It is possible but you will have incredible peak broadening due to longitudinal molecular diffusion unless you use small lenght column (ie 100mm), smaller particules size and low void volume (from the unions especially).
Best Regards,
Laurent
DEAR NASSER:
SALAAM AND GREETINGS. YOU CAN USE 3 COLUMS IN SERIES PROVIDED THEY ARE SHORT COLUMNS e.g. 5cm in length or may two 5cm columns + one 15 cm column to make the total length 25 cm or other combinations in length so it does not exceed 25 to maxium 30cm other wise you will have peak broadening. Also it is wise to use small particle size columns 3 micron and /or preferably solid core columns.
I would not recommend this approach at all and not just because of back pressure concerns. With most chiral columns, they either work to resolve your sample apart or they do not. They do not all contribute to the separation as is the case with some other types of columns. Coupling different chiral columns in-series provides no gain in efficiency or performance at all (unlike with SEC/GPC columns). Each column will only add dispersion to the sample and make it harder for the next column to show any separation (if there is any). The result will be that you will miss finding all possible chiral separation methods. With NP/RP chiral separations, it is best to use one chiral column at a time (largest column you can get to maximize the available chiral selector present).
Nasser: I am not sure what your specific application was to ask about placing several chiral columns in-series, but if your goal was to improve separation (much as we do in SEC applications), then I would like to add one more related comment.
One other option to improve the resolution of difficult chiral racemates involves recycling the primary peak over the same column, again and again, to improve resolution. Re-injecting the partially resolved sample gives it another chance to separate further. This technique is referred to as "Recycle Chromatography" (Note: the sample is recycled, not the mobile phase). The technique can be applied to chiral and achiral samples.
Recycle Chromatography: The idea is to extend the existing column's length to provide unlimited time and therefore interaction with the support to resolve the two enantiomers apart. Recycle chromatography is a very popular technique used in Preparative chromatography applications where the column volumes used are HUGE (>1L). Large column volumes allow you to recycle the peak from the column outlet, back through the pump head and onto the column head again (and again). The peak dilution from this cycle is very small when large columns are used. I have used the technique many times to clean up batches of materials on a very large scale without having to spend large amounts of money to purchase larger columns. However, this technique can not be successfully applied to analytical scale separations as the peak would be diuted and all separation lost on the first recycle. The trick to actually doing this on an analytical scale was developed by me in the 1990's. I created some fancy plumbing and valving to manipulate the peak back onto the column without running it back through the pump head. I found that adding a second identical column allowed me to pass the sample from one column to another, in sequential fashion, by leap frogging the columns over each other again and again until the desired separation is achieved. The effect created is that you have a chiral column of unlimited length without the associated backpressure problems. Now, this only works IF the column being used provides a partial resolution to start with. If the column chosen does not show any signs of separating the compound, then making it ten times longer is not likely to improve the separation at all. With chiral separations it is critical to first identify the column that does shows signs of resolving the sample first, then the mobile phase is fine tuned.
The analytical scale "Leap Frogging System" is commercially know as the Model CS3080, "HPLC SAMPLE PEAK RECYCLER™". You can read more information about it at this link: http://www.hplctools.com/peakrecycler.htm *BTW: Please accept my apologies in advance as I did not want to make this post appear to be a commercial one, but the device was created for this exact purpose so wanted to share the info in case someone was not aware of it.
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