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Type of Analytic Method

Type of Analytic Method. Radadiya Shraddha Kevadiya Pinal Goti Priyanka Thummar Vaishali. The End Point Method. The reaction : Substrate + Reagent (specific) → Colored Complex It required time until reaction end. Completely substrate get utilized

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Type of Analytic Method

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  1. Type of Analytic Method Radadiya Shraddha Kevadiya Pinal Goti Priyanka Thummar Vaishali

  2. The End Point Method • The reaction : Substrate + Reagent (specific) → Colored Complex • It required time until reaction end. • Completely substrate get utilized • More time required (Approx. 30 min) = Called “Incubation Time” • Incubation time is depend on “temperature” • At the end of reaction, Optical Density ( OD ) is taken. • At completion of reaction, The amount of PRODUCTἁ SUBSTRATE

  3. GOD • Glucose + O₂ + H₂O Gluconic acid + H₂O₂ • H₂O₂ + Phenol + 4amino-antipyrine Quinoneimine + 2H₂O • Incubation time 30 min at 37°C temperature. POD

  4. Kinetic Method • Concentration of substrate is high in compare to enzyme; Rate of reaction ἁ concentration of enzyme • so, at given time period, the amount of product is proportional to the concentration of enzyme present. • OD is taken in time period of 1 min.(delta OD) ΔOD = OD60sec – OD0sec • Most of examination are UV kinetic • Measured by UV filter (340 nm) • Delay time / Incubation time = For Equilibrium between Substrate & Enzyme • Reading time = Measuring time , when change of O.D. is measured.

  5. Type of Kinetic Method • Decreasing Kinetic Method • As reaction progress, Optical density decrease • As instrument measure, substrate utilization • E.g. ALT • Increase Kinetic Method • As reaction progress, Optical density increase • As instrument measure, product formation • E.g. Alkaline Phosphate

  6. ALT • Principle of ALT: • ἁ - Ketoglutarate + Alanine L- Glutamate + Pyruvate Pyruvate +NADH Lactate + NAD⁺ • Measuring NADH utilization. • Decrease kinetic. GPT dfLDH

  7. Alkaline Phosphatase • P-nitrophenol production (Yellow Colour Product = 420 nm) • Increasing kinetic.

  8. FIXED TIME KINETIC METHOD • In Kinetic method, analyte is playing role as enzyme in reaction • In Fix Time Kinetic method, analyte is playing role as substrate in reaction • So substrate is utilized as reaction is progress. • Reaction velocity is decrease as reaction is progress towards end. • Delta OD should be measured in initial phase of reaction. (With in Fix Time Period) • So It is called Fixed Time Kinetic.

  9. Principle of Creatinine Picrate + NaOH Alkaline Picrate Complex • Alkaline Picrate Complex + Creatinine Janovaski Complex (Red Orange Colour) Non-Enzymatic Non-Enzymatic

  10. Principle of urea

  11. THANK YOU

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