Introduction
Liquid chromatography (LC) was the first type of chromatography to be discovered and, in the form of liquid-solid chromatography (LSC) was originally used in the late 1890s by the Russian botanist, Tswett to separate and isolate various plant pigments. The colored bands he produced on the adsorbent bed evoked the term chromatography (color writing) for this type of separation. Initially the work of Tswett was not generally accepted, partly due to the original paper being in Russian and thus, at that time, was not readily available to the majority of western chemists and partly due to the condemnation of the method by Willstatter and Stoll in 1913. Willstatter and Stoll repeated Tswett's experiments without heeding his warning not to use too "aggressive " adsorbents as these would cause the chlorophylls to decompose. As a consequence, the experiments of Willstatter et al. failed and their published results, rejecting the work of Tswett, impeded the recognition of chromatography as a useful separation technique for nearly 20 years.
In the late 1930s and early 1940s Martin and Synge introduced a form of liquid-liquid chromatography by supporting the stationary phase, in this case water, on silica gel in the form of a packed bed and used it to separate some acetyl amino acids. They published their work in 1941 (3) and in their paper recommended the replacement of the liquid mobile phase with a suitable gas which would accelerate the transfer between the two phases and provide more efficient separations. Thus, the concept of gas chromatography was born. In the same paper in 1941, Martin and Synge suggested the use of small particles and high pressures in LC to improve the separation which proved to the critical factors that initiated the development of high performance liquid chromatography(HPLC).
"Thus, the smallest H.E.T.P. (the highest efficiency) should be obtainable by using very small particles and a high pressure difference across the column".
The statement made by Martin in 1941 contains all the necessary conditions to realize both the high efficiencies and the high resolution achieved by modern LC columns. Despite his recommendations, however, it has taken nearly fifty years to bring his concepts to fruition. Activity in the field of liquid chromatography was eclipsed in the 1950s by the introduction of gas chromatography and serious attempts were not made to improve LC techniques until the development of GC neared completion in the mid 1960s.
The major impediment to the development of LC was the lack of a high sensitive detector and it was not until the refractive index detector was developed by A. Tiselius and D. Claesson (4) in 1942 could the technique be effectively developed.
Tswett's original LC consisted of a vertical glass tube, a few centimeters in diameter and about 30 cm high, packed with the adsorbent (calcium carbonate). The plant extract pigments was placed on the top of the packing and the mobile phase carefully added to fill the tube. The solvent percolated through the packing under gravity, developing the separation which could be seen as different colored bands at the wall of the tube. The simple apparatus of Tswett contained all the essentials to achieve a chromatographic separation.
The contemporary chromatograph, however, is a very complex instrument operating at pressures up to 10,000 p.s.i providing flow rates ranging from a few microliters per minute to 10 or 20 ml/minute depending on the type of LC that is carried out. Modern detectors can detect solutes at concentration levels of 1x10-9 g/ml and an analysis can be completed in a few minutes with just a few micrograms of sample.
The Basic Liquid Chromatograph
The basic liquid chromatograph consists of six basic units. The mobile phase supply system, the pump and programmer, the sample valve, the column, the detector and finally a means of presenting and processing the results. A block diagram of the basic liquid chromatograph is shown in figure.
The Mobile Phase Supply System
The mobile phase supply system consists of number of reservoirs (200 ml to 1,000 ml in capacity). At least two reservoirs would be necessary and are usually constructed of glass or stainless steel and contain an exit port open to air.
Stainless steel, however, is not considered satisfactory for mobile phases buffered to a low pH and containing certain materials that can cause corrosion. Each reservoir is usually fitted with a gas diffuser through which helium can be bubbled. Many solvents and solvent mixtures (particularly aqueous mixtures) contain significant amounts of dissolved nitrogen and oxygen from the air. These gasses can form bubbles in the chromatographic system that cause both serious detector noise and loss of column efficiency. As helium is very insoluble in most solvents, it purges the oxygen and nitrogen from the solvent but does not produce bubbles in the system itself. Applying a vacuum to the reservoir is not a permanent solution to dissolved air as, on releasing the vacuum to allow the solvent to pass to the pump, air again dissolves in the solvent.
The solvent is filtered through a stainless steel or sintered glass filter to remove any solid contaminants. Depending on the type of solvent programmer that is employed, the supply from each reservoir may pass either to a pump or to a valved blending device. Solvent reservoirs are not usually thermostatted but, when necessary, the solvent can be brought to the column temperature by the use of an appropriate heat exchanger. The solvent containers are often situated in an enclosure that protects the user from toxic solvent vapors such as chloroform or aromatic hydrocarbons. Such enclosures also isolate the solvents from atmospheric moisture.
The Gradient Programmer
The High Pressure Programmer
There are two basic types of solvent programmer. In the first, the solvent mixing occurs at high pressure and in the second the solvents are premixed at low pressure and then passed to the pump. The high pressure programmer is the simplest but most expensive as each solvent requires its own pump. Theoretically, there can be any number of solvents involved in a mobile phase program, however, most LC analyses require only two solvents, nevertheless, up to four solvents can be accommodated.
The layout of a high pressure gradient system is shown in figure below and includes, as an example, provision for three solvents to be mixed by appropriate programming.
Column Ovens
The effect of temperature on LC separations is often not nearly so profound as its effect in GC separations, but can be critical when closely similar substances are being separated. In LC a change in temperature will change the free energy of the solute in both phases, (generally in a commensurate manner) and so the net change in the free energy difference with temperature, which controls the magnitude of the absolute retention, can be relatively small. Its effect on relative retention, however, can be very significant and, in fact, be the determining factor in achieving a satisfactory resolution. (5-7) The effect of temperature on diffusivity will be similar in both GC and LC. An increase in temperature will increase the diffusivity of the solute in both phases and thus increase the dispersion due to longitudinal diffusion and decrease dispersion due to resistance to mass transfer. As a result, at the optimum velocity, the efficiency of both the LC and GC column will be largely independent of temperature, however, the optimum velocity will be higher at higher temperatures and provide the potential for faster analyses. Due to the lesser effect of temperature on solute retention in LC (compared to that in GC), temperature is not nearly so critical in governing absolute retention time but is often essential in achieving adequate resolution, particularly between closely eluting solutes such as isomers. In contrast to the GC column, the thermal capacity of an LC column is much higher as the specific heats of liquids are much greater than those of a gas. As a consequence, a high heat capacity thermostatting fluid is necessary and if retention measurements need to be precise, air ovens would not ideal for thermostatting LC columns. On the other hand, liquid thermostatting media are rather messy to use and tend to make column changing difficult and lengthy. However, if accurate data is required, good temperature control may be essential. If precise retention measurements are not required, an air thermostatting oven might be a reasonable compromise.
Detectors
A large number of LC detectors have been developed over the past thirty years based on a variety of different sensing principles. However, only about twelve of them can be used effectively for LC analyses and, of those twelve, only four are in common use. The four dominant detectors used in LC analysis are the UV detector (fixed and variable wavelength) the electrical conductivity detector, the fluorescence detector and the refractive index detector. These detectors are employed in over 95% of all LC analytical applications. These four detectors will be described and for those readers requiring more information on detectors are referred to Liquid Chromatography Detectors. The subject of detector specifications will not be discussed here but will also be dealt with in detail there. Detector sensitivities and detector linearity will, however, be given for each of the four detectors.
The UV Detector
The UV detector is by far the most popular and useful LC detector that is available to the analyst at this time. This is particularly true if multi-wavelength technology is included in this class of detectors. Although the UV detector has some definite limitations (particularly for the detection of non polar solutes that do not possess a UV chromaphores) it has the best combination of sensitivity, linearity, versatility and reliability of all the LC detectors so far developed .
NOTE: Further Information on Chromatography can be obtained from www.chromatography-online.org
1 comment:
Great summary of the Liquid Chromatography process. What do you think of the improvements being made to the speed up high performance liquid chromatography in general?
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