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Columns Facts and Tips
Certificate, Shipment, Installation,
Solvent compatibility, Storage,
Cleaning,
successful GPC/SEC
separation.
Our stationary phases come in stainless steel columns of standard
dimensions that fit any HPLC or LC (GPC, SEC, GFC) instrument .
PSS offers a wide variety of column dimensions and will manufacture custom sizes
upon request.
- Column
Performance/ Certificate
PSS carefully
tests every column in your choice of solvent. A column
quality
certificate that is unique for each column contains the testing
results.
Shipment
Standard
shipment time for columns is 10 to 15 business days. All column
orders are shipped via air.
Unless
otherwise
requested by a customer, the columns suitable for organic solvents ship
in Tetrahydrofurane (THF). Aqueous columns ship in water
containing 0.05% of NaN3 (preservative).
Installation
Hardware:
PSS usually
ships connection fittings with the column. However, you may use
Valco ferrules and nuts to replace such fittings. WARNING:
Make sure that the fitting tubing protrudes a maximum of 1 mm inside
the column head (see
illustration). Use stainless steel tubing
for all organic mobile phases, however, water based mobile phases work
better in other tubing materials compatible with the system.
Refill
Service
PSS will re-fill
your old columns, at a considerable cost saving over new
columns.
Sample Evaluation
for Column Selection:
PSS will perform a GPC test to document
the performance of our
columns on your own samples when you are researching alternative GPC
columns or new products. We will process the sample as an
analytical service charging the customary testing fee. The cost of this
service will be credited to the purchase of the column(s) specified by
the analysis report.
Column Cleaning
When columns
lose efficiency or you suspect the presence of foreign material, remove
the column from the detector and install the column in reverse for
clean-up. Flush the column with a solvent that is fully
compatible with your system, which will remove the suspect
impurities. Change the polarity, the content of salt, the pH, or
the functionality of your solvent.
Solvent
Compatibility
It is imperative
that a sample passing through a column be soluble in the mobile
phase. When the using a solvent different to the mobile phase to
dissolve a sample, the two solvents must be miscible. Warning,
prevent the formation of precipitates inside of the columns.
Column
Installation Tips
- Purge the system
with mobile phase; pump solvent through to remove air from the
capillary to prevent air from entering the column; flush the injection
loop also.
- Use the column
connections supplied to connect columns in series
- Use a middle
porosity as the first column use the highest porosity as the last
- Be aware of column
flow direction (operating the flow rate in the reverse direction
is only part of troubleshooting or operating after a long storage time)
- Thread the column
fittings finger tight into the system (do not over tighten the fitting!
Over-tightening will damage the column and the column head)
- Do not connect the
column to the detector during the installation and the first solvent
exchange
- Flush the column
with 10 times of the column volume at 1/5 of the recommended flow rate:
0.2 ml/min for ID 8 mm
- When the solvent
exchange is completed, connect to the detector
- Slowly increase
the flow rate to the typical operating flow rate which depends on the
column dimensions (see table below)
- When installing
multiple columns):
- Check each
column separately as described above
- Check the
combined plate count for the column series after installation
- Install the
largest porosity in front of the combination
- Install the
smallest porosity in the middle and the medium porosity at the
end and
- Use only
recommended column combinations
- Do
not combine linear and single porosity columns
Flow
Rates vs. Column Dimension
|
Flow
|
ID
8 mm
|
ID
4.6 mm
|
ID
20 mm |
Normal
|
1 ml/min
|
0.33 ml/min |
6.25 ml/min
|
| Reduced |
0.25 ml/min |
0.1 ml/min |
1.5
ml/min
|
Idle
|
0.1 ml/min |
0.03 ml/min |
0.6 ml/min
|
Storage
Recomendations
- Remove
all salt solutions with pure solvents and plug tight with the original
end plugs.
- Store the
columns in the solvent used during shipment; If the column was
shipped and used in THF or CHCl3 you can store the column in toluene as
well. Toluene has the advantage of a very low evaporation rate.
Columns shipped in DMAc or DMF must be stored in DMAc or DMF.
- It is good
practice to keep columns with volatile mobile phases in a refrigerator
(4°C) after use to prevent solvent evaporation. Warning
never let the column temperature fall below the freezing point of the
storage solvent. This can destroy the stationary phase.
- Occasionally
solvent is lost during long-term storage or due to high storage
temperatures. When the expected pressure does not build up or the pump
constantly re-regulates the flow, it is an indication of a partially
evaporated column solvent
Recovering
a partially dry column
To re-wet a
partially dry column, install it in the reverse direction (to
prevent damaging the packing). Using a flow of 0.1 ml/min, fill
the column until no more bubbles appear at the column outlet. Change
the column to the correct flow direction and again use 0.1 ml/min for
1h. Then you can slowly increment the flow rate, systematically, using
0.2 ml/min steps.
Successful
Molecular Size Separation and Column Use
Gel Permeation
Chromatography (GPC), Size Exclusion
Chromatography (SEC), or Gel Filtration Chromatography (GFC) are names
used interchangeably for a liquid chromatography technique to obtain
the molecular weight distribution of a polymer. Contrary to normal HPLC
methods that rely on interactions between sample and stationary phase,
GPC must operate free of interactions to assure separation by size
only. Only the entropy effects should influence the separation.
- Select
an adequate stationary phase (column material)
- Completely
fill the system with mobile phase.
- Make sure
to use only mobile phases that are compatible with the gel.
- Use guard
column to increase the column life.
- Determine
the Plate Count, Asymmetry and Resolution of your column
- Determine
the molecular weight range of the separation with a calibration curve.
- Filter
your sample through a 0.45-micron filter to prevent solids from
entering the column.
- Use
appropriate concentrations and injection volume balance to prevent
column overload. The higher the molecular weight the lower the
sample concentration.
- Maintain
a flow that agrees with the column diameter and viscosity of sample to
prevent shearing and backpressure.
- Use
multiple columns (in series) to improve the resolution and/or expand
the separation range.
- Also available
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