Lecture Notes - Introduction to Bioanalytical Techniques and Bioinformatics - Biotechnology Engineering (BT) PDF Download

 Page 1


NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics 
 
Joint initiative of IITs and IISc – Funded by MHRD                                                            Page 1 of 20 
Module 1 : Introduction 
Lecture 1 Introduction 
Bioanalytical techniques, as the name suggests, are the analytical tools to study the 
biological molecules; non-biological molecules involved with life, such as drugs; and 
biological processes. These tools are routinely used to identify, estimate, purify, and 
characterize the biomolecules. Quantification of molecules in biological samples is at 
the heart of bioanalysis and is routinely used to diagnose various diseases and 
metabolic disorders. For example, estimation of thyroxine and triiodothyronine 
concentrations in blood provides information about the activity of thyroid gland. 
Home pregnancy test kits look for the human chorionic gonadotropin (hCG) hormone 
in the urine, presence of which above a threshold concentration is an indicator of 
pregnancy. Bioanalytical methods are also used to detect drugs and their metabolites 
in biological samples. Initially, nonspecific assays were used to quantify the drugs in 
biological samples. Evolution of the existing assays, advancement in instrumentation, 
and introduction of newer techniques have made it possible to distinguish the drug 
molecules and their closely related metabolites in complex biological specimens.  
Estimation of the analytes 
Identification and 
quantification of analytes is 
perhaps the most common 
application of bioanalytical 
methods. Various diseases and 
disorders including cancers are 
diagnosed by estimating the 
levels of the characteristic 
biomarkers in a particular tissue or organ. Semenogelase, for example, is a biomarker 
for prostate cancer, one of the most frequently diagnosed cancers in human males.  
 
 
 
Biomarker: In disease and diagnostics, a 
biomarker is a molecule, presence of which 
beyond a threshold level is an indicator of the 
biological state. 
In cell biology, a biomarker is a molecule 
characteristic of a cell type or a group of cells 
e.g. Oct-4 is a biomarker for embryonic stem 
cells. 
Page 2


NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics 
 
Joint initiative of IITs and IISc – Funded by MHRD                                                            Page 1 of 20 
Module 1 : Introduction 
Lecture 1 Introduction 
Bioanalytical techniques, as the name suggests, are the analytical tools to study the 
biological molecules; non-biological molecules involved with life, such as drugs; and 
biological processes. These tools are routinely used to identify, estimate, purify, and 
characterize the biomolecules. Quantification of molecules in biological samples is at 
the heart of bioanalysis and is routinely used to diagnose various diseases and 
metabolic disorders. For example, estimation of thyroxine and triiodothyronine 
concentrations in blood provides information about the activity of thyroid gland. 
Home pregnancy test kits look for the human chorionic gonadotropin (hCG) hormone 
in the urine, presence of which above a threshold concentration is an indicator of 
pregnancy. Bioanalytical methods are also used to detect drugs and their metabolites 
in biological samples. Initially, nonspecific assays were used to quantify the drugs in 
biological samples. Evolution of the existing assays, advancement in instrumentation, 
and introduction of newer techniques have made it possible to distinguish the drug 
molecules and their closely related metabolites in complex biological specimens.  
Estimation of the analytes 
Identification and 
quantification of analytes is 
perhaps the most common 
application of bioanalytical 
methods. Various diseases and 
disorders including cancers are 
diagnosed by estimating the 
levels of the characteristic 
biomarkers in a particular tissue or organ. Semenogelase, for example, is a biomarker 
for prostate cancer, one of the most frequently diagnosed cancers in human males.  
 
 
 
Biomarker: In disease and diagnostics, a 
biomarker is a molecule, presence of which 
beyond a threshold level is an indicator of the 
biological state. 
In cell biology, a biomarker is a molecule 
characteristic of a cell type or a group of cells 
e.g. Oct-4 is a biomarker for embryonic stem 
cells. 
NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics 
 
Joint initiative of IITs and IISc – Funded by MHRD                                                            Page 2 of 20 
 
Qualitative versus quantitative analyses 
A qualitative analysis simply tells about the presence or absence of an analyte in a 
sample. An absence of analyte, however, may result due to concentrations below the 
detection level of the bioanalytical technique used. Qualitative analyses are used 
wherein detection of an analyte is sufficient to take further course of action. For 
example, identification of a banned performance-enhancing drug in athletics is 
sufficient enough to determine the qualification of the athlete to participate in the 
event. In certain cases, however, it is important to estimate the concentration of the 
analyte. A quantitative analysis would result in the determination of actual amount of 
the substance present in the sample. Consider a person suspected to be diabetic. A 
qualitative test for glucose is not good enough to ascertain if the person is diabetic. It 
is important to accurately determine the concentration of glucose in the blood to 
arrive at a conclusion. Breath alcohol detectors are used by traffic personnel to 
quantify the breath alcohol level, which in turn is proportional to blood glucose level 
and thereby enable them to identify the drunk drivers.  
 
 
 
 
 
 
 
 
 
 
 
Page 3


NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics 
 
Joint initiative of IITs and IISc – Funded by MHRD                                                            Page 1 of 20 
Module 1 : Introduction 
Lecture 1 Introduction 
Bioanalytical techniques, as the name suggests, are the analytical tools to study the 
biological molecules; non-biological molecules involved with life, such as drugs; and 
biological processes. These tools are routinely used to identify, estimate, purify, and 
characterize the biomolecules. Quantification of molecules in biological samples is at 
the heart of bioanalysis and is routinely used to diagnose various diseases and 
metabolic disorders. For example, estimation of thyroxine and triiodothyronine 
concentrations in blood provides information about the activity of thyroid gland. 
Home pregnancy test kits look for the human chorionic gonadotropin (hCG) hormone 
in the urine, presence of which above a threshold concentration is an indicator of 
pregnancy. Bioanalytical methods are also used to detect drugs and their metabolites 
in biological samples. Initially, nonspecific assays were used to quantify the drugs in 
biological samples. Evolution of the existing assays, advancement in instrumentation, 
and introduction of newer techniques have made it possible to distinguish the drug 
molecules and their closely related metabolites in complex biological specimens.  
Estimation of the analytes 
Identification and 
quantification of analytes is 
perhaps the most common 
application of bioanalytical 
methods. Various diseases and 
disorders including cancers are 
diagnosed by estimating the 
levels of the characteristic 
biomarkers in a particular tissue or organ. Semenogelase, for example, is a biomarker 
for prostate cancer, one of the most frequently diagnosed cancers in human males.  
 
 
 
Biomarker: In disease and diagnostics, a 
biomarker is a molecule, presence of which 
beyond a threshold level is an indicator of the 
biological state. 
In cell biology, a biomarker is a molecule 
characteristic of a cell type or a group of cells 
e.g. Oct-4 is a biomarker for embryonic stem 
cells. 
NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics 
 
Joint initiative of IITs and IISc – Funded by MHRD                                                            Page 2 of 20 
 
Qualitative versus quantitative analyses 
A qualitative analysis simply tells about the presence or absence of an analyte in a 
sample. An absence of analyte, however, may result due to concentrations below the 
detection level of the bioanalytical technique used. Qualitative analyses are used 
wherein detection of an analyte is sufficient to take further course of action. For 
example, identification of a banned performance-enhancing drug in athletics is 
sufficient enough to determine the qualification of the athlete to participate in the 
event. In certain cases, however, it is important to estimate the concentration of the 
analyte. A quantitative analysis would result in the determination of actual amount of 
the substance present in the sample. Consider a person suspected to be diabetic. A 
qualitative test for glucose is not good enough to ascertain if the person is diabetic. It 
is important to accurately determine the concentration of glucose in the blood to 
arrive at a conclusion. Breath alcohol detectors are used by traffic personnel to 
quantify the breath alcohol level, which in turn is proportional to blood glucose level 
and thereby enable them to identify the drunk drivers.  
 
 
 
 
 
 
 
 
 
 
 
NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics 
 
Joint initiative of IITs and IISc – Funded by MHRD                                                            Page 3 of 20 
Accurate and precise determination of analytes 
It is hardly necessary to explain how critical an accurate determination of an analyte 
is. If a breath alcohol detector is not accurate, a drunk driver may be let off risking the 
life of others while a sober one may be detained. Unless the concentration of analyte 
is determined accurately and precisely, it is difficult to make meaningful conclusions. 
So, what exactly do the accuracy and precision mean? Accuracy is the measure of 
how closely the measured values match the true values. Precision tells about the 
reproducibility of the measurement i.e. how closely the measured values are if 
repeated measurements are made on the sample (Figure 1.1).  
 
Figure 1.1 Schematic representations of accuracy and precision. Consider the centre of the concentric circles as the 
true value; the measured values are represented as the black dots. The measured values shown in panel A are close to 
the true value (accurate) as well as to each other (precise). The measured values in panel B are close to each other 
(precise) but far from the true value (inaccurate). The individual values in panel C are far away from the true value 
but randomly distributed about the true value; the average value lies close to the true value (accurate but imprecise). 
Panel D represents inaccurate and imprecise measurements. 
It is easy to imagine the consequences of using an inaccurate equipment; it would give 
inaccurate results. Imprecise equipments, even if accurate, are problematic as a large 
number of measurements are required to arrive close to the true value which may take 
considerable amount of time.  An analytical tool therefore has to be both accurate and 
precise to be used reliably and for faster analysis. 
Page 4


NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics 
 
Joint initiative of IITs and IISc – Funded by MHRD                                                            Page 1 of 20 
Module 1 : Introduction 
Lecture 1 Introduction 
Bioanalytical techniques, as the name suggests, are the analytical tools to study the 
biological molecules; non-biological molecules involved with life, such as drugs; and 
biological processes. These tools are routinely used to identify, estimate, purify, and 
characterize the biomolecules. Quantification of molecules in biological samples is at 
the heart of bioanalysis and is routinely used to diagnose various diseases and 
metabolic disorders. For example, estimation of thyroxine and triiodothyronine 
concentrations in blood provides information about the activity of thyroid gland. 
Home pregnancy test kits look for the human chorionic gonadotropin (hCG) hormone 
in the urine, presence of which above a threshold concentration is an indicator of 
pregnancy. Bioanalytical methods are also used to detect drugs and their metabolites 
in biological samples. Initially, nonspecific assays were used to quantify the drugs in 
biological samples. Evolution of the existing assays, advancement in instrumentation, 
and introduction of newer techniques have made it possible to distinguish the drug 
molecules and their closely related metabolites in complex biological specimens.  
Estimation of the analytes 
Identification and 
quantification of analytes is 
perhaps the most common 
application of bioanalytical 
methods. Various diseases and 
disorders including cancers are 
diagnosed by estimating the 
levels of the characteristic 
biomarkers in a particular tissue or organ. Semenogelase, for example, is a biomarker 
for prostate cancer, one of the most frequently diagnosed cancers in human males.  
 
 
 
Biomarker: In disease and diagnostics, a 
biomarker is a molecule, presence of which 
beyond a threshold level is an indicator of the 
biological state. 
In cell biology, a biomarker is a molecule 
characteristic of a cell type or a group of cells 
e.g. Oct-4 is a biomarker for embryonic stem 
cells. 
NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics 
 
Joint initiative of IITs and IISc – Funded by MHRD                                                            Page 2 of 20 
 
Qualitative versus quantitative analyses 
A qualitative analysis simply tells about the presence or absence of an analyte in a 
sample. An absence of analyte, however, may result due to concentrations below the 
detection level of the bioanalytical technique used. Qualitative analyses are used 
wherein detection of an analyte is sufficient to take further course of action. For 
example, identification of a banned performance-enhancing drug in athletics is 
sufficient enough to determine the qualification of the athlete to participate in the 
event. In certain cases, however, it is important to estimate the concentration of the 
analyte. A quantitative analysis would result in the determination of actual amount of 
the substance present in the sample. Consider a person suspected to be diabetic. A 
qualitative test for glucose is not good enough to ascertain if the person is diabetic. It 
is important to accurately determine the concentration of glucose in the blood to 
arrive at a conclusion. Breath alcohol detectors are used by traffic personnel to 
quantify the breath alcohol level, which in turn is proportional to blood glucose level 
and thereby enable them to identify the drunk drivers.  
 
 
 
 
 
 
 
 
 
 
 
NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics 
 
Joint initiative of IITs and IISc – Funded by MHRD                                                            Page 3 of 20 
Accurate and precise determination of analytes 
It is hardly necessary to explain how critical an accurate determination of an analyte 
is. If a breath alcohol detector is not accurate, a drunk driver may be let off risking the 
life of others while a sober one may be detained. Unless the concentration of analyte 
is determined accurately and precisely, it is difficult to make meaningful conclusions. 
So, what exactly do the accuracy and precision mean? Accuracy is the measure of 
how closely the measured values match the true values. Precision tells about the 
reproducibility of the measurement i.e. how closely the measured values are if 
repeated measurements are made on the sample (Figure 1.1).  
 
Figure 1.1 Schematic representations of accuracy and precision. Consider the centre of the concentric circles as the 
true value; the measured values are represented as the black dots. The measured values shown in panel A are close to 
the true value (accurate) as well as to each other (precise). The measured values in panel B are close to each other 
(precise) but far from the true value (inaccurate). The individual values in panel C are far away from the true value 
but randomly distributed about the true value; the average value lies close to the true value (accurate but imprecise). 
Panel D represents inaccurate and imprecise measurements. 
It is easy to imagine the consequences of using an inaccurate equipment; it would give 
inaccurate results. Imprecise equipments, even if accurate, are problematic as a large 
number of measurements are required to arrive close to the true value which may take 
considerable amount of time.  An analytical tool therefore has to be both accurate and 
precise to be used reliably and for faster analysis. 
NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics 
 
Joint initiative of IITs and IISc – Funded by MHRD                                                            Page 4 of 20 
Identification and characterization of molecules 
Researchers involved in the discovery of novel bioactive natural products often have 
to identify the bioactive component present in the crude sample; for example, 
isolation of novel antibiotics and antimicrobial peptides from various organisms. 
Individual components in the crude sample are isolated based on the differences in 
their physical and chemical properties. The bioactive component is identified by 
testing the activities of these isolated compounds. The bioactive compound is then 
characterized using various spectroscopic methods to arrive at its structure and 
function(s). Bioanalytical techniques can typically be classified as shown in Figure 
1.2.  
 
Figure 1.2 Various bioanalytical methods 
Spectroscopic tools such as infrared spectroscopy, circular dichroism spectroscopy, 
and nuclear magnetic resonance spectroscopy can provide structural information 
about the molecules which in turn provides insights into their functional aspects. 
 
Studying biological processes 
Life is an outcome of the complex interplay of biological molecules. These involve 
interactions between macromolecules (e.g. protein-protein interactions and DNA-
protein interactions, RNA-protein interactions); interactions of biomolecules with 
small molecules (glucose channels, water channels, ligand-binding) and ions (K
+
 
channel, Na
+
 and K
+
 pump, Ca
2+
 channels); and interaction of molecules with light 
Page 5


NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics 
 
Joint initiative of IITs and IISc – Funded by MHRD                                                            Page 1 of 20 
Module 1 : Introduction 
Lecture 1 Introduction 
Bioanalytical techniques, as the name suggests, are the analytical tools to study the 
biological molecules; non-biological molecules involved with life, such as drugs; and 
biological processes. These tools are routinely used to identify, estimate, purify, and 
characterize the biomolecules. Quantification of molecules in biological samples is at 
the heart of bioanalysis and is routinely used to diagnose various diseases and 
metabolic disorders. For example, estimation of thyroxine and triiodothyronine 
concentrations in blood provides information about the activity of thyroid gland. 
Home pregnancy test kits look for the human chorionic gonadotropin (hCG) hormone 
in the urine, presence of which above a threshold concentration is an indicator of 
pregnancy. Bioanalytical methods are also used to detect drugs and their metabolites 
in biological samples. Initially, nonspecific assays were used to quantify the drugs in 
biological samples. Evolution of the existing assays, advancement in instrumentation, 
and introduction of newer techniques have made it possible to distinguish the drug 
molecules and their closely related metabolites in complex biological specimens.  
Estimation of the analytes 
Identification and 
quantification of analytes is 
perhaps the most common 
application of bioanalytical 
methods. Various diseases and 
disorders including cancers are 
diagnosed by estimating the 
levels of the characteristic 
biomarkers in a particular tissue or organ. Semenogelase, for example, is a biomarker 
for prostate cancer, one of the most frequently diagnosed cancers in human males.  
 
 
 
Biomarker: In disease and diagnostics, a 
biomarker is a molecule, presence of which 
beyond a threshold level is an indicator of the 
biological state. 
In cell biology, a biomarker is a molecule 
characteristic of a cell type or a group of cells 
e.g. Oct-4 is a biomarker for embryonic stem 
cells. 
NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics 
 
Joint initiative of IITs and IISc – Funded by MHRD                                                            Page 2 of 20 
 
Qualitative versus quantitative analyses 
A qualitative analysis simply tells about the presence or absence of an analyte in a 
sample. An absence of analyte, however, may result due to concentrations below the 
detection level of the bioanalytical technique used. Qualitative analyses are used 
wherein detection of an analyte is sufficient to take further course of action. For 
example, identification of a banned performance-enhancing drug in athletics is 
sufficient enough to determine the qualification of the athlete to participate in the 
event. In certain cases, however, it is important to estimate the concentration of the 
analyte. A quantitative analysis would result in the determination of actual amount of 
the substance present in the sample. Consider a person suspected to be diabetic. A 
qualitative test for glucose is not good enough to ascertain if the person is diabetic. It 
is important to accurately determine the concentration of glucose in the blood to 
arrive at a conclusion. Breath alcohol detectors are used by traffic personnel to 
quantify the breath alcohol level, which in turn is proportional to blood glucose level 
and thereby enable them to identify the drunk drivers.  
 
 
 
 
 
 
 
 
 
 
 
NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics 
 
Joint initiative of IITs and IISc – Funded by MHRD                                                            Page 3 of 20 
Accurate and precise determination of analytes 
It is hardly necessary to explain how critical an accurate determination of an analyte 
is. If a breath alcohol detector is not accurate, a drunk driver may be let off risking the 
life of others while a sober one may be detained. Unless the concentration of analyte 
is determined accurately and precisely, it is difficult to make meaningful conclusions. 
So, what exactly do the accuracy and precision mean? Accuracy is the measure of 
how closely the measured values match the true values. Precision tells about the 
reproducibility of the measurement i.e. how closely the measured values are if 
repeated measurements are made on the sample (Figure 1.1).  
 
Figure 1.1 Schematic representations of accuracy and precision. Consider the centre of the concentric circles as the 
true value; the measured values are represented as the black dots. The measured values shown in panel A are close to 
the true value (accurate) as well as to each other (precise). The measured values in panel B are close to each other 
(precise) but far from the true value (inaccurate). The individual values in panel C are far away from the true value 
but randomly distributed about the true value; the average value lies close to the true value (accurate but imprecise). 
Panel D represents inaccurate and imprecise measurements. 
It is easy to imagine the consequences of using an inaccurate equipment; it would give 
inaccurate results. Imprecise equipments, even if accurate, are problematic as a large 
number of measurements are required to arrive close to the true value which may take 
considerable amount of time.  An analytical tool therefore has to be both accurate and 
precise to be used reliably and for faster analysis. 
NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics 
 
Joint initiative of IITs and IISc – Funded by MHRD                                                            Page 4 of 20 
Identification and characterization of molecules 
Researchers involved in the discovery of novel bioactive natural products often have 
to identify the bioactive component present in the crude sample; for example, 
isolation of novel antibiotics and antimicrobial peptides from various organisms. 
Individual components in the crude sample are isolated based on the differences in 
their physical and chemical properties. The bioactive component is identified by 
testing the activities of these isolated compounds. The bioactive compound is then 
characterized using various spectroscopic methods to arrive at its structure and 
function(s). Bioanalytical techniques can typically be classified as shown in Figure 
1.2.  
 
Figure 1.2 Various bioanalytical methods 
Spectroscopic tools such as infrared spectroscopy, circular dichroism spectroscopy, 
and nuclear magnetic resonance spectroscopy can provide structural information 
about the molecules which in turn provides insights into their functional aspects. 
 
Studying biological processes 
Life is an outcome of the complex interplay of biological molecules. These involve 
interactions between macromolecules (e.g. protein-protein interactions and DNA-
protein interactions, RNA-protein interactions); interactions of biomolecules with 
small molecules (glucose channels, water channels, ligand-binding) and ions (K
+
 
channel, Na
+
 and K
+
 pump, Ca
2+
 channels); and interaction of molecules with light 
NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics 
 
Joint initiative of IITs and IISc – Funded by MHRD                                                            Page 5 of 20 
(chlorophyll, photoreceptors). Interactions of the molecules with their 
receptors/ligands, both in vitro and in vivo, are usually studied using various 
spectroscopic and microscopic tools. Fluorescence spectroscopy and microscopy are 
among the most commonly employed tools to study the biological processes. 
Discovery of the green fluorescent protein (GFP) and subsequent development of its 
analogs with different spectral properties have revolutionized the area of cellular 
research. Before discussing in detail the various tools that have gained importance in 
bioanalytical research, it is worthwhile to take a pause for very quickly reviewing the 
important structural aspects of major classes of biomolecules.  
 
Features of major biomolecules 
Classification of biomolecules is largely based on their chemistry. There are four 
major classes of biomolecules: proteins, nucleic acids, carbohydrates, and lipids. 
Amino acids and proteins 
Proteins constitute the functional machinery in the living systems by carrying out 
most of the biological reactions. They are the unbranched polymers of L-a-amino 
acids. D-amino acids do exist in nature, but such molecules are rare. The structure of a 
typical amino acid is shown in Figure 1.3A. 
 
Figure 1.3 Structures of amino acids and proteins: structure of a typical L- a-amino acid (A); peptide bond showing the 
partial double bond character (B); primary and secondary structures (C); and tertiary and quaternary structures (D) 
formed by proteins.