Int. J. Pharm. Sci. Rev. Res., 74(2), May - June 2022; Article No.
03, Pages: 23-29 ISSN 0976 – 044X
Based on the elution technique 4. Sample preparations
Gradient separation and isocratic separation 5. Method optimization
Based on modes of operation 6. Method validation 15
Normal phase chromatography and reverse-phase 1. Understanding the Physicochemical Properties of the
chromatography.9 drug molecule
A. Normal phase chromatography: The physicochemical qualities of a therapeutic molecule
are critical in method development. To develop a method,
The mobile phase in normal phase chromatography is non-
one must first evaluate the physical properties of the drug
polar, whereas the stationary phase is polar. As an
molecule, such as solubility, polarity, pKa, and pH. A
outcome, the polar analyte is retained by the station
compound's polarity is a physical property.16 It supports an
phase.10 The increased polarity of solute molecules
analyst in evaluating the solvent and mobile phase
improves adsorption capacity, resulting in a longer elution
composition. The polarity of molecules can be used to
time. In this chromatography, a stationary phase of
explain molecular solubility. Polar solvents, such as water,
chemically modified silica (cyanopropyl, aminopropyl, and
and nonpolar solvents, such as benzene, do not combine.
diol) is used 11. As an example, A typical column has an
In general, like dissolves like, which means that elements
interior diameter of around 4.6 mm and a length ranging
with comparable polarities dissolve in one other. The
from 150 to 250 mm. Polar compounds in the mixture that
analyte's solubility is used to select diluents.17 The pH value
are passed through the column will stick to the polar silica
is commonly used to define a substance's acidity or
for a longer period than non-polar compounds. As a result,
basicity. Choosing the correct pH for ionizable analytes
the non-polar ones will go quickly through column 12
frequently results in symmetrical and crisp peaks in HPLC.
B. RP-HPLC (Reversed-phase HPLC): The pH value is defined as the negative of the logarithm to
base 10 of the hydrogen ion concentration18
The stationary phase of RP-HPLC is non-polar, and the
mobile phase is polar or moderately polar. The notion of pH = - log10[H3O+].
hydrophobic interaction underpins RP-HPLC 13. The non-
Selecting an appropriate pH for ionizable analytes
polar stationary phase will hold analytes that are
frequently results in symmetrical and sharp peaks in HPLC.
comparatively less polar in a combination of components
In quantitative analysis, sharp, symmetrical peaks are
for a longer period than those that are substantially more
required to achieve low detection limits, low relative
polar. As a result, the most polar component elutes the
standard deviations between injections, and predictable
first14
retention durations 19.
METHOD DEVELOPMENT ON HPLC
2. Selection of chromatographic conditions
During the early stages of method development, a set of
beginning conditions (detector, column, and mobile phase)
is chosen to generate the sample's first "scouting"
chromatograms. These are typically based on reversed-
phase separations on a C18 column with UV detection. At
this point, a choice should be taken between establishing
an isocratic or a gradient method. 20
Selection of column
The first and most significant stage in method
development is the selection of the stationary phase or
column. It is impossible to develop a robust and
reproducible procedure without the availability of a stable,
high-performance column. Columns must be stable and
reproducible to avoid difficulties caused by irreproducible
sample retention during technique development. A C8 or
Figure 2: Steps involved in HPLC Method development C18 column made of particularly purified, less acidic silica
and specifically intended for the separation of basic
Method development involves the following steps: chemicals is generally suitable for all samples and is
1. Understanding the Physicochemical Properties of strongly recommended 21.
the drug molecule. The key ones include column diameters, silica substrate
2. Selection of chromatographic conditions. qualities, and bonded stationary phase characteristics. Due
to a variety of physical properties, silica-based packing is
3. Developing the approach of analysis. preferred in the majority of today's HPLC columns. The
International Journal of Pharmaceutical Sciences Review and Research
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