Lecture 07

Added: 2026-05-12

[00:00:03][music] [music] Hello everyone. Welcome back to the second lecture of the week two of the MOO's course on advanced analytical techniques. In the previous lecture, we have discussed the different components of the atomic absorption spectrophotometer in detail.

[00:00:37]In this lecture, we'll talk about the possibility of various interferences which causes difficulties in analyzing the spectrum or which creates some confusion in the spectral analysis of the given analyte.

[00:00:54]The interferences in atomic absorption spectrophotometer are divided into two categories. We have spectral interferences as well as non-spectral interferences. Spectral interferences mainly related to the spectrum or some new peaks that belongs to some impurities or some molecular absorption or emission peaks. While nonspectral interferences are further divided into three categories. one is the matrix interferences, the chemical interferences as well as the ionization interferences. We'll discuss them one by one. So as I said the interferences refers to the occurrence of a new peak that belongs to an impurity or the peak belong to a molecule which is not completely converted into individual atom and that and that if that molecule peak is present if that molecular peak is present then that means those atoms that are present in the molecules or those elements that are present in the molecule they have not been converted into vaporized individual atoms and thus their characteristic peaks will be missing in the absorption spectrum.

[00:02:05]Therefore, while working with atomic absorption spectrophotometer, we have to keep this thing in mind. We have to minimize the interferences in order to get reliable and reproducible results.

[00:02:18]So these interferences are broadly divided into two categories spectral as well as nonspectral interferences. The spectral interferences refers to an extra peak in the spectrum of atomic absorption. Sometimes this peak belongs to an impurity that is being present in this sample matrix or there's also a possibility that since we know that since we know that automizer basically converts this molecule present in the sample or the compound into individual vaporized atom. So sometimes because the the thermal stability of the molecule is high or if the flame is not stable or the temperature provided by the flame especially in direct aspiration is not sufficient to automize that particular molecule then that molecule exist in its original form and it also absorbs some radiations resulting into a molecular peak instead of the individual elemental peaks and that peak is sometimes very close to the target analyte creating a difficulty or ambiguity in the analysis of the given sample. So we have to avoid these interferences by certain measures. As I said that these spectral interferences are caused by either the molecular iron peaks or by some impurities that belongs to some other sample or sometimes what will happen if for example we are working on a flame automizer that flame automizer uses oxygen gas with acetylene because a mixture of acetylene and oxygen in the previous lectures I have I told you that a mixture of acetylene and oxygen gas provides a very high temperature.

[00:04:10]Further, the burning velocity is also quite good. So if we are maintaining a good flow rate, there is a very good balance between the flow rate and the burning velocity giving high temperature and effective thermal energy. So if you are working with that direct aspiration using flame and acetylene and oxygen is present there's a possibility that some metals some metal react with oxygen to form a metal oxide and metal oxides of some metals are thermally very stable once they are formed they do not atomize easily and they also absorb certain radiation giving a extra peak in the spectrum which sometimes very close to our target analy peef further there are certain absorption which are called as background absorption that are mainly caused by undissociated molecule the same thing that I have just explained you that undissociated molecule means the molecule that has not been converted into its individual atom so when this is the case we have some extra peaks that creates ambiguity in the analysis These background absorptions might be due to tiny solid particles that may not be evaporated or sometimes even the solvent droplets because of low flame temperature or instability of the flame. The solvent droplets has not been completely evaporated and the sample is still wet. Though those solvent droplets may also give an absorption resulting into some new peaks in the spectrum.

[00:06:04]And sometimes these peaks are away from our sample. But in some cases these peaks are very close to the target analyte. And then it becomes very difficult to resolve the target analyte peak properly means to separate these two two peaks becomes quite challenging.

[00:06:27]So when such a problem occur we have to minimize these problems and these problems can be minimized or overcome by certain by taking certain measures. One of them is by measuring and subtracting the background absorption from the total measured absorption to determine the true atomic absorption. What does it mean? It means that first of all we go for a blank run in which we use the same solvent matrix without the target analyte and then calculate the absorption and then we go for a second run in which this same matrix contains the analyte as well in which the analyte is also present in the same matrix. Then again we go for the absorption and from this absorption we subtract the absorption of the blank or what is also possible that we first of all just like in UV spectroscopy we put a sample even if the sample is little bit colored we press zero to make it blank and then we put an aite and do the analysis again. Similarly in AS also we first do a dry run label it as blank so that when we go for the next run containing the target analyte the the instrument automatically substract the previous radiation and the data that is being displayed in the computer is automatically free from the background radiations because we have already performed a blank run. The second possibility of minimizing such interferences is that as you might have seen in the previous lecture I have explained that a single element may have more than one peaks. For example, a s in a single element we have for example we have a ground state E not and then we have different excited state E1 we have E2 and E3. Suppose upon giving light radiation we have excitation of electron from E not to E1 and we also have an excitation from E not to E2. Of course the since the energy gap between E 0 and E2 is high. We have high energy radiation required here high energy which means low lambda shorter wavelength light will cause this transition. While in this case since the energy gap is low we need a longer wavelength light.

[00:09:01]So we can in this spectrum we can have two peaks.

[00:09:08]The peak is at shorter wavelength represents the transition from E not to E2 while the peak at longer wavelength means lower energy is for E not to E1.

[00:09:25]Now if our background radiation is very close to this particular peak which represents the transition from grounded state to first excited state. Then in order to avoid this confusion instead of using this peak as a characteristic peak of the target analyte we use the second peak because this peak also represents the transition of electron for the same element. The only difference is that here in this particular case the transition of electron or the promotion of electron is taking place from the ground state to the second excited state. While in this former case the transition is taking place from the ground state to the first excited state. So if the background radiation or the radiation due to some impur is getting very close to transition of targeted and light from E 0 to E1 then we do not use it as a characteristic peak but we go for the other characteristic peak of the same element which corresponds to the transition of electron from ground state to the second excited state. So in this way we avoid this spectral interferences.

[00:10:42]Now comes to the nonspectctral interferences.

[00:10:46]They are of three types. The metric interferences. Matrix refers to the sample which we are using for analyzing a particular analyte. This matrix could be a river water. If we are analyzing a river water for a particular type of element. That matrix could be a tab water. It may also be blood, urine or any other sample that can be used for detection of target analyte. Then there are after matrix interferences there are certain chemical interferences which are due to the formation of different compounds. And then finally we have ionization interferences. These ionization interferences are due to ionization of the atoms. And I told you in the previous few lectures that in case of atomic absorption spectroscopy when we automize the sample the main purpose is to convert the molecule into individual vaporized atoms in ground state only. We do not convert the molecules into individual atoms in excited state because if we excite them they also start emitting light radiation. So we will get an emission spectrum but we are focusing here on atomic absorption spectrum. Therefore the energy the thermal energy which we are providing should not be very high. It should only be sufficient enough to vaporize the sample as well as to dissociate into its individual atoms. But this energy should not be too high which causes the excitation as well because if excitation occur it will form an emission spectrum as well. So these ionization interferences refers to the auto excitation. So we'll discuss them one by one. These matrix interferences occurs when the sample matrix is quite different from the standards. What does it mean? For example, we are analyzing a particular metal for example iron and for the quantification of iron we have prepared certain standards.

[00:13:04]Now those standards we have prepared in deionized water.

[00:13:10]While the sample that is being coming in the laboratory for analysis it is blood or maybe serum sample whose viscosity is quite different from the standard sample. So due to difference in viscosity highly viscous sample are very difficult to be dissolvated and to be dissociated into individual atoms. So when we are dealing with standard in this case since it is prepared in deionized water dissolvation will occur smoothly followed by dissoci vaporization and dissociation. But when we are using the real sample for example blood or serum or any other sample because of its high viscosity it cannot be easily dissolvated and vaporized and finally dissociated. And if less number of atoms are produced by dissociation ultimately the peak height will be low and we do not get the exact concentration value which we are expecting. So in this case the data or the result is not reliable. Further there is also a possibility that the results are not reproducible because when you do the run second time since the viscosity is not constant the sample and standard are not same. when you do the second run then even then the results are again different from the first run. So in this case matrix interferences affect the reliability as well as reproducibility of the results.

[00:14:44]And if you want to minimize these interferences we have to keep the standard prepared in the same solution in a similar solution in which we are expecting our samples to be.

[00:15:01]The chemical interferences uh occur due to the formation of some stable compounds during the process of automization. As I said earlier that when we are heating the sample at high temperature and sometimes oxygen is also present chemical interferences is quite common in direct aspiration technique means when we are using the flame automizers. So in this direct aspiration we have oxygen present as well. So this oxygen reacts with the component present in the sample and at high temperature they form oxides. Some of them are very stable thermally and then they do not dissociate and in that case we these these oxides absorb some radiations and the transmitted light radiation and then the intensity of the transmitted light would be very low. But this loss of intensity is not because of the sample uh the target is not because of the target and light. But this loss of intensity is because of the undesired molecular absorption which is formed due to the oxidation of the target analyte atoms. So these interferences should also be avoided and the best way to avoid these uh interferences is to use a higher flame temperature because if we are using a higher flame temperature even if those oxides are formed because we are using this higher flame temperature even if those oxides are formed they are immediately uh dissociated because of the high thermal availability because of the high thermal energy they are immediately dissociated into individual components and then those atoms absorb light radiations resulting into reliable results. So by keeping the flame temperature high we can minimize these chemical interferences. Then comes the third and the last type of nonspectral interferences which are called as the ionization interferences.

[00:17:03]And these ionization interferences occur when our sample molecule while undergoing automization through the process of dissolvation, vaporization because there are three steps in the automization and dissociation.

[00:17:35]Sometimes what will happen that there is another step added after dissociation when the molecules are converted into vaporized individual atoms.

[00:17:47]These atoms undergo excitation due to this thermal energy because of the very high temperature and these excitation causes electrons to move to higher energy level not beau by the absorption of light but by the absorption of excessive but by the absorption of extra amount of heat extra amount of thermal energy they goes to the excited state and when they return they emit light and this light radiation then in then interfere with the transmitted light and ultimately it will spoil our analysis. In this in such a case it will give rise to an emission spectrum while we are working on absorption spectrum. So ionization interferences must be minimized. So before moving on to give you a detailed idea about ionization interferences. I think I should discuss about the chemical interferences that what kind of metals are um usually give chemical interferences. For example, refractive refractory elements like titanium, tungsten, zirconium, malibdinum and aluminium. They are quite sensitive to the formation of oxides. And when when they are present in the sample they form they react with oxygen forming oxides and their oxides are thermally very stable which and therefore they do not dissociate and in that case the atoms which is required to absorb light radiation giving a spectrum so that its quantity or its concentration can be determined. That atom has now been converted into an oxide. And since atom has been converted into thermally stable oxide, it will no longer remain available for absorption of light and ultimately there will be very less number of atoms. Therefore, the correct information will not be obtained. So we have to either dissociate this thermally stable metal oxide or we have to prevent its formation.

[00:19:43]So this can be possible if we use a as I said before if we use a flame with higher temperature and that is possible if we are using nitrous oxide and acetylene flame and we are not using oxygen gas because oxygen acetylene also provides high temperature but in case of oxygen there's a possibility of formation of oxide. So if we are avoid using oxygen gas as an oxidant along with acetylene fuel and we are using nitrous oxide as an oxidant with acetylene then we can achieve high temperature and we can also minimize the possibility of the formation of these thermally stable oxides. So in this way we can minimize or completely prevent these chemical interferences.

[00:20:34]Similarly uh there's another uh uh interesting case of uh these chemical interferences when we are working with uh calcium. If you are analyzing calcium present in a sample, calcium when is heated in the automizer, it reacts and forms sometime it sometimes forms a phosphates calcium phosphate which is quite stable and this calcium phosphate which is formed and this calcium phosphate will reduce the absorption of the calcium because now calcium ions are not free. they have been converted into calcium phosphate and since some of the calcium ion has been converted into calcium phosphate so they will not absorb light radiation and the result will not be the exact value the values that are being obtained in this case they are not very reli reliable the results are not correct so we have to reduce the formation of this calcium phosphate for that we need to use lenum Because if phosphate ions are present this lenthnanum form for lenthnum phosphate thereby preventing the possibility of formation of calcium phosphate and when calcium phosphate is not formed all the calcium ion all the calcium atoms absorb light radiation resulting into reliable resulting into reliable data.

[00:22:07]Now the ionization interferences as you um as I just explained that ionization interferences are possible when the flame temperature or the graphite furnace temperature is very high that it not only causes the automization of the sample but it also causes excitation of the sample.

[00:22:30]So it causes along with automization it causes excitation of the sample as well. And when excitation also occur the electrons of those atoms are excited by this thermal energy only to the excited state or maybe to the second excited state from the ground state. And when they return back they emit light radiation giving an emission spectrum while our main target was the absorption spectrum. So we have to uh avoid these ionization interferences. Most of the time easily ionizable species like alkaly metal or alkaline earth metal they undergo ionization interferences because they ionized easily they excited they excites easily and then they give some light emissions which interferes with our absorption spectrum.

[00:23:29]So in order to avoid in ionization interferences we have to keep the temperature low. We have to use a little bit cooler flames.

[00:23:37]We can reduce the temperature of the flame. We can use little bit cooler flame so that the chances of these ionization would be minimum or there is an other way out in which we use salts of elements like copper, rubidium and cesium. When we use salts of these elements they produces excessive electrons as well as kines which suppresses the ionization of our target analyte. So the ionization interference of the target analyte can be minimized can be reduced by using c other by using some other metals belonging to its own group like rubidium, cesium or potassium by using so that they ionize easily giving more number of electrons and in this way they suppress the ionization of the target analyte. So in this way we can get away with uh so in this way we can get rid of these ionization interferences resulting into a robust and reliable absorption spectrum. Then coming on to the application of atomic absorption spectroscopy. This is very useful technique for the analysis of trace elements present in water or waste water sample like calcium, magnesium, mercury and lead. You all know that all these metals are responsible for causing hardness in water. So with this technique we can check the hardness of the water. Further these elements like these further these metals like mercury and lead if they are present they are very toxic as well. Although these calcium and magnesium are not toxic but they contributes towards the hardness of the water. While on the other hand these mercury and lead they causes hardness but on the other hand they are extremely toxic for us. Therefore we must not drink the water which is contaminated with these two and various other heavy metals. So it is always essential to do the proper analysis of water samples and atomic absorption spectroscopy is one of the finest and simple technique for such analysis. For such analysis then we can analyze the fly ash. Fly ash is obtained from thermal power plant and because when we process coal for generating steam and then that steam ultimately runs the turbine producing electricity. Since the coal contains various other heavy metals which may be toxic. Therefore analysis of the fly ash is very essential and we can do the analysis of this fly ash by using atomic absorption spectroph photometer and of course we do not directly use this fly ash. We digest it into some acid so that those metals comes into liquid state and then they can be injected into the nebulizer.

[00:26:34]Then determination of sodium and potassium in the OS.

[00:26:39]You all have used these OS samples or OS sache when you are sick. Uh during dehydration they are being suggested by our doctors. OS means oral rehydration sache. They are used during dehydration to maintain proper water prop to maintain requisite amount of water as well as salts in the body. So this oral rehydration sache the amount of sodium and potassium present in this OS can be easily determined with the help of this AAS technique. Then pharmaceutical analysis various drugs contains metals like for calcium deficiency we have been suggested by doctors to use some calcium tablets. Similarly we use various tablets that contains magnesium, zinc or various other metals. So the analysis of those uh pharmaceutical ingredients is also possible with the help of this atomic absorption spectrophotometer and the technique is simple as I said and the data given is quite reliable.

[00:27:49]Further to check the toxicity of a heavy metal in blood and serum sample, we can analyze those blood and serum sample and then identify the concentration or the amount of that toxic metal present in the blood through which we can estimate its toxicity.

[00:28:07]Even alloys and their composition can be tested with the help of this atomic absorption spectrophotometer. The advant the advantages or the benefits of atomic absorption spectrophotometer are it is one of the simplest and the first and foremost elemental analytical technique introduced it was introduced in the year 1950. It was introduced and first and foremost elemental technique introduced for analysis of heavy metals and for analysis of metals present in different media. It is still continues to be widely used across many industries like pharmaceutical industries, prochemicals, paints, even in research centers industries for the treatment of water they also used and even in thermal power plant fly we use this technique.

[00:29:04]The key advantages of this technique, the key advantages of atomic absorption spectroscopy is its simplicity, reliability of the data as well as its low cost. These three parameters makes it one of the most widely used technique.

[00:29:22]Despite its age means it's quite old technique. Now there are numerous techniques with which are very advanced.

[00:29:30]Still it delivers precise results and accurate results and therefore it is is still in use and preferred by research centers as well as industries.

[00:29:43]This is again a brief idea about the use about the advantages or benefits of using a flame automizer.

[00:29:51]It has a low capital cost making the cost of the equipment quite affordable.

[00:29:56]The technique is simple and it can be operated by a you user which is not very much trained. Uh even after a short training a person can operate this machine.

[00:30:15]We get reliable results. The sample throughput is very good with flame atomic absorption spectrophotometers. It can measure sample over a wide concentration range. We which means that we can measure up to low ppm low ppm level as well as high ppm level can be tested.

[00:30:36]The results are quite precise and accurate.

[00:30:40]we can operate it manually but if the sample number is very high we can install an autosampler in the machine to make things easier for us. Similarly the graphite furnace atomic absorption spectrophotometer if we compare it with our flame automizers they are quite robust giving very reliable data and reproducible data. They can be used even for high dissolved solid contents and for very low content as well. They can go up to ppb level easily or even up to ppt level in some cases. The the technique is again cost effective not very high as the cost is not very high as compared to our [clears throat] flame automizers and again it is simple in operation ideal for detection of trace elements in aqua sample fly ash blood or serum and if uh although ICPMS is a wonderful technique so we can have this machine and we can perform similar analysis This is in a limited budget. So with this our atomic absorption spectroscopy is complete. Its principle it instrument its principle its instrumentation working and the different types of interferences possible in atomic absorption spectroscopy as well as the ways to minimize those interferences.

[00:32:05]Now in the next lecture we'll discuss about the atomic emission spectroscopy.

[00:32:10]Till then thank you very much.

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