for carbohydrate rings, but it also shortens their retention times. The overall resolution of these types of com-pounds is still primarily analyte dependent.

100RT: 0.0025.00

0.69 2.85 7.47

7.677.77

9.99

9.99

7.725.362.270.73

0.75 2.29 6.71 9.59 13.81

16.69

19.36 20.49 23.99

24.0120.82

19.38

17.6313.9311.3610.026.010.76

0.68

0.69 2.85 6.66 9.95

9.97

11.19

15.01

12.42 16.22

16.24 19.64

20.87

18.18

19.52 22.09

22.726.685.344.110.71

2.94 4.17

7.67

9.63 11.58 13.74 16.31 19.09 24.13

2.20

TG

GG

GTG

GDG

G

DG

4A, TIC

9.88 16.4817.87

18.1818.38 20.87

21.2822.72

23.6619.4516.6711.43

50

0100

50

0

50

100

0100

50

0100

50

0100

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0100

50

00 5

Relat

ive ab

unda

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10 15Time (min)

20 25(a)

FIGURE11.25 HILIC-MS/MS chromatograms of G, DG, TG, GG, GDG, and GTG under HILIC condition on a Hypersil silica column, 100 1 mm, particle size of 3 m with mobile phase of 25% water and 75% ACN and 0.4% formic acid in both solvents. Column tem-perature was at 80C for (a), 30C for (b), and 5C for (c). (Adapted from Hao, Z. etal., J.Chromatogr. A, 1147, 165, 2007. With permission.)

(continued)

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100

RT: 0.0026.00

0.75 4.405.61

5.82

6.035.92

7.52 12.16

12.47 14.0714.37

7.52

9.067.003.400.73

0.75 3.94 6.92 9.908.15 15.2514.5314.55

18.75 22.45 23.68

25.621.2318.0416.0912.189.715.700.76

2.02

0.70 4.40 7.59 8.72

9.35

12.63

12.33

14.07

16.24

20.05

21.78 25.5119.5220.03

20.56 24.817.504.410.71

4.38

5.82

8.917.16 11.17 13.74 16.83 18.78 21.56 22.69

4.36

TG

GG

GTG

GDG

G

DG

4B, TIC

16.9616.24 20.03

20.56 22.83

22.33 23.7718.3214.4112.45

12.47

50

0100

50

0100

50

0100

50

0100

50

0100

50

0100

50

00 5

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10 15Time (min)(b)

20 25

FIGURE11.25(continued)Dow

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(c)

100

RT: 0.0026.00

0.754.59

4.894.79

9.69

9.59

9.8011.56

14.39

5.97

5.97

9.787.212.270.73

0.75 3.83 5.89

9.80

8.36 15.76 18.54 21.62 23.99

24.8322.0515.47 19.7913.00

11.56

10.026.830.76

0.78

1.31 2.96 5.94 9.54

10.9812.01

8.841.64 3.69

13.04 15.6114.08

13.47

16.7615.42

21.16 24.917.67

20.87 24.266.78

2.32

4.895.29

8.396.23 9.73 12.09 15.80 18.88 21.45 24.02

2.20

TG

GG

GTG

GDG

G

DG

4C, TIC

16.24

12.21

20.87 22.10

20.68 22.8417.4914.3111.73

12.5711.65

50

0100

50

0100

50

0100

50

0100

50

0100

50

0100

50

00 5

Relat

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unda

nce

10 15Time (min)

20 25

FIGURE11.25(continued)Dow

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ACKNOWLEDGMENTS

The author would like to thank Mark Storton and Kate Jackson from our Global Analytical Science Department for their helpful comments on my manuscript!

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Acknowledgments11.1 Introduction11.2 Ionic Functional Groups in HILIC Separation11.3 Nonionic Polar FunctionalGroups in HILIC SeparationReferences

polar functional groups for the hilic method

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