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Lab Exercise 0ne

Lab Exercise 0ne. Carbohydrate Analysis Lab A.1 Page 26. Biochemical Assay. Biochemistry deals with the identification and quantification of bio-molecules from a variety of living systems

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Lab Exercise 0ne

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  1. Lab Exercise 0ne Carbohydrate Analysis Lab A.1 Page 26

  2. Biochemical Assay • Biochemistry deals with the identification and quantification of bio-molecules from a variety of living systems • Rely on the chemical reactivity and physical properties of bio-molecules to make identification and quantification. • Primary tool is the spectrophotometer • Uses absorption of mono chromatic light

  3. Spectrophotometer

  4. Measure quantity • Some bio-molecules have properties which allow direct measurement. • proteins have aromatic amino acids (280nm) • Nucleic acids have unsaturated ring structures (260nm) • Other molecules have chemical properties which can be used in indirect measurement.

  5. Introducing concept of standard curve • Uses dilutions of a solution of known concentration to determine concentration of unknown

  6. Standard Curve • Assumes that unknown will respond in assay the same as the known • Valid in todaysassay as they (the reactive groups. glucose) are the same • Problem in other assay as they may not contain same amount of reactive groups • Protein assays (have to choose) • But usually close

  7. Our model carbohydrate is the sugar glucose We will exploit its ability to reduce other compounds to produce a product which can be measured optically

  8. Requirement placed on sugar • Must be an aldehyde • Ketones and hemiacetalconfigurations are not reducing • Conditions of reactions favor conversion to aldehyde by lowering aldehyde concentration

  9. Sugars as Reducing Agents Equilibrium between hemiacetal and open chain is driven to open chain as oxidation to acid form takes place. This ensures a quantitative conversion with time and a stoicheometric production of reduced copper.

  10. Nelson Assay (a two step Rx) • In the Nelson assay Cu+2 is reduced to Cu+1 by the reducing activity of the sugar (step 1) • Cu+1 is oxidized to Cu+2 by addition of arsenomolybdic acid (colorless) (step 2) • Results in blue (reduced) arsenomolybdous acid • Amount is directly related to [CU+1] • Will detect any reducing sugar (concentration of sugar must be limiting factor)

  11. 3,5-dinitrosalicylic acid (DNS) assaySection A1 pages 33-49 • Sugar reduces the organic DNS which absorbs maximally at yellow wave length • Results in change (shift) in absorption spectrum from yellow to red/brown at 540nm • Different from Nelson reaction • Measured at 540nm • Unreacted DNS not seen at this wavelength • Amount of absorbance directly related to amount of reducing sugar

  12. The DNS reagent • From the MSDS: • LABEL PRECAUTIONARY STATEMENTS TOXIC (USA) HARMFUL (EU) HARMFUL BY INHALATION, IN CONTACT WITH SKIN AND IF SWALLOWED. IRRITATING TO EYES, RESPIRATORY SYSTEM AND SKIN. IN CASE OF CONTACT WITH EYES, RINSE IMMEDIATELY WITH PLENTY OF WATER AND SEEK MEDICAL ADVICE. • 3,5-dinitrosalicylic acid is reduced to 3-amino,5-nitrosalicylic acid

  13. The DNS assay • Experimental design and flow charts page 36 &37 • Be sure to read “Hazards” page 37 • Protocol on page 38 • Data analysis page 42

  14. Today's Experiment • Measure the concentration of glucose by detecting the reducing end of the monosaccharide. • This group converts the oxidized form of 3,5-dinitrosalicylic acid, DNS, to reduced form which absorbs at 540nm. • Amount of reduced DNS proportional to amount of glucose.

  15. What are we doing today?

  16. Important: See data table page 39 • Pipetting technique is critical to accuracy and to preventing cross contamination of samples • Read Micropipette operation (8 to12) • Pipettes have two stops • First to take up selected volumes • Second to deliver • Choose pipette “in the range” that you need.

  17. You will create a standard curve • You are provided a stock solution which contains 1.2 mg/ml • You will dilute this stock solution in a specified manner always producing a 4 ml solution (See table A1-2) • After reacting with DNS you will read the absorbance of each solution at 540 and plot vs concentration • You will compare the A540 of unknown to standard curve

  18. Standard curve • Uses dilutions of a solution of known concentration to determine concentration of unknown

  19. Protocol Page 38 • Steps 8,9,10 • Critical for uniform reaction rates • 100C accelerates the reaction • Cool samples in Ice water bath for 10 to 15 seconds • Rapidly brings the sample to low temp which slows the reaction • Carefull too long in ice bath will cause condensation on the cuvettes

  20. Important • Careful handling of Cuvettes is essential for accuracy and prevent contamination • Handle only with gloves • Touch only the areas not in the light path • Rinse carefully with DH2O after each use • Always go from lowest concentration to highest concentration. • Wipe clear surface if necessary with “Kimwipe”

  21. Extremely Important • Put cuvette into Spec slot that is in the beam path • Be certain that clean panes face the beam path • Measure only with the lid closed • Always set the spec with a blank(line 1 table A.1-2, page 39) • Contains all components of reaction except that which is to be measured • Always use same cuvette

  22. PLEASE DO NOT SLAM THE SPEC LIDS

  23. Important • 1. Wear Gloves and Safety Glasses • 2. Record the code number of your unknown • 3. Be certain that test tubes are clean • 4. Water/H2O always means distilled water • 5.Have TA initial your data before you leave. See lab exit requirements page

  24. Application quizAddress in your report • What does the portable glucometers used by diabetics measure? • How do they measure it?

  25. Reminder • Lab Reports are PERSONAL

  26. Grading for This Experiment • Number of lab periods = 1 • Lab Report = 10 points • Pre lab= 3 points • Total = 13 points

  27. Clean up (Please)before you go • See page 46. Waste Disposal & Clean up • Return pipettes to rack

  28. Next Lab: Enzyme Kinetics Lab C1Page 73-92. Read carefully • Due next time: Jan 27,28. • Prelab assignment for Enzyme Kinetics 1 • Lab report for Carbohydrate Analysis • See Report Requirements page 47-48

  29. Constructing Lab Reports BCH 452-001

  30. 5 Components • (Cover Page) • Abstract • Introduction/Background • Methods • Results • Discussion • (References)

  31. Cover Page • Lab Title • Name • Date • Lab partners • Instructor and TA’s

  32. Abstract • Theory (background/intro and methods summary) • Results

  33. Introduction • Conceptual Theory • Experimental Theory

  34. Methods • Protocol with general description • “In a beaker, 5ml of reagent X was mixed with 2ml of reagent Y…” • “1) Obtain gloves, lab coat, four micropipettes and a clean beaker . 2) Set a micropipette to 1000μl….”

  35. Results • Properly labeled data tables and graphs • Captions and descriptions • Sample calculations (with units!) • Other requirements? (Percent error)

  36. Graph Example The following graph shows standard curve of glucose concentration. Absorbency readings were taken at 540 nanometers of 5 samples with known glucose concentration. R2 value of .9688 indicates a fit linear correlation. The slope of this graph was used to calculate glucose concentration in unknown samples (Fig 4). Fig 3: Graph of concentration of “standard” glucose vs. absorbancy at 540 nm for tubes 1-5.

  37. Discussion • Explain why the experiment was run and what information was gained • Answer questions posed in lab manual- look at lab report requirements • Results • Sources of error

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