I've just recently read an article about the importance of priming injections and how it minimizes the first injection from having either a large or small peak areas from the subsequent ones. However, I don't understand why would the last injection produce the lowest or higest area? The last injecion could be 10 to 30% higher or lower than the previous injections these variations affect system suitability. Can you explain what is happening?

My opinion is that if you need to use priming injections you should probably go back the drawing board and figure out what exactly is wrong with the method and correct it. Peak areas that get larger or smaller with succeeding injections generally indicate some type of adsorbtion onto the column packing material, although I have seen this phenomenon occur due to drastic differences between the sample solvent and mobile phase. Also, there could be mechanical causes (e.g. air in the injector lines) that causes the first or second or third injection, etc. to have a significantly different area than the others.

Priming injections should be a last resort to obtain acceptable results with a problematic method and, if required, their use should be justified on the basis of experimental data. I certainly wouldn't recommend using them with every method as a matter of course.

Michael Hinsberg
Hinsbar Laboratories, Inc.
www.hinsbarlabs.com

The phenomenon of increasing response with injection number in not unfamiliar to many HPLC users in terms of "carry over", where a residue from the previous injection on the needle or transrer tubing causes an increase in the response of a component in the next injection. This will usually be seen (where it occcurs) in related substances tests where the difference between sample and standard concentrations is at least an order of magnitude.

The increase in a component's response with respect to injection number is not particularly dificult to explain. However... I am currently trying to transfer an assay method from a third party to my company where the peak area response of one of the two components decreases with respect to injection number, until a steady response in obtained around injection 7 - 8. On leaving the HPLC system overnight, the same pattern is observed then next day. Can anyone explaine this? There's a beer in it for the most convincing explanation,(honest!).

Response to Dave about decreasing response with respect to number of injections

The explanation to your problem is probably that the decreasing components molar absorption is extremely metal sensitive and the other component is not. The component form metal complex with metal (iron-)-ions leaking from the HPLC-system. Every injection washes metal-ions out of the system, and as you wrote after 7-8 injections the leaking of ions is constant resulting in a steady response.
To check this theory you could after a nights shot down of the system inject 20-100 ul of a 1% EDTA solution in water. Observe the EDTA peak and inject the EDTA solution several times until you see a steady response for the EDTA peak , decreasing or rising area of the EDTA peek indicate that loose metal ions is removed from the system, and steady declining respons indicate that the system gets cleaner after every injections (the retentiontime of the EDTA peek will probably be 1,5-3 minutes).
Next inject the component of interest and see if the response still decreases. If you get a steady response at once the theory is right.
If the theory is right you might solve the problem by adding 100-200 mg EDTA ( or another metal eating agent) to 1 liter of mobile phase.


lej@.nycomed.com