1. Separation & Purification (2) CHROMATOGRAPHY
Dwi Koko Pratoko, M. Sc., Apt.
Pharmacochemistry Dept.
Faculty of Pharmacy – Univ. of Jember

2. Content : Planar Chromatography Column Chromatography Preparative HPLC
Preparative GC
Ion-Exchange Chromatography
Counter current Chromatography

3. Planar Chromatography
Planar chromatography utilizes the separation of mixtures of organic compounds on thin-layers of adsorbents which are in most cases coated on glass, plastic, or aluminum sheets.
The most widely used form of planar chromatography is thin-layer chromatography (TLC) which is the easiest and cheapest technique for the isolation of natural products.
Procedure :
Separation by TLC is effected by the application of a mixture or an extract as a spot or thin line on to a sorbent which has been applied to a backing plate
The plate is then placed into a glass developing tank with sufficient suitable solvent just to wet the lower edge of the plate/sorbent but not suffi cient to wet the part of the plate where the spots were applied (the origin).
The solvent front then migrates up the plate through the sorbent due to capillary action and this process is known as development

4. TLC equipment & Development procedure
R f values are always ratios, are never greater than one, and vary depending on sorbent and/or solvent system. These values are sometimes quoted as h R f , i.e., relative to solvent front = 100, h R f = R f × 100 (in our case h R f = 82)

5. In the case of adsorption chromatography , where the sorbent is silica, polar compounds (e.g., artemisinin)  higher affinity for the sorbent (stationary phase)  will “stick” to the sorbent and move slowly up the plate as the solvent (mobile phase) migrates  These compounds will have relatively small Rf values
As a consequence of development, compounds of a mixture will separate according to their relative polarities.
Polarity is related to the type and number of functional groups present on a molecule capable of hydrogen bonding
Solvent strengths are also measured in terms of polarity and generally dielectric constants are of use to quantify their relative strengths.
A high dielectric constant indicates a polar solvent with a strong power of elution and a low dielectric constant indicates a nonpolar solvent with a lower ability to elute a component from a sorbent.
This elution strength applies to normal-phase adsorption chromatography.

6. Dielectric constant

7. Mechanism of Chromatography
Adsorption Chromatography
Most common sorbent : silica & alumina
Separation occurs when one compound is more strongly adsorbed by the sorbent than the other components
Partition Chromatography
The most commonly used reverse phase sorbent is silica which has been reacted with a straight chain 18 carbon alkyl unit to form an octadecasilyl (ODS) phase (RP-TLC)
Separation is effected by compounds having different rates of partition between the stationary phase and mobile phase

8. Mechanism of Chromatography
Size Inclusion Chromatography
The most commonly used size inclusion sorbents are the dextran gels, particularly the lipophilic versions, such as Sephadex LH-20
Compounds may be separated by their relative sizes and by their inclusion (or exclusion) into a sorbent
As compounds migrate with the solvent through the gel, small molecules become included (stuck) into the gel matrix and larger molecules are excluded and migrate at a faster rate
Ion Exchange Chromatography
the sorbent is usually a polymeric resin which contains polar charged groups and mobile counter ions which may exchange with the ions of a component as the mobile phase migrates through the sorbent
Separation is achieved by differences in affinity between ionic components in the mixture and the stationary phase

9. Preparative TLC
PTLC does not require expensive equipment, separations can be effected rapidly and the amount of material isolated generally falls into the 1 mg to 1 g range which is certainly sufficient for structure elucidation purposes
PTLC is nearly always used as a fi nal purifi cation step in an isolation procedure
The number of compounds that can be separated on a prep plate will ultimately depend on how those compounds behave on a
particular system but as a rule the separation of no more than a mixture of three major components should be attemptedThe number of compounds that can be separated on a prep plate will ultimately depend on how those compounds behave on a particular system but as a rule the separation of no more than a
mixture of three major components should be attempted

10. Advantages PTLC
PTLC is cost-effective compared to the instrumental methods, for example HPLC or CCC.
A simple technique that requires little training or knowledge of chromatography to use
An analytical method may be easily scaled up to a preparative method
Ability to isolate natural products quickly in the milligram to gram range
Flexibility of solvent and stationary phase choice, i.e., the solvent system can be changed quickly during a run
The separation can be optimized readily for one component, i.e., it is relatively easy to “zero in” on a particular product of interest
Methods are quickly developed
Almost any separation can be achieved with the correct stationary phase and mobile phase
A large number of samples can be analyzed or separated simultaneously

11. Disadvantages PTLC Poor control of detection when compared to HPLC
Poor control of elution compared to HPLC
Loading and speed are poor compared to vacuum-liquid chromatography
Multiple development methods to isolate grams of material may be time consuming
Commercially available plates are restricted to simple sorbents, such as silica, alumina, cellulose, and RP-2samples can be analyzed or separated simultaneously

12. Column Chromatography
In column chromatography a mobile phase is allowed to flow through a densely packed adsorbent.
Different separation mechanisms can be applied depending on the choice of packing material and mobile phase selected
Mechanism :
Adsorption
Size Exclusion

13. Separation Process
The separation takes place through selective distribution of the components between a mobile and a stationary phase
there are a number of factors, related to the physical and chemical nature of the mobile and stationary phases as well as the solutes controlling the various interactions between the solutes and two phases, involved in the separation process.
The number of possible interactions between the solutes and the stationary phase (adsorbent)depends on the particle size of the stationary phase; the greater the surface area of the stationary phase, the greater the number of interactions.
A stationary phase with a high surface area tends to give enhanced separations. The equilibrium between the solute and the stationary phase is termed the distribution constant , which is dependent on the chemical nature of the system.

14. Type of Separation Process 1. Adsorption
The analyte molecules are retained by their interaction with the surface of the stationary phase.
Therefore, the separation is based mainly on the differences between the adsorption affinities of the analyte molecules for the surface of the stationary phase.
The extent of adsorption of analyte molecules on to the stationary phase is Van der Waal forces, dipole–dipole interactions, acid–base properties, complexation, and charge transfer.

15. Type of Separation Process 2. Size Exclusion
This chromatographic technique, where molecules in solution are separated by their size
The stationary phases used are non- adsorbing porous particles with pores of approximately the same size, as the effective dimensions in solution, of the molecules to be separated.
Various types of materials are used to make beads with various pore sizes.
Large molecules cannot enter the matrix, intermediate size molecules can enter part of the matrix, and small molecules freely enter the matrix.
There is no interaction between the solute and the stationary phase, and separation is on the basis of molecular size and shape of the analyte molecules.
size exclusion chromatography
(SEC) or gel permeation chromatography (GPC)

16. Stationary phase 1. Silica gel (Adsorption)
The silica gel surface consists of exposed silanol groups, and these hydroxyl groups form the active centers
The hydroxyl groups can potentially form strong hydrogen bonds with various compounds.
In chromatographic terms, the stronger the hydrogen bond, the longer the compound will be retained on the silica gel

17. Stationary phase 2. Bonded-Phase Silica gel (Adsorption)
Silica gel can be chemically modified in a variety of ways to alter both its physical properties and chromatographic behavior.
The main purpose of surface modification is shielding of the active silanol groups and attachment to the accessible adsorbent surface organic ligands which are responsible for specific surface interactions

19. Stationary phase 3. Alumina (Adsorption)
Alumina is a porous polymer of aluminum oxide (Al2O3)
can be produced with an acidic, basic, or neutral surface based on the pH of the final wash of the synthetic absorbent
The acidic alumina (pH ~ 4.0) is useful for the separation of carboxylic acids;
The basic alumina (pH ~10.0) for basic compounds such as alkaloids,
while the neutral alumina (pH ~7.0) is appropriate for the separation of nonpolar compounds such as steroids

20. Stationary phase 4. Polyacrilamide (Size Exclusion)
Copolymerization of acrylamide and N,N’ -methylene-bis- acrylamide leads to the formation of porous polyacrylamide beads,
it can be used as a stationary phase to carry out purification of macromolecular natural products, e.g., carbohydrates, peptides, and tannins

21. Stationary phase 4. Carbohydrate (Size Exclusion)
For the chromatography of labile natural products, one of the most commonly used materials is an inert polymer of carbohydrates
Cross linking of polysaccharides produces threedimensional networks, which can be converted to beads ideal for column chromatography
These highly specialized gel filtration and chromatographic media are composed of macroscopic beads synthetically derived from the polysaccharide, dextran
Sephadex® is prepared by cross-linking water soluble dextran with epichlorohydrin. These gels swell in water
One of the most extensively used gels in natural products separation (especially nonpolar or intermediate polarity compounds) is Sephadex® LH-20, a hydroxypropylated form of Sephadex® G-25
The useful fractionation range of LH-20 is approximately 100–
4,000 amu and is particularly ideal for the removal of chlorophyll
from plant extracts

23. Column Operation Selection of Stationary Phase Column Packing
The stationary phase is normally introduced into the column dry or in slurry using a suitable solvent.
Sample Application
Column Development

24. Preparative HPLC
The term “preparative” in the context of separation or isolation of compounds from a complex matrix generally refers to a “largescale” process.
Preparative high performance/pressure liquid chromatography (prep-HPLC) usually implies large columns, large sample loading, and high flow rates in an HPLC system with the aim to purifying or isolating compounds in large quantities
Prep-HPLC is a robust, versatile, and usually rapid technique by which compounds can be purifi ed from complex mixtures

25. Modes of Separation and Stationary Phases
Normal-Phase Prep-HPLC
Use a polar stationary phase (usually silica) and less polar (non aqueous) eluting solvent
Normal phase prep-HPLC is best suited to the separation and isolation of lipophilic compounds, long-chain alkane derivatives or where the mixture of interest is sparingly soluble in aqueous conditions
Reversed-Phase Prep-HPLC
this technique is the reverse on normal phase prep-HPLC whereby the stationary phase is more nonpolar than the eluting solvent
Other Modes
GPC (also called size exclusion chromatography) is predominantly used for fractionating and purifying proteins and oligosaccharides but has been used in some cases for separating lower molecular weight molecules
Ion exchange chromatography uses an anionic or cationic stationary phase for the separation of acids and amines

27. Solvent Solvents used in prep-HPLC typically have to be
high purity to maintain the integrity of the system and sample,
compatible with the detector and not interfere with the observation of your target compounds, i.e., “transparent,”
compatible with the sample (solubility and nonreactive),
low viscosity to keep system back pressure low, and
reasonably priced (a typical prep-HPLC run may use a liter or more of solvent each time).
the solvents need to be “degassed” to remove dissolved oxygen, which comes out of solution to form microscopic bubbles under the high pressures seen in the system

30. Fraction Collection
Fraction collectors can be programmed in three ways: collect by time, collect by peak threshold, or collect by peak gradient.
To collect “time fractions,” the fraction automatically switches tube after a set time period, e.g. , 30 s.
This allows the collection of all components and is useful if wanting to create many fractions to form a library of fractions or for bioassay-guided fraction when the identity of the active is unknown.
The fractions collected may not be pure, but this method helps narrow the field down to a particular region of the active mixture.
Collecting by peak threshold involves the collection of peaks over a set threshold, e.g. , 10% of total detector response.
This allows the collection of the major peaks within a mixture but results in the loss of the minor ones.
Collecting by peak gradient results in the fraction collector measuring the upslope of peaks and when the upslope is high enough the fraction collector begins collecting.
When the peak has been reached, the fraction collector measures the downslope, and stops collecting when the gradient of the peak becomes shallow