Unit III - Purification and Chromatographic Techniques

General methods of separation and purification of organic compounds - solvent extraction, soxhlet extraction and
Pressurized liquid extraction, fractional crystallization, membrane dialysis.

Chromatography - classification of chromatographic techniques. Ion exchange, Column, Planar, and Size Exclusion.
Chromatography. Ion Chromatography- Principle, Instrumentation-Eluent generation techniques, anionic and
cationic suppressors, Detectors-CD, ECD, UV-Vis. Applications.
HPLC-Principle, Instrumentation and detectors – RI and UV-Vis detectors, preparative HPLC - methods and
applications. Gas chromatography – principle, instrumentation, and detectors – FID, ECD, NPD, MS detectors.
Methods and applications.

• Separations are extremely important in synthesis, in industrial chemistry, in the biomedical sciences, and in chemical analyses
• A substance that affects an analytical signal or the background is called an interference or an interferent
• Separations isolate the analyte from potentially interfering constituents
• lists several separation methods that are in common use including (1) chemical or electrolytic precipitation, (2) distillation, (3)
solvent extraction, (4) ion exchange, (5) chromatography, (6) electrophoresis, and (7) field-flow fractionation.

Separation by Precipitation
• Separations by precipitation require large solubility differences between the analyte and potential interferents
Separation of Species by Distillation
• Distillation is widely used to separate volatile analytes from nonvolatile interferents
• Distillation is based on differences in the boiling points of the materials in a mixture
• There are many types of distillation. Vacuum distillation is used for compounds that have very high boiling points.
Lowering the pressure to the vapor pressure of the compound of interest causes boiling and is often more effective for
high boilers than
• raising the temperature.
• Molecular distillation occurs at very low pressure (,0.01 torr) such that the lowest possible temperature is used with the
least damage to the distillate.
• Pervaporation is a method for separating mixtures by partial volatilization through a nonporous membrane.
• Flash evaporation is a process in which a liquid is heated and then sent through a reduced-pressure chamber. The
reduction in pressure causes partial vaporization of the liquid.
Soxhlet Extraction
• Soxhlet extraction is one of the most popular techniques for extraction of analytes from solid materials
• Soxhlet technique has been routinely applied in almost every analytical laboratory. Up to this day, Soxhlet extraction technique
remains a standard technique to which the performance of modern extraction techniques is compared.
• Soxhlet extraction is a continuous process of extraction with a hot organic solvent.
• Typically, Soxhlet extraction is used when the desired compound has a limited solubility in a
solvent, and the impurity is insoluble in that solvent.
• A Soxhlet extractor has three main sections:
1. A percolator (boiler and reflux) which circulates the solvent.
2. A thimble (usually made of thick filter paper/cotton) that retains the solid to be extracted.
3. And a siphon mechanism, which periodically empties the thimble.
• Thimble: The source material containing the compound to be extracted is placed inside the thimble.
The thimble is loaded into the main chamber of the Soxhlet extractor.
• Distillation flask or Round Bottom Flask (RBF): The extraction solvent to be used is placed in a
distillation flask.
• Heating mantle: The flask is placed on the heating element.
• Reflux condenser: A reflux is placed atop the extractor.
• Water inlet & outlet: Attached in the inlet & outlet opening of condenser.
Working
• Solid material placed in thimble.
• Soxhlet extractor is placed onto a flask containing the extraction solvent. Soxhlet equipped
with a condenser.
• The solvent is heated to reflux.
• The solvent vapour travels up a distillation arm and floods into the chamber housing the
thimble of solid.
• Solid material in chamber slowly fills warm solvent.
• Desired compound dissolves in the warm solvent
• When the Soxhlet chamber is almost full, the chamber is emptied by the siphon. The solvent
running back down to the distillation flask.
• This cycle may be allowed to repeat many times, over hours or days.
• During each cycle, a portion of the compound dissolves in the solvent.
• After many cycles (72 hours) the desired compound is concentrated in the distillation flask.
Pressurized Liquid Extraction
• Pressurized liquid extraction (PLE) is a technique for extracting a
sample using conventional solvents at high temperatures and
pressures. It is also known as accelerated solvent extraction or
pressurized fluid extraction.
• When the temperature increases the solubility increases
• In that physical region the high temperature enables high solubility
and high diffusion rate of lipid solutes in the solvent, while the high
pressure, in keeping the solvent below its boiling point, enables a
high penetration of the solvent in the sample. Thus, PFE permits a
high extraction efficiency with a low solvent volume (15-40 ml) and a short extraction time (15-20 min).
Separation by Extraction
The extent to which solutes, both inorganic and organic, distribute themselves between two immiscible liquids differs
enormously, and these differences have been used for decades to separate chemical species
Principles
• The partition of a solute between two immiscible phases is an equilibrium process that is governed by the distribution law
• If the solute species A is allowed to distribute itself between water and an organic phase, the resulting equilibrium may be
written as

• Ideally, the ratio of activities for A in the two phases will be constant and independent of the total quantity of A so that,
at any given temperature,

• The equilibrium constant K is known as the distribution constant.

• Generally, the numerical value for K approximates the ratio of the solubility of A in each solvent.
• Distribution constants are useful because they permit us to calculate the concentration of an analyte remaining in a
solution after a certain number of extractions.
• They also provide guidance as to the most efficient way to perform an extractive separation
Extracting Inorganic Species
• The processes of equilibration and separation of phases in a separatory funnel are less tedious and time consuming than
conventional precipitation, filtration, and washing.
Separating Metal Ions as Chelates
• Many organic chelating agents are weak acids that react with metal ions to give uncharged complexes that are highly
soluble in organic solvents such as ethers, hydrocarbons, ketones, and chlorinated species (including chloroform and
carbon tetrachloride)
• Most uncharged metal chelates, on the other hand, are nearly insoluble in water. Similarly, the chelating agents themselves
are often quite soluble in organic solvents but of limited solubility in water
Solid-Phase Extraction
• Liquid-liquid extractions have several limitations. With extractions from aqueous
solutions, the solvents that can be used must be immisicible with water and must not form
emulsions
• Solid-phase extraction, or liquid-solid extraction, can overcome several of these
problems. Solid-phase extraction techniques use membranes or small disposable syringe-
barrel columns or cartridges
• A hydrophobic organic compound is coated or chemically bonded to powdered silica to
form the solid extracting phase
• The compounds can be nonpolar, moderately polar, or polar. For example, an octadecyl
(C18) bonded silica (ODS) is a common packing
• The functional groups bonded to the packing attract hydrophobic compounds in the sample
by van der Waals interactions and extract them from the aqueous solution.
• The sample is placed in the cartridge and pressure is applied by the syringe or from an air or
nitrogen line.
• Organic molecules are then extracted from the sample and concentrated in the solid phase. They can later be displaced from the
solid phase by a solvent such as methanol.
• By extracting the desired components from a large volume of water and then flushing them out with a small volume of solvent,
the components can be concentrated.
Separating Ions by Ion Exchange
Ion-Exchange Resins
• Synthetic ion-exchange resins are high-molecular-mass polymers that contain large
numbers of an ionic functional group per molecule
• Cation-exchange resins contain acidic groups, while anion-exchange resins have basic
groups
• Strong-acid-type exchangers have sulfonic acid groups (-SO3-H+) attached to the
polymeric matrix and have wider application than weak-acid-type exchangers, which
owe their action to carboxylic acid (-COOH) groups.
• Similarly, strong-base anion exchangers contain quaternary amine [--N(CH3)3+OH-]
groups, while weak-base types contain secondary or tertiary amines.
• Ion-exchange separations are usually performed under conditions in which one ion predominates in both phases. Thus,
in the removal of calcium ions from a dilute and somewhat acidic solution, the calcium ion concentration will be much
smaller than that of hydrogen ion in both the aqueous and resin phases,

Chromatographic Separations
• We have in common the use of a stationary phase and a mobile phase. Components of a mixture are carried through
the stationary phase by the flow of a mobile phase, and separations are based on differences in migration rates among the
mobile-phase components
• Chromatographic methods are of two basic types. In column chromatography, the stationary phase is held in a narrow
tube, and the mobile phase is forced through the tube under pressure or by gravity.
• In planar chromatography, the stationary phase is supported on a flat plate or in the pores of a paper, and the mobile

phase moves through the stationary phase by capillary action or under the influence of gravity.
Elution in Column Chromatography
• Elution is the separation process in which solute molecules are washed through a stationary phase by the movement of
mobile phase
• With the first introduction of fresh mobile phase, the eluent, the portion of the sample contained in the mobile phase
moves down the column, where furtherpartitioning between the mobile phase and the stationary phase occurs (time t1).
• Partitioning between the fresh mobile phase and the stationary phase takes place simultaneously at the site of the original
sample.
• the average rate at which a solute migrates depends on the fraction of time it spends in that phase. This fraction is small
for solutes that are strongly retained by the stationary phase
• Ideally, the resulting differences in rates cause the components in a mixture to separate into bands, or zones, along the
length of the column
• Isolation of the separated species is then accomplished by passing a sufficient quantity of mobile phase through the
column to cause the individual bands to pass out the end (to be eluted from the column), where they can be collected or
detected

Chromatograms
• If a detector that responds to solute concentration is placed at the end of the column during elution and its signal is plotted
as a function of time (or of volume of added mobile phase), a series of peaks is obtained. Such a plot, called a
chromatogram, is useful for both qualitative and quantitative analysis
Retention Times
• The time tM after sample injection for this peak to
appear is sometimes called the dead or void time
• The time required for this zone to reach the detector
after sample injection is called the retention time
and is given the symbol tR. The analyte has been
retained because it spends a time tS in the stationary
phase.
• The retention time is then

• Retention time depends upon the nature mobile phase and temeperature ,
• Retention time of an eluent will be constant if same concentration and same temperature
Band Broadening and Column Efficiency

A Quantitative Description of Column Efficiency

(1) plate height, H, and
(2) plate count or number of theoretical plates, N.