High performance liquid chromatography
High performance liquid chromatography ( english high performance liquid chromatography , HPLC in the early days of this technique also -) high pressure liquid chromatography ( english high pressure liquid chromatography called) - is an analytical method in chemistry . HPLC is a liquid chromatography process that not only separates substances, but also identifies and quantifies them using standards (determining the exact concentration). In contrast to gas chromatography , which is a very good separation method for vaporizable substances, non- volatile substances can also be analyzed using HPLC . HPLC can also be used preparatively.
HPLC was developed in the 1960s. The pioneers include Joseph Jack Kirkland , Josef Franz Karl Huber , Csaba Horváth and John Calvin Giddings .
functionality
It is a chromatographic separation process in which the substance to be examined is pumped through a so-called separation column , which contains the stationary phase , together with a mobile phase (also called "eluent" or "eluent") . A separation column in an HPLC device is between 18 and 300 mm long and mostly has an internal diameter of 2 to 4.6 mm in the case of analytical HPLC systems. The separating power of HPLC is about 100 times greater than that of column chromatography. For economic reasons, a so-called guard column or a column filter is often installed upstream. This is a short column or a filter disk made of the same material as the separation column, in order to keep contaminants from the main column. HPLC is also used for the purification of substances as (semi-) preparative HPLC. The inside diameter can be considerably larger, as purification can be carried out up to the production scale. Preparative columns for the laboratory scale have a diameter of 10 or 25 mm.
A = eluent reservoirs
B = electromagnetic mixing valves with double
-
action piston pump C = 6-way valve D = pressure compensation loop to equalize pump impulses
E = mixing chamber
F = manual injection valve
G = separation column
H = HPLC unit
I = detector unit (e.g. UV spectrometer)
J = computer interface
K = PC
L = printer for outputting the results
The structure of a typical HPLC apparatus, reduced to the essential elements, can be seen in the adjacent figure. After the separation principle in normal phase, a distinction (engl. Phase normal , NP), reverse phase (engl. Reverse phase , RP), ion exchange (IEC) and size exclusion chromatography (engl. Size exclusion chromatography , SEC) and enantiomer separation ( chiral chromatography ) .
If a component of the substance to be examined interacts strongly with the stationary phase, it remains in the column for a relatively long time. On the other hand, if it interacts weakly with the stationary phase, it leaves the column earlier. Depending on the strength of these interactions, the constituents of the substance appear at different times (the retention times ) at the end of the separation column, where they can then be detected with a suitable detector. For the reverse phase, the retention time of a substance depends on the length of time it remains in the stationary phase (solvent film around the alkyl chains of the modified silica gel ). The step that determines the rate is "dissolution" ( desorption ) into the mobile phase.
A polar stationary phase (e.g. silica gel / silica gel ) is used in NP-HPLC . The strength of the elution power of the mobile phase generally depends on the polarity . The various solvents are arranged in the elutropic series according to increasing polarity . The more polar a mobile phase, the faster a substance is eluted. Polar molecules are adsorbed / retarded (retained) longer on the column than non-polar molecules and therefore leave the column later. The normal phases have the disadvantage that you can usually only work with organic solvents and not with aqueous eluents. Hydrophilic interaction chromatography (HILIC) offers a way out of this problem . In the HILIC, polar stationary phases are used, analogous to NP selectivity, but they work as eluent with aqueous buffer systems. In contrast to RP chromatography, however, water is the strongest eluent in HILIC.
RP-HPLC is the most common method in practice. About 70% of all analytical HPLC separations are RP separations. A non-polar stationary phase is used here, and the elution power of the mobile phase decreases with increasing polarity. The stationary phase is produced by reacting silanes that have been substituted with long-chain hydrocarbons with silica gel. The polar surface of the silica gel particles is coated with a non-polar layer of alkanes, i.e. the polarity is reduced. Mixtures of water or buffer and acetonitrile or methanol are usually used as the mobile phase . With isocratic separations, the composition of the mobile phase remains the same throughout the entire time. In the case of gradient separations, the polarity of the solvent mixture is changed during the analysis.
The RP-HPLC is particularly used for the separation of polar analytes, which would have too long retention times on normal phases. A C18 column ( i.e. an octadecylsilane as a derivatizing reagent for the silica gel, hence the common name ODS column) is usually used for this. The detection is mostly carried out using a UV or fluorescence detector. Combinations with simple mass spectrometers (MS) or tandem mass spectrometers are used more and more frequently.
Practical implementation and applications
A chemical compound can only be identified to a limited extent using HPLC by comparing the retention time of the unknown substance with that of a standard (a known substance) ( external standardization ). If the retention time is the same, you can add a little standard to the sample with the unknown substance and examine whether only one peak is visible in the chromatogram , whether a "double peak" has arisen or whether the chromatogram two separate “peaks” with very similar retention times become visible ( internal standardization ). If only one tip is visible after adding the standard to the sample, it cannot be assumed that the chemical compound in the sample and in the standard are identical. Another parameter would be the comparison of the UV spectrum when using a diode array detector or the mass trace with a coupled mass spectrometer . However, the retention time, UV spectrum and MS spectrum for isomers often only offer insufficient identification features. In practice, this technology can only facilitate efficient identification by excluding other possibilities.
Alternatively, this experiment can be carried out under two different separation conditions (e.g. HPLC separations with two different columns). So you can identify unknown chemical compounds with a certain degree of certainty.
If you want to determine the concentration of a chemical substance (e.g. vitamin E in a vegetable oil), you can do this by producing standards of this chemical substance with known concentrations and the peak area of the standards with the peak areas the substance in the samples. As with any analytical method, care must be taken to ensure that the recovery rate is included in the calculation of the concentration when the sample is processed beforehand .
Chromatographic separations are not used exclusively for analytical purposes, but also for preparative purposes in the laboratory and in the chemical industry, in order to purify a product (e.g. proteins ) or to separate very similar substances (e.g. enantiomers ) from one another. Columns with a diameter of up to one meter are used here.
Method development
The separation of the substances depends on many parameters, including
- Type of substances
- Type and dimensions of the separation column,
- Composition of the mobile phase,
- Temperature ,
- pH and
- Mobile phase flow rate
To achieve a complete and reproducible separation, a separate method usually has to be developed for each more complex mixture of substances, especially with the gradient method. Even slight deviations from a method can mean a change in selectivity. The aim of a method development is a chromatogram in which all peaks are completely separated, but appear at a minimal distance from one another in order to keep the duration of a separation process as short as possible. The parameter is not time consuming and costly by trial and error (Engl. Trial and error have to be determined), to chemical, pharmaceutical and food industries often use a simulation software, based on experimental data or molecular structures of the samples, appropriate methods can predict.
Current developments: UHPLC
In recent years the trend has been towards ever higher sample throughput with ever smaller sample volumes. The abbreviation UHPLC (short for Ultra High Performance Liquid Chromatography ) is suitable as a neutral term for HPLC with greatly increased performance . This designation is to be understood analogously to " High Frequency " (HF) and " Ultra High Frequency " (UHF) or " High Temperature " (HT) and " Ultra High Temperature " (UHT). Different manufacturers of HPLC systems, on the other hand, have coined different abbreviations and terms:
- RRLC: Rapid Resolution Liquid Chromatography
- RSLC: Rapid Separation Liquid Chromatography
- UFLC: Ultra Fast Liquid Chromatography
- UPLC: Ultra Performance Liquid Chromatography
What all techniques have in common is that particles with a diameter of 2.2 to 1.7 μm are used as column material. This can significantly improve the speed and efficiency of a chromatographic separation. 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 µl being used. But there are also established methods with an analysis time of less than a minute. To carry out a separation with such a column material, a significantly higher working pressure of up to 1000 bar is required, which cannot be achieved by conventional HPLC systems. Both the columns and the other components such as injection gauges, pumps, valves and connecting elements must work reliably under these conditions.
With these methods, the analysis time is shortened by optimized system components (e.g. fast, low-carryover samplers, smaller capillaries, sensitive detectors, low gradient delay).
The first UHPLC system was brought onto the market under the name UPLC ™ by Waters Corporation in 2004. UHPLC has now become a standard method and is in the process of largely replacing classic HPLC. This is largely due to the increasing spread and the simple transfer of methods.
Simulated moving bed (SMB) technology
Another highly effective separation and purification process, especially in natural substance or drug analysis. The principle of continuous countercurrent distribution is used.
Detectors
-
UV / VIS detector
- Diode array detector (DAD)
- Multi-wavelength detector (MWD)
- Light scattering detector
- Fluorescence detector
- Refractive index detector
- mass spectrometry
- Conductivity detector
- Electrochemical detector : amperometry , coulometry
- Radioactivity detector
- Nitrogen selective detector
See also
literature
- Gaby Aced, Hermann J. Möckel: Liquid chromatography - apparatus, theoretical and methodological foundations of HPLC , VCH, Weinheim 1991, ISBN 3-527-28195-9
- Heinz Engelhardt (Ed.): Practice of High Performance Liquid Chromatography. Applications, Equipment and Quantitative Analysis. Springer, Berlin et al. 1986, ISBN 3-540-12589-2 .
- Hans Henke: Liquid Chromatography. Analytical and preparative separations. Vogel-Buchverlag, Würzburg 1999, ISBN 3-8023-1757-2 .
- Henk Lingeman, Willy JM Underberg (ed.): Detection-Oriented Derivatization Techniques in Liquid Chromatography (= Chromatographic Science. Vol. 48). Marcel Dekker Inc., New York NY et al. 1990, ISBN 0-8247-8287-9 .
- Reinhard Matissek, Reiner Wittkowski (Eds.): High Performance Liquid Chromatography in Food Control and Research. Behr's Verlag, Hamburg 1992, ISBN 3-86022-029-2 .
- Veronika R. Meyer: Pitfalls and sources of error in HPLC in pictures. Hüthig GmbH, Heidelberg 1995, ISBN 3-7785-2417-8 .
- Lloyd R. Snyder, Joseph J. Kirkland, John W. Dolan: Introduction to Modern Liquid Chromatography. 3rd edition. John Wiley & Sons, Hoboken NJ 2010, ISBN 978-0-470-16754-0 .
Web links
hplc-säule.de : Information about HPLC columns and their use
Individual evidence
- ↑ Derek Lowe, Das Chemiebuch, Librero 2017, p. 426
- ↑ HPLC for newcomers (PDF, 296 kB)
- ↑ Lucie Nováková, Ludmila Matysová, Petr Solich: Advantages of application of UPLC in pharmaceutical analysis . In: Talanta . tape 68 , no. 3 , p. 908-918 , doi : 10.1016 / j.talanta.2005.06.035 .
- ^ Faria RP, Rodrigues AE: Instrumental aspects of Simulated Moving Bed chromatography. , J Chromatogr A. 2015 Nov 20; 1421: 82-102, PMID 26341597
- ↑ Caes BR, Van Oosbree TR, Lu F, Ralph J, Maravelias CT, Raines RT: Simulated moving bed chromatography: separation and recovery of sugars and ionic liquid from biomass hydrolysates. , T. ChemSusChem. 2013 Nov; 6 (11): 2083-9, PMID 23939991