Chromatography Forum

I work in a small university lab and I have recently set up a new HPLC. I am trying to write a procedure to fully qualify my instrument. I would like to know how others qualify their gradient pumps. My thought is to use some type of flow restricter in place of the column and then to measure the absorbance change as I change the mobile phase ratios. However, I'm not sure of the specifics such as what to use for a flow restrictor, best mobile phase choices, etc. Any help would be greatly appreciated.

Thanks,

Karen

Any length of small bore tubing should do - a meter (or less) of 0.005mm ID peek, for example. There are also back pressure devices available from any number of LC accessories vendors.

The flow restrictor sounds like a good idea. Could then add some acetone to the organic phase, run the gradient and monitor an appropriate absorbance for acetone. Will show if gradient performing properly and the offset in the gradient requested vs that actually noted showing the dead volume in the system.

Dionex sells a kit for instrument OQ/PQ. Unfortunately, it assumes you are using Dionex software. The general process for gradient qualification is:
A= water
B= 0.1% acetone in water
UV 265 nm
Flow 2.0 mL/min
Restrictor 13 meter of 0.13 mm i.d. tubing producing about 80 bar
Gradient steps at 100% A, 99% A, 50% A, 1% A, 0% A holding for 4 min each.

Run gradient in triplicate. Compare expected versus observed absorbance.

Of course this requires the detector to first be qualified as linear, which requires the injector to be qualified as reliable, which requires certified standards. See why it is a kit?

Other vendors have similar products that are agreeable with their software packages. Ask your local representative.

the 2 major parameters that you must check in the pump are the flow rate and the gradient composition.

you cannot check the gradient composition if you have not checked the flow rate first.

the flow rate is generally checked with water for easier calculations.
you can check at more then 1 flow rate and more then once each rate but that depends on your decision. one check is also acceptable. As follows:

flow rate: 1 ml/min (05 ml/min and 2ml/min as well if you wish to check flow rate range).
solvent: water

turn the pump on for at least 10 min. collect the water coming out of it. after ten min. have past weight the amount of water collected and transfer it volume units.
acceptable specifications are +- 2%

after the flow rate check move on to the gradient check. in this check you must make sure that you check the proportions for all channels. this check is very varied in the way it is performed. but the most important parameters to check are the minimum gradient change and also around 50% composition (many pumps show a weakness between 45 to 55% change)

proceed as follows:

solvents:
channel a: water
remaning channels: 1% acetone in water, or 1% caffeine, or 1% methylparaben. all are used for this procedure.

UV 250 nm.
Flow 2.0 mL/min
Restrictor 13 meter of 0.13 mm i.d. tubing producing about 80 bar

set the gradient program as said by Mark Tracy, just make sure to test channels C and D if it is applicable for yur pump. remember you must check all channels.
As with the flow rate check, it is acceptable also to check all the parameters once and not sevral times. this is your decision if more then one test is necessary for each procedure. don't forget that all of this is also time consuming.
acceptable specifications for the gradient test are +- 1% for each composition compare to 0% A.