HPLC: Principle and Maintenance with Application

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ISSN No: 2456
International
Research
HPLC: Principle and Maintenance with Application
Yogesh Kumar
Research Scholar,
Delhi Pharmaceutical Sciences
ABSTRACT
High performance liquid chromatography (HPLC) is a
significant qualitative and quantitative technique,
usually used for the estimation of pharmaceutical and
biological samples. The chromatography term is
derived from the Greek words namely chroma
(colour) and graphein (to write). The chromatography
is very accepted technique and it is mostly used
analytically. It is the most resourceful, safest, reliable
and fastest chromatographic technique for the quality
control of drug components. This technique involves
2 phases’ stationary and mobile phases. There are
different types of chromatographic techniques. The
separation of constituents is based on the variation
between the partition coefficients of the two phases.
This article is primed with an aim to review different
aspects of HPLC, such as principle, types,
instrumentation and application with maintenance.
KEYWORD: HPLC; Instrumentation; maintenance;
application; chromatography
I. INTRODUCTION
Analytical chemistry is widely used in determining
the qualitative and quantitative composition of typical
mixtures. These two methods are the main aspects to
understand the sample materials. Generally, analytical
chemistry divides into two branches i.e. q
and quantitative analysis. High performance liquid
chromatography also is known as High Pressure
Liquid Chromatography. HPLC is an analysis
technique used for separation, identification and
quantification of typical mixtures such as organic,
inorganic, biological, ionic and polymeric materials.
HPLC is a type of column chromatography in which
solvent flows with high pressure, so that sample can
be separated into different constituents divided into
different types based on modes of separation, the
@ IJTSRD | Available Online @ www.ijtsrd.com | Volume – 2 | Issue – 5 | Jul-Aug 2018
ISSN No: 2456 - 6470 | www.ijtsrd.com | Volume
International Journal of Trend in Scientific
Research and Development (IJTSRD)
International Open Access Journal
Yogesh Kumar, Sayed Md Mumtaz, Mustaq Ahmad
search Scholar, Pharmacology Department,
Delhi Pharmaceutical Sciences and Research University Delhi, Delhi, India
High performance liquid chromatography (HPLC) is a
significant qualitative and quantitative technique,
usually used for the estimation of pharmaceutical and
chromatography term is
derived from the Greek words namely chroma
(colour) and graphein (to write). The chromatography
is very accepted technique and it is mostly used
analytically. It is the most resourceful, safest, reliable
echnique for the quality
control of drug components. This technique involves
2 phases’ stationary and mobile phases. There are
different types of chromatographic techniques. The
separation of constituents is based on the variation
fficients of the two phases.
This article is primed with an aim to review different
aspects of HPLC, such as principle, types,
instrumentation and application with maintenance.
HPLC; Instrumentation; maintenance;
Analytical chemistry is widely used in determining
the qualitative and quantitative composition of typical
mixtures. These two methods are the main aspects to
understand the sample materials. Generally, analytical
chemistry divides into two branches i.e. qualitative
and quantitative analysis. High performance liquid
chromatography also is known as High Pressure
Liquid Chromatography. HPLC is an analysis
technique used for separation, identification and
quantification of typical mixtures such as organic,
rganic, biological, ionic and polymeric materials.
HPLC is a type of column chromatography in which
solvent flows with high pressure, so that sample can
be separated into different constituents divided into
different types based on modes of separation, the
principle of separation, elution technique, scale of
separation and based on the type of analysis
Detector is the heart of this instrument and the
efficiency of this system depends upon detecting
techniques. Many types of HPLC detectors such as
UV-Vs, refractive index detector, photodiode
detector, fluorescence, electrical conductivity
detector.
A. Chromatography:
Chromatography is an analytical method in which
compounds are physically separated prior to
measurement. The main purpose of chromatography
is to separate and quantify the target sample in the
matrix. The first developer of
Tswett.
B. Scope of HPLC:
HPLC is used in various fields like in
pharmaceuticals, biochemicals, food products,
industrial chemicals, forensic chemistry,
environmental field, clinical medicine for analysis for
typical mixtures like antibiotics, amino acids, fatty
acids, drugs, poisons, inorganic ions, urine extracts,
oestrogens [2, 3].
C. Need of HPLC
The use of HPLC are increased day by day across the
world due to its unique properties like high
resolutions, high sensitivity (ppm
repeatability, small sample size, moderate analysis
condition, no need to vaporise the sample as in the gas
chromatography, easy to fractionate the sample and
purify [4, 5].
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, India
principle of separation, elution technique, scale of
separation and based on the type of analysis [1]. The
Detector is the heart of this instrument and the
efficiency of this system depends upon detecting
techniques. Many types of HPLC detectors such as
Vs, refractive index detector, photodiode
detector, fluorescence, electrical conductivity
Chromatography is an analytical method in which
compounds are physically separated prior to
measurement. The main purpose of chromatography
is to separate and quantify the target sample in the
matrix. The first developer of chromatography was M.
HPLC is used in various fields like in
pharmaceuticals, biochemicals, food products,
industrial chemicals, forensic chemistry,
environmental field, clinical medicine for analysis for
ntibiotics, amino acids, fatty
acids, drugs, poisons, inorganic ions, urine extracts,
The use of HPLC are increased day by day across the
world due to its unique properties like high
resolutions, high sensitivity (ppm-ppb), good
repeatability, small sample size, moderate analysis
condition, no need to vaporise the sample as in the gas
atography, easy to fractionate the sample and

International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470
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D. Instrumentation:
The HPLC instrumentation involves pump, injector,
column, detector, and integrator and display system.
 Solvent reservoir: In HPLC the mobile phase or
solvent is a mixture of polar and non-polar liquid
components. Depending on the composition of the
sample, the polar and non-polar solvents will be
varied [6].
 Pump: The pump suctions the mobile phase from
the solvent reservoir and forces it to the column
and then passes to the detector. 42000 KPa is the
operating pressure of the pump. This operating
pressure depends on column dimensions, particle
size, flow rate and composition of mobile phase
[7].
 Sample Injector: An injector for an HPLC
framework should give an infusion of the fluid
specimen inside the scope of 0.1 mL to 100 mL of
volume with high reproducibility and under high
pressure (up to 4000 psi).
 Columns: Columns are typically made of cleaned
stainless steel, are somewhere around 50 mm and
300 mm long and have an inward distance across
of somewhere around 2 and 5 mm. They are
generally loaded with a stationary phase with a
molecule size of 3 μm to 10 μm [8].
 Detector: The HPLC detector, situated toward the
end of the column distinguishes the analytes as
they elute from the chromatographic column.
Regularly utilized detectors are UV-spectroscopy,
fluorescence, mass spectrometric and
electrochemical identifiers [9].
 Data Collection Devices or Integrator: Signals
from the detector might be gathered on graph
recorders or electronic integrators that fluctuate in
many-sided quality and in their capacity to
process, store and reprocess chromatographic
information. The PC coordinates the reaction of
the indicator to every part and places it into a
chromatograph that is anything but difficult to
interpret [10].
Fig.1. Flow diagram of HPLC
II. TYPES OF HPLC-
A. BASED ON MODE OF SEPARATION:
 Normal phase Chromatography: In this method,
separation is based on polarity. The first developer
used CaCo3 as separation column and Petroleum
ether as developing solvent. The combination of
stationary phase and mobile phase called normal
phase chromatography. The stationary phase is
polar, like silica gel type, cyanotype, amino type
and mobile phase is non-polar example organic
solvents, hexane, benzene [11].
 Reversed phase Chromatography: It is the
reverse of the normal phase chromatography in
which stationary phase is non- polar and mobile
phase is polar. The example of the mobile phase
are organic solvents (methanol, acetonitrile),
buffer (phosphate buffer)[10, 12].
B. Based on the principle of Separation:
 Absorption Chromatography-
In the absorption chromatography solute molecules
bond directly to the surface of the stationary phase
[13]. The component which has more affinity towards
mobile phase elutes first and the component which
has less affinity towards stationary phase elutes later.
No two components have the same affinity towards
mobile phase and stationary phase.
 Ion- exchange chromatography-
Ion exchange chromatography is a process that allows
the separation of ions and polar molecules based on
their charge. It can be used for almost any kind of
charged molecules including large proteins, small
nucleotides and amino acids. Retention is based on
the attraction between solute ions and charged sites
bound to the stationary phase. Ions of the same
charged are excluded. The use of resin (the stationary
solid phase) is used to covalently attach anions or
cations onto it. Solute ions of the opposite charge in
the mobile liquid phase are attracted to the resin by
electrostatic forces [10].
C. Ion- Pair chromatography-
It is a form of chromatography in which ions in
solution can be “paired” or neutralized and separated
as an ion pair on a reversed- phase column. Ion-
pairing agents are usually ionic compounds that
contain a hydrocarbon chain that imparts a certain
hydrophobicity so that the ion pair can be retained on
a reversed-phase column [14].
 Gel permeation chromatography-
This type of chromatography lacks an attractive
interaction between the stationary phase and solute.

The liquid or gaseous phase passes through a porous
gel which separates the molecules according to its
size. The pores are normally small and exclude the
larger solute molecules, but allow smaller molecules
to enter the gel, causing them to flow through a larger
volume. This causes the larger molecules to pass
through the column at a faster rate than the smaller
ones [15].
 Affinity chromatography-
This is the most selective type of chromatography
employed. It utilizes the specific interaction between
one kind of solute molecule and a second molecule
that is immobilized on a stationary phase. For
example, the immobilized molecule may be an
antibody to some specific protein. When a solute
containing a mixture of proteins are passed by this
molecule, only the specific protein reacted to this
antibody, binding it to the stationary phase. This
protein is later extracted by changing the ionic
strength or pH [16].
 Chiral chromatography-
It involves the separation of stereoisomers. In the case
of enantiomers, these have no chemical or physical
differences apart from being three- dimensional
mirror images. Conventional chromatography or other
separation processes are incapable of separating them.
To enable chiral separations to take place, either the
mobile phase or the stationary phase must themselves
be made chiral, giving differing affinities between the
analytes [17].
D. Based on elution technique-
 Isocratic elution:
A separation in which the mobile phase composition
remains constant throughout the procedure is termed
isocratic elution. In isocratic elution, peak width
increases with retention time linearly with the number
of theoretical plates. This leads to the disadvantage
that late-eluting peaks get very flat and broad. Best for
simple separations. Often used in quality control
application those supports and are in close proximity
to a manufacturing process [18].
 Gradient elution-
A separation in which the mobile phase composition
is changed during the separation process is described
as a gradient elution Gradient elution decreases the
retention of the later-eluting components so that they
elute faster, giving narrower peaks. This also
improves the peak shape and the peak height. Best for
the analysis of complex samples. Often used in
method development for unknown mixtures. Linear
gradients are most popular [19].
E. Based on a scale of operation-
 Analytical HPLC: No recovery of individual
components of a substance.
 Preparative HPLC: Individual components of
substance can be removed.
F. Based on the type of Analysis-
 Qualitative analysis: Analysis of a substance in
order to ascertain the nature of its chemical
constituents and we can separate individual
components but cannot assess the quantity in this
analysis.
 Quantitative analysis: Determining the amounts
and proportions of its chemical constituents and
the quantity of the impurity and individual
components can be assessed.
Separation Mechanism: Compound are separated
because the molecules are moves at different rates in
the column.
Fig.2. Separation mechanism
Due to different interactions between stationary phase
and different sample, the molecules move to different
rate, therefore separation can be done [20].
Fig.3. Separation mechanism
III. DIFFERENT TYPES OF HPLC
DETECTORS:
Detectors in HPLC is placed at the end of the
analytical column. The function of the detector is to
examine the solution which is eluting from the
column. An electronic signal is proportional to the
concentration of individual components of the
analyte.

HPLC detector has particular characteristics such as
excellent linear response as a function of the good
sensitivity works approximately in the range of 0.01-
100 µg of the compound in elutes.
A. UV-Visible- Ultraviolet Visible detector: It is the
most commonly used detector in the HPLC. Most
of the organic compounds absorb light in the
region of UV (190-400nm) and in the visible
region (400-750nm). It is based on Beer-Lambert
law; deuterium and high-pressure xenon lamp are
the sources of the UV. It has various advantages
and disadvantage [21].
 Advantages: It has high sensitivity, relative
robust to temperature, compatible with gradient
elution.
 Disadvantages: only compounds with UV or
visible absorption could be detected.
B. Refractive index detectors: Refractive index is a
bulk property of column eluent. In this detector,
detection depends on solute modifying the overall
refractive index of the mobile phase. Bulk
property detectors have an inherently limited
sensitivity. For the detection of the non-ionic
compounds refractive index can be very useful
and non-ionic compounds are not absorbed in the
UV region and in fluoresce [22]. Various types of
RI detectors are the following [23].
 Christiansen effect detector
 Interferometer detector
 Thermal lens detector
 Dielectric constant detector
 Advantages:
1. Responds to nearly all solutes
2. Unaffected by the flow rate
 Disadvantages:
1. Not as sensitive as most other types of detectors
2. Could not be used with a gradient elution
C. Photodiode Array detector (PAD), Diode Array
detector: Photodiode arrays (semiconductor
devices) are used in the detection unit. A DAD
detects the absorption in UV to VIS region. While
a UV-VIS detector has only one sample-side light-
receiving section, a DAD has multiple (1024 for
L-2455/2455U) photodiode arrays to obtain
information over a wide range of wavelengths at
one time, which is a merit of the DAD [24].
 Advantages:
1. .PDA Detector could analyze a sample
simultaneously at many different wavelengths
2. UV visible spectra are useful for compound
identification, checking peak purity, as well as
finding the optimum absorbance for the
compounds.
3. UV visible spectra of many compounds could be
stored in the spectrum libraries, which are used for
compound identification.
4. Relatively robust to temperature and flow rate
fluctuations
5. Compatible with gradient elution.
 Disadvantages:
1. Slightly less sensitive than the UV-Visible
detector.
D. Fluorescence Detector: It is the most sensitive,
specific detector among all existing HPLC
detector. It is possible to detect the presence of
single analyte molecule in the flow cell. The
sensitivity of this detector is 10-1000 times higher
than UV detector [25]. Various types of
Fluorescence detector are:
 The single wavelength excitation Fluorescence
detector
 Multi wavelength Fluorescence detector
 Laser-induced Fluorescence detector
Advantages:
1. Sensitivity is higher than UV-Vis detector
2. Selectivity is high because of relatively few-
compounds fluorescences.
3. Compatible with gradient elution
Disadvantages:
1. Difficult to predict fluorescence
2. Greatly affected by environment, solvent, pH,
temperature, viscosity, ionic strength, dissolved
gas.
E. Electrical Conductivity Detector: It provides
universal, reproducible, high-sensitivity detection
of all charged species such as anions, cations,
metals, organic acids. These detectors measure the
conductivity of the total mobile phase hence
categorized in bulk density detectors. Electrodes
of this detector are usually made up of platinum,
stainless steel or some other noble metal [26].

 Advantages:
1. Respond to ionic compounds and suitable for ion
chromatography.
2. High sensitivity for low concentration range.
 Disadvantages:
1. Sensitive to the fluctuations in the solvent flow
and mobile phase composition
2. Not compatible with gradient elution.
IV. SELECTION OF DETECTOR:
Table1. Selection of Detector [27]
S.NO DETECTORS Type of compounds can be
detected
1. UV-Vis
&PDA
Compounds with
chromophores, such as
aromatic rings or multiple
alternating double bonds.
2. RF Fluorescent compounds,
usually with fused rings or
highly conjugated planar
system.
3. CDD Charged compounds, such as
inorganic ions and organic
acids.
4. ECD For easily oxidized
compounds like quinones or
amines.
V. BASIC MAINTENANCE OF HPLC:
A. Daily maintenance (pre-analysis check):
 Filter mobile phase
 Wait for mobile phase to reach room temperature
 Purge all the flow lines with mobile phase
 Replace mobile phase as necessary
 If the solvent tubing is not completely filled with
mobile phase, purge the flow line
 After periods of disuse or changes in ambient
temperature, purge the flow line.
 If the buffer is used as mobile phase, wash the
back of plunger seal.
 If auto sampler is used, verify the auto sampler
rinse the bottle fully.
 Purge the autosampler rinse solution
 Checks for leaks
 Checks the pump pressure
 Perform a baseline check.
B. Daily maintenance (post-analysis checks):
 Wash the column
 If the column is not going to be used for a long
time, take off it from the system after washing,
and cap both ends of the column, store it in a cool
and dark place.
 Clean the flow line or the rest of the instrument, as
necessary
 Store the solvent reservoir filters in rinse solvent
to prevent them from drying out
 Solvent Reservoir Maintenance: The material of
containers is glass which is generally used and
plastic which has high sensitivity inorganic
analysis.
1. Always cover the containers
2. Prevent the evaporation of solvents
3. Prevent the dust enter the mobile phase
4. Reduce the vapours in the room
5. Do not make containers as this may lead to the
creation of partial vacuum and pump system.
6. Do not expose the bottle to direct sunlight/wind
7. Always keep reservoir above solvent delivery
system, ensures good siphon feed to pump and
prevents starving pumping system.
8. Always use a suction filter, prevent the
particulates from reaching pumps and clean the
suction filter by sonicating in isopropanol or 1N
nitric acid and water.
9. Good practice in the lab to label all bottles with
composition inside as well as the date of
prescription.
 Mobile Phase: If the solvent has background
absorption, the linear range will be smaller, the
noise will be bigger and baseline stability,
especially during gradient, runs will be poor.
1. The use of pure water systems is ideal as both
inorganic and organic contaminants are removed.
2. Modern pure water systems give TOC values as
indicated of total organic content in water.
3. Ghost peak will appear in gradient elution if water
is bad quality.
VI. PRECAUTIONS IN HANDLING SOME
HPLC GRADE ORGANIC SOLVENTS.
1. Analytical grade THF contains butylated
hydroxytoluene (BHT) as an oxidation inhibitor.
As BHT has strong UV absorption, it is not added
for HPLC grade THF.
2. As such, HPLC grade THF must be stored in cool
and dark places to reduce oxidation to form
peroxides. Accumulation of a large amount of
organic peroxides may lead to explosions.

A. Use of buffer solutions:
 Buffer solutions must be filtered before use.
 Buffer solutions must not be left in the system to
avoid crystallization.
 Effect on the pump- damage plunger and seal
 Effect on the column- the creation of column
voids
 Effect on flow line- corrosion of stainless steel
lines
 Possible bacterial growth, especially phosphate
buffers good medium for bacterial and fungus
growth.
 Ideally, solutions should be prepared fresh every
day [28].
B. Premixing of Solvents: For isocratic systems,
different solvents are premixed.
Example: to prepare a 1000ml of 50:50 mixture of
water/methanol.
 Measure out 500 ml of water and 500ml of
methanol separately, and then mix them together.
 Measure 500ml of water and top up to the 1000ml
mark of measuring cylinder/ volumetric flask with
methanol.
 Measure 500ml of methanol and top up to the
1000ml mark of measuring cylinder/ volumetric
flask with water.
All three methods will give different compositions,
whichever method selected must be used consistently
for the repeatable result.
C. Pump maintenance:
 Clean behind plunger seal when using buffer
solution-
 Automatic rinse kit for LC-10Avp and LC_20A
series.
 Useful spares to be maintained- plunger seals,
plunger, check valves, filter frit for line filter.
 Most’s tools required for maintenance work come
with the pump accessories kit- do not lose them.
D. Sample treatment: Always filtered sample with
0.2µ m or 0.45µ m filters before injection. If the
sample contains a lot of solid, centrifuge it, and
the filter the solution.
 Select suitable solvent to dissolve the sample.
Normally dissolve sample in the mobile phase.
But do not select a high soluble solvent as a
sample solvent.
E. Injector maintenance:
Manual injector:
Precautions:
 Do not use pointed or beveled needle tip. Use only
flat and end type.
 If pH higher than 10 is to be used, Delrin rotter
seal is required.
 When both manual and auto-injector are present in
the system, auto sampler is always connected first,
followed by manual.
 Manual injector always set to load position when
auto sampler is used and to inject position
otherwise.
F. Auto-Injector: General Maintenance
 Purge flow line at least once a day.
 Change purge liquid frequently.
 Never used the salt solution as a purge liquid.
 Used septums for sample vial recommended by
the manufacturer only.
 Septums should not be reused.
 Do not fill vials to the brim.
G. Detector maintenance:
 UV-Vs detector: Check D2 lamp energy- Check
the energy of reference cell. Change D2 lamp if
the energy is lower than 800.
(Note- do not touch the surface of D2 lamp by hand.)
 Waste bottle maintenance:
 Remember to empty waste bottle regularly.
 Do not mix concentrated nitric acid with an
organic solvent.
 Do not immerse the drain tubing in the waste
liquid as this may create back pressure on the flow
cells [30].
 Advantages of HPLC-
 High Resolution
 Good repeatability
 High Sensitivity (ppm-ppb)
 Small sample size
 Moderate analyses condition
 No need to vaporize the sample like GC
 Easy to fractionate the sample and purify
 Non-Destructive
 Separations fast and efficient (High Resolution
Power)
 Continuous monitoring of the column effluent
 It can be applied to the separation and analysis of
very complex mixtures.
 Accurate quantitative measurements

 Repetitive and reproducible analysis using the
same column
 Adsorption, partition, ion exchange and exclusion
column separations are excellently made
 HPLC is more versatile than GLC in some
respects because it has the advantage of not being
restricted to volatile and thermally stable solute
and the choice of mobile and stationary phases is
much wider in HPLC.
 Both aqueous and non- aqueous samples can be
analyzed with little or no sample pre-treatment
 A variety of solvents and column packing are
available, providing a high degree of selectivity
for specific analysis.
 It provides a means for determination of multiple
components in a single analysis.
 Application-
HPLC is one of the most widely applied analytical
separation techniques-[29]
 Pharmaceutical:
 Tablet dissolution of pharmaceutical dosages.
 Shelf life determination of pharmaceutical
products.
 Identification of counterfeit drug products.
 Pharmaceutical quality control.
 Environmental:
 Phenols in drinking water
 Identification of diphenhydramine in sediment
samples.
 Bio monitoring of PAH pollution in high-altitude
mountain lakes through the analysis of fish bile
 Estrogens in coastal waters- The sewage source
 Toxicity of tetracyclines and tetracycline
degradation products to environmentally relevant
bacteria
 Assessment of TNT toxicity in sediment.
 Forensics:
 A mobile HPLC apparatus at dance parties- on-
site identification and quantification of the drug
Ecstasy.
 Identification of anabolic steroids in serum, urine,
sweat and hair
 Forensic analysis of textile dyes
 Simultaneous quantification of psychotherapeutic
drugs in human plasma
 Clinical:
 Quantification of DEET in Human Urine.
 Analysis of antibiotics
 Increased urinary excretion of aquaporin 2 in
patients with liver cirrhosis
 Detection of endogenous neuropeptides in brain
extracellular fluids.
 Food & Flavour:
 Ensuring soft drink consistency and quality
 Analysis of vicinal dike tones in beer
 Sugar analysis in fruit juices
 Polycyclic aromatic hydrocarbons in Brazilian
vegetables and fruits.
 Trace analysis of military high explosives in
agricultural crops
 Stability of aspartame in the presence of glucose
and vanillin [2, 10].
VII. CONCLUSION
In this review, we include all fundamental aspects of
HPLC technique. We describe the general technique
of HPLC method and maintenance of the instrument.
The general approach for these all point makes the
precise quality of techniques. This method is widely
accepted in the laboratory as well as in the clinical
and forensic science lab due to the accuracy and
precession and specificity with the HPLC. The only
disadvantage of HPLC is high cost and complex
methodology.
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