So in the last class I started discussing about advanced spectroscopic technique and I elaborated you on the different basic principles which are used for these techniques to remind you that we normally use these techniques for spectroscopic analysis of different inorganic and organic compounds. So whenever an isolated molecule suppose is subjected to any kind of radiations and it is excited from a state suppose if I write it properly e1 to e2 it undergoes transition by application of any kind of radiation does not matter what is the wavelength of the radiation and in such a case the molecule or the species rather will come back from the high energy state e2 to u1 by emitting certain radiation and if you know what is this frequency of the radiation we can relate this as this one like this that means the emitted energy of the radiation h nu is equal to the modulus of u1-e2 right. So therefore I can always write this one as hc mu bar equal to mod u1-e2 so where mu bar is nothing but the inverse of frequency that is c by lambda sorry 1 by lambda and that means it can be written as hc by lambda equal to mod u1-e2. So therefore depending on the type of interactions the radiation will have on the molecule on the species we can either have absorptions or we can have otherwise what is known as the emission so either we can have absorptions spectroscopy or emission spectroscopy. Depending on the kind of energy levels if u1 suppose is greater than e2 then we can have we will have absorption that means if the molecule is going to the high energy state to low energy state by absorbing certain amount of energy then it is absorption on the other hand that means this is absorption on the other hand if the molecule is going to high energy state to from the low energy state by absorbing the radiation and it comes back that is if u1 is less than e2 we call it emission. So we can measure the spectroscopic measurement you can do either in absorption state or in emission states that is the way normally the spectroscopic techniques are done. Now the kind of radiation which will be emitted from the absorb by the species will be depending on the what is the input radiation we are applying. So therefore depending on input radiations different kind of situations can be possible I have shown this slide last class I am showing you again if you have suppose very high frequency like 10 to the power 6 when the power 8 or rather we can have if you have radio frequency like a blend starting from 10 meter to 100 centimeter we will have normally change of the electronics in the change of nucleus pin okay can be changed nothing will happen to this molecule or the atomic species present on the other hand if the energy levels are little increased form as a frequency level from 10 to the power 10 to 10 to the power 12 harsh that is obviously corresponding to energy level increase that is if you go to microwave regions and then we have change of orientation the molecules okay or change of configuration of the molecule in for it can also lead to change of configuration of the molecule and in the visible the ultimate region you have change of electronic distributions or rather you can always have some kind of rotations or transition between the different electronic states which can which can be inclusive of the rotations finally in the X-ray regions tension can occur which can ionize or dissociate to certain molecules that is what is shown here in the X-ray and if you apply a very high energy that is gamma ray okay our frequency to power 18 very high frequency then you can have change of nuclear configuration itself like you can have the nuclear pieces can undergo transitions so by using a host of radiation studying from radio waves to the gamma rays one can get a large number spectroscopic studies done in the students lecture what I am going to do is we are going to analyze or look at it the most simplest one and the most you know widely used and the oldest technique in the spectroscopies called eb visible that is if we use the radiation sociabilites are in the range of visible or near what is called visible but in the ultraviolet range so I have shown the whole spectrum starting from gamma ray to the radio frequency 10 to the power minus 10 to the power 20 other 20 by 18 to 10 to the power 4 second or hard and you be visible comes very small 10 to the power you can see this frequency in the past 16 of it 14 that means the wavelength should be a couple of 100 nanometers it will be something like 180 to 460 or 70 nanometers in that range and visible we know it consisting of a large number of lengths like red to what is called red to violet lead orange yellow green blue indigo and violet seven colors are possible that took a very simplistic perspective of this ultraviolet and the eb visible spectroscopy I will start from very basic thing you know the obvious difference between many compounds is color right to give an example chlorophyll is what is called green on the other hand very complex ones like say 2 4 dry nitro phenyl hydrogen or derivatives of any alkyne ketones are basically right yellow kind of on the other hand Q in is yellow so this is these are the general difference of the color of different compounds now obviously the you have already learned in your different courses that why different materials are different chemicals show different kinds of color well that is basically that is our eye perceives or I basically sees an object and then see determine the color sky is blue sun is yellow like that so why it is so because the light reflected on the surface of it is solid basically passing through a liquid it comes back to our eyes and we see the particular wavelength of light kind form a particular substance and that wavelength are really corresponding to different colors like red or maybe yellow or maybe your violet in the whole visible spectrometer this is what we know so that means our eye is basically spectrometer acting as a spectrometer to be to be frank our eye can actually distinguish different colors come from different objects so this probably the simplest spectrometer one can always tell that I can be of many things it is basically a camera is basically a as called phenol camera of we can I can always tell that from the spectroscopic point of view this is a spectrometer simply spectrometer so and then there are many compounds which are colorless correct there is no color so that means well light falls on them no light basically comes to our eyes in the wavelength range in the visible so that we can do not see any color so most important aspect will be those objects which are which looks colorless they are emitting large emitting radiations whose wavelength does not fall in the normal spectrum it the spectrum range like red to violet so they may be coming in the near-alpha red alpha ultraviolet regions so so we do not see them or we do not see their colors we rather see them colorless now when a light white light white light consisting of all kinds of this wavelength starting from red different colors studied it to the pilot is passing through are basically getting reflected by any color substance and we know that a basically characteristic portions of the mixed wavelengths okay all this mixed wavelengths 1200 to 300 to maybe 600 nanometers is absorbed remaining portions of a light the main light will assume the complementary color obviously whatever will be absorbed will not be seen and what about does those call the light those wavelengths will not come to our eyes so whatever will not be absorbed by that color substance will come back to the eyes so we say the complementary colors so that means we can say that if something is absorbed in the to 420 to 430 nanometers it will render your law like something absorbs light in the wavelength 500 to 520 nanometers it will be looking red higher lens rates that we know why that's why the red signals are used in the traffic lights and and therefore there are many so on okay so early human beings actually seen these different colors and that is the rest of the story actually goes and use this for the decorative purposes you know many of these are organic minerals in fact we we know the many stones which are used for jewelry purposes are colorful because of the color there are many organic substance like dyes they also color so that question is now these are all well known to all kinds of students even the school students also they know question is that how can I use these concepts as I said these using lights to determine molecular or electronic conditions in the in the real what's called scientific purpose and so that can be done you know by using common feature of these color compounds whichever displays whatever I have told you told you is basically a system which is extensively conjugated pi electrons you know probably the electronic structure there are sigma months pi months and other bonds so these are the pi electrons which absorb the radiations in the visible range and that is why you see a different colors so this is all very typical of many spectroscopic techniques and this is how we will discuss and I will discuss different parts of that now as I showed you that I will show you the visible spectrum in detail manner alternate pulse in the 190 to 400 meters valid is 400 to 420 Indigo is 420 to 440 and read very high wavelength 620 to 770 nanometers so that means read will have very smallest frequency from ultraviolet to rate so what are the different things you visible spectroscopy can do well you will be specific spectroscopy as I said at the outset can be used for that for determining the different molecular transition or electronic transition rather in the organic molecules like outer electron binding transitions conjugations which will discuss in detail or visible ones can be used for metal or ligand studies solutions like the orbital transitions and the end of this lecture if I will discuss about the instrumentation also so to give you a bit better idea you will spectroscopy is routinely used in many of this analytical chemistry labs if you just walk in many of the crystal labs people uses nowadays in fact in metal science also those who people study different kind of nanoparticles and solutions we use extensively visual spectroscopy and we can actually do both quality qualitative study and the quantitative study like tennis and metal science we know that they so different kind of colors because of the D-electron transitions and then similarly there are many others so solutions transitions solutions of transition model science are colored okay and that means they absorb visible lights because electrons with these metal atoms can be excited for one electronic student to one electronic student that is for sure so that because we know that the color is because of the electronic transitions from electron transition from the from the lower energy to the higher energy so that means we can actually determine what is the exact electronic state and these can be basically affected by different and then you know the different this color also can be affected by different pieces like different ligands okay to the example copper sulphate CuS4 and 5H2O this compound basically is very light blue we know that we all of you use copper sulphate in the school days for doing experiments if we add ammonia to it in H3 it becomes in a color is more light or rather the blueness of the copper sulphate increases and that means there is a change in the wavelength where the absorption maximum happens so we can actually if you plot the absorption versus wavelength a versus wavelength so at a very high absorption of length lambda max will see high absorption and these lambda max will tell us they look on tensions similarly organic compounds like DNA RNA those are very high conjugations absorb light in the UV region or may be visible regions solvent of these determinations are often water or water solution we know that ethanol can be also be used so that means the absorption in the ultramarlet and near ultramarlet in the visible spectroscopy region visual wavelength region can be used to study this kind of features and to kill you the exactly the ultramarlet absorption process let us look at that so we know that as I told you there are different kinds of all sigma 1 pi mons so we can have sigma from bonding orbital to the anti-bonding orbital or sigma to anti-bonding pi orbital transitions they are very high energy only accessible in vacuum ultramarlet that is when wavelengths are less than 150 nanometers not usually observed in the ultramarlet visible spectroscopy on the other end to sigma star we will discuss about a and sigma pi in detail in a moment's time or spy to sigma star transitions they are actually bonding to non-bonding transitions all the stars trans corresponding to non-bonding transitions that is lone pair electrons normally wavelengths where these absorption is maximum pulse in 250 to 250 nanometer region or you can have likes n to 6 pi star or pi to pi star transitions most common observed in organic molecule these transitions and they are observed with the lone pair component lone pair and multiple bonds normally in a little higher wavelength 200 to 6 nanometers any of this requires incoming photons to be matched in energy the gap corresponding to transitions from the gown state to the excited states like from n to the pi star energies correspond to a one photon of 300 nanometer slides are basically 250 now visible region of spectrum obviously corresponds to the energy levels from 36 to 72 kilo calorie per mole right now the near middle regions it will be high so that energy will be near with this is visible the near visible it will be little high so this will be approximately 143 kilo calorie per mole and in that case you know this this energies or the energies don't these are all sufficient enough to promote or excite a molecular transitions this is a very high sufficient enough to promote this kind of transitions and observe and so therefore we normally can use this as a transition so to give you some better idea let us know that molecules have quantized energy levels from quantum mechanics you have already studied I know then you have energy and there are different energy levels and if I apply certain energies like UV or visible spectrum UV or visible range then you can see this transition happened from this level to this level okay so whenever this excited state will go back to the gown state there will be emission and emission will correspond to certain objects and sometimes you know each electronic levels can be thought of as such with many vibration energy levels like this these are vibration energy levels you within each you can see the different vibration energy bands okay and they are studied in IR infrared spectroscopy which we will discuss after some time and you can have rotation energy level of electronic electronic state can get rotated also so therefore one can determine this this transitions so what are the nature of this absorption that is now I told you like one example is sigma 2 by pi to pi is the transitions from ethylene at about 170 and it can be calculated actually using this software and like this this is the homo pi you homo means homo-lomo as you know high occupied molecular orbital and I can have a lumos lowest occupied molecular orbital and you can see it is going from bonding molecular orbital to the anti-bonding molecular orbital that is pi to pi star if I apply 170 millimeter photon at this transitions can be easily detected this is apsop so one can actually show that in different charts and like they can see their pi pi pi n then the high anti-bonding orbitals so you can have different transitions from pi to this pi or pi to n or vice versa and then n to pi star or you can have this type also like reversible transitions or you can have n to pi star others are reversible so all kinds of different transitions possible this happens at lambda max of 218 this happens at lambda max of 320 and so this is in a shell what happens in ethylene molecule ethylene is we know that this is CCH2H2 that is CHC2H4 so there is a double bond here and this is what is shown there well so if I consider the total internal energy of the molecule to be simplistic it at me consisting of e-tanz that is electron transitions are e-electrons that is select sorry e-transitions is the transitions then electron transition elective the vibration transitions rotational transitions and nuclear transitions so normally electronizations are determined by UV and X-ray vibration also are infrared and are this that is I have just told also and this are different as level I have shown you so in our UV and visual spectroscopy this is the interaction of the molecules and absorption of proton results in electron transitions I have also told what is most important thing is that this ones are used to detect the presence of comaspia like dynes aromatics or polythene so conjugate kittens itself we will also discuss what is these okay so there are as I said I will discuss about this different electron extra the theta absolute electronization which can happen actually one is p s and n electrons and transition silver bomb basically charge transfer and transition involving d and f electrons are also possible in metal ions like tension metals absorptions of these lights like the ultra water visible radiation organica molecular is stick to certain functional groups like homosphere that contain valid electrons of low electronic excitation that means if you want to have a transitions in the UV visual spectroscopy for p s and n electrons the excitation energy should be as low as possible otherwise because UV and visible not very high energy so it will not happen this is again shown here this like sigma 2s sigma from bonding to anti bonding pi 2p to pi 2p star bonding to anti bonding sigma 2p to sigma 2p star bonding to anti bonding orbitals and if I want to show in a detail this is what I shown here this is the sigma bonding orbital pi bonding orbital or n non-bonding orbital then pi star anti bonding sigma first anti-bonding energy level increases this way so transitions are like this from n to pi star is this one which is shown here left one sigma a pi to pi star is this one shown there this can be detected by UV visible okay now you can also have potential like n to pi sigma star like non-bonding to anti bonding or you can have pi bonding orbitals to sigma anti bonding orbitals or you can have sigma to pi star you can sigma to sigma star so and you know that these transitions require different energy levels so depending on these energies available we can basically use different wavelengths of light or UV to have this transition possible so that is what I said in a nutshell in a UV visible spectroscopy you actually got to know the exact transitions happening from different electronic orbitals and these are the different electronic orbitals possible and again this is shown in detail manner okay so that you can you can even look at it this is I think this is the new bar and this is lambda so 100 200 300 400 nanometers up to 800 and this is visible from 400 to 800 and then 400 200 near ultraviolet 200 200 is for ultraviolet or vacuum maybe which are normally you not used in a visual spectroscopy so we will talk about from 200 to about 800 or 700 nanometers and you can have this kind of transitions in 200 turbulence you have a these are actually supply to pi star n to sigma star possible and you have in 300 level is n to sigma pi star and very highly visible lens you can have n to pi star so therefore if you use this turbulence we can determine these tangents very easily now let me tell you each of these tangents sigma to sigma is a transition and electron in a bonding s orbital bonding s orbital s is one of these spd orbitals they are in a in an atom so electrons in electron in a bonding orbital s is excited to corresponding anti-bonded orbital energy required is obviously very high because s is the lowest energy level so you have to take it from the s energy state to the anti-bonding state is very high so that when a methane like you have CH4 in a methane saturated compound only CH bonds are there so I can actually write down there there are 4 CH bonds in a methane molecule and these bonds are actually all saturated you know that and can only undergo this sigma to sigma star tangents that means electron can be excited from s a bonding orbital add to the anti-bonding orbital and maximum absorption for this is at about 125 nanometers so these transitions are not seen because on 25 is you know far IV not seen in the visible spectroscopy range they have to be used we have to use for a V or back of a V and if you have a n to sigma star tension that is non-bonding to anti-bonding sigma star saturated compounds like contain atoms like lone pair non-mini electrons they are able to these transitions this time usually need less energy than the sigma to sigma star sigma to sigma star obviously because because you are going from bonding to anti-bonding sigma of transitions saturated they cannot be initiated by light whose wavelength range of 150 to 250 number of organic functional groups with n the star peaks in the region is very small because there as range is 150 to 250 that changes has to come so that it is small now if you want to look at n to see pi star or pi to pass transition they are the most once most is easily detectable in UV visible most of the spectroscopy organic compounds is basically based on the transition from n or pi electrons to the pi star excited state transitions fall in the experimentally common regions of spectrum like 200 to 700 that is visible visible to UV this transitions are needed and saturated group in the molecule provided provide this pi that is the ethylene ethylene has unsaturated this bond double bond and it has also pi electrons which can undergo transitions from bonding to anti-bonding. So these are the things which are called comosphere CC double bond CO double bond this is ketone NO double bond or you can have CXX can be promenium and CC double bond times you will normally have pi to pi star transitions and like in hexane solvent if this is the maximum wavelength possible absorption can happen you can have n to pi star sigma or n to pi star transitions pi to pi star at these two wavelengths for ketones for nitrous group you can have a different wavelength regions these absorption maxima and for this kind of CX bonds you can have n to sigma star at this non-bonding to anti-bonding sigma star or transition that is at these wavelengths possible in all cases except nitrous bonds we use a skull hexane in this case we see tunnels of solvent and this is the in a big this is a table which is obtained from this book Lambert and verb it cooks stout schrubel organic structure analysis from manual publications what is showing you this different comospheres and the present in different compounds solvent use absorption maxima and one can actually molar absorptivity values can be calculated from the Lambert we have slow rather which again I will discuss and for those of you by not have understood exactly what is a sigma bond sigma bond is basically suppose you have two nucleus and this is how the electron distribution happens in a sigma bond okay single bonds usually too high and you know it because very high excertions we want this to go to the sigma star a transition sigma to sigma star transition that is bomb sigma bonding to anti-bonding transitions is a very high we have seen that also so we need vacuum UV and then you can have that this is the anti-bonding so you break this bond between them and form this kind of tensions so this is low to high from bonding 20 bonding sigma pi is the orbital is like this you can have electrons distribution in this kind of geometry and if you want to excite it by using of certain energy it will go to the anti-bonding state of pi star where there are different states different thing this is easily accessible by usable spectroscopy and non-bonding electrons do not take part any bonds actually they are neutral energy levels to give an example like in a formal dehyde it very classical aldehyde compounds you can have carbon there are two oxygens and there is a double bond here and carbon so this is like this I can write down so there are two electrons two electrons here therefore electrons here two are morning and two are others and then in oxygen as four electrons so we can clearly write this black dots filled once are basically sigma cross are basically pi and these are actually non-bonding time so you can have in this molecule itself if I apply energy there are different kind of transition possible I hope I have given you enough idea of what kind of tensions so in a ethylene if I want to write it down properly ethylene you can see that this is the pi and this is sigma and different energy levels I am putting and these are these sigma pi star and sigma star anti-bonding and if I apply energy levels all has the tension probably has happened because there is a double bond between the two carbon atoms so this commosphere is cc double bond so it can undergo tension from pi to sigma is a pi star and giving this much of energy and this happens at this wavelength so this is all known classically so one can use in determined this very easily similarly in a ketone these are the non-bonding electrons which I told you and there is a double bond and there are pi sorry there is a pi and then other electrons so you can have different kind of tension like pi to pi into n star or pi to pi star sigma to sigma star by putting energy level and when n to pi star tensions is actually at longer wavelength but not as strong as pi to pi star so therefore n to pi station is not normally forbidden in acetone this n to sigma star tension happens at 188 nanometers and n to pi star to happen at 279 nanometers with the molecular absorptivity given by this value which can be calculated or measure and I can actually go on tell different kinds of bonds this is saturated carbon-carbon bonds whatever you can see as the only possible thing is sigma to sigma star is very high energy not possible any visible spectroscopy CC single bond pi to pi star is also not possible at the end to sigma star in this case is very weak so maybe possible 183 UV and then you can have wage group is alcohol wage all group you can have sigma to pi to pi star n to sigma star n to pi star tensions were weak the same thing is possible in this to give a ketone situations you can see that there are spikes in the wavelengths at 188 when you put absorption busses wavelength and 279 so these two are telling you said two kinds of transitions possible one at 279 is basically corresponding to n to pi star and 180 it cost me to n to sigma star transitions well now these are all qualitative discussions now what we can do is that we can basically use Bias law to quantify different kinds as you know that when light passes through a molecule is passes like subject to the light or a particular wavelength of light is passing through a solution when the molecules are there they can undergo transitions which have shown him and obviously some light is getting absorbed some light is used this absorb light is used to promote this kind of transitions so optical spectrometer basically records the wavelengths at which the absorption happens light is also a ice also an optical spectrometer it also do the same stuff and then the spectrum which is presented as a so new in terms of a versus wavelength absorption versus wavelength and it gives you speaks so absorption usually ranges from very small by like 0 to 99% possible and it can be precisely determined by spectrometer so because the absorption of a sample is basically proportional to their number of absorbing molecule present in the solutions the molar concentration basically determines the number of absorption present in the sample and then that one needs to correct its absorptions by different considering different kinds of parameters like optional parameters and to exactly obtained the amount of light or amount of radiation absorbed by the molecules present in the solutions the corrected absorption is obviously called as molar light absorbing functions okay so the molar absorptivity and they can be used to determine to basically this is determined molar absorptivity is determined using this formula which is given here a by BC where a is this absorption and B is this path length through which the light is of the radius is passing through and C is known as the concentrations so this can be done so suppose I will give you an example if I take a isoprene isoprene is rubber all of you know this natural level is isoprene okay which is obtained from the tree and it is used for many kinds of purposes so and you know this isoprene actually in a dilute exchange solutions if you have like C is equal to 100-5 moles per liter and if the path length P is basically 1 centimeter and then we can use this formula and get sigma to be about 20,000 sorry 20,000 amount so by knowing this epsilon so not sigma epsilon we can actually quantify the different kinds of different kinds of absorption behavior by the molecules and it is can be even formulated by this way suppose I 0 is the initial radiation initial intensity of the radiation and this is these B is this path length I is the final which is coming out so I by 0 is DI by D0 is basically KC by DB or you can say this case is a constant and then one can get this kind of formula and the epsilon which is the molecular absorptivity can be written by K by 2.303 which come basically from log and transmission as I said can vary from 0 to 100 and opposite is the absorptions absorptions can also vary from 0 to 2% in this way so one can actually get idea now depending the path lengths obviously if you have 0 100% transmissions if you have 0.2 so one can get percent absorption versus path length this kind of behavior or in this kind of behavior and then one can actually use this external standard and calibrate this curve so this is the absorption this is the concentration you can see it follow the linear law where R is very high so the R is the confidence levels of the fitting which is almost 99% or 99.86% so standard addition method is standard addition must be used whenever you have a matrix because you have lot of other factors which determine this absorptivity and so slope of this working curve the standard made with the distilled water is different from the same working curve so therefore there are many ways how do you prepare the standards the standards can be prepared in different ways like this is stock solution which constantly known then you can dilute it and get different concentrations and that is why you can prepare the standards the constant volume the stock solutions should be chosen to increase the concentration of the unknown by about 30% in each flag that is this is the known this is the unknown so you add unknown quantity different concentrations and then add up make it up to maximum 30% and this is the response one can see that if you plot CSA and response this is a straight line it passes from these two points so using this one can actually find out this is the unknown constant in CX as you know a is given by sigma BC C is this concentration and B is the path length so we can write the sigma BCX by BT and this is the known this is the unknown and this is what is known as K is obviously written by sigma epsilon B by VT and VT is the total volume and so therefore Y is going to BX B plus CX AX is the KVS CS is known and basically and X is the CX and so therefore when A goes to 0 KVX is minus KV CS and CX is going to that is why you have to do before I go to the real instrumentation how the experiments are done let me show you some spectra so this is one spectrum electronic spectrum basically shown here you see there is nothing in the visible range nothing in the visible range in a sense these there is nothing and absorbed in the visible range 0 one can what has happened is that there are two peaks in the UV range for this whatever compounds abused and they are coming at different values of the lambda and so the one can actually determine this the epsilon that is more absorb it is by using this two different compounds of first of one can find out from this speaks what is the exact conditions so what is normally done is that normally if you want to apply BR sloper quantification we need to use very small concentrations and as I said UV bands are broader than the photo strength and so therefore this is vibrational this is normally superimposed that is now people say well what solvents normally used for UV like water or you can use this very high epsilon you can use CH3 CN okay which has 210 C6 H12 is hexane 210 etha 210 very small we have a little ethyl alcohol 210 hexane also 210 methyl alcohol 210 dioxane 220 then you have CH2 Cl2 235 CHCl3 245 carbon tetrachloride 265 benzene 280 acetone 300 these are the different sol energy cutoffs okay so now what you can calculate can you calculate the UV spectroscopy actually so one can actually calculate by looking at the local structure so this is suppose the local structure of certain compound Nacinol and Acidol and then one can actually calculate the absorption happening you can see absorption happening at this two wavelength one is this is 2 and 3 and 232 and 40244 45 and this about 275 similarly this is another one this is same molecule okay this is sort of change is the rotation this is the transition we are talking about it from this to this and this to this here so one can determine this spectroscopic things if I have an orbital involves like here you have a orbital transitions which are shown here atoms with molecule orbitals contributed most of the bands like one this one can be considered to be this tensions this one considered to be these tensions these are all Nacinol and then you can have this tension corresponding to this one or you can have this tension corresponding to sorry this one corresponding to this a molecules going to sigma 2 pi tensions so one can determine that and quantification also I just told you so I do not need to discuss so let me state I go to the spectrometer principles or the how these instrumentations are there well as you can see that this is the the in a nutshell this is the schematic picture what I can tell you that you have basically a reference and we are sample Kuwait is the sample kusable which is used okay now you have a light source like this you can have light source from BV a light source from visible and then falls on the mirror it passes through the slit and then falls on a diffraction getting passes through another slit it can be filter also because you want to use probably the exact wavelengths which is required and then it can basically good one of this can basically go to the difference beam can go to the different sample and it will be then focus by the lens one to a detector and intensity will be recorded other part you can go to the sample and then falls on the lens and then it will focus by the detector and gets the intensity I and then once you know the absolute value of the intensity is then you can use the Lambert's law BS Lambert's law to get the value of the molar absorptivity for the particular sample well normally we used quarch as a create samples obviously are to be very small concentration like to the minus 5 molar and it has to be protected all these tubes has to be protected from steroids which I discussed in the first class where the steroids are bad and normally D2 lamp is used for UV and tungsten lamp is basically used for visible and double beam actually is made makes a different different techniques now there are different kinds of instruments can be used one is fixed well in the instruments and scanning a little instrument or diode instruments the most recent one is diode array so in a fixed element instrument you have LED light emitting diode as a source LEDs there are green LED blue LED all kinds of visible LEDs are possible so this LED is actually can see red yellow green and blue so this LED is actually emit a particular light of wavelengths of light and then falls on a sample and no monochromat has needed because LED is actually emits very is in a precise monochromatic light and then it falls on a sample and then whatever getting out are detected by a photodiode this is how they fixed well instrument work normally there are four LEDs to be used to the four elements can be chosen by this now a scanning instrument we do not do that what we do is that your stanchion filaments were visible and your day to lamp that lamp per UV this is G2 O okay D2 D2 lamp and this is the tungsten I showed so both of them actually comes and passes to the slit and then you have a monochromator here how the mark water works is basically as crystals basically a backs law I will tell you how it works so there are crystals and then from which you can orient this can this move this one so that it can fall and when you scan this one only there is a slit here so only a particular lens will pass through the slits and it will form the sample quivet and then your photo multibular tube which will determine the whatever radiations coming out very simple so how this monochromator really works okay as I said there are different lamps can be used you can use general lamps also and monochromatic basically applies these Bragg's law okay and this is the Bragg's law n lambda equal to d sin theta d sin I plus sin R you can write it in it as a angular dispersions given by 2 I by T as n by d cos R R is this basically reflection and I is the incident beam so many times we write n lambda is 2 d sin theta and sin theta is basically the instant the angle but here you can split into like this d sin theta sin I plus d sin R so that is nothing if R and I are different then one has to write like this and illusion is obviously given by lambda by delta lambda okay or in a n is extended can be actually extend by concave mirrors in this monochromators this is what is done here you have this crystals which are sitting there they are actually reflecting the lights at different directions so if you scan it out only a fixed element will pass through this gap and holder sample holder this is also very important for instrumental purpose you have a visible for visible use plastic or glass for UV you must use watch these are all and examples you can have single beam double beam normally single beams are used double beams are normally used for single beam can be used but we have a source flicker the last one is diode arrangements in which we have both the lamps tungsten and the deuterium for the deuterium lamps for the UV and tungsten for the visible and then it goes to a slit and falls in mirror and mirror focuses it on to the sample and then this falls on a monochromator this is opposite to what you have seen in case of the the techniques scanning techniques and then this is by this monochromator actually moves and then different turbulence of lights falls on 328 detectors which each one is a 320 detectors each one is a diode and diodes measure the amount of the radiation which is coming out well so the advantage segment is for the both scanning and the diode array scanning has low spectral resolutions high spectral resolution sorry lambda by lambda delta lambda is very high but it has a long rate acquisition time which can be several minutes that is sometime may be problem for different solutions and it has a low through output through put so that array has a very fast acquisition time because it is that is it is basically determined the intensity of this radiation which is coming at different turbulence after it passes to the sample and normally it is couple of second encompasses several minutes and is compatible with online problem that is computer compatibility is there a very high throughputs there is no slits and it is a but it has problem is very low resolution so if you want to go for high resolution you have to use scanning instruments you want to go for high fast and the what is called high throughput instruments you have to go for diode array normally people have both in the labs normally and this is the extended view of diode array I am showing you these are the 328 diodes which have slid there and after the sample radiation comes up on sample it is a detected there well so this that is all in the next class will I will show you some more spectrum spectra from the UV visible results in my lab and some of the things which I gather from the literature and then we move on to the next techniques that is the photo luminescence.