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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
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
Spectrophotometer
Measure quantity
Some bio-molecules have properties
which allow direct measurement.
proteins have aromatic amino acids
(280nm)
Nucleic acids have unsaturated ring
structures (260nm)
A540
m = y/x
b
(may or may
not equal 0)
[glucose(red)]
0
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
Requirement placed on
sugar
Must be an aldehyde
Ketones and hemiacetal configurations
are not reducing
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
Measured at 540nm
Unreacted DNS not seen at this wavelength
Amount of absorbance directly related to
amount of reducing sugar
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.
Standard curve
Uses dilutions of a solution of known
concentration to determine
concentration of unknown
A540
m = y/x
b
(may or may
not equal 0)
[glucose(red)]
0
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
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
Extremely Important
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
Application quiz
Address in your report
What does the portable glucometers
used by diabetics measure?
How do they measure it?
Reminder
Clean up (Please)
before you go
Constructing
Lab Reports
BCH 452-001
5 Components
(Cover Page)
Abstract
Introduction/Background
Methods
Results
Discussion
(References)
Cover Page
Lab Title
Name
Date
Lab partners
Instructor and TAs
Abstract
Theory (background/intro and
methods summary)
Results
Introduction
Conceptual Theory
Experimental Theory
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
1000l.
Results
Properly labeled data tables and
graphs
Captions and descriptions
Sample calculations (with units!)
Other requirements? (Percent error)
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).
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