Sugars, Organic Acids, Alcohols

[HPLC_Help]

Hi, all -

I'm entering a new field and have no experience with the chromatography being run.

We're using a Bio-Rad Aminex resin-based column to separate carbohydrates, organic acids and alcohols. Mobile phase is sulfuric acid, and detection is RI.

Does anyone have some links to articles/application notes/etc for this type of multi-mode chromatography, or any competitors columns, etc.?

Also - any tips, hints, advice, etc are welcome.

Thanks!!

H_H

[Vlad Orlovsky]

Here is separation of sugars, acids and amino acids:
http://www.sielc.com/application_183.html

All compounds are retained by combination of HILIC and cation- and anion-exchange for trimodal interaction.

[Bruce Hamilton]

BioRad website offer several good bulletins on how to use these ion-exclusion columns.

Operating Instructions
http://www.bio-rad.com/cmc_upload/Liter ... LIT42D.PDF

Guide to Aminex HPLC columns
http://www.bio-rad.com/LifeScience/pdf/ ... n_1928.pdf

There are heap sof freely available publication abstracts on different samples in the literature - you don't even have to purchase the articles, as the abstracts give enough information.

Bruce Hamilton

[HPLC_Help]

Thank you, all! I did find the Bio-Rad info previously, and it was very helpful.

I will do a lit search, as well.

Anything else is welcome, too.

H_H

[Koen Hollebekkers]

Shodex has good alternatives for this. Check their website.

[mbicking]

I have attempted to help several labs with this separation, and it can be a bit mysterious in its behavior. But most of the problems have been related to operational aspects of the separation.

If you are new to chromatography, then make sure you get some training on basic GCP (Good Chromatography Practices).

Here are some things to remember:

1. These columns are not as stable as a traditional C18 column. They have much lower pressure limits. Set your instrument max pressure level so that you don't damage the column.
2. Filter your samples before you inject them. (It is obvious to most of us experienced users, but if you are injecting fermentation samples, for example, the particulates will damage your column.)
3. Dissolve your sample in mobile phase (dilute sulfuric). If you can't do that, at least dilute it with mobile phase and then filter.
4. Many labs are using this method outside the linear range of their RI detector, and don't realize it. You will usually get better results by diluting (with mobile phase).
5. If you have messy samples, some regular column cleaning will be necessary. Check the documentation.

Labs who don't follow these guidelines may have to replace the column every four to six weeks (at $1000/column). With the above changes, the labs tell me the columns can last for up to 2000 injections.

[Bruce Hamilton]

As well as the excellent advice from Merlin, a couple more points.

1. For fermentation monitoring, we used to get 5 year column life ( about 8 - 10,000 injections ) by diluting samples before microcentrifuging ( rather than filtering, which can get expensive with lots of samples, and also can lose some compounds , such as metabolites - if you are looking for them ).

If you are looking for trace components, you may not be able to dilute, and may need to filter or concentrate.

2. Temperature control is critical, we used to run the columns in a 35C temperature-controlled waterbath with 40C RI setting. The higher temperatures produce sharper peaks, shorter run times, and greatly reduce junk retention on column. Constant temperature and flow greatly enhances retention repeatability as well, we kept ours running 24/7.

3. Mobile phase is about the cheapest you can get, but use really good quality Milli-Q water and concentrated acid. Don't allow the column to stand with just water present ( if you switch from the current acid mobile phase ), wildlife can grow and quickly kill it.

4. Use a guard column - not just a prefilter, but a guard column, which is virtually mandatory if using a silver-based column, unless you really want to keep meeting the local column-selling representative.

Guard columns are especially important if samples are from research fermentations with unusual media. Large-scale defined media fermentations are much "cleaner" than small-scale research broths, which can produce diverse snot that is difficult to process and shortenens column life.

Another company that makes alternatives, and has some good free information, is Phenomenex with their Rezex range.

Please keep having fun,

Bruce Hamilton

[mbicking]

Bruce added in some more excellent points.

... and a few more that I forgot about ...

There was a time when the Bio-Rad columns had a problem that limited their lifetime. I think it is fixed, but can't verify. I have had good luck with the Supelco columns as well.

As Bruce mentioned, you are dealing with an active microbiological sample. If you are near the plant, your lab environment is also "active." Cleanliness of the mobile phase is critical - not just the solvent, but also the bottle, and the bottle cap. I saw a situation where the inside of the mobile phase reservoir cap was contaminated with mold (or algae, or some back, slimy thing), so even though they changed the solution and bottle, they just re-inoculated the solution when they screwed the cap back on. (I have a picture if you want to see it.)

[HPLC_Help]

Does anyone have any experience with the Bio-Rad Fermentation Monitoring column? I found a method online with a 10 min run time using it. It looks like it could be a huge improvement, but it seems like there must be a catch. From the looks of it, I think it's typically used in-line at the back end of a fermentation tank for in-process testing, but I don't know why it couldn't be used in a lab.

H_H