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1 Principles and Applications of NMR spectroscopy Professor Hanudatta S. Atreya NMR Research Centre Indian Institute of Science Bangalore Module 1 Lecture No 05 Welcome back! In the last class we looked at different aspects of NMR spectrometer and what are its different components. Given a NMR spectrometer, once we record the data, we will now onwards focus on how actually we analyze the data and how do we interpret the data; This will be from a chemist point of view because NMR spectroscopy is a very important tool for chemistry. We will look at some of the chemicals and the simple NMR experiments which can be done on chemical compounds, and we will look at how to analyze them. (Refer Slide Time: 1:03)

2 The next few classes are going to focus on chemical shifts and what is called spin-spin coupling or J-coupling. Let us start from chemical shifts. Typically a NMR spectrum consists of the following four parameters: chemical shifts, peak integral, J-coupling and linewidth. The first thing what we look at is chemical shifts. This lies at the heart of NMR spectroscopy. Chemical shifts are directly related to the structure of the molecule and also give information about different functional groups present such as Alkyl, Alkene, Benzyl, Aldehyde etc. They are measured in the units called ppm ; we will shortly see as to how the ppm scale is generated. The next parameter to look for in a NMR spectrum is the integral or the area of the peak. If you recall, we mentioned a few classes back that the area of the peak is directly related to the number of hydrogen atoms or number of protons giving rise to that peak. Therefore integral is important if you want to quantify or measure the number of hydrogens. Another parameter is called spin-spin splitting also known as J coupling or scalar coupling. The J coupling takes place between hydrogen atoms which interact with each other through intervening bonds. We will look at this shortly. Coupling constants also give information about the structure/arrangement of atoms. The J-coupling pattern can deciphered by just looking at the peak structure but the value of the coupling constant gives you information about the structure of the molecule. Let us look at what are chemical shifts, from where do they actually originate. Every molecule has atoms, which have nucleus and the nucleus is surrounded by an electron cloud.

3 (Refer Slide Time: 3:23) Shown here is an electron cloud. This electron cloud, which surrounds the nucleus generates a small magnetic field opposite to the main magnetic field because of the motion of electrons. Remember electrons are also charged particles. They are negatively charged; when they move around they generate a magnetic field similar to the external magnetic field, which may or may not be in the same direction as the main magnetic field. So, the effective magnetic field around this nucleus is no longer B 0, but reduced/increased slightly because of the additional magnetic field. This phenomenon is called shielding. Shielding means protecting, so essentially these electrons which are around the nucleus try to protect this nucleus or shield and it is basically denoted by these parameter/symbol:. The extent of shielding is small and results in a very small deviation of the order of 10-6 from than the main magnetic field. The notion of ppm (parts per million) typically comes from here because it is a very small deviation from the main magnetic field. (Refer Slide Time: 5:55)

4 Let us see how does this chemical shift vary for different atoms. Look at this structure now CH3-CH2-OH, which is ethanol, a standard compound. What happens is that the shielding effect for proton of OH is slightly different from the shielding caused by the electrons around CH2 hydrogen, which is again different from the extent of shielding caused around the nucleus of the CH3 hydrogen. The extent of shielding depends on the local structure such as presence of electron withdrawing or electron donating groups. Now recall from the previous slides that resonance frequency, = B0. So, if the effective B0 is different for each hydrogen the for each hydrogen also differs. The is basically the frequency of Larmor precession or the energy difference between the alpha and beta states. So, we can say that the protons of CH3 have a different resonance frequency compared to CH2 hydrogens and compared to the proton of the OH moiety. We can see that there is a gradual change of the energy gap and therefore the resonance frequency varies. So the point in this slide is to see that each type of hydrogen in a given molecule has a different local magnetic field. Thus, when we talk about shielding we talk about the local magnetic field. Why local? Because the main magnetic field is same for all of them but locally around each nucleus, there is shielding which is different from atom to atom. That is how the chemical shifts are exploited to look at the structure of molecules. If all these hydrogens, if all of them had no or same shielding, then all of them would look alike and there would a single peak; that would make NMR very uninteresting and of no use. The reason why NMR is so popular today is because the local magnetic field changes between

5 different hydrogens in the same molecule and that change is small but good enough to separate them into different positions and that gives information about the structure of the molecule. (Refer Slide Time: 8:47) Now let us see how the ppm scale is calculated. For understanding this one should keep in mind that chemical shifts is always based on a reference. Why do we use a reference? the reference is used because if we record an NMR spectrum at some particular spectrometer, let us say, we have a 300 Megahertz spectrometer in the laboratory and we take the same molecule or sample and go to a 700 Megahertz spectrometer in some other laboratory we should not get any difference in chemical shifts. Because chemical shifts are inherently related to the structure of the molecule and since structure of a molecule is given and does not change from one spectrometer to another, chemical shifts should also not change. Therefore to preserve this uniformity of chemical shifts we use a reference and with respect to reference the chemical shifts are calibrated. This is a very important point that chemical shifts do not change with the field strength. So, now see let us see how it is calculated. Look at this equation given on the slide below. Suppose we have a reference molecule, that particular molecule will have a proton which has a resonance frequency, which can denote as ref. We can set the shielding factor of the reference proton, ref=0 so that ref= B0. The proton of our interest has some shielding factor, which we can denote as. So, what we do is we then subtract the two equations shown. So, if

6 you do a simple mathematical operation of subtraction and rearrange the equation, the can be written like as shown. (Refer Slide Time: 11:26) When we put in these values we see that will be of the order of 10-6 ok and it is unitless. This is very important because what we see from these formula is that we subtract two frequencies and divide by another frequency. When you divide the two parameters which have the same same units, we get a parameter which is unit less. So, the shielding factor is parts per million because it is of the order of (Refer Slide Time: 12:51)

7 Now let us say that we want do a simple calculation. Let us say we ask the question if I have a difference in chemical shift of two nuclei which are one ppm difference how can we calculate the difference in terms of Hertz. This is a very important conversion, which we do routinely in NMR (that we often need to convert ppm scale into Hertz scale and vice versa). Conversion of a ppm into Hertz is very simple. We can go through the algebra and what basically happens is the following. Let us say we have two chemical shifts, two peaks which differ in chemical shifts by one ppm; remember one ppm means one into 10-6 parts. If you want to convert this in Hz scale, all we have to do is we want to simply subtract the two equations shown in the slide above and multiply with the reference frequency, which is the spectrometer frequency ( = B0). Let us suppose that we are working at 500 Megahertz, when we multiply 1 ppm with 500 MHz, we get 500 Hertz. So, what this means is that if I have a spectrometer operating at 500 Megahertz in my laboratory and I record a proton spectrum and I get two peaks which are separated by one ppm, the separation between those two peaks in Hertz scale will be 500 Hertz. Let us say we have two ppm difference. If the difference is two ppm, then two ppm will become 1000 Hertz. So one has to simply take the spectrometer frequency without the Megahertz (e.g., 500 for 500 Megahertz) and multiply it by the difference in the ppm value (without considering the 10-6 factor) and that will give you the difference in the Hertz scale. So, what it means is that the difference in ppm value does not change between two peaks, but their difference in Hertz scale changes, because the spectrometer frequency is required to be multiplied with. If I go to 700 Megahertz two peaks with 1 ppm difference will be separated in by 700 Hertz. If I go to 1 GHz, then it will be 1000 Hertz. (Refer Slide Time: 16:24)

8 This increased separation of peaks with increasing magnetic field is actually what helps you in increasing the resolution. This is what is shown in this slide below. If you see what is shown is that the chemical shift value or difference in ppm does not change with the field strength but the precessional frequency changes between two peaks. So, why is that so important? The point here is that when I increase the separation between the peaks, basically what I am doing is I am separating the peaks more and more, which means two peaks which are close by (or overlapped) at 400 MHz can be separated better or resolved at 800 MHz. If you go from 300 to to 600 MHz you keep increasing the resolution between the peaks. Therefore it is important to go to higher magnetic fields if in case the resolution is not sufficient at lower field strengths. Typically for chemists and those working in the area of peptide NMR, up to 600 Megahertz is more than sufficient. But when we study biomolecules such as proteins and nucleic acids and carbohydrates, we need high resolution. That is why those working in the area of biomoleculer NMR prefer to use high magnetic field like 800 Megahertz or one Gigahertz compared to the organic chemists, for whom 600 MHz and below is sufficient for most practical purposes. (Refer Slide Time: 18:39)

9 This is what is shown in this slide. So, if I start from 0 Mega Hertz and increase the spectrometer frequency to 250 Mega Hertz, 500 Mega Hertz, 750 Mega Hertz or 900 Mega Hertz you see the peaks get well separated. However, if we convert this into into ppm scale, we subtract the frequencies and divide by again a frequency and what you get is that the peak separation does not change in the ppm scale, in the Delta scale. Whether you go to 250 Megahertz or you look at the spectrum at 900 Megahertz you get the same ppm value. However, if you look at the Hertz scale, frequency value changes. This is the important concept which has to be kept in mind. (Refer Slide Time: 20:05)

10 The next point in NMR spectrum, which we saw, is the area of the peak. So, that is what is shown here. It is the area which determines the number of protons. This is also called as integration. So, we integrate a peak to determine the area under the peak. This is shown in this particular diagram here. (Refer Slide Time: 20:29) This picture is taken from a book called Spectroscopy by Pavia et al. Here you can see that a peak (we will look more closely later at the reason for the particular position of these peaks; that comes under topic of chemical shift analysis). But, in this slide the point to take home is that there are 3 hydrogens in the methyl group and there are five hydogens in the aromatic system. Therefore the area of the aromatic portion should be five units, two units for CH2 and three units for CH3. Hence, we should have the integral ratio as 5:2:3. So, what typically we do is we measure the area under the peak and the area s ratio is taken with respect to each other or a reference and the ratio is what is presented in the spectrum. Ratio is an important parameter to know because we then come to know from the ratio how many hydrogens are there in a particular peak, without which it is very difficult to analyze the structure.

11 (Refer Slide Time: 21:52) This slide again shows this particular height for benzyl acetate, the same molecule that we looked at to get the height in the ratio of 5:2:3 from the three different type of protons. (Refer Slide Time: 22:07) We will continue with the chemical shifts now and look at how the chemical shifts are influenced by various factors. Next, what is important to know here is that typically when you look at a NMR spectrum, we use the word downfield and upfield.

12 Typically, in the spectrum the reference is always at the right hand side of the spectrum. So, the reference molecule, you will see in the next few slides, is always kept at the right hand side, that is at the high field end. So, down field is less shielded or de-shielded; the de-shielded ones come on the left side which implies their ppm value goes higher or larger in magnitude. If you look at the spectrum the ppm starts at zero somewhere on the right and we then read from right to left. The lower ppm value is denoted as up field shifted and the higher ppm value is denoted as the down field shifted. (Refer Slide Time: 23:44) This chemical shifts typically we see for different functional groups fall in particular zones shown. The scale is marked from zero onwards towards the positive side and typically this is what happens in all organic compounds; we choose zero ppm as a reference. That means anything more than the 0 ppm is shifted down field with respect to the reference. Amides hydrogen resonate typically between 6 to 8 ppm. The aldehyde comes very much down field; the aromatics come somewhere between 7 to 8 ppm. Methyls are CH3 groups and are always up field shifted to somewhere between 0 to 2 ppm. Methyl peaks are very prominent in NMR spectroscopy and they can be very easily recognized because they always appear somewhere between ppm. The reason for protons from these functional groups to come at such different positions is what we will focus in the next class, where we will see what are the factors which affects the

13 chemical shifts and how we can figure out the functional groups present based on the chemical shift value.