Reversible Negative Staining of Protein on Electrophoresis Gels
43
tioned in place with a drop of preheated epoxy resin at the junction and heated
until the epoxy is set using a hot-air gun. The epoxy is preheated to minimize
movement of the epoxy into the column by capillary action. Once the epoxy has
set, the column is ready to be slurry packed under high pressure. First, the column is connected to a column packer (Shandon, U.K.) via a slurry reservoir (SOmm x 2-mm LD) by the use of a reducing union with finger tight nuts and graphite ferrules (Moritz et aI, 1996a. The column is then pressure tested to 300 Bar
using n-propanol. A slurry of reversed-phase silica in n-propanol (40mg/200!!1) is
prepared by sonication for 10 mins and then pippeted into the empty slurry reservoir, and rapidly packed into the prefilled fused-silica column at 300 Bar with
n-propanol as the packing solvent for 20 mins. Once packed, the fused-silica column is conditioned in SO % methano1!SO % water at 300 Bar for a further 20 mins
and then allowed to depressurize slowly. The fused-silica column is then carefully
dismantled and installed into the modified HPLC for gradient micro-high performance chromatography.
2.13
Modification of Standard HPLC for Operation as Capillary HPLC
Flow generation. To achieve accurate flow rates (OA-20!!I/min) and reproducible
gradient formation for microcolumn operation, a standard HPLC pumping system is used with a preinjection solvent split (l/16-in tee) installed in the flow line
that directs ~9S-99 % of the flow through a length (~ 300mm) of 0.07S-mm
LD. x 0.320-mm LD. fused-silica tubing to waste with the remainder directed to
the microcolumn. Fused-silica capillary columns are connected directly to a Rheodyne Model812S injector fitted with a SO!!1 sample injection loop. The flow rate
through the column was measured with a S!!l Hamilton syringe connected to the
exiting fused-silica tubing of the micro column with a teflon tubing union and
accurately timing the advancing meniscus. Once the flow rate was accurately
measured, it could be adjusted by either changing the length of the fused-silica
tubing on the split tee or by adjusting the primary pump flow rate (100-S00!!1!
min) to achieve low flow rates in the range of OA-20!!1!min.
UV detection. Column effluent detection is achieved by modifying the original
detector by replacing the standard flow cell with low volume (:::; O.S!!I) flow cell
with pathlengths of comparable size (S-10mm). This can be achieved with flow
cells constructed from either fused-silica tubing (LC Packings, Netherlands) or
fused quartz blocks (Hewlett-Packard, Germany). With the latter, these flow cells
can be directly installed into diode-array detectors (DAD) with performance levels of signal to noise ratios close to standard flow cells (Moritz et ai, manuscript
in preparation). For capillary LC/MS, the eluent from the UV detector was connected directly to the electrospray inlet via a SO-cm length of O.OS-mm LD. x 0.19
O.D. fused-silica tubing. This tubing, which replaced the standard stainless steel
electro spray needle, extended to the tip of the electrospray needle assembly.
43
tioned in place with a drop of preheated epoxy resin at the junction and heated
until the epoxy is set using a hot-air gun. The epoxy is preheated to minimize
movement of the epoxy into the column by capillary action. Once the epoxy has
set, the column is ready to be slurry packed under high pressure. First, the column is connected to a column packer (Shandon, U.K.) via a slurry reservoir (SOmm x 2-mm LD) by the use of a reducing union with finger tight nuts and graphite ferrules (Moritz et aI, 1996a. The column is then pressure tested to 300 Bar
using n-propanol. A slurry of reversed-phase silica in n-propanol (40mg/200!!1) is
prepared by sonication for 10 mins and then pippeted into the empty slurry reservoir, and rapidly packed into the prefilled fused-silica column at 300 Bar with
n-propanol as the packing solvent for 20 mins. Once packed, the fused-silica column is conditioned in SO % methano1!SO % water at 300 Bar for a further 20 mins
and then allowed to depressurize slowly. The fused-silica column is then carefully
dismantled and installed into the modified HPLC for gradient micro-high performance chromatography.
2.13
Modification of Standard HPLC for Operation as Capillary HPLC
Flow generation. To achieve accurate flow rates (OA-20!!I/min) and reproducible
gradient formation for microcolumn operation, a standard HPLC pumping system is used with a preinjection solvent split (l/16-in tee) installed in the flow line
that directs ~9S-99 % of the flow through a length (~ 300mm) of 0.07S-mm
LD. x 0.320-mm LD. fused-silica tubing to waste with the remainder directed to
the microcolumn. Fused-silica capillary columns are connected directly to a Rheodyne Model812S injector fitted with a SO!!1 sample injection loop. The flow rate
through the column was measured with a S!!l Hamilton syringe connected to the
exiting fused-silica tubing of the micro column with a teflon tubing union and
accurately timing the advancing meniscus. Once the flow rate was accurately
measured, it could be adjusted by either changing the length of the fused-silica
tubing on the split tee or by adjusting the primary pump flow rate (100-S00!!1!
min) to achieve low flow rates in the range of OA-20!!1!min.
UV detection. Column effluent detection is achieved by modifying the original
detector by replacing the standard flow cell with low volume (:::; O.S!!I) flow cell
with pathlengths of comparable size (S-10mm). This can be achieved with flow
cells constructed from either fused-silica tubing (LC Packings, Netherlands) or
fused quartz blocks (Hewlett-Packard, Germany). With the latter, these flow cells
can be directly installed into diode-array detectors (DAD) with performance levels of signal to noise ratios close to standard flow cells (Moritz et ai, manuscript
in preparation). For capillary LC/MS, the eluent from the UV detector was connected directly to the electrospray inlet via a SO-cm length of O.OS-mm LD. x 0.19
O.D. fused-silica tubing. This tubing, which replaced the standard stainless steel
electro spray needle, extended to the tip of the electrospray needle assembly.