Introduction to Chromatography (Column chromatography)

Introduction to:
Column chromatography
Silica gel columns
RP-silica gel columns
Ion exchange chromatography
Sephadex LH-20
TLC
Paper Chromatography

1. CHROMATOGRAPHY
INTRODUCTION-I
Ahmed M. Metwaly
Associate Professor at Faculty of Pharmacy, Al-Azhar University: Cairo, Egypt
Associate Professor at City of Scientific Research and Technological Applications (SATR), Egypt
Research Scientist at Liaoning University of Traditional Chinese Medicine -Dalian Campus: Dalian, Liaoning,
China (2-18-2019).
Research Scientist at National Centre for Natural Products Research, University of Mississippi, USA (2012-2014).

2. WHAT IS CHROMATOGRAPHY?
Chromatography is a technique for separating a mixtures into their components
in order to identify, quantify and/or purify component/s.
Chromatography, from Greek :chroma, colour and :"graphein" to write.

3. HISTORY OF CHROMATOGRAPHY
Mikhail Tswett's 1900 experiment aimed to separate plant pigments, specifically
chlorophyll, using a method called adsorption chromatography. In this process, a light
petroleum extract of green leaves slowly percolated through a vertical glass tube filled
with powdered calcium carbonate. The pigments, such as xanthophylls and chlorophylls
that were more strongly adsorbed, formed distinct colored bands near the top of the
column. Conversely, the pigments with weaker adsorption, like carotenes, accumulated
lower down in the column.

4. PRINCIPLE
The separation process involves spreading the component/s between a moving
(mobile) phase and a stationary phase.
The component/s that favor the moving phase pass through the chromatographic
system more quickly than those that prefer the stationary phase.
As a result, the substances are released from the chromatographic system in the
opposite order of their distribution coefficients concerning the stationary phase.
Separate
•Identify
• Purify
• Quantify
ComponentsMixture

6. USES FOR CHROMATOGRAPHY
Chromatography is used by scientists to:
•Identify: determine component/s comparing known component/s
•Quantify: determine the amount of the component/s
•Isolate/Purify: separate component/s for further study

7. APPLICATIONS OF CHROMATOGRAPHY
•Pharmaceutical Companies
• Hospitals: detect drug, toxicant or alcohol level in a patient’s blood stream
• Law Enforcement: to compare a sample found at a crime scene to samples from suspects
• Environmental Agencies: determine the level of pollutants in the water supply

8. CLASSIFICATION OF THE CHROMATOGRAPHY
According to the mobile phase:
1.Liquid chromatography, in which the mobile phase is a liquid.
2.Gas chromatography, in which the mobile phase is a gas.
According to the stationary phase:
1. Liquid-solid chromatography, as in column chromatography and thin layer chromatography [TLC].
2. Liquid-liquid chromatography, as in column partition chromatography, thin layer partition
chromatography and paper chromatography [PC].
3. Gas-solid chromatography [GSC].
4. Gas-liquid chromatography [GLC].
According to method of holding the stationary phase:
1. Planer chromatography as PC and TLC.
2. Columnar chromatography as CC.
According to the purpose of use:
1. Analytical chromatography: aiming at identification (qualitative chromatography) or
determination of the amounts (quantitative chromatography) of the sample components.
2. Preparative chromatography: aiming at the isolation of the sample components.

9. SEPARATION MECHANISM
▪Adsorption
▪Partition
▪Size Exclusion
▪Ion -Exchange & Ion -Interaction
▪Affinity (antibody-antigen interactions; chemical
interaction; attraction)

10. MECHANISM OF SEPARATION
Adsorption Partition Ion exchange

11. MECHANISM OF SEPARATION
Affinity Size Exclusion

12. ADSORPTION
▪Thecomponentswithinthemixtureunderanalysisareinfluencedbytheirselectiveretention,or
adsorption,bythesorbent(stationaryphase).Themigrationofthesecomponentsisdrivenbyanon-
selectiveforceexertedbythesolvent.Thesolventinducesacompetitionbetweenthecompound
adsorbedandtheactivesitesontheadsorbent.Separationoccurswhenthereisastrongeraffinity
betweenthesolventandtheadsorbentcomparedtotheaffinitybetweenthecompoundandthe
adsorbent.
▪Silicaandaluminaarecommonlyusedporousadsorbentsinadsorptionchromatography.These
adsorbentshavesurfacesterminatedwithpolarhydroxylgroups,facilitatingsurfaceinteractionswith
solutemolecules.
▪Inseparation,it'sessentialtomatchthesample'spolaritywiththeadsorbentanduseasolventwith
theoppositepolarityforflushingout.Weexploitslightdifferencesinhowsubstancessticktothe
movingandstationaryphasestoseparatethem.
▪Forexample,onapolaradsorbentlikesilica,twopolarsubstancescanbeseparatedusingaless
polarsolvent.Butifaverypolarsolventisused,bothsubstancesmaymovethroughthecolumn
withoutgettingseparated.
▪Organiccompoundscanbegroupedbyhowwelltheysticktotheadsorbent.Acidsandbases
behavedifferentlyfromalcoholsandthiols,followedbyaldehydesandketones,halogen-containing
compounds,esters,steroidsandhydrocarbons.
▪Theattachmentofhydrocarbonstotheadsorbentgetsstrongerwithmoredoublebonds.

13. TYPES OF ADSORBENTS (STATIONARY PHASE)
▪1) Silica, Silica gel
▪This is one of the most widely used adsorbent in both column and thin-layer.
▪◊Silica gel is a porous material, with very large surface areas. The average particle size
60-200 mesh, 75-250 µm (for open column)
▪◊The chemical nature of the surface of silica gel consists of silanol groups.
▪◊These OH groups are the active centres and potentially can form strong H-bonds with
the compounds which are chromatographed.
▪◊Thus, the stronger the H-bonding potential of a compound, are strongly adsorbed on
silica gel e.g. polar compounds (containing COOH, NH2 or amide groups) are strongly
adsorbed on silica gel; while Non-polar compounds (terpenes &
hydrocarbons, lacking polar functional groups) are poorly adsorbed on silica gel.
The mesh size: refers to the mesh number and its relationship
to the size of the opening in the mesh. The mesh number is in
fact the number of openings in one linear inch of screen. The
higher this number, the smaller the particles are because the
size of the openings narrows down.

15. Advantages
High surface area: allowing direct interactions with solutes or after bonding of variety of ligands
for versatile interactions with the sample molecules, leading to better separations.
Chemical and thermal stability and inertness: Silica gel is chemically stable, as it usually does
not react with either the solvents of the mobile phase nor the compounds being separated.
Wide applicability: Silica gel is versatile and can be modified with various functional groups,
making it suitable for a wide range of analytes and applications.
Efficient separation: The unique properties of RP-silica gel particles, combined with their high
surface area and controlled average particle diameter pore size, facilitate efficient and precise
separation of compounds
Reproducibility: Silica gel particles can offer high batch-to-batch reproducibility, which is crucial
for consistent and reliable HPLC analyses throughout decades.
Particle diameter and pore size control: Silica gel can be engineered to have specific pore sizes,
enabling precise control over separation based on molecular size.
Cost-effectiveness: Silica is the most abundant element on earth, hence its gel is a cost-effective
choice.
Disadvantages
•Some oxidizing potential
•Used for one time only
•Reduce the sample amount

16. Neutral alumina (pH ~ 7)
Is useful for the separation of relatively non-polar
compounds, e.g. Steroids.
Alumina a porous polymer of aluminum oxide
Acidic alumina (pH ~ 4)

Is useful for the separation of acidic compounds e.g. Carboxylic acids.
Basic alumina (pH ~ 10)
Is useful for the separation of basic compounds, e.g. Alkaloids. Disadvantage of alumina as adsorbent:
Alumina can catalyze both inter-and intramolecular reactions especially with
compounds which are base sensitive due to its alkaline nature.

17. Reversed phase silica (RP- Silica, C-8 and C-18)
It’s a non-polar type of silica resulted from the linking of
•Alkyl chain length of C-8 and C-18 are the most commonly used for RP
silica.
•The mobile phases are mixtures of water and polar organic solvents, the vast
majority of which are methanol and acetonitrile. These mixtures
usually contain various additives such as buffers (acetate, phosphate, citrate)
Bonded normal phase silica
•The main advantage of bonded normal phase supports over unmodified silica
gel is the increased stability to polar solvents including water, which allows
the separation of very polar compounds that would be highly retained on
silica gel.
The most popular column in this classification is an octadecyl carbon chain (C18)-bonded silica This is
followed by C8-bonded silica, pure silica, cyano-bonded silica (CN) and phenyl-bonded silica.
Advantages
•High efficiency (mostly used in HPLC)
•Totally inert
•Doesn't reduce the sample amount
•Used for several times
Disadvantages
•Expensive
•Needs pressure

18. Adsorption Normal Phase Reversed phase (RP)

19. ▪2. Partitionmechanism,liquidstationaryphases.Thesolutesofthemixture
distributesbetweenthemobileandthestationaryphasesproportionallyaccordingto
theirpartitioncoefficient,andtherelativepositionofanyonesoluteisdeterminedby
itsrelativesolubilityinthetwophases.Thosesubstanceshavingagreateraffinityforthe
stationaryphasemovemoreslowlythanthosehavingagreateraffinityforthemobile
phase.
▪Theliquidstationaryphaseisadsorbedonaninertsupport,whichmaybeeither
packedinachromatographictube(ColumnPartitionChromatography)orlayeredona
glassplate(PartitionTLC)orintheformofsheetsofpaper(PC).

20. ION EXCHANGE MECHANISM
▪Ionexchangemechanism,isaformofchromatographythatseparatesionsand
ionizablepolarmoleculesbasedontheiraffinitytotheionexchanger.
▪Itworksonalmostanykindofchargedmolecule—includingsmallinorganic
anions,largeproteins,smallnucleotides,andaminoacids.However,ion
chromatographymustbedoneinconditionsthatareonepHunitawayfromthe
isoelectricpointofaprotein.
▪Thetwotypesofionchromatographyareanion-exchangeandcation-exchange.
▪Cation-exchangechromatographyisusedwhenthemoleculeofinterestis
positivelycharged.

22. Size exclusion or molecular sieving or gel permeation mechanism,
for separation of a large molecular weight compound from smaller one, the small molecular size particles will pass through the
pores of the gel, while largest one will be excluded.
Sephadex
◊ polymers of carbohydrates, manufactured from the cross linking of water soluble dextran with epichlorohydrin.
➢ Are useful for gel chromatography of water soluble products such as carbohydrates and small peptides.
➢ Water, DMF, DMSO and aq. Methanol can are used as mobile phase.
Sephadex LH-20
◊ Is a hydroxypropylated Sephadex G-25.
◊ Lipophilic gel.
◊ Lipophilicity allows this gel to swell in organic solvents, makes LH-20 the preferred gel for the fractionation of
organic-soluble natural products.
Advantages
•High efficiency
•Totally inert
•Doesn't reduce the sample amount
•Used for several times

24. AFFINITY MECHANISM
Affinity mechanism, for separation of biological substances. This is the most
selective type of chromatography.
This technique depends on the bioselective attraction of a biomolecule
(substrate) for the bioligand which connected to the packing material, this
bioselective attraction forces occurs such as between antigen-antibody, and
between enzymes and their substrates and inhibitors like a lock and key.

26. COLUMN CHROMATOGRAPHY
Apparatus:
A vertical glass tube, constricted at the bottom end, into which the adsorbent has
been packed, a small plug of glass wool at the base of the tube. A height-diameter
ratio of 10 or 20 to one is employed. Long column having ratio 100 t0 one.
The solid stationary phase:
The solid stationary phase (adsorbent or sorbent), should be
1. insoluble in the solvent used as mobile phase.
2. inert to the adsorptives (solutes), unless otherwise required.
3. colorless, especially when used to separate colored substances.
4. have a suitable particle size with great surface area to allow more efficient
adsorption; but, not so fine as to slow the rate of percolation.Classification of the adsorbents
Classification according to strength of adsorption
Weak adsorbents
e.g. sugar & talc
Strong adsorbents
e.g. silica, alumina & charcoal.
Classification according to polarity
Polar adsorbents
e.g. Metal oxides
Non-polar adsorbents
e.g. charcoal.
Classification according to pH
Acidic adsorbents
e.g. Silica
Basic adsorbents
e.g. Alumina

27. The mobile phase:
Mobile phase is the solvents which are used to transport the sample
components through the column to the outside. The choice of the
suitable solvent (s) depends mainly on the elution power of the
solvent and the relative adsorption of the class of compounds, which
are to be separated.
Less polar solvents are used for saturated hydrocarbons and more
polar solvents are used for unsaturated hydrocarbons or
hydrocarbons with functional groups.
Less polar solvents are weak eluents, so increase adsorption, while
more polar solvents are strong eluents, so decrease adsorption.
Eluotropic series:
The solvents are arranged in series in order of increasing polarity,
called elutropic series as follow;
Petroleum ether or hexane, cyclohexane, carbon tetrachloride,
benzene, chloroform, ether, ethyl acetate, acetone, ethanol, methanol,
water and acidified water.

28. Packing of the column:
1.Slurry method, the adsorbent is suspended with the first solvent to be used (usually
a non polar solvent) and added to the column continuously.
The solvent level is maintained above the packing material at all times.
Avoid drying of the column to prevent cracking.
2. Dry method, the adsorbent is added with vibration or tamping, the column is then
washed with the first solvent.
Application of the sample to the column:
1.Dry Method: The sample is mixed with an inert matter
2.Wet Method: The sample is dissolved in the first solvent and introduced into the
column in the smallest volume.
When the sample is not soluble in the first solvent, it is dissolved in any other solvent,
then add a small amount of adsorbent and mixing, allow to dry, then added or
introduced into the column. Small amount of the packing material or a piece of cotton is
introduced on to the sample to prevent any interruption of adsorbent during the
development. The amount of solute relative to the adsorbent is 1 to 20 or 1 to 50.

29. ELUTION AND DETECTION
Elution analysis and the gradient elution analysis.
A. Isocratic elution analysis: by using an eluent of the same composition throughout the
elution analysis i.e. until the separation is completed. Only one or a mixture of solvents can
be used. This is most suitable when the components have similar affinities for the stationary
phase and therefore eluted rapidly.
Drawback of isocratic elution analysis: The strongly adsorbed and retained components
travel very slowly and the substance is said to be tailing or trailing through the column.
B. Gradient elution analysis: the composition of the eluent is changed by the gradually
addition to the non polar solvent an organic solvent (modifier) which is more powerful in the
eluting strength or by using a mixture of solvents with increasing elution power. Gradient
elution analysis results in the decrease of tailing.
Organic modifiers which are added to the mobile phases are dichloromethane, chloroform,
ether, ethyl acetate and methanol.
Detection of the sample:
A. Detection on the column. Examination in either visible light for colored compounds or
UV light for fluorescent compounds.
B. Detection in the fractions. The eluate is collected in fractions and tested for the separated
compounds in each fraction by using TLC.

30. Factors affecting column efficiency:
1. Particle size of the adsorbent. The decrease in the particle size of the adsorbent, will increase
column sorption, hence separation is improved.
2. Column dimension. Column efficiency improves as the length to width ratio of the column is
increased. The more uniform packing obtained in narrower columns.
3. Uniformity of packing of the column. If the column is not packed uniformly, uneven and
irregular moving solvent front will occur and less uniform zones are formed.
4.Column temperature. Liquid column chromatography is usually carried out at room temperature.
The elevation of the temperature will reduces the adsorption and speed up the elution. Also
affects the viscosity of the solvent, the higher the temperature, the lower the viscosity and may
affect the flow rate. This is a poor alternative to using a stronger eluent.
5.Solvent flow rate. A uniform and low flow rate give more uniform zone formation than a fast flow
rate. The optimum flow rate is 20 drops per minute.
6. Constancy of flow. The solvent flow should be continuous, to avoid zones diffusion.
7. Adsorbent activity. Adsorbents are activated by removal of adhering or blocking impurities
such as water. Such impurities will occupy the active sites of adsorbent and can greatly decrease the
adsorbing power of the adsorbents.
8. Selection of solvents. The flow rate is inversely proportional to the viscosity; therefore, solvents
of low viscosity give a suitable flow rate and hence a high efficiency of separation will results. The
arrangement of bands of the components of a sample mixture may be affected by the use of different
solvents. The presence of impurities in the solvents often changes the order of the separated zones.
9. Effect of concentration. Highly concentrated samples move more rapidly through the column.

31. Terminology:
1.Stationary Phase….
2.Mobile Phase…..
3.Solvent….
4.Solute…..
5.Eluent….
6.Eluate or fraction: the solvent that contain the eluted
components.
7.Retention or retardation volume: the volume of pure
solvent that passing out the column before the
substance is eluted. Therefore, the more adsorbed
substance having a higher retention volume than the
less adsorbed one.

34. ▪Flashchromatographyisapurificationtechniquespecificallydevelopedforrapidseparation.
Unlikeslowandinefficientgravity-fedchromatography,flashchromatographyutilizesair
pressuretoachievefasterandmoreefficientseparation.Thetechniqueemployedinthis
methoddeviatesfromtheconventionalcolumntechniquebyutilizingsilicagelparticlesthat
areslightlysmallerinsize,alongwiththeapplicationofpressurizedgasatarangeof50to
200poundspersquareinch(psi).Flashchromatographycolumnsarecommonlyusedin
chemicalseparationsandaredesignedasprepackedplasticcartridgescontainingsilicagel
particleswithsizesrangingfrom40to60μm.
▪Particleswithasizelessthan25μmarerecommendedtobeexclusivelyemployedin
conjunctionwithmobilephaseswithextremelylowviscosity.Thisisduetothefactthat,
otherwise,theflowratewouldbesignificantlydiminished.

35. SOLID-PHASE EXTRACTION (SPE)