Bio analytical instruments

1. BIOANALYTICAL INSTRUMENTATION
Dr. M.S.DINESH
Department of Biotechnology
1

2. BIOANALYTICAL INSTRUMENTATION
INTRODUCTION
M.S.DINESH
Department of Biotechnology
2

3. Introduction to Chromatography techniques

4. Introduction to Chromatography technique

5. Introduction to Bio analytical instrumentation
PRINCIPLE OF COLUMN CHROMATOGRAPHY
The most powerful methods for fractionating proteins make use
of column chromatography.
It takes advantage of differences in protein charge, size,
binding affinity, and other properties.
A porous solid material with appropriate chemical properties
(the stationary phase) is held in a column, and a buffered
solution (the mobile phase) percolates through it.

6. Introduction to Chromotography
Stages of separation of proteins in Column Chromatography

7. Introduction to Bio analytical instrumentation
PRINCIPLE OF ION EXCHANGE CHROMATOGRAPHY
(IEX)
IEX is based on the reversible interaction between a charged
protein and an oppositely charged chromatography medium.
Biomolecules with even small differences in net surface
charge can be separated, and very high resolution is obtained
by choosing the optimal ion exchanger and separation
conditions.

8. Introduction to Ion exchange chromatography

9. Introduction to Ion exchange chromatography

10. Introduction to Size exclusion chromatography
PRICIPLE OF SIZE-EXCLUSION CHROMATOGRAPHY (SEC)
Also known as Gel Filtration (GF) is the simplest and mildest
of all chromatography techniques and separates molecules
based on differences in size.

11. Introduction to size exclusion chromatography

12. Introduction to affinity chromatography
PRINCIPLE OF PAFFINITY CHROMATOGRAPHY
This is based on the binding affinity of a protein.
The beads in the column have a covalently attached chemical
group.

13. Principle of affinity chromatography

14. Introduction to High Performance Liquid
Chromatography (HPLC)
High performance liquid chromatography is now one of the most
powerful tools in analytical chemistry.
It has the ability to separate, identify, and quantitate the compounds
that are present in any sample that can be dissolved in a liquid.
Compounds in trace concentrations as low as parts per trillion [ppt]
may easily be identified.
HPLC can be, and has been, applied to just about any sample,
Pharmaceuticals, food, nutraceuticals, cosmetics, environmental
matrices forensic samples, and industrial chemicals.

15. COMPONENTS OF HPLC

16. Introduction to Gas chromatography
The principles of gas chromatography (GC) are
similar to those of HPLC
The apparatus is significantly different.
It exploits differences in the partition coefficients
between a stationary liquid phase and a mobile gas
phase of the volatilised analytes.

17. Components of Gas Chromatography

18. Introduction to Spectroscopy
Spectroscopy is a scientific measurement technique.
It measures light that is emitted, absorbed,
or scattered by materials and can be used to study,
identify and quantify those materials.

19. Introduction to UV Visible Spectroscopy
Examples: Proteins, Polymers
Molecular (sub-)structures
responsible for interaction with
electromagnetic radiation are
called chromophores. In
proteins, there are three types
of chromophores relevant for
UV/Vis spectroscopy:
•peptide bonds (amide bond);
•certain amino acid side
chains (mainly tryptophan and
tyrosine); and
•certain prosthetic groups and
coenzymes (e.g. porphyrine
groups such as in haem).

20. Introduction to UV Visible Spectroscopy
Beer’s law tells us that absorption is proportional to the number of absorbing molecules –
ie to the concentration of absorbing molecules (this is only true for dilute solutions) –
And
Lambert’s law tells us that the fraction of radiation absorbed is independent of the
intensity of the radiation. Combining these two laws, we can derive the Beer-Lambert Law:
where Io = the intensity of the incident radiation
I = the intensity of the transmitted radiation
ε = a constant for each absorbing material, known as the molar absorption coefficient
(called the molar extinction coefficient in older texts) and having the units mol-1 dm3 cm- 1,
but by convention the units are not quoted
l = the path length of the absorbing solution in cm
C = the concentration of the absorbing species in mol dm-3

21. Introduction to Circular
Dichroism spectroscopy
Circular Dichroism, an absorption spectroscopy, uses
circularly polarized light to investigate structural
aspects of optically active chiral media.
It is mostly used to study biological molecules, their
structure, and interactions with metals and other
molecules.

22. Principle of CD Spectroscopy

23. Infra-red Spectroscopy or IR Spectroscopy
An invaluable tool in organic structure determination and verification involves the
class of electromagnetic (EM) radiation with frequencies between 4000 and 400
cm-1 (wavenumbers).
Absorption of IR is restricted to
compounds with small energy
differences in the possible vibrational
and rotational states.

24. Components of Infra-red Spectroscopy or IR
Spectroscopy

25. Principle of Microscopy
Microscopes constitute the very basic requirement for cell
biologists, thereby facilitating the deciphering the fine
details of intracellular components.
The first light microscope was developed in 1590 by Z.
Janssen and H. Janssen. During the next century, many
microscopic observations were reported, notably those of
Robert Hooke (who observed the first cells) and Antonie van
Leeuwenhoek (who provided first glimpse of internal cell
structure through improved microscopes).

26. Principle of Microscopy
There are fundamentally two different types of microscopes:
1.The light microscope and
2.The electron microscope.
Components of light microscope

27. Comparision of LM, TEM and SEM

28. Applications
Surface characterization of the atoms or molecules
Dimensions of the molecules
Internal cell organelle characterization
Imaging structural components of small specimens, such as cells
Conducting viability studies on cell populations (are they alive or dead?)
Imaging the genetic material within a cell (DNA and RNA)
Viewing specific cells within a larger population with techniques such as FISH
Large polymers structural characterization

29. THANK YOU
29
dineshms@pes.edu
+91 80 2672 6672 Extn. 349
Dr. M.S.Dinesh
Department of Biotechnology