Beruflich Dokumente
Kultur Dokumente
: M.Sc. Chemistry
YEAR
FINAL
PAPER
VB
TITLE OF PAPER
: ANALYTICAL CHEMISTRY
PAPER-
DISTANCE EDUCATION
SELF LEARNING MATERIAL
2013
UNIVERSITY
PROGRAMME
M.Sc. Chemistry
TITLE OF PAPER -
ANALYTICAL CHEMISTRY
BLOCK .1
UNIT WRITER-
EDITOR-
(2) (Name)
(Address)
(Name)
DR.MAHESH SRIVASTAVA,D.Sc.
(Address)
COORDINATION -
COMMITTEE-
Consultant-
BLOCK 1:--
VB-ANALYTICAL CHEMISTRY
UNIT I
UNIT II
UNIT III
UNIT I V
UNIT V
(TITLE) INTRODUCTION
(TITLE) ERRORS AND EVALUATION
(TITLE) FOOD ANALYSIS
(TITLE) ANALYSIS OF WATER POLLUTION
(TITLE)ANALYSIS OF SOIL FUEL ,BODY FLUIDS AND DRUGS
BlockIntroduction
ANALYTICAL CHEMISTRY
2012
UNIVERSITY
PROGRAMME
PAPER
V-B
TITLE OF PAPER
Analytical Chemistry
BLOCK NO.
UNIT WRITER
UNIT-I, II, III & IV
UNIT-V
EDITOR
BLOCK-I
UNIT-I
ANALYTICAL CHEMISTRY
Role of analytical chemistry. Classification of analytical methodsclassical and instrumental. Types of instrumental analysis. Selecting an
analytical method. Neatness and cleanliness. Laboratory operations and
practices. Analytical balance. Techniques of weighing, errors. Volumetric
glassware cleaning and calibration of glassware. Sample preparations dissolution and decompositions. Gravimetric techniques Selecting and
handling of reagents. Laboratory notebooks. Safety in the analytical
laboratory.
UNIT-I
ANALYTICAL CHEMISTRY
1.0 Introduction
1.1 Ojectives
1.2 Analytical Chemistry and its Role.
1.3 Classification
of
Analytical
Methods
Instrumental Methods.
1.3.1 Classical Methods.
1.3.2 Instrumental Methods.
1.3.3 Advantages of Instrumental Methods.
1.4 Types of Instrumental Analysis.
1.4.1 Volumetric Analysis.
1.4.2 Gravimetric Analysis.
1.4.3 Optical Methods.
1.4.4 Separation Methods.
1.4.5 Electrical Methods.
1.5 Selecting an analytical Methods.
1.6 Methods & Cleanliness.
1.7 Laboratroy Note Books.
1.8 Safety in the Analytical Laboratory.
1.9 Laboratory operations & practices.
1.9.1 Filtration.
Classical
and
1.9.2 Drying
1.9.3 Measuring Volume
1.9.4
Graphs.
1.9.5
Concentration.
1.9.6
Percentage Solute.
1.9.7
Activity.
1.9.8
Standards.
1.9.9
Sampling.
1.9.10 Drying
1.9.11 Weighting
1.9.12 Precipitation
1.10 Analytical Balance .
1.10.1
Technique of weighing.
1.10.2
Weighing Errors.
1.10.3
Electronic Balance.
Leaning of Glassware.
1.11.2
Calibration of Glassware.
UNIT-I
ANALYTICAL CHEMISTRY
1.0 Introduction
Chemistry could be divided into five main areas analytical, biochemical
inorganic, organic and physical. The analytical chemistry it is necessary for growth
and development of science and technology and it must be integrated with other
scientific and chemical disciplines.
1.1 Objectives
Analytical chemistry may be defined as the science and art of determining the
composition of materials in terms of the elements or compounds contained in them.
Analytical chemistry is the science of chemical identification and determination of the
composition of substances and materials their chemical structure, analytical aim can
be achieved, analytical methods can be divided into identification or detection
methods, methods for measuring the content of an element in a sample and
methods of determining the molecules composition of materials.
environmental,
forensic,
manufacturing,
metallurgical,
and
(1)
(2)
(3)
Environmental
quality
is
often
evaluated
by
testing
for
suspected
(5)
1.3.1Classical Methods
These methods generally involved measurement of mass of a substance or
the volume of resulting solution.
These are also called chemical methods and according to quantity measured,
are classified as gravimetric and volumetric methods.
(i)
(ii)
In this method, the equivalence point, the moment when the amount of
standard solution added is equivalent to that of the substance being
determined, should be detected correctly.
(i)
Physicochemical Methods
(ii)
Physical Methods
Physicochemical Methods
These methods are concerned with the measurement of certain physical
parameters of a chemical system that are dependable on the nature of the system
components and vary in the process of the reaction. The parameters include:
(a) Potential value in potentiometry, and (b) optical densities of coloured
complexes in spectrophotometry etc.
The optical methods are based on the relation between optical properties of a
system and its composition. The electrochemical methods are based on the
interdependence of electrochemical properties and composition of the system.
In addition to these groups, radiometric, mass spectral number is fast
growing day by day.
(ii)
Physical Methods
These methods do not use chemical reactions. The composition of a
Analytical
methods
based
on
measurement of property
1. Mass
Gravimetric.
2. Volume
Volumetric.
3.
Electrical
potential,
conductance,
electrical
electrical Conductometry,
current, voltammetry,
potentiometry
chronopotentiometry,
polarography,
amperometry
and
coulometry,
(electrometric
or
electroanalytical methods).
4. Absorption of radiation
Spectrophotometry
Visible,
IR,
absorption,
resonance
(X-ray,
colorimetry,
nuclear
and
UV,
atomic
magnetic
electron
spin
resonance).
5. Emission of radiation
Emission
spectroscopy,
photometery,
flame
fluorescence,
radiochemical methods
6. Scattering of radiation
Specroscopy
7. Refraction of radiation
Refractometry, interferometry
8. Rotationof radiation
Polarimetry, interferometry
9. Diffraction of radiation
Mass spectrometry
1.4
1.
Volumetric analysis
2.
Gravimetric analysis
3.
Optical methods
4.
Separation methods
5.
Electrical methods
For example, acid base titration involve a neutralization reaction. Thus acids
are determined by titration or neutralization with a standard base solution and
based by titration with a standard acid solution. For example,
In precipitation titrations, a stoichiometric amount of titrant asprecipitating agent is added and volume required for the complete reaction is
measured. The amount of desired constituent is then calculated because the
precipitating agent is used as standard solution.
Ag+ + NO-3 + H+ + Cl- AgCl +H+ + NO-3
CH2CO2H
N - CH2 - CH2N
Na+O-2CCH2
CH2CO-2Na+
Cu EDTA + 2Na+ + 2H+
of the
substances
in
solution are
determined by
the
(2)
Filtration
(3)
Drying
(4)
(2)
(3)
(4)
These measurements are made making use of instruments which involve the
use of lenses, mirrors, prisms and gratings. There are some other instruments
which are included in this general classification but do not have optical parts
and still depend on electromagnetic radiation. The most important techniques
are nuclear magnetic resonance, electron spin resonance and mass
spectrometry.
all the components. Separation methods are used in many industrial processes of
isolating metals, organic compounds and other metals.
1.4.5 Electrical Methods
Electrical methods involve electronic instruments that are used to measure or
produce electrical phenomena. Current flow as a function of time, potential
developed or required, ability to pass a current and resistance, etc., are the
important properties which are related to the reaction taking place or/are causing a
reaction to take place. The fundamental measurements then are resistance, current,
potential and time.
1.5
determination. This will require careful consideration of the following features :(1)
(2)
(3)
(4)
(5)
(6)
The time required to complete the analysis, this will be particularly relevant
when the analytical results are required quickly for the control of a
manufacturing process. This may mean that accuracy has to be a secondary
rather than a primary consideration as it may require the use of expensive
instrumentation.
(7)
(8)
(9)
(10)
1.6
(1)
The bench must be kept clean and a bench-cloth must be available so that
any spillage of solid or liquid chemicals can be removed immediately.
(2)
The container vessels or bottles must be labeled, so that the contents can be
readily identified.
(3)
(4)
Bark corks should not be used to cover the vessels because they invariably
tend to shed some dust.
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
1.7
the source for reports, publications and regulatory submission the note book is a
record of original ideas.
Same good rules are given below for a well-maintained note book.
(1)
A hard covered note book of A-4 size must be used for recording
experimental observations as they are made.
(2)
(3)
(4)
(5)
Never tear out pages. If page is not used, put a line through the page.
(6)
(7)
(8)
All the data should be recorded on the same day, when they are obtained.
(9)
(10)
The record must conclude with the calculation of the result of the analysis
and in this connection the equations & reactions involved in the determination
should be shown together.
(11)
(12)
(13)
1.8
(1)
(2)
(3)
(4)
One should neutralize the acid spills with sodium bicarbonate and alkali spills
with boric Acid.
(5)
(6)
Analyst should clean up the mercury thoroughly becok mercury vapours from
five droplets are highly toxic.
(7)
(8)
(9)
(10)
One should perform only the authorized experiments, and should not work
alone in the laboratory.
(11)
When working with volatile chemicals, as when heating acids or when using
organic solvents, use the fume hood.
(12)
One should use a safety shield, when working with potentially dangerous
reactants.
(13)
(14)
Many chemicals are inflammable and many have been identified as acute or
chronic toxic substances frequently carcinogenic. Use rubber gloves when
possible and avoid breathing in fumes.
1.9
(B)
(c)
(d)
Asbestos
(e)
1.9.2
Filter Membranes
Drying
For drying purpose desicator is used. It allows to cool the hot crucible in a
dry atmosphere, before desicator provides a dry and moisture free atmosphere for
storing a sample.
1.9.3
Measuring Volume
In analytical chemistry solutions are measured by graduated glass wares such
as volumetric flaks, pipette, burette etc. People handling and use of glass wares in
volumetric analysis needs an accurate reading of the liquid a solution level.
1.9.4
Graphs
In analytical chemistry, the experimental observations are analysed and
Concentration
In stoichionetry reactions, concn is generally defined in terms of
(i)
(ii)
(iii)
Molarity
Moles
Volune of solution (in lt )
Formality
Normality
percentage by
weight, it means the parts of the total solution weight that is participated by the
solute.
1.9.7 Activity
Ideal concentration like normality molarity, or formality, corresponds to what
was originally added and true or effective concn. which makes into account the
various interaction, is called the activity.
a = c
1.9.8
Standards
For all types of determent of a standard or references point is necessary.
Standard
Primary Standard
Secondary Standard
Statistical Sampling
(b)
Random Sampling
1.9.10
Drying
After getting the sample, it is imp. to know whether sample was to be used as
such, or it has to be dried. Some samples are hygroscopic in nature a may contain
moisture. Hence there are 2 types :
(a)
Received Basis
(b)
Dried Basis
1.9.11 Weighing
Samples are weighted after the drying process. Generally are analytical
balance is used for employed in triplicate.
1.9.12 Precipitation
If the main goal is quantitative precipitation, the whole process is known as
gravimetry. In this procedure the measurement is weight or weight change.
(a)
(b)
(c)
(d)
Technique of Weighing
Weighing Errors
While using an analytical balance, there are five main sources of errors. These
are :
(a)
Static Errors
These can be imparted to the balance from the operator or from the objects
placed on the pan. Semimicro and microbalances of equal arm type are particularly
susceptible to this type of error.
(b)
Defective Balance
Errors due to balance construction or operation of the weights are possible.
These errors are difficult to discover in single pan balance, where the working parts
are enclosed. Defects may also occur because of corrosion, chipped knife edges,
dust and magnetic damping errors.
(c)
Temperature Effects
These may be caused by temperature gradient in the balance case, because
(d)
Operative Errors
Occur as a result of carelessness and improper handling of the balance and
Spillage of chemicals may lead to etching of the pan or other parts of the
balance.
(2)
Sudden jarring or adding or removing weights or the sample from the pan can
damage the knife edges in the balance.
(3)
Buoyancy Effects
Buoyancy effects arise when an object is placed on the balance pan, the net
downward forces on the pan is due to the mass of the object minus the force due to
the buoyancy of air on the object. At balance.
m0 = mw + (Buoyancyo - Buoyancyw)
1.10.3
Electronic Balance
(2)
In this, pan sits on the arm of a movable hanger & this movable system is
compensated by a constant, electromagnetic force.
(3)
(4)
(5)
(6)
(7)
A single control bar is used to switch the balance on and off, to set the
display to zero, and to trace a container automatically on the pan.
(8)
Electrochemical quartz balances are available with 100 g range that can
detect ng (10-9g) changes.
1.11
Decon 90
Many commercial detergents are available which are suitable for this purpose.
Teepol
Teepol is relatively mild and inexpensive detergent which may be used for
cleaning glassware.
(3)
CARE
A method which is frequently used, consists of filling up the apparatus with
with the supply of distilled deionized water, so that they acquire the temp. of the
room.
Graduated Flask
In the clean dry flask a small filter funnel is inserted into the neck and distill
water is added slowly. The funnel is then removed and then using a dropping tube
later is added dropwise until the meniscus stands on the graduation mark. Now the
flask is weighted and the temperature of water is noted.
Pipette
The pipette is filled with distilled water above the mark. Remove the excess of
water the pipette is then allowed to discharge into a clean weighted stopped flask.
The receiving flask is weighted & the temp. of the water is noted.
Burette
The calibration of a burette is similar to the procedure for a pipette, except
that several volumes will be delivered.
To calibrate a burette, first make satisfaction about its (i) leakage and (b)
delivery time.
1.12
Liquid reagents should be poured from the bottle, a pipette should never be
inserted into the reagent bottle.
(2)
(3)
(4)
It there is any doubt about the purity of the reagents used, they should be
tested by standard methods for the impurities, which right the cause of errors
in the determination.
(5)
(6)
Special grades to solvents for special purpose should be used e.g., spectral
grades or chromatographic grades.
(7)
Whenever possible, pick the smallest bottle that will supply the desired
quantity.
(8)
(9)
(10)
Keep the reagent shelf and the laboratory balance clean & neat clean up spills
immediately, even though some are waiting to use the same chemical or
reagent.
(11)
1.13
SAMPLE PREPARATIONS
1.13.1
of the analytic.
It is very difficult to select a people reagent and technique for decomposing
and dissolving an analytical sample particularly when refractory substance is involved
or the sample is present in trace quantity.
There are four types of common methods of decomposing & dissolving an
analytical sample.
(1)
(2)
(3)
(4)
HCl
(b) HNO3
(c)
H2SO4 (d)
(e)
(b)
Microwave Decomposition
Perchloric Acid
Hydrofluoric acid
in an open dish or crucible once a flame so that all the carbonaceous matter gets
oxidized and converted into volatile components follows dissolution of the residual
solid. In addition, voltaic metallic compounds may be lost during the ignition
process. Although dry ashing is the simplest method of decomposing organic
compounds, it is often the least reliable.
(d)
Procedure of Fusion
(1)
(2)
(3)
(4)
QUESTIONS
1. What is analytical chemistry ? Classify the analytical methods and describe the
factors affecting the choice and selection of an analytical.
2.
3.
(b)
(c)
(d)
(e)
(f)
(g)
4.
5.
(b)
Analytical balance
(c)
(d)
(e)
BLOCK-I
UNIT-II
ERRORS AND EVALUATION
UNIT-II
ERRORS AND EVALUATION
2.0 Introduction.
2.1 objectives.
2.2 Mean.
2.3 Median.
2.4 Precision.
2.4.1 Standard Deviation.
2.4.2 Variance.
2.4.3 Co-Efficient of Variation.
2.5 Accuracy.
2.5.1 Absolute Error.
2.5.2 Relative Errors.
2.6 Types of Errors.
2.6.1 Determinate Errors.
2.6.2 Indeterminate or random Errors.
2.6.3 Gross errors.
2.7 Reporting of Analytical Data.
2.8 Statistical Evaluation of Data.
2.9 The uses of Statistics.
UNIT-II
ERRORS AND EVALUATION
2.0 Introduction
When numerical data and numerical result are measured with the greatest
Exactness that the instrument method and observer are capable of, it has been
observed that the results of successive determination differ among themselves to a
great or lesser extent. The average value of a series of measurements is accepted as
the most probable value.
2.1 Objectives
In all method the reliability of the result depends upon the magnitude of the
difference between the average value and the true value. In some cases difference
may be small, and in others it may be so large that the result is unacceptable. All the
measurement from the true value. The average value of these observations is then
considered to be the most probable value. The difference a between most probable
value and the true mean value the absolute error. The absolute error in the
measurement may be beyond the permissible limits and the probable average value.
2.2
Mean
Mean (Arithmetic Mean of ungrouped data) x1,x2,x3 ..xn, are n values of a
variable x1 then the arithmetic mean or simply the mean of there value is denoted
by x is defined as. The arithmetic mean of a set of observation is equal to their sum
divided by he total number of observations.
Ex.
If the mean of 6, 7, 4, and 10 is 8. Find the value of .
8
6 4 7 10
, 40=27+
5
13 =
Arithmetic Mean of grouped Data. In a discrete frequency distribution the
arithmetic mean may be computed by the any one of the following methods.
(1)
Direct Method
(2)
Shortcut Method
(3)
only.
Direct Method
f i xi
, i =1 and N = fi =f1+f2+f3 ..fn
N
Ex.
Find the mean of the following distribution.
x4
10
15
f 5 10
10
Soln
Calculate the Arithmetic Mean
xi
fi
fixi
20
10
60
10
90
10
70
15
120
N=fi=40
N=fi=360
Mean x
2.3
fi xi 360
9
fi
40
Median
The median is the middle result when replicate data are arranged according
to increasing or decreasing. For on odd no. of result the median can be evaluated
directly. For an even number, the mean of the middle pair is used.
Median of an ungrouped Data
Precision
The precision the responsibility of the result accuracy without precision is
impossible.
Precision describe the reproducibility of measurement in their words the
closeness of results that have been obtained in exactly the same way. Three terms
are used to describe precision.
(i)
Standard deviation
(ii)
Variance
(iii)
Co-efficient of variance
( xi ) 2
n
xi = (Mn-M)
Mn = No. of observation
M = Mean
n
Number of measurements.
( xi ) 2
(n 1)
(n-1) is the No. of independent deviations. From the mean which arises prove
in determination.
2.4.2 Variance
The square of standard deviation is called varience. It is devoted by S2.
2.4.3 Co-efficient of Variation
The co-efficient of varience is an accurate measure of the precision.
CV
S 100%
S = Standard deviation
= data set mean value.
called relative
standard deviation.
S
x
2.5
s
100 ppt
x
Accuracy
The accuracy indicate the closeness of the measurement to the true or
accepted value and is expressed by the errors. Accuracy is often more difficult to
determine because the true value is usually unknown.
Accuracy is expressed in terms of
(i)
Absolute error
(ii)
Relative error
E=xi - xt
xi xt
100
xi
Types of Errors
(1)
(2)
(3)
Groos errors
(4)
Errors in measurements
(5)
Other errors
Personal errors
(b)
Operational errors
(c)
(d)
Methodic errors
(e)
(a)
Personal Errors
These are due to the factors for which the individual analyst is responsible
and are not connected with the method or procedure.
1.
2.
3.
4.
5.
6.
7.
8.
(b)
9.
10.
Errors in calculations.
Operational Errors
These errors are mostly physical in nature and occur when sound and proper
(c)
(2)
(3)
(4)
The
attack
of
reagents
upon
glassware
(d)
(5)
(6)
Methodic Errors
resulting
in
the
(e)
(b)
Erratic Errors
(a)
xl (OH)3 to constant weight, an analyst may obtain. Successive values which vary
without a definite trend. This variation could be due to varying amount of water in
the weighed residue to xl2O3.
(b)
Erratic Errors
The analyst has no control over erratic errors weighing with a sensitive
(2)
magnitude
are
(3)
Narrow packed curve with steep slope indicate a relatively high degree
of precision.
(4)
2.7
(1)
(2)
(3)
kept in mind :
(1)
Eliminate all digits that are not significant. Never retain more than one
doubtful digit.
(2)
(4)
(5)
1.
2.
3.
4.
5.
6.
2.9
can prevent judgment being mode on the basis of limited information in addition
there is rapiding developing subject of cheneometries which may be broadly defined
as he application of Mathematical and statistical methods to design or to appomise
measurement procedure and to prove the chemical information by analyzing relevant
data.
QUESTIONS
1.
(b)
(c)
(d)
(e)
2.
3.
4.
5.
BLOCK-I
UNIT-III
FOOD ANALYSIS
UNIT-III
FOOD ANALYSIS
3.0 Introduction
3.1 Objectives
3.2 Moisture Analysis in Foods.
3.2.1 Form of water in Foods.
(a)
Free Water.
(b)
Absorbed water.
(c)
Water of hydrogenation.
Drying Method.
(b)
(c)
(d)
(e)
(f)
Coulmetric Tirtation.
(g)
Physical Methods.
3.11.3
3.11.4
contamination.
3.12 Microscopic Examination of food.
3.13 Analysis of organophosphats in food by HPLC.
3.14 Gas Chromatography for organophosphate in food.
3.15 Thin Layer Chromatography for chlorinated pesticides in
food.
UNIT-III
FOOD ANALYSIS
3.0
Introduction
Chemical analysis of food is done to determine be acceptability, nutritive
value equality, composition and authencity of the food products. Major steps in the
analysis included
(i)
(ii)
(iii)
3.1
Objectives
The food materials contain organic as well as in organic constituents. The
organic compounds comprise carbohydrates, proteins, fats, oils, and
nitrogenous compounds. The food analysis involves the determination of
percentages of moistures, ash, crude fat or ether extract, crude protein,
sugars and crude fibers etc. all of these important of our life.
3.2
(1)
Moisture is used as a quality factor for Jams, filling, sugar syrups & it is a
quality factor in the preservation of food products.
(2)
(3)
Free Water : Free water acts as the dispersing agent for caller and the
solvent for salts.
(b)
(c)
monohydrate.
3.2.2 Procedures for Moisture Analysis
(a)
Drying Method's
The dry matter that remain after moisture removal is referred to as total
solids. The food samples can be dried in forced draft oven, vaccum oven or
microwave oven etc.
(b)
content of the sample. The moisture content value obtained is highly dependent on
the type of oven used, condition in the oven, time and temperatures of drying.
% Moisture ( w / wt )
% Moisture ( wt / wt )
% Total Solids ( wt / wt )
(c)
wt of dry sample
100
wt of wet sample
food sample with a high boiling point solvent that is immiscible in water. Measuring
the volume of water. Distillation methods casue less thermal decomposition of some
foods than oven drying at high temperature. Water in measured directly in the
distillation procedure but reading the meniscus of a receiving tube to determine the
volume of water is less accurate than a weight measurement.
(d)
vegetables roasted coffee coils, fats, sugar or protein. The records involve reduction
of I2 by SO2 in the presence of water.
2H2O+SO2+I2 H2SO4+2HI
Titration procedure
Iodine and SO2 are added to the sample in a closed chamber protected from
atmospheric moisture. Excess of iodine can't react with H2O can be determined
visually.
The colour in red brown.
Karl Fischer Reagent (KFR) is added directly as the titrant if the water in the sample
is accessible. If water in the solid sample is in accessible to the reagent, the
moisture extracted from the food with Methanol. The Methanol extract is then
titrated with KFR.
Determination of KFR water Equivalence (KF Req.)
The KFReq. value represents the equivalent amount of water that reacts with
1 ml of KFR. The KFReq. can be established with pure water, a water-in-Methanol
standard or Sodium tartrate dehydrate.
KF Re q. (mgH 2O / mL)
KFR eq. = KFR water equivalence, S=wt of sodium titrate dehydrate (g) A=ml of
KFR required for titration of sodium tartrate dehydrate.
% moisture content
KF Re q Ks
100
S
2.
3.
4.
(f)
Coulmetric Titration
It is ideal for samples with very low levels of moisture, from 0.03% to ppm
Physical Methods
(1)
Electrical Method
(a)
(b)
(c)
Hydrometry :
a refractometer, or by
gravimetric means.
Refractive index, n
3.3
Ash Analysis
Ash refers to the inorganic residues remaining after complete oxidation of
may
Sample Preparation
(i)
Fat and Sugar Products : Animal products, species and syrups require
treatment prior to cashing because of high fat and moisture or high sugar
content (forming) result in loss of sample. Bean, Sugars and syrups need to
be evaporated to dryness.
(ii)
Plant Materials: Plant materials are dried prior to grinding. The sample may
be used for multiple determination. Fresh stem and leaf tissues should be
dried in two stages to prevent afrifact lignin.
Dry Ashing
Principle
Procedure
-
Open the door of muffle furnance carefully to avoid leasing ash that may be
fulfill.
Quickly transfer the crucible to a desiccator for cooling and weigh it.
Calculation
Wet Ashing
Wet ashing or wet digestion is a procedure for dissolving minerals and
oxidizing substances with high fat content (meatsite) using oxidizing agents.
Procedure
-
Add 3 ml of 60% HClO4 and heat upto 3500C until fronting stops and HNO3 is
almost evaporated.
Continue boiling until per chloric reaction occurs. Place watch glass on beaker.
Sample should be colourless.
Cool the beaker wash watch glass with minimum devonised water. Add 10 ml
50% HCl.
The oxidation time is short and requires a hot plate, food, tongs and safety
equipments.
Disadvantages
-
3.
4.
ashing method whereby foods are oxidised in a partial vaccum by nascent oxygen
formed by radio frequencies electromagnetic field generator.
Instrumentation
Procedure
The ground material is inserted into individual glass boats which separate
glass chamber. The chamber is sealed and a vaccum is applied. A small flow of O 2 or
air is introduced into the system maintaining minimum vaccum. The frequency
generator is less than 14 MHZ and adjusted by the amount of wattage applied (30200 ks)to control undation.
Advantages
- There is less change of lasing trace elements by volatilization.
- The low temperature (1500C) used with plasma as hers keeps the microscopic and
crystalline structure unaltered.
Disadvantages
The major disadvantages are small sample capacity, expense of the equipment and
operator's time.
Other Ash Measurements
1.
Filter on ashless filter paper and rinse with hot distilled water five to six times.
2.
Boil for 5 min. filter on ashless filter paper and wash with hot distilled water.
3.4
Analysis of Protein
Nutritive value
Procedure
Dilute 200 ml milk to one litre with distilled water in a 26 beaber.
Add 1g glacial acetic acid when a white precipitate settles down.
Decent off the aqueous layer and wash the precipitate with water.
Grind the precipitate with a little 0.1% NaOH solution to neutralise the acid.
Add its the filterate by adding glacial acetic acid so that the solution contains
0.1% of it.
Wash the precipitate obtained from decanted water neutralist with 0.1%
NaOH solution and filter.
Again filter the precipitate with alcohol and then with to remove fats.
Kjeldahl Method
Sample Preparation- Solid foods are ground to pass a 20 mesh screen.
Sample should be homogenous.
Procedure
Add H2SO4 and catalyst (HgO, Cu3 in 3:1) for complete breakdown of organic
matter. During digestion, protein N is liberated to form NH 4+ ions H2SO4 oxidises
organic matter and combines with ammonium formed carbon
Pr otein ( N )
H 2 SO4
( NH 4 ) 2 SO4
Catalys
Calculations
Moles HCl = Moles NH3= Moles Ni
The sample and reagent blank should be run to subtract reagent N from the
sample N.
% N NHCl
100
g of sample
Mole
Principal
At 280mm due to tryptophan and tyrosine resides in proteins. Since the
content of tryptophan and tyrosine in each protein is constant the absorbance at
280mm could be used to estimate the concentrate of protein, using Beer's laws.
Procedure
Protein are solublised in alkali or Buffer. Absorbance of protein solution in read at
280nm again a reagent blank.
Advantages
-
The method is rapid, sensitive non destructive and used widely in part-column
detection of protein.
Disadvantages
-
Analysis of Fat
Fats are esters of fatty acids with teracyleglycerols. An accurate analysis of
lipids in foods is important for nutritional labeling to determine whether the food
with the standard of identify and is uniform.
Methods
1.
2.
3.
4.
Instrumental method.
5.
Calorimetric method.
6.
First Extraction
-
Add 1.5 ml NH4OH and shake vigaroulsy. NH4OH neutralizes acidic and
dissolves protein.
Add 10 ml of 93% ethanol to prevent gel formation and shake for 1 minutes.
Add 25 ml petroleum ether and shake. It remove moisture from the ethyl
ether extract and dissolves more nonpolar lipid.
Decant ether solution from the flaks into the previously wughed mojonner fat
disk.
Second Extraction
-
Third Extraction
-
Add 15ml ethyl ether and 15mL petroleum shake for 60S.
Centrifuge for 30S at 600 rpm and decent solution into the same mojonnier
disk.
Dry the disk and fat to a constant weight in a forud air oven at 1000C.
Calculation
% Fat = 100 x [(wt. of disk+fat)] - [(wt. of disk)] - ( av wt. of blank reside)
(wt. of sample)
Add 10 ml HCl heat the beaker at 800C in water bath with stirring for 30
minutes for hydrolysis.
Add 10ml alcohol and cool. The acid hydrolysed flour is extracted by a
mixture of ethyl ether and petroleum ether as described in the Mojonnier
method for milk fat.
Procedure
-
Heat the bottle in a water bath at 600C for 5 min. Read the fat content from
the graduations on the bottle neck.
Applications
Gerber method is simpler, faster and has wider application to a variety of
dairy products.
(ii)
Principle
Free Sugar and lipids are extracted with ethanol and hexane starch is
removed by enzymatic digestion and insoluble fibre is separated from soluble fibre.
Fibre protections are hydrolysed with H2SO4.
Procedure
1.
2.
3.
4.
Extracted residues are dried and weighed to determine Sugar and lipid loss.
5.
6.
7.
8.
Filtrate
(a)
(b)
(c)
Wash with ethanol, acetone and dry overnight under vaccum (400C)
(b)
(c)
(d)
3.7
ANALYSIS OF CARBOHYDRATES
Carbohydrates play an important role in human nutrition. Carbohydrtes
analysis of raw materials and processed foods can be used to provide a wealth of
information. The fingerprint oligosaccharide patterns can be used to detect food
adulteration.
3.7.1 Methods of Analysis
1. Chemicals methods for the analysis of Monosaccharides and oligasaccharides.
(i)
Upon heating, water is driven off and copper oxide is converted to cuprous
oxide.
Gravimetric Method.
(ii)
electrodes.
is
(iv)
Run down 1% honey solution from the Burette into the mixture of conical
flask till the solution terms brick red in colour.
Again add honey solution till the end point is indicated by the colour change
from blue to red.
Calculation
Total Reducing Sugar
0.5 x
100 10
Conversion factor from glucose (mol. wt. 180) to starch (mol. wt 162).
10 parts of glucose are equal to 9 parts of starch.
162 9
0.9
180 10
Add 2 ml glacial acetic acid and heat to bail keep it for 2 hours.
Calculation
Reducing Sugar (after inversion)
(i)
to product. The amount of substrate in the sample is than determined from one total
change in the sample either as substrate disappearance or as product formation.
(ii)
Modern
Methods
afford
accurate
HPLC
analysis
of
structurally
similar
3.8
(ii)
Microscale HPCL
(iii)
(iv)
(v)
(vi)
(vii)
Determination of Calcium
Reagent Required
-
Acetic Acid volume of glacial acetic acid diluted with 2 volumes of water.
Bromo cresol green indicator solution Grind 0.1g bromo 10 Cresol green with
14.3 mL of 0.01N NaOH is an agate mortor.
Procedure
-
Digest the ash in dish with Dil. HCl Evaporates to tryness. Treat the residue
with dilute HCl and again evaporate to dryness on a water bath. Treat the
residue with 10 ml Conc. HCl
beaker.
-
Wash to the residue with not water and collect the washing in the same
beaker.
Add to the solution in the beaker 0.5 ml of bromocresol green indicator and
then NH4OH till the colour changes to blue.
Filter the solution wash with hot water Called the washing in the same beaker
and heat to boil.
Digest for 3 hours filter. Through the some filter paper. Wash with hot water
until it is chloride free.
Perforate the apix of the of the filter Con. Wash the filter paper with not
dilute H2SO4 and titrate with standard KMno4 solution at 700C.
Calculation
Calcium percent by mass
2.8 NV
where
M
for
titration.
M
Ca mg/100g
3.9
Principle
Potassium in solution is atomized into an oxyhydrogen flame. The flame
execitesatoms of potassium causing them to must radiation limited is measured on a
spectrophotometer.
Reagent
(1)
KCl stock solution Dissolve 1.90 g of KCl in Distilled water and make up the
volume to 1 litre.
(2)
Standard solutions measure 150 ml stock standard solution and 5ml HCl into a
flask and make solution to 1 litre. In order to compensate for minute
Procedure
Dilution aliquotes of ash solution so that it contains less than 150 ppm
potassium. Add HCl so that the concentration of acid is same as that in the standard
solution. Atomise the diluted extract in a calibrated flame photometer with the
wavelength dial set at 768 nm and transmittance set at 100% for the top standard
solution of potassium.
Calculations
Kng/100g
Pure Ghee- Vanaspati Ghee, Animal Fat, Rancid Stuff, excess moisture.
Vanaspati Ghee- Animal body fat, rancid fat, argemon oil, seami oil,
prohibited colours as well as flavour.
Vegetable oils- Minerals oil, rancid oil, argemone oil, rubber seed oil, tea,
seed oil, watermelon seed oil.
Butter- Animal fat, starch excess moisture, rancid stuff, Vanaspati ghee,
prohibited colours.
Wheat flour- Atta, sujji, maida,sand dirt, soapstone, excess bram, chalk
powder.
Vinegar- mineral acid, coal tar dyes, Synthetic Vinegar sold as malt,
or
Coriander- Cow dung, saw dust, house dung, powered bran, foreign starch,
Chilles- Brick powder, coloured saws dust talcum powder, foreign starch,
powdered bran.
Coffee powder used coffee chicory roasted date seeds, tamarind husk starch
The consumption of adulterated foods has a slow pairing effect. The victims
of adiable oil adulterated with argemone oil show epidemic lepopsy.
(2)
(3)
(4)
The use of culture and non-permitted colours in dals, sweets or tea leaves.
(5)
(6)
(2)
Flat sour
(ii)
Thermophilic anaerobes
(iii)
Putrefractive anaerobes
(ii)
Butyric anaerobes
(iii)
(iv)
Lactobacilli
(v)
Yeasts
(vi)
Moulds
Bacillus stero thermophilic cause flat saur spoilage in low acid foods (ph 5.3)
like peas corn, potatoes.
Cocciymould and yeasts casue frothy permentation in liquars and brines due
to leakage.
B. polymyxa group (B. macerans) contaminate citrus juices, prunes jack fruit
and peach etc.
1.
Plate Method
Adding enriched food samples to a micro titrate plate a highly specific
2.
strength, and is prepared by dissolving 2.15 g NaCl, 0.075 g KCl, 0.12 g CaCl2/
dry/and 0.5g Na2S2O3.5H2O in 1000 mL of Distilled water. Methods Shake 100 g of
the vegetables with 200 mL of Ringer solution. Allow it to sand for 20 min and
decant. Test positive tubes for the presence of coli.
3.
ingredients are 10g tryptone, 5g dextrose, 0.04g bromo cresol purple 12g and 1 l
water. Steam the ingredients until dissolved.
Procedure
Add 1g of inoculum to the sterlises petridish and pour 10mL of melted agar
medium. The growth of organisms in the broth is indicated by the change of colour
from purple to yellow. The colonies appear on the surface with a typical spot in the
centre. Count total bacteria growing at 70C in 5 days also count lactic acid bacteria,
moulds and yeasts. Slime usually contains large number leuconostocs which gives
them a yellow appearances but sometimes heavy growth by coryneforms produces
ammonia and this neutralizes the acid formed by Luconostoc count on frozen
vegetables are low (106/g).
4.
In fruit juices, lactic acid and acetic acid bacteria may grow at pH4. Fruit
juices of oranges, lemon and grapes fruit used for soft drinks and beverages
must be sterile and give a negative result.
5.
with a sterile inoculating loop stain with methylene blue and gram stain. The
presence of gram positive rod suggests underprocessing while E. cocci and yeasts
etc.
Suggests Leakage Spoilage
Pesticides Analysis in food product chromatographic techniques used for the
analysis of pesticides in food stuffs are HPLC, GLC and TLC.
Reagents
(i)
H2SO4
(ii)
Acetone
(iii)
Hexane
(iv)
Dichlaromethane
(v)
Experimental Techniques
The column of the HPCL is packed with 5m c-8 bonded phase particles.
The width and height dimension of column are 4.5 x 250 mm.
The flow rate of eluting solvent method is maintained of 2mL per minute.
Process
The organophosphates are separated by using the gradients elution system of
mobile phase and the monitored the UV detector at 254mm chromatogram showing
the peaks of various. Organophosphates in food sample.
Calculations
Calculations are based on peak height measurement of the sample and the
standard for determing the concentration of species.
Analytical Steps
Sample preparation
Anhydrous salt (NaCl or Na2SO4) can be added to absorb water or water can
be added so that
For matrices like Butter fats and animal tissue sufficient water is added to the
sample to obtain at total of 100g of water.
Capillary column pached column megabore column may be used. The column
of GC is packed with chronosurb WHp capilay GC columns are fabricated from
fused silica.
The temperature of the gas chromatograph kept at 2000C for 10 minutes the
temperature of the injector is maintained at 2250C.
N2 is used as carrier gas and its flow rate is maintained at 25ml per minutes.
An
autoinjector
chromatograph.
can
deliver
present
volume
of
sample
to
the
HTPLC
High performance thin layer chromatography is a new techniques in which
TLC plates are coated with smaller, more uniform particles of controlled porosity.
This permits better detection of organochlorines in food stuffs.
QUESTIONS
1.
2.
What do you know about the moisture content of food ? how will you
determine moisture in vegetable oils and spices?
3.
How will you analyse total ash, water-insoluble ash and acid-insoluble ash in a
food sample?
4.
5.
What are the common food adulterants? How will you analyse crude fibers as
adulterant in food products.
BLOCK-I
UNIT-IV
ANALYSIS OF WATER POLLUTION
Origin of waste water, types, water pollutants and their effects. Source of
water pollution-domestic, industrial, agricultural soil and radioactive wastes as
sources of pollution. Objectives of analysis-parameter for analysis-colour turbidity,
total solids, conductivity, acidity, alkalinity, hardness, chloroide sulphate, fluoride,
silica, phosphates and different forms of nitrogen. Heavy metal pollution-public
health significance of cadmium, chromium, copper, lead zinc, manganese, mercury
and arsenic. General survey of instrumental technique for the analysis of heavy
metals in aqueous systems. Measurements of DO, BOD, and COD. Pesticides as
water pollutants and analysis. Water pollution laws and standards.
UNIT-IV
4.8.1 Ammonia
4.8.2 Nitrite
4.8.3 Nitrate
4.9
UNIT-IV
ANALYSIS OF WATER POLLUTION
4.0
Introduction
Pollution:- The term pollution has been derived from the Latin word 'Pollution',
meaning 'defilement from polluere' to soil or defile (make dirty). In recent times the
word pollution is used to denote the contamination of water soil or air.
4.1 Objectives
Water pollution is harmful for our life. Water pollution may be divided into ground
water pollution , surface water pollution ,river water pollution, lake water pollution ,
sea water pollution. Water is an indispensable need of life. It is extremely necessary
to develop a suitable technology to protect at lest, the quality of drinking water not
only against biological hazardou pollutants and biodegradable organics.
4.2
Water Pollutions
1.
Water pollution means the presence of any substance in water which affects
temporarily or permanently the quality of its usefulness.
2.
3.
Water is polluted when its basic properties are changed it becomes harmful
for human health.
4.
5.
6.
7.
Any unusual activities of man, which make water unfit for all living being
directly or indirectly, causes water pollution.
8.
4.3
Water Pollutants
2.
Physical factors:- Among the physical pollutants, heat and radiation are
important which have a marked effect on organisms.
3.
Classification of pollutant
1.
2.
3.
4.
5.
6.
4.4
The origin of waste water can be traced to its natural occurrence on the earth
formation by transformation and concentration of natural substances.
The generation of sewage and waste containing agrochemicals, certain
pesticides and surfactants. Many chemicals do not occur in nature and pollution
caused by them is entirely man-made. For ex. synthesis of various pesticides,
surfactants, radionuclide.
4.5
1.
2.
3.
Industrial wastes:- The Industrial waste have the greatest potential for
polluting the recipient water. The nature and composition of industrial waste
depends upon the raw materials, processes and operational factors. Metal
plating industries, release substantial quantities of heavy metals and cyanide
in their wastes. The chemical industries release wastes with highly variable
composition which are often acidic or alkaline in nature.
4.
The wastes from atomic reactors, hospitals etc. are most dangerous because
their radioactivity can't be destroy easily at human level. These wastes destroy the
aquatic plant and animals to a great extent. They generally cause gene mutationionization of body fluids and chromosomal mutation.
4.7
Water Analysis
4.7.1 Colour
Pure water has no colour. The presence of humic acids, fulvic acids, metallic
ions, industrial effluents may give colour to natural water. Colour can be detrermined
by following two methods:
1.
2.
(blue).
Colour Standard:- Dissolve 1 gm of crystalline cobalt chloride and 1.245 gm of
potassium chloro platinate in a small quantity of water. Then add 100 ml of conc.
then diluted to 1 litre. This solution has a colour value 500 colour unit. Prepare
standard by tilting 5.0, 1, 1.5, 2.0 etc. of above solution with distilled water to 50 ml
in standard nessler tubes. This solution has colour value of 5, 10, 15 respectively.
Indicate the colour value on each tube and protect them.
Methods:1.
Centrifuge the sample at high speed to remove suspended matter.
2.
Fill standard nessler tube with sample to the same level as that of standard
(50 ml)
3.
Compare the colour of sample with that of various standard tubes held
vertically above a white surface and find one standard with same colour as
that of sample and read the colour value.
Note: If the sample shows a colour more than 70 units it should be diluted
with distilled water and the colour estimated is multiplied with dilution factor.
2.
Fill the sample in clean tube and compare its colour with 22 mixtures of the
forel-use colour scale.
4.7.2 Turbidity
Turbidity in water is caused by suspended matter like clay, silt, organic
matter, phytoplanptons and other micro scopic organism.
Turbidity when caused (i) Largely because as phytoplankton, is considered as
an index of productivity, put on the contrary, when caused (ii) because of suspended
matter other than phytoplankton, it restricts the light penetration in water resulting
in reduced primary production (photosynthesis).
Material
1.
Nephelometer (Turbiditymeter)
2.
3.
Dilute 10 ml of above stock solution (of 400 NTU) to 100 ml with distilled
water. This standard solution has 40 NTU and can be stored for a week.
Method
1.
2.
Shake the sample thoroughly and let the air bubbles subside.
3.
Take the sample in nephelometer sample tube and find out the value on
scale. If the sample has turbidity more than 40 NTU, dilute it, so that its
turbidity can be read on the same scale.
4.7.3 Conductivity
The cell of the conductivity meter or salt bridge is filled with water sample
and the electrical conductivity (EC) is measured. It is expressed as Ds/m at 250C,
and mhos/Cm at 250C.
The Relationship between conductivity and salt concentration varies some
what depending on the ionic composition of the solution the electrical conductivity
provides a rapid and resorably accurate estimate of solute concentration.
Conductance is the reciprocal of resistance unit is ohm-1 or mhos or siemens(s).
The EC is directly proportional to the area (surface area) and inversely proportional
to the length (distance).
EC [Electrical conductivity] and a/l
EC= k a/l
a=area, l=lengtn
k= proportionality constant called specific conductance.
In case a=1 cm2, l-1cm / then conductivity =k
Measurement of Electrical conductivity
The instrument consist of an AC salt bridge or electrical resistance bridge and
conductivity cell having electrodes coated with platinum black.
Principle: A simple wheatstone bridge circuit is used to measure EC by null method.
The bridge consist of two known and fixed resistance r1,r2 and variable standard
resistance r4 and the unknown r3. The variable resistance r4 is adjusted until a
minimum or zero current flows trough the AC galvanometer. At equilibrium,
r1 r3
r2 r4
OR
r3
r1
r4
r2
2.
3.
4.
Rinse the conductivity cell with distilled water and then with the sample.
5.
6.
Connect the conductivity cell to meter and dip in the sample. Pass the current
and adjust the current by rotating the dial in such a way that maximum
sensitivity is obtained.
7.
Read the conductivity value in ds/m. Direct reading may be obtained in digital
types of meters. Observed values of EC are multiplied by the cell constant
(usually given on conductivity cell) and a temperature factor to express
results at 250C, if instruments are provided with temperature compensations
in which reading directly comes at 250C. Operating manual must be read
before the operation of the instrument.
Sometimes water shows acidity due to presence of uncombined CO2, Salts of strong
acids and weak bases and mineral acids. For determing the acidity of water, it is
titrated with standard solution of strong base by using a suitable indicator.
Apparatus :
1.
Conical flask
2.
Pipette
3.
Burette
Reagents :
1.
2.
Procedure :
1.
2.
2.
3.
Standard Sulphuric Acid: Dil. 2.8 ml of Conc. H2SO4 to 1 litre with distilled
water, dilute 200 ml of this solution to again 1 litre for getting .02 NH2SO4
standardize this solution.
Method :
1.
2.
3.
To the colourless from this titration (or to the original sample of water its
there was no colour with phenopthalein)
methyl red indicator are added and the titration continued till the colour
changes from yellow to rose red.
4.
A 1000
ml of sample
(b)
When A
1
B
2
B 1000
ml of sample
B 1000
carbonate
ml of sample alkalinify
1
2
(b) When A B
B 1000
ml of sample
2A 1000
Carbonate alkalinity, (mg/litre)=
ml of sample
Total alkalinity, (mg/litre)=
(c)
When A=0
Total alkalinity of Bicarbonate =
B 1000
ml of sample
4.7.5 Hardness
(a)
(b)
Reagents
1.
2.
Ammonia Buffer: Take 16.9g of NH4Cl in 143 ml. of liquor ammonia and dilute
to 250 ml with distilled water. In presence of metallic ions, use borate buffer.
Take 20g of barax (Na2B4O7 10H2O) in 400 ml of distilled water. Dissolve 5g
of NaOH and 2.5g of sodium supplied in 50 ml of distilled water, mix with
borex solute on and dilute to 500ml with distilled water.
3.
Method:
1.
Take 100 ml of sample in a conical flask and add 1 ml. of ammonia buffer and
2 drops of Erichrome black T indicator. Shake the solution well.
2.
Titrate it with standard (0.01 N) EDTA. The colour Charge is from wine red to
blue or bluish green.
3.
4.
Note the volume used for EDTA solution from burette. This is the end point
reading.
2.
0.02N silver nitrate 3.40g of silver nitrate is dissolved in double distilled water
and made up to one litre. This to be standardized against the standard NaCl
solution and stored in amber (brown) coloured bottle away from light.
3.
Methods :
1.
2.
3.
Titrate with standard AgNO3 solution with stirring till the first brick red tinge
appears.
4.
Chlorides in gram/litre
4.7.7 Sulphate
Sulphates are generally found in hard water. Suphate can be determined
gravimetrically, Colourimetrically and Turbdionetrically. The procedure given here is
based on EDTA titration described by Jackson (1973) .
Reagents
1.
0.02N MgCl2, BaCl2 and EDTA AR grade BaCl2 (2.44 g/l) can be directly
weighed out and dissolved. MgCl2 being highly hygroscoic, the required
quantity of AR magnesium metal of MgCo3 may be dissolved in a little excess
of dilute HCl and made up to the volume. The EDTA solution must be
standardized against 0.02N CaCl2.
2.
Buffer solution : This consist of 8.25g of NH4Cl plus 5 ml of NH4OH (Sp. gr.
0.88) in a litre. the amount of NH4OH may be so adjusted that 10 ml of this
solution added to 50 ml of water sample can give pH of 10.
3.
4.
Method:
Take 100 ml of sample and add a few drops of methyl orange indicator and
slightexcess of HNO3. Boil the mixture to remove dissolved CO2. Add 10 ml of
standard CO2. Add 10 ml of standard BaCl2 solution in the boiling solution. Allow to
Cool down and make the volume up to 150 ml of clean supernatant liquid into a
beaker. Add 1 ml of Buffer solution and some amount of Erichrome Black T indicator
titrate with EDTA solution until a permanent blue colour is produced indicating end
point. Calculation: Suppose 25ml of sample is taken for precipitation of sulphates
then the following formula can be used.
10ml of BaCl2 solution = 10gm of CaCo3 = 10ml EDTA solution.
SO-24 (mg/litre)=
of BaCl2 used
Titre value insulphate 0.98 1000
25
determination
Note:
If the sulphate in water is more than 100 mg/litre then volume of barium
chloride should be in creased.
4.7.8 Fluorides
Fluorides are more commonly found in ground water than in surface water.
The main source of fluoride in water are apatite and mica. The maximum permissible
limit of fluoride in drinking water is recommended to be 1.5 mg/kg by WHO.
Materials and Reagents:
1.
2.
3.
Method:
1.
Take 100 ml of sample in a flask and add 5 ml each of alizarin red solution
and zirconyl acid solution.
2.
Wait for 1 hour and then note the absorbance on spectrophotometer at 520
nm.
3.
2.
3.
4.7.9 Silica
Silica in water is present as silicate. In concentration in natural water is
considerably high. Silica is an important structural constitunt for diatoms and many
sponges. The assimilation of silica and subsequent sedimentation by diatoms is the
major source of silica in water.
2.
3.
Oxalic acid solution (10%) : Dissolve 20g of oxalic acid in distilled water
and make the volume up to 200 ml.
4.
Method
1.
2.
Wait for about 10 min and add 1.5 ml of oxalic acid solution.
3.
4.
5.
6.
4.7.10 Phosphates
Materials and Reagents :
1.
Spectrophotometer.
2.
3.
4.
NaOH Solution (1 N): Dissolve 4g NaOH in distilled water and make volume
to 100ml.
5.
6.
7.
Methods:
1.
2.
3.
Heat the flask gently, so that the content be comes colourless. Cool and add
10 ml distilled water and 2 drops of phenolphthalic in indicator.
4.
Titrate against sodium hydroxide solution until pink colour appears. Make up
the volume to 25 ml by adding distilled water.
5.
6.
Make the volume to 50 ml with distilled water and let the blue colour develop.
Wait for 30 minute and record the absorbance on spectrophotometer at 660
nm.
7.
Calculation : P mg/l =
4.8
mg P in 50ml 1000
Volume of sample
Colorimetrically as well
Colormetric Method
Material and reagents :
1.
Spectrophotomer.
2.
3.
4.
Method:
1.
2.
3.
4.
5.
6.
Volumetric Method:
Materials and Reagents
Micro-Kjeldhal distillation assembly.
1.
Hydrochloric acid (0.01N): Dilute 8.34 ml of 12N conc. HCl with a distilled
water to prepare 100ml of 1.0 N HCl. Dilute 10ml of this HCl with Distilled
water to prepare 1 litre of 0.01 HCl.
2.
3.
Method
1.
2.
Put 5 ml of Boric acid cum indicator solution in a conical flask. Place it below
the condensor so that the dip of outlet of condenser is dapper in contents of
conical flask.
3.
4.
5.
Remove the conical flask having distillate which turns blue due to dissolution
on of ammonia.
6.
Titrate the distillate in conical flask against 0.01N hydro chloric acid. turning
of blue colour to taint pink brown indicates the end point.
7.
4.8.2 Nitrite
Nitrites can be determined colourimentrically by EDTA method and
sulphonilamide method. The EDTA method in the followings.
Materials photometer.
1.
2.
Spectrophotometer.
EDTA solution: Dissolve 0.5g of disodium salt of EDTA in distilled water to
prepare 100 ml of solution.
3.
Take 50 ml filtered sample in an Erelenmeyer flask and add 1ml of each EDTA
Soln Sulphonic acid and naphthylamine HCl one after the other appearance
of wine red colour indicates the presence of nitrites.
2.
3.
4.
Run standard nitrite solution in similar way and record the absorbance for
different conc of standard solutions and deduce the nutrite nitrogen content
of sample by comparing its absorbance with the standard curve.
4.8.3 Nitrate
Phenol desulphonic acid method
Materials and Reagents:
1.
Spectrophotometrs
2.
3.
Phenol disulphide acid: Dissolve 25g as white phenol in 150 ml of Conc. H2So4
then again add 85 ml of conc. H2So4. That it for about 2 hours on a water
bath, cool and keep the solution in a dark bottle.
4.
5.
Method:
1.
2.
Add 3 ml of phenol disulphuric acid and dissolve the latter by rotating the
dish.
3.
After 10 minute 15 ml of distilled water is added and stirred with a glass rod.
4.
Add ammonia (1:1) slowly with mixing till the solution is alkaline as indicated
by the development of yellow colour due to the presence of nitrate. Then add
another 2ml of ammonia and the volume made up (100 ml) with distilled
water.
5.
6.
2.
3.
4.
Boric acid solution : Dissolve 1g of boric acid in distilled water to make 100 ml
of solution.
5.
Mixed Indicator : Prepare 0.1% methyl red solution and 0.5% Bromo cresol
green solution in 95% ethyl alcohol. Mix the methyl red and bromo cresol
green solution in 1:2 ratio.
6.
Hydrochloric acid (0.01N): Dilute 8.3 ml of conc. HCl with distilled water to
prepare 100ml of 1.0 N HCl. Dilute 100ml of this 1.0 NH4 with distilled water
to prepare 1 litre of 0.1N HCl. Take 100ml of 0.1N HCl and dilute to 1 litre
with distilled water to get 0.01 HCl.
Method:
1.
2.
Add 4ml of digestion mixture to the residue and dissolve it in about 20ml of
distilled water.
3.
4.
Transfer the digest to micro kjeldahl distillation assembly and add about 3-5
ml of hypo solution.
5.
Take 5ml of Boric acid solution containing 2-3 drops of mixed indicator in a
conical flask and place the flask below the condensor that the tip of outlet of
the condenser is dipped in contents of conical flask.
6.
Heat the Kjeldahl flask: Continue distillation for about 10 mm. Remove the
concial flask having. destillate.
7.
Titrate the distillate against HCl end point is determined by change of blue
colour to pink colour.
8.
(T B) N 1000 14
Volume of aliquot
Where
T= Volume of titrant HCl used against sample (ml)
B= Volume of titrant (HCl) used a against blank (ml)
N= Normality of titrant (0.0)
The atomic weight of N is 14
4.9
Water pollution problem exists every where in the country and are increasing
day-by-day around inertial and urban countries.
Metal and their toxicity: The power of toxicity varies from metal to metal. The
term toxicology can be defined as a branch of science which deals with the study of
adverse and harmful effect of chemical agents on any biological system.
Types of Toxicology:
(a)
Clinical Toxicology
(b)
Industrial Toxicology
(c)
Forensic Toxicology
(d)
Environment Toxicology
(e)
Economic Toxicology
1.
Cadmium (Cd): Cadmium does not exist free in nature and there is no
specific are from which it can be obtained. Cadmium is obtained from refining
of zinc, and copper as by product.
Uses:
1.
2.
3.
4.
5.
(b)
(c)
3.Copper
Occurance:In nature, copper occurs in sulphide ores copper is present in liver and
brain in blank pepper its is present as at 53 ppm whereas in oysters its quantity is
137 ppm. toxicity and disease copper is an industrial health nazard. More than 470
mg copper in human body is toxic and causes many disorders in the body.
4.
Lead
Occurance: ores are galena, and its sulphide. Lead is present in the bones the
concentration of lead increases with age lead is not an essential metal for
mammalians. Lead is usually deposited in bones and some soft tissues. It is also
retained by animal in lever. Kidney muscle etc.
Under some Specific conditions lead becomes Stimulatory and enhances (a)
protein synthesis (b) DNA Synthesis (c) cell replication. Toxicity and Disease: About
800 mg lead creates toxicity inhuman beings resulting in lead poisioning. Due to lead
paisioning a number of body disorders are caused.
Generally, lead toxicity is due to the concentration of diffusion of Pb in soft
tissues Another possible common mechanism. For Pb toxicity is formation of
metallotheonic.
5.
Zinc
Occurance: Zinc is not found in free form, it is present in ores. These are
(a)Sulphide (b) Silicate ore.
In human body
(i)
(ii)
Toxicity and diseased : More than 165 mg zinc causes some disorders in human
body. It cause Vomitting, cramps, Nausea.
6.
Manganese
Magnatite
(c)
Braunite
(d) tephroite
Manganese is also found in:1.
2.
Mn is less toxic but Mn is toxic when its in concentrate form. it is more than
100 ppm in human body it causes following disorder and Blindness, fever etc.
7.
Mercury
Occurance: Mercury occurs as native metal mixed with its ores. The human body
contain about 13 mg mercury 70% of which present in muscles tissues. In human
body mercury is found in kidney, lever, Intestinal and colon walls, brain, heart and
lungs. Toxicity and disease mercury and its salts are severe health hazards. It is
toxic more than 100mg causes body disorder. Inorgonic form of mercury is also
injurious to health.
Mercury is converted into methyl mercury salt and dimethyl mercury by micro
organisns, which escape into atmosphere. Blood serum protein from complex are
responsible for killing of fish in rivers and oceans.
From Industries, CH3Hg Settle as sediment at the bottom of water bodies
from where fish trap. Mercury salts. If such types of mercury contaminate fish are
used in food they cause neutrotoxic minanata disease.
Body tissue retention is greater for CH3Hg+ than for Hg2+ salts. The toxic
action is due to crowding of Hg2+ ions around the immediately available thiol groups
of proteins and delay in distribution of these ions among rest of thiol group
throughout the body.
8.
Arsenic
(b)
(c)
(d)
50
Copper
1000
3000
Manganse
5000
15000
Zinc
200
Iron
300
1000
4.10 Dissolved Oxygen (DO)
Dissolved Oxygen (DO) in water is an index of physical and Biological
processes going on non-polluted surface waters are generally saturated with
dissolved oxygen.
1.
2.
2.
Electrical stirrer
3.
Methods
Read the operation manual carefully and adjust the instrument accordingly.
Dip the Do probe in 5% Sodium sulphate solution with constant stirring. Now dip the
D.O. probe in water. Sample being constantly stirred and record the dissolved
oxygen in mg/litre from the scale.
Winkler's Method
Principal : Oxygen combines with Mn(OH)2 and form higher hydroxides which on
subsequent acidification in the presence of iodine, liberate iodine in an amount
equivalent to the original dissolved oxygen content of the sample.
Materials and reagents:
1.
2.
3.
Alkaline potassium iodine solution. Weight 50g of KI, and 100g of potassium
hydroxide. Dissolve the chemicals in 200ml of previously boiled distilled water.
4.
5.
Starch Indicator: Dissolve 1g starch in 100 ml warm distilled water and add a
few drops to toluene as preservative.
6.
Method :
1.
Take a glass stoppered BOD bottle of known volume (100-300ml) and fill it
with sample avoiding any bubbling. No air should be trapped is bottle after
the stopper is placed.
2.
Open the bottle and pour is each 1 ml of manganese sulphate and alkaline
potassium Iodide solution using separate pipettes. If the volume of sample is
over 200 ml add 2 ml of each reagent instead of 1 ml.
3.
A precipitate will appear. Place the stoper and shake the bottle thoroughly.
Sample at this stage can be stored for a few days, if required.
4.
5.
Calculation :
(i)
(ii)
V1N 8 1000
V2 V3
V1 N 8 1000
V
V4 (V2 3 )
V2
is 8.
2.
3.
B.O.D. free water: Pass the deionized glass distilled water through a column
of activated carbon and redistilate.
4.
5.
6.
7.
distilled water.
8.
Sulphuric acid (1N): Add 2.8 ml of conc. sulphuric acid to 100 ml of BOD free
distilled water.
9.
10.
Method
1.
2.
3.
Fill two sets of BOD Bottles and add 1 ml of Aruglthiourea solution to each
bottle. As for as possible avoid entrapping air bubbles in BOD bottles.
4.
5.
Determine the dissolved oxygen content. Take out the battles after 5 days
and determine immediately their dissolved oxygen content (Ds) Calculations :
BODs (mg/l) = (D.O-Ds)Dilution factor Dissolve oxygen after 5 days sample
factor.
4.12 Chemical Oxygen Demand (COD) The amount of oxygen required for
oxidation of organic compound which are present in water by means of reaction.
Substances such as potassium dichromate and potassium permangate, potassium
dichromate ion is most suitable oxidant.
Principle : Organic matter decomposes poses and produces carbon dioxide and
water when it is boiled with a mixture of potassium dichromate and sulphuric acid.
Amount of potassium dichromate in the sulphuric acid medium and the excess
dichromate is titrated against ferrous ammonium sulphate (FAS).
COD reflux unit consisting of flat bottom flask with ground glass Mouth and
leibig's condenser.
2.
3.
4.
5.
Conc. H2SO4.
6.
7.
Normality of FAS =
Methods :
1.
10 ml of
Attach to the moush of flask and heat the flask on a hot water bash or
heating mantle for at least 2 hours to reflux the contents.
3.
Cool the flask, detach from unit and dilute its contents to about 150 ml by
adding distilled water.
4.
Add 2-3 drops of forroin indicator solution and titrate against ferrous
ammonium sulphate solution. At the end point blue green colour of contents
changes to reddish blue.
Calculation : COD (mg/Le)=
(B T) N 1000 8
Volume of sample (mL)
QUESTIONS
1.
What is the origin of waste water ? Describe the water pollutants and their
effects.
2.
What are the sources of water pollution? Give details about industrial wastes
as source of pollution.
3.
4.
5.
BLOCK-I
UNIT-V
ANALYSIS OF SOIL, FUEL, BODY FLUIDS AND
DRUGS
(a)
silica, lime
Fuel Analysis : Solid, liquid and gas. Ultimate and proximate analysis
heating values-grading of coal. Liquid fuels-Flash, aniline point octane number and
carbon residue. Gaseous fuels-producer gas and water gas-calorific value.
(C) Clinical Chemistry : Composition of blood-collection and preservation of
samples. Clinical analysis. Serum electrolytes, blood glucose, blood urea nitrogen, uric,
albumin, globulins, barbiturates, acid and alkaline phosphatases. Immunoassay :
principles of radio immunoassay (RIA) and applications. The blood gas analysis - trace
elements in the body.
(D) Drug Analysis : Narcotics
Screening
by
measurements.
gas
and
thin-layer
chromatography
and
spectrophotometric
UNIT-V
ANALYSIS OF SOIL FUEL, BODY,FLUIDS AND
DRUGS
5.0 Introduction
5.1 Objective
5(a) Analysis of Soil
5a.1 Introduction
5a.2 Determination of moisture
5a.3 pH measurement
5a.4 Analysis of total nitrogen
5a.5 Analysis of phosphorus
5a.6 Analysis of silica
5a.7 Analysis of lime
5a.8 Analysis of Magnesium
5a.9 Analysis of Manganese
5a.10 Analysis of Sulphur
5a.11 Analysis of Alkali-salts
5(b) Fuel Analysis
5b.1 Solid, Liquid & Gaseous Fuels
5b.2 Gaseous Fuels
5b.2.1
Producer Gas
5b.2.2
Water Gas
5d
Drug Analysis
5d.1 Narcotics and dangerous drugs
5d.2 Classification of drugs
5d.3 Screening of drugs by gas and thin-layer
chromatography and spectrophotometric
measurements.
5c.3.1
Spectrophotometric determination
5c.3.2
TLC determination
5c.3.3
UNIT-V
ANALYSIS OF SOIL FUEL, BODY FLUIDS AND
DRUGS
5.0
Introduction
Soil, fuel, body fluids and drugs are non-separable parts of human-beings &
their livlihood since time immortal. Without body fluids existence of human body is
not feasible. Soil forms the base upon which we live. Food we eat is produced on the
soil. Fuels are the need for cooking our food, for providing energy to us, for the
transportation, for generating electricity. Fuels are needed to heat boilers for the
industries. Drugs cure ailments occurring in body. Studies in these fields are a must.
5.1
Objective
Clinical analysis are needs to check proper functioning of body. Any imbalance
Analysis of soil
5a.1 Introduction
Soil is as much essential as the food. We draw our food either from plants or
from animals. Plants grow in soil, and animals also depend upon plants and
indirectly draw their food from soil. Main components of soil are moisture, nitrogen,
phosphorus, silica, lime, magnesia, manganese, sulphur and alkali metal salts etc.
Thus soil may be acidic or basic. A brief study of origin of soil may reveal its
Formation of Soil
Soil might have formed mainly by three important process occurring in rocks :
1.
1.
Physical weathering
2.
Chemical weathering
3.
Biological weathering
Physical Weathering
Physical weathering i.e. disintegration of soil exerts mechanical effect on the
rocks as a result of which rock might have broken down into particles of smaller size.
Physical weathering does not bring any chemical transformation of rock minerals.
This process occurs in deserts, at high altitudes, high latitudes and in topographic
relief as well as the rocks where vegetation is poor. The temperature, water, ice,
gravity and winds are some climatic factors responsible for physical weathering. e.g.
temperature may bring about the break down of heterogenous rocks. Water may be
responsible for mechanical weathering of rocks by rain water. Freezing and melting
of ice causes weathering of rocks by frost action and glacier formation.
Landslides and rock slippages due to earthquake fall in the category of
gravitational weathering where rocks are disintegrated by abrasion and forces of
impact. Stromy winds carry suspended sand particles & may cause abrasion of
exposed rocks.
2.
Chemical Weathering
Exposed rocks undergo chemical weathering which causes chemical
(i)
Formation of solution
Water soluble minerals like lime stone and gypsum etc., get weathered by the
solvent action of water which is enhanced in presence of CO2 and organic acids.
Later come into existence by the decay of organic remains of animals and plants.
Solutions of these minerals are either absorbed on the surface of negatively charged
colloidal particles or are removed by leaching.
(ii)
Hydrolysis
Hydrolysis involves chemical action of water over strong bases and produces
Hydrolysis releases Na, K, Ca, Mg and silicates into the solution. These are
responsible for the growth of plants.
(iii)
Carbonation
Carbonation involves the combination of CO2 and H2O to carbonic acid which
H2CO3
(iv)
Oxidation
Oxidation involves reaction of minerals with oxygen leading to the formation
of oxides which get dissolved in water & weaken the rock. This process bring about
the weathering of rock e.g.
4 FeO + O2 2 Fe2O3
Oxides and sulphides of iron, manganese and aluminium are easily oxidized and lead
to chemical weathering of rocks.
(v)
Reduction
Fe2O3 (red ferric oxide) can be reduced to FeO (grey ferrous oxide).
Reduction generally occurs in deep zones of earth crust.
2Fe2O3 4 FeO + O2
(vi)
Hydration
During hydration water gets attached to rock material as water of
Biological Weathering
Micro-organisms like bacteria, fungi, protozoa, lichens and mosses transform
rocks into a dynamic system where energy is stored & organic material is
synthesised. This process changes physical structure as well mineral composition of
rocks.
From the process of soil formation it is clear that chief constituents of soil are
moisture, oxides, hydroxides, silicates, lime, salts of magnesium, aluminium, alkali
metals etc. It contains organic compounds & may be rich in nitrogen & sulphur.
Moreover, soil may be acidic as well as basic.
5a.2 Determination of moisture
Methods generally used for the determination of moisture contents in soil are
:
(i)
Gravimetric method
(ii)
Volumetric method
(iii)
Electrical conductivity
(iv)
(v)
Neutron scattering
(vi)
(vii)
(1)
Volumetric Method
Volumetric determination of moisture contents in soil may be done by (i) Soil
By Soil-core
Requirements:
(1)
Sample tube
(2)
Physical balance
(3)
Moisture boxes
(4)
Drying oven
Procedure:
1.
Place the sample of soil in sampling tube whose volume (v) is known.
2.
3.
Dry it in oven to constant weigh (B) at 105C and again weigh it.
Calculation:
Moisture percentage ( Mv)
A B
100
dw V
For better results take more than 50 C.C. in size and greater than 200 g of
soil.
2.
(ii)
Bulk-Density Method
Moisture percentage on volume basis (Mv)
= Mw Bd
Gravimetric Method
For gravimetric estimation of moisture content soil sample is kept at 105 is
oven and is dried to constant weight. Difference in weight of moist soil and dry soil
gives wt of moisture.
Formula used:
% moisture
Loss in weight
100
dry weight soil
Requirements:
(1)
Sample auger
(2)
Physical balance
(3)
Moisture cans
(4)
(5)
Desiccator
(6)
Drying agent.
Procedure:
1.
2.
Transfer about 100 g of sample in the sample can with the help of auger.
3.
4.
5.
Remove the caps of moisture can and heat in oven at 105 to constant
weight. It takes about 45 hrs.
Precaution:
After each heating cool the moisture cans in desiccator containing drying
agent.
Observations:
(i)
(ii)
(iii)
Calculations:
Moisture contents in soil = Y - Z
Wt of dry soil = Z - X
Percentage of moisture
(Y Z )
100
(Z X )
Result:
Soil sample under investigation contains _______ % of moisture.
5a.3 "pH Measurement"
pH is negative logarithm of hydrogen ion concentration.
pH =-log [H+]
pH of soil ranges from 0 to 14 accordingly soil may be acidic or alkaline. Soil
with pH 1 to 7 is acidic, with exact 7 is neutral and soil with pH range 7 to 14 is
alkaline. Word pH derives it origin from French word" pouvoir hydrogen" mean
hydrogen power. pH of soil provides important information about properties of soil.
It affects solubility of soil as well as plant growth as later depends upon activity of
micro-organisms. Nitrogen fixing bacteria of soil responsible for growth of legumes
are not very active in acidic soil. Bacteria causing decomposition of organic matter
of soil are ineffective in strong acidic medium. However, fungi survive in acidic
medium.
Soil of pH range 4-5 have toxic concentrations of manganese and aluminium.
Tea, blue berries, Green berries, few confers as well as rhododandrons grow is
strongly acidic soil. But beans, alfa, beets and barley grow in slightly acidic to
moderately basic soil on account of high calcium demand and little tolerance of
aluminium. Minerals are soluble in acidic soil as compared to basic or neutral soil.
Measurement of pH
pH - meter method
(2)
Colorimetric method
(1)
is generally used.
pH-Meter Method
For measuring pH of soil pH meter used which is a glass electrode pH meter
with calomel reference electrode with salt-bridge. Modern digital pH meters are
single electrode pH meters which require calibration before use with buffer solutions
of known pH. A glass surface in contact of H+ ions pH of which is to be measured
acquires an electrical potential & gives H+ ion concentration i.e. pH of solution.
Requirements:
(i)
(ii)
Beaker
(iii)
Glass rod
(iv)
(v)
Determination of pH of soil:
pH of soil can be determined in three forms of soil solutions:
(i)
1.
Prepare the paste of the soil in small amount of distilled water in a beaker
with the help of a spatula.
2.
3.
After preparing saturated paste cover it & allow it to stand for approximately
four hours.
4.
Now there should be no free water on the surface of soil and the paste should
not stick or loose its glistening. From this paste pH can be determined.
5.
(ii)
to 40g of soil in 250ml Erlenmeyer flask & mixing the contents on reciprocating
shaker for 1 hr. During the process as well as after preparing the suspension flask
should be stoppered.
(iii)
1.
2.
3.
Now leave the contents so that CaCl2 solution get absorbed by the soil. It is
worth mentioning do not stir the contents during absorption process. But
after the absorption has taken place stir thoroughly for 10 seconds with the
help of glass rod.
4.
5.
Procedure:
1.
2.
Set the temperature compensating knob of pH-meter and also ensure if the
electrode is completely filled with saturated KCl solution. Let the pH-meter to
warm for 15 minutes so that asymmetric potential of instrument get
eliminated.
3.
4.
Now read the pH of soil paste or 1:2 soil-water or 1:2 soil-CaCl2 suspension
with the pH - meter calibrated between 7.0 and 9.2 in step-3.
5.
After step-4 again electrodes/electrode are washed with distilled water & then
immersed in distilled water. To maintain electrodes in working condition they
should be kept immersed in distilled water.
Theory:
Soil sample is first digested with H2SO4 so that organic nitrogen get converted
in NH+4 ions. N-N and N-O nitrogen can't be converted completely by Kjeldhahl
digestion. However, in soil very little nitrogen is in this form. But, it contains nitrities
and nitrates.
Requirements:
1.
Digestion block [20- place block digester with tractor auto temperature
controller].
2.
3.
4.
Procedure:
1.
Place ~ 2g of mineral soil with low content of nitrogen (60 mesh) into
digestion tube after accurate weighing and add 10 ml. of conc. H2SO4 with
shaking.
2.
Now heat the digestion tube in digestion block until very black [for about 30
minutes]. Digestion block should be loaded in fume-cupboard to avoid
irritation.
3.
Add one Kjeltab to the contents. Heat till Kjeltab dissolves at 200C which
takes about 15 minutes.
4.
5.
Now, let the temperature rise to 375C and continue heating till sample turns
turquoise which may take upto 45 minutes.
6.
Take out digestion tube from block and allow to cool for 5 minutes. Cooling
should not be done in heating block, because ammonia formed from
ammonium sulphate formed during the digestion may be lost by the action of
heat.
7.
Mix the sample with 50 ml of water till sample goes into the solution.
8.
Alternative Method
In Kjeltc Auto 1030 Analyses method ammonia liberated by the distillation of
digest with strong alkali is absorbed in unstandardized H3BO3 to get Ammonium
borate. Borate is titrated back to H3BO3 by titration against standard acid (HCl).
Requirements:
1.
2.
Distillation apparatus
3.
Titration Apparatus
4.
5.
6.
Procedure:
1.
2.
3.
4.
Calculations:
% of nitrogen in sample =
Notes:
(a)
(b)
Compare your answer with those given at the end of the unit.
(i)
(ii)
(iii)
(i)
_________________________________________________
(ii)
_______________________________________________
(iii)
_________________________________________________
Requirements:
(i)
(iv)
(v)
(vi)
make up the volume to 1 lt. with D.W. This solution is 100 ppm stock solution of
phosphorus 5ml of it are diluted to 100 ml in volumetric flask to get 5 ppm
phosphrous solution.
(x)
Spectrophotometer.
Procedure:
1.
2.
3.
Now add 50 ml of NaHCO3 solution at 25C and shake the contents for 30
minutes.
4.
5.
6.
7.
Add 8 ml Murphy - Riley solution & make up the volume to 50 ml with D.W.
Now, keep the contents form 15 minutes at R.T. & record the absorbance of
blue coloured solution on spectrophotometer or colorimeter at 730 mm.
Calculations:
Wt of soil sample = 2.5 g
Volume of NaHCO3 extractant added = 50 ml.
Vol. of extract taken to develop colour = 1o ml.
Reading of colorimeter or spectrophotometer = X
Concentration of phosphorus in 50 ml extract
= C/10g/ml.
In 1 g of soil concentration of phosphorus
50
g / ml
10 2.5
C 50
2.24
10 2.5
some extent other plants also contain silicon. Analysis of total silicon contents
involves following steps.
1.
Digest soil with perchloric and nitric acids followed by heating for 15 minutes.
2.
Dilute the digest and filter silica through whatmann no. 41 filter paper. Wash
it with warm and dil HCl (1:20) as well as with D.W.
3.
Evaporate the filtrate [containing washings as well] to dryness and heat the
residuce at 110C for an hour. This step recovers small amount of silicic acid
present in solution.
4.
Now extract the residue with dil HCl, filter and wash as given as step 2.
5.
Ignite the combined residue along with filter paper in a weighed platinum
crucible in muffle furnace till constant weight and find out the weight of Crude
silica. From the weight of silica amount of Si present in soil can be determined
by multiplying with the factor 0.4672.
Analysis of water soluble Silicon : Quantity of water soluble silicon in soil
can be determined by molybdenum blue method.
Requirements:
1.
2.
3.
0.25 M-HCl
4.
5.
Citric acid.
Procedure:
1.
2.
3.
4.
Mix the contents well and record optical durity (O.D.) at 650 mm exactly 1
minutes after adding reductant.
5.
Standard curve needed to estimate Si can be prepared to cover the range 0-5
g/ml of Silicon.
Requirements:
(i)
p-Nitrophenol - 1.8 g
(ii)
(iii)
K2CrO4 - 3.0 g
(iv)
(CH3COO)2Ca - 2.0 g
(v)
CaCl2.2H2O - 40.0 g
To prepare Buffer dissolve above salts in 800 ml of D.W. and adjust pH at 7.5
with the help of dil HCl or NaOH solution. Dilute it to 1 litre with D.W.
Procedure:
1.
2.
Requirements:
(i)
EDTA - 0.01 N.
(ii)
(iii)
(iv)
2% NaCN solution.
(v)
Carbamate
Procedure:
1.
Pipette out in a 150 ml conical flask 25 ml aliquot containing not more than
0.1 ml of Ca+ Mg. If solution in more concentrated it should be diluted.
2.
3-4 drops of
Titrate the solution in conical flask with 0.01N EDTA till colour charges from
wine red to blue or green.
Calculations:
If N1 & V1 are normality and volume of aliquot taken and N2 & V2 are
normality & volume of EDTA used.
Then,
N1V1 = N2V2
N1
or
N1
N 2V2
N1
Importance of Mg:
Mg is known as secondary micronutrient for plant growth & is required from
the beginning in small amount. It is found dissolved in water as soluble salts. Mg is
integral part of chlorophyll and is also involved in enzymatic reactions. It helps to
increase vitamin, sugar, starch and insulin in root crops. Its deficiency causes
chlorosis.
Requirements (Apparatus)
(i)
(ii)
20 ml pipette
(iii)
(iv)
(v)
Funnels.
(vi)
(vii)
(viii)
Analytical balance.
Reagents Required:
(i)
Extracting Solution
Stock Solution
Standard stock solution should be prepared by dissolving 0.1 g foil or wire of
to 100 ml volumetric flask and diluting each to mark with DTPA extracting solution.
These solutions contain 0, 1, 2, 3, 4, 6, & 8 g/ml (ppm).
Procedure:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Calculations:
Wt of soil taken = 10 g.
Vol of DTPA solution added = 20 ml.
Dilution = 2 times.
Absorbance shown by AAS = A.
Concentration of micro nutrient (Mn) read from standard curve against Absorbance A
= C.
Mn is soil sample = C 2 mg/kg or ppm or g/ml.
N.B. Standard curve is drawn between readings of absorbance & concentrations of
Mn in ppm on graph paper.
Importance of Mn
It is a constituent of enzyme & is involved in protein synthesis. It increases
translocation of sugars through stems and leaves of cotton plant to improve the fibre
strength of cotton. Mn also favours the accumulation of ascorbic acid in grape leaves
and of sugar in grapes. Excess of Mn leads to chlorosis and nacrosis of leaves.
5a.10 Analysis of Sulphur
For the determination of S, heat soluble sulphur, 0.15% CaCl2 extractable
sulphur and phosphate extractable sulphur methods are used. Heat soluble sulphur
method is more common.
Requirements:
(i)
1% NaCl solution
(ii)
(iii)
(iv)
(v)
3% H2O2.
Procedure:
1.
Place 5.0 g accurately weighed soil in a silica basin and add 20 ml D.W. to it.
2.
Keep the basin in gently boiling water and evaporate to dryness. Then, heat it
in hot air oven at 102C for 60 minutes.
3.
Cool the contents and transfer the soil in 50 ml centrifuge tube. Extract it with
33 ml of 1.0% NaCl solution.
4.
5.
Cool the residue and add to it 25 ml D.W., transfer again to centrifuge tube
and centrifuge to remove suspended particles. From this aliquot sulphur is
determined.
6.
7.
Now, add 2.5 ml stabilizing solution, 0.2 g BaCl2 crystals by a spatula to the
standard and extract. Shake the flask for 1-minute.
8.
After about 3 minutes measure the turbidity in colorimeter using blue filter or
in spectrophotometer at 340 nm.
9.
Calculations:
SO4 - S mg/Kg
Procedure:
1.
Pipette out 5 ml of 0.1 N Barium chromate in 150 ml volumetric flask. Add 1.2
ml 5N-NH4OH to it which reduces free acidity to 0.05N. This solution should
be clear.
2.
3.
Now add 1 ml NH4OH to precipitate unreacted barium chromate & make upto
100ml with D.W.
4.
Stopper the flask and make upside down 3-4 times. Filter the contents
through whatmann - 42 filter paper. Reject first few ml fitrate.
5.
N.B.
1.
2.
Important Information
S is present in soil in both organic as well as inorganic forms, but only a
fraction is available to plants. Inorganic sulphur is in the form of SO4--.
5a.11. Analysis of Alkali-Salts
Alkali salts make the soil saline or alkaline. In aqueous solution they are
present in their ionic concentrations. Water soluble alkal-salts are determined in Soil
by:
(1)
(1)
Saturation extracts
(2)
Procedure:
1.
2.
Take it in weighed flask and add sufficient D.W. to it with mixing to prepare
saturated solution.
3.
Allow to stand for 4 hrs. If soil has stiffness and does not glistens, add more
D.W. and mix well.
4.
5.
SP
6.
Now transfer the soil to Buchner funnel with highly retentive filter paper &
collect the extract - by applying vacuum till air passes through filter. Add one
drop of 1.0% (NaPO3)6 solution per 25 ml of extract to check the precipitation
of CaCO3.
7.
(2)
Procedure:
1.
2.
3.
Filter the suspension through whatman No. 1- filter paper & collect the
filtrate.
4.
N.B.
(1)
The cations [Ca2+, Mg2+, Na+, K+] are normally determined by compleximetric
titration, AAS or atomic emission spectrometric methods.
(2)
Primary anions in soil extracts are normally SO42-, CO2-2, Cl-, NO3- and HCO3-.
These are determined by titrimetric or Atomic emission spectrometric
methods.
Notes:
(a)
(b)
Compare your answer with those given at the end of the unit.
(i)
(ii)
(iii)
(i)
_________________________________________________
(ii)
_________________________________________________
(iii)
_________________________________________________
5b.
"FUEL-ANALYSIS"
In homes for cooking & other heating purpose. Fuels like LPG, Wood, Coal,
Kerosene, Cow-dung & Charcoal etc., are used for this purpose.
2.
For transporting men and material from are place to other through motor
cars, trains and aeroplanes etc. Petrol and diesel are used for this purpose.
3.
Fuels used in industry are coal, fuel oil or natural gas which are used for
heating the boilers.
4.
For generating electricity coal or natural gas are used in thermal power
plants.
5.
In rockets used for exploring space: Special fuels known as propellants are
the fuels for this purpose. For instance, synthetic rubber and liquid hydrogen
are rocket fuels.
Most of the fuels are carbon compounds containing hydrogen. Therefore,
when a fuel is burnt, it combines with O2 of air and produces CO2, water vapours
and a lot of heat. Some fuels like Coke and Charcoal are mainly carbon. Three main
sources of natural fuels are; coal, petroleum and natural gas.
Classification of Fuels
Fuels can be classified in following ways.
1.
2.
3.
(I)
1.
2.
3.
Solid Fuels
Liquid Fuels
Gaseous Fuels
Wood
Coal
Coke
Charcoal
Paraffin Wax
Kerosene
Petrol
Diesel
Alcohol
Liquid hydrogen
Liquid fuels
Natural gas
LPG
Coal gas
Producer gas
Gobar gas
Acetylene
Hydrogen gas
No residue is left after burning liquid & gaseous fuels, but solids leave behind
ash.
2.
3.
Ignition temperatures of gaseous and liquid fuels are low, therefore, they
burn easily as compared to solid fuels.
4.
Liquid & solid fuels have high calorific values, hence, produce more heat on
burning than equal amounts of solid fuels.
5.
6.
Handling of liquid and gaseous fuels is easier than solid fuel. For instance, it is
easier to handle kerosene than coal.
(II)
Fuels that are used in the same form as they occur in nature are called
natural fuels or raw fuels. e.g., Wood, Coal, natural gas, petroleum,
animal drug and agricultural wastes.
On the other hand, fuels which are prepared by natural fuels by various
physical and chemical processes are known as manufactured or processed
fuels e.g. fuels like charcoal, coke, petrol, diesel, kerosene, coal gas, producer
gas & water gas etc. Charcoal is prepared by heating natural fuel wood by
heating in absence of air.
Fuels which are used directly to produce heat are known as primary fuels
e.g., Wood, Coal, Petroleum, natural gas, agricultural wastes etc. Infact,
primary fuels are raw fuels.
The fuels which are prepared from primary fuels are known as secondary
fuels. Infact, they are processed fuels e.g., producer gas, coal gas and water
gas etc.
Choice of fuel depends upon factors like cost, calorific value, convenience,
side effects like pollution as well as availability. Most of the fuels we use come from
bio-mass and fossils and are known as Bio-mass fuels and fossil fuels,
respectively. It is worthnoting waste materials of living objects (like cattle dung) and
dead parts of living objects (like animals, plants and trees) are Bio-mass. But,
fossils are remains of pre-historic animals or plants, buried under the earth, millions
of years ago.
(i)
"Solid Fuels"
Ingredients of solid fuels are to two types: (i) Combustible organic matter
(2)
(3)
(1)
Wood
Coal
Natural
Solid
Fuesl
Lignite
Bagasse
Peat
(i)
Wood
Wood is the fuel found in plenty in India. It contains cellulose, lignin, resin,
inorganic material and water [to the extent of 60%]. Approximate composition of
dry wood is C=50%, O=35%, N=70%, H=6%, & ash=2% Calorific value of Wood
(dried) varies from 4000 - 6400 BTU/pound. Following products are obtained by the
destructive distillation of dry wood.
Product
Percentage by weight
1.
Charcoal
23.5%
2.
Pyroligneous acid
44.3%
3.
Wood tar
5.3%
4.
26.9%
Dry wood is better fuel on account of its low ignition temperature and low ash
contents.
Advantages of wood as a fuel are:
1.
2.
It is easily available.
3.
4.
5.
Because, wood gives smaller amounts of ash and soot it can be used in some
industries like glass, firing of porcelain etc.
High moisture contents keep its calorific value low. So, it is rarely used in
industry.
2.
(ii)
Coal
Coal is complex mixture of compounds containing carbon, hydrogen, oxygen
and also contain some free carbon. Some nitrogen & sulphur compounds are also f
ound in coal. Coal is found in deep coal mines beneath the surface of earth.
India is rich in coal in states like Bihar, West Bengal, Orissa and Madhya Pradesh.
Jharia and Bokaro are rich coal mines found in Bihar, whereas Ranigang is in west
Bengal. Coal is still an important source of energy in our country, therefore, it is
regarded as backbone of energy sector of our national economy. Coal can be
converted into other sources of energy like : electricity, synthetic petrol, coal gas
etc. It is a rich source of organic compound which can be converted into dyes,
drugs, polymers like fibres, explosives, detergents and paints etc.
Original of Coal
It is thought that coal was formed millions of year ago by the decomposition
of large land plants and trees buried deep inside earth. Forests might have been
buried under the surface of earth due to Volcanoes and earthquakes etc and in due
course they were covered with sand, clay and water. At high temperature and
pressure and in absence of air inside the earth, wood might have been converted
into coal. This slow chemical process of conversion of wood into coal is
named carbonization. Carbonization is very slow process and may have taken
thousands of years to take place. As carbonization is slow process several
intermediate products of carbonization exit. They are: anthracite, bituminous, lignite
and peat. Anthracite is last step of coal formation. These products differ in
percentage of carbon. Anthracite is richest in carbon.
Type of Coal
Based upon the concentration of carbon following variation of coal are formed
depending upon the extent of carbonization:
Type of Coal
Percentage of Carbon
a.
Peat
60%
b.
70%
c.
80%
d.
90%
(a)
Peat
Peat is formed by the gradual decomposition of plant materials under moist
conditions. This is the initial state of transformation during the coal formation. Peat
is jelly like and is brown colored. Calorific value of peat is low (6000-9000 BTU)
hence, it is not of any economical value. In dry peat ash contents are 2.5-6.0%.
Peat is sometimes used as fertilizer as well as packing material. Deposits of peat are
found in England, Scotland, Iceland, Northern Europe, Germany, Sweden & Russia
etc. In India peat reserves are found in Nilgiri Hill, Palani Hills, in west coast of Tamil
Nadu and in Kashmir state. In dry condition composition of peat is
C
57%
6.1%
34.9%
Bituminous Coal
Formation of Bituminous Coal is third stage of coalification. Bituminous coal is
of two types:
(i) Sub-bituminous coal (ii) Bituminous coal
(i)
Sub-Bituminous Coal
Sub-bituminous coal is also known as black lignite as it is black is colour. Its
(ii)
Bituminous Coal
It is hard, black & dense. Bituminous coal is of two types: low volatile coal &
high volatile coal. Fuel ratio of low volatile at coal is < 2. Bituminous coal burns with
yellow smoky flame. High volatile variety have long flames and find use in gas
industry, coal-tar distillation and in the manufacture of glass. Light grade bituminous
coal is known as Super-bituminous coal. Its calorific value range is 12000-15000
BTU. Super-bituminous coal have higher fuel ratio as compared to bituminous coal &
have best heating power among all coals. Bituminous coal can be converted to coke.
(d)
Anthracite
Anthracite contains highest percentage of carbon [~ 90%]. It is hard,
compact & black with sub-metallic lusture and is of fossil origin. Anthracite is last
product of carbonization process. It is sulphur-less is low-ash coal & burns with
smokeless flame. Calorific value range of anthracite is 14000-15000 BTU and fuel
ratio is 9.0-10.2. Anthracite is used in metallurgy, in slow combustion stoves as well
as for domestic purpose.
(e)
Pulverised Coal
Pulverised coal is obtained after grinding the fossil coal. It burn quickly & its
combustion temperature is very high. Burning capacity of this coal depends upon the
fineness of particles. Pulverised coal has low ignition temperature because of
presence of volatile material in it. The pulverization procedure includes crushing,
drying & grinding of coal.
(iii)
Bagasse
Bagasse (the residuce of sugar industry) is used as fuel for generating steam
in the boilers of sugar industry as well as in some other industries like paper.
(2)
Briquette
Artificial
(a)
Briquette
Briquette is compressed fuel. It is prepared from coal, peat, lignite or coke in
Charcoal
Charcoal can be prepared by the destructive distillation of wood or heating
the wood in limited supply of air. It can also be made in Kilns and in India charcoal
is prepared generally by this method. Generally hard wood is carbonised and gas, tar
and aqueous liquors are obtained, in addition to charcoal. Yield of charcoal is 3035%. Gases obtained are 20-23%; tar and oil are
5-7%. Aqueous distillate contains acetone, methanol and acetic acid. Charcoal
contain litte volatile matter hence gets easily ignited & burns with non-smoky flame.
It is good fuel containing small amount of sulphur and phosphorus. It is used in
metallurgy as well as for domestic purpose & also for making carbon disulphide. Its
caloric value is 6000-8000 Cals/Kg.
(c)
Coke
Coke can be prepared by the destructive distillation of coal in coke-oven at
(3)
Fossil coal
Peat
Industrial
Fuels
Furnace
Feunace Slags
Slags
Oil Shales
Metallurgical Coal
2.
Liquid Fuels
(i)
Kerosene
The composition of Kerosene oil is between C10 to C12 hydrocarbons with the
boiling range 170-250C. It is used as a domestic fuel in stoves for cooking and for
lighting purpose in lanterns. Kerosene oil is also used for making gaseous fuel " The
carburetted water gas."
(ii)
Petrol
It is also known as gasoline. Petrol is a mixture of C5 to C10 hydrocarbons and
its boiling range is 40C to 170C. Petrol is a fuel for motor cars, motor cycles,
scooters and other light vehicles. Petrol is also a good solvent & is also used in drycleaning as well as in making petrol gas. Its octane No. ranges 60-70.
(iii)
Diesel
Diesel is a mixture of C13 to C15 hydrocarbons and its boiling range is 250-
350C. It is a fuel for heavy vehicles like trucks, buses, ships and diesel enzines etc.
Diesel is also used to run water pumps for irrigation as well generators which
generate electricity on small scales for houses, agriculture and industry.
(iv)
Fuel oil
Fuel oil contains hydrocarbon containing 15-18 carbon atoms. Boiling range of
(v)
Alcohol
Power alcohol is used as fuel when blended with petrol in internal combustion
enzymes. Ethanol can't be used as such as a fuel but is used as an additive. Octane
number of alcohol is 95. When alcohol is mixed with petrol it increases its Octane No
by 0.9 unit for percentage volume of alcohol. Therefore, it is blended with petrol by
using blending reagents like benzene, ether and tetralin. Blending can't be done
without blending reagent. Alcohol is blended because of its anti-knocking property &
can be used in enzines with higher compression ratio. However, alcohol reduces
calorific value of petrol.
Natural Gas
Producer Gas
Gaseous
Fuels
Coal gas
Blue Water gas
Semi water gas
Carburetted Water Gas
Ethyene
Aectylene
(i)
Natural Gas
Natural gas consists of mainly methane with small quantities of ethane &
propane. It contains about 95% of methane. Natural gas occurs deep under the
crust of earth either alone or alongwith oil above the petroleum deposits. Thus,
some well dug into the earth produce only natural gas while others produce natural
gas as well s petroleum oil. In latter case, natural gas is by-product of petroleum
mining. In India, we have a number of gas fields. Some recent ones are located in
Tripura, Jaisalmer, off-shore area of Bombay and in Krishna Godvani delta. Natural
gas is formed under earth by the decomposition of vegetable matter lying under
water. Decomposition occurs through anaerobic bacteria in absence of oxygen.
Natural gas obtained from oil wells is of two types:
(a)
(a)
(b)
Natural gas being a complete fuel in itself can be used directly for heating
purposes in home & industries. Nothing needs to be added to it.
2.
It is a good quality fuel with high calorific value. It leaves behind no residue &
also burns with smokeless flame. Moreover, gases produced by burning it are
not poisonous.
3.
A great advantage of natural gas is that it can be supplied directly from the
gas well to homes and factories for burning through undergrounds pipelines.
This eliminate the need for additional storage and transport.
The construction of pipelines for natural gas was earlier thought to be
expensive, but it is economical in long run. Once, a pipeline has been laid, there is
no need to spend money on the construction of storage tanks for gas or for
transporting the gas by rail or road. Earlier, pipeline for natural gas was constructed
in Baroda city.
Uses of Natural gas
1.
2.
Strong
CH 4 C 2H 2
heating
[Present in natural gas]
Hydrogen gas thus obtained in used to prepare ammonia which reacts with
acids to give fertilizers.
5b.2.1
(ii)
Producer Gas
Producer gas is a mixture of carbon monoxide and nitrogen. It can be
obtained from low grade coal, hence is a cheaper gas. Wooden wastes, peat, spent
tar bark and other carbonaceous fuel , are used to prepare it; but fuel generally
used are coke and coal. For its preparation restricted supply of air is allowed through
burning solid carbonaceous fuel in a special type of furnace known as producer.
Temperature of the furnace is kept at 1000-1400C. Producer gas obtained from coal
has a calorific value of
145 BTU/cubic ft. Another gas produced with producer gas is semi-water gas.
Combined Manufacture of Producer Gas and Semi-Water Gas
Two types of manufacturing units are used for this purpose:
(i)
(i)
In pressure plants air is pumped through hot fuel. A steam-air mixture can be
forced through hot fuel bed by steam pressure.
(ii)
Pressure type plant has a diameter of 10 feet and its height in 12ft.
2.
3.
4.
5.
In pressure type plant steam- air mixture is forced through fuel with the help
of pipe at the bottom & semi-water gas or producer gas passes out from the
top through outlet pipe.
Composition of producer gas and semi water gases are:
(a)
(b)
0.4%
C2H4
0%
H2
13.2%
CO
25.3%
CO2
5.4%
N2
55.2%
O2
0.5%
Calorific value
2.5%
C2H4
0.4%
H2
12.0%
CO
27.0%
CO2
2.5%
N2
55.2%
(c)
O2
0.3%
Calorific value
2.0%
C2H4
0.0%
H2
34.0%
CO
27.0%
CO2
8.0%
N2
29.0%
O2
0%
Calorific value
2.
At higher temperature CO2 reacts with hot carbon to give carbon monoxide
gas.
C + CO2 2 CO + 38,700 cals.
3.
4.
5.
6.
Some CH4 and C2H4 are formed by the destructive distillation of coal.
7.
For the formation of semi-water gas 7 volumes of air and 1 volume of steam
is passed.
N.B.
(1)
(2)
(3)
(ii)
(i)
produced by cracking kerosene oil. Oil gas contains considerasle amount of ethylene.
It's calorific value is 444 BTU/cubic ft. Manufacturing unit for carburetted water gas
consists of :
(ii)
(a)
Generator
(b)
Carburetter
(c)
(d)
Super heater
(e)
Purifiers
(f)
Scrubbers
(g)
Condensers.
water gas can be prepared by passing steam through hot bed of coke at 1000C1400C. The reaction is endotheremic, hence temperature falls down from 1400C.
Little carbon dioxide is also formed and its amount becomes considerable at 1000C.
Note:
(a)
(b)
Compare your answer with those given at the end of the unit.
(i)
(ii)
(iii)
(i)
_________________________________________________
(ii)
_________________________________________________
(iii)
________________________________________________
Ultimate Analysis
2.
Proximate Analysis
Ultimate Analysis
Estimation of carbon, hydrogen, nitrogen, sulphur and ash in the coal is
carried out in the ultimate analysis of coal. This analysis is needed to calculate the
heat balances in any process for which coal is used a fuel.
1.
purpose. It is burnt in a combustion apparatus which converts C & H into CO2 and
H2O, respectively. CO2 is absorbed in pre-weighed KOH tube & H2O in pre-weighed
anh. CaCl2 tube. Percentage of C & H is calculated from the increase in weight of
absorption tubes.
C + O2 CO2
12
44
H2 + O2 H2O
2
18
% age of C
(ii)
% age of H
2.
Estimation of Nitrogen
heated to convert
nitrogen into ammonium salt. The sample solution in than made alkaline with NaOH
and liberated ammonia is distilled into definite volume of standard acid solution, in
which it gets absorbed. Unused acid is determined by titration with standard NaOH
solution. From the volume of acid used with ammonia liberated is used to calculate
percentage of nitrogen.
% of Nitrogen
3.
Estimation of Sulphur
Sulphur is estimated from the washings of known mass of coal used in Bomb
% age of sulphur
4.
Wt of BaSO4 obtained
32
100
Wt of coal and in bomb colorimete r 233
Estimation of Ash
For the determination of ash 1-2 gm powered coal is taken in platinum
crucible & are heated carefully on a Bunsen burner. Then lid is removed & strong
heating is done to burn any tarry material. The lid is kept in desiccator. Heating of
the coal in crucible is continued at 800C in muffle furance till coal is completely
burnt leaving behind ash only. Crucible is now cooled is desiccator & weighed to
constant weight.
% age of Oxygen
5.
Wt of ash left
100
Wt of coal taken
Estimation of Oxygen
%age of oxygen = 100 - [%age of C+H+N+S+ash]
S.
Type of Coal C
No.
1
Peat
60
06
32
1.5
0.5
Caloric Value
8000
BTU/
Pound
2
Anthracite
94
03
02
01
00
15000
BTU/
Pound
2.
Proximate Analysis
It records moisture, volatile carbon matter, ash & fixed carbon percent of
Determination of Moisture
1-2 gm of air-dried powder of coal sample is taken in a silica crucible which
has been heated to constant weight. This crucible is then heated in electric oven for
1 hr at 107 2C. Crucible is cooled in desiccator and weighed again. Amount of
weight loss is equal to amount of moisture.
% age of moisture
2.
Loss in wt
100
Wt of coal taken
Muffle furnace at 925 2C for 7 minutes. Now, crucible is taken out and cooled first
in air, then in desiccator and is weighed again. Loss in weight gives volatile matter.
Determination of Ash
As given in ultimate analysis.
4.
matter fixed carbon + ash. Fixed carbon is left behind after destructive distillation of
coal. Weight of ash is detected from weight of residue & % age of fixed carbon can
be calculated as
If moisture contents of coal are low, it is good quality fuel. Moisture contents
are lower the calorific value of fuel. Because, it takes some of the liberated
heat in the form of latent heat of evaporation. 10% moisture contents
produce uniform fuel bed & less fly - ash.
2.
3.
4.
Type of coal
No.
Moisture
Volatile
Fixed
matter %
carbon %
Ash %
1.
Peat
20
50
21
03
2.
Anthracite
01
08
88
03
N.B.
Calorific value of peat & anthracite are 7700 & 15,000 BTU/pound.
Grading of Coal
Different qualities of coal have following grading on the basis of (i) Ultimate
analysis (ii) Proximate analysis (iii) Calorific value determination;
(1)
coal (4)
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
heated on water both. Cover (lid) of the metallic cup is opened from time to time &
vapous of oil are ignited by tiny flame introduced momentarily by the means of an
automatic device. The temperature at which the oil vapor burn with slight explosion
is noted which is the flash - point.
The heating of the sample is done at the rate of 5F/minute and speed of
stirring paddle is kept approximately one revolution per second. As the temperature
of the oil reaches within 15F of probable flash point first application of the flame is
made by pulling sliding shutter outwords when the test flame drips into the central
opening in the lid & comes in contact
T1 T2 0
F.
2
Precautions
1.
Moisture affects the flash point, hence, all parts of the cup & its accessories
should be properly dried before filling oil in it.
2.
No oil should be left between sliding and fixed plates before convering the
cup.
3.
Oil should be filled so as it just prevents wetting of the cup above the pointed
tip.
4.
For oil with low flash point sample as well as cup may be cooled in melting ice
before filling.
5.
For the determination of flash point fresh oil should be used each time. Used
oil gives higher flash point.
6.
7.
While applying flame, slide should be drawn open slowly & closed quickly.
8.
2.
Liquids having flash point < 140F are known as flammable & those with >
140F are combustible.
3.
4.
Flash & fire points are used to detect the solvent contamination and to
determine the approximate extent of dilution of lubricating oils.
the lowest
For it equal volumes of sample & aniline are heated till dissolution and are
then cooled under controlled condition. The temperature at which two phases
separate i.e. cloudiness appears throughout the solution is regarded as the aniline
point of the sample.
Methodology
1.
2.
5-10ml of pure aniline [dried over KOH pellets, filtered & freshly distilled] &
exactly same volume of sample [dried over one Na2SO4] are taken in 2.5 x
15cm test tube of heat resistant glass.
3.
Test tube is corked with Cork carrying stirrer and thermometer of suitable
range. The bulb of thermometer is kept 5mm above the bottom of test-tube.
4.
This tube is inserted into air-jacket [4x175cm] which is also made up of heat
resistant glass.
5.
6.
7.
Precautions
1.
2.
Aniline is hygroscopic. Hence, water should not be used even in hot or cold
baths.
3.
4.
5.
2.
Low aniline point i.e. high aromatic content make oil attack rubber gaskets,
seals etc.
3.
Lubricants with high aniline point are useful for systems where rubber seals
are used.
4.
CH2
|
CH3
H3C-C-CH2-CH-CH3
|
CH3
CH3
Iso-octane
(ii)
(iii)
different colours which indicate their value. Their octane rating is expressed as:
Octane No
N.B.
Power No. is proportional to power supplied by engine.
Check your progress-IV
Notes:
(a)
(b)
Compare your answer with those given at the end of the unit.
(i)
(ii)
(iii)
(i)
_________________________________________________
(ii)
_________________________________________________
(iii)
_________________________________________________
5(b).7
"CALORIFIC VALUES"
Calorific value
Calorie
It is unit of heat in C.G.S. system & is defined as amount of heat that
Kilo Calories
Kilocalories is the amount or heat require to raise the temperature of 1 Kg of
by 1C.
1 K. Cal.
4.
2.2. CHU.
3.968 B.t.u.
by 1F (60-61F).
1 B.T.U.
1 K. Cals.
252 Cals
1.054 Joule
3.968 B.t.u.
0.252 K. Cals
Combustion Bomb :
It is made up of chromium - nickel mohybdenum steel and is resistant to
acids as well as corrosion. It weighes approx - 3 Kg. and its capacity in 250-300 ml.
Bomb is provided with an air- tight screw cap to which a couple of stainless steel
electrodes and release valve is attached. To electrode is attached a small ring that
supports crucible containing fuel.
2.
Copper Calorimeter
Copper calorimeter is a vessel that is polished outside and it can contain 2-
litre of water when bomb is placed inside. Bombs should be sub-merged in water. A
space should be there between water and cover.
3.
4.
A Mechanical Stirrer
Mechanical stirrer is fitted for stirring water in calorimeter to stir at uniform
rate.
5.
A Beckman thermometer
6.
A Crucible
It is made up of nickel, stainless steel or fused silica.
Procedure
Place weighed quantity of fuel in crucible. Keep the crucible containing fuel at
its position supported over the ring. A fine Pt- wire is tightly stretched across the
pole piece of the bomb and one end of the piece of sewing cotton thread is tied
around the wire. Loose end of cotton thread is so arranged to be in contract of with
the fuel (e.g. coal). Now, 10 ml of distilled water is placed inside the bomb by the
means of pipette to absorb vapours of H2SO4 and HNO3 during combustion.
Oxygen is forced into the bomb till a pressure of 25 atm is reached. Now,
bomb is placed inside the copper calorimeter that contains a known amount of water
in which bomb is submerged, but terminal remains above the water level. Make the
electrical connection and place the lid and start stirring through mechanical stirrer.
Note down initial temperature of water by thermometer. Connect the electrodes
with battery & complete the circuit so that sample burns and heat is liberated.
Uniform stirring of water is done and maximum temperature attained is noted by
Beckman thermometer. Now, bomb is left for at least one hr. so that all the acid
mist formed settles down. Dismantle the assembly & bomb is placed on holder.
Release the oxygen from bomb carefully. Open the bomb to examine internal portion
if any unburnt fuel content. If combustion is complete wash the contents with
distilled water quantitatively & transfer the washing in beaker. HNO3 and H2SO4 can
be estimated from these washings (which are formed during combustion) which are
used for corrections.
( w w)(t 2 t1 t c ) (CS C N C F CC )
m
Here ;
G.C.V. = Gross calorific Value
w
t1
t2
CS
CN
CF
CC
Net calorific value or lower calorific value, L.C.V, can also be calculated by
L.C.V. = G.C.V. (0.09 x 587) cals/gm or K.Cals/Kg.
Hence,
Corrections :
1.
Acid Correction :
Fuels that contain sulphur and nitrogen get oxidized to H2SO4
HNO3
H2SO4 + Heat
2N + 2H + 3O2
2HNO3
These acids can be estimated from the washing of bomb through titration.
For each ml of N/10 - H2SO4, 3.6 cals should be subtracted and for each ml of N/10 HNO3 1.43 calories are minused from the actual value of G.C.V. or L.C.V.
2.
fuse wire burnt in the combustion experiment. Heat liberated for each fuse wire
burnt in the experiment is to be subtracted from total heat evolved as per the
instructions of fuse wire manufacturer .
4.
Cooling correction
Time for cooling of water from maximum temperature to R.T. should be
recorded . From the rate of cooling and actual time/min (t/min) the cooling
correction of dt x t is added to the rise is temperature. Hence
Caloric Value
=
2.
3.
Before filling with water as well as after filling with water weight of beaker
should be taken. Difference of there two readings is mass of water (m)
4.
5.
Now candle is burnt below the beaker as a result beaker of water get heated
& temperature gradually increase. When temperature of water increases by
150 or 200C candle is extinguished to stop heating & exact temperature (t2) is
recorded immediately .
7.
Extinguished candle is weighed (w2). The difference is weight of candle (w1w2) is the weight of wax burnt.
W1gm
1. wt of burnt candle =
W1-W2gm
2. Mass of water
mgm
W2gm
3.(i)
t1
(ii)
t2
t2-t1
4.
M x S x (t2-t1)
=
Here, S
Heat produceed
Man of wax burnt
m s (t 2 t1 )
Joules / gm / 0 C
w1 w2
Result :
N.B. :
(i)
(ii)
(iii)
(iv)
(v)
5.C
"CLINICAL CHEMISTRY"
Blood Plasma
(2)
Cellular elements.
[1]
Blood Plasma
Blood plasma is the liquid part of circulating blood. It contain (a) serum and
(b) Fibrinogen. Later is responsible for the clotting of blood. Upon clotting of blood
fibrinogen is removed along with cells & serum is left behind. Most of the blood
tests are performed on serum.
-
Blood
(i)
4 - 6 Mg/dL
(ii)
Ammonia
40 - 125 g/dL
(iii)
Non-Protein nitrogen
25-40 mg/dL
(B)
(iv)
65-90 mg/dL
(v)
Urea nitrogen
up to 20 mg/dL
Serum
(i)
Albumin
4-5 g/dL
(ii)
Amylase
(iii)
Bilrubin
(iv)
Calcium
4.5-5.5 meq/L
(v)
CO2
25-32 meq/L
(vi)
Chloride
100-108 meq/L
(vii)
Total Cholesterol
140-250 mg/LB
(viii)
Cholesterol (ester)
50-65% of total
(ix)
Creatinine
0.7-1.7 mg/dL
(x)
Lipase
(xi)
Lipids (Total)
350-800 mg/L
(xii)
Fatty acids
200-400 mg
(xiii)
Globulins (Total)
2.5 - 3.5g/dL
-1 ..
-2 ..
..
=
=
(xiv)
Iron
50-190 g/dL
(xv)
Mg
1.5-2.5 meq/L
(xvi)
Phosphatase (Acid) =
(xvii) Phospholipids
100-250 mg/dL
(xix)
Phosphorus (inorganic)
3 - 4.5 mg / dL
(xx)
K (Potassium)
(xxi)
Protein (total)
3.5-8.0 g/dL
(xxiii) Sodium
upto 40 units.
3 - 6 mg/dL
Water
91 - 92%
(B)
Other Solids
8% - 9%
(I)
(i)
Organic Substances
(ii)
Inorganic substances
(iii)
Respiratory gases.
Organic Substances
These are 7.1 - 8.1%
They include
(i)
(a)
Albumin
(b)
Globulin
(c)
Fibrinogen
(d)
Prothrombin
(iii)
(iv)
(v)
(vi)
Inorganic Substances
NaCl, Ca, K, iodine, iron, bicarbonates etc.
[2]
Cellular Elements
Following types of cells are suspended in liquid portion of blood:
(a)
(a)
(b)
(c)
Platelets (Thrombocytes)
R.B.C.
These are also known as erythrocytes and are produced in bone marrow of
flat bones, ribs, vertebrae, heads of humerus and femur. Their size is 7.2 micron
diameter. They contain no nucleus. R.B.C. contain red pigment haemoglobin & their
life is 120 days. R.B.C. are responsible for the transportation of O 2 & CO2 from one
tissue to other.
(b)
W.B.C.
These are also known as Leucocytes and are of five types :
(i)
Neutrophil polymorph
(ii)
Eosinophil
(iii)
Basophil
(iv)
Lymphocytes
(v)
Monocytes.
(i)
to (iii) are also called granulocytes. (iv) and (v) are non-granular
leucocytes.
There is only one W.B.C. for every 500 R.B.C. In adults they count 5,00010,000 per cm. of blood. They do not contain pigment.
They constitute defence systems of body against infections by phagocytes.
Their function is also production of antibodies by lymphocytes.
(c)
Platelets
These are non-nucleated parts of cytoplasm & are also called thrombocytes.
[Thrombo = Clottings, cyte=cell], they cause clotting of blood & are produced in
spleen and bone-marrows. Size of platelets is 2-4 micron (diameter) and they count
2,50,000 to 5,00,000/cc of blood.
Functions of Blood
(1)
It transports food from one tissue to other & provides connection link
between individual cells of distant organs and tissues.
(2)
It carries oxygen from lung to other parts by combining with haemoglobin &
removes CO2 from tissues through lungs to air.
(3)
For the analysis of blood samples for glucose, cholesterol, uric acids, lipid
profiles, R.B.C., W.B.C., platelets etc collection and preservation of blood samples is
needed. Blood for some distinct purpose is collected after fasting over night.
Collection of Blood
The blood is collected aseptically from the median cubital vein, in front of the
elbow, into a sterile container containing an anti-coagulant solution. During
collection the bottle is shaken gently to ensure that the blood and anti-coagulant are
mixed well, therefore, preventing the formation of fibrin-clots. Not more than 420 ml
of blood is taken at single attendance. Now container is sealed immediately and is
cooled to 4-60C. For taking blood the equipment used is made up of plastic & is
disposable. Container is also made up of plastic. In America plastic bags are used.
Anti-Coagulants
(1)
Citrates
The solution most often used as anticoagulant of blood is Acid-citrate
2.10 - 2.5 g
Dextose
3.0 g
Heparin
Heparin is naturally occurring blood anti-coagulant made by the mast cells of
Disodium Edetate
It is preferred when preservation of platelets are necessary. This is a
chelating agent because of strong affinity for divalent metal like Ca.
Collection of Blood of an HIV Infected Patient
The collection & handling of blood requires great care as some transferable
disease constituents like HIV or AIDS may be present in it. To collect blood of such
patient gloves and masks should be used. Such persons should not be handled
without expert advise.
Collection of Blood for Plasma or Whole Blood Analysis
Blood for this purpose is collected in tubes containing anti-coagulant. Anticoagulants used have already been discussed. However, potassium oxalate (about
1mg/ml blood) may also be used as anti-plasma coagulant. Pot. oxalate precipitates
blood calcium which is required in clotting. Potassium oxalate causes R.B.C. to shrink
& intracellular water diffuses into plasma.
Collection of blood for serum Analysis
Blood for this purpose is also collected in dry & clean tube to prevent
haemolysis & contamination. Haemolysis is destruction of R.B.C. & release of
haemoglobin etc into serum or plasma. As haemolysis occurs, serum becomes red
instead of normal straw colour. Therefore, blood sample is centrifuged as soon as
possible to separate serum or plasma from cells.
Collection of Blood for Glucose Analysis :
NaF is widely used as a preservative for the samples to be analysed for
glucose alongwith anti-coagulant. NaF is enzyme inhibitor & prevents enzymatic
break-down of glucose. 1 mg of NaF/ml of blood is enough.
Collection of Blood for CO2 Analysis
Blood collected for this purpose should be kept anaerobically & mineral oil
must be added to collection tube which being ligther covers the blood. A rubber
stopper should not be used to such sample which swells up.
Storage of Samples of Blood
Apart from period of transport and examination which much not exceed 30
minutes, blood should be stored at 4-6C until required for use. Even, at his
temperature some deterioration may occur. Leucocytes disintegrate in few hrs and
platelets in few days. R.B.C. show fall in ATP and other organic phosphates, a
reduction in oxygen carrying capacity and partrial loss of lipid from their membranes
& increased fragility. Storage at R.T. even for a day seriously reduces posttransfusion survival
1.
Sample in solution form is introduced in the form of fine continuous spray into
non-luminous gas using either acetylene, propane or butane gas. The light is
emitted by the use of colour filter or diffraction grating.
2.
3.
Range
(i)
(ii)
3.6-5.6
mEq./lt.
Inferences from Na+ and K+ion concentrations
-
Sodium ions maintain acid-base balance in the body as well as the smotic
pressure.
(ii)
(iii)
Ionized calcium.
Physiological functions of calcium are due to ionic calcium. But in routine work
total calcium is estimated. Commonly used method for the estimation of
calcium is Clark & Collip Method.
Principle
Ammonium Oxalate 4%
Prepared by the dissolution of 4g ammonium oxalate in water & made up to
2% Ammonia
2 ml of H2SO4 diluted to 100 ml with distilled water.
(iii)
1N-H2SO4
28 ml of H2SO4 diluted to 1000 ml with distilled water
(iv)
2.
3.
4.
5.
6.
Keep the tube in boiling water-bath with occasional shaking till precipitate
completely dissolves.
7.
Titrate the warm solution with 0.01N-KMnO4 till pink colour develops and
persists at least for one minutes.
8.
9.
Take a blank reading with 2 ml of 1N-H2SO4 by titrating it against 0.01 NMKnO4. Let volume used is Y ml.
Calculation
Serum calcium [mg/dL]
(X Y)
0.2
100
2
= (X-Y) 10
Range
Range of calcium is serum varies from 9-11 mg/DL in a healthy person.
Serum
calcium
polycytharmia,
increases
infantile
in
hypervitaminosis-D,
hypercalcemia,
sarcoidosis,
hyperparathyroidism,
multiple
myeloma,
Chloride ion gets converted into un-ionized mercuric chloride when mercuric
nitrate solution is added to them.
2Cl- + Hg (NO3)2 HgCl2 + 2NO3Endpoint is indicated by the appearance of bright blue - violet colour in
presence of diphenyl carbazone indicator. Titration can be done directly with serum.
But highly coloured or jaundiced sample, may suffer from clarity of end point. For
such sample, a Folin Wu tungstate filtrate is prepared and titrated.
Requirement
(i)
(ii)
2/3 N-H2SO4
(iii)
and
(NO3)2 in
make
up
the
volume to 1 litre.
(iv)
[Indictor].
(v)
Methodology
1.
diphenyl carbazone to it. Now titrate it with Hg (NO3)2 till colour change occurs. Note
down the volume used.
2.
Titration of Sample
Taking 1 ml of serum or plasma or blood and add 7 ml of D.W. to it. Add 1 ml
of 10% sodium tungstate & 1 ml of 2/3 N-H2SO4 also. Stir the contents and filter.
Taking 2 ml of this filtrate in 50 ml conical flaks titrate it with Hg (NO 3)2
solution. Note down the volume used.
Calculation
Volume of serum has been diluted 10 times. If the concentration of Cl ion in
serum is C, then
10
10 vol. of Hg(NO 3 ) 2 used with standard
or C m Eq/l
Range
Rage of Cl- ion
(i)
in Serum plasma
98-105 m Eq/lt
(ii)
in whole blood
78-85 mEq/lt
Decrease in concentration of Cl- ions also occurs during Addison disease &
pneumonia.
(ii)
(iii)
Anti foam A
(iv)
tube.
2.
3.
Keep the tube anaerobically, for which stopper the sample tube to keep CO2
out. Sample should be prepared at the time of analysis only.
4.
5.
Now add 2-3 drops of phenol red indicator to it, stopper the flask and mix
gently. End-point of this titration is in the pH range of 8.4-6.7 by the colour
change from yellow to red.
6.
7.
8.
Now add 2-3 drops of indicator and titrate with 0.01M-NaOH till pink colour
appears.
Calculation
0.1ml of blood sample was taken & 0.26 ml of 0.01 M-HCl might have been
consumed if the normal value of calcium [26 m Eq/l] was present in blood. As 1 ml
of 0.01 M-HCl was taken, 0.74 ml of it should have been left un-reacted & back
titration might has consumed about 0.7 ml of 0.01M-NaOH. From latter reading (X
ml) amount of HCO3- ion in blood can be calculated and expressed in meq/lt.
Conc of HCO3- in meq/lt = 1 ml of 0.01 M-HCL - X ml of 0.01M-HCl
Check your progress-V
Notes:
(a)
(b)
Compare your answer with those given at the end of the unit.
(i)
(ii)
(iii)
(i)
_________________________________________________
(ii)
_________________________________________________
(iii)
_________________________________________________
blood.
Chemicals Required
(i)
Albumin reagent
It is prepared by mixing following chemicals in 900 ml of distilled water:
(a)
(b)
(c)
Sodium azide =
100 mg
Brij 35
4.0 ml
(d)
pH of the solution is adjusted to 4.1 using NaOH and the volume is made up to 1
litre.
(ii)
(i)
Albumin reagent
(ii)
(iii) Albumin
Standard
(1:10
dilution)
Test tube
Standard
Blank
(1)
(2)
(3)
(5)
5.0
5.0
5.0
5.0
0.5
0.5
5.0
0.5
(4)
0.5
0.5
(iv)Distilled water
Calculations :
Serum albumin (g/tube) [0.05 ml of serum]
ODt
0.002 [Conc. of standard tube]
ODs
ODs
0.05
ODt
4
ODs
Clinical Diagnosis
Higher value of serum albumin is noted during dehydration due to
hemoconcentration. Decrease in serum albumin is noted during severe malnutrition,
renal diseases, pregnancy, liver inefficiency and burn.
However, serum globulin is increased during chronic infections, autoimmune
diseases, multiple myeloma & macroglobulinemia.
Estimation of serum protein by Biuret method of Reinhold.
Principle
Biuret method (most commonly used method) of determination of serum
protein is based on the principle that subrtances which contain two-CONH2 groups
or more groups joined together or those containing peptide linkages give purple
colour with alkaline CuSO4. One Cu-atom complexes with four molecules of biuret &
central nitrogen is involved in linkage. The amount of colour varies with different
proteins and carbohyhate contents in complex protein.
Chemicals required
(i)
approx. 400 ml of 0.2 N-NaOH and adding 15 gms of CuSO4 to it with continuous
stirring. 5.0 gms of KI is also added to it and volume is made upto 1 litre with 0.2 NNaOH.
(ii)
(iii)
Biuret Reagent
200 ml of stock reagent is diluted to 1 litre with 0.2 N-NaOH containing 5.0
gm of KI/litre.
Methodology of estimation
In 6 dry & clean test tubes different solutions are added in the quantities
given in following Table:
Reagent (ml)
(i)
Serum (1 : 10 dilution)
(ii)
(iii)
Distilled water
(iv)
Biuret reagent
Test
Standard
Blank
(1)
(2)
(3 (4)
(5)
(6)
1.0
1.0
2.0
3.0
2.0
3.0
1.0
_
1.5
2.0
3.0
3.0
Mix the solution in each test tube; keep on water-bath at 370C for 10 minutes
& record the absorbance at 540mm calorimetrically using green filter.
Calculations
Draw calibration curve & find out total protein present in serum by reading
absorbance of sample from the curve.
Normal value of serum protein is between normal 6-8%. An increase in
normal value is noted due to dehydration and decrease is due to excess of water
intake.
Hyporproteinemia:
Is due to increased globulin synthesis, chronic liver fever, chronic infections
like Kalazar, bone disease like multiple myeloma, collagen discease etc.
(i)
Principle
Astor and king method (1954) of estimation of glucose is based upon the fact when
blood is kept into isotonic sodium sulphate-copper sulphate solution no further
copper sulphate is required. Upon addition of sodium tungstate to it copper
tungstate if formed & proteins as well as non-glucose reducing substances are
preipitated. Results obtained by this method are very close to glucose concentration.
From the filtrate concentration of glucose is determined by the use of its property to
reduce alkaline cupric (Cu2+) ions producing Cu2O (cuprous oxide). Cu2O reduces
phosphomolybdic acid to molybdenum blue which can be measured photometrically.
Reagents required
(i)
Reagent (ml)
Test
(1)
(2)
Standard
Blank
(3)
(5)
(4)
(i)
Supernatant
(ii)
Standard glucose
(iii)
DW
(iv)
Isotonic
(v)
Alkaline tartarate
1.0
1.0
1.0
2.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
2.0
2.0
1.0
2.0
Mix the solution & heat on water - bath for 10 minutes. Cool, add to test tube
3.0 ml g phosphomolybdic acid. Mix & record absorbance after 5 minuts at 680 nm.
Calculations
mg of glucose in test/0.25 ml of blood/plasma/serum
ODt
0.02(conc of standard/tube).
ODs
Blood/serum/plasma glucose (mg/ml)
ODt 0.02
ODt
100
80
ODs 0.025
ODs
Normal values (fasting)
Venious or capillary whole blood = 60-100 mg/dl
Renal threshold for glucose
= 180 mg/dl
Plasma or serum
= 70-110 mg/dl
Clinical diagnosis
Blood
sugar
increases
during
Diabetes
mellitus,
hperthyroidism,
Principle
Uric acid gets oxidised with phophotungstic acid in alkaline medium. In the
process phosphotungstic acid gets reduced into a blue complex which can be
determined calorimetrically between 650-700nm.
Requirements
1.
2.
3.
4.
5.
6.
Methodology
1.
Dilute 1ml of plasma with 8 ml of D.W., 0.5 ml of 10% sodium tungstate and
0.5 ml of 2/3 N-H2SO4. Mix the contents well and centrifuge . Uric acid is
tested from the supernatant.
2.
To test uric acid take 5 test tubes and lebel 1-5; 1 & 2 contain sample;
standard (0.04mg/tube) are in 3 & 4 and t.t. 5 is for blank reading.
Compositions in test tubes 1-5 are as given in following table :
Reagents (ml)
Sample
Standard
Blank
t.t.1
t.t.2
t.t.3
t.t.4
t.t.5
1. Supernatant
3. D.W.
4. Na2CO3 Solution
5.Phosphotungstic Reagent
3.
Mix the contents of each tube thoroughly allow and the tubes to stand at R.T.
for 15 minutes.
4.
Calculation
mg of Uric acid/tube i.e. 0.2 ml of blood
=
ODT
0.04(Conc. of sandard / t.t. )
ODS
OD T
OD S
ODT
20
ODS
0.04
100
0.2
Range
Range of uric acid in serum of a healthy pession in 2-6 mg./dL.
Inference from the concentration of Uric acid
-
Elevation in uric acid in serum level may also be seen in the case like
leukemia, lobar pneumonia, large abcesses, toxemia of pregnancy etc.
Exercise 1
Determination of Phenobarbital (Luminal)
Methodology
Dissolve in a conical flask accurately weighted 0.1g of sample in 5ml of
pyridine and add 0.25 ml of thymolphtalein solution as well as 10 ml of AgNO3
Pyridine reagent to it. Titrate the contents with 0.1 N-NaOH (alcoholic) till blue color
appears.
Calculation
1.0 ml of 0.1 N-NaOH - 0.011610g of Phenobarbital.
Exercise -2
Determination of Cyclobarbitone calcium.
Methodology
as in Ex-1
Calculation
1 ml of 0.1 N- NaOH - 0.02553 g. of cyclobarbitone Calcium.
Exercise -3
Determination of Quinobarbitone sodium.
Methodology
Same as Ex -1
Calculations
1ml of 0.1 N- NaOH = 0.02603g of Quinobarbitone sodium.
5C.9 Estimation of Serum Alkaline Phosphatase
Phosphates are the enzymes that catalyse the removal of phosphates form
certain monophosphoric esters. This enzyme is most active at the pH-10.
Phosphatases are intercellular but are also present in smaller amounts in plasme
certain diseases their amount increases in plasma. Most popular method to
determine concentration of alkaline phosphatases in plasma in King and King
(1954) method.
At a pH of 10, phenyl phosphate is hydrolysed to phenol when incubated with
alkaline phosphatases. The phenol formed condenses with 2-amino antipyrine. The
product formed, after the oxidation with alkaline potassium ferrocyanide gives red
colored complex which can be estimated at 520 nm.
Requirements
1.
2.
3.
4.
5.
6.
7.
Methodology
1. Four test tubes are lebelled as 1,2,3 & 4. They contain sample, control,
standard and blank. They are filled with following solution:
S.No. Reagents
1.
1.0
1.0
2.
0.01N-Carbonate buffer
1.0
1.0
1.1
1.1
0.1
Serum
4.
1.0
5.
D.W.
1.0
0.5N-NaOH
0.8
0.8
0.8
0.8
7.
Serum
0.1
8.
0.5 N-NaHCO3
1.2
1.2
1.2
1.2
9.
4-Amino antipyrine
1.0
1.0
1.0
1.0
10.
K4[Fe(CN6)]
1.0
1.0
1.0
1.0
2.
Calculation
Serum alkaline phosphatases
(In king Armstrong unit)
=
O.D.T O.DC
10
O.DS
2.
Serum alkaline phosphatases activity also increases during disease of liver &
bialary tract : like all types of jaundice except homolytic jaundice.
3.
&
cretinism.
5C.10 "Immunoassay"
Immunoassay is a technique for the determination of drugs, hormones and
vitamins and some other compounds at nanogram and still smaller scale. It involves
a reaction between analyte antigen and a specific antibody to give a precipitate or
complex.
1.
Antigen :
Antigen is a foreign substance that can induce formation of antibody in the
body and is capable of combining with it. Antigen is a macromolecule like protein,
hormone etc.
2.
Antibody:
Antibody is a protein which is capable to recognize a foreign substance & can
have stereospecific association with it. e.g. Virus and bacteria. Globulin protein with
high mol. wt of about 1,50,000 is an antibody. This protein exhibiting activity of
antibody in known as immunoglobulin (Ig) . Five main immunolglobulins in human
blood are IgA, IgD, IgE, IgG and IgM. But, IgG is most abundant. The Ig consists of
three polypeptide clains. Two are of 240 amino acid residues and third of 430 animo
acid residues which are linked through disulphide bridge.
When Ig is treated with papain (an enzyme) three fragments (each of mol. wt
approximately 50,000) are formed. Two are identical and can combine with antigen
and are referred to as Fab Fragment, antigen binding. Third can't combine with
antigen and is fragment crystallizable [Fc]
It is worth- noting all antibodies are similar in structure but have variable
antigen binding proportions of fab.
Antigen-Antibody association
Antibodies are produced in organism only after organism has at least one
exposure to antigen through vaccination etc. For immunoassay antibody is produced
by injecting antigen into animal and by recovering the serum that contains resultant
antibody. This serum is known as antiserum. Antigen- Antibody reaction gives
antigen-antibody complex. Forces binding them are called affinity. Infact, affinity is
intrinsic association constant between antibody and univalent antigen. Here, is
another term avidity which refers to the overall binding energy between antibodies
and a multivalent antigen.
Overall binding reaction can be written as :
Ab + Ag
Ab.Ag
[Ab.Ag ]
[Ab ][ Ag ]
weak Vender waal force, electrostatic and hydrophobic force. The bonds are broken
by the addition of salts, increasing temperature, pH or polarity of solvent.
5.c10.1
Antibody solution (Ab antiserum) labelled antigen (Ag)* and serum sample
containing unlabelled antigen sample to
reaction vessel.
2.
3.
After incubation, bound antigens are separated from free antigens & labelled
portion of either or both phases is measured by measuring radio-activity or
fluorescence in order to determine the percent bound to Ag*.
4.
Drawbacks of RIA
125
I and
131
1.
Both
2.
Application of RIA
It is an important technique for clinical laboratories & is used in :
1.
b.
Hormones
c.
2.
3.
Blood banking.
4.
Diagnosis of allergies.
5.
the amount of O 2
actually combined with haemoglobin to the total amount of O2 which can combine
with haemoglobin.
CO2 dissolves in blood to form bicarbonate and carbonic acid that maintain pH
of blood at which cellular functions go on normally. Lungs and kidneys maintain
balance of carbonic acid and HCO3 in blood, respectively.
Normal Ranges of Blood gas Analysis :
1.
75-100 mm Hg.
2.
35-45 mm Hg.
3.
Oxygen content
15-23 %
4.
Oxygen saturation
94-100%
5.
HCO-3
6.
pH
22-26 mEq./lt.
=
7.35-7.45
Abnormal results are also seen is disorders like anaemia which affects O2
carrying capability of blood & gives abnormally low O2 contents.
Copper
2.
Chromium
3.
Cobalt
4.
Iron
5.
Selenium
6.
Zinc
7.
Iodine
1.
Copper (Cu)
Cu is essential part of some enzymes which inactivate free radicals. Thus, it
2.
Chromium (Cr)
It is needed by cells in the glucose intake process. It's deficiency leads to
increased blood sugar as well as increased cholesterol & triglyceride levels. Yeast,
cheese & meet are chief source of Cr. Its recommended dietary allowance is 50200lg. Sometimes, its deficiency is supplemented through multivitamins which
contain 15-100 g of it. Cr is used up in exercise, infection & injuries etc.
3.
Cobalt [Co]
It is a part of Vitamins - B12 .
4.
Iron (Fe)
It is part of RBC and have anti-oxidant activity. Fe is frequently used in the
body to prevent its use by bacteria as a source of fuel. Iron gets attached to proteins
& reaches to other parts of body through them. Vitamin-C promotes its functions.
Deficiency of Fe leads to anaemia. Chief sources are liver & red metals.
Recommended dietary allowance is 7-14 mg./day.
5.
Selenium (Se)
Se is anti oxidant. It marks immune body function which are like functions of
vitamin-E. Deficiency of Se leads to heart diseases and anaemia. Its deficiency have
also been found in HIV affected persons. Recommended dietary allowance of Se is
approx 50 g from one to four times a day.
6.
Zinc (Zn)
Functions of Zn involve the wound healing & maintenance of membranes. It is
also involved in anti-body production. During stess it is shifted from blood to tissues.
Hence, plasma levels do not reflect their actual concentrations. Its deficiency impairs
immune response and causes hair loss.
7.
Iodine(I)
Characteristics of Drug
1.
The action of an ideal drug should be localized at the site where it is desired
to act. However, there is hardly any drug that have this activity.
2.
It should be non-toxic.
3.
4.
5.
6.
It should not make the host cells resistant to drug after its use for some time.
However, most of the drugs do not possess this characteristic.
Route of Administration
1. Orally
2. Intravenous (though injections)
3. transdermally (skin patches)
Complications
1. Hepatitis
2. Skin, lungs and brain abcesses
3. Endocarditis
4. AIDS etc.
5. They are habit forming. A chronic drug user needs more & more doses to
attain desired effect like decrease in sedation, euphoria etc.
Withdrawal Symptoms
Withdrawal symptoms from morphine & /or morphine like addictions are
experienced before the time of next scheduled dose e.g.
1. Watering of eye
2. Running nose
3. Yawning & sweating
4. Irritability
5. Restlessness.
6. Loss of appetite
7. Increased heart beat & B.P.
8. Vomiting
9. Tremors and severe sneezing
10. Pain in muscles & back bone
11.Severe depression
Uses of Narcotics
These are used in the treatment of :
1. Pain, acute diarrhoea & cough supression.
2. Reduces tension, anxiety and agression & hence produces sense of well
being.
Dangerous Drugs
Dangerous drugs are the drugs that are used illegally. Thay are dangerous in
the sense that they cause addition as well as some harmful chemical and physical
effects. Dangerous drugs can cause dangerous behaviour. They are good for none
but are most dangerous for kids and teenagers who are at growing stage. These
drugs can damage brain, heart and other important organs. For example, cocaine
can cause heart attack even to kids & teenagers. Some examples, of most
dangerous drugs are : (i) Methamphetamines (ii) Heroine (iii) cocaine (iv)
Barbiturates (v) LSD (vi) Benzodiazepine (vii) Marijuana (viii) opium (ix) caffiene &
nicotine (x) Hashish.
5d.2 "Classification of Drugs"
Most important classification of drugs is on the basis of their therapeutic
actions. On this basis they are classified into two main classes:
1. Chemotherapeutic agents
2. Pharmacodynamic agents
(1)
Chemotherapeutic agents
These drugs are used to cure specific diseases like microbial infections,
maleria, tuberculosis, syphillis etc. Chemotherapeutic agents attack and destroy the
invading organisms without any adverse effect on infected organism. Chemotherapy
is defined as the treatment of diseases by chemicals which selectively inhibit the
growth of infecting organisms. First chemotherapeutic agent was an anti-syphillis
agent discovered by Ehrlich (1910). This class has been divided into following types :
(i)
Antibacterials
(ii)
Antimalarials
(iii)
Antiprotozoals
(iv)
Antifungals
(v)
Antiseptics
(vi)
Anthelmintics
(2)
(vii)
Antineoplastic agents
(viii)
(ix)
Organometallic therapeutics
Pharmacodynamic agents
These drugs have characteristic action on infected organisms but are not
specific remedies for particular disease. These drugs are further classified into
following classes:
1.
Anaesthetics
2.
3.
Tranquillisers.
4.
Antipsychotic drugs.
5.
Anti-anxiety drugs.
6.
7.
Pschotogenic drugs.
8.
9.
Anti coagulants
10.
Adrenergic stimulants
11.
12.
Diureties
13.
14.
Antispasmodics
15.
Anti-histamines
16.
Anti-inflammatory drugs
The medicinal value of a drug is expressed in "therapeutic index" which is the ratio
of amount necessary to kill the patient i.e. maximum tolerated dose to maximum
curative dose.
Therapeutic index
The therapeutic index of 10 indicates that 10 times of the dose used for
curative purpose will kill the patient as well as to the parasite. Some important
classes of both the major classes are being discussed here:
(1)
(A)
Sulpha drugs
(B)
Antibiotics
etc.
Some
examples
are:
sulphathiazole,
sulphate
acetamide,
sulphapyridine etc.
(B)
(9)
(A)
(B)
Hyprnotics are CNS depressants that produce sleep resembling the natural
sleep. e.g. Luminal
Sedatives are CNS- depressants that reduce nervous tension and promote
relaxations without producing sleep. e.g. Diazepam
(12) CNS-Stimulants : These drugs stimulate central nervous system and are
anti-depressants. e.g. Amphetamine.
(13) Psychotogenic drugs : They induce behaviourial abnormalities resembling
psychosis. e.g. LSD [Lysergic acid diethyl amide] & mescaline [3,4,5trimethoxyphenyl ethylamine].
(14) Hypo and Hypertensive agents : Hypotensive agents increase B.P. &
hypertensive elvevate it e.g. methyldopa is used in hypertensions & reduces
B.P.
-
(15)
(16)
(17)
(18)
(19)
(20)
Spectrophotometric determinations
(ii)
(iii)
450-500 nm
Green 500-570 nm
Yellow
570-590 nm
Orange
590-620 nm
Red
620-760 nm
I0
kI
It
or
It = IO. e-KI
or
It
10 KI
I0
It
is the fraction of incident light transmitted by thickeness l of the medium
Io
as is known as Transmittance(T).
I0
= opacity
It
A log
I0
It
It
= Io. 10-acl
or
log
I0
= acl
It
I0
cl
It
or = A/cl
Instrumentation
Instrumentation of U.V.-visible spectroscopy is discussed ahead. Essential
Features of simple filter colorimeter are : (i) Light source (ii) filter to select suitable
wave length (iii) cuvettes (iv) photocell to receive transmitted light (v) a meter to
measure the response of photocell.
log
Io
) is plotted against the concentration & a straight line is obtained if Beer's Law
It
(ii)
(iii)
For inorganic substances water is the best solvent. But, for organic
estimations organic solvent is needed. Polar organic solvents like alcohols, ketones,
esters water etc suffer from drawback that they obliterate fine structure of spectrum
due to vibrational effects. Therefore, hydrocarbons like cyclohexane (e.g. in case of
phenol) give better spectrum with three sharp peaks as compared to water in which
a single broad band is obtained. Wavelengths of absorption of some good solvents
are given below:
Solvent
nm
Solvent
nm
Water
190
Cyclohexene
212
Hexane
199
Dichloromethene
247
Heptane
200
Chloroform
257
Diethylether
205
Benzene
280
Ethanol
207
Pyridine
306
Methanol
210
Acetone
331
contaminated may be cleaned with detergents like teapot followed by proper drying.
(II)
Preparation of Solutions
All the solutions needed should be prepared accurately. In UV-instruments
direct measurements may be made without adding reagents. Solutions which are
unstable should be prepared the moment, they are to used. The test solution should
be dilute to the extent that absorbance lies is the region 0.2-1.5, otherwise they
should be further diluted.
Experiment
Estimation of phenacetin, caffeine and Aspirin in the given mixture of drugs.
Principle
A mixture of phenacetin, caffeine and Aspirin may be analysed quantitatively
by UV spectrophotometric method. Phenacetin shows absorption maxima ( max) at
250 nm, caffeine at 275 nm and aspirin at 277 nm. First they are separated & then
analysed spectrophotometrically :
Chemicals Required :
(i)
(ii)
(iii)
Conc. HCl
(iv)
1M H2SO4
Methodology
Accurately weigh
CH2Cl2 and transfer the solution to 60 ml separatory funnel. Extract aspirin from
CH2Cl2 extract with two 10ml portions of chilled 4.0% NaHCO3 solution. Add to
extract 5 ml water containing two drops of HCl. Wash the extract with three 10 ml
portions of CH2Cl2 & add the washings to original CH2Cl2 layer. Leave the aqueous
extract is separatory funnel.
Now filter the CH2Cl2 solution through the filter paper impregnated with
CH2Cl2 in a 50 ml volumetric flask & make up the volume.
Acidify NaHCO3 layer with 6 ml 1M-H2SO4 solution to avoid hydrolysis of
aspirin. pH at this point should be 1-2. Extract this acidified solution with eight
separate 10 ml portions of CH2Cl2 and filter through filter paper already impregnated
with CH2Cl2 in a 100 ml volumetric flask and make up volume upto the mark. Now,
dilute 5 ml of this solution to 25 ml with CH2Cl2 in a volumetric flask [Ca: 25 ml].
Record the absorbance of this solution on at 277 nm. Absorbance of standard
solutions of aspirin is recorded under exactly same conditions at 277 nm from which
concentration of aspirin in given mixure of drugs can be calculated. Concentration of
aspirin is used to calculate its percentage.
Similarly, concentrations and hence percentages of phenacetin and caffiene in
the tablet (mixture of drugs) can be determined at 250 and 275 mm, respectively.
Result
Given
tablet
is
mixutine
of
drugs
.......................%
Aspirin;
open
column
chromatography
or
surface
chromatography.
TLC Technique
Infact TLC is adsorption chromatography and is based upon the difference in
adsorption of the components of mixture on the adsorbent. Distribution coefficients
of components between stationary and mobile phase play an important role in TLC
separations. Stationary phase in TLC is adsorbent coated on glass plate and mobile
phase is the solvent system which moves on adsorbent thin layer. On the basis of
difference in adsorbitivity different components get adsorbed at different distances
and are hence separated. The process by which different components move upward
through continuous flow of mobile phase is known as elution and solvent is known
as eluents. The components which move upward are known as eluates. Entire TLC
technique can be studied under following steps:
(1)
(i)
Selection of adsorbent
(ii)
(iii)
Choice of solvent
(iv)
Application of sample
(v)
Development of plates
(vi)
Location of spots
(vii)
Determination of Rf.
Selection of Adsorbent:
While selecting adsorbent polarities of sample to be separated or purified and
Before coating glass plates with adsorbent they should be properly cleaned
with laboratory detergents using test-tube brush to remove adhering particles and
washed thoroughly with distilled water. Plates should be allowed to drain and dried
in oven. Plates should be handled from edges or the side which is not to be coated.
Otherwise a mechanically unstable layer is formed. If glass plates have some greasy
material over them, they should be cleaned with chromic acid. Generally 205 cm
glass plates or microscopic slides are used to prepare chromatogram.
First, glass plates of even thickness are selected. A slurry of adsorbent in
suitable solvent is prepared. For example, 25g silica gel containing 13% plaster of
paris is made into slurry in 50 ml of distilled water by slow addition and stirring with
glass rod or spatula. Then a thin coating of this slurry is applied on glass plates by
means of spreader. The slurry is transfered immediately to spreader which is pulled
across with uniform motion over row of glass plates of even thickness. Thickness of
thin layer of adsorbent is controlled with the help of clearance by the means of two
screws present. Generally a layer of 0.25 mm thickness is suitable.
Alternatively, slurry may be prepared in dichloromethane also. For instance,
30g of adsorbent (usually silica gel or alumina) are slowly added to 100 ml of dry
dichloromethane contained in wide mouth bottle with continuous stirring and bottle
is capped. For preparing the plates pair of microscopic slides is held together and
dipped in slurry, then slowly taken out and allowed to drain for a while over the
mouth of bottle. Slides are separated carefully, placed horizontally and allowed to
dry for 10 minutes. Adsorbent lying on the edges of slides is removed wih the help
of a razor.
After coating and drying plates are activated in oven at 1100C for half an
hour. Cellulose and polyamide plates are allowed to dry at room temperature and
stored in dust-free cabinet; they don't need activation.
(3)
Choice of Solvent
Choice of solvent depends upon the nature of sample to be separated as
Generally, polarities of sample and solvents are matched and then choice is
made. More polar solvents make more migration and give better separation. A
combination of two solvents also gives better separation as compared to single
solvent. Best position of the spot of compound is half way between line and solvent
front.
(4)
Application of Sample
1-3 mg of substance is dissolved in 0.1-0.6 ml of a solvent in which it
Finish line
Adsorbent
Base line
Glass plate
(5)
Development of Plates
Chromoplate of 20 5 cm. size may be developed in cylinderical glass jar.
Lacation of Spots: Following locating agents can be used for the the
visualization of spots :
(i)
(ii)
(iii)
It has been found that sulphuric acid mixed with aromatic aldehydes,
powerful oxidising agents like potassium permanganate, chromic acid,
nitric acid and ceric sulphate also give good results.
(iv)
(7)
Determination of Rf
As distance moved by solvent (i.e. solvent front) is always more than that travelled
by component, Rf is always lesser than one. It is worth noting that Rf is constant for
each component under identical conditions. Following factors affect Rf value :
(i)
(ii)
(iii)
(iv)
Quality of solvent
(v)
(vi)
Spot should not be too concentrated otherwise spot will smear out. If this
happens spotting solution should be diluted.
2.
Spots should not be too close to each other otherwise spots start bleeding
into each other and it becomes difficult to tell which spot came from which
origin.
3.
(b)
True/False
(ii)
(iii)
(i)
............................................
(ii)
............................................
(iii)
............................................
............................................
............................................
............................................
5.2
Let us Sum up
Our objective was analysis of various components of soil, fuel, body liquid as
well study and identification of various drugs as they all are integral part of our life.
Study can be summarized through following points:
Soil is a natural medium for the growth and development of plants & it
provides
mechanical
support
to
vegetation.
Hence,
presence
of
essential
Characterization of fuel is necessary to choose good quality fuel for which (i)
Proximate analysis & (ii) Ultimate analysis are used. Furthermore, fuel can be
solid, liquid or gaseous. Among solid fuels coal like anthracite & charcoal are
widely used. Important liquid fuels are diesel & petrol. Liquid fuels are
characterized by the determination of calorific value, flash point and aniline
point.
Gaseous fuels like LPG, natural gas, producer gas and water gas are widely
used.
For proper metabolism which is essential for good health all ingredients of body
should be in normal range. Their concentration in blood, plasma, serum etc are
determined chemically and spectroscopically. Different methods of analysis which
help is the diagnosis have been described in unit 5(c). Preservation and collection
methods related to blood, plasma and serum have been described. For preservation
blood anticoagulants like heparin are added. Immunoassay and principle of
redioimmunassay have been discussed.Later is based on antigen-antibody reaction
to give complex which can be estimated spectrophotometrically.
Blood gas analysis is done to check respiratory & kidney diseases. It also
indicates pH of blood is 7.33-7.45.
Subunit 5d. deals with the study and screening of drugs. Dangerous and
narcotic drugs have been differentiated. Classification of different drugs have been
done on the basis of their therapeutic actions into
(i)
Chemotherapeutic agents.
(ii)
Pharmacodynamic agents.
21-sub-classes of these two main classes have been described with examples
of each. Screening of drugs through spectrophotometric and chromatographic
methods (Gas & TLC) have been described.
5.3
(I)
(i)
Yes
(ii)
False
(iii)
Lime
(i)
Olsen method
(ii)
(iii)
Chlorolysis
(i)
True
(ii)
CO + H2
(iii)
It will be consumed.
(i)
(ii)
(iii)
High.
(II)
(III)
(IV)
(V)
(VI)
(i)
Oxy-haemoglobin
(ii)
(iii)
(i)
True
(ii)
Cancer
(iii)
drugs]
5.4
References
1.
India (P)
Ltd,
Meerut (1999)
2.
Jackson, M.L; Soil chemical Analysis, Prentice Hall of India, Pvt. Ltd., New
Delhi (1973)
3.
4.
Hesse, P.R., Soil Sampling and Methods of Analysis, CBS- Publisher &
distributors, New Delhi (1994).
5.
6.
Gartley, K.L., Recommended Soil Testing Procedures for the North Eastern
united state (2011).
7.
8.
9.
10.
11.
Samson Wright's Applied Physiology, Revised by Keele, C.A. and Neil, E.,
ELBS.
12.
Cooper & Gunn's Tutorial Pharmacy, Ed. by S.J. Carter, CBS Publishers &
Distributors, New Delhi (2000)
13.
Kar, A., Medicinal Chemistry, New Age International (P) Ltd., New Delhi
(2000)
14.
15.
Books Suggested
1.
2.
Fundamentals of Analytical Chemistry, D.A. Skoog, D.M. West and F.J. Holler,
W.B. Saunders.
3.
4.
5.
Principles of Instrumental Analysis, D.A. Skoog and J.L. Loary, W.B. Saunders.
6.
7.
Quantitative Analysis. R.A. Day, Jr. and A.L. Underwood, Wiley Eastern.
8.
9.