ANALYTICAL BIOCHEMISTRY

A Report

Submitted in partial fulfillment of the requirement of the award of the degree

of

MASTER OF BIOCHEMISTRY

By

RAHEEMA TASLEEM Z

232033225249992

NATIONAL P.G. COLLEGE-Lucknow

PGBR-01N

SCHOOL OF SCIENCE

U.P. RAJARSHI TANDON OPEN UNIVERSITY PRAYAGRAJ 211013

UTTARPRADESH 2023
 PERFORMA FOR THE APPROVAL OF REPORT PROPOSAL

Enrollment no. - 232033225249992

Name of the Student - RAHEEMA TASLEEM Z

Title of the Project/Dissertation - ANALYTICAL BIOCHEMISTRY

Name of the Guide - Dr. VIKAS SINGH

Teaching experience of the Guide -

Signature of the student Signature of the Guide

Date - Date -
 U.P. RAJARSHI TANDON OPEN UNIVERSITY

PRAYAGRAJ 211013

SCHOOL OF SCIENCE

CERTIFICATE

This is to certify that the report “ANALYTICAL BIOCHEMISTRY” is bona fide

work of RAHEEMA TASLEEM Z bearing Enrollment No. 232033225249992
submitted to partial fulfillment of the requirement the award of degree of MASTER OF
SCIENCE in BIOCHEMISTRY from U.P. Rajesh Tondon Open University, Prayagraj.

Name of Guide - Dr. VIKAS SINGH

External Examiner-

Date:
 ABSTRACT

The combination of analytical biochemistry to measure the metabolic complement with

sophisticated informatics, bioinformatics, and statistics makes up the newest of the ‘omics fields
called metabolomics. Metabolites are characterized by a diverse chemistry and therefore require
the application of numerous analytical approaches for their extraction, separation, detection, and
quantification. In the past decade, the technologies have improved substantially allowing the
analysis of thousands of compounds simultaneously. However, this has led to the current
bottleneck in metabolomics that is how to extract information from raw data of many different
analytical platforms and the subsequent appropriate analysis in a biological context. In this article,
we aim to summarize the state of the art of metabolomics technologies from both an analytical and
a bioinformatics point of view. We present the challenges currently faced by metabolomics
researchers and provide the readers with potential approaches to address those challenges. This
review synthesizes recent developments in analytical biochemistry, providing an overview of
innovative methodologies and their applications in diverse research domains. Emphasizing
advancements in analytical techniques, such as Mass spectrometry, Centrifuge, Electrophoresis,
Chromatography, Spectroscopy, Nuclear magnetic resonance, imaging technique Microscopy,
Enzyme Linked Immune Sorbent Assay and Polymerase Chain Reaction we explore their role in
elucidating molecular structures, quantifying bimolecular, and unraveling intricate biochemical
pathways. The integration of omics technologies and data analytics has propelled precision and
depth in bimolecular analysis.
 ACKNOWLEDGEMENT

Our first and irrefutable thanks to that internal and innate power that give us strength not only to
starts this project but also to accomplish the target

This project report bears a vital contribution by many people, and it becomes my
pleasant duty to express heartiest gratitude towards them

We are deeply obliging and acknowledge …………………………………………

For this encouragement and kind support in the journey of completing this report. We want to
express our since gratitude to all the faculty members and lab supports staff of the Biochemistry
department for their support, without which we were in capable of bringing this report to its
completion.
 DECLARATION

I hereby declare that the project / dissertation entitled “ANALYTICALBIOCHEMISTRY “done,

this report has not been in any case duplicated to submit to any other university for the award of
any degree.

The report is done in partial fulfillment of the requirement for the award of degree of MASTER
OF SCIENCES in BIOCHEMISTRY to be submitted as 1st semester report as part of our
curriculum.

Name – RAHEEMA TASLEEM Z

Signature of student
 TABLE OF CONTENTS

1. Introduction……………………………………………………………………………….00-01
2. General Principle of Analytical Biochemistry……………………………………………02-09
3. Spectroscopy………………………………………………………………………………10-30
4. Chromatography………………………………………………………………………….31-42
5. Electrophoresis……………………………………………………………………………43-48
6. Centrifugation…………………………………………………………………………….49-57
7. Automation……………………………………………………………………………….58-62
8. Conclusion………………………………………………………………………………..63
9. Reference…………………………………………………………………………………64
 Page |1

INTRODUCTION

Analytical Biochemistry investigates and employs equipment and processes familiarized separate,
classify, and calculate material. Identification, separation, preparation and evaluation may organize
the perfect analyze and in conjunction with some other technique. Analytical Biochemistry is a
branch in which we can analyze any chemical and define its properties. Analyses of separation
isolates Analyses are identified by analysis. The numerical amount or attentiveness is determined
by the measure. Analytical Biochemistry is the study of acquiring, dispensing, and interacting with
information regarding matter's arrangement and structure. To put it another way, it's science and art
of figuring out what case is and how much of outlets. It is unique in one of the most widely studied
topics of research for chemists.

Under analytical chemistry separation, identification, and qualification of natural and synthesized
chemical components substances is complete it is a two types qualitative analysis & quantitative
analysis. Analysis identifies the components present throughout a sample &measurement regulates
the quantity of this components. The term analysis in chemistry was first utilized by chemist for
the strategy of finding the arrangement of substance. Again, analytical methods are often divided
into two parts classical & instrumental. The classical analytical method is additionally called wet
chemistry. The analyzed also measured the look of chemical experiments the event of latest
instrument of chemistry & measurement etc. analytical chemistry is used in bio-analysis, clinical
analysis, environmental analysis, and substance analysis are all types of forensic analysis. The
results of the analysis also provide information related to the structure of matter. Due to the
acquaintance of micro analysis method in analysis, micro analysis has a got special place in
laboratory additionally to the compounds obedient from chemical laboratories for research purpose
in other research work were the substantial in available in very small quantifies the help of micro
analyses is prerequisites within the analysis. Analytical or what we also call analysis such as a
technique which is used in every branch which is developing and increasing every day.
 Page |2

General Principle of Analytical Biochemistry

• Principle of Analytical biochemistry

• The selection valid method of analysis
• Quality Control
Topics • Calibration
• Hazards from Dangerous Chemicals

Analytical biochemistry involves the use of laboratory methods to determine the composition of
biological samples and it has applications in many widely differing areas of biological science. The
information gained from an analysis usually presented as a laboratory report, which may simply
say what substances are present (a qualitative report) or may specify the precise amount of a
substance in the sample (a quantitative report).
A qualitative report will often indicate whether a particular
substance or group of substances is present without commenting on the complete composition of
the sample. In many cases the report will also specify the individual member of that group of
substances. It might, for instance, name only the different carbohydrates present although the
sample contained other substances, e.g. lipids and proteins. It is possible, when using some
qualitative methods, to compare the amount of substance in the sample with the amount in a
reference sample and to report the presence of either increased or decreased quantities. Such a
report is said to be semi-quantitative. Chromatographic and electrophoresis methods often give
results which can be interpreted in this way.
A quantitative report will state the amount of a particular
substance present in the sample and it is important that the units of measurement are meaningful
and appropriate in order to prevent subsequent misunderstandings. When reporting quantitative
results, it is desirable to indicate their reliability, a feature which can often be assessed statistically.
In practice it may not be necessary to present this information with each report but it should be
readily available for reference.
The selection of valid method of analysis
In order to be able to choose a suitable analytical method it is essential to know something about
the chemical and physical properties of the test substance like proteins, carbohydrates, lipid,
elements, amino acids etc. Because the relationship between the property and the amount of
 Page |3

substance is not always a simple one, some methods are only suitable for the detection of the
substance (qualitative) while others may be quantitative. For any method it is important to
appreciate the nature of the substance.
Physical properties that can be measured with some degree of precession

Mass
Extensive volume

Mechanical Specific Gravity

Viscosity
Surface Tension
Spectral Abortion
Emission
Fluorescence
Turbidity
Rotation
Electrical
Conductivity
Current/Voltage
Half-cell Potential

Nuclear Radioactivity

Instrumental methods
The most convenient methods are those that permit simultaneous identification and quantitation of
the test substance. Unfortunately, these are relatively few in number but probably the best
examples are in the area of atomic emission and absorption spectroscopy, where the wavelength of
the radiation may be used to identify the element and the intensity of the radiation used for its
quantitation. If a compound does not show an easily detectable characteristic, it may be possible to
modify it chemically to produce a compound which can be measured more easily. In the early part
of this century, this approach to analysis led to the development of many complex reagents
 Page |4

designed to react specifically with particular test substances. Generally, these reagents resulted in
the formation of a color which could be measured using visual comparators. Most of these reagents
have been superseded by improved instrumental methods but some very reliable ones still remain
in use. Interference occurs when other substances, as well as the test compound, are also detected,
resulting in erroneously increased values. Occasionally interference effects can result in
suppression of the test reaction. For any method it is important to be aware of substances that may
cause interference and to know if any are likely to be present in the sample. If interference is a
major problem the sample must be partially purified before analysis. This breaks the analysis into
preparatory and quantitative stages. In order to reduce the technical difficulties resulting from such
two-stage methods much work has gone into the development of analytical techniques such as gas
and liquid chromatography in which separation and quantitation are effected sequentially.

QUALITY CONTROL

Quality control refers to an internal scheme that will give a warning when unforeseen factors cause
a reduction in the analytical performance of the method. This allows an immediate decision to be
made on whether the test results are acceptable or must be rejected. This is usually done by the
analysis of a control sample with each batch of tests.
It is important to recognize that laboratory investigations are liable to certain errors which may
mislead the clinical assessment. The most common cause of a single abnormal biochemical value
is probably an error of the laboratory. The study of the sources of variation for which laboratory is
responsible and procedures used to recognize and minimize them is defined as quality control
(QC). The sources of variation include all sources which arise within the laboratory, from the
receipt of the specimen to the dispatch of reports. The main emphasis of QC is to monitor the
precision and accuracy of the performances of analytical methods. Besides being important for
patient care, QC is designed to give the laboratory staff confidence in their methods. Results from a
laboratory that do not maintain quality control cannot be relied upon. So to maintain a good
standard of accuracy, laboratory should monitor its own performance continuously by keeping in
mind a few things and by applying one or more quality control systems.

TO ASSESS THE VALIDITY OF RESULTS ON PATIENT SPECIMENS

Regulatory agencies require the use of quality control (QC) materials to assess the validity of
 Page |5

results in a laboratory. This is achieved by assaying and evaluating at least two levels of control
material every 24 hours. Most laboratories are required to establish their own means and standard
deviations for the parameters tested and reported on patients. The published assay range for a given
control is the range in which a laboratory's mean must fall to be considered acceptable.

EVALUATION OF QC RESULTS

QC results are evaluated by calculating standard deviation and coefficient of variation as follows:

Standard Deviation (SD):

Standard deviation is a measure of the dispersion of a set of data from its mean. The more spread
apart the data, the higher the deviation. For the individual x-values the SD or S can be calculated as
follows:
𝑥𝑖−𝑥)2
SD= √(∑( ))
(𝑛−1)

X is the mean value of observations and the denominator n stands for the size of the sample.

Coefficient of Variation (CV):

CV refers to the coefficient of variation, which describes the standard deviation as a percentage of
the mean, as shown in the following equation: CV%= (SD/Xx) 100
Where SD is the standard deviation, x is the mean, and the multiplier of 100 is used to convert the
SD/x ratio to a percentage.
The CV also provides a general feeling about the performance of a method, CVs of 5% or less
generally give us a feeling of good method performance, whereas CVs of 10% and higher sound
bad.
One should look carefully at the mean value before judging a CV. At very low concentrations, the
CV may be high and at high concentrations the CV may be low. For example, a bilirubin test with
an SD of 0.1 mg/dL at a mean value of 0.5 mg/dL has a CV of 20%, whereas an SD of 1.0 mg/dL
at a concentration of 20 mg/dL corresponds to a CV of 5.0%.

Monitoring QC Data
• QC results are usually monitored by Levy- Jennings charts (L-J charts) or following
Westguard rules.
 Page |6

• Plot control values each run, make decision regarding acceptability of run
• Monitor over time to evaluate the precision and accuracy of repeated measurements
• Review charts at defined intervals, take necessary action, and document.
Levy-Jennings plot for recording daily quality control values: Many laboratories take either the
first quality control value of each daily run or averages of the values obtained for each quality
control pool for each analyte for each day and plot that on a quality control chart. Such charts, e.g.
Levy- Jennings graph Gives visual presentation of the data. It is a graphical method for displaying
control results and evaluating whether a procedure is in-control or out-of-control.
In Levy-Jennings graph control values are plotted versus time. Average +2 USD is drawn on the
Y-axis and the days of the month are indicated on the X-axis. Trends or shifts from the average
target values are known as biases. If the performance of the analytical method remains unchanged,
succeeding points will be evenly scattered on either side of the midline indicating no change in
accuracy. If the bias becomes severe and the value falls outside 2 SD limit it is called warning limit
and if value falls outside 3 SD limit it is called action limit. Change in accuracy should be
detectable before the graph passes beyond warning limits. If a result falls beyond the action limit,
the chances of true error are much higher and there is risk that all results are erroneous. Control
samples should be randomly inserted amongst patients' samples and analyst should not know that
sample is a control sample otherwise results are unconsciously biased.

Westgard Rules

The individual Westgard rules are defined

below.

Westgard-12s rule: 12s refers to the control

rule that is commonly used with a Levy-
Jennings chart when the one of two control
results falls outside +2SD. This rule is used as
a warning rule to trigger careful inspection of the control data by the following rejection rules. This
alerts technical staff to possible problems. It is not a cause for rejecting a run but one must then
evaluate the 13s rule.
 Page |7

Westgard- -13s rule: 13s refers to a control rule that is commonly used with a Levy-Jennings chart
if either of the two control results falls outside of ±35D. A run is rejected when a single control
measurement exceeds the mean plus 3s or the mean minus 3s control limit.

Westgard -22s rule: 22s Rule is violated when 2 consecutive control values for the same level fall
outside f\pm2SD~in~t same direction, or both controls in the same run exceed ±2SD. This requires
corrective action. Reject the run. Patient results cannot be reported.

Westgard –R4s rule: R4s Rule is violated when one control exceeds the mean by the other control
exceeds the mean by +25D. The range between the two results will therefore exceed 4SD. Test run
must be rejected.

Westgard-41s rule: 41s Reject when 4 consecutive control measurements exceed the same mean
plus 1s or the same mean minus 1s control limit.

Westgard 10x rule: 10x When 10 consecutive control measurements fall on one side of the mean,
10, Rule is violated. Reject the run.

Warning Rule
In manual applications, a t_{25} rule should be used as a warning to trigger application of the
other rules, thus anytime a single measurement exceeds a 2s control limit, one responds by
inspecting the control data using the other rules
Rejection Rules
Other control rules can be used to inspect the control points.
a. Rules that can be applied within a run. Stop if a single point exceeds a 3s limit.

• Stop if two points in a row exceed the same 2s limit.

• Stop if one point in the group exceeds a plus 2s limit and another exceeds a minus
2s limit.

b. Rules that can be applied across runs.

Often the 41s and 10, must be used across runs in order to get the number of control measurements
needed to apply the rules.
 Page |8

• A 41s violation occurs whenever 4 consecutive points exceed the same 1s limit. These 4
may be from one control material or they may also be the last 2 points from a high level
control material and the last 2 points from a normal level control material, thus the rule may
also be applied across materials
• The 10, rule usually has to be applied across runs and often across materials.
Some alternative control rules-are more suitable when three control materials are analyzed. which
is common in hematology, coagulation, and immunoassays.
Steps for rejection rule for out-of-control values:
• Stop testing
• Identify and correct the problem
• Repeat testing on patient samples and controls
• Do not report patient results until problem is solved and controls indicate proper
performance.

CALIBRATION AND CALIBRATION MATERIALS

Calibration in analytical biochemistry involves setting the relationship between instrument
readings and known concentrations of a substance. Regularly calibrate instruments such as
spectrophotometers or chromatographs using certified reference materials. Create calibration
curves by analyzing standard solutions with known concentrations. Periodically re-calibrate to
account for changes in instrument performance. Calibration ensures accurate and precise
quantification of analytes in your samples, enhancing the reliability of your analytical
measurements.
Accuracy
Accuracy in analytical biochemistry refers to the closeness of measured results to the true values.
Achieving accuracy involves proper calibration, using high-quality standards, and minimizing
systematic errors. Regularly calibrate instruments, validate methods, and employ quality control
measures. Compare results against certified reference materials to verify accuracy. Ensuring
accurate measurements is essential for reliable data interpretation and drawing meaningful
conclusions in analytical biochemistry research.
Precision
Precision in analytical biochemistry refers to the consistency and reproducibility of measurements.
It is the ability of a method to generate similar results under consistent conditions. Enhance
precision by optimizing experimental conditions, maintaining a stable environment, and using
 Page |9

precise equipment. Conduct replicate analyses and calculate measures like standard deviation and
coefficient of variation to assess precision. Reliable precision minimizes random errors and
increases confidence in the reproducibility of your analytical results.
Hazards from Dangerous Chemicals
Explosive
In general laboratories there are some compounds that are potentially explosive, e.g. picric acid. It
is important that such substances are stored under suitable conditions, e.g. under water, and that
they are regularly inspected in order
to maintain these conditions. The use of such substances should be carefully controlled and only
small amounts used.
Flammable
Flammable liquids and solids are subdivided according to their flash points and many oxidizing
substances can cause fire when in contact with combustible materials. Storage, handling and
disposal are obviously major features to be considered when using such substances.
Toxic
Substances are graded in toxicity and by the route, e.g. inhalation, swallowing or contact, the latter
being particularly important in the design of the working environment. In some instances, it is
possible to specify exposure limits and in
such cases monitoring of the environment may be essential.
Corrosive and irritant
The result of skin contact with corrosive substances is usually obvious but the effects of irritants
are less obvious and hence are potentially likely to be treated more casually. Protective clothing is
essential when using toxic or irritant
substances.
Radioactive
These substances are subject to very strict control and laboratories must be approved to handle the
different categories of radioactivity.
 P a g e | 10

Spectroscopy

• Properties of electromagnetic Radiation

• Atomic Absorption Spectroscopy
Topics • U-V Visible Spectroscopy
• Nuclear Magnetic Resonance

Spectroscopy is a powerful analytical technique in biochemistry that involves studying the

interaction between matter and electromagnetic radiation. It helps identify and quantify substances
based on their unique spectral patterns. In analytical biochemistry, common types include UV-
Visible spectroscopy, infrared spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy.
These methods provide valuable insights into molecular structures, concentrations, and chemical
environments, contributing significantly to various fields such as protein analysis, drug discovery,
and environmental monitoring. Spectroscopy is the study of the absorption and emission of
radiation by matter. The most easily appreciated aspect of the absorption of radiation is the color
shown by substances that absorb radiation from the visible region of the spectrum. If radiation is
absorbed from the red region of the spectrum, the transmitted or unabsorbed radiation will be from
the blue region and the substance will show a blue color. Similarly, substances that emit tradition
show a particular color if the radiation is in the visible region of the spectrum. Sodium lamps, for
instance, owe their -yellow light to the specific emission of sodium atoms at a wave length of 589
nm.
Measurements of the intensity and wavelength of radiation that is either absorbed or emitted
provide the basis for sensitive methods of detection and quantitation. Absorption spectroscopy is
most frequently used in the quantitation of molecules but is also an important technique in the
quantitation of some atoms.

Properties of Electromagnetic Radiation

Electromagnetic radiation is generated when electrically charged particles, such as electrons, move
through a medium or empty space. These particles carry energy as they travel, creating a wave-like
pattern of electric and magnetic fields.
 P a g e | 11

Electric and Magnetic Fields: Electromagnetic radiation consists of electric and magnetic fields.
These fields are intimately connected and perpendicular to each other, with a 90-degree angle. As
the combined waves propagate through space, they move perpendicular to the electric and
magnetic oscillations occurring during the disturbance.

Diverse Forms of Electromagnetic Radiation: Electromagnetic radiation encompasses a vast

spectrum of energy, ranging from low-energy radio waves to high-energy gamma rays. Some
common forms of electromagnetic radiation include microwaves, television waves, radio waves,
infrared radiation, visible light, ultraviolet radiation, X-rays, and gamma rays. Each form has

different wavelengths and energy levels, resulting in unique properties and applications.
Transverse Waves: Electromagnetic radiation travels as transverse waves, meaning that the
oscillations occur perpendicular to the direction of wave propagation. This characteristic allows
electromagnetic radiation to traverse space, enabling it to propagate through a vacuum, unlike
other types of waves.

Photons: Electromagnetic radiation is emitted as discrete packets of energy called photons. These
photons carry light energy and travel at the constant speed of light.
Wavelength: Electromagnetic radiation is grouped into different categories based on its
wavelength within the electromagnetic spectrum. Each category has distinct properties and
applications.
Perpendicular Electric and Magnetic Fields: Electromagnetic waves consist of perpendicular
electric and magnetic fields. These fields oscillate in directions that are perpendicular to each other
and to the direction of wave propagation.
Wavelength (λ) represents the distance between successive crests or troughs of a wave. It is the
length of one complete cycle of oscillation. Wavelength and frequency (ν) are related by the
equation c = λν, where ‘c’ is the speed of light. Shorter wavelengths correspond to higher
frequencies and higher energy levels.
Amplitude refers to the maximum displacement of a wave from its equilibrium position. It
represents the distance from the middle of the wave to the crest or trough. Amplitude provides
information about the brightness or intensity of a wave relative to other waves. Larger amplitudes
indicate higher energy levels, while smaller amplitudes correspond to lower energy levels.
 P a g e | 12

Frequency is the number of wave cycles that occur per second, measured in Hertz (Hz) or cycles
per second. It determines how many times a wave oscillates within a given time frame. Frequency
(ν) and energy (E) is directly proportional, as indicated by the equation E = hν, where ‘h’ is
Planck’s constant (6.62607 x 10-34). Higher frequencies correspond to higher energy levels.
Period (T) represents a wave’s total time to complete one full cycle or travel one wavelength. It is
the reciprocal of frequency, given by T = 1/ν. The period provides information about the time
characteristics of a wave.
Velocity
In electromagnetic radiation, velocity is expressed as the product of wavelength and frequency,
given by Velocity = λν. For electromagnetic waves propagating in a vacuum, the velocity is equal
to the speed of light, which is approximately 186,282 miles per second or 2.99 x 108 meters per
second (m/s).

Types of Electromagnetic Radiation

Gamma rays have the highest frequency and photon energy in the electromagnetic spectrum. They
have extremely short wavelengths and are associated with nuclear processes and high-energy
particle interactions.
X-rays have frequencies and photon energies slightly lower than gamma rays. They are widely
used in medical imaging, security screening, and industrial applications due to their ability to
penetrate materials and reveal internal structures.
Ultraviolet rays have higher frequencies and photon energies than visible light. They are emitted
by the Sun and play a crucial role in biological processes, such as vitamin D synthesis and
triggering chemical reactions in the atmosphere.
Visible light is the portion of the electromagnetic spectrum visible to the human eye. Visible light
is essential for vision. It has a range of frequencies and photon energies that allow us to perceive
different colors.
Infrared rays have lower frequencies and photon energies than visible light. They are associated
with thermal radiation and are commonly used in night vision, remote sensing, and infrared
spectroscopy applications.
Radio waves have the lowest frequencies and photon energies in the electromagnetic spectrum.
They are used for communication, broadcasting, and radar systems. Different portions of the radio
 P a g e | 13

wave spectrum are allocated for various applications, including AM and FM radio, television, and
mobile communication.
Microwaves have slightly higher frequencies and photon energies than radio waves. They are
commonly used in microwave ovens, satellite communication, and wireless technologies.

Electromagnetic radiation interaction with matter:

We know the EMR a noted type of radiations and various interactions with matter and produce
result. The electronic radiations have both properties of wave and particles. Albeit having a mass
of zero. As particle, when EMR interact with matter transferring its energy E.

𝐸 =h𝑐/𝜆 = h as c = υλ (1.1)

where h is plank’s constant = 6.62 x 10-34 joule-second.

In order for a transition the energy must be absorbed. The energy change ΔE needed is defined in
quantum terms by the difference in absolute energies between the final and the starting state. The
energy change (ΔE) is measured in KJ/mole by following equation.

Δ𝐸 = 𝐸𝑓𝑖𝑛𝑎𝑙 − 𝐸𝑖𝑛𝑡𝑖𝑎l (1.2)

From eq. 1.1 & 1.2 the energy will be

Δ𝐸 = h𝑣 (1.3)
Electrons in either atoms or molecules may be distributed between several energy levels but
principally reside in the lowest energy level (ground state). In order for an electron to be promoted
to a higher energy level (excited state), energy must be put into the system. If this energy E¼h_ is
derived from electromagnetic radiation, this gives rise to an absorption spectrum,
and an electron is transferred from the electronic ground state to into the first electronic excited
state. If we consider the matter in diatomic molecule, then rotational and vibration level possess
discrete energies that only manage into a continuum at very high energy. Each electronic state of a
molecule possesses its own set of rotational and vibrational levels. Electron either in atom or
molecule may be distributed between several energy levels. Knowing Δ𝐸 = ℎ𝑣is derived from
 P a g e | 14

electromagnetic radiation this gives rise, to a spectrum and electron is transferred from lower
energy level to higher excited state (S1). The molecule will also be in an excited vibration and
rotation state. Subsequent relaxation of the molecule into the vibrational ground state of the first
electronic excited state will occur. The electron can then revert back to the electronic ground state.
The plot of absorption probability against wavelength is called absorption spectrum. Single atom
gives the line spectra while molecules produce band spectra.

Relation between wavelength, Frequency and wave number:

Wavelength (λ) and frequency υ are related as follows

𝑐 = 𝜐. 𝜆

Since c is constant (c = 3 x 1010 cm/sec in vacuum. The above relation

may be express as proportional 1/𝜆

Reciprocal of wavelength is called wave number

1/𝜆 = 𝜐
The unit of wave number is cm-1
Atomic Absorption Vs Atomic Emission Spectroscopy

Atomic absorption Spectroscopy Atomic Emission Spectroscopy

Atomic absorption Spectroscopy is used to find Atomic Emission Spectroscopy is used to find
the concentration of metals atom in a solution out the concentration of the analyte by emission
of light
A fixed amount of energy is absorbed by the The discrete energy emitted during excitation
electrons of an atoms
From ground state to excited state From excited state to ground state
Electromagnetic radiation is absorbed Electromagnetic radiation is emitted
It cannot be used for direct analysis of solid It can be used for direct analysis of solid sample
sample
 P a g e | 15

The spectrum obtained is dark lines are gaps Color spectrum obtained
It require a light source It does not require a light source
It depend upon a number of Ground state Atoms It depend upon a number of exited state Atoms

ATOMIC SPECTROSCOPY

We know that theory of electromagnetic radiation that says the molecules give rise to band spectra
whereas atom gives line spectra. Thus, the study of atomic level, the atomic spectroscopy is very
useful to characterization of elements. The atomic spectroscopy leads to both atomic absorption
and atomic emission spectra of an atom. The atomic emission occurs due to light of particular
wavelength (color) converse. But in atomic absorption spectra occurs when the black line can be
observed against a bright light. In general, atomic spectroscopy is not carried out in solution. In
order for atoms to emit or absorb monochromatic radiation, they need to be volatilized by exposing
them to high thermal energy. Usually, nebulizers are used to spray the sample solution into a flame
or an oven. Alternatively, the gaseous form can be generated by using inductively coupled plasma
(ICP). The Absorption spectroscopy, as per name is based on the absorption of electromagnetic
radiations by matter. Electron presents in an atom, absorb energy from electromagnetic radiations
which fall on it and jump from ground state to excited state. This phenomenon is called absorption
and spectroscopy related to it is termed as absorption spectroscopy. An absorption spectrum is
measured as a function of wavelength. The absorption spectrum of an atom or molecule depends
on its energy level structure and useful for identification of compounds.

ATOMIC ABSORPTION SPECTROSCOPY (AAS)

As it is clear from the name, atomic absorption spectroscopy(AAS) that this spectroscopy deals
with the absorption of electromagnetic radiation of specific wavelength by atom. The first AA
Spectrometer was built by scientist Alan Walsh at the Commonwealth Scientific and Industrial
Research Organization (CSIRO) in 1954, Division of Chemical Physics, in Melbourne, Australia.
Atomic absorption spectroscopy is a spectra-analytical technique. The absorption wavelength is
associated with transition that requires a minimum of energy change. The electronic transition in
atom are limited by the availability of empty orbital, because one orbital can be occupied
 P a g e | 16

with maximum of two electrons and spin of electron need to be paired inanity parallel fashion. The
analyte molar concentration is determined from the amount of absorption. The sample used in
atomic absorption spectroscopy, needs to volatile by using the higher energy. Thus, the tested
sample either liquid should be converted into vapor form. Atomic absorption is very technique for
detecting specific metals and its concentration present in the sample at ppm. Concentration
analysis by atomic absorption spectroscopy is carried out by comparison with calibration standard.
It finds extensive applications in the analysis for trace metals in biological serum and drinking
water. Some examples of elements detected by AAS are as given below.

Principle:
In AAS the free atoms (gas) generated in an atomizer can absorb radiation at specific frequency.
Atomized element absorbs energy of a wavelength that is peculiar to that element. In the process of
atomization, the hallow cathode lamp is used as a light source which emits light of wavelength that
is peculiar to that element. When a beam of electromagnetic radiation of a particular wavelength is
passed through the vaporized atom present in the flame, the atoms absorb the radiation and extent
of radiation will be directly proportional to the number of ground state atoms presented in the
flame. Atomic-absorption spectroscopy quantifies the absorption of ground state atoms in the
gaseous state. The atoms absorb ultraviolet or visible light and make
transitions to higher electronic energy levels. The analyte concentration is determined from the
amount of absorption of electromagnetic radiation. The atomic absorption is a very common
technique for detecting metals and metalloids in environmental samples.

Instrumentation:
The Atomic absorption spectroscopy has simple instrumentation. Every absorption spectrometer
must have the three basic component requirements (1) a light source; (2) a sample cell; and (3) a
means of specific light measurement. But, unlike other spectroscopy methods, it has two additional
requirements. These include a specially designed lamp to produce light of a desired wavelength
and a burner to prepare the sample for absorption of light radiation. Additionally, the instrument
also sprays the sample in the solution state over an atomizer(burner). This leads to evaporation of
the solvent and leaves a fine dry residue. This residue has neutral atoms in the ground state. The
sample of interest is aspirated and atomized into the flame. If that metal is present in the sample, its
atoms will absorb some of the light, thus reducing its intensity. This decrease in intensity of the
 P a g e | 17

light is the process of atomic absorption. The instrument measures the change in intensity. A
computer data system converts this change into an absorbance.

i. Only liquid samples can be analyzed

ii. Nonmetal cont. be analyzed
iii. It may be used for quantitative analysis under special circumstances.

An atom absorbs light at discrete wavelengths. So, it is necessary to use a light source, which emits
the specific wavelengths which can be absorbed by the atom. The two most common light sources
used in AA are the ‘‘hollow cathode lamp’’ and the ‘‘electrode less discharge lamp. The
light should be stable and have sufficient intensity and should produce a narrow spectrum with
little background noise. Hollow cathode lamps (HCL) are the most common radiation source in
AAS. It contains a tungsten anode and a hollow cylindrical cathode made up of metal to be
determined. For instance, if sodium is to be analyzed from the sample, a cathode coated with
sodium is used. These are sealed in a glass tube filled with an inert gas like argon or neon which is
ionized by an electric arc. The ions get attracted toward cathodes and strike it leading to excitation
of metal ions. This leads to the emission of radiation with a characteristic wavelength of analyte
metal. The advantage of hollow cathode lamp is that it provides radiation with a bandwidth of
0.001 to 0.01nm. So these lamps give highly specific radiation. The disadvantage of this hollow
cathode lamp is that for every metal different cathode lamp has to be employed.

Nebulizer sucks up the liquid sample at controlled rate and creates fine aerosol spray that mixes
with fuel and oxidant and where it utilized by exposing them to higher thermal energy and
introduce into the flame. The nebulizer uses the combustion flames to atomize and introduce the
sample into the light path. More small the size of the droplets produced, more high will be the
sensitivity of the element tested. Alternating, the gases form can be generated by using induced
coupled plasma (ICP). Atomization is a separation of particles into individual molecules and
breaking molecules into atoms. This is done by exposing the analyte to high temperatures in a
flame or graphite furnace. The (spectroscopic)flames and electro thermal (graphite tube) atomizers
are the two most common atomizers used nowadays. Other atomizers, such as glow discharge
atomization, hydride atomization, or cold-vapor atomization might be used for special purposes.
 P a g e | 18

Flame atomizers is the oldest and most commonly used atomizers. In this atomizer air-acetylene
flame with a temperature of about 2300 °C or air-nitrous oxide flame with a temperature of about
2700 °C are used. Liquid or dissolved samples are typically used with flame atomizers. Inflame
AAS; a steady-state signal is generated during the time period when the sample is aspirated. This
technique is typically used for determinations in the mg L−1 range, and may be extended down to a
few μg L–1 for some elements.

Electro thermal atomizer uses graphite coated furnace to vaporize the sample. The graphite tubes
are heated using a high current power supply. In ET AAS a transient signal is generated, the area of
which is directly proportional to the mass of analyte (not its concentration) introduced into the
graphite tube. This technique has the advantage that any kind of sample, solid, liquid or gaseous,
can be analyzed directly.

Monochromator is a very important part of an AA spectrometer used to select the specific

wavelength of light from the lines emitted by the Hollow cathode lamp and transmit it to the
detector. It not only selects the specific analytical line, but excludes also all other interfering lines
in that region. The selection of specific light allows the determination of the selected elements in
the presence of others.

Schematic diagram of working Atomic

Absorption Spectroscopy

Detector detects the intensity of radiation absorbed by the elements. The detector consists of a
photomultiplier tube or simple photocell. The PMT determines the intensity of photons of the
analytical line exiting the Monochromator. The processing of electrical signal is fulfilled by a
signal amplifier. The signal from the PMT is converted to digital format by a transducer for read-
out, or further fed into data station for printout by the requested format. The unknown
 P a g e | 19

concentration of the element is then calculated from the calibration curve. The absorbance of each
known solution is measured and after that calibration curve of absorbance is plotted against
concentration.

Application:

AAS has both qualitative and quantitative application in different areas. The modern emission
spectrophotometers allow determination of about 20 elements in biological samples, the most
common being calcium, magnesium and manganese. Absorption spectrophotometers are usually
more sensitive than emission instruments and can detect less than 1 p.p.m. of each of the common
elements with the exception of alkali metals. The relative precision is about 1% in a working range
of 20–200 times the detection limit of an element.

• Clinical analysis: This spectroscopy is helpful to analyze metal present in biological fluids such
as blood and urine.
• Environmental analysis: AS has numerous applications in monitoring our environment such as
finding out the levels of various elements in rivers, seawater, drinking water, air, soil, petrol.
• Pharmaceuticals: This technique helpful to detect the impurities in drugs because in some
pharmaceutical manufacturing processes, minute quantities of a catalyst used in the process
(usually a metal) are sometimes present in the final product. By using AAS the amount of catalyst
present can be determined. Now a day, AAS is used to detect the amount of heavy metal present in
synthetic drugs.
• Industry: Many raw materials are examined and AAS is widely used to check that the major
elements are present and that toxic impurities are lower than specified. For example, in concrete,
where calcium is a major constituent, the lead level should be low because it is toxic.
• Mining: Testing the concentration of valuable substances in potential mining areas can be done
by using AAS, such as gold in rocks can be determined to see whether it is worth mining the rock
to extract the gold.

• Food industries: determination of calcium, iron and many other elements present in drinks such
as wine, beer and fruit drinks and quality assurance and contamination testing for food materials
can also be performed with the help of AAS.
 P a g e | 20

UV-VISIBLE SPECTROSCOPY

Ultraviolet–visible spectroscopy refers to absorption spectroscopy or reflectance spectroscopy

concern with ultraviolet and the visible spectral regions of electromagnetic radiation. Means this
technique utilizes visible and adjacent region for analysis work and research into biological
probe. The absorption or reflectance in the visible region affects the observed color of the chemical
involved. In this region of the electromagnetic spectrum, atoms and molecules undergo electronic
transitions. There are four possible transition (𝑛 → 𝜋∗, 𝜋 →𝜋∗, 𝑛 → 𝜎∗ 𝑎𝑛𝑑𝜎 → 𝜎∗) occurs but
only two transition states (𝑛 → 𝜋∗ 𝑎𝑛𝑑𝜋 → 𝜋∗) excited with light form the UV/Vis spectrum for
some biological molecules. The electromagnetic transition into molecules can be classified
according to the participating molecular orbital. UV-Vis spectroscopy is used for both the
quantitative and qualitative determination of different analytes, such as transition metal ions,
highly conjugated organic compounds, and biological macromolecules. In qualitative manner, UV-
VIS spectroscopy is used to identify the functional group or confirm the identity of compound by
matching the absorbance spectrum. The UV extends from 100–400 nm and the visible spectrum
from400–800 nm. The 100–200 nm range is called the deep UV. Light source for deep UV range is
more difficult so it is not commonly used for UV-V is measurements. Spectroscopy analysis is
commonly carried out in solutions but solids and gases may also be studied. The molecular
structures which are responsible for interaction with electromagnetic radiation are called
Chromophore. Some molecular structure such as protein has three types of chromophore, related to
UV/VIS spectra.
• Peptide bond (amide)
• Certain amino acids (tryptophan, tyrosine)
• Certain prosthetic group (porphyrin group in heme)

Principle:

When a photon hits a molecule, it is absorbed by molecules, given by an extinction with itself
depend on the wavelength λ of photon. The photon is promoted into a more excited energetic state.
UV-visible light has enough energy to excite the electrons from ground state to a higher electronic
state. The energy difference between the lower to high energy level is called the band gap. The
energy of the photon must exactly match the band gap for the photon to be absorbed. When the
 P a g e | 21

incident light intensity (Io) passes through a sample with appropriate transparence through the path
length (thickness) l, then the change in intensity occurs this is represented as Observed Intensity
(I). The characteristic absorption parameter for the sample is the extinction coefficient a, yielding
the correlation I= Io. The ratio T= I/Io is called transmission. Thus, molecules with different
chemical structures have different energy band gaps and different absorption spectra. The larger
the band gap between the energy levels, the greater the energy required to promote the electron to
the higher energy level, ultimately resulting in light of higher frequency, and therefore shorter
wavelength, being absorbed.

Absorption Law:

We know the biological samples mainly comprise aqueous solutions; the detection of substance
present in sample is measured in molar concentration c. The transition phenomenon in solution is
governed by Lambert- Beer’s law. The absorption of light by any absorbing material is governed
by two empirical laws. Lambert’s Law states that “when abeam of monochromatic radiation is
passed through the absorbing medium, then the decrease in the intensity of radiation will be
directly proportional to the thickness (path length) of the vessel containing solution. Beer’s Law
states that “when a beam of monochromatic radiation is passed through the absorbing medium the
decrease in the intensity of radiation will be directly proportional to the concentration of solution.”

Thus the combination of Lambert’s law and Beer’s law is

log(Io/I) =Ecl

log10 (Io/I) is the absorption, a (optical density), ℇ is molecular absorptivity (extinction

coefficient), c is concentration of the solution indm-3 and l path length in cm.

Factor affecting UV-Vis absorption:

The Uv- visible spectrum gets affected by the nature of solvent used such as water act as native
solvent for some of compounds like protein and peptide; feels comfortable in aqueous solution for
detection. But the wavelength, about 700-200nm i.e. spectrum of water does not show band
 P a g e | 22

and thus act as silent component of sample. The chromophore partially determines by it chain
structure in absorption spectroscopy because of some factors:

• Protonation/deprotonation (pH, Redox);

• Solvent polarity (dielectric constant of the solvent); and
• Orientation effects.

Beside those factors, the immediate environment of chromophore can be probed by assessing their
absorption that is following:
• Due to bathochromic effect: a wavelength shifts to higher values is called red shift or
bathochromic effect.
• Due to hypochromic effect: when a wavelength shift to lower wavelengths is called blue
shift or hypochromic effect
• Due to increase in hyper chronicity (more color’)
• Due to decrease in absorption (‘less color’)
In addition, the solvent polarity also affects the difference between the ground and excited states.
The orientation effect such as increase in order of nuclei single standard to double standard DNA
leads to different absorption behavior.

Instrumentation:

Ultraviolet- visible spectroscopy involves the spectroscopy of photons in the UV-visible region.
There is an interaction between UV visible light and sample to be analyzed. As a result of this
interaction, some photons (photons of UV-Vis EMR) are absorbed and this absorption of UV
visible is measured by an instrument named UV visible spectrophotometer. It measures the
intensity of light passing out through a sample (I), and compares it to the intensity of light before it
passes through the sample (Io). The ratio (I⁄Io) is called the transmittance, and is usually expressed
as a percentage (%T). From the transmittance (T), the absorbance can be calculated as, A=-logT.
 P a g e | 23

Fig. Schematic diagram of working spectrophotometer

The components of spectrophotometer are as follows.

Light source: Light source is the basic part of spectrophotometer. It must be stable and provide
continuous radiation. Light source must be of the sufficient intensity for the transmitted energy to
be detected at the end of the optical path. Most commonly radiation source used in UV-V is
spectroscopy are as follows.
Deuterium lamp: Deuterium arc lamp, which is continues over the ultraviolet region (190–400
nm). The intensity of radiation of deuterium lamp is 3-5 times than the hydrogen lamp.
Hydrogen discharge lamp: Hydrogen discharge lamp consists of two electrodes contain in
deuterium filled silica envelope. This lamp covers a range from 160-375 nm. These lamps are
stable, robust and widely used.
Tungsten lamps: It is similar to house hold lamp in construction and provide a supply of radiation
in wave length ranging from 320-2500 nm.
Xenon discharge lamp: These are enclosed in a glass tube with quartz or fused silica and xenon
gas is filled under pressure, contains two tungsten electrodes separated by a distance. An
intense arc is formed between electrodes by applying high voltage.
Mercury arc lamps: In mercury arc lamp, mercury vapor is stored under high pressure and
excitation of mercury atom is done by electric discharge.
Monochromator: It is used to remove the radiation of desired wavelength from the wavelength of
continuous spectra. Following types of monochromator are used.
• Filters
 P a g e | 24

• Prisms
• Gratings
Sample compartment: Cells or cuvettes are used for holding liquid sample. Sample holder should
be transparent to the wavelength region to be recorded. Quartz or fused silica cuvettes are required
for spectroscopy in the UV region. Cells may be rectangular in shape or cylindrical with flat ends
generally having 1 cm thickness.
Detector: Detectors convert light energy into electrical signals that are display on read out device.
It measures the absorption of an analyte via the intensity of transmitted light. Mainly three types of
photosensitive devices are used.
• Barrier layer cell / Photovoltaic cell
• Phototubes / Photo emissive tube
• Photomultiplier tube
Recorder: Signals from the detector are finally received by the recording device.

Application of UV-Vis Spectroscopy:

UV-Vis is used in many chemical analyses. It is used to quantitative the amount of protein in a
solution, as most proteins absorb strongly at 280 nm such an example spectrum of cytochrome C,
which has a high absorbance at 280 and also at 450 nm because of a heme group. UV-Vis is also
used as a standard technique to quantify the amount of DNA in a sample, as all the bases absorb
strongly at 260 nm. RNA and proteins also absorb at 260 nm, so absorbance at other wavelengths
can be measured to check for interferences. Specifically, proteins absorb strongly at 280 nm, so the
ratio of absorbance at 280/260 can give a measure of the ratio of protein to DNA in a sample. In
addition, other applications of UV Visible spectroscopy are as follows.

Impurities Analysis:

This spectroscopy helps to determine the impurities in organic molecules. Additional peak in UV-
Vis spectra is due to the presence of impurities in the sample and can be compared with that of
standard raw material. By measuring the absorbance at specific wavelength, impurities can be
detected. For example, benzene appears as common impurity in cyclohexane which can easily
detected as benzene shows absorption at255nm. UV-Vis spectroscopy is useful in elucidation of
structure of organic compounds, presence and absence of unsaturation and heteroatoms. Common
 P a g e | 25

applications for difference UV spectroscopy include the determination of the number of aromatic
amino acids exposed to solvent, detection of conformational changes occurring in proteins,
detection of aromatic amino acids in active sites of enzymes, and monitoring of reactions involving
‘catalytic’ chromophores (prosthetic groups, coenzymes).

Qualitative Analysis:

The qualitative analysis is carried out by the UV-Vis spectroscopy when the atom or molecules
absorb UV radiation and identification is done by comparing the absorption spectra with the
spectra of known compound. Qualitative analysis is done in UV/Vis regions to identify certain
classes of compounds both in the pure state and in biological mixtures (e.g. protein-bound). The
application of UV/Vis spectroscopy to further analytical purposes is rather limited, but possible for
systems where appropriate features and parameters are known. Most commonly, this type of
spectroscopy is used for quantification of biological samples either directly or via colorimetric
assays. In many cases, proteins can be quantified directly using their intrinsic chromophores,
tyrosine and tryptophan. The characteristic picks in protein spectrum are a band at278/280 nm and
another at 190 nm. The region from 500 to 300nmprovides valuable information about the
presence of any prosthetic groups or coenzymes. Protein quantification by single wavelength
measurements at 280 and 260nm only should be avoided, as the presence of larger aggregates
(contaminations or protein aggregates) gives rise to considerable Rayleigh scattering.

Difference spectra:
Difference spectrum is obtained by subtracting one absorption spectrum from another. Difference
spectra can be obtained in two ways: either by subtraction of one absolute absorption spectrum
from another, or by placing one sample in the reference cuvette and another in the test
cuvette. Difference spectra have three distinct features as compared to absolute spectra

• difference spectra may contain negative absorbance values;

• absorption maxima and minima may be displaced and the extinction coefficients are
different from those in peaks of absolute spectra;
• there are points of zero absorbance, usually accompanied by a change of sign of the
absorbance values. These points are observed at wavelengths where both species of related
 P a g e | 26

molecules exhibit identical absorbance’s and which may be used for checking for the
presence of interfering substances

NUCLEAR MAGNETIC RESONANCE

OVERVIEW

NMR stands for Nuclear Magnetic Resonance it is another form of absorption spectroscopy
because under magnetic condition the sample absorbs certain wavelength of electromagnetic
radiations. NMR is an analytical tool to determine the structure and purity of a sample as well as its
molecular structure. Most studies in organic chemistry involve the use of 1H, but NMR
spectroscopy with 13C, 15N and 31P isotopes is frequently used in biochemical studies. The
resonance condition in NMR is satisfied in an external magnetic field of several hundred mT, with
absorption occurring in the region of radio waves (frequency 40 MHz) for resonance of the 1H
nucleus. However, in the NMR the magnet involved is not electron but nuclei of atom of element.
Once the basic structure is known, NMR can be used to determine molecular conformation in
solution in which the studying physical properties such as conformational exchange, phase
changes, solubility, and diffusion are determined. The nuclei of molecules give rise to spectrum
which absorbs electromagnetic radiation under magnetic condition. Most of the study shows in
organic chemistry involve the use of 1H NMR but NMR study with 13C, 15N and 31P is
frequently used in biochemical study. While the proton of nuclei is put under magnetic conditions.
The proton present in nuclei shows spin due to absorption of radio wave and act like small magnet.
Resonance in this all magnate show spin and process is called NMR in other word we can say that,
the nuclear magnetic resonance is the phenomenon of nucleolus in which proton and neutron spin
about the axis due to electromagnetic
radiation. All nuclei carry a charge. In some nuclei charge “spin” on the nuclear axis and it
circulation of nuclear charge generates a magnetic dipole along the axis. The angular moment of
spinning charge can be describing in term of quantum spin numbers I; these have number values
of0, ½,1, 3/2 and so on. The intrinsic magnitude of generated dipole is expressed in term of
magnetic moments μ. The H-nucleus is the most commonly studied by NMR spectroscopy because
of its high natural abundance of 99.985% and its presence in the majority of organic compounds.
NMR studying 1H atom is called Hydrogen or proton NMR spectrum. The proton NMR spectrum
 P a g e | 27

gives the information about the number of different types of protons and also the chemical
environment around it.

13C NMR

Carbon forms the backbone of the organic molecules therefore CNMR is also important and gives
valuable information about the molecule. The 13C NMR is generated in the same fundamental
principle such as proton PNMR spectrum. Only 1.1 % of naturally occurring carbon is 13Cand
actually an advantage because of less coupling. In 13C NMR spectrum is occurs directly due to
present of carbon skeleton in the molecule and the proton present in molecules does not play
direct role. The 12C do not absorb radio frequency energy but the other isotope i.e. 13C absorbs
the energy but the 13C has a natural abundance of only about 1%. Therefore, the sensitivity of 13C
is less than the 1H NMR and requires longer time to record

There is some point regarding 13C NMR as follows:

• The number of signals tell us how many different carbons or set of equivalent carbons
• The splitting of a signal tells us how much hydrogen is attached to each carbon. (N+1 rule)
• The chemical shift tells us the hybridization (sp3, sp2, sp) of each carbon.

PRINCIPLE

The principle behind NMR is that many nuclei have charge “spin “when they have odd number of
proton in their nuclei. When an external magnetic field applied and the nuclear spin occurs, the
energy transfer is possible between the base energy to a higher energy level. The energy
transfer takes place at a wavelength that corresponds to radio frequencies and when the spin returns
to its base level, energy is emitted at the same frequency. The signal that matches this transfer is
measured in several ways and processed in order to yield an NMR spectrum. The molecular
environment of proton governs the value to applied external field at which the nucleuses resonate.
This gives recorded as chemical shift. The chemical field rises from the applied field including
secondary field of about 0.15 – 0.2 mT at proton by interacting with adjacent bonding electron.
Nuclear magnetic moment μ = gNuN[I (I + 1)]1/2
 P a g e | 28

Where I is angular momentum

Also nuclear spin angular momentum = [I(I + 1)]1/2 h/2􀀀
ΔE = hv guNBo (bore-Einstein)
gN = Nuclear g factor, characteristic nucleus
μN = nuclear magnetron = 5.05X 10-27 J T-1
B o = External magnetic field in tesla

Schematic diagram of
NMR

INSTRUMENTATION

In NMR the samples in solution are contained in sealed tubes which are rotated rapidly in the
cavity to eliminate irregularities and imperfections in sample distribution. In solid samples, the
number of spin–spin interactions are greatly enhanced due to intermolecular interactions that are
absent in dissolved samples due to translation and rotation movements. As a result, the resonance
signals broaden significantly.

However, high-resolution spectra can be obtained by spinning the solid sample at an angle of 54.7˚.
Advanced computer facilities are needed for operation of NMR instruments, as well as analysis of
the acquired spectra.

APPLICATIONS
NMR spectroscopy is the use of NMR phenomena to study the physical, chemical, and biological
properties of matter. Chemists use it to determine molecular identity and structure. In each carbon
 P a g e | 29

the multiplicity of the signal depends upon how many protons are attached to it. The 13C-13C
coupling is not possible while 1H-13C coupling is possible.

Molecular structure determination:

The NMR is very useful method to structure determination for organic compounds. When the
proton or carbon exhibit in the similar chemical environment it gives similar signal. In this case the
chemical shift provides a clue about the environment of a particular proton or carbon, and thus
allows conclusions as to the nature of functional groups. Spin–spin interactions allow conclusions
as to how protons are linked with the carbon skeleton. For structure determination, the fine
structure usually is the most useful information because it provides a unique criterion while
chemical shifts of some groups can vary over an extended range. The structures of proteins up to a
mass of about 50 kDa can be determined with bimolecular NMR spectroscopy. The development
of magnets with very high field strengths (currently 900 MHz) continues to push the size limit.
Heteronuclear multidimensional NMR spectra need to be recorded for the assignment of all
chemical shifts (1H, 13C, 15N). For inter proton NOEs,13C- and 15N-edited 3D NOESY spectra
are required.

Magnetic resonance imaging:

The NMR spectroscopy is very useful in the imaging of live samples because the proton is one of
the more sensitive nuclides and is present in all biological systems abundantly. The 1H NMR
spectroscopy has significance role in the imaging of live samples. The most important compound
in biological samples in this context is water. It is distributed differently in different tissues, but
constitutes about 55% of body mass in the average human. In NMR, the resonance frequency of a
particular nuclide is proportional to the strength of the applied external magnetic field. If an
external magnetic field gradient is applied then a range of resonant frequencies are observed,
reflecting the spatial distribution of the spinning nuclei. Magnetic resonance imaging (MRI) can be
applied to large volumes in whole living organisms and has a central role in routine clinical
imaging of large-volume soft tissues. The number of spins in a particular defined spatial region
gives rise to the spin density as an observable parameter. This measure can be combined with
analysis of the principal relaxation times (T1 and T2). The imaging of flux, as either bulk flow or
 P a g e | 30

localized diffusion, adds considerably to the options available. Inters of whole-body scanners, the
entire picture is reconstructed from images generated in contiguous slices. MRI can be applied to
the whole body or specific organ investigations on head, thorax, abdomen, liver, pancreas, kidney
and musculoskeletal regions. The use of contrast agents with paramagnetic properties has enabled
investigation of organ function, as well as blood flow, tissue perfusion, transport across the blood–
brain barrier and vascular anatomy. Resolution and image contrast are major considerations for the
technique and subject to continuing development. The resolution depends on the strength of the
magnetic field and the availability of labels that yield high signal strengths.
 P a g e | 31

CHROMATOGRAPHY

• Paper chromatography and Thin Layer Chromatography

• Column Chromatography
• Gas Chromatography
Topics • High Performance Liquid Chromatography
• Ion exchange chromatography
• Affinity Chromatography
• Gel Filtration Chromatography

The word chromatography is derived from the Greek words for color and write. It quite literally
means ‘to write with color’. Chromatography is based on the broad field of separation sciences.
Very simply, this field of science focuses on how to separate the chemicals contained within a
mixture. chromatographic techniques focus on identifying the components of a mixture and
determining how much of each component exists within a sample.

Chromatography is an analytical technique that can be used for qualitative or quantitative analysis.
It can both identify components in a mixture and determine how much of each component is
present.

Chromatography has a wide range of uses, including in:

• pharmaceuticals science – identifying elements and molecules in drugs as well as measuring the
purity of medicines
• environmental science – analyzing water quality and gases in the air and the impact they may
have on Earth
• police work – analysis such as breathalyzers and drug detection
• forensic science and archaeology – analyzing material such as blood and hair samples
 P a g e | 32

• biomedical research – analyzing material such as proteins in cancer research

• food science – analyzing the nutritional quality of food and food spoilage.

Stationary and mobile phases

All chromatography uses two phases to separate a mixture. These are the:

• Mobile phase, which is a solvent, that moves over or through the stationary phase

• Stationary phase, which is either a solid with a high surface area or a liquid coated onto a
solid support. It always stays still.

The mobile phase moves through and carries a sample (solute) over the stationary phase.
Components of the sample are more attracted to either the mobile or the stationary phase,
depending on their intermolecular forces. This makes the components move at different rates and
separates them. Several key terms are used to describe this attraction.
• Affinity: components of a sample are attracted, by intermolecular forces, to the mobile
or stationary phase. High affinity means that the component interacts strongly with the
mobile or stationary phase.
• Adsorption: components of the sample adsorb onto the stationary phase from the mobile
phase if they are attracted to, or have an affinity for, the stationary phase.
• Desorption: components of the sample desorb off the stationary phase into the mobile
phase if they are attracted to, or have an affinity for, the mobile phase.

PAPER CHROMATOGRAPHY AND THIN –LAYER CHROMATOGRAPHY

In paper chromatography and thin- layer chromatography (TLC), a sample containing

a mixture of substances is applied to the stationary phase as a spot. The spot is carried up the
stationary phase by the mobile phase. The resulting chromatogram is usually qualitative in
nature – it can only be used to identify substances; not determine how much is present.
 P a g e | 33

Paper chromatography

In paper chromatography, the stationary phase is a thin strip of absorbent paper, which is cut
to fit inside a container that holds the mobile phase. A pencil line is ruled across
the bottom of the paper to mark where the sample will be placed – this is called the origin.
Pen or ink should not be used to mark the origin because inks will separate and contaminate
the sample. The sample is placed on the paper and standards of known identity can also be
added to help identify the substances in the sample.

The mobile phase is added to the

container so that it sits below the
origin line. The paper is
then placed into the container
and left to adsorb the mobile
phase. The sample travels up the
piece of paper, separating into
its components.

Components of the sample that have a strong affinity for the stationary phase move slowly.
On the resulting chromatogram, they have not moved far from the origin. Components that have a
higher affinity for the mobile phase move faster. On the resulting chromatogram, they have moved
further and are located closer to the solvent front.
The mobile phase is water and the stationary phase is filter paper. Paper is a derivative of
cellulose, which contains many polar – OH groups. However, very few of the intermolecular forces
of paper extend beyond its network of fibers (and any surface coating).
Water is also a polar molecule, but more polar than the paper. Therefore, any component of
the sample that has whole or partial charges, such as ionic or polar substances, will have a
higher affinity, or attraction, for the water mobile phase. These components move further
from the origin. Any component of the sample that experiences dispersion forces has a higher
affinity for the stationary phase and will not move as far from the origin.
 P a g e | 34

Retardation factor (Rf ) calculations

Once the separation is completed, the resulting chromatogram is analyzed to identify the
substances in the sample and to determine the purity of the sample. Although we can simply
look at a chromatogram to judge whether two substances are identical, it is more precise to
calculate the retardation factor (Rf) of a substance. This is the ratio of the distance moved
by a substance to the distance moved by the solvent, or mobile phase.
This can be simplified to:
Rf = distance solute moves from origin = distance of sample spot/distance of mobile phase
distance solvent moves from origin
Each atom, ion or molecule has a unique Rf depending on the properties of the mobile and
stationary phases. Increasing the polarity of the mobile phase increases its affinity to charged
particles within the sample. This makes the substances move further up the paper.
All Rf values must be expressed as a decimal (never a fraction) and cannot be greater than or
equal to one. An Rf of 1 indicates that the substance has not separated from the mobile phase and
therefore cannot be identified. The analysis must be run again under different mobile or stationary
phase conditions.

Thin-layer chromatography

TLC works by the same principles as paper chromatography with one major change that
makes the process more efficient (faster and more sensitive). In TLC, the support for the
stationary phase is a piece of glass or plastic, which is coated in the stationary phase,
consisting of silica gel, aluminum oxide or cellulose. This coating is a thin layer spread on
the surface of the plastic or glass; hence, the name ‘thin- layer’ chromatography.
The components within the sample are separated, as with paper chromatography,
according to their affinity for the mobile or stationary phase. It is important to note that both paper
chromatography and TLC are not limited to colored compounds. Fluorescent TLC plates can be
used under UV light to see the components of a sample that would not otherwise be visible. These
are shown in the darker areas of the plate where the sample blocks the fluorescence of the plate.
 P a g e | 35

Two-dimensional paper or thin- layer chromatography

Occasionally, a chromatography technique will not separate some components sufficiently
within the sample. This is because the components interact the same amount with the
mobile and stationary phases. Rather than repeating the analysis, chemists will rotate the
chromatogram by 90°, so that the sample is on the bottom. They rule a new origin line and
run the analysis again, using a different mobile phase with different properties.
The chromatogram is analyzed by using Rf.

Column chromatography

Column chromatography is a more advanced form of separation science than paper and thin- layer
chromatography. It uses the same basic principles. But the stationary phase is contained within a
tube, open at both ends. The simplest form of column chromatography uses a glass column, similar
to a burette, which contains a stationary phase of aluminum oxide or silica. The stationary phase
resembles finely ground sand. The stationary phase must be packed tightly to minimize pore spaces
(gaps). Otherwise, the sample will move too fast and will not interact with the stationary phase.
This means that less separation will occur.

The mobile phase is poured into the top of the column and allowed to run slowly down through the
stationary phase until it reaches the tap at the bottom. Once the column is soaked in the mobile
phase, the sample is added to the top of the column and the tap at the bottom is opened. This allows
the mobile phase to run through the column with the sample. The sample separates into its various
components depending on their affinities for the mobile or stationary phases. A higher affinity for
the mobile phase means that a component spends longer interacting with it. As the mobile phase
flows through the column, the component moves with it and it is collected earlier
 P a g e | 36

.
Schematic diagram of Column chromatography

The various components of the sample are collected in separate beakers as they elute, or
come out of, the column. Once the components are separated, further techniques are used to
identify and quantify them. There are several types of column chromatography. The two most
commonly used are gas chromatography and high-performance liquid chromatography (HPLC).

Gas chromatography
In gas chromatography, the mobile phase is an inert gas (carrier gas), such as nitrogen(N2). The
sample is vaporized and injected into the mobile phase, which carries the sample through a very
small (1– 2 mm diameter) and very long (up to 60 m or longer) column containing the stationary
phase. The stationary phase can be either a solid, packed into the column, or a liquid that
coats the inside of the column (Figure 3). Both stationary phases must be able to withstand very
high temperatures because the column is contained within an oven to ensure that the sample
remains vaporized throughout the separation. The sample separates as each component interacts
with the stationary phase. There is no attraction to the mobile phase because it is an inert gas; its
purpose is to move the sample through the column. The components with a low affinity for the
stationary phase elutes from the column first – they are not retained on the column for long.
The components with a high affinity for the stationary phase are retained on the column
for a longer period of time. The resulting chromatogram is obtained when the detector recognizes
the components as they elute from the column. Each component forms a peak in the chromatogram
as it elutes.
 P a g e | 37

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

HPLC stands for high-performance liquid chromatography. As its name suggest, high performance
means that this analytical technique used for proper separation, identification and for quantification
of components from the mixture. HPLC is an instrumental form of liquid chromatography and
gives high performance due to the small particle size of the stationary phase. The particle size of
stationary phase is 3.5 to 10 micrometer. Due to smaller size, surface area of the particle is high
and ultimately HETP (height equivalent to theoretical pressure) increased and thereby help in
achieving more efficient separation of the components of the mixture than those used in
conventional liquid chromatography.

In this chromatography particle size of stationary phase is small and due to small size, packing of
stationary phase will be high. Due to tight packing flow rate of mobile phase is reduced. So that to
increase the flow rate or to increase efficiency or to increase the separation of the mixture high
pressure is applied. The applied pressure is about 1000-4000psi. Because of the use of high
pressure in this technique, it is sometimes also known as high pressure liquid chromatography. So
we can say HPLC is modern application of liquid chromatography. HPLC has the ability to
separate, and identify compounds that are present in any sample that can be dissolved in a liquid in
trace concentrations as low as parts per trillion. Because of this versatility, HPLC is used in a
variety of industrial and scientific applications, such as pharmaceutical, environmental, forensics,
and chemicals.
Principle:
HPLC is highly automated and extremely sensitive technique as compared to column
chromatography because the components of a mixture are separated from each other due to their
different degrees of interaction with the absorbent particles. This causes different elution rates for
the different components and leads to the separation of the components as they flow out from the
column. Solvents are used as mobile phase. In general organic compounds are analyzed using
HPLC and these organic compounds are soluble in polar solvents. Some commonly used solvents
as mobile phase are methanol, acetonitrile and water. Acidifiers or basifies or buffer solution are
added to the solvent to achieve better separation. As these neutralize the ionized analytes or
compounds present in the column. If analyte or compounds present in column will be ionized so
affinity with stationary phase will be lost and proper separation does not take place. Means every
 P a g e | 38

analyte immediately come with the mobile phase. Particle size of stationary phase ranges 3.5 to 10
micrometer. There are two types of stationary phase. i) Normal stationary phase for example silica
gel is used and ii) Reversed stationary phase for example octal decyl silane silica gel etc. There are
generally two types of columns are used. Normal phase column and reversed phase column.

Schematic Diagram of HPLC

Application:

• In Environment: HPLC can be use in detection of phenolic compounds and other

contaminants present in drinking water. It also behaves as bio-monitor of pollutants.
• In Forensics Science: This separation technique is used to determine quantify of steroids,
cocaine and other drugs in blood, urine etc. Forensic analysis of textile dyes can also be
performed by using this technique.
• In Food and Flavor: It helps in measurement of quality of soft drinks and water, sugar
analysis in fruit juices, polycyclic compounds analysis in vegetables. Preservative analysis
can also be done by this technique.
• In clinical diagnosis and health industry: HPLC is used in Urine analysis, antibiotics
analysis in blood. Many disorders related to body metabolism, those related to endocrine
and exocrine gland secretion, alteration in body fluids are diagnosed by HPLC analysis of
concerned fluids. For example, detection of bilirubin, biliverdin in hepatic disorders and
endogenous Neuropeptides in extracellular fluid of brain can be performed by using HPLC.
 P a g e | 39

ION EXCHANGE CHROMATROGRAPHY

As it name suggest ion exchange chromatography, means ion exchanger resin will be there and due
to presence of that resin ion exchange will take place i.e. sample is ionized and that sample will
exchange with counter ion present in stationary phase and ultimately separation will take place.
This technique separates charged or polar molecule in a mixture. Only hydrophilic molecules can
be separated out from this technology. In this type of chromatography separation
occurs as a result of formation of ionic or electrostatic bond between the charged group of
biomolecules and an ion exchange resin bearing opposite charge. Ions exist in a state of
equilibrium between the mobile phase and stationary phases which give rise two possible formats,
anion and cation exchange are referred to as counter ion (Fig. 2.1). These exchangeable matrix
counter ions may include protons (H+), hydroxide groups (OH-), single charged mono atomic ions
(Na+, K+, Cl-), double charged mono atomic ions (Ca2+, Mg2+), and polyatomic inorganic ions
(SO42-, PO43-) as well as organic bases (NR2H+) and acids (COO-). Cations are separated on cation
exchange resin column and anions on an anion exchange resin column. It is one of the most
important adsorption chromatography, performed for separation of peptides, proteins, nucleic acids
and related biopolymers which have different molecular sizes and molecular nature with electronic
charge. Advantage of using ion chromatography is that only one interaction involved during the
separation as compared to other separation techniques; therefore, ion chromatography may have
higher matrix tolerance.

Principle:

Ion exchange chromatography is based on the reversible electrostatic interaction of charged species
with the ion exchange matrix and ultimately separation takes place. On the basis of ions separated,
the ion exchange chromatography can be divided into two categories.

a. Anion Exchange Chromatography:

Regin-OH- + A Regin-A + OH
(in solution) (in eluting solution)
 P a g e | 40

When molecule of interest is negatively charged then anion exchange chromatography is used. In
this process anion in a mobile (liquid) phase exchanges with another anion that is previously bound
to a positively charged solid support or matrix. Commonly used anion exchange resins are Q-resin,
a Quaternary amine; and DEAE resin, Diethylaminoethane. Anion exchange chromatography is
used both for preparative and analytical purposes and may also be used chromatographically, to
separate anions and medicinally to remove an anion from gastric contents or bile acids in the
intestine.

b. Cation Exchange Chromatography:

Regin- H+ + M+ Regin- M+ + H+
(in solution) (in eluting solution)

Cation exchange chromatography is used when the desired molecules to separate are cations in
mobile phase. Positively charged molecules are attracted to a negatively charged solid support. S-
resin, sulfate derivatives; and CM resins, carboxylate derived ions are commonly used as cation
exchange resins. This type of chromatography is used both for preparative and analytical purposes
and can separate a large range of molecules from amino acids and nucleotides to large proteins.

AFFINITY CHROMATOGRAPHY

Affinity chromatography is also called bio-affinity chromatography. It is one type of liquid

chromatography that depends upon the reversible adsorption of biomolecules in a biochemical
mixture through bispecific interaction on the ligands. This type of interaction may take place
between antigen and antibody, enzyme and substrate, receptor and ligand, or protein and nucleic
acid. These interactions which are used for purification by placing one of the interacting molecules,
referred to as affinity ligand, onto a solid matrix to create a stationary phase while the target
molecule is in the mobile phase. The high selectivity of affinity chromatography is take place by
allowing the desired molecule to interact with the stationary phase and be bound within the column
in order to be separated from the undesired material which will not interact with ligand and elute
first.
Affinity chromatography is one of the most powerful and unique technique used for the
purification and isolation of biological molecules on the basis of its biological function or
 P a g e | 41

individual chemical structure. As a result, purifications that would otherwise be time consuming
and complicated, can often be easily achieved with affinity chromatography.
This technique offers high selectivity, resolution, and capacity in most protein purification
schemes. This chromatography can be used to purify and concentrate a substance from a chemical
mixture into a buffering solution, reduce the amount of unwanted substances in a mixture, identify
the biological compounds binding to a particular substance, purify and concentrate an enzyme
solution. The molecule of interest can be immobilized through covalent bonds. In bio affinity
chromatography the stationary phase is solid modified resin and the mobile phase is liquid
containing sample mixture or buffer is used.

Principle:

The principle of affinity chromatography involves highly specific biological or chemical

interaction between an immobilized ligand and a desired target molecule, which can bind
selectively and interact with the target even in complex solutions containing many other
components. So due to this specific interaction others molecules or contamination present in
mixture are eluted while target molecules are separated out from all other molecules that cannot
bind the ligand.

Schematic diagram of Affinity

Chromatography

GEL FILTRATION CHROMATOGRAPHY

Gel filtration chromatography is a type of size exclusion chromatography, which separate molecule
according to their size and shape. With some exceptions, the separation of the components in the
 P a g e | 42

sample mixture correlates with their molecular weight. In these cases, gel filtration

chromatography can be worked as analytical tool to find out the molecular weight of an
uncharacterized molecule. It is also an important preparative technique which can be used to
separate protein, peptides and oligonucleotides on the basis of size. In gel filtration
chromatography separation is achieved by physical means, which make it differ from other types
of chromatography. As gel filtration chromatography does not involve any interaction of the
sample or the solvent with the matrix in a column. Aqueous solution or buffer solution used as
mobile phase in gel filtration chromatography. Gel consisting of porous beads or carbohydrates
cross linking agents is used as stationary phase. Most commonly used gels are dextrin (sephadex),
agarose(sepharose) and polyacrylamide (Bio Gel).

Principle:

Separation in gel filtration chromatography is based on the differences in sizes from biomolecules
as they pass through a column packed with a chromatographic medium or stationary phase, which
is a gel. So the larger size molecules separate out first from the sample solution, after which
smaller size molecules separated.
 P a g e | 43

ELECTROPHORESIS

• Principle of Electrophoresis
• Gel Electrophoresis
Topics • SDS-Polyacrylamide Gel Electrophoresis
• Native Page
• Agarose Gel Electrophoresis

Electrophoresis is the process of moving charged molecules in solution by applying an electric

field across the mixture. Because in an electric field move with a speed dependent on their charge,
shape, and size. Electrophoresis has been extensively developed for molecular separations. The
electrophoresis is useful in the determination of the number, amount and mobility of components
in a given sample, to obtain information about the electrical double layers surrounding the
particles. It is also useful into determination of molecular weight of proteins and DNA sequencing.
The electrophoresis of different types as mentioned in

GENERAL PRINCIPLE OF ELECTROPHORESIS

Electrophoretic literally means running in the electric field. The charge molecule moves their
counter charge electrode but electric field is removed before it reaches the electrode. Charge
 P a g e | 44

species movement in an electric field gives differential mobility to the sample based on the charge
and consequently resolves them. The moment of charged particles is retarded with the
addition of a polymeric gel so that a different time is available for resolving the sample. Polymeric
gel is inert, uncharged and does not cause retardation by different size (depending on the
concentration of polymer) and sample pass through the pore and as a result their electrophoretic
mobility is reduced.

If migration velocity is 𝜐and E is applied electric field strength them electrophoretic mobility μ, Μ
is positive or negative while neutral
species have on mobility.
μ = υ/E
υ = EQ/ηπ

where Q= charge of molecule; E = magnetite of applied potential, η = viscosity of medium; π =

shape of molecule in terms of radius. Μ is +Ve and, –Ve while neutral species have no mobility.
Different types of electrophoresis techniques are designated depending upon whether it carried out
in the presence or absence of a supporting media.

GEL ELECTROPHORESIS

Principle of gel electrophoresis:

When we place any charged molecules in an electric field, they move toward the positive or
negative pole according to the charge they are having. Proteins do not have any net charge whereas
nucleic acids have a negative charge so they move towards the anode when electric field is applied.

Factors affecting gel electrophoresis:

• How much charge the particles have

• What is the molecular weight?
• Secondary structures (i.e., its shape).
• pH of solution
• Electric field
• Solution viscosity
• Temperature
 P a g e | 45

Gel electrophoresis is a technique used for the separation of Deoxyribonucleic acid (DNA),
Ribonucleic acid (RNA) or protein molecules according to their size and electrical charge using an
electric current applied to a gel matrix. Gel is a cross linked polymer whose composition and
porosity is chosen based on the specific weight and porosity of the target molecules. Types of gels
used are as

i) Polyacrylamide gel:

Polyacrylamide gel electrophoresis (PAGE) is a technique widely used in biochemistry, forensic

chemistry, genetics, molecular biology and biotechnology to separate biological macromolecules,
usually proteins or nucleic acids, according to their electrophoretic mobility.
ii) Agarose gel:

Agarose is a polysaccharide extracted from seaweed (Gelidium)and contains many agarobiose

subunits. During solidification, agarose form a network of polymers and its pore sizes can be
determined by its concentration. It is usually used at concentrations of 0.5 to 2%. Stiffer gel
means the agarose concentration is higher. Heat agarose with buffer to prepare gel and after it
cools down it is poured in to the tray called as casting tray. These gels are not toxic unlike
acrylamide gels. Range of separation in agarose gels is higher but resolving power is low.

SDS-POLYACRYLAMIDE GELELECTROPHORESIS (SDS-PAGE)

SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) is the most widely used method for
analyzing protein mixtures qualitatively. It is particularly useful for monitoring protein
purification. The SDS-PAGE method is based on the separation of proteins according to their size;
it can also be used to determine the relative molecular mass of proteins. When proteins are
separated by electrophoresis through a gel matrix, smaller proteins migrate faster due to less
resistance from the gel matrix. Other influences on the rate of migration through the gel matrix
include the structure and charge of the proteins. In SDS-PAGE, the use of sodium dodecyl sulfate
(SDS, also known as sodium lauryl sulfate) and polyacrylamide gel largely eliminates the influence
of the structure and charge. SDS is an anionic detergent. Samples to be run on SDS–PAGE are
firstly boiled for 5 min in sample buffer containing b-mercaptoethanol and SDS. Each protein in
 P a g e | 46

the mixture is therefore fully denatured by this treatment and opens up into a rod-shaped structure
with a series of negatively charged SDS molecules along the polypeptide chain. On average, one
SDS molecule binds for every two amino acid residues. The original native charge on the molecule
is therefore completely swamped by the negatively charged SDS molecules. By heating the protein
sample between 70-100°C in the presence of excess SDS and thiol reagent, disulfide bonds are
cleaved, and the protein is fully dissociated into its subunits. In the presence of SDS and a reducing
agent that cleaves disulfide bonds critical for proper folding, proteins unfold into linear chains with
negative charge proportional to polypeptide chain length. Polymerized acrylamide
(polyacrylamide) forms a mesh-like matrix suitable for the separation of proteins of typical size.
Protein is separated based on their polypeptide chain length by electrophoresis in a polyacrylamide
gel with an appropriate size.
The negatively charged protein–SDS complexes move towards the anode because they have the
same charge per unit length, they travel into the separating gel under the applied electric field with
the same mobility. However, as they pass through the separating gel the proteins separate, owing to
the molecular sieving properties of the gel.

Application

• Measuring molecular weight.

• Peptide mapping.
• Estimation of protein size.
• Determination of protein subunits or aggregation structures.
• Estimation of protein purity.
• Protein quantization.

• Monitoring protein integrity.

• Comparison of the polypeptide and composition of different
samples.
• Analysis of the number and size of polypeptide subunits.
• Post-electrophoresis applications, such as Western blotting.
• Staining of Proteins in Gels with Coomassie G-250 without
Organic Solvent and Acetic Acid.
 P a g e | 47

• Pouring and Running a Protein Gel by reusing Commercial

Cassettes.

NATIVE PAGE

Native PAGE, separates acidic water-soluble and membrane proteins in a polyacrylamide gradient
gel. In native PAGE, proteins are separated according to the net negative charge, size, and shape of
their native structure. Due to present of net negative charge in alkaline running buffers the protein
molecules are electrophoretic migrate. The electrophoretic migration depends on the negative
charge density. If protein has high negative charge density, then it will migrate faster than less
negative charge density. At the same time, the frictional force of the gel matrix creates a sieving
effect, regulating the movement of proteins according to their size and three-dimensional shape.
Small proteins face only a small frictional force, while larger proteins face a larger frictional
force. Thus native PAGE separates proteins based upon both their charge and mass. However, the
no denaturants are used in native PAGE, subunit interactions within a multimeric protein are
generally retained and information can be gained about the quaternary structure. In addition,
some proteins retain their enzymatic activity (function) following separation by native PAGE.
Thus, this technique may be used for preparation of purified, active proteins.

AGAROSE GEL ELECTROPHORESIS FOR DNA

Agarose gel electrophoresis is performed in a continuous fashion with both electrodes and gel
cassette submerged within the buffer. The electrophoreses chamber has two platinum electrodes
placed on the both ends connected to the external power supply from a power pack which supplies
a direct current or DC voltage. The tank filled with the running buffer and the gel casted is
submerged inside the buffer. There are additional accessories needed for casting the agarose gel
such as comb, spacer, gel, caster etc.

The purpose of each reagent used in horizontal gel electrophoreses are

1. Agarose – Polymeric sugar used to prepare horizontal gel for DNA Analysis.
2. Ethidium bromide – for staining of loading dye for horizontal gel to the DNA.
3. Sucrose-for preparation of loading dye for horizontal gel
 P a g e | 48

4. Tris HCl- The component of the running buffer.

5. Bromophenol blue – Tracking dye to monitor the progress of the electrophoresis.
The agarose powder is dissolved in a buffer and heated to melt the agarose. Hot agarose is poured
into gel cassette and allowed in to set. A comb can be insert in to the hot agarose to cast the well
for loading the sample. In some cases, Ethidium bromide is added within the gel so that it stains the
DNA while electrophoresis.

Different steps in casting of the agarose gel for horizontal electrophoresis

The gel cassette is placed in the electrophoresis tank submerged completely and DNA loaded in to
the well with the help of pipet and run with the constant voltage. DNA runs from negative to
positive end and Ethidium bromide present in the gel stain the DNA Observing the agarose
gel in a UV chamber shown the DNA stained with ether as organ color fluorescence.

Observation of DNA stained with etbrina U.V. Chamber

For DNA agarose gel electrophoresis can be determined by comparing the size of the known DNA
molecules. The DNA of known size is resolved on 0.8 % agarose along with the unknown sample.
The value of the relative migration (R f) of each DNA band is calculated from the agarose gel, the
values of relative migration (R f) and size of the DNA is used to draw the calibration curve to
calculate the size of the unknown DNA sample. DNA protein introduction – DNA is negatively
charged molecule and it interact with positively charged protein to form DNA – protein complex.
The size and hydrodynamic volume changes. DNA + Protein DNA-Protein The DNA protein
interaction, a fixed amount of DNA is incubated with the increasing concentration of protein. Due
to the formation of DNA protein complex. The hydrodynamic volume of the complex increases
and a shift in band is observed. The DNA has an extended structure and it provide docking site for
several protein molecules such as single stranded binding protein. Gradual shift in DNA band will
be observed until the DNA binding site is not saturated with the protein molecules. The DNA
proteins interaction will be able to several aspects-Whether protein–X has a affinity for DNA and
interaction is specific in nature.
 P a g e | 49

CENTRIFUGATION

• Principle of Centrifugation
Topics • Differential Centrifugation
• Ultracentrifugation

The centrifugation is the method of separating two things with different density, by rapid rotating it
in a circular motion which eventually, separates the less dense and denser particles. It forces less
dense particles to come out on the surface of the mixture. The centrifugation force is play
significance role to separate different density particles over the time. In practice, centrifugal force
is necessary to separate most of the particles. The rate of separation in a suspension of
particles by means of gravitational force mainly depends on the particles, size and density.
Particles of higher density or large size typically travel at a faster rate and at some point will be
separated from particles less dense or smaller. This sedimentation rate of particles, which describes
the movement of a sphere in a gravitational field, is shown in equation 1, which calculates the
velocity of sedimentation.

𝜐 = 𝑑2(𝑝−𝐿) 𝑔/18 𝑛

𝜐= sedimentation rate or velocity of the sphere

d = diameter of the sphere
p = particle density
L = medium density
n = velocity of medium
g = gravitational force

PRINCIPAL OF CENTRIFUGATION

A centrifuge is a device for separating particles for the solution according to their size, shape,
density, velocity of medium and rotor speed. In a solution, particles whose density is higher than
 P a g e | 50

that of the solvent sink and particles that are lighter than it floats to the top. However, those
particles which have size more than 5 μm are considered in sedimentation at the bottom due to
gravitational force. If the size of particles is less than 5 μm they undergo Brownian motion. The
centrifugation involves principle of sedimentation, where the acceleration at centrifugal force
causes denser substance to separate out along the radial direction at the bottom of tube.
Considering a body of mass m rotating in a circular path of radius r at a velocity υ. The force
acting on the body in a radial direction is given by

F = mυ2/r

Where
F = centrifugal force
m = mass of body
υ = velocity of the body
r = radius of circle of rotation

The gravitational force acting upon the body; G=mg, where G=gravitational force, g = acceleration
due to gravity.
However, the rate of sedimentation is dependent upon the applied centrifugal field (cm s-2), G, that
is determined by the radial distance, r, of the particle from the axis of rotation (in cm) and the
square of the angular velocity, ω of the rotor.

𝐺= ω2𝑟
The sedimentation rate of a given particle will be zero when the density of the particle and the
surrounding medium are equal RCF (relative centrifugal force), which is the ratio of the centrifugal
acceleration at a specified radius and the speed to the standard acceleration of gravity. RCF is
measured in force x gravity or g-force. This is the force exerted on the contents of the rotor,
resulting from the revolutions of the rotor. RCF is dependent on the speed of rotation in rpm and
the distance of the particles from the center of rotation. Where the speed of rotation is given in rpm
(θ) and the distance (r) is expressed in centimeters, RCF can be calculated by using the formula in
equation.

RCF = 4π2(rev min−1)2r/360 X 981 = G/g

 P a g e | 51

RCF units are therefore dimensionless (denoting multiples of g) and

revolutions per minute are usually abbreviated as r.p.m.: RCF =1.12X10-5
r.p.m

TYPES OF ROTOR CENTRIFUGES

A centrifuge rotor is the rotating unit of the centrifuge, which has fixed holes drilled at an angle.
Test tubes are placed inside these holes and the rotor spins to aid in the separation of the materials.
There are three types of centrifuge rotors: swing-bucket, fixed-angle and vertical rotors.
Companies usually name rotors according to their type of design, the maximum allowable speed
and sometimes the material composition. Depending on the use in a simple low-speed centrifuge, a
high-speed centrifuge or an ultracentrifuge, different centrifugal forces are encountered by a
spinning rotor.

Swing-Bucket Rotors: It is usually supports samples ranging in volume from 36 mL to 2.2 mL.
Swing-buckets can support two types of separations: rate-zonal and isopycnic. Swing-buckets are
preferred for rate-zonal separations.
Fixed-Angle Rotors: It has cavities range from 0.2 mL to 1 mL and used for pelleting applications
to either pellet particles from a suspension or remove the excess debris. The most important aspect
in deciding to use a fixed-angle rotor is the K factor.
Vertical Rotors: Vertical rotors are highly specialized and has very short to run. It is typically
used to band DNA in cesium chloride. Vertical rotors have very low K factors, which is useful if
the particle must only move a short distance until it pellets.

TYPES OF CENTRIFUGE
Centrifugation techniques take a central position in modern biochemical, cellular and molecular
biological studies. Depending on the particular application, centrifuges differ in their overall design
and size. However, a common feature in all centrifuges is the central motor that’s pins a rotor
containing the samples to be separated. Particles of biochemical interest are usually suspended in a
liquid buffer system contained in specific tubes or separation chambers that are located in
specialized rotors.
 P a g e | 52

Many different types of centrifuges are commercially available including:

• large-capacity low-speed preparative centrifuges

• refrigerated high-speed preparative centrifuges
• analytical ultracentrifuges
• preparative ultracentrifuges
• large-scale clinical centrifuges
• small-scale laboratory microfuges

The low speed centrifuge has a maximum speed of 4000-5000rpm. This instrument usually
operates at the room temperature with no means of temperature control. It is mainly used in the
sedimentation of red blood cells unit. In this techniques the particles are tightly packed into a pellet
and the suspension is separated by decantation. The low speed centrifuge has following types of
rotor such as fixed angle and swinging bucket.

The high speed centrifugation has a maximum speed of 15,000-20,000 rpm. The operator of this
instrument can be carefully control speed and temperature which is require for sensitive biological
sample. The high speed centrifuge has following types of rotor such as fixed angle, swinging
bucket and vertical rotors.

Ultracentrifuge has maximum speed of 65,000 rpm. Intense heat is generated due to high speed
thus the spinning chambers must be refrigerated and kept a high vacuum. It is used both in
preparative and analytical work.

DIFFERENTIAL CENTRIFUGATION

Differential centrifugation is simplest form of separation deferential palliating. It separates

components of a cell on the basis of their densities and mass. The cell membrane is first ruptured to
release the cell’s components by using a homogenizer. The resulting mixture is referred to as the
homogenate. The homogenate is centrifuged to obtain a pellet containing the densest organelles.
Compounds that are the densest will form a pellet at lower centrifuge speeds while the less dense
 P a g e | 53

compounds will likely remain in the liquid supernatant above the pellet. Different pelleting is
commonly used for harvesting or producing crude sub cellar fractions for tissue homogenizing, for
example a rat liver hematogenate containing nuclei, mitochondria, lysosomes and membrane
vesicles centrifuge at low speed for a short time will pellet mainly the larger and denser nuclei.
Equilibrium sedimentation uses a gradient of a solution to separate particles based on their
individual densities (mass/volume). The sedimentation rate can be increased by using centrifugal
forces. Different densities or size of particles will sediment at different rates with
Largest and most dense particles sedimenting the fastest followed by less dense and smaller
particles. Subsequent centrifugation at a higher centrifugal force will pellet particles of the next
lower order of size e.g. Mitochondria and so on. It is usually for more than four differential
centrifugation cycles. For a normal tissues damage due to the heterogeneity in biological particles,
differential centrifugation suffers from contamination and poor recoveries. The contamination by
different particles types can be addressed by resuspension and repeating the centrifugation steps.

Two Types

• Density gradient centrifugation

• Rate-zonal centrifugation

DENSITY GRADIENT CENTRIFUGATION

It is the preferred method to for purification of sub cellular organelles and macromolecules.
Density gradient can be generated for placing layer after layer of gradient media such as sucrose in
a tube with the heaviest layer at the bottom and the lightest at the top in either a discontinuous
mode. The cell function to be separate is placed on top of the layer and centrifuged. Density
gradient separation can be classified into two categories such as Rate-zonal centrifugation and
Isopycnic centrifugation.

RATE-ZONAL CENTRIFUGATION

The problem of cross contamination of particles of different sedimentation rates may be avoided by
layering the sample as a narrow zone on top of density gradient in rate-zonal centrifugation. In this
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way the faster sediment particles are not contaminated by the slower particles as occurs in
differential centrifugation. However, the narrow load zone limits the volume of sample that can be
accumulated on the density gradient. The gradient stabilizes the bands and provides a medium of
increasing density and viscosity. As the particles in the bond more down through the density
medium, zone containing particles of similar size the faster sedimenting particles move a head of
the slower ones. Because the density of the particles is greater than the density of the gradient, all
the particles will eventually form a pellet if centrifuged is long enough. Rate-zonal centrifugation
was initially used mainly for analytical separations such as the analysis of the size distribution of
samples of polysomes or of RNA. Although very soon after the introduction of the technique
Thomson and his colleagues used rate sedimentation to separate mitochondria and lysosomes.

ULTRACENTRIFUGATION

An important tool in biochemical research is the centrifuge, which through rapid spinning imposes
high centrifugal forces on suspended particles or even molecules in solution and cause separations
of such matter on the basis of difference in weight. For example, red blood cells may be separated
from plasma of blood, nuclei from mitochondria in cell homogenates and one protein from another
in complex mixture. Proteins are separated by ultra-centrifugation at very high speed spinning with
appropriate of the protein layers as they form in the centrifugal field. It
is possible to determine the molecular weight of the proteins. The ultracentrifuge is a centrifuge
optimized for spinning a rotor at very high speed, capable of generating acceleration as high as
19600Km/ S2 (around 5000 rpm).
Biological centrifugation is a process that uses centrifugal force to separate and purify mixture of
biological particles in a liquid medium. The smaller the particle higher the g- force required for the
separation. It is a key technique for isolating and analyzing cells, subcellular fractions,
supramolecular complexes and with high g-forces instruments or ultracentrifuges (up to 60000
revolutions per minute corresponding to 200000g) isolated macromolecules such as proteins or
nucleic acids. Such high speed devices require a vacuum to avoid overheating.
.
TYPES OF ULTRACENTRIFUGATION
• Analytical ultracentrifugation
• Preparative ultracentrifugation
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ANALYTICAL ULTRACENTRIFUGATION

Analytical ultracentrifugation (AUC) refers to the analysis of a macromolecular solution by its

subjection to gravitational forces up to300000-fold greater than gravity. Three optical systems are
available for the analytical ultracentrifuge (absorbance, interference and fluorescence), that permit
precise and selective observation of sedimentation in real time. Some of the most attractive
features of AUC are:

Versatility: A wide variety of samples can be examined by AUC, including molecules ranging in
size from sucrose to virus particles.
Rigor: AUC experiments are directly interpreted in the context of thermodynamic and
hydrodynamic theory, so it is not necessary to run standards to calibrate each experiment.
Convenience: The data analysis methods have made AUC are much more convenient and
accessible to the general biochemistry and polymer science communities. In contrast to earlier
instruments, experiments are easy to set up and centrifugation parameters and data acquisition are
all under computer control.
Working: As a rotor turns, the images of the cell (proteins) are projected by an optical system on
to film or a computer. The concentration of the solution at various points in the cell is determined
by absorption of a light of appropriate wavelength. This can be accomplished either by measuring
the degree of blackening of a photographic film or by the deflection of the recorder of the scanning
system, fed into a computer. This allows the operator to observe the separation of the sample
concentration verses the axis of rotation. As a result of the applied centrifugal field two kinds of
experiments are commonly performed on these instruments: sedimentation velocity experiments
and sedimentation equilibrium experiments.
Sedimentation velocity experiments aim to interrupt the entire time course of sedimentation and
report on the surface and molar mass of the dissolved macromolecules as well as their size-
distribution. Sedimentation velocity experiments can also be used to study reversible chemical
equilibria between macromolecular species by monitoring the number and molar mass of
macromolecular complexes.
Sedimentation equilibrium experiments are concerned only with the final steady state of the
experiments where sedimentation is balanced by diffusion opposing the concentration gradients,
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resulting in a time independent concentration profile. Analytical centrifugation involves measuring

the physical properties of the sedimenting particles such as sedimentation coefficient or molecular
weight.

PREPARATIVE ULTRACENTRIFUGATION

Preparative ultracentrifuges are available with a wide variety of rotors suitable for a great range of
experiments. Most rotors are designed to hold tubes that contain the samples. Swinging bucket
rotors allow the tubes to hang on hinges so the tubes reorient to the horizontal as the rotor initially
accelerates. Fixed angle rotors are made of a single block of meta land hold the tubes in cavities
bored at a predetermined angle. Zonal rotors are designed to contain a large volume of sample in a
single central cavity rather than in tubes. Some zonal rotors are capable of dynamic loading and
unloading of samples while the rotor is spinning at high. They can also be used for gradient
separations, in which the tubes are filled from top to bottom with an increasing concentration of a
dense substance in solution.

• The Preparative ultracentrifuges are also used in biology for pelleting of fine particulate
fractions, such as cellular organelles such as mitochondria, microsomes, ribosomes and
viruses.
• Used for gradient separations. Gradients of sucrose for separation of cellular organelles.
• Gradients of calcium salts for separation of nucleic acids.
• After the spun at high speed for sufficient time, allow the rotor to come to a smooth stop
• Gradient is gently pumped out of each tube to isolate the separated components.

APPLICATION OF CENTRIFUGATION
• A centrifuge is used to separate two miscible substances:
▪ Cells
▪ Sub-cellular components
▪ Proteins -Nucleic acids
• Centrifugation is basis the of size, shape and density of particles
• It utilizes density difference between the particles/macro molecules and the medium in which
these are dispersed
• To analyze the hydrodynamic properties of macromolecules and purification of mammalian cells
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• To Separation of urine components and blood components in forensic and research laboratories.
• It use to separate fraction of sub-cellular organelles and fractionation of membrane vesicles
• A tube of anti-coagulated whole blood left standing on a bench top will eventually separate into
plasma, red blood cell and white blood cell fractions.
• It utilizes density difference between the particles/macromolecules and the medium in which
these are dispersed. Dispersed systems are subjected to artificially induced gravitational fields.
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AUTOMATION

Automation can be defined as the self-moving or mechanical transfer of a specimen within the
complex. In clinical laboratory, it is a process by which analytical instruments perform many tests
with the least involvement of an analyst. Although the term automation applies to processing of
large number of samples but the principles in automation can be applied to the analysis of a single
sample also. For example, in case of automated blood gas analyzer a single sample is analyzed. In
this chapter, the reasons for automation and the ways to achieve it are discussed.

Automated instruments enable laboratories to process a much larger workload without a relative
increase in manpower. Automation may initially incur high costs for procurement of the
equipment’s but is economical in the long run due to the reduction in the manpower required to
perform the tasks.

Classification of Analysers

These are available in the range of 1.5-3.0lac. In the instrument, sample and reagents are mixed
and fed manually. The unit performs the chemical reaction and gives the results. The setting of the
unit is automatic with regards to wavelength time and temperature, etc.

Batch Analyzer
In this sample, reagent mixture is mixed and fed automatically. One reagent is stored in the
machine at a time. So one batch of particular chemistry tests, e.g. urea or glucose can be set up and
performed automatically.

Random Access Auto analyzers

More than one reagent can be stored in such units. Patient samples are kept in the machine. Then
computer is programmed to carry out any number of selected tests on individual samples. Any
particular sample and reagent can be selected, mixed, incubated as per command to the processing
system in or out of sequence and without regard to their initial order.
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Depending on the specific requirements and workload, laboratories opt for a combination of
automatic, semiautomatic and manual mode of analyses. Different models may be suitable to
different hospitals depending on their needs. So while selecting a particular model, the primary
objective of the laboratory is to be defined. A particular model may have an advantage in economy
whereas another may be a better choice for other features, e.g. speed and convenience. While
comparing two models, the cost analysis on reagent, etc. should also be done. So depending upon
the speed, accuracy, reliability and economy, either of the above mentioned analyzers can be
purchased. Generally, a medium sized laboratory can meet its work load through a semiautomatic
analyzer (about 100 tests per day).

PROFILES OF ANALYZERS
In auto analyzers, various steps are similar to those in the traditional manual method of carrying
out the analysis. The early development of automation by Skeggs in clinical biochemistry has led
to the introduction of auto analyzers. In this system, the sample to be analyzed passes sequentially
through the same analytical pathway in a continuous stream of fluid. This concept of continuous
flow analysis has been modified and extended.

In analyzer, all samples and reagents pass along plastic and glass tubes. The reagents flow
continuously but the samples are introduced at intervals, usually separated by wash fluids. Mixing
occurs when tubes join to form a common pathway and all components of the reaction mixture
have been added. The degree of mixing is influenced by the different velocities of flow across the
cross section of the combined stream. A series of air bubbles are introduced into the liquid stream
to segment this in small lengths. Mixing within the segment is achieved by repeatedly inverting
them. Purging effect of the air bubbles modifies differential flow rates.

The different functions in analyzer are carried out by modules. The analytical system is a collection
of linked modules, arranged in such a way that analysis of a series of specimens, for one of more
constituents may be performed. A single channel system will analyse for one substance in each
specimen. In multiple channel system each sample is subjected to multiple analytical processes so
that a set of test results is obtained from it. Over the years, analytical performances and sensitivity
of auto analyzers have improved. This has made available multichannel analysis with automatic
preparation of printed reports.
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Different modules involved in the analytical system of auto analyzers are as follows:

Sampler
This module consists of a base containing a motor, on which is mounted a circular tray with
equally spaced holes, into which plastic cups are placed. A probe connected by plastic tubing to the
manifold, enters the sampler serially and allows the contents which is water, standard or test to be
aspirated. The plate then rotates to a distance, which is sufficient to allow the tube to dip into the
next cup. From the sample, the probe passes into a wash reservoir so that while probe is out of the
sample, air is not drawn in, which makes a break between samples. The sampler is also fitted with
a vibrator which is used to ensure that when sampling blood, the cells are evenly distributed. In the
sampler loss of water by evaporation from the surface of sample cups, lead to an error up to 5%.
This can be reduced by placing a cover over the sampler.
Proportioning Pump
This module replaces the use of pipettes of different sizes used in manual methods. The pumping
technique involves the peristaltic action, produced by a series of rollers passing along an array of
parallel plastic pump tubes. Each roller compresses all tubes, so that the rate of flow in each tube
depends on the square of the pump tube diameter. A series of flexible plastic tubes (the manifold)
coming from the sampler, from reagent bottles or those simply drawing in air, is placed lengthwise
along the plate. As the roller passes along the tubes, it pushes the various liquids and
air forward, ahead of it in each tube. But when it eventually loses contact with the manifold,
backflow is prevented by one or more other rollers behind, already forcing liquid forward. The
volume of the sample and reagents taken up, is determined by the internal diameter of the tube.
Since the proportions of the various reagents are fixed by the tube size, no measurements are
needed. One form of proportioning pump can work two speeds-a slow one for ordinary working,
and a quicker one for filling the system with reagents before the run, and for washing with water
after the run. For each determination a separate manifold is required.
Dialyzer
In some tests, it is necessary to separate small and big molecules. This is achieved by allowing the
stream of liquid to pass through a semi permeable membrane. The continuous channel is then
divided to pass through a semi permeable membrane. The continuous channel is then divided into
two halves by the membrane, across which dialysis occurs. As the donor stream passes along the
upper half, diffusible substances including one being determined, passes through the membrane
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into the recipient stream of reagents, in the lower half. The rest of the sample stream may go waste
or be used for the determination of another constituent in a multiple analysis system.
Mixing Coils
When two or more fluid stream join, these coils bring about adequate mixing. They are multiturn
glass coils. If the two liquids are immiscible, solvent extraction occurs while the liquid traverses
the coil. Some mixing coils are jacketed with water to allow rapid cooling of the reaction mixture.
For the method where time delays are required, shorter delays are achieved by inserting one or
more mixing coils in the circuit but longer delays are better achieved using a delay coil. Tube for
mixing coil is either of glass or polythene.
Heating or Incubator Bath
On leaving the dialyser, the recipient stream may be joined by one or more additional reagents
before passing to a heating bath. This module maintains the reaction mixture at a constant
temperature. Most water baths are set at 37°C or 90°C but some are adjustable to 120°C or even
higher. Therefore, passage through the heating coil depends on the coil volume and the flow rate.
Colorimeter and Recorder
Finally, the stream of fluid passes through the optoelectronic instrument. This unit monitors the
above reaction thereby establishing the exact amount of substance. Originally, the analyzers were
used with colorimetric methods. But colorimeters have been replaced by devices as
spectrophotometers, fluorometers, nephelometers, flame photometers or radioactive counters.
A number of instruments are now in common use in the hospital laboratories. Complete
information about the instrument is supplied by the manufactures.

The steps in the automated systems:

1. Sample identification: The tube containing each of the samples is labeled at the time of
sample collection. On reaching the lab, the sample is recorded by computerized procedure
after which the samples are processed.
Bar coding: Bar coding technology for sample identification of blood and other products may
allow for reduction of medical error and increase patient safety. A bar coded label is placed onto
the sample containers and is read by the bar readers placed at key positions in the analytical train.
The information that is read by the reader is transferred to and processed by the system software.
2. Sample preparation: Sample preparation includes clotting of blood, centrifugation, and
transfer of serum to work benches.
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3. Sample handling, transport, and delivery: The containers holding the samples are kept
covered till the time of analysis so as to avoid evaporation or spillage. For analysis, the
sample is loaded on loading zone of the analyzer.

4. Sample processing: Automation helps in testing a large number of samples quickly and
efficiently
5. Reagent handling: Reagents should be stored in 4°C refrigerator till the assay as per
requirement. On board cooling is also there in automated instruments.
6. Chemical Reaction: In the presence of appropriate reagent and optimum temperature the
samples undergo the chemical reaction.
7. Measurement, signal processing and micro processing: The measurement and output
signals are automatically processed and result are made available in the form of
reading/graphs as per the requirements input initially. The demand for the increased
efficiency and cost effectiveness in health care has led to the production and commercial
availability of number of sophisticated automated analyzers to analyte blood, urine and csf
samples.
8. As compared to the colorimeter, the analyzers are considered more expensive. But in
general the reagents volume used per test is very less.

So, the above advantages along with saving on reagents costs, time costs, glassware and workspace
justify higher investment on the instruments.
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Conclusion

The field of Analytical biochemistry leverages a diverse array of techniques including

Spectroscopy, Centrifugation, Chromatography, Electrophoresis, and Automation to unravel the
complexities of biological systems. Automation plays crucial role in streamlining processes
enhancing efficiency, and increasing throughput in analytical workflows, thereby accelerating
research and discovery. By combining these advanced analytical methods with automation,
scientists are empowered to delve deeper into the molecular intricacies of biological phenomena,
driving innovation and progress in various fields such as medicine, biotechnology and
environmental sciences
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