There
are numerous reports in the literature describing the construction of microcolumns
(<0.5-mm ID) from such diverse materials as teflon tubing, glass-lined
stainless steel tubing, and polyimide-coated fused-silica tubing.
We prefer to construct microcolumns from the latter. Compared to
the other materials mentioned above, polyimide-coated fused-silica offers
a number of distinct advantages. Uppermost of these are -(i) its
flexibility and ease of handling, -(ii) its transparency, -(iii) the commercial
availability of this tubing in a wide range of ID and OD, and -(iv) its
relatively low cost.
The
following step-by-step procedure for constructing 0.1 - 0.32-mm ID fused-silica
microcolumns is outlined using the construction of a 250-mm x 0.20-mm ID
column as an example. First, two lengths of polyimide fused-silica
tubing were cut, one 250-mm length (0.200-mm ID x 0.340-mm OD) to provide
the column body and another 200-mm length (0.050-mm ID x 0.190-mm OD) to
provide the column exit tubing.
[Note:
(i) It should be noted that thin-walled capillary tubing has a tendency
to fracture easily when subjected to high pressures (~400 bar) during the
slurry-packing of chromatographic supports. For instance, in our
experience, fused-silica tubing of 0.320-mm ID x 0.540-mm OD and 0.200-mm
ID x 0.340-mm OD can withstand packing pressures of up to 450 bar, while
tubing of 0.320-mm ID x 0.450-mm OD fractures readily at these high pressures.
In the latter case, slurry-packing pressures should not exceed 50 bar.
For the above reasons, safety glasses should be worn and recommended safety
precautions adhered to, during column packing. Finally, it should
be noted that superior column efficiencies can be achieved by packing at
the higher pressures afforded by using the thick-wall fused-silica tubing.
(ii) To avoid jagged ends and to obtain square cut capillaries, a sapphire
knife (Supelco cat. no. 2-3740, Bellefonte, PA, USA) was used instead of
the more common ceramic squares. Tubing with a jagged end will
result in imprecise frit positioning. (iii) Teflon tubing
is used to connect lengths of fused-silica tubing (see Fig. 1). Since
there are slight variations in the OD of fused-silica tubing from different
manufacturers, to obtain tight-fitting teflon connectors, it is often necessary
to fabricate such connectors. This is readily achieved by stretching
a slightly oversized ID piece of teflon tubing by hand with two pairs of
plyers.]
A variety
of porous membranes can be used as column frits to retain the column packing.
These include Zitex membrane, glass-fiber filter paper and hydrophilic
polyvinylidene difluoride (PVDF) (see Table 1 for further details).
For the columns described in this study, we used 0.45-m porosity hydrophilic
PVDF. The PVDF frit was cut from a large sheet of PVDF membrane using
a short length (50-mm) of fused-silica silica tubing (0.32 (or 0.20)-mm
ID x 0.45 (or 0.34)-mm OD) as a disc cutter. This is accomplished by placing
the PVDF sheet on a silicon rubber septum and punching the frit.
The column exit tubing was used to insert the fabricated PVDF frit into
the microcolumn body to a depth of approximately 10-mm. The microcolumn
body and the column exit tubing were permanently positioned by placement
of a small drop of pre-cured epoxy resin at the junction of the two pieces
of tubing.
[Note:
(iv) The epoxy resin (24 h curing-time variety) is partly polymerised
(pre-cured) with a heat gun to increase its viscosity so that it will not
seep into the capillary tubing and block the frit.]
Once
the PVDF frit had been positioned and the fused-silica capillary column
constructed, a slurry-packing procedure was employed to pack the microcolumn.
A slurry reservoir was manufactured from an empty 50 mm x 2 mm ID glass-lined
stainless-steel tube (SGE, Ringwood, Victoria, Australia) with standard
male liquid chromatography (0.5 mm ID flow-through ports) 1/4-in column
end-fittings (Alltech, Dearfield, IL, USA). The fused-silica microcolumn
was connected to the standard 1/4-in liquid chromatography end-fitting
by vespel/graphite ferrules (Alltech, cat no. 100/0.4-VG1) and fingertight
nuts (Swagelock cat. no. 1102-IK, Solon, OH, USA).
A slurry
of reversed-phase silica in n-propanol (40-mg/200-l) was sonicated for
15 min in a 1.5-ml polypropylene tube. Prior to packing, the capillary
column was filled with the packing solvent (n-propanol) under high pressure
(400 bar) using a standard liquid chromatography column packing pump (Shandon,
Cheshire, UK). This step pre-filled the microcolumn with packing
solvent and also allowed the system to be checked for tight seals.
The slurry reservoir was disconnected from both the column packer and the
microcolumn, emptied, replaced into position and filled with 150 l of the
prepared slurry mixture. The column was packed at a constant pressure
of 400 bar for 20 min, consolidated with 50% (v/v) aqueous methanol for
a further 20 min at the same pressure and then allowed to depressurise.
The column was carefully dismantled and sealed by inserting both the top
and bottom ends of the column into silicon rubber septa.
JPSL
Construction of Capillary Columns for HPLC
