Lab Exercise 0ne 2008

7/31/2019 Lab Exercise 0ne 2008

1/31

Lab Exercise 0ne

Carbohydrate Analysis

Lab A.1(Page 28)


2/31

Biochemical Assay

Biochemistry deals with the identification andquantification of bio-molecules from a varietyof living systems

Rely on the chemical reactivity and physicalproperties of bio-molecules to makeidentification and quantification.

Primary tool is the spectrophotometer Uses absorption of mono chromatic light


3/31

Spectrophotometer


4/31

Measure quantity

Some bio-molecules have properties whichallow direct measurement.

proteins have aromatic amino acids (280nm)

Nucleic acids have unsaturated ring structures(260nm)

Other molecules have chemical propertieswhich can be used in indirect measurement.


5/31

Introducing concept of standard curve

Uses dilutions of a solution of known

concentration to determine concentration of

unknown

A540

[glucose(red)]

b

m = y/x

0

0

(may or maynot equal 0)


6/31

Standard Curve

Assumes that unknown will respond in assay

the same as the known

Valid in todaysassay 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


7/31

Our model carbohydrate is the

sugar glucose

We will exploit its ability to reduce other

compounds to produce a product whichcan be measured optically


8/31

Reducing Sugars

Have aldehyde group

Can be oxidized to

acid

Reduces another

compound


9/31

Requirement placed on sugar

Must be an aldehyde

Ketones and hemiacetalconfigurations are not

reducing

Conditions of reactions favor conversion to

aldehyde by lowering aldehyde concentration


10/31

Sugars as Reducing Agents

Equilibrium between

hemiacetal and open chainis driven to open chain asoxidation to acid form takesplace. This ensures a

quantitative conversion withtime and a stoicheometricproduction of reducedcopper.


11/31

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 ofsugar must be limiting factor)


12/31


13/31

3,5-dinitrosalicylic acid (DNS)

Sugar reduces the organic DNS which absorbsmaximally 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 ofreducing sugar


14/31

The DNS reagent

From the MSDS: LABEL PRECAUTIONARY STATEMENTS TOXIC (USA)

HARMFUL (EU) HARMFUL BY INHALATION, IN CONTACTWITH SKIN AND IF SWALLOWED. IRRITATING TO EYES,

RESPIRATORY SYSTEM AND SKIN. IN CASE OF CONTACTWITH EYES, RINSE IMMEDIATELY WITH PLENTY OF WATERAND SEEK MEDICAL ADVICE.

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


15/31

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


16/31

Today's Experiment

Measure the concentration of glucose bydetecting the reducing end of themonosaccharide.

This group converts the oxidized form of 3,5-dinitrosalicylic acid, DNS, to reduced formwhich absorbs at 540nm.

Amount of reduced DNS proportional toamount of glucose.


17/31

What are we doing today?


18/31

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.


19/31

You will create a standard curve

You are provided a stock solution whichcontains 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 tostandard curve


20/31

Table A.1-2. DNS Assay Components

Tube Number Water Volume

(ml)

Glucose

Standard

Volume(ml)

Unknown

Volume

(ml)

DNS

(ml)

A540 Amount

(mg)

[Glucose]

(mg/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


21/31

Standard curve

Uses dilutions of a solution of known

concentration to determine concentration of

unknown

A540

[glucose(red)]

b

m = y/x

0

0

(may or maynot equal 0)


22/31

Important

Careful handling of Cuvettes is essential foraccuracy 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 highestconcentration.

Wipe clear surface if necessary with Kimwipe


23/31

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 isto be measured

Always use same cuvette


24/31

PLEASE DO NOT SLAM THE SPEC LIDS


25/31

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


26/31

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!


27/31

Application quiz

Address in your report

What does the portable glucometers used by

diabetics measure?

How do they measure it?


28/31

Reminder

Lab Reports are PERSONAL


29/31

Grading for This Experiment

Number of lab periods = 1

Lab Report = 15 points

Pre lab= 4 points

Total = 20 points


30/31

Clean up (Please)before you go

See page 44. Waste Disposal &

Clean up Return pipetts to rack


31/31

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.

Lab Exercise 0ne 2008

Documents

Transcript of Lab Exercise 0ne 2008