I'm running HPLC and I'm using methanol to wash the column. Usually the absorbance should be around 0. Last time, it goes up to 10. This time, it's around 25. I don't know why although it's very stable. We had another problem for HPLC and I'm not sure whether they are related. The injector was broken three months ago and one of our PhD student replaced it by himself. Our sample loop is 10uL, when I injected 30uL, I should see liquid coming out from the back tube. However, I never saw the liquid coming out from the back tube. Instead, sometime I will see the liquid coming out from the hole of the injector when I inject the samples.
You need a stronger solvent to wash your column. Try unbuffered 95% Acetonitrile. I am going to suggest the student inserted the wrong loop into the injector (sounds 100 uL or larger). Solvent should not come out of the injector (something wrong with gasket).
I see a fairly stable baseline around 30 units of abs with no noise and no drift, that could be only a problem of (auto)zeroing it. Could you tell me what happens when you leave it run with methanol for long time like half an hour or more? It drift up?
Concerning the High base line, three things can be involve: Contamination in mobile phase, Mobile base not in Equilibrium with the column and Contamination bleed in the system.
1. CONTAMINATION IN MOBILE PHASE
If you are using a reversed-phase method, you are likely to use an aqueous mobile phase with methanol, acetonitrile or tetrahydrofuran (THF) as the organic modifier. For any one of the organic solvents you need to make sure that they are HPLC-grade. If other grades are used, they often contain small amounts of miscellaneous chemicals that can influence the detector-background of the solvent (especially for VWD). But there are other ingredients of reversed-phase mobile phases that can create background problems. In many reversed-phase methods, buffers are used to control the pH of the mobile phase. Most inorganic buffer salts like phosphate are of sufficient purity that an increase in the UV background is not very likely.
2. MOBILE BASE NOT IN EQUILIBRIUM WITH THE COLUMN
For this all you need to do is to change either the solvent or the column in used.
3. CONTAMINATION BLEED IN THE SYSTEM
You need a stronger solvent to wash your system. Try unbuffered 95% Acetonitrile as suggested by Bruce Neagle.
Concerning the sample injector problem, two things can be involve: Wrong sample loop or Blockage of the sample loop.
1. WRONG SAMPLE LOOP
As suggested by Bruce Neagle, The student might have installed a wrong sample loop. So you may need to confirm the volume capacity of the loop and change if wrong.
2. BLOCKAGE
The sample loop might have been block by debris. For this you might need to get a syringe with higher volume capacity (may be 5ml) and inject a solvent or blank under high pressure and see if the loop will be through or not. If not the you might need to change your sample loop.
What is the model of you HPLC (a snapshot will be highly appreciated)?
Thanks
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