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May/June 2011


HILIC After the Hype: A Separation Technology here to Stay


by Petrus Hemström, Tobias Jonsson*, Patrik Appelblad, Wen Jiang Merck SeQuant AB, Box 7956, 90719 Umeå, Sweden *corresponding author: tobias.jonsson@merckgroup.com


Today Hydrophilic Interaction Liquid Chromatography (HILIC) is by far the fastest growing HPLC separation mode, currently being used by almost 20% of the HPLC instrument owners [1]


HILIC is yet another technology-hype on the separation science scene, and that the interest will soon vanish? We believe not.


This paper reviews the reasons behind the remarkable rise of HILIC and based on that, tries to predict future development within this separation technology. For an introduction to the basic separation requirements and mechanisms, we refer to previous texts [2]


HILIC History


The story about modern HILIC started with a publication [3]


more than 20 years ago:


“Hydrophilic-Interaction Chromatography for the Separation of Peptides, Nucleic Acids and other Polar Compounds”


The HILIC separation technique had actually by then been used for about 15 years for sugar analysis on aminopropyl silica columns, but it was with his landmark paper in 1990 that Andrew Alpert showed HILIC to be a universal separation mode, potentially applicable to separation of all types of polar compounds. The publication went by relatively unnoticed by the HPLC community, and for the next 12 years the number of people using HILIC were rather limited [2]


, many of whom did not even use the term HILIC.


By 2002 the number of publications in the scientific literature using HILIC was still only a handful, but the dormant phase of HILIC was now passed and within two years the scene changed dramatically. The small company SeQuant, a spin-off from Umeå University in northern Sweden, launched a new zwitterionic HILIC phase (the ZIC®


-


HILIC), Waters launched their plain silica Atlantis®


their TSKgel®


HILIC column and Tosoh re-braded Amide-80 column as a HILIC


column. The number of HILIC publications (and with some delay also column sales) have since then been increasing by 30-40% each year and today scientific papers on new HILIC stationary phases are published in unmatched numbers and every HPLC column manufacturer have at least one HILIC stationary phase in their program.


Success of Selectivity What have been the key ingredients in this tremendous success of HILIC? Naturally its perfect fit to mass spectrometry (MS) and the lack of need for investments in any new HPLC equipment have played a part by providing the soil for this growth, but what really has been the key driving force is the fact that HILIC focuses on selectivity. With HILIC it is now possible to separate compounds previously impossible to retain on HPLC columns. The direction towards selectivity is in sharp contrast to the development in reversed phase (RP) over the last decades, where UHPLC, with sub-2 µm, core-shell particles or monolithic approaches, all have been attempts to optimize the separation physics. At the same time, selectivity was minimized by ever better synthesis schemes and end capping protocols, trying to make all C-18 columns similar. What HILIC thus has brought is an alternative view of what is important in HPLC separations, away from physics and plate counts, towards chemistry and selectivity. A quick glance at the equation for chromatographic resolution [4]


shows that this


is a wise path; increasing selectivity is the most effective way to improve a separation (Figure 1).


With its alternative selectivity, HILIC addresses a range of compounds that have been problematic to separate for many years; polar and hydrophilic compounds. Such molecules are found in many different application areas. Consequently, a continuous expansion of the application base has followed which will keep on increasing as the HILIC separation technology reaches new audiences.


on the subject. , although the relative number of applications still is considerably fewer. Does this mean


Figure 1. Relative impact of selectivity (α), efficiency (N) and retention (k) on chromatographic resolution (Rs). Graphs were obtained by plotting the resolution equation [4]


one parameter while keeping the others fixed at the intersection point values (α=1.05, N=5000, k=5).


Metabolomics and Clinical Analysis HILIC columns can easily be interfaced with single and tandem MS, electrochemical detectors (ECD), inductive coupled plasma (ICP), and nuclear magnetic resonance spectroscopy (NMR) for high sensitivity and specificity. This makes HILIC very well suited for the detection and quantification of low- level biomarkers targeted in metabolomics [5] and clinical analysis [6]


for diagnosis and


for physiological levels of methyl malonic acid (MMA), which is an important biomarker for vitamin B12 deficiency.


disease monitoring. Many of these new clinical assays require only protein precipitation as sample preparation, use of isotopically labeled internal standards, and an isocratic chromatographic method, as exemplified by the 3-min HILIC-MS assay (see Figure 2) [7]


against


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