Waters Protein-Pak Hi Res HIC Column and HIC Protein Standard - Manual (Page 4)

4
[ CARE AND USE MANUAL ]
Protein-Pak Hi Res HIC Column and HIC Protein Standard Test Mix Care & Use
IV. USEFUL FUNCTIONAL T EST
It is very valuable to benchmark the column for the first use to obtain a useful measure of the physical state of the packed bed
and chemical integrity of the bonded phase. Once this is established, it is recommended to use this same test on a regular basis.
The HIC Protein Standard Test Mix (P/N 186007953) was specially designed to provide a relevant standard for this type of testing.
This standard comes with each new column purchase and can be reordered to continue to benchmark and monitor the column
efficiency and lifetime. It is also a very valuable tool for method development and troubleshooting. The standard contains 6
lyophilized intact proteins that provide good chromatographic representation throughout the example gradient. It is package
in a Waters TruView™ Vial for direct solubilization which can be dissolved in distilled water or initial gradient conditions.
Protein Concentration/Vial
Bovine Ribonuclease A 0.05 mg
Horse Cytochrome c 0.025 mg
Horse Myoglobin 0.05 mg
Chicken Lysozyme 0.03 mg
Yeast Enolase 0.10 mg
Alpha-chymotrypisinogen A 0.05 mg
Table 1: HIC Protein Standard Test Mix Contents
a. Sample Preparation
Prior to injection of your sample onto your Protein-Pak Hi Res HIC Column, it is important to determine the appropriate salt
concentration to dissolve the sample without having it precipitate out of solution yet sufficiently high enough to allow the proteins
to bind to the hydrophobic butyl groups contained on the non-porous, 2.5 μm particles. While results will vary depending on proteins
to be analyzed, we suggest dissolving your sample in 1M (NH
4
)
2
SO
4
containing 50 mM NaH
2
PO
4
/ Na
2
HPO
4
, pH 7 since the proteins
should remain in solution yet be sufficiently “hyrdophobic” to bind to the column.
*Note Be sure to adequately flush the LC eluent lines and system with water to remove any organic solvent to prevent HIC solvent
precipitation (i.e., salt precipitation if exposed to high organic solvent concentration).