Unit-4

Gas Chromatography
 Introduction
• Chromatography is a physical method of separation of the
components of a mixture by distribution between two phases, of
which one is a stationary bed of large surface area and the other a
fluid phase that penetrate through or along the stationary phase.
• The stationary phase is the phase that doesn't move and the mobile
phase is the phase that does move.
• The mobile phase moves through the stationary phase picking up the
compounds to be tested.
• As the mobile phase continues to travel through the stationary phase
it takes the compounds with it.
• At different points in the stationary phase the different components of
the compound are going to be absorbed and are going to stop moving
with the mobile phase.
• This is how the results of any chromatography are gotten, from the
point at which the different components of the compound stop moving
and separate from the other components.
 Classification of Chromatography
• Super critical fluid chromatography
• Liquid chromatography (HPLC, TLC)
• Gas chromatography

Courtesy: A Handbook of Analytical Instruments by R.S.Khandpur,3 rd Edition

• The process of chromatographic separation involves
transport of a sample of the mixture through a column.
• For this purpose, the mixture may be in the liquid or
gaseous state.
• The stationary phase may be a solid adsorbent or liquid
partitioning agent.
• The mobile phase is usually a gas or a liquid and it
transforms the constituents of the mixture through the
column.
• During such transport, the material in the column
(stationary phase) exercises selective retardation on the
various components of the sample.
• This retardation may be due to adsorption, solubility,
chemical bonding, polarity or molecular filtration of the
sample.
• Therefore, the components of the mixture tend to
move through at different effective rates, and thereby
result in tending to segregate into separate zones or
bands.
• In general, all chromatographic procedures isolate,
detect and characterise these bands at some point,
usually the column exit.
• From the column, the gaseous phase immediately
enters a detector attached to the column.
• At this place, the individual components register a
series of signals, which appear as successive peaks
above a baseline on the recorded curve, called
chromatogram.
• The area under the peak gives a quantitative
indication of the particular component and the time
delay between injection and emergence of the peak
serves to identify it.
 Peaks represents concentration

Retention time
Chromatogram
• Retention Time:The time between sample injection and an
analyte peak reaching a detector at the end of the column
is termed the retention (tR).
• The time taken for the mobile phase to pass through the
column is called tM.

Figure: Illustration of ‘Retention Time’

• Dead Time: The dead time (tm) is the time a non-retained
compound spends in the mobile phase which is also the
amount of time the non-retained compound spends in the
column. Dead time is generally reported in minutes.
Block diagram of Gas Chromatography

Courtesy: A Handbook of Analytical Instruments by R.S.Khandpur,3 rd Edition

Internal diagram of gas chromatography
 Parts of gas chromatography

The various parts of a gas chromatographic system

are:
• Carrier gas supply or mobile phase
• Sample injection system and the size of the
sample
• Chromatographic column
• Thermal compartment or thermostat
• Detection system
• Recorder
 Carrier gas supply or mobile phase
• Mobile phase is formed by the continuous supply of a
carrier gas in which they are stored at pressures up to 2500
lb/sq.in.
• Carrier gas supply system comprises a needle valve, a flow
rate, a pressure gauge and a few feet of metal capillary
restrictions.
Types of gases, Purity of gases, gas flow rate
• Hydrogen, helium, nitrogen, argon and carbon dioxide can
be used as carrier gas.
• Carrier gas may effect the performance of column and
detector.
• Helium and hydrogen are preferred when thermal
conductivity detection system is used.
• Argon is used with argon ionization detectors.
• Nitrogen is used where separating power is more
important than high detector response.
• The presence of contaminants in the carrier gas may
effect column performance and detector response
particularly when ionization detectors are used.
• The rate of gas flow to be used in analysis depends
upon column diameter.
• Generally in the range of 10-400ml/min-very low and
very high flow rates.
• Flow rate should be controlled within 1% in order to
reduce analytical errors.
• Flow can be maintained constant by inserting a
capillary before the column.
 Sample injection system and the size of
the sample
• The purpose of sample injection system is to
introduce a reproducible quantity of the sample to be
analysed into the carrier gas stream.
• Samples can introduce in their gaseous, liquid or solid
states.
• The choice of method of injection depends upon the
pressure in the column at the point of introduction,
the type of detector to be used and the source of the
sample.
• The injector is basically a hollow, heated, glass lined
cylinder wherein the sample is introduced into GC.
• The temperature of the injector is controlled so that
all components in the sample will be vaporized.
 Liquid Samples
• To inject the liquid samples a micro
syringe method through a silicon
rubber septum.
• Syringes are employed for injection
of samples between 0.1 and 10µl.
• A sample is injected into hot zone of
the column, so that the liquid gets
transferred into gaseous phase.
• The metal block containing a
capillary is heated by a controlled
resistance heater.
• The sample is vaporized and carried
in to the column by carrier gas.
• Care must be taken to insert, inject
and remove the needle.

Courtesy: A Handbook of Analytical Instruments by R.S.Khandpur,3 rd Edition

 Gas Samples
• Gas samples are injected by a gas light
syringe suitable for delivering 0.1-10ml of
sample.
• The other method of injecting gaseous
samples is the bypass system, also called a
stream splitter.
• The principle of the system is to fill a loop of
known volume with the sample.
• By operating a valve, the loop is placed
directly in the carrier gas line.
• The valves were earlier made of glass, but
they have now been replaced by
polytetrafluoroethylene designs.
• The arrangement consists of three Figure: By-pass system for
stopcocks, between two of which there is injecting samples
standard volume in which the gas sample is
enclosed.
• Gas from bypass capillary loop is introduced Courtesy: A Handbook of
into column, by rotating or sliding valve, so Analytical Instruments by
that the loop is connected with the stream of R.S.Khandpur,3rd Edition
the carrier gas.
 Solid samples
• Solid samples may also be injected by using solid
injection syringes, where the sample is deposited on
the end of the plunger and withdrawn inside the
needle.
• After piercing the injection septum, the plunger is
extended to place the solid in the hot zone of the
column, where it is vaporized.
• Alternatively, the solid is deposited in a glass tube
from solution.
• After the evaporation of the solvent, the sample
holder is dropped into the column, thus making the
injection.
• Another method is to dissolve the solids in volatile
liquids by exposure to IR heat.
 Pyrolysis
• Pyrolysis offers a technique for injection of certain
types of materials, which are low or non volatile but
may be thermally decomposed in an inert atmosphere
to offer a qualitatively and quantitatively reproducilble
mixture of volatile fragments.
• The pyrolysis products are transferred to a
chromatographic column and separated.
 Chromatographic Column
• The column is the heart of GC, where the fundamental
process of separation takes place.
• When the sample is introduced into a column, it
spreads by molecular diffusion to yield a concentration
profile.
• As sample moves through the column, additional
spreads takes place but the band maintains its
general shape, which is detected and recorded as
chromatographic peak.
• The degree of peak broadening with respect to time
and column length is an indication of column
efficiency.
 Types of columns
Two types of columns commonly used
• Packed columns
• Capillary column or open tubular columns
Packed Columns:
• It is a tube packed with a suitable material, which performs
separations.
• Glass, stainless steel or copper are the materials used for
making columns.
• For moderate temperature PVC tubing is satisfactory.
• Internal diameter of column is in between 4 and 8mm
• Length may be in between 1 and 50m
• Columns longer than 3m is more difficult to pack uniformly.
• The column is made in the form of a U or helix or it may be
straight.
• Straight and U shaped columns can be repacked more
easily.
• A helical column is normally difficult to fill.
• Copper tubing is preferred.
• T he helix type column is a having a diameter of 50-250mm
and length of 2m.
Capillary columns:
• Capillary columns are open tube columns of tubing
approximately 0.25mm in diameter or even 30 to 300m.
• Very high efficiencies have been achieved with capillary
columns.
• Capillary columns contain no packing and the stationary
phase is coated directly on the inside of tubing.
• Capillary columns cannot handle samples more than 0.1 ml.
Larger samples are handled by the use of inlet splitters.
• Capillary columns are of two types: Wall-coated open
tubular (WCOT) or support-coated open tubular
(SCOT).
• Wall-coated columns consist of a capillary tube whose
walls are coated with liquid stationary phase.
• In support-coated columns, the inner wall of the
capillary is lined with a thin layer of support material
such as diatomaceous earth (source of silica), onto
which the stationary phase has been adsorbed.
• SCOT columns are generally less efficient than WCOT
columns.
• Both types of capillary column are more efficient than
packed columns.
• A new type of WCOT column called the Fused Silica
Open Tubular column.
• These have much thinner walls than the glass
capillary columns, and are given strength by the
polyimide coating.
• These columns are flexible and can be wound into
coils.
• They have the advantages of physical strength,
flexibility and low reactivity.
• The average capillary column (30 m long) has
approximately 1,00,000 theoretical plates while the
average packed column (3 m) has only 2,500 plates.

Self study topic: Difference between packed and capillary columns

 The stationary phase
• The separation of the sample into its components is achieved
by a partition process involving the stationary phase and the
moving carrier gas phase.
• The stationary phase is either liquid or solid.
• The following are the two possible methods with the gas as the
mobile phase:
- Gas-liquid chromatography
- Gas-solid chromatography
• Gas-Liquid Chromatography: Here, the stationary phase
is liquid, which is distributed on a solid support material. The
stationary phase must be involatile at all temperatures, at
which the column will be operated for the analysis and should
be coated as a thin even film on to the support. It is chosen
for the selective retention characteristics of components in the
sample that it will be used to separate.
• Gas-Solid Chromatography: The stationary phase in this
type is a solid material with surface active properties.
• The separating principle is based on the variation in the
extent to which constituents of a mixture are adsorbed on
the adsorbent packed in the column.
• Therefore, the separation is obtained because of the
different adsorption affinities which the column packing has
towards the sample components.
• This type of chromatography is used in the analysis of
inorganic gases and low-molecular weight hydrocarbon
gases.
• Among the most commonly used adsorbents in gas-solid
chromatography are silica gel (SiO2), alumina (Al2O3),
charcoal and molecular sieves (sodium or calcium
aluminium silicates).
 Thermal Compartment
• The column is not operated in normal room temperature.
• The column has to be kept at constant temperature b’coz the
quantitative response of the detector is affected by column
temperature.
• The column is housed in an oven, whose temperature is controlled to
an accuracy of 0.10C.
• Various methods are vapor jackets, electrically heated air baths, liquid
baths or metal blocks.
• The temperature of the oven may be controlled accurately by using a
proportional temperature controller with Platinum resistance
Thermometer as a sensing element.
• When the column temperature is kept constant, it is difficult to
analyse samples having components
• of a wide boiling range. This difficulty can be overcome by using
programmed heating of
• the column, so that its temperature is not kept at a constant
temperature, but is subjected to an
• exactly controlled temperature rise, while a separation is in progress
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 Detection Systems
• The detector is placed at the exit of the column.
• It is employed to detect and provide a quantitative
measurement of the various constituents of the
sample, as they emerge from the column in
combination with the carrier gas.
• The detector acts as a transducer and converts the
changes in some physical property to changes in an
electrical signal, which can be conveniently recorded.
The choice of a particular type of detector is governed by the
following factors:
• The detector should have high sensitivity.
• It should also permit the use of lower column
temperatures.
• It is desirable to be able to measure components from the
fractional ppm to almost 100% in one sample.
• The response of the detector should be linear over the
whole range.
• A small internal volume ensures that the resolution of
components, which are separated by the column, is not lost
and that the shape of peaks is not distorted by the detector.
• Detector temperature should be such that appreciable
amount of the eluted vapors does not condense in it.
• The detector should be insensitive to changes in the rate of
flow of the carrier gas.
• The detector should give good reproducibility of base line.
 Types of Detectors
• Flame Ionization Detector
• Argon Ionization Detector
• Photo Ionization Detector
• Electron Capture Detector
• Nitrogen Phosphorous Detector
• Atomic Emission Detector
• Flame Photometric Detector
• Mass Spectrometry Detector
 Flame Ionization Detector

• FID makes use of an oven, wherein a flame is

produced by burning hydrogen gas in presence of
oxygen or air.
• Effluent from the column is directed into a
air/hydrogen flame.
• A potential difference is maintained between the two
electrodes with the help of a series of batteries.
• Amplifier and recorder record chromatograms.
 Flame-Ionisation Detector

Courtesy: A Handbook of Analytical Instruments by R.S.Khandpur,3 rd Edition

 Operating Principle
• In this detector, the effluent from the column is led
into an oxy-hydrogen flame.
• An electrical potential is applied across two electrodes
placed in a stainless steel housing.
• The hydrogen flame burns at the tip of a capillary,
which also functions as the cathode and is insulated
from the body by a ceramic seal.
• The collector electrode consists of a loop of platinum
and is located at about 6 mm above the burner tip.
• The current across the electrodes remains constant,
when only the inert carrier gas passes the flame.
• The vapour molecules are broken into ions by the hot
flame.
• These ions result in the ionisation current and there
would be a consequent change in the current flowing
across the electrodes.
• The magnitude of the variation in current would be
directly proportional to the number of ions or
electrons formed in the flame gases, which in turn
would be proportional to the carbon content of the
organic molecules in the vapour.
 Advantages
• Cost: Inexpensive to acquire and operate.
• Low maintenance requirements: Apart from cleaning
or replacing the FID jet, these detectors require no
maintenance.
• Rugged construction: FIDs are relatively resistant to
misuse.
• Linearity and detection ranges: FIDs can measure
organic substance concentration at very low and very
high levels, having a linear response of 107.
• The FID exhibits a high sensitivity.
• Exhibits high output impedance.
 Disadvantages
• Flame ionization detectors cannot detect inorganic
substances.

• FID flame oxidizes all compounds that pass through it.

• It destroys sample.

• It requires additional Gases and controllers.

• For this reason, FIDs tend to be the last in a detector

train and also cannot be used for preparatory work.
 Applications

• FID responds only to substances that produce charged

ions when burned in a hydrogen/air flame.
• Satellite manufacture pure gas analysis.
• Analysis of impurities in closed cycle refrigerator
compressor gas.
• High purity hydrogen analysis in steel mill reducing
processes.
• Semiconductor process gas supply.

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 Argon Ionization Detector
• Argon gas is used as carrier gas.
• The detector contains two electrodes placed parallel to
each other and a potential difference is applied across
them.
 Operating Principle
• Under normal conditions, the carrier gas emerging out
of the column, no current passes across the
electrodes, as the gas is non conductor.
• A radio active source (tritium) is placed near the
electrodes so that the rays emitted by it excite the
argon atoms and electrons are produced by this
bombardment action.
• These electrons are accelerated under the influence of
a potential of about 1000V and upon collision with
other argon atoms, raise them to the metastable
state.
• Such metastable argon atoms collide with organic
molecules of the sample emerging from the GC
column, resulting in these molecules becoming ionized
and conducted.
• This results in flow of proportionate current
across the electrodes and produces signals,
magnitude of which depends on the quantity of
organic samples passing through the detector.
• AID responds to most of organic and inorganic
compounds and is inert to water vapour, oxygen,
methane, carbondioxide and oxygen.
• Sensitivity is 0.08µg/ml and linear dynamic range
is 105.
• AID can measure organic molecules over wide
range of concentrations.
 Photo Ionization Detector
• This detector is mainly
used for selective
determination of
aromatic hydrocarbons
or organo-heteroatoms.
• It uses UV light to ionize
an analyte exiting from
GC column.
• The ions produced by
this process are collected
by electrodes
• The current generated is
a measure of analyte
concentration.
Courtesy: A Handbook of Analytical Instruments by R.S.Khandpur,3 rd Edition
• If amount of ionization is reproducible for a given
compound , pressure and light source, then the
current collected at the PID’s reaction cell electrodes
is reproducibly proportional to the amount of that
compound entering the cell.
• Ionization potentials are within the range of
commercial available UV lamps, so hydrocarbons or
heteroatoms are used.
Advantages:
• Only a small fraction of the analyte molecules are
actually ionized in PID chamber, thus this can be
considered as a non destructive GC detector.
• The exhaust port of PID can be connected to another
detector in series with PID.
• So that data from two detectors can be taken
simultaneously.
 Electron Capture Detector

Courtesy: A Handbook of Analytical Instruments by R.S.Khandpur,3 rd Edition

 Working principle
• This was invented in year 1954. It is a radioactive
decay-based detector
• Works on the principle that the ionization current set
up by certain radioactive sources like Ni63 or H3 gets
reduced when an electron capturing compound is
introduced in the cell.
• The ECD measures the loss of signal due to the
recombination phenomenon rather than measuring a
positively produced electrical current.
• The detector consists of two electrodes across which a
potential difference of 10 to 100 V can be applied.
• A radiation source of β rays (tritium) is mounted on a
tantalum wire saturated with the radioactive isotope
of hydrogen, so that the emitted β rays encounter the
effluent from GC column.
• As the carrier gas(nitrogen) flows through the
detector, β particles from tritium source ionize the
nitrogen molecules and form slow electrons.
• These slow electrons migrate to the anode under fixed
voltage.
• When these electrons are collected at the collector
electrode, they produce a steady current, which
provides a base line on the recorder.
• Halogen, nitrogen and phosphorus have the property
of capturing electrons, resulting in a variation in the
number of electrons reaching the collector electrode,
which producing proportionate signals in the detection
device.
• Detector discrimination can be regulated through a
potential applied to the collector electrode.
• Extremely sensitive and linear range is limited to less
than 103.
• Nitrogen and hydrogen are best carrier gases with this
type of detectors.
• Hydrogen should be used in caution, otherwise there
be an explosion.
Advantages:
• useful for environmental testing
detection of chlorinated pesticides or herbicides
detection of polynuclear aromatic carcinogens
detection of organometallic compounds
• Highly sensitive towards compounds of halogen- (I, Br, Cl,
F), nitro-, and sulfur-containing compounds.
• detects polynuclear aromatic compounds, anhydrides and
conjugated carbonyl compounds.
• Does not alter the sample.
Disadvantages:
Non linear response unless potential across the detector is
pulsed.
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Thank you
LIQUID CHROMATOGRAPHY
 Outline
• Introduction
• Principle of operation
• Types of liquid chromatography
• Detection systems
- Fluorescence detector
- Refractive index detector
- Thermal detector
• Applications
 Introduction
• LC is an analytical chromatographic
technique which is useful for separating ions
or molecules that are dissolved in a solvent.
• Earlier separations were performed by using a
bed of solid powder absorbent such as
alumina or charcoal through which the
sample was passed in a stream of solvent.
• Later developments: Liquid/liquid partition
chromatography, paper chromatography, Ion
exchange chromatography, Gel permeation
chromatography and thin layer
chromatography.
• The mixture to be separated is loaded onto the
top of the column at different rates due to
differences in their portioning behavior between
the mobile liquid phase and stationary phase.
• The compounds are separated by collecting
samples from the whole of the column liquid
sewage as a function of time.
• Stationary phases are: solids(adsorption), ionic
groups on a resin(ion exchange), liquids on an
inert solid support(partitioning) and porous inert
particles(size exclusion).
Types of Liquid Chromatography
Classification of Liquid column
chromatography
 Adsorption chromatography
• A solid adsorbent is in powder form
is stationary phase through which a
mobile liquid phase carrying the
mixture to be analyzed is allowed to
percolate.
• Adsorption chromatography is
carried out in columns with the
adsorbent supported by a plug of
glass or cotton wool or by a sintered
glass filter.
• The stationary phase consists of
silica or alumina particles.
• Analytes are separated due to their
varying degree of adsorption onto
the solid particles.
 Partition chromatography
• The separation of the components
from the sample mixture is carried
out by the process of partition of
the components between 2 phases.
• Both phases are in liquid form. In
this process, the immiscible solid
surface coated with the liquid
surface on the stationary phase is
in the mobile phase.
• The liquid surface is immobilized by
a stationary phase which results in
making it as a stationary phase.
• The mobile phase moves from the
stationary phase and components
get separated.
• The separation depends on
different partition coefficient.
Ion exchange chromatography

Figure: Ion exchange configuration

• When a sample is introduced at the top of an
ion-exchange column, the ions exchange
rapidly with the ions in the resin.
• If a mobile phase is used, the sample ions are
displaced into the solution again and then re-
exchange on to the resin.
• This process continues until the sample ions
emerge from the end of the column.
• If the various sample ions are held on to the
resin to different extents, then the time taken
for them to pass through the column will be
different and a separation will be achieved.
 Gel permeation chromatography
• The separation is based on
molecular size and shape.
• The gel permeation column is
packed with a stationary phase in
the form of a gel which contains
pores of a specific size.
• As the sample is carried through
the column bed by the carrier
liquid, the sample molecules
penetrate the pores in the
packaging gel, depending upon
the size and shape of the
molecules.
• Large molecules do not penetrate the gel and
are quickly eluted.
• Elution takes place in inverse order of their
degree of gel permeation and in decreasing
molecular size.
 Thin-layer chromatography
• Thin-layer chromatography (TLC) is a
chromatographic technique that is useful for
separating organic compounds.
• It is often used to monitor the progress of
organic reactions and to check the purity
of products.
• In TLC, the stationary adsorbents are applied to a
planar glass or plastic surface and the solvent
flows over them.
• TLC consists of a stationary phase immobilised on a
glass or plastic plate, and an organic solvent.
• The sample, either liquid or dissolved in a volatile
solvent, is deposited as a spot on the stationary phase.
• The bottom edge of the plate is placed in a solvent
reservoir and the solvent moves up the plate by capillary
action.
• When the solvent front reaches the other edge of the
stationary phase, the plate is removed from the solvent
reservoir.
• The separated spots are visualised with ultraviolet light.
• The different components in the mixture move up the
plate at different rates due to differences in their
portioning behaviour between the mobile liquid-phase
and the stationary phase.
 Paper Chromatography
• It is a simplified version of column chromatography,
which makes use of strips or hollow cylinders of filter
paper to hold both the solid and liquid phases.
• The drops of the solutions containing unknown
mixtures are applied to a number of parallel strips
from the end of each test paper and allowed to dry.
• The strips of paper are placed in a chromatography
camber with a saturating and equilibrium vapor and
hung from a solvent reservoir, so that the downward
movement of the solvent can be timed and the
relative partition of the different substances are
measured.
 High Pressure Liquid
Chromatography

Figure: Block diagram of a liquid chromatograph

Physical layout of HPLC system
 High Pressure Pump System

• Commonly used methods for solvent delivery are

gravity feed system but not able to deliver at
high pressure.
• Various types of pump systems are piston
pumps, peristaltic pumps, diaphragm pumps and
syringe pumps are used in HPLC systems.
• Several types of pumps are available with HPLC
analysis which includes constant flow pumps and
constant pressure pumps.
 Constant flow pumps
• Reciprocating piston pumps consist of a small
motor-driven piston which moves rapidly back
and forth in a hydraulic chamber that may vary
from 35–400 µL in volume.
• On the back stroke, the separation column valve
is closed and the piston pulls in solvent from the
mobile-phase reservoir.
• On the forward stroke, the pump pushes solvent
out to the column from the reservoir.
• A wide range of flow rates can be attained by
altering the piston stroke volume during each
cycle, or by altering the stroke frequency.
Principle of single-piston
reciprocating pump
• Here, a rotating eccentric cam forces the piston
to eject liquid through a one-way valve, called the
check valve.
• The purpose of the check valve is to assure that
liquid moves only in one direction.
• These pumps deliver a series of pulses of the
mobile phase, which may disturb the detector.
• It is thus necessary that these pulses may be
eliminated.
• Dual and triple head pumps consist of identical
piston-chamber units which operate at 180° or
120° out of phase.
 Constant pressure pumps

• In these pumps, the mobile phase is driven

through the column with the use of pressure from
a gas cylinder.
• A low-pressure gas source is needed to generate
high liquid pressures.
• The valving arrangement allows the rapid refill of
the solvent chamber and provides continuous
mobile phase flow rates.
• Generally used pumps are the piston type, which provide
very high solvent pressures.
• The flow rate can be set to the desired rate by adjusting the
pump stroke length and the motor speed.
• The pressure is observed as a dependent variable.
• The flow of solvent from a piston pump is usually in the
form of a series of pulses.
• This type of ripple in the flow is likely to affect column
resolution and detector stability.
• To smooth out the ripple, a long nylon tube of about 1.5
mm diameter may be used between the pump and the
chromatographic column.
• Ripple can also be reduced by using bellows, restrictors or
multi-piston pumps, where the action of the individual
pistons is arranged at regular intervals of a complete stroke
cycle.
• A flow control system for HPLC, which maintains constant
flow irrespective of differing solvent viscosities and
compressibilities, utilises a hydraulic capacitor which
smoothens the high-pressure pump pulsations, normally
encountered in piston operated pumps.
• It consists of a rigid vessel filled with fluid of known
compressibility.
• A small fraction of the space is separated from the
compressible fluid by an impermeable membrane.
• The solvent mixture passes through this separated space.
 Gradient Elution or Solvent
Programming
• In LC, a single substance may be
used as a mobile phase during an
analysis of the mixture of two or
more substances to properly
adjust the characteristics of the
phase.
• Also, one may maintain a
constant mobile-phase
composition during analysis or
change it.
• The first mode is called the
Isocratic operation (mixture of
mobile phase is consistent entire
testing time), while the second is
called the gradient
elution(altering the composition
of mobile phase)
• Gradient elution is required to resolve complex
mixtures, especially those containing components with
significantly different chromatographic behaviour.
• A solvent programmer helps to control the
composition of the mobile phase according to a
predetermined programme as the analysis proceeds.
• Solvent programming is generally carried out by
continuously adding a more polar solvent to the
mobile-phase feed reservoir, thereby increasing the
polarity of the eluent as a function of time.
• The technique involves the use of separate pumps,
feeding different solvents or solvent mixtures
concurrently into the column and programming the
output of each pump.
 Sample Injection System
• There could be several methods for the introduction of
the sample on the top of a LC column.
• One method is to disconnect the solvent supply, to
add the sample in solution and reconnect the solvent
supply to the column.
• More recently developed methods fall into two
categories namely, the syringe injection method and
the injection valve method.
• Both these methods enable the sample to be
introduced directly into the column packing, without
interrupting the solvent flow.
 Syringe injection method
• Samples are injected into the HPLC via an injection
port.
• The injection port of an HPLC commonly consists of an
injection valve and the sample loop.
• The sample is typically dissolved in the mobile phase
before injection into the sample loop.
• The sample is then drawn into a syringe and injected
into the loop via the injection valve.
• A rotation of the valve rotor closes the valve and
opens the loop in order to inject the sample into the
stream of the mobile phase.
• Stopped-flow injection is a method whereby the
pump is turned off allowing the injection port to
attain atmospheric pressure.
• The syringe containing the sample is then
injected into the valve in the usual manner and
the pump is turned on.
• For syringe type and reciprocation pumps, flow in
the column can be brought to zero and rapidly
resumed by diverting the mobile phase by means
of a three-way valve placed in front of the
injector.
• The method can be used up to very high
pressure.
• Syringe injection method basically involves the
insertion of the syringe needle through a rubber
septum at the top of the column.
• This method cannot be used for the injection of
large sample volumes into high-pressure solvent
systems.
• At pressures greater than about three
atmospheres, the pressure has to be reduced by
turning off the solvent supply before the injection
of the sample can be carried but.
Syringe injection method
 Injection valve method
• In this method, the injection valve containing sample
loops are connected in the solvent supply pipe work at
the top of the column.
• The sample loop can be introduced into the solvent
stream when desired, without turning off the solvent
flow.
• After sufficient flushing of the loops with solvent has
taken place, the sample gets completely carried to the
column.
• The loop can then be removed from the solvent
stream for refilling with the next sample.
• Injection valves can be used for sample introduction
into very high solvent pressure systems.
• By changing the volume of the sample loop, the
sample size can be easily varied.
• Also, this method can be conveniently automated for
automatic injection of the samples.
 The Column
• The column is the most important part of any chromatographic
system, since the ultimate performance of the chromatograph is
determined largely by the column.
• Most of the early analysis work, which was carried out by LC,
made use of large columns with an internal diameter of 1 cm or
more.
• With the development of highly sensitive detection systems, it has
become possible to analyse minute quantities of sample and to
reduce the column diameter.
• Reduction of sample size and the column diameter result in an
improvement in separation efficiency.
• There is another factor which necessitates the use of small
diameter columns.
• A large contribution to the band broadening in the chromatographic
peaks is known to be due to large-scale unevenness of flow, which
becomes worse as the column diameter increases.
• The columns in current use are generally in the 0.1–2.0 cm internal
diameter range.
• Glass columns are usually preferred on account of their inert nature
and the facility of being able to observe the packing visually.
• HPLC columns are stainless steel tubes, typically of 10–30 cm in
length and 3–5 mm inner diameter, short, fast analytical columns and
guard columns, which are placed before an analytical column to trap
junk and extend the life time of the analytical column, are 3–10 cm
long.
• In most of the separations, LC columns are operated under ambient
temperature conditions.
• Columns are therefore placed inside ovens capable of operation up to
25°C.
• The temperature of these ovens is controllable to a high degree of
constancy.
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Interpretation of chromatogram
• https://www.youtube.com/watch?v=qXmSb6X
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 Ideal Properties of a Detector
The detectors used in HPLC should have following ideal properties:

❑ High sensitivity.
❑ Good stability and reproducibility.
❑ A linear response to solute.
❑ Negligible base line noise.
❑ Should be inexpensive.
❑ Capable of providing information on the identity of the solute.
❑ A short response time independent of flow-rate.
❑ High reliability and ease of operation.
❑ The detector should be non-destructive.
❑ Responses independent of mobile phase composition.
❑ A temperature range from room temperature to at least 400 0 C
 Types of detectors
• Refractive index (RI) detectors
• Ultraviolet (UV)
• Fluorescent detectors
• Radiochemical
• Electrochemical
• Near-infrared (Near-IR)
• Mass spectroscopy (MS)
• Nuclear magnetic resonance (NMR) and
• Light scattering (LS)
• Thermal detectors
Fluorescence Detector
Fluorescence Detector
• A fluorescence detector excites the sample with
excitation light and breaks up the emitted
fluorescence light with a fluorescence
monochromator.
• It extracts the required fluorescence wavelengths
and measures the intensity with a
photomultiplier.
• In a fluorimeter, the presence of fluorescence
emitting substance is measured.
• The emitting substance is only present
occasionally in LC and that its emission of energy
is detected by a highly sensitive photomultiplier.
 Working principle
• A spectrofluorimeter designed specifically for LC
applications offer continuously selectable
monochromatic excitation energy over the entire UV-
visible spectrum, utilising a highly stabilised
deuterium or tungsten-halogen lamp.
• The monochromator makes use of a grating system.
• Excitation energy from the monochromator enters the
cuvette and emission from a 5 µl cavity is collected
and directed towards the photomultiplier.
• A set of easily interchangeable filters is provided to
select the emission spectra of interest.
• The filter set contains filters of wavelengths 370, 389,
418, 470, 550 and 580 nm.
• Transmittance is greater than 0.9 in these areas
compared to less than 10−5 below the cut-off point
and because of this high efficiency, virtually no
emission energy gets absorbed before reaching the
photomultiplier.
• In normal fluorimeters which utilise standard cuvette
volumes of several cc’s are illuminated by larger
excitation light beams of at least 1 cm2 .
• The emitting material acting as secondary light source
to be detected, originates from a much larger area or
volume than what is available in the micro-litres type
cuvette of fluorimeters used in HPLC.
 Steradian cuvette used in
fluorescence detector

• Since emission occurs in all

directions from an excited
sample surrounding this
sample with an efficient
optical collector is a must.
• 2 Steradian cuvette
provides a solution to this
problem.
• This cuvette is of stainless
steel and is flow-through
type.
 Applications
• It can be used to detect any compound absorbing and emitting light at the
given excitation and emission wavelength.
• Fluorescence can be used to selectively detect any compound that absorbs and
emits light at the chosen set of excitation and emission wavelengths
- Relatively few compounds undergo fluorescence
- high selectivity, low background signal
- limits of detection for a fluorescence detector are ~ 10-10 M
- Typical applications
- drugs
- food additives
- environmental pollutants
- any compound that can be converted to a fluorescent derivative:
alcohols, amines, amino acids and proteins
- Can be used with gradient elution
- requires extremely pure mobile phases
- trace impurities can affect background signal or quench the
fluorescence of solutes
 Refractive Index Detector
• Measures the overall ability of the mobile phase and
its solutes to refract or bend light.
• Refractive index detector measures the molecule’s
ability to deflect light in a flowing mobile phase in a
flow cell relative to a static mobile phase contained in
a reference cell.
• The amount of detection is proportional to the
concentration of the solute in the mobile phase.
• The common RI detectors are based on refraction,
reflection or interference of light beams.
• Most RI detectors, light proceeds through a bi-
modular flow cell to a photo detector.
• Refraction type RI detectors depends on snell’s law at
the interface between the cell in glass and the flowing
liquid to deflect a light beam.
• Changes in RI are monitored at far field by a
position sensor, or as an intensity change on a
small area photodiode.
• Since the effect is produced at the interface, very
small volumes are possible if the proper cells are
made.
• Reflection type RI detectors are based on
Fresnel’s laws at the interface between the cell
(glass) and the liquid, which has a smaller RI.
• Sensitivity will increase as the incidence angle
approaches the critical angle.
Fresnel Type Refraction Index Detector
 Working principle
• The Fresnel refractometer which measures the change in
the fractions of reflected and transmitted light at a glass-
liquid interface as the refractive index of the liquid changes.
• In this detector both the column mobile phase and a
reference flow of solvent are passed through small cells on
the back surface of a prism.
• When the two liquids are identical there is no difference
between the two beams reaching the photocell, but when
the mobile phase containing solute passes through the cell
there is a change in the amount of light transmitted to the
photocell, and a signal is produced.
• The smaller cell volume in this detector makes it more
suitable for high-efficiency columns but, for sensitive
operation, the cell windows must be kept scrupulously
clean.
• Here, the light beam is focused on and reflected
from both the liquid-prism cell interfaces and a
polished back plate (which forms the near surface
of both sample and reference cells) onto the
detecting photocell.
• As the intrusion of sample into the one cell
causes light to be refracted at a different angle,
this light changes in intensity rather than
position, with the unbalance once again being
detected as a change in electrical energy.
• This difference between sample-cell signal and
reference-cell signal is given to a recorder.
 Advantages

• Higher potential sensitivity

• Ability to operate at extremely low flow rates,
with very low-volume flow cells
• Easy cell accessibility and low cost.
• It is suitable for detecting all components.
• It provides a direct relationship between the
intensity and analyte concentration.
 Disadvantages
• Need to change prism to accommodate either
high or low RI solvents.
• The limit of detection is limited by fluctuations in
the RI of the LC effluent.
• Solvent delivery systems must be pulse-free to
avoid pressure effects.
• Also, thermostated cells must be used to avoid
temperature effects.
 Thermal detectors
• Thermal detectors are also known as micro-
adsorption detectors.
• The principle of operation of these detectors
depends on temperature changes taking place
due to the heat of adsorption on an active solid
source.
• Generally, a portion of the adsorbent column is
contained in a small chamber at the column
outlet.
• A second chamber filled with an inert material
such as glass micro-beads is used to achieve a
reference signal.
• This assembly is carefully thermostated.
• Both chambers contain matched thermistors, which
constitute the measuring arms of a Wheatstone
bridge.
• As the eluted sample is adsorbed on the solid, a local
temperature change takes place, which initiates a
signal.
• These detectors can be used in applications involving
liquid-solid, liquid-liquid, ion-exchange and gel-
permeation chromatography.
• They are non-destructive.
• However, they are subject to other thermal effects
such as thermal conductivity and heat capacity of the
solvent.
• They require accurate calibration before use.
 Applications of liquid
chromatography
• Pharmaceutical analysis
• Metabolomics analysis
• Clinical analysis is mainly for detection, extraction, enrichment of
targeted components in biofluids.
• Proteomics analysis, It is used for the separation of different
colors of ink.
• It is also used to identify and separate the preservatives and
additives added in the food items.
• It is also used in DNA fingerprinting and bioinformatics.
• It is also used to separate different amino acids, proteins etc.
• Food Industry: Food spoilage and additive detection. In addition it
also helps in determining nutritional quality of food.
• Forensic Science: Gas Chromatography has been widely used in
forensic pathology and crime scene testing like analysing blood
and hair samples of crime place.
Thank you
 y&S
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Journal of Chromatography
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ISSN: 2157-7064 Zhu and Chen, J Chromatogr Sep Tech 2017, 8:2
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DOI: 10.4172/2157-7064.1000358
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Mini Review Open Access

Development and Application of Liquid Chromatography in Life Sciences

Bo Zhu* and Ying Y Chen
Faculty of Health Sciences, University of Macau, Taipa, Macao SAR, China
*Corresponding author: Bo Zhu, Faculty of Health Sciences, University of Macau, Taipa, Macao SAR, China, Tel: 8822 2734; E-mail: [email protected]
Received date: April 27, 2016; Accepted date: March 13, 2017; Published date: March 17, 2017
Copyright: © 2017 Zhu B, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

Liquid chromatographic approaches cover a wide range of applications today. It is closely relevant in scientific
studies, which provides a powerful identification of certain components that will be effective for curing specific
diseases. Especially important, LC methods can assist us for isolation and purification of therapeutic drugs that
benefit for the development of medical industry. As the development of liquid chromatography, the single effective
component within a drug can be separated and purified under extreme purity requirements from a biological system
with great complexity and abundance. However, improvement of LC methods is still urgently needed for better
application of this method to benefit our lives.

Keywords: Analytical methods; Application; Development; Life Two decades later, Kuhn and Brockmann, the two German scientists
sciences; Liquid chromatography recognized that adsorbents are reproducible and selective adsorbents
are needed. Therefore, the improved requirements of stationary phases
Introduction trigger the production of materials with different adsorption strengths,
which is the first attempt to reproduce separations for individual
This review focuses on liquid chromatography development history, components.
current state of development, in particular, its application in the life
sciences. After this early liquid chromatography, between 1960 and 1970, the
application of LC appeared faster due to the development of high-
Currently, liquid chromatography is developing into a widely performance liquid chromatography (HPLC) [3]. The benefits of
applied analytical method. LC is becoming an increasingly important improved-column LC method over the other common column LC due
tool that helps us discover the underlying of specific phenomenon in to several factors as follows, firstly particle size of the adsorbent
life sciences. Actually, LC is the method essential for the development reduced, then the small particles were packed into pressure-stable
of biomedicine industry, especially for purification of drug columns, and high and constant velocities were required for operation
components, recombinant proteins and antibodies, which cannot be of the separation [4]. Soon after the manufacture of mechanically
used for application without help from LC. stable adsorbents like microparticulate silica which was packed into
This review aims to provide information that covers the history of stainless-steel column densely, further improved the efficiency of
LC development, especially, to highlight its applications in the study of HPLC [5]. The combination of improvements in the adsorbents,
life sciences. Commonly, the “life sciences” has broader definition that proper column, and corresponding system development became
sciences study live organisms, including microbes, plants, animals, and typical innovations during the past 50 years for exploration phase of
humans, and it can also be grouped by application differences, HPLC.
including diagnostics and therapeutic in clinic, contaminants and The modern LC methods are based on three foundations, including
pollutants analysis, and other aspects. Since the 1950’s, it has process LC, analytical HPLC, and bio-separation, and they have been
continuous improvements for the discovery, development, and discussed one by one as follows.
production of drug for the following decades. As the coupling of LC to
mass spectrometry, it supports us in advanced analytical platform Process LC
which facilitates peptide and proteins identification and purification,
and separation of other small molecules. As the continuous progress of chromatography, the invention of
stimulated moving-bed (SMB) technology, together with materials
Development history of liquid chromatography optimization for specific separation tasks contributed to high efficiency
for application in petrochemical area [6]. Actually, the concept of SMB
Back to 19th century, the accurate separation of small size chemicals chromatography was not adapted until 1990s; further the technology
seems impossible although adsorptive separations were already applied was transferred to the production of pharmaceuticals [7]. It is great
in the 19th century [1]. The term “chromatography” was first adopted important to develop several types of highly selective adsorbents in
in 1903 for isolation of chlorophyll constituents [2]. The key difference 1980s, the resolution is for analytical HPLC application [8], which
between “chromatography” and previous methods is the use of suitable allows precisely separation of components from complex products. In
and sufficiently well characterized adsorbents. The finding of suitable particular, the combination of HPLC methods and SMB technology
adsorbents provides a possibility for invention of novel methods, liquid with the assist of dynamic axial compression (DAC) [9], facilitates the
chromatography, which needs huge background knowledge for the production of large amounts of pure products.
selection of materials and liquid-solid interactions.

J Chromatogr Sep Tech, an open access journal Volume 8 • Issue 2 • 1000358

ISSN: 2157-7064
Citation: Zhu B, Chen YY (2017) Development and Application of Liquid Chromatography in Life Sciences. J Chromatogr Sep Tech 8: 358. doi:
10.4172/2157-7064.1000358

Page 2 of 4

LC is considered as a great powerful method for analysis, currently, methods to overcome pore-diffusion effects trigger the introduction of
it acts as the only technique that deals with the separation and hydrophobic interaction chromatography (HIC) and ion-exchange and
identification of compounds from complex biofliud sample at the level affinity chromatography [17] due to retention, which occurs not inside
of femtomolar amounts. Furthermore, LC is also applied for the of microparticles but at the outer surface. Actually, the development of
purification and isolation of industrial products at high amount level. robust adsorbents for process-scale chromatography has been
increased fast from the late 1960s. However, the challenge was still to
Analytical HPLC overcome diffusion-induced mass-transfer limitation. Many factors
can contribute this limitation, including time of retention, size and
Analytical HPLC was widely applied for analysis of composition for porosity of bead, stationary phases, and matrix morphology.
drugs and chemicals. The most revolutionized developments for HPLC
in the 1970s were the application of small particle silica as adsorbent, Since 2000, Protein A adsorbents have been widely applied for
with its diameters smaller than 10 µm, which was packed into affinity chromatography, especially for the dominant application for
stainless-steel columns [10]. The past several decades, the diameters of purification of biological components from biofluid mixture, such as
adsorbents for packing is significantly reduced from 10 µm to 5 µm or monoclonal antibodies. The benefits of this Protein A adsorbents-
3 µm, and further to less than 2 µm to improve the efficiency of based affinity chromatography were improved stability to alkaline
column, reduce time for analysis, and enhance the sensitivity [11]. cleaning, and high specificity of binding capacities against individual
antibody. The bottleneck for biopharmaceutical development mainly
The breakthrough of column development occured around 1980, it locates at the downstream processing [18]. The cost for processing
is the first application of fused-silica capillary columns with a coat of chromatography is the most expensive part among downstream
polyethyleneimine. Special micro- and nano- LC instruments are technologies [19]. However, it is still the dominant powerful tool for
required to operate these capillary columns which results in generation biopharmaceutical industry, since affinity chromatography showed a
of flow rates in the range of µl min-1 to nl min-1 [12]. Furthermore, the unique advantage over other technologies, its selectivity. Recently, the
efficiency for analysis is dramatically improved by capillary columns. techniques for purification of biological products, especially for
For example, the capillaries packed with microparticulate silica can recombinant proteins, have been improved dramatically, such as
analyze plate over the speed of 100000 plate min-1 [13]. continuous multicolumn counter-current solvent gradient purification
The great development of LC triggers it as a common tool of processes [20].
analysis in life sciences research for several benefits. The most obvious In summary, the methods for bioseparation are comprised of several
advantages include extremely low amount of sample required, low different modes according to selectivity differences, including
consumption of solvent, and extremely high level of sensitivity for hydrophobic interaction chromatography (HIC), hydrophilic
reduced sample dilution required. However, the operating of HPLC interaction chromatography (HILIC), ion-exchange chromatography
systems is more difficult than conventional LC and high demand of (IEC), the variants of affinity chromatography (AC), and size-exclusion
knowledge and skills are needed for the operator. chromatography (SEC) [21], which are widely applied either for
The most important breakthrough in the development of HPLC was scientific research or product purification in industry.
its coupling to mass spectrometry (MS). Thus, MS coupled HPLC can
act as both the detector and separator; this great advancement occured LC in the new era
during the 1990s [14]. It is well known that mass spectrometry can be
After the completion of human genome project in April, 2003, it was
divided two classes, electrospray ionization (ESI) and matrix-assisted
found that human proteome with higher level of complexity than
laser-desorption/ionization (MALDI) techniques. The advantages of
genome consisted of a protein system that randomly combined 20
LC-MS used for analysis of environmental pollutants, drug
different amino acids. The dynamic protein system has a wide range of
components, etc. are improvement of accuracy for detection of in low-
posttranslational modifications, huge concentration difference of
mass molecules, improvements of sensitivity level, reduced limits for
abundance. Therefore, proteomics has a much higher level of
detection, etc. Furthermore, the low rates of flow can be produced after
complexity than genomics.
coupling micro- and nano-LC systems to MS.
All in all, analytical HPLC provides a wide range of opinions for The traditional 2D gel-based followed by MS analysis has its
selection of various efficient silica columns, and the columns provide limitation as follows:
us an effective platform for precise analysis in the industry of
pharmaceuticals, such as the discovery and development for drugs. The major defect is the missing for detection of small proteins and
peptides, and few low-abundance proteins and peptides can be
Bio-separations identified due to the loading limitation. Furthermore, the capacity for
separation of proteins and peptides is also limited. Moreover, the
Bio-separations originate the application of cross-linked dextrans procedure for accurate identification is time consuming since various
which are used as packing materials for size-exclusion by researchers posttranslational modification of the same protein might disperse over
around 1950 [15]. The defects of the packing gels were not stable as the entire gel.
rigid silicas to pressure. Since the hydrophilic polymers showed
swelling properties, they were named as soft gels, for example, agarose Considering the limitations of gel-based proteomics for protein
gel was classified as one of soft gels. identification, liquid chromatography provides the possibility for
separation with high flexibility and selectivity. Different substances can
Since the disadvantage of LC packing materials were intrinsic, it is be monitored and separated according to differences of quantities by
difficult for diffusion in the pores, therefore, around 1980, small size of adjusting the column size. The sensitivity can be improved by using
silicas that with relative large pores were used as packing materials to column with reduced internal diameter. Furthermore, high separation
achieve more efficient and faster separation than before [16]. Other capacities can be achieved by using multidimensional LC. It is difficult

J Chromatogr Sep Tech, an open access journal Volume 8 • Issue 2 • 1000358

ISSN: 2157-7064
Citation: Zhu B, Chen YY (2017) Development and Application of Liquid Chromatography in Life Sciences. J Chromatogr Sep Tech 8: 358. doi:
10.4172/2157-7064.1000358

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to quantify the abundance of proteins with extreme abundance acts as equipment for identification, while liquid chromatography is
differences by LC-based approaches [22]. Therefore, the combination considered as a device for sample clean-up. Currently, LC with
of suitable columns chosen and processing of multidimensional LC, reversed-phase silica columns is dominantly preferred for
allows us to trace a number of components in a complex sample. pharmaceutical analysis [24].
Normally, some well-known proteins in biofluids that have high
abundance, such as albumin, increase the difficulty for detection of Metabolomics analysis
low-abundance proteins and peptides. Therefore, it is necessary to kick Metabolomics aims in identification and quantitation of metabolites
these proteins out before digestion of samples followed by separation. smaller than 1kDa in the metabolome [25]. Metabolomics is also
One of the most highly specific approaches is to load the total protein considered as the process of detecting and measuring metabolites
sample into affinity column that has specific adsorption of these high changes responding to different status, such as diseases, environmental
abundance proteins, such as albumin. However, the specificity of the or genetic perturbation etc. It was reported that at least 200000
affinity column requires highly concentrated samples. Furthermore, metabolic compounds in plants, which acted as a pool for searching
antibody-based affinity column also can be applied for reducing the new products for medical and nutritional usage [26]. Importantly, the
abundance of albumin in biofluids. However, the affinity development of human comparative metabolomics triggers greatly
chromatography can be only used to reduce the amount of known improved diagnostic power and enables individualized treatment for
high-abundance proteins, and is not useful for handling low- disease [27].
abundance targeted proteins.
Traditional approaches for metabolomics investigation have their
It is well known that sample clean-up is essential for further analysis limitations due to the large differences of abundance for different
by LC system. To obtain highly reproducible data, on-line sample metabolites. LC system is compatible for analysis, for it’s multiple
clean-up procedures are mandatory. The development of column with selectivity. The improvements of mass spectrometry equipment further
restricted access materials (RAMs) packed that has hydrophobic promote the development of metabolomics. As the advancements of
interior but coated by some hydrophilic material [23]. Therefore, only highly selective columns and MS technique, coupled LC to MS is
the small molecules can pass the column, however, the large molecules becoming the dominant technique for quantitative and qualitative
have to be excluded from pore by a size-excluding mechanism. analysis in metabolomics.
Furthermore, RAM columns that have strong cation-exchange groups
on surface, which facilitate extraction of positively charged peptides LC in clinical analysis
from biofluids. At present, two dimensional LC system is dominantly
applied in biological research, especially for separation of endogenous The objective of clinical analysis is mainly for detection, extraction,
peptides from biofluids. Normally, strong cation-exchange enrichment of targeted components in biofluids. Removing target
chromatography can increase mass load capacity, and it is considered pathogens and toxins out of biofluids is the major goal of therapy.
as a primary separation technique. Normally, the reversed-phase LC is Although the wide application of LC technique for analytical purposes
applied as secondary separation technique due to its function of salts- is still a long way for its selective cleans of pathogenic and toxic
clean. More importantly, it is compatible to couple to mass compounds in biofluids. There are several important parameters which
spectrometry equipment. Actually, the particular benefits of this need to be considered for application of LC columns, especially for
multidimensional LC system lay on its high reproducibility and high packing materials, including parameters which refer to the specificity,
separation capability. capacity, particle size, toxicity, ligand stability etc. Safety and efficiency
are the priority for LC clinical application. Furthermore, the
In summary, LC in new era has some features that are essential for
characteristics of column surface should be biocompatible to in vivo
application in life sciences. Firstly, LC system can do the cleanup for
cellular environment to reduce cellular response to minimal, either for
selective sample; even quantify its content in an automated mode.
acute or chronic treatment.
Secondary, LC system has a number of phases for selection to analyze
individual samples, such as HIC, HILIC, SEC, AC. Thirdly, Although it is still a long distance for LC technique to be widely
multidimensional process can be integrated into LC system, especially applied for clinical application, highly selective chromatographic
valuable for high concentrated samples. packing materials can be used for treatment of a number of diseases.
We have confidence to believe that LC-based technology shows great
Application of Liquid Chromatography in Life Science potential for clinical applications as more and more scientists equally
have both medical skills and skills in separation sciences.
Pharmaceutical analysis
Proteomics analysis
Pharmaceutical analysis is important for the discovery and
development of drugs, also essential for drug quality control. Such Liquid chromatography which is currently a widespread application
analysis cannot be performed without the development of LC, in proteomics study is normally coupled to MS detection after protein
especially HPLC technique. There are high requirements of digestion. The sample cleanup is priority that can affect the data
pharmaceutical analysis, including higher efficiency and sensitivity, quality. At present, various clean-up methods prior to sample
and faster analysis. Furthermore, the biggest difficulty for separation have been rapidly developed with the assist of
pharmaceutical analysis is low abundance of partial active components multidimensional LC technique, however, it still needs further
in a complex mixture. Therefore, LC is a better choice than gel-base development for application. LC technique is powerful for proteomics
proteomics for detection of low abundance components which can also research, its application in proteomics can be considered as milestone
relatively reduce the complexity of mixtures. After separation of due to the skip of gel-based digestion, which also boost the perspective
different components in biofluids by LC, mass spectrometry usually of biological industry. Many biological companies have achieved

J Chromatogr Sep Tech, an open access journal Volume 8 • Issue 2 • 1000358

ISSN: 2157-7064
Citation: Zhu B, Chen YY (2017) Development and Application of Liquid Chromatography in Life Sciences. J Chromatogr Sep Tech 8: 358. doi:
10.4172/2157-7064.1000358

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benefits by using LC in proteomics application, for example, diagnostic 10. Neue UD (1997) HPLC columns-Theory, Technology and Practice.
and pharmaceutical companies. Wiley-VCH, New York, pp: 235-250.
11. Mazzeo JR, Neue UD, Kele M, Plumb RS (2005) A new separation
technique takes advantage advantage of sub-2-µm porous particles. Anal
Conclusion and Perspectives Chem 77: 460-465.
The history of LC development is over a century since 1903, LC 12. Rapp E, Tallarek U (2003) Liquid flow in capillary
technique shows tremendous improvements during the past century. (electro)chromatography: Generation and control of micro- and
nanoliter volumes. J Sep Sci 26: 453-470.
Meanwhile, the advantages of LC mentioned above cannot be easily
replaced by other methods for sample isolation and purification. 13. Mayer-Rosenkranz J, Kutter JP, Neue UD, Grum bach ES, Kele M, et al.
(2006) D. Sievers in Miniaturization in HPLC made to measure: A
Especially, the benefit of analytical LC for small molecules analysis is practical handbook for optimization (Kromidas S). Wiley-VCH,
very significant, including applications involved in analysis of Weinheim, pp: 467-505.
pharmaceutical, food, environmental materials etc. At present, it is well 14. Niessen WMA (2006) Liquid Chromatography-Mass Spectrometry. In:
known that the application of HPLC for small molecules analysis in Taylor and Francis Group, LLC, pp: 600.
pharmaceutical and metabolomics has reached a mature level. 15. Hjerten S, Issaq HJ (2000) A century of separation science. Marcel
Dekker, New York, pp: 421-452.
However, LC chromatography technique in life sciences still needs
to keep developing. Sometimes, LC deals with some unstable target 16. Regnier FE, Issaq HJ (2002) A century of separation science. Marcel
Dekker, New York, pp: 265-276.
molecules, worse still, these unstable target molecules might have
extreme wide range of concentration. Therefore, the progress in 17. Liapis AI, Xu Y, Grosser OK, Tongta A (1995) Perfusion chromatography.
The effects of intraparticle convective velocity and microscope size on
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J Chromatogr Sep Tech, an open access journal Volume 8 • Issue 2 • 1000358

ISSN: 2157-7064
Comparison of Gas Chromatography and High Pressure
Liquid Chromatography

HPLC GC
Separate, identify and Separating and
quantify compounds analyzing compounds
Method
depending on the that can be
interactions between the vaporizedwithout
stationary phase, the decomposition
molecules being analyzed
and the solvent(s) used.
Acronym HPLC
GC
organic molecules
Analytes Organic, Inorganic
biomolecules ionspolymers Must be volatile
High pressure pumps
Sample Carrier Gas (i.e Hellum,
mobility Nitrogen)
method
UV/VIS Photodiode FID - Flame
Optional array (PDA) Mass Ionization TCD –
detectors spectrometry based Thermal
Conductivity
Selective
detectors:
GC/MS, ECD, FPD,
PID
medical (e.g. detecting Hydrocarbons
vitamin D levels in blood separations from
serum), legal (e.g. detecting oil/plants, Oil quality
Applications
performance enhancement evaluation, Solid
drugs in urine), research drug dose (pre-
(e.g. separating the consumption form)
components of a complex Identification and
 biological sample, quantification, Arson
manufacturing (e.g. during investigation, Paint
the production process of chip analysis,
pharmaceutical and Toxicology cases,
biological products) Quantify various
biological specimens
and crime-scene
evidence

Parts
diagram
Differences between of LC and GC
The key differences between liquid and gas chromatography are
tabulated below.

S.N Liquid Chromatography Gas Chromatography

o.

1. Mobile phase is a liquid Mobile phase is a gas

2. Separation is based on Separation is primarily based

interaction of solute with the on the boiling points of solute
chromatography medium molecules

3. Can be performed in a sheet or Can be carried out only in a

a column column

4. Can be used to separate any Can be applied in the

soluble compound, e.g. amino separation of volatile
acids, proteins, drugs, nucleic compounds and gaseous
acids, lipids, antioxidants, mixtures
carbohydrates, and natural and
artificial polymers

5. Usually carried out at room Performed at higher

temperature so heat-sensitive temperatures so thermally
compounds can be safely labile substances might get
analyzed using the technique denatured

6. Solute retention here is based Separation is based on the

on the interaction of solutes with boiling points of the solute
the mobile and stationary molecules so it is not very
 phases so it is easy to optimize flexible in terms of optimizing
results separation

7. This is a relatively slower The analysis is faster and

technique usually measured in minutes,
although it can take as little as
a couple of seconds

8. Usually gives a greater peak or Provides comparatively better

broader band resulting in lower resolution
resolution

9. Uses polar solvents like water or Uses any solvent that vaporizes
methanol