Lecture 2b

by

Sherif S. Ebada, Ph.D.

Instrum. Pharm. Analys.

Contact information:

Assoc. Prof. Dr. Sherif S. Ebada

Pharmaceutical Chemistry Dept.

Office hours:

Mondays and Tuesdays: 12:00–14:00 pm

E-mail: ss_ebada@mutah.edu.jo

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Course outline

Introduction.

Adsorption (CC & TLC), Flash CC,

Chromatotron etc.

Partition (Paper).

Gel Chromatography.

Ion exchange Chromatography.

Affinity Chromatography.

Electrophoresis.

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Chromatography

Lecture 2b

Partition

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Partition Chromatography

Partition chromatography, or liquid-liquid chromatography is a

chromatographic technique in which solute are separated based on their partition

between a liquid mobile phase and a liquid stationary phase coated on a

solid support.

The support material used in partition chromatography is usually silica.

The solid support should have the following properties:

Uniform spherical particles

Porous structure

Diameters in the range from 3-10 μm

High surface area

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Countercurrent chromatography (CCC) or Partition Chromatography

Liquid-liquid chromatography techniques?

In contrast, standard column chromatography uses a solid stationary

phase and a liquid mobile phase.

Normally, it comprises three phases Mixing, Settling, Separating.

Any pair of immiscible solutions can be used in liquid-liquid

chromatography, and most instruments can be operated in standard or

reverse-phase modes.

Advantages of CCC

Solvent costs are also generally cheaper than for HPLC.

The cost of purchasing and disposing of solid adsorbents is

completely eliminated.

The experiments conducted in the lab can easily be scaled up to

industrial volumes.

When GC or HPLC is done with large volumes, resolution is lost due to

issues with surface-to-volume ratios and flow dynamics; this is

avoided when both phases are liquid.

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Droplet Countercurrent Chromatography (DCCC)

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Droplet Countercurrent Chromatography (DCCC)

Droplet CCC is the oldest form of CCC.

It uses only gravity to move the mobile phase through the stationary phase.

In descending mode, droplets of the more dense mobile phase and sample

are allowed to fall through a column of the lighter stationary phase using only

gravity.

Ascending mode If a less dense mobile phase is used it will rise through

the stationary phase.

The eluent from one column is transferred to another; the more columns that

are used, the more theoretical plates can be achieved.

Disadvantages of DCCC

Flow rates are low, and poor mixing is achieved for most binary solvent

systems, which makes this technique both time-consuming and inefficient.

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Advantages of the CCC separation methods

Absence of the irreversible adsorption.

Recovery of all components of a mixture. This feature is priceless when it

comes to the bioactivity-directed isolation of unknown compounds as it

prevents an unexpected loss of activity from fractionation.

High load acceptance; 1L-rotor can accept a sample of up to 30 grams and

typical fractionation will consume 3 to 5 liters of a solvent system.

CPC can widely separate the compounds that have the same retention

times on the solid phase. Good examples of the highly similar co-occurring

in nature compounds that co-elute on silica gel, but can be purified by CPC,

are the pairs of rotenone – Deguelin.

Applicable to a wide range of polarity of chemical compounds.

Applicable to the water-sensitive compounds. Several non-water

containing solvent systems.

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Centrifugal Partition Chromatography (CPC)

•It is also known as Hydrostatic Countercurrent Chromatography.

•It is an automated liquid-liquid extraction process permitting hundreds of automatic successive extractions.

•This refers to the rotary movement of the column which rotates around one single rotation axis.

•One of the liquid phases of the two-phase system is used as a stationary liquid phase: it is fed into the column (the rotor) while the latter is spinning at moderate rotational speed.

•The stationary phase is retained inside the rotor by the centrifugal force generated

•The second phase of the two-phase system is used as the mobile phase containing the solutes to be extracted. It is fed under pressure into the rotor and pumped through the stationary phase.

•Both phases are mixed together. It is at that time that the exchange of molecules between the two phases occurs.

•The separation of the solutes is achieved as a function of the specific partitioning coefficient (Kd) of each solute between the mobile and stationary phases.

•The mobile phase then decants at each cell outlet thus entering the next cell.

•The eluted fractions of the mobile and stationary phases are collected over a period of several minutes to several hours. These fractions, or eluates, will contain the individualpurified solutes.

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Advantages of the CCC separation methods

• No column to replace, no silica to recycle

• Low solvent consumption

• High flow rate for low run time

• High performances. Purity > 99%, recovery > 90%

• No sample losses

• No denaturation, no irreversible adsorption of the sample

• Huge application fields from petroleum extract to proteins.

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Supercritical Fluid Extraction

(SFE) is the process of separating one component (the extractant) from another

(the matrix) using supercritical fluids as the extracting solvent.

Extraction is usually from a solid matrix, but can also be from liquids.

SFE can be used as a sample preparation step for analytical purposes, or on a

larger scale to either strip unwanted material from a product (e.g. decaffeination)

or collect a desired product (e.g. essential oils). Carbon dioxide (CO2) is the most

used supercritical fluid, sometimes modified by co-solvents such as ethanol or

methanol.

Extraction conditions for supercritical CO2 are above the critical temperature of

31°C and critical pressure of 74 bar.

Addition of modifiers may slightly alter this.

Compression of a gas results in change of state to liquid.

However when a gas is heated beyond a particular temperature, it can not be

liquefied regardless of any further increase in the pressure.

This particular temperature is called the "critical temperature (Tc)" and the

corresponding pressure on the pressure-temperature diagram is called the

"critical pressure (Pc)".

The region on the Pressure-Temperature diagram above Tc - Pc is called the

"super critical region".

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Supercritical Fluid Extraction

•Compression of a gas results in change of state to liquid.

•However when a gas is heated beyond a particular temperature, it cannot be liquefied regardless of any further increase in the pressure.

•This particular temperature is called the "critical temperature (Tc)" and the corresponding pressure on the pressure-temperature diagram is called the "critical pressure (Pc)".

•The region on the Pressure-Temperature diagram above Tc - Pc is called the "super critical region”.

•The "fluid" exhibits many of the properties of both gas and liquid.

•The state of the fluid is called the "super critical" state or "dense gas" state.

•The closeness to gaseous state helps in faster diffusion-almost two orders of magnitude higher than that of other liquids while the proximity to the liquidstate helps in better solubility of the components to be extracted.

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Supercritical Fluid Extraction•The super critical carbon dioxide is called as SCO2 (the most favored solvent ) because:

•It is an inert gas,

•Easily available at low cost,

•Odorless, tasteless,

•Eco-friendly and GRAS solvent.

•Beyond a specific temperature 31.1 ᵒC and pressure 73.8 bar (the critical point) carbon dioxide becomes a supercritical fluid, a state that is neither a gas nor a liquid, but has properties of both.

•Preparative SFC is particularly interesting for the purification of:

•Classes of compounds that not readily amenable to either gas chromatography (GC) or liquid chromatography (LC). These compounds include species that are non-volatile or thermally labile and in addition, contain no chromophore that can be used for spectrophotometric detection.

•Chiral compounds.

•Actives or intermediates from complex mixtures.

•Lipophilic compounds.

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Liquid CO2 is pumped by a high-pressure pump through a super heater to

achieve supercritical state prior to entering the extractor containing the material

to be extracted.

The SCO, carrying the desired extract, is taken to the separator and with the

help of pressure reduction valve-by controlling temperature and pressure, the

solute is precipitated in the separator while the CO2 is recycled into the extractor

via a condenser. The beauty of the process is that at the time of separation of

solute from the solvent, the temperature is lower than the extraction

temperature.

It doesn't degrade or `Volatize' the heat sensitive components present in the material resulting in closest to the natural end product.

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Advantages of the Process using Super Critical CO2

Non-toxic, odorless, tasteless, colorless, easily removable solvent. No Hydrolysis or destruction of temperature sensitive active ingredients. Products that is closest to the aroma and other properties of the original

botanical material. Longer shelf-life for end product.Why should one use CO2 Extracts when they are "Expensive"?

Nearest to natural botanical raw materials!

No solvents!

No Heavy metals!

No microbial contamination!

Difficult to "duplicate" or "imitation"

Economy benefits of 15-25% in final application!

Applications

Natural Flavors.

Pure ingredients for Herbal Medicines and Cosmetics.

Excellent Fragrance for Perfumery.

Unaltered ingredients for Nutraceuticals and Functional Foods.

Active ingredients for Aromatherapy formulation.

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Preparative Supercritical Fluid Chromatography (SFC)

Preparative SFC is more and more used in research and development

laboratories and pilot plants of the pharmaceutical and fine chemical industries.

SFC is particularly interesting for the purification of:

Chiral compounds.

Actives or intermediates from complex mixtures.

Lipophilic compounds.

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Paper chromatography

• It is a form of planar chromatography.

• The stationary phase consists of a sheet of paper, the water carried on

the cellulose paper fibers a matrix act as the stationary phase and are

able to retain a liquid stationary phase.

• The mobile phase solvent moves up the paper by capillary action

carrying the solute molecules over the paper fibers.

• When the organic solvent flows carrying the sample on the paper,

partition occurs between the mobile phase and the stationary aqueous

phase bound by cellulose.

• The organic portion of the solvent flows on, carrying with it some of the

solute. The amount left in the aqueous portion is a function of the

partition coefficient of the solute.

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Paper chromatography

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Paper chromatographyGood results will be reproduced, if the following rules are considered:

• Use sufficient solvent and an appropriate chamber, so that the composition

of the solvent will remain constant throughout development.

• Maintain constant temperature.

• Employ solvents in which the components to be separated have a low but

definite solubility.

Mobile phase

The most satisfactory solvents are those which are partially miscible with

water, such as phenol, n-butanol and amyl alcohol.

Sample

No high concentration of highly corrosive ingredients e.g. mineral acids.

Free from inorganic salts, since they cause streaking, discoloration or other

distortions on final chromatogram.

Sample as small spot less than 5 mm diameter and uniform, in preparative

scale sample applied as a streak.

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Paper chromatography

Equilibration of the paper

A dish containing some of the water saturated with the mobile phase is put

on the floor of the jar to saturate the atmosphere of the jar with vapors of the

solvent to prevent evaporation of the solvent from the paper.

Development

The range of development techniques available in paper chromatography is

similar to those employed in TLC, namely, ascending, descending, radial,

two-dimensional, multiple and stepwise.

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Detection (visualization or location of the spots)

A- Physical detection methods:

• These include the detection of the spots or bands colour, size, shape on the chromatogram in ordinary light or detection of their visibility and fluorescence in ultraviolet light. This is usually done with comparison with similar known substances.

B- Chemical detection methods:

• These include the use of spraying agents but not corrosive, example of suitable reagents are :

Iodine solution or vapour,

Modified Dragendroff's reagent for alkaloids,

Ferric chloride for phenolics,

Alkali for anthraquinone,

Antimony trichloride in chloroform for steroids, etc.

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Chromatographic artifacts in PC

A Major Difficulty In Paper Chromatography Is The Formation Of Several Spots From A Pure Substance. The Appearance Of Multiple Spots May Be One Or More Of The Following Causes:

1. Polymerization of the substance during development.

2. Oxidation may, in certain cases, occur between application of the sample and development with the solvent.

3. Isomerization of the substance during development of the chromatogram.

4. Denaturation of proteins during drying of the paper at room temperature and this is avoided by drying at 10°C.

5. If the paper is overloaded or some substances of the applied sample react with the cellulose of paper, the spot will remain at the origin.

6. The presence of very minute amounts of impurities in the paper may aid catalytic changes in the components producing artifacts.

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Factors affecting separation in PC and TLC:

1. Layer thickness:

Thick layers retard the migration of the spot thus lower the Rf value. Thickness of TLC (0.25-2 mm ). Paper chromatography of normal thickness or thick paper can be used Whitman No. 1, 3.

2. Degree of activation (TLC) and saturation (equilibration) in PC:

In TLC more active adsorbent results in retardation in migration of the spot and lower Rf value. In PC degree of saturation of paper; fully equilibrated paper contain up to 20% of aqueous stationary phase also lower Rf.

3. Concentration of the spotting solution:

Increase the concentration of the spotting solution results in apparent increase in Rf value.

4. Temperature:

It should be constant to get constant results.

5. Purity of the solutes:

Impurities in solute retard the migration of the spot, thus sample (solute) should be purified especially in PC

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