Chromatography Forum

hi, I have worked 2 year in Pharmaceutical industry. my question is ( can ı test my hplc for gradient test in house application.) ı read some articles about gradient performance test; for example %0.5 acetone in channel A and %100 H20 in channel B; 265 nm, 2ml/dk.)

thanks for your answer.

Yep sounds good to me

Usually you'd have the low absorbing mobile phase (water) as A and the high absorbing (water + acetone) as B.
Gradient test might consist of running a step gradient from 100% A to 100% B (e.g. 0%B, 10% B, 50% B, 90% B, 100%B, 0% B again; be creative ) and measuring the plateau heights. Decent HPLC systems should get +/- 1% accuracy.

Call FOX for that..[ just kidding]

Usually you'd have the low absorbing mobile phase (water) as A and the high absorbing (water + acetone) as B.
Gradient test might consist of running a step gradient from 100% A to 100% B (e.g. 0%B, 10% B, 50% B, 90% B, 100%B, 0% B again; be creative ) and measuring the plateau heights. Decent HPLC systems should get +/- 1% accuracy.

Thanks. but what is my reference value %100 acetone (container B) plateau of height?
the answer is ok example my %100 B (ACETONE) plateau of Height is 101 cm=%100 B ( OR %100 HEİGT)
İS İT CORRECT WAY TO SOLVE THİS?

AND MY SECOND QUESTİON İS DWEEL VOLUME OF HPLC HP 1200 SYSTEM?
HOW TO MEAUSURE İT ?

THANKS.

Here's the procedure from our web site: http://www.lcresources.com/resources/TSWiz/hs410.htm

Gradient tests are typically used as part of instrument qualification. They are used to demonstrate several aspects of the system such as relative pump flow, composition accuracy, gradient linearity, dwell volume, high absorbance noise effects, etc. The purpose is generally to test the relative volumetric accuracy of multiple pumps in a binary pump or the low pressure mixer performance in a quaternary pump.

Pointers
You will want to run any of these tests with the same solvent in both sides; one doped with 0.5% acetone. Don't use different base solvents or you will face non-linearity due to reduced mixing volumes and refractive index differences of the binary solvents. The abs max of acetone is 273 nm which will give about 1 AU for a 10mm cell at this concentration.

If you are using water as the base solvent set the compressibility compensation for water on both pumps. [many binary systems come with the left pump set for water and the right set for an organic [about 2-3x the compression per stroke]. This will slightly alter flow rates.

I have had some irregularity running the acetone doped solution through a degasser. Just sonicate for a few minutes, plumb directly to the pump head and results will be fine.

Run gradients without a column.

Flow equivalence requires first running gradient test with absorber in pump 1 then running it in pump 2 and comparing results.

The step gradient described by mirac_han gives compositional accuracy. Plot the slope of each plateau vs a 100% water blank. Correlation > 0.999x and intercept appropriately near zero. Also can compare each step level to absorbance of 100% absorber level to get accuracy per step. Gradient plateaus can be evaluated fro detector noise at various background absorbances.

Finally a linear gradients are run from 0% to 100% at user speced conditions. Linearity is generally from 5 points taken from the absorbance plot [e.g. 10,30,50,70,90%]. Correlation coeff > 0.999x.

Tom Jupille has described dwell volume.

Good luck

I am performing a gradient composition test on my HPLC and unfortunately my absorbance ratios are incorrect. I am trying for 100%, 90%, 50%, and 10% using Water on Line A and 30mg/L Caffeine on Line B, but I am getting 95%, 57%, and 5%. I have sonicated the check valves, there are no clogs in the lines, the pressure is fine, and I've replaced the Mobile Phase twice. Any suggestions?

when you say the pressure is fine, what is it? And what hplc are you using? Some, particularly low-pressure-mixing (typical quaternary) systems, don't pump accurately unless they are pumping against a reasonably high pressure.

You've probably done this already, but make sure you purge the lines really, really thoroughly before you do the test. Also your concentration of caffeine looks sensible to me (I can't remember its molar extinction coefficient, I'm just guessing!) but do make sure that you're working within the linear range of your detector. I don't think this is your problem because the errors aren't behaving as they would with a systematic deviation from linearity at high conc.

The document at the below link shows the conditions and set-up used for an Agilent system

http://www.micannalytics.com/validation ... istics.pdf

To answer your question I am using a High Pressure quartenary pump. I've repeated the test multiple times going from 0-100 and 100-0 and I cant seem to fix the problem. Any suggestion you have would be helpful.

I meant to mention my pressure is about 200 psi.

Once you have eliminated the "common" causes (e.g., air bubbles, clogged inlet lines, etc.), on a low-pressure-mixing system (which is what you have if it will do quaternary gradients), the remaining possibilities are:
- the gradient proportioning valve (sticking?)
- the compressibility compensation system (faulty transducer, or incorrect settings in the software).

Thanks! I will check those.

I couldn't find anything on a "gradient proportioning valve" but this is what I have in my system http://www.environmental-expert.com/pro ... w-comments . Could this be the problem?