My diastereomer peak is coming just after my main peak and also the main peak has tailing in it and the diastereomer peak is comin with the tailing in reverse phase HPLC system.I have attached the chromatographic conditions and the chromatogram of my sample where there is main peak and diastereomer peak.
Kindly suggest some methods to separate these two peaks.
I would change selectivity of your system. There are two ways, a) change stationary phase and b) change the mobile phase. However, you have to change one thing at a time. Example, keep the same mobile phase condition and use different columns. Next use the same column and modify the mobile phase. Say if you are using methanol, change it to acetonitrile. The ratio of mobile phase will change the retention (k') time but would not help the selectivity.
Dear Rakesh Samal
many thanks for your question. In our laboratories we successfully used a "C30" column to separate structural isomers.
You might want to check the Acclaim C30 columns manual under the following link:
https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FCMD%2Fmanuals%2Fman-065416-acclaim-c30-columns-man065416-en.pdf&title=QWNjbGFpbSBDMzAgQ29sdW1ucyAmbmRhc2g7IFByb2R1Y3QgTWFudWFsIChSZXZpc2lvbiAwMyk=
I hope this helps.
Good luck with your research,
Detlef.
Many 'reverse phase' stationary phases have a 'slight' separation between diastereomers. In order to increase the baseline separation between them, you can try adding a chiral component to your mobile phases.
Bruce Neagle Thankx but what chairal component can be added in our mobile phases
Dear Rakesh Samal
What is the purpose of your separation? Is it only analytical or also preparative? If it is only analytical, you can change the detection system. If this is not possible, I will go for another solvent system or column. For example, I had cases where changing the organic solvent from ACN to MeOH could help to resolve diastereomers.
Good luck then!
Jean-Pierre Mufusama.
I would change selectivity of your system. There are two ways, a) change stationary phase and b) change the mobile phase. However, you have to change one thing at a time. Example, keep the same mobile phase condition and use different columns. Next use the same column and modify the mobile phase. Say if you are using methanol, change it to acetonitrile. The ratio of mobile phase will change the retention (k') time but would not help the selectivity.
Rakesh,
The answer depends on the structure and chemistry of your diastereomer. I have used in the past D or L-Citric Acid but it absorbs in the UV and will raise your baseline. However most common HPLC grade reagents are racemic mixtures that contain both chiral compounds.
Rakesh,
You can also use L-Alanine the amino acid since it has achiral center.
Hello Rakesh,
Just studied the provided chromatographic conditions, Since you are using columns as Acquity CSH-C18, 100*2.1mm, 1.7µ, you can try using the column with higher diameter and it will help in resolution, or try changing the particle size as the particle size though works in providing resolution it also facilitates mass transfer and thus the separation of diastereomer would be difficult.
Hope this helps!!!!!
Hi Rakesh,
I don’t know compound structure. But to increase the resolution you can; either increase column length or column diameter, increase buffer strength i.e. from 0.01M to 0.02M, using Methanol instead of Acetonitrile in diluent may also help to sharpen peak hence increasing resolution or change the stationary phase from C18 to alternative eg. Biphenyl. Also using core shell stationary phase (column) reduce bend spreading of compound on column and help to increase resolution.
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