HILIC and Its Applications for Biotechnology, Part I

Dec 01, 2013
Volume 31, Issue 12, pg 998–1007

What is hydrophilic interaction liquid chromatography (HILIC)? How does it operate? What types of columns are now commercially available? What sort of mobile phases are commonly used for biopharmaceuticals? What detection modes are most compatible with its operations? What are the advantages or disadvantages of using HILIC for this class of analytes?

The intent of this column is to provide an up-to-date summary of the specific applications, already described in the scientific, technical, or commercial applications literature, that are relevant for using hydrophilic interaction liquid chromatography (HILIC) in biopharmaceutical applications and characterizations. Such applications could be saccharides (glycans; mono-, di-, oligo-, and polysaccharides) and glycoproteins, glycoprofiling, intact biopharmaceuticals (proteins, peptides, antibodies), peptide mapping, oligonucleotides, amino acids, polar basic compounds, small molecules or drugs, antibiotics, glycopeptides, metabolites, marine toxins, and related topics. It is our intention to emphasize only those specific applications relevant to the biopharma industry and biotechnology. As such, we will not discuss topics wherein HILIC does not provide serious advantages over other, current high performance liquid chromatography (HPLC) separation modes for those applications. Thus, HILIC has no obvious advantages in analyzing intact antibodies or very large proteins, even if these are heavily glycosylated. On the other hand, HILIC has some very clear advantages in comparison with reversed-phase HPLC when analyzing for low-abundance molecules such as free glycans derived from various biopharma samples. It has also proven useful in glycomics studies and in the analysis of smaller glycopeptides — for example, when one is trying to resolve them from other nonglycopeptides in a complex peptide digest from a large protein. So, why use HILIC for biopharma applications?

Some Questions About HILIC

In this section we address some questions regarding HILIC: What is HILIC? How does HILIC operate? What types of columns are now commercially available? What mobile phases are commonly used for biopharmaceutical related analytes? What detection modes are most compatible with HILIC operations? What are the advantages or disadvantages of using HILIC for biotechnology-derived analytes or applications?

HILIC is a subset of conventional HPLC (including ultrahigh-pressure liquid chromatography [UHPLC]) that has been around for several decades. Only within the past decade or so has HILIC gained a lot of followers and commercial development of new and novel stationary phases (1–3). Indeed, several companies introduced UHPLC and superficially porous HILIC columns in 2013 as high efficiency columns, designed for retention of extremely polar analytes and having orthogonal selectivity versus C18 columns (1–8). Some of these columns comprise solid-core particles, 1.6 µm in diameter with a thin shell of porous, bonded phase of an undisclosed nature, by and large. There are already a large number of pharmaceutical and biopharmaceutical applications available at the vendors' web sites (Agilent Technologies, Thermo Fisher Scientific, Nacalai Tesque, Merck SeQuant, PolyLC, Waters, and others). There are a number of review articles, books, and individual scientific or technical articles that are useful for learning the basics and that describe advanced packing materials, many useful for biopharmaceutical applications (9–14). Finally, the number of papers appearing per year, determined as of November 2012 using the American Chemical Society's (ACS) SciFinder Scholar software, showed a constant increase from 1991 to 2012 to about 300.

Wikipedia defines HILIC as follows: Hydrophilic interaction chromatography (or hydrophilic interaction liquid chromatography, HILIC) is a variant of normal-phase liquid chromatography that partly overlaps with other chromatographic applications such as ion chromatography (IC) and reversed-phase liquid chromatography (12). HILIC uses hydrophilic stationary phases with reversed-phase eluents (water-miscible organic solvents, in the presence of a minor amount of water). The name was suggested by Dr. Andrew Alpert in his 1990 paper on the subject (15). He described its chromatographic mechanism as liquid–liquid partition chromatography, where analytes are eluted in order of increasing polarity, a conclusion supported by a review and re-evaluation of published data (16). As such, HILIC is clearly seen as a region of the HPLC partition universe, applied to compounds with a negative Log P value, whereas reversed-phase LC (without pairing agents) is suitable for those compounds with a positive Log P value. In other words, HILIC separates by polar differences and reversed-phase LC separates by nonpolar differences.

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