HILICON is the manufacturer of the iHILIC columns.
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- Charge modulated hydroxyethyl amide HILIC columns
- Complementary selectivity for polar analytes
- Excellent durability and ultra-low bleeding for LC-MS
- Available in 1.8, 3.5, 5 μm and 1, 2.1, 3.0, 4.6 mm i.d.
- Also available in SPE cartridges
These columns are designed for the separation and purification of polar and hydrophilic compounds using HILIC (Hydrophilic Interaction Liquid Chromatography).
The packed stationary phases are charge modulated hyrdroxyethyl amide silicas which are covalently bonded with neutral, positively charged and negatively charged hydrophilic functional groups.
Therefore, the separation mechanism with iHILIC columns is based on a combination of hydrophillic interaction, hydrogen bonding and weak electrosttic interactions. They have unique separation selectivity and high separation efficiency. iHILIC columns are available in two different surface chemistries, providing complementary selectivity.
iHILIC - Fusion - HILICON
i HILIC - fUSION (+)- HILICON
i HILIC FUSION ( P )HILICON
i SPE HILICON
The iHILIC columns are available in both PEEK and in SS. The column end fittings have 10-32 UNF thread and a Parker port design.
The silica based iHILIC columns can be used between pH 2 to 8, max operating temperature is 60 degrees.
The max column operation back pressure is
- PEEK HPLC columns packed with 3.5 and 5 um particles : < 350 bar at roomtemperature
- SS HPLC columns packed with 3.5um particles : <450 bar at roomtemperature
- SS UHPLC columns packed with 1.8um particles : < 650 bar at roomtemperature
For HILIC separations, water and buffer solutions are strong eluents and organic solvensts are weaker.
Acetronitrile is the most favorable solvent. The relative solvent strength for HILIC is : THF < Acetone < Acetonitrile < Isopropanol < Ethanol < Methanol < Water. Contrary to reversed phase chromatography, polar compouns have increased retention when increasing the proportion of organic solvent in mobile phase.
Buffers and Additives
Solutions of 2-50 mM ammonium format and ammonium acetate are the best buffers for HILIC with MS dtetection. Their pH can be adjusted by adding formic acid, acetic acid or ammonia. Phosphate buffers should be used with extra caution at lower concentrations due to the lesser solubility of sodium, potassium, and phosphate in HILIC mobile phases, which contain high concentrations of organic solvent. TFA and ion-pair reagents will change the selectivity of HILIC separations and often interfere with MS detection. Thus they should be avoided or used consciously to reduce the polarity of amines and the protonation of carboxyl groups for a specific purpose (i.e. isolation of glycopeptides from peptide digests).
It's highly recommended to do sample preparations with mobile phase or solutions with similar ionic strength and concentration of organic solvent. These would help to get sharp peaks and avoid tailing or split peaks. More organic solvent in the sample solution will sharpen the peak due to peak compression effects. Complex samples such as plasma or urine should be treated with a high proportion of organic solvent to precipitate proteins ans salts, and filtered with 0.45 or 0.22um syringe filters that are compatible with organic solvents.
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