peak separation

[mk12]

I have trying to separate two peak..which i have no idea what kind of molecule are those..casue its unknown from bacterial culture. and i do not have enough to do nmr to know what they are ,..mass spec is not very help ful..i tried c8, c18 and phenyl columns..but they wont separate used acn and water gradient with formic acid..

what should be my next step?,..any advise

[mardexis]

Why do you say 'two peaks' if you can't get anything to separate?

If you have access to GCMS I would try shooting it in there. The EI spectral libraries are very good for identigying unknown compounds and if they will fly the separation is generally much better anyway.

[mk12]

I mean two peak because i can separate them very little and they both have different uv max one has 257 and other one has 242 as uv max

[Alex Buske]

Trying different columns is a good way. As you don't know anything about the molecules next tries should be:
- different organic solvent, e.g. methanol
- different pH
Additionally you try to find isocratich conditions. Temperature could also help, especially low temperatures.

Alex

[Gerhard Kratz]

I also would recommend to try another column, but also another chromatographic mode. My first choice would be a HILIC column. HILIC will not work every time, but if one of the 2 compounds is neutral and the other charged, you will get a baseline separation. Hope that will help. Gerhard

[ym3142]

There aren't too many options. Excuse me to repeat:

stationary phase: C18, Phenyl, hydrophilic-embedded, HILIC, IEC, NP, mixed-mode, polymer-based, monolith, hydride silica based, zirconium-based, B1 silica, etc.(sorry, not well categorized)

organic Mobile: ACN, MeOH, THF, acetone (for RP), etc.

Other conditions: temperature, Flow, Ion pair, pH, smaller particle size, longer column, etc.

[unmgvar]

at around what % ACN do they come out?
can you give some details of the column you are using, length particle size?

what is the pH of your formic acid solution?
at what concentration? is it formic acid because you use MS/MS?

[SIELC_Tech]

If your compounds are ionizable (basic or acidic) you best bet is mixed-mode chromatography, where compounds are separated by two mechanisms - reversed-phase and ion-exchange. Here how mixed-mode columns are different from RP or ion-exchange. You are using 2-D approach on a single column:
http://www.sielc.com/Technology_2D_Properties.html

With mixed-mode you can separate structural isomers which are very similar in properties:
http://www.sielc.com/compound_215.html
http://www.sielc.com/compound_308.html
http://www.sielc.com/compound_037.html