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The Theory of HPLC

Hydrophilic Interaction Chromatography

Aims and Objectives

Aims and Objectives

Aims

Introduce Hydrophilic Interaction Chromatography (HILIC) as a novel mode of chromatography

Present advantages and limitations of the HILIC separation mode

Introduce Electrostatic Repulsion Hydrophilic Interaction Chromatography (ERLIC) as a new HPLC option for hydrophilic analytes.

Present concepts of ion-exchange chromatography and its separation potential for certain ionic samples

Objectives

At the end of this Section you should be able to:

Understand the benefits and limitations of using HILIC for certain applications

Recognise some of the most important parameters that can be used to alter the separation in HILIC separation mode

Demonstrate an awareness of the benefits and limitations of HILIC over normal and reversed phase chromatography

Understand the benefits and limitations of using ERLIC for certain hydrophilic samples

Recognise some of the most important parameters that can be used to alter the separation in an ERLIC separation

Demonstrate an awareness of the limitations of the ERLIC separation mode

© Crawford Scientific www.chromacademy.com

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Content Introduction 3 General Considerations 4 Normal Phase Chromatography 5 HILIC Separation Mechanisms 6 Applications 9 Stationary Phases 15 Columns 17 Eluent Systems 17 Buffers and Additives 19 Gradient 19 ERLIC 21 Ion Exchange Chromatography 23 Anion-Exchange Mixed Modes of Separation I 25 Anion-Exchange Mixed Modes of Separation II 26 Anion-Exchange Mixed Modes of Separation III 28 ERLIC Vs. HILIC 29 References 30


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Introduction Chromatography is the collective name for a set of techniques used for the separation of a mixture of components. In any mode of chromatography, the separation of components is promoted by their difference in affinity for, or solubility in, two different phases – the so called Stationary and Mobile phases. The chromatographic separation of hydrophilic compounds (highly water soluble, usually more polar), has traditionally been regarded as difficult: In Reversed Phase HPLC, the highly aqueous eluents required to gain any retention of polar compounds are known to cause problems such as non-reproducible retention times and low separation efficiencies, even with so-called ‘Aqueous’ stationary phase types.

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Hydrophilic interaction chromatography (HILIC) is a type of chromatography based on a mixed mode of retention mechanisms. It describes a type of normal-phase chromatography that employs a polar stationary phase (like silica or a polar bonded phase) and an aqueous–organic mobile phase, in which the aqueous content is the ‘strong’ solvent (typical initial eluent composition is 98% organic / 2.0% water).[1, 2] Despite being regarded as a new chromatographic method, the origins of HILIC separation modes date back to the 1970s; however, the term HILIC was coined by Alpert only in 1990.[2,3] Because of the relative polarities of the mobile and stationary phase, HILIC chromatography is sometimes known as ‘Reverse Reversed Phase Chromatography. General Considerations In HILIC mode, the stationary phase is hydrated with a slow moving layer of water, where hydrophilic compounds are preferentially retained compared with hydrophobic ones. To a first approximation, the retention order of a series of analytes in HILIC is the opposite of that in reversed-phase chromatography. HILIC can be used in certain situations where reversed phase chromatography fails or is not efficient:[1, 2]

Samples with limited solubility in water or highly aqueous mobile phases

Samples that contain very polar compounds that are not retained adequately in reversed phase

Hydrophilic water-soluble analytes, which are intractable to reversed-phase and/or ion-exchange chromatography

HILIC presents the added advantage of using acetonitrile, which has low UV absorbance (for better detection sensitivity) and low viscosity (for high chromatographic efficiency). Drawbacks of the technique are related to the increasingly higher prices of acetonitrile, its variable supply and the increasing focus on ‘green’ chromatography where the use and disposal of organic solvents is being driven down. Some of the general approaches to reduce consumption of acetonitrile in HILIC include:

Reduce content of acetonitrile in the mobile phase

Replace of acetonitrile with alcohols, THF or other solvents

Use columns with smaller internal diameter The use of ethanol and higher alcohols as alternative solvents, as well as the use of reduced dimension chromatography columns continues to be the foundation of efforts to reduce acetonitrile consumption.


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The HILIC-MS process, oversimplified A mobile phase composed of a low fraction of water and a large fraction of organic solvent (like acetonitrile) provides an ideal composition for efficient desolvation in electrospray ionisation mass spectrometry. Normal Phase Chromatography In order to fully understand the nature of HILIC chromatography – let’s first look at the nature of Normal phase chromatography in order that we can distinguish between Normal Phase and HILIC modes. Normal phase chromatography was, chronologically, the first Liquid Chromatographic technique. Tswett used this mode to separate plant pigments using a calcium carbonate stationary phase with a petroleum ether mobile phase.[4] By definition, normal-phase HPLC utilises a stationary phase that is more polar than the mobile phase. Typical stationary phases include bare silica as well as cyano, diol, and amino bonded phases. Typical mobile phase constituents include organic solvents such as hexane and ethyl acetate. One of which is ‘non-localising’ (the weak solvent), whilst the other so called ‘localising’ solvent competes for surface retention sites with the analyte and therefore acts as the strong solvent. The retention mechanism in normal phase HPLC is based on polar adsorption of either the solvent molecules or the analyte onto the polar stationary phase surface. If the solvent molecules are ‘localising’ they will be adsorbed onto the stationary phase surface, displacing the analyte and hence effecting analyte elution. The order of elution is least polar first, followed by increasingly polar (less hydrophobic) analytes.

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The normal phase chromatographic process (oversimplified)

A dynamic competition for sites on the stationary phase between the analyte (phenol) and eluent (acetonitrile) molecules is established, and when the eluent concentration increases, its presence on the stationary phase becomes dominant and analyte molecules are displaced. HILIC Separation Mechanisms As was previously described, HILIC describes a type of normal-phase chromatography that employs a polar stationary phase but uses aqueous–organic mobile phases, as opposed to normal phase modes that we have seen use organic solvents only. In contrast to reversed-phase chromatography, the aqueous component of the mobile phase (for example, water or buffer) serves as the strong solvent, and the organic component (for example, acetonitrile) is the weak solvent. Analytes are eluted in order of increasing hydrophilicity.[1] Due to the polar nature of the stationary phase, water molecules concentrate at the surface, and a water enriched layer is thus created at the silica surface. It has been proposed that in HILIC mode retention occurs as the analyte partitions between the bulk mobile phase and the water-enriched layer which hydrates the hydrophilic stationary phase. This is in contrast to retention in conventional normal-phase chromatography, which occurs by adsorption at the polar stationary phase surface.[5,6]

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Some authors have suggested that the HILIC mechanism is similar to that in normal phase chromatography. Several attempts have been made to establish whether the HILIC mechanism involves partitioning or adsorption; however, more research is required for absolute clarity and the debate still continues.[7]

HILIC separation mechanism (partition in the aqueous mobile phase)

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HILIC separation mechanism (interaction with the stationary phase)

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Applications HILIC can be used to address certain limitations of reverse phase chromatography:


Samples that contain very polar compounds that are not retained adequately in reversed phase mode

To list the full range of HILIC application areas is prohibitive, its flexibility makes it suitable to a multitude of application types. Examples of some interesting applications are shown below: Agrochemistry:[8] Chlormequat and mepiquat are quaternary ammonium compounds used as plant growth regulators to reduce unwanted longitudinal shoot growth without lowering plant productivity.

Full-scan HILIC LC–MS chromatograms of chlormequat and mepiquat standard solutions. Column: bare silica 150mm×2.1mm, 3μm. Eluent system: The gradient elution started with a 0.5 min isocratic step at 60% of solvent A (acetonitrile) and 40% solvent B (50mM formic acid–ammonium formate buffer solution at pH 3.75), followed by a linear gradient of solvent A down to 40% in 0.5 min and followed by an isocratic step of 2.5 min at 40% of solvent A. Eluent flow rate: 400μL/min


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Bioanalysis:[9] Sildenafil citrate, is used to treat erectile dysfunction and pulmonary arterial hypertension.

HILIC-MS determination of sildenafil citrate in human plasma.

Column: bare silica 50mm×5.0mm, 3μm. Eluent system: acetonitrile–water–TFA–acetic acid (92:8:0.025:1, v/v/v/v). Eluent flow rate: 0.3mL/min Cosmetics:[10] Due to its moisturizing and keratolytic properties, allantoin as an active ingredient widely used in the preparation of cosmetics.

HILIC determination of allantoin from a commercial lotion

Column: triazol-bonded silica column 4.6mm ID ×250mm, 5μm. Eluent system: acetonitrile and water in the ratio 90:10 ammonium acetate buffer (2.0 mM, pH 4.0) Eluent flow rate: 1.0mL/min


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Environmental analysis:[11] Estrogens are a group of strong endocrine-disrupting chemicals that can cause the feminization of male fish at trace level concentrations, estriol-3-sulfate is one of such compounds

MRM chromatogram of estriol-3-sulfate from a water river sample. Detection ESI negative

ion mode. Column: Zwittterionic HILIC mixed mode 100mm ×2.1mm ID, 5μm. Eluent system: Mobile phase A consisted of acetonitrile/aqueous ammonium acetate (5mM, pH 6.80) (5/95, v/v) and mobile phase B consisted of acetonitrile/aqueous ammonium acetate (5mM, pH 6.80) (95/5, v/v). The step gradient started at 20% B for 2.0 min; 30% B for 8.0 min; 80% B for 8.0 min; and 20% B for 7.0 min. Eluent flow rate: 0.15mL/min Food analysis:[12] Ascorbic acid and its derivatives are of overriding importance in human diet.


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HILIC chromatogram of a standard mixture of ascorbic acid derivatives.

Column: Diol 4.6 i.d.×250 mm, 5μm. Eluent system: acetonitrile and water (85:15, v/v) 66.7mM ammonium acetate. Eluent flow rate: 0.7mL/min Forensic analysis:[13] Saxitoxin, one of the most potent natural toxins known. It is naturally produced by certain species of marine species. Human toxicity and mortality can occur after ingestion of these substances.

HILIC–MS analyses of an Alexandrium tamarense (dinoflagellate) extract

Column: Cyano 250mm×2.0mm or 4.6mm i.d. Eluent system: acetonitrile and water (95:5, v/v) with 10.0mM ammonium acetate buffer (pH=2.5). Eluent flow rate: 0.8mL/min


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Metabolomics:[14] The metabolome represents the collection of all metabolites in a biological organism, which are the end products of its gene expression.

HILIC-MS determination of citric acid.

Column: bare silica 4.6 i.d.×100 mm, 5μm. Eluent system: The separation was done at 30 oC with a linear gradient elution of mobile phases A and B at a flow rate of 0.3 mL/min. Solvents A, B were acetonitrile and a 10mM ammonium formate buffer with pH adjusted to 2.5 by formic acid, respectively. B was kept at 25% for 3 mins, then it was changed from 25 to 50% in 25 mins. Eluent flow rate: 0.3mL/min Pharmaceutical chemistry:[15] Analysis of highly polar pharmaceutical products.


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HILIC separation of anthracyclines (antitumor antibiotics used for the treatment of cancer

–chemiotherapy). Column: C18 250mm×4.6mm i.d. 5μm. Eluent system: acetonitrile and water (90:10, v/v) with 20.0mM sodium formate buffer (pH=2.9). Eluent flow rate: 0.7mL/min Pharmacokinetics:[16] Zanamivir has been used in preventing, controlling, or rapidly reducing certain types of influenza.

HILIC-MS determination of zanamivir from rat plasma.

Column: bare silica 50mm×2.1mm, 3μm. Eluent system: 80% acetonitrile and 20% ammonium acetate (10mM) with 1% methanol Eluent flow rate: 0.3mL/min


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Proteomics:[17] Fetuins are blood proteins that are made in the liver and secreted into the blood stream.

MALDI TOF mass spectra obtained from the fetuin tryptic digest after HILIC separation.

Column: Zwittterionic HILIC mixed mode C18, 50mm ×2.1mm ID, 3.5μm. Eluent system: 80% acetonitrile and 20% of an aqueous solution of formic acid at 0.5%. Eluent flow rate: 0.3mL/min Stationary Phases In essence HILIC requires a hydrophilic stationary phase to adsorb a water layer for the partitioning process to take place, however there are several ways in which to achieve this and also to ‘fine tune’ the HILIC separation process. The separation mechanism in HILIC is not well understood, but it is known that parameters such as eluent pH and analyte-stationary phase interactions will affect the separation to some extent. Modern HILIC stationary phases are either neat silica or use ionic (or ionisable) ligands bonded to the silica surface. The use of ligands capable of undergoing electrostatic interactions can add an extra dimension to the separation when analysing ionisable compounds. Accordingly, HILIC stationary phases can be divided into three main categories:[18,19] Neutral: polar surface with no electrostatic interactions Charged: strong electrostatic interaction -the stationary phase contains anionic or cationic functional groups Zwiterionic: weak electrostatic interaction -the stationary phase contains both positive anionic and cationic functional groups


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In HILIC, different mixed retention modes can occur according to the analyte and column

functionality.

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Columns A huge variety of different types of polar stationary phase have been used in HILIC, including bare silica, aminopropyl, diol and zwitterionic phases bonded to silica or polymer matrices; nonetheless, bare silica is by far the most widely used stationary phase. HILIC columns from selected manufacturers are presented below.

Eluent Systems Mobile phase strength is one of the important parameters in HILIC. Unlike reverse phase chromatography, water in HILIC is the stronger eluting solvent. In a typical HILIC mobile phase, acetonitrile is used as a weak eluent component and water or aqueous buffer as a strong component. Due to the recent shortage of acetonitrile, alcohol had been considered as an alternative to replace acetonitrile; however, the efforts to use alcohol as a weak eluent in HILIC had often fail due to insufficient retention and quality of the separation.[8] Mobile phase pH plays an important role in the HILIC separation. Acidic or neutral mobile phase pH is commonly used due to the instability of silica-based columns at high pH.[20] Solvent strength (from weakest to strongest) for HILIC is in generally as follows: THF < acetone < acetonitrile < isopropanol < ethanol < methanol < water. Unfortunately, this is not always true, and sometimes is not possible to predict solvent strength. The interactive experience presented opposite, illustrates a separation where the solvent strength is as follows: acetonitrile < THF < isopropanol < methanol, slightly different to what is expected. Very strong solvents, such as dimethylformamide or dimethylsulfoxide, will usually result in poor peak shapes and are not recommended. These solvents will generally have to be

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diluted with a weaker solvent, such as acetonitrile, before satisfactory peak shape can be obtained.

HILIC separation of anthracyclines (antitumor antibiotics used for the treatment of cancer –chemiotherapy) on a porous silica column. Mobile phase: sodium formate buffer (20 mM, pH 2.9)

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Buffers and Additives HILIC uses buffers and additives to achieve high column efficiency and reproducibility. In general terms, the buffer concentration should be in the range of 10 – 20 mM and additives should be used in concentrations usually not exceeding 1.0%. Due to their poor solubility in high organic mobile phases, phosphate buffers are not recommended for HILIC applications. Phosphate buffers are also incompatible with MS detection. Buffers or additives above pH 6 usually are not recommended for HILIC applications because they may enhance the slow dissolution of the silica support; however, with the advent of new HILIC stationary phases, modern columns are capable of dealing with severe conditions of pH (up to about pH of 10).[21]

Buffer/Additive pKa Used for HILIC? Further information

TFA 0.3 YES Ion pair additive, can suppress MS signal. Used in the 0.01 – 0.1% range

Formic Acid 3.75 YES Used in the 0.1 – 1.0% range

Acetic Acid 4.76 YES Used in the 0.1 – 1.0% range

Formate 3.75 YES Used in the 1.0 – 10.0 mM range

Acetate 4.76 YES Used in the 1.0 – 10.0 mM range. Sodium or potassium salts are not volatile.

Phosphate 2.15, 7.2

NO Will reduce column lifetime.

To maintain maximum HILIC separation performance, always use high quality eluent, buffers and additives, and use good eluent preparation practice:

Filter all aqueous buffers prior to use

Particulate solvents will generally clog the column

Bacterial growth can be prevented by adding small amounts of organic modifier to the buffer system

Degas all solvents before use

Use freshly prepared eluent systems wherever possible Gradient In reverse phase chromatography, highly hydrophilic compounds will elute close to hold-up time, which may adversely affect their resolution and reliable quantification. The increased retention of highly polar, ionisable or ionic compounds in the HILIC mode effectively overcomes this problem.[22] Retention in HILIC decreases with mobile phase polarity (which in practical terms means that retention decreases with the water content). In HILIC gradient applications, the water composition is increased during the analysis. The gradient usually begins with only 2–10 percent water and can reach values in the order of 60-70% or even above. As with reversed-phase separations, the solvent used to inject the sample should, as closely as possible, resemble the strength and type used in the eluent starting composition, i.e. solute injections should be carried out in the mobile phase or in a weaker


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solvent (that is, containing less water) to prevent loss of efficiency. An alternative for samples not very soluble in high concentrations of organic is to use small sample volumes (<10μL). The use of gradients in HILIC should take into consideration the extended equilibration times sometimes required to establish the initial gradient composition and equilibrate the column.[23]

The time required for a non-retained particle to pass through the column is known as the

hold-up time (to).

HILIC separation of peptides on a HILIC reverse phase silica column.

Column: C18, 200 μm ID×40 cm. Mobile phase: gradient; A: aqueous solution of 0.2% formic acid, B: Acetonitrile+0.2% formic acid. Gradient A 10-90% linear gradient in 20 min Solutes: 1. Leu-Enkefalin; 2. VGSE; 3. EH, 4. DSDPR


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ERLIC Electrostatic Repulsion Hydrophilic Interaction Chromatography (ERLIC) is a new mode of chromatography in which an ion-exchange column is eluted with a predominantly organic mobile phase. Here a water enriched layer (which hydrates the polar stationary phase) permits hydrophilic analytes to partition and gain retention (even if they have the same charge as the stationary phase.).[5,24]

Under ERLIC conditions, analytes and stationary phase functional groups present the

same charge Under ERLIC conditions ionic analytes and functional groups from the stationary phase present the same charge, and as a consequence analyte retention is increased by hydrophilic interactions (partition of ionic molecules in the aqueous phase) and selectively antagonized (decreased) by electrostatic repulsion with functional groups.

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Very basic and very hydrophilic species exhibit strong retention in HILIC, and, when present in a multi-component mixture of less polar analytes, require gradient elution for separation within a reasonable time frame. ERLIC provides an alternative for dealing with complex mixtures with components at the extreme of retention (poorly vs. highly retained).

Under HILIC conditions ionic analytes and functional groups from the stationary phase present affinity by each other, and as a consequence analyte retention is increased by hydrophilic interactions (partition of molecules in the aqueous phase) and probably by a normal phase like retention mechanism on the stationary phase.

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As previously explained, in HILIC mode, the stationary phase is hydrated with a slow moving layer of water, where hydrophilic ions are:

Preferentially partitioned in the aqueous phase, i.e. their retention is increased by hydrophilic interactions

Retained on functional groups from stationary phase (retention is increased by a normal phase like mechanism)

A similar situation takes place under ERLIC conditions, where ionic analytes are:

Preferentially partitioned in the aqueous phase, i.e. their retention is increased by hydrophilic interactions

Repelled by electrostatic interactions with functional groups (of the same charge) present on the stationary phase i.e. retention is decreased

Ion Exchange Chromatography In order to fully explain the HILIC and ERLIC modes of chromatography we first need to study the simple mechanisms of Ion Exchange HPLC. This mode of chromatography is based on the different affinities of the analyte ions for oppositely charged ionic centres in the stationary phase or adsorbed counter-ions in the hydrophobic stationary phase.[25] Ion-exchange chromatography uses a stationary phase that possesses electrical charges on its surface. Ionic groups such as 3SO , 2CO , 3NH , etc, are incorporated into a resin

or gel.

Anion exchange

Cation exchange

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Ionic sample molecules compete with counter ions present in the mobile phase for binding at such stationary phase charged sites. This process is illustrated opposite. Ion-exchange chromatography is useful for the separation of ionic or ionisable species and resolves solutes based on the strength of their ionic interactions with ionic functional groups on the stationary phase. Ion-exchange chromatography uses an aqueous mobile phase (usually buffered to a certain pH) to separate analytes of opposite charge to the stationary phase. The mobile phase contains counter-ions with the same type of charge as the analyte that actively compete with analyte ions for ion pair interactions with the stationary phase.

Oversimplified separation mechanism of cationic analytes (K+ in this example) on a cation-

exchange resin column

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HKSOKHSO 33 sinResinRe

Continued elution of the column with an acidic eluent (H+) introduces competition of H+, and K+ for the exchange sites 3SO causing the K+ to move down the column.

Anion-Exchange Mixed Modes of Separation I Hydrophilic interaction can be superimposed as a mixed mode on an ion-exchange column in the presence of high concentrations of an organic solvent. The important feature of this combination is the independence of hydrophilic interaction and electrostatic effects;[1,3,5] or in other words, with sufficient organic solvent in the mobile phase, hydrophilic interaction dominates the separation. In this way, the behaviour of charged solutes can be controlled to address extremes of retention. For anion-exchange chromatography, three important situations can be considered:


Samples that contain very polar compounds that are not retained adequately in reversed phase

Hydrophilic water-soluble analytes, which are intractable to reversed-phase and/or ion-exchange chromatography

The first situation is the late elution of acidic analytes on an anion-exchange column; in this case, the pH of the eluent system can be used to control analyte retention. Let’s consider a reduction of the mobile phase pH below the pKa of certain analyte of interest; in this case, the analyte will remain as a neutral compound and its interaction with the stationary phase is minimised and as a consequence, its retention is reduced. As expected, if the pH is raised above the pKa, then the analyte will remain in the anionic state, the electrostatic interaction with the stationary phase will be maximised and its retention will increase.


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Anion-Exchange Mixed Modes of Separation II The second important case of mixed mode of separation in anion-exchange chromatography is related to the strong retention of basic analytes. As previously described,[1,3] with sufficient organic solvent in the mobile phase, hydrophilic interaction dominates the chromatographic separation even under anion-exchange conditions. The positively charged basic analytes partition into the aqueous phase and despite the electrostatic repulsion with functional groups (also positively charged) present on the stationary phase, analytes are strongly retained.

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This is the same mechanism that accounts for analyte retention under HILIC conditions; however, note that hydrophilic stationary phases for HILIC are neutral and not positively charged that is the case in anion-exchange chromatography.

Note how analytes ( 3NHR in the present case) presenting the same polarity than the

stationary phase and are precluded from accessing certain regions within the stationary phase, from which their eluting time is reduced compared with other species. In some cases this reduction is so dramatic than their eluting time become shorter than the void volume (to).

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Anion-Exchange Mixed Modes of Separation III The third important case of mixed mode of separation in anion-exchange chromatography is related to the poor retention (prior to the void volume) of basic analytes. As previously described,[1,3] with sufficient organic solvent in the mobile phase, hydrophilic interaction dominates the chromatographic separation even under anion-exchange conditions; however, when the aqueous content exceeds a certain value (application dependent), the electrostatic repulsion between the analytes and positively charged functional groups (from the stationary phase) become important. HILIC retention is counteracted by electrostatic repulsion. In this case, the two superimposed modes antagonize each other's extremes of retention and can permit isocratic elution of complex mixtures of analytes (as mixtures of peptides). This last case forms the basis for Electrostatic Repulsion Hydrophilic Interaction Chromatography (ERLIC) of analytes. In the example opposite, vasopressin has a net positive charge, is repelled electrostatically, and is eluted before the void volume since it is excluded from the pore system (note that neutral solvent molecules are not affected by the positively charged stationary phase, they are not precluded from reaching any site within the stationary phase).

ERLIC separation of selected peptides.

Mobile phase: 10.0 mM sodium methylphosphonate (pH=2) plus 10%Acetonitrile; Column: anion-exchange. In the present example, the determination of vasopressin was performed under ERLIC conditions and vasopressin is the only compound that elutes before the hold-up time. As far as all peaks within the chromatogram (even the ones eluting before the hold-up time) are well resolved and well shaped then the separation is OK.


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ERLIC Vs. HILIC Under anion exchange conditions certain analytes can elute before the void volume. In the example shown opposite, vasopressin (a polypeptide) holding a net positive charge, is repelled electrostatically, and is eluted before the void volume since and is excluded from the pore system. The comparative performance of HILIC and ERLIC to resolve a mixture of peptide standards is also shown. In HILIC mode, a concentration of acetonitrile that promotes retention of the basic peptides 15 and 17 affords inadequate retention of acidic and neutral peptides. In the ERLIC mode, electrostatic repulsion of peptides 15 and 17 allows the acetonitrile concentration to be increased to a level where all peptides can be retained and well resolved.[1] Very basic and very hydrophilic species exhibit strong retention in HILIC, and, when present in a multi-component mixture of less polar analytes, require gradient elution for separation within a reasonable time frame. The technique of ERLIC provides an alternative strategy for dealing with complex mixtures with components at the extreme of retention (poorly vs. highly retained).

HILIC vs ERLIC separation of selected peptides


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References 1. Tim Wehr. “Electrostatic Repulsion Hydrophilic Interaction Chromatography” LCGC North America. Mar 1, 2009 2. David V. McCalley. “Hydrophilic Interaction Chromatography”. April 1, 2008. Chromatography Online.com The Global Website of LC/GC 3. Andrew J. Alpert. “Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds” Journal of Chromatography A, Volume 499, 19 January 1990, Pages 177-196 4. Normal Phase (absorption) Chromatography. Theory of HPLC, from the HPLC Channel. 5. Eric S. Grumbach, Diane M. Wagrowski-Diehl, Jeffrey R. Mazzeo, Bonnie Alden, and Pamela C. Iraneta. “Hydrophilic Interaction Chromatography Using Silica Columns for the Retention of Polar Analytes and Enhanced ESI-MS Sensitivity” LCGC NORTH AMERICA VOLUME 22 NUMBER 10 OCTOBER 2004 6. Eugene P. Kadar , Chad E. Wujcik, David P. Wolford, Olga Kavetskaia. “Rapid determination of the applicability of hydrophilic interaction chromatography utilizing ACD Labs Log D Suite: A bioanalytical application” Journal of Chromatography B, 863 (2008) 1–8 7. Tatsunari Yoshida. “Peptide separation by Hydrophilic-Interaction Chromatography: a review” Journal of Biochemical and Biophysical Methods. 60 (2004) 265–280 8. X. Esparza, E. Moyano, M.T. Galceran. “Analysis of chlormequat and mepiquat by hydrophilic interaction chromatography coupled to tandem mass spectrometry in food samples” Journal of Chromatography A, 1216 (2009) 4402–4406 9. Wilson Z. Shou, Weng Naidong. “Simple means to alleviate sensitivity loss by trifluoroacetic acid (TFA) mobile phases in the hydrophilic interaction chromatography–electrospray tandem mass spectrometric (HILIC–ESI/MS/MS) bioanalysis of basic compounds” Journal of Chromatography B, 825 (2005) 186–192 10. Takahiro Doi, Keiji Kajimura, Satoshi Takatori, Naoki Fukui, Shuzo Taguchi, Shozo Iwagami. “Simultaneous measurement of diazolidinyl urea, urea, and allantoin in cosmetic samples by hydrophilic interaction chromatography” Journal of Chromatography B, 877 (2009) 1005–1010 11. Feng Qin, Yuan Yuan Zhao, Michael B. Sawyer, Xing-Fang Li. “Column-switching reversed phase–hydrophilic interaction liquid chromatography/tandem mass spectrometry method for determination of free estrogens and their conjugates in river water” analytica chimica acta 627 (2008) 91–98

Where: Tryptic Peptides Acidic Peptides Basic Peptides (1) Thr-Tyr-Ser-Lys (2) Asp-Leu-Trp-Gln-Lys (3) Tyr-Gly-Gly-Phe-Leu-Arg (4) Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg (5) Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Arg (6) Val-Gln-Gly-Glu-Glu-Ser-Asn-Asp-Lys

(7) Asp-Val (8) Val-Asp (9) Glu-Ala-Glu (10) Asp-Ala-Asp-Glu-(pTyr)-Leu (11) Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu (12) [isoAsp

5]-DSIP

(13) [phosphoSer5]-DSIP.

(14) ACTH (15) Arg-Lys-Arg-Ser-Arg-Lys-Glu (16) Lys-Arg-Gln-His-Pro-Gly-Lys-Arg (17) Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro


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12. Akihiro Tai, Eiichi Gohda. “Determination of ascorbic acid and its related compounds in foods and beverages by hydrophilic interaction liquid chromatography” Journal of Chromatography B, 853 (2007) 214–220 13. Carmela Dell’Aversano, Philipp Hess, Michael A. Quilliam. “Hydrophilic interaction liquid chromatography–mass spectrometry for the analysis of paralytic shellfish poisoning (PSP) toxins” Journal of Chromatography A, 1081 (2005) 190–201 14. Yanping Lin, Duanyun Si, Zongpeng Zhang, Changxiao Liu. “An integrated metabonomic method for profiling of metabolic changes in carbon tetrachloride induced rat urine” Toxicology 256 (2009) 191–200 15. Ruiping Li, Junxiong Huang. “Chromatographic behavior of epirubicin and its analogues on high-purity silica in hydrophilic interaction chromatography” Journal of Chromatography A, 1041 (2004) 163–169 16. Todd M. Baughman, Wayne L. Wright, Kathryn A. Hutton. “Determination of zanamivir in rat and monkey plasma by positive ion hydrophilic interaction chromatography (HILIC)/tandem mass spectrometry” Journal of Chromatography B, 852 (2007) 505–511 17. Cosima D. Calvano, Carlo G. Zambonin, Ole N. Jensen. “Assessment of lectin and HILIC based enrichment protocols for characterization of serum glycoproteins by mass spectrometry” Journal of Proteomics. 71 (2008) 304 – 317 18. Petrus Hemström. “Hydrophilic Separation Materials for Liquid Chromatography” ISBN 978-91-7264-406-9. Print och Media : 2003591 Umeå University, UMEÅ. 2007 19. “A PRACTICAL GUIDE TO HILIC. A Tutorial and Application Book” ISBN 978-91-631-8370-6. http://www.sequant.com/hilicguide 20. Min Liu, Emily X. Chen, Ruthie Ji, David Semin. “Stability-indicating hydrophilic interaction liquid chromatography method for highly polar and basic compounds” Journal of Chromatography A, 1188 (2008) 255–263 21. “ZIC®-pHILIC HYDROPHILIC POLYMER PHASE FOR LIQUID CHROMATOGRAPHY” http://www.sequant.com/ 22. LLOYD R. SNYDER and JOHN W. DOLAN. “HIGH-PERFORMANCE GRADIENT ELUTION. The Practical Application of the Linear-Solvent-Strength Model” Pp 361-364. Copyright © 2007 by John Wiley & Sons, Inc. All rights reserved 23. J.C.Valette, C. Demesmay, J. L. Rocca, E. Verdon. “Separation of Tetracycline Antibiotics by Hydrophilic Interaction Chromatography Using an Amino-Propyl Stationary Phase” Chromatographia 59, January (No. 1/2), Pp 55-60. 2004. 24. Andrew J. Alpert. “Electrostatic Repulsion Hydrophilic Interaction Chromatography for Isocratic Separation of Charged Solutes and Selective Isolation of Phosphopeptides” Analytical Chemistry 2008, 80, 62-76 25. “Ion Exchange Chromatography. Principles and Methods” Amersham Biosciences. ISBN 91 970490-3-4

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