Lab Exercise 0ne

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Lab Exercise 0ne

• Carbohydrate Analysis
• Lab A.1(Page 28)

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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

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Spectrophotometer
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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.

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Introducing concept of standard curve

• Uses dilutions of a solution of known
  concentration to determine concentration of
  unknown

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Standard Curve

• Assumes that unknown will respond in assay the
  same as the known
• Valid in todays assay 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

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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

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Reducing Sugars

• Have aldehyde group
• Can be oxidized to acid
• Reduces another compound

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Requirement placed on sugar

• Must be an aldehyde
• Ketones and hemiacetal configurations are not
  reducing
• Conditions of reactions favor conversion to
  aldehyde by lowering aldehyde concentration

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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.
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Nelson Assay (a two step Rx)

• In the Nelson assay Cu2 is reduced to Cu1 by
  the reducing activity of the sugar (step 1)
• Cu1 is oxidized to Cu2 by addition of
  arsenomolybdic acid (colorless) (step 2)
• Results in blue (reduced) arsenomolybdous acid
• Amount is directly related to CU1
• Will detect any reducing sugar (concentration of
  sugar must be limiting factor)

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We will do the DNS assaySection A1 pages 37-39

• Is a direct assay
• Measures the reducing capability of glucose
• Uses a color conversion reaction from yellow to
  red brown _at_ A540
• Conversion of moles of DNS equals moles of
  glucose.

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3,5-dinitrosalicylic acid (DNS)

• Sugar reduces the organic DNS which absorbs
  maximally at yellow wave length
• Results in change (shift) in absorption spectrum
  from red/orange 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

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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

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The DNS assay

• Experimental design and flow charts page 36
• Be sure to read Hazards page 37
• Protocol on page 38
• Data analysis page 41

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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.

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What are we doing today?
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Important See data table page 38

• Pipetting technique is critical to accuracy and
  to preventing cross contamination of samples
• Pipetters have two stops
• First to take up selected volumes
• Second to deliver
• Choose pipetter in the range that you need.

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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
• You will read the absorbance of each solution at
  540 and plot vs concentration
• You will compare the A540 of unknown to standard
  curve

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Standard curve

• Uses dilutions of a solution of known
  concentration to determine concentration of
  unknown

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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

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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 38)
• Contains all components of reaction except that
  which is to be measured
• Always use same cuvette

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PLEASE DO NOT SLAM THE SPEC LIDS
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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

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Lab reports for this class(see Report
construction Page 44)

• Abstract. Statements regarding
• WHAT you are doing (-gt procedure)
• WHY you are doing it (-gt your hypothesis)
• WHAT you hope to accomplish (-gt also hypothesis)
• Cf. purpose/goal in a good lab notebook! Might
  think of it as a very short introduction
• Background information and theory
• Results/Data/Data Analysis
• Discussion MUST relate data analysis to
  hypothesis!

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Application quizAddress in your report

• What does the portable glucometers used by
  diabetics measure?
• How do they measure it?

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Reminder

• Lab Reports are PERSONAL

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Grading for This Experiment

• Number of lab periods 1
• Lab Report 15 points
• Pre lab 4 points
• Total 20 points

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Clean up (Please)before you go

• See page 44. Waste Disposal Clean up
• Return pipetts to rack

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Next Lab Enzyme KineticsPage 65

• Due next time January 25th, 26th and 27th.
• Prelab assignment for Enzyme Kinetics
• Lab report for Carbohydrate Analysis
• Abstract
• Data table, graph with best-fit line, calculate
  average concentration (avg conc) of unknown and
  standard deviation (std dev) in average.
• Discussion linear relationship? Can also use
  Thought questions (page 45) as topics for
  discussion.

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Good luck with your lab !!!