HPLC Products

Normal

                                            

Problem                                                        Problem

Unlike the previous two sections, this can only result from variations in the injection volume from one injection to the next. The size of the variations may give a clue as to the cause.

No air gap set in the Autosampler Configuration. The sample is pulled into the autosampler tube by the syringe. But the syringe is a long way from the sample, and in between is a volume of wash solution. It is normal to have a small air gap between the sample and the wash solution to prevent diffusion and mixing. If no air gap is set, it is normal for there to be noticeable differences in peak height from one injection to the next.

Very Small Injection Volume. If injecting very small volumes such as 1ul, it is very hard for the autosampler to give good reproducibility. In this situation, some level of variation is normal, and it is only significant because it is now a bigger percentage of the injection volume. The solution is to use an internal standard.

Air Bubble in the Autosampler Syringe. An air bubble tends to move around and each time an injection is made, different volumes of sample are injected. Eventually the air bubble will clear itself. To preven air, ensure that sample vials are filled up to around 75% with sample, so no air can be drawn in, and ensure that the wash solution is properly degassed.

Blockage in the needle. This can be a total blockage, cause by a piece of rubber septum, but is can be a partial blockage. In this case, the injection volume achieved is at best imprecise. The needle must be cleared (using needle wire to push the blockage out the end it came in) or replaced.

If the first injection is always smaller than the second, but after that they are consistent. This is caused by the needle being washed before each sample rather than before each injection. For the first injection, the needle is wet with wash solution, but for the second it is wet with sample, giving a slightly larger peak. To stop this, the needle should be washed before each injection, rather than before each sample.

If the first injection is always bigger than the second, which is bigger than the third etc. This is caused by the syringe in the autosampler becoming old and unable to pull against a vacuum. If the vial septum is resealing (eg rubber or silicone septum) and the vial is full, the vacuum in the vial increases with each injection, resulting in less and less sample being withdrawn. To test for this, run with a PTFE disc as septum, and th problem should go away. To stop the problem, either use PTFE septa all the time, or replace the syringe, or try filling the vial only 75% full. This should be more than enough, and the reduction in pressure from one injection to the next should be much less.

Sample Degradation. This indicates that sample degradation is excessive. To test, inject the same sample over and over. There is no immediate solution but to redevelop the method, using a faster run time, chilled sample tray, possibly amber vials, and a small number of samples. A clue to this will be a curve instead of a straight line for the calibration. The highest sample will be run last, and will experience the most noticeable degradation.

Tray Chilling without allowing samples to attain the chilled temperature. The temptation is to set the tray to 4oC, load the samples, press Start and go home. However as the tray chills, the volume decreases, which increases the concentration, and hence the peak height increases. If using a chilled tray, allow enough time for the samples to acquire the new temperature. To establish how long, load the tray and make injections every 10 minutes until constant peak heights are obtained.