UNIVERSITY OF WAH
Application of HPLC
in Pharmaceutical
Industry
Submitted by: Laraib Zulfiqar
Submitted to: Mam Sana Javaid
Registration number: UW-20-CHM-BS-057
Course code:
Section: BS 6th B
3/17/2023
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Contents
1 Chromatography ..................................................................................................................... 2
2 High Performance Liquid Chromatography ........................................................................... 2
3 (HPLC) .................................................................................................................................... 2
3.1 History: ............................................................................................................................. 2
3.2 Working principle: ........................................................................................................... 2
3.3 Instrumentation: ............................................................................................................... 3
4 Pharmaceutical Industry.......................................................................................................... 3
4.1 Role of HPLC in Pharmaceutical Industry:...................................................................... 3
4.1.1 Drug analysis: ........................................................................................................... 3
4.1.2 Analysis of difficult components: ............................................................................. 4
4.1.3 Quantification of drug: .............................................................................................. 4
4.1.4 Identification of active ingredients: .......................................................................... 5
4.1.5 Quality control: ......................................................................................................... 5
4.1.6 Determination of impurities: ..................................................................................... 5
4.1.7 Evaluation of stability of pharmaceutical products: ................................................. 6
4.1.8 Determining shelf life of drugs: ................................................................................ 6
4.1.9 HPLC and biopharmaceuticals ................................................................................. 6
4.1.10 Toxicology and HPLC: ............................................................................................. 6
5 References: .............................................................................................................................. 7
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1 Chromatography
Chromatography is the technique for the separation, purification, and testing of compounds. The
term “chromatography” is derived from Greek, Chroma meaning, “color,” and graphein
meaning “to write.”
In this process, we apply the mixture to be separated on a stationary phase (solid or liquid) and a
pure solvent such as water or any gas is allowed to move slowly over the stationary phase,
carrying the components separately as per their solubility in the pure solvent.
2 High Performance Liquid Chromatography
3 (HPLC)
High-performance liquid chromatography (HPLC), formerly referred to as high-pressure liquid
chromatography, is a technique in analytical chemistry used to separate, identify, and quantify
each component in a mixture.
3.1 History:
In 1903, the Russian botanist Mikhail Tsvet invented the chromatographic technique when he
reported separations of different plant pigments into a series of colored bands on a packed
column. He called this technique „chromatography‟.
The first commercial HPLC was manufactured by Waters Corporation in 1969, and was known
as the ALC100 HPLC. Then in 1980‟s, HPLC was commonly used for the separation of
chemical compounds. New techniques improved separation, identification, purification and
quantification far beyond previous techniques, while computers and automation provided
convenience. Improvements in reproducibility were made as techniques such as micro-columns,
affinity columns, and fast HPLC emerged. HPLC gradually developed over the years, more by
evolution than revolution. Incremental improvements combined to generate an extremely
powerful tool capable of high precision and reproducibility.
3.2 Working principle:
The purification takes place in a separation column between a stationary and a mobile
phase.
The stationary phase is a granular material with very small porous particles in a
separation column. The mobile phase, on the other hand, is a solvent or solvent mixture
which is forced at high pressure through the separation column.
Via a valve with a connected sample loop, i.e. a small tube or a capillary made of
stainless steel, the sample is injected into the mobile phase flow from the pump to the
separation column using a syringe.
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Subsequently, the individual components of the sample migrate through the column at
different rates because they are retained to a varying degree by interactions with the
stationary phase.
After leaving the column, the individual substances are detected by a suitable detector
and passed on as a signal to the HPLC software on the computer.
At the end of this operation/run, a chromatogram in the HPLC software on the computer
is obtained. The chromatogram allows the identification and quantification of the
different substances.
3.3 Instrumentation:
4 Pharmaceutical Industry
The pharmaceutical industry discovers, develops, produces, and markets drugs or pharmaceutical
drugs for use as medications to be administered to patients, with the aim to cure them, vaccinate
them, or alleviates symptoms.
4.1 Role of HPLC in Pharmaceutical Industry:
HPLC is one of the most useful analytical methods in the development and manufacture of
pharmaceuticals. Its applications are not confined to just one area and it is instrumental in a
number of critical steps necessary for robust pharmaceutical analysis. Mentioned below are some
of the most important applications of HPLC in Pharmaceutical industry.
4.1.1 Drug analysis:
HPLC is the most important analytical method for identification and quantification of drug.
HPLC can provide results that are precise and reliable. The results acquired can be used to
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analyze finished drug products. This is achieved through separation, quantification and
identification of components of a mixture. Its applications are not confined to just one area and
are widespread.
4.1.2 Analysis of difficult components:
Most importantly, the method lends itself to the analysis of difficult components, including high
molecular weights, thermal instability and compounds which are difficult to volatilize. This
makes it suitable for the analysis of drugs in both their pure form and dosage form, such as pills,
drinks, powders, injections or inhalers.
4.1.3 Quantification of drug:
HPLC is a separation technique and components of sample on the basis of difference in nature of
components. Composition of drug in given sample can be found out using HPLC.
4.1.3.1 Quantity of drug determination from pharmaceutical dosage forms:
RP-HPLC is a method for simultaneous estimation of drug composition in tablets.
4.1.3.1.1 RP-HPLC:
Reversed-phase high-performance liquid chromatography (RP-HPLC) involves the separation of
molecules on the basis of hydrophobicity. The separation depends on the hydrophobic binding of
the solute molecule from the mobile phase to the immobilized hydrophobic ligands attached to
the stationary phase, i.e., the sorbent. RP-HPLC uses more polar mobile phase and less polar
stationary phase.
Example:
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It is selective, rapid, and economical method for simultaneous estimation of paracetamol and
acectofenac from paracetamol dosage form.
4.1.3.2 Quantity of drug determination from biological fluids:
We can also determine contents or amount of drug in body fluids i.e. Blood plasma from HPLC.
In this case peak areas of chromatogram give quantitative calculations.
Example:
Rapid detection of metformin in human plasma using ion pair HPLC.
4.1.4 Identification of active ingredients:
HPLC can be used for ensuring the consistency of active pharmaceutical ingredients (API). It
can provide quantitative analysis of selective molecules, so you can confirm the correct dosage
of active ingredients.
For Quality assurance Quality control testing, HPLC can be useful in ensuring critical quality
attributes such as strength/concentration, content uniformity, the detection and quantification of
impurities, and the quality and identity of raw materials.
4.1.5 Quality control:
Quality control within the pharmaceutical industry is imperative, especially through the
conduction of tests, experiments, and analyses. This often includes using various physical or
chemical separation techniques to determine the composition and purity of pharmaceutical
products.
One common method of solution separation is high-performance liquid chromatography
(HPLC). HPLC is a technique used to identify different compounds found in solutions by
separating and quantifying each mixed component. The process also reveals how different
compounds behave or react when mixed with different liquids.
4.1.6 Determination of impurities:
One of the main benefits of HPLC is its ability to determine the quantities of impurities in
pharmaceutical formulations. Impurities can pose a serious safety risk to patients, and their
detection and identification is often facilitated by the use of HPLC. Standard HPLC techniques
may be combined with highly efficient detection methods (such as UV detection) to provide a
complete and accurate impurity profile. Doing so gives developers a better idea of a drug‟s
properties, with each component‟s quantities affecting the overall performance and strength of a
product.
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4.1.7 Evaluation of stability of pharmaceutical products:
In a similar vein, HPLC can be vital in evaluating the stability of pharmaceutical products. The
composition of formulations can alter over time due to a variety of environmental factors, such
as exposure to humidity, oxygen, heat, and light. HPLC can assist in the identification of
degradation products as well as determine the extent of change over time.
4.1.8 Determining shelf life of drugs:
Indeed, Byrd notes that HPLC can also be useful in determining shelf life; “for example, some
bio therapeutics are sensitive to aggregation over time, or if not stored properly, and HPLC can
be used to monitor this aggregation.”
4.1.9 HPLC and biopharmaceuticals
Another area of pharmaceuticals for which HPLC is used, is the characterization of
biopharmaceuticals. Put simply, these are drug products made within and potentially extracted
from living organisms like bacteria or blood cells. It‟s a growing area of pharmaceuticals, with
many large companies even shifting their focus to biopharma. But it does come with a unique set
of characteristics that need to be analyzed in the development and production process.
Again, this is an area where HPLC can lend a hand. One example of this is reverse-phase HPLC,
which can be used to reveal characteristics at protein level. It separates proteins based on
hydrophobicity with solvophobic and electrostatic interactions.
4.1.10 Toxicology and HPLC:
HPLC is one of the separation techniques most frequently used in forensic toxicology. In
comparison to other chromatographic methods, it has particular advantages in cases of
nonvolatile, thermally sensitive, and high-molecular-weight substances that can be analyzed
under mild conditions and without derivatization.
In forensic toxicology, the identification and quantification of illegal and therapeutic drugs,
pesticides, and other organic poisons from human body fluids and tissue samples are the
dominant tasks. Since its invention in the mid-1970s, a steady development of HPLC techniques
has occurred with respect to general performance parameters, such as separation efficiency
(increasing plate number), sensitivity, reproducibility, robustness, automation, and applicability
to a wide range of analytes and matrices, as well as to adaptation for special analytical problems.
4.1.11 HPLC Characterization of Phytochemicals:
Traditionally, people gathered plant and animal resources for food, health care, and shelter. In
developing countries, millions of people derive a substantial part of their daily needs and
livelihood from animal and plant resources. There is high demand for wild species of plants due
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to interest in their medicinal properties. Currently, the overexploitation of natural resources,
particularly the wild species of plants, has attracted the attention of conservationists, and certain
species are threatened to go extinct in the near future if their overuse is not controlled. In the
modern world, medicinal plants are considered the backbone of pharmaceutical industries due to
their valuable phytochemical constituents.
HPLC can be used for determination of components in phytochemicals in plants.
4.1.12 Monitoring progress of therapy:
HPLC can be used to track progress of therapy and effectiveness of a drug.
Example:
It is a sensitive and precise method for the identification of Hb A2, Hb F and abnormal
hemoglobin. It has become the method of choice for thalassemia screening because of its speed
and reliability.
5 References:
1. https://www.chromatographytoday.com/news/hplc-uhplc/31/breaking-news/how-is-hplc-
used-in-the-pharmaceutical-industry/56929
2. https://www.europeanpharmaceuticalreview.com/article/116263/how-is-liquid-
chromatography-used-in-the-pharmaceutical-industry/
3. https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-
biology/reverse-phase-high-performance-liquid-chromatography
4. https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/abs/10.1002/jssc.202200064
5. https://pubmed.ncbi.nlm.nih.gov/15629016/
