9
Revision2.0
1/1/02
8.0PACKINGTECHNIQUES:
After thepacking is finished, thepackingdevice isdisconnected from thepumpand theupper
tube (slurry container) is screwedoff from the column tube.Make sure the residual systempres-
sure is relaxedbyopening the fittingat thecolumnoutlet.Becarefulwhen installing theadjust-
ablepiston:There shall not beanyparticlesbetweenpiston sealingandglass tube.Remove the
column tube from thepackingassemblyand thoroughlyplace it into thecolumnholder.The
cartridge is then lockedwith theupper devisable screw lock. Reconnect the column to thepump
and start pumpingwith a low flow rate. Increase thepump flow slowlyuntil thepressure limit of
the column is reached.Due to thehigh flow rate small deadvolumebetweenupper piston andgel
bedmayoccur.Remove this deadvolumeby screwingdown the adjustablepiston:Make sure the
pump is switchedoff and slowly turn thedevisabe screwcounter clockwiseuntil thedeadvolume
isdiminished.
The column cannowbe equilibrated to the required chromatographic conditions and is ready for
use.
Checking thecolumnperformance:
We strongly recommend to test the columnperformancewith a suitable test substance toobtain
HETPandpeak symmetry.By repeating this test regularly thedeteriorations of the columnpack-
ingcaneasilybedetermined.
Numberof theoretical plates:
N=5,54* (T1/W1/2)
T1:Retention time (min)
W½: Peakwidth (min) at halvepeakheight
HETP=L/N
L:Column length inm
Peaksymmetry:
S=W½,re/W½ li
W1/2, re: Peakwidth, right of peakmedian
W½, li: Peakwidth, left of peakmedian
9.0CLEANING INSTRUCTIONSFORPACKEDCOLUMN (CIP):
Acleaningprocedure includes the three following, steps: regenerationof thechromatographic
support, sterilizationanddepyrogenation.
Regenerationeliminatesmineral andorganiccontaminants,whicharenon-specifically fixedon
thechromatographic resin.Thesemoleculesaremost often lipid substances (including
pyrogens), proteinaggregates, pigments, polyphenolsormetal complexes.These substances
decrease theperformanceof thepackingmaterial (capacity, resolution, purityandyield).
