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Chromatography i anu

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Chroma tography -I Presenter: DR ANURAG YADAV Moderator: DR AVINASH S S 1 D r A n u r a g Y a d a v
Transcript

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Chromatography-I

Presenter: DR ANURAG YADAVModerator: DR AVINASH S S

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The word derived from Greek:

Initial described by Mikhail Tswett in 1903.

• colorChroma

• To writegraphein

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CHROMATOGRAPHY “Chromatography is a technique for

separating mixtures into their components in order to analyze, identify, purify, and/or quantify the mixture or components.”

Separate

• Analyze

• Identify

• Purify

• QuantifyComponentsMixture

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TERMINOLOGIES:

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.

Stationary phase - Immobilized phase

Immobilized on the support particles or on the inner wall of the

column tubing.

Examples : Silica layer - Thin Layer Chromatography

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TERMINOLOGIES:

Mobile phase

Moves in a definite direction. Liquid (LC), Gas (GC).

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) :Substance analyzed in chromatography.

Solvent : Any substance capable of solubilizing another

substance.

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Chromatogram

Visual output of the chromatograph.

Separation - Different peaks or patterns on the chromatogram

correspond to different components of the separated mixture.

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CLASSIFICATION:

1. Based on supporting medium

2. Based on mobile & stationary phase

3. Based on mechanism of separation

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1. BASED ON SUPPORTING MEDIUM

Supporting medium

planar column

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2. BASED ON MOBILE & STATIONARY PHASE:

MOBILE AND STATIONARY PHASE

GC

GLC GSC

LC

LLC LSC

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DEPENDING ON PHYSICAL PROPERTIES OF STATIONARY PHASE

LC – flat methods ( Paper chr , HPTLC , TLC ) - Column methods(open column LC, HPLC)

GC – packed column - wall coated column ( capillary )

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3. BASED ON MECHANISM OF SEPARATION:

MECHANISMION

EXCHANGE

PARTITION

ADSORPTION

AFFINITY

SIZE EXCLUSION

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PLANAR CHROMATOGRAPHY:

Based on principle of the partition Chromatography.

“The differential distribution of solute between two immiscible liquids on plane.”

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partition chromatography a process of separation of solutes utilizing the partition of the solutes between two liquid phases, namely the original solvent and the film of solvent.

When substance mixed with immiscible

solvent,it will distribute such that, At equilibirum the

ratio of its conc In two phase is constant

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This ratio is termed as partition coefficient & is characteristic of a particular substance for a given pair of solvent.

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TYPES OF PARTITION CHROMATOGRAPHY

Normal phase Reverse phase: Ion suppression & Ion pair

chromatography

Normal phase LC, stationary phase is polar & mobile phase is non-polar. water is the stationary phase; hexane, benzene, chloroform or butanol form the mobile phase.

Reverse phase LC, stationary phase is non-polar (eg. octadecyl silane packing in a column) and mobile phase is polar (solvents like methanol, acetonitrile used in column mode of chromatography).

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ION-SUPPRESSION CHROMATOGRAPHY

Ionic character of weakly acidic/basic→suppressed(by

modification of mobile phase PH →solutes become less

polar →interact with nonpolar stationary phase

→reverse phase chromatography

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ION-PAIR CHROMATOGRAPHY

Counter ion of analyte → added to mobile phase →ionic

pair with analyte →neutralysed analytes are separated by

reverse phase chromatography

Uses : separation of therapeutic drugs & metabolites.

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PAPER CHROMATOGRAPHY :

Paper chromatography is a variant of partition

chromatography procedure in which the cellulose

support is in the form of a sheet or paper

Cellulose contain a large amount of bound water

even when extensively dried

Partitioning occurs between the bound water and

the developing solvent

In paper chromatography the mixture to be

separated is spotted onto the paper and dried

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TYPES :

Paper chromatography. Ascending paper. Descending paper. Ascending-descending. Radial paper. Two-dimensional paper.

Thin layer chromatography.

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Paper Chromatography has different types or modes:

Ascending chromatography: As the name indicates, the chromatogram ascends. Here the development of paper occurs due the solvent movement or travel in upward direction on the paper.Descending chromatography: Here the development of paper occurs due to solvent travel downwards on the paper.

Ascending- descending mode: Here solvent first travels upwards and then down wards on the paper.

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Radial mode: Here the solvent travels from center(mid point) towards periphery of Circular chromatography paper.

Two dimensional chromatography: Here the chromatogram development occurs in two directions at right angles.

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INSTRUMENTATION

Chromatography jar

Capillary tube

Stationary phase (liquid impregnated paper)

Mobile phase

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INSTRUMENTATION

Chromatography jar:It is made of glass and has a lid on it. Jar maintains proper environment that is required for separation.

Capillary tube:It is used to apply sample mixture.

Stationary phase:liquid impregnated paper

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INSTRUMENTATION

Mobile phase:Mobile phase may be a single liquid or a mixture ofliquids.Commonly used mobile phases are;

Methanol Ethanol Ethyl acetate Diethyl ether Acetone Chloroform

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HOW TO PERFORM PAPER CHROMATOGRAPHY?

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1. PREPARING THE PAPER STRIPS

• Cut the filter paper into 5 x4 measurement.

• Draw a line 0.5 cm above the bottom edge of the strip with the pencil.

• Label each strip with its corresponding solution.

• • Place a spot from each

pen on your starting line.

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2. DEVELOPING THE CHROMATOGRAMS

• Place the strips in the beakers.

• Make sure the solution does not come above your start line.

• Keep the beakers covered.

• Let strips develop until the ascending solution front is about 2 cm from the top of the strip.

• Remove the strips and let them dry.

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More polar!

Less polar!

solvent frontoriginmixture

solvent front

component B

component A

origin

solvent front

component B

component A

origin

Increasing Development Time

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VISUALIZATION OF CHROMATOGRAPHY

If the sample is separated into colored components, thenthe location is dried in ordinary light. But in case ofcolorless components following are used;

Uv lamp

Iodine crystals

Spraying agents: Ninhydrin for aminoacids and

proteins , sulfuric acid for phospholipids ,

diphenylamine for sugars

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DOCUMENTATION

Storage of chromatogram.

Calculating Rf values

Calculate Rf value & interpret.

Defined : “as the ratio of the distance travelled by the substance & the distance travelled by solvent front”

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RF VALUE IMPORTANCE:

Ratio of distance travelled by the solute to

the distance travelled by the solvent

Rf value is constant for a particular solvent

system at a given temperature

Spots of the unknown substance can be

identified by comparing those of the pure

standards

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APPLICATIONS

It is used for separation and identification of;

Amino acids

Carbohydrates

Tannins

Glycosides

Alkaloids etc.

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THIN-LAYER CHROMATOGRAPHY (TLC)

“ The technique which involves flowing of mobile phase over a thin layer of adsorbent, applied on solid support, where separation of components occur by differential migration which occurs when solvent flows along fine powder spread on glass plates, is called thin –layer chromatography.”

Silica / Alumina layered over a glass plate ----- uniform thin layer(0.2mm)

Procedure same as that for paper

chromatography. Better resolution HPTLC: particle size-4.5μm.

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Instrumentation: Chromatography jar Capillary tube Thin layer chromatography plate Stationary phase Mobile phase

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Chromatography jar: It is made of glass and has a

lid on it. Jar maintains proper

environment that is required for separation.

Capillary tube: It is used to apply sample

mixture on TLC plate.

TLC plate: Borosilicate glass plates are

preferred. Most commonly used

sizes are; 20 X 20cm 20 X 10cm 20 X 5cm 

Mobile phase:

Mobile phase may be a

single liquid or a mixture of

liquids.

Commonly used mobile

phases are;

Methanol

Ethanol

Ethyl acetate

Diethyl ether

Acetone

Chloroform

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Procedure

Location Of Separated Components

Documentation

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APPLICATION:

It is used for separation and identification of;

Amino acids

Peptides and proteins

Alkaloids

Carbohydrates

Fats and fatty acids

Antibiotics

Narcotic analgesics

Glycosides

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ADVANTAGES OF TLC Simple Rapid Ability to process large number of samples in minimal

time Low cost in terms of reagent & equipment.

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APPLICATIONS OF CHROMATOGRAPHY.

• In clinical diagnosis : detection & estimation of amino

acids, metabolites, sugars, mucopolysaccharides in urine

& blood.

• Useful for screening and diagnosis of inborn metabolic

disorders : Aminoacidurias, hemoglobinopathies,

mucopolysaccharidoses, etc.

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APPLICATIONS OF CHROMATOGRAPHY

In clinical diagnosis

Paper chromatography,TLC –qualitative

HPLC, GC –For quantitation

In clinical diagnosis

Assay of Hormones, drugs, vitamins, metabolites ---HPLC and GC

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APPLICATIONS OF CHROMATOGRAPHY

Chromatography in protein research –

1) Purification –adsorption, ion exchange ,affinity, gel

filtration chromatography.

2) Sequencing – ion-exchange chromatography.

3) Mol.wt. determination – gel filtration chromatography.

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TROUBLESHOOTING

It seems to be easy procedure, but error

do occur like;

- Compound runs as streak rather than

spot (sample was overloaded)

- Sample run as smear/upward crescent

: compound possess strongly acidic or

basic group- add few drops of ammonium

hydroxide or acetic acid to the solvent.

- Sample runs downward crescent-

adsorbent was disturbed during spotting.

streak

More polar

Cross placed

Less sample

Normal

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- Plate solvent front runs

crookedly: either the adsorbent has

flaked off the sides or the side of plate

are touching sides of container.

- Many random spots

- No spots are seen on plate

- Blur blue spots on plate : use of ink

pen instead of pencil to mark the

origin.

streak

More polar

Cross placed

Less sample

Normal

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ADSORBENTS: “ An adsorbent is a substance, usually porous in nature and with a high surface area that can

adsorb substances onto its surface by intermolecular forces.”   AN IDEAL ADSORBENT: The Ideal adsorbent must fulfill the following requirements:   Insoluble in mobile phase Inert to solutes (adsorptive) Colorless especially when work with colored mixtures Suitable particle size enough to give good separation and reasonable flow rate COMMON ADSORBENTS: Hydrated silica gel Silica gel G Silica gel S Silica gel GF 254 Silica gel H Silica gel N Silica gel HF 254 Silica gel PF 254 Modified silica gel Alumina Kieselghur (Diatomaceous earth) Cellulose MN 300 Cellulose microcrystalline

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THIN LAYER CHROMATOGRAPHY (TLC)

In TLC, any substance that can be finely divided and formed into a uniform layer can be used.

Both organic and inorganic substances can be used to form a uniform layer for TLC.

Organic substances include: cellulose, polyamide, polyethylene

Inorganic: silica gel, aluminum oxide and magnesium silicate

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THIN-LAYER CHROMATOGRAPHY: A TWO-COMPONENT MIXTURE

More polar!

Less polar!

solvent frontoriginmixture

solvent front

component B

component A

origin

solvent front

component B

component A

origin

Increasing Development Time

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APPLICATIONS

1. Separation of carbohydrates:

Mobile phase: acetonitrile : water (85:15)

Detection: sulfuric acid : methanol (1:3)

heat for 10 min at 110 C to see brown spots

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Separation of Total Lipid into different Classes

Mobile Phase: hexane: diethyl ether: formic acid (80:20:2)

Cholesteryl esters

TAG

Free fatty acids

Cholesterol

1,3-DAG

1,2-DAG

Monoacyl glycerols

Phospholipids

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Separation of Triacylglycerols

Mobile Phase: Pet ether: diethyl ether: acetic acid (90:9:1)

Tristearin

2-oleodistearin

1-stereodiolein

Triolein

Trolinolein

With HUFA


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