Transcript Lecture 2: Carbohydrate analysis

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)
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
copper.
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 3,5dinitrosalicylic 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)
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
Data collection
Table 1. DNS Assay Calculations
Tube
Water Volume
Glucose
Glucose
DNS Volume
Number
(ml)
“Standard”
“Unknown”
(ml)
Volume (ml)
Volume (ml)
Measured A540
[Glucose]
Glucose
(mg/ml)
Total Amount
(mg)
1(blank)
3.000
0.000
-
1.000
2
2.750
0.250
-
1.000
3
2.500
0.500
-
1.000
4
2.250
0.750
-
1.000
5
2.000
1.000
-
1.000
6
2.750
-
0.250
1.000
7
2.500
-
0.500
1.000
8
2.000
-
1.000
1.000
PLEASE DO NOT SLAM THE SPEC LIDS
Important
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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
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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: September: 12 & 13.
– 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
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(Cover Page)
Abstract
Introduction/Background
Methods
Results
Discussion
(References)
Cover Page
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Lab Title
Name
Date
Lab partners
Instructor and TA’s
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…” Important statement
• “1) Obtain gloves, lab coat, four micropipettes
and a clean beaker . 2) Set a micropipette to
1000μl….”Too much detail, nobody cares
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).
Concentration of Standard Glucose vs Absorbency at
540 nm wavelength
0.5
y = 0.0015x - 0.0396
R² = 0.9688
0.4
A540
0.3
0.2
Series1
0.1
0
0
-0.1
50
100
150
200
250
300
350
Standard Glucose Concentration (ug/mL)
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