Chromatography Forum

Hi everybody,

I am a HPLC-beginner and have some questions regarding our new project: We want to perform quantitative analysis of water- and fat-soluble vitamins by HPLC.

Hopefully, you can give me some background information about some topics:
In the first step we have to create calibration curves. Is it possible to create such a curve with a mixture of different vitamins? For example: Can we use a mixture of vitamin B6, vitamin B2 and vitamin D3 to measure the calibration curve for vitamin B6? Or do we have to use a pure substance for measurement?

Is it correct, that HPLC columns can be used for approximately 2000 injections?

Does somebody already have experiences with vitamin analysis? Is it also possible for HPLC beginners to analyse mixtures of vitamins?

Thanks a lot?

It should be possible to create a calibration curve for B6 by using a mixture of vitamins in the calibration standards if the peaks are well separated and do not contribute to each other.

A HPLC column's lifespan is determined by what you run on it, if you use a pre-filter/pre-column, pressure etc. You can't really claim that a column has a certain lifespan without knowing a bit or two about what's being performed on it.

I would say that it is fully possible for beginners to perform this type of analysis. If you have the background of chromatographic theory and some knowledge in HPLC and columns, you are off to a good start. The tricky part is to be able to get retention on the water soluble B-vitamins and still be able to elute D3 within reasonable time. But you should try to search a bit on the web for this type of analysis, there should be a lot of information out there for these types of analyses.

I agree with Perreman.

In addition:
2000 injections?? Even people doing HPLC for several years I saw killing a column with 1 injection or even without any injection or run. Others got more than 5000 injections on a column.

Several column manufacturers have published over the few past years applications to run water and fat soluble vitamins in one run.
Get 2 or 3 application notes and than you should be able to do your analysis.
Good luck

Here we go with a nice application:
http://www.scielo.br/scielo.php?script= ... 3000700020 and

http://www.sepscience.com/Sectors/Food/ ... Vegetables

That will give you a good starting point.
Good luck

Carefully filter your sample extracts and do sample cleanup where possible. We run both water and fat soluble vitamins in one run and have many thousands of injections on one column. We make sure we keep the pre-column filters clean and a good pre-column in line. The biggest problem we routinely run into is particulates in the sample; thus the careful filtering.