Chromatography Forum

Dear All,

I am trying to separate polar isomeric compounds using LC-MS/MS;

I prepared a standard mixture of polar isomeric compounds ( configuration chemical group is -OH) of with known concentration in MeOH and optimized a method development

The compounds m/Z values after dansyl chloride derivatization are;

504/171
506/171
522/156
530/171 (Internal Standard)
536/171 and 534/171
753/170 and 755/521
520/171

The method is:
SP: C18; 25 cmx4.6 mm, 5um BECKMANN COULTER
MP: A: 95%Water/5% ACN with 0.1 % FA and

B is 95% Acetonitrile/ 5% Water with 0.1% FA

Flow rate: 800 uL/min

Gradient Programme is:

Time %B

At 0 min 0
5 min 40
8 min 50
12 min 55
19 min 65
25 min 100
31 min 0
35 min 0


PROBLEM IS:

All the compounds are eluting after 100% B only i.e., starting at 24 min, and with almost same RTs (Very little difference)


Any Idea how can get a good separation method with a shorten the analysis time given the current information.


-------------------
Raj

Hi
Isomers or stereoisomers?

For stereoisomers separation- selection C18 column is a bad variant. Need use stereisomer phase , OVM or dextrane.
For optimize isomer separation , should we know configuration chemical groups (Phenyl, -NH2, =0, -OH , -COOH, others) in isomeric centres.
PS
This may be a trade secret - then we will "pass" as they say in patience (in a card game)
)

Before worrying about selectivity, you need to get reasonable retention. If your compounds are all eluting at the end of the gradient, then you should try a shorter-chain column. Possibly a C8 or even a C4 or cyano column. *Then* you can start looking at selectivity if necessary.

That said, be sure that you are accounting for the gradient delay time when estimating the %B at elution.

Why do you use a complicated gradient starting at 0%, when your analytes elute with 100% B?
Without further knowledge I would do a 98% B run (2 % buffer just for ionisation) and the run gradient90%B to 98% B in 25 min. 80% B to 98% B in 25 min and so on.

Alex

Maybe I am missing something but why are you derivatizing if your molecules are hydrophobic and you end up doing mass spectrometry? Are your compounds really isomers/isobars (before derivatization), meaning the same MW?