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This comprehensive HPLC Analysis Manual Text Book serves as an essential resource for both novice and experienced professionals in the field of chromatography.

It meticulously covers the principles and applications of High-Performance Liquid Chromatography (HPLC), providing in-depth explanations of various techniques, methodologies, and instrumentation.

Each chapter is designed to enhance your understanding of HPLC, featuring detailed illustrations, practical examples, and step-by-step protocols that facilitate hands-on learning.

The text also includes troubleshooting tips and best practices to optimize your analytical results, making it an invaluable reference for laboratory settings. Whether you are conducting research, quality control, or method development, this manual equips you with the knowledge and skills necessary to excel in HPLC analysis, ensuring accurate and reliable outcomes in your work.

HPLC Analysis – Training Manual

INDEX

1.0     Introduction of  Chromatography :

Chromatography was originally used by the Russian botanist Mikhail Tswett (1872-1919), who is recognized for isolating plant pigments such as xanthophylls and chlorophylls in 1906. He passed them through a glass column filled with calcium carbonate. Because of the color of these pigments, the method was termed after the Greek terms “chroma,” which means “color,” and “graphein,” which means “to write.” This explains why the approach’s current usage doesn’t seem to be significantly influenced by its name.

What is of Chromatography?

Chromatography is the process of separating components in a mixture by measuring how long it takes for each component to pass through a stationary phase when a mobile phase passes through it.

The following forms of chromatography are produced by the potential phase mixes.

The mobile phase passes through the stationary phase, which can be a solid, a liquid adsorbed on a solid, or an organic species (such a C18 alkyl chain) bound to a solid surface. The stationary phase is locked in place in a column, which is a hollow tube composed of an appropriate material.

High Performance Liquid Chromatography (HPLC):

High Performance Liquid Chromatography (HPLC) is an analytical method used to determine the composition of drug-related substances.

The information gathered might be quantitative, giving the precise amount of compound in the sample, or qualitative, showing which compounds are present in the sample.

HPLC is employed frequently in drug manufacturing as well as at every stage of the development of a new medication.

The sample’s characteristics and developmental stage will both influence the analysis’s goal. Since HPLC is a chromatographic technology, an understanding of chromatography fundamentals is required to comprehend how it operates.

How Does High Performance Liquid Chromatography Work?

The solvent, often known as the mobile phase since it moves, is kept in a reservoir.

A fixed flow rate of mobile phase, usually milliliters per minute, is generated and measured by a high-pressure pump [solvent delivery system].

The sample can be introduced [injected] into the constantly flowing mobile phase stream that transports the sample into the HPLC column using an injector [autosampler or sample management].

The chromatographic packing material required to accomplish the separation is contained in the column. Because the column hardware holds this packing material in place, it is known as the stationary phase. because it is held in place by the column hardware.

As the separated compound bands elute from the HPLC column, a detector is required to view them because most compounds are colorless and invisible to the naked eye.

After leaving the detector, the mobile phase can either be collected or disposed of. HPLC makes it possible to extract the portion of the eluate that includes the purified component for additional analysis when the mobile phase has a separated compound band. This is known as preparative chromatography, which is covered in the HPLC Scale section.

It should be noted that the conduit for the mobile phase, sample, and separated compound bands is formed by connecting the pump, injector, column, and detector components using high-pressure tubing and fittings.

The computer data station, a part of the HPLC system that records the electrical signal required to create the chromatogram on its display and to determine and measure the concentration of the sample constituents, is connected to the detector.

Many kinds of detectors have been created since sample chemical properties can vary greatly. For instance, a UV-absorbance detector is used to determine whether a substance is capable of absorbing ultraviolet radiation. A fluorescence detector is employed if the substance exhibits fluorescence. A more general kind of detector, like an evaporative-light-scattering detector [ELSD], is employed if the chemical lacks either of these properties.

The most powerful approach is the use multiple detectors in series. For example, a UV and/or ELSD detector may be used in combination with a mass spectrometer [MS] to analyze the results of the chromatographic separation. This provides, from a single injection, more comprehensive information about an analyte. The practice of coupling a mass spectrometer to an HPLC system is called LC/MS.

HPLC System Operation

The mobile phase travels through the particle bed after entering the column from the left and leaving at the right. Green arrows indicate the direction of the flow.

The top image, which shows the column at time zero [the moment of injection], when the sample enters the column and starts to form a band, should be examined first.

This sample, which is a blend of red, blue, and yellow dyes, shows up as a single black band at the column’s inlet. [In practice, anything that dissolves in a solvent may be used as this sample; usually, the compounds would be colorless and the column wall would be opaque, so we would need a detector to see the separated compounds as they elute.]

We can observe that the individual dyes have moved in distinct bands at varying speeds after a few minutes [bottom image], during which the mobile phase flows continuously and steadily past the packing material particles. This is because the stationary phase and the mobile phase compete with one another to draw in each dye or analyte. Observe that the yellow dye band is on the verge of leaving the column and is moving the fastest. Compared to the other colors, the yellow dye prefers [is drawn to] the mobile phase.

As a result, it travels more quickly and approaches the mobile phase. The packing material is preferred by the blue dye band over the mobile phase. It moves much more slowly due to its higher attraction to the particles. Stated otherwise, it is the component that is most maintained in this mixture of samples. The red dye band travels through the column at an intermediate pace because of its intermediate attraction to the mobile phase. We can separate the dye bands chromatographically because they all travel at different speeds.