INTRODUCTORY BIOCHEMISTRY

TO7 –C211  LAB MANUAL

David Blicq A425M dblicq@rrc.mb.ca  (update 01/04/2010)  DIRECTORY I BIO I NOTICE BOARD

Table of Contents

Laboratory Safety

Laboratory Records

Lab 1 - Buffers and Titration Curves

Lab 2 - Saponification of Triglycerides

Lab 3 - Iodine Number of Triglycerides

Lab 4 - Carbohydrate Testing

Lab 5 - Hydrolysis of Starch by Amylase

Lab 6 - Selective Precipitation / Fractionation

Lab 7 - Determination of Protein Concentration

ATTENTION!

These laboratory exercises have been created for the students of the Chemical and Biosciences Technology program at Red River College, Winnipeg Manitoba. By design, the exercises contain selected errors and problems intended for the student to troubleshoot and remedy in the course of the activities.

Laboratory Safety  In chemical experimentation there are always hazards present due to materials, equipment, and the reaction of materials. There is a simple rule of thumb: WHAT YOU DON’T KNOW CAN HURT YOU! By following basic safety precautions you can minimize the probability and consequences of an accident.

PROTECT YOUR EYES!
1. Wear safety goggles when there is a risk of splattering, when working with
corrosive chemicals, and when working with apparatus under reduced or increased pressure.
2. Never have your eyes over a vessel opening! Look at a vessel but never into it.

DON'T POISON YOURSELF!
1. Regard all chemicals as poisons.
2. Do not eat in the laboratory.
3. Wear gloves when using chemicals that pose a health hazard by rapid adsorption through intact skin.
4. Handle obnoxious chemicals in a fume hood.
5. Deal with all spills immediately.
6. When finished working, wash hands thoroughly.

GUARD AGAINST FIRE

1. Regard all organic liquids as flammable.
2. Solvents are to be stored only in closed containers: never in an open beaker.
3. Familiarize yourself with the location of Fire extinguishers, eye-washes, safety showers and first aid equipment in your laboratory!

NOTIFY YOUR INSTRUCTOR IMMEDIATELY OF ANY / ALL ACCIDENTS!

LABORATORY PREPARATION

Students are expected to attend and prepare for all laboratories by completing the following:
1. Experiments are to have been read and an outline written up prior to each lab session.
2. Potential hazards and handling considerations for each experiment are to written up in your “Lab Log” before the laboratory session.
 

Evaluation:

1. Mark Weights: Adept = 10 marks; Average = 7 marks; Poor = 4 marks. Marks totaled and delivered as a percentage of 20 possible course marks.
2. Total course scoring: 25% Mid-term, 25% Final; 30% Laboratory Notebook, 20% Outcome-based.


Student score (out of 20):_____________

Authorized Signature:____________________________ Date:________________
 

Laboratory 1

 Buffers and Titration Curves

Introduction

                 A buffer system is defined as a system that resists change in pH as a result of the addition of acid or base. The use and preparation of buffers is a key tool in the BioSciences industry: buffers are used to stabilize solutions of proteins, carbohydrates, in microbial fermentation’s and for many other uses. Mathematically, the Henderson-Hasselbach equation is used to describe the relationship between pH, the concentration of acid and conjugate base and the pK_a:

                                                                 [Conjugate base]

pH = pK_a  +  log( --------------------------)

                         [Acid]

The dissociation of an acid can be described as:

 AH  Û  H+   +   A^-

And this balance is described as:

                                                                [H+]  [A-]

K_a =   -----------------

                                                                    [HA]

where K_a is the “dissociation constant”.

                                    and:

     pK_a  = -log(K_a)

 The pK_a  is critical, since it  represents the point at which 50% dissociation occurs,  (i.e. half H+, half A^-), so the addition of more acid or base has the least effect.  This represents the “buffering range” of the acid.   When a “Titration Curve” for an acid is plotted (by measuring pH as base is added to an acid solution) the following events occur:

·        Initially the acidic (HA⁺) state predominates, and there is a net positive charge. The pH value gradually increases as more base is added.

·        As the pK_a is reached there is a period where little or no change in pH is seen. This is represented by a flat area on the titration curve, and represents the “buffering capacity” of the acid.

·        As more base is added, the (A^-) state predominates and the pH increases.

 Objective: In this lab you will prepare four acetic-acid / sodium acetate buffer / solutions  and determine the pK_a values by plotting titration curves (measured pH vs. ml of base added) for each buffer solution.

 Method:

 1. Prepare four tubes with the following concentrations of acid and base:

 2. Measure the pH for each buffer mixture.

 3. Titrate each buffer with 0.2M NaOH.  At regular intervals (0.5 pH units) measure and record pH as well as the amount of base added. Record this information in tabled format:

Results:

1.      Plot each titration curve on a graph (measured pH vs. ml NaOH added).

2.      Identify the pK_a for each buffer preparation (where possible).

3.      Draw the predominant ionic state for each area of the curves (+), (-),  or (0).

 Questions:

1.      Which buffer system offers the greatest buffering capacity and why?

2.      Identify three alternative buffer systems common to research / industry.

Laboratory 2

  Saponification of Triglycerides

Introduction

 Triglycerides are composed of three fatty acids linked to glycerol by fatty acyl esters (-O-CO-R). The fatty acids may be saturated (no C=C double bonds) or unsaturated.   Liquid triglycerides are oils, while solid triglycerides are fats. 

 Saponification: By heating a triglyceride in aqueous potassium hydroxide (KOH) the fatty acyl esters can be cleaved off (hydrolysis) leaving behind glycerol and the potassium salt of the fatty acid. The process is called “saponification” (or soap formation) since the potassium salts of fatty acids are in fact “soaps”.

The “saponification number” is used as an indicator of fatty acid chain length in triglycerides. The value is simply a measurement of the ml of KOH required to complete the hydrolysis of one gram of fat or oil.

Triglycerides containing long fatty acids will have a lower saponification number than triglycerides with shorter fatty acids. 

Objective:

            In this laboratory you will evaluate one of several triglycerides to determine the saponification number.

Method:

1.      Select one of the following: margarine, corn oil, sunflower oil, lard, or linseed oil.

2.      Place 1.0 g of the sample triglyceride in to a small beaker and dissolve in 4 ml of solvent (solvent is 1:1  ethanol / ether).

3.      Transfer dissolved triglycerides to a small distillation flask and wash the beaker twice with 1 ml of solvent (1:1 ethanol/ether) to collect all residual material. Add the “wash” to the distillation flask.

4.      Add 25 ml of 0.5M KOH /ethanol solution WARNING: KOH is a very strong corrosive agent and can cause serious burns on contact. Use appropriate eye / hand protection during this procedure.

5.      Measure the exact volume of your mixture.  Set up a second system as a “Control” (or reference) with 25 ml of the 0.5M KOH/ethanol solution plus additional 1:1 ethanol/ether solvent for a final volume identical to your experimental solution.

6.      Set up a reflux condenser on each flask and place in boiling water bath for 30 minutes. The hydrolysis will occur during this period.

7.      Allow flasks to cool.  Add three drops of indicator solution (phenolphthalein, 10 g/L) to both flasks and titrate with 0.5M HCl solution.

8.         The molar difference between the amount of 0.5M HCl  required to neutralize the “Control” and the amount of HCl required to neutralize the test sample equals the amount of 0.5M KOH used in the saponification process.

Results:

1.      Calculate the weight (mg) of KOH used to saponify the 1 g  sample.  Determine the saponification number for tested samples. One approach would be to use: ( ml HCl x 0.5 M x F.W. KOH).

2.      Obtain the final results from the other groups for each sample and prepare a table to summarize the results.

 Questions:

1.      What is the relationship between saponification and phase (liquid / solid) of a triglyceride.

2.      Why do triglycerides with longer fatty acids have a lower saponification number than those with shorter fatty acids?

3.      Why is the difference in the molar amount of HCl used to neutralize the control and the amount of HCl used to neutralize the sample equivalent to the molar amount of KOH used to saponify the test sample?

4.      Why do soaps disperse grease?

 Laboratory 3

 Iodine Number of Triglycerides

Introduction:

                 Fatty acids (component of triglycerides) can be saturated (no C=C double bonds) or unsaturated (C=C double bonds are present). Treatment of a triglyceride (or fatty acid) with a halogen will result in the addition of the halogen at the C=C double bond site: 

HC=CH  + HI  ®   HIC-CH₂

The result is simple:  more double bonds mean more iodine (or other halogens) will be absorbed.  The Iodine Number is the number of grams of iodine absorbed by 100 g of fat or oil.

 Objective:

                 The objective of this laboratory is simple: to determine the iodine number for a test sample of fat or oil.

 Method:

 1.      Select one of the following: margarine, corn oil, sunflower oil, lard, or linseed oil.

2.      Dry two Erlenmeyer flasks by rinsing with ethanol and then rinsing with chloroform. The flasks are for the sample and control. Label each flask.

 3.      Weigh out 0.5 g of the selected test fat /oil into the “test” flask. Accurately measure the precise weight of the sample.

 4.      Add 10 ml of chloroform to both flasks.

 5.      Add 25 ml 0f 0.2M Iodine monochloride (ICI) to each flask. Shake each flask and place in dark (lab cabinet) for 1 hour. During this incubation period the iodine will add to the C=C double bonds in the unsaturated fatty acids.

6.      Add 50 ml of distilled water to each flask. Add the water to rinse the walls of the flask and the stopper.

 7.      Add 10 ml of potassium iodide (KI) solution (100 g/L). This will react with the iodine     monochloride  (ICI) to produce “free” iodine and hydrogen chloride.

 i.e.    KI  +  ICI  ®  KCl  +  I₂

8.      Measure the amount of free iodine in the “test” and “control/blank” solutions. The measurement is conducted by titration with 0.1 M sodium thiosulfate (Na₂S₂O₃). The reaction of the titration is:  

2 Na₂S₂O_{3 +} I₂   ®   Na₂S₄O₆  + 2 NaI

9.      At the end point of the titration the solution will turn a clear straw-colour. At this point, add 1ml of 10 g/L starch solution. The solution will turn blue.

10.  Continue titrating (with continuous mixing) until the solution clears.

11.  The amount of free iodine produced is equal to amount of iodine initially added as iodine monochloride (ICI) minus the amount of iodine reacting with the sample. Calculate the amount of iodine added as iodine monochloride and you should measure this value in the blank.

12.  The calculation of the iodine number is as follows:

                                                         Volume Titrant Control - Volume Titrant Sample

                             Iodine Number  = -----------------------------------------------------

                                                                                          0.5

Results:

1.      Calculate the iodine number for your sample. 

2.      Obtain the final results from the other groups for each sample and prepare a table to summarize the results.

 Questions:

 1.      What is the role of starch in the titration?

2.      What is the practical use of the iodine number?

3.      How do saponification number and iodine number help characterize a commercial triglyceride product?

4.      Why not titrate with sodium hydroxide?

5.      What other ways are there of characterizing triglycerides?

Laboratory 4

  Carbohydrate Testing

Introduction:

 There are simple colourmetric tests which have been developed for a variety of carbohydrates. This laboratory exercise will examine three such tests: Benedict’s test, Barfoed’s test, and the Iodine test.

Objective:

             In this laboratory you will characterize three unknown carbohydrates after first performing the above tests on known carbohydrate samples and determining the results.

 Method:

 1.      You will be given three known samples:   glucose,  sucrose, and starch.  Perform the following tests on all three samples and carefully record the results.

Benedict’s Test: place 5 ml of Benedict’s reagent in each of three tubes. Add 1 ml of each known carbohydrate solution (20 mg/ml). Place in boiling water bath for three minutes.

Barfoed’s Test: place 4 ml of Barfoed’s reagent into a series of three tubes. Add 1 ml of each test carbohydrate (20mg/ml). Place in boiling water bath and note time of colour reaction over a 15 minute period. (Time is the important factor)

Iodine Test: place 5ml of each test carbohydrate solution (20mg/ml) into a separate tube. Add four drops of 2% iodine solution and mix. Note colour reaction.

2.      Repeat the above test procedures for the three unknown samples.

  Results:

 1.      Prepare a table of results for the three known samples, detailing the reactions. (You will use your actual results on your control samples to characterize your unknown).

2.      Prepare a table for the three unknown samples and identify them using your table of results.

 Questions:

1.      Describe four alternative carbohydrate test procedures.

Laboratory 5

Hydrolysis of Starch by Amylase

Introduction:

             Starch is a polysaccharide made of repeating glucose units that are linked by a(1®4) bonds.  Each repeating disaccharide (two sugar) unit is “a-amylose”.   As well, there are branches in starch molecules caused by random formation of a(1®6) bonds. 

            When starch is consumed, it is broken down into progressively smaller units, eventually becoming “maltose” (the two glucose molecule linked by the a(1®4) bond) and “glucose”.  This hydrolysis of starch is brought about by the enzyme amylase.

             Saliva contains a-amylase, which randomly cleaves starch into smaller residues. The pancreas secretes b-amylase which cleaves starch residues into maltose, maltotriose (three glucose molecules linked a(1®4)) and dextrin’s (which contain branched residues). 

 Objective:

             In this laboratory we will examine the kinetics of a-amylase as found in saliva.

 Method:

 1.      Prepare a stock solution of starch containing 1.0 mg/ml in 100 ml of distilled water.

 2.      Preparation of Standard Curve of absorbance (@  590 nm) vs. concentration of a starch/iodine mixture:

i. The standard curve will use different concentrations of starch

    (0.01 mg/ml – 1.0 mg/ml) mixed with iodine solution and distilled water.

ii. The volumetric proportions of the starch / iodine / water solution will be 8:1:1, (with the starch being added at different appropriate concentrations).

iii. Preparation of Starch Solutions: from the stock solution (1.0 mg/ml):       prepare dilutions of 0.01, 0.025, 0.05, 0.1, 0.3, 0.5, 0.7, and 1.0 mg/ml from the starch stock solution. You will require two sample at each starch concentration of at least 8 ml.

iv. Preparation of Iodine Solution: add 5 g potassium iodide to 100 ml water. Once the potassium iodide has dissolved, add 1 g of iodine and stir until completely dissolved.       

v.       Mix each standard curve sample (starch dilution, iodine and distilled water) in the volumetric proportions of: 8:1:1.

vi.     Perform a wavelength scan of the absorption spectrum to determine the lmax (absorption maximum wavelength, in nm) for the iodine-starch mixture.   This wavelength will be used for all readings.

3.      Hydrolysis of starch: prepare 10 ml of saliva.  You will determine the rate of hydrolysis of starch by applying a constant volume of saliva while varying the starch concentration.

 i.         To a series of tubes containing 8 ml of the various starch concentrations (0.01, 0.025, 0.05, 0.1, 0.3, 0.5, 0.7, and 1.0 mg/ml starch) add 1 ml of saliva.

ii.       Incubate each sample @ 35°C for 10 minutes.

iii.      After 10 minutes incubation (and hydrolysis) add the iodine solution. Place the tubes in an ice bath to stop the hydrolysis.

iv.     Measure the absorbance of each tube.

 Results:

 1.      Calculate starch concentration for each sample after hydrolysis (C_E) through use of the standard curve. The initial starch concentration (C₀) is already known.

2.      The velocity (rate of digestion) of the reaction for each sample can be calculated as:

V  =   DC/Dt   =  (C₀  -  C_E)  / 10 minutes

3.      Prepare a table showing rate of hydrolysis (V) at different the starch concentrations.

4.      Prepare a graph of 1/starch concentration (x-axis) vs. 1/rate of digestion (y-axis). This type of reciprocal graph displaying enzyme kinetics is a Lineweaver-Burke plot. 

The y-intercept of the Line-weaver Burke plot is the reciprocal of the maximum velocity of the reaction (V_max).  The x-intercept  is the negative reciprocal of the Michaelis constant. (K_m).

Questions:

 1.      Why is a-amylase in saliva when the bulk of digestion is later in the digestive system?

2.      What is significance of K_m and V_max?

3.        Would the saliva of a vegetarian or primary meat-eater contain more a-amylase?

4.        What is difference between an a(1®4) and a(1®6) linkage?

Laboratory 6  

Selective Precipitation / Fractionation

Laboratory 7

Determination of Protein Concentration

Introduction:

             Determination of protein in an unknown sample is a common analytical procedure. In the case of relatively pure protein solutions it may be determined indirectly, through measuring absorbance at 280 nm (A₂₈₀) and dividing by the “extinction coefficient” to get an approximate protein concentration. This provides an approximate value but if a higher degree of accuracy is required, or if there are a mixture of proteins present then a more precise method is required.  

A simple analytical method for measuring total protein is the Folin-Lowry method. This method relies on the formation of a colour due to the reaction of the alkaline copper with the protein and the reduction of phosphomolybdate by tyrosine, cysteine, and tryptophan present in the protein. The overall intensity of colour produced depends on the amount of these aromatic amino acids present and thus will vary for different proteins.

Objective:

            The objective of this research is to use the Folin-Lowry assay to determine the protein concentration in an unknown sample, after first preparing a standard curve using a standard protein (albumin).

Method:

1.        Prepare the following materials:

i. alkaline carbonate solution (20 g/L Na₂CO₃) in 0.1M NaOH.

ii. Copper-sulfate-sodium tartrate solution (5g/L CuSO₄^.5H₂O)

iii. Alkaline Solution: prepare fresh on day of use: 50 ml of (i) and 1 ml of (ii)

2.        Folin-Ciocalteau reagent: dilute the commercial reagent 1:1 with water on the day of use. (This solution contains sodium tungstate and sodium molybdate in phosphoric and hydrochloric acids.)

 3.        Standard Protein: prepare an albumin solution of 0.2 mg/ml. You are to prepare a series of dilutions of the “standard proteins” that will allow you to plot an accurate standard curve (i.e. prepare a series of dilutions from 0.001 to 0.2 mg/ml)

 4.         Protein Testing: Add 5 ml of the “Alkaline solution”  to  1 ml of the test mixture. Mix thoroughly and allow to stand at room temperature for 10 minutes or longer.  Add 0.5 ml of the Folin-Ciocalteau reagent rapidly with immediate mixing.  Repeat this procedure for  all samples in the standard curve.

 5.        After 30 minutes read the absorbance at 750 nm (A₇₅₀) against an appropriate blank.     

 6.        Repeat the procedure in (4.) for your unknown sample(s).

 Results:

 1.        Plot a standard curve of A₇₅₀ vs. standard protein concentration.

 2.        Use the A₇₅₀ readings obtained for your unknown(s) to estimate protein concentration.

 Questions:

 1.        Under what conditions would this methodology not be appropriate?

 2.        Describe (in brief) three alternative methods of determining protein concentration.

 3.        What is an “appropriate blank” and why?