I am using a gradient method consisting of phosphate buffer and Acetonitrile/H20 as the mobile phases. The column is a Hypersil ODS. The run time of the method is about 45 min. The retention time of the analyte in subsequent injections is always decreasing by about 0.6% from the previous injection. The retention time never stabilizes. The column is being thermostatted. Any comments?

Questions:
Is this a new or established method?

Is the re-equilibration time sufficient?

Are you possibly leaving something on the column with each injection or with a contaminated mobile phase? To find out, wash the column with a strong solvent and see if retention is restored.

Is your pump OK? How about your gradient proportioning valve?

Are your peak areas constant?

Is your analyte neutral? (I hope so w/ an unbuffered MP)

Are you absolutely sure there is no temperature variation? How far above /below ambeint are you running? How are the temperature fluctuations in your lab?

Fill us in and we can help alot more.

pH?

This is a fairly new method.

I believe the re-equilibration time is sufficient. It's 1.5 times the column length.

Pump and proportioning valve are working properly.

Peak areas are constant.

The phosphate buffer is pH'd to 3.0.

The column is thermostatted at 30C.
The temperature in the lab does not fluctuate much.

Re-equlibration time should be 3X System volume plus 5X the column volume....

Re-equilibration time is probably fine if you're flowing at 1 ml/min on a 4.6mm column. It's actually re-equilibration volume in relation to the column volume that's important .

I think you may be hanging some unknown material up on the column, which is causing the column to be less retentive. I've seen this before many times...What does it do when you rinse the system with a strong solvent. Is there any difference in behavior between samples and standards?