HPLC -problem with solvent peak - (Jul/20/2009 )
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
mimosa on Jul 23 2009, 09:00 PM said:
because i using c8 column
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.
mdfenko on Jul 23 2009, 03:59 AM said:
mimosa on Jul 22 2009, 09:01 AM said:
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.
mimosa on Jul 25 2009, 02:13 AM said:
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.
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.
Hi mimosa...You may also wish to visit Chromatography Forum
mdfenko on Aug 1 2009, 02:26 AM said:
mimosa on Jul 25 2009, 02:13 AM said:
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.
according to u paper, the mobile phase that you use was ACN:MeOH:chloroform (47:47:6).
i extract the fat soluble vitamin by solid-liquid extraction. aceton:chroroform was used to extract the fat soluble vitamin
the problem here is the my sample contain allmost 65% fat.
will the fat affect the separation of fat soluble vitamin?
or better doing saponification to reduce the volume of fat?but the vitamink will degrade under alkaline condition.
thank you
(I just hope I'm not violating any laws...)
From: "Current Protocols in Food Analytical Chemistry"
<snip>
Mobile phase (all HPLC grade):
25:22:3 (v/v/v) methanol/acetonitrile/methylene chloride for reversed phase
(...)
High-performance liquid chromatograph (HPLC) with fluorescence detector
(excitation 290 nm, emission 330 nm) or UV detector (295 nm)
<snip>
SUPPORT PROTOCOL 2
SAMPLE PREPARATION FROM CRUDE OIL AND FAT PRODUCTS
<snip>
1. Weigh 0.1 g (exact to 0.001 g) sample and 0.05 g ascorbic acid into a 16 × 125–mm test tube.
2. Add 5 ml of 90.2% ethanol and 0.5 ml of 80% KOH to the test tube and vortex 30 sec.
3. Flush the test tube with nitrogen and cap the tube.
4. Incubate in a 70°C water bath for 30 min, vortexing periodically.
5. Place in an ice bath for 5 min.
6. Add 3 ml deionized water and 5 ml hexane and vortex 30 sec.
7. Centrifuge 10 min at 1000 × g, room temperature.
8. Transfer the hexane layer to another test tube.
9. Add 5 ml hexane to the residual and aqueous layer and vortex 30 sec to re-extract. The residual is the solid phase that consists of aggregated sample particles on the bottom of the test tube. The aqueous layer is the aqueous phase above the sample particles. They are extracted together by hexane a second time.
10. Centrifuge 10 min at 1000 × g, room temperature.
11. Transfer hexane layer to the test tube containing the previous hexane layer (see step 8).
12. Evaporate hexane from the tube under nitrogen flow.
13. Add 1 ml (exact to 0.01 ml) mobile phase and vortex 30 sec to redissolve the extract.
14. Transfer extract to an HPLC sample vial.
(There's also protocol for refined oil, meat, cereals and nuts - PM me if you need them.)
mimosa on Aug 4 2009, 10:54 AM said:
i extract the fat soluble vitamin by solid-liquid extraction. acetone:chloroform was used to extract the fat soluble vitamin
the problem here is the my sample contain almost 65% fat.
will the fat affect the separation of fat soluble vitamin?
or better doing saponification to reduce the volume of fat?but the vitamink will degrade under alkaline condition.
thank you
as long as the fat remains soluble it should separate on the column along with the vitamins.
we originally used this procedure (including sample preparation) with blood samples and for more than just vitamin e. fat in the sample did not seem to be a problem.
as long as the fat remains soluble it should separate on the column along with the vitamins.
we originally used this procedure (including sample preparation) with blood samples and for more than just vitamin e. fat in the sample did not seem to be a problem.