 Hello everyone. In the previous lecture, I discussed about different radiochemical practices. Like, you know, you have good laboratory practices, GLPs for a chemistry laboratory. We have good radiochemical practices, GRPs for a radiochemical laboratory. I also discussed some of the radiochemical separation techniques. In this present lecture, I will discuss the radio analytical techniques which employ radio isotopes at tracers to study different chemical processes and also some of the applications in many areas. So, let us first discuss the radio tracer concept. The father of radio tracer concept is George Habeci in 1920s, early 1920s, George Habeci developed this tracer concept and there is a very nice story about George Habeci. How he first time used radio tracers, you know, I would like to share with you. George Habeci was actually staying as a paying guest in a house and the paying guest, you know, means the house landlady or landlord will give you not only the accommodation but the food also. So, one day they realized that their landlady is serving the leftover of the previous day to them for breakfast. And so, he wanted to do experiments to confirm, scientifically prove that they are being served the leftovers. So, what he did? He took a tracer of that time, there was no artificial activity. So, we have lead 210, so lead compounds contain lead 210 and lead 210 was, in fact, he just added that lead 210 in the meals. And next day, when he was served the food, he took that portion to the laboratory and he counted it for beta activity and he found that lead 210 was presented in those samples. So, lead 210 is a 20-year supply and you can separate it from the uranium series. So, then he conclusively proved that his landlady is serving the stair food to them. So, he actually traced the path of food using 210 lead as a tracer. That is what is the meaning of radio tracer. So, the concept is like radioisotopes have the same chemistry as their stable isotopes. So, you can use them to trace the path of an element. You have, for example, you have a sulfur, a process where sulfur is being used. You can have radioactive sulfur, sulfur 35 mixed with the reactants and you can know where the radiated sulfur is going. Outside, you can trace the path by radiations. So, they can use it in industry, environment, medicine, agriculture. You can use them to trace the path of an element. You can use them in diagnosis of diseases by imaging because you can make them to go to a particular organ and then they are emitting radiation. From outside the human body, you can take an image of like X-ray you take. Similarly, you can take the gamma image using gamma ray counters. They are used in vitro for determination of different compounds like hormones in our body. I will discuss that. And you can also do the trace element determinations of metal ions, biological molecules using radiocese techniques. So, some of them I will discuss in this video lecture. Radioceses have a lot of advantages and in fact, you will notice that many of them over a period of time when modern techniques not based on activity are being developed, many of them have become obsolete also. But sometimes you will find that they are indispensable. Some of the experiments you will find you can do only with the radioactriotases. So, one of the advantages of this is they are very sensitive. Suppose you are doing a counting of radioacetop, emitting a particular gamma ray, then it is very sensitive because that gamma ray is unique to that particular isotope. That will be simple. Some of the detectors like GM counters are very low cost. So, you can carry out even research using this. So, low cost instrumentation is one of the important advantages of radiocese techniques. Of course, it is said that you should use radioactivity for your any experiment or research or any problem solving when there is no alternative. So, you must there should be an advantage in terms of manpower, time, cost and so on. So, either you save time, you save money, you save manpower. So, then only you should be able to justify the use of radioactive isotopes in a research or in solving any problem. So, you have to take care. You take precautions like mass effect means because of the very low concentrations of these isotopes. Some of them are carrier free. So, there is a chance that they may get absorbed onto the wall of the container. So, you have to like I was telling you some carriers to see to increase the mass so that they are not lost. So, the low concentrations have their associated difficulties and challenges. When you produce a radio isotope, it may be produced in a particular oxygen state, you do not know. So, you have to verify that you what is the oxygen state and then you make sure all the isotopes, all the atoms are present in that particular process. So, you need to adjust the oxygen state by using oxidation, sterilizing, reducing agents. Similarly, you are using some isotopes, stable isotopes to like if you are using a tracer of an element, then you have to make sure that the tracer and this stable isotope have the same oxygen state. Otherwise, we cannot trace the path of the element in the process. So, suppose tracer is in the iodide form and the iodine present is in the iodate form. So, you cannot trace the path of iodate using iodide. So, you make sure both of them are in the same oxygen state. Then many times, the radio isotope may decay to another isotope which is also radioactive as we discussed in radioactive decay chain and that may interfere in your separation. So, you make sure that if you have a doctor product, then you should also be conscious about interpreting your data. You must know that the doctor product is also radioactive and you have to factor that into your annual controls. Okay. So, let me discuss some of the radio analytical techniques which are commonly used and many of them you will find have become obsolete because there are now much better techniques available. So, you may not one may not be needing it, but it is very interesting to see and how people have developed those techniques and it is so unique about radioactive based techniques. One of them is radiometric titrations. So, you as a radio tracer, you do the norms for example, precipitation. So, you normally in radiometric type you can do, you can precipitate, you can take the precipitate weight and find out the concentration. So, you go on adding the reagent and you will find at some point of time the equivalent point is reached. So, how do you track that? So, for example, you want to determine chloride concentration in a solution like you can have sodium chloride or beryl chloride and chloride salt and you want to do a titration using a precipitation reaction. So, silver chloride is insoluble. So, you take silver nitrate as a titer and add go on adding silver nitrate. This silver nitrate you tag with 110 silver and silver chloride will precipitate 110 and you are left with sodium nitrate in the solution. So, what you do? In the puret you take labeled silver nitrate and in the solution you have the chloride ions, chloride salt solution and now you go on adding drop by the titrant and then record the activity in the supernatant. So, the precipitate will settle at the bottom from the supernatant you take an aliquot go on taking the aliquots and you find the activity. So, what is happening as you add the reagent radio tracer labeled reagent silver nitrate solution initially when silver nitrate concentration is low all of it will be precipitated and you will see the background in the counter. When all the chloride is precipitated as agCl then at the end point 110 silver activity will begin to rise and therefore because they are in the excess of silver activity in the solution. So, you can then find out the end point what is the concentration of silver nitrate that is sufficient to neutralize to precipitate all the chloride. So, beyond this this silver nitrate is not precipitating it is going up and up because you are just adding the one solution to another solution. So, that is how you can find out the equivalent point and a very simple experiment based on did you test the counting can be done to do the determination of chloride concentration. Same thing you can do for thiocyanate silver ions tungstate magnesium and so on. So, you can use the proper reagent for thiocyanate you can use silver 110 or silver chlorhexate for tungstate you can use cobalt 60 for magnesium you can use phosphate ions. So, you can use the suitable radio tracer labeled titrating agent and do the radiometric titrations. But you will find that nowadays none this this is not very common these days because there are much better techniques you can use them for these experiments. Salvatore extractions are still very very useful very commonly used and they are used for separating the different metal ions or different organic molecules. One of the applications is determination of microgram quantities of metal ions like mercury, lead, cobalt, zinc, and nickel by salvatore extractions with a reagent called tithyzoin. So, you can do salvatore extractions and you can use the radio tracers of these elements to see the distribution in a quiescent organic phase. So, that is the beauty of radio tracers. You can determine the distribution ratio. So, suppose you have used mercury. So, you use 203 mercury as a radio tracer. So, in the aqueous solution you have the radio tracer mercury 233. After the salvatore extraction some mercury will go in the organic phase and you can take aliquots from aqueous and organic 100 microliters or 50 microliters and count the activity of 203 mercury in the two phases and you can find out the distribution coefficient. Suppose you are having multiple elements in the same solution you can find out the D value for different elements and you can find out the separation factors. So, this is a for radio tracers salvatore extraction very ideal system. You can use you can even there are now complexing reactions you can study the equilibrium. So, how they know as a first of acid bay acid concentration how the distribution equilibrium is shifting as a first of ligand concentration how the equilibrium is shifting all those studies can be done using radio tracers. So, much so even you can determine the stability constant of metal ligand complexes. For example, if the metal ligand is going in the organic phase. So, you can do as a first of ligand concentration you do salvatore extraction and then find out what is the time what is the concentration of the ligand at which you will see the metal ligand complexes getting into the extraction phase and then you can set up the equations for finding out the basically what you need this stability constant k equal to concentration of ml upon concentration of metal into concentration of ligand. Now, this is a free metal ion this is a complex metal ion this is the free ligand. So, you can find out this ratio metal ion free will be in aqueous phase and complex metal ion will be the agronic phase. Once you know this and the ligand concentration is nothing but free ligand plus metal complex concentration total ligand is known you can find out the free ligand and therefore, you can find out the stability constant. So, this is how you can use erotases for salvatore extraction. Similarly, the ionic state reasons ionic sense separations are union erotases very frequently. In fact, I give you some very interesting example of even the discovery of trans amyretium I can say see amyretium amyretium. If you see the actinide element after plutonium, plutonium can exist in 4, 6 and so on. After plutonium amyretium curium, bercilium, cationium, anesthenium, permium and other elements they all elements are present in the plus 3 state called actinides. Now, in the early late 1940s and early 1950s in the different cyclotron experiments this heavy the trans plutonium elements will be produced and they will be discovered. So, how to discover a new element it was known in 1944 by Seawork's actinide concept that beyond amyretium all trans actinides are predominantly present in the plus 3 states. They are similar to erots, they are lighter homologs. So, they set up it was already known that individual lanthanides can be separated by a cationic sense resin. You can load the solution of cations from a dilute acid into a dou x 50 cross 4 cationic resin and then elute with alpha hydroxy isometric acid which I explained sometime back. One by one you will see the heavier lanthanides come first and followed by the later lanthanides. Similarly, these actinides also follow the same trend as the lanthanides. And so, the different lanthanide actinide ions can be seen in the profile of little tracers of the different elements. Now, you do not have the little tracers of actinides. You can use little tracers of lanthanides to see the position of individual actinides. And that is how this element mandilibium z equal to 101 was discovered by G. R. Gragini chopping, the student of the D. C. Bohr by analogy with their lighter lanthanide homologs and their position in the ion chromatography system was useful discovering new elements. So, this is how the power of ionic cell separations you can you can calibrate the concentration of alpha hydroxy butyric acid to separate individual actinides and lanthanides as elements of the and see their position uniquely in the as a function of the drop number in the elution. That is what in the advantage of better tests. In fact, we have done experiment we have the tracers of each lanthanide is a lanthanide ion at a element and you can mix them and do the chemistry and you can see beautiful profile of individual results. Now, I will discuss some of the applications of radioactivity techniques like isotope dilution analysis. This is a beautiful radioactivity techniques determine concentrations of some analytes and isotope dilution, you don't have to do a quantitative surface 100 percent surface is not needed. So, you essentially when you get the isotope dilution means you dilute the specific activity of a radio isotope. So, you what you essentially do is you have a standard condition of radio taser which for which the specific activity is known. The specific activity means activity per unit mass. Now, suppose you have a quantity m. So, a specific activity means per gram this is the activity this is the amount of the substance. So, total is total activity m into s m into s is the total activity. Now, you want to find out the concentration of an unknown analyte mx. So, what you do? You add this unknown analyte with this standard solution of the same element, but tag with the radio taser and then you measure the specific activity. So, you have now the standard solution and the unknown solution total you have your quantity mx plus m which will have a reduced specific activity because we have added some inactive metalite. So, this total activity specific activity in the amount is same as the initial activity activity into concentration. So, from this equation conservation of the total activity we can find out mx equal to can solve this m into s by sx minus 1. So, m is known what was the initial amount of standard solution, what is the specific activity of the standard solution, what is the specific activity after mixing the two and you can find out the concentration in the unknown solution. That is what is called the isotope dilution by diluting the specific activity by what factor the isotope diluted tells you what is the concentration of analyte. Just to give you an example to illustrate this point more clearly. Suppose you have a solution of cobalt in 10 ml solution you want to know what is the amount of cobalt in this solution. So, just give some numbers you have a standard solution of cobalt 60 7.5 milligram cobalt, cobalt 60 may be very small it may be picogram quantity, but amount amount of cobalt total cobalt in that solution is 7.5 gram you count that solution and you get 340 counts per minute. So, the specific activity of that will be 340 by 7.5 will be 45.3 cpm per gram. So, counts per minute per milligram not gram per milligram because it contains 7.5 milligram of cobalt not cobalt 60 total cobalt it is cobalt total cobalt. Now, you add to this solution 10 ml of the unknown solution for which you want to find out the cobalt concentration. Then you have to now do separation. So, you take separate out cobalt by you can do separate extraction or you can do electrode deposition you separate cobalt and mind you cobalt 60 and the cobalt. So, both the solution should have the state oxygen state of cobalt. Oxygen state of cobalt. So, by separation you got 10.3 milligram of cobalt. So, it is not necessary to have quantitative separation. Whatever the amount you got and you take the activity of that and you got 178 counts per minute. So, the specific activity will be 178 upon 10.3 means 17.3 counts per minute per milligram of the separated cobalt fraction. So, we can put in this equation the value. So, s upon s x minus 1 into 7.5 will be 12.1 milligram of cobalt in the unknown solution total amount of cobalt. So, the concentration of cobalt in that way 12.1 by 10 ml to be 1.2 milligram per ml. So, this is how so, of course, you can say that today you do not have to do reductive set technique because you can put it in a ICP AES ICP MS. So, there are no modern techniques based on spectrophotometry, auto-optical emission spectrometry and so on. So, such techniques sometimes you may not be useful, but I will give you an example this one and you will really appreciate the application of isotope dilution technique. Blood volume means human being, human body. Suppose you want to know what blood is there in a particular human being. The simplest experiment you can take 1 ml of your patient's blood, take out 1 ml, add sodium-24 activity 14 hours supply and in that 1 ml solution and find out the specific activity. You can count the gamma ray activity of sodium-24 and suppose you got 20,000 counts per minute in 1 ml of that solution. Now, this 1 ml you inject intravenously into the human patient and after 15 minutes take out 1 ml. So, what has happened that this 20,000 counts per minute activity is got now distributed in the human entire body, whatever volume we have is included with that in 15 minutes. And now you take out 1 ml and measure the activity of sodium-24 and let us say you got 4 counts per minute in that 1 ml which you got later. So, now you can again put the birth of the total activity initial activity 20,000 counts per minute per ml into 1 ml this is what you added and finally, the volume of the blood is not known in the human body. So, V plus 1 ml we added V plus 1 ml into 4 cpm per ml and you can find you can find out the V that is equal to 5000 ml. So, this is a total volume. So, you can say you can take 1 ml and do that so that the volume of the blood you can know total body is very difficult experiment. So, this is the very high-stability experiment can tell you without much difficulty the concept the volume of blood in there. So, you have to find out the ingenuity in their experiments. So, there are some experiments we will find reductisers offer a unique advantage. Another such techniques is radioimmuno SA. The radioimmuno SA is a technique developed by Berson and Rosalind Yalov in 1950s and Rosalind Yalov received the Nobel Prize for this in 1977. So, this radioimmuno SA is actually used to determine minute quantities of substances of clinical interest in biological systems. So, if your body has got certain hormones like P3, T4 this high right hormones or you can have many other you know in our body there are methiroglobulin many antigens are available and you want to know their concentration as a part of the diagnosis. So, this is based on this again the you see like the these isotope dilutions for sub-stockometry sub-stockometric isotope dilution. Sub-stockometric means there are two reagents one is the antigen and one is the antibody and one of them is in excess other one is in very dilute low concentration. So, that they are not stoichiometrically being added. So, the concept behind is that suppose you want to determine the concentration of this antigen it is not silver it is called antigen like P3, T4, TSH or prostate specific antigen in the body different glands are excreting different hormones and you want to determine their concentrations. So, we do not know we take out a sample from a person and you add to that a labeled antigen. So, suppose you have thyroid hormones they contain iodine you can take a iodine pressure 125 iodine and make sure that they have the same oxygen state of iodine in the body. So, you have now you have a small quantity of this antigen you add the two and now you react them with the antibody. So, the antibodies are specific to a particular antigen, but this antibody is a very small concentration to respect to antigen. So, the radioactive and the inactive antigen will compete with each other to bind this antibody and so later on you will find that you will have a mixture of so, you can now label you can form a complex which you can centrifuge and count. So, what you are doing now you are counting the labeled antigen with antibody and find out the concentration of antigen that is labeled. So, what you do this bound activity this concentration. So, you can find the in that total actually you this is total no. So, in the total what fraction of what fraction of antibody is bound that is called the bound activity and as a function of increasing concentration of antigen. So, you have to do multiple experiment. So, you do this is like a calibration graph you take different concentrations of the antigen and generate this graph as you increase the concentration of antigen the activity bound will be reducing because it increasing the fraction of inactive antigen will decrease the fraction of labeled antigen and this is now a calibration graph. So, for example, you have an unknown sample you find out the fraction of unlabeled labeled antigen and then you find out what is the concentration from this calibration graph. So, it is a sub-stopometric antigen antibody is in a very small concentration with respect to antigen and then the decrease in the concentration of the antigen leveled antigen tells you what is the concentration of antigen in the unknown sample. So, antigen leveled with redotation 125 i.d. you can use and then you can concentrate find out concentration of different hormones in the hypothesis. The applications of radio even know SA are like for example, thyroid related hormones and hormones of reproductive system protein molecular steroids, differential diagnosis of hypothyroidism, neonatal hypothyroidism, but you can screen the newborns for their treatment very quickly you can do experiments and there are two more markers such as thyroid globulin, prostate specific antigen, alpha, petro, protein and carcinoma embryonic antigen for diagnosis of cancer and screening of vaccines. In fact, nowadays radio even know SA actually has taken a back seat because there are no chemiluminescence fluorescence based techniques which can also reach the same sensitivity as RIA. The RIA can tell you concentrations in nanograms, nanomodes and so now the techniques are advancing other techniques and they are competing vigorously with the radio you know the radioactive based techniques. So, you will find that these techniques are also slowly slowly being pays out because if you can do the same experiment with the non-addictive technique with the same sensitivity then and same cost of course then this is there is no need for such techniques but still many laboratories follow up this way. And lastly, I want to tell you the applications of radioactive traces using in dating. Dating means finding out the age of an object one of the object like for example the rocks geochronology. So, you want to find out the when that particular rock was formed and this is based on the natural radioactive series 238 uranium decaying by alpha and beta to 206 lead. So, you want to know so you find out the uranium content and the lead content in the rock. So, initially let us assume that there was only or even if there was some lead then we will know because all isotopes are not found by uranium decay. So, you can find out what was the initial uranium lead present in the rock. So, the uranium present in today can be given in terms of initial concentration of uranium exponential decay e raise to minus lambda t this is the time which we want to find out. Lead present today is uranium initial present minus uranium present today. So, this is initial uranium minus present uranium is the lead present today you can find out lead by mass spectrometry. And by solving these two equations you can find that time when this rock was formed using this expression. So, if you measure the ratio of 238 uranium to 230 206 lead you can find out the age of the when that particular geological sample was formed. And lastly the carbon dating carbon protein has half life of 5730 years and it is being formed in the atmosphere by cosmic ray neutron bombarding nitrogen 14 and this carbon is reacting with oxygen to form carbon dioxide by photosynthesis green plants assimilate this carbon protein. And so much so that all living organisms between our body contain a specific activity of 15.3 GPM per gram this integration is per minute one gram of any living organism will give you this much specific activity. But once the organism dies then this activity is no more being equally accumulated and so there will be a decay and from that decay you can find out what is that time. So, you have suppose the activity is a today and initially till it was life was 15.3 by the same similar equation you can find out what was the time that this particular organic and the living organism died. So, this is being used for carbon dating of hostiles, food samples, leaves, leather, charred bones, clothes, paper so on. And since the half life of carbon 14 is 5700 years about 4 half life of carbon 14 20,000 years and about half a half life of carbon 14 or 2000 years this is the time zone which you can scan using carbon dating you want to go more than 30,000 their particulates. Similarly, tritium half life is 12 years. So, tritium dating can be used for water bodies whether they are in contact with the rainwater or not their isolated water bodies will have less percentage of tritium. So, these three isotopes are used for dating of different applications. So, this was just a glimpse of applications of different different techniques. I will stop here, take up the next lecture next time. Thank you.