Tip#
107: Chiral HPLC and SFC Column Screening Strategies:
You should always have a clear strategy when developing an automated
HPLC or SFC chiral method development screening system to separate a racemic sample. Here
are a few points to ponder.
(1)
Make sure the chiral sample is as
“chemically” pure as possible before you start. By “chemically” pure I
mean it should only contain the two complementary enantiomers and not other impurities,
starting materials or chemicals which might interfere with the identification or
separation of the racemate. Often, many of the intermediate chemicals used in the
synthesis of a compound have similar absorbance or retention characteristics. When this
happens, you can be fooled into thinking you have resolved your chiral component apart
when in fact you have simply resolved a non-enantiomer apart from the racemate. Chiral
columns are very poor at discriminating the racemate from other non-chiral species. As
such, you may find that the impurities or other components make it difficult to determine
the actual HPLC/SFC purity of your chiral sample. Try and remember this phrase: “The
chiral purity of a sample is only as good as the chemical purity”. So start with as
clean a sample as possible when developing a chiral method.
(2)
Column choices are very important when using a fully
automated Chiral Screening System such as the LC Spiderling™ Column Selection
System. Here are some popular questions we are asked on this topic.
How do you know how many and which types of columns to include in
your chiral screening system?
Keep the goal of creating a "screening system" in mind.
Don't lose sight of this basic strategy. You want to select the smallest number of
different columns which have the widest specificity for your expected sample types. Often
five (5) to nine (9) different types of chiral columns will do the job. Screening more
columns than that is often a waste of time. Leave a “test” position in your
screening system so you can evaluate new columns all of the time. The key is to identify
the best ones first.
Normal or Reverse Phase Columns?
Most chiral screening is performed in the normal phase mode (NP
provides easy solvent removal for scale-up and there are a wide range of quality columns
available which can be used to generate useful data). You can mix reverse phase and normal
phase columns in the same system as long as you incorporate a bypass and flush step
between the different methods to wash out the old solvent and bring in the new solvent
safely. For this brief discussion, we will assume you have a dedicated normal or reversed
phase screening system.
How many different mobile phase systems should I use?
Quick answer is as few as possible. The concept of using a screening
system is to quickly identify the column (1st) and mobile phase (2nd)
which baseline resolves the racemate. Select three or four different mobile phase or
modifier systems which span the range of polarity needed to increase your chance of
retaining the sample on the column, but for no longer than 30 minutes.
Which types of chiral columns are best?
Well, if you ask the different column suppliers this question, then
they will most likely answer that the columns “they sell” are the best. At last
count, there are over two hundred different chiral columns advertised on the market today.
Most are advertised to be the best, but in fact they all can not be… In reality, you
should evaluate as many different kinds as possible with your own samples to determine
which the “best” are. Do not be fooled by examples of the column separating out
very simple compounds such as racemic trans-Stibene oxide as this is one of the easiest
compounds to separate even if 90% of the chiral stationary phase is missing! Consider also
if the column type can separate compounds without the use of fancy modifiers or complex
mobile phase mixtures. Simple is better and usually more easily reproduced too. Some of
the most commonly used types of chiral columns are the: cellulose/amylase, Protein, Pirkle type and cyclodextrin based columns. All
these columns have different preferred mobile phase choices (Most protein columns are run
in reverse phase, while all of the others mentioned have versions which can be run in
either normal or reverse phase). You should consult with the appropriate manufacturer
about how to best use these columns. Do not necessarily select a column because the column
has been “reported in the literature to be used by the largest number of
people”. Who cares how many people use the column. We routinely see people who
develop methods on a particular column and publish their results with it. That alone does
not mean that their method or column choice was a good one. Invest some time learning
about and evaluating the different columns with your own samples to find out which are
best. Ignore the potentially sales biased recommendations and select a chiral column based
on your own scientific evaluation and testing.
Can you provide some free advice as to which columns are the
“best”?
OK, we have tried them all (but not for your samples), but here are
two of our favorites (in no order):
The Pirkle based Whelk-O 1 (and/or Whelk-O 2) is the only Pirkle
based column we have ever found which can produce a significant quantity of racemic
separations using simple mobile phase systems. No other Pirkle based chiral column has
ever proven to be as good as this one in real world chiral pharmaceutical drug method
development. Every other one tried has disappointed us, but this one has been responsible
for a number of successful separations in normal and reversed phase modes.
The coated polysaccharide chiral stationary phase made by Daicel,
Chiralcel OD (OD-H) is one of the best chiral columns on the market. Note that this is the
non-covalently bound coated version of the column. It has a broad range of selectivity not
seen in any other types of columns available. At this time, the covalently bound versions
just do not measure up to this one.
> Bill Letter,
09/20/10
