Operation of the Packed Column
|
| 3.1 |
Start Up of the Packed Column
The packed column is connected to the HPLC pump as well as a suitable
injection system. It is recommended that the tubing is purged before
connecting to the detector to prevent any possible contamination
of the detector flow cell and subsequent blockage by fines from the
packing material. This may be especially prevalent with certain irregular
materials especially if they have been mishandled previously.
Important:
Maximum operating pressure of 200 bar (3000PSI) for the column
tube should NOT be exceeded.
Column inlet is on piston side whereas connection to detector
is on flange for column cover side. |
The sorbent bed is stabilized by compressing the column piston to
the appropriate pressure using the hydraulic cylinder.
Important:
Safety reasons require NOT to exceed a maximum hydraulic
pressure of 100 bar (1500PSI). |
This piston is fixed in its position by screwing in the piston safety
stop until it meets the piston.
RP-sorbents are directly conditioned with an appropriate eluent.
For buffered systems, buffer salt precipitation must be avoided (rinsing
with unbuffered eluent).
Si-sorbents are rinsed according to following procedure with
2-propanol to achieve an optimized column bed.
The column is now operated for approximately 1 minute at maximum possible
elution speed. The hydraulic cylinder and piston safety stop are then
re-adjusted and the packing is again stablized at high flow.
This procedure should be repeated 3-4 times to achieve maximum bed
stability.
The packing is now tested for efficiency and peak symmetry according
to manufacturer's data for the packing material.
|
| 3.2 |
Testing the Integrity of the Packing
General information regarding efficiency and symmetry testing of Merck
KGaA packing materials:
Since Merck KGaA, Darmstadt, Germany, LiChrospher® and LiChroprep®
packing materials are characterized by the use of extensive selectivity
tests at the time of manufacture, further testing for reproducibility
of packing selectivity is not necessary.
With all other materials it is recommended that you perform a selectivity
test using suitable test substances (similar to the substance mixture
to be separated) in addition to the efficiency test, especially for
critical separations.
For efficiency testing
for RP-sorbents: a mixture of phthalates is used, which are
tested in methanol/water 80/20 (v/v) at high flow (approximately
40ml/min).
for Si-sorbents: a mixture of anisoles is used which are tested
in n-heptane/1,4-dioxane 90/10 (v/v) at high flow (approximately 40ml/min).
Flow rates of approximately 40ml/min correspond with a flow of 1ml/min
in an analytical 4mm ID column and should enable a side by side comparison
to be made.
|
| 3.3 |
Operation of the Column "in the Packing System" (Hydraulic Packing
Stand)
Operation of the column "in the packing system" is outlined according
to the instructions above.
|
3.4 |
Operation of the Column "Remote from the Packing System" (Hydraulic
Packing Stand) as a Conventional Column
To operate the column without the stand, the piston is fixed in its
final position using the piston safety stop, the 3 domed nuts are loosened,
and the column is removed from the hydraulic stand. As a replacement
for the draw rods, 3 screws M10x45 are installed and screwed together
with the 3 domed nuts.
Important:
Operation of the column without these additional 3 screws is
not recommended and can lead to column damage ! |
Using as a stand alone column without the hydraulic packing stand is
now possible.
If efficiency should descrease during operation of the column, the column
can be reinstalled in the hydraulic packing stand by reversing the steps
described above, and the packing can be restablized as described in
3.1.
The hydraulic packing stand itself can thus be utilized for the packing
of additional columns.
|
| Column Storage After Use |
|
As a general rule, normal phase silica columns are stored in hexane
or heptane. Reversed-phase silicas can be stored in 100% acetonitrile
or methanol. Aqueous mixtures of acetonitrile and methanol are also
commonly used but not all silicas can be stored with water. However,
the final criterion for storage solvent will be the recommendations
of the silica gel manufacturer.
|
| 4.1 |
Following are some tips which are easily forgotten when hurried (at the end of the day
or the week):
Before removing the column from the system, all buffers should be rinsed out. Some buffers
are an ideal growth medium for microorganisms which in turn could increase the packing
volume and thus the back pressure. The silica may then become irreversibly contaminated.
Also, leaving buffer salts in a column could cause corrosion of the metal. The salts may
also crystallize or precipitate leading to high back pressures. In either case, the column
may have to be unpacked and the silica discarded.
If sample components have become adsorbed to the silica at the column inlet, much time
and solvent can be saved by disconnecting the outlet line from the detector and backflushing
the column with solvent strong enough to dislodge the adsorbed material. This will also
flush any particulate matter that has accumulated on the frit. Keep the original inlet line
disconnected and let the solvent go to waste. Never backflush through the detector flow
cell.
To determine a suitable solvent for cleaning the column, the raw sample can be applied to a
TLC plate (such as a 5x10mm or 5x7.5mm Aluminum backed plate) and eluted with various solvents.
The solvent that causes the total sample spot to move with the solvent front is the solvent
which is strong enough to clean the sample and maxtrix components from the column.
Always maintain sample and buffer salt solubility when changing eluents. Also, eluent changeovers
should be done in a manner that maintains miscibility of organic solvents with aqueous eluents and
of organic solvent with each other. By doing so, back pressure problems associated with
precipitation of sample or salts and immiscible solvents will be avoided.
|
| Emptying the Column |
| 5.1 |
Removing the Sorbent from the Packed Column
Removal of the column packing should only be carried out using the packing stand. To remove
the packing, proceed as follows:
| a) |
Turn off the eluent pump, remove eluent inlet and outlet lines from the column |
| b) |
Release the piston safety stop with the hydraulic pump using slight pressure and
unscrew it from the inlet flange |
| c) |
Remove the outlet flange for column cover in reverse order as described in 1.2.6 "Assembly
of the Column Head" |
| d) |
Install the packing reservoir onto the column as described in 2.1.3 |
| e) |
Expel the packing by operating the hydraulic pump and pushing the packing material and piston
assembly into the packing reservoir. As soon as the hydraulic cylinder has reached the
piston safety stop, packing extrusion rods are installed and packing is completely emptied.
Important:
It is imperative that the PEEK capillary is guided into the groove in the piston
stop thread of the inlet flange or the capillary will be damaged. |
|
| f) |
The expelled packing can be discarded or repurified for later reuse.
|
|
| 5.2 |
Preparation for Re-Packing
| a) |
The piston baody if carefully removed from the column and cleaned using compressed air. |
| b) |
It is recommended that the frit holder is unscrewed from the piston body after 3-4 packings
and the frit cleaned using ultrasound or rinsing with a 1N NaOH solution or other suitable
method (depending on the type of packing material, samples and eluents). A similar procedure
is recommended for the frit.
Important:
The even permeability of the frits is very important for the optimal efficiency
of the system. |
|
| c) |
After thorough cleaning of the column tube and associated components, the system can be used
immediately for repacking.
Important:
The life of the seals is increased significantly if all solid particles are
carefully removed from the surface of the seals and ALL items they contact. |
|
|
