instruments in biochemistry.pptx

for first MBBS and DMLT

1. CENT R IF U GE

2. INTRODUCTION WHAT IS CENTRIFUGE? Centrifuge is a device for separating particles from a solution according to there size, shape, density, viscosity of the medium. WHAT IS CENTRIFUGATION? Centrifugation is a process which involves the use of the centrifugal force for the sedimentation of heterogeneous mixtures with a centrifuge .

3. Basic Principle

4.  The centri f uge i n v o lve s prin c i p l e of c en tri f u gati on, where t he revolution causes denser substances to separate out along the radial direction at the bottom of the tube.

5. In a solution, particles whose density is higher than that of the solvent sink (sediment), and particles that are lighter than it float to the top. The greater the difference in density, the faster they move.

6. A p p li c ati o n s Remove cellular elements from blood to provide cell free serum or plasma Conc entrating c ellular element s for microscopy. Remove protein precipitate from analytic sample. Isolation of macromolecules such as DNA, RNA, proteins, or lipids Use in haematology lab for PCV determination.

7. p H meter

8. A pH meter provides a value as to how acidic or alkaline a liquid is. The basic principle of the pH meter is to measure the concentration of hydrogen ions. Acids dissolve in water forming positively charged hydrogen ions (H+). The greater the concentration of hydrogen ions, the stronger the acid is.

9. A pH meter measures essentially the electro-chemical potential between a known liquid inside the glass electrode (membrane) and an unknown liquid outside

10. When the p H electrode is dipped in a solution, two things can happen: if the H+ conc outside is greater there is movement of H+ ions into the electrode If the concentration of H+ in the inner solution is more it will move towards the solution The p H electrode has a very delicate glass membrane with pores that exactly fit only H+ ions

11. This movement of H+ ions creates a potential difference that can be measured using Nernst Equation

12. Nernst equation

13. Applications of p H meter To measure the p H of biological fluids like blood, urine, gastric fluid, pleural, peritoneal fluids p H meter is an integrated part of blood gas analyzer To determine the p H of buffer solution To determine the p H of soil In food industry

14. Autoanalyzers

15. What is automation Automation in clinical laboratory is a process by which analytical instruments perform many tests with the least involvement of manpower

16. Advantages Instruments can use very small amounts of samples and reagents subsequently allowing less blood to be drawn from each patient. The use of small amounts of reagents decreases the cost of consumable.

17. To minimize the variation in results from one individual to another

18. Applications For biochemical analysis : estimation of glucose, urea, creatinine, total protein, albumin, uric acid, liver function tests Lipid profile Cardiac enzymes: CK T,CKMB Hb A1c Urinary protein, CSF sugar and protein

19. SEMI AUTOANALYZER

20. Here the samples and reagents are mixed and read manually . Based on colorimetry principle. Disadvantages are:  More amount of sample is needed  Time consuming  Need technical expertisation

21. uses Same as autoanalysers Semiautoanalyzers are cheaper and easier to handle than fully auto analyzer

22. It is prepared by polymerizing acryl amide monomers in the presence of methylene-bis-acrylamide to cross link the monomers. Polyacrylamide gels are tougher than agarose gels. It is thermostable, transparent, strong and relatively chemically inert. Gels are prepared in a variety of pore sizes. Proteins are separated on the basis of charge to mass ratio and molecular size, a phenomenon called Molecular sieving. POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE) 15

23. Advantages Gels of different pore size can be formed. Simple and separation speed is good comparatively

24. Uses of Electrophoresis Separation of serum proteins Separation of serum lipoproteins to diagnose Hyper / Hypo lipoproteinemias Separation of Hemoglobin variants – Hemoglobinopathies

25. 4. Separation iso Enzymes - LDH, CK 5. purification of proteins: SDS PAGE 6. Detection of mutations : RFLP test 7. In blotting techniques 8. Detection of cancers by DNA fingerprinting 9. DNA sequencing 10.testing the purity of thyroid hormones by zone electrophoresis

26. Electrolyte analyzer

27. Electrolyte analyzers Electrolytes present in body fluids like Na, K , chloride are estimated routinely in clinical biochemistry lab Previously methods like flame photometry was used for electrolyte estimation The modern day electrolyte analyzers use Ion selective electrodes

28. Basic principles An ion-selective electrode ( ISE ), is a transducer (or sensor) that converts the concentration of a specific ion dissolved in a solution into an electrical potential, which can be measured by a voltmeter

29. An ISE operates o n exactly the same principles as a p H electrode In fact, a p H electrode is a type of ion selective electrode sensitive to hydrogen ion.

30. An ideal I.S.E. consists of a thin membrane across which only the intended ion can be transported. The transport of ions from a high conc. to a low one through a selective binding with some sites within the membrane creates a potential difference.

31. Uses of ISE Ion-selective electrodes are used in determining the concentrations of various ions like sodium, potassium, chloride, calcium ions in biological fluids . To calculate anion gap in ABG analyzer

32. Lithium estimation for patients receiving lithium therapy Urea estimation Ammonia estimation Ionised calcium estimation

33. Chemiluminescence Immunoassay (CLIA) Technique

34. Principle: -same as ELISA -uses chemiluminescent substrate, hydrogen peroxide, enhancers -stopping reagent is not required -Incubation period is small