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Similaire à Proteins and amino acids; Brief introduction of methods for quantification of types of proteins and amino acids
Similaire à Proteins and amino acids; Brief introduction of methods for quantification of types of proteins and amino acids (20)
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Proteins and amino acids; Brief introduction of methods for quantification of types of proteins and amino acids
- 1. PROTEINS AND AMINO ACIDS METHODS FOR IDENTIFICATION AND QUANTIFICATION UMAIR BIN IRFAN 1001851413
- 2. WHAT ARE PROTEINS ? • Building blocks of amino acids • Workhorses of life • Present in every single cell • Structure and texture to food • Maillard reaction
- 3. Protein Analysis – Significant ?? • Healthy food • Biological activity indicator • Chemical reactions
- 4. SOME METHODSTO DETERMINE PROTEIN CONTENT • ENZYME HYDROLYSIS AND DERIVATISATION WITH O- PHTHALDEHYDE • CAPILLARY ELECTROPHORESIS
- 5. ENZYME HYDROLYSIS AND DERIVATIZATION WITH O-PHTHALDEHYDE • Based on the concept of enzyme hydrolysis of proteins • Derivatization of peptides • Partially automated
- 6. ENZYME HYDROLYSISAND DERIVATIZATIONWITH O-PHTHALDEHYDE • Chemicals Needed: Samples to analyze: Buffer salts Enzymes O-Phthaldehyde Heptane Acetic acid Apparatus : => Pancreatic enzyme column => Centrifuge =>Vortex mixer Fresh full-fat and low-fat milk Skimmed milk powder Fresh standard and low-fat creams Whey protein isolate
- 7. ENZYME HYDROLYSISAND DERIVATIZATIONWITH O-PHTHALDEHYDE PROCEDURE Preparation of pancreatic enzyme column • Immobilization • Incubation for 17 hours • Washing in blocking buffer • Packed column Sample Preparation • Heptane addition • Mixing withVortex mixer • Centrifugation -- defatting • Dilution and denaturation Enzyme Hydrolysis • Sample injection • On & Off Buffer • OPA reagent • Fluorescence detector
- 8. ENZYME HYDROLYSISAND DERIVATIZATIONWITH O-PHTHALDEHYDE FLOW DIAGRAM
- 9. ENZYME HYDROLYSISAND DERIVATIZATIONWITH O-PHTHALDEHYDE
- 10. ENZYME HYDROLYSISAND DERIVATIZATIONWITH O-PHTHALDEHYDE PROTEIN CONTENTS (STANDARD)
- 11. ENZYME HYDROLYSIS AND DERIVATIZATION WITH O-PHTHALDEHYDE ADVANTAGES DISADVANTAGES Partially automated High Cost No loss of tryptophan Time consuming Good accuracy and precision OPA sensitive to moisture Avoid underestimation of proteins Cysteine instability may occur. Fast chemical reactivity of OPA with amino acids Accurate determination of protein with enzyme hydrolysis Avoid sample contamination, using immobilized enzymes
- 12. CAPILLARY ELECTROPHORESIS (CE) • Electrophoresis is a differential movement of ions in an electric field • Separation of milk proteins based on their charge to mass ratio • Quality of dairy products at commercial level
- 13. CAPILLARY ELECTROPHORESIS (CE) • Chemicals and Reagents Sample: Protein standards (Bovine milk) Camel Milk (20 Liters) Dialysis sacks Bovine Milk Distilled water Sodium hydroxide Apparatus: Hydrochloric acid Centrifuge, Freeze dryer, CE-DAD.
- 14. CAPILLARY ELECTROPHORESIS (CE) Sample Defatting Casein –Whey Proteins separation CAPILLARY ELECTROPHORESIS PROCEDURE
- 17. CAPILLARY ELECTROPHORESIS (CE) MAJOR PROTEIN CONTENT
- 18. ADVANTAGES DISADVANTAGES Simplicity of method Negative effects of pH and Temperature on molecular charge and flow Quick and reliable High injection volume suffers separation Low operating cost Tiny peaks Fully Automated Low sensitivity CAPILLARY ELECTROPHORESIS (CE)
- 19. COMPARISON BETWEEN BOTH METHODS: Enzyme hydrolysis and derivatization with o- phthaldehyde Capillary Electrophoresis Advantages Advantages Partially automated Simplicity of method No loss of tryptophan Quick and reliable Good accuracy and precision Low operating cost Avoid underestimation of proteins Fully Automated Fast chemical reactivity of OPA with amino acids Disadvantages Disadvantages High Cost Negative effects of pH and Temperature on molecular charge and flow Time consuming High injection volume suffers separation OPA sensitive to moisture Tiny peaks Cysteine instability chances Low sensitivity
- 20. CONCLUSION: • Enzyme Hydrolysis and derivatization with O-phthaldehyde method shows various merits in terms of precision and accuracy.The main purpose of protein analysis is to know the accurate quantity of food proteins in order to describe food more purposely. But High cost and more time consumption are the two main draw backs of this method. However, chance of cysteine instability cannot be neglected as it is important for protein synthesis. Overall, this method can be improved by lowering the operating cost as well as reducing the chances of cysteine instability. • Capillary Electrophoresis technique has good command in automation, simplicity and low operating costs but this technique is low sensitive as well as shows tiny peaks. However derivatization process can make this technique superior than the previous one. I preferred first method as it provides accurate quantification of proteins in foods.
- 21. AMINO ACIDS • Basic components of proteins • Source of energy • Impact on nutritional value of proteins • Essential and Non essential amino acids
- 22. SOME METHODSTO DETERMINE AMINO ACIDS IN FOOD SYSTEM • CAPILLARY ELECTROPHORESIS WITH UV-DETECTION • REACTION FLOW CHROMATOGRAPHY WITH POST COLUMN DERIVATISATION
- 23. CAPILLARY ELECTROPHORESIS WITH UV DETECTION • Separation on the basis of charge to mass ratio • Used pre column derivatization to enhance the detectability of amino acids • NBD-Cl is used as derivatization agent
- 24. CAPILLARY ELECTROPHORESISWITH UV DETECTION CHEMICALS • Amino acids analytical standards • Ethanol • Acetonitrile • Boric acid • NaOH • Ultra-pure water SAMPLES • SUDANESE Samples were used: • Potato • Eggplant • Chickpeas • SoftWheat flour • Sorghum durra flour APPARATUS • Centrifuge • Vortex mixer • Thermomixer comfort • CE System with PDA detector
- 25. Preparation of Electrolytes and standard solution Sample Preparation Pre Column Derivatization with NBD-Cl CAPILLARY ELECTROPHORESISWITH UV DETECTION PROCEDURE
- 26. • Capillary Electrophoresis: Separation was carried out on fused silica capillaries of 40 cm total length with internal diameter of 50 m. The temperature of capillary was kept constant at 25℃. Before each injection of sample, capillary was preconditioned with 0.1M NaOH, water and the running buffer for 3 min. Samples were injected at a pressure of 0.5 psi for 10 sec, and separation were performed under 25kV positive high voltage. The data was collected and processed by software developed by Beckman. CAPILLARY ELECTROPHORESISWITH UV DETECTION
- 27. CAPILLARY ELECTROPHORESISWITH UV DETECTION
- 28. CAPILLARY ELECTROPHORESISWITH UV DETECTION
- 29. ADVANTAGES DISADVANTAGES Less expensive Sometimes Less sensitive Short analysis time Short preparation time Powerful separation technique NBD-Cl for pre column derivatization NBD-Cl Produce low number of bio products Precision and accuracy CAPILLARY ELECTROPHORESISWITH UV DETECTION
- 30. REACTION FLOW CHROMATOGRAPHY • Fast and more sensitive approach forAA analysis • Use fluorescamine for post column derivatization • Detect derivatized and underivatized samples simultaneously
- 31. REACTION FLOW CHROMATOGRAPHY • Chemicals: Acetonitrile, Water, acetone, fluorescamine, ultrapure water ammonium acetate, ammonia and amino acids. • Sample: Espresso Coffee – Nestle • Apparatus: Multi Solvent delivery LC System with pumps and detectors
- 32. • Buffer Solution and Standard Solution preparation • Sample Preparation • Reaction flow (RF) Chromatography - Post column Derivatization (PCD) REACTION FLOW CHROMATOGRAPHY PROCEDURE
- 33. • Reaction flow (RF) – PCD: • The standards were separated in isocratic mode using a premixed mobile phase of ratio 95:5 (H2O: ACN) at a flow rate of 0.7 ml/min. • The eluent was then combined with ammonium acetate buffer (0.45 ml/min) at a zero dead volumeT-piece, passing through a reaction coil. • The fluorescamine reagent was then introduced at the multiport outlet of RF column through one peripheral port at a flow rate of 0.1 ml/min, one peripheral port was blocked and the derivatized eluent containing the derivatized solutes exited the RF end fitting from third peripheral port to the detector at 390 nm. • Mobile phase carrying underivatized solutes eluted from the radial central exit port and this was directed to a second detector at 280 nm. • Same procedure was applied for coffee samples (spiked with 500 ppm amino acids) and chromatographic separation for all four derivatized amino acids was shown in figure. REACTION FLOW CHROMATOGRAPHY
- 36. ADVANTAGES DISADVANTAGES Multiplexed detection Expensive Fluorescamine rapid reaction Time consuming High sensitivity Excellent separation performance REACTION FLOW CHROMATOGRAPHY
- 37. COMPARISION BETWEEN BOTH TECHNIQUES: Capillary Electrophoresis with UV-Detection Reaction flow Chromatography- PCD Advantages Advantages Less expensive Multiplexed detection Short analysis time Fluorescamine rapid reaction Short preparation time High sensitivity Powerful separation technique Excellent separation performance NBD-Cl for pre column derivatization Disadvantages NBD-Cl Produce low number of bio products Expensive Precision and accuracy Time consuming
- 38. CONCLUSION • Capillary electrophoresis with UV detection as well as derivatized by low cost reagent shows numerous benefits for the analysis of amino acids in terms of less expensive, less time consuming and with high precision and accuracy. But some times for complex samples, it shows difference in migration times with respect to the standards. It can be modified with high usage of derivatization reagent to overcome this problem. I prefer this technique superior than RF-PCD for analysis of amino acids. • Advantages of RF-PCD cannot be neglected in terms of sensitivity and accuracy, but sometimes cost and time are the factors that contribute to neglect this technique for amino acid analysis.