Spectrophotometry in clinical chemistry

spectrophotometry, spectrophotometer, beer lamberts law, monochromator, cuvet, cuvette,

1. SPECTROPHOTOMETRY Dr. Ofonmbuk U moh IN CLINICAL CHEMISTRY

2. OUTLINE Introduction Operating Principle Methodology Instruments Applications Conclusion References

3. INTRODUCTION Photometry is defined as the measurement of light ; Spectrophotometry is defined as the measurement of the intensity of light at selected wavelengths. Spectrophotometric analysis is a widely used method of quantitative and qualitative analysis in Clinical Chemistry. Spectrophotometer measures the intensity of the light entering a sample and the light exiting a sample and compares the two intensities.

4. …introduction contd It does this by : - diffracting the light beam into a spectrum of wavelengths - Directing it un to an object - receiving the light reflected or returned from the object - detecting the intensities with a charge-coupled device - displaying the results on the detector and then the display device

5. Spectrophotometer was invented in 1940 by Arnold Beckman and Howard H Cary at National Technologies Laboratories This discovery streamlined and simplified chemical analysis, by allowing researchers to perform a 99% accurate biological assessment of a substance within minutes as opposed to the weeks required previously for results with only 25% accuracy. …introduction contd

6. …introduction contd

7. Spectrophotometry operates on the principles of Beer-Lambert’s Law . Beer’s Law states that the Concentration of a substance is directly proportional to the amount of light absorbed, or inversely proportional to the logarithm of the transmitted light. The  Beer-Lambert law (or Beer's law)  is the linear relationship between absorbance and concentration of an analyte . The general Beer-Lambert law is usually written as: A = a b c where A is the measured absorbance, a is the wavelength( λ )- dependent absorptivity coefficient,  b  is the light’s path length in cm, and  c  is the analyte concentration in moles per litre . Beer’s Law OPERATING PRINCIPLE of Spectrophotometry

8. The linearity of the Beer-Lambert law is limited by chemical and instrumental factors. Causes of nonlinearity include High Concentration: deviations in absorptivity coefficients at  high concentrations (>0.01M)  due to electrostatic interactions between molecules in close proximity S cattering of light due to particulates in the sample F luoresecence or phosphorescence of the sample C hanges in refractive index at high analyte concentration S hifts in chemical equilibria as a function of concentration N on-monochromatic radiation, deviations can be minimized by using a relatively flat part of the absorption spectrum such as the maximum of an absorption band S tray light Limitations of the Beer-Lambert’s Law

9. …operating principle The intensity of transmitted light passing through a solution containing an absorbing substance (chromogen) is decreased by the absorbed fraction. This fraction is detected, measured and used to relate the light transmitted or absorbed to the concentration of the analyte in question. If an incident light beam with intensity I o , passes through a square cell containing a solution of a compound that absorbs light of a certain wavelength, λ, given that the intensity of the transmitted light beam is I s , then transmittance (T) of light is defined as: T = I s /I o . Percentage Transmittance %T = I s /I o . X 100 …and the amount of light absorbed is expressed as Absorbance, and related to Transmittance by the equation A = -log T

10. A portion of the incident light, however, may be reflected by the surface of the cell or may be absorbed by the cell wall or solvent. To focus attention on the compound of interest, elimination of these factors is necessary. This is achieved using a reference cell identical to the sample cell, except that the compound of interest is omitted from the solvent in the reference cell. The transmittance (T) through this reference cell is I R divided by I O ; and the transmittance for the compound in solution then is defined as I s divided by I R . …operating principle

11. …operating principle In practice, the light beam is blocked and the detector signal set to zero transmittance, then a reference cell is inserted and the detector signal adjusted to an arbitrary scale reading of 100 (corresponding to 100% transmittance), followed by the cell containing the sample to be measured, and the percentage transmittance reading is made on the sample. Modern absorption instruments can usually display the data as either transmittance, %-transmittance, or absorbance. An unknown concentration of an analyte can be determined by measuring the amount of light that a sample absorbs and applying Beer's law. If the absorptivity coefficient is not known, the unknown concentration can be determined using a working curve of absorbance versus concentration derived from standards.

12. METHODOLOGY Turn the switch at ‘ON’ and “Warm up” the instrument for about 15 min. Zero adjust to read infinite absorbance (Zero % transmittance). With no cuvette in the chamber , a shutter cuts off all light from passing thru the cuvette chamber. Adjust wavelength control to read the wavelength at which the test is desired. Blank, Control & Standard adjustments: fill the blank cuvette with the solvent used to dissolve specimen. Polish to clean, insert into the cuvette chamber , aligning mark to front of light source. Close chamber cover. Sample 1 & 2: Then, fill the cuvette with the desired specimen, insert into the cuvette chamber, aligning mark to front of light source. Close chamber cover.

13. INSTRUMENTATION

14. …i n s truments Light source: provide a sufficient source of light which is suitable for marking a measurement. T he light source typically yields a high output of polychromatic light over a wide range of the spectrum . Types of light source INCANDESCENT LAMPS: Tungsten light, Hydrogen light, Deuterium light LASER light – extremely intense, focused & nearly non-divergent beam of monochromatic light

15. Light spectrum

16. …i n s truments Monochromator : Accepts polychromatic input light from a lamp and puts out monochromatic light . Monochromator consists of these parts: Entrance slit Collimating lens or mirror III.Dispersion element IV.Focusing lens or mirror V. Exit slit 9

17. …i n s truments 10 Dispersion devices: A special plate with hundreds of parallel grooved lines. The grooved lines act to separate the white light into the visible light spectrum. The more lines the smaller the wavelength resolution .

18. …i n s truments Focusing devices: Combinations of lenses, slits, and mirrors. relay and focus light through the instrument . 11

19. …i n s truments Cuvettes : (also cuvet ) designed to hold samples for spectroscopic experiments. made of Plastic, glass or silica should be as clear as possible, without impurities that might affect a spectroscopic reading .

20. …i n s truments Detectors : Convert radiant energy (photons) into an electrical signal. The photocell and phototube are the simplest photodetectors, producing current proportional to the intensity of the light striking t hem .

21. …i n s truments Display devices: The data from a detector are displayed by a readout device, such as an analog meter, a light beam reflected on a scale, or a digital display , or LCD . The output can also be transmitted to a computer or printer . 14

22. Single & Double Beam Spectrophotometer

23. SINGLE BEAM; Advantage include: low cost, high throughput and hence high sensitivity because of optical system is simple. Disadvantage include: An appreciable amount of time elapses between taking the reference and making the sample measurement such that there can be problems with drift . DOUBLE BEAM: this is designed to eliminate drift by measuring blank and sample virtual simultaneously. Advantage: High stability because reference and sample are measured virtually at the same moment in time. Disadvantages include : Higher cost, lower sensitivity as throughput of light is poorer because of the optical complexity.

24. APPLICATION of Spectrophotometry Spectrophotometry has a wide range of application in Clinical Chemistry

25. …a pplications 1. Concentration measurement – – Prepare samples Make series of standard solutions of known concentrations

26. Set spectrophotometer to the λ of maximum light absorption Measure the absorption of the unknown, and from the standard plot, read the related concentration . …a pplications

27. 17 2. Detection of Impurities UV absorption spectroscopy is one of the best methods for determination of impurities in organic molecules . Additional peaks can be observed due to impurities in the sample and it can be compared with that of standard raw material. …a pplications

28. 3. Structure elucidation of organic compounds. From the location of peaks and combination of peaks UV spectroscopy elucidate structure of organic molecules: o the presence or absence of unsaturation …a pplications

29. 4. Chemical kinetics Kinetics of reaction can also be studied using UV spectroscopy. The UV radiation is passed through the reaction cell and the absorbance changes can be observed . …a pplications

30. 5. Detection of Functional Groups Absence of a band at particular wavelength is regarded as an evidence for absence of particular group …a pplications

31. 6. Molecular weight determination Molecular weights of compounds can be measured spectrophotometrically by preparing the suitable derivatives of these compounds. For example, if we want to determine the molecular weight of amine then it is converted in to amine picrate . …a pplications

32. CONCLUSION Spectrophotometric analysis is a widely used method of quantitative and qualitative analysis in Clinical Chemistry. It is a veritable diagnostic technique for rapid turnover of analytes

33. THANK YOU FOR LISTENING

34. R E F E R ENCES . Tietz Textbook of Clinical Chemistry & Molecular Medicine 6 th Edition 2014 Clinical Chemistry – Principles, Techniques, Correlations; Bishops; 7 th Edition 2015 . Fundamentals of UV-visible spectroscopy, Tony Owen, 1996 4. UNIT : Spectrophotometry, Clinical Chemistry Lab Manual