Schedule

Experiment #

1
2
3
4
5
6
7
8
9
10

Activity or Experiment Title

Orientation & Check-in of lockers
Review of Buffers
Preparation of Buffers for Chem 40.1 Experiments
Extraction and Characterization of Proteins
Stability of Proteins
Enzymatic Catalysis
Isolation of Nucleic Acids
Hydrolysis of Nucleic Acid
Post-lab Discussions
Long Exam 1
Extraction and Characterization of Polysaccharides
Hydrolysis and Analysis of Carbohydrates
Determination of Saponification and Iodine numbers
of Some Lipids
Total Serum Cholesterol
Digestion
Demo Experiment: Electrophoresis of Proteins
Post-lab Discussions
Check-out of Lockers
Long Exam 2

Session(s)
1
2
3
4,5
6,7
8
9, 10, 11
12, 13
14, 15, 16
17
18
19, 20
21
22
23, 24, 25
26, 27
28, 29, 30
31
32

Attendance
1. Students who come to class 15 mins after scheduled class time are marked late
and are given appropriate demerit points. Those who come in 30 mins late are
considered absent and will be given ZERO for the pre-lab report. An absence
also means a grade of ZERO for the individual performance for that experiment.
No make-up experiments are allowed for both excused and unexcused
absences. However, for excused absence, the student may submit post
laboratory report using the data of his/her group mates.
2. Students who have incurred more than 6 unexcused absences are advised to
drop the course. Otherwise, the student will be given a grade of 5.0.

Requirements
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Required materials per group: cleaning rags, detergent, hand soap, scalpel or blade,
labeling paper or masking tape, old newspapers, pencil,
pentel or labeling pen, plastic/small garbage bags, ruler,
sponge, pair of scissors, a roll of tissue paper, gloves,
used plastic containers (preferably 1/2 and 1-gal ice
containers), cheesecloth
Required materials per student: scientific calculator, laboratory manual, lab gown, safety
glasses or goggles (as per teachers instruction for
some experiments)
Reports
Pre-lab Report
This is accomplished individually and presented to the instructor for checking before
the start of the designated experiment.
Post-lab Report
This is accomplished individually and submitted to the instructor on the next
laboratory session after the completion of an experiment.
Formal Report
A formal report will be required per student for four (4) experiments.
1. The formal report is submitted a week after the experiment has been finished.
2. Late reports will merit a grade of ZERO.
3. Plagiarism is a major offense, cases of which are subjected to sanction or
disciplinary action.
4. The formal report should be hand-written (except for tables and graphs that can be
computer- generated) using blue/black pens on an 8.5 x 11 bond paper with 1- in
margin on all sides.
5. Use ink only.
6. Cross out mistakes with a single line. Never use correction fluid.
7. All measurements must include units.
8. Graphs are titled and axis labeled.
9. Show all work for every calculation.
10.Record all data and report derived values in significant figures.
11.Data must be in tabular form and properly titled.
12.The prescribed format for the report is as follows:
Name of Student
Section and Group #

Date Due
Date Submitted
EXPERIMENT NO.
EXPERIMENT TITLE

Abstract
A one-paragraph condensed version of the entire report/paper. Summarize the four essential aspects of the
report, written in the past tense without use of personal pronouns. It should give an idea of the scope of the
2

study. It is recommended that the abstract is written after the final draft of the paper is complete. Remember
that detailed description of methods should not be included here.
Introduction
A one- page discussion about the experiment, its purpose and objective(s), background information and
literature review
Summary of the methodology
Results (descriptive or numerical)
Discussion of data and results
Most important part of the report. It should provide a description and explanation of the materials and
methods used; analyses and interpretation of the data and results. The data should be presented (organized
into tables, figures, graph, etc.) as you write the discussion. Do not separate the data and results from their
respective discussions. Include also a comparison of the expected results or theoretical information with
those obtained and account for the differences, if there are any.
Major conclusions
Draw conclusions based on the results and provide applicable recommendations.
References
State only reference materials that were actually used.
There must be at least two book references. There should only be minimal internet-based sources.
Attach photocopies (at least two pages) of the exact reference materials to the formal report.

Results Sheet
This is a group requirement and is submitted at the completion of each experiment.
Results can be descriptive or numerical. Data should always be in a table format.
Include titled and labeled graphs when necessary. When presenting your data, it is
advisable to include ALL data points. Only remove a data point if you can justify its
removal by some known error. Your observations should also include error/s committed,
attempts at correcting the error/s, and any change/s made in the protocol.
The group members will decide on what format to use but it should always include the
following information:
Students Names (exclude absent members):
Section:
Experiment No.
Experiment Title.

Date Completed:
Group No.

Laboratory Folder
The prescribed folder is a properly-labeled, 8.5 x 11 folder plastic cover. All graded
pre-lab/post-lab and formal reports should be placed in the folder. The first page should
be a listing of all pre-lab/post-lab and formal reports and the corresponding points
earned in each paper. This is to be submitted to the instructor on or before the second
long examination. Any missing report will be marked ZERO even if it has been
previously earned some points.
Laboratory Policies
Proper laboratory attire must be followed. NO LAB COAT, NO EXPERIMENT.
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Read the protocol in advance. Understand clearly the instructions in using

laboratory apparatus or equipment. Consult the lab instructor, if the instructions
are unclear.
NO CHEATING (exams, laboratory reports). If caught, a grade of 5.0 will be
automatically given and possibly a recommendation for expulsion.
Observe cleanliness and orderliness in the laboratory.
Always clean up spills immediately and always leave your work area clean at the
end of the lab period.
Work carefully and follow precautionary measures.
Avoid conducting unauthorized experiments.
No eating and playing inside the lab.
Cellular phones and other gadgets are not allowed during the lab sessions.
Visitors must not be entertained inside nor at the door of the laboratory room.
Always ask permission from the instructor if you are to leave the laboratory.

Duties of Monitors
Monitors are assigned for every experiment. The duties of the monitors include the
following:
o Submit a list of the reagents and equipment that will be needed a week
before the laboratory exercise.
o Bring any fresh/live specimens needed by the class as indicated in the
procedure.
o Sign out and return any equipment (i.e., hot plate, weighing balance, etc.,)
needed by the class.
o Make sure all the working areas are clean after the experiments, stoppers
of reagent bottles are replaced, and all electronic materials are
unplugged.
Laboratory Precautions
What are Carcinogenic, Teratogenic, and Mutagenic Materials?
Carcinogens are substances which are known to induce cancer; teratogens are
physical/chemical agents that may interrupt or alter the normal development of a fetus
leading to birth defects while mutagens are substances which are to known to cause an
increase in the frequency or extent of gene mutation.
Flammable Liquids- Why not to use a Bunsen burner all the time?
Flammable liquids are defined by the National Fire Protection Association as
those with a flash point below 60 oC and a vapor pressure not exceeding forty pounds
per square inch absolute at 38 oC.
The flash point of a liquid is the temperature at which it gives off sufficient vapors
to form an ignitable mixture with the air near the surface of the liquid. Since the ignition
temperature of a substance is the minimum temperature required to initiate self4

sustained combustion, some substance can burn and cause fire without external ignition
simply by heating above their ignition temperature.
The following table lists some flammable liquids commonly used in laboratories:
LIQUID
FLASH POINT
IGNITION TEMPERATURE (OC)
2-propane (acetone)
-18
538
Carbon disulfide*
-30
100
Cyclohexane
-17
----Ethanol
+13
371
Diethyl ether
-56
246
For a fire to happen, therefore, a flammable liquid, an oxidizing atmosphere
(e.g., air) and an ignition source are commonly required. You will be working with
flammable liquids and under most laboratory conditions you will encounter here, air will
be present. Flammable vapors, therefore, should be kept as far away as possible from
any ignition source, the most readily adjusted parameter. It is safer to use a hot plate or
heating mantle as the heat source than a Bunsen burner.
When distilling a flammable liquid, a flask with a ground-glass joint and which has
been cooled in an ice-water bath should be used in order to minimize the escape of the
flammable vapor into the air. The open part of the system at the receiving adapter must
be properly vented. Finally, illuminating gas which fires burners is also flammable and
toxic. Thus, gas jets must be completely turned off when you have finished.
Safety Rules
Appropriate Outfit: clothing should be comfortable, nonexpensive and cover most of the
body. A laboratory coat or a rubber apron is highly recommended. Shoes should be
comfortable and should cover the whole foot. Sandals (with exposed toes, etc.) and
high-heeled shoes should not be worn in the laboratory. When handling concentrated
acid or corrosive liquids, rubber gloves should be worn.
Safety equipment: Know the locations of ventilation hood, fire extinguishers, fire alarm
boxes, safety showers and eye fountains and the medical kit for emergency purposes.
Study also the exit plan of the laboratory room and where the nearest exit is from your
work bench.
Emergency Instructions
Fire: Any fire is dangerous. If there is fire, report it to the instructor immediately. In
putting out a fire, the extinguisher should be aimed at the base of the flame and a backup extinguisher held ready. Students should not overestimate their ability to put out a
fire. It may spread more rapidly than imagined. Individuals not involved in fire should
quickly, but calmly, leave the area for a safer place.
If Your Clothing Is on Fire: Get under the safety (deluge) shower. Avoid running.
Running will intensify the flame and cause you to inhale more deeply. If a shower is
5

unavailable or if the safety blanket is closer, wrap yourself in a safety blanket to smother
the flame. If neither is available, roll on the floor and smother the fire with the coat or
use a fire extinguisher. To treat a minor burn, immerse the burned area cannot be
immersed in ice water, cover the area with crushed ice. This treatment will relieve and
reduce the severity of the burn. Then, apply ointment and sterile gauze pad. For an
extensive burn, get medical help at once and keep the victim as calm and warm as
possible while waiting for help. DO NOT LEAVE A BURN VICTIMSEND FOR HELP.
Acid and Alkaline Burns: Wash the burned area immediately with copious quantities of
running water. Do not try to neutralize the area. If the burn area is on the face or head,
DO NOT remove eye goggles until the face and the head have been thoroughly flushed.
Cover the area with sterile gauze and contact a physician.
Bromine Burns: wash the burned area with the large amount of running water. Next
soak the burned area in a 10% sodium thiosulfate solution pads for a few hours. Wash
the area again with water and apply sterile pad. If the burned area is extensive, seek the
aid of a physician.
Chemicals in the EYE: Flush the eye or eyes immediately (eye fountain) and continue
flushing for at least 15 minutes while a physician is notified. During the flushing, eyelids
should be forcibly held up so that the entire eye can be thoroughly irrigated. A physician
should be notified immediately. Tell the physician the names of the chemicals involved.
Cuts: After washing the wound with soap and water, cover with a sterile bandage. If
deep and freely bleeding, cleanse the wound as above to remove foreign bodies and
apply pressure at the wound with sterile gauze until the bleeding stops. For deep cuts
and cuts in which glass or other foreign bodies are lodged, professional medical help
should be sought at once. The victim should not be permitted to seek such help alone.
Shock may set in at any time after the trauma.
Disposal of Wastes
Improper disposal of chemicals is not only hazardous to the person involved and
housekeeping personnel, but also can result in environmental pollution. Follow waste
disposal instructions that are provided in each experimental protocol.
Always dispose broken glassware in especially-designated trash bin. Do not
throw other waste in this bin.
Do not throw filter paper or solid materials into the water troughs or sinks.
Additional Basic Safety Rules
Work in the laboratory is prohibited unless an instructor is present.
Students must study each laboratory assignment before coming to class and
may not perform experiments that may have not been assigned.
Arrange apparatus neatly and compactly, with the taller objects at the rear of the
work area. Assemblies consisting of several pieces of apparatus must be approved by
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the instructor. Keep all books except the laboratory manual and your notebook off the
laboratory workbench.
Read the labels on bottles carefully. Do not take reagent bottles form the shelves
to the workbench. Remove only enough material from the bottle to do the experiment.
Any excess reagent should not be returned to the bottle, but placed in the trash can
(solid) or in the appropriate waste bottle (liquid).
When heating a substance in the test tube do not point it at anyone. The hot
contents may bump and be thrown from the tube. Bumping or sudden boiling is due
to localized heating and formation of a large vapor bubble beneath the surface of the
liquid.
When testing for odor of gases being generated in test tubes, fan the gas toward
the nostrils with the hand. Do not breathe deeply.
Do not accumulate flammable or hazardous substance at the workbench. All
chemicals are dangerous if inhaled or ingested in quantity.
DO NOT EAT OR DRINK IN THE LABORATORY. Wash your hands with the
soap and water if they come in contact with any chemical. Clean up spills promptly.
At the end of the laboratory period, clean off your work space with a sponge or
wet paper towels. Check to see that the gas and water have been turned off. You are
responsible for keeping the area neat. Repeated failure to do so may result in loss of
credit.
Material Safety Data Sheet
Always be on alert in handling toxic and hazardous materials as denoted in their
MSDS. Always consult MSDS references online when in doubt as to the nature of the
reagent.

Review of Buffers
7

The pH of living cells is tightly regulated to allow cells to maintain their structural
integrity and for cellular processes to function since the H+ concentration affects the
conformation of biomolecules as well as the concentrations of molecular and charged
species in the solutions. To keep living cells or fluids in an organism at the proper pH, a
solution of special compounds or a buffer is used. This solution tends to resist changes
in pH, so that biochemical reactions, which often produce or use up acids, can proceed
without producing wild fluctuations in cellular pH. In this way, the whole cell is protected
from high concentrations of acid, but critical acidbase reactions can still proceed in the
cell. Humans are equipped with remarkably efficient mechanisms to maintain the
intracellular H+ concentration. These mechanisms include (1) the buffer systems of the
body, (2) the excretory function of the kidneys by which acids and bases are flushed out
in the urine, and (3) the respiratory mechanism by which the H + concentration in bodily
fluids is regulated by the rate of carbon dioxide elimination in the lungs.
Since pH is so critical to biological molecules, any biochemical experiment that involves
studying proteins or cellular systems needs to be performed in a buffered solution to
keep the molecules in their natural, or native, conformation. Thus, an understanding of
buffers is essential in biochemistry.
A buffer is a solution that resists drastic changes in pH when small amounts of acid or
base are added. The solution is made by mixing a large volume of a weak acid or weak
base together with its conjugate. A weak acid and its conjugate base can remain in
solution without neutralizing each other. The same is true for a weak base and its
conjugate acid. In view of the different combinations of weak acid/base and conjugates
that can be mixed, buffers can be classified as follows:
Buffer Type
1. solutions of weak acid and its
salt (or conjugate base)
2. solutions of a primary salt and a
secondary salt

Example
Acetic acid Sodium acetate
Monosodium phosphate Disodium
phosphate
KH2PO4 -- K2HPO4
Ammonia Ammonium chloride

3. solutions of a weak base and its

salt (or conjugate acid)
4 . solutions of ampholytes

Amino acids and proteins

Table 1. General Classes of Buffers

Buffer Action
The ability of a buffer solution to resist changes in pH on the addition of small amount of
an acid or a base is known as buffer action. The buffering action of a solution of a weak
acid and its salt is due to the ability of the salt component to neutralize any added acid,
and the ability of the acid component to neutralize any added base. To illustrate this,
consider an acidic buffer such as a solution containing equimolar amounts of acetic acid
and sodium acetate. The solution contains a large number of sodium ions (Na +), acetate

ions (CH3COO-) and also a large number of undissociated acetic acid molecules as
shown in the equations below.
CH3COONa (aq) CH3COO- (aq) + Na+ (aq)

Eq. 1

CH3COOH H+ (aq) + CH3COO- (aq)

Eq. 2

When a few drops of HCl are added to this buffer solution. The concentration of
hydrogen (H+) ions will be increased. These additional H + ions would combine with the
large reserve of CH3COO- ions to form undissociated acetic acid molecules with the
equilibrium in Eq. 2 favoring the reverse direction. The additional H + ions are neutralized
by CH3COO- ions in the solution, hence there will be no change in the pH of the
solution. The reserve basicity of the solution is due to acetate ion which is the conjugate
base of the weak acid, CH3COOH.
When two or more buffers are present, the effects are additive so that the buffering
action occurs over a wider range. In a similar fashion, the buffer action of a weak base
and its salt is due to the ability of the base component to neutralize the added acid, and
the ability of the salt component to neutralize the added base. However, buffer solutions
of weak bases and their salts are impractical to use due to the volatility and instability of
the bases.
The buffering action of solutions consisting of a primary salt and a secondary salt can
be elucidated using the equations pertinent to the acid-base reactions of KH 2PO4
and K2HPO4 as shown in Eqs. 3 and 4.
OH- (aq) + H2PO4- (aq)

H2O

acid (salt)

H+ (aq)

HPO42- (aq)

Eq. 3

conjugate base (salt)

+ HPO42- (aq) H2O

base (salt)

H2PO4- (aq)

Eq. 4

conjugate base (salt)

Some organic buffers with good buffering capacity in the physiologically important pH
range of 6 to 8 are used in the biochemistry laboratory. These buffers are amino acids,
mainly N-substituted taurines, or N-substituted glycines. Amino acids are ampholytic
since they are typically zwitterionic (ions that contain both a negative and a positive
charge) and can therefore react as an acid and as a base.
The Henderson-Hasselbalch equation, also known as the buffer equation is employed
to calculate the pH of a buffer solution made up of known concentrations of a weak acid
and its salt (conjugate base).
Buffer pH is obtained from the expression

pH = pKa +

log

[salt]
[acid]

For the weak acid HA whose ionization can be written as:

HA (aq)
acid
the buffer equation is:

H+ (aq)

A- (aq)
salt (conjugate base)

pH = pKa + log ([A-]/[HA])

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The pH of a buffer solution after the addition of an acid or a base may be calculated
using the general formulas:
pH = pKa + log [S a]
[A + a]

or

pH = pKa + log [S + b]
[A - b]

where
\

S = number of moles of salt component in the buffer

A = number of moles of acid component in the buffer
a = number of moles of acid added
b = number of moles of base added
Factors Affecting pH
In addition to the actual quantities of acid and its salt, the other factors that influence
the pH of a buffer are the following:
1. The addition of a neutral salt changes the pH due to a change in ionic strength
2. Dilution decreases the reserve acidity or alkalinity of the buffer due to decrease
in the ionic strength.
3. An increase in temperature generally increases the pH of a buffer.
To be suitable for a particular application, the buffer must meet the following criteria:
1.
2.
3.

Buffer pH must be within 1 unit of the pKa value of the acid or the base.
Buffer components must appreciably be soluble in water.
Buffer components (i.e., ions or molecules present in solution) should not
react with each other.

General Steps in the Preparation of Buffers

1.

2.

3.

Choose an appropriate buffer system. The weak acid component should

have a pKa closest to the desired buffer pH to ensure maximum buffer
capacity. Refer to a table of Ka or pKa values to select the appropriate weak
acid/s.
From the Henderson-Hasselbalch equation, calculate the ratio of salt to
weak acid required to obtain the desired pH. The Henderson-Hasselbalch
equation is most satisfactory in the pH range 4 - 10.
a) If both acid and salt (base) forms are available as concentrated and/or
stock solutions or as solid, mix the two forms based on the calculated
moles in step 2 and the molarity or molar mass, whichever is applicable,
of the two substances.
Dilute to a volume just less than the desired final volume of the buffer
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(e.g. 10 ml less than the desired final volume). Verify the pH using a pH
pH meter. If necessary, adjust the pH by adding a small amount of 1 M
solution of HCl or NaOH until the desired pH is attained, then add enough
distilled water to obtain the desired final volume.
b) If only the base form is available, you will have to generate the acid form
first. Do this as follows:
i. Calculate the amount of base (salt) corresponding to the total number
of moles of buffer needed, using the following equation:
moles base(salt) = moles buffer
= (desired conc. of buffer) x (desired vol. of buffer)
Compute for the amount of base(salt) you will measure out using the
calculated mole of base(salt) and its molarity or molar mass,
whichever is applicable.
ii. Slowly add small amounts of strong acid to the measured amount of
base until the desired pH is attained.
iii. Adjust to the required volume by dilution with distilled water. Adjust the
pH, if necessary, as described in 3a.
c) If only the acid form is available, follow the procedure as in 3b to
determine the amount of weak acid to be used. Add a strong base in
step (b)ii instead of a strong acid
The resulting concentration of a buffer is the sum of the concentrations of the weak acid
and its conjugate base. Thus, a 0.10 M acetate buffer may contain 0.050 mole
CH3COOH and 0.050 mole CH3COONa, or 0.025 mole CH3COOH and 0.075 mole
CH3COONa in a liter of the buffer.
Sample Calculations:
A 100 ml 0.50 M HA buffer at pH 3.5 needs to be prepared. Show the calculations for a
scenario where no salt of HA (conjugate base) is available. (K a of HA = 1.8 X 10-4,
MM = 50)
1. Compute ntotal:
ntotal = nHA + nsalt = 0.50 mmole/ml X 100 ml = 50 mmoles
2. Use the HHE to compute for nHA and nsalt
pH = pKa log nHA 3.5 = log (1.8 X 10-4) log x where x = nHA
nsalt
50-x
The calculations will yield: x = nHA = 18.3 mmoles and 50-x = nsalt = 31.7 mmoles
3. Determine the quantities of HA and the strong base that should be reacted to obtain 31.7
mmoles salt and 18.3 mmoles HA after the reaction.
Assume the use of NaOH (MM = 40) as the strong base:
NaOH
+
HA

NaA + H2O
31.7 mmoles
31.7 mmoles 31.7 mmoles
(+18.3 mmoles excess)
massNaOH = 31.7 mmoles X 40 mg/mmole = 1268 mg X 1g/1000 mg = 1.268 g
mass HA = 50.0 mmoles X 50 mg/mmoles) = 2500 mg X 1 g/1000 mg = 2.500 g

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Sample Problems
1.What is the pH of a solution containing 0.10 M chloroacetic acid (K a = 1.4 x 10-3) and
0.15 M sodium chloroacetate?
2. Show the calculations involved and describe how you will prepare 100 ml 0.10 M
CH3COOH-NaCH3COO buffer with a pH of 5.00 from 0.20 M acid and 0.30 M salt
stock solutions.
3. Repeat problem 2 but this time the conjugate base is solid KCH 3COO.
4. (a) Calculate the pH of a buffer prepared by mixing 1.21 g NaH 2PO4 and 1.43 g
Na2HPO4 in enough water to obtain 200 ml buffer.
(b) What is the M of the resulting buffer solution in 4(a)?
(c) What would be the pH if 5.0 ml of 0.25 M HCl is added to 95.0 ml of the buffer in
4 (a)?
5. The buffers listed below will be needed for a number of Biochemistry experiments.
Show calculations pertinent to the preparation of each buffer taking into consideration
the availability of the materials and the forms in which they exist (solid/stock solution).
Refer to a table of Ka values for the pertinent ionization constants of the weak acids.
Consult the lab instructor for the buffer volume that you will use in the computations.
(a) 0.10 M acetate buffer, pH 5
(b)0.10 M acetate buffer pH 4.7
(c) 0.14 M Tris-HCl buffer pH 8.0
(d) 0.10 M phosphate buffer, pH 7
(e) 0.014 M sodium citrate buffer in 0.15 M NaCl, pH 7.4
(f) 0.10 M acetate buffer pH
(g) 2.0 M Tris-HCl, pH 8.8
(h) 0.625 M Tris-HCl, pH 6.8
6. Use the next session to prepare the buffer assigned to your group. Keep the buffer
solution that you prepared in a clean and dry reagent bottle labeled with the following
information:
Name and concentration of buffer
pH of buffer
Date prepared
References
Biochemistry Laboratory Manual. Quezon City: UP Diliman, 2007.
Boyer, R. F. Modern Experimental Biochemistry. CA, USA: Benjamin Cummings, 1993.
Zubay, G.L., W.W. Parson and D.E. Vance. Principles of Biochemistry. IA, USA: W.C.
Brown Communications, 2003.

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