High-performance liquid chromatography

Performance liquid chromatography ( engl. high performance liquid chromatography, HPLC ) - in the early days of this technology also high pressure liquid chromatography ( engl. high pressure liquid chromatography ) called - is an analytical tool in chemistry. The HPLC is a liquid chromatography method with which not only separates substances, but this also identify on standards and quantify ( the exact concentration determined) can. In contrast to gas chromatography, which is a very good method for separating vaporizable materials, and non-volatile substances can be analyzed by HPLC. HPLC can also be used preparatively.

Operation

Is a chromatographic separation process, wherein the examined substance is used together with a mobile phase, the mobile phase (also called " eluent " or " mobile phase " referred to ) is pumped through a so-called the separation column containing the stationary phase. A separation column in an HPLC device is 18 to 300 mm long, and usually has an inside diameter of 2 to 4.6 mm in the case of analytical HPLC systems. The separation capacity of a HPLC is about 100 times greater than in the column chromatography. Frequently, for economic reasons, a so-called pre-column or a column filter is installed upstream. This is a short column or a filter disc of the same material as the separation column in order to prevent contamination of the main column. HPLC is also used for the purification of substances as a (semi-) preparative HPLC. The inner diameter can be much larger, since up to the production scale, a purification may be carried out. Preparative column for the laboratory scale, having a diameter of 10 or 25 mm.

The reduced to the essential elements of building a typical HPLC apparatus can be found in the adjacent figure. A distinction is made according to the principle of separation in normal phase ( engl. normal phase, NP ), reversed-phase (English reverse phase RP), ion exchange (IEC) and size exclusion chromatography (german size exclusion chromatography, SEC ) and separation of enantiomers (chiral chromatography).

Does changing one part of the test substance strongly with the stationary phase, it remains a relatively long time in the column. Does he change is weak with the stationary phase, he leaves the column earlier. Depending on the strength of these interactions appear to the components of the substance at different times ( retention times ) at the end of the column where they can then be detected with a suitable detector. For the reverse phase, the retention time of a substance depends on the residence time in the stationary phase ( solvent film to the alkyl chains of the modified silica gel ). The rate-determining step is the "back solution " (desorption) in the mobile phase.

In the NP- HPLC a polar stationary phase is used (eg, silica gel / silica gel). The strength of the elution of the mobile phase depends on the polarity in general. The various solvents are arranged in order of increasing polarity in the eluotropic series. A more polar mobile phase, the faster a substance is eluted. Polar molecules are adsorbed on the column longer / retarded ( retained ) than non-polar molecules and therefore leave the column later. The normal phase have the disadvantage that you can usually only work with organic solvents and not with aqueous eluents. One way out of this problem provides the hydrophilic interaction chromatography (HILIC ). In the HILIC polar stationary phases are analogous to NP- selectivity used, but work with aqueous buffer systems as eluent. In contrast to RP chromatography, however, is the strongest eluent in HILIC water.

The RP- HPLC is the most common method in practice. About 70 % of all analytical RP- HPLC separations are separations. Here, a non-polar stationary phase is used, and the elution of the mobile phase decreases with increasing polarity. The stationary phase is prepared by mixing, is allowed to react with silica, silanes, which have been substituted with long-chain hydrocarbons. In this case, the polar surface of the silica gel particle is coated with a layer of non-polar alkanes, that reduces the polarity. The mobile phase mixtures of water or buffer and acetonitrile or methanol are used mostly. Isocratic separations the composition of the mobile phase during the entire period is the same. For gradient polarity of the fluid mixture is changed during the analysis.

Particular application is the RP-HPLC for the separation of polar analytes, which would have to be normal phase high retention times. For this is usually a C18 column (ie, a octadecylsilane as a derivatization reagent for silica gel, hence the common name ODS column ) are used. The detection is carried mostly by UV or fluorescence detector. More and more frequently combinations with simple mass spectrometers (MS) or tandem mass spectrometers are used.

Practical implementation and applications

A chemical compound can be identified only partially by means of HPLC by the retention time of the unknown sample with those of a standard ( a known compound ) compares ( external standards). If the retention time is equal, can the sample with the unknown substance enforce and investigate something standard, whether the chromatogram still only one peak is (English peak) visible if a " double - peak " has been or whether the chromatogram are visible two separate " peaks " with a very similar retention time ( internal standardization ). If, after the addition of standard only one peak is visible to the sample, one can not assume that the chemical compound is the same in the sample and the standard. An additional parameter would be the calibration of the UV spectrum when using a diode-array detector, or ground track in a coupled mass spectrometer. However, retention time, UV spectrum and MS spectrum at isomers often provide insufficient identification features. Here, this technique can only facilitate in practice, the efficient identification by excluding other possibilities very well.

Alternatively, leads to this experiment two different separation conditions (for example, using two different HPLC separation column ) through. So one can thus identify unknown chemical compounds with some degree of certainty.

If one wants to determine the concentration of a chemical substance (for example, vitamin E in a vegetable oil ), it is possible to do so by preparing standards of known concentrations of the chemical substance and the peak area of ​​the standards with the peak area of the substance in the samples compared. As with any analytical method is to ensure that at a previous sample preparation, the recovery rate is included in the calculation of concentration.

Chromatographic separations are not used solely for analytical purposes, but also for preparative purposes in the laboratory and in the chemical industry to purify a product ( eg, proteins ) or very similar substances (eg enantiomers ) from each other. These columns come with up to a meter in diameter for use.

Method development

The separation of substances is dependent on many parameters, including

• Nature of the substances
• Nature and extent of the separation column,
• The composition of the mobile phase,
• Temperature,
• Flow rate of mobile phase

To achieve a complete and reproducible separation usually has its own method for each complex mixture of substances, especially in the gradient method are developed. Even small deviations from a method may mean a change in selectivity. Aiming a development of methods is a chromatogram in which all peaks are completely separated, but occur at a minimum distance from each other to keep the duration of the separation operation to a minimum. Having to determine the parameters are not time consuming and costly trial and error (English trial and error ), make use of chemical, pharmaceutical and food industries often a simulation software which can based on experimental data or molecular structures of the samples predict appropriate methods.

Current developments: UHPLC

In recent years the trend has been towards ever higher sample throughput with smaller and smaller sample volumes. As a neutral term for HPLC with greatly enhanced performance UHPLC is the abbreviation (short for engl. Ultra High Performance Liquid Chromatography ) suitable. This term is to be understood in analogy to " High Frequency" ( RF ) and " ultra high frequency " ( UHF) or "High Temperature" (HT) and "Ultra High Temperature " (UHT). Different manufacturers of HPLC systems, however, have influenced the use of abbreviations and terms:

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

All techniques have in common that in this case particles having a diameter from 2.2 to 1.7 microns are used as the column material. This allows the speed and efficiency of a chromatographic separation can be considerably improved. A typical UHPLC analysis, including gradient elution and subsequent equilibration takes 5 to 10 minutes ( rule of thumb 1/10 of the HPLC), with sample volumes of 0.5 to 2 ul be used. However, there are established methods, with an analysis time of less than a minute. To perform a separation with such a column material, a significantly higher operating pressure is required, which can not be achieved by conventional HPLC systems. Both the column and the other components, such as injection timer, pumps, valves and joints must operate reliably under these conditions.

In these methods, the analysis time is shortened by optimizing plant components (eg, fast, low carryover autosampler, smaller capillaries, sensitive detectors, low gradient delay ).

The first UHPLC system was launched under the name UPLCTM from Waters Corporation launched in 2004. Meanwhile, the UHPLC is a standard method and to replace the classical HPLC in many parts of the term. This is largely in the growing distribution and easy method transfer.

Detectors

• UV / VIS detector Diode array detector (DAD)
• Multi-wavelength detector ( MWD)