How to Perform TLC

Most people who are new recruits to the research labs learned their basic skills for carrying out reactions in an undergraduate laboratory. Inevitably, most of the organic chemistry undertaken in these lab classes involves following recipes, which have been well tried and tested. Therefore the conditions and the time taken for the reactions to reach completion arc well established, and work-up can be carried out after a preset time. Unfortunately, the idea that you can guess the time it takes for a reaction to reach completion is a very bad habit to carry over into a research environment.

Every reaction you carry out should be monitored, and one of the first things you should do before starting any reaction is to decide on a suitable method for monitoring its progress. Even if you are following a literature procedure, reaction monitoring is still essential and it will usually save you time as well as giving you confidence about what is happening. Carrying out a reaction without monitoring its progress is like trying to thread a needle with your eyes closed!

The simplest and most universal method of reaction monitoring is thin layer chromatography (tic) and this will be discussed first of all, but it is not always the best or only method, and sometimes you may have to use a little ingenuity to find an appropriate reaction monitoring technique.

1 Thin Layer Chromatography (TLC)

Tic is a simple, but extremely powerful analytical tool. However it may take a little time before your expertise reaches a consistently high level since a certain element of intui-tion is always involved in choosing the appropriate solvent system, spotting the correct amount of sample, etc. Once you have gained experience and confidcnce in the use of tic, you will find it extremely useful for a variety of purposes.

1. 1 The Main Uses of TLC

Tic is normally the simplest and quickest way to monitor a reaction and the reaction mixture should be chromatographed against starting materials (and a co-spot). This allows you to follow how the reaction is progressing, and to assess when is the best time to work it up. In all cases a record of the tic should be made in your lab book.

Tic can be used to indicate the identity of a compound, by comparing the unknown sample, with a known material. In general each substance is spotted separately and also together (co-spot). Caution should be applied as co-running on tic is not definitive proof of identity. Of course, substances that do not co-run are definitely not the same.

TLC usually gives a good indication of the purity of a substance. Diastereoisomers can usually (but not always), be distinguished.

For flash chromatography, tic is first used to determine the solvent system and quantity of silica required, and secondly to monitor the column fractions.


USES OF ANALYTICAL HPLC

Finding a tic system and running a sample can be done very quickly and for this reason
tic is the normal method of choice for routine reaction monitoring. However, there are occasions when it is worth spending the time to set up an hplc system for reaction monitoring, especially if, as in many modern synthetic labs you have a system close to hand. One reason to use hplc is that the compounds in which you are interested do not separate very well on tic. The other common reason is that you require a quantitative technique. This may be the case if you are trying to optimize a reaction to maximize the quantity of one product over another, and for this type of extended study it is well worth the time it takes to set up the system. For most synthetic purposes it is the relative rather than the absolute proportions of substances which are important, and if that is the case a simple comparison of integrated peak areas may be all that is required. If accurate quantification is needed then a calibration is required and this can be done using an internal standard (see below). Another common use of hplc is for identification of a compound by comparison with a known substance. Under a specific set of conditions (solvent, flow rate and quantity applied) any compound will have a specific retention time and this can therefore be used as a characteristic of the compound. However, just as a mixed spot should be always be run when comparing substances on tic, so with hplc a single enhanced peak should be observed when the comparison substance and the unknown are injected as a mixture. Again caution should be used, since a single peak is not absolute proof that compounds are the same.

Preparative hplc is now becoming widely used in organic chemistry for separating compounds with, very similar polarity. Before committing all your material to a preparative column it is always best to run a small quantity of the sample on an analytical column, in order to work out the best conditions. Indeed, columns are produced in various sizes which are directly comparable with one another.

If you are monitoring a reaction by hplc and you want to know the identity of one or more of the productsyou can often separate a few milligrams from a few runs on the analytical column, which is enough to get a full range of spectral data. On simple hplc systems this can be done manually by collecting the effluent from the column when the peak of interest is coming off, and repeating several times. On more sophisticated systems a fraction collector is often incorporated and in some cases injections can be made automatically, so that the system can be set up to collect a particular peak or peaks over a large number of runs.

Several methods are now available for coupling an hplc system to a mass spectrometer, so that a mass spectrum is produced for each peak thus providing some structural information.


HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

There is a broad range of hplc techniques, and many different types of equipment. It is beyond the scope of this book to describe in great detail the methods for operating the equipment. This section will therefore focus on some of the ways that hplc can be used to aid the synthetical organic chemist.

Description of hplc

The general arrangement of an hplc system is fairly simple.

Solvent is pumped from a reservoir through a piston pump, which controls the flow rate. From the pump the solvent passes through a pulse damper, which removes some of the pulsing effect generated in the pump and also acts as a pressure regulator. Between the pulse damper and the column there is an injection valve which allows the sample to be introduced into the solvent stream. In the ‘load’ mode the solvent by-passes a sample loop, into which the sample is injected from a syringe. On switching to 4 inject, the solvent stream is diverted through the load loop, introducing a very accurately measured volume of the sample solution onto the column. Components are separated on the hplc column in exactly the same way as they would be on a tic plate? The less polar compounds running faster and coming through first. The effluent from the column passes into a detector (usually an ultraviolet or refractive index) which produces a signal on the chart recorder when a component is present.

The time at which the compound comes off the column is characteristic of that particular material, and is referred to as the retention time.

The area under any peak on the chart recorder is proportional to the quantity of that component and the method is therefore quantitative.



Recent Posts

Recent Comments

    Archives

    Categories

    Meta