Base line increase

[hammad]

I am analyzing the vegetable oil for glycerides estimation on HPLC - C18 column.
Solvent system: A-methanol. B (50% hexane + 50%Isopropanol).
Gradient of 0 min - 90%A + 10% B to 15min - 50%A + 50% B.
Detector is UV/Vis at 212nm (the only detector available in my Lab.)
At 5-6 min of run the baseline increase very much to no peak can be seen after this area.
Please guide/help

[Gerhard Kratz]

No Water in the Mobile Phase?????
What you see is probably the UV cut off from your B mobile phase. Maybe a higher wavelength than 210nm is recommended.

[hammad]

No Water in the Mobile Phase?????
What you see is probably the UV cut off from your B mobile phase. Maybe a higher wavelength than 210nm is recommended.

I doubt about the water contamination in isopropanol. The recommended wavelength is 205nm but below 210nm the UV/Vis detector gives "A/Z out of range error" therefore I am using 212 nm.

[tom jupille]

The nominal UV cutoffs for MeOH and iPrOH are both 205 nm. However:
- UV cutoff is the wavelength at which the absorbance goes to 1.0 AU, which is high for HPLC.
- The slopes of the UV spectra for those alcohols is fairly shallow in thst region, so that you can still have significant absorbance in the 210-215nm range.
- The quality of the solvents can vary significantly.
- Essentially, at 212 nm, you are on the ragged edge of usability.

If this were my problem, I would run UV spectra of both the A and B solvents to see what's going on. If the absorbances look OK, it may be time for a new detector lamp and/or flow cell cleaning and alignment.