HPLC Products

Normal

Problem

To be clear, the problem we are describing here is when we run a standard, where we already know the concentration, and the peak heights are all smaller than we expect; smaller than we would normally get.

The first question is:

Is the void volume peak smaller too, or is it its noraml size?

If the void volume peak is smaller too, the answer is that for some reason, we injected less sample.

There are several possible reasons for this:

A smaller loop was fitted. This only applies to manual injection valves using filled loop injection, and to fixed volume autosamplers, where the injection volume is the loop volume. Clearly if you work on your own and don't suffer from Alzheimers Disease, you would hopefully know if you changed the loop. But in a multi-user lab, it can happen.

The waste outlet pipe from the valve might be partially blocked. This restricts the flow through the loop, and results in a smaller than expected injection volume. The waste pipe may need to be replaced if blocked.

For a manual valve, it may be that the user is not injecting at least three times the loop volume. See the Rheodyne website or the valve instructions for a discussion on this. Eddy Diffusion is the culprit. At least 3 x the loop volume must be injected for filled loop injection.

Again for manual valves, the needle may not be engaging in the PTFE sleeve in the rotor seal, and sample is leaking back around the needle.

The valve may not have been turned completely to the load position. Hence relatively little sample can pass through the loop.

Finally for a manual valve, the valve may not have been mounted horizontally, or the waste pipes may be ending lower than the valve body. See the instructions. A valve must be mounted horizontally, and if the waste pipes hang don from the back of the valve, a syphon action pulls air into the valve and allows the sample to drain out.

Air in the autosampler syringe. This can come from insufficient sample in the vial, or from not degassing the wash solution. Run the wash routine a few times to clear the air.

Restriction in the tubing from the needle to the valve or the valve to waste. It is common for autosamplers to use PTFE tubing because of its flexibility, which helps when the needle must move long distances. However if it becomes snagged and is pulled, it will stretch quite a lot and become thinner, restricting the liquid flow, and resulting in very small injections.

Blocked needle. An autosampler needle can suffer from coring, where some of the septum becomes lodged in the needle. This restricts flow, giving small peaks or no peaks.

Sample level too low in the vial. If not enough sample is present or the wrong type of vial is used, the needle struggles to pick up the sample, and air is sucked ionto the line. If more sample is available, fill the vial up to 75% full. If very little sample is available, use smaller vials or vial inserts.

Needle depth set too high. The autosampler needle depth is settable from the method to allow for the use of different vials, or the use of vials and inserts. If the needle depth is set too low, it makes a hole in the bottom of the vial. But if it is too high, air is drawn into the syringe.

If the void volume peak is normal size, then a normal injection volume has been made, and we are looking for a reason for a lower sample concentration or lower detector response.

Incorrect sample dilution. This error could have come from the weighing (usually the error will be a factor of 10) or from making the sample up in 100ml instead of 10ml etc. If it is a small difference in peak size, suspect that the dilution was made when the lab was cold. Hence more sample solvent will have been used to make up to the mark.

Sample degradation. If this is the answer, it may be an old sample. But otherwise the method may need to be changed to allow for this. Samples should then be stored in the fridge, possibly in amber flasks, and a stability trial undertaken to quantify the rate of degradation and establish a shelf life.

Incorrect wavelength used. This can cause some peaks to be smaller and some to be larger, But it could result in all peaks being smaller.

Incorrect Range setting. eg 2 AUFS instead of 1AUFS. This tends to give peaks which are smaller by a factor of 2.

Lamp Energy. If the lamp is old and losing energy at an appreciable rate, the peak size will decrease noticeably, and at an increasing rate. The clue will be a very noisy baseline, often with spikes.

Changed flow cell path length. Suspect this if the flow cell has been changed. The longer the pathlength, the larger the peaks, the greater the low end sensitivity, and the lower the maximum concentration which can be used.

10mv Recorder channel connected instead of 1V Integrator Channel.