Table of Contents
Advertisement
3. Locate the tubing which exits the right side of the
instrument through a union located near the bottom of the
instrument. Attach this to the outlet of the column and
feed the tubing into the slot of the lower column mount.
4. Lower the column fitting into the space in the column
mount.
5. Locate the tubing connected to the guard column outlet (or
inject valve if no guard column is used). Connect this
tubing to the preparative column inlet fitting and feed the
tubing through the slot of the column mount.
6. Lower the top column mount over the fitting. The tubing
passes through slots in the upper and lower mounts
allowing the tubing fittings to be captured in the holes in
the upper and lower mounts.
5.5.2 Sample Preparation
The HPLC sample must be prepared as a liquid. To prevent
sample loss to waste, the sample injection must not exceed ½
of the injection loop volume of the EZ Prep. (Standard loop
volume is 5 ml. 1 and 10 ml loops are available.) The solvent
used to prepare the sample should be similar to the stating
conditions of the gradient if possible. If the sample is dissolved
in a solvent that is too strong, it can have negative effects on
the chromatography. The sample should be filtered with a 2
µm filter or finer to prevent clogging of the HPLC column.
The loop will contain solvent from the previous separation.
Typically, reverse phase separations end with an organic
concentration between 50 and 80%. If your sample elutes at a
low percentage of organic, this remaining fluid in the loop can
affect the quality of your separation. This can be addressed by
flushing the high organic solvent from the loop before loading
your sample. Flushing the loop with at least 2 loop volumes of
water or minimal organic solvent will result in improved
separations in this situation. This also has the benefit of
rinsing any residue remaining in the injection port from
previous injections.
After injection of your sample, ~50µl of injection fluid will
remain in the lower port. To maximize sample recovery, a
small amount of suitable solvent should be used to push the
sample into the injection loop. An alternative injection port
fitting is available from Teledyne Isco. This alternative port
Section 5 Operation
5-17
Advertisement
Table of Contents
