Sunday, June 19, 2011
Some of the common terms used in chiral chromatography often need to be reviewed. Here are some of the most common ones we encounter in the chromatography lab each day. There are many detailed chemistry books on the topic, but I will try and present some simple explanations for each here.
"Structural Isomers": When two compounds have the same chemical formula, but different structures, then they are referred to as structural isomers. AKA "Constitutional isomers".
"Stereoisomers": When two compounds have the same chemical formula and are connected to the same molecules, then they are referred to as stereoisomers.
"Enantiomers": When two compounds have the same chemical formula and are connected to the same molecules (they are stereoisomers), but are also non-superimposable mirror images of each other, then they are referred to as chiral enantiomers. *This is another class of stereoisomer.
"Diastereomer": When a stereoisomer compound has two or more chiral centers which are not mirror images of each other, then the compound is a diastereomer.
"Meso Compounds": When a stereoisomer appears chiral due to the presence of more than two chiral centers (but is in fact not chiral), but is superimposable on its mirror image, then it is a meso compound. These are interesting forms and they are optically inactive because the two opposites cancel each other out (no +/- signal).
Wednesday, June 1, 2011
We are experts in chiral separations and have learned a great deal about how to efficiently and quickly identify the best conditions to resolve a chiral sample by HPLC and SFC. You should always have a clear strategy in mind when developing an automated HPLC or SFC chiral method development screening system to separate racemic samples. Here are a few key points to focus on.
(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 or 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 types, with and without 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. Keep it simple! The goal is to retain the sample, hold, then elute it.
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 used a particular column. This is not a scientifically valid argument that the column is useful. We routinely read published papers which describe a "novel" chiral method run on a specific column which clearly shows worse results than could have been obtained with another commercial column using a simpler mobile phase. Many try and force a column to resolve a sample apart because it is the only chiral column they have. The purpose seems to be directed at publishing a paper and not at developing high quality chiral HPLC or SFC methods at all. Just because someone tried it before does not mean that their method or column choice was a good one. Remember, very few chromatographers have practical experience developing CHIRAL methods. It takes special training to be successful at it. Invest some time learning about and evaluating the different chiral column types with your own samples to find out which ones are most applicable. Just as with achiral analysis, make sure the sample is fully soluble in the selected mobile phase before analysis. Select a chiral column based on your own scientific evaluation and testing. Start with a "full sized" columns to maximize the amount of stationary phase the sample comes in contact with. We do not use or recommend the short "scouting" columns available from some suppliers for screening purposes as they will often cause you to miss identifying a column type that works.Don't make this novice mistake (with a chiral columns, it is a good strategy, but chiral MD works very differently).
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 used 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 are good, but just do not measure up to the high success rate this one has.