2.  Laboratory technique for the Separation of
mixtures
 Chroma -"color" and graphein - "to write”.
 Colour bands - separation of individual
compounds
 Measured or analysed.

3.  Analytical
 Determine Chemical composition of a sample
 Preparative
 Used to purify sufficient quantities of a
substance

4.  Chromatograph - equipment that enables a
sophisticated
separation
EX. Gas chromatography or Liquid
chromatography
 Eluent - Fluid entering column/ solvent that
carries the analyte.
 Eluate - Mobile phase leaving the column.

5.  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)- Substance analyzed in
chromatography.
 Solvent - Any substance capable of solubilizing
another substance.

6.  Techniques by Chromatographic bed shape
› Column chromatography
› Planar chromatography
 Paper chromatography
 Thin layer chromatography
 Techniques by Physical state of mobile phase
› Gas chromatography
› Liquid chromatography
 Affinity chromatography
› Supercritical fluid chromatography

7. Mobile Phase Stationary Phase
Solvent Bonded Phase
Separation is based on the analyte’s relative solubility
between two liquid phases
Partitioning

8. Normal Phase.
- Polar stationary phase and non-polar solvent.
Reverse Phase.
- Non-polar stationary phase and a polar solvent.

9. 1) Solvent reservoirs,
2) Solvent degasser,
3) Gradient valve,
4) Mixing vessel for
delivery of the
mobile phase,
5) High-pressure pump,
6) Switching valve in
"inject position"
Switching valve in
"load position",
7) Sample injection
loop,
8) Pre-column (guard
column),
9) Analytical column,
10) Detector (i.e. IR,
UV),
11) Data acquisition,
12) Waste or fraction
collector.

12. Ion Exchange
Gel Filtration
Affinity
Charge
Size
Conformatio
n

13. Partition chromatography uses a retained solvent, on the surface or
within the grains or fibres of an "inert" solid supporting matrix as
with paper chromatography; or takes advantage of some additional
coulombic and/or hydrogen donor interaction with the solid
support. Molecules equilibrate (partition) between a liquid
stationary phase and the eluent. Known as Hydrophilic Interaction
Chromatography (HILIC) in HPLC, this method separates analytes
based on polar differences. HILIC most often uses a bonded polar
stationary phase and a non-polar, water miscible, mobile phase.
Partition HPLC has been used historically on unbonded silica or
alumina supports. Each works effectively for separating analytes by
relative polar differences, however, HILIC has the advantage of
separating acidic, basic and neutral solutes in a single
chromatogram.

16. Ion-exchange chromatography is a process that allows the separation
of ions and polar molecules based on their charge. It can be used for
almost any kind of charged molecule including large proteins, small
nucleotides and amino acids. The solution to be injected is usually
called a sample, and the individually separated components are called
analytes. It is often used in protein purification, water analysis, and
quality control
Ion exchange chromatography retains analyte molecules on the
column based on coulombic (ionic) interactions. The stationary phase
surface displays ionic functional groups (R-X) that interact with
analyte ions of opposite charge. This type of chromatography is further
subdivided into cation exchange chromatography and anion exchange
chromatography. The ionic compound consisting of the cationic species
M+ and the anionic species B- can be retained by the stationary phase.

17. A charged solid phase or matrix
Liquid phase contains molecules
of different charges
Solutions (eluant) of different
charges to influence interactions
between liquid and solid phases

18.  1. A cation exchanger:
› Matrix negative charge
› Exchanges cations
 2. An anion exchanger:
› Matrix positive charge
› Exchanges anions
Solid matrix exchangers :-

21. Size exclusion chromatography (SEC), also known as gel
permeation chromatography or gel filtration chromatography,
separates particles on the basis of size. It is generally a low
resolution chromatography and thus it is often reserved for the
final, "polishing" step of purification. It is also useful for
determining the tertiary structure and quaternary structure of
purified proteins. SEC is used primarily for the analysis of large
molecules such as proteins or polymers. SEC works by trapping
these smaller molecules in the pores of a particle. The larger
molecules simply pass by the pores as they are too large to enter
the pores. Larger molecules therefore flow through the column
quicker than smaller molecules, that is, the smaller the molecule,
the longer the retention time.

22.  Gel permeation(GPC), gel filtration(GFC)
chromatography
 Technique applicable to separation of
high-molecular weight species
 Rapid determination of the molecular
weight or molecular-weight distribution
of larger polymers or natural products
 Solute and solvent molecules can diffuse
into pores -- trapped and removed from
the flow of the mobile phase

23.  Specific pore sizes-average residence time
in the pores depends on the effective size
of the analyte molecules
› larger molecules
› smaller molecules
› intermediate size
molecules

26. This is the most selective type of chromatography
employed. It utilizes the specific interaction between
one kind of solute molecule and a second molecule that
is immobilized on a stationary phase. For example, the
immobilized molecule may be an antibody to some
specific protein. When solutes containing a mixture of
proteins are passed by this molecule, only the specific
protein is reacted to this antibody, binding it to the
stationary phase. This protein is later extracted by
changing the ionic strength or pH.

27. Affinity Chromatography
Surface bound with
Epoxy, aldehyde or aryl ester groups
Metal Interaction Chromatography
Surface bound with
Iminodiacetic acid + Ni2+/Zn2+/Co2+
Affinity Chromatography

28. Affinity Chromatography
Binding Capacity (mg/ml) medium
12mg of histag proteins (MW= 27kDa)
Depends on Molecular weight
Degree of substitution /ml medium
~15mmol Ni2+
Backpressure ~43psi
Change the guard column filter

31. Pump
Injector
Column
Detector
Gradient
Controller
•

32. Restek® ULTRA C-18 and CN Columns (250mm x 4.6mm, 5µ),
Mobile Phase: (1:1 Methanol:Water), 1.5 mL/min.

33. A Supelcosil LC-PAH Columns B
Conditions: A: 150mm x 4.6mm, 5µ.
Flow Rate: 1.5 mL/min
Conditions: B: 50mm x 4.6mm, 3µ.
Flow Rate: 3.0 mL/min