Chromatography Forum

Dear All
Greetings!!!!!

i am using agilent HPLC for the related substances of ampicillin, since past 1 week i have observed that all of a sudden baseline is going in negative or positive then again will go in straight line......

is it the problem with D2 lamp or something else?


Pls help.

Regards
mohammad......

What model?
What detector?
What wavelength?
What mobile phase?
Isocratic or gradient?
Is the baseline excursion always "up and then down", or is it sometimes up and sometimes down?
Does the excursion occur at a consistent time in the run or is it random?

If this happens near the "dead volume", then typical. Unretained substances can make detector go up, and the solvent from the injection dilutes the mobile phase a tad which then has lower response than mobile phase alone, resulting in a negative.

In gradient, the new mobile phase may have lower or higher response than the mobile phase at which the zeroing occurred.

hello,

Make: Agilent
Model : 1200 series
Detector: DAD (G1315D)
Product: Ampicillin Trihydrate.
Wavelength: 254nm
Flow: 1.00ml/min (Gradient) as per EP 7.0.
Mobile Phase :
A: 0.5ml of dil. acetic acid, 50ml of 0.2M KH2PO4 & 50ml ACN, dilute to 1000ml with water.
B: 0.5ml of dil. acetic acid, 50ml of 0.2M KH2PO4 & 400ml ACN, dilute to 1000ml with water.
Run time : 65 min.

The baseline excursion is sometimes up & sometime down between 10 to 35 minutes & it occurs randomly...

Regards
mohammad

Hi Mohammad

This does sound like a detector issue in the absence of any other clues.
I would be tempted if you have ChemStation software or the hand-held controller to check the lamp hours and run a lamp intensity test and a cell intensity test. The lamp hours for a DAD lamp should be less than 2000hours, the lamp intensity test will give a pass/fail and the cell test should give a result ~0.6.

Hope this helps

That elminates a lot of possibilities. A lamp problem is certainly one of the remaining suspects. Another is intermittent flow problems. That can be confirmed or denied by recording the pressure and then going back to see if any pressure anomalies coincide with the baseline problem.

Actually seeing the chromatogram might suggest something else. Instructions for posting images are here: viewtopic.php?f=1&t=2617

i have chemstation software...........

Hi Mohammad

Try running the tests I mentioned (or ask the person responsible for the LC systems). They should be in the diagnosis section of the ChemStation software. The built in help gives all the details for running the tests.