Biochemistry Lab Protocol

Experiment No: Page No:
Biochemistry Laboratory (FT 491)

Department of Food Technology
Vision: Department of Food Technology envisions to be a lead centre of academic and research resource, for
creating competent professionals to ensure global food security and safety.
Mission:
i. To impart quality education through excellent Teaching Learning Process and infrastructural facilities for
producing globally competent food technologists capable of extending technological services.
ii. To inculcate/imbibe qualities of lifelong learning, team spirit, leadership, entrepreneurship and
communication skills with social responsibilities.
iii. To engage and promote cutting edge research practices through multi-institutional and industry institute
collaborations.
Vision: Department of Food Technology envisions to be a lead centre of academic and research resource, for creating competent professionals to
ensure global food security and safety.
Details:
Name of the Student:
Roll No:
Registration No:
Academic Year: 20 - 20
Laboratory Schedule:
Day Time Room No. Group
Laboratory Faculty/Instructor:
Faculty I
Faculty II
Technical Assistant I
Technical Assistant II
Signature of Student:
Signature of the Faculty(s): Signature of the Technical Assistant(s):
Read the following instructions carefully
Dos & Don’ts:
1. Wear an apron.
2. Be familiar with your lab assignment before you come to lab. Follow all written and verbal instructions
carefully. Observe the safety alerts in the laboratory directions. If you do not understand a direction or
part of a procedure, ask the teacher before proceeding.
3. When entering the lab/classroom, do not touch any equipment, chemicals, or other materials without
being instructed to do so. Perform only those experiments authorized by the instructor.
4. No student may work in the laboratory without an instructor present. Work only with your lab partner(s).
Do not venture to other lab stations for any reason.
5. Do not wear bulky or dangling clothing.
6. Never eat or drink in the laboratory. Don't chew on the end of a pen which was lying on the lab bench.
7. Wash acid, base, or any chemical spill off of yourself immediately with large amounts of water.
8. If chemical substances get in your eye, wash the eye out for 15 minutes. Hold your eye open with your
fingers while washing it out.
9. If you take more of a chemical substance from a container than you need, you should not return the
excess to the container. This might cause contamination of the substance remaining. Dispose of the
excess as your teacher directs.
10. When weighing never place chemicals directly on the balance pan. Never weigh a hot object.
11. Never smell anything in the laboratory unless your teacher tells you it is safe. Do not smell a substance
by putting your nose directly over the container and inhaling. Instead, waft the vapors toward your nose
by gently fanning the vapors toward yourself.
12. Do not directly touch any chemical with your hands. Never taste materials in the laboratory.
13. If you burn yourself on a hot object, immediately hold the burned area under cold water for 15 minutes.
Inform your teacher.
14. Observe good housekeeping practices. Work areas should be kept clean and tidy at all times. Only lab
notebooks or lab handouts should be out on the table while performing an experiment. Books and book
bags should not be on the lab table.
15. Always replace lids or caps on bottles and jars.
16. If your Bunsen burner goes out, turn the gas off immediately.
17. Constantly move a test tube when heating it. Never heat a test tube that is not labeled Pyrex and never
point the open end at anyone.
18. Always add acid to water and stir the solution while adding the acid. Never add water to an acid.
19. Report all accidents to your teacher.
20. Absolutely no running, practical jokes, or horseplay is allowed in the laboratory.
21. Thoroughly clean your laboratory work space at the end of the laboratory session. Make sure that all
equipment is clean, and returned to its original place.
Lab Safety Protocols:
1. Familiarize yourself with the location of safety equipments in the lab (e.g., eye-wash station, fire
extinguisher, biological safety cabinet, first aid kit, gas valve, etc).
2. In case of accidents keep calm, prioritize your own safety first and alert the authorities immediately.
3. In case of chemical spills identify the area of the chemical spill and inform your laboratory co-workers of
the spill. Evacuate the location and areas surrounding the spill if the substance is hazardous.
4. Chemical spills onto the body wash off all chemicals spilled on a body immediately using a safety shower
for at least 15 min. If clothes are saturated with spilled chemical, remove clothing immediately.
5. If the spill splashed into eyes, use an eyewash right away for at least 15 min. Open the eyes to allow
complete washing. Only attempt to remove contact lenses after eye washing has commenced.
6. If the spilled chemical is a strong acid, wipe out the residues first before washing to avoid excessive or
painful burning.
7. Remove contaminated clothing immediately to avoid further exposure to chemicals.
8. Call local responders or EHS for emergency assistance and alert people in the vicinity of the spill.
9. In case of a lab fire or explosion, ensure your safety first and call emergency responders immediately for
help. Evacuate the building safely and pull fire alarms or notify nearby people, if possible.
10. Don't use elevators. Use stairs and locate the nearest exit.
11. If possible, shut down the electric power before evacuating.
12. Use a wet towel to cover the mouth and nose, if there is heavy smoke.
13. In case of a small fire, use a proper fire extinguisher and make sure an easy exit is available if you fail in
extinguishing the fire.
14. In case of a fire involving an individual's clothing, do not run since it might accelerate the fire.
15. Stop, drop onto the ground with hands covering the face, and roll to extinguish the fire. If possible, use
the water to extinguish the fire.
16. Be safe first and then help others if possible.
17. Be aware of re-entering the affected area a second fire or explosion might follow.
18. In case of personal injuries or sudden deterioration of health of a person assess the situation before taking
any actions.
19. Ask the person what happened to them first, if they are conscious. Look for possible signs of injury if the
person is unconscious and/or unresponsive.
20. Call local emergency responders immediately if the person is in danger.
21. Don't move the injured personnel unless imminent danger is present.
22. If an individual has received an electrical shock, shut down the power first, if possible. Do not touch the
person with bare hands. Use non-conductive material such as wood, glass, or rubber to pull the person
away from the electric contact.
23. If bleeding from minor cuts, flush with water to avoid contamination and treat with first aid supplies. If
cuts are more serious, call for medical assistance.
24. Initiate first aid to help, if possible.
Syllabus
1. Separation of amino acids/sugars by Ascending Paper Chromatography.
2. Separation of sugars/amino acids by Thin Layer Chromatography.
3. Separation and isolation of proteins/amino acids by Paper Electrophoresis.
4. Determination of BOD5 and COD of a sample of waste water.
5. Preparation of cell-free extract: Bacterial cell by sonication, Chicken liver by
homogenization.
6. Assay of enzyme activity — (a) Phosphatase assay [Chicken liver] (b) Protease
assay
7. Study of an enzymatic reaction.
Course Outcomes:
1. Identify biomolecules ( sugar, aminoacid) relevant to food composition
2. Distinguish biomolecules from their mixture of similar compunds
3. Assess pollution level in any sample in terms of BOD and COD
4. Demonstrate methodology for isolation, recovery and activity - determination
of biocatalyst
Contents Page:
Exp Name of Experiment Date of Date of Marks (40)* Signature of
No. Performance Submission evaluator
1 Separation of amino acids/sugars by
Ascending Paper Chromatography
2 Separation of sugars/amino acids by
Thin Layer Chromatography.
3 Separation and isolation of

proteins/amino acids by Paper
Electrophoresis
4 Determination of BOD5 and COD of
a sample of waste water.
5 Preparation of cell-free extract:
Bacterial cell by sonication, Chicken
liver by homogenization
6 Assay of enzyme activity — (a)
Phosphatase assay [Chicken liver] (b)
Protease assay
7 Study of an enzymatic reaction
* Observation 10; Result & Discussion 10; Troubleshooting & Precautions 5; Pre-Experiment Evaluation + Post Experiment
Evaluation/Assignment Question 15.
Feedback on the Course Outcomes based on each Experiment: Score between 1-3 (3 – Highest;
2 – Medium; 1 – Lowest)
Exp No. CO1 CO2 CO3 CO4 CO*

1
2
3
4
5
6
7
* Average score of all the COs.
Rubrics: Rubrics for Lab Performance Assessment
Each week, students will be assessed on their participation and performance in lab. The points each week will be
totalled and combined with other assessments/report writing.
Objectives:
Outcomes:
Overall Lab Performance (End of the Semester)

Lab participation – 40% of internal marks (i,e 16)
Assignments/Report Submission –60% of internal marks (24)
A: Lab performance is excellent with the majority of assessments rated as proficient. The student has
attended all labs.
B: Lab performance is good with most assessments at the adequate level (with no more than 2 substandard)
or above. At most, the student has one lab absence.
C: Lab performance is fair with most assessments at the Adequate and Substandard levels.The student may
have been absent 2-3 times.
D: Lab performance is barely adequate with less than half of assessments at the Adequate level or higher.
The student may have been absent up 40% of total experiments.
E: Lab performance is not sufficient to pass since 50% of assignments were not completed (or
unacceptable) and/or the student missed more than 50%
Quality/Score Excellent Good Fair Poor Marks

Criteria (4) (3) (2) (1)
Lab Participation Student demonstrates an Student Student Student

(Following accurate understanding arrives on unpreparedn was absent
Procedure +Lab of the lab objectives and time to lab, ess makes it from lab
Techniques+ concepts. The student but may be impossible or did not
Subject can correctly answer underprepar to fully participate
Knowledge + questions and if ed. participate. . There
Contribution) appropriate, can explain Answers to If able to was no
concepts to fellow questions participate, attempt to
classmates. Student is are basic student has make prior
eager to participate and and difficulty arrangeme
assists when needed. superficial explaining nts to
suggesting key lab make up
that concepts. the lab.
concepts
are not fully

grasped.
Lab Report Student demonstrates an Student has Student has Student
accurate understanding of a basic problems turns in
the lab objectives and knowledge with both lab report
concepts. Questions are of content, the graphs late or the
answered completely and but may and the report is so
correctly. Graphs are neat,
lack some answers. incomplet
creative and include
understandi Student e and/or so
complete titles and accurate
ng of some appears to inaccurate
units. Errors, if any are concepts. have not that it is
minimal Questions fully unaccepta
are grasped the ble
answered lab content
fairly well and the
and/or graph(s)
graphs possess
could have multiple
been done errors
more
neatly,
accurately
or with
more
complete
information
Interaction with Very good participation Good Minimal No
Group (Team with a good leadership participatio participatio participati
work) quality; is respectful of n; appears n; Shows on; sits on
others and their point of interested; little the
view; makes sure that enthusiastic interest; sidelines
everyone gets a turn; but talks doesn't pay with no
conscious of time over attention to interaction
teammates; other group ;
try to help members; disinterest
group may argue ed;
complete to get point No stake
tasks; across; in time
somewhat helps group manageme
conscious only when nt
of time asked; little
emphasis on
time
Safety Proper safety Proper Proper Proper

precautions are safety safety safety
consistently followed. precautions precautions precaution
are are often s are
generally missed, consistentl
followed y missed;
Seldom
warned
Cleaning/Rearra Placed all the Proper Needs to be Proper
nging after components as per clean-up reminded clean-up
experiment instruction after procedures more than procedure
experiment; Keep generally once during s are
experiment station followed. the lab to seldom
generally neat and clean Station use proper used.
Consistently uses proper generally clean-up
clean-up procedures; left clean. procedures. Requires
Reminds others of their May need other’s
responsibility. reminding help to
occasionall complete
y. clean-up
Note: This document is a general guide line for a Particular Laboratory course. It will be kept with the
Laboratory manual. Teacher will complete the Annexure after evaluation of each and every laboratory
report and to be kept with student Laboratory file
*Attach one copy of the rubrics sheet before beginning of each experiment
Experiment No. 1.
IDENTIFICATION OF AMINO ACIDS BY ASCENDING PAPER
CHROMATOGRAPHY
Pre-experiment Evaluation Questionnaire (MCQ):

1)Name three essential amino acids.
2) What is general formula of an amino acid? What is a peptide bond?
3) Name some common separation processes for soluble/insoluble components.
4) What do you mean by partition phenomena?
Experiment Protocol:
Aim: To identify unknown amino acid(s) in a sample
Theory: Chromatography is a major technique of separation and estimation of components of a sample. All
chromatographic techniques have two phases-a stationary phase and a mobile phase.In paper chromatography,
when a strip of filter paper is suspended vertically with the lower end dipping into a mixture of water and organic
solvent, such as n-butyl alcohol, n-propyl alcohol, phenol etc; the mixture of water and organic solvent moves up
the filter paper. Cellulose in the form of paper sheets makes an ideal support medium where the water is adsorbed
and strongly held between the cellulose fibers and thus the hydrated cellulose forms a stationary hydrophilic
solvent phase; while the organic solvent for which the paper has little attraction represents a mobile phase. The
paper and solvent mixture are enclosed in a covered glass cylinder or other container so that evaporation loss does
not occur; and the temperature is maintained constant, so that the solvent moves up the paper in an atmosphere
saturated with the vapour of both water and organic solvent at the temperature used. The solute is spotted on the
paper close to the end dipping in the solvent, dried and the chromatogram develops as the solvent moves up the
strip and over the solute by capillary action. In this process, the solute gets partitioned between the water and
organic phase. This is known as Ascending Paper Chromatography. The solvent is allowed to run almost till the
upper end of the paper (1cm below the upper end),the solvent front marked after drying the paper and the positions
of the compounds present are visualized by using a suitable staining reaction.
However, when the strip of filter paper is suspended vertically with its upper end dipping into a mixture of water
and an organic solvent kept in a boat or a trough mounted on the glass chamber, the mixture of water and organic
solvent moves down the filter paper carrying with it the sample spotted close to the solvent dipped end, it is known
as Descending Paper Chromatography.
Under a given set of carefully controlled conditions like temperature, nature of solvent system, solvent
concentration and pH, the movement of a particular component in a mixture with respect to the movement of the
solvent front will remain constant. The relative movement of the solute w.r.t the solvent is expressed as retardation
factor Rf.
Rf = Distance covered by solute/distance covered by solvent; varies from 0 to 1. Rf values for a given component
and solvent system are used for the identification and characterization of the components of a sample.
The amino acid dissolved in a solvent and spotted on the paper and run. The spots are then visualized by ninhydrin
staining reaction. Ninhydrin (2,2-dihydroxyndan-1,3-dione) is a powerful oxidizing agent and oxidatively
decarboxylates α-amino acids to carbon dioxide, ammonia and RCHO with 1C less, than the parent amino acid.
The reduced ninhydrin then reacts with the liberated NH3, forming a blue to purple complex that maximally
absorbs light of λ=570nm.This purple color is given by all amino acids and peptides having a free α-amino group;
whereas praline and hydroxyproline, in which the α-amino group is substituted, yield derivatives with a
characteristics yellow color. This blue color forms the basis of a quantitative test for α-amino acid and can detect
amino acid as little as 1µg.However,only qualitative detection is attempted in this experiment.
Apparatus/Equipments:
Chromatographic chamber, measuring cylinder, spraying bottle, capillary tube, Hot air oven, Whatman 1
Chromatography paper
Material & Methods:

Materials/reagents – Solvent mixture (Butanol: Glacial acetic acid:water = 4:1:1)
ninhydrin, unknown amino acid, standard amino acid
The papers are cut into appropriate size depending on the chamber size.
1. The paper is marked where the sample has to be spotted; usually 1cm above the bottom end of the
paper.
2. Spot around 20µl of the amino acid solution with a thin capillary on the sheet. Dry the spot in a current
of hot air using hair dryer before respotting. Care should be taken to avoid spreading of the sample
spot to prevent tailing.
3. Spot all amino acids side by side in a straight line leaving sufficient gap in between.
4. Place the paper in the chamber previously saturated with the mobile phase (for atleast 30min).Care
should be taken to see that the sample spots do not dissolve in the solvent while placing the paper. The
solvent level in the chamber should be below the sample spots.
5. The solvent is allowed to run till it reaches 1cm below the top end of the paper.
6. Remove the paper, mark the solvent immediately and air dry the paper rapidly.
7. Spray ninhydrin reagent on the paper using the spray bottle and allow the spots to develop in the oven
at 105°C for 2-3min.
8. Note the color of each amino acid and calculate Rf value for each amino acid spotted.
Note:
Iso-propanol or a 1:1 mixture of acetone/butanol may be used in lieu of ethanol in preparing the ninhydrin reagent
Observation:
Result & Discussion:
Troubleshooting & Precautions:
(Attach extra sheets for observation, result & discussion and troubleshooting & precautions)
Post- experiment Evaluation.

1) What precautions did you take while performing the experiment?
2) Why do we saturate the chamber before the experiment?
3) Can retardation factor (Rf) be more than one? Justify.
Evaluators Remarks:
Experiment No. 2.
IDENTIFICATION OF SUGARS BY ASCENDING PAPER CHROMATOGRAPHY

1) Write 3 main properties of carbohydrate.
2) What is a reducing sugar? Name one disaccharide which is a reducing sugar and is available in common food.
3) What are the functional groups present in sugar?
Aim: To identify unknown sugar compound(s) in a sample
Theory: Chromatography is a major technique of separation and estimation of components of a sample. All
chromatographic techniques have two phases-a stationary phase and a mobile phase.
In paper chromatography, when a strip of filter paper is suspended vertically with the lower end dipping into a
mixture of water and organic solvent, such as n-butyl alcohol, n-propyl alcohol, phenol etc; the mixture of water
and organic solvent moves up the filter paper. Cellulose in the form of paper sheets makes an ideal support
medium where the water is adsorbed and strongly held between the cellulose fibers and thus the hydrated cellulose
forms a stationary hydrophilic solvent phase; while the organic solvent for which the paper has little attraction
represents a mobile phase. The paper and solvent mixture are enclosed in a covered glass cylinder or other
container so that evaporation loss does not occur; and the temperature is maintained constant, so that the solvent
moves up the paper in an atmosphere saturated with the vapor of both water and organic solvent at the temperature
used. The solute is spotted on the paper close to the end dipping in the solvent, dried and the chromatogram
develops as the solvent moves up the strip and over the solute by capillary action. In this process, the solute gets
partitioned between the water and organic phase. This is known as Ascending Paper Chromatography. The solvent
is allowed to run almost till the upper end of the paper (1cm below the upper end),the solvent front marked after
drying the paper and the positions of the compounds present are visualized by using a suitable staining reaction.
However, when the strip of filter paper is suspended vertically with its upper end dipping into a mixture of water
and an organic solvent kept in a boat or a trough mounted on the glass chamber, the mixture of water and organic
solvent moves down the filter paper carrying with it the sample spotted close to the solvent dipped end, it is known
as Descending Paper Chromatography.
Under a given set of carefully controlled conditions like temperature, nature of solvent system, solvent
concentration and pH, the movement of a particular component in a mixture with respect to the movement of the
solvent front will remain constant. The relative movement of the solute w.r.t the solvent is expressed as retardation
factor Rf.
Rf = Distance covered by solute/distance covered by solvent; varies from 0 to 1
Rf = values for a given component and solvent system are used for the identification and characterization of the
components of a sample.
The retention of a compound often differs considerably between experiments due to variations of the eluent, the
stationary phase, temperature, sample matrix and the setup. It is therefore important to compare the retention of
the test compound to that of several standard compounds under absolutely identical conditions. Rf value for a
given component and solvent system are used for the identification and characterization of the components of a
sample.
1. Chromatography chamber
2. Oven at 105deg C
3. Hair dryer
4. Spray bottle
5. Glass capillary
6. Whatman 1 Chromatography paper
Material/reagents & Methods:

1. Solvent mixture (Butanol: Glacial acetic acid:water = 4:1:1)
2. H2SO4 solution ( sulphuric acid: methanol :acetone – 1:8:1)
Procedure:
1. The papers are cut into appropriate size depending on the chamber size.
2. The paper is marked where the sample has to be spotted; usually 1cm above the bottom end of the paper.
3. Spot around 20µl of the sugar solution with a thin capillary on the sheet. Dry the spot in a current of hot air
using hair dryer before respotting. Care should b taken to avoid spreading of the sample spot to prevent tailing.
4. Spot all sugars side by side in a straight line leaving sufficient gap in between.
5. Place the paper in the chamber previously saturated with the mobile phase (for atleast 30min).Care should be
taken to see that the sample spots do not dissolve in the solvent while placing the paper. The solvent level in the
chamber should be below the sample spots.
6. The solvent is allowed to run till it reaches 1cm below the top end of the paper.
7. Remove the paper, mark the solvent immediately and air dry the paper rapidly.
8. Spray H2SO4 solution on the paper using the spray bottle and allow the spots to develop in the oven at 67°C
for 2-3min.
9.Note the color of each sugar and calculate Rf value for each sugar spotted.
Observation:

1) Which property of carbohydrate you have written early before the experiment helped you to spot it during the
experiment?
2) Why did we saturate the chamber before the experiment?
3) What is the major precaution to take in this experiment?
Evaluators Remarks:
Experiment No. 3
IDENTIFICATION OF SUGARS BY THIN LAYER CHROMATOGRAPHY

1. Name two sources of mono-saccharides and di-saccharides in our regular diet.
Aim: To identify unknown sugar compound(s) by thin-layer chromatography
Theory: Separation of compounds on a thin layer is similar in many ways to paper chromatography but has the
added advantage that a variety of supporting media can be used so that separation can be by adsorption and
partition chromatography depending on the nature of the medium employed. The method is very rapid and many
separations can be detected at a lower concentration than on paper, as the spots are very compact.
1. Chromatography chamber 2. Oven at 105deg C 3. Hair dryer 4. Spray bottle 5. Glass capillary 6. Whatman 1
Chromatography paper
Reagents/Material & Methods: Silica gel, ethanol, standard sugar solution, unknown sugar solution, Solvent
mixture (Butanol: Glacial acetic acid: water = 4:1:1), H2SO4 solution (sulphuric acid: methanol :acetone – 1:8:1)
1. Clean the glass plate with ethanol.
2. Prepare a slurry of silica gel (40% w/v of silica gel G in water)
3. Pour silica slurry on glass plate and spread uniformly using a glass rod.
4. Activate the plate in the oven at 105°C for at least 30min to 1hour and allow to cool in air.
5. Spot around 20µl of the sugar solution with a thin capillary and dry the spot using hair drier.
6. Place the plate in the chamber saturated with the mobile phase and allow the solvent to run almost to
the top of the plate.
7. Remove the plate, immediately mark the solvent front.
8. Air dry the plate and develop the spots by spraying the sugar locating reagent using a spray bottle.
9. Heat the plate at 105°C for 2-3min in oven.
10. Note the color of the sugar spot and calculate Rf value for each sugar spotted.
Observation:

1) What precautions did you take while performing the experiment?
2) If during experiment you noticed that your friend has spilled conc H2SO4 in his hand, what you will do?
3) Compare the spots observed by paper chromatography and TLC.
Evaluators Remarks:
Experiment No. 4
IDENTIFICATION OF AMINO ACIDS BY THIN LAYER CHROMATOGRAPHY

1 Name two food sources from where a vegetarian can get proper amino acids.
Aim: To identify amino acid(s) present in sample by thin layer chromatography….
Theory: Separation of compounds on a thin layer is similar in many ways to paper chromatography, but has the
added advantage that a variety of supporting media can be used so that separation can be by adsorption, ion
exchange, partition chromatography or gel filtration depending on the nature of the medium employed. The
method is very rapid and many separations can be detected at a lower concentration than on paper, as the spots
are very compact.
Apparatus/Equipments: 1. Chromatography chamber 2. Oven at 105deg C 3. Hair dryer 4. Spray bottle 5. Glass
capillary 6. Whatman 1 Chromatography paper
Material & Methods:

Material/Reagents: Solvent mixture((Butanol: Glacial acetic acid:water = 4:1:1), ninhydrin, unknown amino acid,
standard amino acid
1. Clean the glass plate with ethanol.
2. Prepare a slurry of silica gel (40% w/v of silica gel G in water)
3. Pour silica slurry on glass plate and spread uniformly using a glass rod.
4. Activate the plate in the oven at 105°C for at least 30min to 1hour and allow to cool in air.
5. Spot around 20µl of the sugar solution with a thin capillary and dry the spot using hair drier.
6. Place the plate in the chamber saturated with the mobile phase and allow the solvent to run almost to the
top of the plate.
7. Remove the plate, immediately mark the solvent front.
8. Air dry the plate and develop the spots by spraying the sugar locating reagent using a spray bottle.
9. Heat the plate at 105°C for 2-3min in oven.
10. Note the color of the sugar spot and calculate Rf value for each sugar spotted.
Observation:

1) What differences did you notice between Paper Chromatography and TLC?
2) What is Rf and how does it help to identify the amino acids present in a sample?
Evaluators Remarks:
Experiment No. 5
TO DETERMINE THE COD OF A SAMPLE OF WASTE WATER

1) Define COD.
2) What is the requirement of COD determination?
3) How is it different from BOD?
Aim: To assess quality of water sample with respect to its organic and inorganic waste
Theory: COD or Chemical Oxygen Demand is an index of the organic content of water (oxygen demanding
substances in water) and is an important water quality parameter. It is defined as the amount of oxygen required
to oxidize all the organic material in a sample of impure water such as sewage effluent and is expressed in terms
of milligrams of oxygen required per liter of water, mg L-1.It is more scientific than the traditional empirical
concept of BOD. The test is based on the chemical oxidation of material in water by potassium dichromate in
acidic medium. The analysis of the impure water is done in parallel with a blank determination of pure, double
distilled water.
Most type of organic matters is destroyed by a boiling mixture of chromic acid and sulphuric acids. A sample is
refluxed with known amount of potassium dichromate and sulphuric acids. The excess dichromate is titrated with
ferrous ammonium sulphate. The amount of oxidizable organic matter is proportional to the amount of dichromate
consumed.
Unstable samples should be tested without delay sample containing settable solid should be homogenized by
suitable means for cause of representative sampling. Initial dilutions in volumetric flask should be made on those
samples having a high oxygen consuming value in order to reduce the error, which arises in measuring small
sample volumes. Chlorides are quantitatively oxidized by this method when silver sulphate is not used as a
catalyst. In this case, a correction should be applied
by determining chlorides on separate sample and subtracting the calculated oxygen consumption of the chlorides
from the result.Since,1mg/l of chloride will consume 0.23mg/l of oxygen the correction is 0.23X 1 mg/l chloride.
Equipments: Reflux apparatus consisting of a 300ml round bottom flask with ground neck 24/40 and a
Friedrich’s condenser.
1. Standard potassium dichromate solution (0.25 N)Dissolve 12.259gm potassium dichromate (previously
dried at 105degreeC for 2hrs.) in DW and dilute to 1000ml
2. Concentrated sulphuric acid
3. Standard ferrous ammonium sulphate hexahydrate solution, Mohr’s Salt(025N).Dissolve 98gm of Mohr’s
Salt in DW. Add 20ml concentrated sulphuric acid, cool and dilute to 1000ml.This solution must be
standardized against standard potassium dichromate.
4. Ferroin indicator solution:Dissolve 1.485gm of 1,10 phenanthroline monohydrate together with 0.695 gm
FeSO4.7H2O in DW and dilute to 100ml. Silver sulphate crystals.
Procedure:
1. Place a 50 ml sample or an aliquot diluted to 50ml with DW in a rod bottom flask(RBF)
2. Also run a sample blank taking 50 ml DW in a RBF.
3.Placing the RBF in an ice bath , add 25 ml standard dichromate solution and carefully add 75 ml of conc. H2SO4
down the sides of the flask mixing after each addition. Add glass beads or pumice stones to the reflux mixture to
prevent bumping during heating.
Caution: The reflux mixture must be thoroughly mixed before heat is applied. If this is not done local heating
occurs at the bottom of the flask and the mixture may be
Observation:

1. Questionnaire (MCQ)
2. Assignment
Evaluators Remarks:
Experiment No. 6 Determination of Biological Oxygen Demand of waste water sample

1) What is the need of dissolved oxygen for marine life? What is the DO level in water?
2) Comment on the nature of food-industry waste.
Aim: To assess quality of waste water with respect to organic waste.
Theory: Any marked increase in decomposable materials in a stream or lake is an indication of pollution . These
organic materials are consumed by various microflora naturally present in the stream. It has been found that as
microorganisms consume the materials, there is a proportional decrease in the amount of free oxygen present in
the stream. So the measurement of the oxygen consumption during a microbial degradation process gives an
indication of the amount of biodegradation material present. Thus the amounts of decomposable material may be
monitored at various stages of treatment of sewage by use of biochemical oxygen demand (BOD) test. BOD is a
water quality parameter for organic matter in water, which is empirical in nature.
BOD is a measure of the amount of oxygen necessary for the decomposition ort stabilization of a given amount
of waste by aquatic microorganism and is expressed quantitatively as parts of oxygen per million parts of sewage.
The BOD test consists of the incubation of a waste water sample in a sealed bottle followed by the titration of
residual oxygen in the bottles following incubation. From the difference between the amount of oxygen titrated
and that present in the original water (diluted or neat sample), the BOD of the waste water is calculated. This is a
semi quantative method of estimation.
The dissolved oxygen (DO) in the sample oxidizes MnSO4 to Mn(OH)2 which in turn oxidizes iodide to free
iodine in the acidic medium. The liberated iodine is then determined by titration. The method of determination of
amount of oxygen in water by iodometric titration was developed by winkler. The DO level in water in streams
and lakes is around 5 mg/L. for stagnant water such as from ponds, DO level may be 1-2 mg/L. normally domestic
sewage may have a 5-day BOD of around 200 mg/L and industrial sewage may have a BOD of several thousands
of mgs per liter.
• The method of estimation of BOD was developed abroad taking into consideration the fact that water
bodies are mostly at 20° C.
• The value of 5 days BOD at 20° C is to a reasonable extent comparable to that obtained at 4 days at 30°
C and 3 days at 35° C. for better accuracy of results, it is customary to estimate DO at a lower temperature
with longer period of incubation.
Reaction:
MnSO4 + 2 NaOH = Mn(OH)2 + Na2SO4
4 Mn(OH) 2 + O2 + 2H2O → 4 Mn(OH) 3 ↓ (brown)
4[( Mn(OH) 3 + I- + 3 H+ → Mn+2 +I2 + 3 H2O)]
2(I2 + I- → 𝐈3-)
2(I3- + 2 S2O3-2 → 6 S 4O 6-2 + I-
2(I2 + 2 S2O3-2 → S4O6-2 + 2 I-
4 mole Thiosulphate ≅ 1 mole O2
i.e. 1000 ml 4 N thiosulphate = 32 gm O2
BOD bottles, pipettes, burettes, 250 ml Erlenmeyer flasks BOD incubator
Material & Methods:

Reagents:
• MnSO4
Per liter: MnSO4. 4 H2O,480 gm.
Filter and dilute to 1 lt. The solution should liberate not more than a trace of iodine when added to an acidified
solution of KI.
• Alkaline- iodied- azide reagent:
Per liter: NaOH- 500 gm; NaI- 135 gm, NaN3 - 10 gm.
Dissolve 500 gm NaOH and 135 gm of NaI or 150 gm of KI in distilled water and dilute to 1 L. (The reagent
should not give a colour with starch solution when diluted and acidified). Dissolve 10 gm of sodium-azide in 40
ml of distilled water and add to 950 ml of the above. Then make up the volume to 1 L.
• Concentrated H2SO4
• 0.025 N sidium thiosulphate.
• Starch indicator
Per liter: starch- 5 gm.
Weigh 5 gm of starch in a 250 ml beaker and add little water to it. Stir with a glass rod to make slurry. Boil 900
ml of water in a 1000 ml beaker and add the starch slurry to the boiling water in small aliquots. Stir to dissolve.
Allow the solution to become clear on boiling. Make up the volume to 1 L in 1000 ml volumetric flask.
Procedure
1. Filter the sample of wastewater through glass wool or cotton to remove larger pieces of suspended matter. If
the wastewater sample is turbid, a dilution of the same is necessary.
2. Take eight 300 ml BOD bottles. Label them 1/100, 1/50, 1/33 and B for blank and determine the volume of
each bottle accurately. Prepare 1/100, 1/50 and 1/33 dilutions by adding 3,6 and 9 ml aliquots of the sample
to appropriately labeled bottles.
3. Fill each bottle with enough water that upon insertion of the glass stopper the water level will rise above the
ground glass joint, creating a liquid seal. To the bottle labeled B. only water is added.
4. Sonicate the bottles in a sonicator to degas the samples. This will ensure that the filled bottle with 0.025 N
thiosulphate as described below to know the initial oxygen content of each sample. The dissolved oxygen
content of the sample before incubation should be approximately 3 mg/lt or preferably lower.
Measurement of oxygen:
1. Remove the stopper from the bottles and into each pipette 2 ml of MnSO4 reagent followed by 2 ml of alkaline
–iodide-azide reagent. When adding each reagent, place the tip of the pipette below the surface of the liquid
in the bottle. After replacing stopper, rinse the top of the bottle with water.
2. Vigorously shake each bottle for about 30 sec to distribute the precipitate uniformly throughout the bottle.
Repeat the shaking after the precipitates begin to settle.
3. Let the bottles stand until the precipitate has settled about half way down. Unstopper and acidify each bottle
by adding 2 ml of concentrated H2SO4, allowing the acid to run down the inside of the neck of the bottle.
After restoppering and rinsing the top of each bottle, immediately shake for 30 seconds.
4. Titrate a 200 ml volume of each sample with 0.025 N thiosulphate. While gently agitating the sample ,
continue the titration until a faint straw yellow colour develops. Add 1 ml of starch indicator solution, the
colour will become deep blue. Slowly continue the titration (adding the thiosulphate drop by drop) until the
blue colour disappears and the sample is colourless. Record the amount of thiosulphate used.
5. Standardize 0.025 N thiosulphate against 0.025 N K2Cr2O7.(The procedure for the same has been discussed
in a separate sheet. )
Observation:
Table for determination of DO and BOD.
DF Day Volume titrated Thio Titer value DO (mg/lit) DO0- BOD
(ml) V1 strength (ml) [= (VN × 8000 DO5 [={( DO0-
(N) V )/ V1] DO5)- CB}
×1/DF]
1/33
1/50
1/100
blank
• DO (mg/lit) = [(VN × 8000 )/ V1]

• BOD = [{( DO0- DO5)- CB} ×1/DF]
• CB = (DO0 blank – DO5 blank)

1. What precautions you took while performing the experiment?
2. How BOD is related to Industrial pollution –explain in max 50 words.
1. Why we need to standardize sodium thiosulphate again on Day 5 during the experiment?
Evaluators Remarks:

Biochemistry Lab Protocol

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Experiment No: Page No:

Biochemistry Laboratory (FT 491)

Name of the Student:

Day Time Room No. Group

Signature of the Faculty(s): Signature of the Technical Assistant(s):

Read the following instructions carefully

Dos & Don’ts:

Lab Safety Protocols:

3 Separation and isolation of

Exp No. CO1 CO2 CO3 CO4 CO*

Rubrics: Rubrics for Lab Performance Assessment

Overall Lab Performance (End of the Semester)

Quality/Score Excellent Good Fair Poor Marks

Lab Participation Student demonstrates an Student Student Student

are not fully

Safety Proper safety Proper Proper Proper

Pre-experiment Evaluation Questionnaire (MCQ):

Aim: To identify unknown amino acid(s) in a sample

Material & Methods:

Post- experiment Evaluation.

IDENTIFICATION OF SUGARS BY ASCENDING PAPER CHROMATOGRAPHY

Pre-experiment Evaluation Questionnaire (MCQ):

Aim: To identify unknown sugar compound(s) in a sample

Material/reagents & Methods:

Post- experiment Evaluation.

IDENTIFICATION OF SUGARS BY THIN LAYER CHROMATOGRAPHY

Pre-experiment Evaluation Questionnaire (MCQ):

Aim: To identify unknown sugar compound(s) by thin-layer chromatography

Post- experiment Evaluation.

Pre-experiment Evaluation Questionnaire (MCQ):

Aim: To identify amino acid(s) present in sample by thin layer chromatography….

Material & Methods:

Post- experiment Evaluation.

Pre-experiment Evaluation Questionnaire (MCQ):

Post- experiment Evaluation.

Experiment No. 6 Determination of Biological Oxygen Demand of waste water sample

Pre-experiment Evaluation Questionnaire (MCQ):

BOD bottles, pipettes, burettes, 250 ml Erlenmeyer flasks BOD incubator

Material & Methods:

• DO (mg/lit) = [(VN × 8000 )/ V1]

Troubleshooting & Precautions:

Post- experiment Evaluation.

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