Chrom-Ed Series
R. P. W. Scott

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HPLC Supplement

1. Principles and Practice of Chromatography

   Chromatography, although primarily a separation technique, is mostly employed in chemical analysis. Nevertheless, to a limited extent, it is also used for preparative purposes, particularly for the isolation of relatively small amounts of materials that have comparatively high intrinsic value. Chromatography is probably the most powerful and versatile technique available to the modern analyst. In a single step process it can separate a mixture into its individual components and simultaneously provide an quantitative estimate of each constituent. Samples may be gaseous, liquid or solid in nature and can range in complexity from a simple blend of two entantiomers to a multi component mixture containing widely differing chemical species. Furthermore, the analysis can be carried out, at one extreme, on a very costly and complex instrument, and at the other, on a simple, inexpensive thin layer plate.

   
   
2. Gas Chromatography

   Chromatography, in one of its several forms, is the most commonly used procedure in contemporary chemical analysis and the first configuration of chromatography equipment to be produced in a single composite unit and made commercially available was the gas chromatograph. Gas chromatography was invented by A. J. P. Martin who, with R. L. M. Synge, suggested its possibility in a paper on liquid chromatography published in 1941. Martin and Synge recommended that the liquid mobile phase used in liquid chromatography could be replaced by a suitable gas. The basis for this recommendation was that, due to much higher diffusivities of solutes in gases compared with liquids, the equilibrium processes involved in a chromatographic process would be much faster and thus, the columns much more efficient and separation times much shorter.
   
   Derivatization
   GC samples are usually derivatized to render polar materials sufficiently volatile so that they can be eluted without thermal decomposition. Examples of such materials that need to be derivatized are the organic acids, polyols, amino acids etc.

   
   
3. Liquid Chromatography

   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 (1) 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 (2) 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.

   
   
4. Gas Chromatography Detectors

   A chromatography detector is a device that locates in the dimensions of space and time, the positions of the components of a mixture that has been subjected to a chromatographic process and thus permits the senses to appreciate the nature of the separation.

   
   
5. Liquid Chromatography Detectors

   Although chromatography was discovered late in the 1890s its development was almost negligible until the 1940s and this was largely due to the lack of an inline sensitive detector. The first, effective inline liquid chromatography (LC) detectors were the refractive index detector reported by Tiselius and Claesson (1) in 1942 and the conductivity detector described by Martin and Randall (2) in 1951. These two devices should have evoked a growth in LC development, but, in the early fifties, gas chromatography (GC) was invented which completely eclipsed the development of LC. It was not until the early 1960s that the renaissance of LC took place, initially based on the use of the refractive index of Tiselius and Claesson. Although a significant number of GC detectors were developed over two or three years, the development of LC detectors was much slower, largely due to the fact that low concentrations of solute in a liquid do not change the properties of a liquid nearly as much as they do a gas. In fact, the development of LC detectors was gradual and arduous.

   
   
6. Plate Theory and Extensions

   The first and most important aspect of chromatography theory that needs to be initially understood is summed up as follows.

    

   The dynamic and thermodynamic effects that result in a chromatographic separation are straightforward and easy to understand.

    

   In the words of Einstein

    

                             "first order effects are simple".

    

   Only when second order effects are considered does the theory and accompanying mathematics become more complex.

   
   
7. The Mechanism of Chromatographic Retention

   A separation is achieved in a chromatographic system by moving the peaks apart and constraining their dispersion so that they are eluted discretely. To move the peaks apart each solute must be retained to a different extent, which (as discussed in Plate Theory and Extensions) means that their distribution coefficients must differ or they must interact with different volumes of stationary phase. Retention is therefore controlled by regulating the magnitude of the distribution coefficient or modifying the quantity of stationary phase available to each solute. The former is employed in interaction chromatography and the latter in exclusion chromatography. In practice, it is rare that either procedure is exclusive in any given separation as both retentive processes are usually present to some extent, particularly in liquid chromatography (LC). Retention in gas chromatography (GC), using coated open tubes, is, perhaps, the exception, as, in this distribution system, exclusion processes (aside from chiral phases) are normally absent and, consequently, retention control is purely interactive. Interaction and exclusion processes, when they do occur together, act independently. Although, together they determine overall the retention of a solute, the exclusion properties of a stationary phase do not effect the magnitude of any of its interactive properties.

   
   
8. The Thermodynamics of Chromatography

   The retention of a solute in a chromatographic system is determined firstly, by the magnitude of the distribution coefficient of the solute between the two phases and secondly, by the amount of stationary phase available to the solute for interaction. This is fully discussed in book 6 of this series. In addition, the mechanism of distribution has been considered exclusively on the basis of molecular interactions in book 7. However, the distribution coefficient in chromatography is an equilibrium constant and, consequently, it can be treated rationally by conventional thermodynamics.

   
   
9. Dispersion in Chromatography Columns

   The separation of a solute pair in a chromatographic system depends on moving the peaks apart in the column and constricting their dispersion so that the two solutes are eluted discretely. The factors that control retention have been discussed in Book 7 and in this book the processes of peak dispersion will be considered together with the means by which peak dispersion can be minimized.

   
   
10. Extra Column Dispersion

    Columns with very low plate heights and corresponding high efficiencies can be easily and reproducibly prepared using modern packing techniques (for capillary columns precise coating procedures) in conjunction with well designed stationary phases and supports. Columns having very high efficiencies, however, produce very narrow peaks with very small peak volumes and, thus, the impressive advances in column technology have been accompanied by a continual reduction in peak size. As a consequence, all dispersion processes that takes place outside the column, once relatively unimportant, have now become far more significant and potentially more deleterious to chromatographic performance.

11. Preparative Chromatography

    Preparative chromatography can be a very ambiguous term and its meaning will often depend on the raison d'être for its use. To the forensic chemist, preparative chromatography may mean the isolation of only a few micrograms of material for structure elucidation by subsequent spectroscopic examination. To the biochemist, it may mean the isolation of a few milligrams of a substance required for assessing its physiological activity.In contrast, to the organic chemist, preparative chromatography will often mean the isolation of 5 or perhaps even 50 g or more of a pure intermediate for subsequent synthetic work (this can be particularly important in the separation of chiral mixtures). Thus, the amount of material that is separated does not necessarily determine whether the separation can be classed as preparative or not. However, all preparative separations involve the actual collection of an eluted component and does not merely comprise peak profile monitoring for quantitative estimation and elution time measurement.

Method Map 1: Methods preview 8 to 15.

Method Map 2: Methods preview 16 to 23.

Method Map 3: Methods preview 24 to 31.

Method Map 4: Methods preview 32 to 40.