Brief Introduction of HPLC

Liquid Chromatography Introduction

The history of Liquid Chromatography (LC) began in the early 20th century. In 1906, a Russian botanist Mikhail Tswett invented LC in order to separate various plant pigments. He injected the plant extract and petroleum ether through a glass column packed with calcium carbonate.

Since this was done on a glass column, he was able to observe the changes inside the column. In the beginning, there is only one layer of pigment on the top of the column. But as time passes by, the pigment is separated into four different colored-layers. The later research discovered that those four-layers were bluish-green; chlorophyll-a, yellowish-green; chlorophyll b, yellow; xanthine, and orange; carotene.

The whole process of separation took several hours and thus it was not a very practical method. This long analysis time was a part of the reason that LC did not become a popular analytical tool until the 1970s, a half-century after Mikhail Tswett’s invention.

*Chromatography is a separation technique and the word chromatography originated from chroma meaning “color” and graphy meaning “write”. Chromatography is the separation equipment and chromatogram is an out-put chart obtained from the analysis.

HPLC Introduction

HPLC stands for High-Performance Liquid Chromatography. Before HPLC was available, LC analysis was carried by the gravitational flow of the eluent (the solvent used for LC analysis) thus required several hours for the analysis to be completed. Even the improvements added in later time we're able to shorten the analysis time slightly. Those classical/initial LC systems are called “low-pressure chromatography” or “column chromatography”.

In the 1970s in the US, Jim Waters founded Waters Corporation and started to sell HPLC instruments. This promoted the use of HPLC in practical analysis areas. The LC systems that Waters Corporation developed used a high-pressure pump that generates rapid-flow of eluent, and thus resulted in a dramatic improvement in the analysis time. Compared to the “low-pressure chromatography” the newer types were called “high-pressure liquid chromatography”. Therefore it was used to be thought that HPLC stands for High-Pressure Liquid Chromatography, however, nowadays it is a common agreement that HPLC stands for High-Performance Liquid Chromatography. Another big change from Tswett’s date was the data acquisition methods. Instead of observing the changes of layers by eyes, the detector system was coupled to the LC and out-put was recorded on a paper chart. If we were to demonstrate Tswett’s analysis result on a chart (chromatogram).

Initially, the HPLC system was referred to as Waters Corporation’s system. Still now, Waters Corporation is the HPLC pioneer, but there are several other companies that manufacture and sell HPLC systems. Representation of Tswett’s LC analysis.

Technically speaking, the word LC represents all the Liquid Chromatography, including low-pressure LC, however, most LC systems are HPLC thus often the word LC is used as comparable as HPLC.

Lactoferrin purification HPLC introduction

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