If we are asking an autosampler to inject the same sample several times to check reproducibility,
we would expect that an automated machine which just runs programs should
be able to do this fairly easily. However sometimes this is not the case. Before blaming the
autosampler, do check that its not an air bubble in the pump, or integration problems. But
assuming the problem lies within the autosampler, here’s a few places to look:
Not enough sample. The needle barely dips into the sample, so air is drawn in.
Needle depth too high. Same as above.
Blocked needle. It causes a resistance to flow, and varying amounts of sample get in
each time.
Sample too viscous, or syringe speed too fast for the sample viscosity. The syringe
generates a vacuum and the sample follows more slowly. Slow down the syringe
speed, or use a less viscous sample solvent.
Worn syringe. If the syringe cannot suck hard enough, and resealing vial septa are
being used, there is a reduction in pressure in the vial after each injection, making it
progressively harder for the autosampler to withdraw sample. Replace the syringe,
or use PTFE septa.
Air in the syringe. It can only meter sample accurately in the total absence of air. See
the earlier section on dealing with air in the syringe.
Wash routine. Sometimes it is found that the first injection gives a lower result than
subsequent injections, and the reason is that for the first injection, the syringe starts
wet with wash solution, but for subsequent injections it is wet with sample. If this is
a problem, the solution is to wash before each injection, not just before each sample.
Sample solvent evaporating. This occurs if the sample solvent is volatile, and PTFE
septa are used (they do not reseal) allowing solvent evaporation between injections.
Hence the concentration of sample is gradually increasing, so the results will increase
with each injection (unlike the previous case where only the first injection is
low).
Sample not down to temperature. If working at (eg) 4oC, the samples must be allowed
to reach temperature before the run is started. Reducing temperature reduces
the volume, and hence increases the apparent concentration in a 20ul injection. It
can take at least 30 minutes for sample temperature to stabilise.
No air gap left between sample and eluent. When a sample is withdrawn from the
vial, it is kept separate from the eluent by a tiny (1-2ul) air gap. Failure to do this
allows it to diffuse into the eluent that is pushing it around the autosampler, and
this causes a variation in peak height from injection to injection.
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