Download lecturers Notes, word doc on chromatography and more Lecture notes Chemistry in PDF only on Docsity!
Chromatography
THIN LAYER CHROMATOGRAPHY, TLC
Chromatography is used to separate mixtures of substances into their components. All forms of chromatography work on the same principle. They all have a stationary phase (a solid, or a liquid supported on a solid) and a mobile phase (a liquid or a gas). The mobile phase flows through the stationary phase and carries the components of the mixture with it. Different components travel at different rates. We'll look at the reasons for this further down the page. Thin layer chromatography is done exactly as it says - using a thin, uniform layer of silica gel or alumina coated onto a piece of glass, metal or rigid plastic. The silica gel (or the alumina) is the stationary phase. The stationary phase for thin layer chromatography also often contains a substance which fluoresces in UV light - for reasons you will see later. The mobile phase is a suitable liquid solvent or mixture of solvents. Producing the chromatogram For example: To show that a particular dye is a mixture of simpler dyes.
A pencil line is drawn near the bottom of the plate and a small drop of a solution of the dye mixture is placed on it. Any labelling on the plate to show the original position of the drop must also be in pencil. If any of this was done in ink, dyes from the ink would also move as the chromatogram developed. When the spot of mixture is dry, the plate is stood in a shallow layer of solvent in a covered beaker. It is important that the solvent level is below the line with the spot on it. The reason for covering the beaker is to make sure that the atmosphere in the beaker is saturated with solvent vapour. To help this, the beaker is often lined with some filter paper soaked in solvent. Saturating the atmosphere in the beaker with vapour stops the solvent from evaporating as it rises up the plate. As the solvent slowly travels up the plate, the different components of the dye mixture travel at different rates and the mixture is separated into different
before it has a chance to evaporate. These measurements are then taken: The Rf value for each dye is then worked out using the formula: For example, if the red component travelled 1.7 cm from the base line while the solvent had travelled 5.0 cm, then the Rf value for the red dye is: If you could repeat this experiment under exactly the same conditions, then the Rf values for each dye would always be the same. For example, the Rf
value for the red dye would always be 0.34. However, if anything changes (the temperature, the exact composition of the solvent, and so on), that is no longer true. What if the substances you are interested in are colourless? There are two simple ways of getting around this problem. Using fluorescence The stationary phase on a thin layer plate often has a substance added to it which will fluoresce when exposed to UV light. That means that if you shine UV light on it, it will glow. That glow is masked at the position where the spots are on the final chromatogram - even if those spots are invisible to the eye. That means that if you shine UV light on the plate, it will all glow apart from where the spots are. The spots show up as darker patches.
along with a few iodine crystals. The iodine vapour in the container may either react with the spots on the chromatogram, or simply stick more to the spots than to the rest of the plate. Either way, the substances you are interested in may show up as brownish spots. Using thin layer chromatography to identify compounds Suppose you had a mixture of amino acids and wanted to find out which particular amino acids the mixture contained. For simplicity we'll assume that you know the mixture can only possibly contain five of the common amino acids. A small drop of the mixture is placed on the base line of the thin layer plate, and similar small spots of the known amino acids are placed alongside it. The plate is then stood in a suitable solvent and left to develop as before. In the diagram, the mixture is M, and the known amino acids are labelled 1 to 5. The left-hand diagram shows the plate after the solvent front has almost reached the top. The spots are still invisible. The second diagram shows what it might look like after spraying with ninhydrin.
There is no need to measure the Rf values because you can easily compare the spots in the mixture with those of the known amino acids - both from their positions and their colours. In this example, the mixture contains the amino acids labelled as 1, 4 and 5. And what if the mixture contained amino acids other than the ones we have used for comparison? There would be spots in the mixture which didn't match those from the known amino acids. You would have to re-run the experiment using other amino acids for comparison. How does thin layer chromatography work? The stationary phase - silica gel
silica gel or alumina on a glass, metal or plastic plate. Column chromatography works on a much larger scale by packing the same materials into a vertical glass column. Various sizes of chromatography columns are used, and if you follow a link at the bottom of the page to the Organic Chemistry section of the Colorado University site, you will find photographs of various columns. In a school lab, it is often convenient to use an ordinary burette as a chromatography column. Using the column Suppose you wanted to separate a mixture of two coloured compounds - one yellow, one blue. The mixture looks green. You would make a concentrated solution of the mixture preferably
in the solvent used in the column. First you open the tap to allow the solvent already in the column to drain so that it is level with the top of the packing material, and then add the solution carefully to the top of the column. Then you open the tap again so that the coloured mixture is all absorbed into the top of the packing material, so that it might look like this: Next you add fresh solvent to the top of the column, trying to disturb the packing material as little as possible. Then you open the tap so that the solvent can flow down through the column, collecting it in a beaker or flask at the bottom. As the solvent runs through, you keep adding fresh solvent to the top so that the column never dries out.
perhaps even has the ability to hydrogen bond. You can tell this because the blue compound doesn't travel through the column very quickly. That means that it must adsorb more strongly to the silica gel or alumina than the yellow one. The less polar yellow one spends more of its time in the solvent and therefore washes through the column much faster. The process of washing a compound through a column using a solvent is known as elution. The solvent is sometimes known as the eluent. What if you want to collect the blue compound as well? It is going to take ages to wash the blue compound through at the rate it is travelling at the moment! However, there is no reason why you can't change the solvent during elution. Suppose you replace the solvent you have been using by a more polar solvent once the yellow has all been collected. That will have two effects, both of which will speed the blue compound through the column. The polar solvent will compete for space on the silica gel or alumina with the blue compound. Any space temporarily occupied by solvent molecules on the surface of the stationary phase isn't available for blue molecules to stick to and this will tend to keep them moving along in the solvent. There will be a greater attraction between the polar solvent molecules and the polar blue molecules. This will tend to attract any blue molecules sticking to the stationary phase back into solution. The net effect is that with a more polar solvent, the blue compound
spends more time in solution, and so moves faster. So why not use this alternative solvent in the first place? The answer is that if both of the compounds in the mixture travel quickly through the column right from the beginning, you probably won't get such a good separation. What if everything in your mixture is colourless? If you were going to use column chromatography to purify the product of an organic preparation, it is quite likely that the product that you want will be colourless even if one or more of the impurities is coloured. Let's assume the worst case that everything is colourless. How do you know when the substance you want has reached the bottom of the column? There is no quick and easy way of doing this! What you do is collect what comes out of the bottom of the column in a whole series of labelled tubes. How big each sample is will obviously depend on how big the column is - you might collect 1 cm 3 samples or 5 cm 3 samples or whatever is appropriate. You can then take a drop from each solution and make a thin layer chromatogram from it. You would place the drop on the base line alongside a drop from a pure sample of the compound that you are making. By doing this repeatedly, you can identify which of your samples collected at the bottom of the column contain the desired product, and only the desired product. Once you know this, you can combine all of the samples which contain your pure product, and then remove the solvent. (How you would separate the solvent from the product isn't directly relevant to
detection methods which can be used. These methods are highly automated and extremely sensitive. The column and the solvent Confusingly, there are two variants in use in HPLC depending on the relative polarity of the solvent and the stationary phase. Normal phase HPLC It isn't the most commonly used form of HPLC. The column is filled with tiny silica particles, and the solvent is non-polar
- hexane, for example. A typical column has an internal diameter of 4.6 mm (and may be less than that), and a length of 150 to 250 mm. Polar compounds in th e mixture being passed through the column will stick longer to the polar silica than non-polar compounds will. The non-polar ones will therefore pass more quickly through the column. Reversed phase HPLC In this case, the column size is the same, but the silica is modified to make it non-polar by attaching long hydrocarbon chains to its surface
- Typically with either 8 or 18 carbon atoms in them. A polar solvent is used - for example, a mixture of water and an alcohol such as methanol. In this case, there will be a strong attraction between the polar solvent and
polar molecules in the mixture being passed through the column. There won't be as much attraction between the hydrocarbon chains attached to the silica (the stationary phase) and the polar molecules in the solution. Polar molecules in the mixture will therefore spend most of their time moving with the solvent. Non-polar compounds in the mixture will tend to form attractions with the hydrocarbon groups because of van der Waals dispersion forces. They will also be less soluble in the solvent because of the need to break hydrogen bonds as they squeeze in between the water or methanol molecules, for example. They therefore spend less time in solution in the solvent and this will slow them down on their way through the column. That means that now it is the polar molecules that will travel through the column more quickly. Reversed phase HPLC is the most commonly used form of HPLC. A flow scheme for HPLC
For a particular compound, the retention time will vary depending on: the pressure used (because that affects the flow rate of the solvent) the nature of the stationary phase (not only what material it is made of, but also particle size) the exact composition of the solvent the temperature of the column That means that conditions have to be carefully controlled if one is using retention times as a way of identifying compounds. The detector There are several ways of detecting when a substance has passed through the column. A common method which is easy to explain uses ultra-violet absorption. Many organic compounds absorb UV light of various wavelengths. If one have a beam of UV light shining through the stream of liquid coming out of the column, and a UV detector on the opposite side of the stream, one can get a direct reading of how much of the light is absorbed. The amount of light absorbed will depend on the amount of a particular compound that is passing through the beam at the time.
Do the solvents used don't absorb UV light? They do! But different compounds absorb most strongly in different parts of the UV spectrum. Methanol, for example, absorbs at wavelengths below 205 nm, and water below 190 nm. If one is using a methanol-water mixture as the solvent, one would therefore have to use a wavelength greater than 205 nm to avoid false readings from the solvent. Interpreting the output from the detector The output will be recorded as a series of peaks – Each one representing a compound in the mixture passing through the detector and absorbing UV light. As long as the conditions on the column were carefully controlled, the retention times can be used to identify the compounds present - provided, one had already measured them for pure samples of the various compounds under those identical conditions. But one can also use the peaks as a way of measuring the quantities of the
