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HPLC -problem with solvent peak


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20 replies to this topic

#1 mimosa

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Posted 20 July 2009 - 08:36 AM

Hi,

i got problem with HPLC.
my sample was dissolved in hexane and this solvent was came out in the middle of those compound.
is that possible the solvent peak come out in the middle of analayze compound?
but the peak separation is okay. the only problem is the solvent peak!

thank you very much

#2 nightingale

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Posted 20 July 2009 - 09:57 AM

hello Mimosa,
what i know is that the solvent peak appears at the beginning of the run...
what are the remaining compoonds ???
and what's the coloumn used ???
" The more you learn, the more you realize how little you know ... "

#3 swanny

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Posted 20 July 2009 - 04:17 PM

Hi,

i got problem with HPLC.
my sample was dissolved in hexane and this solvent was came out in the middle of those compound.
is that possible the solvent peak come out in the middle of analayze compound?
but the peak separation is okay. the only problem is the solvent peak!

thank you very much

Try injecting solvent alone and see where it comes.
Heart disease kills more women than breast cancer, but heart attack symptoms differ from men's symptoms. Get to know your heart... it could save your life.

#4 T C

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Posted 21 July 2009 - 01:03 AM

Hey,

I doubt if its the solvent peak. Did you wash the column nicely before using...maybe some left over by the previous user or some reaction product [something new ;)]. However, running the colvent alone as Swanny suggests will resolve the issue.

Best,
TC

Hi,

i got problem with HPLC.
my sample was dissolved in hexane and this solvent was came out in the middle of those compound.
is that possible the solvent peak come out in the middle of analayze compound?
but the peak separation is okay. the only problem is the solvent peak!

thank you very much



#5 mimosa

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Posted 21 July 2009 - 06:56 AM

thank you for u all suggestion.

i injected and confirm that peak was solvent peak.
i used hexane to dissolve sample and the mobile phase is methanol.
i figure out that hexane not miscible with methanol.
i wondering that the solvent peak in middle of those compound will affect the quantitation?

thank you very much

#6 mdfenko

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Posted 21 July 2009 - 08:04 AM

i wondering that the solvent peak in middle of those compound will affect the quantitation?

it probably will. integration software will "solvent skim" to try to offset the effect of a solvent peak but the solvent peak and the sample peak shape will have to be consistent from one run to the next.

talent does what it can
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i used to do what i got paid to do


#7 DRT

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Posted 21 July 2009 - 07:05 PM

Is there anything stopping you drying off the hexane and redissolving your sample in methanol (or at least something more compatible with your mobile phase)?

#8 mimosa

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Posted 22 July 2009 - 05:01 AM

why the solvent peak in middle will affect the quantitation?
is that better the solvent peak elute at the beginning?

i try to dissolve the sample in methanol.
but the sample not totally dissolve because i could see the fat globule.

thank you

#9 mdfenko

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Posted 22 July 2009 - 11:59 AM

why the solvent peak in middle will affect the quantitation?
is that better the solvent peak elute at the beginning?

i try to dissolve the sample in methanol.
but the sample not totally dissolve because i could see the fat globule.

thank you

sorry, i thought you meant that the solvent peak came out with the other components (rising from the solvent peak). if the solvent peak is discrete then your quantitation will be unaffected (unless you are looking at percent of all peaks eluted, then i would turn off integration during the solvent elution).

what would worry me is that the sample is not completely solubilized by the mobile phase (methanol). i would expect the fat globules to form in the column as they separate from the hexane during the run.

can you run the analysis in hexane instead of methanol or, at least, add hexane to the mobile phase?

talent does what it can
genius does what it must
i used to do what i got paid to do


#10 mimosa

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Posted 23 July 2009 - 06:30 AM

hexane can use as mobile phase for reverse phase column?
because i using c8 column

#11 T C

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Posted 24 July 2009 - 12:56 AM

I guess no...it may affect the column. Look at the column manufacturers guidelines. Besides since it absorbs, you would have very high background and won't help in yr case.

Best,
TC

hexane can use as mobile phase for reverse phase column?
because i using c8 column



#12 mdfenko

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Posted 24 July 2009 - 01:58 PM

i wouldn't use it alone but in combination with methanol and/or acetonitrile and/or water and/or chloroform or some other combination that allows you to separate your components without any of them coming out of solution.

talent does what it can
genius does what it must
i used to do what i got paid to do


#13 mimosa

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Posted 24 July 2009 - 10:13 PM

why the solvent peak in middle will affect the quantitation?
is that better the solvent peak elute at the beginning?

i try to dissolve the sample in methanol.
but the sample not totally dissolve because i could see the fat globule.

thank you

sorry, i thought you meant that the solvent peak came out with the other components (rising from the solvent peak). if the solvent peak is discrete then your quantitation will be unaffected (unless you are looking at percent of all peaks eluted, then i would turn off integration during the solvent elution).

what would worry me is that the sample is not completely solubilized by the mobile phase (methanol). i would expect the fat globules to form in the column as they separate from the hexane during the run.

can you run the analysis in hexane instead of methanol or, at least, add hexane to the mobile phase?


the sample will solubilized in fat or not?i running the fat soluble vitamin analysis.which solvent better used to dissolved besides hexane?
because the hexane will shorten the column life.

#14 mdfenko

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Posted 31 July 2009 - 10:26 AM

the sample will solubilized in fat or not?i running the fat soluble vitamin analysis.which solvent better used to dissolved besides hexane?
because the hexane will shorten the column life.

we ran fat soluble vitamins on a c-18 column. i can't get my hands on the paper at the moment but the reference is:

Malik, M.N., M.D. Fenko, A.M. Sheikh and H.M. Wisniewski, “Isolation of a-Tocopherol (Vitamin E) from Garlic”, J. Agric. Food Chem., 45, 817-819 (1997)


it includes the extraction technique, solubilization, and the hplc conditions for fat soluble vitamins.

talent does what it can
genius does what it must
i used to do what i got paid to do


#15 K.B.

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Posted 01 August 2009 - 12:21 AM

HPLC method for tocopherols I've found for my buddy uses C18 column and isocratic elution with mobile phase of 25:22:3 (v/v/v) methanol/acetonitrile/methylene chloride. After the extraction tocopherol is dissolved in hexane, then is exaporated to dryness and dissolved in mobile phase.




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