Waters Protein-Pak Hi Res HIC Column and HIC Protein Standard - Manual (Page 5)

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b. HIC Protein Mixture Performance Test Example
Column: Protein-Pak Hi Res HIC, 2.5 µm
4.6 x 100 mm (P/N 176003576)
Sample: HIC Protein Standard Test Mix (P/N 186007953)
Dissolved in 100 µL 50%A / 50%B
Mobile phase A*: 2 M (NH
4
)
2
SO
4
in 50 mM NaH
2
PO
4
/Na
2
HPO
4
, pH 7
Mobile phase B*: 50 mM NaH
2
PO
4
/Na
2
HPO
4
, pH 7
*Note it is recommended to filter all Mobile Phases with 0.2 µm GVWP filters
Gradient: 0 – 100%B in 15 mins
Flow rate: 0.6 ml/min
Detection: 220 nm
Temperature: 30 °C
Sample: 2 µL injection
Figure 1: Example Protein Test Mixture Chromatogram.
AU
0.00
0.03
0.06
0.09
0.12
Minutes
0.0 5.0 10.0 15.0
1
2
3
4
5
6
1. Cytochrome C
2. Myoglobin
3. Ribonuclease A
4. Lysozyme
5. Enolase
6. Alpha-chymotrypsinogen A
TIPS AND T RICKS
■ ■
Equilibrate at the initial condition for 10 column
volumes before running the sample set.
■ ■
After running HIC application, be sure to purge the
LC eluent lines and system with water to prevent
salt precipitation that could occur when system is
not being used.