Calibration of HPLC equipment

[oscarBAL]

Hello
I need to know the procedure to calibrated an HPLC equipment. I do not refer to the validation procedure. Thanks for your help!!!!!

[philippem]

Hi oscar ,
what do you want to calibrate: flow , temperature, injection volume???
do you have citeria to test against ?
calibraton is done against a reference

philippe

[wanda50]

If you have never calibrated your instrument, it might be worth your while to have an external company perform the first one.

As an alternative, here are the basic experiments. Some instrument manuals and/or repair manuals might have these in more detail.

Pumps:
flow: pump thourgh a column at 1.5 mL/min, collect effluent for 30 min. Criteria wil be +/- a few tenths of a mL.
Dwell volume - this was discussed earlier this month on this forum.
Gradient - water with 3% acetone and plain water. Run gradient with specific jumps. measure heigth and compare to water "control".

Detector: although there is a procedure with potassium dichromate, we prefer the caffiene method. You make readings at specific wavelenghts and do linearity.

Injector:
Reproducibility and linearity - inject a test mix at 5 volumes to perform lineaterity. make 10 shots at one of the volumes for RSD.

Heater:
measure temp at at least one value. criteria is +/- the desired temp.

I did not give ranges and criteria since your ocmpany has to decide what is allowable.

[juddc]

Have a look at the specifications for your instrument(s), then use those as guidelines for what to do. Many manufacturers also publish IQ/OQ/PQ procedures with acceptability criteria for their machines.

For UV detector wavelength accuracy, I find that erbium perchlorate works well.

As an aside, I just ran an abbreviated OQ and full PQ on one of my systems today after one of the valves in the injector was replaced and was delighted to find that the linearity and repeatability results were actually better than those from when the instrument was first purchased ~18 mos / 9000 injections ago: Repeatability % RSD's were in the 0.15-0.30% range and the R-squared for the injector was between 0.999992-1.000000 for all of 4 analytes.

What I found somewhat amusing was the apparent fact that for a 10-ul injection volume, there was no precision advantage in switching to a 25-ul syringe from a 250-ul unit. I ran syssuits for both on the same PQ sample and % RSD's were unchanged. The average peak areas for all analytes were within ~1% of one another regarless of which size syringe was used, too.

I guess I can go forth an validate my method in peace and tranquility...

[Alfred88]

Dear oscarBAL:
If you are asking about PQ procedure for HPLC, you may search for old inquiry in this forum (Perhaps under FAQ).

I just want to add two references here. The first one is truly a good ref.

1. Performance Qualification of LC Systems. Hall, Gerald and Dolan, John. LC Resources. October 2002. http://www.lcgceurope.com/lcgceurope/da ... rticle.pdf

2. Committeeing to Calibrate. Michael Dong. ACS. Feb 2001, Vol. 10, No. 02, p 42-44. http://pubs.acs.org/subscribe/journals/ ... 2dong.html.

What is the brand of your LC's?

Alfred.