HIGH PERFORMANCE

LIQUID CHROMATOGRPHY

Written Report in
Technical Analysis

Prepared for
DR. MA.LOURDES C. ALVAREZ

Preapred by
Jessa M. Villanueva

May. 2,2019
OBJECTIVES:

1. Introduction of HPLC

2. Enumerate the parts and its function

3. Application of HPLC

4. Hplc instrumentation

Introduction

Liquid Chromatography was iitially discovered as an analytical technique in the

early 20th cenntury and was first used as method of separating colored compounds.This
is the where chromatography means color and write. It was derived from the two
Greek words chroma means ”color” and graphein means to” write”. In 1903, Mikhail S.
Tswett a Russian botanist used a rudimentary form of chromatographic separation to
purify mixtures of plant pigment into the pure constituents. There was physical method
in chromatography in which separation of components takes place between two phases,
they are stationary phase and the mobile phase. In stationary phase, this is the region
over which the compounds gets separated while mobile phase is the solvent system
which carries the mixture to be separated.
 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

High Performance Liquid Chromatography is a form of column chromatography

that pumps a sample mixture or analyte in a solvent(known as the mobile phase) at
high pressure through a column with a chromatographic packing material (stationary
phase). It is used biochemistry and analytical chemistry to separate, identify, and
quantify components dissolved in a liquid solvent with a high analytical resolution.
PUMP

The role of pump is to force a liquid (called a mobile phase) through the liquid
chromatography at a specific flow rate, expressed in milliliters per minute (mL/min).

INJECTOR

The injector serves to introduce the liquid sample into the flow stream of the
mobile phase for analysis. It is equipped with six port valves so that a sample can be
injected into the flow path at continuous pressure.
COLUMN

Considered the “heart of chromatogram” the column`s stationary phase

separates the sample components of interests using various physical and chemical
properties.

DETECTOR

The function of detector in HPLC is to monitor the mobile phase as it merges

from the column.

COMPUTER

This is called data system, the computer not only controls all the modules of the
HPLC instrument but it takes the signal from the detector and uses it to determine the
time of elution(retention time) of the sample components(qualitative analysis) and the
amount of sample(quantitative analysis).

Solvent reservoir

Holds the solvent, which is refered to as the mobile phase because it moves.
There are two reservoir ina system, which each holding up to 1000cc of solvent and
usually fitted with agas diffuser through which helium can be bubbled.

Degasser

The commonly used degasser is a Vacuum degasser. It is high-effeciency, inline

module that removes dissolved gasses from HPLC solvent.
 Real- life Applications for HPLC

 Pharmateutical company- determine the the amount of each chemical found in

new product.
 Hospital- detect blood or alcohol levels in a patient`s blood stream.
 Law Enforcement- to compare a sample found at a crime scene to samples from
suspects.
 Environmental Agency- determine the level of pollutants in the water supply.
 Manufacturing Plant- to purify a chemical needed to make a product.
 Food Analysis- quality control, additives, stability testing, analysis of sub-micron
levels of aflatoxins and sugar analysis in fruit juices.

INSTRUMENTATION

The solvent is needed to create a mobile phase. To create the mobile phase, we
need a pump. The pump moves the solvent continuously from the flask to the waste
can. To measure the sample, an injection system is needed to inject the samples into the
mobile phase. The identification and quatification of the components happens in the
column. When the sample reaches the column, it leaves the mobile phase and enters in
the stationary phase. The stationary phase is the substance fixed in placed for the
chromatography procedure. This is where separation takes place. After leaving the
stationary phase, the sample moves through a detector. The detector sends the signal
to the computer which converts in a chromatogram.