Lab Exercise 0ne

Carbohydrate Analysis
Lab A.1(Page 28)
2010
 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
 Reducing Sugars

• Have aldehyde group

• Can be oxidized to
acid
• Reduces another
compound
 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)
 We will do the DNS assay
Section A1 pages 37-39

• Is a direct assay
• Measures the reducing capability of glucose
• Uses a color conversion reaction from yellow to red
brown @ A540
• Conversion of moles of DNS equals moles of glucose.
 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
 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
 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
 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 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.
 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
 Table A.1-2. DNS Assay Components

Tube Number Water Volume Glucose Unknown DNS A540 Amount [Glucose]
(ml) “Standard” Volume (ml) (mg) (mg/ml)
Volume (ml)
(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

 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
 Important
• Careful handling of Cuvettes is essential for
accuracy and prevent contamination
– Handle only with gloves
– Touch only the frosted area
– 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 clear 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
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
 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!
 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= 2 points
• Total = 12 points
 Clean up (Please)
before you go

• See page 44. Waste Disposal &

Clean up
• Return pipetts to rack
 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.