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BASICS OF RP-HPLC AND HILIC

GEETHAANJALI VIJAYAKUMAR 13/07/2015

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CHROMATOGRAPHY

• What is it? Physical separation method based on differential

migration of analytes in mobile phase as they move along a stationary phase.

• How does it work? Mixture – flushed through column – differential

migration over adsorptive material – separation by desorption.

Types of chromatography : column and planar.

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RP - HPLC

• Mode of separation of components – primarily based on differences in hydrophobicity.

• Separation depends on the hydrophobic interaction of solute molecule from mobile phase to immobilized hydrophobic ligands (stationary phase) attached to a support.

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• Solute – initially applied to sorbent (hydrophobic stationary phase) – eluted by increasing organic solvent concentration in mobile phase.

• Eluted in the order of increasing molecular hydrophobicity.

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IMPORTANT REVERSE PHASE PARAMETERS

• Solvent (mobile phase ) Strength• Choice of Solvent• Mobile Phase pH• Silanol Activity• Mobile phase additives – generally TFA

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Binding of peptide(A) and protein (B) to an RP-HPLC sorbent.

Ref: Aguilar, M. I. and Hearn, M. T. W. (1996) High resolution reversed phase high performance liquid chromatography of peptides and proteins.

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RP-HPLC

ADVANTAGES• Widely used due its

flexibility and high resolution.

• Separation of proteins and peptides – both analytical and preparative.

DISADVANTAGES• High affinity towards the

protein, so that elution requires extreme conditions – denaturation of proteins.

• Very polar compounds – low retention – elute with aqueous eluent – unfavourable for MS.

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HILIC

• Hydrophilic interaction chromatography – technique used to separate polar and/or basic components.

• RP HPLC – used for wide variety of components – but difficult for the analysis of small polar molecules. Here’s when HILIC is used.

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• HILIC combines the characteristics of the 3 major methods in liquid

chromatography – reversed phase (RPC), normal phase (NPC) and ion chromatography (IC).

• Stationary phases (adsorbents) are mostly polar modifications of silica or polymers (SiOH, Amino, Diol etc) – like in NPC.

• Mobile phases (eluents) are mixtures of aqueous buffer systems and organic modifiers like acetonitrile or methanol – like in RPC.

• Fields of application include quite polar compounds as well as organic and inorganic ions – like in IC.

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HILIC is NP chromatography of polar and ionic compounds under RP conditions.Ref : Hydrophilic interaction liquid chromatography (HILIC)—a powerful separation technique Bogusław Buszewski and Sylwia Noga

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• Polar phase – when used with a mobile phase high in organic concentration, the more polar water preferentially adsorbs on the surface creating a semi-stagnant, water-rich stationary phase and a water depleted mobile phase.

• Polar analytes can then partition in to aqueous-enriched phase.

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MECHANISM

• More polar nature of the eluent – No issues with the solubility of polar analytes.

• HILIC retention mechanism works on the basis that water adsorbs onto the stationary phase surface - becomes immobilised - analyte partitioning takes place between this and the bulk mobile phase. (liquid/liquid extraction system).

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• Stationary phase for HILIC – silica• Side groups attached – silanol – acidic and

hydrophilic.• Interact with basic analytes through hydrogen

bonding/electrostatic interactions.

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HYDROPHILIC PARTITIONING :AN AQUEOUS LAYER ALLOWS PARTITION BETWEEN HYDROPHILIC STATIONARY PHASE AND HYDROPHOBIC ELUTION BUFFER

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SECONDARY INTERACTION :ELECTROSTATIC INTERACTION WITH CHARGED STATIONARY PHASE

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ADVANTAGES OF HILIC

ADVANTAGES• Retention of hydrophilic

compounds that are difficult to retain by Reversed Phase Liquid Chromatography (RPLC).

DISADVANTAGES• Reliance on ACN – acetone

can be used as an alternative but UV detection – not possible at low wavelenghths.

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ADVANTAGES• Low viscosity of eluents –

desirable for analysis – possible with HILIC where RP requires elevated temperatures.

• Compatibility of buffers used in LC- ESI- MS (presence of water as an eluting solvent favors ionization).

DISADVANTAGES• Solubility issues with some

analytes.

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REFERENCES

• Aguilar, M. I. and Hearn, M. T. W. (1996) High resolution reversed phase high performance liquid chromatography of peptides and proteins.

• Advantages and Disadvantages of HILIC; a Brief Overview by James Heaton and Norman W. Smith.

• HILIC Chromatography: Theory and Method Development Practices David S. Bell.

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QUESTIONS!!!!!!

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