Chromatography Forum

Hi
I am tring to seperate and identify some organic acids produced by microorganisms.
In some refernces, they used H2SO4(20mM) as mobile phase.
But from what I have heard from the column manufactures, that H2SO4 is not suitable for their column
(Synergi Hydro-RP C 18 column)
In their data sheet they used 20mM KH2PO4 as mobile phase for seperating organic acids.
(All the refernce I have looked used H2SO4....)
Can anyone give me some advice on this?


*If there is anyone who is working with similar work that I am doing, please help me with this also. PLEASE
-How do you prepare HPLC samples from supernant of your bacterial or fungal culture?
Here is how I prepare my sample
1. take 1ml of culture supernant
2. filter through 0.2 micro filter
3. analyize

However, whenever I do this some of the media component(e.g. glucose, lactose, maltose)
seems to show up as a peak on my result. Always giving me hard time to quantify the actual acid I'm looking for.
So my question will be 'How do you get rid of unwanted components of media?'


Thanks in advance for any adivces.

what is the retention time of your products on the chromatogram
which acid is the one co-eluting with the sugars?
is the column a 250mm lenght?

(All the refernce I have looked used H2SO4....)
Can anyone give me some advice on this?

Dilute sulfuric acid is the standard mobile phase for "ion-exclusion" columns. These are typically PS-DVB sulfonate cation exchange resins in the hydrogen form. These are *not* silica-based C18 columns.

Sugars are retained to some extent on these columns, and they *can* be interferences. You can tweak the selectivity by adjusting the acid concentration and/or by adding ACN as a modifier.

You can clean up your samples using anion exchange SPE cartridges to retain the acids while passing neutrals through. The organic acids can then be "bumped" off the cartridge with a low-pH buffer (actually, the H2SO4 mobile phase should work just fine).

you will find information here on these types of columns
like Tom said these are ion exchange columns
and they can separate both sugars and acids
http://www.sepax-tech.com/Carbomix.php


your column, Synergi Hydro-RP C 18 is a c-18 with an added polar group to regain some of the hydrophilic interactions lost with modern columns and the end-capping done to them.

what is the retention time of your products on the chromatogram
which acid is the one co-eluting with the sugars?
is the column a 250mm lenght?

Sorry for the late late very late reply
It's D-gluconic acid that co-elutes with Glucose.
retention time of these are about 2.7 min~2.78 min
and the length of my column is 150mm

Thank you

Just wanted to reiterate- the H2SO4 mobile phase is not for C18 type columns. In fact, many times you need to be careful and avoid pH's below 2.5. Some manufacturers like Restek offer specific reverse phase columns designed for analysis or organic acids. A buffer like phosphate is typically used for those.