Chromatography

HILIC PHASES (Part 1 of 2)

A Useful Complement to Conventional HPLC Phases

The methodology behind the more efficient separation of medium-polar and polar compounds applications on HILIC phases has been more frequently published as of late. The advantages and differences of HILIC from NP, RP and ion exchanger phases are described below.

HILIC: What´s that?
The term HILIC (Hydrophilic Interaction Chromatography) was introduced by Alpert in 1990 [Alpert, A.J., J. Chromatogr. A, 499, 177-196 (1990)].

When talking about NP chromatography, polar stationary phases are used in HILIC chromatography. On the other hand, mobile phases are associated with the buffer systems of RP chromatography.
The organic proportion (60 – 97 %) is higher than the aqueous one (40 – 3 %).
Several materials that have different selectivity and reproductivity can be used as stationary phases. Besides neutral polar phases (e.g. diol phases) and charged phases (unmodified silica gels, amino phases, polysulfoethyl cation exchanger), zwitterionic phases with quaternary ammonium and sulphonic acid groups have been qualified for HILIC chromatography.
Mainly volatile buffers like ammonium acetate or formate are used as aqueous buffer systems in the mobile phase. Because of the application of ionic additives; pH, ionic strength and thus the retention behavior can be controlled.

Separation principle of HILIC phase

HILIC-interaction-EN
The separation principle is based mainly on the distribution of polar analytes between the water-rich, partially immobilized boundary layer and the organic phase.

HILIC in contrast to NP, RP and ion exchange chromatography

In Normal Phase Chromatography (NPC) polar phases (silica gel, amino or cyano phases) are used with non-polar, lipophilic eluents (hexane, heptane etc.) with an addition of up to 20 % alcohols (isopropanol, ethanol etc.). NP chromatography is suited for the separation of non-ionic, non-polar and medium-polar substances. Disadvantages are the long equilibration time and often appearing tailing effects, especially at higher loading.

Reversed Phase Chromatography (RPC) has been the method of choice, because of its reproductibility and robustness. The non-polar phases (several concepts of base deactivated C18, C8, C6H5 phases etc.) are applied with polar, aqueous eluents with an addition of acetonitrile or methanol.
However with highly polar and charged compounds they show limits in retention capacity. Polar-embedded RP phases could not really produce relief, because they show an elevated bleeding in UV detection and thus are not applicable in trace analysis.

In Ion Exchange Chromatography (IEC) strong and weak cation or anion exchangers
(SCX, SAX phases etc.) are used partially with highly concentrated buffers for the separation of ionic and highly polar compounds. These buffers are improper for increasingly applied detection systems like LC-MS, LC-MS/MS or light scattering detectors.

In HILIC Chromatography many substances show an inverse retention behavior compared to RP chromatography. This is shown for the example of the separation of polar uracil and non-polar naphthalene.

Retention sequence on HILIC phases

Comp-Pyramid-HILIC
Separation of uracil and naphthalene Columns: 125 x 4 mm NUCLEODUR® C18 Pyramid, 3 µm NUCLEODUR® 100-3 HILIC Eluent: acetonitrile – water (90:10, v/v) Flow rate: 1.0 ml/min Temperature: 25 °C Detection: UV, 254 nm Peaks:
1. Uracil
2. Naphthalene

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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