URGENT help needed in negative peak in HPLC chromatogram

[clint]

hi, i recently work on beta-carotene in palm oil
i have encounter a problem in my work that is negative peak occur at the initial state(about 2-3 minute)of the peak...

mobile phase: ACN 75%, MEOH 20%, dichloromethane 5%

diluent: acetone

an aditional question: will trace amount of water remain in the sample cause negative peak??

Any help is appreciated!

Thanks

[John]

Other method parameters please; Column etc.
Could well be water. Try injecting a small amount of water and see where it elutes.
J

[dave.cheng]

You need add main information: detector wavelength!
I remember that the UV wavelength is 340nm for beta-Carotene. the negative peak mainly produced by setting mistake detector wavelength.
good lucky!

[clint]

thank a lotz john and dave for your kindly help...
the column i use is C18, 250 x 4.6mm...
although the recommended is C30 but it is not available in the lab...
and the wavelength i use is 450nm because beta-carotene show show the highest absorbance in spectrophotometry (from previous article)...
thx again 4 ur help

[ym3142]

if the flow is 1ml/min, then your void is around 3 and the neg you saw is the front.

[clint]

thx ym3142....
my flow rate is 1ml/min and the negative peak come out arround 2.5 minute...

[Mark Tracy]

That is indeed the void peak. If you have an important component that elutes that close to void, you need very much to improve your method. Otherwise, you will be cursed with problems just like you have described. For this mobile phase, you should be using the highest %C type of column you can find; that is why the C30 was in the original method. If you really are stuck with only the columns available in your lab, you will need to adjust the mobile phase so that the earliest peak of interest comes out no earlier than 5 min.

[clint]

thx mark....
i will try to run several sample today to see if the if it work..

[clint]

hi.....
today i find out that the negative peak in my chromatogram may be due to the diluent i use...acetone...
because when hplc analysis of acetone also show the negative peak at the same retention time...