1. What is the difference between HILIC and Normal Phase Chromatography?
HILIC (Hydrophilic Interaction Liquid Chromatography) is not a new mode of separation, but rather a variation of normal phase liquid chromatography (NPLC), in which sample components distribute between a polar stationary phase and a less polar mobile phase. The stationary phase is usually comprised of silica or alumina either modified by adsorption with a polar liquid or converted by chemical reaction to form a polar bonded phase.
Compounds elute in order of decreasing hydrophobicity or increasing polarity. The major differences between HILIC and NPLC are in both the mobile phase make-up and the mechanism of separation. Traditionally NPLC utilizes a 100% organic solvent or a blend of miscible organic solvents, while HILIC uses organic mobile phases that are water miscible. When using a HILIC gradient, increasing proportions of water are used as the elution solvent. In addition, various salt buffers may be added to the mobile phase in order to keep the analyte in a single ionic form.
HILIC’s mechanism of action is thought to be mostly liquid-liquid partitioning created by the formation of a water layer on the surface of the stationary phase. Retention is also influenced by differences in hydrogen bonding and electrostatic forces.
2. What is the chemical structure of the TSKgel® Amide-80 column?
The TSKgel® Amide-80 resin consists of a spherical silica base material to which carbamoyl functional groups are covalently bonded. The polar functional groups on the sample molecule hydrogen bond to the amide groups on the carbamoyl functionality and are retained.
3. How much sample can I load on the TSKgel® Amide-80 column 4.6 mm ID × 25 cm?
Sample loading capacity increases with decreasing polarity of the mobile phase. For example, the highest loading capacity for mannitol (200 µg) occurs in 75:25 acetonitrile:water but decreases to <100 µg in 65:35 acetonitrile:water. The maximum injection volume is 50 µL.
4. What kinds of mobile phase are recommended for the TSKgel® Amide-80 column?
The pH range is 2.5 to 7.5 with a maximum salt concentration of 100 mmol/L. The column is stable in up to 100% organic solvents.
5. How do you control elution volume in normal phase liquid chromatography?
Elution volume is controlled by the polarity of the mobile phase. By decreasing the mobile phase polarity (increasing the organic component) the sample is retained longer on the column. For example, oligosaccharides are very polar and require 40-50% water in the mobile phase in order to elute.
6. What is the temperature range of the TSKgel® Amide-80 column?
The TSKgel® Amide-80 column can be run up to 80 °C.
7. What endfitting do I use with my particular TSKgel® Normal Phase/HILIC column?
Please visit the HPLC Columns Accessories section of the product catalog for the available fittings to be used with your TSKgel® column. Question #10 within the General FAQ category includes additional information on fittings and connectors.