YMC Glass Columns
- Lab scale
- Low / medium pressure (pressure limit 10-30 bar)
- Easy to use
- Extended pressure rating up to 80 bar
- Lab scale to prep
- Easy to use
Packing Techniques for Glass Columns
Packing the column with rigid media
Introduce a few mL of solvent or buffer (see instructions on packing material) into the packing device, so that the lower frit is moistened and free from air bubbles. Shake the slurry carefully until it has a uniform consistency and quickly pour it into the packing device without introducing any air bubbles. The slurry container must not have any air bubbles in it, if necessary, it should be topped up with solvent. The packing device is then sealed and packed as quickly as possible using a pump. This means that the flow rate should be set at the pressure limit of the column and if necessary packing is carried out at the pump’s maximum flow rate. The flow rate during packing should always be considerably higher (>20%) than the flow rate needed for later use.
A narrow PEEK capillary at the column outlet may improve packing quality, as it will act like a back-pressure regulator and prevent the slurry entering the column too quickly at first. Pumping must continue at least until a constant pressure is reached. After packing, the filling tube is unscrewed. Care must be taken when opening the column outlet so that any remaining pressure is released completely.
The piston is introduced carefully, without allowing any particles of packing material to get between the glass and the piston seal. The column is now re-connected to the pump. The pump is started at low flow and gradually increased to the pressure limit of the column. At this point dead volume may occur between the variable piston and the column bed, which can be removed as follows:
- The column must not be under pressure, i.e. the pump must be turned off and the column inlet opened.
- The dead volume is removed by moving the variable piston towards the column bed.
- This procedure may need repeating to eliminate all dead volume
Finally the column is conditioned with the relevant eluents and is ready to be used.
Packing the column with soft gel
Only degassed and filtered solvents or buffers may be used when packing chromatography columns. The lower frit is first dampened and covered with approx. 1 cm solvent. The slurry is then introduced carefully and quickly, ensuring that no air bubbles occur. The column outlet should be open while the column is being filled; the solvent can also be sucked from the column outlet with a peristaltic pump at the same time. When all the slurry has been poured in, the gel must be allowed to settle and the solvent drain to approx. 0.5 – 1 cm above the packing level in the gel bed. The gel bed must not be allowed to run dry. The column outlet is closed or the peristaltic pump stopped. Next the variable piston is inserted whilst not connected to any solvent lines, etc. Care must be taken not to allow particles to come between the seal and the column body. By turning the lock slowly, the piston can be moved towards the gel bed. At the same time, all the air above the gel bed should be forced out of the column inlet. It is essential that the gel bed is not compressed when moving the piston towards it. Now the column can be equilibrated with the appropriate buffer or solvent.
Dead volume can occur between the gel bed and the piston during normal use, but this can be removed by moving the piston gently towards the gel bed.
We recommend that you determine the plate count and peak symmetry of the column after packing using with a suitable (non-adsorbent) test substance. By repeating this test frequently, the quality and durability of the packing material can be recorded efficiently.
|Amount of theoretical plates (N):
N = 5.54 x (T1 / W1/2)²
|T1: retention time (sec)
W1/2: peak width (sec) at half peak height
|HETP = L / N
||L: column length in mm
|Peak symmetry (S): S = A / B
||A: peak width to the right of the peak median
B: peak width to the left of the peak median