Principles and Practical Aspects of Preparative Liquid Chromatography
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5. Disperse the material in packing solvent in the ratio 1 g <strong>of</strong> dry media to<br />
2 mL <strong>of</strong> packing solvent. Packing solvent is usually isopropanol (IPA).<br />
To ensure the media fully disperses <strong>and</strong> is free <strong>of</strong> lumps, the packing<br />
slurry should be shaken not stirred, or bottled-rolled for approximately<br />
five minutes. As with all HPLC media, do not use a magnetic stirring<br />
bar as this grinds the particles <strong>and</strong> produces fines. It is essential that<br />
the slurry be well mixed. Since there are significant differences in the<br />
characteristics <strong>of</strong> stationary phases (for example, particle size, shape,<br />
nature <strong>of</strong> functional groups, <strong>and</strong> so on), you should consult the resin<br />
manufacturer to determine the optimal slurry solvent <strong>and</strong> compression<br />
pressure. As you gain experience in column packing with a given<br />
resin, the packing conditions can be adjusted to optimize results.<br />
The packing slurry is now ready for use.<br />
6. Take the homogenous, free-flowing slurry <strong>and</strong> pour quickly into<br />
the assembled column in one continuous action.<br />
7. Complete the assembly <strong>of</strong> the column <strong>and</strong> operate the packing<br />
station according to the instructions supplied. Mechanical pressure<br />
<strong>of</strong> approximately 1000 psi is recommended for reversed-phase media<br />
with 100-Å pore size. Hence a hydraulic pressure <strong>of</strong> 1500 psi is<br />
required. Make sure that the hydraulic pressure has been set using<br />
the correct ratio for the combination <strong>of</strong> column id <strong>and</strong> packing station<br />
being used.<br />
8. Once column packing is complete, the flow <strong>of</strong> packing solvent<br />
has ceased <strong>and</strong> the pump has stopped, allow the column to<br />
st<strong>and</strong>/equilibrate for 10 minutes. The column is ready to be transferred<br />
to the chromatographic eluent. If required, the column plunger can be<br />
locked in the compressed position so that the column can be operated<br />
in SAC mode.<br />
9. The packed column is now ready for use. It can be used while still<br />
assembled on the packing station or it can be undocked for use in<br />
a purification facility.<br />
The typical column efficiency for 10-micron reversed-phase media<br />
in a bench-top Load & Lock column is 30,000 plates/meter.<br />
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