Fluorimetry Research

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1 ● Fluorimetry Spectroscopy and Its Applications ● ● Department of Analytical Chemistry Faculty of Pharmacy Minia University ● Introduction Some organic or inorganic compounds liquids or solids molecular or ionic crystals whether pure or in solution emit light when they are excited by photons from the visible or the near ultra-violet regions. Among the analytical applications of this phenomenon known as photoluminescence is fluorimetry a selective and highly sensitive method for which a wide range of measurements are accessible. The intensity of the fluorescence is proportional to the concentration of the analyte and the measurements are made with the aid of fluorometers or spectrofluorometers. The extremely rapid extinction of the light intensity when excitation ceases is the object of analysis. By contrast phosphorescence is characterized by a more gradual diminution during time. Fluorescence is equally employed as the basis of detectors used in liquid chromatography. Although of different origin chemiluminescence which comprises the emission of light during certain chemical reactions has also received several applications in analytical chemistry. Spectrofluorimetry is the study of a specific mode of light emission by molecules called fluorescence. It can be used both for quantitative and qualitative analysis for research purpose like the determination of the effect of different halides on the quenching of a molecule. Absorption of UV-visible radiation causes transition of electrons from ground state low energy to excited state high energy. As excited state is not stable excess energy is lost by ● Collisional deactivation ● Emission of radiation Photo Luminescence The phenomenon of fluorescence is not only used for chemical analyses but is equally applied to substances such as washing powders where a fluorescing agent that sticks to the textile fibers is added. This type of compound absorbs solar radiation in the non-visible part of the spectrum and re-emits at longer wavelength in the blue spectral region of the spectrum visible to the eye which makes clothing appears „whiter‟.

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2 ● Fluorimetry Spectroscopy and Its Applications ● ● Department of Analytical Chemistry Faculty of Pharmacy Minia University ● 1. Molecular Spectroscopy Overview Molecular spectroscopy is the study of absorption or emission of light by molecules. Absorption and emission of light corresponds to absorption and emission of energy by the electrons of the molecule. The range of those energetic transitions is very wide so that absorption and emission can be observed in any part of the electromagnetic spectrum. Hence molecular spectroscopy can be divided in three different techniques defined by the range of the spectrum observed: ● UV-Visible ● Infrared ● Nuclear magnetic resonance Fluorimetry is part of UV-Visible spectrometry. To understand the in and out of molecular spectroscopy one must know that the energy levels of electrons in molecules are quantified. This is why each molecule can only absorb/emit discrete wavelength corresponding to transitions from one level to another. Selection rules established through quantum mechanics calculation allow telling which transition is possible and which is not. Figure 1.0 Transitions in a diatomic molecule Figure 1.0 shows several transitions and the resulting kind of radiation. The intensity of radiation absorbed/emitted is related to the change in dipole moment of the molecule induced by the transition in other words the more electrons there is to undertake the transition the more intense it is. In the case of absorption the energy is not stored indefinitely but re-emitted using different processes depending on the quantity of energy to emit. Transitions to first excited state are usually done through a non radiative process collision whereas transition from this state to ground state occurs within a radiative process. If the emission of light is in the same direction

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3 ● Fluorimetry Spectroscopy and Its Applications ● ● Department of Analytical Chemistry Faculty of Pharmacy Minia University ● than the excitation radiation it is called transmission. If the emission of light is not in the same direction than the excitative radiation it is called scattering. Another distinction is made between scattering process with respect to the excitation: ● Rayleigh scattering: is the result of an elastic relaxation which means that the molecule gives back all the energy it has received form excitation. In term of wavelength the wavelength of excitation and emission are the same. ● Stockes scattering: refers to an inelastic relaxation which means that the molecule gives back more or less energy than what it has received form excitation. In term of wavelength the wavelength of excitation and emission are different.  Notice: for the purpose of this research anti-Stokes scattering will be considered part of Stockes scattering. 2. Fluorescence and Phosphorescence Many compounds when excited by a light source in the visible or near ultraviolet regions absorb energy which is nearly instantaneously re-emitted in the form of radiation. According to Stokes‟ law the maximum of the spectral emission band is located at a longer wavelength than that of the original excitation light. These are fluorescent compounds. After excitation the light intensity decays extremely rapidly according to an exponential law. The following expression links the intensity of fluorescence It and the time passed t since the excitation: I t I 0 ·exp −kt This emission can be classified as fluorescence which has a very rapid decrease in intensity or phosphorescence where emission decay is much slower. The difference between the two is characterized by the value of the constant k which for fluorescence is much greater than for phosphorescence. The lifetime of fluorescence to is defined using the rate constant k by to 1/k. At the instant to the intensity It will become according to expression 36.8 per cent of the initial intensity Io. In other words a fluorescent

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4 ● Fluorimetry Spectroscopy and Its Applications ● ● Department of Analytical Chemistry Faculty of Pharmacy Minia University ● compound corresponds on the microscopic scale to a population of individual species of which 63.2 per cent have relaxed to a non-emissive state after this brief period of time. Fluorescence lifetimes are only a few nanoseconds. To facilitate the measurements of fluorescence current instruments operate in a stationary regime which is the source of excitation is maintained illuminated though with the obligatory discrimination between the light from the source and that due to the fluorescence. Fluorescence can be resolved over time. The use of very short pulsed light sources picosecond lasers and laser diodes has rendered accessible graphs of fluorescence decay as a function of the time. New applications based upon a greater knowledge of the lifetime are under development although they are still not used much in chemical analysis. 3. UV-Visible Spectroscopy Ultraviolet and visible wavelengths are associated with transitions between molecular orbitals usually between the ground and first excited electronic state. Figure 2.0 gives a view of the transitions available between those two energy levels: Figure 2.0 Schematic energy-level diagram The Franck-Condon principle states that in representation like figure 1.0. The absorption can be represented as vertical lines as it occurs more quickly than any vibrational or rotational motion of the molecule. 3.1. Qualitative Analysis As it has been mentioned above the intensity of an

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5 ● Fluorimetry Spectroscopy and Its Applications ● ● Department of Analytical Chemistry Faculty of Pharmacy Minia University ● emission relies on the population of electrons transferred by the excitation. Thus it is clever to choose an excitation wavelength which will generate the most important emission so as to have a signal easy to detect especially in case of trace analysis. Such a value is usually chosen from the absorption spectrum of the molecule from the values the absorbance of which is minimal. Obviously exciting a molecule will result in an emission which is a superposition of transmission and scattering. Hence if only the scattering should be considered the detection must not occur in the excitation direction. Detection perpendicular to incident light ensures that neither total reflection nor refraction will interfere with scattering emission. If the exciting wavelength is monochromatic the scattering radiation will be composed of a Rayleigh ray and several Stokes rays which can be considered a signature of the molecule. Figure 3.0 shows such a spectrum. The central peak is the Rayleigh ray and the satellite peaks are the Stokes rays. This can be used as a qualitative method to identify a fluorescent species in a sample providing others species or the solvent will not interfere. Figure 3.0 Signature spectrum 3.2. Quantitative Analysis Quantitative analysis with spectroscopic methods is based on the Beer-Lambert Law. This law links the intensity of light before and after a sample with the concentrations of absorbing species:  Where: A is the absorbance of the sample I and Io are the transmitted and incident intensities Several factors may create deviations to Beer-Lambert law: ● Concentrations superior to 0.01M may alter the value of ε.

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6 ● Fluorimetry Spectroscopy and Its Applications ● ● Department of Analytical Chemistry Faculty of Pharmacy Minia University ● ● Species involved in chemical equilibriums have their ε dependent of the displacement of those equilibriums acid/basic species ε is pH dependent. ● Negative deviation may occur if the excitation radiation is polychromatic. Which makes sampling a bit tricky as it is sometimes necessary to separate components of the mixture prior to any analysis.  Notice: This quantitative analysis also referred as colorimetry or spectrophotometry is only applied to transmission not scattering. Beer-lambert law is applicable to measurements in all the electromagnetic spectrum but in only used extensively in UV-Visible and IR spectroscopy. The usual procedure is to plot a calibration curve from a series of standards using an incoming wavelength that is the maximum of absorbance for the component studied to improve sensitivity and minimize the last deviation factor effect. 3.3. Instrumentation Several designs of spectrophotometers are available to fit the needs of the analyst. Anyway they all share several common structural elements: source monochromator beam doubler sample reference cell and detectors. It is mainly the type of those elements and their organization that sets the differences between spectrophotometers. Figure 4.0 shows a diagram of a UV-visible spectrometer. It is slightly different from the one used during the lab session as it is designed for transmission measurement. A single beam instrument is usually favored for quantitative measurements at a precise wavelength as it is often the case in biochemistry as an example. Special attention must be paid to the choice of the cell. Glass does not absorbs visible radiations or at least it did not last time I looked through a window and is thus a good material for the cell used at visible wavelength. However it does absorb at UV wavelength and is therefore unusable for

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7 ● Fluorimetry Spectroscopy and Its Applications ● ● Department of Analytical Chemistry Faculty of Pharmacy Minia University ● measurements at such wavelengths. Quartz cell does not absorb neither in UV nor visible but they are actually more expansive and should be handled with more care than glass cells. Figure 4.0 Schematic diagram of a scanning spectrometer 4. Spectrofluorimetry As illustrated by figure 2.0 fluorescence is an emission of radiation between the first excited state to the ground level from and to any vibrational levels of those states. If an inter system crossing as occurred moving electrons to a triplet state the phenomenon is called phosphorescence. Fluorescence emission is a scattering so it could be measured in any direction from the sample which is very convenient on an experimental point of view as it will be explained later. 4.1. Studying of fluorescence Rayleigh and Stokes fluorescence scattering are possible. Thus two kind of spectrum can be observed: ● Emission spectrum: almost identical in shape to the molecule‟s absorption spectrum the emission spectrum results from the Rayleigh fluorescence. It indicates the relative efficiency of a given wavelength to cause fluorescence. It is obtained by plotting relative intensity of emitted radiation at the same wavelength as the source. ● Excitation spectrum: resulting from the Stockes fluorescence its shape and intensity are independent of the exciting radiation wavelength. Its overall appearance is an approximate mirror image of the emission spectrum moved to longer wavelength. It is obtained by plotting the intensity of the emission on all the range of wavelength any excitation wavelength will fit providing it

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8 ● Fluorimetry Spectroscopy and Its Applications ● ● Department of Analytical Chemistry Faculty of Pharmacy Minia University ● can generate the ground  first level transition. Depending of the objective of the analysis the experiment should be conducted in one or two times. For a qualitative analysis an emission spectrum of the sample is plotted and used as a “fingerprint”. For a quantitative analysis both spectrums are plotted and their maximums are kept. The purpose of getting those values is simple: by exciting at the wavelength of maximum emission we make sure to have the greatest emitted intensity possible and by measuring at the excitation maximum we ensure the best sensitivity. Intensity Only a few species are able to relax by fluorescence mainly rigid organic aromatic structures for which radiationless relaxation mechanism are comparatively slow. For those species the intensity emitted is expressed as a function of the quantum yield Φƒ: Q I o 2.303 ε C l Φƒ  Where: Q and Io are the transmitted and incident intensities ε is the molar absorption coefficient C is the concentration of the fluorophore and l is the length of the cell. The quantum yield is highly linked with the rigidity of the fluorescent structure as seen on figure 5.0 Figure 5.0 Difference in quantum yield Such relation makes it possible to establish calibration curves in the same way than Beer- Lambert law application. Factors affecting intensity In addition to all factors mentioned previously affecting the validity of Beer-Lambert law quenching can lower the fluorescence intensity. Quenching is a general name for all phenomenons which decreases intensity by generating in reactions of the excited state with its surrounding. Quenching is rare and its causes are known. ● Temperature quenching: increasing temperature increases the probability of collisional energy dissipation

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9 ● Fluorimetry Spectroscopy and Its Applications ● ● Department of Analytical Chemistry Faculty of Pharmacy Minia University ● thus “robbing” fluorescence of its energy. The most common source of temperature is the lamp of the spectrometer which needs to be isolated. ● Oxygen quenching: dissolved O2 at 10 −3 M reduce fluorescence by 20 ● Concentration quenching: at high concentration all the excitation is absorbed and therefore no emission is possible. ● Impurity quenching: impurity can cause quenching by collisional effect energy or charge transfer or inner-cell effect. It is the case of quenching that is studied in the experimental part of the lab session. Relationship between fluorescence and concentration At each point of the solution the intensity of fluorescence is different because a part of the excitation radiation is absorbed before reaching the point being considered and because a part of the emitting radiation light finds itself trapped before it can exit the cell. Globally the fluorescence received by the detector corresponds to the sum of the fluorescence emerging from each of the individual small volumes constituting the space delimited by the entrance and exit slits. This is why the calculation of the absolute fluorescence intensity emission If for the sample is difficult. The phenomenon of radiation damping called internal quenching is due to the partial overlap of the absorption and emission spectra color quenching and is increased by transfers of energy from excited species to other molecules or ions through collisions or complexes formation chemical quenching. That is why the presence of oxygen can cause an underestimation of fluorescence. For solutions the quantum yield of fluorescence Φf between 0 and 1 inclusive which is independent of the intensity emitted by the light source is defined by the ratio of the number of photons emitted to the number of photons absorbed this latter being equivalent to the ratio of fluorescence intensity If over that absorbed Ia. Φf If / Ia Accepting that Ia I0 − It

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10 ● Fluorimetry Spectroscopy and Its Applications ● ● Department of Analytical Chemistry Faculty of Pharmacy Minia University ● It representing the transmitted light intensity the following reasoning allows relating If to the concentration C of the compound: If Φf I0 − It Φf · I0 · 1 − It / I0 Knowing that the absorbance A is equal to log I0 / I becomes: If Φf · I0 · 1 − 10 -A 10 -A If the solution is diluted the term A is close to 0 and the term 10 −A is therefore close to 1− 2.3A it can then be simplified becoming: If 2.3 · Φf · Io · A I f I o · 2.303 · ε · C · l · Φƒ With I0 the intensity of the excitation radiation C the molar concentration of the compound ε is molar absorption coefficient l is the cell thickness and Φf is the fluorescence quantum yield. This last expression reveals that the intensity of fluorescence depends upon the concentration C the experimental conditions l Io and of the compound ε Φf. If all the parameters due to the instrument and most due the compound are factored into a global constant K then the following equation can be used for weak concentrations A0.01: If K · Io · C Measurements by fluorimetry make use of several classic methods employing one or more standards single point calibration or calibration curve or methods of addition but to obtain the best results the solutions must be very dilute. Above a given limit fluorescence is no longer directly proportional to concentration. This arises because of the non- linearity of Lambert–Beer law. The excitation is proportionally weaker and association complexes appear between excited molecules and those in ground state. This leads to the apparently paradoxical result that the fluorescence can diminish even though analyte concentration increases. If Φ· K· Io· 10 - ε 1 l 1 + ε 2 l 2 · 1- 10 -ε l c 2

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11 ● Fluorimetry Spectroscopy and Its Applications ● ● Department of Analytical Chemistry Faculty of Pharmacy Minia University ● Rayleigh scattering and Raman bands When the wavelengths of excitation and of emission are close together then confusion becomes possible between the fluorescence of the sample and two artifacts due to the solvent: Rayleigh scattering and Raman diffusion. The limits of detection in fluorescence are often governed by the ability to distinguish the analyte fluorescence from these additives interferences. ● Rayleigh scattering: is the re- emission by the solvent in which the compound is dissolved of a small fraction of the absorbed excitation light in all directions at the same wavelength. The Rayleigh scattering intensity depends upon the polarizability of the solvent molecules. ● Raman diffusion: is 100 to 1000 times weaker than that of Rayleigh scattering is produced by the transfer of a part of the excitation radiation energy to the solvent molecules in the form of vibrational energy. The solvent molecules re-emit photons of less energy than those having served to excite them. Compared to Rayleigh scattering the Raman emission band is shifted towards longer wavelengths. For each solvent the difference in energy between the absorbed photons and the re-emitted photons is constant. Therefore it is possible by modifying the excitation wavelength to displace the shift in nanometers between the position of the Raman and Rayleigh bands. The Raman scattering of water serves as a sensitivity test for fluorometers. This consists of measuring the signal/noise ratio of the Raman peak with a cell filled with water for example at 397 nm if the excitation wavelength is fixed at 350 nm as a result of the specific shift of 3380 cm −1 for this solvent and to compare with the background signal. 4.2. Instrumentation Before we have used a scanning spectrofluorimeter that is a spectrometer with two monochromators for independent scanning of both emission and excitation spectrum. Filter fluorimeters are single beamed single monochromator instruments used for quantitative analysis only.

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12 ● Fluorimetry Spectroscopy and Its Applications ● ● Department of Analytical Chemistry Faculty of Pharmacy Minia University ● We have mentioned earlier that the fact that fluorescence is a scattering was especially interesting on an experimental point of view. The reason is that by measuring the emission perpendicularly to the excitation we ensure to have no transmitted intensity. 5. Time-Resolved Fluorimetry This time-resolved fluorescence technique allows a measure of the time dependence of fluorescence intensity after a short excitation pulse. It consists of obtaining a spectrum measured within a narrow time window during the decay of the fluorescence of interest. The usefulness of this technique is now well proven for biochemical assays and immunoassays. Lanthanide chelates have luminescence decay times over 600 s which allows time-gated fluorecence detection with a complete rejection of other fluorescence signals. For these quantitative applications the primary source is generally a quartz lamp associated with a splitter. There are several ways to perform time-resolved fluorescence measurements. Since the time dependence of fluorescence emission is typically on a picosecond to nanosecond time scale it is very difficult to achieve. To overcome this difficulty either a frequency domain method or the single photon counting approach is used. For studying fluorescent compounds per se lifetime spectrometers are used. A highly diluted solution of the compound to be studied is submitted to a pulsed source laser diode for example repeatedly for several picoseconds at frequencies of several megahertz. During these successive cycles when the source is switched off the detector measures the time until a photon of fluorescence reaches it. Then this event is stocked in the memory channel reserved for the corresponding time. After having accumulated a large number of photons the graph of the decay is finally constructed by creating a histogram of the contents of all of the memory channels.

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13 ● Fluorimetry Spectroscopy and Its Applications ● ● Department of Analytical Chemistry Faculty of Pharmacy Minia University ● Normal fluorescence is useful as a highly selective and sensitive non-invasive probe. However better chemical information can be gained from the same experiment. While normal fluorescence spectroscopy is useful as a highly selective and sensitive non- invasive probe better chemical information can often be gained from the same experiment by exploiting the time-dependent nature of fluorescence. Time-resolved fluorescence provides more information about the molecular environment of the fluorophore than steady state fluorescence measurements. Since the fluorescence lifetime of a molecule is very sensitive to its molecular environment measurement of the fluorescence lifetimes reveals much about the state of the fluorophore. Many macromolecular events such as rotational diffusion resonance- energy transfer and dynamic quenching occur on the same time scale as the fluorescence decay. Thus time-resolved fluorescence spectroscopy can be used to investigate these processes and gain insight into the chemical surroundings of the fluorophore. It is important to remember that the fluorescence lifetime is an average time for a molecule to remain in the excited state before emitting a photon. Each individual molecule emits randomly after excitation. Many excited molecules will fluoresce before the average lifetime but some will also fluoresce long after the average lifetime. Fluorescence lifetimes are generally on the order of 1-10 nsec although they can range from hundreds of nanoseconds to the sub-nanosecond time scale. Figure 6.0 Time-resolved fluorescence of Cs atoms in solid He

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14 ● Fluorimetry Spectroscopy and Its Applications ● ● Department of Analytical Chemistry Faculty of Pharmacy Minia University ● 6. Applications 6.1. Fluorimetry in Medicine and Biology The use of fluorimetry in the fields of medicine and biological samples is well established. In fact fluorescence technique has got a big boost with its applications in Biological sciences. Today highly selective and sensitive biochemical determinations can be accomplished by fluorimetry. Spectrofluorimeters tailored to suit the need of specific sample to be analyzed are available in the market. Interphasing fluorimeter with chromatograph has also yielded good results as it facilitates analysis of mixtures without resorting to tedious separations. In clinical situations many times we need quick analysis of the clinical samples as the diagnosis and treatment options depend on the outcome of such determinations. For example the enzyme released after heart attack must be analyzed within 15 minutes so as to ascertain the future course of action. In these life-saving situations the fluorescence methods find favor as these are simple rapid selective and sensitive. The cleanup separation and determination of the analyte are the important steps involved in the analysis of clinical samples. For example the removal of red blood cells from blood is necessary before one undertakes fluorescence analysis. In some of the biological samples we need to undertake deproteination as the latter quenches fluorescence. In case a direct determination of the analyte faces too many interference it is advisable to use a suitable agent and convert the analyte into a fluorinating derivative. Therefore many non- fluorescent compounds are converted into fluorinating derivatives prior to their analytical determination. Fluorescence and phosphorescence are particularly useful in physical Biochemistry since they provide much information with regard to the interaction of molecular complexes with their environment. Molecular rotation and reorientation in biochemical systems can be conveniently studied as they have similar

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15 ● Fluorimetry Spectroscopy and Its Applications ● ● Department of Analytical Chemistry Faculty of Pharmacy Minia University ● lifetimes to the excited singlet and triplet states. 6.1.1. Analysis of Amino Acids and Proteins Quantification of total protein content in a sample is common to many applications in basic science and clinical research. Over the years many different absorbance based colorimetric methods to quantify protein have been developed. These methods work well however these are subject to interference by many compounds. Several methods based on fluorescence measurements have been developed for protein estimation. Fluorescamine and o-phthalaldehyde have been used with success to quantify protein content of samples. As regards the protein samples three amino acids constituting them exhibit natural fluorescence. These are tyrosine tryptophan and phenylalanine other amino acids however need a suitable agent to convert them to fluorescent derivatives. Fluorescamine a heterocyclic dione is very useful agent for the analysis of the non- fluorescent amino acids. It reacts with the primary amino group of the amino acid to form a fluorescent product. The fluorescence of a solution containing protein and fluorescamine is found to be proportional to the quantity of free amino groups present. Therefore this reaction forms the basis of fluorimetric assay of the proteins and amino acids. Fluorescamine has also been used in labeling casein the milk protein so that it can be used as a substrate for measuring protease activity. Another very important reagent is o-phthalaldehyde OPA. It is probably the most widely used reagents and has sensitivities in the femtomole range. The reagent reacts with the primary amino group at alkaline pH ranges in the presence of certain thiols such as mercaptoethanol or ethanethiol. The reaction is quite

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16 ● Fluorimetry Spectroscopy and Its Applications ● ● Department of Analytical Chemistry Faculty of Pharmacy Minia University ● quick and is essentially complete within a minute. 6.1.2. Analysis of Blood Glucose The analysis of blood glucose is most critical for diagnosis of diabetic patients. The people with diabetes mellitus need to constantly monitor their blood glucose levels in order to detect and control fluctuations in glucose level that could lead to hyperglycemia high blood glucose levels or hypoglycemia low blood glucose levels. It is therefore necessary to have a simple specific and rapid test for the concentration of glucose in the blood. Here again fluorescence method can be exploited. Nowadays blood glucose levels are measured by a procedure based upon the enzyme named glucose oxidase. Since an enzyme is used it is specific for only D- glucose and will not be subject to interferences from other molecules in the blood. The determination of blood glucose is based on the following reactions. ● Step 1: The enzyme glucose oxidase catalyzes the oxidation of b-D-glucose to form D- glucono-15-lactone and hydrogen peroxide. ● Step 2: D-glucono-15-lactone then spontaneously gets hydrolyzed to produce gluconic acid. D-Glucono-15 –lactone + H2O  D-Gluconic acid As the a-D-glucose is rapidly converted to the beta form therefore all of the glucose is measured at one time. This glucose oxidase catalyzed reaction can be monitored by fluorescent detection of the consumption of oxygen or by monitoring the production of hydrogen peroxide. The amount of hydrogen peroxide produced is determined by using a fluorophore named luminol. The mechanism of fluorescence emission by the dianion formed above is as follows:

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17 ● Fluorimetry Spectroscopy and Its Applications ● ● Department of Analytical Chemistry Faculty of Pharmacy Minia University ● The detection limit of glucose by this method is 50 μg/mL. This reaction can be used for analysis of fructose or sucrose also. Nowadays some biosensors are also being developed to measure blood glucose levels. These biosensors are based on sensitive fluorescence measurements which work by monitoring changes in the intrinsic FAD flavin adenine dinucleotide fluorescence of glucose oxidase. FAD is the cofactor of the enzyme. 6.1.3. Analysis of Blood Serum In order to facilitate diagnosis the serum from human blood is analyzed for the presence of different ions or metabolites. Here too fluorescence measurement plays an important role and the leukocytes in blood are analyzed. For this excitation and emission wavelength are 465 nm and 475 nm respectively. 6.2. Fluorimetry in Analysis of Inorganic Substances Several minerals and alloys contain metals and many of such metals are analyzed by fluorescence spectroscopy. In view of emphasis on the fluorimetric analysis of elements in this section we will take an overview of analysis of inorganic compounds including metals non-metals minerals and alloys by fluorescence spectroscopy. While fluorescence spectroscopy is widely used in the qualitative and quantitative analysis of inorganic compounds phosphorescence spectroscopy does not find many applications. Phosphorescence measurement is used mainly in the area of inorganic phosphors. Further though several inorganic substances are fluorescent or phosphorescent in the solid state the majority of analyses are performed in solution phase.

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18 ● Fluorimetry Spectroscopy and Its Applications ● ● Department of Analytical Chemistry Faculty of Pharmacy Minia University ● Aluminium was the first element to be analyzed by fluorimetry using morin reagent. Today a large number of ions are being analyzed using fluorescence the number of cations analyzed being much larger as compared to anions. The analytical determinations of inorganic species by fluorimetric methods involve different types of chemical reactions or methodologies. Before taking up the applications of fluorimetry to inorganic substances let us learn about these reactions. Chemical Reactions Producing Fluorescence There are several kinds of reactions which lead to generation of fluorescence. These are binary or ternary complexation ion association substitution reactions redox reactions enzymatic reactions kinetic methods extractions etc. ● Binary Complexation: it contains one central ion and one ligand only e.g. Al combines with alizarin to produce fluorescent binary complex. Similarly several derivatives of 8- hydroxyquinoline azo dyes and Schiff‟s bases also show fluorescence by the formation of binary complexes with metal ions. ● Ternary Complexation: in it a central ion is coupled with two ligands. These complexes are called as ion association complex e.g. interaction of Hg or Sn with Rhodamine in the presence of chloride or bromide ions. ● Substitution Reactions: in some cases an anion is made to react with cation central atom of a metal-organic compound complex. This undergoes a substitution reaction and as a result the organic ligand is set free. The analysis of F - CN - or sulphide ions is based on such substitution reactions. Some ions are determined by quenching or generation of non-fluorescence organic cation complex. ● Redox Reactions: in this case the reaction is between an organic reagent and the inorganic species. The anions like Br - PO4 3- or cations like CeIV FeIII HgII and VV are analyzed by these kinds of reactions. For example chloramine T and nicotinamide react with cyanide to give fluorinating cyanogen chloride.

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19 ● Fluorimetry Spectroscopy and Its Applications ● ● Department of Analytical Chemistry Faculty of Pharmacy Minia University ● ● Enzymatic Reactions: some fluorimetric determinations involve the use of enzymes. The inorganic species reacts with enzymes but the methods are not selective. For example in the analysis of arsenic the enzyme glyceraldehyde-3-phosphate dehydrogenase is used. ● Kinetic Reactions: some reactions need heating therefore are categorized under the kinetic methods. For example aluminium reacts with oxine or its sulpho derivative to form fluorescent complex the initial rate being directly proportional to concentration of the aluminium species. ● Extractive Fluorimetry: in many fluorimetric determinations the interferences are too serious to be ignored. These interfering species need to be removed before taking up the determination. The ion to be determined is extracted out of the sample leaving the interferences behind. Solvent extraction is one such method that facilitates the removal of interferences e.g. TlIII is first extracted in benzene with crystal violet which in turn is substituted by butyl Rhodamine- B. Like extractive photometry which is so extensively used in analytical chemistry extractive fluorimetry is getting very popular e.g. BiIII is analyzed with dibenzoylmethane at 1 μg level or niobium Nb with oxine can be determined at 0.18 ppb level. Beryllium can be analyzed by dibenzoylmethane with pyridine and as small as 0.0004 μg/ml of Be can be conveniently analyzed by extractive fluorimetry. Having talked about different types of reactions that aid in the fluorimetric determination we can now take up the applications of fluorimetric determinations of inorganic substances. In a broad way the applications of fluorescence in the analysis of inorganic species have been grouped under three heads as given below: ● Inorganic substances showing luminescence ● Fluorescence with inorganic reagents ● Fluorescence with organic reagents The first group pertains to direct analysis whereas the other two exploit indirect methods. Let us begin with inorganic substances

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20 ● Fluorimetry Spectroscopy and Its Applications ● ● Department of Analytical Chemistry Faculty of Pharmacy Minia University ● having intrinsic fluorescence i.e. they are fluorescent in nature. 6.2.1. Inorganic Substances Showing Luminescence As mentioned earlier only lanthanides and uranium compounds show fluorescence in solution hence these ions are analyzed directly without use of any of the organic or inorganic reagents. Within lanthanides Ce Pr Nd show fluorescence with electronic transitions from 5 d to 4 f shells e.g. for CeIII ex. 260 nm em. 350 nm. In dilute solutions the spectrum is very sharp due to transitions of f-electrons. The uranium compounds show natural fluorescence at 520-620 nm. UVI is extracted with tri-butyl phosphate and is back washed with Na3PO4 and the resulting fluorescence is measured. In presence of H3PO4 the intensity of fluorescence is enhanced. 6.2.2. Fluorescence with Inorganic Reagents Most of the metals show fluorescence in the presence of suitable reagents. In other words these can be analyzed by the indirect method. For example certain metals like Tl Sn Pb As Sb Bi can show fluorescence at low temperature on reaction with acids like HCl HBr. Most of the p- Block elements in presence of HCl/HBr show fluorescence with excitation wavelength in ultraviolet and emission wavelength in the visible region. For example arsenic has ex. 350 nm and em. 690 nm. The detection limit varies from 0.002-1.0 ppm. Apart from acids Na2WO4 promotes fluorescence for lanthanides at pH 4.5. The analysis of UO2 SO4 in H3PO4 by TlI ions in the range of 0.1-80 ppm is done with fluorescence quenching. Unfortunately several of the ions show strong interference and reduce fluorescence intensity. 6.2.3. Fluorescence with Organic Reagents The inorganic substances may be analyzed using organic reagents for the complex formation. The formation of fluorescent complex between the organic compound and the metal ion can be exploited in two ways. These may readily be

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21 ● Fluorimetry Spectroscopy and Its Applications ● ● Department of Analytical Chemistry Faculty of Pharmacy Minia University ● used for the determination of metal species using organic reagent. Alternatively the organic compound may be determined using metal ion as the reagent. These are called fluorescent probes. However the former has far more applications than the later. In fact the latter is currently an important research area. The most effective organic reagents are the ones that can form a chelate or a ring with the metal ion by binding with it at more than one place. This in turn requires that the organic compound has two or more functional groups. Further these functional groups should be so placed in the molecule that on chelation they form 5- or 6- membered ring. While Mg Cd Zr Zn are analyzed by complex formation with organic reagent few of the metals can be analyzed by methods based on phenomena of quenching the most favored metals being Ga Al and Be. The transition elements rarely form fluorescent chelates however copper is one exception to this rule. Amongst anions borate has maximum number of methods available for analysis. Then we have fluoride and to some extent sulphide anion which is generally analyzed by substitution reaction. 6.3. Fluorimetry in Analysis of Minerals Several minerals like calcite fluorite rubies and zircon on exposure to UV radiation start emitting fluorescence. The only requirement being that the substances inhibiting and quenching fluorescence must be absent. e.g. gypsum CaSO4.2H2O one of the common minerals in sediment environments exhibit red fluorescence on exposure to UV radiation. Similarly the phosphate mineral e.g. Zircon shows fluorescence on exposure to UV radiation. The minerals containing silicon uranium and molybdenum respectively show bright fluorescence. Amongst fluorites the mineral willemite shows fluorescence in the present of S block metals. Some crystalline materials absorb light in the UV region and emit in the visible region. In fluorescent lamps the ultra-violet radiation from low-

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22 ● Fluorimetry Spectroscopy and Its Applications ● ● Department of Analytical Chemistry Faculty of Pharmacy Minia University ● pressure mercury arc Hg vapor emit light at 253 and 184 nm is converted to visible light by calcium halo-phosphate phosphor Ca10F2P6O24. The crystalline materials emitting fluorescence are referred to as crystallophosphors. Many vanadate oxyhalides oxides etc. show fluorescence as matrices. Table 6.5 lists application of crystallophosphor for analysis of metal ions. Polycrystalline substances containing traces of ionic activators of luminescence are also known. They contain crystalline imperfections a large sized oxide ion being predominant in them. Xenon lamp lasers cathode rays x-rays are used for exposure. The technique is mainly used for the analysis of lanthanide elements. 6.4. Fluorimetry in Analysis of Pharmaceutical Drugs In addition to the drugs of abuse there have been included in this section a number of pharmaceutical products for which fluorimetric methods exist. Drug detection and analysis usually involve an extraction procedure as a wide range of sample matrices are found. For example LSD is sometimes taken on sugar cubes and cannabis resin found on vegetable matter. Liquid samples are predominantly body fluids such as blood or urine. Many of the assays first published in the sixties and seventies 192-196 have been modified to include liquid chromatography and the use of derivatizing reagents has contributed significantly to the number of compounds detected by fluorescence. In addition photochemical derivatization has provided the analyst with a highly sensitive and specific means of drug identification for example cannabinol chlordiazepoxide and clomiphene. The use of fluorescence immunoassay FIA for drug analysis has been reviewed by Jolle and by Quattrone and Putna. In particular therapeutic drug monitoring by FIA is a fast growing technique and is now routine in many clinical laboratories.

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23 ● Fluorimetry Spectroscopy and Its Applications ● ● Department of Analytical Chemistry Faculty of Pharmacy Minia University ● 6.4.1. Analysis of Acetylsalicylic Acid Acetylsalicylic acid is the analgesic pain reliever which is found in aspirin tablets. In addition to acetylsalicylic acid some aspirin tablets contain other ingredients such as binders and buffering agents. In the experiment a portion of an aspirin tablet is dissolved in water and converted to salicylate ions by the addition of sodium hydroxide. The salicylate ion strongly fluoresces at about 400 nm after it has been excited at about 310 nm. A series of standard solutions of the salicylate ion are prepared the fluorescence of the standards and the samples are measured and the working curve method is used to determine the concentration of salicylate ion in the sample solutions. The concentration is used to calculate the percentage of acetylsalicylic acid in the aspirin. 6.4.2. Analysis of Morphine Several procedures exist for estimating morphine in urine. One of them involves the formation of a fluorophore with an excitation of 395 nm and an emission of 424 nm after treating the sample with concentrated sulphuric acid. Potassium ferricyanide is used in another procedure to form the fluorescent pseudomorphine which when used in an automated assay can give a detection limit of 6 μg/mL in urine. Two procedures have been released using liquid chromatography to separate morphine and other opiates. In a method to determine codeine in plasma hexane and dichloromethane are used to extract the drug from an alkaline sample. After washing with dilute alkali the organic layer is evaporated to dryness reconstituted with water- methanol and injected on a reverse phase column. The native fluorescence of the codeine is measured at an excitation of 213 nm with an emission of 350 nm.

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24 ● Fluorimetry Spectroscopy and Its Applications ● ● Department of Analytical Chemistry Faculty of Pharmacy Minia University ● 6.4.3. Analysis of Quinine and Quinidine Quinine and its stereoisomer quinidine have been determined by fluorescence for many years. Quinine is frequently used as an adulterant of morphine and heroin and therefore its detection could be used as a screening procedure for these drugs. Broussard determined quinidine in serum by extracting an alkaline sample with benzene followed by back extraction of the organic layer into dilute sulphuric acid. The fluorescence excitation is at 360 nm with an emission of 450 nm. Because of the objections raised against benzene a possible carcinogen Horvitz suggested the use of toluene as a substitute. A liquid chromatography assay for quinine and its metabolites in biological fluids has been described by Rakhit et al. Proteins are precipitated by acetonitrile the supernatant evaporated to dryness and the reconstituted residue separated on a reverse phase column. As little as 1 μg/L can be quantitated.

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