1. Carbohydrate Analysis Lab A.1 Page 26
Lab Exercise 0ne
Carbohydrate Analysis
Lab A.1
Page 26

2. 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

3. Spectrophotometer

4. 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.

5. Introducing concept of standard curve
Uses dilutions of a solution of known concentration to determine concentration of unknown

6. 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

7. 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

8. Reducing Sugars Have aldehyde group Can be oxidized to acid
Reduces another compound

9. 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

10. 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.

11. 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)

12. 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

13. 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

14. 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

15. 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.

16. What are we doing today?

17. 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.

18. 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

19. Standard curve
Uses dilutions of a solution of known concentration to determine concentration of unknown

20. 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

21. 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”

22. 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

23. PLEASE DO NOT SLAM THE SPEC LIDS

24. 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

25. Application quiz Address in your report
What does the portable glucometers used by diabetics measure?
How do they measure it?

26. Reminder
Lab Reports are PERSONAL

27. Grading for This Experiment
Number of lab periods = 1
Lab Report = 10 points
Pre lab= 3 points
Total = 13 points

28. Clean up (Please) before you go
See page 46. Waste Disposal & Clean up
Return pipettes to rack

29. 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

30. Constructing Lab Reports
BCH

31. 5 Components (Cover Page) Abstract Introduction/Background Methods
Results
Discussion
(References)

32. Cover Page
Lab Title
Name
Date
Lab partners
Instructor and TA’s

33. Abstract
Theory (background/intro and methods summary)
Results

34. Introduction
Conceptual Theory
Experimental Theory

35. 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 1000μl….”

36. Results Properly labeled data tables and graphs
Captions and descriptions
Sample calculations (with units!)
Other requirements? (Percent error)

37. 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 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.

38. 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