Chromatography Forum

Please forumers kindly help me to find a parameters to analyze betacarotene in fruit concentrate by HPLC. I have C18 Column Zorbax and I sued ACN:MeOH as mobile phase and diluent but still I cant find a good peak in my standard. I used the VWD at 450 nm wavelength. Please give me idea. My blank is showing peaks and noise... I need it please I want he result next week huhuhuhuh...

Nelis WJCF, Deleenher AP (1983) Isocratic non aqueous reversed phase liquid Chromatography of carotenoids. Anal Chem 55: 270.

Weissenberg, M.; Levy, A.; Schaeffler, I.; Menagem, E.; Barzilai, M., 1997: Chromatographia 46(7/8): 399-403

Hart DJ, Scott KJ (1995) Development and evaluation of an
HPLC method for the analysis of carotenoids in foods, and the
measurement of the carotenoid content of vegetables and fruits
commonly consumed in the UK. Food Chem 54:101–111
doi:10.1016/0308-8146(95)92669-B

Below is an application for fat soluble vitamins (including beta-carotene) on various reversed phase columns:
http://www.imtaktusa.com/site_media/fil ... TI225E.pdf

Looks like for ODS, you should try acetonitrile / THF

Also, you said your blank just "is showing peaks and noise". What do you mean by that? Can you post a picture of your baseline with 'peaks and noise'? Also, can you post some more specific parameters of your analysis, like mobile phase composition, column dimensions, mobile phase proportions, injection volume, etc?

hi Chemron,

would you please give detailed information on your HPLC parameters (dimensions of the column, MP composition, if there exist gradient program injection volume and the concentration of your standart) I hope you will find an answer soon.

Hello folks,

Thank you so much for your reply regarding my query. regarding the parameters of my analysis: Mobile phase is ACN:MeOH 50:50 with diluent of 60:40 ACN:MeOH and my column is zorbax rx c8 4.6x150 5 micron under ambient temp of 25 degree celcius. run time is 20 minutes with 10uL injection volume. Stock solution was prepared by dissolving it in 10ml CHCl3 and aliqouts are taken to prepare working stds by using diluent. Detector is variable wavelength detector at 450 nm but when i scan the sample using the spectro it shows two peaks one is 252 bigger peak and 452 nm wavelength.

Blank (diluent) shows some peak on the first run and is peak retention time is not identical on the 2nd injection.

Kindly share your idea about my query.

Thanks and Merry xmas...

Hi chemron,
First I will advice to use no stop time till being sure about your standart peak has come. then try once again by not diluting your solution with the mentioned diluent, instead dry your standart which is in CHCl3 then solve it in MeCN:water. if those advices not work maybe giving your standart into different column would be a solution.

Best wishes

hi rindtr,

Thank u so much for your advice.. I will try that one but my mobile phase is ACN:MeOH so u mean ill dry the CHCl3 then ill use the MeOH:H2O as diluent? but i think ill measure it to 452 nm because when i scan the standard it shows two peak..highest peak at 252 and then 452 next... what do u think?

Hi Chemron,

Sorry for missunderstanding I sgould write ACN:MeOH instead of MeCN:H2O sorry again )

Ok chemron, I'm not sure you took a look at Bryan's application note, or the articles I posted. I don't think your elution solvents are strong enough. You need a non-polar solvent, or a solvent that beta-carotene is soluble in (like dichloromethane or hexane or THF). Increase your proportion of acetonitrile, or switch to acetonitrile/THF. See Bryan's application note, posted above.

Hi there,

Thanks for your reply. I will try also to change the composition of my mobile phase instead ill make it 88:10:2 ACN:MeOH:ethyl acetate mixture and I will use Zorbax RX C8 as my column. I will let you know th result.

Thanks...

If it were me, I would only change one part of the system at a time (e.g. - keep the ODS column you were using, but change the mobile phase; or, change the column, but use the original ACN:MeOH mobile phase).

I think another more important question is: Are you just doing method development right now, so you get to change parameters of the analysis without repercussion, or are you operating under an existing method and are having problems with it?

If you're doing method development, you have some freedom to change parameters. I probably would have just copied the mobile phase parameters from Bryan Evans' app. note, then adjusted the mobile phase proportions to optimize the separation for my column.

If you're operating under an existing method, and good results have been achieved before, then the question should be why your analysis is not working now, not what can you change to make it work.

Hi Bisnetrj2,

Thanks for your reply. Actually, im developing a method for our HPLC for the analysis of betacarotene in food products e.g. fruit concentrate, juice.... We dont have existing method at the moment for betacarotene.

chemron:

Then I'll post it again - here's an article that I think should be of interest to you:

Hart DJ, Scott KJ (1995) Development and evaluation of an HPLC method for the analysis of carotenoids in foods, and the measurement of the carotenoid content of vegetables and fruits commonly consumed in the UK. Food Chem 54:101–111 doi:10.1016/0308-8146(95)92669-B

http://tiny.cc/WdM6s

chemron:

Any luck with the mobile phase changes?