Kumar and Kumar, IJPSR, 2012; Vol.

3(12): 4626-4633 ISSN: 0975-8232

IJPSR (2012), Vol. 3, Issue 12 (Review Article)

Received on 25 August, 2012; received in revised form 07 October, 2012; accepted 20 November, 2012

IMPORTANCE OF RP-HPLC IN ANALYTICAL METHOD DEVELOPMENT: A REVIEW

Sanjay Kumar D and D.R. Harish Kumar*

Department of Pharmaceutical Analysis, Krupanidhi College of Pharmacy, Sarjapura Main Road, Carmelaram
post, Bangalore-560 035, Karnataka, India

ABSTRACT
Keywords:
Chromatography, although primarily a separation technique, is mostly
HPLC, RP-HPLC, Analytical methods,
Chromatographic parameters
employed in chemical analysis in which High-performance liquid
chromatography (HPLC) is an extremely versatile technique where analytes
Correspondence to Author:
are separated by passage through a column packed with micrometer-sized
Dr. D.R. Harish Kumar (M. Pharm, Ph. D) particles. Now a day reversed-phase chromatography is the most commonly
used separation technique in HPLC. The reasons for this include the
Assistant Professor, Department of
simplicity, versatility, and scope of the reversed-phase method as it is able to
Pharmaceutical analysis, Krupanidhi
College of Pharmacy, Bangalore, India handle compounds of a diverse polarity and molecular mass. Reversed phase
chromatography has found both analytical and preparative applications in
Email id: [email protected] the area of biochemical separation and purification. Molecules that possess
QUICK RESPONSE CODE some degree of hydrophobic character, such as proteins, peptides and
IJPSR: nucleic acids, can be separated by reversed phase chromatography with
ICV (2011)- 5.07
excellent recovery and resolution. This review covers the importance of RP-
Website: HPLC in analytical method development and their strategies along with brief
www.ijpsr.com knowledge of critical chromatographic parameters need to be optimized for
an efficient method development.

INTRODUCTION: Chromatography is probably the most Now a day reversed-phase chromatography is the most
powerful analytical technique available to the modern commonly used separation technique in HPLC due to
chemist. Its power arises from its capacity to its broad application range. It is estimated that over
determine quantitatively many individual components 65% (possibly up to 90%) of all HPLC separations are
present in mixture by single analytical procedure 1, 2. carried out in the reversed-phase mode. The reasons
High-performance liquid chromatography (HPLC) is a for this include the simplicity, versatility, and scope of
chromatographic technique that can separate a the reversed-phase method as it is able to handle
mixture of compounds and is used in biochemistry and compounds of a diverse polarity and molecular mass 5,
6, 7
analytical chemistry to identify, quantify and purify the .
individual components of the mixture3. Reversed phase
chromatography has found both analytical and Theory of Reversed Phase Chromatography: Reversed
preparative applications in the area of biochemical phase chromatography has found both analytical and
separation and purification. Molecules that possess preparative applications in the area of biochemical
some degree of hydrophobic character, such as separation and purification. Molecules that possess
proteins, peptides and nucleic acids, can be separated some degree of hydrophobic character can be
by reversed phase chromatography with excellent separated by reversed phase chromatography with
recovery and resolution 4. excellent recovery and resolution 8.

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 Kumar and Kumar, IJPSR, 2012; Vol. 3(12): 4626-4633 ISSN: 0975-8232

The separation mechanism in reversed phase aqueous which indicates a high degree of organised
chromatography depends on the hydrophobic binding water structure surrounding both the solute molecule
interaction between the solute molecule in the mobile and the immobilised ligand. As solute binds to the
phase and the immobilised hydrophobic ligand, i.e. the immobilised hydrophobic ligand, the hydrophobic area
stationary phase. The actual nature of the hydrophobic exposed to the solvent is minimised. Therefore, the
binding interaction itself is a matter of heated debate 9 degree of organised water structure is diminished with
but the conventional wisdom assumes the binding a corresponding favourable increase in system entropy.
interaction to be the result of a favourable entropy In this way, it is advantageous from an energy point of
effect. The initial mobile phase binding conditions used view for the hydrophobic moieties, i.e. solute and
in reversed phase chromatography are primarily ligand, to associate 10 (Figure 1).

FIGURE 1: INTERACTION OF A SOLUTE WITH A TYPICAL REVERSED PHASE MEDIUM

Water adjacent to hydrophobic regions is postulated to Separations in reversed phase chromatography depend
be more highly ordered than the bulk water. Part of on the reversible adsorption/desorption of solute
this ‘structured’ water is displaced when the molecules with varying degrees of hydrophobicity to a
hydrophobic regions interact leading to an increase in hydrophobic stationary phase. The majority of
the overall entropy of the system. reversed phase separation experiments are performed
in several fundamental steps as illustrated in Figure 2.

FIGURE 2 : PRINCIPLE OF REVERSED PHASE CHROMATOGRAPHY WITH GRADIENT ELUTION

Choice of Separation Medium: The proper choice of 2) The molecular weight, or size of the sample
reversed phase medium is critical for the success of a components.
particular application. This choice should be based on
the following criteria: 3) The hydrophobicities of the sample
components.
1) The unique requirements of the application,
including scale and mobile phase conditions. 4) The class of sample components.

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Analytical method development using RP-HPLC: Sample collection and preparation: The sample should
Methods of analysis are routinely developed, ideally be dissolved in the initial mobile phase. If this is
improved, validated, collaboratively studied and not possible due to stability or solubility problems,
applied. Compilations of these developed methods formic acid, acetic acid or salt can be added to the
then appear in large compendia such as USP, BP and IP, sample to increase solubility. These additives do not
etc. In most cases as desired separation can be usually effect the separation so long as the volume of
achieved easily with only a few experiments. In other the sample loaded is small compared to the column
cases a considerable amount of experimentation may volume. The only effect when large sample volumes
be needed. However, a good method development are applied may be an extra peak or two eluting in the
strategy should require only as many experimental void volume after sample injection.
runs as are necessary to achieve the desired final
result(s). The development of a method of analysis is Sample preparation is an essential part of HPLC
usually based on prior art or existing literature using analysis, intended to provide a reproducible and
almost the same or similar experimentation. The homogenous solution that is suitable for injection onto
development of any new or improved method usually the column. The aim of sample preparation is a sample
tailors existing approaches and instrumentation to the aliquot that,
current analyte, as well as to the final need or  Is relatively free of interferences,
requirement of the method.
 Will not damage the column, and
Method development usually requires selecting the
method requirements and deciding on what type of  Is compatible with the intended HPLC method that
instrumentation to utilize and why. In the HPLC is, the sample solvent will dissolve in the mobile
method development stage, decisions regarding choice phase without affecting sample retention or
of column, mobile phase, detectors, and method resolution 12
quantitation must be considered. So development
involves a consideration of all the parameters Sample preparation begins at the point of collection,
pertaining to any method. extends to sample injection onto the HPLC column and
encompasses the various operations summarized in
Therefore, development of a new HPLC method table 1. All of these operations form an important part
involves selection of best mobile phase, best detector, of sample preparation and have a critical effect on the
best column, column length, stationary phase and best accuracy, precision, and convenience of the final
internal diameter for the column 11, 12. The analytical method 12.
strategy for HPLC method development contains a
number of steps 13, as shown in figure 3. Measurement: The measurement of a given analyte
can often be divided into a separation step and a
detection step.

Separation: Analytes in a mixture should preferably be

separated prior to detection. Simple LC consists of a
column with a fritted bottom containing the stationary
phase in equilibrium with a solvent. The mixture to be
separated is loaded on to the top of the column
followed by more solvent. The different components in
the column pass at different rates due to difference in
their partitioning behavior between mobile liquid
phase and stationary phase 13, 14.
FIGURE 3: A TYPICAL STRATEGY FOR HPLC METHOD
DEVELOPMENT

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TABLE 1: SAMPLE PRETREATMENT OPTIONS

S. no. Option Comment
1. Sample collection Obtain representative sample using statistically valid processes
Use appropriate inert, tightly sealed containers; be especially careful with
2. Sample storage and preservation volatile, unstable, or reactive materials; biological samples may require
freezing.
Sample must be in a form for more efficient sample pretreatment (e.g.,
3. Preeliminary sample processing drying, sieving, grinding, etc.); finer dispersed samples are easier to dissolve
or extract
Take necessary precautions for reactive, unstable, or biological materials;
4. Weighing or volumetric dilution
for dilution, use calibrated volumetric glasswares.
5. Alternative sample processing methods Solvent replacement, desalting, evaporation, freeze drying, etc.
6. Removal of particulates Filtration, solid-phase extraction, centrifugation.
7. Sample extraction Different methods used for liquid samples and solid samples
Used mainly to enhance analyte detection; sometimes used to improve
8. Derivatization
separation.

Detection: It is essential to use reagents and solvents and selectivity usually changes as either %B or solvent
of high purity to ensure minimum detection limits for type is varied.
optimum sensitivity. All organic solvents and many
additives, such as ion pairing agents, absorb in the UV It is possible to separate many regular samples just by
range and the detection limit is related to the varying solvent strength and type. Therefore, RPC
wavelength 15. A large number of LC detectors have method development for all regular samples (both
been developed over the past thirty years based on a neutral and ionic) can be carried out initially in the
variety of different sensing principles for detecting the same way 11, 12.
analytes after the chromatographic separations. The column/Stationary phase: Selection of the
However, only about twelve of them can be used stationary phase/column is the first and the most
effectively for LC analysis and, of those twelve, only important step in method development. The
four are in common use. The four dominant detectors development of a rugged and reproducible method is
used in LC analysis are the UV detector (fixed and impossible without the availability of a stable, high
variable wavelength), the electrical conductivity performance column. To avoid problems from
detector, the fluorescence detector and the refractive irreproducible sample retention during method
index detector. These detectors are employed in over development, it is important that columns be stable
95% of all LC analytical applications. The choice of and reproducible. A C8 or C18 column made from
detector depends on the sample and the purpose of specially purified, less acidic silica and designed
the analysis 16. specifically for the separation of basic compounds is
Critical Parameters in Reversed Phase generally suitable for all samples and is strongly
Chromatography: recommended 5, 12, 13, 17, 18. Some important factors
need to be considered while selecting column in RP-
Classifying the sample: The first step in method HPLC are summarized in table 2.
development is to characterize the sample as regular
or spherical. Regular samples are a mixture of small The column is selected depending on the nature of the
molecules (<2000 Daltons) that can be separated using solute and the information about the analyte.
more or less standardized starting conditions. Reversed phase mode of chromatography facilitates a
Separations in regular samples respond in predictable wide range of columns like dimethyl silane (C2),
fashion to change in solvent strength (%B) and type butylsilane (C4), octylsilane (C8), octadecylslane (C18),
(Acetonitrile, methanol) or temperature. A 10% base deactivated silane (C18) BDS phenyl, cyanopropyl
decrease in %B increases retention by about threefold, (CN), nitro, amino, etc. Generally longer columns
provide better separation due to higher theoretical

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plate numbers. As the particle size decreases the with 5-µm particle size give the best compromise of
surface area available for coating increases. Columns efficiency, reproducibility and reliability.
TABLE 2: FACTORS AFFECTING COLUMN EFFICIENC
Factor(s) Effect on column efficiency
*Choose longer columns for enhanced resolution
Column length *Choose shorter column for shorter analysis time, lower back pressure and fast equilibration and
less solvent consumption
*Choose wider diameter column for greater sample loading
Column internal diameter
*Choose narrow column for more sensitive and reduced mobile phase consumption
*Choose spherical particles for lower back pressure, column stability and greater stability
Particle shape
*Choose irregular particles when high surface area and high capacity is required
*Choose smaller particle (3-4 µm) for complex mixture with similar components
Particle size *Choose larger particle (5-10 µm) for sample with structurally different compounds
*Choose very large particle (15-20 µm) for preparative separation
*Choose a pore size of 150?or less for sample with molecular weight less than 2000
Pore size
*Choose a pore size of 300?or less for sample with molecular weight greater than 2000
*Choose end capped packing to eliminate unpredictable secondary interaction with the base
materials
Surface area
*Choose non-end capped phase for selectivity differences for polar compounds by controlling
secondary interaction
*Choose high carbon loads for greater column capacities and resolution
Carbon load
*Choose low carbon loads for fast analysis

The column should provide, but is limited by its high viscosity which results in lower
column efficiencies and higher backpressures.
 Reasonable resolution in initial experiments,
Both acetonitrile and methanol are less viscous than
 Short run time, isopropanol. All three solvents are essentially UV
transparent. This is a crucial property for reversed
 An acceptable pressure drop for different
phase chromatography since column elution is typically
mobile phases 12.
monitored using UV detectors. Acetonitrile is used
Mobile phase: In many cases, the colloquial term used almost exclusively when separating peptides. Most
for the mobile phases in reversed phase peptides only absorb at low wavelengths in the ultra-
chromatography is “buffer”. However, there is little violet spectrum (typically less than 225 nm) and
buffering capacity in the mobile phase solutions since acetonitrile provides much lower background
they usually contain strong acids at low pH with large absorbance than other common solvents at low
concentrations of organic solvents. Adequate buffering wavelengths.
capacity should be maintained when working closer to
Ion suppression: The retention of peptides and
physiological conditions.
proteins in reversed phase chromatography can be
Organic solvent: The organic solvent (modifier) is modified by mobile phase pH since these particular
added to lower the polarity of the aqueous mobile solutes contain ionisable groups. The degree of
phase. The lower the polarity of the mobile phase, the ionisation will depend on the pH of the mobile phase.
greater its eluting strength in reversed phase The stability of silica-based reversed phase media
chromatography. Although a large variety of organic dictates that the operating pH of the mobile phase
solvents can be used in reversed phase should be below pH 7.5. The amino groups contained
chromatography, in practice only a few are routinely in peptides and proteins are charged below pH 7.5. The
employed. The two most widely used organic modifiers carboxylic acid groups, however, are neutralised as the
are acetonitrile and methanol, although acetonitrile is pH is decreased. The mobile phase used in reversed
the more popular choice. Isopropanol (2-propanol) can phase chromatography is generally prepared with
be employed because of its strong eluting properties, strong acids such as trifluoroacetic acid (TFA) or ortho-
phosphoric acid. These acids maintain a low pH

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 Kumar and Kumar, IJPSR, 2012; Vol. 3(12): 4626-4633 ISSN: 0975-8232

environment and suppress the ionisation of the acidic It is important to maintain the pH of the mobile phase
groups in the solute molecules. Varying the in the range of 2.0 to 8.0 as most columns does not
concentration of strong acid components in the mobile withstand to the pH which are outside this range. This
phase can change the ionisation of the solutes and, is due to the fact that the siloxane linkage area cleaved
therefore, their retention behaviour. below pH 2.0; while at pH valued above 8.0 silica may
dissolve 12, 19.
The major benefit of ion suppression in reversed phase
chromatography is the elimination of mixed mode Absorbance: An UV-visible detector is based on the
retention effects due to ionisable silanol groups principle of absorption of UV visible light from the
remaining on the silica gel surface. The effect of mixed effluent emerging out of the column and passed
mode retention is increased retention times with through a photocell placed in the radiation beam. UV
significant peak broadening (Figure 4). detector is generally suitable for gradient elution work.
Most compounds adsorb UV light in the range of 200-
350 A°. The mobile phase used should not interfere in
the peak pattern of the desired compound hence it
should not absorb at the detection wavelength
employed 20.

Selectivity: Selectivity (α) is equivalent to the relative

retention of the solute peaks and, unlike efficiency,
depends strongly on the chemical properties of the
chromatography medium.

The selectivity, α, for two peaks is given by;

α= k2´ /k1´ = V2 - V0/V1 – V0 = V2/V1

Where V1 and V2 are the retention volumes, and k2´

/k1´ are the capacity factors, for peaks 1 and 2
respectively, and V0 is the void volume of the column.
Selectivity is affected by the surface chemistry of the
FIGURE 4: TYPICAL EFFECTS OF MIXED-MODE RETENTION. Peaks reversed phase medium, the nature and composition
are broader and skewed, and retention time increases.
of the mobile phase, and the gradient shape (Figure 5).
pH: pH plays an important role in achieving the
chromatographic separations as it controls the elution
properties by controlling the ionization characteristics.
Reversed phase separations are most often performed
at low pH values, generally between pH 2-4. The low
pH results in good solubility of the sample components
and ion suppression, not only of acidic groups on the
sample molecules, but also of residual silanol groups
on the silica matrix. Acids such as trifluoroacetic acid,
heptafluorobutyric acid and ortho-phosphoric acid in
the concentration range of 0.05 - 0.1% or 50 - 100 mM
are commonly used. Mobile phases containing
ammonium acetate or phosphate salts are suitable for
use at pH’s closer to neutrality. Note that phosphate
buffers are not volatile.

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spectrometry detector and the UV detector (fixed and

variable wavelength). These detectors are employed in
over 95% of all LC analytical applications 21-22.

The detector selected should be chosen depending

upon some characteristic property of the analyte like
UV absorbance, fluorescence, conductance, oxidation,
reduction, etc. Characteristics that are to be fulfilled by
a detector to be used in HPLC determination are:

 High sensitivity, facilitating trace analysis

FIGURE 5 : THE EFFECT OF SELECTIVITY AND EFFICIENCY ON
RESOLUTION  Negligible baseline noise to facilitate lower
detection
Both high column efficiency and good selectivity are
important to overall resolution. However, changing the  Low drift and noise level
selectivity in a chromatographic experiment is easier
than changing the efficiency. Selectivity can be  Wide linear dynamic range (this simplifies
changed by changing easily modified conditions like quantitation)
mobile phase composition or gradient shape.
 Low dead volume (minimal peak broadening)
Viscosity: Solvent of lowest possible viscosity should
be used to minimize separation time. An added  Cell design that eliminates remixing of the
advantage of low viscosity is that high efficiency separated bands
theoretical plate (HETP) values are usually lower than  Insensitivity to changes in type of solvent, flow
with solvents of higher viscosity, because mass transfer rate, and temperature
is faster. Viscosity should be less than 0.5 centipoise,
otherwise high pump pressures are required and mass  Operational simplicity and reliability
transfer between solvent and stationary phase will be
reduced.  Tunability, so that detection can be optimized
for different compounds
Temperature: Temperature can have a profound effect
on reversed phase chromatography, especially for low  Large linear dynamic range
molecular weight solutes such as short peptides and  Non destructive to sample
oligonucleotides. The viscosity of the mobile phase
used in reversed phase chromatography decreases APPLICATIONS:
with increasing column temperature. Since mass
transport of solute between the mobile phase and the  Designing a biochemical purification
stationary phase is a diffusion-controlled process,
 Purification of platelet-derived growth factor
decreasing solvent viscosity generally leads to more
(PDGF)
efficient mass transfer and, therefore, higher
resolution. Increasing the temperature of a reversed  Purification of cholecystokinin-58 (CCK-58) from
phase column is particularly effective for low molecular pig intestine
weight solutes since they are suitably stable at the
elevated temperatures 8.  Purification of recombinant human epidermal
growth factor
Detectors: A large numbers of detectors are used for
RP-HPLC analysis. However, among these the five  Process purification of inclusion bodies
dominant detectors used in LC analysis are the
electrical conductivity detector, the fluorescence CONCLUSION: Analytical methods development plays
detector, the refractive index detector, mass important roles in the discovery, development and

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 Kumar and Kumar, IJPSR, 2012; Vol. 3(12): 4626-4633 ISSN: 0975-8232

manufacture of pharmaceuticals. RP-HPLC is probably 8. Amesham Biosciences: Reversed Phase Chromatography.

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How to cite this article:

Kumar SD and Kumar HDR: Importance of RP-HPLC in Analytical Method Development- A Review: A Review. Int J Pharm Sci Res. 3(12); 4626-
4633.

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