Dilip,

I am attaching for reference the manual pdf for this column. I do not think you did anything wrong, but it can be any of the many factors typically affecting chiral HPLC.

Did you use the same batches of heptane and IPA (trace water content is a very important parameter, and it can very from batch to batch, not only in IPA but also in heptane!)

Is the temperature of the separations quite different? (ChiralPak columns sometimes show high sensitivity to temp., with much better behavior at 20 degC than at 27degC)

Is it possible that the concentration of your injected sample changed? generally, the lower detectable conc. the better the separation.

Do you now have (traces) of any admixtures that were not present in the original racemate sample? Sometimes this has a drastic effect.

Let us know how it went...

with regard sir,

i forwarded my every step with each and every factor mentioned by you, i.e. Same Reagent, Temperature(Room, sample), Concentration,composition,Pressure and i tried even same batch, which analyzed earlier.

Please tell me is there any procedure to increase resolution in chiral chromatography.

Thanks and Regard

Dilip,

Regarding work with ChiralPak and ChiralCel stationary phases:

1) For the hexane-IPA phase people use buffers like 0.1%v/v diethylamine (for basic compounds or slightly basic compounds, it works also for some carbamates); or 0.1% acetic acid (or 01% formic acid) for some acidic compounds; however, for neutral molecules oftentimes a buffer (modifier) will not improve your separation, it can make it much worse.

2) A column must be well equilibrated with the phase (never use a gradient for these phases, use an isocratic flow). Importantly, if the instrument is not dedicated for chiral phases, and it has been switched from RP mode - make sure that you use proper protocols to fully reconfigure the system (remove traces of old solvents from lines, needle, injector, seals, etc). Chiral separation require much more work/preparation time, and more attention to cleanness of the system.

Good luck!

Jacek

P.S . By "much more work" I meant that suitable composition of the mobile phase has to be found, and that process sometimes requires a few days of work for a single compound. Interestingly, often a close analog will require meaningfully (if not drastically!) different conditions.

To my knowledge, there is no systematic way to model the composition of mobile phase needed for a chiral separation. Possibly, this is so difficult because the stationary phase is coated with sugar oligomers that are very conformationally labile, which means that it does not "take much" to change the molecular shape (and the properties) of the stationary phase.

Working with multiple chiral separations is fun!

One of the tricks to using these columns well is that they must be washed down with a more polar solution. Anytime you use high percentages of non-polar mobile phase (i.e. Hex. IPA, Hep), you will load up and foul the column with material. A stronger solution is needed and 100% ethanol (100% MeOH too) usually works very well in this respect. Just like any column (chiral or achiral), they all need to be washed off on a regular basis. Always use the highest grades of solvent too (esp with n-Hexane) and filter samples before injecting.

In your case, you would start by increasing the IPA concentration in the mobile phase wash to 40% (60% Hex), then slowly switch over to 100% Ethanol and wash the column with about 30x column volumes of solution. Re-test for performance.

Warning: for washing ChiralPak AD and AD-H columns DO NOT use 100% MeOH (se the pdf brochures posted on the web).

While I strongly suggest that users follow the column manufacturer's guidelines and recommendations, we routinely develop excellent methods using Chiralpak AD (the coated version) with isocratic mobile phases consisting of:

 100% MeOH (usually at lower flow rates of 0.500 ml/min);

 100% EtOH;

 100% Butanol;

 100% IPA (usually at lower flow rates of 0.500 ml/min);

 100% 2-Butanol (usually at lower flow rates of 0.500 ml/min);

 100% ACN (Yes, it results in some very interesting separations, but usually inferior to all of the other mobile phase choices);

We have even run methods on AD with water such as 80/20 mixtures! No damage to the columns.

I presented a talk at one of the HPLC conferences many years ago on this very topic and will have to see if I still have any of the chromatograms used. I teach the use of these and many other unusual mobile phases in my chiral method development courses as well.

Great, I just would recommend to new users that they fully obey the manuals/pdf brochures posted by the makers/vendors of a given column. In case of Chiral Tech AD and AD-H columns, it is not recommended to use 100% MeOH, UNLESS you want to keep running this solvent afterwards. Other than that, fully agreed.

As follow up to my earlier post where I mentioned that I presented data on the use of Chirapak columns with many non-traditional 'straight' alcohol mobile phases, here is the reference: "Comparison of the Chiral Selectivity Differences Seen with Different Alcohol mobile phase solutions on Chiralpak AD Column",  DOI: 10.13140/RG.2.1.3172.5928. 

NOTE: The data presented in the above article was obtained by me in 1994/1995 and was originally presented in 1996. Re-printed here for informational purposes. *We have learned a great deal about these and many other chiral phases since then (that was 20 years ago!).