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High-Performance Liquid Chromatography The Reason Why HPLC is the Most Used Chromatography Method

Author / Editor: Myles Davis / Ahlam Rais

The abbreviation ‘HPLC’ stands for High-Performance Liquid Chromatography. In a previous article, we already reported on chromatography, what it is and how it can be performed. One of these options, as described there, is the HPLC variant. In this article, we will talk about how it is performed, what it is used for and what makes it different from normal liquid chromatography.

Related Company

HPLC.
HPLC.
(Source: www.hplc.com)

As described in the previous basic article, chromatography is a separation process in which a mixture of substances is separated using two phases, a stationary phase and a mobile phase. When liquid chromatography is referred to, the stationary phase is solid, and the mobile phase is a liquid. It is referred to as the eluent. If you want to know more about general chromatography, you can read all the information about it in our Basic article.

HPLC

The difference to normal liquid chromatography is the high separation performance, which is achieved by very small, pressure stable packing particles (<10µm), low pulsation pumps, high pressures (up to 400bar), appropriate injection systems and miniaturized detectors. The absolute number of possible separable compounds is usually further limited to about 25. However, the P in the abbreviation HPLC has also been given other humorous interpretations, e.g. ‘price’ because of the initial high acquisition costs.

Where is HPLC being used?

This chromatography variant is used for analytical purposes as well as in the chemical industry. For example, to separate proteins from impurities. HPLC is also a frequently used analytical tool in biochemistry. Especially when identifying mixture components such as nucleic acids, amino acids, peptides, or enzymes.

Other typical applications include the analysis of polymers, active ingredients and contaminants, purity controls or quantity determinations, e.g. of active ingredients in biological samples. HPLC analysis is also used as a standard in monitoring drinking water today.

How it works

The structure of a HPLC.
The structure of a HPLC.
(Source: Vogel Communications Group (Myles Davis))

Supply tanks (1) contain the substances to be separated. These are transported by a pump into a mixing chamber (2). The mixing ratio can be determined by means of a valve. From the mixing chamber, the solvent mixture moves to the injector (3). Via a separate capillary, the so-called sample loop, the injector feeds the sample to be analyzed via a valve, which is entrained by the flow of the running medium. The solvent together with the sample now flows on and hits the HPLC pillar (4).

The contents of the pillar consist of closely packed particles, which are equipped with different end groups depending on the type of pillar. During transport through the pillar, the sample components interact to varying degrees with the end groups and adhere to them to a greater or lesser extent. To allow the sample components bound to the pillar to float along, the mixing ratio can be changed. The steady stream of solvent then passes through to the detector (5), which detects the various dissolved sample components.

The simplest would be a UV/Vis detector that measures the absorption of the sample constituents at an input wavelength. The measurement is displayed graphically using a computer program (6) and a so-called chromatogram is obtained. After detection, the flow of solvent is transferred to a suitable waste canister (7).

UHPLC - Next step in HPLC

In UHPLC (Ultra High-Performance Liquid Chromatography) the main difference is that the particles, which are <10µm in HPLC, are now smaller than 3µm. The backpressure is therefore also greater. With UHPLC the pressure is between 1000 - 1500 bar. The pressure with regular HPLC is only up to approx. 400 bar.

Due to these changes, a much higher efficiency and separation performance is achieved. The time of analysis can thus be drastically reduced. However, the higher efficiency leads to an increase in the already high acquisition costs. Maintenance costs are also higher with UHPLC machines.

Not all companies name their more powerful HPLC, Ultra High Performance Liquid Chromatography. Due to that fact, there are multiple synonyms for UHPLC such as:

  • UFLC – Ultra Fast Liquid Chromatography
  • RRLC – Rapid Resolution Liquid Chromatography
  • UPLC – Ultra Performance Liquid Chromatography
  • RSLC – Rapid Separation Liquid Chromatography

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