Here we describe novel application areas in which hydrophilic interaction liquid chromatography (HILIC) has vast importance
and utility, including peptides, glycopeptides, two-dimensional liquid chromatography in proteomics, oligonucleotides, lipids
and lipidomics, glycan analysis, proteins, glycoproteins, phosphoproteins, and related biopolymers. We also describe some
specific HILIC conditions that have proven useful for these classes of biologically active analytes and provide typical chromatograms
from the recent literature, both scientific and commercial.
In part I of this two-part series on modern hydrophilic interaction liquid chromatography (HILIC) applications of relevance
to the biotechnology industry, we discussed the fundamental principles and mechanisms suggested from the literature for this,
now very widely accepted and practiced form of modern high performance liquid chromatography (HPLC) and ultrahigh-pressure
liquid chromatography (UHPLC). We also discussed just a few of the common applications already described for simple and complex
glycans often derived from glycopeptides or glycoproteins or for synthetic, standard glycans. We discussed the current roles
of HILIC in glycoprofiling and glycan analysis from any source, but in particular by deglycosylation of proteins and peptides.
In part II, we discuss modern usage and specific applications of HILIC for two other major classes of analytes important in
biotechnology today: glycopeptides and peptides, and glycoproteins and proteins. Other types of proteins, such as phosphoproteins
and their applications, are also discussed. Finally, we propose some possible future applications in which HILIC can prove
itself useful, including complex glycans, glycomics, oligo-drugs (for example, heparins), glycosaminoglycans, and others.
HILIC stationary phases were developed with the aim of providing a tool for the analysis of hydrophilic compounds, which are
poorly retained in reversed-phase liquid chromatography (LC) and, therefore, are not ideally resolved (1). Although the elution
order of analytes in HILIC is generally reversed compared to that in reversed-phase LC (that is, the most hydrophobic compounds
are eluted first in HILIC and the most hydrophilic are eluted last), it has been recognized that the retention order is not
inversely proportional (2). Because of various interactions in the HILIC mode that contribute to the separation and selectivity
in reversed-phase LC and HILIC, the techniques to a large degree are orthogonal (3,4).
Besides the analysis of small molecules, many early HILIC applications investigated the separation of peptides. Some of these
include those of Alpert (1), Yoshida (5), and Mant and colleagues (6). More recently, Gilar and Jaworski (3) compared three
HILIC stationary phases and concluded that for the separation of charged species, such as peptides, the interactions with
the charged silanols on silica significantly contributed to the retention mechanism.
The separation orthogonality of HILIC is often used for the analysis of compounds that are difficult to resolve in reversed-phase
LC. For example, Zhu and colleagues (2) have used both reversed-phase LC and HILIC for analyses of peptide maps of erythropoietin
and found that the methods were complementary. HILIC was more suitable for the detection and quantification of small or hydrophilic
peptides and also glycosylated peptides that were not adequately resolved in reversed-phase LC. Moreover, HILIC peptide maps
contained some unique, very hydrophobic peptides that were not detected in reversed-phase LC.
HILIC was also applied to the quantitative bioanalysis of therapeutic peptides and proteins. This class of biological therapeutics
holds great potential; however, the analysis has been challenging. Biological fluids contain many peptides, especially when
the analysts perform proteolysis of serum proteins before liquid chromatography–mass spectrometry (LC–MS). Two-dimensional
liquid chromatography (2D LC), reversed phase × HILIC, combined with multiple reaction monitoring mass spectrometry (MS),
was used for the quantitation of peptides of interest. Limits of detection below 10 ng/mL were achieved (7).
As stated above, HILIC and reversed-phase LC separation modes are orthogonal, and they can be utilized for 2D LC. D'Attoma
and colleagues (8) adopted HILIC as the second dimension of 2D LC for the separation of protein digests. They reported an
effective peak capacity of 2600 in a 180-min analysis. A HILIC × reversed-phase 2D LC setup was also evaluated by Di Palma
and colleagues (10) for the separation of complex proteomic samples. The authors observed a favorable separation of peptides
on zwitterionic HILIC in the first dimension compared to the more typical, first dimension — strong cation-exchange chromatography.
Importantly, the authors noted a potential for enrichment of phosphopeptides in HILIC × reversed-phase 2D LC (9,10). McNulty
and Annan (11) also investigated the potential of HILIC × reversed-phase 2D LC for proteomic analysis (11). Similar to previous
reports, they observed significant advantages of HILIC for phosphoproteomic analysis. Because of charge–charge interactions
with the zwitterionic stationary phase, or simply because of the more hydrophilic nature caused by phosphate groups, the phosphopeptides
were eluted in a later elution time window when compared to general peptides (9–11). Phosphopeptides also appeared to be better
recovered and more detectable in the HILIC × reversed-phase 2D LC method (11). A similar observation was made by Alpert (12),
who proposed that the solute's interaction with the charged, hydrophilic sorbent may affect their elution patterns. This separation
mode was then termed electrostatic repulsion hydrophilic interaction chromatography (ERLIC), and it can be used for the selective isolation of phosphopeptides from a protein digest. To further this observation,
Sze (13,14) has used ERLIC–weak anion-exchange approaches (HILIC on weak anion-exchange surfaces) for selectively retaining
phosphopeptides, free of other, acidic peptides, by operating at pH 2, where the phosphate group retains a negative charge
and peptide carboxyls are neutral.