I synthesised a compound with two stereogenic centres and have tried running chiral HPLC to determine enantioselectivity and diastereomeric ratios. For most molecules with two stereogenic centres, I usually get 4 peaks (as its typical for the 2^n rule). For this particular compound, I could see the diastereomeric pair of protons on HNMR, however, on chiral HPLC, after trying a wide range of columns, varying mobile phase ratios and flowrate, my best separation showed only 3 peaks. I tried synthesising a derivative with a varied functionality, but still ended up with 3 peaks. Is it possible to have 2 stereogenic centres but less than 4 peaks in chiral HPLC? If yes, what could be the reason for this? Does anyone know an example of such molecules? If no, I will be glad if you could recommend a possible solution.
If your compound has two chiral centres, you end up generally with 4 stereoisomers. Out of these four, two will be diastereomeric pair and the other two will be enantiomeric pairs. Let me ask you few important questions first.
1. What are the ratio of the 3 peaks that you obtained so far?
2. Did you run RP HPLC/LCMS of the same compound?
To better understand the case, remember this:
1. Diastereomeric pairs can be separated in RP HPLC and it can be of any ratio, i.e. 50:50 or 60:40 or 80:20 depending on the type of compound you have.
2. Data correlation is very necessary here. You get 3 peaks in Chiral HPLC. Is your compound 100% pure by RP HPLC? How will you confirm that your third peak is isomer/impurity?
3. If your compound is 100% pure, check the ratio of all 3 peaks and correlate with RP HPLC method. You may then confirm whether the fourth isomer is not separated or not eluted, etc.
In my experience, 2 stereocentres never give 3 isomers. It is either 4 or 2 (In case one centre is specific R or S).
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