ABOUT CHROMATOGRAPHY TECHNIQUES AND ITS TYPE FOR PROTEIN PURIFICATION IN INDUSTRIAL MICROBUOLOGY

1. CHROMATOGRAPHY
BY:DARAKHSHAN SALEEM
M.S MICROBIOLOGY

2. According to Britannica
Chromatography
Technique for separating the components,
or solutes, of a mixture.

3. On the basis of
Moving fluid stream,
called the Mobile Phase.
Contagious stage
or Stationary Phase
Either a Solid or LiquidEither a Gas or Liquid

4.  Laboratory technique for the Separation of
mixtures
 Chroma -"color" and graphein - "to write”.
 Colure bands - separation of individual compounds
 Measured or analyzed.
CHROMATOGRAPHY

5. PURPOSE OF
CHROMATOGRAPHY
PreparativePreparative:
Used to purify sufficient quantities of a
substance
AnalyticalAnalytical :
Determine Chemical composition of a
sample

6. TSWETT EXPERIMENT

7. TERMS
• Chromatograph - equipment that enables a
sophisticated separation.(Gas chromatography or
Liquid chromatography)
• Eluent - Fluid entering column/ solvent that carries
the analyte.
• Eluate - Mobile phase leaving the column.
• Stationary phase - Immobilized phase
 Immobilized on the support particles or on the inner

8. • MOBILE PHASE
• The mobile phase moves through the chromatography column
(the stationary phase) where the sample interacts with the
stationary phase and is separated.
• Retention time : Time takes for a particular analyte to pass
through the system (from the column inlet to the detector)
under set conditions.
• Sample (Anylate): Sample analyzed in chromatography
• Solvent : Any substance capable of solubilizing another
substance.

9. Visual output of the chromatograph.
Separation - Different peaks or patterns on the
chromatogram correspond to different components
of the separated mixture.
CHROMATOGRAM

10. Important Steps In Chromatography
Pack Column - Column is packed with material
(resin) that can absorb molecules based on some
property (charge, size, binding affinity, polarity)
 Equilibrate Column - Column is washed with
several column volumes of buffer

11.  Load sample - apply sample mix to column
 Wash column - Molecules washed through the
column with buffer
 Collect fractions - Fractions are taken, at
some point your molecule will elute

13. The techniques can be broadly divided into
Planar
Columnar.
CHROMATOGRAPHY TECHNIQUES

14. In Planar type the stationary phase is a plane surface i.e
(two dimension surface where only length and breadth are
taken as area) on which chromatograms are formed.
This method is adopted in techniques like
1. Using Paper.
2. Thin layer chromatography.
3.High performance thin layer

15. here is use of a column on whose walls lies a stationary
phase and the mobile phase is flushed through the column.
The techniques which employ this method are
1. Column Chromatography.
2. Gas Chromatography.
3. HPLC
4. Size Exclusion.
5. Ion Exchange.
COLUMNAR TYPE

16.  Planar techniques have the advantages
 like faster separation,
 visualization of formed chromatograms or spots and
are less expensive.
 But they are not useful for preparative purposes.
PROS AND CONS

17. In Columnar techniques, the advantages like better
or effective separations of even complex mixtures,
possibility to yield large amount of compounds by
separation of mixtures i.e preparative mode..
PROS AND CONS
But they have disadvantage of being expensive,
time consuming and cumbersome (heavy).

18. is a chromatographic technique used to
separate the components of a mixture using a
thin stationary phase supported by an inert
backing.
THIN LAYER CHROMATOGRAPHY
(TLC)

19.  Routinely used by researcher in the field of phyto-
Chemicals, Biochemistry,Pharma and Food Industry
 To identify the components in a compound mixture like
alkaloids, phospholipids, amino acids etc..
 It is a semi quantitative method of analysis
APPLICATION:

20. TLC SYSTEM
TLC Plate
FILTER PAPER
TLC Chamber
Mobile phase

21. Simple, Rapid and Cheap
Allow for quantification
Better separations
Choice between different adsorbents.
Better resolution

22. High Pressure Thin Layer Chromatography.
This is a sophisticated advancement in Thin Layer
Chromatography (TLC).
HPTLC
High Performance Thin layer Chromatography

23. The injection of a small volume of liquid sample into a tube
packed with tiny particles (3 to 5 micron (µm) in diameter
called the stationary phase)
where individual components of the sample are moved
down the packed tube
(column) with a liquid (mobile phase) forced through the
column by high pressure delivered by a pump.
HPLC IS A SEPARATION TECHNIQUE

24. These components are separated from one another by the
column packing that
involves various chemical and/or physical interactions
between their molecules and the packing particles.
• These separated components are detected at the exit of
this tube (column) by a
flow-through device (detector) that measures their amount.

25.  Detecting very small amounts
 High resolution
 Rapid analysis
 Speed, efficiency, sensitivity and ease of operation
 High degree of versatility
 Easily separate a wide variety of chemical mixtures
 400 atmospheres.
 PUMP PRESSURE 1000 atmosphere

26.  Protein separation
 Insulin purification
 Plasma fractionation
 Enzyme purification
APPLICATION OF HPLC

27. Column chromatography is the actual or the basic
type of chromatography procedure which was
developed during early stages of chromatography

28. When a mixture of mobile phase and sample to be
separated are introduced from top of the column, the
individual components of mixture move with different
rates. Those with lower affinity and adsorption to
stationary phase move faster and eluted out first while
those with greater adsorption affinity move or travel
slower and get eluted out last.
The solute molecules adsorb to the column in a
reversible manner

29.  Solid materials (Adsorbents) – Ability to hold
the molecules at their surface
 Attractive forces (Vanderwalls & Hydrogen )
 Functional groups (Hydroxyl/ Aromatic)
 Silica

30. The stationary phase material is suitably moistened with mobile
phase and packed sufficiently in the column with a cotton or asbestos
pad at the bottom. The extract material or sample to be separated is
placed on the top of packed stationary phase with a second cotton or
asbestos pad in between.
The mobile phase is poured into the column over the sample. A
collecting beaker is placed at the bottom of column near the end to
collect the elute.
PROCEDURE:

31. • Gas-Liquid chromatography, (GLC)
• Mobile phase – Gas (Helium) Carrier Gas Pressure = 4
kg/cm2
• Stationary phase - Column, which is typically
"packed" or "capillary".
• The stationary phase is adhered to the inside of a
small-diameter glass tube (a capillary column) or a
solid matrix inside a larger metal tube (a packed
column).
GAS CHROMATOGRAPHY

32. • High sensitivity,
• High Resolution,
• High speed
• High Accurasy,
• Highly Quantitative
ADVANTAGES

33. Size Exclusion Chromatography (SEC) is the separation technique based
on the molecular size of the components. Separation is achieved by the
differential exclusion from the pores of the packing material, of the
sample molecules as they pass through a bed of porous particles. The
principle feature of SEC is its gentle non-adsorptive interaction with the
sample, enabling high retention of biomolecular activity.

34. Separate by size
Column packed with porous beads
As wash with buffer:
Small molecules enter the beads
Large molecules move between the beads
Elution:
Large proteins elute first
small proteins last
Size Exclusion (Gel Filtration)

35.  SEC can be used to guide cell-line selection.
 It can also discriminate between aggregate forms that may be more
or less difficult to remove during downstream purification steps
 SEC can also be used extensively to guide the development of the
purification process for biopharmaceuticals
 not recommended for separating proteins with only a small
difference in molecular weight.

36. Separate by charge
Column packed with a charged resin
Use a charged buffer
• Like charged proteins flow through with buffer
• Oppositely charged proteins bind to column
Elute protein
• Increase salt or pH to elute protein of interest
ION EXCHANGE CHROMATOGRAPHY

37.  Separate by specificity
 Column packed with: Molecules (ligands) that interact
strongly with protein of interest
 As wash: Molecules of interest bind to column, other
proteins flow through
 Elution: Bound proteins eluted by adding high
concentration of ligand
AFFINITY CHROMATOGRAPHY

39.  Affinity chromatography is widely used in the pharmaceutical
industry to purify and extract molecules of interest from complex
mixtures.
 These molecules tend to be enzymes, proteins or amino acids, but
other biological species can be selectively retained.
 Once isolated, these biological species can be selectively amplified
to produce larger quantities, although at large concentrations.