Introduction
Diagnostics
The art of identifying and correlating the results of multiple tests to determine
system condition and status and to generate the input required for optimal
maintenance.
Isolates
suspicious sample further require molecular techniques - confirm identification or
diagnosis.
A diagnostic test is any kind of medical test performed to aid in the diagnosis or
detection of disease.
It may be used to diagnose diseases or measure the progress or recovery from
disease or confirm that a person is free from disease.
Diagnostic tests are parts of a simple physical examination which require only
simple tools in the hands of a skilled practitioner.
LIMITATIONS OF CONVENTIONAL DIAGNOSTIC AIDS
Does not give information about:
Cause of the conditions
Patient’s susceptibility to disease
Progressing (or) Remission.
Response to therapy: positive (or) negative.
No reliable markers for disease activity
No reliable criteria for identifying at risk individuals.
Inaccurate measurements
There is need to overcome these limitations of conventional methods
Advanced diagnostic techniques
However, with advancements in health care, a large array of diagnostic tests
have become available.
Various modern diagnostic modalities are often routinely used in medical world
Classification:
The various modern diagnostic technologies can be classified as
Nucleic Acid Based Methods
Immunological Based Methods
Techniques For Detecting Antibodies and Antigens
Molecular Biology
Hematology
Radiology
Miscellaneous.
Advantages of ADT
Rapidity
Sensitivity
Specificity
Chromatography
Chromatography (from Greek chroma "color and graphein "to write") is
the collective term for a set of laboratory techniques for the separation of
mixtures.
chromatography provides a way to identify unknown compounds and
separate mixtures.
History:
Chromatography, literally "color writing", was first employed by Russian
scientist Mikhail Tsvet in 1900
He continued to work with chromatography in the first decade of the 20th
century, primarily for the separation of plant pigments such as chlorophyll,
carotenes, and xanthophylls.
Since these components have different colors (green, orange, and
yellow,respectively) they gave the technique its name.
Definition:
Chromatography may be defined as
'A method of separating a mixture of components into individual components
through equilibrium distribution between two phases’.
OR
‘A technique by which a mixture is separated into its components on the
basis of relative ability of each component to be moved along/through a
stationary phase by mobile phase’ One of these phases is a mobile phase and
the other is a stationary phase.
The substances must interact with the stationary phase to be retained and
separated by it.
Porous medium through which the mobile phase migrates is called the support.
The technique of chromatography is based on the differences in the rate at which
the components of a mixture move through a porous medium under the influence
of some solvent or gas.
PRINCIPLE:
Chromatography usually consists of mobile phase and stationary phase.
The mobile phase refers to the mixture of substances to be separated
dissolved in a liquid or a gas.
The stationary phase is a porous solid matrix through which the sample contained
in the mobile phase percolates.
The interaction between the mobile phase and the stationary phase results
in the separation of the compound from the mixture.
Chromatography is a nondestructive procedure for resolving a multi-
component mixture of minor or major constituents into its individual
fractions.
It can be applied for both qualitative and quantitative studies as it is a separation
technique.
STEPS
1. Adsorption or retention of substances on the stationary phase
2. Separation of the adsorption of substances by the mobile phase
3. Recovery of the separated substances by a continuous flow of the mobile
phase; the method being called elution
4. Qualitative and Quantitative analysis of the eluted substances.
Chromatographic terms
The analyte is the substance to be separated during chromatography.
Mobile phase or carrier: solvent moving through the column
Stationary phase or adsorbent: substance that stays fixed inside the
column.
The eluent is the solvent that carries the analyte i.e Fluid entering a column
The eluate: Fluid exiting the column. The eluate is the mobile phase leaving
the column.
Elution: The process of passing the mobile phase through the column.
The detector refers to the instrument used for qualitative and quantitative
detection of analytes after separation.
A chromatogram is the visual output of the chromatograph.
Uses of Chromatography:
Chromatography is used by scientists to:
Analyze – examine a mixture, its components, and their relations to one
another
Identify – determine the identity of a mixture or components based on
known components.
Purify – separate components in order to isolate one of interest for
further study
Quantify – determine the amount of a mixture and/or the components
present in the sample.
Applications of Chromatography:
The chromatographic technique is used for the separation of amino acids,
proteins & carbohydrates.
It is also used for the analysis of drugs, hormones, vitamins.
Helpful for the qualitative & quantitative analysis of complex mixtures.
The technique is also useful for the determination of molecular weight of
proteins.
Real life example uses of Chromatography:
A few application of chromatography in different industries are:
Chemical Industry
Contaminants’ detection in pesticides and oils
Water sample testing
Air quality checks
Numerous applications in life sciences
Pharmaceutical Industry
Taking element composition and its molecular weight to separate
compounds
During the process of drug development
Detecting chemicals or trace elements in various samples with proper
analysis
Examination of mixture purity
Identification of unknown compounds
Forensic Industry
Blood and hair sample analysis under crime scene testing
Forensic pathology
Food Industry
Identifying the food products’ nutritional values and quality
Detection of additives and food spoilage
Molecular Biology Studies
The fuel industry, biochemical processes, and biotechnology use HPLC for
purification and fractionation process
Metabolomics and proteomics study through specific chromatography
techniques
Nucleic acid research with specific chromatography methods
These are only the popular applications of chromatography. The technique is
used in various other places and industries.
Mechanism of separation of different components
Differential affinities (strength of adhesion) of the various components of
the analyte towards the stationary and mobile phase results in the differential
separation of the components.
Affinity, in turn, is dictated by two properties of the molecule: ‘Adsorption’
and ‘Solubility’.
Adsorption: as the property of how well a component of the mixture sticks
to the stationary phase, while
Solubility is the property of how well a component of the mixture dissolves
in the mobile phase.
Higher the adsorption to the stationary phase, the slower the molecule
will move through the column.
Higher the solubility in the mobile phase, the faster the molecule will move
through the column.
The interplay between these two factors determines the differential rates at
which the different components of the analyte will move through the
column.
Adsorption and solubility of a molecule can be manipulated by choosing the
appropriate stationary phase and mobile phase.
the question arises why do different compounds possess different affinities
towards the stationary and mobile phases?
Polarity” of the compounds dictates their affinities towards the stationary and
mobile phases.
Example:
Suppose we have a mixture of two molecules A and B, where ‘A’ is a protein and
‘B’ is a lipid
Our column is packed with silica, which is polar in nature; our mobile
phase is hexane, which is non-polar in nature.
What do you think will happen when we load this mixture of A and B onto
this column?
A’, being polar in nature, will adsorb on to the polar stationary phase
(silica).
B’ being non-polar in nature, will readily dissolve in the non-polar mobile
phase (hexane) without adhering to silica, and will thus elute out of the
column with hexane.
Recovery
Recovery
Once B is eluted out, the mobile phase will be changed to something polar like
acetonitrile
By doing so we will now force A to detach from the silica and dissolve in the
polar solvent, acetonitrile, and get eluted out of the column with acetonitrile.
Classification
There are several types of chromatography, each differing in the kind of
stationary and mobile phase they use.
The underlying principle though remains the same: differential affinities of
the various components of the analyte towards the stationary and mobile
phases results in the differential separation of the components.
Again, the mode of interaction of the various components with the stationary
and mobile phases may change depending on the chromatographic technique
used.
The commonly used chromatographic techniques are tabulated below.
Based on mechanism of separation
I. Adsorption chromatography
II. Partition chromatography
Based on phases
I. Solid phase chromatography
i.
Solid-liquid chromatography
ii. Solid-gas chromatography
II. Liquid phase chromatography
i. Liquid-liquid chromatography
ii. Liquid –gas chromatography
Based on shape of chromatographic bed
I. Planner chromatography
i. Paper chromatography
ii. Thin layer chromatography
Column chromatography
i. Packed column chromatography
ii. Open tubular column chromatography
Adsorption chromatography
IT is process of separation of components in a mixture introduced into
chromatography system based on the relative difference in adsorption of
components to stationary phase present in chromatography column.
Adsorption chromatography is one of the oldest types of chromatography.
The equilibration between the mobile and stationary phase accounts for the
separation of different solutes.
It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface
of a stationary solid phase.
Partition Chromatography:
Chromatography in which separation is based mainly on differences between
the solubility of the sample components in the stationary phase or on
differences between the solubility of the components in the mobile and
stationary phases.
This form of chromatography is based on a thin film formed on the surface
of a solid support by a liquid stationary phase.
GAS-liquid Chromatography;
Gas chromatography employs an inert gas as the mobile phase
The mobile phase is a gas, often nitrogen, but sometimes helium,
hydrogen or occasionally another gas. It is called the "carrier gas".
Common solids are charcoal, a synthetic zeolite called "molecular
sieve", or a combination of the two.
Separation depends on the relative partial pressures of the sample
components above the stationary phase.
Gas-solid chromatography is relatively rare, but it is used to separate
atmospheric gases
Solid-Liquid Chromatography:
Liquid chromatography (LC) is a separation technique in which the mobile
phase is a liquid.
The preferred mobile phase is a nonpolar or slightly polar...
Liquid chromatography can be carried out either in a column or a plane
In liquid-solid chromatography the porous adsorbent is polar.
Popular adsorbents are Silica and Alumina.
Liquid-Gas Chromatography
The mobile phase is an unreactive gas, such as nitrogen (the carrier gas)
The stationary phase comprises of a small amount of liquid held on a
finely-divided inert solid support.
Gas-liquid chromatography is very sensitive and can be used to detect small
quantities of substances
it is often used in forensic tests.
Stationary phase used in (LGC)
Dimethyl Polysiloxane (350oC)
Hydrocarbons, Polynuclear aromatics
Poly(phenyl methyl) siloxane (250oC)
Steroids, Pesticides, Glycols
Liquid-Liquid Chromatography
Liquid-liquid chromatography is a chromatography separation technique in
which the mobile phase is a liquid and the stationary phase is also a liquid.
The system is unstable
● The first liquid-liquid system was reported by A. J. P. Martin who used
water supported on silica gel as the stationary phase and n-heptane as
the mobile phase
● The system is unstable, as the stationary phase will always have some
solubility in mobile phase
Planner Chromatography:
Planar chromatography is a separation technique in which the stationary
phase is present on a plane.
The plane can be a paper, infused by a substance as the stationary bed
(paper chromatography) or a layer of solid particles spread on a support
such as a glass plate (Thin layer chromatography).
Different compounds in the sample mixture travel different distances
according to how strongly they interact with the stationary phase as compared to
the mobile phase.
The specific Retention factor (Rf) of each chemical can be used to aid in the
identification of an unknown substance.
Column chromatography is a separation technique in which the
stationary bed is within a tube.
The particles of the solid stationary phase or the support coated with a liquid
stationary phase may fill the whole inside volume of the tube (packed column) or
be concentrated on or along the inside tube wall leaving an open, unrestricted path
for the mobile phase in the middle part of the tube (open tubular column.
● Differences in rates of movement through the medium are calculated to
different retention times of the sample
