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Lab Exercise 0ne: Carbohydrate Analysis Lab A.1 (Page 28)

This lab document outlines procedures for measuring glucose concentration using a 3,5-dinitrosalicylic acid (DNS) assay. Students will create a standard curve by measuring absorbance of glucose standards and use it to determine the concentration of an unknown glucose sample. The assay exploits glucose's ability as a reducing sugar to reduce DNS into a product measured colorimetrically at 540nm, correlating product amount to initial glucose concentration.

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Goh Kae Horng
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51 views

Lab Exercise 0ne: Carbohydrate Analysis Lab A.1 (Page 28)

This lab document outlines procedures for measuring glucose concentration using a 3,5-dinitrosalicylic acid (DNS) assay. Students will create a standard curve by measuring absorbance of glucose standards and use it to determine the concentration of an unknown glucose sample. The assay exploits glucose's ability as a reducing sugar to reduce DNS into a product measured colorimetrically at 540nm, correlating product amount to initial glucose concentration.

Uploaded by

Goh Kae Horng
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPT, PDF, TXT or read online on Scribd
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Lab Exercise 0ne

Carbohydrate Analysis Lab A.1(Page 28)

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 0

[glucose(red)]

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:

3,5-dinitrosalicylic acid is reduced to 3-amino,5nitrosalicylic acid

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.

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 3,5dinitrosalicylic 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 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 (ml)

Glucose Standard Volume (ml) 0.000

Unknown Volume (ml) 0.000 1.00

DNS (ml)

A540

Amount (mg)

[Glucose] (mg/ml)

3.000

2.750

0.250

0.000

1.00

2.500

0.500

0.000

1,00

2.250

0.750

0.000

1.00

2.000

1.000

0.000

1.00

2.750

0.000

0.250

1.00

2.500

0.000

0.500

1.00

2.000

0.000

1.000

1.00

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 0

[glucose(red)]

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 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 = 15 points Pre lab= 4 points Total = 20 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.

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