HPLC
High performance liquid chromatography (HPLC) is a chemical analysis method for the chromatographic separation of dissolved substances.
The chemical-analytical process of HPLC is used to separate, identify and quantify semi-volatile and non-volatile compounds in samples under high pressure. HPLC is used particularly in pharmacy, biochemistry, environmental analysis and in the food and beverage industry, as it is easy to use and can detect trace elements.
TABLE OF CONTENTS
HOW DOES HPLC WORK?
In HPLC, the analyte (the substance to be analysed) is pumped under high pressure through a separation column containing the stationary phase by means of an eluent, which serves as the mobile phase. As this is liquid chromatography, the eluent can also be water or a buffer solution. Depending on their polarity, the individual components of the analyte have a higher affinity for the stationary phase or the eluent and remain in the stationary phase for a longer or shorter period. When leaving the separation column, the dissolved components of the sample are detected. A variety of different detectors are available for this purpose. This method is used, for example, to determine the purity of drugs.
THE HPLC AT QUALITY ANALYSIS
At Quality Analysis, we not only have many years of experience in the use of HPLC and the evaluation of test results, but also the expertise and the necessary technical equipment to combine this procedure with a variety of detection methods. If required, we can apply further analytical methods to provide you with a comprehensive analysis of your samples.
We are specialised in customers from the industrial and commercial sectors. Accordingly, our analyses are practice-oriented and provide concrete answers to your questions. Together with you, we identify your problem and find the best method to quickly and reliably arrive at a solution that supports you in your daily work.
HOW TO DETERMINE SUBSTANCES WITH THE HPLC?
The individual components of the sample generate a certain signal intensity in a suitable detector. This is called the peak and is typical for the respective substance. In combination with the retention time, this gives a peak area that can be compared with known standards. The retention time is the time from the injection of the sample until it leaves the separation column. By comparing the peak area in the standards with that in the sample, the concentration of the identified substance can also be deduced.
DETERMINATION OF UNKNOWN SUBSTANCES USING HPLC
The identification of unknown substances using HPLC is not straightforward. This requires a combination with other analyses, such as mass spectroscopy. For this purpose, there are HPLC systems that can be coupled directly with a mass spectrometer.
PRACTICAL IMPLEMENTATION AND RELEVANCE OF THE HPLC
In addition to its use in research, HPLC plays a important role in analysing plastics and pharmaceuticals. However, it has also become an important method for monitoring the quality of drinking water and is used for medical purposes, for example to determine the content of certain vitamins in the blood. The quantitative analyses carried out using HPLC are not only extremely precise, but can also be automated to a high degree, which makes this method particularly suitable for economical quality control.
QUALITY CONTROL WITH HPLC
HPLC plays a major role in quality control, particularly in the production and processing of plastics and in the pharmaceutical industry. It can be used to analyse the components of polymers (see polymer chemistry). In the pharmaceutical industry, on the other hand, the focus is on determining the purity of drugs. The purity of the actual active ingredient as well as other drug components can be analysed, or the presence of undesirable substances can be detected. HPLC is also used in the food industry. There it plays a role in the detection of dioxins in particular.
HPLC FOR RECOVERY AND PURIFICATION OF SUBSTANCES
HPLC is also used on an industrial scale for the extraction and/or purification of substances. For example, the chemical industry uses HPLC to purify proteins or to separate enantiomers. The columns used for this are many times larger than those used in laboratory equipment. They have a diameter of up to one metre.
SEPARATION METHODS AT THE HPLC
There are various methods of separation in HPLC, which must be selected depending on the application. In normal phase chromatography (normal phase or NP-HPLC), the stationary phase is polar and consists of silica or silica gel, for example. In practice, however, reversed phase chromatography (also known as RP chromatography) is the most important method. In this method, the mobile phase is polar and the stationary phase is non-polar. The retention time is usually shorter with this method than with NP-HPLC. It is therefore particularly suitable for analytes with a long retention time in the column.
NEW DEVELOPMENTS AND METHODS IN HPLC
However, reverse phase chromatography is not the only method that can help speed up the separation process. The current trend is generally towards HPLC methods that enable faster analyses and require ever smaller sample quantities. This is achieved using significantly higher pressures (up to 1,500 bar instead of the usual 300 bar) and the use of ever smaller particles in the column material (down to a size of just 1 µm).
This technological progress means that the separation of substances can be significantly accelerated. There are currently several competing methods in this field, such as Ultra High Performance Liquid Chromatography (UHPLC), Rapid Resolution Liquid Chromatography (RRLC) and Ultra Fast Liquid Chromatography (UFLC). It is currently not foreseeable whether UHPLC or another of these methods will be able to establish itself as the standard in the future.
BRIEFLY SUMMARISED: HPLC
HPLC or high performance liquid chromatography is a chemical analysis method used to separate the individual substances in a sample. In combination with a detector, the separated substances can be detected as they leave the column and identified using standards. It is a liquid chromatography, as the eluent does not necessarily have to be a solvent but can also be water or a buffer solution.
