Colorimeter and spectrophotometer, Mass Spectrometer

It contains a brief knowledge on Introduction, Principle, Laws, Flow representation, Instrumentation, Applications
and Mass spectrometer
- Principle
- Instrumentation

1. Colorimeter , Spectrophotometer and Mass Spectrometer. By : Mr. Prachand Man Singh Rajbhandari . BSc Medical Biochemistry (Nobel College, Pokhara University, Nepal) MSc Medical Biochemistry (JN Medical College, KLE University, Belgaum)

2. Outlines Introduction Principle Laws Flow representation Instrumentation Applications Mass spectrometer - Principle - Instrumentation

3. INTRODUCTION COLORIMETER Colorimeter is instrument which is used in the measurement of the luminious intensity of light. Invented by Louis Jules Duboscq in 1870.

4. PRINCIPLE COLORIMETER Involves the quantitative estimation of colors. The difference in color intensity results in the difference in the absorption of light. The intensity of color is directly proportional to the concentration of the compound being measured.

5. CONTD. The amount of light absorbed or transmitted by a colored solution is in accordance with two laws: Beer’s law Lambert’s law

6. LAWS Beer’s law : A α C Lambert’s law : A α L

7. I o I

8. Derivation of the Formula Combining the two laws A α CxL OR A= KxCxL Let A T =absorbance of the test solution C T =concentration of the test solution A S = absorbance of the standard solution C S = concentration of the standard solution 2/2/2015 5:54 PM

9. 2/2/2015 5:54 PM A T A S KxC T x L KxC S x L = A T A S C T C S = C T = A T A S X C S A S = KxC S x L A T = KxC T x L

10. 2/2/2015 5:54 PM C T = A T A S X C S Concentration of TEST solution Absorbance of TEST Absorbance of STANDARD Concn of STANDARD X = Concentration of TEST /100ml Absorbance of TEST Absorbance of STANDARD Concn of Std X 100 X = Xml

11. 2/2/2015 5:54 PM Concentration of TEST /100ml Absorbance of TEST Absorbance of STANDARD X = Xml Concn of Std X 100 Concentration of TEST /100ml O.D of ‘T’- O.D of ‘B’ O.D of ‘S’- O.D of ‘B’ X = Volume of ‘T’ Amount of ‘S’ X 100 Concentration of TEST /100ml T - B S - B X = Volume of ‘T’ Amount of ‘S’ X 100

12. Flow representation of colorimeter

13. Parts of the colorimeter Light source Slit Condensing lenses Filter Detector (photocell) Output : INSTRUMENTATION

14. Complementary filters for coloured solutions. The selected filters has the color to the complementary to that of the color of unknown solution

15. Cuvette are rectangular cell , square cell or circular one Made up of optical glass for visible wavelength. Common one is square,rectangular to avoid refraction artefacts . dimension of cuvette is 1cm. Cuvette (sample holder)

16. cuvettes

17. For estimation of biochemical samples , like plasma, serum, cerebrospinal fluid ( csf ) , urine. Ex. Determination of blood glucose, blood urea, serum creatinine , serum proteins, serum cholesterol, serum inorganic phosphate, urine creatinine & glucose in CSF, etc. T hey are used by the food industry and by manufacturers of paints and textiles . APPLICATION OF COLORIMETRIC ASSAY

18. SPECTROPHOTOMETER

19. Introduction compounds absorb light radiation of a specific wavelength. the amount of light radiation absorbed by a sample is measured. The light absorption is directly related to the concentration of the compound in the sample. As Concentration increases, light Absorption increases, linearly, As Concentration increases, light Transmission decreases, exponentially. 19

20. Introduction Spectrophotometer: a) Single-beam. b) Double-beam [4] 20

21. Beer-Lambert law Light Absorbance : ( A) = log (I / I)= ƐLC Light Transmission (T) = I/I = 10-ƐCL I : Light Intensity entering a sample I: Light Intensity exiting a sample C: The concentration of analyte in sample L: The length of the light path in glass sample cuvette Ɛ: a constant for a particular solution and wave length 21 [5]

22. Flow representation of spectrophotometer 22

23. Parts of spectrophotometer Light source: . 23 INSTRUMENTATION

24. Parts of spectrophotometer Monochromator : Accepts polychromatic input light from a lamp and outputs monochromatic light . 24

25. Parts of spectrophotometer 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. 25 The more lines the smaller the wavelength resolution .

26. Parts of spectrophotometer Focusing devices: Combinations of lenses, slits, and mirrors. relay and focus light through the instrument. 26

27. Cuvettes : designed to hold samples for spectroscopic experiments. made of Plastic, glass or optical grade quartz should be as clear as possible, without impurities that might affect a spectroscopic reading. 27

28. Parts of spectrophotometer 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 Them . 28

29. Applications Concentration measurement Prepare samples Make series of standard solutions of known concentrations 29

30. Applications Measure the absorption of the unknown, and from the standard plot, read the related concentration 30

31. Applications 2. 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. 31

32. MASS SPECTROMETER (Principle and Instrumentation)

33. PRINCIPLES Technique involves - Creating gas phase ions from the analyte atoms or molecules - Separating the ions according to their mass-to-charge ratio (m/z) - Measuring the abundance of the ions

34. PRINCIPLES Technique can be used for - Qualitative and quantitative analysis - Providing information about the mass of atoms and molecules - Molecular structure determination (organic & inorganic) - Identification and characterization of materials

35. PRINCIPLES - Separates gas phase ionized atoms, molecules, and fragments of molecules - Separation is based on the difference in mass-to-charge ratio (m/z) m = unified atomic mass units (u) 1 dalton ( Da ) = 1 u = 1.665402 x 10 -27 kg z = charge on the ion (may be positive or negative)

36. PRINCIPLES - Analyte molecule can undergo electron ionization M + e - → M ●+ + 2e - - M ●+ is the ionized analyte molecule called molecular ion - Radical cation is formed by the loss of one electron - Permits easy determination of molecular weight of analyte

37. General Structure of Mass Spectrometer

38. References Text book of biochemistry, DM Vasudevan and U. Satyanaarayana Principle and techniques of biochemistry and molecular biology, wilson and walker. Hand book of Biomedical Instrumentation. R S Khandpur Clinical chemistry. Bishop . Wikipaedia .

39. THANK YOU..