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Welcome to the Chromatography FAQ (Frequently Asked Questions) Wiki
ChromFAQ started in 2005 as an offshoot of Chromatography Forum (http://www.chromforum.com) and of HPLC training courses presented by LC Resources (http://www.lcresources.com). As you might expect, the same questions keep coming up again and again. Rather than "reinvent the wheel", we decided to gather the answers to the most frequently asked questions in one place.
Chromatography Basics
What is Chromatography?
In total, "chromatography" is far and away the most widely used technique (perhaps "family of techniques" would be more appropriate, because there are several types of chromatography) in analytical chemistry. There is a good reason for this: if you want to analyze for a particular compound in a more-or-less complex matrix, the best way to do it is to separate that compound from all the rest of the stuff in the matrix. Separation is what chromatography is all about: chromatography is a chemical separation technique. The separation is accomplished with a system consisting of two immiscible phases. One phase (called, appropriatedly enough, the stationary phase) is fixed in place while the second phase (the mobile phase) flows through the system.
In the simplest case (to describe), both phases would be liquids. If we add a sample compound to the system, it will partition or distribute between the two phases, with some sample molecules spending some time in each phase. How much time a sample molecule spends in which phase is determined by the relative solubility in the two phases. Samples which have relatively high solubility in the mobile phase will be swept through the system quickly. Samples which have a relatively high solubility in the stationary phase will be swept through the system slowly. Therefore, if we introduce a mixture of two (or more) compounds (with differenc relative solubilities) into one end of a chromatographic system, they will be swept through the system at different speeds and will be separated, emerging from the outlet of the system at different times.
Why is it called "Chromatography"?
The name "chromatography" is a concatenation of the Greek words for "color" and "writing". The term was first used at the beginning of the 20th century by Mikhail Semyonovich Tswett (Михаил Семенович Цвет, also spelt Tsvett, Tsvet, Tswet, Zwet, and Cvet), a Russian botanist working in Warsaw. He had developed a technique for separating plant pigments in a glass tube filled with powdered chalk. A few drops of plant extract were placed at the top of the tube and then washed down the tube with petroleum ether (http://ptcl.chem.ox.ac.uk/MSDS/PE/petroleum_ether.html). Although supposedly inspired by the colorful aspect of the resulting bands of orange and yellow pigments against the while chalk backgound, Tsvett took the opportunity to name the technique after himself, since the Russian word "tsvett" can be translated as the English "color".
In the event, the vast majority of compounds separated or anlyzed using chromatography are colorless, and color is not involved in the process. So the next time you tell a non-chemist what you do, and they nod and say something like "Oh, yeah, I do a lot of color photography myself!", just smile politely and inquire about the weather.
Where can I find a glossary of chromatography terms?
There are actually a lot of them available, most as part of on-line courses or tutorials. Here are links to a few:
- Getting Started in HPLC Glossary (http://www.lcresources.com/resources/getstart/section0.htm) from the LC Resources Inc. on-line short course.
- Chromatography Glossary (http://www.lcsupport.com/glossary.htm) from LC Support
- Basic Liquid Chromatography (http://hplc.chem.shu.edu/BOOK/glossary/define.html) from the Kazakevich & McNair on-line text.
- German-English Chromatography Glossary (http://www.dyerlabs.com/chemistry/glossary.html) from Dyer Laboratories
- TLC Glossary (http://www.chromatography.co.uk/TECHNIQS/tlc/gloss.htm) from VWR International
- Vocabulary of Chromatography (http://www.uft.uni-bremen.de/chemie/Chromatography/glossary.htm) from University of Bremen
- Glossary of Terms (http://www.upchurch.com/TechInfo/glossary.asp#) from Upchurch Scientific
How many kinds of chromatography are there?
Because of the wide range of technology used, and the corresponding wide range of applicability, chromatography is subdivided into a bewildering array of specialized categories, with a great deal of mutual overlap.
The most basic distinction is based on the kind of mobile phase used. If the mobile phase is a gas, then the technique is called gas chromatography (GC). If the mobile phase is a liquid, then the technique is called liquid chromatography (LC). The technologies involved in controlling a flowing gas versus a flowing liquid are sufficiently different that these are usually considered as distinct techniques, and each warrants its own detailed page. A third, less widely used technology uses a fluid above its critical temperature as the mobile phase. Under these conditions, there is no distinction between liquid and gas. Supercritical fluid chromatography (SFC) is in many respects, intermediate between GC and LC.
LC is, by a large margin, the more widely used technique. GC is a distant second. SFC is used primarily in specialized applications where both LC and GC have problems.
Subcategories of Gas Chromatography (GC)
- On the basis of the physical stationary phase.
- If the stationary phase is a solid adsorbent, the technque is called Gas-Solid Chromatography (GSC).
- If the stationary phase is liquid (usually as a film coated on support particles or on the wall of a capillary tube), the technique is called Gas-Liquid Chromatography (GLC). GLC is by far the more widely used technique, because it is applicable to a wide range of volatile organic compounds. GSC is used primarily for very low molecular weight species such as atmospheric gases.
GC separations are carried out in one of two ways:- Isothermal GC keeps the temperature constant during the course of the separation.
- Programmed-temperature GC gradually increases the temperature during the course of the separation.
Subcategories of Liquid Chromatography (LC)
- On the basis of the physical stationary phase.
- If the stationary phase is a solid adsorbent, the technique is called Liquid-Solid Chromatography (LSC) or, less commonly Liquid-Solid Adsorption Chromatography (LSAC)
- If the stationary phase is a liquid, the techique is called Liquid-Liquid Chromatography (LLC) or, less frequently, Liquid-Liquid Partition Chromatography or simply Partition Chromatography. The latter two are uncommon enough that they really have never picked up an acronym or abbreviation.
With the advent of HPLC, in which the typical stationary phase consists of a non-polar layer covalently bonded to a solid support, the distinction between LSC and LLC has blurred to the point where these acronyms are encountered only infrequently today. - If the stationary phase consists of a sheet of paper, it is called Paper Chromatography (PC). Paper chromatography was a major breakthrough when it was invented in 1942 but it has been superseded by other techniques and is used today primarily as a teaching tool.
- If the stationary phase consists of a thin layer of very small particles stuck on a glass or plastic plate, the technique is called Thin-Layer Chromatography (TLC). Because measuring the amount of sample on a TLC plate is difficult, and because TLC does not lend itself to computer control, the technique is used primarily as a screening tool for qualitative analysis of large numbers of samples.
- If the stationary phase consists of a bed of relatively small particles in a glass or plastic tube, with the mobile phase flowing as a result of gravity, vacuum, or low applied pressure (less than about 1 atm), then the technique is called Column Chromatography, Open-column Chromatography, or Classical Column Chromatography. A variant on this technique uses small packed cartridges for sample cleanup and preparation; it is called Solid-Phase Extraction (SPE).
- If the stationary phase consists of a bed of very small particles in a column with the mobile phase pumped through under pressure, then the technique is referred to as High Performance Liquid Chromatography or High Pressure Liquid Chromatography (HPLC). This increasingly becoming the dominant form of liquid chromatography. HPLC separations are carried out in one of two ways:
- On the basis of separation chemistry
- If the stationary phase is more polar than the mobile phase, the technique is called Normal-phase Chromatography (NP)
- If the mobile phase is more polar than the stationary phase, the technique is called Reversed-phase chromatography (RP or RPC)
- A variant on Reversed-phase chromatography adds an ionic surfactant (called an "Ion-pair Reagent) to the mobile phase to allow interaction with charged analyte molecules. This technique is referred to as Ion-pair Chromatography (IP or IPC)
- If the stationary phase consists of ionic groups attached to a support, and the mobile phase consists of a buffer or salt solution, then the technique is called Ion-exchange chromatography (IEX or IEC).
- If the stationary phase consists of an inert matrix (i.e., no chemical interaction with the analyte) with a pore size distribution which allows limited penetration of anlyte molecules based on their size, the technique is called Size-exclusion chromatography (SEC). A further distinction is often made based on the type of analyte and its solubility:
- For organic-soluble polymers, the technque is typically called Gel Permeation Chromatography (GPC)
- For proteins or water-soluble polymers, the technique is typically called Gel Filtration Chromatography (GFC).
Questions about Gas Chromatography
Answers to Frequently Asked Questions about Gas Chromatography (GC) can be found here.
Questions about Liquid Chromatography
For answers to frequently asked questions about LC, click here.
