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Chromatographic Interfacing
Electrospray ionisation can be routinely interfaced to reversed phase and normal phase
chromatographic separations. Depending on the LC pumping system, chromatography
column and setup, there are some basic options:
•
Microbore and capillary chromatography separations employing 1 mm diameter
(and smaller) columns can be interfaced directly to the electrospray probe.
Typical flow rates for such columns may be in the region of 3-50 µl/min. It is
suggested that a syringe pump is used to deliver these constant low flow rates
through a capillary column. Alternatively, accurate pre-column splitting of
higher flow rates from reciprocating pumps can be investigated.
In all cases, efficient solvent mixing is necessary for gradient elution separations.
This is of paramount importance with regard to low flow rates encountered with
capillary columns. HPLC pump manufacturers’ recommendations should be
heeded.
•
2.1mm diameter reversed phase columns are gaining popularity for many
separations previously addressed by 4.6mm columns. Typically flow rates of
200 µl/min are used, allowing direct coupling to the electrospray source. The
increased sample flow rate requires increased source temperature and drying gas
flow rate.
A UV detector may be placed in-line to the Quattro Ultima probe. However,
ensure that the volume of the detector does not significantly reduce the
chromatographic resolution. Whenever a UV detector is used, the analog output
may be input to MassLynx NT for chromatographic processing.
•
The interfacing of 4.6mm columns to the electrospray source can be achieved
either by flow splitting or by direct coupling. In both cases an elevated source
temperature and drying gas flow rate are required. In general, the best results are
obtained by splitting after the column using a zero dead volume tee piece so that
200-300 µl/min is transferred to the source.
Conventional reverse phase and normal phase solvent systems are appropriate for
LC-electrospray.
Involatile buffers may be used but prolonged periods of operation are not
recommended. When using involatile buffers the probe should be moved as far away
from the sample cone as possible. This may reduce sensitivity slightly, but also
reduces the rate at which involatile material is deposited on the sample cone.
Trifluoroacetic acid (TFA) and triethylamine (TEA) may be used up to a level of
0.05%. If solvents of high aqueous content are to be used then tuning conditions
should be appropriate for the solvent composition entering the source.
Higher source temperatures (150°C) are also recommended for high aqueous content
solvents. Tetrahydrofuran (THF) should
not be used with peek tubing.
Electrospray
Page 157
Quattro Ultima
User's Guide