A] HPLC

Liquid chromatography
Liquid chromatography is a separation technique that involves:
• the placement (injection) of a small volume of liquid sample
• into a tube packed with porous particles (stationary phase)
• where individual components of the sample are transported along the packed tube
(column) by a liquid moved by gravity.
The components of the sample are separated from one another by the column packing
that involves various chemical and/or physical interactions between their molecules and
the packing particles. The separated components are collected at the exit of this column
and identified by an external measurement technique, such as a spectrophotometer that
measures the intensity of the color, or by another device that can measure their amount.

High Performance Liquid Chromatography (HPLC)

HPLC is an abbreviation for High Performance Liquid Chromatography. High
Performance Liquid Chromatography (HPLC) was developed in the late 1960s and
early 1970s. It has been around for about 35 years and is the largest separations
technique used.

HPLC is a separation technique that involves:

• the injection of a small volume of liquid sample
• into a tube packed with tiny particles (3 to 5 micron (μm) in diameter called the
stationary phase
• where individual components of the sample are moved down the packed tube
(column) with a liquid (mobile phase) forced through the column by high pressure
delivered by a pump.
In principle, LC and HPLC work the same way except the speed efficiency, sensitivity
and ease of operation of HPLC is vastly superior. These components are separated from
one another by the column packing that involves various chemical and/or physical
interactions between their molecules and the packing particles.
These separated components are detected at the exit of this tube (column) by a
flow-through device (detector) that measures their amount. An output from this
detector is called a “liquid chromatogram”.

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Major HPLC components and their functions

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1. Pump: The role of the pump is to force a liquid (called the mobile phase) through
the liquidchromatograph at a specific flow rate, expressed in milliliters per min
(mL/min). Normal flow rates in HPLC are in the 1-to 2-mL/min range. Typical pumps
can reach pressures in the range of 6000-9000 psi (400-to 600-bar). During the
chromatographic experiment, a pump can deliver a constant mobile phase composition
(isocratic) or an increasing mobile phase composition (gradient).
2. Injector: The injectorserves to introduce the liquid sampleinto the flow stream of
the mobile phase. Typical sample volumes are 5-to 20-microliters (μL). The injector
must also be able to withstand the high pressures of the liquid system. An autosampler
is the automatic version for when the user has many samples to analyze or when manual
injection is not practical.

3. Column: Considered the “heart of the chromatograph” the column’s stationary phase
separates the sample componentsof interest using various physical and chemical
parameters. The small particles inside the column are what cause the high
backpressureat normal flow rates. The pump must push hard to move the mobile
phasethrough the columnand this resistance causes a high pressurewithin the
chromatograph. Matrices and stationary phases
Two main forms of matrix/stationary phase material are available, based on a rigid
solid structure. Both forms involve approximately spherical particles of a uniform
size to minimise space for diffusion and hence band broadening to occur. They are
made of chemically modified silica or styrene/divinylbenzene copolymers.
The two forms are:
• Microporous supports: In which micropores ramify through the particles that are
generally 5_10 mm in diameter.
• Bonded phases: In which the stationary phase is chemically bonded onto an inert
support such as silica.

4. Detector: The detectorcan see (detect) the individual molecules that come out (elute)
from the column. A detector serves to measure the amount of those molecules so that
the chemist can quantitatively analyze the sample components. The detector provides
an output to a recorder or computer that results in the liquid chromatogram(i.e., the
graph of the detector response).

5. Computer: Frequently called the data system, the computer not only controls all the
modules of the HPLC instrument but it takes the signal from the detector and uses it to
determine the time of elution (retention time) of the sample components (qualitative
analysis) and the amount of sample (quantitative analysis).

Mobile Phase
The choice of mobile phase to be used in any separation depends on the type of
separation to be achieved. Isocratic elution may be made with a single pump, using a
single eluent or two or more eluents premixed in fixed proportions. Gradient elution
generally uses separate pumps to deliver two eluents in proportions predetermined by
a gradient programmer. All eluents for use in HPLC systems must be specially
purified because traces of impurities can affect the column and interfere with the
detection system. This is particularly the case if the detection system is based on the
measurement of absorbance changes below 200 nm. Pure eluents for use in HPLC
systems are available commercially, but even with these a 1_5mm microfilter is
generally introduced into the system prior to the pump. It is also essential that all

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eluents be degassed before use otherwise gassing (the presence of air bubbles in the
eluent) tends to occur in most pumps. Gassing, which tends to be particularly bad for
eluents containing aqueous methanol and ethanol, can alter column resolution and
interfere with the continuous monitoring of the eluate. Degassing of the eluent may be
carried out in several ways – by warming, by stirring vigorously with a magnetic stirrer,
by applying a vacuum, by ultrasonication, and by bubbling helium gas through the
eluent reservoir.

Applications
Separation and analysis of non-volatile or thermally-unstable compounds:
HPLC is optimum for the separation of chemical and biological compounds that are
non-volatile. Typical non-volatile compounds are: Pharmaceuticals like aspirin,
ibuprofen, or acetaminophen (Tylenol), Salts like sodium chloride and potassium
phosphate, proteins like egg white or blood protein, Organic chemicals like polymers
(e.g. polystyrene, polyethylene), Heavy hydrocarbons like asphalt or motor oil ,many
natural products such as ginseng, herbal medicines, plant extracts, Thermally unstable
compounds such as trinitrotoluene (TNT), enzymes.

The identification(ID) of individual compounds in the sample:

The most common parameterfor compound ID is its retention time(the time it takes for
that specific compound to elute from the column after injection). Depending on the
detector used, compound ID is also based on the chemical structure, molecular weight
or some other molecular parameter.

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B] GAS CHROMATOGRAPHY

Principle
The principles of gas chromatography (GC) are similar to those of HPLC but the
apparatus is significantly different. It exploits differences in the partition coefficients
between a stationary liquid phase and a mobile gas phase of the volatilised analytes as
they are carried through the column by the mobile gas phase. Its use is therefore
confined to analytes that are volatile but thermally stable. The partition coefficients
are inversely proportional to the volatility of the analytes so that the most volatile elute
first. The temperature of the column is raised to 50-300 oC to facilitate analyte
volatilisation. The stationary phase consists of a high-boiling-point liquid material
such as silicone grease or wax that is either coated onto the internal wall of the column
or supported on an inert granular solid and packed into the column. There is an
optimum flow rate of the mobile gas phase for maximum column efficiency (minimum
plate height, H). Very high resolutions are obtained hence the technique is very useful
for the analysis of complex mixtures. Gas chromatography is widely used for the
qualitative and quantitative analysis of a large number of low-polarity compounds
because it has high sensitivity, reproducibility and speed of resolution. Analytically, it
is a very powerful technique when coupled to mass spectrometry.

Apparatus
The major components of a GC system are:
• a column housed in an oven that can be temperature programmed;
• a sample inlet point;
• a carrier gas supply and control; and
• a detector, amplifier and data recorder system .
Columns
These are of two types:
Packed conventional columns: These consist of a coiled glass or stainless steel column
1_3m long and 2_4mm internal diameter. They are packed with stationary phase coated
on an inert silica support. Commonly used stationary phases include the polyethylene
glycols (very polar), methylvinylsilicone (non-polar respectively), Apiezon L (non-
polar) and esters of adipic, succinic and phthalic acids. The most commonly used
support is Celite (diatomaceous silica). Capillary (open tubular) columns: These are
made of high-quality fused quartz and are 10_100m long and 0.1_1.0mm internal
diameter. They are of two types known as wall-coated open tubular (WCOT) and
support-coated open tubular (SCOT), also known as porous layer open tubular (PLOT)
columns, for adsorption work. In WCOT columns the stationary phase is thinly coated
(0.1-5 mm) directly onto the walls of the capillary whilst in SCOT columns the support
matrix is bonded to the walls of the capillary column and the stationary phase coated
onto the support. Commonly used stationary phases include polyethylene glycol (CP
wax and DB wax, very polar) and methyl and phenyl-polysiloxanes (BP1, non-polar;
BP10, medium polar). They are coated onto the supporting matrix to give a 1% to 25%
loading, depending upon the analysis. The capacity of SCOT columns is considerably
higher than that of WCOT columns. The operating temperature for all types of column
must be compatible with the stationary phase chosen for use. Too high a temperature
results in excessive column bleed owing to the phase being volatilised off,

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contaminating the detector and giving an unstable recorder baseline. The working
temperature range is chosen to give a balance between peak retention time and
resolution.
Application of sample
The majority of non-and low-polar compounds are directly amenable to GC, but other
compounds possessing such polar groups as -OH, -NH2 and -COOH are generally
retained on the column for excessive periods of time if they are applied directly.
Poor resolution and peak tailing usually accompany this excessive retention. This
problem can be overcome by derivatisation of the polar groups. This increases the
volatility and effective distribution coefficients of the compounds. Methylation,
silanisation and perfluoracylation are common derivatisation methods for fatty acids,
carbohydrates and amino acids. The test sample is dissolved in a suitable solvent such
as acetone, heptane or methanol. Chlorinated organic solvents are generally avoided as
they contaminate the detector. For packed and SCOT columns the sample is injected
onto the column using a microsyringe through a septum in the injection port attached
to the top of the column. Normally 0.1 to 10mm3 of solution is injected. As there is
only a small amount of stationary phase present in WCOT columns, only very small
amounts of sample may be applied to the column. Consequently a splitter system has
to be used at the sample injection port so that only a small fraction of
the injected sample reaches the column. The remainder of the sample is vented
to waste. The design of the splitter is critical in quantitative analyses in order to
ensure that the ratio of sample applied to the column to sample vented is always
the same. It is common practice to maintain the injection region of the column at
a slightly higher temperature than the column itself as this helps to ensure rapid and
complete volatilisation of the sample. Sample injection is automated in many
commercial instruments as this improves the precision of the analysis.
Mobile phase
The mobile phase consists of an inert gas such as nitrogen for packed columns or
helium or argon for capillary columns. The gas from a cylinder is pre-purified by
passing through a variety of molecular sieves to remove oxygen, hydrocarbons and
water vapour. It is then passed through the chromatography column at a flow rate of
40_80 cm3 min–1. A gas-flow controller is used to ensure a constant flow irrespective
of the back-pressure and temperature of the column.
Detectors
Several types of detector are in common use in conjunction with GC:
- Flame ionisation detector (FID): This responds to almost all organic compounds.
It has a minimum detection quantity of the order of 5× 10–12gs -1, a linear range of 107.
A mixture of hydrogen and air is introduced into the detector to give a flame, the jet of
which forms one electrode, whilst the other electrode is a brass or platinum wire
mounted near the tip of the flame. When the sample analytes emerge from the column
they are ionised in the flame, resulting in an increased signal being passed to the
recorder. The carrier gas passing through the column and the detector gives a small
background signal, which can be offset electronically to give a stable baseline.
Modern GC systems are controlled by dedicated microcomputers capable of automating
and optimising the experimental conditions, recording the calibration and test retention
data and carrying out statistical analysis of it and displaying the outputs in colour
graphics in real time. They are capable of carrying out both qualitative and
quantitative analysis on a similar basis to that of LC

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