Praktikum Biokimia

BASIC BIOCHEMISTRY
LABORATORY MANUAL
Faculty of Biotechnology, Assumption University

Third Edition : ISBN 974-615-203-3
Arunee Leksakorn
Trilert Chaicherdsakul
EDITORS:
ARUNEE LEKSAKORN
Project Analyst,
Technology Management Center (TMC),
National Science and Technology Development Agency (NSTDA),
111 Thailand Science Park, Paholyothin Road, Klong 1, Klong Luang,
Pathumthani 12120 THAILAND
Tel. (66) 2-564-7000 ext. 1338
arunee@nstda.or.th
TRILERT CHAICHERDSAKUL
Full time Lecturer,
Lecturer Office: 9th floor Q-Building
Huamak Campus, Ram Kamhaeng 24, Bangkapi
Bangkok 10240. THAILAND
Tel: (66) 2-300-4553-62 ext. 3790, 3796
Fax: (66) 2-719-1515 ext. 3792
trilertChc@au.edu
+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
BASIC BIOCHEMISTRY LABORATORY MANUAL
ASSUMPTION UNIVERSITY PRESS
Assumption University Press, Bangna Campus

88 Moo 8, Bangsaothong District, Bangna-Trad Road
Samutprakarn, Thailand
Published in the Thailand

by Assumption University Press Inc., Thailand
Leksakorn A. and Chaicherdsakul T., 2006
Copyright 2006 by Faculty of Biotechnology, Assumption University
Huamak Campus, Ramkhamhaeng 24 Bangkok, Thailand
http://www.biotech.au.edu
All rights reserved.
First published 2003

Second Edition (with correction) 2004
Third Edition (additional contents) 2005: ISBN 974-615-203-3

(revised April 2006):
All rights reserved. No part of this publication may be reproduced, stored in a retrieval
system, or transmitted, in any form or by means, without the prior permission in writing of
University Press. Within the Thailand, exceptions are allowed in respect of any fair dealing for
the purpose pf academic, research or private study, or criticism or in the case of reprographic
reproduction in accordance with the terms of licenses issued by the Copyright Licensing Agency.
Enquiries concerning reproduction outside those terms and in other countries should be sent to
the Rights Faculty, Assumption University Press, at the address above.
Table of Contents
Laboratory Safety and Manner Issues
Laboratory Report
Basic Instruments and Glasswares in Biochemistry Laboratory
10
Experiment 1:
pH and Buffer
25
1.1 Property of acids used in buffer
34
1.2 Property of buffer in different concentrations
35
UV-Visible Absorption Spectrophotometry
40
2.1 Study of the absorption spectrum of syrup solution
50
2.2 Determination of unknown sample concentration
51
Amino acids and Proteins I
54
Experiment 2:
Experiment 3:
Part A: Qualitative analysis of amino acids and proteins
Experiment 4:
3.1 Qualitative analysis of free -amino group by Ninhydrin test
64
3.2 Qualitative analysis of peptide bonds by Biuret test
65
3.3 Qualitative analysis of R group reaction by Sulfur test
67
Amino acids and Protein II
69
Part B: Isoelectric property for protein precipitation

4.1 Isoelectric property for protein precipitation
77
Part C: Protein denaturation

4.2 Protein denaturation by chemical agent
78
Part D: Quantitative analysis of amino acids and proteins
Experiment 5:
Experiment 6:
4.3 Determination of protein concentration by Lowry method
79
Lipid
82
5.1 Solubility property
85
5.2 Test of degree of unsaturation
86
5.3 Properties of soap and detergent
87
Nucleic acid
90
Isolation of high molecular weight DNA
92
Experiment 7:
Carbohydrate
94
Part A: Qualitative analysis of carbohydrates

7.1 Qualitative test for carbohydrate by furfural forming
property (Molischs test)
104
7.2 Qualitative test for carbohydrate by furfural forming

property (Seliwanoffs test)
106
7.3 Qualitative test for carbohydrate by reducing property

(Benedicts test)
7.4 Qualitative test for polysaccharide by Iodine test
107
109
Part B: Quantitative analysis of carbohydrates

7.5 Determination of reducing sugars using 3, 5-dinitrosalicylic
acid (DNS)
Experiment 8:
110
Enzyme
114
8.1 The enzyme kinetic of -amylase
119
8.2 pH influencing the rate of enzymatic reaction
120
Experiment 9:
Enzyme purification
124
Experiment 10:
Glucose Fermentation by yeast
129
Experiment 11:
Introduction to Bioinformatics
132
Appendix
135
References
142
Basic Biochemistry Laboratory
LABORATORY SAFETY
AND MANNER ISSUES
A lab can be dangerous place if you are not careful.
This potential danger
comes from several sources. First, the very nature of the chemicals and equipment
used could be hazardous. Some equipment can be dangerous due to moving parts,
heat or potential electric shock.
Only by using the equipment correctly, as
instructed, will you be sure that you are safe.

Second, glassware used in a lab can always be considered dangerous
because it breaks when dropped or mishandled.
Flying glassware can come from
anywhere, so you may not be the one who makes the mistake, but you may be the
one who pays for it. Proper clothing and eye protection is the only sensible way to
protect yourself against most common lab accidents.
Third, most accidents are caused by carelessness.
A student who is
mentally prepared to undertake the lab, has studied the material and understands
the procedures is much less likely to make a mistake that could injure someone.
Simple mistakes like careless washing of glassware can be dangerous if water ends
up on the floor and isnt clean up.
Please note and observe the following lab
procedures :
THINGS STUDENTS SHOULD DO

Always use some form of protective eyewear especially in dangerous
experiment.
Be aware of what chemicals you will be using. Wear gloves when using toxic
chemicals.
Remember that although you are not using a dangerous
chemical, the student next to you on the bench may have spilled one.
This book is Authors property and cannot be distributed or duplicated without proper authorization.
Wear proper clothing in the lab.
The lab is a good place for long sleeves,
long pants, closed-toed shoes.
Dont even think about going into a lab
barefoot!
Always clean up your lab area and any equipment and glassware you used.
The next class may need to use the same stuff. The job is not over until the
lab is clean and the equipment is ready for the next class.
THINGS STUDENTS SHOULD NEVER DO

Never eat, drink or smoke in the lab.
Never use mouth suction on glass pipets to draw up a solution. Even if you
think the solution is a harmless buffer, the pipet may be contaminated with
something hazardous. Use pipet pumps and bulbs to draw up solutions.
Never work alone. You should always have someone else in lab with you
when you are working.
Never stick your personal pipets into a community reagent bottle. It reflects
your responsibility and your manner. If your pipet is dirty, you will have just
contaminated the supple for the whole class. If you need 10 ml of a reagent
and there is a 1-liter bottle in a community reagent area, take a small beaker
over and pour in about 10 ml. That way, if your beaker is dirty, you will have
contaminated only your own supply.
Never take a community reagent back to your own bench. One of the most
frustrating things that can happen in a lab is to not be able to find something
you need.
Never use more of the chemical than you need. It seems that half of the
students start taking chemicals from the community area before they have
the slightest clue about how much they need. They seem to look at the size
of the community reagent bottle and try to guess from there. Just because
the reagent id out in a 1-liter bottle doesnt mean you should take 100 ml of it.
You might only need 2 ml.
3
The 1-liter bottle may be the total supply for all
sections of the lab for the week.
LABORATORY REPORT
A laboratory report is a formal document consisting of about 6 sections,

each identified with a heading and similar in format to those in a scientific paper, but
with some simplifying features. Like a scientific paper, a lab report should describe
your work and the results you obtained, in sufficient detail to allow the reader to
understand and duplicate your experiments. It should be written or typed, using A4
papers. Proper spelling, grammar and syntax are an important and integral part of
any written document and will be considered in its evaluation. While every lab report
is an individual effort, it may be appropriate to acknowledge and credit co-workers or
others who contributed to the final product. These contributions may run the gamut
from the manual aid of a lab partner to ideas for analysis or suggested
improvements from a colleague or other investigator.
FORMAT
1. Title page
-
Title of the experiment
Name and ID number of the student and lab partners
Date of the experiment
Name of this course
2. Abstract
This is a precise of the experiment or exercise. It should briefly (~100 words)
state what was done, the major result(s), and the major conclusion(s).
3. Introduction
This section compose of 2 part
-
Objective and purpose :
Theory :
the statement of point of the experiment.
gives the hypothesis and rationale behind the experiment, and as
briefly as possible, the minimum necessary background material to understand the

hypothesis and rationale. If a new technique or instrumental method is introduced,
give a brief description of the method. Include chemical or biochemical reactions
when appropriate
4. Experimental (Materials and Methods)

This section is a careful description of the procedures, protocols, materials
and reagents used in the experiment (Not Flow charts in lab notebook!). The
information should be in sufficient detail for the informed reader to understand and
duplicate your experiment. Statistical methods used to analyze the data should also
be reported. Be sure to specify the quantities of materials used and their units.
5. Results
This section presents the findings of the experiment, in narrative form
supplemented when appropriate with figures showing tables, diagrams, graphs,
illustrations, etc. All figures must have complete legends, including a descriptive
title and the information necessary to comprehend the figure.
There are several options available in the presentation of experimental data.
Undoubtedly the two most popular formats are tables and graphs, since both allow a
great deal of material to be presented in a concise manner.
Tables
Remember that a data table should be designed to present a set of data as
clearly and concisely as possible.
All columns and rows within the table should be
clearly defined with respect to the identity and units associated with each value. In
addition, the table should be titled to allow the reader to determine quickly what
features of the table are relevant to the study or experiment.
The example in the
box below presents a poorly designed and a properly designed (well-designed) data
table. Note that the poorly designed table has no title, is very redundant, is cluttered
and is presented in a manner that does not allow the reader to easily see differences
between (to compare) trials of the same experiment. In contrast, the well-designed
data table features the experimental values and quickly draws the readers attention
to differences between the values.
Examples of a poorly designed and a well-designed data table
A poorly designed data table
Absorbance
Absorbance
Absorbance
values
values
values
for reaction # 1
for reaction # 2
for reaction # 3
at 360, 420, and
at 360, 420, and
at 360, 420, and
540 nm
540 nm
540 nm
0.876 (360 nm)
0.885 (360 nm)
0.823 (360 nm)
0.253 (420 nm)
0.250 (420 nm)
0.244 (420 nm)
0.164 (540 nm)
0.163 (540 nm)
0.157 (540 nm)
A well-designed data table

Table 1 : Absorbance values for three trials of the experiment at 360, 420,
and 540 nm.
Trial #
A360
A420
A540
0.876
0.253
0.164
0.885
0.250
0.163
0.823
0.244
0.157
Graphs
As with data tables, graphs are good tool for presenting a large amount of
experimental data in a concise manner. The most commonly used is the line graph.
Line graphs should always be designed with the controlled variable as the abscissa
(x-axis) and the experimentally observed variable as the ordinate (y-axis). As with
the data table, the axes of ant graph should be clearly labeled with respect to identity
and units.
6. Discussion
This section contains a careful analysis and interpretation of the data
presented in the Results section. It is often useful to give a re-statement of the
hypothesis of the experiment, coupled with an assessment of it in light of the
experimental findings. The data obtained should also be compared to those
reported in the literature, and sources of error, as well as possible further studies
discussed. A description of the relevance of the results to larger questions in the

field may be appropriate here as well.
7. References
This section is a listing of all the references used in the report. Whenever
possible, citations should be of the authors of the original work rather than of the
authors of review articles. References serve several roles; they acknowledge the
sources of information or ideas used in the paper, and so help to identify what is
your work. They also provide a connection to other work in the area, and a
permanent intellectual history of the topic. References should be presented "in a
form acceptable to the discipline" (Garrett-Goodyear et. al., 1986), which is usually
the form used by the most commonly cited periodical in the discipline.
Examples of reference styles acceptable in chemistry or biochemistry .

(Use only one style in any report.)
For books
Garrett-Goodyear, J., Harries, J., Patey, D., Shook, M. Writing Papers: A Handbook
for Students at Smith College, 2nd Ed.. Littleton, MA: Sundance Publishers Inc.,
1986.
Clark, J. M., and Switzer, R. L. 1977. Experimental Biochemistry. San Francisco :
W. H. Freeman and Company.
For scientific journal papers

Clark, J. & Nicklas, W. 1970. "The metabolism of rat brain mitochondrial
preparations." J.Biol.Chem. 245: 4724 - 4731.
For internet sources

Name of the author(s). Topic. Available from: name of website. [year, date of
information taken]
Groot, M. N. Conversion of phenylalanine to benzaldehyde. Available from:
http://www.ftns.wau.nl/imb/research/phenylalanine.html. [2001, Oct 2]
H
The NutraSweet company. Aspartame sheet fact. Available from:

http://www.nutrasweet.com [2001, Dec 15]
H
10
BASIC INSTRUMENTS AND GLASSWARES

IN BIOCHEMISTRY LABORATORY
Erlenmeyer Flasks and Beakers
Erlenmeyer flasks and beakers are used for mixing, transporting, and
reacting, but not for accurate measurements. The volumes stamped on the sides are
approximate and accurate to within about 5%.
Graduated Cylinders
Graduated cylinders are useful for measuring liquid volumes to within about
1%. They are for general purpose use, but not for quantitative analysis. If greater
accuracy is needed, use a pipet or volumetric flask.
11
Volumetric Flask
A volumetric flask is calibrated to contain (TC) a given volume at a specified

temperature, usually at 20 to 25oC.
It is used to make up a solution of fixed volume very accurately. This volumetric flask
measures 500 mL 0.2 mL. This is a relative uncertainty of 4 x 10-4 or 400 parts per
million. To make up a solution, first dissolve the solid material completely, in less
water than required to fill the flask to the mark.
After the solid is completely dissolved, very carefully fill the flask to the 500 mL mark.
Move your eye to the level of the mark on the neck of the flask and line it up so that
the circle around the neck looks like a line, not an ellipse. Then add distilled water a
drop at a time until the bottom of the meniscus lines up exactly with the mark on the
12
neck of the flask. Take care that no drops of liquid are in the neck of the flask above
the mark. After the final dilution, remember to mix your solution thoroughly, by
inverting the flask and shaking.
Buret
A
buret
is
calibrated
to
deliver
volumes
indicated
between
graduations. Delivery is completed only after the drop of liquid adhering to the
burette tip is removed (by touching the tip to the inside of a glass vessel). A buret is
used to deliver solution in precisely-measured, variable volumes.
Burets are used
primarily for titration, to deliver one reactant until the precise end point of the
reaction is reached.
13
USING A BURET
(a)
To fill a buret, close the stopcock at the bottom and use a funnel. You may
need to lift up on the funnel slightly, to allow the solution to flow in freely.
(b)
You can also fill a buret using a disposable transfer pipet. This works better
than a funnel for the small, 10 mL burets. Be sure the transfer pipet is dry or
conditioned with the titrant, so the concentration of solution will not be
changed.
Before titrating, condition the buret with titrant solution and check that the
buret is flowing freely. To condition a piece of glassware, rinse it so that all
surfaces are coated with solution, then drain. Conditioning two or three times will
insure that the concentration of titrant is not changed by a stray drop of water.
(c)
14
Check the tip of the buret for an air bubble. To remove an air bubble, whack
the side of the buret tip while solution is flowing. If an air bubble is present
during a titration, volume readings may be in error.
(d)
Rinse the tip of the buret with water from a wash bottle and dry it carefully.
After a minute, check for solution on the tip to see if your buret is leaking. The
tip should be clean and dry before you take an initial volume reading.
(e)
15
When your buret is conditioned and filled, with no air bubbles or leaks, take
an initial volume reading. A buret reading card with a black rectangle can help
you to take a more accurate reading. Read the bottom of the meniscus. Be
sure your eye is at the level of meniscus, not above or below. Reading from
an angle, rather than straight on, results in a parallax error.
(f)
Deliver solution to the titration flask by turning the stopcock. The solution
should be delivered quickly until a couple of mL from the endpoint.
16
The endpoint should be approached slowly, a drop at a time. Use a wash

bottle to rinse the tip of the buret and the sides of the flask.
Pipets
A pipet is used to measure small amounts of solution very accurately. A pipet

bulb is used to draw solution into the pipet.
Pipets are calibrated according to the various types:
a.
Volumetric or transfer pipets (this type has a large bulb) are designed to
deliver a fixed volume of liquid. Proper measurement is done by filling the pipet to
17
about an inch above the calibration mark, and the upper end of the pipet is held
closed with the forefinger. The liquid is then allowed to drain slowly until the bottom
of the meniscus is level with the mark, and the tip is touched to the inner side of a
clean beaker or test tube (not the receiving vessel). Transfer is completed by
allowing the liquid to flow into the receiving vessel by gravity until flow ceases and
the drop adhering to the tip is removed. Do not blow this type of pipet although a
small amount of liquid still remains in it after complete transfer.
b.
Ostwald-Folin pipets are similar to volumetric pipets, but have their bulb
closer to the deliver tip. They are used for measuring viscous fluids such as blood or
serum. The Ostwald-Folin pipet is a blow out type, indicated by an etched ring near
the mouthpiece; however it is blown out only after the fluid has drained to the last
drop in the delivery tip.
c.
Mohr pipets are graduated pipets but the graduations do not extend to
the tip. They are never blown out in making quantitative delivery.
d.
Serological pipets are graduated to the tip and for complete delivery of
an indicated volume, they must be blown out.
e.
Blood pipets are calibrated to contain a given volume and delivery is
completed only after it is blown out and rinsed out a few times.
f.
Pasteur pipets are not used for measuring volume but for convenient
transfer of fluid from vessel to vessel especially when the volume of fluid is
small. For quantitative transfer, the vessel from which the fluid is transferred and the
pipets must be rinsed well. Pasteur pipets are used with rubber bulbs.
18
Using a Pipet
(a) Start by squeezing the bulb in your preferred hand. Then place the bulb on the
flat end of the pipet.
(b) Place the tip of the pipet in the solution and release your grip on the bulb to pull
solution into the pipet. Draw solution in above the mark on the neck of the pipet.
If the volume of the pipet is larger than the volume of the pipet bulb, you may
need to remove the bulb from the pipet and squeeze it and replace it on the pipet
a second time, to fill the pipet volume completely.
(c) Quickly, remove the pipet bulb and put your index finger on the end of the pipet.
Gently release the seal made by your finger until the level of the solution
meniscus exactlylines up with the mark on the pipet. Practice this with water until
you are able to use the pipet and bulb consistently and accurately.
19
Top-loading Balance
Use a top loading balance to weigh solid material when a precision of 0.1 g is
adequate. For more accurate mass measurements or small amounts, use an
analytical balance.
Using a Top-loading Balance

(a)
Check if the balance is turned on. If not, press the on/off button and wait until
the display reads 0.0 g.
(b)
Place a container or large, creased weighing paper on the balance pan. Push
tare button to zero the balance.
(c)
20
Carefully add substance to the container or paper. Record mass.
Clean-up
Use the brush provided to clean any spills. Discard any disposable tare containers,
weighing paper, or Kimwipes in the nearest wastebasket.
21
Analytical Balance
An analytical balance measures masses to within 0.0001 g.
Use these
balances when you need this high degree of precision.

Using an Analytical Balance
(a)
Turn the balance on by pressing the control bar. The display lights up for
several seconds, then resets to 0.0000.
(b)
Place creased, small weighing paper on the balance pan.
(c)
Close the sliding glass doors. Wait for the green dot on the left to go out. This
is the stability indicator light, indicating that the weight is stable.
(d)
22
Press the control bar to cancel out the weight of the container or paper. The
display will again read 0.0000.
(e)
Carefully add the substance to be weighed up to the desired mass. Do not

attempt to reach a particular mass exactly.
(f)
Before recording the mass, close the glass doors and wait until the stability
detector lamp goes out. Record mass of solid.
23
Note
Don't pick up tare containers with bare hands since your fingerprints add mass. Use
Kimwipes or tongs to prevent this.
Don't lean on the bench while weighing.
Do record the mass of your container, if you will need it later.
Do check the level indicator bubble before weighing. The two rear balance feet serve
as leveling screws.
Clean-up
Use the brush provided to clean spills in the weighing chamber. Discard any
disposable tare containers, weighing paper, or Kimwipes in the nearest wastebasket.
24
Quantitative Transfer
Precise and accurate measurements of volumes are essential if reliable

experimental results are to be obtained. Burettes and pipettes must be absolutely
clean to deliver accurately because otherwise liquid may adhere to the wall leaving
droplets behind during drainage. This may also happen if the burettes or pipettes
are allowed to drain too rapidly. It is therefore advisable to allow slow drainage, in
fact the rate should be considerably less than "wide open" in a burette, and, with a
pipette, especially one with a wide tip, the forefinger only partially released. It is also
essential to observe whether the volumetric glassware in use is a to contain or to
deliver type so that quantitation can be appropriately accomplished. To deliver
glassware may or may not need blowing after drainage, this will be indicated by an
etched ring or sandblasted area near the mouthpiece.
25
EXPERIMENT 1
pH AND BUFFER
Objective
1. To understand the composition and functioning system of buffer.
2. To know how to prepare and calculate the pH as well as concentration of buffer
solution.
Introduction
1. pH
1.1 Definition of pH
pH is a measure of the concentration of protons (H+) in a solution and,
therefore, its acidity or alkalinity. The concept was introduced by S.P.L. Sorensen in
1909. The p stands for the German potenz, meaning power or concentration, and
the H for the hydrogen ion (H+).
In layman's terms , the "pH" value is an approximate number between 0 and
14 that indicates whether a solution is acidic (pH < 7),basic (pH > 7) or neither (pH =
7).
1.2 Measurement of pH
1.2.1 Liquid pH indicator
A pH indicator is a chemical compound that is added in small amounts to a
solution so that the pH (acidity or alkalinity) of the solution can be determined easily.
Hence a pH indicator is a chemical detector for protons (H+). Normally, the indicator
causes the color of the solution to change depending on the pH.
26
pH indicators themselves are frequently weak acids or bases. When

introduced into a solution, they may bind H+ (Hydrogen ion) or OH- (hydroxide) ions.
The different electronic configuration of the bound indicator causes the indicator's
color to change.
Because of the subjective determination of color, pH indicators are
susceptible to imprecise readings. For applications requiring precise measurement
of pH, a pH meter is frequently used.
pH indicators are frequently employed in titrations in analytic chemistry and
biology experiments to determine the extent of a chemical reaction.
Tabulated below are several common laboratory pH indicators. Indicators
usually exhibit intermediate colors at pH values inside the listed transition range. For
example, phenol red exhibits an orange color between pH 6.6 and pH 8.0. The
transition range may shift slightly depending on the concentration of indicator in
solution and on the temperature at which it is used.
Table 1-1 Examples of pH indicators
Color at low pH
Indicator
(Acid condition)
Transition pH
range
(approximate)
Color at High pH
(Base condition)
Blue-violet
0.0-1.6
Yellow
Red
1.2-2.8
Yellow
Red
2.9-4.0
Yellow
Yellow
3.0-4.6
Violet
Congo red
Blue
3.0-5.2
Red
Methyl orange
Red
3.1-4.4
Yellow
Litmus
Red
4.5-8.3
Blue
Bromocresol purple
Yellow
5.2-6.8
Violet
Bromothymol blue
Yellow
6.0-7.6
Blue
Phenol red
Yellow
6.6-8.0
Red
Methyl violet
Thymol blue (first
transition)
Methyl yellow
Bromophenol blue
Thymol blue (second
27
Yellow
8.0-9.6
Blue
Phenolphthalein
Colorless
8.2-10.0
Pink
Thymolphthalein
Colorless
9.4-10.6
Blue
Alizarin Yellow R
Yellow
10.1-12.0
Red
transition)
Universal Indicator is a blend of different indicators that exhibits several smooth

color changes over wide range of pH values. It is one of the most commonly used
indicators. It is made from a mixture of pH-sensitive dyes to create the spectrum of
colours it exhibits across a range of different pH values.
For examples,
In very acidic conditions, universal indicator is red, and in less acidic

conditions it is orange or yellow.
In neutral conditions the indicator appears green.
In alkaline or basic conditions, the colour is blue, and in very basic conditions
purple.
Anthocyanins are a class of compounds (pigments) that occur in many
different plants; they appear red in acidic solutions and blue in bases. Extracting
anthocyanins from red cabbage leaves to form a crude acid-base indicator is a
popular introductory chemistry demonstration.
1.2.2 pH indicator papers
The convenience of using indicator papers for the rapid determination of pH
values has led to many applications in laboratories and industry. These inexpensive
pH indicator papers have been available for decades in booklets or in the form of
rolls. They consist of high value filter paper soaked with indicators or indicator
mixtures. They are ideal for pH measurements in solutions where no problems are
expected.
Another higher version of pH indicator papers is pH indicator strips.
These versatile indicator strips contain special indicator dyes which are covalently
28
bonded to the cellulose of the reagent paper. They have many excellent properties
such as
Clear colour difference between pH gradient steps
Instant pH readings and Convenient and portable for field use
No bleeding of dyestuff (i.e. the colours do not run and therefore
cannot contaminate the medium under examination).
Measurement in weakly coloured or turbid liquids is possible.
Figure 1-1 Various version of commercial pH indicator papers and stripes
1.2.3 pH meter
A pH meter is a specific type of voltmeter with a very high impedance of the input
channels. The high impedance is a necessary part of the equipment because of high
resistance of the pH glass electrode typically used with pH meters (usually between
20 and 100 M).
First commercial pH meters were built around 1936 by Dr. Arnold Beckman in the
US and by Radiometer in Denmark. Dr. Beckman's invention helped him launch the
successful Beckman Instruments company.
29
The common pH meter has several inputs: for indicator (ion-sensitive or redox) and
reference electrodes and temperature sensors such as thermoresistors or
thermocouples. Cheaper models require external temperature measurements
entered using knob or keyboard.
The pH scale of the device should be calibrated by at least two buffer solutions.
Usually one of the buffers used for calibration has pH 7.00 and the second is
selected depending on the range where the measurements are to be taken - 10.00
for basic solutions and 4.01 for acidic solutions. This correlates the measured
potential of the indicator electrode with the pH scale.
(Please see APPENDIX 1 : Procedure in pH measurement by using pH meter)
2. BUFFER
The proper functioning of biological systems requires pH control.
Cells
maintain a certain pH rang partly by active process (i.e. energy) and partly by a
complex mixture of buffering agents.
additions of acids and bases.
Buffers allow only small changes in pH upon
An appropriate buffer from some system must meet
specifications such as pH and total buffer concentration or ionic strength or

concentration of certain species.
2.1 Buffer definition
A buffer may be defined as a solution, which resists large changes in pH that
might be expected to occur upon the addition of small amounts of acid (H+) or base
(OH-) to the solution. To put in another way, a buffer helps maintain a near constant
pH upon the addition of small amounts of H+ or OH- ions to a solution.
2.2 Buffer components
Common buffer mixtures contain two substances, a conjugate acid and a
conjugate base.
Acidic buffer
30
contains a weak acid and a salt of the weak acid (conjugate
base). For instance, acetate buffer (CH3COOH + CH3COO- )

Basic buffer contains a weak base and a salt of the weak base (conjugate
acid). For instance, ammonium buffer (NH4Cl + NH3)
2.3 Buffer action

In case of Acidic buffer (the weak acid (HA) and its salt (M+A-)).
- If we add some hydrogen ions (H+) in the form of a strong acid ; the anion A- from
the salt component of the buffer will combine with these added H+ to form the weakly
dissociated water molecule.
Hence the pH does not become alkaline as would
otherwise occur.
A- + H+
HA
If we add some hydroxyl ions (OH -) in the form of a strong base ; the weak acid
component of the buffer will combine with these added OH- .
HA +
OH-
A-
+ H2O
2.4 Buffer and Henderson-Hasselbalch equation

The pH of any mixture of a weak acid and its salt can be calculated from the
Henderson-Hasselbalch equation, which is readily derived as follows : in any
solution of a weak acid, whether its salt is present or not, the usual dissociation
constant equation holds true,
31
[H+] [A-]
Ka
--------------[HA]
Ka is dissociation constant of weak acid.
The dissociation of the average
weak acid is so small that the term [HA] can be replaced with the actual, total
molarity of the acid dissolved in the solution.
Similarly, when the salt of the acid is
present, practically all of the anion A- in the solution can be regarded as coming
from the salt, so that the term [A-] can be replaced by the molarity of salt added.
1
[H+]
[salt].
Ka
[acid]
Taking logarithms of both sides of the equation

log
log
[H+]
1
Ka
log
[salt].
[acid]
or
pH
pKa +
log [salt]
[acid]
From which one can calculate the pH of any buffer system from the pK of the
acid involved and the salt-acid ratio.
It may be pointed out that, when the salt and
acid are present in equal concentrations, the pH of the solution is numerically equal
to the pK of the acid, since the logarithmic term becomes zero.
Buffers are most efficient at pHs near their pKs. The effective range of a
buffer system is generally two pH units, centered at the pKa value.
32
Effective pH range for a buffer = pKa 1
2.5 Buffer capacity

The ability of a buffer to resist changes in pH is referred to as the Buffer Capacity
(). Buffer capacity can be defined in two ways ;
Buffer capacity in the acid direction

The number of moles of H+ that must be added to one litre of
the buffer in order to decrease the pH by 1 unit.
Buffer capacity in the alkaline direction

The number of moles of OH- that must be added to one litre of
the buffer in order to increase the pH by 1 unit.
33
Blood Buffering: the carbonate system

The pH of human blood is about 7.4
If pH falls outside range 6.8 to 7.8, you are dead.
The main blood buffer is carbonate system (but phosphate and protein systems
help)
The pKa for carbonic acid is 6.1
H2CO3 HCO3- + H+
At physiologic pH, find ratio of carbonate to carbonic acid
pH = pKa + log [A-]/[HA]
7.4 = 6.1 + log [A-]/[HA]
1.3 = log [A-]/[HA]
Therefore: [A-]/[HA] = 20.
Given total buffer is about 28 mM, [HCO3-] = 26.6 mM and [H2CO3] = 1.4 mM
This seems like a poor buffer BUT infinite CO2 reservoir gives huge buffering
capacity and MOST assaults on system are metabolic acid production.
CO2 (gas) CO2(aqueous) + H2O H2CO3 HCO3- + H+
In clinic, conditions that effect MAINLY [HCO3-] are metabolic, and conditions
that effect MAINLY [H2CO3] are respiratory
Metabolic acidosis means excess H+: often seen with diabetic conditions
Metabolic alkalosis is loss of H+: may indicate vomiting or poisoning with base
Respiratory acidosis: High CO2 yields high [H2CO3]. May indicate poor CO2
clearance from lungs.
Respiratory alkalosis arises from very rapid removal of CO2, which is indicative of
hyperventilation.
Ex. A patient comes in comatose. Blood gas analyzer shows [HCO3-] = 50 mM and
[H2CO3] = 1.4 mM.
pH = 6.1 + log 50/1.4 = 7.67
metabolic alkalosis, maybe ingested too much bicarbonate (a Tums addict!)
34
Experiment
Experiment 1.1 : Property of acids used in buffer
Principle :
Strong acids for instance HCl usually are totally dissociated into H+ and Cl+
when dissolved in water.
HCl
H+
While weak acids for example H3PO4
Cl+
are partly dissociated in form of
positive ion and negative ion.

H3PO4
H+
H2PO4-1
Ka1
H2PO4-1
H+
HPO4-2
Ka2
1.6 x 10-7
Ka3
5 x 10-13
HPO4-2
H+
HPO4-3
The value of Ka1 , Ka2 and Ka3 are dissociation constant.
1.1 x 10-2
The experiment
aims to compare the titration profile of pH changing between strong acid, HCl and
weak acid, H3PO4 after titrated with NaOH.
Materials :
1. 0.1 M HCl
2. 0.01 and 0.05 NaOH
3. 0.05 M H3PO4
4. pH meter
Methods :
A. Titration of HCl with NaOH
1. Pipet 2 ml of 0.1 M HCl into 18 ml of distilled water in a small beaker and
then mix well.
2. Measure pH by using pH meter.
35
3. Then titrate the acid solution by pipeting 0 to 30 ml of 0.01 M NaOH (2 or

4 ml intervals, see in data note) into the acid solution and mixed well.
4. Measure pH of each steps of NaOH addition by pH meter.
5. Plot the titration curve between pH changing against volume (ml) of NaOH
addition.
B. Titration of H3PO4 with NaOH
1. Pipet 20 ml of 0.05 M H3PO4 into a small beaker.
2. Measure pH by using pH meter.
3. Then titrate the H3PO4 solution by pipeting 0 to 62 ml of 0.05M NaOH (2
or 4 ml intervals, see in data note) into the H3PO4 solution and mixed well.
4. Measure pH of each steps of NaOH addition by pH meter.
5. Plot the titration curve between pH changing against volume (ml) of NaOH
addition.
Experiment 1.2 : Property of buffer in different concentrations
Objective :
To compare the buffering capacity of phosphate buffer in different concentrations.
Materials :
1. 0.2 M Disodiumhydrogenphosphate (Na2HPO4)
2. 0.2 M sodiumdihydrogenphosphate (NaH2PO4)
3. 0.2 M HCl
4. 0.2 M NaOH
Methods :
1. Preparing 0.2 M phosphate buffer
Mixed 30 ml of 0.2 M Na2HPO4 with 30 ml of 0.2 NaH2PO4. This buffer
is contained the ratio of salt and acid as equal concentration of 0.2 M.

36
Take 20 ml of 0.2 M phosphate buffer to dilute with 20 ml of water to get

the final concentration of 0.1 M, in final volume of 40 ml.
Take 10 ml of 0.2 M phosphate buffer to dilute with 30 ml of water to get
the final concentration of 0.05 M , in final volume of 40 ml.
4. Prepare 8 test tubes and add the following solution as shown in Table in
Data note and then measure the pH of each tube.
Add 0.2 M HCl into
tube no. 1-4 and 0.2 M NaOH into tube no. 5-8 (1 ml intervals for 5 times).
Mix well before measuring the pH. Record the pH reading. Note that tube
no. 1 and 5 are distilled water (used as blank).
Questions
1. Is there any difference in pattern between titration curve of HCl and NaOH, and
titration curve of H3PO4 and NaOH ? How ? and Why?
2. From titration curve, how can you determine the pKa value of phosphoric acid
(H3PO4)?
3. Compare the buffering capacity of phosphate buffer in different concentrations
and give reason why the buffer concentration affects on pH of solution when
adding acid or base in to the buffer.
4. Show how to prepare 0.15 M Acetate buffer pH 4.9 by using HendersonHasselbalch equation in calculation. (pKa of acetic acid = 4.76) Please try !!
37
DATA NOTE
Experiment 1.1 : Property of acids used in buffer
A. Titration profile of strong acid, HCl
0.01 N NaOH
12
16
18
20
22
26
30
added (ml)
pH
measurement
B. Titration profile of weak acid, H3PO4
0.05 N NaOH
12
16
18
20
24
28
32
34
36
38
42
46
18
50
54
58
62
added (ml)
pH
measurement
0.05 N NaOH
added (ml)
pH
measurement
38
Experiment 1.2 : Property of buffer in different concentration
1. Adding 0.2 M HCl into phosphate buffer at different concentrations.
Tube
no.
concentration of 10 ml
0 (water)
0.05
0.1
0.2
pH
reading
phosphate buffer (M)
Volume of 0.2 N HCl added (ml)

(1 ml intervals for 5 times)
0
1
2
3
4
5
39
2. Adding 0.2 M NaOH into phosphate buffer at different concentrations.
Tube
no.
concentration of 10 ml
0 (water)
0.05
0.1
0.2
pH
reading
phosphate buffer (M)
Volume of 0.2 N NaOH added (ml)

(1 ml intervals for 5 times)
0
1
2
3
4
5
40
EXPERIMENT 2
UV-VISIBLE ABSORPTION SPECTROPHOTOMETRY
Objectives
1. To understand the principle of spectrophotometry
2. To study the spectrum of substances
3. To apply the spectroscopic technique to the determination of substances
in the solution
Introduction
Light and Spectrum profile
Light can be classified according to its wavelength. Figure 1 shows the
relationship between the wavelength of light and the common types of
electromagnetic radiation. As you can see, those regions where the wavelength is
very short correspond to the types of radiation that you know are powerful and often
harmful, such as X rays, rays and ultraviolet radiation.
wavelengths of 200 to 400 nm is referred to as ultraviolet (UV).
Light in the short

Light in the longer
wavelengths of 700 to 900 nm is referred to as near infrared (near IR).
Figure 2.1:
Wavelength regions of light
41
Visible light falls between the wavelengths of 400 and 700 nm.
Within this
range of wavelengths is found all of the colors visible to the human eye.
Any
solution that contains a compound that absorbs light in the visible region will appear
colored to the eye.
The solution is colored because specific wavelengths of light
are absorbed as they pass through the solution. Then, the only light that the eye will
perceive are the wavelengths of light that are transmitted (not absorbed). In Figure
2-2, the solution looks green to us because green light (blue and yellow) is
transmitted while the red light is absorbed.
A solution may contain many compounds that absorb at many different
wavelengths, but if compound we are interested in absorbs at a unique wavelength,
we can determine its concentration even in a solution of other compounds.
Figure 2-2: Absorption of light by a solution

Another example is illustrated as in the absorption spectrum for riboflavin
(Figure 2-3).
An absorption spectrum is a plot representing the absorbance of a
solution at a number of wavelengths.
Why does a solution of riboflavin appear
yellow to the eye? As shown in Figure 2-3, riboflavin absorbs light at 450 nm, which
is in the blue region of visible light, Because of this, red and yellow light will be
transmitted through the solution and detected by the eye. Figure 3 also shows that
riboflavin absorbs light strongly at 260 and 370 nm. Although this will not influence
the apparent color of the solution (since these wavelengths lie outside the range of
42
visible light), these absorption events in the UV range can be detected with a
spectrophotometer.
Spectrophotometric analysis is the most widely used method of quantitative and
qualitative analysis in the chemical and biological sciences; it is an accurate and
very sensitive method which can analyze quantities as small as micrograms. The
method depends on the light absorbing properties of either the substance being
analyzed or a derivative of its. The basis of spectrophotometry is simple: the
intensity of the light which is transmitted through a solution containing an absorbing
substance (chromogen) is decreased by that fraction which is absorbed, and this
fraction can be detected and measured photoelectrically, generated spectrum
profile as shown in Figure 2-3.
Figure 2-3: Absorption spectrum of riboflavin

(condition : 22M in 0.1 M sodium phosphate, pH 7.06, in 1-cm light path)
43
The Beer-Lambert law

When a ray of monochromatic light of initial intensity Io passes through a
solution in a transparent vessel, some of the light is absorbed so that the intensity of
the transmitted light I is less than Io.
There is some loss of light intensity from
scattering by particles in the solution and reflection at the interfaces, but mainly from
absorption by the solution.
In addition, this law describes that each unit layer of a
solution will absorb an equal fraction of the light passing through it. For example, if
the intensity of light incident (Io) upon a solution is 1.00 and each unit layer absorbs
10% of the light passing through it, then the light transmitted through the solution will
be diminished by 10% per unit layer (1.00, 0.90, 0.81, 0.73, 0.66,.).
Figure 2-4: The absorption of light by a solution
The Beer-Lambert law states that the amount of light absorbed is proportional
to the number of molecules of absorbing substance in the light path; i.e. absorption
is proportional both to the concentration of the chromogen in solution and to the
length of the light path through the solution. This relationship can be expressed as
follows:
44
- log I / Io = kcl
where
I is the intensity of transmitted light in the presence of the chromogen
(incident light)
Io is the intensity of transmitted light in the absence of the chromogen
(emergent light)
c is the concentration of the chromogen
l is the length of the light path through the solution
k is a constant, characteristic for each absorbing substance at a specific wavelength
of light and in a specified solvent or often called as extinction coefficient
The ratio I / Io is called the light transmission and in usually measured in
percent. The absorbance (Abs), or optical density (OD), is the quantity more
frequently used, and is given by
A (or OD) = - log I / Io

On substitution of this definition into the Beer-Lambert law;
A (or OD) =
kcl
Spectrum is a plot of the absorbance of a solution of an absorbance

substance over a range of wavelengths.
To calculate the concentration of
substance in the solution, after measurement the absorbance while the length of the
light path through the solution is 1 cm ( l = 1). Therefore,..
c = A/k
45
In this form the Beer-Lambert law states that doubling of either the
concentration of the absorbing substance or doubling of the depth of solution leads
to a doubling of the absorbance. The quantitative measurement of the amount of
light
absorbed
spectrophotometer.
is
accomplished
with
an
instrument
called
Spectrophotometers detect photoelectrically and compare
electronically the difference in the amount of light transmitted through solutions

containing differing concentrations of an absorbing substance. The difference is
expressed on a dial of the instrument as percent transmission or optical density.
In the expression for the Beer-Lambert law, the proportionally constant k
depends on the wavelength of light and the nature of the absorbing substance. This
relationship is the one used in spectrophotometric work with precise instruments. In
this case, that portion of the spectrum used is so narrow that it can be considered
monochromatic (a single wavelength).
In additon, carefully made solution
containers, called cells or cuvettes, with plane parallel sides are used so that the
light path is the same through all portions of the cell and is accurately known.
If the concentration (c) is expressed in moles per liter (molar ; M) and the light
path in cm, then the proportionality constant k is called the molar extinction
coefficient (given symbol E1M1cm or ).
If If the concentration (c) is expressed in gram%
(gram in 100 gram of
solution) and the light path in cm, then the proportionality constant k is called the
gram percent extinction coefficient (given symbol E1%1cm or ).
And since absorbance (A) and optical density (OD) values are unitless, the k
is most often expressed in units of inverse concentration times inverse pathlength
(i.e., M-1 cm-1 , or mM-1 cm-1 , or (g/l)-1 cm-1 , or (mg/ml)-1 cm-1).
The extinction coefficient (k) may depend strongly on both the ionic strength
and the pH of solution in which the solute resides.
The greater k under aparticular
condition (solvent and wavelength), the greater the amount of light absorbed.
This
idea becomes important when considering the sensitivity assocuiated with

quantifying a compound by measuring the absorbance of a solution at a particular
46
wavelength. For example, since riboflavin has a greater extinction coefficient at 260
nm than at 450 nm, an absorbance reading taken at 260 nm would be much more
useful in attempting to determine the concentration of a dilute solution of this
compound.
Qualitative Measurements
The absorption may occur when light of the energy (wavelength) required to
excite certain of the bonding electrons strikes the absorbing molecule. The intensity
and position in the electromagnetic spectrum of the absorbed light is related in a
complicated way to the electronic structure of the molecule and its constituent atoms
and is a unique optical characteristic of the substance. A plot of the absorbance of a
solution of an absorbing substance over a range of wavelengths is referred to as the
absorption spectrum of the compound and is useful for the qualitative identification
of unknown compounds. Since the concentration of the chromogen is constant for
such a plot the curve describes the dependence of k upon wavelength. Values for k
are largest in regions referred to as absorption peaks or absorption bands. The
wavelength, or (lambda), of maximum absorption of a particular compound
is usually referred to as the max.
Quantitative Measurements
Although the extinction coefficient (k) is a constant value for each substances,
in many cases the condition of measurement and the quantitative measurement
cannot be controlled or get the correct k value.
47
The quantitative measurement of concentration of an absorbing substance

can be accomplished using such a plot, called a standard curve (Figure 5). The
curve is constructed by preparing a series of standards of the substance to be
analyzed in a graded series of known concentrations. The absorbance of each
standard is then determined and a plot constructed of the absorbance on the
abscissa (X-axis). The absorbance of a solution of unknown concentration is then
determined under identical conditions and the value of the concentration simply read
from the plot.
Figure 2-5:
Standard curve of riboflavin
Quantitative Measurement by turbidity assays
When light passes through a suspension of particles of fairly large size, i.e.,
molecular weight of 50,000 up to the size of unicellular organisms several microns in
length, some of the light interacts with the individual atoms of each particle and,
though not absorbed, is directed away from its normal path of transmission. This
"scattered" light is what allows us to see dust grains in the beam from a movie
projector and causes the sky to be blue and the sunset red. When particles
suspended in a solvent scatter light sufficiently to give the suspension a milky cast,
48
we refer to the suspension as being "turbid". As a result of scattering the light

transmitted straight through a turbid suspension is less than that transmitted through
the solvent alone. Thus turbidity can be measured as optical density and is related
to the total mass of the scattering particles.
Quantitative Measurement by colorimetric assays
The principles of quantitative spectrophotometric assays that we have

disscuss
Construction of spectrophotometers
Figure 2-6: Operational diagram of a spectrophotometer
49
Operating Procedure for the Spectronic genesys 5
The spectrophotometer used in this lab is the spectronic genesys 5.

1. Turn the instrument on by pressing the on/off switch.
2. The instrument will check all programs itself automatically.
Wait until ready is
shown on the monitor of instrument.

3. Select the desired wavelength by entering the number of wavelength (eg, if
desired wavelength is 340 nm, then press 340), and follow by pressing the GO
TO WL key.
Then, the instrument will run to the desired wavelength and it will
be shown on the monitor.

4. Setting blank by inserting the reference (blank) cuvet in to the cuvet holder and
close the lid.
Pressing the AUTO ZERO key. The absorbance of 0.000 will
be appeared on the monitor.

5. Remove the reference cuvet, replace it with the cuvet containing the sample
solution and again close the lid. Pressing the ENTER key. The absorbance of
sample will be shown on the monitor.
50
Precautions
1. Keep the lid cover down while making measurements.
2. Wipe tubes clean of finger marks and stains before inserting into the holder.
3. Do not attempt to make measurements on solutions that are turbid (unless
turbidity measurements are desired). Also do not make measurements if air
bubbles adhere to the wall of the cuvet or if the outside walls are coated with
condensation.
4. Do not spill liquids in the cuvet holder. If spillage occurs inadvertently, inform an
instructor. Wipe off the instrument immediately.
5. Always be sure that the solutions are well mixed before measurements are
made.
Experiment
Experiment 2.1
Study of the absorption spectrum of red syrup solutions
Principle :
The chromophoric compounds are able to absorb the light intensity at the
visible region ( the wavelength between 300-800 nm ).
However, the ability of the
light absorbing compound depends on characteristic of substances or its color,

which is corresponded to the color of the visible light region. The objective of this
experiment is to study the spectrum profile and determine the maximum wavelength
of the red solution.
Materials :
1. Stock solution of red syrup
2. Distilled water
3. Cuvet
4. Spectrophotometer
51
Methods :
1. Dilute stock solution of red syrup into 1:20 with distilled water by adding
9.5 ml of distilled water in 0.5 ml of red syrup solution and mixed well.
2. Take 1 ml of the diluted red solution into the cuvet and use 1 ml of distilled
water as a control or blank.
3. Determine the spectrum of the diluted red solution by measuring the
absorbance at 50 nm intervals from 420 nm to 670 nm against blank in
every wavelength.
4. Plot a spectrum profile with wavelength as the abscissa (x-axis) and the
absorbance as the ordinate (y-axis).
5. Determine the maximum absorption wavelength (max) of the red
solution.
Experiment 2.2
Determination of unknown sample concentration
Principle :
Since the red syrup solution is composed of many substances, this makes it
difficult to calculate the concentration of the sample using the Beer-Lambert
equation.
A = kcl
When l (which is light path) = 1 cm,
c=A/k
Where A is absorbance, c is concentration and k is standard constant.
The
value of k is still unknown we cannot calculate the concentration of the sample. An

alternative ways is to construct the standard curve and the slope of the curve is
represented the k value of the compounds.
The absorbance of a solution of
unknown concentration is determined under identical conditions followed by

calculation as the equation as well as the value of the concentration simply read
from the plot.
52
Materials :
1. Diluted red syrup solution (as prepared in experiment 2.1)
2. Distilled water
3. Spectrophotometer
4. Unknown sample
Methods :
1.
Prepare a set of 5 serial dilution of diluted red syrup as the following and
mix well every step.
And these solutions are used as standard
solutions.
Tube no.
Red syrup solution
Distilled water
% Red solution
10 ml
10 ml
50
10 ml from tube 1
10 ml
25
10 ml from tube 2
10 ml
12.5
10 ml from tube 3
10 ml
6.25
10 ml from tube 4
10 ml
3.125
2.
Set up the spectrophotometer at maximum absorption wavelength (max)

obtained from the experiment 2.1 and adjust the zero absorbance by
using distilled water as blank.
3.
Measure the absorbance of each standard tube number 1 to 5 and then

measure the absorbance of unknown sample.
4.
Plot a standard curve with the % of standard solution as the x-axis and
the absorbance as the y-axis.
5.
Calculate % of unknown solution and calculate the E%1cm value.
53
DATA NOTE
Experiment 2 : UV-Visible absorption Spectrophotometry
2.1 Study of the absorption spectrum of red syrup solutions

Wavelength (nm)
Absorbance
420
470
520
570
620
670
2.2 Determination of unknown sample concentration

Tube no.
% of standard solution
Absorbance at ____ nm
Blank
50 %
25 %
12.5 %
6.75 %
3.125 %
Unknown no. ____
54
EXPERIMENT 3
AMINO ACIDS AND PROTEINS (Part I)
Objective
To study some basic properties of amino acids and proteins
Introduction
Proteins are about 50% of the dry weight of most cells, and are the most
structurally complex macromolecules known.
unique structure and function.
Each type of protein has its own
Proteins compose of the building blocks amino
acids linked together by peptide bonds.

H
Amino acids are built from a central carbon bonded to four different groups.
Each amino acid has an amino group and a carboxyl group , joined by a single
Carbon atom. In addition, each amino acid has a characteristic "side chain" (often
called the -R group).
Each amino acid (except glycine) can occur in two isomeric forms, L- and D-,
because of the possibility of forming two different enantiomers (stereoisomers)
around the central Carbon atom. Only L-amino acids are found in proteins in all
organisms.
Protein is one of the most biological importance that has many types and
functions such as the protein hormones, protein structures, protein connections,
protein transports, protein catalysts or enzyme and etc.
Figure 3-1:
Figure 3-2:
55
The structure of amino acid
The formation of peptide bond
56
Proteins are of crucial importance in more or less all organs and processes in
organisms. Fibrous proteins are the major components of muscles as well as of skin,
bones, tendons, blood-vessels, teeth, and hair. Blood contains various proteins that
are responsible for the storage and transport of biologically important substances
such as oxygen, metal ions, glucose, lipids, and many other molecules.
Furthermore, proteins called enzymes participate in all the different chemical
reactions that an organism performs to generate energy from the diet and to
synthesize its building blocks, which are summarized with the term metabolism.
Also, the regulation of the metabolism as well as of growth and development is
performed by proteins: hormones and their receptors. Proteins are involved in the
acquisition of sensory information and in the generation and transmission of nerve
impulses. The protection of the organism against external invasion is mediated by
the proteins of the immune system.
All those different proteins are composed of only 20 monomeric units known
as amino acids. Dietary proteins are cleaved to short chains of amino acids
oligopeptides and amino acids during digestion. A major difference between amino
acids and most of the other building blocks of the body is that amino acids contain
nitrogen. Nitrogen from the air can be converted to metabolically useful forms only
by a few strains of bacteria that live in symbiotic relationships with certain plants.
Humans and animals, however, have to take in metabolically useful nitrogen with
their food. The intake of protein with the diet is therefore essential and the amino
acids that are released from those proteins are not only a source of energy to the
organism but deliver the building blocks for the synthesis of proteins and serve as
precursors for all nitrogen containing compounds.
In addition to amino acids,
nitrogen containing compounds include nucleotides (the building blocks of the

genes), some hormones and neurotransmitters, various intermediates in metabolic
processes, as well as heme, a complex molecule that is involved in the oxygenbinding property of red blood cells and in energy generation by the respiratory chain.
57
Proteins, like all the components of living cells, are constantly turning over.
They are broken down to their constituents and rebuilt. The function of this process
is twofold. Abnormal molecules whose accumulation would be harmful to the cell
are eliminated. And the processes in the body can be regulated by the speed with
which enzyme and other regulatory molecules are broken down and rebuild.
Proteins have life spans that range from as short as a few minutes to weeks or
more. Cells continuously synthesize proteins from and degrade them to their
component amino acids.
Figure 3-3
Importance of proteins
Amino acids
Proteins are composed of 20 standard amino acids, organic molecules
with an amino group as well as a carboxylic acid group at the a-C-atom.
Figure 3-4
58
Forms of amino acid
Of the 20 standard amino acids, 10 are essential. Humans and mammals

cannot synthesize them by themselves and must obtain them in sufficient quantities
from their diet. The essential amino acids occur in dietary proteins of animal as well
as of plant origin.
The 20 amino acids vary considerably in their physico-chemical properties, which
gives rise to the great range of properties and functions of proteins. Commonly, they
are classified into three groups according to the polarities, i.e. the distribution of
electrons, of their side chains (residues):
1. amino acids with non-polar side chains
2. amino acids with uncharged polar side chains
3. amino acids with charged polar side chains (negative / positive charge)
After a protein has been synthesized it has to fold in a specific way to achieve its
characteristic properties. The structure of a protein is determined by the side chains
or residues of its amino acids. Non-polar side chains prefer non-aqueous
environments and are found usually inside the protein whereas polar and charged
side chains are found on the outside where they interact with the water molecules of
their liquid environment.
59
Besides the standard amino acids also nonstandard amino acids are found in
proteinsp1.htm. They result from the specific modification of a standard amino acid
residue after the polypeptide chain has been synthesized. Examples are 4hydroxyproline and 5-hydroxylysine found in collagen and carboxyglutamic acid
found in proteins involved in blood clotting.
Other nonstandard amino acids and derivatives of standard amino acids are not
components of proteins but have various biologically important functions:
as building blocks of the genes: the nucleotides
as chemical messengers (hormones and neurotransmitters) in the
communication between cells, e.g. aminobutyric acid (GABA), dopamine,
epinephrine, histamine, thyroxine
as important intermediates in various metabolic processes, e.g. citrulline
and ornithine (urea cycle), homocysteine (amino acid metabolism), Sadenosylmethionine (biological methylating reagent), creatine (energy
reservoir in muscles), carnitine (fatty acid metabolism), glutathione
(detoxification reactions)
as oxygen-binding substance in red blood cells and the electron-transport
chain: heme
AMINO ACIDS WITH NON-POLAR SIDE CHAINS
60
AMINO ACIDS WITH UNCHARGED POLAR SIDE CHAINS
AMINO ACIDS WITH CHARGED POLAR SIDE CHAINS
61
62
63
Structure of proteins
Figure 3-5
Levels of protein structure
64
Experiment
Part A : Qualitative analysis of amino acids and proteins
Experiment 3.1 Qualitative analysis of free -amino group by Ninhydrin test

Principle :
Ninhydrin (Triketohydrindene hydrate) undergoes an oxidation-reduction
reaction with free amino groups, oxidatively deaminating them to carbonyl groups
and ammonia.
The reduced form of ninhydrin couples with ammonia and the
residual ninhydrin to give rise to a blue-violet dye.

This is the essence of the ninhydrin color reaction, which is positive for all free
amino groups, whether in amino acids, peptides, or proteins.
By the way, the ninhydrin reagent also reacts with imino acids such as praline
to yield a yellow product.
Materials :
1.
1% glycine
2.
1% urea
3.
1% cysteine
4.
1% casein
5.
Diluted egg white solution in 0.85% saline (1:10)
6.
0.1 M sodium acetate buffer pH 5.0
7.
Ninhydrin solution : 4.0 % ninhydrin in 95% ethanol
8.
Water bath, 100o C controlled
65
Methods :
Prepare 6 test tubes and add the following solution (using pipet).
Solution (ml)
Tube no.
1
1.0
1% glycine
1.0
1% cysteine
1.0
1% urea
1.0
1% casein
1.0
Diluted egg white solution
1.0
2.0
2.0
2.0
2.0
2.0
2.0
1% Ninhydrin solution
1.0
1.0
1.0
1.0
1.0
1.0
Distilled water
in 0.85% saline (1:10)
Mix well , then boil in water bath at 100 oC for 5 minutes.

Observe and report the results (compare with Tube no. 1).
(Tube 1 = control tube)
Experiment 3.2 Qualitative analysis of peptide bonds by Biuret test

Principle :
When substances containing too or more peptide bonds (e.g. Proteins) react
with the biuret reagent, copper sulfate in alkaline condition, a purple color complex is
formed.
The colored product is the result of coordination of Cu2+ with nitrogen
atoms peptide (Cu2+ reacts with the peptide bond). This complex can also absorbs
66
light maximally at 550 nm. The amount of product formed depends on the
concentration of protein.
Figure 3-6 The purple complex of Biuret test

Materials :
1.
1% glycine
2.
1% urea
3.
1% cysteine
4.
1% casein
5.
6.
7.
Biuret reagent :
weigh 1.5 g of CuSO4.5H2O

and 6.0 g of sodium potassium tartrate
(NaKC4H4O6) and solve in 500 ml of distilled
water. Then add slowly 300 ml of 10% NaOH
and stirr gently all the time and make the final
volume of the solution to 1 L by distilled water.
(Note : Add 1.0 g of KI if precipitated Cu2O is
presented.)
Methods
67
Solution (ml)
Tube no.
1
1.0
1% glycine solution
1.0
1% cysteine
1.0
1% urea
1.0
1% casein
1.0
1.0
4.0
4.0
4.0
4.0
4.0
4.0
Distilled water
in 0.85% saline (1:10)

Biuret reagent
Mix well , then leave at room temperature for 5 minutes.

Experiment 3.3 Qualitative analysis of R group reaction by Sulfur test

Principle :
Sulfur test is use to test the side chain of amino acid reaction especially with
amino acid which is sulfer containing side chain .
Materials :
1.
1% glycine
2.
1% methionine
3.
1% cysteine
4.
1% casein
5.
6.
40% NaOH
68
Methods :
Solution (ml)
Tube no.
1
0.5
1% glycine solution
0.5
1% cysteine
0.5
1% methionine
0.5
1% casein
0.5
0.5
2.5
2.5
2.5
2.5
2.5
2.5
drop
drop
drop
drop
drop
drop
Distilled water
in 0.85% saline (1:10)

Distilled water
40% NaOH
Mix well and add 2 drops of Lead acetate in every tube.

Mix well , then boil in water bath at 100 oC for 5 minutes.
69
EXPERIMENT 4
AMINO ACIDS AND PROTEINS (Part II)
Introduction
Folding behaviour of proteins
The folding of proteins to yield their native conformations is favored under
physiological conditions.
The interactions which stabilize protein conformation
include H-bonds, disulfide bridges, electrostatic interactions and complex formation

with metal ions (such as myoglobin and hemoglobin). A further stabilizing factor of
outstanding importance is the hydrophobic effect. Soluble proteins in water fold so
that the majority of the nonpolar (apolar) amino acid side chains lie as a pocket
within the molecule (and being hydrophobic environment) , whereas the polar side
chains face toward the solvent. (as summary, nonpolar in, polar out)
Figure 4-1 Folding behavior of proteins

2, 4, 6, 9 represent as nonpolar amino acids
3, 5, 8, 10 represent as polar amino acids
1 and 7 represent as amino acids containing
sulfhydry groups
70
Protein denaturation
Three-dimensional structure of a protein represents a delicate balance of
covalent and noncovalent interactions.
We must take care when isolating and
studying proteins that this balance is not disturbed and that we do not alter the
structure of the molecule in any way.
Even a slight structural alteration may result
in a significant change in the proteins properties and a decrease or loss of its

biological activity. We refer to such alterations in, or loss of, the native structure of a
protein as denaturation (the unfolding of a protein).
reversible or irreversible.
Denaturation may be
Under proper experimental conditions, the disrupted
structure can then be completely recovered. This reversal of denaturation is called

renaturation (the refolding of a protein).
Many factors can bring about denaturation. The factor that disrupts either a
covalent or noncovalent bond critical for the structure of the molecule may cause
partial or complete unfolding of the protein.
1. Heat (high temperature)
An increase in temperature favors vibrations within the molecule and the
energy of these vibrations can become great enough to disrupt the tertiary structure.
This increase in temperature disrupts H-bonds and at higher temperatures,
hydrophobic bonds as well. Heat denaturation usually results in eventual protein
precipitation as a result of destruction of the secondary structure and formation of
random aggregates.
2. Extremes of pH (Acid or Alkali)
Changes in pH alter the charges of specific functional groups in amino acid
side chains and thus affect electrostatic interactions that would normally stabilize the
protein structure. These lead to denaturation.
71
3. Ionic strength
Changing the ionic strength may break ionic interactions among amino acid
side chains. Added ions interact with oppositely charged groups on the protein and
thereby disrupt intraprotein ionic interactions.
4. Chemical agents
Detergents such as soap tend to disrupt hydrophobic interactions.
If a
detergent is charged (ionic detergent eg., sodium dodecyl sulphate (SDS)), it

can disrupt electrostatic interactions within the protein, as well.
(Note : detergents are amphiphilic molecules with a hydrophilic head region
and a long hydrophobic tail)
Denaturants (Chaotropic agents) such as guanidine hydrochloride and urea
are stronger in denaturation than detergents. They can form very strong Hbonds to the protein and can also disrupt hydrophobic interactions.
Reducing
agents
such
as
mercaptoethanol
(HS-CH2-CH2-OH)
and
dithiotreiotol or DTT (HS-CH2-CH2-SH) have ability to reduce disulfide bridges

(-S-S-) to two sulfhydryl groups (-SH). In addition, urea is usually added to
the reaction mixture to facilitate unfolding of the protein and to increase the
accessibility of the disulfides to the reducing agent. If experimental conditions
are properly chosen, the netive conformation of the protein can be recovered
when both mercaptoethanol and urea are removed.
5.
Enzymatic action
Proteinases present in crude or unpurified protein preparations often catalyze
the breakdown of proteins by hydrolyzing the peptide bonds of proteins. Since these
enzymes act more slowly at low temperatures, crude protein solutions are frequently
kept cold (0-2 oC) during the early stages of purification.
6.
Physical factors
Many physical factors can be involved in denaturation of proteins such as
mechanical agitation, sonication, UV light, pressure and so on.

72
Measurement of Protein Solution

Biochemical research often requires the quantitative measurement of protein
concentrations in solutions. Several techniques have been developed ; however,
most have limitations because either they are not sensitive enough or they are
based on reactions with specific amino acids in the protein. Since the amino acid
content varies from protein to protein, no single assay will be suitable for all proteins.
In four of the methods discussed in this section, chemical reagents are added to
protein solutions to develop a color whose intensity is measured in a
spectrophotometer. A standard protein of known concentration is also treated with
the same reagents and a calibration curve is constructed. The other assay relies on
a direct spectrophotometric measurement. None of the methods is perfect because
each is dependent on the amino acid content of the protein. However, each will
provide a satisfactory result if the proper experimental conditions are used and/or a
suitable standard protein is chosen. Other important factors in method selection
include the sensitivity and accuracy desired, the presence of interfering substances,
and the time available for the assay.
Biuret Assay
When substances containing two or more peptide bonds react with the biuret
reagent, alkaline copper sulfate, a purple-blue complex is formed which can be
measured at 540-550 nm.
The color of product is the result of coordination of
peptide nitrogen atoms with Cu2+. The amount of product formed depends on the
concentration of protein.
This assay eliminates the problem of UV absorption variability. This reaction
is dependent in part on peptide bonds and not solely on amino acid moieties.
The biuret assay is simple and gives a linear correlation between protein
concentration and absorbance; however, it lacks sensitivity.
In addition, the biuret
demonstrates lsee protein-to-protein variability than UV absorbance, and it requires

the use of relatively large sample sizes. Because large amounts of material are not
73
always available, the Lowry assay, which uses the Folin reagent to increase
sensitivity, was developed.
Lowry Assay
The Lowry method relies on two different reactions. The first is the formation
of a copper ion complex with amide bonds, forming reduced copper in alkaline
solutions. This is called a "Biuret" chromophore. The second is the reduction of
Folin-Ciocalteu reagent (phosphomolybdate and phosphotungstate) by tyrosine and
tryptophan residues.
The reduced Folin-Ciocalteu reagent is blue and thus
detectable with a spectrophotometer in the range of 500-750 nm.
The Biuret
reaction itself is not all that sensitive. Using the Folin-Ciocalteu reagent to detect
reduced copper makes the assay nearly 100 times more sensitive than the Biuret
reaction alone.
The assay is relatively sensitive, but takes more time than other assays and is
susceptible to many interfering compounds. The following substances are known to
interfere with the Lowry assay : detergents, carbohydrates, glycerol, Tricine, EDTA,
Tris,
potassium
compounds,
sulfhydryl
compounds,
disulfide
compounds,
magnesium and calcium. Most of these interfering substances are commonly used
in buffers for preparing proteins. This is one of the major limitations of the assay.
The Lowry assay is sensitive to variations in the content of tyrosine and tryptophan
residues. If the protein you are assaying has an unusual content of these residues,
an appropriate substitute standard is required. The standard curve is linear in the 1
to 100 g protein region. The absorbance can be read in the region of 500 to 750
nm.
Most researchers use 660 nm, but other wavelengths also work and may
reduce the effects of contamination (e.g. chlorophyll in plant samples interferes at

660 nm, but not at 750 nm).
74
Figure 4-2 Typical Standard Curve for a Lowry Assay
Bradford Assay
The many limitations of the biuret and Lowry assays have encouraged
researchers to seek better methods for quantitation of protein solutions.
The
Bradford assay, based on protein binding of a dye (perhaps to arginine residues),

provides numerous advantages over other methods.
The Bradford assay is a very popular protein assay method because it is
simple, rapid, inexpensive and sensitive. The Bradford assay works by the action of
Coomassie brilliant blue G-250 dye (CBBG). This dye specifically binds to proteins
at arginine, tryptophan, tyrosine, histidine and phenylalanine residues in acidic
solution. It should be noted that the assay primarily responds to arginine residues
(eight times as much as the other listed residues) so if you have an arginine rich
protein, you may need to find a standard that is arginine rich as well. CBBG binds to
these residues in the anionic form, which has an absorbance maximum at 595 nm
(blue). The free dye in solution is in the cationic form, which has an absorbance
maximum at 470 nm (red).
75
The assay is monitored at 595 nm in a
spectrophotometer, and thus measures the CBBG complex with the protein.
The choice of standard for this assay is very crucial to the success of the
assay. Many investigators have noted abnormalities of using various standards with
the Bradford assay. BSA was the original standard of choice, and is standard you
are most likely to receive with the assay if purchased as a kit. However, it has been
noted that BSA has a double than normal response in the assay and may not
always be suitable. Several researchers therefore use Imunnoglobulin G (IgG) as
the preferred standard for the assay.
The CBBG dye used in the assay binds to quartz cuvettes quite strongly.
Therefore, glass or plastic cuvettes should be utilized.
Since this assay has a
general tendency to bind to cuvettes, it is highly recommended to use disposable

plastic cuvettes. This is by no means critical to the assay, but it does make cleaning
the cuvettes much more convenient.
Figure 4-3 Chemical Structure of Coommassie Brilliant Blue G-250
76
BCA Assay
The BCA assay is based on chemical principles similar to those of the biuret
and Lowry assays. The protein to be analyzed is reacted with Cu2+ (which produces
Cu+) and bicinchoninic acid (BCA). The Cu+ is chelated by BCA, which converts the
apple-green color of the free BCA to the purple color of the copper-BCA complex.
Samples of the unknown protein and relative standard are treated with the reagent,
the absorbance is read in a spectrophotometer at 562 nm and concentrations are
determined from a standard calibration curve.
This assay has the same sensitivity
level as the Lowry and Bradford assays. The BCA assay is advantageous in that it
does not interact with as many substances as the Folin-Ciocalteu reagent, especially
detergents and buffers. This assay is limited in that it interacts with most reducing
agents and copper chelators.
In general, these are not critical components of
buffers and can be easily eliminated prior to assay.
Figure 4-4 The Structure of BCA
UV Spectrophotometric Assay
Most proteins have relatively intense UV absorption centered at 280 nm. This
is due to the presence of tyrosine and tryptophan residues in the protein that can
absorb UV light at 280 nm. However, the amount of these amino acid residues
varies in different proteins so it is still only use for an estimate of protein
concentration.
77
Part B : Isoelectric property for protein precipitation

Experiment 4.1 Isoelectric property for protein precipitation
Principle :
Casein is the main protein found in milk and is present at a concentration of

about 35 g/litre. It is actually a heterogeneous mixture of phosphorus containing
proteins and not a single compound.
Most proteins show a minimum solubility at their isoelectric point (pI) and this
principle is used to isolate the casein by adjusting the pH of the milk to its pI.
Casein is also insoluble in ethanol and this property is used to remove unwanted
fat from the preparation.
Materials :
1.
0.1 M sodium acetate
2.
0.1 M acetic acid
3.
0.01 M acetic acid
4.
Fresh milk
Methods :
78
Solution (ml)
Tube no.
1
0.1 M sodium acetate
1.0
1.0
1.0
1.0
1.0
1.0
0.1 M acetic acid
4.0
2.0
1.0
0.01 M acetic acid
5.0
2.5
1.25
Distilled water
2.0
4.0
5.0
1.0
3.5
4.75
pH (the estimated value)
4.1
4.4
4.8
5.1
5.4
5.7
Milk
1.0
1.0
1.0
1.0
1.0
1.0
Mix (shake) well , then observe and compare the amount of precipitated form.
Note : The protein in this experiment is casein. What is the isoelectric point of
casein ? You can get the answer from your experiment. Please answer this
question in your report.
Part C : Protein denaturation
Experiment 4.2
Protein denaturation by chemical agent
Principle :
Proteins in their natural form are called native proteins. Any change in the
structure of a protein from its native state is called denaturation.
Materials :
1.
10 % White egg in 1% NaCl solution
2.
5 % Lead acetate solution
3.
5 % Trichloroacetic acid (TCA)
79
Methods :
Solution
Tube no. 1
Tube no. 2
Tube no. 3
2 ml
2 ml
2 ml
5 % Lead acetate
5 drops
5 % TCA
5 drops
10 % White egg
Mix well and observe the changes in color of solutions and the amount of the
precipitates in each tube. Report the results.
Part D : Quantitative analysis of amino acids and proteins
Experiment 4.3
Determination of protein concentration by Lowry method
Principle :
The Lowry (or Folin-Ciocalteu) assay is a modification of the biuret reaction to
give a more sensitive determination of protein. It has been widely used.
The
principle behind color development is identical to that of the biuret assay except that
a second reagent (Folin-Ciocalteu) is added to increase the amount of color
development.
Two reactions account for the intense blue-green color that develops :
(1)
the coordination of peptide bonds with alkaline copper (biuret reaction)

and
(2)
the reduction of the Cu2+ (cupric) ions in the reagent to Cu1+ (cuprous) by
the tyrosine and tryptophan residues in protein. The Cu1+ (cuprous) ions in
turn
reduce
the
Folin-Ciocalteu
reagent
(phosphomolybdate-
phosphotungstate) to form and intense blue-green complex .
80
Because proteins have varying contents of these two amino acids (tyrosine
and tryptophan), the amount of color development changes with different proteins,
including the bovine serum albumin (BSA) standard. Because of this, the Lowry
assay should be used only for measuring changes in protein concentration, not
absolute values of protein concentration. For example, the method is very useful for
following changes in protein content, as during purification of a protein. This assay
is used in most of the experiments because it is convenient and inexpensive. The
obvious advantage of the Lowry assay is its sensitivity, which is up to 10 times
greater than that of the measurement of absorbance at 280 nm (A280 method) and
up to 100 times greater than that of the biuret assay.
However, more time is
required for the Lowry assay.

The procedure to determine protein concentration is similar to that of the
biuret assay. A standard curve is prepared with bovine serum albumin (BSA) or
other pure protein and the concentration of unknown protein solutions is determined
from the graph.
Materials :
1.
Standard protein solution

the standard protein used in this experiment is Bovine Serum Albumin (BSA) 0.2 mg/ml
2.
Diluted Folin reagent

Dilute the commercial reagent with an equal volume of water on the
day of use (dilute from stock 1:1 with distilled water). This is a
solution of sodium tungstate and sodium molybdate in phosphoric
and hydrochloric acids.
3.
Alkaline CuSO4 solution

Prepare freshly on the day of use by mixing 50 ml of (3.1) and
1 ml of (3.2)
3.1)
81
Alkaline sodium carbonate solution

(20 g/litre Na2CO3 in 0.1 M NaOH)
3.2)
Copper sulphate-sodium potassium tartrate solution

(5 g/litre CuSO4.5H2O in 10 g/litre sodium potassium tartrate)
Methods :
Prepare 8 test tubes and add the following solution (using pipet). And run by this
protocol. The absorbance measured in this method is at 750 nm.
Solution (ml)
Tube no.
1
0.2 mg/ ml BSA
0.2
0.4
0.6
0.8
1.0
Unknown
0.2
0.5
Distilled water
1.0
0.8
0.6
0.4
0.2
0.8
0.5
Alkaline CuSO 4
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
0.5
0.5
Mix well and leave at room temperature for 10 minutes

Diluted Folin reagent
0.5
0.5
0.5
0.5
0.5
0.5
Immediately mix and then leave at room temperature for 30 minutes

A750
Protein (g)
-
Tube 1 is blank .
Make a standard curve (A750 vs [protein] in unit g) from tube no. 1-6.
Determine [protein] of unknown (tube 7, 8) by using standard curve and the

formula A = k.c.l
82
EXPERIMENT 5
LIPID
Objectives:
1. To study some properties of lipids and detergent
2. To understand the principle and quantitative analysis of lipids.
Introduction:
Lipid or fat is a biomolecule, which differs from the carbohydrates and the
proteins in being insoluble in water and soluble in certain organic solvents (ether,
benzene, chloroform etc.). The fat depots of animals contain mainly neutral fat, by
which is meant esters of glycerol with three fatty acid molecules. In contrast, most
cells other than adipose tissue contains much less fat; their lipids consist largely of
phospholipids and chloresterol. The classification of lipids is divided into 3 groups.
1. Simple lipids are ester of fatty acids and alcohol. There are two types of
simple lipid.
1.1 Glyceride is ester of fatty acid and glycerol. The storage lipid of animals
and a high percentage of the lipid material in their diet consist of
triglyceride or neutral fats. Fat is glyceride, which is solid form at room
temperature, but glyceride that is liquid at room temperature is called oil
83
Figure 5-1 : Triglyceride

Most of the glycerides found in nature are mixed, that is R1, R2, and R3 are all
different, or only two of the three are the same. Fatty acids found in nature almost
always have straight chains and the number of carbon atoms is about 4-30 atoms.
The low fatty acids are soluble in water, but the property decreases with increasing
chain-length as well as type of bonding such as number of single bond (saturated
fatty acids) or number of double bond (unsaturated fatty acids) are soluble in water,
but the property decreases with increasing chain-length as well as type of bonding
such as number of single bond or number of double bond.
1.2 Waxes consist of fatty acids esterified to alcohols other than glycerol.
One of the most important of these alcohols is cholesterol, a number of the
steroid series. The formula for cholesterol, a common wax found in blood
plasma is:
Figure 5-2 : Cholesterol
84
Chloresterol esters occur only in small amounts in animal tissues under

normal conditions, except in the adrenal glands and liver.
2. Compound lipid
There are 3 major types of compound lipid that are:
a. Phospholipids
b. Sphingolipids
c. Lipoprotein
Figure 5-3 : Phospholipid

3. Derived lipid
Steroids and fat-soluble vitamins are classified as derived lipid since they can
be inactivated by saponification reaction, the structure therefore is not ester
type but it is an alcohol type.
85
Experiment 5.1 Solubility property

Principle:
The lipid material occurring in nature is insoluble in water and soluble in
certain organic solvents. In water the effect of oil on rough water is a consequence
of a large reduction in the waters surface tension as emulsion form. But in some
lipid can aggregate to form micelles, bilayer and liposome structure.
Figure 5-4 : Form of lipids
Materials:
1. Animal oil
2. Plant oil
3. Chloroform
4. Benzene
5. Ethanol
6. 0.05 M NaOH
7. 0.05 M HCl
86
Methods:
1. Pipitte 2 ml of each organic solvent, alkaline and acid solution into three
separated test tubes (prepare 2 sets)
2. Add 2 drops of each oil in to each solvent and solution in 1
3. Mix well and report the solubility in each solvent and solution.
Experiment 5.2 Test of degree of unsaturation

Principle:
Since halogens react by addition to linkages (or double bond), the iodine
number is a measure of the degree of unsaturation of fat. For triglycerides consisting
largely of C18 fatty acids.
Materials:
1. Animal oil
2. Plant oil
3. Chloroform-Ethanol (1:1) mixture
4. 1 M Acetic acid
5. 10% KI solution
Methods:
1. Pipette 1 ml of chloroform-ethanol mixture into 3 test tubes.
2. Add 1-2 drops of distilled water into tube 1, animal oil into tube 2 and plant oil
into tube 3 then mix well.
3. Add 2 ml of acetic acid and 1 drop of 10% KI then mix and leave at room
temperature for 5 min.
4. Compare the iodine color in each tube using number 1 as control or blank.
87
Experiment 5.3 Properties of soap and detergent

Principle:
Soap is potassium or sodium salts of fatty acid that is prepared by
saponification of triglyceride.
Salt of fatty acid or soap is amphiphatic molecule which consist of polar
portion (hydrophilic) and non-polar portion (hydrophobic).
glycerol
soap
Figure 5-5 : Saponification

The amphiphatic structure can aggregate to form micelle, which the polar
portion localizes at the outer phase while the non-polar portion is in the inner phase.
This molecular arrangement eliminates unfavorable contacts between water
and the hydrophobic tails of lipid part that increase the solubility of the molecule.
Although sodium and potassium soaps are soluble in water, magnesium and calcium
soaps are not. Therefore in hard water contain high concentration of Ca2+ , Mg2+
ions, the amphiphatic structure of soap is not effective compare to sodium or
potassium soap. Detergent is more effective in calcium and magnesium solution,
since calcium or magnesium detergent is soluble in water.
88
Materials:
1. 1% soap solution
2. 1% detergent solution
3. 0.5% CaCl2 solution
4. 0.5% MgCl2 solution
5. 0.5% FeCl3 solution
Methods:
1. Take 25 ml of soap into 4 beakers and 25 ml of detergent into another 4
beakers.
2. Add 5 ml of 0.5% CaCl2 into the first soap beaker and detergent beaker,
mix well.
3. Add 5 ml of 0.5% MgCl2 into the second soap beaker and detergent
beaker, mix well.
4. Add 5 ml of FeCl3 into the third soap beaker and detergent beaker, mix
well.
5. Add 10 drops of oil into the forth soap beaker and detergent beaker, mix
well.
6. Observe and report the characteristics of soap and detergent in each
beaker.
89
DATA NOTE
Experiment 5 : Lipids
5.1 Solubility property

Type of lipids
Type of solvent
Animal oil
Plant oil
Distilled water
Chloroform
Ethanol
Benzene
0.05 M NaOH
0.05 M HCl
5.2 Test for degree of unsaturation
Sample
Colors / Degree of unsaturatation
Animal oil
Plant oil
5.3 Property of soap and detergent

Test
Soap
Detergent
0.5% CaCl2
0.5% MgCl2
0.5% FeCl3
Oil
90
EXPERIMENT 6
NUCLEIC ACID
Objectives:
To isolate nucleic acid material (deoxyribonucleic acid, DNA)
Introduction:
The nucleic acids are high polymer of strongly acidic character. Their
monomeric components are mononucleotides, consisting of base, sugar, and
phosphoric acid. Two series of bases are involved (purines and pyrimidines) and two
pentose sugars (ribose and deoxyribose) and that can be devided into 2 types;
deoxyribonucleic acid (DNA), ribonucleic acid (RNA). DNA is confined to the nucleus
of the cell, whereas RNA is found in both the nucleus and the cytoplasm, in the latter
location it occurs in the mitochondria, microsomes and particle-free fraction.
Figure 6-1 : Structure of nucleic acid

91
Figure 6-2 : Structure of purines and pyrimidines
Figure 6-3 : DNA double helix
The quantitative and qualitative analysis of DNA can be performed in many

ways such as spectrophotometric measurement, gel electrophoresis, chemical
reaction, etc.
92
Experiment : Isolation of high molecular weight DNA

Principle :
Nucleic acids occur naturally in a complex form with proteins. By using an
ionic detergent and concentrated salts, proteins can be dissociated from nucleic
acids. Chelating agents are often added to remove polyvalent metal and the pH is
adjusted to a mild alkaline condition where proteins have lowered electrostatic
interaction with nucleic acids. The released proteins can be removed by precipitation
with saturated salt (NaCl) or can be extracted with phenol. The deproteinized DNA
can be precipitated in ethanol that exhibits fibrous precipitated form.
Materials :
1. Chicken red blood cells
2. Lysis buffer (10 mM Tris HCl, 400 mM NaCl, 2mM EDTA)
3. 10% SDS
4. Protease solution (100 mg/ml)
5. 6 M NaCl
6. TE buffer (10 mM Tris HCl, 1mM EDTA)
7. Ethanol
Methods :
1. Transfer 0.5 ml of chicken red blood cells into flask.

2. Add 12 ml lysis buffer and mix well.
3. Add 0.9 ml 10% SDS and mix by vigorous for 10 min.
4. Add 0.2 ml protease solution, mix well and incubate at 37 C for 30 min.
5. Add 4.5 ml 6 M NaCl and mix well.
6. Transfer the supernatant into beaker and measure the volume of

supernatant.
7. Add 2 volume of ethanol and mix well.
8. Transfer the precipitated DNA by using stirring rod then dissolves the
DNA in 10 ml TE buffer.
93
94
EXPERIMENT 7
CARBOHYDRATE
Objectives
1. To study the chemical reaction of various types of carbohydrate.
2. To learn how to test and detect each type of carbohydrate.
3. To understand the principle quantitative and qualitative analysis of
carbohydrate.
Introduction
Carbohydrates are carbon compounds that contain large quantities of
hydroxyl groups. The simplest carbohydrates also contain either an aldehyde moiety
(these
are
termed
(polyhydroxyketones).
polyhydroxyaldehydes)
All
carbohydrates
can
or
be
ketone
classified
as
moiety
either
monosaccharides, oligosaccharides or polysaccharides. Anywhere from two to ten

monosaccharide units, linked by glycosidic bonds, make up an oligosaccharide.
Polysaccharides are much larger, containing hundreds of monosaccharide units.
The presence of the hydroxyl groups allows carbohydrates to interact with the
aqueous environment and to participate in hydrogen bonding, both within and
between chains.
Moreover, derivatives of the carbohydrates can contain nitrogens, phosphates
and sulfur compounds. Carbohydrates also can combine with lipid to form glycolipids
or with protein to form glycoproteins.
95
Carbohydrate Nomenclature
The predominant carbohydrates encountered in the body are structurally
related to the aldotriose glyceraldehydes and to the ketotriose dihydroxyacetone.
All carbohydrates contain at least one asymmetrical (chiral) carbon and are,
therefore, optically active. In addition, carbohydrates can exist in either of two
conformations, as determined by the orientation of the hydroxyl group about the
asymmetric carbon farthest from the carbonyl. With a few exceptions, those
carbohydrates that are of physiological significance exist in the D-conformation.
The mirror-image conformations, called enantiomers, are in the L-conformation.
D-Glyceraldehyde
L-Glyceraldehyde
Figure 7-1 Structures of glyceraldehyde enantiomers
96
Classification of carbohydrates
Carbohydrate is classified into 3 groups.
1.
Monosaccharide or sugar
It is a smallest carbohydrate which cannot be broken down to simple
substances by acid hydrolysis.
There are many types of monosaccharides
can be found in nature depending on number or length of the carbon atoms

and position of hydroxyl (-OH) generating types of aldehyde or ketone, the
isomer forms such as triose (3C), tetrose (4C), pentose (5C), hexose (6C)
etc., for instance, glucose is an aldohexose but fructose is a ketohexose.
Anyway, the major monosaccharides contain four to six carbon atoms.
Table 7-1 Monosaccharide classifications
Number of carbons
Category name
Relevant examples
Triose
Glyceraldehyde,
Dihydroxyacetone
Tetrose
Erythrose
Pentose
Ribose, Ribulose, Xylulose
Hexose
Glucose, Galactose,
Mannose, Fructose
Heptose
Sedoheptulose
Nonose
Neuraminic acid
(also called Sialic acid)
97
Figure 7-2 Monosaccharides : Glucose, Fructose, Galactose and Mannose
The aldehyde and ketone moieties of the carbohydrates with five and six
carbons will spontaneously react with alcohol groups present in neighboring carbons
to produce intramolecular hemiacetals or hemiketals, respectively. This results in
the formation of five- or six-membered rings. Because the five-membered ring
structure resembles the organic molecule furan, derivatives with this structure are
termed furanoses. Those with six-membered rings resemble the organic molecule
pyran and are termed pyranoses.
Such structures can be depicted by either
Fischer or Haworth style diagrams. The numbering of the carbons in carbohydrates

proceeds from the carbonyl carbon, for aldoses, or the carbon nearest the carbonyl,
for ketoses.
Cyclic Fischer Projection of

-D-Glucose
98
Haworth Projection of
-D-Glucose
Figure 7-3 Types of glucose structure diagrams

The rings can open and re-close, allowing rotation to occur about the carbon
bearing the reactive carbonyl yielding two distinct configurations ( and ) of the
hemiacetals and hemiketals. The carbon about which this rotation occurs is the
anomeric carbon and the two forms are termed anomers. Carbohydrates can
change spontaneously between the and configurations-- a process known as
mutarotation. When drawn in the Fischer projection, the configuration places the
hydroxyl attached to the anomeric carbon to the right, towards the ring. When drawn
in the Haworth projection, the configuration places the hydroxyl downward.
The spatial relationships of the atoms of the furanose and pyranose ring
structures are more correctly described by the two conformations identified as the
chair form and the boat form. The chair form is the more stable of the two.
Constituents of the ring that project above or below the plane of the ring are axial
and those that project parallel to the plane are equatorial. In the chair conformation,
the orientation of the hydroxyl group about the anomeric carbon of -D-glucose is
axial and equatorial in -D-glucose.
99
Figure 7-4 Chair form of -D-Glucose
2.
Oligosaccharide
Covalent bonds between the anomeric hydroxyl of a cyclic sugar and
the hydroxyl of a second sugar (or another alcohol containing compound) are
termed glycosidic bonds, and the resultant molecules are glycosides. The
linkage of two monosaccharides to form disaccharides involves a glycosidic bond.
Several physiogically important disaccharides are sucrose, maltose and lactose.
Sucrose: prevalent in sugar cane and sugar beets, is composed of
glucose and fructose through an -(1,2)-glycosidic bond.
Maltose: the major degradation product of starch, is composed of 2
glucose monomers in an -(1,4) glycosidic bond.
Lactose: is found exclusively in the milk of mammals and consists of
galactose and glucose in a -(1,4) glycosidic bond.
100
Figure 7-5 Many types of disaccharides
3.
101
Polysaccharide
Most of the carbohydrates found in nature occur in the form of high molecular
weight polymers called polysaccharides. The monomeric building blocks used to

generate polysaccharides can be varied; in all cases, however, the predominant
monosaccharide found in polysaccharides is D-glucose. When polysaccharides are
composed
of
single
monosaccharide
building
block,
they
are
termed
homopolysaccharides. Polysaccharides composed of more than one type of

monosaccharide are termed heteropolysaccharides.
Starch is the major form of stored carbohydrate in plant cells. Starch is a
polymer of glucose bonded by -linkages. Its structure is identical to glycogen,
except for a much lower degree of branching (about every 20-30 residues).
Unbranched starch is called amylose (amylose consists of a linear chain of
several hundred glucose molecules) and branched starch is called amylopectin
(amylopectin is a branched molecule made
of several thousand of glucose units)
Starch is insoluble in water. It can be digested by hydrolysis catalyzed

by enzyme called amylase, which can break the -linkages. Humans
and
other
animals have amylases, so they can digest starches. Potato, rice, wheat, and maize
are major sources of starch in the human diet.
Cellulose is a long-chain polymer polysaccharide carbohydrate, of glucose. It forms the primary structural component of plants and is not digestible by
humans. Humans and many other animals lack an enzyme to break the -linkages,
so they do not digest cellulose. Certain animals can digest cellulose, because
bacteria possesing the enzyme are present in their gut.
102
Glycogen is the storage form of glucose in animals. It is a branched

polymer of glucose. This crucial molecule is a homopolymer of glucose in -(1,4)
linkage ; it is also highly branched, with -(1,6) branch linkages occurring every 8-10
residues. Glycogen is a very compact structure that results from the coiling of the
polymer chains. This compactness allows large amounts of carbon energy to be
stored in a small volume, with little effect on cellular osmolarity.
Figure 7-6 Many types of polysaccharides

103
General property of carbohydrate

There are 2 chemical reactions that can be used to detect carbohydrate in solution ;
Furfural-forming property and Reducing property.
1.
Furfural-forming property
Carbohydrates, in the presence of non-oxidizing acids, undergo
dehydration to form furfural or hydroxyfurfural since these aldehydes will condense

with aromatic amines and phenols to give intensely colored compounds, furfural
formation can be used as a qualitative or quantitative test for carbohydrates.
By
employing vigorous conditions all carbohydrates can be made to react, the test then
becomes general. Under milder conditions only certain classes of compounds will
react, thus giving rise to more specific tests.
In the presence of strong sulfuric acid all of the sugars and their
polymers give positive reactions.
When -naphthol is the coupling reagent
the test is known as the Molischs reaction.
Orcinol and resorcinol also
give similar reactions and have been used for quantitative estimations so
called Bials test and Seliwanoffs test, respectively.
These tests show positive for all carbohydrates.
give
Monosaccharides
a rapid positive test. Disaccharides and polysaccharides react
slower.
2.
Reducing property
In the natural state, the aldehydes and ketones group exists in a
condensed form, as a hemiacetal, i.e. furanose (6-ring) or pyranose (5-ring).
However, in the water solution the ring structure is open to be open-chain
structure producing free form of aldehydes or ketone.
In case of aldose
sugar, aldehydes (-CHO) has reducing property that is able to reduce
104
oxidizing agent such as cupric ion (Cu2+) to form precipitated product of

cuprous oxide (Cu2O).
In case of ketose sugar, for instance, fructose in
alkaline condition can be changed and generated glucose and mannose

which are aldose structure as shown in the figure below.
This phenomena is
called tautomerism.
The most commonly used test for the detection of reducing sugars
involves heating the sample with an alkaline reagent containing cupric copper
held in solution by some type of complexing agent.
Potential aldehyde or
ketone groups are oxidized and the copper is reduced to the red cuprous
oxide.
Experiment
PART A : Qualitative analysis of carbohydrates

Experiment 7.1
Qualitative test for carbohydrate by furfural forming

property (Molischs test)
Principle :
The test reagent dehydrates pentoses to form furfural (top reaction) and
dehydrates hexoses to form 5-hydroxymethyl furfural (bottom reaction). The furfurals
further react with -naphthol present in the test reagent to produce a purple product.
A positive test is indicated by the formation of a purple product at the interface of the
two layers
105
Materials :
1. Molischs reagent ( -napthol in 95% ethanol)
2. Sulfuric acid
3. 1% ribose
4. 1% glucose
5. 1% fructose
6. 1% lactose
7. 1 % sucrose
8. 1 % glycogen
9. 1 % starch
Methods :
1. Pipette 1 ml of each carbohydrate solution for the test into each test
tube.
2. Prepare 1 test tube as a control by adding 1 ml of distilled water instead of
carbohydrate solution.
3. Add 2-3 drops of Molischs reagent into each tube.
106
4. Pour the solution slowly into a tube containing 2 ml of concentrated

sulfuric acid, so that two layers form. Be careful of the conc. acid !! and
please do it in the hood.
5. Observe the formation of purple ring at the interface of two layers.
Experiment 7.2
Qualitative test for carbohydrate by furfural forming

property (Seliwanoffs test)
Principle :
Ketoses, when heated with hydrochloric acid and resorcinol, produce a bright
red color, while aldoses produce a red color slower than ketoses. The Seliwanoffs
test, therefore, is suitable for distinguish ketose sugars from aldose sugars.
Under the condition of this test, any ketoses which may be bound in
glycosidic linkanges, will be liberated to give a positive reaction, eg. the fructose
portion of sucrose.
Materials :
1. Seliwanoffs reagent (0.05 % resorcinol in 3 M HCl)
2. 1 % ribose
3. 1 % glucose
4. 1 % fructose
5. 1 % lactose
6. 1 % sucrose
7. 1 % glycogen
8. 1 % starch
9. water bath, 100oC controlled
Methods :
1. Pipette 1 ml of each carbohydrate solution for the test (1%ribose, 1%
glucose, 1% fructose,1% lactose,1% sucrose,1% glycogen and 1%

starch) into each test tube.
107
2. Prepare 1 test tube as a control by adding 1 ml of distilled water instead of

carbohydrate solution.
3. Add 4 ml of Seliwanoffs reagent into each tube and mix well.
4. Boil these solutions in water bath at 100oC
5. Every 1 minute, observe the color in every tube and record the data
6. Follow the color reaction until 5 minutes.
Experiment 7.3
Qualitative test for carbohydrate by reducing property

(Benedicts test)
Principle :
Ketoses and aldoses can react with Benedicts reagent in alkaline condition to
produce the precipitate cuprous oxide. The positive reactions of those sugars are
called reducing sugar.
If a suspension of copper hydroxide in alkaline solution is heated, then black
cupric oxide is formed ;
Cu(OH)2 CuO + H2O
However, if a reducing substance is present, then rust-brown cuprous
oxide is precipitated ;
2 Cu(OH)2 Cu2O + 2 H2O + O2
Carbohydrates with a free or potentially free aldehyde or ketone group have
reducing properties in alkaline solution.
In addition, monosaccharides act as
reducing agents in weakly acid solution.

The Benedicts reagent is also sensitive to other aldehyde compounds, formic
acid, hydrozobenzene, phenols, phenylhydrazine, pyrogallol and uric acid.
Therefore the Benedicts test is not a very specific test, however the test can be led
to the reducing property of sugar.
108
Materials :
1. Benedicts reagent
(Dissolve 173 g of sodium citrate and 100 g sodium carbonate in about 800
ml of warm water.
Filter through a fluted filter paper into a 1000 ml
measuring cylinder and make up to 850 ml with water. Meanwhile, dissolve

17.3 g of copper sulphate in about 100 ml of water and make up to 150 ml.
Pour the first solution into a 2 litre beaker and slowly add the copper
sulphate solution with stirring)
2. 1 % ribose
3. 1 % glucose
4. 1 % fructose
5. 1 % lactose
6. 1 % sucrose
7. 1 % glycogen
8. 1 % starch
9. water bath, 100oC controlled
Methods :
1.
Pipette 1 ml of each carbohydrate solution for the test (1%ribose, 1%

glucose, 1% fructose,1% lactose,1% sucrose,1% glycogen and 1%
starch) into each test tube.
2.
Prepare 1 test tube as a control by adding 1 ml of distilled water

instead of carbohydrate solution.
3.
Add 5 ml of Benedicts reagent into each tube and mix well.
4.
Boil these solutions in water bath at 100oC for 5 minutes.
5.
Observe and record the color in every tube .
Experiment 7.4
109
Qualitative test for polysaccharide by Iodine test
Principle :
Certain polysaccharides give characteristic colors when treated with iodine
solution.
The amylose fraction of starch, for example, gives a deep blue color,
whereas the reaction with amylopectin is red to purple. Although the usual sample
of starch handled in the student laboratory contains more amylopectin that amylose,
the latter substance combines with much more iodine and produces such an intense
color that the typical reaction to the iodine test for starch is the appearance of
intense blue color. Other polysaccharides react to a lesser extent with iodine to form
a red-brown or reddish-purple color. Glycogen gives a red brown color with iodine,
and cellulose does not react.
Mono- and disaccharides are too small to trap the
iodine molecules, so they do not show a color change in the presence of iodine.
Hydrolysis of starch proceeds through a series of dextrin of presumably
decreasing molecular wieght, reacting in the iodine test to give color changing from
blue through violet to red-brown (erythrodextrins) to colorless (achrodextrins).
Materials :
1.
Iodine solution (0.005 M I2 in 3 % KI)
2.
1 % glycogen
3.
1 % starch
4.
1 % cellulose
5.
1 % glucose
6.
1 % sucrose
7.
1 % unknown solution
Methods :
1.
Pipette 3 ml of each carbohydrate solution and unknown solution for

the test into each test tube.
2.
Prepare 1 test tube as a control by adding 3 ml of distilled water

instead of carbohydrate solution.
110
3.
Add 1 drop of iodine solution into each tube and mix well.
4.
Observe and record the color in every tube .
5.
Add 3-5 drops of diluted HCl into each tube, mix well and then boil in
the water bath for 5-10 minutes (Acid and heat hydrolysis). Observe
the changes in color and discuss the results.
PART B : Quantitative analysis of carbohydrates
Experiment 7.5
Determination of reducing sugars using

3, 5-dinitrosalicylic acid (DNS)
Principle :
Several reagents have been employed which assay sugars by using their
reducing properties. One such compound is 3, 5-dinitrosalicylic acid (DNS) which in
alkaline solution is reduced to 3-amino-5-nitrosalicylic acid.
The chemistry of the reaction is complicated since standard curves do not

always go through the origin and different sugars give different color yields. The
method is therefore not suitable for the determination of a complex mixture of
reducing sugars.
Materials :
1.
Sodium potassium tartrate.

(Dissolve 300 g of this salt in about 500 ml of water)
2.
3, 5-Dinitrosalicylic acid
(Dissolve 10 g of this reagent in 200 ml of 2 M NaOH)
3.
111
Dinitrosalicylic acid reagent

(Prepare this fresh by mixing solutions (1) and (2) together
and mixing up to 1 litre with water)
4.
2 M of Sodium hydroxide (NaOH)
5.
Stock sugar standards

(glucose, fructose and maltose 1 g/litre solutions in
saturated benzoic acid)
6.
Working sugar standards

(glucose, fructose and maltose stock solutions diluted 1 in 4
before use to give solutions containing 250 g/ml)
7.
Some sugar solutions of unknown concentration
8.
Boiling water baths
9.
Marbles
Methods :
Prepare the fresh DNS reagent just before use by mixing the stock solution as
indicated and add 1 ml of the reagent to 3 ml of the sugar solution in a test tube.
Prepare a blank by adding 1 ml of the reagent to 3 ml of distilled water. Cover each
tube with a marble and place in a boiling water bath for 5 minutes, cool to room
temperature and read the absorbance at 540 nm against the blank. Please note that
all the tubes must be cooled to room temperature before reading since the
absorbance is sensitive to temperature.
Prepare standard curves of the sugars provided and use them to estimate the
concentration of the unknown provided. Comment on the results.
112
DATA NOTE
Experiment 7 : Carbohydrate
Experiment 7.1 and 7.2
Qualitative test for carbohydrate by furfural forming property
Solution
Results of
Results of
Molischs test
Seliwanoffs test
Distilled water
1 % ribose
1 % glucose
1 % fructose
1 % lactose
1 % sucrose
1 % glycogen
1 % starch
Experiment 7.3
Qualitative test for carbohydrate by reducing property (Benedicts test)
Solution
Results
Distilled water
1 % ribose
1 % glucose
1 % fructose
1 % lactose
1 % sucrose
1 % glycogen
1 % starch
113
Experiment 7.4
Qualitative test for polysaccharide by Iodine test
Solution
Color after dropping
Color after dropping of
of iodine solution
diluted HCl and boiling
Distilled water
1 % glycogen
1 % starch
1 % cellulose
1 % glucose
1 % sucrose
1 % unknown
114
EXPERIMENT 8
ENZYME
Objectives
1. To understand kinetic and enzyme catalytic activity
2. To study the factors involved in enzymatic reaction
Introduction
The energy evolved in many processes is stored, and utilized by the cells for
those of many functions, the totality of which we call life. This is accomplished by
degrading chemical compounds of relatively high potential energy to products of low
potential energy. Certain limitations are placed upon the chemical reactions of the
body, owning to the necessity of preserving a physiological environment in the
tissues.
In the body, such a reaction goes on smoothly and rapidly under the
restricted conditions enumerated above.
This remarkable state of affairs is
explained by the presence in the body of a group of powerful catalysts, the enzymes.
The enzymatic activities in the living system require many factors for their activities
that are enzyme concentration, substrate specificity, co-factors and environmental
conditions such temperature, pH etc. In practical, determining of product increasing
or substrate decreasing with time can follow rate of enzyme activity, so called
kinetic.
They study of enzyme kinetic of -amylase is one of the simply reaction
which is followed the zero-order of kinetic equation. -Amylase is an enzyme which

hydrolyse only the -1,4 glycosidic bond of polysaccharide.
Starch and glycogen
are polysaccharide which most of the linkages are -1,4 type while cellulose is
consist of -1,4 type. The enzyme activity therefore hydrolyses starch and glycogen
but do not hydrolyse cellulose.
115
This enzyme is found in salivary and pancreases
that are called salivary -amylase and pancreatic -amylase.
Initial velocity (vo)

The rate of any reaction could be determined from 2 ways.
1.
The disappearance of substrate as the reaction proceeds

(Substrate decreasing).
2.
The appearance of product (Product formation).
However in general, it is considered a better technique to measure the

appearance of something from an initial value of nothing rather that the
disappearance of something from an initial large value. So, it is easier to measure
rate of reaction by monitoring the formation of product .
The rate of an enzyme-catalyzed reaction is often called its velocity.
Enzyme velocities are normally reported as initial velocity (vo).
A typical plot of
product formed [P] against time for an enzyme-catalyzed reaction shows an initial
period of rapid product formation which gives the linear portion of the plot . This is
followed by a slowing down of the enzyme rate as substrate is used up (all the
reactant(s) have been converted into products or ,more commonly, a chemical
equilibrium has been set up, with the rate of the forward reaction now being equal to
the rate of the back reaction). vo is obtained by drawing a straight line through the
linear part of the curve, starting at the zero time-point. The slope of this straight line
is equal to vo.
116
Michaelis Menten model

In 1913, L. Michaelis and Menten proposed the mechanism of enzymatically
catalyzed reactions that The enzyme (E) combines with its substrate (S) to form
an enzyme-substarte (ES) complex. The ES complex can dissociate again to form E
+ S, or can proceed chemically to form E and the product (P). The MichaelisMenten model uses the following concept of enzyme catalysis
k1
E+S
k2
ES
E+P
k-1
where the rate constants k1, k-1, k2 describe the rates associated with each step of
the catalytic process. It is assumed that there is no significant rate for the backward
reaction of E + P being converted to ES complex (because it is rate determination
step)
At low substrate concentrations , vo is directly proportional to [S], while at high
substrate concentration, vo tends towards a maximum velocity (Vmax), that is the rate
becomes independent of [S].
The Michaelis-Menten equation
vo
= Vmax [S]
Km + [S]
Where Km = (k-1 + k2) / k1

The equation describes a hyperbolic curve of vo against [S] .
The Michaelis constant or Km
-
the sum of the rates of breakdown of the ES complex over its rate of
formation or (k-1 + k2) / k1.
117
For many enzymes, k-1 is much greater than k2.
Under these
circumstances, Km becomes a measure of the affinity of an enzyme for

its substrate since its value depends on the relative values of k1 and k-1
for ES formation and dissociation, respectively.
A high Km
indicates weak substrate binding
A low Km indicates strong substrate binding

(enzyme show high affinity to substrate)
Km can be determined experimentally (from the graph) by the fact that its
value is equivalent to the substrate concentration at which the velocity is
equal to half of Vmax. (Km = Vmax/2)
118
Lineweaver-Burk plot (Double reciprocal plot)

Since it is quite not accurate to estimate Vmax ( and hence Km )
from a
hyperbolic plot , Vmax and Km can be determined experimentally by measuring vo at

different substrate concentrations.
Then a
Lineweaver-Burk plot
or Double
reciprocal plot of 1/ vo against 1/[S] is made. This plot is a derivation of the M-M
equation :
1
vo
Vmax
Km
Vmax
[S]
which gives a straight line, with the intercept on the y-axis equal to 1/ Vmax
and the intercept on the x- axis equal to 1/Km.
The slope of the line is equal
to Km / Vmax. . This plot is also a useful way of determining how an inhibitor binds to
an enzyme.
119
Experiment
Experiment 8.1
The enzyme kinetic of -amylase
Principle :
The amylases are importance in mammalian metabolism, since their
substrates include starch and glycogen. Salivary and pancreatic amylases belong to
the group of -amylases.
These enzymes attack the interior of polysaccharide
chains, forming products of the configuration. Although some reducing sugar and
fermentable substances are produced throughout the reaction, the main course of
events is the breakdown of the polysaccharides first to dextrin, followed by a slower
phase of maltose formation. Determination of the enzyme kinetic is usually carried
out by measurement the amount of substarte decreasing with time.
In case of
starch is the substrate for -amylase. The starch solution behave as suspension or
turbid which exhibit the scattered light effect that is a beam of light projected through
such systems become visible to the observer owing to the scattering of light from the
turbid particles. The light scattering can be measured in term of absorbance using
the spectrophotometry at wavelength 400 or 650 nm.
After enzymatic digestion of
substrate (starch), the molecule is breakdown to be a smaller molecule which

decreases the colloidal property of substrate and that will be increased the
absorbance of light.
Materials :
1. 2 % Starch solution
2. -amylase enzyme (1 mg / ml)
3. 0.05 M Tris-HCl buffer pH 6.8
4. Spectrophotometer and cuvet
5. Timer
120
Methods :
1. Set up wavelength of the spectrophotometer at 650 nm and using 0.05 M
Tris-HCl pH 6.8 as blank to adjust the zero.
2. Pipette 4 ml of 2 % starch solution into test tube.
3. Add 0.5 ml of 0.05 M Tris-HCl pH 6.8 buffer into starch solution and
mix well.
4. Add 0.5 ml of -amylase enzyme and mix well and start timer
5. Measure the absorbance every 15 second until 3 minutes of the reaction
(Note : mix the solution before every measuring)
6. Plot the absorbance as the ordinate (y-axis) and time of the reaction as
the abscissa (x-axis)
7. Calculate the rate of reaction (or initial velocity) from the slope of the plot
Experiment 8.2
pH influencing the rate of enzymatic reaction
Principle :
-Amylase enzyme being protein cannot withstand the action of strong acid or
base.
However, even over the pH range in which inactivation does not occur,
enzyme exhibit optima in its activity. Optimum pH is the pH at which an enzyme

shows maximum activity. Many enzyme exhibit a bell shaped curve when enzyme
activity is plotted against pH.
pH affects to enzyme in 2 ways :
1. Effect of pH on ionization of active site
The active site of enzyme contains amino acids and the important parts of amino
acids in binding with substrate are their side chains. If pH change, the ionization
state of amino acid side chains change and then lead to an effect on binding with
substrate.
2. Effect of pH on enzyme denaturation
Extremes of pH can leads to denaturation of enzymes because the structure of
active enzyme depends on the ionic character of amino acid side chains.
121
Materials :
1. 2 % Starch solution
2. -amylase enzyme (1 mg / ml)
7. Spectrophotometer and cuvet
8. Timer
Methods :
1. Prepare 4 test tubes and add the following solutions (using pipette)
Solution (ml)
Tube 1
Tube 2
Tube 3
Tube 4
2 % starch solution
Tris-HCl buffer pH 5.0
-amylase (see note)
Absorbance (every 15 sec. until 3
min)
Note :
Add -amylase to each of the reaction tubes before measuring the
absorbance.
2. Plot the absorbance and time of each reaction and calculate the rate of
reaction (or initial velocity) from the slope of the plot.
3. Plot the pH as the abscissa (X-axis) and the rate of reaction (or initial
velocity) of each pH reaction as the ordinate (y-axis) to find out the
optimum pH of the enzyme.
122
DATA NOTE
Experiment 8 : ENZYME
Experiment 8.1
The enzyme kinetic of -amylase

Time (second)
A650
0
15
30
45
60
75
90
105
120
135
150
165
180
Then, Plot the absorbance as the ordinate (y-axis) and time of the reaction as the
abscissa (x-axis) and Calculate the rate of reaction (or also called initial velocity of
enzyme) from the slope of the plot.
Experiment 8.2
123
pH influencing the rate of enzymatic reaction
Time (sec)
A650
pH 5.0
pH 7.0
pH 8.5
pH 10.0
0
15
30
45
60
75
90
105
120
135
150
165
180
1. Plot the absorbance (Y-axis) and time of each reaction (X-axis) and calculate the
rate of reaction (or initial velocity) from the slope of the plot.
pH
Enzyme activity
5.0
7.0
8.5
10.0
2. Plot the pH as the abscissa (X-axis) and the initial velocity of each pH reaction as
the ordinate (y-axis) to find out the optimum pH of the enzyme.
3. Report the optimum pH of -amylase enzyme.
124
EXPERIMENT 9
SPECIAL REPORT ON ENZYME PURIFICATION
Objectives
1. Learn how to search international scientific journals from electronic sources.
2. Learn how to read and understand international papers.
3. Learn the method on purification of enzyme used in the real research.
Scientific journals are the papers where scientific research are published.
What do you need to do for this special report ?

1.
Search your journal paper from electronic sources (internet).

Some online-journals are available for free, but some are not !
So, choose free online-journals.
For example,
http://aem.asm.org/
- Applied and Environmental Microbiology

- Journal of Bacteriology
http://jb.aem.org/
H
- Journal of Biological Chemistry
http://www.jbc.org/
- Online Abstract and Full Text Articles

http://highwire.standford.edu/lists/freeart/dtl
http://www.ncbi.nlm.nih.gov
http://www.elsevier.com
http://www.sciencedirect.com
2.
Key the word purification
3.
Key the period of time : Jan 2000-May 2006

(But mostly, No free journals are available for the year 2006)
4.
125
Print your journal paper on PDF version only !!!

(So, your computer need Acrobat program)
5.
Choose a best paper (in your opinion) which is the main point / main
idea is about enzyme purification.
(Note : your paper must contain a purification table and at least 3 methods of
purification)
6.
Organization of topics in your report is shown in the report example.
7.
Make sure that you know what you are doing on the report. You need to
understand your paper in the aspect of purification well and deeply because
it absolutely will be on the lab examination.
8.
Please let instructor see your paper before doing if you are not sure.
126
Examples:
1.
Topic on enzyme purification

Purification and Characterization of an Arginine Aminopeptidase from
Lactobacillus sakei
2.
Authors
Yolanda Sanz and Fidel Toldra
3.
Journal
Applied And Environmental Microbiology, Vol. 68, No. 4 ,
April 2002, page 1980-1987
4.
Enzyme and Sources of enzyme

Arginine aminopeptidase (EC 3.4.11.6) from Lactobacillus sakei
(isolated from sausages)
5.
What kind of reaction that this enzyme catalyzed / or What the functions
of this enzyme are.
Arginine aminopeptidase catalyses the hydrolysis of the amino (N) termini of
peptide substrates to basic amino acids
6.
Application of this enzyme

Meat industry
-
This enzyme generates small peptides and free amino acids, which
are flavor compounds, during meat processing.
127
This enzyme promotes the growth of lactic acid bacteria during

meat fermentation.
7.
8.
Summary from purification table

Yield of pure enzyme
4.2 %
Purification fold
500.0 fold
Overview of purification step

Step 1 :
Crude enzyme extraction

( break cells by lysozyme and ultrasonic treatments)
Step 2 :
Ammonium sulfate fractionation
Step 3 :
Hydrophobic interaction chromatography

(Phenyl-Sepharose Fast Flow column)
Step 4 :
Gel filtration chromatography

(Sephacryl 200 HR column)
Step 5 :
Anion-exchange chromatography
(Resource Q anion-exchange column)
128
(Choose 2 methods to explain)

1. Hydrophobic interaction chromatography
(Phenyl-Sepharose Fast Flow column)
Principle and theory :
(at least 2 pages with pictures)
2.
Gel filtration chromatography

(Sephacryl 200 HR column)
Principle and theory :

(at least 2 pages with pictures)
129
EXPERIMENT 10
GLUCOSE FERMENTATION BY YEAST
Objective:
To study the glycolysis pathway of carbohydrate metabolism.
Principle :
The fermenting yeast cell degrades glucose, a compound of relatively high
potential energy, to CO2 and ethanol, compounds of low potential energy.
C6H12O6
2C2H5OH + 2CO2
The fermenting yeast system is a useful system for studying the carbohydrate
metabolism, since it is simple, easily to follow the CO2 production and it is not
expensive. Moreover, it is applied in many of food industry such as bakery, brewery
or alcohol production.
130
Figure:10-1: Glycolysis pathway

Materials :
1. Bakers yeast (10% in buffer)
2. Buffer : 0.1 M sodium phosphate pH 6.6
3. 1% glucose solution in buffer
4. 1% fructose solution in buffer
5. 1% sucrose solution in buffer
6. Test tubes with the same size
7. Serum cap connected with glass rod or tubing
131
Methods :
1. Set up the 2 test tubes with cork and nylon tube.
2. Start the reaction by adding 5 ml of yeast solution and 5 ml of glucose
solution, mix well and close the tube. (use buffer solution alone as a
control)
3. Count the number of gas within 20 min.
4. Repeat the experiment by using fructose and sucrose.
DATA NOTE
Glucose fermentation by yeast
Solution
Number of gas
Buffer alone
Glucose
Fructose
Sucrose
132
EXPERIMENT 11
INTRODUCTION TO BIOINFORMATICS
Objectives
1. To study the fundamental knowledge and definition of Bioinformatics
2. To analyze biological data by using statistical and computing methods
Bioinformatics Definition
General view
Bioinformatics derives knowledge from computer analysis of biological data.
These can consist of the information stored in the genetic code, but also
experimental results from various sources, patient statistics, and scientific literature.
Research in bioinformatics includes method development for storage, retrieval, and
analysis of the data. Bioinformatics is a rapidly developing branch of biology and is
highly interdisciplinary, using techniques and concepts from informatics, statistics,
mathematics, chemistry, biochemistry, physics, and linguistics. It has many practical
applications in different areas of biology and medicine.
Roughly, bioinformatics describes any use of computers to handle biological
information. In practice the definition used by most people is narrower;
bioinformatics to them is a synonym for "computational molecular biology"- the
use of computers to characterize the molecular components of living things.
Organization / committee
Bioinformatics definition by bioinformatics definition Committee, National
Institute
of
Mental
Health
released
on
July
17,
2000
(source:
http://www.bisti.nih.gov/)
(1) The NIH Biomedical Information Science and Technology Initiative
Consortium agreed on the following definitions of bioinformatics and computational
biology recognizing that no definition could completely eliminate overlap with other
133
activities or preclude variations in interpretation by different individuals and

organizations.
Bioinformatics: Research, development, or application of computational tools and
approaches for expanding the use of biological, medical, behavioral or health data,
including those to acquire, store, organize, archive, analyze, or visualize such data.
Computational Biology: The development and application of data-analytical and
theoretical
methods,
mathematical
modeling
and
computational
simulation.
Techniques to the study of biological, behavioral, and social systems.

The National Center for Biotechnology Information (NCBI 2001) defines
bioinformatics as "Bioinformatics is the field of science in which biology, computer
science, and information technology merge into a single discipline. There are three
important sub-disciplines within bioinformatics: the development of new algorithms
and statistics with which to assess relationships among members of large data sets;
the analysis and interpretation of various types of data including nucleotide and
amino acid sequences, protein domains, and protein structures; and the
development and implementation of tools that enable efficient access and
management of different types of information."
As a discipline that builds upon the life, health, and medical sciences,
bioinformatics supports medical informatics; gene mapping in pedigrees and
population studies; functional-, structural-, and pharmaco-genomics; proteomics, and
dozens of other evolving omics.
As a discipline that builds upon the basic sciences, bioinformatics depends on
a strong foundation of chemistry, biochemistry, biophysics, biology, genetics, and
molecular biology which allows interpretation of biological data in a meaningful
context.
As a discipline whose core is mathematics and statistics, bioinformatics
applies these fields in ways that provide insight to make the vast, diverse, and
complex life sciences data more understandable and useful, to uncover new
biological insights, and to provide new perspectives to discern unifying principles. In
134
short, bioinformaticists bring a multidisciplinary perspective to many of the critical

problems facing the health-science profession today.
Even though the three terms: bioinformatics , computational biology and
bioinformation infrastructure are often times used interchangeably, broadly, the three
may be defined as follows:
Bioinformatics refers to database-like activities, involving persistent sets of
data that are maintained in a consistent state over essentially indefinite periods of
time. Computational biology encompasses the use of algorithmic tools to facilitate
biological analyses; while bio-information infrastructure comprises the entire
collective of information management systems, analysis tools and communication
networks supporting biology. Thus, the latter may be viewed as a computational
scaffold of the former two.
Bioinformatics is currently defined as the study of information content and
information flow in biological systems and processes. It has evolved to serve as the
bridge between observations (data) in diverse biologically-related disciplines and the
derivations of understanding (information) about how the systems or processes
function, and subsequently the application (knowledge). Bioinformatics, as both an
enabling and enabled technology, will be defined differently depending on the
domain of the person who is giving the definition. A computer scientist will give one
definition, a biologist another, a biotechnologist yet another, and an individual from a
pharmaceutical company will provide yet another definition. Each definition is as
good as the other.
Demonstration & Self Study: 1. Usage of BLAST program via NCBI webpage
2. Interpretation of Biological data
http://www.ncbi.nlm.nih.gov
135
APPENDIX
Procedure in pH measurement by using pH meter
Make sure that the instrument has been calibrated before taking pH
measurements.
1.
Switch the instrument on by pressing the ON/OFF switch. The pH meter

automatically defaults to the pH measurement mode.
2.
Submerge the tip of the pH electrode (and the temperature probe if

temperature factor is major concern to the experiment.) into the sample to be
tested. Allow time for the electrode to stabilize.

3.
pH is displayed on the primary display and temperature on the secondary

one.
Note :
If the measurements are taken successively in different samples, it is
recommended to rinse the electrode thoroughly with deionized water or, if not
available, tap water first and then with some of the next sample to condition the
electrode before immersing it in the sample. The reset button should only be used
when the instrument displays erroneous messages due to strong electrical
interference or when the instruments power supply was disconnected before the
meter was switched off.
After pressing reset always recalibrate the unit before
proceeding.
pH calibration
Calibrate the instrument often, especially if high accuracy is required.
The
instrument should be recalibrated :

a) Whenever the pH electrode is replaced by other electrode
b) At least once a week
c) After testing aggressive chemicals
d) After pressing RESET
e) If higher accuracy is required
136
It should be very good if two-point calibration is done for more accuracy in pH

reading. The buffer used to calibrate pH meter is called Standard buffer and is
from chemical company (usually pH4, pH7 and pH10).
It is recommended that
standard buffer pH 7 is chosen as the first point and pH 4 as a second point.

1. Immerse the pH electrode into a standard buffer pH 7.
2.
Press CAL. The CAL and BUF1 is shown and the most common 7.01
buffer will be displayed on the LCD.
3.
If necessary, press oC or oC to select a different buffer value.
4.
The
NOT READY
indication will blink on LCD until the reading has
stabilized.
5.
137
When the reading is stable, READY and CFM will blink.
Press CFM to
confirm the calibration.
6.
If the reading is close to the standard buffer, the meter stores the reading.
The calibrated value is then displayed on the primary LCD and the secondary
LCD will display the second expected buffer value.
( If the value measured by the meter is not close to the standard, WRONG
BUF will blink. In this case, check if the correct standard buffer has been used.
)
7.
After the first calibration point is confirmed, immerse the pH electrode in the
second standard solution and stir gently.
8.
The
NOT READY
indication will blink on LCD until the reading has
stabilized.
9.
When the reading is stable, READY and CFM will blink.
Press CFM to
confirm the calibration.

10.
If the reading is close to the standard buffer, the meter stores the reading and
returns to normal operation mode.
138
139
140
TITRATION
A titration is a method of analysis that will allow you to determine the precise
endpoint of a reaction and therefore the precise quantity of reactant in the titration
flask. A buret is used to deliver the second reactant to the flask and an indicator or
pH Meter is used to detect the endpoint of the reaction.
Titrating with a pH meter
(a) Titration with a pH meter follows the same procedure as a titration with an
indicator, except that the endpoint is detected by a rapid change in pH, rather
than the color change of an indicator.
(b) Arrange the sample, stirrer, buret, and pH meter electrode so that you can read
the pH and operate the buret with ease.
(c) To detect the endpoint accurately, record pH vs. volume of titrant added and plot
the
(a)
titration
curve
as
you
titrate.
(b)
141
(c)
142
REFERENCES
Amersham Pharmacia Biotech. 1999. Protein Purifiction Handbook. Sweden.

Berg, Jeremy M., Tymocko, John L. and Stryer, Lubert, 2002. Biochemistry, 5th
ed. New York: W.H. Freeman
Bollag, D. M., Rozycki, M. D., and Edelstein, S. J. 1996. Protein Method. 2nd ed.
New York : Wiley-Liss, Inc.
Boyer, R. 2000. Modern Experiment Biochemistry. San Francisco: Addison
Wesley Longman, Inc.
Chambers, J. A. A., and Rickwood, D., eds. 1993. Biochemistry Labfax. UK : BIOS
Scientific Publishers.
Clark, J. M., and Switzer, R. L. 1977. Experimental Biochemistry. San Francisco :
W. H. Freeman and Company.
Cooper, T. G. 1977. The Tools of Biochemistry. New York : John Wiley & Sons.
David L. Nelson and Michael M. Cox, 2000 Principles of Biochemistry. 3rd ed. New
York: Worth Publishers.
Eisenthal, R., and Danson, M. J., eds. 1992. Enzyme Assay : A Practical
Approach. New York : IRL Press.
Hames, B. D., Hooper, N. M., and Houghton, J. D. 1998. Instant Notes in
Biochemistry. UK : BIOS Scientific Publishers.
Segal, I. H. 1976. Biochemical Calculations. New York : John Wiley & Sons.
143
Seidman, L. A., and Moore, C. J. 2000. Basic Laboratory Methods for

Biotechnology : Textbook and Laboratory Reference. New Jersey : PrenticeHall, Inc.
Voet, D. and Voet, J. G. 1995. Biochemistry. Canada : John Wiley & Sons.
Whitaker, J. R. 1994. Principles of Enzymology for the Food Sciences. New York
: Marcel Dekker, Inc.

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BASIC BIOCHEMISTRY

Faculty of Biotechnology, Assumption University

BASIC BIOCHEMISTRY LABORATORY MANUAL

ASSUMPTION UNIVERSITY PRESS

Assumption University Press, Bangna Campus

Published in the Thailand

First published 2003

Third Edition (additional contents) 2005: ISBN 974-615-203-3

Laboratory Safety and Manner Issues

Basic Instruments and Glasswares in Biochemistry Laboratory

1.1 Property of acids used in buffer

1.2 Property of buffer in different concentrations

UV-Visible Absorption Spectrophotometry

2.1 Study of the absorption spectrum of syrup solution

2.2 Determination of unknown sample concentration

Amino acids and Proteins I

Part A: Qualitative analysis of amino acids and proteins

3.1 Qualitative analysis of free -amino group by Ninhydrin test

3.2 Qualitative analysis of peptide bonds by Biuret test

3.3 Qualitative analysis of R group reaction by Sulfur test

Amino acids and Protein II

Part B: Isoelectric property for protein precipitation

Part C: Protein denaturation

Part D: Quantitative analysis of amino acids and proteins

4.3 Determination of protein concentration by Lowry method

5.1 Solubility property

5.2 Test of degree of unsaturation

5.3 Properties of soap and detergent

Isolation of high molecular weight DNA

Part A: Qualitative analysis of carbohydrates

7.2 Qualitative test for carbohydrate by furfural forming

7.3 Qualitative test for carbohydrate by reducing property

Part B: Quantitative analysis of carbohydrates

8.1 The enzyme kinetic of -amylase

8.2 pH influencing the rate of enzymatic reaction

Glucose Fermentation by yeast

Basic Biochemistry Laboratory

A lab can be dangerous place if you are not careful.

This potential danger

Only by using the equipment correctly, as

instructed, will you be sure that you are safe.

Flying glassware can come from

Please note and observe the following lab

THINGS STUDENTS SHOULD DO

Remember that although you are not using a dangerous

Faculty of Biotechnology, Assumption University

Wear proper clothing in the lab.

The lab is a good place for long sleeves,

long pants, closed-toed shoes.

Dont even think about going into a lab

THINGS STUDENTS SHOULD NEVER DO

Basic Biochemistry Laboratory

You might only need 2 ml.

sections of the lab for the week.

Faculty of Biotechnology, Assumption University

A laboratory report is a formal document consisting of about 6 sections,

Title of the experiment

Name and ID number of the student and lab partners

Date of the experiment

Name of this course

Leksakorn A. and Chaicherdsakul T., 2006

Basic Biochemistry Laboratory

Objective and purpose :

the statement of point of the experiment.