 Okay, so we are going to continue with the second lecture of this morning It's going to be delivered by professor Latin Franco from the University of Nova, Gorica We'll be working so hello to everybody and Welcome to this second session of this still morning I Will be talking today about a technique that you were introduced to earlier last week by my colleagues Marcano and Professor where is umberto to help me from Mexico? About thermal lens spectrometry This is a technique which is basically complementary to what you have just heard about the fluorescence spectroscopy Yeah, so in thermal lens spectrometry in principle. We don't like fluorescence I will and I'm sure you know already because we are we are losing the absorbed energy through fluorescence We don't like the emission we like the conversion of the absorbed energy into heat and this is where the thermal comes from and by changing the optical properties of our samples, which means the Refractive index we create something which is similar to an a real Optical lens and we use it for highly sensitive detection in chemical analysis So the introduction By my colleagues who work in this field was I I'm sure very very From the physicist point of view I'm an analytical chemist and I will not be so rigorous on some Mathematical expressions that we will use in analytical chemistry. We like to use approximations simplifications and if we get a Straight or linear relation between the concentration of an hour signal We are we are quite happy so but I will I will point point out some theoretical Points today, which are important for understanding More or less the coupling of thermal lens spectrometry to other analytical techniques and To understand this I have listed here some Requirements for analytical methods in what I call here biochemical analysis bio comes from the Title of this winter college, which is something related to bio imaging. Yes, so this is not really analysis in biochemistry, but this An elite chemical analysis related to bio systems. We will see tomorrow applications for the analysis of let's say fluids in the single cells different biological fluids different toxic elements which are relevant for Which are related to health Also to food quality if you want there is a long list of Potential applications in what we call here bio chemical analysis so First of course with advanced advancement in science with Let's say new frontiers. We which are set in front of us. We are very frequently facing the problem of Sensitivity so to make new discoveries usually we need to deal with the with the Limitations in sensitivity of the instrumental technique techniques, which are available to us then secondly very important in Particularly in the respect to biochemical analysis is the selectivity. We must be sure that what we are detecting is not Just something in our sample, but it is really the compound that we are targeting and Finally, I would like to focus to new approaches in in chemicals Analysis which have a lot to do also with the cost efficiency, but mainly they are related to sample throughput and in this regard I would like to Define two terms which are in the past ten years or even more used in the in the chemical analysis and this is so-called rear guard and What we would like better is the vanguard analytical methods and it has to do with the Labor intensive analytical methods, which are usually also costly. These are commercially available and Due to the fact that they need a very laborious Sample preparation. They are also time-consuming Of course the advantage of such methods is that They fulfill all the at least the first two criteria and that they are usually certified So if you want to have a certified analysis you would usually Decide for what we call a rear guard analytical method, but why rear guard? because In the big demand for quick answers and analysis of large number of samples, you know Even sometimes large number of compounds in those samples Usually when my colleagues come to me and brings a sample they say Well, if you have done it yesterday would be even better Yeah, and if it would cost nothing even better So these are the problems that we are facing in everyday analysis Be it environmental analysis where I work a lot food quality and safety Medical diagnostics, can you imagine how many samples are analyzed every day in the different hospitals and healthcare institutions so Not to mention industrial processes there It is very important to have information on time to be able to control the production process in the chemical factory, for example And for that you don't need the most The most precise information so to say sometimes we can settle for a semi-quantitative analysis as we call it or even what we use in a Frequently in vanguard methods so-called yes or no response Yes or no response would mean for example then in a sample of fruit You are looking for a pesticide our pesticide present or not to give you an Practical information From Slovenia where I come from on the average with the very strict con I mean with the with the national Surveys which are regulated and require analysis of certain number of samples We find out that about 10% of samples are problematic But we are analyzing 100% of samples so 90% of samples are analyzed Just because they have to be analyzed and we are Losing time. We are losing money. We are producing on the other side a lot of waste Solvents We are trying to preserve our environment, but we are analyzing water water samples and Finding nothing in those so they are okay, but we are polluting environment because we create waste solvents so this is some kind of Contradiction in this sense. So if we have a reliable vanguard methods We can detect accurately those 10% of problematic samples and Only on those we apply what we call then a rear guard then we go looking back What is actually in that sample and how much it is? Yeah So tomorrow I will show you some more concepts, but Just like like you to understand this concept because they are very much important for understanding of the development In what I will talk about in thermal lens spectrometry, which is basically dedicated to this some high sample throughput analytical methods, which I will be describing later on so just Quickly review some basic processes which are important in Spectrometry so to say transmission mode spectrometry and from here on we will go on to the thermal lens spectrometry. So In the transmission mode spectrometry such as spectrophotometry for example or infrared spectrometry Usually we have a sample and we measure the intensity of incident light here Which we call I zero and the intensity of the transmitted light Yeah, and this is governed by the very famous Bearland-Berts law at least famous to analytical chemists, which says that The absorbance of the sample equals the negative log of transmittance, which is the ratio between the outgoing and income in going light intensity Here you can notice immediately that While physicists prefer to work with the natural logs in analytical chemistry. We work with the the caddock logarithmic system and therefore Frequently you will see this two point three oh three factor in the equations which Derive and we have it here already immediately which derived from the transformation of the Natural log of the the caddock to natural log basically For low absorbance is We can develop The exponential Function equals I zero To the e to the minus what we would call Absorbance yeah, this one could can be developed into Taylor series and for small values of a We can then take just the Linear term of this series and under such conditions we can say that Absorbance which in chemical analysis is defined as a molar extinction coefficient times the Optical interaction length times concentration equals The relative change in the intensities of the incident and outgoing light This is one of the approximations that we will make in our further discussions and of course absorbance this relative change which is given here is multiplied by the Conversion of logarithms Factor two point three oh three so should be on this side. That's why it's divided here so please remember this relation because later on we will try to compare the sensitivity of Thermal length spectrometry through to absorption transmission mode absorption measurements Now how can we improve the sensitivity in this kind of measurement We cannot do it simply by Increasing the intensity of light because the ratio is always determined by the Berlin Burt's law So we cannot use in the transmission mode. We cannot use this Very powerful tools such as lasers Increasing the light intensity which would be given by a laser doesn't help in this respect So what do we do we look for the lost photons see here? There is much less photons than on the incoming side and this was explained by a previous speaker of About the fluorescence. So if we are looking about the Emitted phone top photons after the relaxation process those Photons the intensity of this radiation is directly proportional to the to the incident radiation And this is what makes fluorescence such a highly sensitive analytical technique Similarly, if there is no radiative emission The absorbed energy is converted to heat and this changes the temperature of our sample And of course the change in the temperature is Proportional to the incident radiation more power we put in higher the temperature in principle This is what you will start feeling now when the spring comes out You know you will like to sit on the Sun and in the summer when you will enter your car You will touch your steering wheel your fingers will be burning if the the car is left on the Sun This is exactly the same process. It's the conversion of the absorbed energy into heat And this is what we are exploring in photo thermal techniques particularly in thermal and spectrometry, so Change in temperature of course means Change in the physical properties of materials and in thermal and spectrometry. We will Exploit the change in the refractive index But we will not work with with very high temperature changes usually 10 to the minus 4 10 to the minus 3 Kelvin is sufficient so don't worry that Your samples will be boiling but still the Power densities which we will use are high enough or very high To cause photodegradation of our analytes and this is what we will discuss in more detail later on What we want to generate is a temperature gradient And this one usually has the Maximum temperature on the axis of the excitation beam Because of this the Gaussian profiles of laser beams are preferred, but we also know now About the theories have been developed and the practical Experiments have shown that also the so-called Head-top Beam profile can be can be used In most liquids the resulting refractive index gradient is Diverging has a diverging effect Because the change in the refractive index with temperature is negative in liquids in some solids The change is positive, and I will show some measurements also Further on as a result of this thermal lens the laser beam is defocused and we Observe the change in the beam radius Which is really associated with the change in the intensity at the beam axis which we are actually measuring behind Filter that filters out the pump beam and behind the pinhole which Limits our observation of the beam intensity to the to the axis of the probe beam actually and I'm sure that you have heard a lot About this by professor Marcano So I will not get into detail Have you had the thermal lens experiment in the laboratory already? But you were probably not showing such dramatic effects As I have here on my slide. You see here the thermal lens here is a is a image of the beam spot Projected onto the laboratory wall and when we this is the probe beam and when we excite with the green laser we have of course Compound that is absorbing the green light We immediately see the change in the radius of the of the probe beam Now if we insert the filter which will filter out the green light then we get Such such effect. Yeah Basically, this is a very very strong thermal lens effect and We call it a saturated thermal lens with the maximum change in the probe beam intensity and It to impress some visitors, you know, you can make such nice experiments Which are of course useless in in chemical analysis because with such a strong effect. You cannot get Any meaningful result about the concentration of your analyte I will talk today only about what you have learned as a mode mismatched Thermal lens configuration. Yeah, I think Marcano has spoken about mode matched thermal lens configuration mode mismatch means that We will focus our pump beam into the center of the sample and The temperature gradient will be created as shown by this Let's say changing red color Of course with the maximum temperature developed on the in the center of the sample where we then Shine our probe beam As you can see the probe beam is Focused slightly in front of the sample in this case. This is what we call a mode mismatching and The Gaussian profile in such case of the probe beam Here is the detector plane on the very far right side this profile Will change with the changing radius of the probe beam and this change in the intensity On the beam on its axis is what we take as a measure of our signal To be strict we should speak about the relative change But and this is what students sometimes forget. They just take take the signal from the locking amplifier which is even not Exactly the same value as you should get from an oscilloscope And they forget that if they insert another filter the i0 will drop and Relative change will remain the same while the locking will show you a lower signal and then they come oh We have lost sensitivity. What do we do? Also, if you if you put your detector further The intensity will decrease because the radius increases same story you should always take care of measuring the Initial beam intensity so that you can relate it to the relative change in the in a Beam intensity, which is proportional to the to the concentration We can do a lot of nice images about The beam profile. This is the initial probe beam profile And this is the probe beam profile after the excitation we see the Decrease of intensity on its on its axis and we can even Find different theoretical descriptions which describe the time dependence or the evolution of the Thermal and signal with time during a single excitation As I said in chemical analysis we prefer simple relations and we like to simplify Of course, we must have good arguments to do that But in most cases this theta factor which you find here in this expression and It stands for absorbance times the power of the excitation beam Times the temperature coefficient of refractive index of our sample Divided by thermal conductivity and the wavelength of the probe beam this comes from the diffraction The other two terms come from the thermal effects So in in most cases the absorbance is so low We are measuring absorbance is of on the order of ten to the minus six ten to the minus seven so for Usual concentrations that we measure this theta term becomes much much less than one and therefore we can of course neglect all the Second-order terms in this equation I Would in the continuation. I will also Speak frequently about the so-called steady-state thermal lens steady-state thermal lens would be would mean thermal lens at the very very long excitation times and Long excitation times you see that the the second term here would Limit to zero because this is This part with limit to zero and This will become a constant so we will have a constant signal with time and also What I should mention here for this expression this expression is derived for the optimal position Of the sample with respect to the focus of the probe beam If we go back here see we have placed The sample at a certain position with respect to the focus focus of the probe beam and there is always an Optimal position for this in the mode mismatched case and usually this is Now depends which theory you take but this is either One theory says one confocal distance from the Waste of the probe beam the other one says one confocal distance times square root of three in principle We always determine this experimentally in the laboratory So we don't bother so much with the theory, but of course, it's nice if you can show that your measurements are following some theoretical prediction and I will show you some cases later on not for standard or macroscopic thermal lens spectrometry as we call it but for what we call thermal lens microscopy so to In view of chemical analysis and application of thermal aspect from a tree we should outline some advantages which made this technique Quite popular in chemical analysis And this is of course high sensitivity that I have already outlined stems from the fact that the signal is proportional to the excitation laser power so in principle by exciting increasing the excitation power we can increase the sensitivity as We have seen it also depends on the thermal properties of our sample So the higher the change of refractive index with temperature the high will be the signal lower the thermal conductivity Higher will be the signal and we will see how this reflects in the so-called enhancement factor It has been reported that absorbance is as low as 10 to the minus 7 can be measured And this was demonstrated in many applications The response of the thermal lens is relatively fast. It's on the millisecond Sometimes even microsecond time scale With time constant. Yeah on this range, which is very important for online measurements in chemical analysis Because we will see that we cannot perform in most cases just a Simple batch mode measurement in a standard spectrometric Kuwait And of course because we can focus laser beams very tightly We can measure sub picoliter volumes We can probe sub picoliter volumes and we can perform detection in so-called microfluidic systems now microfluidics is a big expanding field of Also in chemistry and of course it also requires highly sensitive chemical analysis and detection instruments, but While many people are usually trying to impress the audience by advantages of their techniques I Would like to be let's say honest to you and Outline some drawbacks of thermal lens spectrometry or photothermal spectrometry sometimes We still need improvements in sensitivity as I said To do this we can change the solvents We can also increase the laser power But this is not good for photo labile compounds because we will destroy our sample if you imagine that we are using laser powers of Some 10 to 100 milliwatts and we focus this Almost to the diffraction limit sometimes especially in thermal lens Microscopy so that the spot radius of of The pump beam is on the order of one micron or less and this causes extremely high power densities which Can destroy Samples or analytes if they are not for photo stable still we are facing the problem of Laser sources and I'm talking about cost effective analysis. So we cannot afford for a routine analysis. We cannot afford Very Sophisticated for example titanium sapphire laser which has a tunability over reasonably wide range Usually we work with the simple solid-state lasers with just one Wavelength available, of course we can have several solid-state lasers But the problem arises when we need to go Low with the excitation wavelengths when we need to do chemical analysis in the UV range there the availability of lasers is very very limited so we have to We have to perform some coloring reactions and this such reactions usually take time these are derivatizations of different type and they require time and for this we try to reduce the time of This coloring reactions sometimes also Sacrificing the sensitivity of the technique because for example if we need half an hour to complete some reaction Maybe it's sufficient to Do it in five minutes with this of course We lose the concentration, but we can compensate it with that with the laser power or sensitivity of our technique the main disadvantage that I would like to stress here is the selectivity of Thermal and spectrometry and this again stems from the fact that in most cases we do Measurement at the single wavelength. There were some reports, but I remember only Only one by professor Omeneto from ISPRA. He did some spectral studies of lanternite ions in aqueous solution by thermal and spectrometry, but that again required sophisticated die laser pumped with the excimer laser and of course also Quite it was time-consuming and certainly we cannot do Wavelength scanning with thermal and spectrometry when we coupled Thermal and detection to different analytical separation techniques as we call it. Those are usually used to Increase or enhance the selectivity of the analytical methods. We we know high-performance liquid chromatography Ion chromatography, capillary electrophoresis. These are all techniques that require detection online with the flow of the element which is eluting our Analyze from chromatographic column About the photodegradation and speaking about the flowing systems flowing systems usually help in reducing the photodegradation Because the residence time in the detection cell is much much shorter and this decreases the Photodegradation and I will show you some some examples later on now first let's see what thermal length spectrometry Combined with liquid chromatography can do in terms of sensitivity and of course in terms of selectivity Here as you as you see in the title this is analysis of a blood plasma sample for Determination of carotenoids carotenoids are known antioxidants and of course we would like to have More antioxidants in our blood that's Our let's say desire in a certain way what are carotenoids carotenoids are Long organic molecules which have a system of conjugated double bonds and those double bonds are basically Quenching radicals and oxidative species You have probably heard about better carotene like opinion tomatoes Carotene in carrots of course and they are there is a whole series of different Analogs of carotenoids which we want to detect of course they are Colored so they absorb in visible the maximum of absorption is around 480 for Most of the carotenoids for lycopene is closer to 500 nanometers But what you see here is actually a thermal length spectrometer The blue is the excitation beam coming from an old argon laser so 488 line and We combine it with the probe beam coming from this Small helium neon laser with a dichroic mirror here and here you see a flow through cell Which is connected to chromatographic column and here is a chromatographic pump Basically with the chromatographic column we separate different components of our sample and they Come to the detector which in this case is thermal lens spectrometer at different times after the injection So if this is the time scale Injection of the sample here so here we would see some Different peaks which correspond to different Carotenoids this one here. I can tell you based on the analysis of standards This is a beta carotene. This one in front is alpha carotene and these are different different Isomers of lycopene here on this at the longer retention times If we compare this sensitivity to the Conventional diode array detection, which you use will be by with the liquid chromatograph today This is what what you get you hardly see the most intensive peaks in this chromatogram So the analysis is much less sensitive This was the first demonstration one of the first applications that we have done in combination with the liquid chromatography and Since then we are mainly focusing on thermal lens detection in the flowing systems Here I would like to point because in the next step we will Look at some theory But I would like to point at the to the sample requirement and the volume requirements for thermal lens spectrometry what we use here in this case is a What they called a sample of a flow through cell Which basically has Two cubings one is in one is out So our solvent is flowing into this sample cell, which is one centimeter Optical interaction lens and the total volume of this cell is eight micro liters. So nothing special It's very easy to focus our laser beam the Diameter of the aperture here is on the order of one one millimeter you can calculate based on the volume and the length So and also the injection volumes which we use in liquid chromatography here You see the injector injection volumes are on the order of 10 20 microliters in ion chromatography this goes up to 100 microliters So here we don't have very very restrictive requirements about sample volume and the detection volume It will be a different situation in a in a capillary electrophoresis for example Where we have a capillary here and we have to perform detection at a certain point of This capillary so we will be we will be using a probe beam And we will focus our pump beam in this direction and so here we have a Collinear Propagation of the pump and probe beam and here we will speak about the transversal Propagation of the pump and probe beam. I didn't see this on the slides of Of Markano, so we can spend a few minutes on this So here we will Speak about the detection volumes of much much less than one microliter Yeah, of course the concentration sensitivity in this case will be much higher Here we will be able to detect only higher concentrations, but the total Mass in the detection volume will be Substantially decreased and in the thermal lens spectrometry Microscopy the Japanese group of Kitamori has reported less than one molecule per detection volume Sensitivity we will this discuss this is of course disputable. How can you detect less than one molecule? But we will see later on baby tomorrow how this can be done It's an average signal over certain time which shows you Less than one molecule now Speaking about flowing samples. I'm sure that you you have seen this Equation in the derivation of thermal lens theory last week, but for I think you have not seen this Velocity term here, and this is very important because our samples are flowing Flowing through the sample cell or flowing through the capillary and Flowing means that the flow is taking your heat away So you are losing the heat that you were generating through the radiation less the excitation of your analytes So this is something not desired something that we would like to be able to correct for but immediately I have to Tell you that in such collinear cases where the Flow rates are on the order usually on the order of half a milliliter per minute up to one milliliter per minute the loss of the heat because of this flow is Insignificant so to say it's on the order perhaps of a few percent only while here in this case you see that the Interaction length is very very short. So with the similar or even smaller flow rates The heat is displaced very far from the Excitation point in a very short time. So in this thermal Thermal diffusion equation we must take into account the velocity term here is the source term which also Depends on the type of excitation Which we are using and here on this slide you immediately see that the source term is quite different for the pulse excitation in case we use pulse lasers or for the modulated and this describes the modulation frequency with the continuous wave laser and That's why we use here the average power or power better say power density because a squared is the Radius of the pump beam so Okay, let's let's go back now to the very very quickly to the expression of thermal and signal we have seen that the Thermal length signal is defined as the relative change in the probe beam intensity which can be related to the Relative change in the squares of the probe beam radius Before the excitation this is time zero and at a certain time t during the excitation cycle and using seem a simple ray transformation matrix You can very easily come to this expression which relates The square of the probe beam radius at time t to the initial value and to the Focal distance of the thermal lens this f t is actually the focal distance of the thermal lens Which we are creating in our sample of course at time zero there is No focal lens or the focal distance of this lens is infinite At a certain time t then the thermal lens appears and it gets stronger with the excitation about The ray transformation matrix gives us also the dependence on the distances Z1 is the distance between the focal point of the probe into our sample and the z2 is the distance between the sample and the detector Usually we work in the far field configuration so the Both distances z1 and z2 are much much bigger than the cofocal distance of the probe beam which also appears here in this equation and Also the focal distance of the thermal lens is usually bigger than the cofocal distance here at the Also the here is the initial condition as I have described and by using these approximations we come to a very simple Solution for the thermal lens signal which basically says that the thermal lens signal is Proportional to the inverse of the focal distance of the thermal lens and With this we can develop then Different expressions for different configurations and for different excitation modes now if we have a collinear Propagation of the two beams the inverse of the focal Distance would be proportional to the temperature coefficient of refractive index optical interaction length and the Temperature gradient with the respect to the radius of the excitation beam in the case of transversal of course we have to integrate Over the entire over the entire pump beam Because the entire pump beam be will be actually creating The temperature gradient and the temperature gradient is Perpendicular we are looking to the temperature gradient perpendicular to the direction of our observation So this is in this direction Here this is the direction y Perpendicular to this is direction x while usually we take a Z as a Propagation of the probe beam in this in such systems. So here you see because of this integration the This is not dependent on the Optical interaction length because it's only over the profile of the excitation beam with this expressions we can then derive the signals for the Collinear configuration pulsed mode here you see that Very soon after the laser pulse the signal is the strongest and then with time the signal slowly dissipates Because the heat is released into the environment or to the bulk of the sample in the case of continuous wave You see that the signal is the largest at longest Times of excitation What is interesting here is that the signal in the continuous wave goes with the inverse of the square of the Pump beam radius here. We must recall the expression For the time constant which also depends on the square of the pump beam radius. Therefore, we have the fourth power here This is even more important when we speak about the transversal configuration and Here you see we talk about the absorbences in Chemical analytical terms. This is the absorption coefficient basically Multiplied by the optical interaction length absorption coefficient again as I have described it earlier molar absorptivity multiplied by concentration and Here in this case Where we need to probe a very small sample volume We can see and this requires Small beam ready Yeah, small beam radius of the pump as well as probing but particularly with respect to the to the pump we see that Given the fact that time constant is proportional to the square of the pump beam radius we have to the third power and this is Just inversely proportional. So it's much much More desired to use pulse excitation because we gain a lot in terms of sensitivity because of this this dependence Smaller the excitation beam more we gain in terms of sensitivity So these are the simple simple consequences of the modes of excitation and the configuration of the pump and probe beams in internal lens spectrometry, okay, and just graphical presentation of the signals now here you must be You must read it properly. Yeah, this is the signal. It's not the change in the probe beam intensity This one will be just the opposite with the opposite sign the probing intensity will drop And then it will relax back to the initial value. Well, the signal in the pulse case is the highest immediately after the pulse and then It decays with the given time constant in the case of continuous wave It builds up slowly Until it reaches the steady state here if we Break the excitation because we use a usually we use a mechanical chopper for this then the signal will start to decrease to the initial value of the probe beam intensity Here I would like to mention just another Approach which is a combination between pulsed and the continuous wave excitation and we we call it quasi continuous excitation For example, you have a pulse laser with a very very high repetition rate and you would then apply a Mechanical chopper on on this output of such a laser. So, you know, if this is the time profile of the excitation This will show the Open and closed period of the mechanical chopper so here you would have a lot a lot a lot of Single pulses which overall will act as quasi continuous excitation so that the signal development is basically similar to the continuous wave Excitation but you can do it also also with the with the pulse lasers, but with the very very high repetition rate now why We prefer continuous wave excitation There are two reasons Pulse to pulse reproducibility with pulse lasers is not Very best so we get the much better stability of the excitation beam in the continue in the continuous wave Excitation and secondly we can use locking amplification which facilitates very much the The convolution of our signal and the data treatment later on so most of the applications in chemical analysis are in continuous wave mode excitation now another look at the So-called ultra sensitivity of thermal lens spectrometry compared to the transmission mode measurements now regardless the fact which Model we use either parabolic or aberrant model of thermal lens generation and the effect on the problem We can immediately see that this Relative change in the beam intensity Which we have for low absorbance is have Approximated with the 2.3 oh three times absorbance. Yeah, this is in a transmission mode for low absorbance is for the thermal lens Here we see a different relation which includes this what we call the enhancement factor and enhancement factor comes mainly from the thermal properties of the Medium in which we perform the measurements. So dn over dt temperature coefficient of refractive index and thermal conductivity so if we calculate this enhancement factor for One milliwatt of excitation power we come up with the following numbers for different solvents of course water is the most widely used solvent in Nature not only in chemistry in nature And especially when we have to do with the bio Chemical analysis we are very very much Forced to do the measurements in the water But unfortunately as you can see the enhancement factor is of water is relatively low So if we want to have a comparable sensitivity to the transmission mode Spectrophotometry we need at least 10 milliwatts of excitation power This is what this number tells us But when we have 100 milliwatts excitation we already win by a fact of an order of magnitude the factor of 12 according to this It also depends a little bit on the probe beam wavelength But we can see that for organic solvents which have much higher temperature coefficient of refractive index and much lower thermal conductivity Already with the with a single milliwatt of excitation we get almost five times higher sensitivity compared to the spectrophotometry Acetone is somewhere in between But unfortunately such nasty nasty solvents such as benzene Carbon tetrachloride and so on they have the best of the thermal properties, but We have to avoid them now another way of expressing the enhancement factor or The suitability of a solvent is just to take the ratio of the temperature coefficient of refractive index and the thermal conductivity Because all the rest is laser power and laser and probe beam laser wavelength Which you usually keep constant for a given Instrumental setup, but here you can see that we can we can avoid some bad solvents for example Working with supercritical fluids. It's a very very interesting and it gives extremely high extremely high enhancement factors the problem is that We are on the very steep part of the curve in terms of the change of Thermal conductivity or the end of the tea with respect to temperature or pressure So we have to have very very stable conditions to keep this enhancement factors stable otherwise The signal is is quite noisy But other Solvents are also interesting very popular lately ionic liquids. So we have studied several different ionic liquid like metal imidazole metal imidazoleum salts with different anions triflate Borel fluoride and so on and different lengths of the alkyl chains attached to this imidazoleum ring So here you can see that with with some ionic liquids we can reach enhancements comparable to the best organic solvents which are less less much less friendly to us than Ionic liquids or at least from the environmental point of view they create much less problems So the selection of the solvent is very important From the point of view of sensitivity of our measurements, of course we can do mixing of Organic solvent solvents which are mixable with water we can use additions of acetone ethanol Acetonitrile, but when we are going to the biological systems again We have to take care of denaturation and such processes To illustrate the dependence of the thermal lens signal on Thermal properties, I would like to show an interesting Piece of work that we have done quite long ago, but it was the question of determining the maximum of the refractive index of water and about Yeah, let's say 25 years ago. You could still find a very very broad range of temperatures from I think plus two to Negative values and some if you look to Lawrence Lawrence equation some were absolutely wrong from the already from the theoretical point of view But still they were published. So we have done a very nice zero zero point measurement by changing the temperature of water Performing the thermal lens measurement here this lighter part of of this plot demonstrates the opening cycle of Of the thermal lens experiment and we can see that when we go from the room temperature Down we are actually decreasing the dn over dt if we have refractive index as a function of temperature This one follows more or less function of density, yeah You know that the water has a maximum at the four degrees Celsius the density of water and the refractive index Has a very much similar behavior. So at the maximum of the refractive index N over dt equals zero The n over t enters all of the equations that I have shown So if we have a maximum the n over dt is zero we have no signal and this is what we see here So at a certain temperature Which was more or less close to zero degrees Celsius? We observed no signal and then we when we went to super cooled water and this was no ice It was water When you go into the region below the maximum the n over dt Changes from the negative to positive So the sign of your thermal and signal changes and this is what you what you see here instead of Foto thermal defocusing you get photo thermal focusing of your beam Because the n over dt changes from negative to to positive and with this measurement We have we were able to determine with quite high accuracy and precision the Temperature of the maximum in refractive index, which is close to zero degrees Celsius So about four degrees shifted from the maximum of the of the density Another thing that we you must not forget is the contribution of the changing concentration so that In addition to the dn over dt term in all the equations that we have shown so far We should include the dn over dc term And this is important in in systems where you can observe such phenomena like a sorae effect Where you have because of the slight heating you get the diffusion of your molecules into the irradiated area and that concentration Concentrations are relatively high and they can change they can change the the refractive index But they appear at a much a much slower timescale and Diffusion is a long process Thermal relaxation is is much faster process. Okay now We have refreshed a little bit our theory and explained For some particular cases the effects of flows we have not spoken yet about the effects of photo degradation and Here I would like to show you an example Which is also important for from the chemical analysis point of view Determination of hexavalent chromium. This is very toxic carcinogen That's why we are interested in determining very low concentrations in natural waters Different water samples and so on however if you use a very very high laser power what you Get is this line eight here B is the line with basically caused by the absorption of the of the blank There is no photo degrade degradable part in it But if we consider the theoretical description of the evolution of the thermal lens now Yeah, here we have some problem This is what we are showing here is basically the negative thermal lens signal because here This would correspond to the probe beam intensity basically but doesn't really Really matter. It's not so important But what I want to show is that the signal doesn't evolve to the steady state Condition after a certain point it starts decreasing and then it reaches some equilibrium value and The reason for this is that this complex which we form to detect chromium It's a complex with diphenyl Carbazone basically It's a very specific Choloremetric reaction only chromium as a chromium 6 or chromate as a very potent oxidant can Oxidize oxidize the reagent and form the complex which absorbs at around 540 nanometers, but this complex or I should say all the complexes with diphenyl Carbazone Metal complexes with alphanic carbazone are known as a photo lab. I so some would degrade already at the sunlight In the laboratory some like chromium is more stable, but still when using high laser powers the complex degrades and This is reflected that already in this way that already during a single excitation cycle We observe the degradation of this complex and we can even Describe this with the equation Here we have the constant of degradation rate. We have the diffusion constant and The the the two concentrations one is the initial concentration one is the equilibrium concentration here So far we have always considered the concentration during a thermal less experiment Constant yeah, we didn't speak about it was always taken as a constant part of the Thermalness signal now. This is not constant anymore, but we can still Describe and this line here in the curve a follows basically this equation and You see that at a certain point when diffusion of the non-degraded chromium complex into the irradiated area Equals the degradation rate at that time we obtain a much much smaller But a constant thermal lens signal. However, we have to wait much much longer than than here so This this is an example of a photodegradation which we have also confirmed by additional measurements in in batch mode system That shows so if we if we have a standard One by one centimeter cuvette. We measure with we put Let's say two to three milliliters of such solution in such cuvette and we Monitor the signal with time Over over Half a minute approximately you can see that we already lose more than 20% of the compound if we connect this Into a flowing system So if we take this as a reservoir of our solvent and we connect it to such a flowing system And then we put it back You see that in such a system then the residence time of our compound in the irradiation area will be much much shorter So you see that the degradation is much much less expressed so this tells us immediately about the advantages of the flowing systems in in for such photo lab lab I'll analyze and Still with what we call the flow injection flow injection system means injection of our sample into a flowing Medium which brings the analyte into the detection cell The exposure to the high power density is short We can obtain a very very favorable limits of detection, which is on the order of Less than less than 100 picograms per milliliter of chromium 6. This is way below the regulatory limits, but also way below Standard techniques if I go back now to the rear guard techniques rear guard technique for the chromium Metals would be usually atomic absorption spectrometry If you do electro thermal Atomization you could reach limits of detection of the order of probably one nanogram per mill Even higher if you if you do ICP emission optical emission spectrometry Okay Here we are still With the relatively large sample volumes now for such system Which was developed for the purpose of capillary electrophoresis by by fauble and his group quite some time ago. We have said that We are using Pulse laser excitation. Yeah, because the dependence on the pump beam radius is with one Over third power Which gives us high sensitivity, but because of the low pulse-to-pulse reproducibility we prefer to work with the Continuous lasers. So for for such purpose the so-called thermal mass microscopy was introduced for detection of Small volumes. Yeah with usually continuous lasers or quasi continuous pulse mode now what is the secret of thermal lens microscopy basically the Principles are all the same as we described already the only difference is that Both the pump and the probe beam are focused with one and a single lens in the case of conventional or Microscopic thermal and spectrometry. We are focusing the pump and the probe beam with the different lenses to obtain the mode mismatch so To get the mode mode mismatch, of course, you cannot use an acromatic lens Because you will focus the pump and the probe into the same spot, which doesn't work. So you must find especially designed Lens or usually you take a very very old microscope those had Standard lenses no acromatic lenses and what you can because of the difference in the wavelength You will find a mismatch in the focal points of the pump and probe beam Which are on the order of some 10 microns or 100 microns, which is sufficient to obtain the Mismatch in the modes and you can use one As a pump beam and the other as a probe beam Since we were working with the flowing systems we needed not a commercial thermal lens microscope, which is you can buy it from a Spin-off company of Professor Kitamori in Japan, but we have Constructed our own thermal lens microscope, which gave us flexibility in changing the beam ready of the pump and probe beams So here you see this is a probe beam still with the quite bulky laser system with Argon ion and the helium neon And by tilting the mirrors we could also displace the the axis We could offset the axis of the pump and the probe beams Because we wanted to compensate for the heat loss due to the flowing sample because now will not be coaxial With the flow, but we will be Orthogonal or transversal to the flow and this would have much much stronger effects More important from the theoretical point of view was our new Consideration of the thermal lens effect and the thermal lens model Which is based on the fact that the thermal lens is considered usually as a thin lens in in your sample This is not true particularly not true for a highly diverging pump beams and probe beams like in thermal lens Microscopy, so we have we have considered our system as a series of a thin Lenses located at different positions along the propagation of the probe beam so the Total thermal and signal is actually the contribution or the sum of all the individual thermal lenses here and We have shown that this gives a better description of the positional dependence of the thermal lens signal The previous theory Has followed the this dotted line the blue one or the red the red one I will explain in a minute why the blue and why the red But our experimental results have shown the dots Which deviated quite a lot particularly in the in the position of the optimal thermal lens signal I suppose you are already familiar with this bimodal curve that my colleagues have described for the case of mode mismatch thermalized signal when The sample is which says basically the following when the sample is positioned in the focal point of the probe beam You get the zero signal if you move it further along the propagation of the probe beam The signal is what we call a normal thermal lens signal a diverging With a maximum at a certain position and if you move it Further towards the probe beam laser or probe source the signal becomes a focusing thermal lens signal, which is it in our terms a negative thermal lens signal, but so the position of the maximum differs quite a lot and With the new theoretical model we could get a much better description of theoretical data But what was more important here in this work was that we wanted to optimize the pump beam radius To diminish the photodegradation of label analytes and this means That you simply increase the radius of the pump beam With this we have also Observed and we have predicted this also that by optimizing the pump beam radius you can optimize the sensitivity for a particular Sample size or thickness which has not been done so before Here we have an example on the right-hand side for a two micrometer excitation beam radius and We were increasing the sample length with this very simple system. We just had a Wedge Vet handmade so by putting this Placer in between the two Windows of a microscope we could obtain different Technices of our samples so just by measuring at different Positions we had different thicknesses of sample and you can see that with this pump beam radius We can increase our sensitivity up to about 300 micrometers sample thickness all beyond doesn't contribute anymore to the thermal lens signal why because With the thermal and with the microscopic objective lens the pump beam is so tightly focused That it diverges and the power density at 300 microns is so low that it doesn't contribute anymore to the generation of thermal lens so Then we have thought Why don't we optimize the pump beam radius for the different thicknesses of the samples because sometimes you had You have to work with 100 microns sometimes with 200 sometimes more Another observation in this work was that By using the diffraction limit Focusing yeah, this is 700 micrometers radius. This is Almost at the diffraction limit for the used wavelength. We see that we observe a much smaller signal see compared to the Lower power density the signal here is much higher in this case. It's lower now We have of course we had to displace The sample because of the changing focal point with the radius But in a given range we could measure We could we could find a maximum of both signals because it's actually not important whether the signal is positive or negative in the same range of positions we could measure the Highest signal for two microns and the highest signal for the point seven micrometer excitation why this because You can see that for different sample thicknesses we have a Different optimal pump beam radius. So here now I'm showing the measurement points for a hundred micrometer thick sample and For a 300 micrometer thick sample the red line see of course the longer the optical interaction the higher signal of course, but For a single curve you can see that Going from the diffraction limit here By increasing the pump beam radius you get the highest signal and then if you increase it further Of course the power density decreases and just you get you start losing your sensitivity for the thicker sample of course over Almost three point five microns radius of the pump beam gave the highest sensitivity So this shows you that for different sample thicknesses you can optimize this sensitivity by optimizing the pump beam radius and Usually you can calculate it From this equation This is the square root of the excitation wavelength times the thickness of your sample divided by 4 pi This is how you can determine the optimal pump beam radius and From the point of view of photo degradation of your analyte a very important consequence yeah with One tenth or even one twentieth of the power density of The one given at the diffraction limit we can improve the signal by almost 40 percent in In the hundred micrometer tick sample or even two point three times so 230 percent for the 300 micrometer sample so Very important when you want to achieve highest sensitivity in thermal lens microscopy and prevent photo degradation in the past Everything was and also when you buy Commercial thermal lens microscope this one is usually diffraction limit focused and does not allow for such Such optimizations Let's now consider the effect of velocity We can calculate how much a Certain limmer flow velocity will affect your signal which in principle follows the temperature rise in your sample and You can see that at the steady-state sample we get of course a maximum signal But when we start increasing the flow velocity in this direction the maximum the position of the maximum temperature rise is shifted along the along the flow and at the same time the Maximum temperature rise decreases so we get in principle less less thermal lens signal so the idea is shifting or Displacing the axis of the pump and probe beams in the direction of the flow and this is demonstrated here so We have the excitation beam which produces the heat at this point, but the flow Carries downstream Therefore we have to set or position the axis of the probe beam slightly Downstream as it's shown here So you see here that the axis of the pump beam and the probe beam the red one do not coincide and By doing this we compensate for the at least partly for the loss of heat and we can obtain Much better sensitivities This is shown here the red dots and they even show some non linearity, but the calibration curve is the As the the lowest slope Even if we take into consideration this linear part here when we go to the Zero velocity or steady-state sample We have of course much higher sensitivity which is shown with this black calibration curve and when we displace and we place The probe beam at what we call the optimal at least experimentally optimal condition we get a higher Sensitivity We can also calculate the optimal curves for different velocities in in micro channel for different excitation beam radius And we can see that some of them the values These are the measured values do not correspond well to the theoretical values the reason is that We know only the Average flow velocity from the measurement or by setting the velocity, but in the micro channel We have to take into account the that the flow velocity is Distributed from the maximum On the axis or in the center of the capillary to lower velocities on the Walls of the capillaries and this is what contributes to them to the differences In terms of effects on photodegradation We can study this by using by comparing the results of a photo labile analyte to a stable analyte and Here we for example here we we have the ferro in this is iron two complex with 110 financial in very known Choloremetric reagent in analytical chemistry, and we see that with increasing Excitation power which will cause the gradation There is no effect on the calibration line. They are they are linear. They are straight lines Yeah, when we do this for chromium DPC you see very very strong deviation from the Linearity with the increasing laser power, yeah, but as you can see The decrease the relative decrease in the in the case of higher flow rate is less than in the case of Lower flow rate this can be found by comparing the slopes of the two lines for 20 and 15 micro it is per minute which Contribute which consider only the Effect of the flow the flow is taking the heat away That's why that higher flow the slope is lower and in this case Of course, we have to consider only the very very lowest Excitation powers because otherwise we cannot compare the slopes Here we have a ratio of only 1.20 means that the the value at 50 Microliter per minute is higher Which is Cost by less degradation at higher flow rate. Yeah, so if the degradation If there would be no degradation or the degradation would be the same in both cases We would we should observe the similar ratio as here with the with the photo stable compound, but in this case There is less degradation at 50 micro liters per minute, which is normal because there is shorter Residence time less degradation. So this value decreases by about about 8% Now the last five minutes. Do I have five more minutes or gonna two? Okay, everybody's hungry Just to Consider Or maybe we do it tomorrow huh, I Would I would still like to spend five more minutes on this? So let's let's do it tomorrow before I bother you with all all the different possible applications What I would like to show is How can we use? How can we use advantage of a tunable? light sources like a xenon lamp of course with much much lower power but taking The advantage of some phenomena in thermal lens microscopy, which we have this regarded in the macroscopic thermal lens This is the heat losses on the boundaries on the front on the rear mirror Because the large of the large sample we consider the loss of heat negligible or the effects on the thermal and signal negligible in the microscopic world We will see that the temperature change At the boundaries is still considerable and we can use it to enhance actually the thermal and signal But let's let's see how do we do this tomorrow? so at this time I thank you for your attention and if There are any questions of course Don't be shy. Thank you very much