Download - Chromatography i anu
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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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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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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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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