HPLC- Definition, Principle, Parts, Types, Uses, Diagram
What is High-performance liquid chromatography (HPLC)?
High-performance liquid chromatography or commonly known as HPLC, is an analytical
technique used to separate, identify or quantify each component in a mixture.
The mixture is separated using the basic principle of column chromatography and then
identified and quantified by spectroscopy.
In the 1960s, the column chromatography LC with its low-pressure suitable glass columns
was further developed to the HPLC with its high-pressure adapted metal columns.
HPLC is thus basically a highly improved form of column liquid 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.
Principle of High-Performance Liquid Chromatography (HPLC)
● The purification takes place in a separation column between a stationary and a
mobile phase.
● The stationary phase is a granular material with very small porous particles in a
separation column.
● The mobile phase, on the other hand, is a solvent or solvent mixture which is forced
at high pressure through the separation column.
● Via a valve with a connected sample loop, i.e. a small tube or a capillary made of
stainless steel, the sample is injected into the mobile phase flow from the pump to
the separation column using a syringe.
● Subsequently, the individual components of the sample migrate through the column
at different rates because they are retained to a varying degree by interactions with
the stationary phase.
● After leaving the column, the individual substances are detected by a suitable
detector and passed on as a signal to the HPLC software on the computer.
● At the end of this operation/run, a chromatogram in the HPLC software on the
computer is obtained.
● The chromatogram allows the identification and quantification of the different
substances.
Instrumentation of High-Performance Liquid Chromatography (HPLC)
The Pump
● The development of HPLC led to the development of the pump system.
● The pump is positioned in the most upper stream of the liquid chromatography
system and generates a flow of eluent from the solvent reservoir into the system.
● High-pressure generation is a “standard” requirement of pumps besides which, it
should also to be able to provide a consistent pressure at any condition and a
controllable and reproducible flow rate.
● Most pumps used in current LC systems generate the flow by back-and-forth
motion of a motor-driven piston (reciprocating pumps). Because of this piston
motion, it produces “pulses”.
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Sample Injection System:
It is a limiting factor in the precision of HPLC measurement because of reproducibility with
which samples may be introduced onto the column packing. Insertion of the sample into the
column must be through a narrow plug so that peak broadening is minimized and the system
should have no dead volume by itself. Generally, samples are dissolved in a mobile phase
solvent to avoid solvent peak and 10 to 50 µL is introduced through micro sampling valves.
These devices form an integral part of liquid chromatography equipment having
interchangeable loops with a choice of sample size from 5 to 500 µL. The most widely used
method of introduction is based on sampling loop as shown in Fig. 8.4. It is filled by
thoroughly flushing it using a sample solution by means of a microsyringe at pressures up to
7000 psi. A rotation of the valve rotor places the sample filled loop into the high-pressure
mobile phase stream whereby the sample is sent to the column. The system can be located
within a temperature-controlled oven if handling at elevated temperatures is required. Many
HPLC instruments incorporate an auto sampler with an automatic injector that can inject
variable volumes as per requirement. In stopped flow injection method, pump is turned off till
atmospheric pressure is attained, syringe is inserted and the sample injected. The flow of
sample can be brought to zero and rapidly resumed by diverting the mobile phase using a
three-way valve placed before the injector. This method is especially very useful for very
high pressures. For best results, a two to fivefold excess of sample should be passed through
the loop to ensure that previous sample has been purged thoroughly.
Figure: Schematic of Injector valve
Column
● The separation is performed inside the column.
● The recent columns are often prepared in a stainless steel housing, instead of glass
columns.
● The packing material generally used is silica or polymer gels compared to calcium
carbonate.
The eluent used for LC varies from acidic to basic solvents.
● Most column housing is made of stainless steel since stainless is tolerant towards a
large variety of solvents.
Column is the heart of the HPLC instrument where actual separation occurs. Separation
column in HPLC is usually made of heavy wall, glass lined metal or 316 grade stainless
steel tubing, that can withstand high pressure and which is inert to the chemical
corrosion due to mobile phase. The interior of the tubing must be smooth with a
uniform bore diameter. Straight columns that can be operated in vertical position are
preferred.
Column fittings and connectors must be so designed that void volume is zero avoiding
unswept corners. Column length ranges10 to 30 cm with inner diameter of 2 to 5 mm
providing 40,000 to 60,000 plates per inch. However, shorter columns of 3 to 8 cm are
also used for fast separations but in such cases, sample size will become limited. The
length of the column may not only affect the resolution of a given separation –the
longer the column the larger number of plates but also the speed of separation. Standard
lengths vary with the manufacturer but most common values are 30, 25, 15, 12.5, 10
and 7.5 cm. It may be noted that shorter columns are described as high-speed columns.
The columns packed with the finer particles are more expensive than the standard 5 µm
packing.
Guard column: In order to increase the life of analytical column, a short guard
column, also called precolumn, is placed before the main column as shown in Fig. 8.3.
It removes contamination from the solvent. Guard column serves to saturate the mobile
phase with the stationary phase so that losses of stationary phase in the column are
minimized. However, it is essential that the composition of the guard column should be
similar to that of the analytical column but its particle size may be larger to minimize
the pressure drop.
Detector
Several features of HPLC Detectors:
(1) it should response to all compounds in the mixture ( a general detector) or it
should response with known sensitivity (a specific detector).
(2) It should not response to mobile phase.
(3) It should give linear response to solute concentration.
(4) It should be unaffected by variation in temperature and flow rate.
(5) It should not contribute to zone spreading.
Suitable detectors can be broadly divided into the following two classes:
(a) Bulk property detectors which measure the difference in some physical property of the
solute in the mobile phase compared to the mobile phase alone, e.g. refractive index and
conductivity detectors. They are generally universal in application but tend to have poor
sensitivity and limited range. Such detectors are usually affected by even small changes in the
mobile-phase composition which precludes the use of techniques such as gradient elution.
(b) Solute property detectors, e.g. spectrophotometric, fluorescence and electrochemical
detectors. These respond to a particular physical or chemical property of the solute, being
ideally independent of the mobile phase. In practice, however, complete independence of the
mobile phase is rarely achieved, but the signal discrimination is usually sufficient to permit
operation with solvent changes, e.g. gradient elution. They generally provide high sensitivity
(about 1 in 109 being attainable with UV and fluorescence detectors) and a wide linear
response range but, as a consequence of their more selective natures, more than one detector
may be required to meet the demands of an analytical problem. Some commercially available
detectors have a number of different detection modes built into a single unit, e.g. the Perkin-
Elmer '3D' system which combines UV absorption, fluorescence and conductometric
detection.
Some of the important characteristics required of a detector are the following.
a) Sensitivity, which is often expressed as the noise equivalent concentration, i.e. the solute
concentration, Cn, which produces a signal equal to the detector noise level. The lower the
value of Cn for a particular solute, the more sensitive is the detector for that solute.
b) A linear response. The linear range of a detector is the concentration range over which its
response is directly proportional to the concentration of solute. Quantitative analysis is more
difficult outside the linear range of concentration.
c) Type of response, i.e. whether the detector is universal or selective. A universal detector
will sense all the constituents of the sample, whereas a selective one will only respond to
certain components. Although the response of the detector will not be independent of the
operating conditions, e.g. column temperature or flow rate, it is advantageous if the response
does not change too much when there are small changes of these conditions.
CLASSIFICATION OF HPLC DETECTORS
● UV/VIS
- Fixed Wavelength
- Variable Wavelength
- Diode array
● Refractive index
-Deflection Detector
-Refractive Detector (Fresnel refractometer)
● Fluorescence Detector
● Electrochemical Detector
● Evaporative light scattering
● Conductivity Detector
● Mass detector
● IR detector
1 Ultraviolet/visible spectroscopic detectors
Principles
Any chemical compound could interact with the electromagnetic field. Beam of the
electromagnetic radiation passed through the detector flow-cell will experience some change
in its intensity due to this interaction. Measurement of this changes is the basis of the most
optical HPLC detectors.
Radiation absorbance depends on the radiation wavelength and the functional groups of the
chemical compound. Electromagnetic field depending on its energy (frequency) can interact
with electrons causing their excitation and transfer onto the higher energetical level, or it can
excite molecular bonds causing their vibration or rotation of the functional group. The
intensity of the beam which energy corresponds to the possible transitions will decrease while
it is passing through the flow-cell. According to the Lambert-Bear law absorbance of the
radiation is proportional to the compound concentration in the cell and the length of the cell.
Three major regions (IR, visible, and UV) are used in the spectroscopy. In liquid
chromatography, IR spectrophotometers have found only limited use. There are few
transparent polar liquids which can be used as the mobile phase. On the other hand,
spectrophotometers working in the range (200 - 600 nm) are used widely as LC detectors.
UV and visible region of the electromagnetic radiation corresponds to the excitation of the
relatively low energy electrons such as pi-electrons, or non-paired electrons of some
functional groups. For example, n-alkanes could absorb in the UV region below 180 nm. s-
electrons require high energy radiation to get excited and to show absorption of the radiation.
But any compounds which have benzene ring will show absorbance at 205-225 and 245-265
nm. The last corresponds to the excitation of conjugated p-electrons of the benzene ring.
The majority of organic compounds can be analyzed by UV/VIS detectors. Almost 70% of
published HPLC analyses were performed with UV/VIS detectors. This fact, plus the relative
ease of its operation, makes the UV detector the most useful and the most widely used LC
detector.
1.1 Fixed wavelength detectors
HPLC detectors which does not allow to change the wavelength of the radiation called fixed-
wavelength detectors. They are usually very cheap, in the nowadays you probably wont be
able to find that type of the detector on the market.
low-pressure mercury vapor lamp emit very intense light at 253.7 nm. By filtering out all
other emitted wavelengths, manufacturers have been able to utilize this 254 nm line to
provide stable, highly sensitive detectors capable of measuring subnanogram quantities of
any components which contains aromatic ring. The 254 nm was chosen since the most
intense line of mercury lamp is 254 nm, and most of UV absorbing compounds have some
absorbance at 254 nm.
1.2 Diode Array Detector
It is also a UV [Link] from the broad emission source is collimated by an achromatic
lens system so that the total light passes through the detector cell onto a holographic grating.
In this way the sample is subjected to light of all wavelengths generated by the lamp. The
dispersed light from the grating is allowed to fall onto a diode array. The array may contain
many hundreds of diodes and the output from each diode is regularly sampled by a computer
and stored on a hard disc. At the end of the run, the output from any diode can be selected
and a chromatogram produced using the UV wavelength that was falling on that particular
diode. During chromatographic development, the output of one diode is recorded in real time
producing a real time chromatogram. It is seen that by noting the time of a particular peak, a
spectrum can be obtained by recalling from memory the output of all the diodes at that
particular time.
Advantages :
Various advantages are, namely :
(a) A very selective detector which will detect only such solutes that specifically absorb
UV/visible radiation e.g., alkenes, aromatics and compounds having multiple bonds between
C, O, N and S.
(b) The mobile-phase* employed ideally must not absorb any radiation.
2 Refractive index detectors:
These bulk property detectors are based on the change of refractive index of the eluant from
the column with respect to pure mobile phase. Although they are widely used, the refractive
index detectors suffer from several disadvantages - lack of high sensitivity, lack of suitability
for gradient elution, and the need for strict temperature control (±0.001 °C) to operate at their
highest sensitivity. A pulseless pump, or a reciprocating pump equipped with a pulse
dampener, must also be employed. The effect of these limitations may to some extent be
overcome by the use of differential systems in which the column eluant is compared with a
reference flow of pure mobile phase. The detector measures the deflection of a beam of
monochromatic light by a double prism in which the reference and sample cells are separated
by a diagonal glass divide. When both cells contain solvent of the same composition, no
deflection of the light beam occurs; if, however, the composition of the column mobile phase
is changed because of the presence of a solute, then the altered refractive index causes the
beam to be deflected. The magnitude of this deflection is dependent on the concentration of
the solute in the mobile phase.
3 Fluorescence detectors
compounds (solutes) present in the mobile-phase on being passed as column effluent through
a cell irradiated with Xenon or Deuterium source first absorb UV radiation and subsequently
emit radiation of a longer wavelength in two different manners, namely
(a) Instantly-termed as ‘Fluorescence,
(b) After a time-gap-known as ‘Phosphorescence
When a molecule adsorbs light, a transition to a higher electronic state takes place and this
absorption is highly specific for the molecules concerned; radiation of a specific wavelength
or energy is only absorbed by a particular molecular structure. If electrons are raised to an
upper excited single state, due to absorption of light energy, and the excess energy is not
immediately dissipated by collision with other molecules or by other means, light will be
emitted at a lower frequency as the electron returns to its ground state and the substance is
said to fluoresce. As some energy is always lost before emission occurs then, in contrast to
Raman scattering, the wavelength of the fluorescent light is always greater than the incident
light. Detection techniques based on fluorescence affords greater sensitivity to sample
concentration, but less sensitivity to instrument instability, (e.g. sensor temperature and
pressure). This is due to the fluorescent light being measured against a very low light
background (i.e., against a very low noise level). This is opposite to light absorption
measurements, where the signal is superimposed on a strong background signal carrying a
high noise level.
These devices enable fluorescent compounds (solutes)present in the mobile phase to be
detected by passing the column effluent through a cell irradiated with ultraviolet light and
measuring any resultant fluorescent radiation. Although only a small proportion of inorganic
and organic compounds are naturally fluorescent, many biologically active compounds (e.g.
drugs) and environmental contaminants (e.g. polycyclic aromatic hydrocarbons) are
fluorescent and this, together with the high sensitivity of these detectors, explains their
widespread use. Because both the excitation wavelength and the detected wavelength can be
varied, the detector can be made selective. The application of fluorescence detectors has been
extended by means of pre- and post-column derivatisation of non-fluorescent or weakly
fluorescing compounds.
Derivatisation for fluorescence detectors is based on the reaction of nonfluorescent reagent
molecules (fluorotags) with solutes to form fluorescent derivatives; the reagent dansyl
chloride (I) is used to obtain fluorescent derivatives of proteins, amines and phenolic
compounds, the excitation and emission wavelengths being 335-365 nm and 520 nm,
respectively.
4 Electrochemical detectors
The term 'electrochemical detector' in HPLC normally refers to amperometric or coulometric
detectors, which measure the current associated with the oxidation or reduction of solutes. In
practice it is difficult to use electrochemical reduction as a means of detection in HPLC
because of the serious interference (large background current) caused by reduction of oxygen
in the mobile phase. Complete removal of oxygen is difficult so that electrochemical
detection is usually based on oxidation of the solute.
Examples of compounds which can be conveniently detected in this way are phenols,
aromatic amines, heterocyclic nitrogen compounds, ketones, and aldehydes. Since not all
compounds undergo electrochemical oxidation, such detectors are selective and selectivity
may be further increased by adjusting the potential applied to the detector to discriminate
between different electro active species. It may be noted here that an anode becomes a
stronger oxidising agent as its electrode potential becomes more positive. Of course,
electrochemical detection requires the use of conducting mobile phases, e.g. containing
inorganic salts or mixtures of water with water-miscible organic solvents, but such conditions
are often difficult to apply to techniques other than reverse phase and ion exchange
chromatography.8 In short, the amperometric detector is presently considered to be the best
electrochemical detector having the following distinct advantages, such as :
(i) very small internal cell-volume,
(ii) high degree of sensitivity,
(iii) more limited range of applications, and
(iv) excellent for trace analyses as UV-detector lacks adequate sensitivity
5 Evaporative light scattering
Detection is based on the scattering of a beam of light by particles of compound remaining
after evaporation of the mobile phase. this detector is of growing importance; it is a universal
detector and does not required a compound to have a chromophore for detection. Application
include the analysis of surfactant, lipid, and sugar. Unlike the refractive index detector ,which
was formerly used for this analysis , it can be with gradient elution and is robust enough to
function under wide range of operating conditions. however, it can not be used with involatile
material such as buffer in mobile phase or to detect very volatile analytes . typical application
include : analysis of chloride and sodium ions in pharmaceuticals, lipids used as component
in formulations, sugar and sugar polymers. Sensitive to ca 10 ng analyte.
6 The Electrical Conductivity Detector
The electrical conductivity detector measures the conductivity of the mobile phase. There is
usually background conductivity which must be backed-off by suitable electronic
adjustments. If the mobile phase contains buffers, the detector gives a base signal that
completely overwhelms that from any solute usually making detection impossible. Thus, the
electrical conductivity detector is a bulk property detector and will sense all ions, whether
they are from a solute, or from the mobile phase. In order to prevent polarization of the
sensing electrodes, AC voltages must be used and so it is the impedance not the resistance of
the electrode system that is actually measured. From a physical chemistry stand point the
conductivity of a solution is more important than its resistance. However, it is the resistance
(impedance) of the electrode system that determines the current across it.
The electrical conductivity detector has a sensitivity (minimum detectable concentration) of
about 5 x 10-9 g/ml and a linear dynamic range of about 200 where 0.97 < r < 1.03. It is used
in probably over 95% of all analyses involving ion exchange procedures to separate
inorganic and organic ions.
Recorder
● The change in eluent detected by a detector is in the form of an electronic signal,
and thus it is still not visible to our eyes.
● In older days, the pen (paper)-chart recorder was popularly used. Nowadays, a
computer-based data processor (integrator) is more common.
● There are various types of data processors; from a simple system consisting of the
in-built printer and word processor while those with software that are specifically
designed for an LC system which not only data acquisition but features like peak-
fitting, baseline correction, automatic concentration calculation, molecular weight
determination, etc.
Degasser
The eluent used for LC analysis may contain gases such as oxygen that are non-visible to our
eyes.
● When gas is present in the eluent, this is detected as noise and causes an unstable
baseline.
● Degasser uses special polymer membrane tubing to remove gases.
● The numerous very small pores on the surface of the polymer tube allow the air to
go through while preventing any liquid to go through the pore.
Column Heater
The LC separation is often largely influenced by the column temperature.
● In order to obtain repeatable results, it is important to keep consistent temperature
conditions.
● Also for some analysis, such as sugar and organic acid, better resolutions can be
obtained at elevated temperatures (50 to 80°C).
● Thus columns are generally kept inside the column oven (column heater).
HPLC Diagram
Types of High-Performance Liquid Chromatography (HPLC)
1. Normal phase:
Column packing is polar (e.g silica) and the mobile phase is non-polar. It is used for water-
sensitive compounds, geometric isomers, cis-trans isomers, and chiral compounds.
2. Reverse phase:
The column packing is non-polar (e.g C18), the mobile phase is water+ miscible solvent (e.g
methanol). It can be used for polar, non-polar, ionizable, and ionic samples.
3. Ion exchange:
Column packing contains ionic groups and the mobile phase is buffer. It is used to separate
anions and cations.
4. Size exclusion:
Molecules diffuse into pores of a porous medium and are separated according to their relative
size to the pore size. Large molecules elute first and smaller molecules elute later.
Applications of High-Performance Liquid Chromatography (HPLC)
The HPLC has developed into a universally applicable method so that it finds its use in
almost all areas of chemistry, biochemistry, and pharmacy.
● Analysis of drugs
● Analysis of synthetic polymers
● Analysis of pollutants in environmental analytics
● Determination of drugs in biological matrices
● Isolation of valuable products
● Product purity and quality control of industrial products and fine chemicals
● Separation and purification of biopolymers such as enzymes or nucleic acids
● Water purification
● Pre-concentration of trace components
● Ligand-exchange chromatography
● Ion-exchange chromatography of proteins
● High-pH anion-exchange chromatography of carbohydrates and oligosaccharides
Advantages of High-Performance Liquid Chromatography (HPLC)
1. Speed
2. Efficiency
3. Accuracy
4. Versatile and extremely precise when it comes to identifying and quantifying
chemical components.
Limitations
1. Cost: Despite its advantages, HPLC can be costly, requiring large quantities of
expensive organics.
2. Complexity
3. HPLC does have low sensitivity for certain compounds, and some cannot be
detected as they are irreversibly adsorbed.
4. Volatile substances are better separated by gas chromatography.