Analytical chemistry:

The branch of chemistry that deals with the

separation, identification and determination of
components in a sample.
It also traditionally includes coverage of chemical
equilibrium and statistical treatment of data.

Analytical chemistry can be broken down into

two general areas of analysis:
- Qualitative analysis
- Quantitative analysis
Quantitative analysis:
Determining how much of a material present in a
sample.

Qualitative analysis:
Attempting to identify what materials are present
in a sample.
The most common pharmaceutical analysis is
the quantitative measurement of the active
ingredient and related compounds in the
pharmaceutical product.

These determinations require the highest

accuracy, precision, and reliability because of
the intended use of the data for manufacturing
control, stability evaluation, and shelf-life
prediction.
Determination of drugs and their metabolites in
biological samples like plasma or urine, is
important in elucidation of drug metabolism
pathways as well as comparing bioavailability of
different formulas.
Basic steps in an analysis:
Prior to starting an assay, one must concentrate
to several aspects:
Technique to be used
Sampling and sample preparation
Proper application of method
Data analysis and reporting
All of these together comprise an analytical
method.
Sampling:
Sampling is a process by which a sample
population is reduced in a size to an amount of
homogenous material that can be conveniently
handled in the laboratory and whose composition
is representative of the population.

Regardless of the motives the analysts must take

sample for analysis that will give the most
accurate picture of the composition of the whole
batch.
There are three main methods for obtaining
samples:
Continuous sampling:
In continuous sampling, a certain portion of the
material to be analyzed is continuously diverted
to form the gross sample.
For example, a water line may be tapped in
such a way that small portions of water are
continuously removed for analysis.
Intermittent sampling:
In intermittent sampling, every nth unit is set
aside to form the gross sample.
At the production level, this may mean that
In most instances, however, the contents (either
liquid or tablets) have already been analyzed,
and this serves only as a final check on the
product.

Grab sampling:
Grab sampling is usually associated with
homogenous materials (usually liquid).
In small scale preparation, where through
mixing is possible, grab sampling is quite
successful.
But in large scale preparation, where through
mixing is not possible, it is preferable to take
the sample from several depths of container.
Pharmaceutical samples:
Table below lists types of samples that are
typically found in the pharmaceutical analytical
chemistry lab.
Sample type Explanation
Plasma sample Metabolism or bioavailability
Tablet, capsule Solid mixture
Packaging material, rubber gasket, Solid mixture
bottle
Transdermal patch, topical gel Semisolid solution
Elixir Solution
Aerosol Solid in gas
Oral suspension, topical lotion Solid suspended in liquid or
Oral suspension, topical cream liquid–liquid suspension
(emulsion)
Candy lozenge Solid–Solid suspension
rimary concern for a sample preparation
- Range
- Selectivity
- Recovery
- Stability
Range:
The instrumental method must be developed
before the sample preparation method. The
concentration of the prepared sample must be
within the working concentration range of the
instrumental method.
If the sample is too diluteted or concentrated,
then the sample concentration must be
adjusted using an appropriate sample
Selectivity:
The sample preparation method must not only
deliver a measurable amount of sample but the
compounds accompanying the analyte (undergo
analysis) must not interfere with the analysis
process.

In gravimetric assay (methods based on a

measured weight) the detection step has no
discriminating power and the sample preparation
provides all of the specificity.

A common example of gravimetric analysis is the determination of

the amount of water in a hydrate by heating the sample to remove
the water such that the difference in weight is due to the water lost.
Recovery:
The recovery of a sample preparation must be
assessed, because the recovery determines the
accuracy of the analysis.

For drug substance and drug product analysis,

recovery of 100% is generally required to
maintain required levels of accuracy and
precision.

For biological samples, less than quantitative

recovery is generally acceptable if the recovery
is reproducible.
Stability:
After the sample is prepared and is awaiting
instrumental analysis, the analyte must be
stable for a reasonable amount of time. If
necessary, the sample may require one
additional step, such as pH adjustment.

∗∗∗ In general, a pharmaceutical sample for

analysis must be in solution form and should
have following properties:
- Concentration must be in the measurable rage
of instrument
- Sample should be compatible with the system
- Quantitative recovery (desirable)
- The analyte is stable until the sample is
Prevention of segregation of sample
Various methods have been suggested to
prevent physical separation of the components
(segregation):
Direct dissolution of tablet in a suitable
solvent and assay aliquot of solution.
The resieving and regrinding of the
ground tablet and assay sievings.
The grinding of a composite with a
suitable organic solvent and the evaporation of
the solvent and assay residue.
The dissolution of the composite
powdered tablet sample in a solvent and assay
aliquot of solution.
 Direct dissolution in a suitable solvent
usually produces the most accurate and precise
analytical result.
For enteric coated tablets, manual grinding
with a mortar and pestle can lead to erratic
result which are overcome by repeated sieving
and grinding of the particle to a uniformly sized
powder.
Alternatively, removing the tablet coating
with an organic solvent prior to manual grinding
facilitates more uniform grinding of the tablets.
For example, ethinyl estradiol tablets are
powdered and triturated with four 20ml portions
of chloroform, decanted, filtered and analyzed
by TLC.
Fundamental theories controlling
sample preparation techniques
Before designing sample preparation
technique, it is necessary to review are some
fundamental theories that control the
separation process.
A. Physicochemical interactions:
Several types of intermolecular
physicochemical interactions have to consider
during development of sample preparation
methods.
The interactions can be divided into 4
types: ionic, dipole-dipole, hydrogen bonding
and hydrophobic interactions.
 Ionic interactions are interactions of two
charged species of opposite charge. Examples
of sample preparation techniques using ionic
interactions are ion-exchange or ion-pairing
chromatography.
The strength of these interactions depends
on the ionic concentration of the solutions. As
the ionic strength increases, the charge-charge
interaction decreases. The presence of
competing charged species (high ionic strength)
results in weaker attractions of the species of
interest.
The pH of the solution plays a critical role,
since most charged pharmaceutical compounds
are weak acids or bases.
 Dipole-dipole interactions (when two
opposite charge of separate dipole interact)
take place when the adsorption of a solute
occur onto the stationary phase in normal-
phase chromatography.
Hydrogen bonding occurs when a
hydrogen of one molecule interacts with an
electronegative dipole of another molecule or
functional group. Hydrogen-bonding
interactions behave
Hydrophobic similarly toare
interactions dipole-dipole
important in
interactions.
liquid-liquid extractions as well as in reversed-
phase chromatography.
B. Solubility:
Many factors affect solubility and must be
considered. For example, pH, ionic strength, and
temperature can significantly affect solubility.
For example, the aqueous solubility of a
carboxylic acid can be higher at a higher pH.
This is simply due to increase in the ionization
of the molecule.
For aqueous sample preparations, addition
of a water-miscible solvent (organic) such as
acetonitrile or alcohol can be used to enhance
solubility.
For example, the solubility of
acetaminophen in water is approximately 11
mg/mL, but the solubility is doubled by adding
B. Phase equilibrium:
Phase equilibrium theory is the
fundamental basis for many of the separations
techniques used for sample preparation,
including liquid-liquid extraction, solid phase
extraction, solid-phase microextraction, and
HPLC.
When a solute X is exposed to two
immiscible solvents, the partition equilibrium
can be described by the following equation:
X1 : X2
and the partition coefficient
Where,is defined as
Kp =
follows: Kp = partition coefficient
X1 = concentration of X in
X1
solvent 1
 Kp for a liquid-liquid extraction is
influenced by many other variables, such as pH,
temperature, ionic strength of the aqueous
phase and the volumes of the two solvents.

Therefore, method development efforts

must include identification and control of these
critical variables that will influence the
extraction.
Specific sample preparation
techniques
Liquid-solid extraction:
A frequently encountered procedure is the
extraction of a substance from a solid dosage
form sample, such as in the analysis of tablets.
This is relatively a simple procedure
involving the selection of a solvent or solvent
combination which ideally provides good
solubility of the substance of analytical interest
and minimal solubility of the components that
interfere with the analysis.
For the majority of procedures, the first
step requires grinding of the solid matrix into a
fine powder followed by solvent extraction, and
 For semisolid formulations, solvent
extraction is generally performed at elevated
temperatures so as to melt the semisolid and
increase the extraction efficiency.
In developing a method that requires
filtration, adsorption of the analyte onto filter
must be taken into consideration. This is
specially true for dilute solutions, in bulk
formulation filter adsorption is not an important
concern.
Soxhlet extraction
A classical liquid-solid sample preparation
technique is soxhlet extraction (hot extraction).
A soxhlet extractor is a piece of laboratory
apparatus. Typically, a soxhlet extraction is only
required where-
* the desired compound has only a limited
solubility in a solvent, and
* the impurity is insoluble in that solvent.
If the desired compound has a high
solubility in a solvent then a simple filtration
can be used to separate the compound from the
insoluble substances (as in cold extraction).
Fig: A schematic representation
of a soxhlet extractor
1: Stirrer bar 2: Solvent pot 3:
Distillation path 4: Thimble 5: Solid 6:
Siphon top 7: Siphon exit 8:
Expansion adapter 9: Condenser 10:
Cooling water in 11: Cooling water
out
Normally a solid material
containing some of the desired
compound is placed inside a
"thimble" made from thick filter
paper, which is loaded into the
main chamber of the soxhlet
extractor. The soxhlet extractor
is placed onto a flask/pot
containing the extraction
solvent. The soxhlet is then
 The solvent is heated to reflux. The solvent
vapor travels up a distillation arm, and floods
into the chamber housing the thimble of solid.
The condenser ensures that any solvent vapor
cools, and drips back down into the chamber
housing the solid material.
The chamber containing the solid material
slowly fills with warm solvent. Some of the
desired compound will then dissolve in the
warm solvent. When the soxhlet chamber is
almost full, the chamber is automatically
emptied by a siphon side arm, with 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 the
desired compound is concentrated in
the distillation flask. The advantage
of this system is that instead of many
portions of warm solvent being
passed through the sample, just one
batch of solvent is recycled.
After extraction the solvent is
removed, typically by means of a
rotary evaporator, yielding the
extracted compound. The insoluble
portion of the extracted solid remains
in the thimble, and is usually
discarded.
Figure: Extraction in progress
 The soxhlet technique is not often used in
routine pharmaceutical sample preparations for
two reasons:
The high temperature can cause
degradation which is unacceptable for stability-
indicating assays.
The technique is difficult to perform on
multiple samples because of time and space
requirements.