Quick way to Manually Determine Gradient Composition Accuracy

Answer

This Information Applies To: Agilent LC Pumps

Issue

If a user suspects that the gradient composition isn’t accurate and causes poor chromatography such as retention time shifts, late, early, no peaks or merging peaks. This article provides a quick way to determine if the pump can accurately proportion the requested composition. The test is done by manually changing the composition and noting the responses.

Note: This procedure can only be done with a UV detector, not valid for non-UV systems.

Steps to Follow

1. Place 200 mL of LC-grade water into a clean reservoir. For a binary pump, place it in channel A1 and for a quaternary pump, use channel A.
2. Prepare 200 mL of a 0.4% acetone aqueous solution and place it into another reservoir. If using a binary pump, place it in channel B1. If using a quaternary pump, place it in channel B.
3. Set the detector wavelength to 265 nm.
4. If a column is installed, remove it, and replace it with a union.
5. Flush the system thoroughly with water.
6. Balance (zero) the detector and ensure that the baseline is flat and stable. It is important to patiently wait for a stable baseline and the detector may need to be balanced several times.
7. Set the flow at 2.0 mL/min and flush for about 5 minutes. The baseline must be flat and above zero. If not, manually balance the detector.
8. Change the composition to 100% acetone aqueous solution.
9. The baseline will rapidly rise when the acetone solution gets into the detector cell. Wait until it flattens and is stable. Note and record the absorbance. You can zoom on the plateau to determine the height. A good alternative would be to use the Agilent Instant Pilot to accurately determine the absorbance value. If the response is above 1000mAU, reduce the acetone percentage of the solution, and start again from step 2.
10. Now manually change to 50:50 water/acetone composition. The baseline will slowly decrease to an expected response of half the response from step 9. Note and record this response as well.
11. If the response at 100% of acetone solution was 1000 mAU in step 9, then the expected response in step 10 should be around 500 mAU. The composition accuracy for Agilent pumps may vary depending on the specific pump model.
12. You can repeat and test at other compositions, however, avoid testing at below 5% composition of either solvent as this test is not significant at those compositions.

If the test fails, ensure you have followed the procedure carefully then contact your local Agilent service representative.