MAKERERE UNIVERSITY

COLLEGE OF HEALTH SCIENCES

DEPARTMENT OF PHARMACOLOGY
PHARMACOLOGY REPORT
DATE: 24/4/17

AUTHOR: TURINAWE JUSSY

COURSE: MBCHB
SIGNATURE: …………………………….

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HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
OR HIGH-PRESSURE LIQUID CHROMATOGRAPHY

DEFINITION:
Technique in analytical chemistry used to separate, identify, and quantify each component in a mixture.

Or

High Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps a
sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through a column
with chromatographic packing material (stationary phase).

INTRODUCTION
High performance liquid chromatography is basically a highly improved form of column
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. That makes it much faster.

It also allows you to use a very much smaller particle size for the column packing material which gives a
much greater surface area for interactions between the stationary phase and the molecules flowing past
it. This allows a much better separation of the components of the mixture.

The other major improvement over column chromatography concerns the detection methods which can
be used. These methods are highly automated and extremely sensitive.

 It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column
filled with a solid adsorbent material. Each component in the sample interacts slightly differently with
the adsorbent material, causing different flow rates for the different components and leading to the
separation of the components as they flow out the column .

Chromatography can be described as a mass transfer process involving adsorption. HPLC relies on
pumps to pass a pressurized liquid and a sample mixture through a column filled with adsorbent, leading
to the separation of the sample components.

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TYPES/MODES OF CHROMATOGRAPHY

1) Partition chromatography

Partition chromatography uses a retained solvent, on the surface or within the grains or fibers of an
"inert" solid supporting matrix as with paper chromatography; or takes advantage of
some coulombic and/or hydrogen donor interaction with the stationary phase. Analyte molecules
partition between a liquid stationary phase and the eluent.

2) Normal–phase chromatography

 This method separates analytes based on their affinity for a polar stationary surface such as silica,
hence it is based on analyte ability to engage in polar interactions (such as  hydrogen-bonding or dipole-
dipole type of interactions) with the sorbent surface.

3) Displacement chromatography

The basic principle of displacement chromatography is: A molecule with a high affinity for the
chromatography matrix (the displacer) will compete effectively for binding sites, and thus displace all
molecules with lesser affinities

4) Reversed-phase chromatography (RPC)

Reversed phase HPLC (RP-HPLC) has a non-polar stationary phase and an aqueous, moderately polar
mobile phase. One common stationary phase is a silica which has been surface-modified with RMe 2SiCl,
where R is a straight chain alkyl group such as C 18H37 or C8H17. With such stationary phases, retention time
is longer for molecules which are less polar, while polar molecules elute more readily (early in the
analysis).

5) Size-exclusion chromatography

Size-exclusion chromatography (SEC), also known as  gel permeation chromatography or gel filtration

chromatography, separates particles on the basis of molecular size (actually by a particle's  Stokes
radius). It is generally a low resolution chromatography and thus it is often reserved for the final,
"polishing" step of the purification. It is also useful for determining the  tertiary structure and quaternary
structure of purified proteins

6) Ion-exchange chromatography

In ion-exchange chromatography (IC), retention is based on the attraction between solute ions and
charged sites bound to the stationary phase. Solute ions of the same charge as the charged sites on the
column are excluded from binding, while solute ions of the opposite charge of the charged sites of the
column are retained on the column. Solute ions that are retained on the column can be eluted from the
column by changing the solvent conditions (e.g. increasing the ion effect of the solvent system by

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increasing the salt concentration of the solution, increasing the column temperature, changing the pH of
the solvent, etc...).
Continuation of modes of chromatography

7) Bio affinity chromatography

This chromatographic process relies on the property of biologically active substances to form stable,
specific, and reversible complexes. The formation of these complexes involves the participation of
common molecular forces such as the Van der Waals interaction, electrostatic interaction, dipole-
dipole interaction, hydrophobic interaction, and the hydrogen bond. An efficient, bio specific bond is
formed by a simultaneous and concerted action of several of these forces in the complementary binding
sites.

8) Aqueous normal-phase chromatography

Aqueous normal-phase chromatography (ANP) is a chromatographic technique which encompasses the

mobile phase region between reversed-phase chromatography (RP) and organic normal phase
chromatography (ONP). This technique is used to achieve unique selectivity for hydrophilic compounds,
showing normal phase elution using reversed-phase solvents

A flow scheme for HPLC

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Visual components of HPLC

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COMPONENTS OF HPLC

The Pump:

The role of the pump is to propel (force) a liquid (the mobile phase) through
the chromatograph at a specific flow rate, expressed in ml/min. Normal flow rates in HPLC are 1-2
ml/min and typical pumps can reach pressures in the range of 2000 – 9000 psi but in applications
covered under UHPLC mode operating pressure can be as high as 15000-18000 psi. The normal flow
rate stability must be < 1%.

There are two types of pump operation:

Isocratic pump - delivers constant mobile phase composition

Gradient pump – delivers variable mobile phase composition

The Injector:

The sample should be introduced as a plug, without disturbing the column packing. The injector serves
to introduce the liquid sample into the flow stream of the mobile phase. Typical sample volumes are 5-
20 μL. The injector must be able to withstand the high pressure of the mobile phase. A pre column, a
small removable section of tubing containing the same packing material as the column, can be used
ahead of the analytical column to protect the latter from contamination. The pre column also acts as a
buffer to prevent channeling of the packing during injection.

The Column:

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The column is considered the heart of the chromatograph. The success or failure of a particular analysis
depends on the choice of column. The column’s stationary phase separates the sample components
using various physical and chemical parameters.

There are four types of columns used in HPLC:

High performance analytical columns [internal diameter (i.d.) 1.0-4.6 mm; lengths 15 –250 mm] - used
mainly for qualitative and quantitative analysis

Preparative columns (i.d. > 4.6 mm; lengths 50 –250 mm) – used mainly for preparative work

Capillary columns (i.d. 0.1 -1.0 mm; various lengths) 

Nano columns (i.d. < 0.1 mm)

Other components of HPLC

Detector

Detector records the relative concentrations of different components of the test sample with respect to
their retention time. Retention time is the time taken by the compound to elute from the column.
Retention time is calculated from the time of injection until the compound is eluted. Detection is based
on several different physical principals. 

Column Oven

Variation of temperature during the analytical run can result in changes of retention time of the
separated eluting components. A column oven maintains constant column temperature using air
circulation. This ensures a constant flow rate of the mobile phase through the column

HPLC Mobile Phase Reservoirs

These are inert containers for mobile phase storage and transport. Generally transparent glass
bottles are used so as to facilitate visual inspection of mobile phase level inside the container.
Stainless steel particulate filters are provided inside for removal of particulate impurities in the mobile
phase if any.

Data Acquisition & Control

Modern HPLC systems are computer based and software controls operational parameters such as
mobile phase composition, temperature, flow rate, injection volume and sequence and also
acquisition and treatment of output.

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METHOD OF OPERATION
The sample mixture to be separated and analyzed is introduced, in a discrete small volume (typically
microliters), into the stream of mobile phase percolating through the column. The components of the
sample move through the column at different velocities, which are a function of specific physical
interactions with the adsorbent (also called stationary phase). The velocity of each component depends
on its chemical nature, on the nature of the stationary phase (column) and on the composition of the
mobile phase. The time at which a specific analyte elutes (emerges from the column) is called its
retention time. The retention time measured under particular conditions is an identifying characteristic
of a given analyte.

Many different types of columns are available, filled with adsorbents varying in particle size, and in the
nature of their surface ("surface chemistry"). The use of smaller particle size packing materials requires
the use of higher operational pressure ("backpressure") and typically improves chromatographic
resolution (i.e. the degree of separation between consecutive analytes emerging from the column).
Sorbent particles may be hydrophobic or polar in nature. Common mobile phases used include any
miscible combination of water with various organic solvents (the most common
are acetonitrile and methanol). Some HPLC techniques use water-free mobile phases (see Normal-phase
chromatography below). The aqueous component of the mobile phase may contain acids (such as
formic, phosphoric or trifluoroacetic acid) or salts to assist in the separation of the sample components.
The composition of the mobile phase may be kept constant ("isocratic elution mode") or varied
("gradient elution mode") during the chromatographic analysis. Isocratic elution is typically effective in
the separation of sample components that are not very different in their affinity for the stationary
phase. In gradient elution the composition of the mobile phase is varied typically from low to high
eluting strength. The eluting strength of the mobile phase is reflected by analyte retention times with
high eluting strength producing fast elution (=short retention times). A typical gradient profile in
reversed phase chromatography might start at 5% acetonitrile (in water or aqueous buffer) and progress
linearly to 95% acetonitrile over 5–25 minutes. Periods of constant mobile phase composition may be

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part of any gradient profile. For example, the mobile phase composition may be kept constant at 5%
acetonitrile for 1–3 min, followed by a linear change up to 95% acetonitrile.

The chosen composition of the mobile phase (also called eluent) depends on the intensity of
interactions between various sample components ("analytes") and stationary phase (e.g. hydrophobic
interactions in reversed-phase HPLC). Depending on their affinity for the stationary and mobile phases
analytes partition between the two during the separation process taking place in the column. This
partitioning process is similar to that which occurs during a liquid–liquid extraction but is continuous,
not step-wise. In this example, using a water/acetonitrile gradient, more hydrophobic components
will elute (come off the column) late, once the mobile phase gets more concentrated in acetonitrile (i.e.
in a mobile phase of higher eluting strength).

The choice of mobile phase components, additives (such as salts or acids) and gradient conditions
depends on the nature of the column and sample components. Often a series of trial runs is performed
with the sample in order to find the HPLC method which gives adequate separation.

USES/ IMPORTANCE OF HPLC

Manufacturing
As briefly mentioned, HPLC has many applications in both laboratory and clinical science. It is a common
technique used in pharmaceutical development as it is a dependable way to obtain and ensure product
purity. While HPLC can produce extremely high quality (pure) products, it is not always the primary method
used in the production of bulk drug materials. According to the European pharmacopoeia, HPLC is used in
only 15.5% of syntheses. However, it plays a role in 44% of syntheses in the United States
pharmacopoeia. This could possibly be due to differences in monetary and time constraints, as HPLC on a
large scale can be an expensive technique. An increase in specificity, precision, and accuracy that occurs with
HPLC unfortunately corresponds to an increase in cost.

Legal
This technique is also used for detection of illicit drugs in urine. The most common method of drug detection
is an immunoassay. This method is much more convenient. However, convenience comes at the cost of
specificity and coverage of a wide range of drugs. As HPLC is a method of determining (and possibly
increasing) purity, using HPLC alone in evaluating concentrations of drugs is somewhat insufficient. With this,
HPLC in this context is often performed in conjunction with mass spectrometry.[] Using liquid chromatography
instead of gas chromatography in conjunction with MS circumvents the necessity for derivitizing with
acetylating or alkylation agents, which can be a burdensome extra step. This technique has been used to
detect a variety of agents like doping agents, drug metabolites, glucuronide conjugates, amphetamines,
opioids, cocaine, BZDs, ketamine, LSD, cannabis, and pesticides Performing HPLC in conjunction with Mass
spectrometry reduces the absolute need for standardizing HPLC experimental runs.

Research
Similar assays can be performed for research purposes, detecting concentrations of potential clinical
candidates like anti-fungal and asthma drugs. This technique is obviously useful in observing multiple species

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in collected samples, as well, but requires the use of standard solutions when information about species
identity is sought out. It is used as a method to confirm results of synthesis reactions, as purity is essential in
this type of research. However, mass spectrometry is still the more reliable way to identify species.

Medical
Medical use of HPLC can include drug analysis, but falls more closely under the category of nutrient analysis.
While urine is the most common medium for analyzing drug concentrations, blood serum is the sample
collected for most medical analyses with HPLC. Other methods of detection of molecules that are useful for
clinical studies have been tested against HPLC, namely immunoassays. In one example of this, competitive
protein binding assays (CPBA) and HPLC were compared for sensitivity in detection of vitamin D. Useful for
diagnosing vitamin D deficiencies in children, it was found that sensitivity and specificity of this CPBA reached
only 40% and 60%, respectively, of the capacity of HPLC. While an expensive tool, the accuracy of HPLC is
nearly unparalleled.

References

1) Nina Hadden et al., “Basic Liquid Chromatography”, Varian Aerograph, 1971

2) C.A. Dorschel, J.L. Ekmanis, J.E. Oberholtzer  et al. “LC Detectors” Anal. Chem., 61, 951A–968A, 1989

3) C. F. Simpson, “Techniques in Liquid Chromatography” Wiley-Hayden: Chichester, England, 1982

4) L.R. Snyder, J. L Glajch,  J. J. Kirkland, “Practical HPLC Method Development”, Wiley-Interscience: New
York, 1988.

5) V.R. Meyer, “Practical High – Performance Liquid Chromatography”, Wiley, 2010

6) M. McMaster, “LC/MS A Practical User’s Guide”, Wiley-Interscience, 2005

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