Chapter 1

Introduction

Introduction
Beside gaining knowledge from working in laboratories, working in biological laboratories is not
an easy job, working in biochemical laboratories in a precise and concise way requires taking lot
of precautions, a person must have great knowledge of biological and biochemical preparations,
safety rules, good manipulating methods of equipment used in lab, knowing methods of handling
and discarding toxic and biochemical hazard molecules, a person must know how to deal with
burns, cuts swallowing of toxic materials, fire and immediate minimizing human exposure to
loss or injury. So, to minimize such loss, a student must carefully read procedure, and
instructions to equipment used before performing experiment, follow protocol exactly as
provided by instructor, preparations of chemicals according to rules gives right results and
minimize loss to the expensive chemicals.
The laboratory provides students and instructors with an environment conducive to
accomplishing specific scientific tasks. Students must follow proper safety practices in the
laboratory and immediately report any safety hazards or accidents to the instructor.
This lab course is intended to give a brief ideas about different tests and laboratory procedures
performed in biology departments of Palestinian universities, this course reflects and covers the
topics have been taught in theoretical part of biology course like: the microscope, the cell,
chemical basis of life including proteins, carbohydrates, lipids, physical properties of the cell,
cellular organization in plants and animals, cellular respiration, photosynthesis, and many other
topics.

Laboratory requirements and safety rules

Basic requirements
1. Clothes: lab coat or apron, lab coat must be worn and buttoned all the time in the lab.
2. Safety goggles for eye protection.
3. Shoes that give full feet protection, sandals are not allowed.
4. Shield cover in case of radiation.
5. Gloves.
Safety rules
1. Chemicals: treat chemicals as a potential hazard, never taste them, flush water on spilled
chemicals, never touch them by hand, in case of hand, eye, or mouth contamination wash
with plenty of water, inform your instructor immediately to seek medical help. Never
inhale any fume in the lab.
2. Fire: get away from fire source as soon as possible, inform university security to press
fire alarm and evacuate all lab members. Don’t run in fire if your self-contacted with fire,
this will increase fire, stop, calm and don’t panic, to stop fire use fire blanket, seek others
help to open fire extinguisher.
 3. Glass: take care with broken glass, never touch broken glass directly by hands, inform
instructor to call clean agent.
4. Body fluids: like blood, protect yourself from any contamination with body fluids by
wearing lab coat, gloves, and glasses. Dispose specimens in a special biohazard bag.
Wash countertop with 10% disinfectant. Inform instructor for any other spills. All
microorganisms should be treated as potential biohazards, sterilize all equipments used in
handling microorganisms and dispose them in a special protected biohazard bag.

General rules
1. Attend lab on time, never be late.
2. Instructor will give introduction about experiment to be performed in the first 30
minutes.
3. Lab is a place for all students to perform their experiments, so keep in order, keep it
clean before you leave.
4. Avoid toxic chemicals, and all biohazards.
5. Don’t panic in case of fire, follow previous rules, if doubt get out of fire immediately.
6. Never eat or drink in the lab to avoid contaminations.
7. University is not responsible in case of any personal item loss.
8. No lab visits are allowed.
9. Learn to locate first aid box, fire blanket, fire alarm, emergency call numbers in the first
lab period.
10. Never dislocate or try move any unfunctional equipment, inform your instructor.
11. Submit your lab report on time, delaying in submission will reduce your point.
12. Be familiar with the location of safety equipment.
13. Treat all laboratory equipment, such as microscopes, with care, and store the equipment
as instructed.
14. Do not open specimen jars unless instructed.
15. Always wash your hands with soap and water before and after the laboratory experience.
Keep your hands away from your face.
16. Properly dispose of broken glass, slides, and disposable laboratory equipment.

I understand the safety rules as presented above and agree to follow them accurately.

Name: ------------------------------------------------------------- Date: -------------

Laboratory Class: ------------------------------------------------------------------

Laboratory time: ---------------------------------------------------------------------
 Rules for Clinical Chemistry

For clinical/medical students

Clinical biochemistry tests comprise over one third of all hospital laboratory investigation.
Clinical biochemistry is that branch of laboratory medicine in which chemical and biochemical
methods are applied to the study of disease while in theory this embraces all non-morphological
studies, in practice it is usually, though not exclusively, confined to studies on blood and urine
because of the relative ease in obtaining such specimens although analysis are made on other
body fluids such as gastric aspirate and cerebrospinal fluid.

The use of biochemical tests:

Biochemical investigations are involved, to varying degrees, in every branch of clinical
medicine.

 The results of biochemical tests may be of use in diagnosis and in the monitoring of
treatment.
 Biochemical tests may also be of value in screening for disease or in assessing the
prognosis once a diagnosis has been made (fig. 1).
 The biochemistry laboratory is often involved in research into the biochemical basis of
disease and in clinical trials of new drugs.

diagnosis treatment

Biochemistry report
sugar – 110 mg/dl
urea – 62 mg/dl
creatinine – 1.8 mg/dl

screening
prognosis

Fig. 1: How biochemical tests are used?

Preparing Blood:
1. Take all precautionary measures needed while using blood whether it has been collected
from hospital or drawn in the lab.
2. Write all needed information on blood collecting vials: Patients complete name and age,
Identification number etc.
3. The specimen containers should be previously labeled.
4. Position of the blood donor should be in the right way to draw blood in an unpainful way.

Requirement of blood collection

1. Collection tubes.
2. Sterilized syringes and needles.
3. Sprit or 70% ethanol.
4. Cotton.
Blood collection

 Compare the requisition form and labeling the tubes.

 Selecting vein site.
 Applying the tourniquet.
 Cleaning the area.
 Inspecting the needles and syringes.
 Performing the venipuncture.
Serum Separation
1. Allow the blood to clot.
2. Loosen the clot slowly and centrifuge the supernatant fluid.
3. By using a pipette, separate the serum from blood cells and store it in a clean and day test
tube.
Sampling errors
There are a number of potential errors which may contribute to the success or failure of the
laboratory to provide the correct answer to the clinician's question. Some of these problems arise
when a clinician first obtains specimens from thepatients.

 Blood sampling technique. Difficulty in obtaining a blood specimen may lead to

haemolysis with consequent release of potassium and other red cells constituents. Results
for these will be falsely elevated.
 Prolonged stasis during venipuncture. Plasma water diffuses into the interstitial space and
the serum or plasma sample obtained will be concentrated. Proteins and protein-bound
components of plasma such as calcium or thyroxin will be falsely elevated.
 Insufficient specimen. Each biochemical analysis requires a certain volume of specimen
to enable the test to be carried out it may prove to the impossible for the laboratory to
measure everything requested on a small volume specimen.
 Errors in timing. The biggest source of error in the measurement of any analyte in a 24-
hour urine specimen is in the collection of an accurately timed volume of urine.
 Incorrect specimen container. For may analyses the blood must be collected into a
container with anticoagulant and preservative. For example, samples for glucose should
 be collected into a special container containing fluoride which inhibits glycolysis;
otherwise the time taken to deliver the sample to the laboratory can affect the result. if a
sample is collected into the wrong container, it should never be decanted into another
type of tube. For example, blood which has been exposed even briefly to EDTA (an
anticoagulant used in sample containers for lipids) will have a markedly reduced calcium
concentration, approaching zero.
 Inappropriate sampling site. Blood samples should not be taken 'down-stream' from an
intravenous drip. It is not unheard of for the laboratory to receive a blood glucose request
on a specimen taken from an intravenous drip. It is not unheard of for the laboratory to
receive a blood glucose request on a specimen taken from the same arm into which 5%
glucose is being infused. Usually the results are biochemically incredible but it is just
possible that they may be acted upon, with disastrous consequences for the patient.
 Incorrect specimen storage. A blood sample stored overnight before being sent to the
laboratory will show falsely.

Clinical question Biochemical answer

Request form with

clinical data reporting

Patient sampled
interpretation

Transit to lab
collation

Reception and
Quality control

analysis

Fig. 2: Circuit diagram of clinical biochemistry process.

Analyzing the specimen

Once the form and specimen arrive at the laboratory reception, they are matched with a unique
identifying number or bar code. The average lab receives many thousands of requests and
samples each day and it is important that all are clearly identified and never mixed up. Samples
proceed through the laboratory as shown in figure 2. All analytical procedures are quality
controlled and the laboratory strives for reliability.
Once the results are available they are collected and a report is issued. Cumulative reports allow
the clinician to see at a glance how the most recent result (s) compare with those tests performed
previously, providing an aid to the monitoring of treatment.

Methodology of its teaching: - Lecture with few C.D. demonstration.

Evaluation of the session: - Asking queries regarding the lecture.

Results Interpretations
The laboratory report
It can take considerable effort, and expense, to produce what may be seen to be just numbers on
pieces of paper, understanding what these numbers mean is of crucial importance if the correct
diagnosis is to be made, or if the patient’s treatment is to be changed. Usually results of tests
carried out in clinical chemistry are reported in units of concentration or of activity.

Concentration
Unit of concentration contain both units of quantity and units of volume. The amount of
substance present can be expressed in grams, equivalents, or moles or divisions of these i.e.
Milligrams, milliequivalents, millimoles etc. similarly the volume can be expressed in liters
milliliters or deciliters (100 ml).

For many substances the usual units of concentration has been in mg per 100 ml. This form had
replaced the earlier less defined form of mg% which should no longer be used as the use of % to
mean per 100 ml here differs from the general use of (%) to more recently deciliter has been
proposed as a term for 100 ml. Just as a milliliter is one thousandth of liter so a 100 ml is one
tenth of a liter (a deci-liter) so units have changed from mg % to mg/100, ml to mg (dl) without
any numerical change.

Serum electrolytes are usually expressed in milli equivalent per liter and protein and albumin in
grams per 100 ml.

S. I. UNITS
For many purpose e.g., calculation of osmolarity or electrolyte balance it would be convenient to
have all results expressed in the same units, this would also help avoid confusion when results
are compared from one laboratory to another or in the evaluation of results by medical staff are
used to different units, for these reasons standard international units for reporting results have
been recommended. As with many changes there has been resistance to the change over to the
new units especially among the medical profession. But as the newer text books and reviews use
these units. They are gaining international acceptance.

The S.I. (standard international) units apply to clinical chemistry as follows.

1. Where the molecular weight of the substance being measured in known, the units of
quantity should be the mole submultiple of a mole e.g., millimoles and micromoles.
2. The units of volume should be the liter. Units of concentration. Will therefore be
millimoles per liter etc.
 e.g., sodium of 140 mg/l in S.I. units is 140 m mol/l.
glucose of 180 mg/100 ml in S.I. units is 10 m mol/l.
3. When the molecular weight is not known, the for example for serum protein or albumin
determinations the concentration should be expressed in grams per liter i.e. 7.0 g/l.
Units of activity
Usually activities are measured in clinical chemistry as an index of the concentration of certain
enzymes, or enzymatic processes e.g., prothrombin activity.
Activity is a measure of the rate at which a process takes place. Enzymatic activity is usually
estimated by measuring the rate at which a substance is converted to a product. This activity is
affected by many things e.g. temperature, time over which the activity is measured, incubation
conditions etc. All of these must be defined when reporting the activity, and so, units or activity
include fixed values for each variant. As the combination of the variations is large, therefore,
many different such units are in use. International standardization of all these variables is almost
impossible, and thus, the international unit of enzyme activity has not been widely accepted, the
units for each test in the laboratory should be clear to everybody.

Reporting results

There is a certain error in all results. Usually a laboratory will claim 95% confidence in its result,
i.e. plus or minus two standard deviations. Thus a blood sugar report of 100 mg/100 ml with a
standard deviation of 1 mg per 100 ml would really be 100 +/- 2mg/100 ml. for simplicity the
variation is not usually reported with the individual result. The standard deviation for the method
should be indicated and the result given with the understanding that the variation is understood.

Significant figures
From mathematical calculations results may be obtained to many decimal places, but these will
not usually be significant. The final result cannot be accurate to a greater degree than the sum of
the errors the test allows. e.g. error in pipetting sample. Or standard error in reading
spectrophotometer etc. When reporting a result, each figure given should have a meaning. A
blood sugar result would not usually be reported as 100.2 mg/ 100 ml. as 0.2 mg is not a
significant part of the result for most blood sugar methods. Thus result should be rounded off to
the nearest significant figure. Before reporting. What is or is not significant must be established
for each test.
Use of zeros after a decimal point can give misleading information if they are not significant. e.g.
7.0 g means between 6.95 and 7.05 g. 7g means between 6.5 and 7.5 g.

Quality control
Biochemical measurements vary for two reasons. There is an analytical variation and also a
biological variation.

Laboratory analytical performance

A number of terms describe biochemical results. These include:

 Precision and accuracy

 Sensitivity and specificity
 Quality assurance
 Reference ranges.
Sensitivity and specificity
Sensitivity of an assay is a measure of how little of the analyte the method can detect, as new
methods are developed they may offer improved detection limits which may help in the
discrimination between normal results and those in patients with the suspected disease.
Specificity of an assay is related to how good the assay is at discriminating between the
requested analyte and potentially interfering substances.

Quality assurance
Every laboratory takes great paints to ensure that the methods in use continues to produce
reliable results. Laboratory staff monitor performance or assay using quality control to give
reassurance that the method is performing satisfactorily with the patients' specimens. These are
internal quality controls which are analyzed every day or every time an assay is run. The
expected values are known and the actual results obtained are compared with previous values to
monitor performance. In external quality assurance schemes, identical samples are distributed to
laboratories; results are then compared.
in this way, the laboratory's own internal standards are themselves assessed.

Reference ranges

Analytical variation is generally less than that from biological variables. Biochemical test results
are usually compared to a reference range considered to represent the normal healthy state (fig.
3) most reference range are chosen arbitrarily to include 95% of the values found in healthy
volunteers, and hence, by definition, 5% of the population will have a result out with the
reference range. In practice there are no rigid limits demarcating the disease population from the
healthy; however, the further a result is from the limits of the range, the more likely is to
represent pathology. In some situations it is useful to define 'action limits', where appropriate
intervention should be made in response to a biochemical result.
There is often a degree of overlap between the disease state and the 'normal value' (fig. 4) a
patient with an abnormal result who is found not to have the disease is a false positive. A patient
who has the disease but has a 'normal' result is a false negative.
Biological factors affect the interpretation of results and the discrimination between normal and
abnormal results. Results are affected by various physiological factors which must be considered
when interpreting any given result. These include:

 Sex of the patient. Reference range for some analytes such as serum creatinine are
different for men and women.
 Age of the patient. There may be different reference range for neonates, children, adults
and the elderly.
  Effect of diet. The sample may be inappropriate if taken when the patient is fasting or
after a meal.
 Time when sample was taken. There may be variations during the day and night.
 Stress and anxiety. They may affect the analyte of interest.
 Posture of the patient. Redistribution of fluid may affect the result.
 Effects of exercise. Strenuous exercise can release enzymes from tissues.
 Medical history. Infection and/or tissue injury can affect biochemical values
independently of the disease process being investigated.
 Pregnancy cycle. Hormone measurements will vary through the menstrual cycle.
 Drug history. Drugs may have specific effects on the plasma concentration of some
analytes.

Other factors
when the numbers has been printed on the report from, they still have to be interpreted in the
light of a host of variable. Analytical and biological variations have already been considered.
Other factors relate to the patient. The clinician can refer to the patient or to the clinical notes,
whereas the biochemist has only the information on the request form to consult. The
accumulation of biochemistry results is often helpful in patient management.

Biochemical calculations
Accuracy
Accuracy is defined as the degree of closeness of measurement of a quality to the actual value,
the degree of closeness to true value.

Precision
Also called reproducibility or repeatability, is defined as the degree to which repeated
measurements under unchanged conditions show the same results.
Figure 1: Accuracy and precision

Accuracy =

Precision =

International base system of units (SI)

The SI system is found on seven base units for seven base quantities assumed to be mutually
independent as shown in Table 1:

Base quantity Name Symbol

Length Meter M
Mass Kilogram Kg
Time Second S
Electric Ampere A
Thermodynamic temperature Kelvin K
Amount of substance Mole Mol
Luminous intensity Candela Cd

Other units are derived from these basic units, some of derived units are shown in Table 2:

Physical quantity Name Expression in basic unit

Frequency Hertz S-1
Force Newton Kgms-2
Energy Joule Kgm2s-2
Pressure Pascal Kg m-1 s-2
Power Walt Kg m2 s-3
Electric charge Coulomb As
Electric potential Volt Kgm2s-3 A-1
Plane angle Radian m.m-1
Magnetic flux Weber m22.kg-1s-3 A-1
Illuminance Lux m-2.cd

Table 3: shows some metric prefixes:

10n Decimal In words Prefix

1012 1,000,000,000,000 Trillion Tera
109 1,000,000,000 Billion Giga
106 1,000,000 Million Mega
103 1000 Thousand kilo
102 100 Hundred Hector
101 10 Ten Deca
100 1 One -
 10-1 0.1 Tenth Deci
10-2 0.01 Hundredth Centi
10-3 0.001 Thousandth Milli
10-6 0.00001 Millionth Micro
10-9 0.000000001 Thousand millionth Nano
10-12 0.000000000001 Billionth Pico

Units used outside SI

Liter (L)
1Liter = 1dm3 = 10-3 m3
1 milliliter (ml) = 1cm3 = 10-6 m3

1 microliter (l) = 1mm3 =10-9 m3

1000 liters = 1 cubic meter = m3

Time
The basic SI unit for time is second, but the common units of time are minute, hour, and year still
can be used.

Exponential forms

Example1
10,000,000 = 1.0×107

5.0 is the coefficient

10 is the base
7 is the exponent or power

Example2
11,300,000 = 11.3 X 106

= 1.13 X 107
Note: any base raised to the power zero is = 1 = 100 = 1

Example3
0.000041 = 41X 1o-6
= 4.1X10-5
Example4
5X101 + 4.2 X10 2 = ( 5X101 ) + ( 42X 101 )

= 47 X 101 = 470

Example 5
9.6 x 104 -3.0 x 103
96.0 x 103 – 3.0 x 103

93.0 x 103
In multiplication and division

Example6
( 7.3 x 106) x ( 1.0 x 105 ) = (7.3 x 1.0) ( 105+6 )

= 7.3 x1011

Example7

(7.3 x 106 ) ÷ ( 1.0 × 105) =

= 7.3×101

= 73

In simplification

Example8

= 0.304

= 0.304

= 0.304

Logarithms
The logarithm is (log) the exponent which indicates the number of times, the base is the factor in
the product when the coefficient is 1.0.

Example 1
2500 = 2.5 x 103

= 1.0 x 103.39
 = 103.39

Example 2
100 = 102
= log10 = 2

Example 3
1 =100

= log10 = 0

Example 4
1,000,000 =106
= log10 = 6

Example 5
0.01 = 10-2
= log10 = -2

Ratio and proportion

Many biochemical calculations involve setting up ratios, and then establishing correct proportion

Example 1
Express as a ratio and simplify to give concentration of 0.08 mole of glucose in 100ml of
solution?

= 0.8M

Example 2
70g 0f glucose in 1L of solvent

= 7%

Units of mass and weight

Molecular weight
Molecular weight is a number, it’s the relative mass of molecule as compared with the mass of
an atom of carbon ( 12C isotope).
For example, if acetic acid C2H4O2, molecular weight is 60, it means that the molecular of acetic
acid is 5X as heavy as an atom of carbon. Weight of substances expressed in grams numerically
equal to its molecular weight is termed its gram molecular weight or gram molecule or mole.

Molecular weight of volatile substances

Molecular weight of volatile substances is given by the following formula:

Molecular weight =

Where: w is weight or substance

v is volume

Empirical formula and molecular formula

Molecular formula is = ( empirical formula ) n.

n =

Molecular formula: the formula which gives the actual number of atoms of various elements
present in the molecules of a substance. Its either the same as the empirical formula of the
substance, or simple multiple of it.

Example
CH2O is the empirical formula for acetic acid, with empirical formula weight 30, its molecular
weight = 60

n =

= 60/30

=2
and its molecular weight is (empirical formula)2.

Mole
Mole is the molecular weight of a compound in grams, for example mole of glucose is equal to
180g (glucose MW 180).

Molarity
Molarity is the number of moles of solute dissolved in liter of solution, its unit is mole/liter.

Molarity =
Molality
Molality is number of moles solute dissolved in 1kg of solvent.

Molality =

Normality
Normality is used when we deal with reaction and its function of equivalent

Normality =

No. of gram equivalent solute =

Equivalent weight =

Percentage solution
The types of percentage solutions used in biochemistry include: W/W, W/V, V/V.
For example:

2g of acetic acid /100g H2O is W/W.

2g of acetic acid /100ml H2O is W/V.
2ml of acetic acid /100ml H2O is V/V.

Table7: Concentrations used in Biochemistry

Concentration Ratio Abbreviation

Molar Mole solute/liter solution M
Millimolar Millimoles / liter solution mM
Micromolar Micromoles / liter solution µM
Molal Moles of solute / gm solvent m
Normal Equivalent solute /liter solution N
% Solution % W/W gm of solute/100g solvent % Solution
%W/V gm of solute/100ml solvent
%V/V ml of solute/100ml solvent
Parts per million Parts of solute/ 106 parts of solvent ppm
Note

1mg = g = 0.001g
1µg = g = 0.000001g

1g = 100g
1mg = 10-3g

1µg = 10-6g
Mole = 6.02× 1023 molecules of pure substance.

Molecular weight = weight in grams of 6.02× 1023 molecules of that substance.

Example 1
If 40g of NaOH with equivalent weight or mass 40 dissolved in 1L, therefore, Normality= 1. A
solution has 4g NaOH is called 0.1N.

Example 2
Calculate molality of ascorbic acid in a solution prepared from 1.94g of ascorbic acid and 50.1g
water? MW of AA 176.

= 0.0110mol AA

= 0.0501kg H2O

Molality =

= 0.220m in acetic acid.

Example 2
How many molecules are there in 0.12mol of alanine?

X = 0.12×(6.02× 1023) / 1
= 0.722× 1023 molecules.

Example 3
How many moles in exponential form of 0.3mmol?

= 0.3×10-3 mol
= 3×10-4mol.
Example 4
What is the weight in grams of 1.4mmol alanine? (MW75)

= 1.4mmol×10-3×75
= 105 × 10-3g

= 1.05 × 10-2g

Example 5
How many microliters are there in 2.83ml?
= 2.83 × 1000

= 2830µl.

Example 6
How many moles of alanine must be added to 5ml of H2O to give 3M alanine solution?

= 3(0.005)

= 1.0X
X = 0.015mol alanine.

Example 7
Express 3.3×10-5 concentration as µmol/ml?
= 33 µmol/L

= 0.033 µmol/ml.

Construction of standard curve

Standard curves are needed in biochemistry to determine concentration, a standard curve relates
known amount of a substance in solution to some measurable property such as the absorption of
light at a particular wave length. After plotting absorbance as a function of the compounds
concentration, a relationship may become obvious and straight line or curve may be drawn
through the points. This curve is called as standard curve. Each point has x value on x axis and y
value on y axis.
 Example
Construct the standard curve of bovine serum albumin BSA (2mg/ml) obtained from Lowry
method in the lab at wavelength of 540nm?
The following data obtained:

BSA mg/ml Absorbance at 540nm

0.125 0.05
0.25 0.1
0.5 0.2
0.75 0.3
1 0.4
1.5 0.5

0.5

0.4

0.3
Absorbance

Absorbance
0.2

0.1

0.05
0.125 0.25 0.5 0.75 1 1.5
Concentration

Accurate measurements
All measurements are subjected to errors, the source of errors are due to many factors, like
human errors, laboratory errors, and equipment limitations. All these errors should be eliminated
and minimized carefully by using proper clean equipment and proper laboratory conditions.

Standard and Blank

Standard solution is a solution has a precise known concentration. Blank solution is a solution
that does not contain a detectable amount of the analyte of interest, it is used for calibration
purposes. Blank is a solution containing all the reagents needed for analysis of a substance
except the tested substance. To obtain accurate results, standard solution must be included to
minimize errors, in general, only one standard is included for volumetric estimations. Blank
solution should be included in any measurement by replacing same amount of distilled water in
place of tested substance, and the blank treated exactly like test and standard, obtained results
subtracted from standard and test in final calculations.

Normal distribution curve

Normal distribution curve (Gaussian distribution curve) is constructed when a large number of
readings are taken, it is bell shaped and shows the trails and near the average results, but
occasionally deviates by large amounts.

Figure: normal distribution curve.

The center of the curve contains the greatest number of values, and therefore would be the
highest point on the arc of the line. This pointis referred to as the : mean, the concentration in the
center and decreases in either sides. Amount of data deviation measured by standard deviation
which show how much variation from average exists.
√

µ= mean of x

= standard deviation of x

= 3.14159

e=2.71828
Because the formula is complex, instead, tables are used to find probabilities for normal
distribution. The following is the equation for entire population:

∑
√

the standard deviation

Glassware / Plastic ware
The glassware and plastic ware generally used in biochemistry include:
1. Pipettes
2. Micropipettes
3. Burettes
4. Measuring cylinder
5. Volumetric flask
6. Beakers
7. Petri dishes
8. Droppers
9. Parafilm
10. Eppendorf
11. Tips
12. Balances
13. Spatulas
Writing an experiment and lab report
Obtaining good results in practical biochemistry lab is not sufficient, it is important to record off
a lab report in special way in lab notebook or journal. Writing lab report has many advantages:
1. It is considered as a data base and a student information record that he can refer to
during exams.
2. To the instructor that can check how much the student was doing well in the lab.
3. Writing good report is the way to obtain good marks.
4. The student will be familiar with writing scientific articles or papers.
Lab report includes the followings:

 The title and date

 Aim of the experiment
 Introduction and theory or principle of the experiment
 Materials
 Protocol or procedure
 Results
 Discussion
 References
The title page / the title
The title of the experiment should be neatly written in bold and it must be concise and
informative, in the title page the course name, the date of submission, student name, student
number, all these information must be included in this page.

Aim
Aim of the experiment shows the main purpose and objectives in which the student explains why
he performs this experiment.
Introduction/ theory/ principle
This section gives background information about the experiment, the detail hypothesis of the
experiment must be written, the student must show what is relay going on in the experiment
theoretically, the goal of the experiment, what are you planning to accomplish or achieve, and
you must show the reader how much you achieved.

Materials
List all materials used in the experiment, and their source.

Protocol/ procedure
This section describes what a student did in the lab step by step and accurately, it’s simply the
flowchart or procedure of the experiment performed in the lab, procedure must be in a way so
that any biochemist can repeat same steps in simple manner.

Results
Results show what you saw and obtained, results can be in the form of a table, calculations,
figures, or all. In addition, a student must note exact and accurate observation, for example
obtaining blue color is not sufficient, the student must note light blue, dark blue, or bluish color.
Tables and figure must include informative legends.

Result Interpretations: Discussion and conclusion

Here the student discusses experimental findings, a good discussion can be written in such a way
if the student is able to answer the following questions: what you have done? Why you have
done? What you have got? Discussion may include major conclusion points in brief,
recommendations to the advanced research may also be included. Discussion must summarize
findings, discuss expected and unexpected results, answer questions raised, reach conclusion,
flow well in a logic and concise way.

References
Most important references cited must be written down in the end of the experiment in a
scientific way for example:
Jazzar, M. (2011) Aromatherapy in the view of science and Islam. Hebron University journal
vol. 5 pp1-23.
 Chapter 2
Measuring Approximate PH
Aim: To measure approximate pH using different methods

Theory
An acid can be defined as a proton donor, a chemical that increases the concentration of
hydrogen ions in solution, where a base is a proton acceptor, a chemical that reduces the
concentration of hydrogen ions in solution. Acidity and basicity for all materials and solutions
can be determined by measure the concentration of the pH (defined as the negative logarithm of
hydrogen ion activity). Amphoteric: ionic species act as acids and base. Amphoteric compounds
have both acidic and basic groups, like amino acids.
Strong acids/ bases are compounds completely ionized in solution, the concentration of H + is the
same as the concentration of acid, where, weak acids/ bases are molecules dissociate to a limited
extent. Buffers are chemical molecules resist change in pH. In our body bicarbonate is the most
important buffer, beside this there are other buffers in blood regulate body fluids like: citrate,
phosphate, proteins.
Hassel back Henderson equation

HA H+ + A-
Ka = [H+] [A-] / [HA]
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Take - log
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Many methods are used to determine the pH value or just to determine the acidity and the
basicity of the solution:

1. Approximate methods: by use of litmus paper, pH strips, and use of indicators.

2. Accurate methods: by use of pH meter.
litmus paper: not specific, converts acidic molecules to red and basic molecules to blue.

pH test strips: are more accurate compared to litmus paper, less accurate to pH meter, these
strips are widely used in medical labs and hospitals for fast
screening of medical samples.

pH meter: the most accurate method of measuring pH.

Color change and useful pH range of some indicators

Acid Base Useful pH range

Thymol blue red Yellow 1.2-2.8
Bromophenol blue yellow Blue 3-5
Methyl red Red Yellow 4.3-6.1
Bromocresol purple Yellow Purple 5.5-7
Phenol red yellow Red 6.8-8.2
Phenolphthalein colorless Red 8.3-10

Principle
Indicators are organic compounds of natural or synthetic origin whose color changes if the pH of
the solution changes. Indicators are usually weak acids which dissociates in solution.
Indicator = Indicator- +H+

Applying the Henderson-Hasselbalch equation,

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The 2 forms of the indicator have different colors, the actual color of the solution depends on
pKln and this is where the indicator is most useful. For example if a solution has a pH close to 7.5
there for phenol red is the best indicator to use. This color change occurs over a wide range of
pH, so indicators will only give an approximate indication of pH. Indicators are used to
determine the end point of titration, so indicators usually used that changes color at the
equivalent point.

Factors affect indicators:

1. Oxidizing agents.
2. Reducing agents.
3. Salt concentration.
4. Proteins.

Materials and equipment

1. Indicators as given by lab assistant.
2. PH strips.
3. PH meters.
4. Samples to be tested (saliva, orange juice, albumin, ascorbic acid, guava juice, apple
juice, milk, etc.) diluted 1 in 10.
Protocol:
1- take 2 drops of an indicator to 2ml of each sample repeat the same for other indicators.

Indicator/
sample
Water
Milk
Apple
Orange
Guava
Albumin
Ascorbic
acid
Citric acid
Coffee
Tea

Results: write for each sample pH range and color change

Indicator/
sample
Water
Milk
Apple
Orange
Guava
Albumin
Ascorbic acid
Citric acid
Coffee
Tea

2- Take a PH strip and insert it in each of the given solution, notice the color changes and compare to the
guide to report the PH measurement.

sample PH
Water
Milk
Apple
Orange
Guava
Albumin
Ascorbic acid
Citric acid
Coffee
Tea
3- Measuring PH using the PH -meter.

PH meters should be calibrated before use, use two standards to calibrate the PH meter, and then insert
the electrode in each of the given solutions, don’t forget to wash the electrode with distilled water
between and after each measurement. Report your results in the following table.

sample PH
Water
Milk
Apple
Orange
Guava
Albumin
Ascorbic acid
Citric acid
Coffee
Tea

Post Lab Questions

1. Define acid, base, weak acid, weak base, amphoteric compounds?
2. Derive Hasselbalch equation?
3. What are indicators? What factors affect them?
4. What is the advantage of using indicators?
5. What are the normal pH values for blood, urine, saliva, and gastric juice? Which
indicator is suitable for each?