Fluorimetry by Dr. MONIKA SINGH as per PCI Syllabus

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Information about Fluorimetry by Dr. MONIKA SINGH as per PCI Syllabus

Published on October 17, 2020

Author: MonikaSingh43

Source: slideshare.net

1. Dr. MONIKA SINGH (Pharmaceutical Chemistry) 03-10-2020 BP701T Instrumental Method of Analysis Unit-I 1 FLUORIMETRY Dr. Monika Singh BP701T Instrumental Method of Analysis Unit-I 1 CONTENTS Principle Factors affecting fluorescence intensity Instrumentation Applications Conclusion References. BP701T InstrumentalMethod of Analysis Unit-I 2

2. Dr. MONIKA SINGH (Pharmaceutical Chemistry) 03-10-2020 BP701T Instrumental Method of Analysis Unit-I 2 FLUORESCENCE It is a phenomenon of emission of radiation when the molecules are exited by radiation at certain wavelength. FLUORIMETRY:- It is measurement of fluorescence intensity at a particular wavelength with the help of a filter fluorimeter or a spectrofluorimeter. BP701T InstrumentalMethod of Analysis Unit-I 3  PRINCIPLE:- Molecule contains electrons, electrons and non bonding (n) electron.  The electrons may be present in bonding molecular orbital. It is called as highest occupied molecular orbital (HOMO).It has least energy and more stable.  When the molecules absorbs radiant energy from a light source, the bonding electrons may be promoted to anti bonding molecular orbital (LUMO). It has more energy and hence less stable.BP701T InstrumentalMethod of Analysis Unit-I 4

3. Dr. MONIKA SINGH (Pharmaceutical Chemistry) 03-10-2020 BP701T Instrumental Method of Analysis Unit-I 3  The process of promotion of electrons from HOMO to LUMO with absorption of energy is called as excitation.  Singlet Ground state (S0):-a state in which all the electrons in a molecule are paired   Doublet state:- a state in which un paired electrons is present  or   Triplet state (T1) :- a state in which unpaired electrons of same spin present   Singlet excited state (S1) :- a state in which electrons are unpaired but of opposite spin like  (un paired and opposite spin) BP701T InstrumentalMethod of Analysis Unit-I 5 Ground singlet states • Spin: paired • S0 • no net mag.field • Stable Excited singlet state • Spin: paired • S1 • no net mag.field • Unstable • S1 energy is more Triplet state • Spins: unpaired • T1 • net mag.field • More stable • T1energy is less Singlet/ Triplet State BP701T InstrumentalMethod of Analysis Unit-I 6

4. Dr. MONIKA SINGH (Pharmaceutical Chemistry) 03-10-2020 BP701T Instrumental Method of Analysis Unit-I 4 Light emitting at once sourcestarts& stops whensource stops BP701T InstrumentalMethod of Analysis Unit-I 7 Jablonski Energy Diagram explaining the Principle: BP701T InstrumentalMethod of Analysis Unit-I 8

5. Dr. MONIKA SINGH (Pharmaceutical Chemistry) 03-10-2020 BP701T Instrumental Method of Analysis Unit-I 5 FIGURE 1 BP701T InstrumentalMethod of Analysis Unit-I 9  When light of appropriate wavelength is absorbed by a molecule the electrons are promoted from singlet ground state to singlet excited state. once the molecule is in this excited state relaxation can occur via several process. For ex by emission of radiation . The process can be the following 1) Collisional deactivation 2) Fluorescence 3) Phosphorescence. BP701T InstrumentalMethod of Analysis Unit-I 10

6. Dr. MONIKA SINGH (Pharmaceutical Chemistry) 03-10-2020 BP701T Instrumental Method of Analysis Unit-I 6  Collisional de activation :- In which entire energy lost due to collision de activation and no radiation emitted.  Fluorescence:-excited singlet state is highly unstable. Relaxation of electrons from excited singlet to singlet ground state with emission of light.  Phosphorescence:-At favorable condition like low temperature and absence of oxygen there is transition from excited singlet state to triplet state which is called as inner system crossing or Inter state conversion (ISC). The emission of radiation when electrons undergo transition from triplet state to singlet ground state is called as phosphorescence. BP701T InstrumentalMethod of Analysis Unit-I 11 1. Concentration 2. Intensity of incident light 3. Adsorption 4. Oxygen 5. pH 6. Temperature 7. Viscosity 8. Photodecomposition 9. Electron Donating Group 10.Electron Withdrawing Group 11.Scatter 12.Quenchers Factors affecting fluorescent intensity: BP701T InstrumentalMethod of Analysis Unit-I 12

7. Dr. MONIKA SINGH (Pharmaceutical Chemistry) 03-10-2020 BP701T Instrumental Method of Analysis Unit-I 7 CONCENTRATION:- Fluorescence intensity is directly proportional to concentration of substance only when the absorbance is less than 0.02 A=log IoIt or A= abc Io= intensity of incident light It = intensity of transmitted light a= absorptivity of constant b= Pathlength c= concentration BP701T InstrumentalMethod of Analysis Unit-I 13  INTENSITY OF INCIDENT LIGHT: Increase in the Intensity of Incident Light on the Sample, Fluorescence Intensity also Increases (luminescence also increases).  ADSORPTION: Adsorption Of Sample Solution In The Container May Leads ToA Serious Problem. (strong solutions must be diluted)  OXYGEN:- Oxidation of fluorescent species to a non fluorescent species, quenches fluorescent substance.  pH:- Alteration of pH of a solution will have significant effect on fluorescence. Neutral and Alkaline solution show fluorescence. E.g Aniline in alkali medium gives visible fluorescence.  Temperature :- Temperature Increases Can Increase the collisional de activation, and reduce fluorescent intensity.BP701T InstrumentalMethod of Analysis Unit-I 14

8. Dr. MONIKA SINGH (Pharmaceutical Chemistry) 03-10-2020 BP701T Instrumental Method of Analysis Unit-I 8  Viscosity:- If viscosity of solution is more the frequency of collisions are reduced and increase in fluorescent intensity.  Photochemical decomposition:- Absorption of intense radiation leads to photochemical decomposition of a fluorescent substance to less fluorescent or non fluorescent substance.  Electron Donating group: increases fluorescence (Amino group, Hydroxylic group)  Electron withdrawing group: decreases fluorescence (Nitro group, Carboxylic group)  Scatter: Scatter is mainly due to colloidal particles in solution. Scattering of incident light after passing through the sample leads to decrease in fluorescence intensity.BP701T InstrumentalMethod of Analysis Unit-I 15  Quenchers:-  Quenching is the reduction of fluorescence intensity by the presence of substance in the sample other than the fluorescent analyte.  Quenching is following types:-  INNER FLUORESCENT EFFECT:- Absorption Of Incident (uv) Light Or Emitted (fluorescent) Light By Primary And Secondary Filters Leads To Decrease In Fluorescence intensity.  SELF QUENCHING:-At Low Concentration Linearity Is Observed, At High Concentration Of The Same Substance Increase In Fluorescent Intensity Is Observed. This phenomena is called self quenching.  COLLISONAL QUENCHING:- Collisions between the fluorescent substance and halide ions leads to reduction in fluorescence intensity.  STATIC QUENCHING:- This occurs because of complex formation between the fluorescent molecule and other molecules. Ex: caffeine reduces fluorescence of riboflavin. BP701T InstrumentalMethod of Analysis Unit-I 16

9. Dr. MONIKA SINGH (Pharmaceutical Chemistry) 03-10-2020 BP701T Instrumental Method of Analysis Unit-I 9 INSTRUMENTATION BP701T InstrumentalMethod of Analysis Unit-I 17 FIGURE 2 BP701T InstrumentalMethod of Analysis Unit-I 18

10. Dr. MONIKA SINGH (Pharmaceutical Chemistry) 03-10-2020 BP701T Instrumental Method of Analysis Unit-I 10 INSTRUMENTATION • SOURCE OF LIGHT • FILTERS AND MONOCHROMATORS • SAMPLE CELLS • DETECTORS BP701T InstrumentalMethod of Analysis Unit-I 19  1)SOURCE OF LIGHT:-  mercury vapour lamp: Mercury vapour at high pressure give intense lines on continuous background above 350nm.low pressure mercury vapour gives an additional line at 254nm.it is used in filter fluorimeter. FIGURE 3 BP701T InstrumentalMethod of Analysis Unit-I 20

11. Dr. MONIKA SINGH (Pharmaceutical Chemistry) 03-10-2020 BP701T Instrumental Method of Analysis Unit-I 11  Xenon Arc Lamp: It give more intense radiation than mercury vapour lamp. it is used in spectrofluorimeter. FIGURE 4  Tungsten lamp:- If excitation has to be done in visible region this can be used. It is used in low cost instruments. FIGURE 5BP701T InstrumentalMethod of Analysis Unit-I 21  2) FILTERS AND MONOCHROMATORS:-  Filters: these are nothing but optical filters works on the principle of absorption of unwanted light and transmitting the required wavelength of light. In inexpensive instruments fluorimeter primary filter and secondary filter are present. Primary filter:-absorbs visible radiation  and transmit UV radiation. Secondary filter:-absorbs UV radiation and transmit visible radiation. FIGURE 6 BP701T InstrumentalMethod of Analysis Unit-I 22

12. Dr. MONIKA SINGH (Pharmaceutical Chemistry) 03-10-2020 BP701T Instrumental Method of Analysis Unit-I 12  Monochromators: they convert polychromatic light into monochromatic light. They can isolate a specific range of wavelength or a particular wavelength of radiation from a source.  Excitation monochromators:-provides suitable radiation for excitation of molecule .  Emission monochromators:- isolate only the radiation emitted by the fluorescent molecules. FIGURE 7BP701T InstrumentalMethod of Analysis Unit-I 23  3) Sample cells: These are ment for holding liquid samples. These are made up of quartz and can have various shapes ex: cylindrical or rectangular etc.   Barrier layer cell/Photo voltaic cells Photomultiplier cells FIGURE 8  4) Detectors: Photometric detectors are used they are BP701T InstrumentalMethod of Analysis Unit-I 24

13. Dr. MONIKA SINGH (Pharmaceutical Chemistry) 03-10-2020 BP701T Instrumental Method of Analysis Unit-I 13 1. Barrier layer /photovoltaic cell:  it is employed in inexpensive instruments. For ex: Filter Fluorimeter.  It consists of a copper plate coated with a thin layer of cuprous oxide (Cu2o). A semi transparent film of silver is laid on this plate to provide good contact.  When external light falls on the oxide layer, the electrons emitted from the oxide layer move into the copper plate.  Then oxide layer becomes positive and copper plate becomes negative.  Hence an emf develops between the oxide layer and copper plate and behaves like a voltaic cell. So it is called photovoltaic cell..  A galvanometer is connected externally between silver film and copper plate and the deflection in the galvanometer shows the current flow through it. The amount of current is found to be proportional to the intensity of incident light BP701T InstrumentalMethod of Analysis Unit-I 25 BARRIER LAYER CELL FIGURE 9 BP701T InstrumentalMethod of Analysis Unit-I 26

14. Dr. MONIKA SINGH (Pharmaceutical Chemistry) 03-10-2020 BP701T Instrumental Method of Analysis Unit-I 14 2. Photomultiplier tubes:  These are incorporated in expensive instruments like spectrofluorimeter. Its sensitivity is high due to measuring weak intensity of light.  The principle employed in this detector Is that, multiplication of photoelectrons by secondary emission of electrons.  This is achieved by using a photo cathode and a series of anodes (Dyanodes). Up to 10 dyanodes are used. Each dyanode is maintained at 75- 100Vhigher than the preceding one.  At each stage, the electron emission is multiplied by a factor of 4 to 5 due to secondary emission of electrons and hence an overall factor of 106 is achieved.  PMT can detect very weak signals, even 200 times weaker than that could be done using photovoltaic cell. Hence it is useful in fluorescence measurements.  PMT should be shielded from stray light in order to have accurate results. BP701T InstrumentalMethod of Analysis Unit-I 27 FIGURE 10 BP701T InstrumentalMethod of Analysis Unit-I 28

15. Dr. MONIKA SINGH (Pharmaceutical Chemistry) 03-10-2020 BP701T Instrumental Method of Analysis Unit-I 15 INSTRUMENTS The most common types are:-  Single beam (filter) fluorimeter  Double beam (filter )fluorimeter  Spectrofluorimeter(double beam) BP701T InstrumentalMethod of Analysis Unit-I 29  Instruments:- It contains tungsten lamp as a source of light and has an optical system consists of primary filter.  The emitted radiations is measured at 900 by using a secondary filter and detector. Primary filter absorbs visible radiation and transmit uv radiation which excites the molecule present in sample cell.  In stead of 90 if we use 180 geometry as in colorimetry secondary filter has to be highly efficient other wiseboth the unabsorbed uv radiation and fluorescent radiation will produce detector response and give false result. BP701T InstrumentalMethod of Analysis Unit-I 30

16. Dr. MONIKA SINGH (Pharmaceutical Chemistry) 03-10-2020 BP701T Instrumental Method of Analysis Unit-I 16  Single beam instruments are simple in construction cheaper and easy to operate.  FIGURE 11 BP701T InstrumentalMethod of Analysis Unit-I 31 It is similar to single beam except that the two incident beams from a single light source pass through primary filters separately and fall on the another reference solution. Then the emitted radiations from the sample or reference sample pass separately through secondary filter and produce response combinly on a detector. BP701T InstrumentalMethod of Analysis Unit-I 32

17. Dr. MONIKA SINGH (Pharmaceutical Chemistry) 03-10-2020 BP701T Instrumental Method of Analysis Unit-I 17 SPLITTER FIGURE 12BP701T InstrumentalMethod of Analysis Unit-I 33  In spectrofluorimeter:-  In this primary filter in double beam fluorimeter is replaced by excitation monochromator and the secondary filter is replaced by emission monochromator.  Incident beam is split into sample and reference beam by using beam splitter. BP701T InstrumentalMethod of Analysis Unit-I 34

18. Dr. MONIKA SINGH (Pharmaceutical Chemistry) 03-10-2020 BP701T Instrumental Method of Analysis Unit-I 18 FIGURE 13BP701T InstrumentalMethod of Analysis Unit-I 35 APPLICATIONS 1) Determination of Uranium Salt in Nuclear Research 2) Determination of inorganic substances from Fluorescent chelates. Al3+,Li+,Zn2+ 3) Determination of vitamin B1 (Thiamine HCl). 4) Determination of Vitamin B2 (Riboflavin). 5) Detemination of phenytoin. 6) Determination of indoles, phenols, & phenothiazines. 7) Determination of napthols, proteins, plant pigments and steroids. 8) Fluorimetry ,nowadays can be used in detection of impurities in nanogram level better than absorbance spectrophotometer with special emphasis in determining components of sample at the end of chromatographic or capillary column. BP701T InstrumentalMethod of Analysis Unit-I 36

19. Dr. MONIKA SINGH (Pharmaceutical Chemistry) 03-10-2020 BP701T Instrumental Method of Analysis Unit-I 19  Determination of ruthenium ions in presence of other platinum metals.  Determination of boron in steel, aluminum in alloys, manganese in steel.  Determination of boron in steel by complex formed with benzoin.  Estimation of cadmium with 2-(2 hydroxyphenyl) benzoxazole in presence of tartarate .  Respiratory tract infections. BP701T InstrumentalMethod of Analysis Unit-I 37 Identification test as per IP2007 , Vol 3, Page no1173: Dissolve about 20 mg in 10 ml of water, add 1 ml of 2 M acetic acid and 1.6 ml of 1 M sodium hydroxide, heat on a water-bath for 30 minutes and allow to cool. Add 5 ml of 2 M sodium hydroxide, 10 ml of potassium ferricyanide solution and 10 ml of 1-butanol and shake vigorously for 2 minutes. The upper layer exhibits an intense light blue fluorescence, particularly in ultraviolet light at 365 nm. Repeat the test but adding 0.9 ml of 1 M sodium hydroxide and 0.2 g of sodium sulphite in place of the 1.6 ml of 1 M sodium hydroxide; practically no fluorescence is produced. Vitamin B1 (Thiamine) Determination Principle: Thiamin (vitamin B1) is analyzed quantitatively by fluorometric methods. • The method of choice is the thiochrome procedure, which involves treatment of thiamin with an oxidizing agent (ferricyanide or hydrogen peroxide or Potassium permagnate) to form a fluorescent compound (thiochrome). • The thiochrome formed is ex- tracted with isobutanol, and, when irradiated with ultraviolet light, emits a blue fluor- escence which can be read visually or in a fluorophotometer. • The intensity of fluorescence is proportional to the thiamine concentration. Vitamin B1 has neurological benefits. Vitamin B1 is found in many foods including yeast, cereal grains, beans, nuts, and meat. Thiamine is also used for boosting the immune system, digestive problems, diabetic pain, heart disease etc. BP701T InstrumentalMethod of Analysis Unit-I 38

20. Dr. MONIKA SINGH (Pharmaceutical Chemistry) 03-10-2020 BP701T Instrumental Method of Analysis Unit-I 20 Assay as per IP2007 , Vol 3, Page no1043-1044: Weigh accurately about 65 mg and transfer to an amber-glass 500-ml volumetric flask, suspend in 5 ml of water, ensuring that it is completely wetted. Dissolve in 5 ml of 2 M sodium hydroxide. As soon as dissolution is complete add 100 ml of water and 2.5 ml of glacial acetic acid and dilute to 500.0 ml with water. To 20.0 ml of this solution add 3.5 ml of a 1.4 per cent w/v solution of sodium acetate and dilute to 200.0 ml with water. Measure the absorbance of the resulting solution at the maximum at about 444 nm. Calculate the content of C17H20N4O6 taking 328 as the specific absorbance at 444 nm. Vitamin B2 (Riboflavin) Determination Riboflavin (vitamin B2) is usually assayed fluorometrically by measuring its characteristic yellowish green fluorescence. Identification test as per IP2007 , Vol 3, Page no1043-1044: Dissolve about 1 mg in 100 ml of water. The solution has a pale greenish yellow colour by transmitted light and an intense yellowish green fluorescence by reflected light, which disappears on addition of mineral acids or alkalis. It can also be assessed microbiologically, using Lactobacillus casei, where the growth of this riboflavin-dependent microorganism correlates with the amount of vitamin in the sample. The growth response of the organism is measured either by titration or by measuring turbidity. Vitamin B2 helps break down proteins, fats, and carbohydrates. It plays a vital role in maintaining the body’s energy supply. vitamin B2 helps maintain proper eyesight. BP701T InstrumentalMethod of Analysis Unit-I 39 S.No Name of the compound Experimental conditions/ PH Emission wavelength 1 Adrenaline 1 335 2 Cynacobalamine 7 305 3 Riboflavin 6 520 4 Morphine 7 350 5 Hydrocortisone Acidic 520 6 Pentobarbitone 13 440 7 Amylobarbitone 14 410 Table 1 BP701T InstrumentalMethod of Analysis Unit-I 40

21. Dr. MONIKA SINGH (Pharmaceutical Chemistry) 03-10-2020 BP701T Instrumental Method of Analysis Unit-I 21 Conclusion :  Fluorimetric methods are not useful in qualitative analysis ,and much used in quantitative analysis.  Fluorescence isthe most sensitive analytical techniques.  Detection studies will increase the development of fluorescence field. BP701T InstrumentalMethod of Analysis Unit-I 41 References :  SKOOG ,Principles of InstrumentalAnalysis.  Practical pharmaceutical chemistry by A.H. BECKETT& J.B.STENLAKE ,volume 2,  B.K.Sharma Instrumental methods of chemical analysis.  A textbook of pharmaceutical analysis by Dr.S.RAVISANKAR. BP701T InstrumentalMethod of Analysis Unit-I 42

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