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Immobilized Phospholipid Chromatography 299
3.2. HPLC Buffer Preparation
1. Filter all solvents through a 0.22-μm Durapore filter membrane in a filtration
apparatus fitted with vacuum. This removes particulates that could block the
column and the solvent tubing.
2. For HPLC systems without an on-line degassing capability, subject the solvent to
degassing before use in the HPLC instrument.
3.3. Column Equilibration and Blank Run
1. Connect the guard and the separation column to the tubing according to the HPLC
system requirements and equilibrate the column with 100% Buffer A at a flow
rate of 0.5 mL/min until the baseline is stable monitored at 280 nm for 30 min
(see Note 3).
2. Maintain the column temperature at 25 ± 1ºC during the equilibration and the
separation. If the HPLC system is not equipped with a column thermostat, ambient
room temperature is also appropriate for the equilibration and separation. Monitor
the baseline and protein separation at 280 nm.
3. Once the stable baseline is obtained, inject 10 μL of Milli-Q water or Buffer A
either manually or through an autosampler to the column (see Note 4).
3.4. Chromatography
1. Injection volume: 50 μL.
2. Inject the sample at a flow rate of 0.2 mL/min and run for 8 min which facilitates
affinity adsorption between the injected proteins and the immobilized lipid surface.
After protein loading, increase the flow rate from 0.2 to 0.5 mL/min over 2 min.
Maintain this flow rate throughout the whole separation process.
3. After the protein loading, elute the proteins with Buffer A for 10 min. Program a
change in the elution solvent from 100% Buffer A to 100% Buffer B over 10 min
and then maintain 100% buffer B for 25 min. Finally, change the solvent from
100% Buffer B to 100% Buffer C over 1 min and maintain these conditions at
100% Buffer C for 30 min (see Notes 5 and 6).
4. After each chromatographic separation, it is strongly recommended that columns
are washed with 50 mL isopropanol followed by about 50 mL of Milli-Q water
before re-equilibrating the column with aqueous mobile phase column. Owing
to the high viscosity of isopropanol, it is also necessary to avoid the high back
pressure. Adjust the flow rate for column washing with isopropanol to 0.2 mL/min
and wash for 250 min. For Milli Q wash, set the flow rate initially at 0.2 mL/min
for 100 min and then raise it to 0.5 mL/min for 150 min (see Note 7).
5. Store the column at 4ºC in either 100% methanol or 100% acetonitrile.
6. A typical chromatographic result is shown in Fig. 2 for the separation of PLA2.
The UV chromatogram at 280 nm shows two early eluting peaks that do not have
any enzymatic activity. PLA2 elutes at approximately 60 min and is well separated
from contaminating proteins.