YMC HILIC columns

As shown below, HILIC uses a highly polar stationary phase and a non polar mobile phase e.g. functionalised silica with a hydrophilic coating and an acetonitrile/water mixture (80/20). The polar or hydrophilic sample experiences more attraction towards the stationary phase or the more polar and more stationary part of the mobile phase. Polar samples can be eluted by using isocratic or gradient methods, giving the mobile phase a more hydrophilic character or even a higher ionic strength.

In general, reliable applications in HPLC require a minimum retention factor (k’) of 2. In the widely used reversed phase methods polar compounds such as peptides, organic acids or pyrimidines often exhibit little or no retention and therefore low resolution on a conventional C18 phase. The use of additives such as ion-pair reagents is not an alternative since these are not suitable for LC-MS. Also normal phase chromatography fails due to poor solubility of the polar compounds in organic solvents. HILIC fills the gap between reversed phase and normal phase chromatography, because polar compounds can be retained while using e.g. acetonitrile/water eluents and buffers suitable for LC-MS.

Melamine (sometimes confused with the plant and animal compound, melanin or melanine) is an industrial chemical used in the manufacture of plastic products. Despite that, tests showed that a number of milk companies have used it in processing milk to artificially increase the apparent protein content by addition of non-protein nitrogen and so allowing illegal dilution of the product with water with a corresponding increase in sales value. The problem is that melamine forms crystals of melamine cyanurate from aqueous solutions when mixed with cyanuric acid (which forms naturally by hydrolysis of melamine). These low solubility, virtually indigestible crystals concentrate in the renal microtubules and form large numbers of round spoke-like, yellow crystals, damage the renal cells and ultimately kidney failure.

Recently, more and more attention has been focused on melamine in milk and food products (including, for example, chocolate). The most distressing incidents occurred in the summer of 2008 when more than 54,000 babies became seriously ill and four died in China due to suspected milk formula. However, earlier in 2007 the FDA reported that an estimated 8,500 cats and dogs died after eating melamine-contaminated pet food. In order to avoid further accidents involving melamine contamination, it is essential to provide an analytical method, which monitors the level of melamine without compromising resolution due to excessive background signals from the sample matrix.

YMC have developed a fast HILIC method which also can be transferred to LC/MS detection.