instrumentation in Biochemistry lab.pptx

Explains instrumentation in the biochemistry laboratory

1. Instrumentation Medical Biochemical Techniques

2. Colorimeter Spectrophotometer Electrophoresis tank Elisa equipment Analytical balance Precision balance pH Meter Biochemistry Laboratory Equipment

3. Hot air oven Incubator Water bath Vortex Micropipette Autoclave Centrifuge Refrigerato r Biochemistry Laboratory Equipment

4. C olorimetry/Photometry Many chemical substances are coloured in solution The intensity of the colour is proportional to concentration of compound in the solution light emerging from the solution is detected and measured absorbance is directly proportional to the concentration of the coloured compound in the solution

5. Principles of Colorimetry/photometry

6. Colorimeter Use correct cuvette type Ensure optical surfaces are dry, clean, free from scratches Insert filter before switching on Calibrate for each test method Avoid spillages Switch off after use

7. Spectrophotometry A spectrophotometer consists of , - spectrometer for producing light of any selected color (wavelength), - photometer for measuring the intensity of light. The instruments are arranged so that liquid in a cuvette can be placed between the spectrometer beam and the photometer. The amount of light passing through the tube is measured by the photometer.

8. Spectrophotometry… UV or Visible

9. Spectrophotometry… Components: light source, monochromator, sample cell, detector, optical system. monochromator sample cell detector light source slit diffraction grating

10. Spectrophotometry… Transmittance (T) Absorbance (A) (AKA optical density, O.D.) usually given in percent by convention, base 10 logs are used In photometry we measure the intensity of light and characterize its change by and object or substance. This change is typically expresses as percent transmittance or absorbance . Frequently when your primary interest is the light beam Used almost exclusively when your interest concerns the properties of the material

11. Beer-Lambert’s Law Lambert’s and Beer’s Laws are combined to describe the attenuation of light by a solution. It is easy to see how the two standard photometric quantities can be written in terms of this law. Transmittance Absorbance

12. Beer-Lambert’s Law… The change in intensity of light ( dI ) after passing through a sample should be proportional to the following: path length ( l ), the longer the path, more photons should be absorbed concentration ( c ) of sample, more molecules absorbing means more photons absorbed intensity of the incident light ( I ), more photons mean more opportunity for a molecule to see a photon

13. Electric Weighing Balance

14. Electric Weighing Balance… Fragile, precision instrument Handle with care Balance must be located in a vibration-free area, including air currents Balance must be kept clean Periodically calibrated with known weights Use plastic weigh boats or paper to weigh chemicals

15. The load causes the beam to tilt downward A null detector senses the position of the beam An electromagnetic force is applied to return the balance to its full position Restoring force is proportional to weight on pan, is applied through a solenoid or torque motor The current required to produce the force is displayed digitally in a form equivalent to mass on balance. Electric Weighing Balance…

16. The Centrifuge Centrifuge is a piece of equipment, generally driven by a motor, that puts an object in rotation around a fixed axis, applying a force perpendicular to the axis. The centrifuge works using the sedimentation principle

17. Types of Rotors

18. Type of Centrifuge Fixed angle

19. Types of Centrifugation Differential or pelleting Cellular fractionation and/or separating coarse suspension removal of precipitates crude purification step

20. Types of Centrifugation… Preparative or Density gradient centrifugation: Separation of complex mixtures Finer fractionation of cellular components Purification of proteins, nucleic acids, plasmids Characterization of molecular interactions

21. Protein Separation Techniques Proteins are separated and purified by taking advantage of differences in their properties. Proteins can be selectively precipitated by the addition of certain salts. A wide range of procedures makes use of differences in size , binding affinities , charge , and other properties of proteins. These include chromatography ( ion exchange, size-exclusion, affinity, and high performance liquid (HPLC)) and electrophoresis .

22. Chromatography Chromatography is a method of separating a mixture of molecules depending on their distribution between a mobile phase and a stationary phase. The mobile phase (also known as solvent) may be either liquid or gas. The stationary phase (also known as sorbent) can be either a solid or liquid, a liquid stationary phase is held stationary by a solid. The solid holding the liquid stationary phase is the support or matrix. The molecules in the mixture to be separated are the solutes.

23. The separation of compounds by chromatography depends : Partition of a solute between a moving solvent phase and a stationary aqueous phase. The solute moves in the direction of a solvent flow at a rate determined by the solubility of the solute in the moving phase. Thus a compound with high mobility is more attracted to the moving organic phase than to the stationary phase. Chromatography…

24. 2.Ion exchange effect: A ny ionized impurities in the support medium will tend to bind or attract oppositely charged ions (solutes) and will therefore reduce the mobility of these solutes. 3.Temperature: Since temperature can effect the solubility of the solute in a given solvent temperature is also an important factor. Chromatography…

25. Chromatography… 4.The molecular weight of a solute affects the solubility and hence chromatographic performance. 5.Adsorption of compound (solute) onto support medium

26. 6. The composition of the solvent: since some compounds are more soluble in one solvent than in the other, the mixture of solvents used will affect the separation of compounds.   Chromatography…

27. Expression of the results The term "Rf" (relative flow) is used to express the performance of a solute in a given solvent system /support medium. The term Rf value may be defined as the ratio of the distance the compound migrates to the distance the solvent migrates. Rf value is constant for a particular compound, solvent system and insoluble matrix. Rf = Distance of migration of solute Distance moved by solvent

28. Partition chromatography The distribution of solutes between two immiscible phases. The solute will distribute it self between the two phases according to its solubility in each phase, this is called partitioning. The two most common are; thin layer chromatography paper chromatography In both cases the stationary phase is a liquid bound to a matrix. In paper chromatography the stationary phase are water molecules bound to a cellulose matrix.

29. Cont… In TLC, the stationary phase is the solvent added to the support to form the thin layer so the solvent gets bound to the matrix (support). Partition chromatography is mainly used for separation of molecules of small molecular weight.

30. Paper chromatography The cellulose support contains a large amount of bound water. Partitioning occurs between the bound water which is the stationary phase and the solvent which is the mobile phase.

31. Detection of spots Spots in paper chromatograms can be detected in 4 different ways: By their natural color By their fluorescence By their chemical reactions that take place after the paper has been sprayed with various reagents for example: during paper chromatography of amino acids, the chromatograms are sprayed with ninhydrin. By radioactivity

32. Identification of spots The spots are usually identified by comparing of standards of known Rf values.

33. Thin layer chromatography Paper chromatography uses paper which can be prepared from cellulose products only. In TLC, any substance that can be finely divided and formed into a uniform layer can be used. Both organic and inorganic substances can be used to form a uniform layer for TLC. Organic substances include: cellulose, polyamide, polyethylene Inorganic: silica gel, aluminum oxide and magnesium silicate

34. TLC The stationary phase is the solvent used to form a layer of sorbent spread uniformly over the surface of a glass or plastic plate

35. Advantages of TLC over paper chromatography Greater resolving power because there is less diffusion of spots. Greater speed of separation Wide choice of materials as sorbents

36. Electrophoresis

37. Electrophoresis This method is based on the migration of charged proteins in an electric field. Its advantage is that proteins can be visualized as well as separated and also allows determination of crucial properties of a protein such as its isoelectric point and approximate molecular weight . Electrophoresis of proteins is generally carried out in gels made up of the cross-linked polymer, the polyacrylamide gel that acts as a molecular sieve, slowing the migration of proteins approximately in proportion to their charge-to-mass ratio.

38. Electrophoresis…

39. Electrophoretic Mobility The electrophoretic mobility of the molecule, μ , is the ratio of the velocity of the particle molecule, V , to the electrical potential E . Electrophoretic mobility is also equal to the net charge of the molecule, Z , divided by the frictional coefficient, f , which reflects in part a protein’s shape. Thus: μ = V / E = Z / f The migration of a protein in a gel during electrophoresis is therefore a function of its size and its shape .

40. Electrophoresis Set-Up -Different samples are loaded in wells at the top of the polyacrylamide gel. -The proteins move into the gel when an electric field is applied (towards cathode). -The gel minimizes convection currents caused by small temperature gradients, as well as protein movements other than those induced by the electric field.

41. Visualization of Gel -Proteins can be visualized after electrophoresis by treating the gel with a stain such as Coomassie blue , which binds to the proteins but not to the gel itself. -Smaller proteins move through the gel more rapidly than larger proteins and therefore are found nearer the bottom of the gel.

42. Estimation of Molecular Weight A plot of log M r (Molecular Weight) of the marker proteins versus relative migration during electrophoresis is linear, which allows the molecular weight of the unknown protein to be read from the graph.