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Abhilasha Shourie
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Bioanalytical Bioanalytical
Bioanalytical Techniques
Techniques
Abhilasha Shourie
Techniques
Shilpa S. Chapadgaonkar
Abhilasha Shourie
Bioanalytical techniques form an integral part of applied biology
and biomedical sciences. The book provides understanding
of the concept and working principles of various bioanalytical
techniques used in biomedical sciences, environmental studies,
life sciences, pharmaceutical analysis, molecular biology, and
biotechnological research, as well as the various instruments
used in these processes. Divided into 12 chapters, the book
provides a comprehensive account of microscopy, centrifugation,
chromatography, electrophoresis, spectroscopy. It also focuses on
two main topics: radioisotope and immunodiagnostic techniques.
Techniques in molecular biology and recombinant DNA technology
have also been described in detail.
Shourie • Chapadgaonkar
Key Features
• Explains analytical instrumentation in a concise manner
• Provides state-of-the-art sophisticated techniques that would
be beneﬁcial to researchers in various ﬁelds for experimentation
• Encourages reader to analytical thinking and practical
application of the technique
The Energy and Resources Institute

Abhilasha Shourie
The Energy and Resources Institute

© The Energy and Resources Institute, 2015
ISBN 978-81-7993-529-3
All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or
transmitted in any form or by any means, electronic, mechanical, photocopying, recording or
otherwise, without the prior permission of the publisher.
All export rights for this book vest exclusively with The Energy and Resources Institute (TERI).
Unauthorized export is a violation of terms of sale and is subject to legal action.
Suggested citation
Shourie, Abhilasha and Shilpa S. Chapadgaonkar. 2015. Bioanalytical Techniques.
New Delhi: TERI
Published by
The Energy and Resources Institute (TERI)
TERI Press Tel. 2468 2100 or 4150 4900
Darbari Seth Block Fax 2468 2144 or 2468 2145
IHC Complex, Lodhi Road India +91 • Delhi (0)11
New Delhi – 110 003 E-mail teripress@teri.res.in
India Website www.teriin.org
Bookstore https://bookstore.teri.res.in
Printed in India
Preface
Science and technology by far depend upon experimental procedures involving

one or more analytical methods. Experiments are conducted not only to
predict phenomena, but also to validate the results. Analysis of chemical and
biochemical entities is metaphorically the path to achieve the objectives of
research in life sciences. In the past few decades, the field of life science has
witnessed rapid advancements through development of highly sophisticated,
automated, sensitive, and accurate analytical techniques. A vast range of
analytical techniques and respective instruments are available, and therefore
it is imperative to understand the principles, limitations, and alternatives of a
given technique in order to apply it effectively to obtain useful results.
‘Bioanalytical techniques’ is included as a fundamental paper in most courses

in chemistry, biochemistry, biology, pharmaceutical and clinical sciences,
environmental, forensic and materials sciences. In the past 10 years of our
career, we have felt a profound need for a comprehensive yet intensive textbook
on analytical techniques that would serve the purpose of both students and
researchers. This book has been written to fulfil the need for a text-cum-
reference in undergraduate and postgraduate level curriculum, providing
necessary information required to demonstrate the concepts of an analytical
technique in all its guises.
The book emphasizes on imparting profound knowledge that is able to

meet the current throughput screening demands of scientists and researchers.
It consists of 12 chapters, encompassing techniques used for biological and
biochemical separation, purification, identification and quantification, all put
together to construct a compact package. The chapters have been prepared
meticulously using simple yet lucid language. We have included a fairly good
vi Preface
number of state-of-the-art sophisticated techniques that would be beneficial to

researchers in various fields for experimentation.
Many typical analytical procedures appear in the book as boxed features

which give a snapshot of the techniques being widely used contemporarily in
research. Throughout the book, wherever felt required, extended illustrative
examples have been incorporated, which are to be read as part of the respective
chapter. The format also encourages the reader to analytical thinking and
practical application of the technique. This book will certainly prove to be an
invaluable reference tool for students, teachers and researchers in the mentioned
fields.
We express our gratitude to Almighty and all those who have contributed to
the book in any manner. We thank our families for their encouragement and
unconditional support. We also thank the editorial team of TERI Press for
their continuous effort and faith in us.
We solicit constructive suggestions from all the readers for further improvement
of the content of the book.
Dr. Abhilasha Shourie

Dr. Shilpa S. Chapadgaonkar
Contents
Preface v
1. GENERAL PRINCIPLES OF ANALYTICAL INSTRUMENTATION 1
1.1 Introduction 1
1.2 Experimental Studies 2
1.3 Experimental Errors 7
1.4 Statistical Parameters for Validation of an Experiment 7
2. SOLUTIONS AND BUFFERS 11
2.1 Introduction 11
2.2 Units of Concentration 12
2.3 The Concept of pH 16
2.4 Acids and Bases 17
2.5 Henderson–Hasselbalch Equation 19
2.6 Determination of pKa 20
2.7 Buffers 20
3. MICROSCOPY 37
3.1 Historical Background 37
3.2 Nature of Light 39
3.3 Compound Microscope 41
3.4 Image Formation in a Light Microscope 43
3.5 Phase Contrast Microscopy 47
3.6 Fluorescence Microscopy 51
3.7 Electron Microscopy 54
viii Contents
4. CELL DISRUPTION 69
4.1 Introduction 69
4.2 Barriers for Cell Disruption 70
4.3 Methods of Cell Disruption—an Overview 70
4.4 Mechanical Methods of Cell Disruption 71
4.5 Non-Mechanical Methods of Cell Disruption 82
4.6 Combinations of Cell Disruption Methods 86
4.7 Selection of Cell Disruption Methods 88
4.8 Analysis of Cell Disruption 90
5. CENTRIFUGATION 91
5.1 Introduction 91
5.2 Principles of Centrifugation 93
5.3 Centrifuge Machines 102
5.4 Centrifugal Separations 109
5.5 Analytical Ultracentrifugation 117
5.6 Care and Safety of Centrifuges 122
6. CHROMATOGRAPHIC TECHNIQUES 125
6.1 Introduction 125
6.2 Types of Chromatographic Techniques 126
6.3 Planar Chromatography 127
6.4 Column Chromatography 137
6.5 Protein Purification Strategies 178
7. ELECTROPHORESIS 181
7.2 Principles of Electrophoresis 182
7.3 Free Solution Electrophoresis 187
7.4 Paper Electrophoresis 188
7.5 Gel Electrophoresis 190
7.6 Gel Electrophoresis of Proteins 199
7.7 Gel Electrophoresis of Nucleic Acids 212
7.8 Capillary Electrophoresis 218
8. SPECTROSCOPY I 225
8.1 Electromagnetic Radiations 225
8.2 Ultraviolet and Visible Light Spectroscopy 228
8.3 Fluorescence Spectroscopy 238
Contents ix
8.4 Atomic Absorption Spectrometry 243

8.5 X-ray Spectroscopy 246
8.6 Circular Dichroism and Optical Rotatory Dispersion 253
9. SPECTROSCOPY II 259
9.2 Infrared Spectroscopy 259
9.3 Nuclear Magnetic Resonance Spectroscopy 267
9.4 Electron Spin Resonance Spectroscopy 276
9.5 Mass Spectroscopy 282
10. RADIOISOTOPE TECHNIQUES 295
10.1 Introduction and Applications 295
10.2 Structure of Atom and Radioactivity 297
10.3 Types of Radioactive Decay 298
10.4 Interaction of Radioactivity with Matter 301
10.5 Kinetics of Radioactive Decay 303
10.6 Units of Radioactivity 306
10.7 Detection and Measurement of Radioactivity 307
10.8 Safety Issues and Radio-waste Management 320
11. IMMUNOCHEMICAL TECHNIQUES 325
11.2 Production and Purification of Antibodies 329
11.3 Immunoassay Techniques 336
11.4 Advances in Immunochemical Techniques 353
12. MOLECULAR BIOLOGY TECHNIQUES 355
12.2 Isolation of Nucleic Acids 355
12.3 Qualitative and Quantitative Evaluation of Nucleic Acids 364
12.4 Nucleic Acid Hybridization 367
12.5 Restriction Digestion 376
12.6 Nucleic Acid Sequencing 380
12.7 DNA Amplification by Polymerase Chain Reaction 387
Further Readings 393
Index 397
About the Authors 411
1
General Principles of
Analytical Instrumentation
1.1 INTRODUCTION
Biological experimentation and chemical experimentation inevitably require
analytical instrumentation to perform detection, isolation, purification,
identification, and quantification of not only organic and inorganic material,
but also of cells and cellular entities. The utility of these techniques and,
thereby, instruments span from laboratory scale testing and experimentation
to advanced research and commercial applications.
Advances in imaging techniques have enabled visualization of micro–nano
scale features in biological systems, which have proved beneficial for cellular
biology, microbiology, molecular biology, and medical research. Chromatographic
techniques enable efficient separation, identification, and measurement of a
wide array of compounds such as amino acids, proteins, enzymes, hormones,
and drugs on the basis of their biochemical properties. High performance
liquid chromatography (HPLC) and gas chromatography (GC) have become
indispensable tools in analytical chemistry. The demands for faster and efficient
chemical and biochemical separations have led to achieving new horizons in
advanced chromatography.
Identification and quantification of inorganic and organic compounds,
based on their spectral and fluorescent characteristics, can be done with high
efficiency and accuracy using spectrometric techniques. Highly sensitive and
specific measurement of trace metals and minerals, and structure determination
of macromolecules can be done with the help of techniques such as atomic
absorption spectrometry, X-ray diffraction, X-ray photoelectron spectroscopy,
nuclear magnetic resonance (NMR), and electron spin resonance (ESR). Mass
spectrometry (MS) is nowadays an indispensable technique for the molecular
analysis of complex compounds. Electrospray-ion trap, matrix-assisted laser
2 Bioanalytical Techniques
desorption/ionization time-of-flight (MALDI–TOF), and Fourier transform

ion cyclotron resonance (FT–ICR) mass spectrometry are high-resolution
sophisticated techniques that enable structural analysis of complex molecules,
such as proteins, and reveal their secondary and tertiary conformations.
In the past two decades, there have been remarkable improvements in the
analytical methods, which have significantly broadened their applications in
the analysis of biological and chemical molecules. Hyphenated techniques
combining chromatographic and spectral methods have been instrumental in
advancements in analytical sciences. Techniques such as liquid chromatography
with mass spectrometry (LC–MS) and gas chromatography with mass
spectrometry (GC–MS) are being widely used where biochemical separations
and subsequent analysis of separated compounds are required. Capillary
electrophoresis combined with mass spectrometry (CE–MS) and liquid
chromatography combined with nuclear magnetic resonance (LC–NMR) have
extended the use of these techniques to structure elucidation and quantification
of analyte concentrations as low as nano or pico grams with high resolution
and sensitivity.
Bioanalytical procedures and instruments are often selective and sensitive in
nature and are critical for the successful conduct of the intended experimental
analysis. A particular experimental study may require several instruments and
methods to be employed in order to obtain the necessary data. Integration
of many processes and interdisciplinary methods may need some “standard
operating procedures” to define a uniform set of specifications to facilitate
interoperability. Often in biological and chemical sciences, uniform nomenclature
systems and standardized measurement systems, such as standard international
(SI) units, are routinely used. Besides these, it is also a common practice
to standardize the experimental conditions, treatments, controls, and other
experimental parameters under which the intended data is generated with
utmost reliability.
1.2 EXPERIMENTAL STUDIES

In science and technology, both elementary studies and research are essentially
based on experimental methods. Scientific research is a directional process
based largely on keen observations, logical hypothesis formulation, experimental
verification of the prediction, and unbiased inferences. Experimental methods
involve deliberate manipulation of one or more variables in comparison to
other variables or a constant. This approach demands systematic and controlled
experimentation involving field, laboratory settings, or both. Experiments generate
vital data which after thorough analysis lead to inferences and conclusions that
address the scientific query and validate the prediction or hypothesis.
General Principles of Analytical Instrumentation 3
1.2.1 Experimental Design

It is necessary that the experimental procedures and processes be organized
properly into a well-formulated “experimental design” to ensure the collection
of right type and amount of data depending upon the objectives of the study.
A good experimental design, thus, minimizes bias and maximizes reliability of
the data so that it tests exactly what the hypothesis states. An experimental unit
can be an individual or a group under study. If it is a group, it should be fairly
homogeneous because any sort of variations may cause bias and uncertainty
in the results.
The three basic principles of experimental design: control, randomization,
and replication are discussed here at length.
• Control Experimental studies are usually carried out under controlled
conditions where two or more things are compared to observe the causes
or effects of imposed or exposed factors, often termed as “treatment” on
the experimental units. The group that is exposed to all the conditions and
factors of the experiment except the one which is being tested or is under
question is called control group. On the other hand, the group which gets all
of the conditions and factors of the experiment along with the variable that
is being tested through the experiment is called experimental group. Use of
control group eliminates experimental errors and bias. Two types of control
groups are recognized: negative control and positive control. In the negative
control group, there is no effect of the imposed factors on the subjects, that
is, it shows null effect. The positive control group essentially shows an effect
of the experimental treatment, conditions, or factors on the subjects being
investigated in the experiment.
• Randomization It refers to random assignment of experimental units to
an experimental group without any prejudice by the experimenter, so that
every experimental unit has the same chance of receiving a treatment. If
this is done absolutely at random, without imposing any specific selection,
then it is called a completely randomized design. If the experimental subjects
are first divided into homogeneous blocks which are subjected to the same
extraneous variables and then randomly assigned to a treatment group,
it is called randomized block design. Randomization is generally used for
eliminating bias in experiments.
• Replication It is the process of repeating each treatment or measurement
on several experimental units (EU) to determine the actual effects of the
treatment. Replication helps in identifying the variation among units and
eliminating the errors. The statistical inferences obtained by measurements
taken from several replicates enhance the reliability and validity of the
results.
1.2.2 Constants and Variables

The factors or conditions under study in an experiment are categorized as
constants and variables. All those factors that remain same for both the
experimental group and the control group are called constants and all those
factors that vary are called variables. The variable that is varied by the
experimenter in a very precise manner is called an independent variable and
the one that is measured in response to the independent variable is called a
dependent variable or response variable. Each treatment is a combination of
one or more independent variables.
For example, while studying the effect of fertilizer on crop growth, the
amount of fertilizer supplied would be an independent variable, whereas the
growth parameters such as height or weight of the plants would be the dependent
variables. The controlled variables which would remain same for all the values
of independent variable would be the type of plant, the type of fertilizer,
temperature, sunlight, humidity, and so on.
Variables can be classified as categorical and continuous:
• Categorical variables These variables are qualitative and differ in kind,
not in magnitude; therefore, they are also called discrete variables. These can
be further classified as nominal and ordinal variables. Nominal variables have
two or more categories which cannot be quantified and also cannot be placed
in an order. For example, gender and marital status. Ordinal variables are
variables that have two or more categories which can be ordered or ranked,
but the intervals between the scale points may not be even. For example,
educational level and socio-economic status of families.
• Continuous variables These variables have a numerical value and differ in
magnitude, therefore, are also known as quantitative variables. For example,
income and age. These can be further classified as interval and ratio variables.
An interval variable can be measured along a continuum such as temperature
measured in degree Celsius (°C). For example, the temperature of 50°C is
higher than 40°C, and an increase from 30°C to 50°C is twice as much
as the increase from 40°C to 50°C. Ratio variable is a type of interval
variable not measured on a linear scale. In this type, 0 (zero) value of a
measurement means absolute absence of such a variable. For example, mass
and distance.
• Other variables A factor or variable that is not related to the study but
may affect a response variable in the study is called an extraneous variable. If
the effect of the extraneous variable on the response cannot be distinguished,
it is called a confounding variable.
1.2.3 Sampling
The entire population with which the experimental study is concerned, needs
to be examined to draw fair conclusions. However, it is often not feasible
to measure the effects of treatments on every individual subject from each
experimental group, especially when the population under consideration is large.
In such a case, the study variables are measured on some randomly selected
individuals, called “sample”, which are the subsets of that population. The
results from the sample are then used to draw valid inferences about the entire
population. The sampling method is thus a scientific procedure of selecting
those units from which the required estimates can be obtained.
Such results are associated with some degree of uncertainty, as only a part
of the population is actually studied and not the whole. Therefore, sampling
must always be accompanied by measures of counteracting bias such as random
selection of sample and appropriate sample size. The size of the sample depends
on the extent of variability in the population. The greater the variability in a
population and the larger the sample required, the better it is for estimating
characteristics of the population with high degree of accuracy.
The basic types of sampling designs are non-probability, probability sampling,
systemic sampling, stratified sampling, and cluster sampling.
• Non-probability sampling In this type of sampling, units are selected
deliberately or on purpose, therefore, it tends to yield biased results. The
method gives no assurance of giving equal chance of selection to all the
experimental units; hence, it is always associated with high risk of bias.
Sampling error cannot be estimated in this sampling.
• Probability sampling It is also called “random sampling” in which the
selection of sampling units is done without imposing any predictions or
preferences. In this method, every individual has an equal probability of
selection, therefore, it is free of bias.
• Systemic sampling It is done when a starting point is chosen from the
sampling frame at random and thereafter every nth member is chosen. This
method tends to spread the sample more evenly throughout the population.
However, it may possess some hidden bias leading to minor unidentifiable
errors.
• Stratified sampling It is carried out when the population is not fairly
homogeneous and naturally falls into groups such as age groups, income range,
etc. In such a case, the representative sample is obtained by first dividing the
parent population into strata composed of as far as possible homogeneous
units, and then selecting items from each stratum. Stratification makes the
estimates of the population parameters more precise and reliable.
• Cluster sampling It is another type of random sampling in which the

entire population is divided into many small sub-populations, each of which is
again a cluster of further smaller units. Once the clusters are chosen, simple
random sampling can be done. In order to ensure a representative spread
across the entire population, each selected cluster should be as dissimilar as
possible.
1.2.4 Measurement Scales

In an experiment, numeric data are generated by measuring variables using four
different types of scales classified as nominal, ordinal, interval, and ratio.
• Nominal scale It is a system of measurement that places the experimental
units into mutually exclusive and exhaustive categories, such that every
measurement essentially falls into one of the categories. This type of
measurement scale is qualitative type and can be assigned an arbitrary
value so that each experimental unit can take only a single value out of
the given options. For example, nationality, language, and gender. The
measure of central tendency for the nominal scale is “mode”. These rankings,
however, do not reveal much about the quantitative differences between the
subjects.
• Ordinal scale An ordinal scale puts the experimental units in a rank order
with respect to the variable being assessed, usually representing a hierarchy
of levels. However, the ordinal measurements do not have absolute values
and the intervals of the scale may or may not be equal in magnitude. Thus,
the ordinal scale only measures the qualitative phenomena. For example,
ranks of students in a class. The suitable measure of central tendency for
the ordinal scale is “median”.
• Interval scale An interval scale has equal differences between the scale
values, therefore, it provides more quantitative information than the ordinal
scale. However, it does not have a true “zero” point, that is it is not able to
measure complete absence of the character being measured. For example,
the Fahrenheit degree scale used for measuring temperature. Using interval
scale, “mean” is taken as the appropriate measure of central tendency and
“standard deviation” is commonly used as a measure of dispersion.
• Ratio scale It is similar to interval scale as it shows equal differences
between the scale values which are quantitatively meaningful. Ratio scale
has a true zero point which indicates complete absence of the character. For
example, measurement of weight and length.
1.3 EXPERIMENTAL ERRORS

Experimental measurements are often susceptible to errors due to many reasons
such as defect in an instrument, calibration error in an instrument, procedural
error inherent in the adopted method of experimentation, or an unidentifiable
error. Since errors lead to deviation in experimental values from the ideal or
“true” value, usually comparisons are made between some standard value
recognized as “true” value and the experimental value. Therefore, it is important
to determine the accuracy of a particular measurement to ascertain the validity
of the results.
Errors in investigations can be classified as random and systematic errors.
• Random errors These are also called precision errors, intrinsic to all
experiments, and caused by lack of repeatability in the measurements.
These are unpredictable and called indeterminate errors because they cause
inconsistency in the measurements, resulting in a scatter in the data about
the true value, since there is equal probability of producing measurements
that are higher or lower than the “true” value.
• Systematic errors These are also called non-random or determinate errors.
These are consistent and repeatable errors, caused due to factors that bias the
result in one direction such as inherent defect in an instrument, fault in the
method or procedure, etc. These errors can be identified and eliminated to
some extent or completely either by using an alternative method or by using
a standard value of some reference sample.
1.4 STATISTICAL PARAMETERS FOR VALIDATION OF AN EXPERIMENT

Analytical procedures and instruments, which are used for intended analytical
applications, must meet the characteristic requirements of that application.
Moreover, the validation of analysis essentially depends upon the reliability and
reproducibility of the experimental procedure and the instruments used for this
purpose. In compliance to these analytical procedures and instruments with
the intended use, their assessment is done on the basis of certain performance
characteristics such as specificity, linearity, range, detection limit, accuracy,
and precision.
The acceptability of analytical data directly corresponds to the criteria used
for validating the method and requires application of standard statistical tests.
A measurement system is considered valid if it is both accurate and precise;
therefore precision and accuracy are the two fundamental parameters used for
such a validation.
1.4.1 Precision
The precision of an analytical method refers to the repeatability or reproducibility
of measurements of the same quantity on the same sample under the same
conditions. When a measurement is repeated several times and a graph is plotted
between the number of times of occurrence of a value and the set of values
obtained, it is normally bell-shaped with the results scattered symmetrically
about a mean value. This type of distribution is called a Gaussian or normal
distribution (Figure 1.1). In such a case, the precision of the data set gives an
estimate of random error.
A measure of variability is “standard deviation” (SD) which shows how
closely all the values are clustered around the mean in a set of data. One SD
away from the mean in either direction on the horizontal axis (the two shaded
areas closest to the centre axis in Figure 1.1) accounts for somewhere around
68 per cent of the values in a data set. Two SDs away from the mean (the four
areas closest to the centre areas) account for roughly 95 per cent and three
SDs (all the shaded areas) account for about 99 percent values in a data set.
Standard deviation (SDx) is computed as
_____________
_ 2
| ÷ |
n
S_____________
i=1
(xi – x)
sx =
n – 1
where
n is the number of data points
_
x is the mean of xi, and
xi is each of the values of the data.
Figure 1.1 A plot showing Gaussian or normal distribution

Note On x-axis is the value in question and on y-axis is the number of data points for each value on the
x-axis
In the case of systemic errors, the plot is skewed to one side of the mean
value, and then increasing the sample size generally increases the precision. The
precision is given in terms of deviation from a mean value and is measured
as either a “standard error” (SE) of a mean or a “least significant difference”
(LSD). In the case of determining LSD, the significance level used should be
stated; for example, 5 per cent LSD.
1.4.2 Accuracy
The accuracy of an analytical method determines how close is the mean set
of test measurements to the standard or true value for that measurement. The
“true value” can either be obtained by referring to some standard measurement
or by applying population statistics. The population mean is said to be the best
estimate of the true value. Accuracy depends on the desired level of confidence
in the test and an acceptable “confidence interval” (CI) that relates the sample
mean to the population mean. A CI gives a range of values about the sample
mean within which there is a given probability that the population mean lies.
This probability is determined by the confidence level, which implies that if
a large number of random samples are taken from a population and CIs are
constructed for each, then 100 per cent of these intervals are expected to
contain the population parameter. For example, a “level of confidence” (LOC)
of 95 per cent means that, if 100 values are taken from the population, the
true mean accuracy of the measurement located within the CI band will be at
least 95 out of the 100 tests whereas uncertainty will be associated with the
remaining 5 values.
The relationship between the two means is expressed in terms of the SD
of the data set, the square root of the number of values in the data set and a
factor known as Student’s t-test, which can be represented as
| m = x ± ___tsn |
_
÷
__
where
_
x is the measured sample mean,
µ is the population mean,
s is the measured SD,
n is the number of measurements, and
t is the Student’s t-factor.
2
Solutions and Buffers
2.1 INTRODUCTION
Most bioanalytical procedures involve preparation of solutions. These solutions
constitute the growth media, extraction media, purification media, formulation
media, and reagents required for quantification as well as for characterization.
Therefore, it is vital for all the concerned people to understand the preparation
of solutions and express their concentration in appropriate units. Section 2.2
will familiarize us with different units for expressing concentration of solutions.
2.1.1 Solution
A “solution” is formed when a substance becomes dispersed homogeneously
throughout the liquid in molecular form. The substance, called “solute”, is
said to dissolve and the liquid is called a “solvent”. Solutions may consist of
solids, liquids, or gases dissolved in a solvent. The properties of a solution are
uniform throughout the mixture. The presence of a solute in a solvent maybe
indistinguishable from the solvent or it may have colour or odour. Table 2.1
summarizes types of solution and their properties.
2.1.2 Properties of a True Solution

A true solution has the following properties:
• A true solution is a homogeneous mixture of solute and solvent.
• A solute exists as individual molecule or as ion in solution. Typically the diameter
of the solute particle is less than one nanometre (10 –9m).
• The solute and solvent do not separate on standing or by centrifugation.
• True solutions may be colourful but they are always transparent. Light is
not scattered by the solution.
Table 2.1 Types of solutions: true, colloids, and suspensions

S. no. True solution Colloidal solution Suspension solution
1. It is a homogeneous mixture It is a heterogeneous mixture It is a heterogeneous mixture
of two or more substances in which one substance is with large particles suspended
(the solute is extremely dispersed in another. The (greater than 10 –7 m).
small, less than 10 –9 m). particles are larger than in a true
solution (10 –9 –10 –7 m).
2. Solute particles exist as Particles form groups of ions, The particles form large groups
molecules homogeneously atoms, or molecules that are of insoluble particles.
dispersed in solvent. evenly dispersed through
solvent.
3. The solution is clear. Colloidal solution appears The mixture is cloudy.
cloudy.
4. The particles do not settle The particles do not settle under The particles settle under gravity
under gravity. gravity. on standing.
5. It cannot be separated using It can be separated by a semi- It can be easily separated by
filters. permeable membrane, that is, filtering.
cellophane and cell walls.
6. Examples are air and Examples are mayonnaise, Examples are sand in water,
gasoline smog, butter, whipped cream, dust in air, and flour in water
and milk
• Solubility is the ability of the solute to dissolve in a solvent at a particular

temperature. It depends on the nature of the solute and solvent, temperature,
and pressure. At “saturation point”, the solvent can no longer dissolve any more
solute.
• Dissolution of an ionic molecule into water results in the formation of
an “electrolyte” solution. The ions of the solute will separate in water. These
ions are responsible for conduction of electric current in the solution.
• The solution has higher osmotic pressure as compared to solvent and it increases
with increase in solute concentration.
• As the concentration of the solute in the solvent increases, the boiling point of
the solution also increases.
• The melting point of the solution decreases as the amount of solute is increased
in the solution.
• A solution of a solid, non-volatile solute in a liquid solvent shows a decrease in
vapour pressure above the solution as the amount of solute is increased.
2.2 UNITS OF CONCENTRATION

Concentrations of chemicals are routinely expressed in a variety of units. The
choice of measurement units to be used in a given situation depends on the
Solutions and Buffers 13
chemical method to be followed in the experiment. It is, therefore, necessary

that we become familiar with the units used and methods of converting between
different sets of units. To describe quantities that may take on such extreme
values, it is useful to have a system of prefixes that accompany the units. Some
of the most important prefixes are given in Table 2.2.
2.2.1 Molarity
Molarity (M) is the most common unit for expressing the concentration of a
solution in biochemical studies. The molarity of a solution is the number of
moles of the solute dissolved per litre of the solution. Simply it is the amount
of substance equal to its molecular mass in grams. Molarity of a solution can
be calculated as
Mass of solute in g/Gram molecular mass of solute

_______________________________________________
Molarity = 2.1
Volume of solution in litres
For example, a solution labelled as 1 M sodium hydroxide (NaOH) has the
equivalent of 1 gram mole of NaOH dissolved in 1 litre of solution. Notice that litre
is the volume of solution and not of solvent, which is water.
1 M NaOH solution would be made by measuring out the mass of 1 mole of NaOH (40
g) dissolving it in distilled water and making up the volume of the solution to 1 litre.
2.2.2 Molality
Molality (m) is defined as the number of moles of solute per kilogram of solvent.
It has its unit as the lower case letter “m” and is read as “molal”.
Number of moles of solute
Molality = ________________________ 2.2
Mass of solvent in kg
It is primarily used when one is dealing with colligative properties of solutions
such as freezing point lowering of solvents by solutes. 1 molal solution of sodium
chloride (NaCl) is prepared by dissolving 58.5 g (1 mole) of NaCl in 1 kg of
distilled water.
Table 2.2 Prefixes of commonly used units

Prefix Value Symbol
–12
Pico 10 p
Nano 10 –9 n
–6
Micro 10 μ
–3
Milli 10 m
Kilo 103 k
2.2.3 Parts per Million

Parts per million (ppm) is a unit of concentration often used when dealing with
very small amounts of metal ions and other solutes in water, air, or soil.
Grams of solute
Parts per million = ________________ × 1,000,000 2.3
Grams of solution
A solution with a concentration of 1 ppm of lead (Pb2+) is equal to 1 mg
lead (II) ion per litre of water, or 1 µg of lead (II) ion per millilitre of water
(this is because 1 ml water weighs 1 g). A copper salt (Cu2+) solution reported
to be 20 ppm would be equivalent to 20 mg of Cu2+ per litre of water or 20
µg per 1 ml.
Concentrations can also be expressed by using percentages. Three different
types of percentage concentrations are used including mass per cent, volume
per cent, and mass/volume per cent.
2.2.4 Mass Per cent

The mass per cent is used for expressing the concentration of a solution when the
mass of a solute and the mass of a solution are given.
Mass per cent = (Mass of solute)/(Mass of solution) × 100 2.4
2.2.5 Volume Per cent

The volume per cent is used for expressing the concentration of a solution when the
volume of a solute and the volume of a solution are given.
Volume per cent = (Volume of solute)/(Volume of solution) × 100 2.5
2.2.6 Mass/Volume Per cent

The mass/volume per cent is used for expressing the concentration of a solution
when the mass of the solute and volume of the solution are given. Volume per cent
is often used for expressing the concentration of a liquid solute in a liquid solvent
whereas mass per cent is often used for a solid dissolved in a liquid solvent.
Mass/Volume per cent = (Mass of solute)/(Volume of solution) × 100 2.6
2.2.7 Mole Fraction

The mole fraction of a substance in a solution is the fraction represented by
number of moles of that substance divided by the total number of moles of all
substances present in the solution. The sum of mole fractions of each substance
present in the solution equals 1.
If the solution is composed of substances A, B, and C, where NA, NB , and

NC are the number of moles of A, B, and C, respectively, then
X A = Mole fraction of A = (Number of moles of substance A)/(Total number
of moles of all substances in solution)
NA
X A = _____________ 2.7
N A + N B + NC
and
X A + XB + XC = 1 2.8
2.2.8 Mole Per cent

The mole per cent (of substance A) is mole fraction X A represented in
per cent form.
Mole per cent (of substance A) = X A × 100% 2.9
2.2.9 Per cent Saturation

Solubility of a solute in any solvent increases with increase in temperature. A solution
is said to be unsaturated at a given temperature if it can still dissolve more solute
in it at that temperature. A saturated solution is one in which the solute in the
solution is in equilibrium with the pure undissolved solute. A supersaturated solution
contains more solute in solution than it would ordinarily hold at a given temperature.
For example, if in hot water one dissolves all the salt it can possibly solubilize and
cools it slowly, the water would contain all the salt that was previously dissolved in
it resulting in the formation of supersaturated solution. If a small crystal of salt is
introduced into this water, some salt will crystallize out and a saturated solution will
remain. The amount of a substance that is dissolved in a solution compared to the
amount dissolved in the solution at saturation is expressed as a per cent saturation.
Amount of substance that is dissolved
Per cent saturation = _______________________________________________
Amount of substance that is dissolved at saturation
2.10
E2.1 EXERCISE
Rahul dissolved 10 g of sugar in 250 ml water. Find out the molar concentration
of the solution he has prepared?
Solution The molecular weight for sugar C12H 22O11 is 342 g in 1 mol.
For solving chemical problems, the unit mol/l is the most useful. Thus, the
concentration can be calculated as
342 g Æ 1 mol
10 g Æ 10/342 = 0.0923 moles
Therefore,
250 ml Æ 0.02923 moles
1 l Æ 29.23/250 moles
Molarity = 0.1169 M
The concentration of sugar solution is 0.116 M.
2.3 THE CONCEPT OF pH

The dissociation of water into hydroxide and hydrogen ions is a reversible process
and can be represented as
H2O Æ H+ + OH– 2.11

The equilibrium constant (Keq ) of a chemical reaction is given by the ratio
of concentration of products to reactants at equilibrium.
For the dissociation of water, we get
[H+] [OH– ]
Keq = ___________ 2.12
[H2O]
The equilibrium constant for the dissociation of water at 25°C has been
measured as Keq= 1.8 × 10 –16. This number is small because only a small
fraction of water molecules dissociate. The concentration of water can be
determined from the fact that 1 mole of water weighs 18 g and 1 litre of
water weighs 1000 g. Hence, the concentration of pure water [H 2O] is
1000 g/l/18 g/mol = 55.5 mol/l. By substituting these into Equation (2.12), we
get
[H+][OH– ] = 1 × 10 –14 2.13
In pure water, the concentrations of hydrogen and hydroxide ions are about
the same. Hence by taking the square root of 1 × 10 –14 we find that [H+]
and [OH – ] are each about 10 –7 M. This means that 1 litre of pure water
contains about one ten-millionth of a mole of hydrogen or hydroxide ions.
When substances are dissolved in water, the concentrations of H+ and OH–
can change depending upon the ability of the substances to donate or accept
hydrogen ions (H+) that is acidity or alkalinity of substances. Acidity and
alkalinity are measured with a logarithmic scale called “pH”. pH is defined as
the negative logarithm of the hydrogen ion concentration (–log[H+]). The pH

scale ranges from 0 to 14. On the pH scale, the value 7 is considered to be
neutral. Substances that can donate hydrogen ions, thus increasing [H+], are
acids. Strong acids have pH much lower than 7. Molecules that accept hydrogen
ions, thus decreasing [H+], are bases and have pH higher than 7.
pH = – log[H+] 2.14
The pH scale allows biologists to define chemical solutions more conveniently
by abolishing the need for exponential notation. In the simplest terms, the value of
pH simply gives us the value of the exponent of the hydrogen ion concentration.
Each one-unit change in the pH on the pH scale corresponds to a ten-fold change
in hydrogen ion concentration in the solution.
2.4 ACIDS AND BASES

Acids and bases have been defined with different concepts. However, the
definition that is better suited to a given situation is usually applied. We will
discuss the different concepts describing acids and bases briefly.
Figure 2.1 The pH scale

2.4.1 Arrhenius Concept

Arrhenius defined acid as any substance that is capable of providing hydrogen ions
(or protons) in aqueous solution. A base is defined as a substance containing hydroxyl
groups and capable of providing hydroxide ion (OH–) in aqueous solution. According
to this definition, a neutralization reaction combines these two ions to form the
solvent, that is, water and a salt. This can be illustrated as in Equation 2.15 stating a
general reaction between an acid HA and a base BOH.
HA + BOH = BA + H2O 2.15
2.4.2 Bronsted–Lowry Concept

According to the definition, an acid is any substance capable of donating a proton
(H+) in a solution. A base is any substance that is capable of accepting a proton.
A Bronsted acid dissociates into a proton and conjugate base of acid. This can be
observed in Equation 2.16 where A– is acting as the conjugate base of HA.
HA = H+ + A– 2.16
For example, the ionization of hydrochloric acid (HCl) in water can be viewed
as a Bronsted–Lowry acid–base reaction but actually no chemical reaction is
taking place.
The chemical species formed when a base accepts a proton is called a conjugate
acid of the base as illustrated in Equation 2.17 where BH+ is acting as the conjugate
base of B.
B: + H+ = BH+ 2.17
Acids and bases can be classified as strong and weak acids and bases. Strong acids
and bases completely dissociate in water. The others are considered to be weak acids
and bases.
Strong acids
HCl is considered as a strong acid because it has a strong tendency to ionize
resulting in a large concentration of H+ as HCl completely dissociates into H+ and
Cl– contributing to an increase in [H+] in the solution.
For example, HCl – hydrochloric acid, HNO3 – nitric acid, H2SO4 – sulfuric
acid, HBr – hydrobromic acid, HI – hydroiodic acid, and HClO4 – perchloric
acid are strong acids.
Strong bases
Strong bases like strong acids dissociate completely in water. For example,
NaOH – sodium hydroxide, KOH – potassium hydroxide, and Ba(OH)2 –
barium hydroxide.
Weak acids and bases ionize only partially in solutions. Examples of weak acids
and bases are as follows:
Weak acids
HCOOH – formic acid, CH3COOH – acetic acid, CCl3COOH – trichloroacetic
acid, HF – hydrofluoric acid, HCN – hydrocyanic acid, and H2S – hydrogen
sulfide.
Weak bases
NH3 – ammonia, N(CH3)3 – trimethyl ammonium, C5H5N – pyridine, and NH4OH –
ammonium hydroxide.
2.5 HENDERSON–HASSELBALCH EQUATION

Dissociation of weak acid Weak acids or bases do not dissociate completely
in water and the dissociation is represented by equilibrium. For example, let us
study the dissociation of a simple acid (HA). It can be described by the given
chemical reaction (Equation 2.18):
HA Æ H+ + A– 2.18
The equilibrium constant for the dissociation of a weak acid is given by

Equation 2.19:
[H+][A– ]
Ka = ________ 2.19
[HA]
The Henderson–Hasselbalch equation is derived from this by taking the

logarithm of both sides and rearranging to get
[A– ]
– log [H+] = – log Ka + Log _____ 2.20
[HA]
We can rewrite Equation 2.20 by replacing –log [H+] with pH and –log Ka
with pKa to get Equation 2.21:
[A– ]
pH = pKa + log _____ 2.21
[HA]
We can think of pKa as pH at which the number of molecules of conjugate

base [A – ] and weak acid [HA] is equal. The value of log [1] = 0; thus, we
get pKa = pH. We can interpret the above equation in the following way:
pH is a function depending on the constant (pKa) as well as the ratio of the
conjugate base to weak acid ([A– ]/[HA]). Henderson–Hasselbalch equation is
an extremely useful equation from which pH of the solutions of various ratios

of concentrations of conjugate acid and conjugate base form of a substance can
be determined. Conversely, it can also be used to find out the required ratio
of the conjugate acid-base pair of known pKa to obtain a buffer of desired pH.
The concept of buffers is elaborated in the following section.
2.6 DETERMINATION OF pKa

Titration leads to the establishment of pKa values. The free acid form of the
chemical entity is titrated with an appropriate base. The pH of the solution
is monitored as a strong base is added to the buffer solution. This titration
curve is recorded as equivalents of base on abscissa against pH as the ordinate.
Figure 2.2 shows the titration curve for CH3COOH. The point of inflection
indicates the pKa value. Polybasic buffer systems have more than one pKa value.
Figure 2.3 shows the titration curve for phosphoric acid, a tribasic acid. The
titration of phosphoric acid shows five inflection points where three signify
pKa , pKa and pKa , and the other two points indicate where H2PO4 and
1 2 3
HPO4 exist as the sole species.
2.7 BUFFERS
Cells must constantly maintain their pH in order to function properly. Enzymes,
the universal catalysts of living beings, function at an optimal pH and get
inactivated by extremes of pH. In biological systems, control of pH in solutions
is a very important part of the practice while working. The control of pH is
brought about by buffers. Buffers are pairs of related chemical compounds
capable of resisting large changes in pH of a solution caused by addition of
small quantities of acids and bases. A buffer system is composed of a weak
acid and its conjugate base or a weak base and its conjugate acid. The two
components of the system, the buffer pair, complement each other. When small
quantities of hydrogen ions are introduced into the medium, they will react
with the conjugate base or basic member of the buffer pair to form a weak acid.
The weak acid is only slightly ionized. In a similar manner, if small amounts
of hydroxide ions are added to the medium, it will react with the weak acid of
the buffer pair to form water and conjugate base. In this way large changes in
hydrogen ion concentrations are resisted.
Let us take the example of acetate buffer. Acetate buffer consists of
CH3COOH and CH3COONa. In solution, the dissociation of CH3COOH
will be given as
CH3COOH(aq) CH3COO –(aq) + H+(aq) 2.22
Since CH3COOH is a weak acid, it will not dissociate completely; thus, it has
a smaller ratio of ions (CH3COO – and H+) to uncharged acid (CH3COOH) in
this equilibrium and a small pKa value. Therefore, the equilibrium is said to
be shifted towards the left. The added CH3COONa completely dissociates into
ions in the solution. Therefore, according to Le Chatelier’s principle, this will
swing the position of the equilibrium even further to the left. Let us understand
how buffers resist large changes in pH of the solutions by considering the
following situations:
Acid is added to a solution containing acetate buffer The buffer solution
must remove most of the new hydrogen ions. Otherwise, the pH would drop
markedly. Hydrogen ions combine with the acetate ions to make CH3COOH.
Although the reaction is reversible, since CH3COOH is a weak acid, most of
the new hydrogen ions are removed in this way.
CH3COO – (aq) + H+(aq) CH3COOH(aq) 2.23
Since most of the new hydrogen ions are removed, the pH will not change to
a great extent.
When alkali is added to this buffer solution Alkaline solutions contain
hydroxide ions. There are two mechanisms discussed further in this section by
which these hydroxide ions are removed in a buffer solution.
(i) Removal of hydroxide ions by reacting with CH 3COOH In the
solution containing acetate buffer, CH3COOH molecules are available to
react with the added hydroxide ions. They will react to form acetate ions
and water.
CH3COOH(aq) + DH(aq) CH3COO –(aq) + H2O (I) 2.24

(ii) Removal of hydroxide ions by reacting with hydrogen ions In a
buffer solution there are some hydrogen ions present from the ionization
of the CH3COOH.
–
CH3COOH(aq) CH3COO(aq) + H+(aq) 2.25
Hydroxide ions can combine with these to make water. The equilibrium
shifts more towards right to replace them. This keeps on happening until most
of the hydroxide ions are removed.
Figure 2.2 Titration curve for CH3COOH
Figure 2.3 Titration curve for phosphoric acid

Equilibrium moves to replace

the removed hydrogen ions
CH3COOH (aq) CH3COOH–(aq) + H+(aq)
Hydroxide ions combine

with these to make water
Again, to maintain the equilibrium, not all of the hydroxide ions are removed.
Water that is formed re-ionizes to a very small extent to give a few hydrogen
ions and hydroxide ions.
E2.2 EXERCISE
A solution contains 0.05 M CH3COOH and 0.05 M CH3COONa. Calculate
the change in pH when 0.001 mole of HCl is added to a litre of the solution,
assuming that the volume increase upon adding HCl is negligible. Compare
this to the pH if the same amount of HCl is added to a litre of pure water.
Solution Initially, when there is no HCl added to the buffer the hydrogen
ions are contributed by the dissociation of CH3COOH. Since CH3COOH is a
weak acid, it dissociates only to a small extent to give acetate ions and hydrogen
ions. CH3COONa dissociates completely in solution to give acetate ions and
sodium ions. Therefore, according to Le Chatelier’s principle the ionization of
CH3COOH is further reduced due to common ion effect. The acid equilibrium
equation remains the same as follows:
+
[H ][A– ]
________
Ka =
[HA]
Since acetate ions contributed by CH3COONa are much higher than the
acetate ions contributed by CH3COOH, the concentration of acetate ions [A – ]
can be considered to be equal to the concentration of CH3COONa (0.05 M)
and the ions contributed by CH3COOH can be neglected. Moreover, since
CH3COOH dissociates only to a small extent and its dissociation is further
suppressed due to common ion effect, the total concentration of undissociated
CH3COOH [HA] can be considered to be constant (0.05 M).
Let ‘y’ be the hydrogen ion concentration in the buffer solution.
Thus,
Ka = y[0.05]/[0.05]
y = Ka
Thus,
Ka (CH3COOH) = 1.76 × 10 –5 mole/litre
pH = pKa = 4.75
When 0.001 M of HCl is added to the buffer solution, the added protons
from HCl combine with the acetate ions to form more CH3COOH, giving the
balanced equation of
A– + H+ Æ HA
[HA] = 0.050 + [H+]HCl = 0.051 mole/litre
[A– ] = 0.050 – [H+]HCl = 0.049 mole/litre
y(0.049)
Ka = ________
(0.051)
y = 1.76 × 10 –5 × [(0.051)/(0.049)] = 1.83 × 10 –5 mole/litre
pH = 5 – 0.26 = 4.74
In the presence of a buffer, the pH changes from 4.75 to 4.74, a difference

of only 0.01 units. In the absence of the buffer, if 0.001 moles of HCl is added
to 1 litre of water, the pH would be pH of 3. Therefore, it can be said that
the buffer resists large changes in pH.
2.7.1 Preparation of Buffers

Method for preparation of buffers is available as buffer tables in numerous
books. Also many websites have “buffer calculators” that help us to calculate
the quantities of buffering species required to prepare buffers of desired pH
and concentration. Henderson–Hasselbalch equation is the basis of computation in
such tables. The method is straightforward and has been exemplified as follows:
Let us prepare 250 ml of 0.05 M phosphate buffer at pH 7.5.
Remember Henderson–Hasselbalch equation (Equation 2.20)
[A– ]
_____
pH = pKa + log
[HA]
where A– and HA are the concentrations of basic and acidic components of the
buffer system, respectively. So, let us calculate the total ion concentrations of both
the components of the buffer system, that is for phosphate buffer, the components
are monobasic potassium phosphate and dibasic potassium phosphate. For 1000 ml
we require 0.05 moles of total ion concentrations in both the solutions. Therefore,
for 250 ml we would require
250 ml × 0.05 M/1000 ml = 0.0125 M
or 12.5 mM in both the solutions.
According to Henderson–Hasselbalch equation, for the phosphate buffer system

of pH 7.5 concentration ratio is given by
7.50 = 6.86 + log ([K 2HPO4]/[KH2PO4])

Therefore,
[K 2HPO4]/[KH2PO4] = 4.37
Let the concentration of KH 2PO4 in the buffer be ‘a’ molar and the
concentration of K 2HPO4 be ‘b’ molar which is equal to 4.37a. However, the
total ion concentration has to be 0.125 M. Therefore,
0.125 M = 4.37a + a
Converting to target buffer solution concentration and units,
a = 0.0233 moles of KH2PO4 = 0.0233 mol × 136.086 g/mol = 3.17 g
b = 0.1016 moles of K 2HPO4 = 0.1016 mol × 174.176 g/mol = 17.70 g
Thus, we need to weigh 3.17 g of monobasic phosphate and 17.6904 g of
dibasic phosphate in 250 ml of deionized water.
Note: It would be advisable to mix the two solutions in little less than the
desired volume and check the pH. If it is somewhat different, then the pH may
be adjusted by adding acid or base making the buffer of exact pH.
2.7.1.1 Buffer concentration

Buffers are effective when used at adequate concentration. Let us understand
this concept by considering the following example:
Consider two different buffer solutions A and B. Buffer A contains
0.01 M CH3COOH and 0.01 M CH3COONa whereas buffer B contains 0.1 M
CH3COOH and 0.1 M CH3COONa. These two buffer solutions were titrated
with HCl and the change in pH was calculated for a given amount of HCl
(assuming that the volume increase upon adding the HCl is negligible). The
results are tabulated in Table 2.3.
Table 2.3 Effect buffer concentration on buffer capacity

Moles of pH of buffer A [0.01 M CH3COOH and pH of buffer B [0.1 M CH3COOH and 0.1 M
HCl added 0.01 M CH3COONa] CH3COONa]
0.000 4.75 4.75
0.001 4.67 4.75
0.002 4.58 4.74
0.003 4.49 4.73
0.004 4.39 4.72
0.005 4.28 4.71
The pH of buffer A changes more rapidly as compared to the pH of buffer

solution B. Therefore, it can be said that increasing the buffer concentration
improves the ability of the buffer to resist the changes in pH. An adequate
buffer capacity is often reached at concentrations higher than 25 mM. Optimum
concentrations lie between 20 mM and 100 mM. However, ionic strength may
affect enzyme activity, therefore, caution has to be exercised.
2.7.1.2 Buffer capacity

The ability of a buffer system to resist pH changes is its buffer capacity and
indicated by the buffer index (b):
b = DB/DpH 2.26
where
B is the number of moles of strong base added and
DpH is the change in pH.
Buffer capacity is defined as the number of equivalents of strong base
(DB) required for a unit change in pH (DpH) in 1 litre of solution. With
the increase in the buffer capacity, the change in pH decreases when a given
amount of strong acid or base is added to it. Buffer capacity is dependent on
the total concentration of the buffer system and on the HA/A – ratio. The buffer
index number is generally experimentally derived in a manner like titration.
Buffer capacity is maximum when pH = pKa and is acceptable in the range
pH = pKa ± 1.
2.7.1.3 Effect of temperature on pH

Generally when we consider the use of buffers, we make the following two
assumptions:
1. The activity coefficient of the buffer ions is approximately equal to one
over the useful range of buffer concentrations.
2. The value of Ka is constant over the working range of temperature.
However, in real practice one observes that pH changes slightly with change
in temperature. This might be very critical in biological systems where a
precise hydrogen ion concentration is required for reaction systems to operate
with maximum efficiency. The difference might appear to be slight but it has
significant biological importance. Although the mathematical relationship of
activity and temperature may be complicated, the actual change of pKa with
temperature (DpKa /°C) is approximately linear.
2.7.1.4 Effects of buffers on factors other than pH

Buffer ions may interfere with the other components under study. For example,
citric acid and citrates are potential calcium chelators; therefore, citrate buffers
are regarded as unsuitable for studies involving calcium ions. If the system
under study contains phosphates, the phosphates react with calcium resulting in
production of insoluble calcium phosphate precipitates. Phosphate ions in buffers
may inhibit the activity of enzymes, such as carboxypeptidase, fumarease,
carboxylase, and phosphoglucomutase. Tris (hydroxy–methyl) aminomethane
can chelate copper and also acts as a competitive inhibitor of some enzymes.
Tris-based buffers are not recommended when studying the metabolic effects
of insulin. Moreover, buffers like Tris that contain primary amine groups may
interfere with the Bradford dye-binding method of protein assay. Buffers such
as ACES, BES, and TES have a tendency to bind copper. HEPES and HEPPS
buffers are not preferable when a protein assay is performed by using Folin
reagent. Borate buffers are not suitable for gel electrophoresis of protein; they
can cause spreading of the zones if polyols are present in the medium.
2.7.1.5 Contamination in buffers

Contamination in buffers can be prevented by (i) sterilization by filtration or
autoclaving and storage in sterile conditions, (ii) addition of 0.02% w/v sodium
azide, (iii) storage at +4°C, and (iv) use of highly concentrated stock solution.
2.7.2 Criteria for Selecting Buffers

(i) Buffer should possess sufficient buffering capacity in the desired
pH range Generally, buffers are most effective over a range of one
pH unit on either side of their pKa value. For example, Tris that has
a pKa value of 8.3 has an effective pH range of 7–9. If you expect the
pH to drop during the experiment, choose a buffer with a pKa slightly
lower than the working pH. This will permit the buffering action to
become more resistant to changes in [H+] as hydrogen ions are liberated.
Conversely, if you expect the pH to rise during the experiment, choose a
buffer with a pKa slightly higher than the working pH. For best results,
the pK a of the buffer should not be affected significantly by buffer
concentration, temperature, and the ionic constitution of the medium.
The pKa values of some important buffers have been given in Table 2.4.
(ii) Minimum effect of temperature and ionic concentration on
buffer pH The buffer pH should not vary considerably on change in
temperature or ionic concentration during the experiment.
(iii) Inert It should be chemically inert and should not react or bind with
biomolecules. Tris is known to be a biological inhibitor and reacts with
primary amines.
(iv) Purity It should be available in high degree of purity and should not
contain impurities that interfere with the analysis. “Buffers” prepared
from reagent grade chemicals may contain UV absorbing “impurities” that
interfere with UV liquid chomatographic detectors.
(v) Stable It should not undergo enzymatic or hydrolytic degradation. For
example some buffers are not stable with Osmium tetraoxide (OsO4) or
aldehydes at high concentrations.
(vi) Non-toxic Buffer should be tissue compatible if used as a rinse or
to store the tissue for any length of time. Otherwise, toxic effects
will be seen (dead or stressed tissue or autolysis before fixation). Tris
can penetrate biological membranes in its unionized form. Tris buffer
reacts with primary amines, and modifies electron transport and
phosphorylation in chloroplasts. Tris also inhibits respiratory enzymes
in mitochondria. Typical “vital buffers” include PBS, Tris, Hepes, and
Pipes.
(vii) No absortion of light This is very important as absorption of light
would interfere with spectrophotometric analysis.
2.7.3 Commonly Used Buffers

The pKa values of commonly used buffers are given in Table 2.4. The useful
pH range is within pKa ± 1 of the buffer.
2.7.4 Recipe of Commonly Used Buffers

The information provided below is intended only as a general guideline.
We strongly recommend the use of a sensitive pH meter with appropriate
temperature setting for final pH adjustment. Addition of other chemicals, after
adjusting the pH, may change the final pH value to some extent.
The buffer concentrations in the tables below are used only as examples. You
may select higher or lower concentrations depending upon your experimental
need.
1. Hydrochloric acid–Potassium chloride buffer (HCl–KCl); pH range 1.0–2.2
Table 2.4 pKa values of commonly used buffers

Buffer pK a Buffer pK a
H3PO4 / NaH2PO4 (pK a1) 2.12 Bicine 8.35
Glycine (pK a1) 2.34 Glycylglycine (pK a2) 8.40
Citric acid (pK a1) 3.13 TAPS 8.40
CH3COOH 4.75 Bis-Tris Propane (pK a2) 9.00
Citric acid (pK a2) 4.76 Boric acid (H3BO3 / Na2B4O7) 9.24
MES 6.15 CHES 9.50
Cacodylic acid 6.27 Glycine (pK a2) 9.60
H2CO3 / NaHCO3 (pK a1) 6.37 NaHCO3 / Na2CO3 (pK a2) 10.25
Citric acid (pK a3) 6.4 CAPS 10.40
Bis-Tris 6.50 Piperidine 11.12
ADA 6.60 Na2HPO4/Na3PO4 (pK a4) 12.67
Bis-Tris Propane (pK a1) 6.80 PIPES 6.80
ACES 6.90 Imidazole 7.00
BES 7.15 MOPS 7.20
NaH2PO4 / Na2HPO4 (pK a2) 7.21 TES 7.50
HEPES 7.55 HEPPSO 7.80
Triethanolamine 7.80 Tricine 8.10
Tris 8.10 Glycine amide 8.20

(a) 0.1 M KCl: 7.45 g/l (MW: 74.5)

(b) 0.1 M HCl
Mix 50 ml of KCl and indicated volume of HCl. Mix and adjust the final
volume to 100 ml with deionized water. Adjust the final pH using a pH meter.
ml of HCl 97 64.5 41.5 26.3 16.6 10.6 6.7

pH 1.0 1.2 1.4 1.6 1.8 2.0 2.2
2. Glycine–HCl buffer; pH range 2.2–3.6

(a) 0.1 M Glycine: 7.5 g/l (MW: 75)
(b) 0.1 M HCl
Mix 50 ml of glycine and indicated volume of HCl. Adjust the final volume
to 100 ml with deionized water. Adjust the final pH to desired pH using pH
meter.
ml of HCl 44.0 32.4 24.2 16.8 11.4 8.2 6.4 5.0

pH 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6
Citrate buffer; pH range 3.0–6.2

(a) 0.1 M citric acid: 19.21 g/l (MW: 192.1)
(b) 0.1 M sodium citrate dihydrate: 29.4 g/l (MW: 294)
Mix citric acid and sodium citrate solutions in the proportions indicated
and adjust the final volume to 100 ml with deionized water. Adjust the final
pH to desired value. The use of pentahydrate salt of sodium citrate is not
recommended.
ml of citric acid 46.5 40.0 35.0 31.5 25.5 20.5 16.0 11.8 7.2
ml of sodium citrate 3.5 10.0 15.0 18.5 24.5 29.5 34.0 38.2 42.8
pH 3.0 3.4 3.8 4.2 4.6 5.0 5.4 5.8 6.2
4. Acetate buffer; pH range 3.6–5.6

(a) 0.1 M CH3COOH (5.8 ml made to 1000 ml)
(b) 0.1 M CH3COONa; 8.2 g/l (anhydrous; MW: 82) or 13.6 g/l (trihydrate;
MW: 136)
Mix CH3COOH and CH3COONa solutions in the proportions indicated and
adjust the final volume to 100 ml with deionized water.
ml of CH3COOH 46.3 41.0 30.5 20.0 14.8 10.5 4.8

ml of CH3COONa 3.7 9.0 19.5 30.0 35.2 39.5 45.2
pH 3.6 4.0 4.4 4.8 5.0 5.2 5.6
Citrate–Phosphate buffer; pH range 2.6–7.0

(a) 0.1 M citric acid; 19.21 g/l (MW: 192.1)
(b) 0.2 M dibasic sodium phosphate; 35.6 g/l (dihydrate; MW: 178) or
53.6 g/l (heptahydrate; MW: 268)
Mix citric acid and sodium phosphate solutions in the proportions indicated
pH using a sensitive pH meter.
ml of citric acid 44.6 39.8 35.9 32.3 29.4 26.7 24.3 22.2 19.7 16.9 13.6 6.5
ml of sodium 5.4 10.2 14.1 17.7 20.6 23.3 25.7 27.8 30.3 33.1 36.4 43.6
phosphate
pH 2.6 3.0 3.4 3.8 4.2 4.6 5.0 5.4 5.8 6.2 6.6 7.0
Phosphate buffer; pH range 5.8–8.0

(a) 0.1 M sodium phosphate monobasic; 13.8 g/l (monohydrate, MW: 138)
(b) 0.1 M sodium phosphate dibasic; 26.8 g/l (heptahydrate, MW: 268)
Mix sodium phosphate monobasic and dibasic solutions in the proportions
indicated and adjust the final volume to 200 ml with deionized water. The
final pH is adjusted to the desired pH using pH meter.
ml of sodium phosphate 92.0 81.5 73.5 62.5 51.0 39.0 28.0 19.0 13.0 8.5 5.3
monobasic
ml of sodium phosphate 8.0 18.5 26.5 37.5 49.0 61.0 72.0 81.0 87.0 91.5 94.7
dibasic
pH 5.8 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0
Tris–HCl buffer, pH range 7.2–9.0

(a) 0.1 M Tris (hydroxymethyl) amino methane; 12.1 g/l (MW: 121)
(b) 0.1 M HCl
Mix 50 ml of Tris (hydroxymethyl) amino methane and indicated volume of
HCl and adjust the final volume to 200 ml with deionized water. Adjust the
final pH using a pH meter.
ml of HCl 44.2 414 38.4 32.5 21.9 12.2 5.0

pH 7.2 7.4 7.6 7.8 8.2 8.6 9.0
8. Glycine–Sodium hydroxide, pH range 8.6–10.6

(a) 0.1 M glycine; 7.5 g/l (MW: 75)
(b) 0.1 M sodium hydroxide; 4 g/l (MW: 40)
Mix 50 ml of glycine and indicated volume of sodium hydroxide solutions,
pH.
ml of sodium hydroxide 4.0 8.8 16.8 27.2 32.0 38.6 45.5

pH 8.6 9.0 9.4 9.8 10.0 10.4 10.6
9. Carbonate–Bicarbonate buffer, pH range 9.2–10.6

Special note for buffers containing sodium hydrogen carbonate (sodium
bicarbonate): this buffer substance requires a closed system. In aqueous
solutions, sodium hydrogen carbonate degrades into carbon dioxide (CO2)
and sodium carbonate above 20°C. Complete degradation occurs at 100°C.
Solutions containing sodium hydrogen carbonate cannot therefore be
autoclaved, but have to be sterile filtered. When preparing, they should not
be stirred too vigorously and too long. The pH of a freshly prepared 100 mM
solution is 8.3 at 25°C.
(a) 0.1 M sodium carbonate (anhydrous), 10.6 g/l (MW: 106)
(b) 0.1 M sodium bicarbonate, 8.4 g/l (MW: 84)
Mix sodium carbonate and sodium bicarbonate solutions in the proportions
indicated and adjust the final volume to 200 ml with deionized water. Adjust the
final pH.
ml of sodium carbonate 4.0 9.5 16.0 22.0 27.5 33.0 38.5 42.6
ml of sodium bicarbonate 46.0 40.5 34.0 28.0 22.5 17.0 11.5 7.5
pH 9.2 9.4 9.6 9.8 10.0 10.2 10.4 10.8
GOOD’S BUFFERS
Good buffers (Good’s buffers) are 12 buffering agents selected and described
by Norman E. Good and colleagues in 1966. The properties that make them
useful candidates for biochemical research are listed as follows:
• pKa values between 6–8 Since most biological reactions take place at near-
neutral pH.
• Good aqueous solubility and poor non-polar solubility For ease of
handling aqueous biological systems, and poor non-polar solvent solubility
to prevent the buffer from accumulating in cell membranes and other
non-polar compartments of the biological system.
• Membrane impermeability Buffers do not readily pass through cell
membranes and show a selective permeability reducing the accumulation
of the buffer compound within cells.
• Minimal salt effects Highly ionic buffers may cause problems or
complications in some biological systems.
• Well-behaved cation interactions If the buffers form complexes with
cationic ligands, the complexes formed should remain soluble. Ideally,
at least some of the buffering compounds will not form complexes.
• Stability The buffers should be chemically stable, resisting enzymatic and
non-enzymatic degradation.
• Optical absorbance Buffers should not absorb the visible or ultraviolet
spectrum so that it does not interfere with commonly used spectrophotometric
assays.
PHYSIOLOGICAL BUFFERS
All the physiological processes have to be carried out at a specific pH range
or the metabolic processes would be affected. The pH of human blood is
closely maintained at 7.4 and if the blood pH falls below 7 or rises to 7.8, it
may result in death within minutes. pH plays an important role in almost
all biological processes. Small change in pH that is a decrease or an increase
can cause metabolic acidosis or alkalosis. Metabolism is usually associated
with the release of protons (H+) (decrease in pH) or uptake of protons (H+)
(increase in pH). Presence of physiological buffers ensures the maintenance
of physiological pH. Important buffers that are dominant in human body are:
(i) Bicarbonate buffers, (ii) Phosphate buffers, and (iii) Protein buffers.
Contd...
E2.3 EXERCISE
1. Determine the molarity of a solution made by dissolving 20 g of NaOH
in sufficient water to yield a 482 cm3 solution.
2. If the 0.131 M sugar solution has a density of 1.10 g/ml, what is the
concentration in mole percentage?
3. How much NaOH is contained in 25 ml of a solution whose concentration
is 0.1234 M?
4. Neeta dissolved 13 g of NaCl in water to make 2 litre of aqueous solution.
Calculate the molarity.
5. A solid mixture contains 300 kg NaCl and 400 kg KCl. Find the
composition of mixture in (i) mass per cent and (ii) mole per cent.
6. The amount of oxygen dissolved in 1000 g fermentation medium was
determined to be 0.007 g. Calculate the dissolved oxygen concentration
of medium in ppm.
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Figure 79. De Konick’s Apparatus.
467. DeKonick’s Modification of Schloesing’s

Method.—This modification consists in an
arrangement of the gas delivery-tube, whereby the
regurgitation of the water in the measuring burette into
the evolution flask is prevented by a device for sealing
the delivery-tube with mercury.[299] The apparatus is
arranged as shown in Fig. 79. The flask in which the
Figure 80. decomposition takes place is provided with a long neck,
End of
Delivery-
into which a side tube is sealed and bent upwards,
Tube. carrying a small funnel attached to it by rubber tubing.
The piece of rubber tubing carries a pinch-cock, by
means of which the solution containing the nitrate and
hydrochloric acid can be introduced into the flask. The small gas
delivery-tube is arranged as shown in the figure, and carries at the
end next the burette a device shown in Fig. 80. The cork represented
in this device has radial notches cut in it, so as to permit of a free
communication between the water in the burette and in the
pneumatic trough. The open end of the burette, when the apparatus
is mounted ready for use, rests on the notched surface of the cork,
and the end of the delivery-tube is placed in the crystallizing dish
resting on the bottom of the pneumatic trough.
The end of the delivery-tube, as indicated, has fused onto it a
vertical tube open at both ends and six to seven centimeters in
length, and carrying the notched cork already described. The
crystallizing dish in the bottom of the pneumatic trough is filled with
mercury until the point of union of the delivery-tube with the vertical
end is sealed to the depth of a few millimeters. As the gas is evolved
it bubbles up through the mercury into the measuring tube and the
displaced water passes out through the notches in the cork. Should
any back pressure supervene the mercury at once rises in the
delivery-tube which is of such a length as to prevent its entrance into
the flask. The operation can then be carried on with absolute safety.
To make an estimation there are placed in the flask about forty
cubic centimeters of ferrous chlorid solution containing about 200
grams of iron to the liter, and also an equal volume of hydrochloric
acid of one and one-tenth specific gravity. The side tube is also filled
up to the funnel with the acid. The contents of the flask are boiled
until all air is expelled, which can be determined by holding a test-
tube filled with water over the end of the delivery-tube. The solution
containing the nitrate is next placed in the funnel, the pinch-cock
opened and the liquid allowed to run into the flask by means of the
partial vacuum produced by stopping the boiling and allowing the
mercury to rise in the delivery-tube. All the solution is washed into
the flask by successive rinsings of the funnel with hydrochloric acid,
being careful to allow no bubble of air to enter. The contents of the
flask are again raised to the boiling-point and the nitric oxid evolved
collected in the nitrometer. The solution examined should contain
enough nitrate to afford from sixty to eighty cubic centimeters of gas.
Without refilling the flask, from eight to nine determinations can be
made by regenerating the ferrous chlorid by treatment with zinc
chlorid. Care must be exercised not to add the zinc chlorid in excess,
otherwise ammonia and not nitric oxid will be produced. The side
tube and funnel must also be carefully freed from zinc chlorid by
washing with hydrochloric acid.
468. Schmidt’s Process.—In the case of a water, or the aqueous
extract of a soil, according to the content of nitric acid, from fifty to
one hundred cubic centimeters are evaporated to thirty cubic
centimeters, and the residue sucked into the generating flask of the
apparatus, Fig. 81, and, with the rinsings with distilled water,
evaporated again to from twenty to thirty cubic centimeters, and the
flask then connected, as shown in the figure, to a Schliff measuring
apparatus, B.[300] This apparatus is previously filled to i with
mercury, and the bulb g connected with k by a rubber tube.
The apparatus is then
filled with a twenty per
cent, previously boiled
and still warm, caustic
soda solution until the
bulb g is partially filled
when raised a little above
the cock h. Then h is
closed and g held, by an
appropriate support, on
about the same level with
h. The cock at b is then
closed and e opened.
Meanwhile the ebullition
in the flask is continued,
and the air bubbles rising
in the Schliff apparatus
are removed, from time
Figure 81. Schmidt’s Apparatus.
to time, by carefully
opening h and raising g.
When bubbles no longer come over, the cock at e is closed and at b
opened, and the steam issuing at a is conducted through a mixture of
ferrous chlorid and strong hydrochloric acid to free it, as far as
possible, from air. When the contents of the flask have been
evaporated to about five cubic centimeters, b is closed and the lamp
at once removed.
By carefully opening b about ten cubic centimeters of a mixture of
ferrous chlorid and hydrochloric acid are allowed to enter the flask,
when b is closed and the flask slowly heated until the positive
pressure is restored. The pinch-cock e is then opened and the
contents of the flask evaporated nearly to dryness. The cock e is
again closed and the flame removed. Another quantity (fifteen cubic
centimeters) of ferrous chlorid and hydrochloric acid solution is
sucked into the flask and the process of distillation repeated,
whereby the whole of the nitric oxid is collected in h. The nitric oxid
evolved is measured in the usual way and calculated to nitric acid,
one cubic centimeter of nitrogen dioxid being equal to 2.417
milligrams of nitric acid.
469. Merits of the Ferrous Chlorid Process.—The possibility
of an accurate determination of nitrates; by decomposition with a
ferrous salt in presence of an excess of acid, has been established by
many years of experience and by the testimony of many analysts. The
method is applicable especially where the quantity of nitrate is not
too small and when organic matter is present. In the case of minute
quantities of nitrate, however, the process is inapplicable and must
give way to some of the colorimetric methods to be hereafter
described.
In respect of the apparatus modern practice has led to the
preference of that form which does not require the use of carbon
dioxid for displacing the air. Steam appears to be quite as effective as
carbon dioxid and is much more easily employed. That form of
apparatus should be used which is the simplest in construction and
has the least cubical content.
The measurement of the evolved gas is most simply made by
collecting over lye in an azotometer, reading the volume, noting the
reading of the barometer and thermometer and then reducing to
standard conditions of pressure and temperature by the customary
calculations. Where a very strong lye is used the tension of the
aqueous vapor may be neglected. While every analyst should have a
thorough knowledge of the ferrous chlorid method and the principles
on which it is based it can not be compared in simplicity to the later
methods with pure nitrates which are based on the conversion of the
nitric acid into ammonia by the action of nascent hydrogen. In
accuracy, moreover, it does not appear to have any marked
advantage over the reduction methods.
470. Mercury and Sulfuric Acid Method.—This simple and
accurate method of determining nitric acid in the absence of organic
matter is known as the Crum-Frankland process.[301]
The method rests on the principle of converting nitric acid into
nitric oxid by the action of mercury in the presence of sulfuric acid.
The operation as at first described is conducted in a glass jar eight
inches long by one and a half inches in diameter filled with mercury
and inverted in a trough containing the same liquid. The nitrate to be
examined, in a solid form, is passed into the tube together with three
cubic centimeters of water and five of sulfuric acid. With occasional
shaking, two hours are allowed for the disengagement of the gas,
which is then measured.
471. Warington’s Modification.—A graduated shaking tube is
employed which allows the nitrate solution and oil of vitriol to be
brought to a definite volume. The nitrate solution, with rinsings, is
always two cubic centimeters and enough sulfuric acid is added to
increase the volume to five cubic centimeters. The sulfuric acid
should give no gas when shaken with distilled water. Any gas given
off in the apparatus before shaking, is not expelled but is included in
the final result. The persistent froth sometimes noticed where some
kinds of organic matter are present, is reduced by the addition of a
few drops of hot water through the stop-cock of the apparatus. The
nitric oxid is finally measured in Frankland’s modification of
Regnault’s apparatus.
This method, accurate for pure nitrates, unfortunately fails in the
presence of any considerable amount of organic matter.
According to Warington’s observations the presence of chlorids is
no hindrance to the accurate determination of both nitric and nitrous
acids by the mercury method. This simplifies the operation as carried
on by Frankland who directs that any chlorin present, be removed
before the determination of the nitric acid is commenced.
472. Noyes’ Method.—In the analyses made by Noyes for the
National Board of Health, the Crum-Frankland method was
employed.[302] The apparatus used was essentially that which is now
known as Lunge’s nitrometer and it will be described in the next
paragraph. No correction is made by Noyes for the tension of
aqueous vapor in the measurement of the nitric oxid because of the
moderate dilution of the sulfuric acid by the liquid holding the nitric
compounds in solution. The chlorin was not removed from the dry
residue of the evaporated water as its presence in moderate quantity
does not interfere with the accuracy of the process. In order to obtain
the amount of nitrogen in the form of nitrates, the total volume of
nitric oxid must be diminished by that due to nitrites present, which
must be determined in a separate analysis. The method of
manipulation is given in the following paragraph.
473. Lunge’s Nitrometer.—The apparatus
employed by Noyes, in a somewhat more elaborate
form, is known as Lunge’s nitrometer.[303] This
apparatus is shown in Fig. 82. It consists of a burette,
a, divided into one-fifth cubic centimeters. At its
upper end it is expanded into a cup-shaped funnel
attached by a three-way glass stop-cock. Below, the
burette is joined to a plain tube, b, of similar size, by
means of rubber tubing. The apparatus is first filled
with mercury through the tube b, the stop-cock being
so adjusted as to allow the mercury to fill the cup at
the top of a. The cock is then turned until the mercury
in the cup flows out through the side tube carrying the
rubber tube and clamp. The three-way cock is closed,
and the solution containing the nitrate placed in the
cup. By lowering the tube b and opening the cock the
liquid is carefully passed into a, being careful to close
the cock before all the liquid has passed out of the
cup. By repeated rinsings with pure concentrated
sulfuric acid, every particle of the nitric compound is
finally introduced into a, together with a large excess
of sulfuric acid. The total volume of the introduced
liquid should not exceed ten cubic centimeters. The
Figure 82. Lunge’s Nitrometer. mixture of the mercury, nitric compound, and sulfuric
acid is effected by detaching a from its support,
compressing the rubber connection between a and b,
placing a nearly in a horizontal position, and quickly bringing it into a vertical position with
vigorous shaking.
After about five minutes the reaction is complete, and the level of the liquids in the two
tubes is so adjusted as to compensate for the difference in specific gravity between the acid
mixture in a and the mercury in b; in other words, the mercury column in b should stand
above the mercury column in a one-seventh of the length of the acid mixture in a. This
secures atmospheric pressure on the nitric oxid which has been collected in a. The measured
volume of nitric oxid should be reduced to 0° and 760 millimeters barometric pressure. Each
cubic centimeter of nitric oxid thus obtained corresponds to 1.343 milligrams NO; 2.417
milligrams N₂O₅; 4.521 milligrams KNO₃; 1.701 milligrams N₂O₃; 2.820 milligrams HNO₃;
and 3.805 milligrams NaNO₃.
474. Lunge’s Improved Apparatus.—Lunge has lately improved his apparatus for
generating and measuring gases and extended its applicability.[304] The part of it designed to
measure the volume of a gas is the same in all cases. For generating the gas, the apparatus
varies according to the character of the substance under examination.
The measuring apparatus is shown in Fig. 83. It is composed essentially of three tubes,
conveniently mounted on a wooden holder with a box base for securing any spilled mercury.
The support is not shown in the illustration.
The tubes A, B, C, are mutually connected by means of a three-way tube and rubber tubing
with very thick walls to safely hold the mercury without expansion. In the middle of the
measuring tube A, is found a bulb of seventy cubic centimeters capacity. Above and below the
bulb the tube is divided into tenths of a cubic centimeter, and its diameter is such, viz., 11.3
millimeters, that each cubic centimeter occupies a length of one centimeter. The upper end of
A is closed with a glass cock with two oblique perforations, by means of which
communication can be established at will, either through e with the apparatus for generating
the gas, or through d with the absorption apparatus, or the opening be completely closed.
The volume of air under the observed conditions which would
measure exactly 100 cubic centimeters at 0° and 760
millimeters pressure of mercury, is calculated by the formula
V = 100(273 + t)760
273(b − f) ;
where t equals observed temperature, b the barometric pressure

less the correction noted above and f the tension of the vapor of
water under existing conditions. For example:
Figure. 83.
Lunge’s Improved Apparatus.
Let the temperature be 18°

Barometric reading 755
Correction for t 2
Corrected barometer 753
Vapor of water tension 16
Then V = 100(273 + 18)760

273(753 − 16) = 109.9.
This indicates that 109.9 cubic centimeters of air would occupy a volume of 100 cubic
centimeters when subjected to standard conditions.
The tubes A, B, and C are filled with mercury of which about two and a half kilograms will
be required. By means of the leveling tube B, the stopper in C being opened, the mercury in C
is brought exactly to 109.9 cubic centimeters. The stopper in C is then closed, mercury
poured into D, which is then closed with a rubber stopper, carrying a small glass tube as
indicated in the figure.
The leveling tube B serves to regulate the pressure on the gas in A and this is secured by
depressing or elevating it as the case may require.
The tube for reducing the volume to standard conditions of temperature and pressure, viz.,
0° and 760 millimeters of mercury, is shown in C. In its narrow part which has the same
internal diameter as A it is graduated into tenths of a cubic centimeter. The upper end of C is
furnished with a heavy glass neck D surmounted by a glass cup. In the neck is placed a
ground-glass stopper, carrying a groove below, which corresponds to a similar groove above
in the side of the neck whereby communication can be established at will between the interior
of C and the exterior. The joint is also sealed by pouring mercury into D as is shown in the
figure. When the stopper is well ground and greased the reduction tube may be raised or
lowered as much as may be necessary without any danger of escape or entrance of gas. To
determine the position of the reduction tube C the reading of the barometer and
thermometer at room temperature is taken. From the reading of the barometer subtract one
millimeter if the temperature be below 12°, two millimeters at a temperature from 12° to 19°,
three from 20° to 25°, and four above 25°.
When a gas has been introduced into the measuring tube A it is brought to the volume
which it would assume under standard conditions by adjusting the tube C in such a way as to
bring the level of mercury in C and A to the same point and the level of the mercury in C is
exactly at 100 cubic centimeters. The gas in A is then at the volume which it would occupy
under standard conditions and this volume can be directly read. This adjustment is secured
by moving the tubes B and C up or down. If gases are to be measured wet, a drop of water
should be put on the side of the upper part of C, and, if dry, of sulfuric acid, before the
adjustment for temperature and pressure.
475. Method of Manipulation.—By the action of mercury in the presence of sulfuric
acid, the nitrogen in nitrates, nitrites, nitrosulfates, nitroses, nitrocellulose, nitroglycerol, and
the greater number of explosives, may be obtained and measured as nitric oxid. The nitrogen
compounds are decomposed in the apparatus shown in Fig. 84.
To make an analysis, the apparatus is filled with mercury, through F, until the two
openings in the cock and i are entirely occupied with that liquid. The cock h is then closed,
and the nitrogen compound, in solution, introduced through g, care being taken that no air
enters g when F is depressed and h opened to admit the sample. The funnel g is washed
several times with a few drops of sulfuric acid, which are successively introduced into G. The
total liquid introduced should not exceed ten to fifteen cubic centimeters, of which the
greater part should be sulfuric acid. The rubber tube connecting G and F is carefully closed
with a clamp and G violently shaken for a few minutes until no further evolution of nitric oxid
takes place. In shaking, the apparatus should be so held as to prevent the escape of the
mercury from the small tube i by keeping it closed with the finger or drawing over it a rubber
cap.
After the evolution of the gas has ceased, the tube e, Fig. 83, is brought into contact with i,
Fig. 84, and the two are joined by a tight-fitting piece of rubber tubing in such a way as to
exclude any particle of air. The tube F, Fig. 84, is lifted and B and C, Fig. 83, depressed. On
carefully opening the cocks h and b and bringing i and e into union, the gas is passed from G
into A. When all the gas has entered A and the acid mixture from G has reached b the latter is
closed, and also h. The apparatus G is disconnected and removed. The gas in A is then
reduced to normal conditions by manipulating the reduction tube C in the manner already
described.
The gas in A is measured dry by reason of having been generated in presence of rather
strong sulfuric acid. Consequently, for this operation the adjustment of the volume of gas in C
should be made in contact with a drop of strong sulfuric acid. In order to make the readings,
a quantity of material must be taken which will give less than thirty or from 100 to 140 cubic
centimeters of nitric oxid.
The quantities of the different compounds of nitric acid corresponding
to the number of cubic centimeters of nitric oxid, measured under
standard conditions, are shown in the following table:
Figure 84.
Lunge’s Analytic
Apparatus.
CORRESPONDING TO
———————————— ———————————— ————————————
Cubic Weight in N₂O₃ in milligrams. HNO₃ in milligrams. NaNO₃ in milligrams.
centimeters milligrams.
of NO.
1 1.343 1.701 2.820 3.805
2 2.682 3.402 5.640 7.610
3 4.029 5.103 8.460 11.415
4 5.372 6.804 11.280 15.220
5 6.715 8.506 14.100 19.025
6 8.058 10.206 16.920 22.830
7 9.401 11.907 19.740 26.635
8 10.744 13.608 22.560 30.440
9 12.087 15.309 25.380 34.245
476. Utility of the Method.—Where it is desirable that the nitric oxid method be used,
and at the same time heating be avoided, the decomposition of a nitrate by means of metallic
mercury and sulfuric acid affords a convenient and accurate procedure. But, as a rule, there is
no objection to the application of the lamp, and in such cases the mercury method appears to
have no advantage over the ferrous chlorid process. Nevertheless, in the hands of a skilled
worker the results are reliable, and the process is a quicker one, on the whole, than by
distillation with ferrous chlorid and hydrochloric acid. This method, however, can not be
recommended as in any way superior to the reduction methods to be hereinafter described.
ESTIMATION OF NITRIC ACID BY OXIDATION OF A
COLORED SOLUTION.
477. Method of Boussingault.—The process for the estimation of nitric acid by the
decoloration of a solution of indigo is due originally to Boussingault.[305] In this method the
extract, obtained by washing slowly 200 grams of soil until the filtrate amounts to 300 cubic
centimeters, is evaporated until its volume is no greater than two or three cubic centimeters,
and it is transferred to a test-tube, with washings, and again evaporated in the tube until the
volume is not greater than that last mentioned. A few drops of solution of indigo are added,
and then two cubic centimeters of pure hydrochloric acid; the whole is then heated. As the
color of the indigo disappears more is added. When the color ceases to fade, the liquid in the
test-tube is concentrated by boiling. If concentration fail to destroy the blue or green color,
another one-half cubic centimeter of hydrochloric acid is introduced. The reaction is
completed when neither concentration nor fresh addition of hydrochloric acid destroys the
excess of indigo present. The color produced by a small excess of indigo is a bright sap-green;
this tint is the final reaction sought. The small excess of indigo necessary to produce a green
color is deducted in every experiment.
When more than mere traces of organic matter are present, Boussingault advises that the
nitric acid be first separated by distillation and then reduced by the indigo solution. For this
purpose the concentrated solution of the nitrate, two or three cubic centimeters, is placed in a
small tubulated retort with two grams of manganese dioxid in fine powder. The retort is next
half filled with fragments of broken glass, over which is poured one cubic centimeter of
concentrated sulfuric acid. The retort is heated carefully by means of a small flame, which is
kept in motion so as to successively come in contact with all parts of the bottom of the retort.
The distillate is received in a graduated test-tube which is kept cool. The distillation is
continued until the vapors of sulfuric acid begin to appear. The apparatus is allowed to cool,
the stopper of the retort removed, two cubic centimeters of water introduced, and the
distillation again made until fumes of sulfuric acid are again seen. The distillation with water
is made twice in order to remove every trace of nitric acid from the retort. The distillate is
neutralized with a solution of potassium hydroxid and concentrated to two cubic centimeters,
and the nitric acid estimated in the manner already described. The manganese dioxid used
should be previously well washed and the sulfuric must be free of nitric acid.
Preparation of the Indigo Solution.—Fifty grams of indigo in fine powder are digested for
twenty-four hours, at 40°, in a liter of distilled water. The water is then poured off and
replaced with a fresh supply. After the second decantation the residue is treated with 750
cubic centimeters of equal parts of water and pure concentrated hydrochloric acid and boiled
for an hour. After cooling, the undissolved portion is collected on a filter and washed at first
with hot, and afterwards with cold water, until the filtrate is no longer colored and is free of
acid. The dried residue is treated with ether under a bell-jar, or in a continuous extraction
apparatus, until the ether is only of a faint blue tint. The fifty grams of indigo at first taken
will give about twenty-five grams of the purified article, which, however, will still leave a little
ash on combustion.
Solution in Sulfuric Acid.—Five grams of the purified indigo are placed in a flask having a
ground-glass stopper, treated with twenty-five grams of fuming sulfuric acid, and allowed to
digest two or three days at a temperature of from 50° to 60°. From seventy to 200 drops of
the solution thus made are placed in 100 cubic centimeters of water for use in the process.
Standardization of the Indigo Solution.—The solution as prepared above is standardized
by a solution of one gram of pure potassium nitrate in 1,000 cubic centimeters of distilled
water. The oxidation of the indigo solution is accomplished as described above. For this
strength of standard nitrate solution two cubic centimeters are taken corresponding to two
milligrams of potassium nitrate. The indigo solution for this strength should have only
twenty drops of the sulfuric acid solution of indigo to 100 cubic centimeters of water. If
twenty grams of potassium nitrate are taken for 1,000 cubic centimeters of the standard
solution then 200 drops of the sulfindigotic acid should be used to 100 cubic centimeters of
water.
478. Method of Marx.—As usually practiced, the indigo method is conducted according
to the variation described by Marx.[306] There are required for the process the following
reagents and apparatus:
a. A solution of pure potassium nitrate containing 1.8724 grams per liter. One cubic
centimeter of the solution is equivalent to one milligram of nitric anhydrid (N₂O₅).
b. A solution of the best indigo carmine in water which should be approximately
standardized by solution in the manner described hereafter, and then diluted so that six to
eight cubic centimeters equal one milligram of nitric acid.
c. Chemically pure sulfuric acid of specific gravity 1.842, perfectly free from sulfurous and
arsenious acids and nitrogen oxids.
d. Several thin flasks of about 200 cubic centimeters capacity.
e. A small cylindrical measure holding fifty cubic centimeters and divided into cubic
centimeters.
f. A Mohr’s burette divided into tenths of a cubic centimeter.
g. A twenty-five cubic centimeter pipette or another burette.
h. A five cubic centimeter pipette divided into cubic centimeters or half cubic centimeters.
i. A measuring flask of 250 cubic centimeters capacity.
Preliminary Trial.—Twenty-five cubic centimeters of the sample are transferred to a flask;
the fifty cubic centimeter measure is filled with sulfuric acid and the burette with indigo
solution. The sulfuric acid is added to the sample all at once, shaken for a moment, and the
indigo run in as quickly as possible with shaking until a permanent greenish tint is produced.
If the sample do not require more than twenty cubic centimeters of indigo solution of the
above strength, it can be titrated directly, otherwise it must be diluted with a proper quantity
of pure water, and subjected again to the preliminary trial.
The Actual Titration.—(1) Twenty-five cubic centimeters of the sample properly diluted if
necessary, are measured and poured into a flask, and as much indigo as was used in the
preliminary trial, is added; a quantity of sulfuric acid, equal in volume to the liquid in the
flask, is added all at once, the mixture shaken, and indigo solution run in quickly out of the
burette until the liquid remains permanently of a greenish tint.
(2) The last experiment is repeated as often as may be necessary adding to the water at first
half a cubic centimeter less indigo than the total quantity used previously, afterwards
proceeding as in (1) until the final test shows too little indigo used.
(3) From the rough titration of the indigo, calculate the amount of potassium nitrate
solution corresponding with the indigo solution used in (2), multiply the result by ten,
transfer this quantity of the standard nitrate solution to a 250 cubic centimeter flask, fill with
pure water to the mark, and titrate twenty-five cubic centimeters of this fluid with indigo as
in (2). If the quantity of indigo solution used is nearly the same as that required in (2), its
exact value may be calculated, but if it is not, another nitrate solution may be made up in the
250 cubic centimeter flask, more closely resembling the sample in strength, and the titration
with the indigo solution must be repeated.
(4) If the water contain any considerable amount of organic matter, it must first be
destroyed by potassium permanganate. In this case, the estimation of the organic matter and
nitric acid may be conveniently combined.
The use of permanganate in the above case is likely to introduce an error as has been
shown by Warington. The method therefore can not be recommended in the presence of
organic matter.
479. Method of Warington.—The modification of the indigo method as used by
Warington, applicable only in absence of organic matter, is the one chiefly employed in
England.[307]
Instead of the ordinary indigo of commerce, indigotin is used. The normal solution of the
coloring matter is made of such a strength as to be equivalent to a solution of potassium
nitrate containing 0.14 gram of nitrogen per liter. Where large quantities of the coloring
matter are to be used it is advisable to prepare it about four times the strength given above
and then dilute it as required. Four grams of sublimed indigotin will furnish more than two
liters of the color solution.
The solution is prepared as follows:
Four grams of indigotin are digested for a few hours with five times that weight of
Nordhausen sulfuric acid, diluted with water, filtered, and made up to a volume of two liters.
The strength of the indigotin solution is determined with a solution of potassium nitrate of
the strength mentioned above. The process is performed as follows:
From ten to twenty cubic centimeters of the standard nitrate solution are placed in a wide-
mouthed flask of about 150 cubic centimeters capacity. A portion of the indigotin solution is
next added, such as will be deemed sufficient for the process, and the whole is well mixed.
Strong sulfuric acid is next measured out from a burette into a test-tube, in volume equal to
the united volumes of the nitrate solution and indigotin. The whole of the sulfuric acid is then
poured as quickly as possible, into the solution in the flask and rapidly mixed, and the flask
transferred to a calcium chlorid bath, the temperature of which should be maintained at 140°.
It is essential to the success of the operation that the sulfuric acid should be mixed with the
greatest rapidity. It should be poured in at once and the whole well shaken without waiting
for the test-tube containing the acid, to drain. The flask should be covered by a watch-glass
while it is held in the bath. As soon as the larger part of the indigotin is oxidized the flask in
the bath should be gently rotated. With very weak solutions of nitrate it may be necessary
sometimes to keep the flask in the bath for five minutes. When the indigo color is quickly
discharged it shows the presence of nitric acid in considerable excess and a considerably
larger quantity of indigo must be taken in the next experiment. The experiments are
continued until just the quantity of indigo necessary to consume the nitric acid is taken, the
amount of indigo being in very slight excess, not exceeding one-tenth cubic centimeter of the
indigo solution used. The tint produced by the small excess of indigo remaining is best seen
by filling the flask with water. On substances of approximately known strength about four
experiments are usually necessary to determine the amount of indigo to be taken, but with
unknown substances a larger number may be necessary.
Usually in determinations of this kind it is directed to use double the volume of sulfuric
acid mentioned above. In this case not only is the quantity of indigo oxidized much greater
than with a smaller portion of acid, but the prejudicial effect of organic matter is also greater
than when the smaller quantity of acid is employed.
An indigo solution standardized as above is strictly to be used for a solution of nitrate of
the strength employed during the standardization. The quantity of indigo oxidized in
proportion to the nitric acid present diminishes as the nitrate solution becomes more dilute.
Instead of determining this during each series of experiments it may be estimated once for all
and a table of corrections used.
The following table is based upon experimental determinations:
Strength of Indigo Difference Nitrogen Difference Difference in the
niter solution required, between corresponding between the nitrogen values for a
used. cubic amounts of to one cubic nitrogen difference of one cubic
centimeters. indigo, cubic centimeter of values, gram. centimeter in the
centimeters. indigo, gram. amount of indigo, gram.
⁸⁄₆₄ Normal 10.00 0.000035000
⁷⁄₆₄ „ 8.71 1.29 0.000035161 0.000000161 0.000000125
⁶⁄₆₄ „ 7.43 1.28 0.000035330 0.000000169 0.000000132
⁵⁄₆₄ „ 6.14 1.29 0.000035627 0.000000298 0.000000231
⁴⁄₆₄ „ 4.86 1.28 0.000036008 0.000000381 0.000000298
³⁄₆₄ „ 3.57 1.29 0.000036763 0.000000756 0.000000586
²⁄₆₄ „ 2.29 1.28 0.000038209 0.000001445 0.000001129
¹⁄₆₄ „ 1.00 1.29 0.000043750 0.000005541 0.000004295
The table is used as follows:

Suppose that twenty cubic centimeters of water under examination have required 5.36
cubic centimeters of indigo solution for the oxidation of the nitric acid contained therein. By
inspection of the table it is seen that this number is five-tenths cubic centimeter above the
nearest quantity given; viz., 4.86 cubic centimeters. From the last column in the table it is
found that the correction for five-tenths cubic centimeter of indigo solution is 0.000000149
cubic centimeter, being half that for the one cubic centimeter given in the table. This is to be
subtracted from the unit value in nitrogen given in the first “gram” column of the table; viz.,
0.000036008. It is thus seen that the 5.86 cubic centimeters of indigo solution are
equivalent to 0.000035859 gram of nitrogen per cubic centimeter. The water under
examination, therefore, contains nine and six-tenths parts of nitrogen as nitric acid per
million.
Attention must also be paid in standardizing indigo solutions to the initial temperature of
the solutions. A rise in the initial temperature will be attended by a diminution in the
quantity of indigo oxidized. Experiments with a room temperature of 10° and a room
temperature of 20°, being the initial temperatures of the experiments, showed that at the
higher temperature the amount of indigo consumed was about five per cent less when the
strong solutions of nitrate were employed. The indigo solution should, therefore, be
standardized at the same temperature at which the determinations are made.
If twenty cubic centimeters of the standard nitrate solution employed be used in setting the
indigo solution, this standard will enable the operator to determine nitric acid up to 17.5
parts of nitrogen per million in water or soil extracts.
The presence of an abundance of chlorids in the water under examination tends to
diminish the content of nitric acid found, and also tends to introduce an error, which is
sometimes of a plus and sometimes of a minus quantity, according to the strength of the
nitric acid present. The reaction is shortened in weak solutions by the presence of chlorids,
and the quantity of indigo consumed is consequently increased. The error introduced by
chlorids is usually of an insignificant nature.
On account of the interference of organic matters with the reaction of indigo it is not of
much use in the examination of nitrates washed out of soils, although in some cases the
results may be quite accurate. This method must, therefore, be considered as applicable, in
general, to waters or soil extracts which contain little or no organic matter.
In analytical work pertaining particularly to agriculture, the use of the indigo method for
determining nitric acid has been largely employed, both in the analyses of soil extracts and
drainage and irrigation waters. The method, however, can hardly survive as an important one
in such work in competition with more modern and speedy processes of analysis.
DETERMINATION OF NITRIC NITROGEN BY REDUCTION
TO AMMONIA.
480. Classification of Methods.—When nitrogen is present in a highly oxidized state,
e. g., as nitric acid, it may be quickly and accurately estimated by reduction to ammonia. This
action is effected by the reducing power of nascent hydrogen, and this substance may be
secured in the active state by the action of an acid or alkali on a metal, or by means of an
electric current. The processes depending on the use of a finely divided metal in the presence
of an acid or alkali have come into general use within a few years, and are now employed
generally instead of the more elaborate estimations depending on the use of copper oxid or
indigo.
The typical reaction which takes place in all cases is represented by the following equation:
2HNO₃ + 8H₂ = 2NH₃ + 6H₂O.
The method will be considered under three heads; viz., 1. Reduction in an alkaline solution;
2. Reduction in an acid solution; 3. Reduction by means of an electric current.
In the first class of processes the reduction and distillation may go on together. In the
second class the reduction is accomplished first and the distillation effected afterwards, with
the addition of an alkali. In the third class of operations the reduction is accomplished by
means of an electric current and the ammonia subsequently obtained by distillation, or
determined by nesslerizing. These processes may be applied to rain and drainage waters, and
to soil extracts. On account of the ease with which the analyses are accomplished, the short
time required and the accuracy of the results, the reduction methods for nitrates have already
commended themselves to analysts, and are quite likely to supersede all others for practical
use where weighable quantities of nitrates are present. For the minute traces of nitrates
found in rain and drainage waters, and in some soil extracts, the reduction method may also
be applied, but in these cases the ammonia which is formed must be determined by
colorimetry (nesslerizing) and not by distillation. The processes about to be described are
especially applicable to the examination of soils and waters rich in nitrates.
REDUCTION IN ALKALINE SOLUTIONS.
481. Provisional Method of the Association of Official Agricultural Chemists.
[308]
—
Extraction of the Nitrates.—Place one kilogram of the dried soil, calculated to water-free
substance, on a percolator of glass or tin. Moisten the soil thoroughly with pure distilled
water, and allow to stand for half an hour. Add fresh portions of pure distilled water until the
filtrate secured amounts to one liter. If the first filtrate be cloudy before use it may be
refiltered.
Qualitative Test for Nitrates.—Evaporate five cubic centimeters of the soil extract in a
porcelain crucible, having first dissolved a small quantity of pure brucin sulfate therein.
When dry, add to the residue a drop of concentrated sulfuric acid free of nitrates. If the
nitrate calculated as potassium nitrate does not exceed the two-thousandth part of a
milligram only a pink color will be developed; with the three-thousandth part of a milligram a
pink color with reddish lines; with the four-thousandth part of a milligram a reddish color;
with the five-thousandth part of a milligram a distinct red color.
Estimation of the Nitrates.—Evaporate 100 cubic centimeters of the soil extract to dryness
on a steam-bath. Dissolve the soluble portions of the residue in 100 cubic centimeters of
ammonia-free distilled water, filtering out any insoluble residue. Place the solution in a flask,
add ten cubic centimeters of sodium amalgam, stopper the flask with a valve which will
permit the escape of hydrogen, and allow to stand in a cool room for twenty-four hours. Add
fifty cubic centimeters of milk of lime and titrate the ammonia produced by distillation, with
standard acid and estimate as nitrogen pentoxid. Where the amount of ammonia is small,
nesslerizing may be substituted for titration.
Preparation of Sodium Amalgam.—Place 100 cubic centimeters of mercury in a flask of
half a liter capacity; warm until paraffin will remain melted over the surface; drop
successively in the paraffin-covered mercury, pieces of metallic sodium of the size of a pea
until 6.75 grams have united with the mercury. The amalgam contains then 0.5 per cent of
metallic sodium and may be preserved indefinitely under the covering of paraffin.
482. Method of the Experiment Station at Möckern.[309]—The principle of this
reaction is based on the reducing action exercised by nascent hydrogen on a nitrate, the
hydrogen being generated by the action of soda-lye on a mixture of zinc dust and finely
divided iron.
Ten grams of nitrate are dissolved in 500 cubic centimeters of water. Of this solution
twenty-five cubic centimeters, corresponding to one-half gram, are placed in a distillation
flask of about 400 cubic centimeters capacity, 120 cubic centimeters of water added, and
about five grams of well-washed and dried zinc dust and an equal weight of reduced iron. To
the solution are added eighty cubic centimeters of soda-lye of 32° B. The flask is then
connected with the condensing apparatus and the distillation carried on synchronously with
the reduction, the ammonia being collected in twenty cubic centimeters of titrated sulfuric
acid. The distillation is continued from one to two hours, or until 100 cubic centimeters have
been distilled, and the remaining sulfuric acid is titrated in the usual way. Soil extracts and
sewage waters should be concentrated until they have approximately the proportion of
nitrates given above.
483. Method of Devarda.—The inconvenience due to slow action and other causes,
arising from the use of pure metals in the reduction of nitrates to ammonia, has been
overcome, to some extent, by Devarda, by use of an alloy, in a state of fine powder, consisting
of aluminum, copper, and zinc.[310] The alloy consists of forty-five per cent of aluminum, fifty
per cent of copper, and five per cent of zinc. In dissolving, the copper is left in a finely divided
state, which is a great help in distillation in preventing bumping.
The analytical process is carried out as follows: The solution containing the nitrate, in
quantity equivalent to about one-half gram of potassium nitrate, is placed in a flask having a
capacity of about one liter, and diluted with sixty cubic centimeters of water and five cubic
centimeters of alcohol, and then forty cubic centimeters of caustic potash solution added of
specific gravity one and three-tenths. From two to two and one-half grams of the alloy,
described above, are introduced, and the flask attached to a condenser with a receiver
containing standard acid. The connection between the flask and the condenser is made by
means of a tube having on the limb next the flask a bulb filled with glass beads to prevent the
contents of the flask splashing over into the receiver, and on the other limb another bulb to
prevent the acid in the receiver finding its way into the distillation flask, should regurgitation
occur. When the flask has been thus connected with the condenser it is gently heated for half
an hour, at the end of which time the evolution of hydrogen will have slackened or ceased,
and then the distillation is begun, at first cautiously, until the zinc of the alloy has completely
dissolved, and then more vigorously, the time necessary being about twenty minutes from the
time when the contents of the flask begin to boil. The distillate is caught in standard acid and
the ammonia determined by backward titration in the ordinary way. It is to be noted that the
strength of the alkali used is of importance, as if it be too strong, the action on the alloy is
unduly vigorous at the beginning of the operation, and if too weak, the contents of the flask
have to be heated overmuch, the result in both cases being the formation of a fine spray of
caustic solution, which is very difficult to stop, even with complicated washing attachments to
the distilling flask. The test analyses on pure nitrates are satisfactory. This method has been
used with satisfaction in the laboratory of the Department of Agriculture, but does not appear
to have any special advantage over the process of Ulsch, to be described further on.
484. Variation of Stoklassa.—Stoklassa has subjected the method of Devarda to a
comparative test with the following methods:[311]
1. Wagner’s Schloesing-Grandeau method.
2. Lunge’s nitrometer method.
3. Stutzer’s method.
Figure 85. Stoklassa’s Nitric Acid Apparatus.

The reduction takes place in a copper erlenmeyer, A, Fig. 85, in which, in addition to the
solution containing the nitrate, are placed 200 cubic centimeters of water, forty cubic
centimeters of potassium hydroxid solution of 33° B., five cubic centimeters of alcohol, and
finally two and one-half grams of the finely powdered Devarda alloy. The distillate passes
through a tube, B, filled with glass pearls and into the condenser D, through the bulbs, C C′.
After the flask is connected with the distilling apparatus, it is gently warmed and the
reduction is ended in about twenty minutes. The ammonia which is formed is then distilled
into E, containing the standard acid, S, requiring about twenty minutes more. The
comparative results given, show that the Devarda method is equally as accurate as any of the
other methods mentioned, giving practically theoretical results.
In so far, however, as speed of an analysis, is concerned the first place is awarded to the
Lunge nitrometer method, with which a complete analysis can be made in from thirty to forty
minutes. In the second rank, so far as speed is concerned, the Devarda method is
recommended. All the methods give accurate results.
485. Method of Sievert.[312]—Two grams of potassium or sodium nitrate are dissolved
and made up to 1,000 cubic centimeters. Fifty cubic centimeters of the solution are placed in
a 600 cubic centimeter flask and diluted with fifty cubic centimeters of water, and from
eighteen to twenty grams of caustic alkali added. After the alkali is dissolved, seventy-five
cubic centimeters of ninety-six per cent alcohol are added and a few pieces of bone-black to
prevent foaming. From ten to fifteen grams of zinc or iron dust are then added to the flask
which is closed and connected with a ᥩ tube holding about 200 cubic centimeters, which
contains about ten cubic centimeters of normal sulfuric acid. This ᥩ tube is kept cool by
being immersed in water. The whole mixture is now allowed to stand for three or four hours
and then the alcohol is distilled slowly and the ammonia formed by the reduction of the
nitrates is carried over with it. The distillation lasts for about two hours. The contents of the
ᥩ tube are carefully rinsed into a dish and the excess of sulfuric acid titrated with one-fourth
normal soda-lye.
For soil extracts and substances containing unknown quantities of nitric acid, a
preliminary test will indicate approximately the amount thereof, and this will be an
indication for the quantity to be used in the analysis.
The method of Stutzer differs from the foregoing in the substitution of aluminum dust
instead of iron or zinc.[313] The reducing power of aluminum, however, varies greatly
according to the method in which the metal has been prepared. Pure aluminum prepared by
the electric method, reduces the nitric acid much less vigorously than the metal prepared by
the older methods of fusion with sodium. For this reason the method of Stutzer is not to be
preferred to that of Sievert.
REDUCTION IN AN ACID SOLUTION.
486. Variation of the Sodium Amalgam Process.—This method is described by
Monnier and Auriol.[314]
The principle of the operation depends on the reduction of
the dissolved nitrate by titrated sodium amalgam in
presence of an acid, and the estimation of the quantity of
nitric acid present from the deficit in the volume of
hydrogen. The apparatus employed is conveniently mounted
as shown in Fig. 86. The brass vessel A is movable by means
of the cord on the pulley B, in such a way as to be fixed at
any required altitude. It is filled with water and connected
by a rubber tube to the cooling tube D. Within the cooling
tube there is a graduated cylinder open at its lower end. Its
upper end is connected directly with the apparatus C. The
cooling tube D has a small side tube, c, near its upper end,
by means of which the air can enter or escape when the
position of A is changed. The apparatus C, in which the
reaction takes place, is a glass cylinder. Its upper end is
continuous with the ⟙ tube provided with the stop-cocks a
and b. One arm of the ⟙ permits connection with the
graduated measuring tube by means of a rubber union. The
lower end of C is closed with a large hollow ground-glass
stopper, carrying a small receptacle within, so that it forms
two separate water-tight compartments, open at the top.
The sodium amalgam is prepared as follows:
In a clay crucible are heated 400 grams of mercury, and,
little by little, with constant stirring, four grams of dry
Figure 86. Variation of the sodium are added. When cold, the amalgam is placed in a
Sodium Amalgam Process.
burette, having a ground-glass stopper, and covered with
petroleum. The strength of the amalgam is established in the
following manner. A small glass thimble, ground even at the top, is filled with the amalgam
and struck off even with a ground-glass straight edge. In this way the same quantity of
amalgam is taken for each test. This measured portion of the amalgam is placed in the inner
vessel of the glass stopper to C. Ten cubic centimeters of water, containing sixty centigrams of
tartaric acid, are placed on the outer ring of the glass stopper, which is then inserted, well
oiled, in C, closing it air- and water-tight. The tartaric acid solution also carries a piece of
litmus paper, so that its constant acidity may be insured. The vessel A is then fixed in a
position which brings the water in the graduated burette and tube D exactly to the 0 mark.
The cock a is next closed, b opened, and C is inverted until all the amalgam is poured into the
solution of tartaric acid. The evolved hydrogen mixed with the air contained in the apparatus,
is passed into the graduated burette. After fifteen minutes, the reaction is ended. The water
level within and without the graduated tube is restored and the volume of gas evolved noted
and reduced by the usual tables to 0° and 760 millimeters pressure of the barometer.
An amalgam prepared as above will give about three cubic centimeters of hydrogen for
each gram. The thimble should hold from twelve to fifteen grams.
The estimation of nitric acid should be made in a solution containing about one-tenth per
cent of nitrate. Ten cubic centimeters are taken, to which six-tenths gram of tartaric acid is
added, and placed in the outer part of the glass stopper. The rest of the process is conducted
exactly as described above. The deficit in hydrogen is calculated to nitrogen pentoxid.
The reduction by sodium amalgam is not so convenient a form of estimating nitric acid as
many of the other forms of using nascent hydrogen. As practiced by calculating from the
deficit of hydrogen, however, it has some advantages by reason of the fact that no heating is
required. The presence of organic neutral bodies, or even those of an acid nature, like humus,
does not, therefore, interfere with the work. Likewise, mineral bodies in solution, which are
not reduced by nascent hydrogen, do not interfere with the accuracy of the reaction.
487. Method of Schmitt.—In the method of Schmitt forty cubic centimeters of glacial
acetic acid are placed in a flask of 600 cubic centimeters content, and fifteen grams of a
mixture of zinc and iron dust added.[315] To this a quantity of the solution containing the
nitrate, representing about half a gram of the pure nitrate, is added with constant shaking, in
portions which do not evolve hydrogen too rapidly. After about fifteen minutes when the
evolution of nitrogen has somewhat diminished, an additional fifteen grams of the metal dust
are added. If the contents of the flask should become thick they can be diluted with thirty
cubic centimeters of water. The reduction is complete in from thirty to forty minutes. The
contents of the flask are now saturated with enough soda-lye not only to neutralize the excess
of acetic acid, but to keep the zinc hydroxid also in solution. For this purpose about 200 cubic
centimeters of soda-lye of 1.25 specific gravity are necessary. The ammonia is obtained by
distillation into standard acid in the usual way.
488. Method of Ulsch.—In practice the method of Ulsch has come into general use.[316]
For the determination of nitrogen by this method half a gram of saltpeter or four-tenths
gram of sodium nitrate is taken and dissolved in twenty five cubic centimeters of water, in a
flask with a content of about 600 cubic centimeters. Five grams of iron reduced by hydrogen,
and ten cubic centimeters of sulfuric acid diluted with two volumes of water are then added
to the flask. To avoid mechanical losses during the evolution of hydrogen a pear-shaped glass
stopper is hung in the neck of the flask. After the first violent evolution of hydrogen has
passed, the flask is slowly heated until in about four minutes it is brought to a gentle boil. The
boiling is continued for about six minutes when the reduction is complete. About fifty cubic
centimeters of water are then added; also an excess of soda-lye and a few particles of zinc and
the ammonia is distilled and collected in standard acid in the usual way.
The method of Ulsch can also be applied, according to Fricke, to the analysis of nitrates
contained in drinking and drainage waters, and it is regarded by him as one of the best
methods to be employed in such investigations.[317]
The method of Ulsch in this laboratory has given entirely satisfactory results, and is
generally used in preference to other methods in cases where a considerable quantity of
nitrates is present. It is based on the following reactions:
2KNO₃ + H₂SO₄ = K₂SO₄ + 2HNO₃

2HNO₃ + 8H₂ = 2NH₃ + 6H₂O
2NH₃ + H₂SO₄ = (NH₄)₂SO₄.

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Bioanalytical Techniques 1St Edition Abhilasha Shourie Online Ebook Texxtbook Full Chapter PDF

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Bioanalytical Techniques 1st Edition

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The Energy and Resources Institute

Science and technology by far depend upon experimental procedures involving

‘Bioanalytical techniques’ is included as a fundamental paper in most courses

The book emphasizes on imparting profound knowledge that is able to

number of state-of-the-art sophisticated techniques that would be beneficial to

Many typical analytical procedures appear in the book as boxed features

Dr. Abhilasha Shourie

8.4 Atomic Absorption Spectrometry 243

desorption/ionization time-of-flight (MALDI–TOF), and Fourier transform

1.2 EXPERIMENTAL STUDIES

1.2.1 Experimental Design

1.2.2 Constants and Variables

• Cluster sampling It is another type of random sampling in which the

1.2.4 Measurement Scales

1.3 EXPERIMENTAL ERRORS

1.4 STATISTICAL PARAMETERS FOR VALIDATION OF AN EXPERIMENT

Figure 1.1 A plot showing Gaussian or normal distribution

2.1.2 Properties of a True Solution

Table 2.1 Types of solutions: true, colloids, and suspensions

• Solubility is the ability of the solute to dissolve in a solvent at a particular

2.2 UNITS OF CONCENTRATION

chemical method to be followed in the experiment. It is, therefore, necessary

Mass of solute in g/Gram molecular mass of solute

Table 2.2 Prefixes of commonly used units

2.2.3 Parts per Million

2.2.4 Mass Per cent

Mass per cent = (Mass of solute)/(Mass of solution) × 100 2.4

2.2.5 Volume Per cent

Volume per cent = (Volume of solute)/(Volume of solution) × 100 2.5

2.2.6 Mass/Volume Per cent

Mass/Volume per cent = (Mass of solute)/(Volume of solution) × 100 2.6

2.2.7 Mole Fraction

If the solution is composed of substances A, B, and C, where NA, NB , and

2.2.8 Mole Per cent

Mole per cent (of substance A) = X A × 100% 2.9

2.2.9 Per cent Saturation

The concentration of sugar solution is 0.116 M.

2.3 THE CONCEPT OF pH

H2O Æ H+ + OH– 2.11

the negative logarithm of the hydrogen ion concentration (–log[H+]). The pH

2.4 ACIDS AND BASES

Figure 2.1 The pH scale

2.4.1 Arrhenius Concept

HA + BOH = BA + H2O 2.15

2.4.2 Bronsted–Lowry Concept

2.5 HENDERSON–HASSELBALCH EQUATION

The equilibrium constant for the dissociation of a weak acid is given by

The Henderson–Hasselbalch equation is derived from this by taking the

We can think of pKa as pH at which the number of molecules of conjugate

an extremely useful equation from which pH of the solutions of various ratios

2.6 DETERMINATION OF pKa

CH3COOH(aq) CH3COO –(aq) + H+(aq) 2.22

CH3COO – (aq) + H+(aq) CH3COOH(aq) 2.23