Some Stereochemistry Terms (Chirality); Structural Isomers, Stereoisomer, Enantiomer, Diastereomer, Meso

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. Enantiomers rotate the plane of plane polarized light to an equal extent, but in opposite directions.

"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. IOW: Diastereomers are stereoisomers that are not enantiomers

"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). 

Chiral HPLC and SFC Column Screening Strategies for Method Development:

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 (1^st) and mobile phase (2^nd) 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. 

 

A Note about AVOIDING "Short Length" Chiral columns: We do not use or recommend any short "scouting" columns. Using SHORT columns will often result in you miss-identifying a column type that actually works. You will not see retention when in fact you would have if you used a standard sized column. Please don't make this novice mistake with chiral columns. Use chiral columns that are LONG, not short for screening (the goal is to maximize the amount of support the sample comes in contact with). The use of short chiral columns in HPLC / SFC column screening is often a waste of your money and time. If you want to identify which chiral columns will resolve your samples, stay away from short columns.

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, known as "Chiralcel OD" (OD-H) is one of the best chiral columns on the market. Note that these are the non-covalently bound coated versions of the column. This support type has a broad range of selectivity not seen in any other types of columns available. At this time, the more stable covalently bound versions are also very good, but just do not measure up to the high success rate this one has. BTW: It is normal to see different results between the covalently bound and coated versions of the same column. They are completely different supports and if budget allows, you can have both types of columns available for screening.

Power and Surge Protection for Computers & Analytical Instruments (e.g. Uninterruptible Power Supply AKA UPS)

(1) When selecting an Uninterruptible Power Supply (UPS) for your computers and analytical instruments, only purchase one which outputs a true sine wave voltage (just like real AC power). Most of the UPS systems sold at the local office and electronic supply stores output either square wave A/C or pseudo-sine wave (which is not the real thing). Some brands will tell you they offer true sine wave outputs right in the model number or package, but always double-check the specifications (e.g. APC's "Smart-UPS" offer sine wave outputs, but NOT the Smart-UPS SC models). Using a UPS that outputs something other than true sine wave power can damage the power supply of what it is attached to and this can lead to premature failure of system you are trying to protect.

(2) Calculate the maximum load of the system being protected in Volt Amps (Line Voltage x Amps = VA) and purchase a system that can provide power well above that load for the time period you want to protect (*I think 20 or 30 minutes of run time, at load, is a realistic guideline to go by). A 700 VA UPS will usually power most desktop computers and a monitor for that amount of time. Most brownouts last for just seconds, but that is enough time to shut everything down or loose communication. If a power outage is more than five minutes in length, then it may be a while before it is restored. In this case, be sure to safely shut everything down manually before you run out of battery reserve power. Once everything has been turned off, remember to turn OFF the UPS system to stop it from slowly draining the battery (most computers are still turned ON and using power when they are OFF). Doing so will preserve the remaining battery capacity. Just turn the UPS back 'ON'  when power has been restored. 

(3) Unless you want to spend really BIG dollars, just put a high quality UPS system on the computer. A high quality UPS system designed for a single high current draw instrument like a chromatograph may cost thousands of dollars. If the system must be maintained at all times, then a serious back up source of energy should be invested in. High current UPS systems are available from a number of manufacturers so it would be wise to investigate these for your application. For everything else, use a dedicated, high quality power conditioner and surge suppressor on the analytical instrument circuits ($85 and up each, not the $20 models sold at most box stores). The computer system is really what you want to protect first. If the computer gets knocked out you loose communication and control of the analytical system. This usually means it will continue to run on and on without stopping. If the computer is protected by a UPS and the power gets glitched (such as a voltage drop/brown-out) or goes out entirely, then the computer may sense this and abort the run or shut the system down (if it has not been shut down). This is the type of approach we have used in our labs for twenty years and it works great during brown outs and black outs. The computers stay on long enough to properly and safely shut down everything without loosing data (which is the key). The instruments are all connected to individual power conditioners/surge suppressors (Tripp lite brand units in our case) so are protected from brown outs and surges. To protect our equipment from large surges, our electrician installed a high amperage DELTA lightning arrestor (100,000 AMP, 3000 joules per pole, unlimited number of surge capacity) on the breaker panel and individual Delta surge capacitors on each critical 20A circuit that supplies power to laboratory equipment. These extras steps provide protection from direct lightning strikes and surges to the panel and equipment. 

(4) The UPS Batteries: The lead acid batteries in your UPS system have a finite life. Mark them with a date and keep records on battery changes. Depending on the quality, temperature and load over time, they may need to be replaced in as little as one year or perhaps last as long as four years. When the batteries loose their ability to hold a charge your on-battery run time will drop dramatically so replace them early rather than late. The battery pack can be (and should be) tested on a regular basis. To test the UPS refer to the Operator's Manual for the correct procedure [*the test often involves selecting a time when the computer is not running any critical applications or providing any networked services (such as right before you would normally shut it off for the day). Once it is safe to do so, go ahead and remove the UPS’s A/C plug from the wall. This will remove the UPS system’s source of input power just as if a real power failure had occurred. Your computer system should behave as if nothing has happened. Monitor how long the system stays on until the available UPS run time shows 15 to 20% remaining time left and you will know how long the system can handle a complete power outage. The system will need overnight to fully recharge after this test and you should repeat this test a few times each year to monitor the status of the system]. *Many of the modern UPS systems provide a software application that continuously monitors the UPS modules which are connected to your computer or entire network. This can provide a record of the systems used, battery change dates and current charge levels right from your desktop.

Determination of HPLC Column Void Volume / Dead Volume, Dead Time (T zero):

Column Hold-up Volume, Column Dead Time or 'Column Void Volume' (the preferred name) are all different terms we apply to find the internal volume of a packed column  (divided by the flow rate and usually expressed in minutes for the Column Void Time). You must know what this value is BEFORE starting to run an HPLC method or perform liquid chromatography. The value for column void volume changes for different column dimensions and different column support types (e.g. fully porous, superficially porous etc) .

Are you peaks or samples eluting at or near the column void volume? If so, for most modes of chromatography, this implies that no chromatography has taken place and no HPLC method has been developed (SEC/GPC separate based on hydrodynamic volume, so elution at or near the column volume means the sample(s) were excluded from the column). Individuals with little to no chromatography training or experience often make this mistake and create methods which show poor retention. Make sure your methods are designed to retain each sample for a long enough time period on the column (K prime). How do you know how long is long enough? Start by estimating the Column Void Volume (use our table or calculate it for an estimate) then, calculate the K prime value for your sample. The K prime for each peak should be at least 1.5 (>2.0 is the accepted standard for most regulatory authorities) for the method to be useful and selective. *A more accurate value of column void volume will be found by measuring the void volume of your column (please read on).

Knowing the Column Void Volume and the Flow Rate used allows you to calculate the Column Void Time (which is the most useful initial value). Determining  the column void time or T0 ("Tee Zero" as we call it), is necessary to find other important chromatography values such as: the Resolution, Separation Factor and Capacity Factor (K prime aka: "K1") in a chromatography separation. Ideally, it is measured by injecting a sample which is unretained by the column & mobile phase (it passes right through the column support with little to no interaction). It may also be easily estimated for most fully porous, spherical, bare or coated silica supports if you know a few physical specifications of the column and media used. You should first estimate it, then measure it (the two values should be close, +/- 15%). Note: A practical "tip". You can also estimate T0 by noting when the small injector valve switching peak ('blip') appears on the baseline. It results from the change from switching the injection valve from the "load" to "inject" positions. Use a low UV wavelength to observe this deflection on the baseline.

Here is short list of typical HPLC column dimensions and their associated estimated void volumes for fully porous silica supports. At a flow rate of 1.000 ml/min these values would also be the same as the void time in minutes.

COLUMN DIMENSIONS (I.D. x Length (mm))                 VOID VOLUME (ml)

                         2.1 x  50                                                                  0.12
                         2.1 x 100                                                                 0.24
                         2.1 x 150                                                                 0.37
                         2.1 x 250                                                                 0.61
                         2.1 x 300                                                                 0.73

                         4.6 x  50                                                                  0.58
                         4.6 x 100                                                                 1.16
                         4.6 x 150                                                                 1.75
                         4.6 x 250                                                                 2.90
                         4.6 x 300                                                                 3.49

                       10.0 x 100                                                                 5.50
                       10.0 x 150                                                                 8.25
                       10.0 x 250                                                               13.75
                       10.0 x 300                                                               16.49

•  Column Void Volume Equation for Std Sized, FULLY Porous Supports:

Column Volume (ul) = (d^2 *Pi * L * 0.7) / 4 ;

•  Column Void Volume Equation for SUPERFICIALLY Porous Supports (e.g. Fused-Core, Core-Shell etc):

Column Volume (ul) = (d^2 *Pi * L * 0.5) / 4 .

   Note: Column Diameter & Length are in mm. Volumes are estimates (always measure to find the actual value).

[Note: All you need is the column's length and ID to estimate it. For most fully porous supports, use a 'Pore Volume' value of 0.70 in the above equation. This is the most commonly measures pore volume found for non-encapped, fully porous spherical bare silica support (please check with the manufacturer for the actual value of your support). For superficially porous supports, use a value of 0.50. Estimating the value will often get you close to the measured value, but due to the unique chemistries used to prepare supports, it is only an approximation.

Always measure the actual void volume of your specific HPLC column with a compound which is unretained by your column. For RP applications which utilize at least 20% organic, Uracil or Thiourea are often used, but some inorganic salts (e.g. sodium nitrite and sodium nitrate) have also been shown to work as well. When determining the "Column Void Volume", you are really measuring the void volume of the column plus any extra-column volume from the injection volume plus all lines connecting the injection to the column and the column to the flow cell. Note: This is very different from the "System Dwell Volume" which includes the volume from the pump (or gradient valve) to the column head.

A more detailed version of this table with other common HPLC Column Sizes and Tubing Volumes for capillary lines are available at the following links (Link #1) or (Link #2).