JSMU- INSTITUTE OF PHARMACEUTICAL SCIENCES

Table of Contents
INTRODUCTION:...........................................................................................................................................2
THE APPARATUS OF HPLC:.......................................................................................................................2
HPLC SEPARATION:....................................................................................................................................3
HOW TO READ A CHROMATOGRAM:....................................................................................................4
TYPES OF HPLC:...........................................................................................................................................5
NORMAL-PHASE HPLC...........................................................................................................................5
REVERSED-PHASE HPLC........................................................................................................................5
ION EXCHANGE CHROMATOGRAPHY:.................................................................................................5
PARAMETERS RELATED TO HPLC SEPARATION:.............................................................................6
FLOW RATE:..............................................................................................................................................6
RETENTION TIME:...................................................................................................................................6
RETENTION VOLUME:............................................................................................................................6
MIGRATION RATE:..................................................................................................................................6
CAPACITY FACTOR:................................................................................................................................6
EQUILIBRIUM CONSTANT AND PHASE RATIO:..............................................................................6
ADVANTAGE OF HPLC................................................................................................................................7
REFERENCES.................................................................................................................................................8

SUBMITTED TO: DR ERUM ZAHEER Page 1 of 8

JSMU- INSTITUTE OF PHARMACEUTICAL SCIENCES

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

INTRODUCTION:
High-performance liquid chromatography (HPLC), formerly referred to as high-pressure liquid
chromatography, is a technique in analytical chemistry used to separate, find, and quantify specific
components in mixtures. The mixtures can be originated from food, chemicals, pharmaceuticals,
biological, environmental and agriculture, etc., which have been dissolved into liquid solutions.
It relies on high pressure pumps, which deliver mixtures of various solvents, called the mobile phase,
which flows through the system, collecting the sample mixture on the way, delivering it into a
cylinder, called the column, filled with solid particles, made of adsorbent material, called the
stationary phase.
Each part in the sample interacts slightly different with the adsorbent material, causing different
migration rate for each part, leading to their separation, as they flow out of the column into a specific
detector. The output of the detector is a graph, called Chromatogram. Chromatograms are graphical
representations of the signal intensity versus time or volume, showing peaks, which represent
components of the sample, each appears in its respective time, called Retention Time, having area
proportional to its amount.
HPLC is widely used for manufacturing (e.g., during the production process of pharmaceutical and
biological products), legal (e.g., detecting performance enhancement drugs in urine), research (e.g.,
separating the components of a complex biological sample, or of similar synthetic chemicals from
each other), and medical (e.g., detecting vitamin D levels in blood serum) purposes. [1]

THE APPARATUS OF HPLC:

The “Basic Overview of the HPLC process" and its mechanisms have now been covered. Going into
more detail, HPLC consists of a variety of components, including a solvent delivery pump, a
degassing unit, a sample injector, a column oven, a detector, and a data processor. The HPLC flow
diagram and the role of each part.

SUBMITTED TO: DR ERUM ZAHEER Page 2 of 8

JSMU- INSTITUTE OF PHARMACEUTICAL SCIENCES

As for HPLC, the pump delivers the mobile phase at a controlled flow rate(a). Air can easily
dissolve in the mobile phase under the standard atmospheric pressure in which we live in. If the
mobile phase holds air bubbles and enters the delivery pump, troubles such as flow rate fluctuations
and baseline noise/drift may occur. The degassing unit helps prevent this issue by removing air
bubbles in the mobile phase(b). After the dissolved air has been removed, the mobile phase is
delivered to the column. The sample injector then introduces a standard solution or sample solution
into the mobile phase (c). Temperature fluctuations can affect the separation of compounds in the
column. The column is placed in a column oven to keep the temperature constant(d). Compounds
eluted from the column are detected by a detector which is placed downstream of the column(e). A
workstation processes the signal from the detector to obtain a chromatogram to identify and quantify
the compounds(f).

HPLC SEPARATION:
HPLC can separate and detect each
compound by the difference of each
compound's speed through the column.
There are two phases for HPLC: the
mobile phase and the stationary phase.
The mobile phase is the liquid that
dissolves the target compound. The
stationary phase is the part of a column
that interacts with the target compound.
In the column, the stronger the affinity
(e.g., van der Waals force) between the
part and the mobile phase, the faster the

SUBMITTED TO: DR ERUM ZAHEER Page 3 of 8

JSMU- INSTITUTE OF PHARMACEUTICAL SCIENCES

part moves through the column along with the mobile phase. On the other hand, the stronger the
affinity with the stationary phase, the slower it moves through the column. Fig. 3 shows an example
in which the yellow part has a strong affinity with the mobile phase and moves quickly through the
column, while the pink part has a strong affinity with the stationary phase and moves through slowly.
The elution speed in the column depends on the affinity between the compound and the stationary
phase. [2]

HOW TO READ A CHROMATOGRAM:

The word "chromatogram" means a plot obtained via chromatography. An example of a
chromatogram. The chromatogram is a two-dimensional plot with the vertical axis showing
concentration in terms of the detector signal intensity and the horizontal axis standing for the
analysis time. When no compounds are eluted from the column, a line parallel to the horizontal axis
is plotted. This is called the baseline. The detector responds based on the concentration of the target
compound in the elution band. The obtained plot is more like the shape of a bell rather than a
triangle. This shape is called a “peak”.
Retention time (tR) is the time interval between sample injection point and the apex of the peak. The
required time for non-retained compounds (compounds with no interaction for the stationary phase)
to go from the injector to the detector is called the dead time (t0).
The peak height (h) is the vertical distance between a peak's apex and the baseline, and the peak area
(A) colored in light blue is the area enclosed by the peak and baseline. These results will be used for
the qualitative and quantitative analysis of a sample's components. [3]

SUBMITTED TO: DR ERUM ZAHEER Page 4 of 8

JSMU- INSTITUTE OF PHARMACEUTICAL SCIENCES

TYPES OF HPLC:
The two most common variants are normal-phase and reversed-phase HPLC.
NORMAL-PHASE HPLC
The column is filled with tiny silica particles, and a non-polar solvent, for example, hexane. A typical
column has an internal diameter of 4.6 mm or smaller and a length of 150 to 250 mm. non-polar
compounds in the mixture will pass more quickly through the column, as polar compounds will stick
longer to the polar silica than non-polar compounds will.
REVERSED-PHASE HPLC
The column size is the same. The column is filled with silica particles which are modified to make
them non-polar. This is done by attaching long hydrocarbon chains (8–18 C atoms) to its surface. A
polar solvent is used, for example, a mixture of water and an alcohol such as methanol. Polar
compounds in the mixture will pass more quickly through the column because a strong attraction
occurs between the polar solvent and the polar molecules in the mixture.
Non-polar molecules are slowed down on their way through the column. They form varying degrees
of attraction with the hydrocarbon groups principally through van der Waals dispersion forces and
hydrophobic interactions. They are also less soluble in the aqueous mobile phase components
helping their interactions with the hydrocarbon groups. Reversed phase HPLC is the most used form
of HPLC. [4]

ION EXCHANGE CHROMATOGRAPHY:

The ion exchange mechanism is based on electrostatic interactions between hydrated ions from a
sample and oppositely charged functional groups on the stationary phase. Two types of mechanisms
are used for the separation: in one mechanism, the elution uses a mobile phase that holds competing
ions that would replace the analyte ions and push them off the column; another mechanism is to add
a complexing reagent in the mobile phase and to change the sample species from their first form.
This modification on the molecules will lead them to elution. In addition to the exchange of ions,
ion-exchange stationary phases can retain specific neutral molecules. This process is related to the
retention based on the formation of complexes, and specific ions such as transition metals can be
kept on a cation-exchange resin and can still accept lone-pair electrons from donor ligands. Thus,
neutral ligand molecules can be retained on resins treated with the transitional metal ions.
The modern ion exchange is capable of quantitative applications at rather low solute concentrations
and can be used in the analysis of aqueous samples for common inorganic anions (range 10 μg/L to
10 mg/L). Metal cations and inorganic anions are all separated predominantly by ionic interactions
with the ion exchange resin. One of the largest industrial users of ion exchange is the food and
beverage sector to determine the nitrogen-, sulfur-, and phosphorous- containing species as well as
the halide ions. Also, ion exchange can be used to determine the dissolved inorganic and organic ions
in natural and treated waters. [5]

SUBMITTED TO: DR ERUM ZAHEER Page 5 of 8

JSMU- INSTITUTE OF PHARMACEUTICAL SCIENCES

PARAMETERS RELATED TO HPLC SEPARATION:

FLOW RATE:
Flow rate shows how fast the mobile phase travels across the column and is often used for
calculation of the consumption of the mobile phase in a given time interval. There are volumetric
flow rate U and linear flow rate u. where A is the area of the channel for the flow,
U=Au
A = (1/4) πεd2
RETENTION TIME:
The retention time (tR) can be defined as the time from the injection of the sample to the time of
compound elution, and it is taken at the apex of the peak that belongs to the specific molecular
species. The retention time is decided by several factors including the structure of the specific
molecule, the flow rate of the mobile phase, column dimension. And the dead time t0 is defined as
the time for a non-retained molecular species to elute from the column. [6]
RETENTION VOLUME:
Retention volume (VR) is defined as the volume of the mobile phase flowing from the injection time
until the corresponding retention time of a molecular species. The retention volume related to the
dead time is known as dead volume V0.
VR = UtR
MIGRATION RATE:
The migration rate can be defined as the velocity at which the species moves through the column.
And the migration rate (UR) is inversely proportional to the retention times. If only a fraction of
molecules that are present in the mobile phase are moving.
uR = u X Vmo/(Vmo+Vst)
CAPACITY FACTOR:
Capacity factor (k) is the ratio of reduced retention time and the dead time,
K = (tR−t0)/t0 = (vR−v0)/v0
EQUILIBRIUM CONSTANT AND PHASE RATIO:
In the separation, the molecules running through the column can also be considered as being in a
continuous equilibrium between the mobile phase and the stationary phase. An equilibrium constant
K could govern this equilibrium, in which Cmo is the molar concentration of the molecules in the
mobile phase, and Cst is the molar concentration of the molecules in the stationary phase.
K = Cst/Cmo
K = k(V0/Vst)

SUBMITTED TO: DR ERUM ZAHEER Page 6 of 8

JSMU- INSTITUTE OF PHARMACEUTICAL SCIENCES

ADVANTAGE OF HPLC
The most important aspect of HPLC is the high separation capacity which enables the batch analysis
of multiple components. Even if the sample consists of a mixture, HPLC will allows the target
components to be separated, detected, and quantified. Also, under appropriate condition, it is possible
to attain a high level of reproducibility with a coefficient of variation not exceeding 1%. Also, it has
a high sensitivity while a low sample consumption. HPLC has one advantage over GC column that
analysis is possible for any sample can be stably dissolved in the eluent and need not to be vaporized.
With this reason, HPLC is used much more often in the field of biochemistry and pharmaceutical
than the GC column. [7]

SUBMITTED TO: DR ERUM ZAHEER Page 7 of 8

JSMU- INSTITUTE OF PHARMACEUTICAL SCIENCES

REFERENCES

[1] Y. Kazakevich and R. e. LoBrutto, "HPLC for pharmaceutical scientists.," wikipedia, 2007.

[2] V. Koester, "What is HPLC?," Chemistry views, June 20, 2016.

[3] J. W. &. Sons., "Practical HPLC methodology and applications," Bidlingmeyer, 1993.

[4] P. M. V. R. &. A. R., "High Performance Liquid chromatography," OpenStax CNX.

[5] J. W. &. Sons., "The HPLC expert: Possibilities and Limitations of Modern high performance liquid
chromatography.," Kromidas, Stavros, ed. , 2016.

[6] T. Hanai, "a practical guide.," Royal Society of Chemistry, vol. 6, 1999.

[7] Sarah Millar, "The quick and efficient setting up of a column can take years to master. Here are some tips
and tricks to set up the perfect column," ChemistryViews.org , 2012.

SUBMITTED TO: DR ERUM ZAHEER Page 8 of 8