Chromatography was first developed in 1906 by Russian scientist Tswett who separated plant pigments using calcium carbonate columns. The term "chromatography" comes from the Greek words for "color" and "to write". Chromatography separates mixtures based on how their components interact and distribute between a stationary and mobile phase. High performance liquid chromatography (HPLC) uses high pressure to force a mobile phase through a column packed with tiny particles. HPLC provides efficient separation of mixtures and is commonly used in analytical and preparative applications.

1. Chromatography
• Russian scientist Tswett in 1906 used a glass columns
packed with finely divided CaCO3 to separate plant
pigments extracted by hexane. The pigments after
separation appeared as colour bands that can come out
of the column one by one.
• Tswett was the first to use the term "chromatography"
derived from two Greek words "Chroma" meaning color
and "graphein" meaning to write.

2. Definition of chromatography
• Tswett (1906) stated that " chromatography is a method in
which the components of a mixture are separated on
adsorbent column in a flowing system”.
• IUPAC definition (International Union of pure and applied
Chemistry) (1993):
Chromatography is a physical method of separation in which
the components to be separated are distributed between two
phases, one of which is stationary while the other moves in a
definite direction.
The stationary phase may be a solid, or a liquid supported on a
solid or gel, the mobile phase may be either a gas or a liquid.

3. Principles of Chromatography
• Chromatography is a physical process.
• Any Chromatography system is composed of
three Components :
• Stationary phase
• Mobile phase
• Mixture to be separated
We can only control stationary and mobile phase
as mixtures are the problem we have to deal
with.
• Chromatography is a dynamic process in which
the mobile phase moves in definite direction.

5. Classification of Chromatographic methods
• Different methods were attempted for classification of
chromatography:
A – According to mechanism of separation:
The mechanism of separation depends mainly on
the nature of the stationary phase. Based on
separation mechanisms chromatography can be
classified into:

6. 1- Adsorption Chromatography:
It is the oldest and most common type of
chromatography. The stationary phase is a solid with
adsorption power. Mixture components will be
adsorbed on the surface of the stationary phase with
different powers and that account for separation. Silica
gel is the most common stationary phase in adsorption
chromatography.

8. 2- Partition Chromatography:
The stationary phase is a liquid forming a thin film on an inert
solid acts as support. The stationary liquid is usually more polar
than the mobile liquid. The two liquids must be immiscible with
each other. Cellulose powder and wet silica gel are examples of
supports in partition chromatography that carry film of water act
as stationary phase. Partition chromatography is preferable over
adsorption when dealing with polar compounds.
Solid Support Film of the liqiud
stationary Phase

9. 3- Ion Exchange Chromatography:
It is used for separation of charged molecules. The stationary phase
is an ion exchange resin to which a cationic or anionic groups are
covalently bonded. Ions of opposite charges (counter ions) in the
mobile phase will be attracted to the resin and compete with the
components of the mixture for the charged group on the resin. Both
the mixture components and the mobile phase must be changed.
Mixture of Alkaloids (compounds with positive charges) can be
separated on anionic exchanger, while mixture of organic acids
(negative charges) can be separated using cationic exchanger. Both
types are used for desalination of water.

11. 4- Molecular Exclusion
( Size Exclusion ) Chromatography:
• Size-exculsion chromatography (SEC), also called gel filtration
or gel-permeation chromatography (GPC), molecular
sieve chromatography uses porous particles to separate
molecules of different sizes.
• It is generally used to separate biological molecules.
• It is usually applied to large molecules or macromolecular
complexes such as proteins and industrial polymers.

12. 4- Molecular Exclusion
( Size Exclusion ) Chromatography:
very large molecules eluted first without separation
large molecules can enter some pores
very small molecules enter all pores and eluted at last

14. 5- Affinity Chromatography:
It uses the affinity of proteins to specific ligands such
as enzymes. The ligand is attached to suitable
polysaccharide polymer such as cellulose - agarose –
dextran.
Or
Affinity chromatography is a method of separating
biochemical mixtures based on a highly specific
interaction between antigen and antibody, enzyme and
substrate, or receptor and ligand.

16. B- ACCORDING TO MOBILE PHASE:
In this regard chromatography is classified into:
1- Liquid Chromatography (LC):
The mobile phase is liquid. In case of separation by adsorption
the stationary phase is solid so it is called: Liquid-Solid
Chromatography (LSC). If separation occurs through partition the
stationary phase is liquid so it is called: Liquid -Liquid
Chromatography (LLC).
2- Gas Chromatography (GC)
Where the mobile phase is inert gas nitrogen or helium. Again if
the stationary phase is solid it is called: Gas–Solid
Chromatography (GSC). When stationary phase is liquid it is
called: Gas-Liquid Chromatography (GLC).

17. C- ACCORDING TO THE TECHNIQUE (methods of
holding the stationary phase):
1- Planar or Plane Chromatography:
In this type of chromatography the stationary phase is used in the form of
layer. Plane chromatography is further classified into:
a- Thin Layer Chromatography (TLC):
A technique used to separate non-volatile mixtures. It is performed on a sheet
of glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent
material, usually silica gel, aluminium oxide (alumina), or cellulose.
b- Paper Chromatography (PC):
A specific type of papers is used as stationary phase in the form of sheets.
2- Columnar or Column Chromatography (CC):
The stationary phase is held in to a tube made of glass or metal.

19. D- ACCORDING TO PURPOSE OF USE:
Chromatography can be used for analytical work and also to obtain pure
materials from mixtures.
1- Analytical Chromatography:
a- Qualitative Chromatography
In this case Chromatography can be used to:
1- Confirm the absence or probable presence of certain constituent in the
sample under investigation
2- Give an idea about the complexity of the mixture and the least number
of compounds present.
3- Check purity and identity of any compound.
4- Establish a (finger print ) pattern for extracts , volatile oils or
pharmaceutical preparations. These finger prints can be then used to check
the identity and purity in the future.
5- Monitor both column chromatography and organic chemical reactions.

20. b- Quantities Chromatography:
The development of modern instruments enable the use
of chromatography to determine the amount of any
component in a mixture as absolute amount or relative to
another component HPLC/ GC/ HPTLC can be used for
there applications.

21. 2- Preparative application:
This was the first and is the main application of
chromatography. The technique was developed primarily
for this purpose.
Chromatography is used to obtain reasonable quantities of
pure compounds from mixtures.

22. HPLC
• High performance liquid chromatography is basically a
highly improved form of column chromatography.
Instead of a solvent being allowed to drip through a
column under gravity, it is forced through under high
pressures of up to 400 atmospheres. That makes it
much faster.
• It also allows you to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past it.
This allows a much better separation of the
components of the mixture

23. Types of HPLC
• Normal phase HPLC
• separation mode in which the retention material is
polar and mobile phase is nonpolar. Retained sample
components are eluted in ascending order of polarity
• Although it is described as "normal", it isn't the most
commonly used form of HPLC.
• The column is filled with tiny silica particles, and the
solvent is non-polar - hexane, for example. A typical
column has an internal diameter of 4.6 mm (and may
be less than that), and a length of 150 to 250 mm.

24. • Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will. The non-polar
ones will therefore pass more quickly through the
column.

25. • Reversed phase HPLC
• separation mode in which the stationary phase is
nonpolar and mobile phase is polar. Elution order of
components is in decreasing order of polarity. It is
the most commonly used mode of HPLC separations.
• Silica is modified to make it non-polar by attaching
long hydrocarbon chains to its surface - typically with
either 8 or 18 carbon atoms in them. A polar solvent
is used - for example, a mixture of water and an
alcohol such as methanol.

26. • In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column. There won't be as
much attraction between the hydrocarbon chains
attached to the silica (the stationary phase) and the
polar molecules in the solution. Polar molecules in
the mixture will therefore spend most of their time
moving with the solvent.

27. • Non-polar compounds in the mixture will tend to
form attractions with the hydrocarbon groups
because of van der Waals dispersion forces. They will
also be less soluble in the solvent because of the
need to break hydrogen bonds as they squeeze in
between the water or methanol molecules, for
example. They therefore spend less time in solution
in the solvent and this will slow them down on their
way through the column.
• That means that now it is the polar molecules that
will travel through the column more quickly.

28. • Size-exclusion chromatography (SEC),
• also known as gel permeation chromatography or gel
filtration chromatography, separates particles on the
basis of molecular size (actually by a particle's
Stokes radius). It is generally a low resolution
chromatography and thus it is often reserved for the
final, "polishing" step of the purification.

29. • 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.
• This technique is widely used for the molecular
weight determination of polysaccharides

30. . BASED ON ELUTION TECHNIQUE
• 1.Isocratic elution
• A separation in which the mobile phase composition remains
constant throughout the procedure is termed isocratic elution
• Best for simple separations
• Often used in quality control applications that support and are
in close proximity to a manufacturing process

31. • Gradient elution
• A separation in which the mobile phase composition is
changed during the separation process is described as a
gradient elution
• Gradient elution decreases the retention of the later-eluting
components so that they elute faster, giving narrower peaks .
This also improves the peak shape and the peak height
• Best for the analysis of complex samples
• Often used in method development for unknown mixtures
• Linear gradients are most popular

32. Instrumentation and working

33. Instrumentation and working
• A . Solvent delivery system(mobile phase reservoir)
• The mobile phase in HPLC refers to the solvent being
continuously applied to the column or stationary phase
• The mobile phase acts as a carrier to the sample solution
• A sample solution is injected into the mobile phase of an assay
through the injector port
• As a sample solution flows through a column with the mobile
phase, The components of that solution migrate according to
the non-covalent interaction of the compound with the column

34. • The chemical interaction of the mobile phase and sample ,
with the column , determine the degree of migration and
separation of components contained in the sample
• The solvents or mobile phases used must be passed through
the column at high pressure at about 1000 to 3000 psi. this is
because as the particle size of stationary phase is around 5-
10µ, so the resistance to the flow of solvent is high.

35. • B. Pumps
• •The role of the pump is to force a liquid (called the mobile
phase) through the liquid chromatograph at a specific flow
rate, expressed in milliliters per min (mL/min).
• •Normal flow rates in HPLC are in the 1-to 2-mL/min range.
• •Typical pumps can reach pressures in the range of 6000-9000
psi (400-to 600-bar).
• •During the chromatographic experiment, a pump can deliver a
constant mobile phase composition (isocratic) or an increasing
mobile phase composition (gradient).

36. • C. Injector:
• The injector serves to introduce the liquid sample into the flow stream of
the mobile phase for analysis.
• It is equipped with six port valves so that a sample can be injected into the
flow path at continuous pressure
• For a manual injector, the knob is manually operated to deliver the sample
to the column
• The knob is set to LOAD position for sample injection using a syringe , the
sample is injected into the sample loop , which is separated from the flow
path
• The knob is turned to INJECT position and the eluent travels through the
loop from the pump and delivers the sample to the column

37. • •Typical sample volumes for manual injector are 5-to 20-microliters (μL).
• •The injector must also be able to withstand the high pressures of the liquid
system.
• •An autos ampler is the automatic version for when the user has many
samples to analyze or when manual injection is not practical. It can
continuously Inject variable volume a of 1 μL – 1 mL

38. • D. Column
• Considered the “heart of the chromatograph” the column’s
stationary phase separates the sample components of interest
using various physical and chemical parameters.
• It is usually made of stainless steel to withstand high pressure
caused by the pump to move the mobile phase through the
column packing other material include PEEK and glass
• •The small particles inside the column are called the “packing”
what cause the high back pressure at normal flow rates.
• Column packing is usually silica gel because of its particle
shape , surface properties , and pore structure give us a good
separation

39. • Other material used include alumina, a polystyrene-divinyl
benzene
• synthetic or an ion-exchange resin
• – Pellicular particle: original, Spherical, nonporous beads,
• proteins and large biomolecules separation (dp: 5 μm)
• – Porous particle: common used, dp: 3 ~ 10 μm. Narrow size
• distribution, porous microparticle coated with thin organic
film
• The dimensions of the analytical column are usually
• -straight, Length(5 ~ 25 cm), diameter of column(3 ~ 5 mm),
diameter of particle(35 μm). Number (40 k ~ 70 k plates/m)

40. Guard column is used to remove particular matter and
contamination, it protect the analytical column and contains
similar packing its temperature is controlled at < 150 °C, 0.1 °C
As mention before , columns are divided into different types
according to their functions.

41. E . Detector:
•The detector can detect the individual molecules that elute from the
column and convert the data into an electrical signal
•A detector serves to measure the amount of those molecules
•The detector provides an output to a recorder or computer that results in
the liquid chromatogram
•Detector is selected based on the analyte or the sample under detection

42. Commonly used detectors in HPLC
Ultraviolet (UV)
•This type of detector responds to substances that absorb light.
•The UV detector is mainly to separate and identify the principal active
components of a mixture.
•UV detectors are the most versatile, having the best sensitivity and
linearity.
•UV detectors cannot be used for testing substances that are low in
chromophores (colorless or virtually colorless) as they cannot absorb
light at low range.
•They are cost-effective and popular and are widely used in industry

43. Fluorescence
•This is a specific detector that senses only those substances that emit
light. This detector is popular for trace analysis in environmental
science.
•As it is very sensitive, its response is only linear over a relatively
limited concentration range. As there are not many elements that
fluoresce , samples must be syntesized to make them detectable.
Mass Spectrometry
•The mass spectrometry detector coupled with HPLC is called HPLC-
MS. HPLC-MS is the most powerful detector, widely used in
pharmaceutical laboratories and research and development.
•The principal benefit of HPLC-MS is that it is capable of analyzing and
providing molecular identity of a wide range of components.

44. Refractive Index (RI) Detection
The refractive index (RI) detector uses a monochromator and is one of
the least sensitive LC detectors.
•This detector is extremely useful for detecting those compounds that are
non-ionic, do not absorb ultraviolet light and do not fluoresce.
•e.g. sugar, alcohol, fatty acid and polymers.

45. F . Data processing unit (Computer)
•Frequently called the data system, the computer not only controls all the
modules of the HPLC instrument but it takes the signal from the detector
and uses it to determine the time of elution (retention time) of the sample
components (qualitative analysis) and the amount of sample (quantitative
analysis).
•The concentration of each detected component is calculated from the
area or height of the corresponding peak and reported.

46. • Ultra High Performance Liquid Chromatography
(uHPLC):
Where standard HPLC typically uses column particles
with sizes from 3 to 5µm and pressures of around
400 bar, uHPLC use specially designed columns with
particles down to 1.7µm in size, at pressures in
excess of 1000 bar.

47. ApplicAtions
HPLC is one of the most widely applied analytical separation
techniques.
Pharmaceutical:
• Tablet dissolution of pharmaceutical dosages.
• Shelf life determinations of pharmaceutical products.
• Identification of counterfeit drug products.
• Pharmaceutical quality control.

48. Environmental
•Phenols in Drinking Water.
•Identification of diphenhydramine in sediment samples.
•Biomonitering of PAH pollution in high-altitude mountain lakes
through the analysis of fish bile.
•Estrogens in coastal waters - The sewage source.
•Toxicity of tetracyclines and tetracycline degradation products to
•environmentally relevant bacteria.
•Assessment of TNT toxicity in sediment..

49. Forensics
•A mobile HPLC apparatus at dance parties - on-site identification and
quantification of the drug Ecstasy.
•Identification of anabolic steroids in serum, urine, sweat and hair.
•Forensic analysis of textile dyes.
•Determination of cocaine and metabolites in meconium.
•Simultaneous quantification of psychotherapeutic drugs in human
plasma.

50. Clinical
•Quantification of DEET in Human Urine.
•Analysis of antibiotics.
•Increased urinary excretion of aquaporin 2 in patients with liver
cirrhosis.
•Detection of endogenous neuropeptides in brain extracellular fluids.

51. Food and Flavor
•Ensuring soft drink consistency and quality.
•Analysis of vicinal diketones in beer.
•Sugar analysis in fruit juices.
•Polycyclic aromatic hydrocarbons in Brazilian vegetables and fruits.
•Trace analysis of military high explosives in agricultural crops.
Stability of aspartame in the presence of glucose and vanillin

52. ADVANTAGES OF HPLC:
1. Separations fast and efficient (high resolution power)
2. Continuous monitoring of the column effluent
3. It can be applied to the separation and analysis of very complex
mixtures
4. Accurate quantitative measurements.
5. Repetitive and reproducible analysis using the same column.
6. Adsorption, partition, ion exchange and exclusion column
separations are excellently made.
7. HPLC is more versatile than GLC in some respects, because it has the
advantage of not being restricted to volatile and thermally stable
solute and the choice of mobile and stationary phases is much wider
in HPLC

53. 8. Both aqueous and non aqueous samples can be analyzed with little or
no sample pre treatment
9. A variety of solvents and column packing are available, providing a
high degree of selectivity for specific analyses.
10. It provides a means for determination of multiple components in a
single analysis.