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HPLC -problem with solvent peak


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#16 ram

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Posted 02 August 2009 - 05:15 AM

Hi mimosa...You may also wish to visit Chromatography Forum
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.

#17 mimosa

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Posted 04 August 2009 - 06:54 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.


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

#18 K.B.

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Posted 04 August 2009 - 09:54 AM

(I just hope I'm not violating any laws...)

From: "Current Protocols in Food Analytical Chemistry"

Analysis of Tocopherols and Tocotrienols

<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 125mm 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.
[Reagent alcohol (HPLC-grade) contains 90.2% ethanol, 4.9% methanol, and 4.9% isopropanol.]
3. Flush the test tube with nitrogen and cap the tube.
4. Incubate in a 70C 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.)

#19 mdfenko

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Posted 04 August 2009 - 11:47 AM

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. 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.

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


#20 mimosa

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Posted 07 August 2009 - 09:12 AM

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.
[/quote]

the problem of fat that it cannot dissolved in acetonitrile.
still could see the fat globular in acetonitrile.
do the fat globular entrapped those fat soluble vitamin?cause the fat soluble vitamin not dissolved in acetonitrile?
do SPE cartridge, c18 recommended use to clean up the sample?

#21 mdfenko

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Posted 11 August 2009 - 08:50 PM

try dissolving in the complete mobile phase, not just acetonitrile.

i can't answer about spe because i never used it.

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





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