INFRA-RED SPECTRUM

WHAT IS AN INFRA-RED SPECTRUM? This page describes what an infra-red spectrum is and how it arises from bond vibrations within organic molecules. The background to infra-red spectroscopy How an infra-red spectrum is produced You probably know that visible light is made up of a continuous range of different electromagnetic frequencies - each frequency can be seen as a different colour. Infra-red radiation also consists of a continuous range of frequencies - it so happens that our eyes can't detect them. If you shine a range of infra-red frequencies one at a time through a sample of an organic compound, you find that some frequencies get absorbed by the compound. A detector on the other side of the compound would show that some frequencies pass through the compound with almost no loss, but other frequencies are strongly absorbed. How much of a particular frequency gets through the compound is measured as percentage transmittance. A percentage transmittance of 100 would mean that all of that frequency passed straight through the compound without any being absorbed. In practice, that never happens - there is always some small loss, giving a transmittance of perhaps 95% as the best you can achieve. A transmittance of only 5% would mean that nearly all of that particular frequency is absorbed by the compound. A very high absorption of this sort tells you important things about the bonds in the compound. What an infra-red spectrum looks like A graph is produced showing how the percentage transmittance varies with the frequency of the infra-red radiation.
	Note:  The infra-red spectra on this page have been produced from graphs taken from the Spectral Data Base System for Organic Compounds (SDBS) at the National Institute of Materials and Chemical Research in Japan. It is possible that small errors may have been introduced during the process of converting them for use on this site, but these won't affect the argument in any way.
Notice that an unusual measure of frequency is used on the horizontal axis. Wavenumber is defined like this: Don't worry about this - just accept it! Similarly, don't worry about the change of scale half-way across the horizontal axis. You will find infra-red spectra where the scale is consistent all the way across, infra-red spectra where the scale changes at around 2000 cm-1, and very occasionally where the scale changes again at around 1000 cm-1. As you will see when we look at how to interpret infra-red spectra, this doesn't cause any problems - you simply need to be careful reading the horizontal scale. What causes some frequencies to be absorbed? Each frequency of light (including infra-red) has a certain energy. If a particular frequency is being absorbed as it passes through the compound being investigated, it must mean that its energy is being transferred to the compound. Energies in infra-red radiation correspond to the energies involved in bond vibrations. Bond stretching In covalent bonds, atoms aren't joined by rigid links - the two atoms are held together because both nuclei are attracted to the same pair of electrons. The two nuclei can vibrate backwards and forwards - towards and away from each other - around an average position. The diagram shows the stretching that happens in a carbon-oxygen single bond. There will, of course, be other atoms attached to both the carbon and the oxygen. For example, it could be the carbon-oxygen bond in methanol, CH3OH. The energy involved in this vibration depends on things like the length of the bond and the mass of the atoms at either end. That means that each different bond will vibrate in a different way, involving different amounts of energy. Bonds are vibrating all the time, but if you shine exactly the right amount of energy on a bond, you can kick it into a higher state of vibration. The amount of energy it needs to do this will vary from bond to bond, and so each different bond will absorb a different frequency (and hence energy) of infra-red radiation. Bond bending As well as stretching, bonds can also bend. The diagram shows the bending of the bonds in a water molecule. The effect of this, of course, is that the bond angle between the two hydrogen-oxygen bonds fluctuates slightly around its average value. Imagine a lab model of a water molecule where the atoms are joined together with springs. These bending vibrations are what you would see if you shook the model gently. Again, bonds will be vibrating like this all the time and, again, if you shine exactly the right amount of energy on the bond, you can kick it into a higher state of vibration. Since the energies involved with the bending will be different for each kind of bond, each different bond will absorb a different frequency of infra-red radiation in order to make this jump from one state to a higher one. Tying all this together Look again at the infra-red spectrum of propan-1-ol, CH3CH2CH2OH: In the diagram, three sample absorptions are picked out to show you the bond vibrations which produced them. Notice that bond stretching and bending produce different troughs in the spectrum.
	THE FINGERPRINT REGION OF AN INFRA-RED SPECTRUM This page explains what the fingerprint region of an infra-red spectrum is, and how it can be used to identify an organic molecule. Note:  It would be helpful if you first read the introductory page on infra-red spectra if you haven't already done so. What is the fingerprint region This is a typical infra-red spectrum: Note:  The infra-red spectra on this page have been produced from graphs taken from the Spectral Data Base System for Organic Compounds (SDBS) at the National Institute of Materials and Chemical Research in Japan. It is possible that small errors may have been introduced during the process of converting them for use on this site, but these won't affect the argument in any way. Each trough is caused because energy is being absorbed from that particular frequency of infra-red radiation to excite bonds in the molecule to a higher state of vibration - either stretching or bending. Some of the troughs are easily used to identify particular bonds in a molecule. For example, the big trough at the left-hand side of the spectrum is used to identify the presence of an oxygen-hydrogen bond in an -OH group. Note:  Using troughs in this way to identify particular bonds is covered on a separate page. INTERPRETING AN INFRA-RED SPECTRUM This page explains how to use an infra-red spectrum to identify the presence of a few simple bonds in organic compounds. Note:  This page follows directly on from the introductory page on infra-red spectra. If you haven't already done so, you should read that page before you go on. The infra-red spectrum for a simple carboxylic acid Ethanoic acid Ethanoic acid has the structure: You will see that it contains the following bonds: carbon-oxygen double, C=O carbon-oxygen single, C-O oxygen-hydrogen, O-H carbon-hydrogen, C-H carbon-carbon single, C-C The carbon-carbon bond has absorptions which occur over a wide range of wavenumbers in the fingerprint region - that makes it very difficult to pick out on an infra-red spectrum. The carbon-oxygen single bond also has an absorbtion in the fingerprint region, varying between 1000 and 1300 cm-1 depending on the molecule it is in. You have to be very wary about picking out a particular trough as being due to a C-O bond. Note:  If you aren't sure what the fingerprint region is, follow this link before you go on. The other bonds in ethanoic acid have easily recognised absorptions outside the fingerprint region. The C-H bond (where the hydrogen is attached to a carbon which is singly-bonded to everything else) absorbs somewhere in the range from 2853 - 2962 cm-1. Because that bond is present in most organic compounds, that's not terribly useful! What it means is that you can ignore a trough just under 3000 cm-1, because that is probably just due to C-H bonds. The carbon-oxygen double bond, C=O, is one of the really useful absorptions, found in the range 1680 - 1750 cm-1. Its position varies slightly depending on what sort of compound it is in. The other really useful bond is the O-H bond. This absorbs differently depending on its environment. It is easily recognised in an acid because it produces a very broad trough in the range 2500 - 3300 cm-1. Note:  You will never have to remember where these absorptions occur. You will always be given this data in an A'level exam. The infra-red spectrum for ethanoic acid looks like this: The possible absorption due to the C-O single bond is queried because it lies in the fingerprint region. You couldn't be sure that this trough wasn't caused by something else. Note:  The infra-red spectra on this page have been produced from graphs taken from the Spectral Data Base System for Organic Compounds (SDBS) at the National Institute of Materials and Chemical Research in Japan. It is possible that small errors may have been introduced during the process of converting them for use on this site, but these won't affect the argument in any way. The infra-red spectrum for an alcohol Ethanol The O-H bond in an alcohol absorbs at a higher wavenumber than it does in an acid - somewhere between 3230 - 3550 cm-1. In fact this absorption would be at a higher number still if the alcohol isn't hydrogen bonded - for example, in the gas state. All the infra-red spectra on this page are from liquids - so that possibility will never apply. Notice the absorption due to the C-H bonds just under 3000 cm-1, and also the troughs between 1000 and 1100 cm-1 - one of which will be due to the C-O bond. The infra-red spectrum for an ester Ethyl ethanoate This time the O-H absorption is missing completely. Don't confuse it with the C-H trough fractionally less than 3000 cm-1. The presence of the C=O double bond is seen at about 1740 cm-1. The C-O single bond is the absorption at about 1240 cm-1. Whether or not you could pick that out would depend on the detail given by the table of data which you get in your exam, because C-O single bonds vary anywhere between 1000 and 1300 cm-1 depending on what sort of compound they are in. Some tables of data fine it down, so that they will tell you that an absorption from 1230 - 1250 is the C-O bond in an ethanoate. The infra-red spectrum for a ketone Propanone You will find that this is very similar to the infra-red spectrum for ethyl ethanoate, an ester. Again, there is no trough due to the O-H bond, and again there is a marked absorption at about 1700 cm-1 due to the C=O. Confusingly, there are also absorptions which look as if they might be due to C-O single bonds - which, of course, aren't present in propanone. This reinforces the care you have to take in trying to identify any absorptions in the fingerprint region. Aldehydes will have similar infra-red spectra to ketones. The infra-red spectrum for a hydroxy-acid 2-hydroxypropanoic acid (lactic acid) This is interesting because it contains two different sorts of O-H bond - the one in the acid and the simple "alcohol" type in the chain attached to the -COOH group. The O-H bond in the acid group absorbs between 2500 and 3300, the one in the chain between 3230 and 3550 cm-1. Taken together, that gives this immense trough covering the whole range from 2500 to 3550 cm-1. Lost in that trough as well will be absorptions due to the C-H bonds. Notice also the presence of the strong C=O absorption at about 1730 cm-1. The infra-red spectrum for a primary amine 1-aminobutane Primary amines contain the -NH2 group, and so have N-H bonds. These absorb somewhere between 3100 and 3500 cm-1. That double trough (typical of primary amines) can be seen clearly on the spectrum to the left of the C-H absorptions.

Gas-Liquid Chromatography

GAS-LIQUID CHROMATOGRAPHY Gas-liquid chromatography (often just called gas chromatography) is a powerful tool in analysis. It has all sorts of variations in the way it is done - if you want full details, a Google search on gas chromatography will give you scary amounts of information if you need it! This page just looks in a simple introductory way at how it can be carried out. Carrying out gas-liquid chromatography Introduction All forms of chromatography involve a stationary phase and a mobile phase. In all the other forms of chromatography you will meet at this level, the mobile phase is a liquid. In gas-liquid chromatography, the mobile phase is a gas such as helium and the stationary phase is a high boiling point liquid absorbed onto a solid. How fast a particular compound travels through the machine will depend on how much of its time is spent moving with the gas as opposed to being attached to the liquid in some way. A flow scheme for gas-liquid chromatography
	Note:  You will have to imagine the coiled column in its oven. Drawing a convincing and tidy coil defeated me completely!
Injection of the sample Very small quantities of the sample that you are trying to analyse are injected into the machine using a small syringe. The syringe needle passes through a thick rubber disc (known as a septum) which reseals itself again when the syringe is pulled out. The injector is contained in an oven whose temperature can be controlled. It is hot enough so that all the sample boils and is carried into the column as a gas by the helium (or other carrier gas). How the column works The packing material There are two main types of column in gas-liquid chromatography. One of these is a long thin tube packed with the stationary phase; the other is even thinner and has the stationary phase bonded to its inner surface. To keep things simple, we are just going to look at the packed column. The column is typically made of stainless steel and is between 1 and 4 metres long with an internal diameter of up to 4 mm. It is coiled up so that it will fit into a thermostatically controlled oven. The column is packed with finely ground diatomaceous earth, which is a very porous rock. This is coated with a high boiling liquid - typically a waxy polymer. The column temperature The temperature of the column can be varied from about 50°C to 250°C. It is cooler than the injector oven, so that some components of the mixture may condense at the beginning of the column. In some cases, as you will see below, the column starts off at a low temperature and then is made steadily hotter under computer control as the analysis proceeds. How separation works on the column One of three things might happen to a particular molecule in the mixture injected into the column: It may condense on the stationary phase. It may dissolve in the liquid on the surface of the stationary phase. It may remain in the gas phase. None of these things is necessarily permanent. A compound with a boiling point higher than the temperature of the column will obviously tend to condense at the start of the column. However, some of it will evaporate again in the same way that water evaporates on a warm day - even though the temperature is well below 100°C. The chances are that it will then condense again a little further along the column. Similarly, some molecules may dissolve in the liquid stationary phase Some compounds will be more soluble in the liquid than others. The more soluble ones will spend more of their time absorbed into the stationary phase; the less soluble ones will spend more of their time in the gas. The process where a substance divides itself between two immiscible solvents because it is more soluble in one than the other is known as partition. Now, you might reasonably argue that a gas such as helium can't really be described as a "solvent". But the term partition is still used in gas-liquid chromatography. You can say that a substance partitions itself between the liquid stationary phase and the gas. Any molecule in the substance spends some of its time dissolved in the liquid and some of its time carried along with the gas. Retention time The time taken for a particular compound to travel through the column to the detector is known as its retention time. This time is measured from the time at which the sample is injected to the point at which the display shows a maximum peak height for that compound. Different compounds have different retention times. For a particular compound, the retention time will vary depending on: the boiling point of the compound. A compound which boils at a temperature higher than the column temperature is going to spend nearly all of its time condensed as a liquid at the beginning of the column. So high boiling point means a long retention time. the solubility in the liquid phase. The more soluble a compound is in the liquid phase, the less time it will spend being carried along by the gas. High solubility in the liquid phase means a high retention time. the temperature of the column. A higher temperature will tend to excite molecules into the gas phase - either because they evaporate more readily, or because they are so energetic that the attractions of the liquid no longer hold them. A high column temperature shortens retention times for everything in the column. For a given sample and column, there isn't much you can do about the boiling points of the compounds or their solubility in the liquid phase - but you do have control over the temperature. The lower the temperature of the column, the better the separation you will get - but it could take a very long time to get the compounds through which are condensing at the beginning of the column! On the other hand, using a high temperature, everything will pass through the column much more quickly - but less well separated out. If everything passed through in a very short time, there isn't going to be much space between their peaks on the chromatogram. The answer is to start with the column relatively cool, and then gradually and very regularly increase the temperature. At the beginning, compounds which spend most of their time in the gas phase will pass quickly through the column and be detected. Increasing the temperature a bit will encourage the slightly "stickier" compounds through. Increasing the temperature still more will force the very "sticky" molecules off the stationary phase and through the column. The detector There are several different types of detector in use. The flame ionisation detector described below is commonly used and is easier to describe and explain than the alternatives. A flame ionisation detector In terms of reaction mechanisms, the burning of an organic compound is very complicated. During the process, small amounts of ions and electrons are produced in the flame. The presence of these can be detected. The whole detector is enclosed in its own oven which is hotter than the column temperature. That stops anything condensing in the detector.
	Note:  This is simplified for clarity. There obviously has to be some way of lighting the flame. This is done with an electrically heated coil, but including it clutters the diagram.
If there is nothing organic coming through from the column, you just have a flame of hydrogen burning in air. Now suppose that one of the compounds in the mixture you are analysing starts to come through. As it burns, it will produce small amounts of ions and electrons in the flame. The positive ions will be attracted to the cylindrical cathode. Negative ions and electrons will be attracted towards the jet itself which is the anode. This is much the same as what happens during normal electrolysis. At the cathode, the positive ions will pick up electrons from the cathode and be neutralised. At the anode, any electrons in the flame will transfer to the positive electrode; and negative ions will give their electrons to the electrode and be neutralised. This loss of electrons from one electrode and gain at the other will result in a flow of electrons in the external circuit from the anode to the cathode. In other words, you get an electric current. The current won't be very big, but it can be amplified. The more of the organic compound there is in the flame, the more ions will be produced, and so the higher the current will be. As a reasonable approximation, especially if you are talking about similar compounds, the current you measure is proportional to the amount of compound in the flame. Disadvantages of the flame ionisation detector The main disadvantage is that it destroys everything coming out of the column as it detects it. If you wanted to send the product to a mass spectrometer, for example, for further analysis, you couldn't use a flame ionisation detector. 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. As long as you were careful to control the conditions on the column, you could use the retention times to help to identify the compounds present - provided, of course, that you (or somebody else) had already measured them for pure samples of the various compounds under those identical conditions. But you can also use the peaks as a way of measuring the relative quantities of the compounds present. This is only accurate if you are analysing mixtures of similar compounds - for example, of similar hydrocarbons. The areas under the peaks are proportional to the amount of each compound which has passed the detector, and these areas can be calculated automatically by the computer linked to the display. The areas it would measure are shown in green in the (very simplified) diagram. Note that it isn't the peak height that matters, but the total area under the peak. In this particular example, the left-hand peak is both tallest and has the greatest area. That isn't necessarily always so. There might be a lot of one compound present, but it might emerge from the column in relatively small amounts over quite a long time. Measuring the area rather than the peak height allows for this. Coupling a gas chromatogram to a mass spectrometer This can't be done with a flame ionisation detector which destroys everything passing through it. Assuming you are using a non-destructive detector . . . When the detector is showing a peak, some of what is passing through the detector at that time can be diverted to a mass spectrometer. There it will give a fragmentation pattern which can be compared against a computer database of known patterns. That means that the identity of a huge range of compounds can be found without having to know their retention times.
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High Performance Liquid Chromatography

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY - HPLC High performance liquid chromatography is a powerful tool in analysis. This page looks at how it is carried out and shows how it uses the same principles as in thin layer chromatography and column chromatography.
Carrying out HPLC Introduction High performance liquid chromatography is basically a highly improved form of column chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres. That makes it much faster. It also allows you to use a very much smaller particle size for the column packing material which gives a much greater surface area for interactions between the stationary phase and the molecules flowing past it. This allows a much better separation of the components of the mixture. The other major improvement over column chromatography concerns the 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 This is essentially just the same as you will already have read about in thin layer chromatography or column chromatography. Although it is described as "normal", 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 the 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.
	Note:  I have been a bit careful about how I have described the attractions of the non-polar molecules to the surface of the stationary phase. In particular, I have avoided the use of the word "adsorpion". Adsorption is when a molecule sticks to the surface of a solid. Especially if you had small molecules in your mixture, some could get in between the long C18 chains to give what is essentially a solution. You could therefore say that non-polar molecules were more soluble in the hydrocarbon on the surface of the silica than they are in the polar solvent - and so spend more time in this alternative "solvent". Where a solute divides itself between two different solvents because it is more soluble in one than the other, we call it partition. So is this adsorption or partition? You could argue it both ways! Be prepared to find it described as either.
Looking at the whole process A flow scheme for HPLC Injection of the sample Injection of the sample is entirely automated, and you wouldn't be expected to know how this is done at this introductory level. Because of the pressures involved, it is not the same as in gas chromatography (if you have already studied that). Retention time The time taken for a particular compound to travel through the column to the detector is known as its retention time. This time is measured from the time at which the sample is injected to the point at which the display shows a maximum peak height for that compound. Different compounds have different retention times. 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 you are 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 you 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, you 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. You might wonder why 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 you were using a methanol-water mixture as the solvent, you 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 you were careful to control the conditions on the column, you could use the retention times to help to identify the compounds present - provided, of course, that you (or somebody else) had already measured them for pure samples of the various compounds under those identical conditions. But you can also use the peaks as a way of measuring the quantities of the compounds present. Let's suppose that you are interested in a particular compound, X. If you injected a solution containing a known amount of pure X into the machine, not only could you record its retention time, but you could also relate the amount of X to the peak that was formed. The area under the peak is proportional to the amount of X which has passed the detector, and this area can be calculated automatically by the computer linked to the display. The area it would measure is shown in green in the (very simplified) diagram. If the solution of X was less concentrated, the area under the peak would be less - although the retention time will still be the same. For example: This means that it is possible to calibrate the machine so that it can be used to find how much of a substance is present - even in very small quantities. Be careful, though! If you had two different substances in the mixture (X and Y) could you say anything about their relative amounts? Not if you were using UV absorption as your detection method. In the diagram, the area under the peak for Y is less than that for X. That may be because there is less Y than X, but it could equally well be because Y absorbs UV light at the wavelength you are using less than X does. There might be large quantities of Y present, but if it only absorbed weakly, it would only give a small peak.
Coupling HPLC to a mass spectrometer This is where it gets really clever! When the detector is showing a peak, some of what is passing through the detector at that time can be diverted to a mass spectrometer. There it will give a fragmentation pattern which can be compared against a computer database of known patterns. That means that the identity of a huge range of compounds can be found without having to know their retention times.
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COLUMN CHROMATOGRAPHY

COLUMN CHROMATOGRAPHY This page shows how the same principles used in thin layer chromatography can be applied on a larger scale to separate mixtures in column chromatography. Column chromatography is often used to purify compounds made in the lab.
Carrying out column chromatography The column In thin layer chromatography, the stationary phase is a thin layer of 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. The next set of diagrams shows what might happen over time.
Explaining what is happening This assumes that you have read the explanation for what happens during thin layer chromatography. If you haven't, follow the very first link at the top of the page and come back to this point afterwards. The blue compound is obviously more polar than the yellow one - it 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 cm3 samples or 5 cm3 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 this topic and would vary depending on their exact nature - so I'm not even going to attempt a generalisation.)

THIN LAYER CHROMATOGRAPHY

THIN LAYER CHROMATOGRAPHY This page is an introduction to chromatography using thin layer chromatography as an example. Although if you are a beginner you may be more familiar with paper chromatography, thin layer chromatography is equally easy to describe and more straightforward to explain.
Carrying out thin layer chromatography Background 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 We'll start with a very simple case - just trying to show that a particular dye is in fact 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 coloured spots. The diagram shows the plate after the solvent has moved about half way up it. The solvent is allowed to rise until it almost reaches the top of the plate. That will give the maximum separation of the dye components for this particular combination of solvent and stationary phase. Measuring Rf values If all you wanted to know is how many different dyes made up the mixture, you could just stop there. However, measurements are often taken from the plate in order to help identify the compounds present. These measurements are the distance travelled by the solvent, and the distance travelled by individual spots. When the solvent front gets close to the top of the plate, the plate is removed from the beaker and the position of the solvent is marked with another line 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. You have to bear this in mind if you want to use this technique to identify a particular dye. We'll look at how you can use thin layer chromatography for analysis further down the page. What if the substances you are interested in are colourless? There are two simple ways of getting around this problem. Using fluorescence You may remember that I mentioned that 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. While the UV is still shining on the plate, you obviously have to mark the positions of the spots by drawing a pencil circle around them. As soon as you switch off the UV source, the spots will disappear again. Showing the spots up chemically In some cases, it may be possible to make the spots visible by reacting them with something which produces a coloured product. A good example of this is in chromatograms produced from amino acid mixtures. The chromatogram is allowed to dry and is then sprayed with a solution of ninhydrin. Ninhydrin reacts with amino acids to give coloured compounds, mainly brown or purple. In another method, the chromatogram is again allowed to dry and then placed in an enclosed container (such as another beaker covered with a watch glass) 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 is a form of silicon dioxide (silica). The silicon atoms are joined via oxygen atoms in a giant covalent structure. However, at the surface of the silica gel, the silicon atoms are attached to -OH groups.
So, at the surface of the silica gel you have Si-O-H bonds instead of Si-O-Si bonds. The diagram shows a small part of the silica surface. The surface of the silica gel is very polar and, because of the -OH groups, can form hydrogen bonds with suitable compounds around it as well as van der Waals dispersion forces and dipole-dipole attractions.
The other commonly used stationary phase is alumina - aluminium oxide. The aluminium atoms on the surface of this also have -OH groups attached. Anything we say about silica gel therefore applies equally to alumina. What separates the compounds as a chromatogram develops? As the solvent begins to soak up the plate, it first dissolves the compounds in the spot that you have put on the base line. The compounds present will then tend to get carried up the chromatography plate as the solvent continues to move upwards. How fast the compounds get carried up the plate depends on two things: How soluble the compound is in the solvent. This will depend on how much attraction there is between the molecules of the compound and those of the solvent. How much the compound sticks to the stationary phase - the silica get, for example. This will depend on how much attraction there is between the molecules of the compound and the silica gel. Suppose the original spot contained two compounds - one of which can form hydrogen bonds, and one of which can only take part in weaker van der Waals interactions. The one which can hydrogen bond will stick to the surface of the silica gel more firmly than the other one. We say that one is adsorbed more strongly than the other. Adsorption is the name given to one substance forming some sort of bonds to the surface of another one. Adsorption isn't permanent - there is a constant movement of a molecule between being adsorbed onto the silica gel surface and going back into solution in the solvent. Obviously the compound can only travel up the plate during the time that it is dissolved in the solvent. While it is adsorbed on the silica gel, it is temporarily stopped - the solvent is moving on without it. That means that the more strongly a compound is adsorbed, the less distance it can travel up the plate. In the example we started with, the compound which can hydrogen bond will adsorb more strongly than the one dependent on van der Waals interactions, and so won't travel so far up the plate. What if both components of the mixture can hydrogen bond? It is very unlikely that both will hydrogen bond to exactly the same extent, and be soluble in the solvent to exactly the same extent. It isn't just the attraction of the compound for the silica gel which matters. Attractions between the compound and the solvent are also important - they will affect how easily the compound is pulled back into solution away from the surface of the silica. However, it may be that the compounds don't separate out very well when you make the chromatogram. In that case, changing the solvent may well help - including perhaps changing the pH of the solvent. This is to some extent just a matter of trial and error - if one solvent or solvent mixture doesn't work very well, you try another one. (Or, more likely, given the level you are probably working at, someone else has already done all the hard work for you, and you just use the solvent mixture you are given and everything will work perfectly!)