6


May/June 2011


which are polar and hydrophilic [27]. Among these we find algal and cyanobacterial toxins, disinfection by-products, hormones and other endocrine disrupting compounds, drugs of abuse and their metabolites, organometallics, organophosphate flame retardants and plasticizers, pharmaceuticals and personal care products, polar pesticides together with their degradation and transformation products, and surfactants and their metabolites. It would also make sense to add artificial sweeteners to the list.


The overall ongoing trend in environmental analytical chemistry is that contaminants, and in particular their degradation products and metabolites, are more hydrophilic. Consequently they are more easily transported by water from the point of emission into streams and lakes diluting, but not necessarily degrading them. Finding ways of concentrating and analyzing such samples will be an immense task where HILIC absolutely will have a role to play.


Biomolecules: Peptides and Oligonucleotides Separation of peptides has since Alpert´s pioneering paper [3]


been an important


application area for HILIC separations. In particular has HILIC enabled separation and extraction of post translational modified peptides, subjected to glycosylation [28, 29] phosphorylation [30]


. More recently the


complementarily of HILIC and RP have been used to accomplish orthogonal 2D mapping separations of protein digests and thereby demonstrating supreme protein identification capabilities [31, 32]


.


Today the available methods for separating oligonucleotides are either ion-pairing RP or ion chromatography [33]


. With both these


techniques the selectivity is mainly derived from the number of negatively charged phosphate groups, i.e., from the length of the oligonucleotide. The difficulty of achieving good separation between different oligonucleotides of the same length has resulted in numerous attempts to use mass spectrometry for resolution, and although this has had some success, both chromatography and detection is severely influenced by the ion-pairing agent concentration.


Scientists are now turning to HILIC to separate oligonucleotides and the advantages are obvious; no need for ion-pairing reagents and the happy marriage to MS. Because the partitioning process causing retention in HILIC is governed by the overall hydrophilicity of the oligonucleotide, the nucleotide bases will also affect the selectivity and make it possible to separate oligonucleotides of the same size but


and


Future of HILIC columns When so many application areas are relying on HILIC, combined with that the basic requirement on a HILIC stationary phase only is to hold the water layer in place for hydrophilic partitioning, it is not surprising that more than 40 different stationary phases have been tested for HILIC-type separations [2]


.


There is certainly a need for several different selectivities in HILIC, especially considering that one great advantage with HILIC is that changing stationary phase actually does change selectivity, but such immense diversity will most likely only be temporary. Still it would be surprising if a harmonization of HILIC phases to the same extreme as seen in RP (all


manufacturers make very similar C-18 phases) will occur. But naturally, the HPLC society will need tools to better judge what stationary phases are similar and which to select for what type of separation.


Figure 4. HILIC-MS separation of two 20 unit heterogeneous oligonucleotides of same composition but different sequence. Separation on ZIC-HILIC 100x2.1, 3.5 µm, 200 Å HPLC column at 50 °C, operated at 0.4 mL/min using linear gradient elution from 65% to 55% acetonitrile in water during 7.5 minutes with constant 10 mM ammonium acetate (pH 5.8). Courtesy of Lingzhi Gong, University of Oxford, UK [34].


with different composition or base sequence, see Figure 4[34]


.


It is not hard to predict that HILIC will continue to be a very strong contributor to analysis of small biomolecules such as peptides and oligonucleotides. The needs for analysis in these fields will continue to increase, especially for oligonucleotide separations which often is mentioned as the next big challenge [33]


due to the growing


importance of these molecules and the drawbacks of present separation approaches.


Today four major groups of HILIC column chemistries exist on the market; plain silica, and bonded phases with functional groups of zwitterions, amide and diol. A survey of scientific literature from 2009 [35]


confirms the


picture but also show that mixed-mode columns of various types are being tested for HILIC despite their inherently lower hydrophilicity and consequently limited retention window. Although development of new HILIC stationary phases have been the topic for several scientific papers, the range of HILIC stationary phases in actual use is not likely to change dramatically within the next few years, with the exception of a continued decline of plain silica phases.


Plain silica does have a number of inherent drawbacks such as pH-dependent surface charge and thereby hydrophilicity, and strong irreversible adsorption or even reactivity towards certain analytes [36, 37]


. For these


reasons it has been concluded that plain silica is not suitable for biological analysis [36]


.


Although numerous HILIC applications still are performed with plain silica, the reasons for this are mainly a) availability in desired separation format or b) lower cost. It is thus expected that as the knowledge and experience of HILIC increase among users and manufacturers, and column prices are reduced, bonded phases will come to dominate in HILIC.


The lower apparent hydrophilicity [38] of diol


columns will likely limit their usefulness in HILIC since they will require higher content of organic solvent to generate retention. On the other hand there are indications [38]


the


Figure 5. Relative number of published scientific papers using different stationary phase chemistries for HILIC separations. Data from literature survey performed in 2009 [35]


.


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