Introduction to HPLC by Saif Ali Raza
Chromatography
"Chromatography" comes from the Greek words : "chroma" (χρῶμα) meaning
"color" and "graphein" (γράφειν) meaning "to write" It was originally coined to describe the
separation of colored substances.
➢ Chromatography is defined as a set of techniques which is used for the separation of
constituents in a mixture. This technique involves 2 phases stationary phase and
mobile phase.
➢ The separation of constituents is based on the difference between partition
coefficients of the two phases. The chromatography is very popular technique and it
is mostly used analytically.
➢ There are different types of chromatographic techniques namely
• Paper Chromatography,
• Thin Layer Chromatography (TLC),
• Gas Chromatography,
• Liquid Chromatography,
• Ion exchange Chromatography and lastly
• High Performance Liquid Chromatography (HPLC).
References :
• Snyder, L. R., Kirkland, J. J., & Dolan, J. W. (2010). Introduction to Modern Liquid
Chromatography (3rd ed.). Wiley.
• Skoog, D. A., Holler, F. J., & Crouch, S. R. (2017). Principles of Instrumental Analysis
(7th ed.). Cengage Learning.
• Ettre, L. S. (2002). The Origin of the Word Chromatography. LC GC North America,
20(10), 972–974.
High performance liquid chromatography (HPLC)
HPLC stands for High Performance
Liquid Chromatography. The term "High
Performance Liquid Chromatography"
evolved from an earlier term, "High Pressure
Liquid Chromatography", which was used in
the early stages of the technique due to the use
of high pressures to push the mobile phase
through the column. As the technology
improved and the performance (speed,
resolution, and sensitivity) of the system
became the defining feature, the terminology
shifted to emphasize "performance" rather
than "pressure".
The invention of High-Performance Liquid
Chromatography (HPLC) is attributed to Jim R.
H. Giddings, who is often credited with the
Contact # 0323 – 4791765 1
, Introduction to HPLC by Saif Ali Raza
development of high-pressure techniques in liquid chromatography. In the 1960s, Giddings
made a significant contribution to improving the speed and resolution of liquid
chromatography by introducing the use of high-pressure systems to force the mobile
phase through the column, which drastically improved the separation process.
However, the concept of HPLC as we know it today came into its modern form in the
early 1970s when the combination of small particle stationary phases, higher pressures,
and faster analysis times were implemented, moving it beyond conventional
chromatography. The first commercial systems of HPLC were introduced by companies
like Waters Corporation and Perkin-Elmer in the mid-1970s, making the technology widely
accessible.
Reference :
• Giddings, J. R. (1965). "Unified Separation Science". Wiley.
Introduction to HPLC
High Performance Liquid Chromatography (HPLC) is an advanced and highly
efficient chromatographic technique used primarily for the separation, identification, and
quantitative analysis of components in a complex mixture. It operates by passing a liquid
mobile phase containing the sample through a column packed with a finely divided
stationary phase under high pressure (typically 5–400 bar). The separation of analytes is
achieved based on their differential interactions—such as partitioning, adsorption, or size
exclusion—with the stationary and mobile phases. Due to its high resolution, speed, and
sensitivity, HPLC is widely applied in pharmaceuticals, environmental analysis, food testing,
and clinical research.
• Reference :
Snyder, L. R., Kirkland, J. J., & Dolan, J. W. (2010). Introduction to Modern Liquid
Chromatography (3rd ed.). Wiley.
• Skoog, D. A., Holler, F. J., & Crouch, S. R. (2017). Principles of Instrumental Analysis
(7th ed.). Cengage Learning.
Harris, D. C. (2015). Quantitative Chemical Analysis (9th ed.). W. H. Freeman.
Basic Concept of HPLC :
1. Chromatographic Principle : HPLC is
based on the principle of
chromatography, where components
of a mixture are separated due to
differences in their distribution between
a stationary phase (solid or liquid
phase packed inside a column) and a
mobile phase (liquid that moves
through the column). The separation
occurs due to differential interactions
between the analytes and the stationary
phase.
Contact # 0323 – 4791765 2
Chromatography
"Chromatography" comes from the Greek words : "chroma" (χρῶμα) meaning
"color" and "graphein" (γράφειν) meaning "to write" It was originally coined to describe the
separation of colored substances.
➢ Chromatography is defined as a set of techniques which is used for the separation of
constituents in a mixture. This technique involves 2 phases stationary phase and
mobile phase.
➢ The separation of constituents is based on the difference between partition
coefficients of the two phases. The chromatography is very popular technique and it
is mostly used analytically.
➢ There are different types of chromatographic techniques namely
• Paper Chromatography,
• Thin Layer Chromatography (TLC),
• Gas Chromatography,
• Liquid Chromatography,
• Ion exchange Chromatography and lastly
• High Performance Liquid Chromatography (HPLC).
References :
• Snyder, L. R., Kirkland, J. J., & Dolan, J. W. (2010). Introduction to Modern Liquid
Chromatography (3rd ed.). Wiley.
• Skoog, D. A., Holler, F. J., & Crouch, S. R. (2017). Principles of Instrumental Analysis
(7th ed.). Cengage Learning.
• Ettre, L. S. (2002). The Origin of the Word Chromatography. LC GC North America,
20(10), 972–974.
High performance liquid chromatography (HPLC)
HPLC stands for High Performance
Liquid Chromatography. The term "High
Performance Liquid Chromatography"
evolved from an earlier term, "High Pressure
Liquid Chromatography", which was used in
the early stages of the technique due to the use
of high pressures to push the mobile phase
through the column. As the technology
improved and the performance (speed,
resolution, and sensitivity) of the system
became the defining feature, the terminology
shifted to emphasize "performance" rather
than "pressure".
The invention of High-Performance Liquid
Chromatography (HPLC) is attributed to Jim R.
H. Giddings, who is often credited with the
Contact # 0323 – 4791765 1
, Introduction to HPLC by Saif Ali Raza
development of high-pressure techniques in liquid chromatography. In the 1960s, Giddings
made a significant contribution to improving the speed and resolution of liquid
chromatography by introducing the use of high-pressure systems to force the mobile
phase through the column, which drastically improved the separation process.
However, the concept of HPLC as we know it today came into its modern form in the
early 1970s when the combination of small particle stationary phases, higher pressures,
and faster analysis times were implemented, moving it beyond conventional
chromatography. The first commercial systems of HPLC were introduced by companies
like Waters Corporation and Perkin-Elmer in the mid-1970s, making the technology widely
accessible.
Reference :
• Giddings, J. R. (1965). "Unified Separation Science". Wiley.
Introduction to HPLC
High Performance Liquid Chromatography (HPLC) is an advanced and highly
efficient chromatographic technique used primarily for the separation, identification, and
quantitative analysis of components in a complex mixture. It operates by passing a liquid
mobile phase containing the sample through a column packed with a finely divided
stationary phase under high pressure (typically 5–400 bar). The separation of analytes is
achieved based on their differential interactions—such as partitioning, adsorption, or size
exclusion—with the stationary and mobile phases. Due to its high resolution, speed, and
sensitivity, HPLC is widely applied in pharmaceuticals, environmental analysis, food testing,
and clinical research.
• Reference :
Snyder, L. R., Kirkland, J. J., & Dolan, J. W. (2010). Introduction to Modern Liquid
Chromatography (3rd ed.). Wiley.
• Skoog, D. A., Holler, F. J., & Crouch, S. R. (2017). Principles of Instrumental Analysis
(7th ed.). Cengage Learning.
Harris, D. C. (2015). Quantitative Chemical Analysis (9th ed.). W. H. Freeman.
Basic Concept of HPLC :
1. Chromatographic Principle : HPLC is
based on the principle of
chromatography, where components
of a mixture are separated due to
differences in their distribution between
a stationary phase (solid or liquid
phase packed inside a column) and a
mobile phase (liquid that moves
through the column). The separation
occurs due to differential interactions
between the analytes and the stationary
phase.
Contact # 0323 – 4791765 2
