17100493.ppt
- 1. Lecture 3 – Analytical Techniques and Instrumentation
- 2. Learning outcomes At the of this session the student shall be able to: • Apply Beer’s law to calculate the concentration of a substance in solution • Explain the basic concepts and clinical laboratory applications of photometry, fluorescence, nephelometry and turbidimetry • Compare clinical uses of Capillary Microchip, Disc Two-dimensional and Isoelectric focusing electrophoresis • Discuss clinical applications of Ion exchange, Affinity and Adsorption chromatography • Describe clinical applications of mass spectrometry including the hyphenated-MS techniques • Discuss advantage and quality control issues in the use of point-of - care testing (POCT).
- 3. Introduction • Analytic techniques used in clinical laboratory fall into four categories Method Technique Spectrometry spectrophotometry, atomic absorption, and mass spectrometry (MS) Luminescence fluorescence, chemiluminescence, and nephelometry Electroanalytic electrophoresis, potentiometry, and amperometry Chromatography gas, liquid, and thin-layer
- 4. SPECTROPHOTOMETRY AND PHOTOMETRY • Spectrophotometers measure absorption or emission of radiant energy to determine concentration of atoms or molecules. • Energy is emitted when excited valence electron falls back to ground state. I = transmitted energy I0 = incident energy %𝑇 = 𝐼 𝐼0 × 100
- 5. Absorbance • Absorbance (𝐴) = − log 𝐼 𝐼0 where I0 = incident light, I = transmitted light But 𝐼 𝐼0 = %𝑇 100 ∴ 𝐴 = log 100 − 𝑙𝑜𝑔%𝑇 = 2 − 𝑙𝑜𝑔%𝑇
- 6. Beer’s Law • states that: concentration of a substance is directly proportional to the amount of light absorbed A = εlc where ε = molar absorptivity, l = path length, c = concentration A ≈ C (ε and l are constants) • Thus concentration of unknown solution can be determined from a calibration curve of a standards of known concentration.
- 7. A spectrophotometer Component Description Light Source lamps emitting UV-visible light . e.g. tungsten, deuterium and mercury lamp. Monochromator isolate individual wavelengths of light. e.g. prisms and Diffraction gratings Sample cell glass or Quartz (glass not suitable for UV region) Photodetectors convert transmitted radiation into electrical energy Analog to-digital (A/D) converters the signal to a voltage and digital.
- 8. Photodetectors Photodiode array (PDA) use pn-junction diode. less sensitive than PMT, but excellent linearity, speed, and small size give them advantage Photocell on illumination generates current that is proportional to incident radiation. Disadvantage: no amplification. temperature sensitive , nonlinear at very low and very high illumination. Photomultiplier tube (PMT) detects and amplifies radiant energy by dynodes, that produce a successively higher positive voltage. PM T is > 200 times more sensitive than the phototube. cathode Incident light
- 9. Double beam spectrophotometer • Double-beam spectrophotometers permit automatic correction of sample and reference absorbance. The system performs continuous zeroing electronically
- 10. Atomic Absorption Spectrophotometer • In atomic absorption spectrophotometer radiation is detected by atoms • It is routinely used to measure concentration of trace metals in samples
- 11. Fluorometry • It is the measurement of emitted fluorescence light. • Fluorescence occurs when a molecule absorbs light at one wavelength and reemits light at a longer wavelength. • Loss of energy is due to vibrational equilibration • The difference btw max of the excitation light and max of the the emitted fluorescence light is referred to as the Stokes shift.
- 12. Fluorometers Source: Gas-discharge lamps (mercury and xenon). Attenuator controls light intensity. Primary filter selects the wavelength that is best absorbed by the solution. Cuvet sample cell Detector PMT (placed at right angles to prevent incident light from striking the photodetector Secondary filter passes only the longer wavelengths of fluorescent. In spectrofluorometers, the filters are replaced by prisms or grating monochromators.
- 13. Concentration and Fluorescence Intensity =0εlc Where = fluorecence intensity = fuorescence efficiency 0 = initial excitation intensity, ε = molar absorptivity l = path length c = the concentration in mol/L. • In dilute solutions with instrument parameters held constant, fluorescence is directly proportional to concentration
- 14. Fluorescence Polarization Fluorescence polarization, 𝑃 = I𝑣− 𝐼ℎ 𝐼𝑣+ 𝐼ℎ – Where, Iv = intensity of emitted fluorescence in the vertical plane – Ih = intensity of emitted fluorescence in the horizontal plane • A large fluorophor emits polarized light if radiant energy is polarized. • A small molecule can only emits polarized light if bound to large molecule. • fluorescence polarization will change if antibody bound to a fluorescent-labeled analyte is mixed with another analyte competing with the fluorescent ligand • The change is inversely proportional to the amount of analyte contained in a given sample • It can thus be used to determination concentration of the analyte contained in a given sample.
- 15. Advantages of Fluorometry • Advantages – Fluorometry increases specificity by selecting the optimal wavelength for both absorption and fluorescence. – Fluorometry is approximately 1,000 times more sensitive than most spectrophotometric methods • emitted radiation is measured directly and can be increased simply by increasing the intensity of the exciting radiant energy. • Disadvantages • fluorescence can decrease due to quenching due to – Changes in pH (affect availability of electrons) – temperature (loss of energy by collision rather than fluorescence). – Contaminants or a change of solvents (may change the structure). – UV light used for excitation can cause photochemical changes.
- 16. Chemiluminescence • Is a process where excitation event is caused by a chemical reaction, and not by photo illumination. • The excitation event is caused by a oxidation reaction with compound such as luminol, acridinium esters, and dioxetanes
- 17. Instrumentation – Chemiluminescence • are known as luminometers. • The basic components of a luminometer include – sample cell housed in a light-tight chamber – injection system for adding reagents to the sample cell – the PMT detector • Advantages : Fast, ultrasensitive and simple instrumentation. • Disadvantage : impurities can cause a background signal that degrades sensitivity and specificity
- 18. Turbidity and Nephelometry • Nephelometry and turbidimetry are analytical techniques that measure scattered light due to interaction of light with particles in solution. • These techniques determine concentration of particulate matter in a sample such as serum proteins. • The amount of light scattered by a suspension of particles depends on: – wavelength, concentration and size.
- 19. Instrumentation • Instrument operation is the same as for any spectrophotometer. • Common nephelometers measure scattered light at 90o or other angle to take advantage of the increased forward scatter.
- 20. Laser Applications • Light amplification by stimulated emission of radiation (LASER) is produced by interaction of radiant energy and suitably excited atoms or molecules. • Laser has same wavelength, propagation, phase, and plane of polarization as the incident radiation. • Laser light can serve as the source of incident energy in a spectrometer or nephelometer. • Laser powered spectrometers are 3 – 6 orders more sensitive than conventional ones