types chrom
TRANSCRIPT
Types of Chromatography
Classification of chromatographyClassification of chromatography
• According to separation mode:
a) Adsorption chromatography
b) Partition chromatography
c) Ion-exchange chromatography
d) Size exclusion chromatography
e) Affinity chromatography
Classification of chrom. (cont.)Classification of chrom. (cont.)
• According to mobile phase: a) Gas Chromatography i- Gas solid chromatography ii- Gas liquid chromatography b) Liquid chromatography i- Paper chromatography ii- Thin-layer chromatography iii- Column chromatography
Classification of chrom. (cont.)Classification of chrom. (cont.)
• According to form of stationary phase
a) Planar chromatography
i- Paper chromatography
ii- Thin-layer chromatography
b) Column chromatography
i- Gas chromatography
ii- Liquid chromatography (LC/HPLC)
LIQUID-COLUMN CHROMATOGRAPHY
A sample mixture is passed through a column packed with solid particles which may or may not be coated with another liquid.
With the proper solvents, packing conditions, some components in the sample travel through the column more slowly than others resulting in the desired separation.
The basic liquid chromatography modes are named according to the mechanism involved:
1. Liquid/Solid Chromatography (adsorption chromatography)
A. Normal Phase LSC
B. Reverse Phase LSC
2. Liquid/Liquid Chromatography (partition chromatography)
A. Normal Phase LLC
B. Reverse Phase LLC
FOUR BASIC LIQUID CHROMATOGRAPHY
L C used for samples:L C used for samples:
containing large molecules/ioniccontaining substances with low vapor
pressure (non-volatile substances)Substances thermally unstableSubstances can’t be vaporized without
decomposing
LIQUID SOLID CHROMATOGRAPHY
Si - O - H
Normal phase LS Reverse phase LS
Silica Gel
The separation mechanism in LSC is based on the competition of the components of the mixture sample for the active sites on an absorbent such as Silica Gel.
LIQUID SOLID CHROMATOGRAPHY
Si - OH
HEXANE
OH
C-CH3
CH3
CH3- C
CH3
CH3
OH
OH
CH3
CH3
Adsorption chromatographyAdsorption chromatography
Stationery phase is solid and mobile phase is liquid
Distribution between two phases (adsorption and desorption)
Attractive forces (ionic, dipole-dipole, dipole induced dipole)
Good adsorbent has large surface area and more active sites
Equilibration occurs at: surface-solute, surface-solvent, solvent-solute
An adsorption isotherm is a plot of the concentration or amount of analyte on a surface as a function of its concentration in the bulk phase.
In liquid chromatography, the bulk phase is, of course, the mobile phase.
Adsorption isothermsAdsorption isotherms
Adsorption isothermsAdsorption isothermsLinear (k = Cs/Cm) = 1Convex (k = Cs/Cm1/n) where n >1Concave
AdsorbentsAdsorbents
Adsorbing power depends on
1- chemical nature of the surface
2- area available
3- pretreatment
Commonly used adsorbentsCommonly used adsorbents
Alumina, Al2O3 ,(Aluminum oxide). It may be acidic, basic and neutral in nature. Available in various grades
Silica gel (silicon dioxide). It is acidic in nature. Available in various grades.
CaCO3
SucroseStarch cellulose
Adsorbent should have uniform size and large surface area
Weight of the adsorbent should be 20-50 times more than the sample
Solvents & adsorbentsSolvents & adsorbentsSince adsorbents are polar, non-polar elute first.
Usually, the elusion order is as: alkyl halids < saturated hydrocarbons< unsaturated hydrocarbons <ethers < esters < ketones < amines < alcohols < phenols < acids and bases. Polymeric compounds and salts often don’t elute
The solvents in the order of polarity Hexane/Pet ether < CCl4 < toluene < dichloromethane < chloroform < diethyl ether < acetone < ethyl acetate < propanol < ethanol < methanol < acetic acid < water
Columns and packingColumns and packing
Various sizes are availableWet methodDry methodSample loading and running the column
WATER-SOLUBLE VITAMINS
1. Niacinamide 2. Pyridoxine
N
CONH2
N
CH2OH
CH2OH
HO
H3C
3. Riboflavin N
NNH
N
CH2
HOCH
HOCH
HOCH
CH2OH
O
OH3C
H3C
ClN
S
N
NH3C
CH2
NH2
CH3
CH2CH2OH
4. Thiamin
WATER-SOLUBLE VITAMINS
0 5 10 15 20
Column: u Bondapak C18 Solvent: MeOH Sample: Water-Soluble Vitamins
Inject1
2
3
4
LIQUID-LIQUID CHROMATOGRAPHY
ODPN(oxydipropionylnitrile)
Normal Phase LLC Reverse Phase LLC
NCCH3CH2OCH2CH2CN(Normal)CH3(CH2)16CH3 (Reverse)
The stationary solid surface is coated with a 2nd liquid (the Stationary Phase) which is immiscible in the solvent (Mobile) phase. Partitioning of the sample between 2 phases delays or retains some components more than others to effect separation.
Advantages of Partition Advantages of Partition chromatographychromatography
– Advantage over adsorption chromatographyMore reproducible and predictableDistribution coefficient is constant over a much
greater range of concentration yielding sharper and symmetrical peaks
It is also of two types1-Normal phase2-Reversed phase
Supports for stationary phaseSupports for stationary phase
Silica gelKieselguhr/CeliteCellulose
stepssteps
Sample preparationSample loadingElutionDetection: chemical methods; diverse types
of detectors can be used
ION-EXCHANGE CHROMATOGRAPHY SO3
-Na
+
Separation is based on the competition of different ionic compounds of the sample for the active sites on the ion-exchange resin (column-packing).
Ion exchange chromatographyIon exchange chromatography
A process where ions held by solid matrix are exchanged for counter ions in the solution
Synthetic ion exchange resins are used for water purification and separation of ions
MECHANISM OF ION-EXCHANGE CHROMATOGRAPHY OF AMINO ACIDS
SO3-
SO3-
Na+
COO-
H3N+
Na+
COOHH3N
+
pH2
pH4.5
Ion-exchange Resin
H3N
+
SO3-
SO3-
SO3-
SO3-
SO3-
SO3-
H3N+
COOH
OH
COOH
COOH
H3N+
H3N+
OH
COO-
Na+
H3N+
COO-
Na+
Na+
H+ OH
- = H2O
H+ OH
- = H2O
Na+
Na+
pH3.5
Mobile PhaseStationary Phase
Exchange Resin
pH4.5
Chromatography of Amino AcidsChromatography of Amino Acids
Ions exchange resinsIons exchange resinsConsist of three dimensional polymeric
chains, cross linked by short chains, which carry ionisable functional groups.
Based on ions these are of two types– Cation exchangers (weak or strong) – Anion exchangers (weak or strong)
Formation of resinFormation of resinStyrene and divinylbenzeneThe number of cross linkers
determine by the ratio of Styrene : divinylbenzene
Increasing cross linkers increases the rigidity and reduces swelling
Ion-exchange chromatography can be used to perform preparative separation of amino acids
Negatively charged resin binds selectively to positively charged amino acids
Behavior of resinBehavior of resin
Important properties which determine behavior of resin are:
1- size of particles
2- degree of cross linking
3- nature of functional groups
4- number of functional groups
Theoretical principlesTheoretical principlesIon exchange equilibriumdistribution coefficient (KD) indicates affinity of
the resin for ions relative to hydrogenGenerally, if KD is large, resin will incline to
attract the ionPolyvalent ions are more attracted to the resin
compared to mono-valent
In groups where charges are same, the difference between KD is related to the size of the ion
Uses of ion exchange Uses of ion exchange chromatographychromatography
Ion separationConcentration of trace mattersSeparation of alkali and alkali earth metalsSeparation of amino acids, proteins,
peptides, nucleic acid and nucleotidesimmunoglobulin
StepsSteps
Same to that of the othersDetection: conduction measuring
Size exclusion chromatographySize exclusion chromatography
Molecular gel chromatography, gel permeation, molecular sieving or molecular exclusion
Stationary phase serves as molecular sieveSeparate molecules based on size via
sieving or filtrationAdsorption and electrical charge play role
in separation
GelsGelsOpen three dimensional network formed by
cross linking large ploymeric chainsPolar groups absorb water and swellHave an exclusion limit i.e critical size of a
molecule that can just penetrate the interior
Theoretical principlesTheoretical principles
There are 2 kinds of solvent in the gel
Vi = Volume within gel
Vo = volume outside the beads of the gel
Large molecules will not be able to enter or penetrate the pores of the gel, hence their elution volume (Ve ) will be
Ve = Vo
Whereas, smaller molecules must be swept through Vo plus some additional volume which is a fraction of Vi
Ve = Vo + KDVi, where KD = distribution coefficient
KD = Average concentration of solute in gel/ Average concentration of solute outside gel
KD value should be between 0 and 1 If sieving action is the only mechanism of
separation (KD=1) thenVe = Vo + Vi
If K<1, It indicates solvent interacts with the gel (adsorption, hydrogen bonding)
Types of the gelsTypes of the gelsSephadex, dextran gel (classified by the
amount of water regain)Biogel, polyacrylamide gel, inert series of
gels, insoluble in water and common organic solvents
Styragel rigid cross linked polystyrene gel
useful at temperature > 150oC with organic solvents, it can be used under high pressure
Gel Filtration
ApplicationsApplications
Desalting (removal of salts and small molecules from macromolecules)
Concentrating (concentration of dilute solutions of macromolecules with MW> exclusion limit
Fractionation (separation of mixture of closely related molecules having small difference in KD values namely proteins, peptides, nucleic acids, polysaccharides, enzymes and hormones)
MOBILE PHASE LIQUID
Liquid-LiquidChromatography (Partition)
Liquid-SolidChromatography (Adsorption)
Liquid Solid
Normal Phase Reverse Phase Normal Phase Reverse Phase
Mobile Phase - NonpolarStationary phase - Polar
Mobile Phase - PolarStationary phase - Nonpolar
FORMAT
STATIONARYPHASE
Types of ChromatographyTypes of Chromatography
1.1. Ultraviolet DetectorUltraviolet Detector
200-400nm 200-400nm 254 nm254 nm
2.2. Reflective Index DetectorReflective Index Detector
Universal DetectorUniversal Detector
DetectorsDetectors
Affinity chromatographyAffinity chromatography
Separation where surface of inert phase has been modified to selectively bind compounds having specific functional group
Binding force should be strong enough to effect separation but weak enough to get the compound when desired
Properties of Inert matrixProperties of Inert matrix
Mechanically and chemically stableLarge surface areaEasily derivatizedGood flow characteristicsExamples (agarose, controlled pore glass,
cellulose)
Spacer Spacer An arm to move active group away from the bead
so that steric hindrances are at minimumEffectiveness depend on their
– length– stability of the attachment to the bead– hydrophobic nature– presence of fixed charges and their concentration
Affi-gel has spacer arm –O- (CH2)3 NH2 [Oxypropylamine]
Affinity Chromatography
Affinity Chromatography
Selected ligands and their affinity Selected ligands and their affinity compoundscompounds
Ligands
Diazo-NAD-
AMP analogues
Blue dextran 2000
Methotrexate
B12
Affinity compounds
dehydrogenases
NADP- binding proteins
Yeast phosphofructokinase
Dihydrofolate reductase
Transcobalamin I and II
Chromatographic techniquesChromatographic techniques
Classical LC TLC/ paper chromatography Modern LC
Classical chromatography Classical chromatography technoquestechnoques
Glass or plastic columns Need skill Solvent flow (gravity, suction) and individual
samples collected manually Detected using different detectors Detection and quantification achieved by manual
analysis of fractions Results are recorded in the form of chromatogram
(sample concentration vs fraction number)
DisadvantagesDisadvantages
Column packing procedure tediousLow column efficiency, long analysis timeTechnique depends on user Detection of solutes is labor intensive and
takes a lot of time
Plane chromatographyPlane chromatography
Plane surface rather than column2 dimensionalSelective properties (use of two solvents)Include
– Paper chromatography and – thin layer chromatography
PrinciplesPrinciples
Principles are similar to columnSuccessive equilibrations of the analyte
between two phasesNon ideal processes may cause zone
spreadingDegree of retention is Rf
– Ratio between distance traveled by solute/distance traveled by solvent
Relation between RRelation between Rff and K and K
Rf = number of moles of solute in mobile phase/total moles in both phases
= Cm Am/CmAm+ CsAs
Am and As are the cross sectional areas of two phases. By dividing Cm
Rf = Am/Am+AsCs/Cm = Am/Am+ KAs
Paper chromatographyPaper chromatography
Mainly qualitative and semi quantiativeEasy to performMechanisms
1- liquid liquid
2- adsorption
3- hydrogen bonding
4- ion exchange
Nature of the paperNature of the paper
Highly purified celluloseGreat affinity for water and polar solventsPaper may be impregnated with alumina,
silica or ion exchange resin
ProcedureProcedure
Sample application Development
1- ascending Simple and popular Solvent flow through capillary action Slow development Slow rate enhances partition, separation
2- descending2- descending
Flow is downwardPaper folded U shapeSolvent flow capillary and gravityMuch faster
detectiondetection Visible Application of Reagents UV absorbance Florescence IR Radioactivity Chemical tests Bioautography
qualitativequalitative
Based on Rf values
Semi-quantitativeExtraction and spectroscopydensitometry