HPLC:

(High-Performance liquid chromatography)

Working Principle:
Chromatography

Analytes:
organic molecules
biomolecules
ions
polymers

High-performance liquid chromatography (HPLC), formerly high-pressure liquid

chromatography, is a technique in analytical chemistry used to separate, identify, and quantify
specific components in mixtures. The mixtures can be originated
from food, chemicals, pharmaceuticals,[1] biological, environmental and agriculture, etc, which have
been dissolved into liquid solutions. It relies on high-pressure pumps, which deliver mixtures of
various solvents, called the mobile phase, which flows through the system, collecting the sample
mixture on the way, delivering it into a cylinder, called the column, filled with solid particles, made
of adsorbent material, called the stationary phase. Each component in the sample interacts slightly
different with the adsorbent material, causing different migration rates for each component, leading
to their separation, as they flow out of the column into a specific detector. The output of the detector
is a graph, called Chromatogram. Chromatograms are graphical representations of the signal
intensity versus time or volume, showing peaks, which represent components of the sample, each
appears in its respective time, called Retention Time, having area proportional to its amount.
HPLC is widely used for manufacturing (e.g., during the production process of pharmaceutical and
biological products),[2][3] legal (e.g., detecting performance enhancement drugs in urine),[4] research
(e.g., separating the components of a complex biological sample, or of similar synthetic chemicals
from each other), and medical (e.g., detecting vitamin D levels in blood serum) purposes.[5]
Chromatography can be described as a mass transfer process involving adsorption and/or partition.
As mentioned, 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. The active
component of the column, the adsorbent, is typically a granular material made of solid particles
(e.g., silica, polymers, etc.), 1.5–50 μm in size, on which various reagents can be bonded. The
components of the sample mixture are separated from each other due to their different degrees of
interaction with the adsorbent particles. The pressurized liquid is typically a mixture of solvents (e.g.,
water, buffers, acetonitrile and/or methanol) and is called a "mobile phase". Its composition
and temperature play a major role in the separation process by influencing the interactions taking
place between sample components and adsorbent. These interactions are physical in nature, such
as hydrophobic (dispersive), dipole–dipole and ionic, most often a combination.

High-Performance Liquid Chromatography (HPLC) is used extensively in analytical chemistry for

various real-life applications. Here's a specific example:
Pharmaceutical Industry: HPLC plays a crucial role in pharmaceuticals to ensure the quality
and safety of drugs. Pharmaceutical companies use HPLC to analyze and quantify active
pharmaceutical ingredients (APIs), identify impurities, and ensure product consistency. For instance,
when manufacturing a specific medication, HPLC can be employed to confirm that the concentration
of the active ingredient falls within the required limits. If there are impurities present, HPLC can
detect and quantify them, ensuring the final drug product is pure and safe for consumption.

The analysis is typically done by preparing a sample of the drug or formulation, injecting it into the
HPLC system, and separating its components using a suitable column and mobile phase. The
detector then identifies and quantifies the various components. This quality control is critical to
meeting regulatory requirements and producing medications that are both effective and safe for
patients.

In summary, HPLC is used in pharmaceuticals to verify the quality, safety, and effectiveness of drug
products, illustrating its practical application in ensuring public health and safety.

Gas chromatography–mass spectrometry:

Gas chromatography–mass spectrometry (GC–MS) is an analytical method that combines the

features of gas-chromatography and mass spectrometry to identify different substances within a test
sample.[1] Applications of GC–MS include drug detection, fire investigation, environmental
analysis, explosives investigation, food and flavor analysis, and identification of unknown samples,
including that of material samples obtained from planet Mars during probe missions as early as the
1970s. GC–MS can also be used in airport security to detect substances in luggage or on human
beings. Additionally, it can identify trace elements in materials that were previously thought to have
disintegrated beyond identification. Like liquid chromatography–mass spectrometry, it allows
analysis and detection even of tiny amounts of a substance.[2]
GC–MS has been regarded as a "gold standard" for forensic substance identification because it is
used to perform a 100% specific test, which positively identifies the presence of a particular
substance. A nonspecific test merely indicates that any of several in a category of substances is
present. Although a nonspecific test could statistically suggest the identity of the substance, this
could lead to false positive identification. However, the high temperatures (300°C) used in the GC–
MS injection port (and oven) can result in thermal degradation of injected molecules,[3] thus resulting
in the measurement of degradation products instead of the actual molecule(s) of interest.

Environmental Analysis:
Gas Chromatography-Mass Spectrometry (GC-MS) is widely used in environmental analysis to detect
and quantify pollutants, toxins, and contaminants in air, water, soil, and sediments. This technology aids
in monitoring and ensuring the safety of the environment.

Example:
Detection of Volatile Organic Compounds (VOCs): In the analysis of air quality, GC-MS is employed to
detect VOCs such as benzene, toluene, and xylene, which can be emitted from industrial processes,
vehicle exhaust, or natural sources. These compounds can have harmful effects on air quality and
human health. By using GC-MS, environmental scientists can monitor and quantify the concentration of
VOCs, contributing to air quality management and public health.

GC-MS works by first separating the complex mixture of environmental compounds through gas
chromatography. Then, mass spectrometry identifies and quantifies individual compounds based on
their mass and chemical structure. This process is highly sensitive, allowing for the detection of trace
levels of pollutants. In the context of environmental analysis, GC-MS is an essential tool for assessing
and mitigating environmental hazards.

The data generated by GC-MS is critical for policymakers, environmental agencies, and researchers to
make informed decisions regarding environmental protection and regulation. It ensures that our air,
water, and soil remain safe for both human and ecological well-being.

Liquid chromatography–mass spectrometry:

Liquid chromatography–mass spectrometry (LC–MS) is an analytical chemistry technique that

combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass
analysis capabilities of mass spectrometry (MS). Coupled chromatography - MS systems are
popular in chemical analysis because the individual capabilities of each technique are enhanced
synergistically. While liquid chromatography separates mixtures with multiple components, mass
spectrometry provides spectral information that may help to identify (or confirm the suspected
identity of) each separated component.[1] MS is not only sensitive, but provides selective detection,
relieving the need for complete chromatographic separation.[2] LC–MS is also appropriate
for metabolomics because of its good coverage of a wide range of chemicals.[3] This tandem
technique can be used to analyze biochemical, organic, and inorganic compounds commonly found
in complex samples of environmental and biological origin. Therefore, LC–MS may be applied in a
wide range of sectors including biotechnology, environment monitoring, food processing,
and pharmaceutical, agrochemical, and cosmetic industries.[4][5] Since the early 2000s, LC–MS (or
more specifically LC–MS–MS) has also begun to be used in clinical applications.[6]
In addition to the liquid chromatography and mass spectrometry devices, an LC–MS system
contains an interface that efficiently transfers the separated components from the LC column into the
MS ion source.[5][7] The interface is necessary because the LC and MS devices are fundamentally
incompatible. While the mobile phase in a LC system is a pressurized liquid, the MS analyzers
commonly operate under high vacuum. Thus, it is not possible to directly pump the eluate from the
LC column into the MS source. Overall, the interface is a mechanically simple part of the LC–MS
system that transfers the maximum amount of analyte, removes a significant portion of the mobile
phase used in LC and preserves the chemical identity of the chromatography products (chemically
inert). As a requirement, the interface should not interfere with the ionizing efficiency and vacuum
conditions of the MS system.[5] Nowadays, most extensively applied LC–MS interfaces are based on
atmospheric pressure ionization (API) strategies like electrospray ionization (ESI), atmospheric-
pressure chemical ionization (APCI), and atmospheric pressure photoionization (APPI). These
interfaces became available in the 1990s after a two decade long research and development
process.[8][7]

Size-exclusion chromatography:
Size-exclusion chromatography, also known as molecular sieve chromatography,[1] is
a chromatographic method in which molecules in solution are separated by their size, and in some
cases molecular weight.[2] It is usually applied to large molecules or macromolecular complexes such
as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the
sample through the column, the technique is known as gel-filtration chromatography, versus the
name gel permeation chromatography, which is used when an organic solvent is used as a mobile
phase. The chromatography column is packed with fine, porous beads which are commonly
composed of dextran, agarose, or polyacrylamide polymers. The pore sizes of these beads are used
to estimate the dimensions of macromolecules.[1] SEC is a widely used polymer
characterization method because of its ability to provide good molar mass distribution (Mw) results
for polymers.
Size exclusion chromatography (SEC) is fundamentally different from all other chromatographic
techniques in that separation is based on a simple procedure of classifying molecule sizes rather
than any type of interaction[3].

Applications:
The main application of size-exclusion chromatography is the fractionation of proteins and other
water-soluble polymers, while gel permeation chromatography is used to analyze the molecular
weight distribution of organic-soluble polymers. Either technique should not be confused with gel
electrophoresis, where an electric field is used to "pull" molecules through the gel depending on their
electrical charges. The amount of time a solute remains within a pore is dependent on the size of the
pore. Larger solutes will have access to a smaller volume and vice versa. Therefore, a smaller solute
will remain within the pore for a longer period of time compared to a larger solute.[4]
Another use of size exclusion chromatography is to examine the stability and characteristics of
natural organic matter in water.[5] In this method, Margit B. Muller, Daniel Schmitt, and Fritz H.
Frimmel tested water sources from different places in the world to determine how stable the natural
organic matter is over a period of time.[5] Even though size exclusion chromatography is widely
utilized to study natural organic material, there are limitations. One of these limitations include that
there is no standard molecular weight marker;[5] thus, there is nothing to compare the results back to.
If precise molecular weight is required, other methods should be used.

Biochemical applications:
In general, SEC is considered a low-resolution chromatography as it does not discern similar
species very well, and is therefore often reserved for the final step of a purification. The technique
can determine the quaternary structure of purified proteins that have slow exchange times, since it
can be carried out under native solution conditions, preserving macromolecular interactions. SEC
can also assay protein tertiary structure, as it measures the hydrodynamic volume (not molecular
weight), allowing folded and unfolded versions of the same protein to be distinguished. For example,
the apparent hydrodynamic radius of a typical protein domain might be 14 Å and 36 Å for the folded
and unfolded forms, respectively. SEC allows the separation of these two forms, as the folded form
elutes much later due to its smaller size.

Polymer synthesis:
SEC can be used as a measure of both the size and the polydispersity of a synthesized polymer,
that is, the ability to find the distribution of the sizes of polymer molecules. If standards of a known
size are run previously, then a calibration curve can be created to determine the sizes of polymer
molecules of interest in the solvent chosen for analysis (often THF). In alternative fashion,
techniques such as light scattering and/or viscometry can be used online with SEC to yield absolute
molecular weights that do not rely on calibration with standards of known molecular weight. Due to
the difference in size of two polymers with identical molecular weights, the absolute determination
methods are, in general, more desirable. A typical SEC system can quickly (in about half an hour)
give polymer chemists information on the size and polydispersity of the sample. The preparative
SEC can be used for polymer fractionation on an analytical scale.