PAPER CHROMATOGRAPHY

Transcription

1 paper chromatography PAPER CHROMATOGRAPHY This page is an introduction to paper chromatography - including two way chromatography. Carrying out paper 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. In paper chromatography, the stationary phase is a very uniform absorbent paper. The mobile phase is a suitable liquid solvent or mixture of solvents. Producing a paper chromatogram You probably used paper chromatography as one of the first things you ever did in chemistry to separate out mixtures of coloured dyes - for example, the dyes which make up a particular ink. That's an easy example to take, so let's start from there. Suppose you have three blue pens and you want to find out which one was used to write a message. Samples of each ink are spotted on to a pencil line drawn on a sheet of chromatography paper. Some of the ink from the message is dissolved in the minimum possible amount of a suitable solvent, and that is also spotted onto the same line. In the diagram, the pens are labelled 1, 2 and 3, and the message ink as M. 1 of 10 9/16/17, 3:49 PM

2 paper chromatography Note: The chromatography paper will in fact be pure white - not pale grey. I'm forced to show it as off-white because of the way I construct the diagrams. Anything I draw as pure white allows the background colour of the page to show through. The paper is suspended in a container with a shallow layer of a suitable solvent or mixture of solvents in it. It is important that the solvent level is below the line with the spots on it. The next diagram doesn't show details of how the paper is suspended because there are too many possible ways of doing it and it clutters the diagram. Sometimes the paper is just coiled into a loose cylinder and fastened with paper clips top and bottom. The cylinder then just stands in the bottom of the container. The reason for covering the container is to make sure that the atmosphere in the beaker is saturated with solvent vapour. Saturating the atmosphere in the beaker with vapour stops the solvent from evaporating as it rises up the paper. 2 of 10 9/16/17, 3:49 PM

3 paper chromatography As the solvent slowly travels up the paper, the different components of the ink mixtures travel at different rates and the mixtures are separated into different coloured spots. The diagram shows what the plate might look like after the solvent has moved almost to the top. It is fairly easy to see from the final chromatogram that the pen that wrote the message contained the same dyes as pen 2. You can also see that pen 1 contains a mixture of two different blue dyes - one of which might be the same as the single dye in pen 3. R f values Some compounds in a mixture travel almost as far as the solvent does; some stay much closer to the base line. The distance travelled relative to the solvent is a constant for a particular compound as long as you keep everything else constant - the type of paper and the exact composition of the solvent, for example. The distance travelled relative to the solvent is called the R f value. For each compound it can be worked out using the formula: For example, if one component of a mixture travelled of 10 9/16/17, 3:49 PM

4 paper chromatography cm from the base line while the solvent had travelled 12.0 cm, then the R f value for that component is: In the example we looked at with the various pens, it wasn't necessary to measure R f values because you are making a direct comparison just by looking at the chromatogram. You are making the assumption that if you have two spots in the final chromatogram which are the same colour and have travelled the same distance up the paper, they are most likely the same compound. It isn't necessarily true of course - you could have two similarly coloured compounds with very similar R f values. We'll look at how you can get around that problem further down the page. What if the substances you are interested in are colourless? 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. 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 a solution of the mixture is placed on the base line of the paper, and similar small spots of the known amino acids are placed alongside it. The paper 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 position of the solvent front is marked in pencil and 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 4 of 10 9/16/17, 3:49 PM

5 paper chromatography purple. The left-hand diagram shows the paper 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 R f 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. Two way paper chromatography Two way paper chromatography gets around the problem of separating out substances which have very similar R f values. I'm going to go back to talking about coloured compounds because it is much easier to see what is happening. You can perfectly well do this with colourless compounds - but you have to use quite a lot of imagination in the explanation of what is going on! This time a chromatogram is made starting from a single 5 of 10 9/16/17, 3:49 PM

6 paper chromatography spot of mixture placed towards one end of the base line. It is stood in a solvent as before and left until the solvent front gets close to the top of the paper. In the diagram, the position of the solvent front is marked in pencil before the paper dries out. This is labelled as SF1 - the solvent front for the first solvent. We shall be using two different solvents. If you look closely, you may be able to see that the large central spot in the chromatogram is partly blue and partly green. Two dyes in the mixture have almost the same R f values. They could equally well, of course, both have been the same colour - in which case you couldn't tell whether there was one or more dye present in that spot. What you do now is to wait for the paper to dry out completely, and then rotate it through 90, and develop the chromatogram again in a different solvent. It is very unlikely that the two confusing spots will have the same R f values in the second solvent as well as the first, and so the spots will move by a different amount. The next diagram shows what might happen to the various spots on the original chromatogram. The position of the second solvent front is also marked. 6 of 10 9/16/17, 3:49 PM

7 paper chromatography You wouldn't, of course, see these spots in both their original and final positions - they have moved! The final chromatogram would look like this: Two way chromatography has completely separated out the mixture into four distinct spots. If you want to identify the spots in the mixture, you obviously can't do it with comparison substances on the same chromatogram as we looked at earlier with the pens or amino acids examples. You would end up with a meaningless mess of spots. You can, though, work out the R f values for each of the spots in both solvents, and then compare these with values that you have measured for known compounds under exactly the same conditions. How does paper chromatography work? Although paper chromatography is simple to do, it is quite difficult to explain compared with thin layer chromatography. The explanation depends to some extent on what sort of solvent you are using, and many sources gloss over the problem completely. If you haven't already done so, it would be helpful if you could read the explanation for how thin layer chromatography works (link below). That will save me a lot of repetition, and I can concentrate on the problems. Note: You will find the explanation for how thin 7 of 10 9/16/17, 3:49 PM

8 paper chromatography layer chromatography works by following this link. Use the BACK button on your browser to return quickly to this page when yhou have read it. The essential structure of paper Paper is made of cellulose fibres, and cellulose is a polymer of the simple sugar, glucose. The key point about cellulose is that the polymer chains have -OH groups sticking out all around them. To that extent, it presents the same sort of surface as silica gel or alumina in thin layer chromatography. It would be tempting to try to explain paper chromatography in terms of the way that different compounds are adsorbed to different extents on to the paper surface. In other words, it would be nice to be able to use the same explanation for both thin layer and paper chromatography. Unfortunately, it is more complicated than that! The complication arises because the cellulose fibres attract water vapour from the atmosphere as well as any water that was present when the paper was made. You can therefore think of paper as being cellulose fibres with a very thin layer of water molecules bound to the surface. It is the interaction with this water which is the most important effect during paper chromatography. Paper chromatography using a non-polar solvent Suppose you use a non-polar solvent such as hexane to develop your chromatogram. 8 of 10 9/16/17, 3:49 PM

9 paper chromatography Non-polar molecules in the mixture that you are trying to separate will have little attraction for the water molecules attached to the cellulose, and so will spend most of their time dissolved in the moving solvent. Molecules like this will therefore travel a long way up the paper carried by the solvent. They will have relatively high R f values. On the other hand, polar molecules will have a high attraction for the water molecules and much less for the non-polar solvent. They will therefore tend to dissolve in the thin layer of water around the cellulose fibres much more than in the moving solvent. Because they spend more time dissolved in the stationary phase and less time in the mobile phase, they aren't going to travel very fast up the paper. The tendency for a compound to divide its time between two immiscible solvents (solvents such as hexane and water which won't mix) is known as partition. Paper chromatography using a non-polar solvent is therefore a type of partition chromatography. Paper chromatography using a water and other polar solvents A moment's thought will tell you that partition can't be the explanation if you are using water as the solvent for your mixture. If you have water as the mobile phase and the water bound on to the cellulose as the stationary phase, there can't be any meaningful difference between the amount of time a substance spends in solution in either of them. All substances should be equally soluble (or equally insoluble) in both. And yet the first chromatograms that you made were probably of inks using water as your solvent. If water works as the mobile phase as well being the stationary phase, there has to be some quite different mechanism at work - and that must be equally true for other polar solvents like the alcohols, for example. Partition only happens between solvents which don't mix with each other. Polar solvents like the small alcohols do mix with water. In researching this topic, I haven't found any easy explanation for what happens in these cases. Most 9 of 10 9/16/17, 3:49 PM

10 paper chromatography sources ignore the problem altogether and just quote the partition explanation without making any allowance for the type of solvent you are using. Other sources quote mechanisms which have so many strands to them that they are far too complicated for this introductory level. I'm therefore not taking this any further - you shouldn't need to worry about this at UK A level, or its various equivalents. Note: If I have missed something obvious in my research and you know of a straightforward explanation (worth about 1 or 2 marks in an exam) for what happens with water and other polar solvents, could you contact me via the address on the about this site page. Questions to test your understanding If this is the first set of questions you have done, please read the introductory page before you start. You will need to use the BACK BUTTON on your browser to come back here afterwards. questions on paper chromatography answers Where would you like to go now? To the chromatography menu... To the analysis menu... To Main Menu... Jim Clark 2007 (modified July 2016) 10 of 10 9/16/17, 3:49 PM

11 the mass spectrometer - how it works THE MASS SPECTROMETER This page describes how a mass spectrum is produced using a mass spectrometer. How a mass spectrometer works The basic principle If something is moving and you subject it to a sideways force, instead of moving in a straight line, it will move in a curve - deflected out of its original path by the sideways force. Suppose you had a cannonball travelling past you and you wanted to deflect it as it went by you. All you've got is a jet of water from a hose-pipe that you can squirt at it. Frankly, its not going to make a lot of difference! Because the cannonball is so heavy, it will hardly be deflected at all from its original course. But suppose instead, you tried to deflect a table tennis ball travelling at the same speed as the cannonball using the same jet of water. Because this ball is so light, you will get a huge deflection. The amount of deflection you will get for a given sideways force depends on the mass of the ball. If you knew the speed of the ball and the size of the force, you could calculate the mass of the ball if you knew what sort of curved path it was deflected through. The less the deflection, the heavier the ball. Note: I'm not suggesting that you personally would have to do the calculation, although the maths isn't actually very difficult - certainly no more than A'level standard! You can apply exactly the same principle to atomic sized 1 of 9 9/16/17, 3:51 PM

12 the mass spectrometer - how it works particles. An outline of what happens in a mass spectrometer Atoms and molecules can be deflected by magnetic fields - provided the atom or molecule is first turned into an ion. Electrically charged particles are affected by a magnetic field although electrically neutral ones aren't. The sequence is : Stage 1: Ionisation The atom or molecule is ionised by knocking one or more electrons off to give a positive ion. This is true even for things which you would normally expect to form negative ions (chlorine, for example) or never form ions at all (argon, for example). Most mass spectrometers work with positive ions. Note: All mass spectrometers that you will come across if you are doing a course for year olds work with positive ions. Even if a few atoms in a sample of chlorine, for example, captured an electron instead of losing one, the negative ions formed wouldn't get all the way through the ordinary mass spectrometer. But it has been pointed out to me that there is work being done on negative ion mass spectrometers, although they use a different ionisation technique. My thanks to Professor John Todd of the University of Kent for drawing this to my attention. Stage 2: Acceleration The ions are accelerated so that they all have the same kinetic energy. Stage 3: Deflection The ions are then deflected by a magnetic field according to their masses. The lighter they are, the more they are deflected. 2 of 9 9/16/17, 3:51 PM

13 the mass spectrometer - how it works The amount of deflection also depends on the number of positive charges on the ion - in other words, on how many electrons were knocked off in the first stage. The more the ion is charged, the more it gets deflected. Stage 4: Detection The beam of ions passing through the machine is detected electrically. A full diagram of a mass spectrometer Understanding what's going on The need for a vacuum It's important that the ions produced in the ionisation chamber have a free run through the machine without hitting air molecules. Ionisation 3 of 9 9/16/17, 3:51 PM

14 the mass spectrometer - how it works The vaporised sample passes into the ionisation chamber. The electrically heated metal coil gives off electrons which are attracted to the electron trap which is a positively charged plate. The particles in the sample (atoms or molecules) are therefore bombarded with a stream of electrons, and some of the collisions are energetic enough to knock one or more electrons out of the sample particles to make positive ions. Most of the positive ions formed will carry a charge of +1 because it is much more difficult to remove further electrons from an already positive ion. These positive ions are persuaded out into the rest of the machine by the ion repeller which is another metal plate carrying a slight positive charge. Note: As you will see in a moment, the whole ionisation chamber is held at a positive voltage of about 10,000 volts. Where we are talking about the two plates having positive charges, these charges are in addition to that 10,000 volts. Acceleration 4 of 9 9/16/17, 3:51 PM

15 the mass spectrometer - how it works The positive ions are repelled away from the very positive ionisation chamber and pass through three slits, the final one of which is at 0 volts. The middle slit carries some intermediate voltage. All the ions are accelerated into a finely focused beam. Deflection Different ions are deflected by the magnetic field by different amounts. The amount of deflection depends on: the mass of the ion. Lighter ions are deflected more than heavier ones. the charge on the ion. Ions with 2 (or more) positive charges are deflected more than ones with only 1 positive charge. These two factors are combined into the mass/charge ratio. Mass/charge ratio is given the symbol m/z (or sometimes m/e). For example, if an ion had a mass of 28 and a charge of 1+, its mass/charge ratio would be 28. An ion with a mass of 56 and a charge of 2+ would also have a mass/charge ratio of 28. In the last diagram, ion stream A is most deflected - it will contain ions with the smallest mass/charge ratio. Ion 5 of 9 9/16/17, 3:51 PM

16 the mass spectrometer - how it works stream C is the least deflected - it contains ions with the greatest mass/charge ratio. It makes it simpler to talk about this if we assume that the charge on all the ions is 1+. Most of the ions passing through the mass spectrometer will have a charge of 1+, so that the mass/charge ratio will be the same as the mass of the ion. Note: You must be aware of the possibility of 2+ (etc) ions, but the vast majority of A'level questions will give you mass spectra which only involve 1+ ions. Unless there is some hint in the question, you can reasonably assume that the ions you are talking about will have a charge of 1+. Assuming 1+ ions, stream A has the lightest ions, stream B the next lightest and stream C the heaviest. Lighter ions are going to be more deflected than heavy ones. Detection Only ion stream B makes it right through the machine to the ion detector. The other ions collide with the walls where they will pick up electrons and be neutralised. Eventually, they get removed from the mass spectrometer by the vacuum pump. When an ion hits the metal box, its charge is neutralised by an electron jumping from the metal on to the ion (right hand diagram). That leaves a space amongst the electrons in the metal, and the electrons in the wire shuffle along to fill it. 6 of 9 9/16/17, 3:51 PM

17 the mass spectrometer - how it works A flow of electrons in the wire is detected as an electric current which can be amplified and recorded. The more ions arriving, the greater the current. Detecting the other ions How might the other ions be detected - those in streams A and C which have been lost in the machine? Remember that stream A was most deflected - it has the smallest value of m/z (the lightest ions if the charge is 1+). To bring them on to the detector, you would need to deflect them less - by using a smaller magnetic field (a smaller sideways force). To bring those with a larger m/z value (the heavier ions if the charge is +1) on to the detector you would have to deflect them more by using a larger magnetic field. If you vary the magnetic field, you can bring each ion stream in turn on to the detector to produce a current which is proportional to the number of ions arriving. The mass of each ion being detected is related to the size of the magnetic field used to bring it on to the detector. The machine can be calibrated to record current (which is a measure of the number of ions) against m/z directly. The mass is measured on the 12 C scale. Note: The 12 C scale is a scale on which the 12 C isotope weighs exactly 12 units. What the mass spectrometer output looks like The output from the chart recorder is usually simplified into a "stick diagram". This shows the relative current produced by ions of varying mass/charge ratio. The stick diagram for molybdenum looks lilke this: 7 of 9 9/16/17, 3:51 PM

18 the mass spectrometer - how it works You may find diagrams in which the vertical axis is labelled as either "relative abundance" or "relative intensity". Whichever is used, it means the same thing. The vertical scale is related to the current received by the chart recorder - and so to the number of ions arriving at the detector: the greater the current, the more abundant the ion. As you will see from the diagram, the commonest ion has a mass/charge ratio of 98. Other ions have mass/charge ratios of 92, 94, 95, 96, 97 and 100. That means that molybdenum consists of 7 different isotopes. Assuming that the ions all have a charge of 1+, that means that the masses of the 7 isotopes on the carbon-12 scale are 92, 94, 95, 96, 97, 98 and 100. Note: If there were also 2+ ions present, you would know because every one of the lines in the stick diagram would have another line at exactly half its m/z value (because, for example, 98/2 = 49). Those lines would be much less tall than the 1+ ion lines because the chances of forming 2+ ions are much less than forming 1+ ions. If you want to go straight on to how you use these mass spectra to calculate relative atomic masses you can jump straight to that page by following this link rather than going via the menus below. Questions to test your understanding If this is the first set of questions you have done, please read the introductory page before you start. You will need to use the BACK BUTTON on your browser to come back here 8 of 9 9/16/17, 3:51 PM

19 the mass spectrometer - how it works afterwards. questions on how a mass spectrometer works answers Where would you like to go now? To the mass spectrometry menu... To the instrumental analysis menu... To Main Menu... Jim Clark 2000 (last modified February 2015) 9 of 9 9/16/17, 3:51 PM

20 the mass spectra of elements THE MASS SPECTRA OF ELEMENTS This page looks at the information you can get from the mass spectrum of an element. It shows how you can find out the masses and relative abundances of the various isotopes of the element and use that information to calculate the relative atomic mass of the element. It also looks at the problems thrown up by elements with diatomic molecules - like chlorine, Cl 2. The mass spectrum of monatomic elements Monatomic elements include all those except for things like chlorine, Cl 2, with molecules containing more than one atom. The mass spectrum for boron Note: If you need to know how this diagram is obtained, you should read the page describing how a mass spectrometer works. The number of isotopes The two peaks in the mass spectrum shows that there are 2 isotopes of boron - with relative isotopic masses of 1 of 7 9/16/17, 3:52 PM

21 the mass spectra of elements 10 and 11 on the 12 C scale. Notes: Isotopes are atoms of the same element (and so with the same number of protons), but with different masses due to having different numbers of neutrons. We are assuming (and shall do all through this page) that all the ions recorded have a charge of 1+. That means that the mass/charge ratio (m/z) gives you the mass of the isotope directly. The carbon-12 scale is a scale on which the mass of the 12 C isotope weighs exactly 12 units. The abundance of the isotopes The relative sizes of the peaks gives you a direct measure of the relative abundances of the isotopes. The tallest peak is often given an arbitrary height of but you may find all sorts of other scales used. It doesn't matter in the least. You can find the relative abundances by measuring the lines on the stick diagram. In this case, the two isotopes (with their relative abundances) are: boron boron Working out the relative atomic mass The relative atomic mass (RAM) of an element is given the symbol A r and is defined as: The relative atomic mass of an element is the weighted average of the masses of the isotopes on a scale on which a carbon-12 atom has a mass of exactly 12 units. A "weighted average" allows for the fact that there won't be equal amounts of the various isotopes. The example coming up should make that clear. 2 of 7 9/16/17, 3:52 PM

22 the mass spectra of elements Suppose you had 123 typical atoms of boron. 23 of these would be 10 B and 100 would be 11 B. The total mass of these would be (23 x 10) + (100 x 11) = 1330 The average mass of these 123 atoms would be 1330 / 123 = 10.8 (to 3 significant figures) is the relative atomic mass of boron. Notice the effect of the "weighted" average. A simple average of 10 and 11 is, of course, Our answer of 10.8 allows for the fact that there are a lot more of the heavier isotope of boron - and so the "weighted" average ought to be closer to that. The mass spectrum for zirconium The number of isotopes The 5 peaks in the mass spectrum shows that there are 5 isotopes of zirconium - with relative isotopic masses of 90, 91, 92, 94 and 96 on the 12 C scale. The abundance of the isotopes This time, the relative abundances are given as percentages. Again you can find these relative abundances by measuring the lines on the stick diagram. In this case, the 5 isotopes (with their relative percentage abundances) are: zirconium zirconium zirconium of 7 9/16/17, 3:52 PM

23 the mass spectra of elements zirconium zirconium Note: You almost certainly wouldn't be able to measure these peaks to this degree of accuracy, but your examiners may well give you the data in number form anyway. We'll do the sum with the more accurate figures. Working out the relative atomic mass Suppose you had 100 typical atoms of zirconium of these would be 90 Zr, 11.2 would be 91 Zr and so on. Note: If you object to the idea of having 51.5 atoms or 11.2 atoms and so on, just assume you've got 1000 atoms instead of 100. That way you will have 515 atoms, 112 atoms, etc. Most people don't get in a sweat over this, and just use the numbers as they are! The total mass of these 100 typical atoms would be (51.5 x 90) + (11.2 x 91) + (17.1 x 92) + (17.4 x 94) + (2.8 x 96) = The average mass of these 100 atoms would be / 100 = 91.3 (to 3 significant figures) is the relative atomic mass of zirconium. Note: If you want further examples of calculating relative atomic masses from mass spectra, you might like to refer to my book, Calculations in A level Chemistry. The mass spectrum of chlorine 4 of 7 9/16/17, 3:52 PM

24 the mass spectra of elements Chlorine is taken as typical of elements with more than one atom per molecule. We'll look at its mass spectrum to show the sort of problems involved. Chlorine has two isotopes, 35 Cl and 37 Cl, in the approximate ratio of 3 atoms of 35 Cl to 1 atom of 37 Cl. You might suppose that the mass spectrum would look like this: You would be wrong! The problem is that chlorine consists of molecules, not individual atoms. When chlorine is passed into the ionisation chamber, an electron is knocked off the molecule to give a molecular ion, Cl 2 +. These ions won't be particularly stable, and some will fall apart to give a chlorine atom and a Cl + ion. The term for this is fragmentation. If the Cl atom formed isn't then ionised in the ionisation chamber, it simply gets lost in the machine - neither accelerated nor deflected. The Cl + ions will pass through the machine and will give lines at 35 and 37, depending on the isotope and you would get exactly the pattern in the last diagram. The problem is that you will also record lines for the unfragmented Cl 2 + ions. Think about the possible combinations of chlorine-35 and chlorine-37 atoms in a Cl 2 + ion. Both atoms could be 35 Cl, both atoms could be 37 Cl, or you could have one of each sort. That would give you total masses of the Cl 2 + ion of: 5 of 7 9/16/17, 3:52 PM

25 the mass spectra of elements = = = 74 That means that you would get a set of lines in the m/z = 70 region looking like this: These lines would be in addition to the lines at 35 and 37. The relative heights of the 70, 72 and 74 lines are in the ratio 9:6:1. If you know the right bit of maths, it's very easy to show this. If not, don't worry. Just remember that the ratio is 9:6:1. What you can't do is make any predictions about the relative heights of the lines at 35/37 compared with those at 70/72/74. That depends on what proportion of the molecular ions break up into fragments. That's why you've got the chlorine mass spectrum in two separate bits so far. You must realise that the vertical scale in the diagrams of the two parts of the spectrum isn't the same. The overall mass spectrum looks like this: Note: This is based on information from the NIST Chemistry WebBook. NIST is the US National 6 of 7 9/16/17, 3:52 PM

26 the mass spectra of elements Institute of Standards and Technology. Questions to test your understanding If this is the first set of questions you have done, please read the introductory page before you start. You will need to use the BACK BUTTON on your browser to come back here afterwards. questions on the mass spectra of elements answers Where would you like to go now? To the mass spectrometry menu... To the instrumental analysis menu... To Main Menu... Jim Clark 2000 (last modified February 2014) 7 of 7 9/16/17, 3:52 PM

27 mass spectra - fragmentation patterns FRAGMENTATION PATTERNS IN THE MASS SPECTRA OF ORGANIC COMPOUNDS This page looks at how fragmentation patterns are formed when organic molecules are fed into a mass spectrometer, and how you can get information from the mass spectrum. The origin of fragmentation patterns The formation of molecular ions When the vaporised organic sample passes into the ionisation chamber of a mass spectrometer, it is bombarded by a stream of electrons. These electrons have a high enough energy to knock an electron off an organic molecule to form a positive ion. This ion is called the molecular ion - or sometimes the parent ion. Note: If you aren't sure about how a mass spectrum is produced, it might be worth taking a quick look at the page describing how a mass spectrometer works. The molecular ion is often given the symbol M + or - the dot in this second version represents the fact that somewhere in the ion there will be a single unpaired electron. That's one half of what was originally a pair of electrons - the other half is the electron which was removed in the ionisation process. Fragmentation The molecular ions are energetically unstable, and some of them will break up into smaller pieces. The simplest case is that a molecular ion breaks into two parts - one of which is another positive ion, and the other is an 1 of 11 9/16/17, 3:52 PM

28 mass spectra - fragmentation patterns uncharged free radical. Note: A free radical is an atom or group of atoms which contains a single unpaired electron. More complicated break-ups are beyond the scope of A'level syllabuses. The uncharged free radical won't produce a line on the mass spectrum. Only charged particles will be accelerated, deflected and detected by the mass spectrometer. These uncharged particles will simply get lost in the machine - eventually, they get removed by the vacuum pump. The ion, X +, will travel through the mass spectrometer just like any other positive ion - and will produce a line on the stick diagram. All sorts of fragmentations of the original molecular ion are possible - and that means that you will get a whole host of lines in the mass spectrum. For example, the mass spectrum of pentane looks like this: Note: All the mass spectra on this page have been drawn using data from the Spectral Data Base System for Organic Compounds (SDBS) at the National Institute of Materials and Chemical Research in Japan. 2 of 11 9/16/17, 3:52 PM

29 mass spectra - fragmentation patterns They have been simplified by omitting all the minor lines with peak heights of 2% or less of the base peak (the tallest peak). It's important to realise that the pattern of lines in the mass spectrum of an organic compound tells you something quite different from the pattern of lines in the mass spectrum of an element. With an element, each line represents a different isotope of that element. With a compound, each line represents a different fragment produced when the molecular ion breaks up. Note: If you are interested in the mass spectra of elements, you could follow this link. The molecular ion peak and the base peak In the stick diagram showing the mass spectrum of pentane, the line produced by the heaviest ion passing through the machine (at m/z = 72) is due to the molecular ion. Note: You have to be a bit careful about this, because in some cases, the molecular ion is so unstable that every single one of them splits up, and none gets through the machine to register in the mass spectrum. You are very unlikely to come across such a case at A'level. The tallest line in the stick diagram (in this case at m/z = 43) is called the base peak. This is usually given an arbitrary height of 100, and the height of everything else is measured relative to this. The base peak is the tallest peak because it represents the commonest fragment ion to be formed - either because there are several ways in which it could be produced during fragmentation of the parent ion, or because it is a particularly stable ion. 3 of 11 9/16/17, 3:52 PM

30 mass spectra - fragmentation patterns Using fragmentation patterns This section will ignore the information you can get from the molecular ion (or ions). That is covered in three other pages which you can get at via the mass spectrometry menu. You will find a link at the bottom of the page. Working out which ion produces which line This is generally the simplest thing you can be asked to do. The mass spectrum of pentane Let's have another look at the mass spectrum for pentane: What causes the line at m/z = 57? How many carbon atoms are there in this ion? There can't be 5 because 5 x 12 = 60. What about 4? 4 x 12 = 48. That leaves 9 to make up a total of 57. How about C 4 H 9 + then? C 4 H 9 + would be [CH 3 CH 2 CH 2 CH 2 ] +, and this would be produced by the following fragmentation: The methyl radical produced will simply get lost in the machine. The line at m/z = 43 can be worked out similarly. If you play around with the numbers, you will find that this corresponds to a break producing a 3-carbon ion: 4 of 11 9/16/17, 3:52 PM

31 mass spectra - fragmentation patterns The line at m/z = 29 is typical of an ethyl ion, [CH 3 CH 2 ] + : The other lines in the mass spectrum are more difficult to explain. For example, lines with m/z values 1 or 2 less than one of the easy lines are often due to loss of one or more hydrogen atoms during the fragmentation process. You are very unlikely to have to explain any but the most obvious cases in an A'level exam. The mass spectrum of pentan-3-one This time the base peak (the tallest peak - and so the commonest fragment ion) is at m/z = 57. But this isn't produced by the same ion as the same m/z value peak in pentane. If you remember, the m/z = 57 peak in pentane was produced by [CH 3 CH 2 CH 2 CH 2 ] +. If you look at the structure of pentan-3-one, it's impossible to get that particular fragment from it. Work along the molecule mentally chopping bits off until you come up with something that adds up to 57. With a small amount of patience, you'll eventually find [CH 3 CH 2 CO] + - which is produced by this fragmentation: You would get exactly the same products whichever side of the CO group you split the molecular ion. 5 of 11 9/16/17, 3:52 PM

32 mass spectra - fragmentation patterns The m/z = 29 peak is produced by the ethyl ion - which once again could be formed by splitting the molecular ion either side of the CO group. Peak heights and the stability of ions The more stable an ion is, the more likely it is to form. The more of a particular sort of ion that's formed, the higher its peak height will be. We'll look at two common examples of this. Examples involving carbocations (carbonium ions) Important! If you don't know what a carbocation (or carbonium ion) is, or why the various sorts vary in stability, it's essential that you follow this link before you go on. Use the BACK button on your browser to return quickly to this page. Summarizing the most important conclusion from the page on carbocations: Order of stability of carbocations primary < secondary < tertiary Note: The symbol "<" means "is less than". So what this is saying is that primary ions are less stable than secondary ones which in turn are less stable than tertiary ones. Applying the logic of this to fragmentation patterns, it means that a split which produces a secondary 6 of 11 9/16/17, 3:52 PM

33 mass spectra - fragmentation patterns carbocation is going to be more successful than one producing a primary one. A split producing a tertiary carbocation will be more successful still. Let's look at the mass spectrum of 2-methylbutane. 2-methylbutane is an isomer of pentane - isomers are molecules with the same molecular formula, but a different spatial arrangement of the atoms. Look first at the very strong peak at m/z = 43. This is caused by a different ion than the corresponding peak in the pentane mass spectrum. This peak in 2-methylbutane is caused by: The ion formed is a secondary carbocation - it has two alkyl groups attached to the carbon with the positive charge. As such, it is relatively stable. The peak at m/z = 57 is much taller than the corresponding line in pentane. Again a secondary carbocation is formed - this time, by: You would get the same ion, of course, if the left-hand CH 3 group broke off instead of the bottom one as we've drawn it. In these two spectra, this is probably the most dramatic 7 of 11 9/16/17, 3:52 PM

34 mass spectra - fragmentation patterns example of the extra stability of a secondary carbocation. Examples involving acylium ions, [RCO] + Ions with the positive charge on the carbon of a carbonyl group, C=O, are also relatively stable. This is fairly clearly seen in the mass spectra of ketones like pentan- 3-one. The base peak, at m/z=57, is due to the [CH 3 CH 2 CO] + ion. We've already discussed the fragmentation that produces this. Note: There are lots of other examples of positive ions with extra stability and which are produced in large numbers in a mass spectrometer as a result. Without making this article even longer than it already is, it's impossible to cover every possible case. Check past exam papers to find out whether you are likely to need to know about other possibilities. If you haven't got past papers, follow the link on the syllabuses page to find out how to get hold of them. Using mass spectra to distinguish between compounds 8 of 11 9/16/17, 3:52 PM

35 mass spectra - fragmentation patterns Suppose you had to suggest a way of distinguishing between pentan-2-one and pentan-3-one using their mass spectra. pentan-2-one CH 3 COCH 2 CH 2 CH 3 pentan-3-one CH 3 CH 2 COCH 2 CH 3 Each of these is likely to split to produce ions with a positive charge on the CO group. In the pentan-2-one case, there are two different ions like this: [CH 3 CO] + [COCH 2 CH 2 CH 3 ] + That would give you strong lines at m/z = 43 and 71. With pentan-3-one, you would only get one ion of this kind: [CH 3 CH 2 CO] + In that case, you would get a strong line at 57. You don't need to worry about the other lines in the spectra - the 43, 57 and 71 lines give you plenty of difference between the two. The 43 and 71 lines are missing from the pentan-3-one spectrum, and the 57 line is missing from the pentan-2-one one. Note: Don't confuse the line at m/z = 58 in the pentan-2-one spectrum. That's due to a complicated rearrangement which you couldn't possibly predict at A'level. The two spectra look like this: 9 of 11 9/16/17, 3:52 PM

36 mass spectra - fragmentation patterns Computer matching of mass spectra As you've seen, the mass spectrum of even very similar organic compounds will be quite different because of the different fragmentations that can occur. Provided you have a computer data base of mass spectra, any unkown spectrum can be computer analysed and simply matched against the data base. Questions to test your understanding If this is the first set of questions you have done, please read the introductory page before you start. You will need to use the BACK BUTTON on your browser to come back here afterwards. questions on fragmentation patterns answers 10 of 11 9/16/17, 3:52 PM

37 mass spectra - fragmentation patterns Where would you like to go now? To the mass spectrometry menu... To the instrumental analysis menu... To Main Menu... Jim Clark 2000 (modified February 2014) 11 of 11 9/16/17, 3:52 PM

38 mass spectra - the molecular ion (M+) peak MASS SPECTRA - THE MOLECULAR ION (M + ) PEAK This page explains how to find the relative formula mass (relative molecular mass) of an organic compound from its mass spectrum. It also shows how high resolution mass spectra can be used to find the molecular formula for a compound. Using a mass spectrum to find relative formula mass The formation of molecular ions When the vaporised organic sample passes into the ionisation chamber of a mass spectrometer, it is bombarded by a stream of electrons. These electrons have a high enough energy to knock an electron off an organic molecule to form a positive ion. This ion is called the molecular ion. Note: If you aren't sure about how a mass spectrum is produced, it might be worth taking a quick look at the page describing how a mass spectrometer works. The molecular ion is often given the symbol M + or - the dot in this second version represents the fact that somewhere in the ion there will be a single unpaired electron. That's one half of what was originally a pair of electrons - the other half is the electron which was removed in the ionisation process. The molecular ions tend to be unstable and some of them break into smaller fragments. These fragments produce the familiar stick diagram. Fragmentation is irrelevant to what we are talking about on this page - all we're interested in is the molecular ion. 1 of 1 9/16/17, 3:53 PM

39 mass spectra - the M+1 peak MASS SPECTRA - THE M+1 PEAK This page explains how the M+1 peak in a mass spectrum can be used to estimate the number of carbon atoms in an organic compound. Note: This is a small corner of mass spectrometry. It would be a good idea not to attack this page unless you have a reasonable idea about how a mass spectrum is produced and the sort of information you can get from it. If you haven't already done so, explore the mass spectrometry menu before you go on. What causes the M+1 peak? What is an M+1 peak? If you had a complete (rather than a simplified) mass spectrum, you will find a small line 1 m/z unit to the right of the main molecular ion peak. This small peak is called the M+1 peak. In questions at this level (UK A level or its equivalent), the M+1 peak is often left out to avoid confusion - particularly if you were being asked to find the relative formula mass of the compound from the molecular ion peak. 1 of 5 9/16/17, 3:54 PM

40 mass spectra - the M+1 peak The carbon-13 isotope The M+1 peak is caused by the presence of the 13 C isotope in the molecule. 13 C is a stable isotope of carbon - don't confuse it with the 14 C isotope which is radioactive. Carbon-13 makes up 1.11% of all carbon atoms. If you had a simple compound like methane, CH 4, approximately 1 in every 100 of these molecules will contain carbon-13 rather than the more common carbon-12. That means that 1 in every 100 of the molecules will have a mass of 17 (13 + 4) rather than 16 (12 + 4). The mass spectrum will therefore have a line corresponding to the molecular ion [ 13 CH 4 ] + as well as [ 12 CH 4 ] +. The line at m/z = 17 will be much smaller than the line at m/z = 16 because the carbon-13 isotope is much less common. Statistically you will have a ratio of approximately 1 of the heavier ions to every 99 of the lighter ones. That's why the M+1 peak is much smaller than the M+ peak. Using the M+1 peak What happens when there is more than 1 carbon atom in the compound? Imagine a compound containing 2 carbon atoms. Either of them has an approximately 1 in 100 chance of being 13 C. There's therefore a 2 in 100 chance of the molecule as a whole containing one 13 C atom rather than a 12 C atom - which leaves a 98 in 100 chance of both atoms being 2 of 5 9/16/17, 3:54 PM

41 mass spectra - the M+1 peak 12 C. That means that the ratio of the height of the M+1 peak to the M+ peak will be approximately 2 : 98. That's pretty close to having an M+1 peak approximately 2% of the height of the M+ peak. Note: You might wonder why both atoms can't be carbon-13, giving you an M+2 peak. They can - and do! But statistically the chance of both carbons being 13 C is approximately 1 in 10,000. The M+2 peak will be so small that you couldn't observe it. Using the relative peak heights to predict the number of carbon atoms If there are small numbers of carbon atoms If you measure the peak height of the M+1 peak as a percentage of the peak height of the M+ peak, that gives you the number of carbon atoms in the compound. We've just seen that a compound with 2 carbons will have an M+1 peak approximately 2% of the height of the M+ peak. Similarly, you could show that a compound with 3 carbons will have the M+1 peak at about 3% of the height of the M+ peak. With larger numbers of carbon atoms The approximations we are making won't hold with more than 2 or 3 carbons. The proportion of carbon atoms which are 13 C isn't 1% - it's 1.11%. And the appoximation that a ratio of 2 : 98 is about 2% doesn't hold as the small number increases. Consider a molecule with 5 carbons in it. You could work out that 5.55 (5 x 1.11) molecules will contain 1 13 C to every ( ) which contain only 12 C atoms. If you convert that to how tall the M+1 peak is as a percentage of the M+ peak, you get an answer of 5.9% (5.55/94.45 x 100). That's close enough to 6% that you 3 of 5 9/16/17, 3:54 PM

42 mass spectra - the M+1 peak might assume wrongly that there are 6 carbon atoms. Above 3 carbon atoms, then, you shouldn't really be making the approximation that the height of the M+1 peak as a percentage of the height of the M+ peak tells you the number of carbons - you will need to do some fiddly sums! Important! Most syllabuses at this level (UK A level or its equivalent) don't ask for this. Before you get too bogged down in all this, check your syllabus and recent past papers to see whether you need to bother. This is a case where you really need to know what line your examiners are currently taking - for example, how far are they pushing the approximation. As far as I am aware, the only UK based syllabus to want this is Cambridge International (CIE). I have covered this specifically on this page in my notes for the CIE syllabus But to be sure, if you are doing a UK-based exam, and haven't got a syllabus and past papers, follow this link to find out how to get them. Questions to test your understanding I am not asking any questions about this page because I can't find any peak height data for the small simple molecules for which the approximation works. Without that, I can only produce completely trivial questions. If you are doing CIE exams, you will find plenty of examples from their past papers using their slightly more complicated formula. Otherwise, just be aware that the M+1 peak is due to the presence of C-13 atoms. Where would you like to go now? To the mass spectrometry menu... To the instrumental analysis menu... 4 of 5 9/16/17, 3:54 PM

43 mass spectra - the M+1 peak To Main Menu... Jim Clark 2000 (updated August 2014) 5 of 5 9/16/17, 3:54 PM

44 mass spectra - the M+2 peak MASS SPECTRA - THE M+2 PEAK This page explains how the M+2 peak in a mass spectrum arises from the presence of chlorine or bromine atoms in an organic compound. It also deals briefly with the origin of the M+4 peak in compounds containing two chlorine atoms. Note: Before you start this page, it would be a good idea to have a reasonable understanding about how a mass spectrum is produced and the sort of information you can get from it. If you haven't already done so, explore the mass spectrometry menu before you go on. The effect of chlorine or bromine atoms on the mass spectrum of an organic compound Compounds containing chlorine atoms One chlorine atom in a compound Note: All the mass spectra on this page have been drawn using data from the Spectral Data Base System for Organic Compounds (SDBS) at the National Institute of Materials and Chemical 1 of 5 9/16/17, 3:54 PM

45 mass spectra - the M+2 peak Research in Japan. With one exception, they have been simplified by omitting all the minor lines with peak heights of 2% or less of the base peak (the tallest peak). The molecular ion peaks (M+ and M+2) each contain one chlorine atom - but the chlorine can be either of the two chlorine isotopes, 35 Cl and 37 Cl. The molecular ion containing the 35 Cl isotope has a relative formula mass of 78. The one containing 37 Cl has a relative formula mass of 80 - hence the two lines at m/z = 78 and m/z = 80. Notice that the peak heights are in the ratio of 3 : 1. That reflects the fact that chlorine contains 3 times as much of the 35 Cl isotope as the 37 Cl one. That means that there will be 3 times more molecules containing the lighter isotope than the heavier one. So... if you look at the molecular ion region, and find two peaks separated by 2 m/z units and with a ratio of 3 : 1 in the peak heights, that tells you that the molecule contains 1 chlorine atom. You might also have noticed the same pattern at m/z = 63 and m/z = 65 in the mass spectrum above. That pattern is due to fragment ions also containing one chlorine atom - which could either be 35 Cl or 37 Cl. The fragmentation that produced those ions was: Note: If you aren't sure about fragmentation you might like to have a look at this link. Two chlorine atoms in a compound 2 of 5 9/16/17, 3:54 PM

46 mass spectra - the M+2 peak Note: This spectrum has been simplified by omitting all the minor lines with peak heights of less than 1% of the base peak (the tallest peak). This contains more minor lines than other mass spectra in this section. It was necessary because otherwise an important line in the molecular ion region would have been missing. The lines in the molecular ion region (at m/z values of 98, 100 ands 102) arise because of the various combinations of chlorine isotopes that are possible. The carbons and hydrogens add up to 28 - so the various possible molecular ions could be: = = = 102 If you have the necessary maths, you could show that the chances of these arrangements occurring are in the ratio of 9:6:1 - and this is the ratio of the peak heights. If you don't know the right bit of maths, just learn this ratio! So... if you have 3 lines in the molecular ion region (M+, M+2 and M+4) with gaps of 2 m/z units between them, and with peak heights in the ratio of 9:6:1, the compound contains 2 chlorine atoms. Compounds containing bromine atoms 3 of 5 9/16/17, 3:54 PM

47 mass spectra - the M+2 peak Bromine has two isotopes, 79 Br and 81 Br in an approximately 1:1 ratio (50.5 : 49.5 if you want to be fussy!). That means that a compound containing 1 bromine atom will have two peaks in the molecular ion region, depending on which bromine isotope the molecular ion contains. Unlike compounds containing chlorine, though, the two peaks will be very similar in height. The carbons and hydrogens add up to 29. The M+ and M+2 peaks are therefore at m/z values given by: = = 110 So... if you have two lines in the molecular ion region with a gap of 2 m/z units between them and with almost equal heights, this shows the presence of a bromine atom in the molecule. Questions to test your understanding If this is the first set of questions you have done, please read the introductory page before you start. You will need to use the BACK BUTTON on your browser to come back here afterwards. questions on the M+2 peak answers 4 of 5 9/16/17, 3:54 PM