APPLICATIONS OF

CHROMATOGRAPHY
Assignment No. 02

Submitted By
Faraz Nabeel
L1F17BSCH7043

Submitted to
Dr. Anum Tahir

Submission Date
November 24, 2021
 Contents
1 Chromatography Introduction to Chromatography............................................................2

1.1 Working......................................................................................................................2

1.2 Principle......................................................................................................................2

2 Applications of Chromatography.......................................................................................3

2.1 Applications of Liquid Chromatography....................................................................3

2.2 Applications of Gas Chromatography.........................................................................4

2.2.1 Food analysis.......................................................................................................4

2.2.2 Quality control.....................................................................................................4

2.2.3 Research...............................................................................................................4

2.2.4 Forensics..............................................................................................................4

2.2.5 Blood alcohol analysis.........................................................................................5

2.2.6 GCMS..................................................................................................................5

2.2.7 GC-Atomic Absorption detector..........................................................................5

2.3 Applications of Super critical fluid Chromatography.................................................5

2.3.1 SFC in bioanalysis...............................................................................................5

2.3.2 SFC in pharmaceutical analysis...........................................................................6

2.3.3 Food analysis.......................................................................................................6

2.3.4 Hyphenated SFC..................................................................................................6

2.3.5 Future perspective................................................................................................6

3 References..........................................................................................................................7
1 Chromatography Introduction to Chromatography
Chromatography comprises a group of powerful separation methods that allow the
separation, identification, and determination of closely related components of complex
mixtures, many of these separations are impossible by other means. Chromatography finds
applications in all branches of science. Chromatography was invented and named by the
Russian botanist Mikhail Tswett shortly after the turn of the last century. He used the
technique to separate various plant pigments such as chlorophylls and xanthophylls by
passing solutions of these compounds through a glass column packed with finely divided
calcium carbonate. The separated species appeared as colored bands on the column, which
accounts for the name he chose for the method (Greek chroma meaning “color” and graphein
meaning “writing”).[1]

1.1 Working
In all chromatographic separations the sample is dissolved in a mobile phase, which may be a
gas, a liquid, or a supercritical fluid. This mobile phase is then forced through an immiscible
stationary phase, which is fixed in place in a column or on a solid surface. The two phases
are chosen so that the components of the sample distribute themselves between the mobile
and stationary phases to varying degrees. Those components strongly retained by the
stationary phase move only slowly with the flow of mobile phase. In contrast, components
that are weakly held by the stationary phase travel rapidly. Because of these differences in
migration rates, sample components separate into discrete bands, or zones, that can be
analyzed qualitatively and quantitatively.[1]

1.2 Principle
Chromatography is
based on the principle of
separation. According to
separation principle the
components are
separated on the basis of
their relative affinities
with stationary and Figure 1: Separation of ink mixture by chromatigraphy
mobile phase. The components have having higher affinity with mobile phase move faster
and the components having higher affinity with stationary move slowly. [1]

2 Applications of Chromatography
The applications of chromatography have grown explosively in the last half century, due not
only to the development of several new types of chromatographic techniques but also to the
growing need by scientists for better methods for characterizing complex mixtures. Different
types of the chromatographic techniques are utilized for different purposes, while all of them
have a universal application i.e. separation of different components like:

 Separation of amino Acids

 Separation of Inks
 Separation Dyes
 Separation of Food colors
 Separation of Analyte from impurities

As we have three major classes of chromatographic methods based on their differences in

mobile and stationary phase i.e. Liquid chromatography, Gas Chromatography and Super
critical fluid chromatography. Detailed applications of all the classes are given below.

2.1 Applications of Liquid Chromatography

Liquid chromatography is used for much more than testing ink samples, liquid
chromatography is commonly used for environmental analysis, food analysis, quality control,
and cleanliness testing.

 Size exclusion chromatography with low-angle laser light scattering detection

(SEC/LALLS) has been applied to the separation and characterization of a number of
proteins as a function of molecular weight using different SEC columns and elution
buffers. [2]
 High performance liquid chromatography (HPLC) is a well known analytical
technique in the study of crude oil, and it has recently become more integrated within
the field of petroleomics as a first step before high-resolution mass spectrometric
analysis.[3]
  : High performance liquid chromatography (HPLC) coupled with mass spectrometry
(MS) is an extremely powerful and indispensable methodology practiced in virtually
every stage of pharmaceutical discovery and development processes, including
biological target discovery, biological assay for high throughput screening,
characterization of physicochemical properties of drug candidates, and drug
metabolism and pharmacokinetics. With continued advances and innovations in the
areas of column technologies, LC/MS interfaces, and instrumentation, the field of
LC/MS is strengthening and expanding to new disciplines constantly. [4]

2.2 Applications of Gas Chromatography

2.2.1 Food analysis
The food industry relies on the technique of gas chromatography for several applications,
including the quantitative and qualitative analysis of food, the analysis of food additives,
components of flavor and aroma, and the detection and analysis of contaminants such as
environmental pollutants, pesticides, fumigants, and naturally occurring toxins.

2.2.2 Quality control

Manufacturing relies on gas chromatography for quality control, companies that produce
cars, chemicals, and pharmaceuticals, in particular, are big adopters of the technology. The
pharmaceutical industry uses gas chromatography to ensure the purity of the produced
material, analyze compounds to check for trace contaminants

2.2.3 Research
Scientists use gas chromatography to analyze the composition of meteorites that fall to the
earth. This provides vitally important information regarding the nature of life outside of
earth, as well as revealing details about primitive life on earth. Specifically, many studies
have been conducted with gas chromatography to determine the presence of ribose in
meteorites, the building block of RNA.

2.2.4 Forensics
For many years gas chromatography has been used in forensic science. Mostly, it is used to
determine the circumstances of a person’s death, such as whether they ingested poison, or
consumed drugs or alcohol in the hours prior. Scientists take samples of blood and fibers
from the crime scene and analyze them using gas chromatography to help investigators piece
together the facts.

2.2.5 Blood alcohol analysis

Gas chromatography has been used since the 1950s to detect blood alcohol levels. Since then,
it has continued to be used to detect how much alcohol a person has consumed to help gauge
how impaired their normal functioning may be. Also, it has been adopted by forensic science
to determine blood alcohol levels at the time of death.

2.2.6 GCMS
Gas chromatography coupled to high-resolution mass spectrometry is a powerful analytical
method that combines excellent separation power of gas chromatography with improved
identification based on an accurate mass measurement. These features designate gas
chromatography with high-resolution mass spectrometry as the first choice for identification
and structure elucidation of unknown volatile and semi-volatile organic compounds.[2]

2.2.7 GC-Atomic Absorption detector

An atomic emission detector for gas chromatography has been developed which is, in
principle, capable of detecting selectively any element (except helium) which can elute from
a gas chromatographic column. Software methods have been developed so far for 23
elements and on isotope, including all of the organic elements along with mercury, lead, tin,
silicon, and deuterium.

2.3 Applications of Super critical fluid Chromatography

For 30 years, the use of packed column supercritical fluid chromatography (pSFC) has been
rising up in numerous analytical fields because of many physical and chemical advantages of
the fluids used as a mobile phase, in combination with a large range of packed columns
available.[5]

2.3.1 SFC in bioanalysis

In bioanalysis, the application of SFC methodology has extended to the separation and
identification of both endogenous and exogenous molecules including their metabolites.
Endogenous compounds in biological samples involve amino acids, proteins, sugars, fatty
acids, steroids and lipids, while exogenous compounds are mainly focused on pharmaceutical
molecules and their metabolites which will be discussed in the section ‘SFC in
pharmaceutical analysis’.[6]

2.3.2 SFC in pharmaceutical analysis

With the expansion of SFC technology, its application in pharmaceutical analysis has been a
new domain. Pharmaceutical analysis related to SFC methodology focuses on chiral
separations, natural products isolation, pharmaceutical metabolism and fingerprint study.[6]

2.3.3 Food analysis

Recently, supercritical fluid chromatography (SFC) has attracted considerable attention for
their application in food analysis. The use of supercritical CO2 (SC–CO2), as a mobile phase
for SFC, with its low viscosity and high molecular diffusiveness, results in shorter analysis
time and lower consumption of organic solvents as compared to high-performance liquid
chromatography (HPLC). In addition, with recent improvements in its detection system, SFC
has shown satisfactory selectivity and sensitivity.[7]

2.3.4 Hyphenated SFC

It was found in various reports that SFC and its hyphenation with mass spectroscopy (MS),
ultraviolet spectroscopy (UV), high-resolution mass spectrometry (HRMS), photodiode array
detector (PDA), nuclear magnetic resonance (NMR), charged aerosol detector (CAD), etc.,
have made possible to quantify analytes even in ultra-small concentrations in complex
matrices. These techniques have been successfully employed for the quantification of a wide
variety of analytes with excellent accuracy, selectivity, and sensitivity.[8]

2.3.5 Future perspective

Due to the distinct properties of SFC, it has not only been considered as a green approach,
but also a high-throughput, high-performance, sensitive, versatile method for bioanalytical
and pharmaceutical analytical field. At modern times, the specific areas for SFC technique
focus on multidimensional applications and hyphenation with advanced detectors, especially
MS.[6]
3 References
1. Skoog, D.A., F.J. Holler, and S.R. Crouch, Principles of instrumental analysis. 2017:
Cengage learning.
2. Santos, I.C. and K.A. Schug, Recent advances and applications of gas
chromatography vacuum ultraviolet spectroscopy. Journal of separation science,
2017. 40(1): p. 138-151.
3. Heshka, N.E., Applications of High Performance Liquid Chromatography in the
Petroleomic Analysis of Crude Oil: A Mini-Review. Energy & Fuels, 2021.
4. Lee, H., Pharmaceutical applications of liquid chromatography coupled with mass
spectrometry (LC/MS). Journal of liquid chromatography & related technologies,
2005. 28(7-8): p. 1161-1202.
5. Lesellier, E. and C. West, 7. Applications of supercritical fluid chromatography:
Natural products in pharmaceutical, cosmetic, and food applications, in Supercritical
Fluid Chromatography. 2018, De Gruyter. p. 139-172.
6. Chen, J. and C. Lou, Applications of supercritical fluid chromatography technique in
current bioanalysis and pharmaceutical analysis. 2020, Future Science.
7. Liu, L.-x., et al., The application of supercritical fluid chromatography in food
quality and food safety: an overview. Critical reviews in analytical chemistry, 2020.
50(2): p. 136-160.
8. Soni, P., et al., Recent Applications of Supercritical Fluid Chromatography in
Modern Analysis: Updates from 2017 to 2020. Current Analytical Chemistry, 2021.
17(6): p. 857-882.