CHROMATOGRAPHY

It is analytical method in which active constituent in complex mixture is separated. It is

technique is used for separation, purification, Identification and extraction of compound.  It is
method it can consist of two phases stationary phase and mobile phase. Stationary phase is
constant phase or column packaging material. Mobile phase is moveable phase. The basic
principle of chromatography is based on Adsorption and partition chromatography. 

HPLC (High Performance liquid Chromatography)

High performance liquid chromatography (HPLC) is a chromatographic technique used to
separate a mixture of compounds in analytical chemistry and biochemistry with the purpose
of identifying, quantifying or purifying the individual components of the mixture. It is column
chromatography and Liquid Chromatography. It is a modified from of gas chromatography
and is applicable for both Volatile as well as Non volatile compound.
It can mainly divided by two types, Normal phase and Reversed Phase
It is having a high resolution and separation capacity.  It is used as qualitative as well as
quantitative analysis.

Principle
High Performance Liquid Chromatography [HPLC] is principle is based on adsorption as well
as partition chromatography is depending on the nature of stationary phase, if stationary
phase is solid principle is based on adsorption chromatography and if stationary phase is
liquid principle is based on partition chromatography. It is important for determination of
Retention Time (the time is required , after sample injection maximum angle peak reaches to
detector).

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How Does HPLC Work?

The mixture moves through the column at varying velocities and interacts with the sorbent,
also known as the stationary phase. The velocity of each component in the mixture depends
on
i. Its chemical nature,
ii. The nature of the column
iii. The composition of the mobile phase.
The time at which a specific analyte emerges from the column is termed as its retention time.
The retention time is measured under specific conditions and considered as the identifying
characteristic of a given analyte.

Types of High-Performance Liquid Chromatography (HPLC)

1. Normal phase:
Column packing is polar (e.g silica) and the mobile phase is non-polar. It is used for
water-sensitive compounds, geometric isomers, cis-trans isomers, and chiral
compounds.

2. Reverse phase:
The column packing is non-polar (e.g C18), the mobile phase is water+ miscible
solvent (e.g methanol). It can be used for polar, non-polar, ionizable, and ionic
samples.

3. Ion exchange:
Column packing contains ionic groups and the mobile phase is buffer. It is used to
separate anions and cations.

4. Size exclusion:
Molecules diffuse into pores of a porous medium and are separated according to
their relative size to the pore size. Large molecules elute first and smaller molecules
elute later.

 ADVANTAGES
 It is simple, rapid, reproducible.
 High sensitivity.
 High performance.
 Rapid process and hence time saving.
 It is having a high resolution and separation capacity.
 Accuracy and Precision.
 Stationary phase was chemically inert. Wide varieties of stationary phase can be
used.
 Mobile phase was chemically inert. Less requirement of mobile phase in developing
chamber.
 Early recovery of separated component.
 Easy visualization of separated components. It is having good reproducibility and
repeatability.
 Important for validation of product, quality control studies of product.
 It is important for qualitative and quantitative analysis.
 It is used for both analytical and preparative purpose.

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LIMITATIONS
 Despite its advantages, HPLC can be costly, requiring large quantities of expensive
organics.
 HPLC does have low sensitivity for certain compounds, and some cannot be
detected as they are irreversibly adsorbed.
 Volatile substances are better separated by gas chromatography.

Applications
1. In endocrinology and toxicology:
o HPLC-MS has been employed as a selective alternative technique use in clinical
laboratories, especially in endocrinology and toxicology.
o It is used to overcome problems related to the analysis of TDM, steroid hormones,
and vitamins, new-born screening, and immunoassay.
o HPLC-MS s basic features are selectiveness, precision, efficiency, and analysis time.

2. For drug and biomarker analysis:

o HPLC is best suitable for substances that are not readily volatilized, thermally
unstable and have high molecular weight. It can therefore quantify a drug in its pure,
dosage form.
o Most important analytical method for identification and quantification of drugs. 
Either in their active pharmaceutical ingredient or in their formulations during the
process of their discovery, development and manufacturing.
o HPLC-MS is a reliable and effective quantitative method in drug and biomarker
analysis for the purpose of disease diagnosis and assessment.
o For the development of the method, the employment of a universal LC method is
preferred. Reversed-phase mode is the most commonly used for the analysis of a
mobile phase consisting of acetonitrile/methanol and formic/acetic acid, drug, and
biomarker especially in the C18 column and gradient mode.

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o In drug and biomarker analysis, the C18 column and the mobile phase consisting of
acetonitrile/methanol and formic/acetic acid in the gradient mode are most commonly
used ones.
o For example, HPLC analysis of the novel antipsychotic drug quetiapine in human
plasma.

3. Identification of Active pharmaceutical ingredients (API)

o One specific use case is ensuring the consistency of active pharmaceutical
ingredients (API).
o HPLC can provide quantitative analysis of select molecules, so you can confirm the
correct dosage of active ingredients.
o 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.
o Impurities can pose a serious safety risk to patients, and their detection and
identification is often facilitated by the use of HPLC.
o Standard HPLC techniques may be combined with highly efficient detection methods
(such as UV detection) to provide a complete and accurate impurity profile.

4. Evaluating the stability of pharmaceutical products

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o HPLC can be vital in evaluating the stability of pharmaceutical products.
o The composition of formulations can alter over time due to a variety of environmental
factors, such as exposure to humidity, oxygen, heat, and light.
o HPLC can assist in the identification of degradation products as well as determine
the extent of change over time.

5. Determination of shelf-life
o Indeed, Byrd notes that HPLC can also be useful in determining shelf life; “for
example, some biotherapeutics are sensitive to aggregation over time, or if not stored
properly, and HPLC can be used to monitor this aggregation.”

6. Quantity of drug determination from pharmaceutical dosage forms

o For example, HPLC method for simultaneous estimation of paracetamol and
aceclofenac in tablets.
o A simple, selective, rapid, precise and economical reverse phase HPLC method has
been developed for the simultaneous estimation of paracetamol and aceclofenac
from pharmaceutical dosage forms. The retention time of paracetamol, aceclofenac
and etoricoxib was 4.75, 6.44 and 8.83 min, respectively.
o Example HPLC Estimation of Risperidone in Tablet Dosage Forms. The flow rate
was 1.3 ml/min and effluents were monitored at 234 nm. The retention time of
risperidone and clozapine were 2.5 min and 3.3 min, respectively.
o Determination of metformin in human plasma using ion-pair. Metformin is an
antidiabetic agent which used in the treatment of non-insulin -dependent diabetes

References:
https://edubirdie.com/examples/application-of-hplc-in-pharmaceutical-industry/

https://microbenotes.com/high-performance-liquid-chromatography-hplc/#:~:text=High
%2Dperformance%20liquid%20chromatography%20or,identified%20and%20quantified
%20by%20spectroscopy.

https://www.analyticaltoxicology.com/en/high-performance-liquid-chromatography-hplc/

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https://pubmed.ncbi.nlm.nih.gov/3307324/

https://edubirdie.com/examples/application-of-hplc-in-pharmaceutical-industry/

https://www.labmanager.com/product-focus/hplc-in-pharmaceutical-applications-25323

https://www.chromatographytoday.com/news/hplc-uhplc/31/breaking-news/how-is-hplc-
used-in-the-pharmaceutical-industry/56929

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