Decrease in Area of Peak in HPLC

[chandusabini]

Dear,

I have developed an HPLC method in normal phase (80:20 ACN:IPA) which is isocratic and column is YMC diol(150mm x 4.6mm, 5µ). On of the impurity is eluted at negative peak, when i ran to gradinet program, main peak response was get decresed on increasing RT(in oncase it was disappeared).

1.Please clarify that why peak response was decreases.

2. How avoide negative peaks

regards
Chandu

[JGK]

Sounds like that in a gradient mode your column may not be equilibrating fully prior to the next injection.

[vicou_chen]

At first ,the description of your question is very hard to be fully understood
Based on my understanding,you might encounter the problems,such as undissolved baseline,aging occurrence in your detector and so on

[tom jupille]

A bit more detail would help:

What detector? if UV, what wavelength?

[mardexis]

And adding a bit to Tom's query: if UV then what wavelength
but also, what is the reference mehtod or wavelength?

On the Agilent systems the absorbance is ususally measured at two wavelengths, the first being the measured wavelenght and the second being the reference which is usually at 360. If your compound absorbs more at the reference wavelength than the primary wavelength then the peak will be negative.