lab exercise 0ne 2008
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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 andquantification of bio-molecules from a varietyof living systems
Rely on the chemical reactivity and physicalproperties of bio-molecules to makeidentification 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 whichallow direct measurement.
proteins have aromatic amino acids (280nm)
Nucleic acids have unsaturated ring structures(260nm)
Other molecules have chemical propertieswhich 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
A540
[glucose(red)]
b
m = y/x
0
0
(may or maynot equal 0)
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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
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Our model carbohydrate is the
sugar glucose
We will exploit its ability to reduce other
compounds to produce a product whichcan 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 hemiacetalconfigurations 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 chainis driven to open chain asoxidation to acid form takesplace. This ensures a
quantitative conversion withtime and a stoicheometricproduction of reducedcopper.
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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 ofsugar must be limiting factor)
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3,5-dinitrosalicylic acid (DNS)
Sugar reduces the organic DNS which absorbsmaximally 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 ofreducing sugar
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The DNS reagent
From the MSDS: LABEL PRECAUTIONARY STATEMENTS TOXIC (USA)
HARMFUL (EU) HARMFUL BY INHALATION, IN CONTACTWITH SKIN AND IF SWALLOWED. IRRITATING TO EYES,
RESPIRATORY SYSTEM AND SKIN. IN CASE OF CONTACTWITH EYES, RINSE IMMEDIATELY WITH PLENTY OF WATERAND 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 bydetecting the reducing end of themonosaccharide.
This group converts the oxidized form of 3,5-dinitrosalicylic acid, DNS, to reduced formwhich absorbs at 540nm.
Amount of reduced DNS proportional toamount 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 whichcontains 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 tostandard curve
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Table A.1-2. DNS Assay Components
Tube Number Water Volume
(ml)
Glucose
Standard
Volume(ml)
Unknown
Volume
(ml)
DNS
(ml)
A540 Amount
(mg)
[Glucose]
(mg/ml)
1 3.000 0.000 0.000 1.00 -
2 2.750 0.250 0.000 1.00 -
3 2.500 0.500 0.000 1,00 -
4 2.250 0.750 0.000 1.00 -
5 2.000 1.000 0.000 1.00 -
6 2.750 0.000 0.250 1.00
7 2.500 0.000 0.500 1.00
8 2.000 0.000 1.000 1.00
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Standard curve
Uses dilutions of a solution of known
concentration to determine concentration of
unknown
A540
[glucose(red)]
b
m = y/x
0
0
(may or maynot equal 0)
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Important
Careful handling of Cuvettes is essential foraccuracy 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 highestconcentration.
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 isto 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 (-> procedure)
WHY you are doing it (-> your hypothesis)
WHAT you hope to accomplish (-> 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 quiz
Address 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 Kinetics
Page 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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