Chromatography Forum

I am having problem in separating two compounds( pKa 10.2 and 7.7 respectively) using shimadzu HPLC. the chromatograpfic conditions i am using are:
column: Lichrosphere RP C18( 5um)
(250*4.6 id)
Mobile phase: ACN:5 mM ammonium acetate(10:90)
flow: 0.5 mL/min
column oven: 40c
Detector: PDA
peak for both compounds are cuming at Rt 6.1 and 6.7 respectively and they are merging near base. can i try ion pair chromatography
please if u can help me as soon as possible..........

Try adjusting the temperature up a bit and see if things get better or worse. You might also try adjusting the pH of your buffer.

Please try the solution with 20mM K2HPO4 and 2mM NaH2PO4 instead of NH4AC solution. If separation get any better, please let me know and we'll do the further.

The last advice was very good. Ammonium acetate is not a buffer, does not provide pH control, but the buffer suggested by sunht does. Furthermore, sunht's buffer can then be changed (slightly) to manipulate the retention pattern of your analytes. Further improvements can be made as well.

what are the capacity factors of both peaks?

The k's seem to be 0.2 to 0.3 based on the condition provided.

I would improve the retention first, by

1) lower the organic solvent contentto 5% or even 0%

And /or 2) run at high pH, say pH11 using one of the base-resistence phases in the market.

Or 3) consider using a cation-exchange/RP mixed-mode column

If analytes are very hydrophilic, HILIC may work, too.

Try adjusting the temperature up a bit and see if things get better or worse. You might also try adjusting the pH of your buffer.

I had tried temperature between 30-40ºC but there was no significance difference......the best resultbi got was with mobile phase ACN: water(10:90) ......
THankyou..

Please try the solution with 20mM K2HPO4 and 2mM NaH2PO4 instead of NH4AC solution. If separation get any better, please let me know and we'll do the further.

pH of buffer should be adjusted or as such..........
Thankyou

The k's seem to be 0.2 to 0.3 based on the condition provided.

I would improve the retention first, by

1) lower the organic solvent contentto 5% or even 0%

And /or 2) run at high pH, say pH11 using one of the base-resistence phases in the market.

Or 3) consider using a cation-exchange/RP mixed-mode column

If analytes are very hydrophilic, HILIC may work, too.

thanks for ur suggestions but i am having not much of facilities but mobile phase changes i will try....
Tankyou

Please try the solution with 20mM K2HPO4 and 2mM NaH2PO4 instead of NH4AC solution. If separation get any better, please let me know and we'll do the further.

pH of buffer should be adjusted or as such..........
Thankyou

Don't need. A solution with 20mM K2HPO4 and 2mM NaH2PO4 in it is about pH 8.2

1. Cut down further on the MeOH to increase overall retention.

2. Drop the pH in increments. At 8.2 you are already well below the pKa of one of your analytes, so its retention should change relatively little (I'm assuming your compounds are bases). The retention of the other analyte should decrease.

If this doesn't work, then you will have to change columns, either to a base-stable column (and increase the pH), or an embeded-polar-group column (which might give more retention with conditions similar to what you are using now) or to a HILIC column (a form of normal-phase chromatography).

The best pH for maximum separation should be between the two pKs (between 10.2 and 7.7). NH4Ac solution pH~7 is too low and compounds too ionized. So separation is too small and retention too short. I don’t know these two compounds’ retention of molecular state. If molecular states are similar, the best pH should be (10.2+7.7)/2=9

All above consideration is based on compounds’ base states are molecules and acidic states are ions. If different, please let me know, consideration would be different.

The best pH for maximum separation should be between the two pKs (between 10.2 and 7.7). NH4Ac solution pH~7 is too low and compounds too ionized. So separation is too small and retention too short. I don’t know these two compounds’ retention of molecular state. If molecular states are similar, the best pH should be (10.2+7.7)/2=9

All above consideration is based on compounds’ base states are molecules and acidic states are ions. If different, please let me know, consideration would be different.

My two compounds are allopurinol and oxypurinol with pKa values 10.2 and 7.7 respectively...........

parveenqa,

as stated earlier,
see your capacity factor situation.
your compounds have nealy zero retention on your c-18 column and this because they are very polar, because of this you have very small chances of getting a good separation between them, also for robustness and accuracy conditions it is best to be with a greater capacity value. above 2 is preferable at least 1 is good. a good chromatography method will take this into account, from there separation will be easier to achieve

now that we know what they are exactely, i would suggest either trying a polar embeded c-18 or you should move to a HILIC or normal phase condition; preferably HILIC in my view.

Hi,

This is a pdf link, I think this may useful to you.

[url]http://dissertations.ub.rug.nl/FILES/faculties/science/2008/m.k.reinders/04-c4.pdf


Nageshwar[/url]