Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer...

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Chromatography “ writing in color “

Transcript of Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer...

Page 1: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

Chromatography“ writing in color “

Page 2: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

Chromatographic analysis

» Principles and theory.

» Definition.

» Mechanism.

» Types of chromatography.

» Uses of Chromatography.

Outlines of Lecture

Page 3: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

• In 1906 Mikhail Tswett used to chromatography to

separate plant pigments as chlorophylls, xanthophylls

and carotenoids by using calcium carbonate as

adsorbent and petrol ether/ethanol mixtures as eluent.

Mikhail TswettRussian Botanist (1872-1919)

•He called the new technique

chromatography because the

result of the analysis was

'written in color' along the

length of the adsorbent

column

•Chroma means “color” and

graphein means to “write”

Page 4: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

•Definition:

• Is a technique for separating mixtures into their

components in order to analyze, identify, purify,

and/or quantify the mixture or components, by

distribution of its components between a mobile and

stationary phase over time

Separate

• Analyze

• Identify

• Purify

• QuantifyComponentsMixture

Page 5: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

•Concepts:

•Mobile phase (Solvent or Eluent or carrier): The mobile

phase can be a gas (GC), a liquid, or a supercritical fluid

(SCFC), that carries the components.

•Stationary phase (Adsorbent): It is a part that does not

move with the sample

Page 6: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

•Concepts:

•The sample (solute): The sample

then has the opportunity to

interact with the stationary

phase as it moves past it.

Samples that interact greatly,

then appear to move more

slowly. Samples that interact

weakly, then appear to move

more quickly. Because of this

difference in rates, the samples

can then be separated into their

components.

Page 7: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

•How Does Chromatography Work?

Components

Separation

Mixture

Stationary Phase

Mobile Phase

Page 8: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

•Classification of chromatography according to mobile

phase:

• Liquid chromatography (LC): mobile phase

is a liquid (water or organic solvents).

• Gas chromatography (GC): mobile phase is

a gas.

• Supercritical fluid (SCFC): mobile phase is a

supercritical fluid (CO2(

Types of Chromatography

Page 9: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

•Classification according to the packing of

the stationary phase:

1.Thin layer chromatography (TLC): the

stationary phase is a thin layer

supported on glass, plastic or

aluminum plates.

2.Column chromatography (CC):

stationary phase is packed in a glass

column.

3.Paper chromatography (PC): the

stationary phase is a thin film of

liquid supported on an inert support.

Types of Chromatography

Page 10: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

•Classification according to the force of separation:

1.Adsorption chromatography:

Types of Chromatography

Separation based on the relative

differences in adsorption of

components to the solid

stationary phase.

Stationary phase is solid: applies

to only solid-liquid or solid-gas

chromatography

For polar non-ionic compounds

Ex; CC, TLC.

Page 11: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

•Classification according to the force of separation:

2. Partition chromatography:

Solute are separated based on their partition between a liquid

mobile phase and a liquid stationary phase coated on a solid

support.

Stationary phase and mobile phase are liquid in nature.

Types of Chromatography

Phase 2

Phase 1

Phase 2

Phase 1

Reverse – analyte is polar

organic; stationary phase

LESS polar than the mobile

phase

Ex : PC, HPLC, HPTLC, GC

Normal – analyte is nonpolar organic; stationary phase

MORE polar than the mobile phase

Page 12: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

•Classification according to the force of separation:

3. Ion exchange chromatography: Use ionic stationary phase

ions separated on the basis of their tendency to displace counter

ions adsorbed on stationary phase (Depends on charge, hydration,

“solubility”…)

Stationary phase: is a resin or gel matrix consisting of agarose or

cellulose beads with covalently bonded charged functional groups

Mobile phase: buffer, pH and salt concentration-opposite charged

solute ions attracted to the stationary phage by electrostatic force

For ionic compounds: large proteins, small nucleotides and amino

acids

Ex : CC, HPLC

Types of Chromatography

Page 13: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

•Classification according to the force of separation:

3. Ion exchange chromatography:

Types of Chromatography

Anionic exchange chromatography:

stationary phase is anion (Q-resin, a

Quaternary amine; and DEAE resin,

DiEthylAminoEthane), used for

cation separation

Cationic exchange chromatography:

stationary phase is cation (S-resin,

sulfate derivatives; and CM resins,

carboxylate derived ions), used for

anion separation

Page 14: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

•Classification according to the force of separation:

4. Size Exclusion Chromatography:

Separation is a result of “trapping” of molecules in the

pores of the packing material

Very large molecules can’t get into the pores –

unretained

Types of Chromatography

Very small molecules get

hung up in to pores for a long

time - most retained –

longest retention time

stationary phase is a porous

matrix

Ex: CC, HPLC

Page 15: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

Uses of Chromatography

• Chromatography is used by scientists to:

• Analyze: Examine a mixture, its components,

and their relations to one another

• Identify: Determine the identity of a mixture or

components based on known components

• Purify: Separate components in order to isolate

one of interest for further study

• Quantify: Determine the amount of the a

mixture and/or the components present in the

sample

Page 16: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

Uses of Chromatography

• Real-life uses of chromatography:

• Pharmaceutical Company: Determine amount of

each chemical found in new product

• Hospital: Detect blood or alcohol levels in a patient’s

blood stream

• Law Enforcement (forensic): To compare a sample

found at a crime scene to samples from suspects

• Environmental Agency: Determine the level of

pollutants in the water supply

• Manufacturing Plant: To purify a chemical needed to

make a product

Page 17: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

Thin layer chromatography (TLC)

• Definition of TLC:

•TLC: is a method for identifying substances

and testing the purity of compounds.

•The stationary phase: is a thin layer of

adsorbent (usually silica gel) coated on a

plate (glass, metal, or plastic).

•The mobile phase: is a developing liquid

(A solvent of varying polarity), which

travels up the stationary phase, carrying

the samples with it.

Page 18: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

Thin layer chromatography (TLC)

TLC sheet

Sample spot

Base line

Glass tank

Cover

Eluent

Adsorbant

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Thin layer chromatography (TLC)

Page 20: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

Thin layer chromatography (TLC)

Page 21: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

Thin layer chromatography (TLC)

• Interpreting the Data:

• If the spots can be seen, outline them

with a pencil.

• If no spots are obvious, use a UV lamp.

• If no spots are obvious with UV, use

iodine solution or Spray plate with

visualizing agents: Alkaloids:

Dragendorff’s reagent, Cardiac

glycosides: Antimony trichloride, Sugar:

Aniline phthalate, Amino acids:

Ninhydrin, flavonoids: Aluminium

chloride

Page 22: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

Thin layer chromatography (TLC)

• Interpreting the Data:

•Once visible, the Rf (relative

flow OR retention factor)

value of each spot can be

determined.

•The Rf is defined as the

distance from the center of

the spot moved divided by the

distance the solvent front

moved (both measured from

the origin)• Rf value is constant for a particular compound, solvent

system and insoluble matrix.

Page 23: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

Thin layer chromatography (TLC)

• Advantages of TLC:

• Low cost

• Short analysis time

• Ease of sample preparation

• All spots can be visualized

• Uses small quantities of solvents

• Wide choice of materials as sorbents

Page 24: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

Paper chromatography (PC)

• Definition of PC:

•PC: A method of partition chromatography using filter

paper as carrier or inert support.

•The factor governing separation of mixtures of solutes

on filter paper is the partition between two immiscible

phases.

•Stationary phase: usually water adsorbed on cellulose

fibers in the paper (bound water).

•Mobile phase: is the organic solvent flows past the

sample on the paper.

Page 25: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

Paper chromatography (PC)

• Principles of PC:

1. Compound is placed on

stationary phase

2. Mobile phase passes through the

stationary phase

3. Mobile phase solubilizes the

components and carries the

individual components a certain

distance through the stationary

phase, depending on their

attraction to both of the phases

Page 26: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

Paper chromatography (PC)

• Types of PC:1. Mono-dimension PC: Ascending chromatography: The

solvent travel in upward directionon the paper.

Descending chromatography: Thesolvent travel in downwarddirection on the paper. Note: The solvent reservoir is at the top,

The movement of solvent is assisted bygravity besides capillary action.

Radial chromatography: Thesolvent travels from centertowards periphery of paper

Page 27: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

Paper chromatography (PC)

• Types of PC:

2. Two dimensional chromatography

When large numbers of substances are to be

separated on a single chromatogram.

The sample is applied on one corner of a

square piece of paper and after development

with the first solvent, the paper is dried ,

rotated 90o and developed in the second

direction.

Usually, different types of solvents are used

in each direction.

It is essential that the first solvent be

completely volatile

Page 28: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

Paper chromatography (PC)

• Interpreting the Data:

•Spots in paper chromatograms can

be detected in 4 different ways: 1.

By their natural color. 2. By their

fluorescence. 3. By their chemical

reactions. 4. By radioactivity.

•The spots are usually identified by

comparing of standards of known

Rf values.

•By comparing the Rf values of components of a mixture

with the Rf values of known substances under identical

conditions, the compounds present in a mixture can be

identified

Page 29: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

Column chromatography (CC)

• CC is an extremely valuable technique for

purification of synthetic or natural products.

The same mechanism as TLC.

• A variety of adsorbents can be used as the

stationary phase (Organic: cellulose,

polyamide, polyethylene or Inorganic: silica

gel, aluminum oxide and magnesium silicate);

silica gel (which is very polar) is most

commonly used in organic chemistry.

• The mobile phase is a moving liquid or gas.

• There are two techniques based on column

chromatography, high performance liquid

chromatography (HPLC) and gas

chromatography (GC).

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Column chromatography (CC)

Page 31: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

Gas chromatography (GC)

• This is the most sensitive chromatographic technique

• It is capable of detecting as little as 10-12g of a

compound

• It is limited to compounds that can be easily

vaporised without decomposing.

Page 32: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

Gas chromatography (GC)

• Gas chromatography has the following features:

The mobile phase is a gas, usually nitrogen, called

the carrier gas

A small amount of sample is injected into the top of

the column through an injection port

The injection port is heated to a temperature

sufficient to instantly vaporise the sample, which is

then swept into the column by the carrier gas.

The column is in a loop, this is because of the fast

moving gaseous phase. So the column must be

longer than in HPLC to allow for effective interaction

with the stationary phase

The column is mounted in an oven and heated

Page 33: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

Gas chromatography (GC)

• Interpreting Chromatograms:

The time a component takes to pass through the

column is called the retention time, Rt.

The same compound will give the same retention

time if the conditions (temp, mobile phase,

stationary phase, flow rate, pressure etc) remain the

same.

Each component forms one peak, however it is

possible for a number of peaks to coincide and be

indistinguishable.

Page 34: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

Gas chromatography (GC)

Figure. Chromatogram of a reference sample containing a mixture

of butane, 2-methylbutane, hexane, benzene and 2-methylhexane.

Figure . Gas chromatogram of a petrol sample.

Page 35: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

High performance liquid chromatography (HPLC)

• This method is used for pharmaceutical and industrial

analysis.

• It allows extremely sensitive analysis of a wide range

of compounds.

Page 36: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

High performance liquid chromatography (HPLC)

• Advantages of HPLC:

High separation capacity, enabling the batch

analysis of multiple components

Superior quantitative capability and reproducibility

Moderate analytical conditions

Unlike GC, the sample does not need to be

vaporized.

Generally high sensitivity

Low sample consumption

Easy preparative separation and purification of

samples

Page 37: Chromatography techni… · 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary

High performance liquid chromatography (HPLC)

• What is HPLC used for?

Food products: Vitamins, food additives, sugars,

organic acids, amino acids, etc.

Environmental samples: Inorganic ions, Hazardous

organic substances, etc.

Organic industrial products: Synthetic polymers,

additives, surfactants, etc.

Biogenic substances: Sugars, lipids, nucleic acids,

amino acids, proteins, peptides, steroids, amines,

etc.

Medical products: Drugs, antibiotics, etc.