The document discusses various chromatography techniques used to purify biological products from fermentation, including ion exchange chromatography, gel filtration chromatography, and affinity chromatography. It provides details on how each technique works, such as how ion exchange chromatography separates molecules based on surface charge using cation or anion exchangers, and how gel filtration chromatography separates proteins based on size as they diffuse in and out of gel beads of different porosity. The document also discusses formulation of biological products, including drying methods like spray drying and freeze drying which are used to produce stable dry powders.
Ribotyping
Introduction
History
Ribosomes
Ribosomal RNA
Principle of ribotyping
16S rRNA
Procedure of ribotyping
Types of ribotyping
Use of ribotyping
Advantage and disadvantage of ribotyping
Reference
Steps involved in fermentation products producing a viable product output.various steps and process were explained in them. A semester syllabus of undergraduate microbiology student in his/her semester -5 in paper -6 . I think this might be helpful to you and have a good response after reading this .thank you.
Process scale-up is a critical activity that enables a fermentation process achieved in research and development to operate at a commercially viable scale for manufacturing.
Recovery and purification of intracellular and extra cellular productsBangaluru
Product recovery and purification, such as centrifugal, chromatography, crystallization, dialysis, drying, electrophoresis, filtration, precipitation, etc., are essential finishing steps to any commercial fermentation process.
Ribotyping
Introduction
History
Ribosomes
Ribosomal RNA
Principle of ribotyping
16S rRNA
Procedure of ribotyping
Types of ribotyping
Use of ribotyping
Advantage and disadvantage of ribotyping
Reference
Steps involved in fermentation products producing a viable product output.various steps and process were explained in them. A semester syllabus of undergraduate microbiology student in his/her semester -5 in paper -6 . I think this might be helpful to you and have a good response after reading this .thank you.
Process scale-up is a critical activity that enables a fermentation process achieved in research and development to operate at a commercially viable scale for manufacturing.
Recovery and purification of intracellular and extra cellular productsBangaluru
Product recovery and purification, such as centrifugal, chromatography, crystallization, dialysis, drying, electrophoresis, filtration, precipitation, etc., are essential finishing steps to any commercial fermentation process.
The present document provide the information about Methods of extraction of drugs from the biological matrix (protein precipitation method, liquid extraction)
Dr.S.Karthikumar
Asst. Prof., Dept. of Biotechnology
Kamaraj College of Engineering and Technology
S.P.G.C.Nagar, Virudhunagar, Tamilnadu, India
skarthikumar@gmail.com
Gel chromatography, Introduction, Theory, Instrumentation, Applications .pptxVandana Devesh Sharma
Affinity chromatography- Content-Introduction
Theory
Instrumentation
Applications
Gel chromatography is a type of partition chromatography used for separating different sized molecules.
Gel chromatography is also called Gel permeation chromatography or gel filtration or gel exclusion, size exclusion, molecular- sieve chromatography.
The separation is based on the analyte molecular sizes since the gel behaves like a molecular sieve.
In size exclusion chromatography, the stationary phase is a porous matrix made up of compounds like
cross-linked polystyrene, cross-like dextrans, polyacrylamide gels, agarose gels, etc.
The gel structure being used contains pores of different diameters upto maximum size.
1.The test molecules are washed through a gel column and molecules larger than the largest pores in the gel are excluded from the gel structure.
2. Smaller molecules penetrate the gel and the extent of penetration depends on the molecular size----- This delay their movement through the column
This technique is used for the separation of proteins, polysaccharides, enzymes, and synthetic polymers. Instrumentation- A. Stationary phase- It is composed of semi-permeable, porous polymer gel beads with a well-defined range of pore sizes. eg. Dextran, Agarose, Acrylamide. 2. sample size and concentration- sample is applied in small volume (1-5% of the total bed volume).3. Column parameters- use long column, ratio of column diameter to column length (1:20 to :100). The method or steps used for gel preparation. 4. Choice of eluent/mobile phase- Buffers Ex- Phosphate buffer pH 7, NaCl solution, Ammonium acetate (CH3COO-NH4+ ), Ammonium bicarbonate (NH₄HCO₃) ethylenediamine acetate. 5. Effect of Flow rate- maintain with the help of pump. Elution carried out with buffer at optimal flow rate (Eg- 0.25-5ml/min) to give maximum resolution with optimal separation time.6. Separation of components from the sample-
Separation of component from mixture is achieved with the help of column. The retention volume (VR).7. Detection- Using UV absorption detectors. A graph of Elution Volume (ml) Vs Molecular weight. 7. Detection- Using UV absorption detectors. A graph of Elution Volume (ml) Vs Molecular weight. For calibration of the gel in column – Calibrators - (Proteins of known molecular weight. Procedure for gel filtration technique-1. Preparation of column- 2. Washing of the column- 3. Loading of the sample-4. Elution using mobile phase (buffers)5. Detection of compounds . Applications
High Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through a column with chromatographic packing material (stationary phase).
The various stages of processing that occur after the completion of the fermentation or bioconversion stage, including separation, purification, and packaging of the product
Similar to Purification by chromatography and formulation (20)
Coronavirus disease 2019 (COVID-19). Complete information on coronavirus. Introduction, history, symptoms, covid19 structure, S protein of coronavirus, M proteins of coronavirus, spreading variations of coronavirus, vaccines, drugs to control coronavirus.
FOXP2 gene mutated in a speech and language disorder.
In humans, mutation of ‘FOXP2’ gene, results in a severe developmental disorder that significantly disrupts speech and language skills.
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Operation “Blue Star” is the only event in the history of Independent India where the state went into war with its own people. Even after about 40 years it is not clear if it was culmination of states anger over people of the region, a political game of power or start of dictatorial chapter in the democratic setup.
The people of Punjab felt alienated from main stream due to denial of their just demands during a long democratic struggle since independence. As it happen all over the word, it led to militant struggle with great loss of lives of military, police and civilian personnel. Killing of Indira Gandhi and massacre of innocent Sikhs in Delhi and other India cities was also associated with this movement.
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3. Introduction:
The biological products of fermentation (proteins, pharmaceuticals,
diagnostic compounds and research materials) are very effectively
purified by chromatography.
Chromatography usually consists of a stationary phase and
mobile phase.
4. The stationary phase is the porous solid matrix packed in a column
(equilibrated with a suitable solvent) on to which the mixture of
compounds to be separated is loaded.
The compounds are eluted by a mobile phase.
5. The different types of chromatography techniques used for
separation of desired product(mainly proteins):
Chromatography Principle
Ion exchange chromatography Net charge
Gel filtration chromatography Size and shape
Affinity chromatography Net charge or
Biological affinity
and Molecular
recognition
9. It involves the separation of molecules based on their surface
charges.
Ion-exchangers are of two types (cation- exchangers which have
negatively charged groups like carboxymethyl and sulfonate, and
anion- exchangers with positively charged groups like
diethylaminoethyl (DEAE).
10. In ion-exchange chromatography, the pH of the medium is very
crucial.
The ionic bound molecules can be eluted from the matrix by
changing the pH of the elutant buffer.
Ion-exchange chromatography is useful for the purification of
antibiotics, besides the purification of proteins.
11. The resin is packed into a column, and the protein solution is
allowed through the column in a buffer whose composition
promotes the binding of some or all of the proteins to the resin.
Proteins are bound to the resin reversibly and can be displaced
by increasing or changing the ionic strength (or pH) of the
buffer. (which adds small ions to compete with the charged
groups of the macromolecules for sites on the resin).
Proteins are eluted from the column in order from the least
strongly bound to the most strongly bound.
14. For example if a solution consists of two different proteins such
as 75 kD and 120 kDa loaded on top of the colum.
15. Gel filtration separates proteins (or nucleic acids) primarily on the
basis of their effective size.
Like ion-exchange chromatography, the separation material consists
of gel beads that are packed into a column through which the
protein solution slowly passes.
The materials used in gel filtration are composed of cross-linked
polysaccharides (agarose or Sephadex G-150 beads) of different
porosity, which allow proteins to diffuse in and out of the beads.
16. For example if a solution consists of three different proteins such as
120 kDa, 75 kDa and 25 kDa.
To purify 120 kDa protein form mixture, the sample pass through a
column of Sephadex G-150 beads.
When the protein mixture passes through the column bed, the 120
kDa protein is unable to enter the beads and remains dissolved in the
moving solvent phase.
The gel beads allows only the entry of proteins that are less than
about 100 kDa size.
17. As a result, the 120 kDa protein is eluted as soon as the preexisting
solvent in the column (the bed volume) has dripped out.
In contrast, the other two proteins can diffuse into the interstices
within the beads and are retarded in their passage through the
column.
As more and more solvent moves through the column, these proteins
move down its length and out the bottom, but they do so at different
rates.
Among those proteins that enter the beads, smaller species are
retarded to a greater extent than larger ones.
Consequently, the 120-kDa protein is eluted in a purified state, while
the 75-kDa and 25 kDa protein remains in the column.
20. Affinity chromatography:
Affinity chromatography is based on an interaction of a protein with an
immobilized ligand.
Such interactions including hydrogen bonding, ionic interaction,
disulfide bridges, hydrophobic interaction,
The ligand can be a specific antibody, substrate, or an inhibitor or
antigen or enzyme.
The protein bound to the ligand can be eluted by reducing their
interaction. This can be achieved by changing the pH of the buffer.
22. Formulation:
For certain small molecules like (antibiotics, citric acid),
formulation can be done by crystallization by adding salts.
The formulation of low molecular weight products can be achieved
by concentrating them with removal of the water
23. Proteins may be formulated in the form of solutions, or dry
powders.
The sugars (sucrose, lactose), salts (sodium chloride, ammonium
sulfate), glycerol used as stabilizers for protein formulation.
Vaccines are formulated by mixing with fluids (such as saline).
24. Drying (Formulation):
Drying is an essential component of product formulation.
It basically involves the transfer of heat to a wet product for
removal of moisture.
25. These two types of dryers are commercially available.
Spray drying:
Freeze-drying:
26. Spray drying:
Spray drying is a method of producing a dry powder from a liquid.
This method preferred method of drying of many thermally-
sensitive materials such as foods and pharmaceuticals.
stream of hot gas
Spray dryer
27. Spray drying is used for drying large volumes of liquids.
In spray drying, liquid containing the product are passed through a
nozzle directing it over a stream of hot gas. (Liquid pumps in the
form of droplets).
The water evaporates and the solid particles are left behind.
This method used for formation of milk powder.
Formulation of vitamins, enzymes, amino acids, antibiotics.
28. Freeze-drying:
Freeze-drying or lyophilization is the most preferred method for
formulation of a wide-range of products.
Antibiotics, bacteria, viruses are formulated by using these process.
In this method, the product is rapidly frozen at a very low
temperature (-70°C) and then dehydrated by vacuum.
29. • Under these conditions, the microbial cells are dehydrated and
their metabolic activities are stopped;
• as a result, the microbes go into dormant state and retain viability
for years.