24 August / September 2019
HILIC Flash Purification – Separation of Polar Pharmaceuticals on Silica
by Q. Liu, J. Reilly, D. Dunstan, K. Galyan and M. Mcknight Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, USA.
Polar pharmaceuticals are notoriously difficult to separate by analytical and semi-prep HPLC and this research demonstrates how we have generated reproducible separations of polar pharmaceuticals using low-pressure flash purification in ‘HILIC’ mode. These separations are not trivial by HPLC, and often reversed-phase purification is the ‘go-to’ method but can be challenging. We demonstrate the feasibility using standard silica flash columns to open up new possibilities for amino acid, nucleoside and nucleotide separations. Flash chromatography is a common practice in medicinal chemistry laboratories and the ability to switch to ‘HILIC’ mode is a simple operation.
Introduction
Hydrophilic Interaction Chromatography (HILIC) was initially reported by Alpert in 1990 (1). The mechanism being that polar analytes interact with a hydrophilic stationary phase and the elution generated by mixing a predominantly aprotic solvent (usually acetonitrile) with a strong eluting solvent (usually water). It is thought analytes partition into the water-rich layer that is partially immobilised onto the stationary phase, and elute with increasing polar solvent [1]. This mechanism has been scrutinised in detail by an analytical evaluation with many different stationary phases in a comprehensive analysis [2], the conclusion being that HILIC retention is a combination of electrostatic adsorption and hydrogen bonding interactions [2]. An increase in water content in the elution solvent reduces multipoint hydrogen bonding interactions, whilst an increase in electrolyte concentration decreases selectivity of various columns [2]. HILIC has advantages over conventional reversed phase or normal phase liquid chromatography as retention and separation of many polar analytes has proven to be a challenge as often unretained or poorly retained on most conventional reversed- phase stationary phases. Other advantages of HILIC are that
the polarity of samples usually aids solubility into aqueous mobile phases and secondly there is also no requirement for ion pair reagents which is advantageous
for coupling with mass spectrometry techniques. There are many bioanalytical examples of using gradient HILIC LC-MS to retain polar analytes such as studies of urine [3] in a 2D-LC-MS approach with RP-LC for pharmaceutical analysis in plasma [4] monoamine neurotransmitters [5] and metabonomic/metabolomic studies [6]. A comparison study was published which demonstrated the advantages of HILIC over reversed-phase for the separation of polar ephedrine’s [7]. Many advantages for this specific analysis were noted for Heaton’s study, mainly improved peak shape, faster analysis and lower viscosity of mobile phases [7], although in a later study it was noted that it’s critically important to choose optimal sample solvent and lower injection volumes for HILIC to optimise peak efficiency for this methodology for analytical applications [8]. Although there are many analytical evaluations of utilising HILIC there are very few articles which describe reproducible preparative methods. One recent article describes the use of hydrophilic solid phase extraction of glycyrrhizin (GA) in liquorice coupled with reversed-phase liquid chromatography purification, and although this method improved enrichment of GA, HILIC was only employed as a crude extraction procedure [9]. Recently a method describes the use of HILIC for large-scale preparative isolation of capreomycin impurities [10] and highlights the advantages of this method over an ion-pair mechanism. Also, recently, an at-column dilution mechanism was utilised
for preparative HILIC and demonstrates the advantages in compound loadability, whilst preserving peak shape [11]. Our group has previously demonstrated how to successfully develop flash C18 purifications from analytical methods [12] so we were eager to determine whether HILIC silica flash purifications could be predicted using the same approach. To our knowledge, this is the first publication which demonstrates the use of HILC purification using flash chromatography with silica columns in a reproducible and cost effective approach to separate polar pharmaceuticals.
Instrumentation
Waters Acquity Binary system, PDA detector, Waters ZQ Mass Spec
Columns: Waters Atlantis 5µm 4.6x10mm, Kromasil 100-5-Sil 5µm 4.6x10mm, YMC- Pack CN 5µm 4.6x10mm, YMC-Pack NH2 5µm 4.6x10mm, YMC-Pack Diol-120-NP 5µm 4.6x10mm
Flow: 1.0 mlmin-1 UV Diode Array
Reverse Phase Flash Purification columns:
Teledyne ISCO SILICA 12g GOLD (20-40µm) Redisep column 30 mlmin-1
Teledyne ISCO 15g GOLD C18 (20-40µm) Redisep column 30 mlmin-1
UV detection 220 nm and 254 nm collection
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