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
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)
Other molecules have chemical
properties which can be used in
indirect measurement.
Introducing concept of 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
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
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
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
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
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)
3,5-dinitrosalicylic acid (DNS)
assay
Section 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
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,5nitrosalicylic acid
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
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.
What are we doing today?
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.
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
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
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
PLEASE DO NOT SLAM THE
SPEC LIDS
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
Lab Reports are PERSONAL
Grading for This Experiment
Number of lab periods = 1
Lab Report = 10 points
Pre lab= 3 points
Total = 13 points
Clean up (Please)
before you go
See page 46. Waste
Disposal & Clean up
Return pipettes to rack
Next Lab: Enzyme Kinetics Lab C1
Page 73-92. Read carefully
Due next time: Feb:2 & 3.
Prelab assignment for Enzyme Kinetics 1
Lab report for Carbohydrate Analysis
See Report Requirements page 47-48
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).
Fig 3: Graph of concentration of standard
glucose vs. absorbancy at 540 nm for tubes
1-5.
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
