High-performance liquid chromatography

sometimes referred to as high-pressure liquid chromatography, HPLC, is a

chromatographic technique that can separate a mixture of compounds and is used in
biochemistry and analytical chemistry to identify, quantify and purify the individual
components of the mixture. High performance liquid chromatography is basically a
highly improved form of column chromatography. Instead of a solvent being allowed to
drip through a column under gravity, it is forced through under high pressures of up to
400 atmospheres. That makes it much faster. It is a form of liquid chromatography that
utilizes smaller column size, smaller media inside the column, and higher mobile phase
pressures.

The heart of a HPLC system is the column. The column contains the particles that contain
the stationary phase. The mobile phase is pumped through the column by a pump. The
mixture to be separated is injected into the flowing mobile phase by an injector.

When the mobile phase passes through the column that contains the stationary phase, the
molecules that adsorbs most to the stationary phase migrates slowest through the column.
When the mobile phase has passed through the column it enters into the detector that
detects the different molecules as they have pass through it. A signal goes from the
detector to a printer that presents the separation graphically.

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HPLC typically utilizes different types of stationary phases, a pump that moves the
mobile phase(s) and analyte through the column, and a detector to provide a characteristic
retention time for the analyte. Analyte retention time varies depending on the strength of
its interactions with the stationary phase, the ratio/composition of solvent(s) used, and the
flow rate of the mobile phase.

With HPLC, a pump (rather than gravity) provides the higher pressure required to move
the mobile phase and analyte through the densely packed column. The increased density
arises from smaller particle sizes. This allows for a better separation on columns of
shorter length when compared to ordinary column chromatography.

Operation
The sample to be analyzed is introduced, in small volumes, into the stream of mobile
phase. The solution moved through the column is slowed by specific chemical or physical
interactions with the stationary phase present within the column. The velocity of the
solution depends on the nature of the sample and on the compositions of the stationary
(column) phase. The time at which a specific sample elutes (comes out of the end of the
column) is called the retention time; the retention time under particular conditions is
considered an identifying characteristic of a given sample. The use of smaller particle
size column packing (which creates higher backpressure) increases the linear velocity
giving the components less time to diffuse within the column, improving the
chromatogram resolution. Common solvents used include any miscible combination of
water or various organic liquids (the most common are methanol and acetonitrile). Water
may contain buffers or salts to assist in the separation of the sample components, or
compounds such as trifluoroacetic acid which acts as an ion pairing agent.

The choice of solvents, additives and gradient depend on the nature of the column and
sample.

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Types of HPLC
Partition chromatography
. The partition coefficient principle has been applied in paper chromatography, thin layer
chromatography, gas phase and liquid-liquid applications. Molecules equilibrate
(partition) between a liquid stationary phase and the eluent. HPLC has been used
historically on unbonded silica or alumina supports. Each works effectively for separating
analytes by relative polar differences.

The polar analytes diffuse into a stationary water layer associated with the polar
stationary phase and are thus retained. Retention strengths increase with increased analyte
polarity, and the interaction between the polar analyte and the polar stationary phase
(relative to the mobile phase) increases the elution time. The interaction strength depends
on the functional groups in the analyte molecule which promote partitioning but can also
include coulombic (electrostatic) interaction and hydrogen donor capability.
Use of more polar solvents in the mobile phase will decrease the retention time of the
analytes, whereas more hydrophobic solvents tend to increase retention times.

Normal-phase chromatography

Also known as normal-phase HPLC (NP-HPLC), or adsorption chromatography, this

method separates analytes based on adsorption to a stationary surface and by polarity. It
was one of the first kinds of HPLC that chemists developed. NP-HPLC uses a polar
stationary phase and a non-polar, non-aqueous mobile phase, and works effectively for
separating analytes readily soluble in non-polar solvents. The analyte associates with and
is retained by the polar stationary phase. Adsorption strengths increase with increased
analyte polarity, and the interaction between the polar analyte and the polar stationary
phase (relative to the mobile phase) increases the elution time. The interaction strength
depends not only on the functional groups in the analyte molecule, but also on steric
factors. The effect of sterics on interaction strength allows this method to resolve
(separate) structural isomers.

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The use of more polar solvents in the mobile phase will decrease the retention time of the
analytes, whereas more hydrophobic solvents tend to increase retention times. Very polar
solvents in a mixture tend to deactivate the stationary phase by creating a stationary
bound water layer on the stationary phase surface. This behavior is somewhat peculiar to
normal phase because it is most purely an adsorptive mechanism (the interactions are
with a hard surface rather than a soft layer on a surface).

Reversed-phase chromatography (RPC)

Reversed phase HPLC (RP-HPLC or RPC) has a non-polar stationary phase and an
aqueous, moderately polar mobile phase. One common stationary phase is a silica which
has been treated with RMe2SiCl, where R is a straight chain alkyl group such as C18H37
or C8H17. With these stationary phases, retention time is longer for molecules which are
less polar, while polar molecules elute more readily. An investigator can increase
retention time by adding more water to the mobile phase; thereby making the affinity of
the hydrophobic analyte for the hydrophobic stationary phase stronger relative to the now
more hydrophilic mobile phase. Similarly, an investigator can decrease retention time by
adding more organic solvent to the eluent. RPC is so commonly used that it is often
incorrectly referred to as "HPLC" without further specification. The pharmaceutical
industry regularly employs RPC to qualify drugs before their release.

RPC operates on the principle of hydrophobic forces, which originate from the high
symmetry in the dipolar water structure and play the most important role in all processes
in life science. RPC allows the measurement of these interactive forces. The binding of
the analyte to the stationary phase is proportional to the contact surface area around the
non-polar segment of the analyte molecule upon association with the ligand in the
aqueous eluent. The retention can be decreased by adding a less polar solvent (methanol,
acetonitrile) into the mobile phase to reduce the surface tension of water.

Structural properties of the analyte molecule play an important role in its retention
characteristics. In general, an analyte with a larger hydrophobic surface area (C-H, C-C,
and generally non-polar atomic bonds, such as S-S and others) results in a longer
retention time because it increases the molecule's non-polar surface area, which is non-

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interacting with the water structure. On the other hand, polar groups, such as -OH, -NH2,
COO– or -NH3+ reduce retention as they are well integrated into water. Very large
molecules, however, can result in an incomplete interaction between the large analyte
surface and the ligand's alkyl chains and can have problems entering the pores of the
stationary phase.

Retention time increases with hydrophobic (non-polar) surface area. Branched chain
compounds elute more rapidly than their corresponding linear isomers because the
overall surface area is decreased. Similarly, organic compounds with single C-C-bonds
elute later than those with a C=C or C-C-triple bond, as the double or triple bond is
shorter than a single C-C-bond.

Another important component is the influence of the pH since this can change the
hydrophobicity of the analyte. For this reason most methods use a buffering agent, such
as sodium phosphate, to control the pH. The buffers serve multiple purposes: they control
pH, neutralize the charge on any residual exposed silica on the stationary phase and act as
ion pairing agents to neutralize charge on the analyte. Ammonium formate is commonly
added in mass spectrometry to improve detection of certain analytes by the formation of
ammonium adducts. A volatile organic acid such as acetic acid, or most commonly
formic acid, is often added to the mobile phase if mass spectrometry is used to analyze
the column eluent. Trifluoroacetic acid is used infrequently in mass spectrometry
applications due to its persistence in the detector and solvent delivery system, but can be
effective in improving retention of analytes such as carboxylic acids in applications
utilizing other detectors, as it is one of the strongest organic acids. The effects of acids
and buffers vary by application but generally improve the chromatography.

Reversed phase columns are quite difficult to damage compared with normal silica
columns; however, many reversed phase columns consist of alkyl derivatized silica
particles and should never be used with aqueous bases as these will destroy the
underlying silica particle. They can be used with aqueous acid, but the column should not
be exposed to the acid for too long, as it can corrode the metal parts of the HPLC
equipment. RP-HPLC columns should be flushed with clean solvent after use to remove

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residual acids or buffers, and stored in an appropriate composition of solvent. The metal
content of HPLC columns must be kept low if the best possible ability to separate
substances is to be retained.

Displacement chromatography

The basic principle of displacement chromatography is: A molecule with a high affinity
for the chromatography matrix (the displacer) will compete effectively for binding sites,
and thus displace all molecules with lesser affinities. There are distinct differences
between displacement and elution chromatography. In elution mode, the speed at which
any component of a mixture travels down the column in depends on many factors. But for
two substances to travel at different speeds, and thereby be resolved, there must be
substantial differences in some interaction between the biomolecules and the
chromatography matrix. Displacement chromatography has advantages over elution
chromatography in that components are resolved into consecutive zones of pure
substances rather than “peaks”.

Size-exclusion chromatography

Size-exclusion chromatography (SEC), also known as gel permeation chromatography or

gel filtration chromatography, separates particles on the basis of size. It is generally a
low resolution chromatography and thus it is often reserved for the final, "polishing" step
of a purification. It is also useful for determining the tertiary structure and quaternary
structure of purified proteins. SEC is used primarily for the analysis of large molecules
such as proteins or polymers. SEC works by trapping these smaller molecules in the
pores of a particle. The larger molecules simply pass by the pores as they are too large to
enter the pores. Larger molecules therefore flow through the column quicker than smaller
molecules, that is, the smaller the molecule, the longer the retention time.

This technique is widely used for the molecular weight determination of polysaccharides.
SEC is the official technique (suggested by European pharmacopeia) for the molecular
weight comparison of different commercially available low-molecular weight heparins.

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Ion-exchange chromatography

In ion-exchange chromatography (IC), retention is based on the attraction between solute

ions and charged sites bound to the stationary phase. Ions of the same charge are
excluded. Types of ion exchangers include:

 Polystyrene resins – These allow cross linkage which increases the stability of the
chain. Higher cross linkage reduces swerving, which increases the equilibration time
and ultimately improves selectivity.
 Cellulose and dextran ion exchangers (gels) – These possess larger pore sizes and
low charge densities making them suitable for protein separation.
 Controlled-pore glass or porous silica

In general, ion exchangers favor the binding of ions of higher charge and smaller radius.

An increase in counter ion (with respect to the functional groups in resins) concentration
reduces the retention time. An increase in pH reduces the retention time in cation
exchange while a decrease in pH reduces the retention time in anion exchange.

This form of chromatography is widely used in the following applications: water

purification, preconcentration of trace components, ligand-exchange chromatography,
ion-exchange chromatography of proteins, high-pH anion-exchange chromatography of
carbohydrates and oligosaccharides, and others.

Bioaffinity chromatography

This chromatographic process relies on the property of biologically active substances to

form stable, specific, and reversible complexes. The formation of these complexes
involves the participation of common molecular forces such as the Van der Waals
interaction, electrostatic interaction, dipole-dipole interaction, hydrophobic interaction,
and the hydrogen bond. An efficient, biospecific bond is formed by a simultaneous and
concerted action of several of these forces in the complementary binding sites.

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Aqueous normal-phase chromatography

Aqueous normal-phase chromatography (ANP) is a chromatographic technique which

encompasses the mobile phase region between reversed-phase chromatography (RP) and
organic normal phase chromatography (ONP). This technique is used to achieve unique
selectivity for hydrophilic compounds, showing normal phase elution using reverse-phase
solvents.

Isocratic flow and gradient elution

A separation in which the mobile phase composition remains constant throughout the
procedure is termed isocratic (meaning constant composition). The word was coined by
Csaba Horvath who was one of the pioneers of HPLC.

The mobile phase composition does not have to remain constant. A separation in which
the mobile phase composition is changed during the separation process is described as a
gradient elution. One example is a gradient starting at 10% methanol and ending at 90%
methanol after 20 minutes. The two components of the mobile phase are typically termed
"A" and "B"; A is the "weak" solvent which allows the solute to elute only slowly, while
B is the "strong" solvent which rapidly elutes the solutes from the column. In reverse-
phase chromatography, solvent A is often water or an aqueous buffer, while B is an
organic solvent miscible with water, such as acetonitrile, methanol, or isopropanol.

In isocratic elution, peak width increases with retention time linearly according to the
equation for N, the number of theoretical plates. This leads to the disadvantage that late-
eluting peaks get very flat and broad. Their shape and width may keep them from being
recognized as peaks.

Gradient elution decreases the retention of the later-eluting components so that they elute
faster, giving narrower (and taller) peaks for most components. This also improves the
peak shape for tailed peaks, as the increasing concentration of the organic eluent pushes
the tailing part of a peak forward. This also increases the peak height (the peak looks
"sharper"), which is important in trace analysis. The gradient program may include

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sudden "step" increases in the percentage of the organic component, or different slopes at
different times – all according to the desire for optimum separation in minimum time.

In isocratic elution, the selectivity does not change if the column dimensions (length and
inner diameter) change – that is, the peaks elute in the same order. In gradient elution, the
elution order may change as the dimensions or flow rate change.

Parameters

Internal diameter

The internal diameter (ID) of an HPLC column is an important parameter that influences
the detection sensitivity and separation selectivity in gradient elution. It also determines
the quantity of analyte that can be loaded onto the column. Larger columns are usually
seen in industrial applications, such as the purification of a drug product for later use.
Low-ID columns have improved sensitivity and lower solvent consumption at the
expense of loading capacity.

 Larger ID columns (over 10 mm) are used to purify usable amounts of material
because of their large loading capacity.
 Analytical scale columns (4.6 mm) have been the most common type of columns,
though smaller columns are rapidly gaining in popularity. They are used in traditional
quantitative analysis of samples and often use a UV-Vis absorbance detector.
 Narrow-bore columns (1–2 mm) are used for applications when more sensitivity
is desired either with special UV-vis detectors, fluorescence detection or with other
detection methods like liquid chromatography-mass spectrometry
 Capillary columns (under 0.3 mm) are used almost exclusively with alternative
detection means such as mass spectrometry. They are usually made from fused silica
capillaries, rather than the stainless steel tubing that larger columns employ.

Particle size

Most traditional HPLC is performed with the stationary phase attached to the outside of
small spherical silica particles (very small beads). These particles come in a variety of

9
sizes with 5 μm beads being the most common. Smaller particles generally provide more
surface area and better separations, but the pressure required for optimum linear velocity
increases by the inverse of the particle diameter squared.

This means that changing to particles that are half as big, keeping the size of the column
the same, will double the performance, but increase the required pressure by a factor of
four. Larger particles are used in preparative HPLC (column diameters 5 cm up to
>30 cm) and for non-HPLC applications such as solid-phase extraction.

Pore size

Many stationary phases are porous to provide greater surface area. Small pores provide
greater surface area while larger pore size has better kinetics, especially for larger
analytes. For example, a protein which is only slightly smaller than a pore might enter the
pore but does not easily leave once inside.

Pump pressure

Pumps vary in pressure capacity, but their performance is measured on their ability to
yield a consistent and reproducible flow rate. Pressure may reach as high as 40 MPa
(6000 lbf/in2), or about 400 atmospheres. Modern HPLC systems have been improved to
work at much higher pressures, and therefore are able to use much smaller particle sizes
in the columns (<2 μm). These "Ultra High Performance Liquid Chromatography"
systems or RSLC/UHPLCs can work at up to 100 MPa (15,000 lbf/in²), or about
1000 atmospheres.

Stationary Phases (Adsorbents)

HPLC separations are based on the surface interactions, and depend on the types of the
adsorption sites. Modern HPLC adsorbents are the small rigid porous particles with high
surface area.
Main adsorbent parameters are:
 Particle size: 3 to 10 μm
 Particle size distribution: as narrow as possible, usually within 10% of the mean;

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Depending on the type of the ligand attached to the surface, the adsorbent could
be normal phase (-OH, -NH2), or reversed-phase (C5, C8, C 18 CN, NH2 ), and even
anion (CH2NR3 +OH-), or cation (R-SO3 -H+) exchangers.

Instrumentation HPLC system

HPLC instrumentation includes a pump, injector, column, detector and data system. The
heart of the system is the column where separation occurs. Since the stationary phase is
composed of micrometer size porous particles, a high pressure pump is required to move
the mobile phase through the column. The chromatographic process begins by injecting
the solute onto the top of the column. Separation of components occurs as the analytes
and mobile phase are pumped through the column. Eventually, each component elutes
from the column as a narrow band (or peak) on the recorder.
Detection of the eluting components is important, and this can be either selective or
universal, depending upon the detector used. The response of the detector to each
component is displayed on a chart recorder or computer screen and is known as a
chromatogram. To collect, store and analyze the chromatographic data, computer,
integrator, and other data processing equipment are frequently used.
Mobile phases
In HPLC, the type and composition of the eluent is one of the variables influencing the
separation. Despite the large variety of solvents used in HPLC, there are several common
properties:
 Purity
 Detector compatibility
 Solubility of the sample
 Low viscosity
 Chemical inertness
For normal phase mode, solvents are mainly non polar; for reversed-phase, eluents are
usually a mixture of water with some polar organic solvent such as acetonitrile or
methanol.

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Size-exclusion HPLC has special requirements. SEC eluents have to dissolve polymers,
but the most important is that SEC eluent has to suppress possible interactions of the
sample molecule with the surface of the packing material.

Functional description of the instrument

 Mobile phase reservoir, filtering
 Pump
 Injector
 Column
 Detector
 Data system

Mobile phase reservoir, filtering

The most common type of solvent reservoir is a glass bottle. Most of the manufacturers
supply these bottles with special caps, Teflon tubing and filters to connect to the pump
inlet and to the purge gas (helium) used to remove dissolved air. Helium purging and
storage of the solvent under helium is not sufficient for degassing aqueous solvents. It is
useful to apply a vacuum for 5-10 min. and then keep the solvent under a helium
atmosphere.

Retention time
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The time taken for a particular compound to travel through the column to the detector is
known as its retention time. This time is measured from the time at which the sample is
injected to the point at which the display shows a maximum peak height for that
compound.

Different compounds have different retention times. For a particular compound, the
retention time will vary depending on:

 the pressure used (because that affects the flow rate of the solvent)
 the nature of the stationary phase (not only what material it is made of, but also
particle size)
 the exact composition of the solvent
 the temperature of the column

That means that conditions have to be carefully controlled if you are using retention times
as a way of identifying compounds.

Pump

High pressure pumps are needed to force solvents through packed stationary phase beds.
Smaller bed particles require higher pressures. There are many advantages to using
smaller particles, but they may not be essential for all separations.
The most important advantages are: higher resolution, faster analyses, and increased
sample load capacity. However, only the most demanding separations require these
advances in significant amounts. Many separation problems can be resolved with larger
particle packing that requires less pressure.
Flow rate stability is another important pump feature that distinguishes pumps.
Very stable flow rates are usually not essential for analytical chromatography.
However, if the user plans to use a system in size exclusion mode, then there must be a
pump which provides an extremely stable flow rate.
An additional feature found on the more elaborate pumps is external electronic control.
Although it adds to the expense of the pump, external electronic control is a very

13
desirable feature when automation or electronically controlled gradients are to be run.
Alternatively, this becomes an undesirable feature (since it is an unnecessary expense)
when using isocratic methods. The degree of flow control also varies with pump expense.

More expensive pumps include such state of-the-art technology as electronic feedback
and multi headed configurations.
Modern pumps have the following parameters:
 Flow rate range: 0.01 to 10 mL/min
 Flow rate stability: not more than 1%
 For SEC flow rate stability should be less than 0.2%
 Maximum pressure: up to 400 bar
 It is desirable to have an integrated degassing system, either helium purging,
or membrane filtering.
Types of Pumps
Reciprocating pumps are by far the most widely used and practically 100% of the
pumps used in commercial HPLC equipment are of the reciprocating type.
In reciprocating pumps, a motor driven reciprocating piston controls the flow of
mobile phase with the help of two ball check valves that opens and closes with the
piston movement. The flow is thus not continuous and damping of flow is necessary.

14
 This is accomplished using pulse dampers which are a long coiled capillary tube.
Displacement pumps, on the other hand, is composed of a one directional motor
driven plunger that pushes the mobile phase present in a syringe like chamber. The
volume of displacement pumps is limited which lacks convenience. A constant flow
rate is usually obtained with syringe like pumps.
Pneumatic pumps are the simplest where the mobile phase is pushed out of the
mobile phase container by the pressure of a pressurized gas. The flow is dependent on
the back pressure of the column and usually the flow is limited to pressures below
2000 psi.
Injector
Sample introduction can be accomplished in various ways. The simplest method is to use
an injection valve. In more sophisticated LC systems, automatic sampling devices are
incorporated where the sample is introduced with the help of auto samplers and
microprocessors.
In liquid chromatography, liquid samples may be injected directly and solid samples need
only be dissolved in an appropriate solvent. The solvent need not be the mobile phase,
but frequently it is judiciously chosen to avoid detector interference, column/component
interference, and loss in efficiency or all of these.
It is always best to remove particles from the sample by filtering over a 5 μm filter, or
centrifuging, since continuous injections of particulate material will eventually cause
blockages in injection devices or columns.
Sample sizes may vary widely. The availability of highly sensitive detectors frequently
allows use of the small samples which yield the highest column performance. Typical
sample mass with 4.6 mm ID columns range from the nano gram level up to about 2 mg
diluted in 20 ml of solvent. In general, it will be noted that much less sample preparation
is required in LC than in GC since unwanted or interfering compounds, or both, may
often be extracted, or eliminated, by selective detection.
This sample is injected via a manual injector or an auto sampler. Each type is equipped
with six-port valves, so that a sample can be injected into the flow path at continuous
pressure. For the manual injector, the knob is manually operated to deliver the sample to
the column,

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1) The knob is set at the "LOAD" position for sample injection, as shown in the left
image. Using a micro syringe, the sample can be injected into the sample loop, which is
separated from the flow path.
2) The knob is turned to the "INJECT" position. The eluent travels through the loop
from the pump then delivers the sample to the column. The auto sampler can perform
similar work automatically, enabling unmanned continuous operation.

Column
A column is selected to suit both the sample and the purpose of separation. The column
oven is used to maintain a constant column temperature. If the column temperature were
allowed to vary during qualitative or quantitative analysis, the elution time of the
components would change, so that an accurate analysis could not be performed. An
analysis temperature between 25 and 50 C0 is often selected. Typical HPLC columns are
5, 10, 15 and 25 cm in length and are filled with small diameter (3, 5 or 10 μm) particles.
The internal diameter of the columns is usually 4.6 mm; this is considered the best
compromise for sample capacity, mobile phase consumption, speed and resolution.
However , if pure substances are to be collected (preparative scale), then larger diameter
columns may be needed. Packing the column tubing with small diameter particles
requires high skill and specialized equipment.
Types of columns in HPLC:

•Analytical [internal diameter (Internal diameter I.D) 1.0 -4.6-mm; lengths 15 –250 mm]

•Preparative (I.D. > 4.6 mm; lengths 50 –250 mm)

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•Capillary (I.D 0.1 -1.0 mm; various lengths)

•Nano (I.D. < 0.1 mm or sometimes stated as < 100 μm)

Materials of construction for the tubing
•Stainless steel (the most popular; gives high pressure capabilities)
•Glass (mostly for bio molecules)
•PEEK polymer (biocompatible and chemically inert to most solvents)

Detector
Today, optical detectors are used most frequently in liquid chromatographic systems.
These detectors pass a beam of light through the flowing column effluent as it passes
through a low volume (~ 10 μl) flow cell. The variations in light intensity caused by UV
absorption, fluorescence emission or change in refractive index, from the sample
components passing through the cell, are monitored as changes in the output voltage.
These voltage changes are recorded on a strip chart recorder and frequently are fed into a
computer to provide retention time and peak area data.
Detectors in common use include,
 UV Detector The most commonly used detector in LC is the ultraviolet absorption
detector. A variable wavelength detector of this type, capable of monitoring from
190 to 400 nm, will be found suitable for the detection of the majority samples.
The light source is a D2 lamp. This detector is used mainly to detect components
having an absorption wavelength of 400 nm or less in the ultraviolet region.

 UV-VIS Detector A D2 lamp and a W lamp are used as the light source. This
detector is effective in the detection of coloring components such as dyes and
stains because of coverage of the visible light region.

17
  Radio chemical Detector it involves the use of the radio labeled material usually
tritium (H3) or carbon -14(C14). They have sensitivity limit of 10-9 to 10-10gm/ml.
 Fluorescence (FL) Detector these measure the ability of a compound to absorb
then re-emit the light at given wavelengths. Each compound has a characteristic
fluorescence. Fluorescent substances can be detected specifically with high
sensitivity. They have sensitivity limit of 10-9 to 10-11gm/ml.
 Differential Refractive index (RI) Detector Change in the refractive index is
detected. Components absorbing no ultraviolet light can also be detected despite
low sensitivity. The RI detector is universal but also the less sensitive one.
 Conductivity Detector Mainly inorganic ions are detected by monitoring the
conductivity.
 Electrochemical Detectors (EC) they measure compounds that undergo oxidation
or reduction reactions. These are accomplished by measuring the gain or loss of
the electron from migrating samples. They have sensitivity of 10-12 to 10-13gm/ml.
 Mass Spectroscopy Detectors the sample compound or molecule is ionized, it is
passed through mass analyzer and then the ion current is detected. They have
detection limit of 10-8 to 10-10gm/ml.
Nuclear Magnetic Resonance (NMR) Detectors, Light Scattering (LS) Detectors,
evaporative light scattering detector (ELSD), charged Aerosol Detector (CAD), and
Laser- based Scattering Detectors are also used for detection.

Data system
Since the detector signal is electronic, using modern data collection techniques can aid
the signal analysis. In addition, some systems can store data in a retrievable form for
highly sophisticated computer analysis at a later time.
The main goal in using electronic data systems is to increase analysis accuracy and
precision, while reducing operator attention. There are several types of data systems, each
differing in terms of available features. In routine analysis, where no automation (in
terms of data management or process control) is needed, a pre-programmed computing
integrator may be sufficient. If higher control levels are desired, a more intelligent device
is necessary, such as a data station or minicomputer. The advantages of intelligent

18
processors in chromatographs are found in several areas. First, additional automation
options become easier to implement. Secondly, complex data analysis becomes more
feasible. These analysis options include such features as run parameter optimization and
deconvolution (i.e. resolution) of overlapping peaks. Finally, software safeguards can be
designed to reduce accidental misuse of the system.

A flow scheme for HPLC

Applications

HPLC is one of the most widely applied analytical separation technique. A

few application areas have been broken out below.

Pharmaceutical

i. Tablet dissolution of pharmaceutical dosages.

ii. Shelf life determination pharmaceutical products.
iii. Identification of counterfeit drug products.
iv. Pharmaceutical quality control.
v. Chemical analysis which is linked to the pharmaceuticals.
vi. It is used in the determination of the preservatives and anti-oxidants.

19
vii. It is used in the analysis of vitamins.

Environmental

i.Phenol in drinking water.

ii. Identification of diphenhydramine in sediment samples.
iii. Estrogen in coastal water- the sewage source.
iv. Toxicity of tetracyclines and their degradation products to
environmentally relevant bacteria.
v. Assessment of TNT toxicity in sediment.

Forensics

i. A mobile HPLC apparatus on site for identification and quantification of

the drugs.
ii. Identification of anabolic steroids in serum, urine, sweat and hair.
iii. Forensic analysis of textile dyes.
iv. Determination of cocaine and metabolites in meconium.
v. Quantification of psychotherapeutic drugs in human plasma.
vi. A large number of poisons and intoxications have been detected by
HPLC.

Clinical

i. Quantification of DEET (N, N-Diethyl-meta-toluamide, active ingredient

in insect repellents) in human.
ii. Urine analysis of antibiotics.
iii. Increased urinary excretion of aquaporin 2 in patients with liver cirrhosis.
iv. Detection of endogenous neuropeptides in brain extra cellular fluids.
v. Metabolic profiling of normal and diseased subjects.
vi. Study of disease processes

Food and Flavors

i. Ensuring soft drink consistency and quality.

20
ii. Analysis of vicinal diketones in beer.
iii. Sugar analysis in fruit juices.
iv. Polycyclic aromatic hydrocarbons in Brazilian vegetables and fruits.
v. Trace analysis of military high explosives in agricultural crops.

Last but not least:-

 It is also used in the separation of Lipids and steroids.

 It is used in the separation and analysis of number of amino acids, proteins
and carbohydrates
 HPLC is used in the separation of the nucleic acid in physiological fluids
e.g. in the study of regulatory effects of cyclic nucleotides.
 It is used in the separation of coal and oil products.
 It is used for the separation of the inorganic anions and cations.

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HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Muhammad Asad Saeed

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