Extraction in Pharmaceutical

Analysis
 QUIZ !!!!!

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 Let's try a sample problem

You want to separate these two compounds. What will

you do?
Let's try a another sample problem

You want to separate these two

compounds. What will you do?
Let's try a third sample
problem

You want to separate these two

compounds. What will you do?
 Intrduction

Liquid-liquid extraction is a useful method

to separate components (compounds) of a
mixture
 Example

Suppose that you have a mixture of sugar in

vegetable oil (it tastes sweet!) and you want
to separate the sugar from the oil. You
observe that the sugar particles are too tiny
to filter and you suspect that the sugar is
partially dissolved in the vegetable oil.

What will you do?

 Error in Quantitative Analysis

Minimize the number of steps

In the example above, the sample preparation
contributed 75% of the error.
Use appropriate techniques
The goal should be to choose a combination of sample
preparation and analytical instrumentation that reduces
both the number of sample preparative steps and the
RSD.
Measurement Scheme

Sampling Sample Sample

Preservation preparation

Homogenization,
extraction
size reduction

Concentration Clean up Analysis

Merit for Instruments/ Analytical
Methods
Common Techniques used to isolate
analytes from sample matrix
 The Concepts of LLE

Liquid-liquid extraction is based on the transfer of a solute

substance from one liquid phase into another liquid phase
according to the solubility.

In the practical use, usually one phase is a water or water-

based (aqueous) solution and the other an organic solvent
which is immiscible with water.

The success of this method depends upon the difference in

solubility of a compound in various solvents.

For a given compound, solubility differences between

solvents is quantified as the "distribution coefficient"
 Choice Of Solvent

Factors to be considered:
Selectivity
Distribution coefficient
Insolubility of solvent
Recoverability of solute from solvent
Density difference between liquid phases
Interfacial tension
Chemical reactivity
Cost
Viscosity, vapour pressure
Flammability, toxicity
Factors influencing solvent extraction

1. Effect of temperature and inert solutes

2. Effect of pH on extraction
3. Effect of ion-pair formation
4. Effect of synergistic extraction
 Universal Rule in LLE

At a certain temperature, the ratio of

concentrations of a solute in each solvent is
always constant. And this ratio is called the
distribution coefficient, K.

The greater the number of small extractions, the greater the

quantity of solute removed. However for maximum efficiency
the rule of thumb is to extract three times with 1/3 volume
It is more
efficient to carry
out two
extractions with
1/2 volume of
extraction solvent
than one large
volume!
What type of organic compounds can be made water-soluble?

(1) Organic acids include carboxylic acids (strong organic

acids) and phenols (weak organic acids).

(2) Organic bases includes amines

 Weak Organic Acids.

Phenol, the parent compound, is partially

water-soluble (1 g will dissolve in 15 mL of
water),
a weak inorganic base, will not deprotonate
phenols to make it ionic. NaOH, a strong
 Organic Bases (Amines)

Organic Bases (amines) can be converted to

their salt form when treated with an aqueous
solution of an inorganic acid such as HCl
(hydrochloric acid).
You can separate four different classes of compounds from a
mixture based on differing solubility properties.
 Separatory Funnel Extraction Procedure

1. Support the 2. Pour in liquid to 3. Add extraction 4. Add ground glass

separatory funnel in a be extracted solvent Stopper (well greased)
ring on a ringstand.
Make sure stopcock is
closed
 Solid Phase Extraction

Solid Phase Extraction (SPE) is most

commonly used in place of liquid-liquid
extraction but similar methods are used to
trap gases also (as shown in purge and trap
GC)
Can be use for contaminant removal or
analyte trapping
Types of solid phases are similar to stationary
phases used in HPLC
High in recovery solute, ~ 99%
Selection of SPE Sorbent
 Procedure of SPE Technique

1. Clean cartridge with removing solvent,

then sample solvent
2. Apply sample; strongly retained
compounds will remain on stationary
phase, weakly retained/unretained
compounds pass through
3. Rinse cartridge with sample solvent
4. Apply eluting solvent to remove strongly
retained compounds
5. It is possible to Increase solvent strength
to remove compounds in several fractions
Figures of SPE Procedure
 Overview of SPE

Solid Phase
Very Similar to HPLC packing particles
Smaller column
Larger particles (allowing low pressure elution)
Some Types
Silica Based (octadecyl or C18, phenyl,
aminopropyl, etc.)
Ion Exchange (normally charged group on
polymeric solid)
Others (e.g. graphitic carbon)
 Maxi-Clean IC-RP Removes O rganic
Contaminants
5351

Trace Anions in Aniline

1. Chloride
2. Nitrate
3. Sulfate

Column: Anion/S, 250 x 4.1mm

Mobile Phase: 4mM KHP pH 4.5
Flowrate: 2.0mL/min
Detector: Alltech Conductivity
Original Sample After Cleanup with
Maxi-Clean IC-RP
 Post Extraction
The analytes are often diluted in the presence of a
large volume of solvents used in the extraction.
This is particularly true when the analysis is being
done at the trace level.
An additional concentration step is necessary to
increase the concentration in the extract.
 If the amount of solvent to be removed is
not very large and the analyte is
nonvolatile, the solvent can be vaporized
by a gentle stream of nitrogen gas flowing
either across the surface or through the
solution.
If large volume reduction is needed, this
method is not efficient, and a rotary
vacuum evaporator is used instead.
 Sample Cleanup

Sample cleanup is particularly important for

analytical separations many solid matrices,
such as soil, can contain hundreds of
compounds and the analyte is present at a
much lower concentration than the
interfering species.
Another important issue is the removal of
high-boiling materials that can cause a
variety of problems.
GPC, Acid-base partition, SPE and Column
Chromatography
Source of Sample Contamination
 Thank you
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