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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.
The loading of preparative columns is considered both practically and theoretically including the maximum column loading capacity, the maximum sample volume, volume overload, and mass overload. Preparative chromatography apparatus is then described including, solvent reservoirs, pumps, sample valves and the preparative columns themselves. The special requirements of preparative column detectors are also discussed together with the use of fraction collectors. The special packing techniques necessary for both preparative gas chromatography and liquid chromatography columns are described including both the radial and axial compression techniques. Procedures such as recycling, together with the moving bed and simulated moving bed systems are discussed in detail and some examples of the use of the moving bed system included.
The modern gas chromatograph is described and includes gas supplies (air tanks, pure air generators, nitrogen and hydrogen generators), pressure controllers, flow controllers and flow programmers, together with injection devices for both packed and capillary columns. Special sampling techniques are discussed including retention gap sampling and solute focusing. The supports used in packed columns are given together with different packing procedures. The static and dynamic methods of coating capillary columns are also given, including the deposition of chiral stationary phases. The design and function of the four most common GC detectors are described, (the flame ionization detector, the nitrogen phosphorus detector, the electron capture detector and the katharometer). The basic design of the modern data acquisition and data processing systems are discussed including the scaling amplifier and the A/D convertor. General GC quantitative analysis is considered including derivatization procedures. Finally quantitative preparative GC is discussed and a series of application examples included.
Gas Chromatography Detectors
Detector specifications are described including sensitivity, noise, response, pressure sensitivity, flow sensitivity, temperature sensitivity, dynamic range and linearity. Methods for the experimental determination of many of the specifications are included. The early gas chromatography detectors are mentioned and their history given. The detectors described in detail include the katharometer, the gas density balance, the flame ionization detector (with a detailed discussion on its design and fabrication), the nitrogen phosphorous detector, ionization detectors (includion different argon and helium ionization detectors) and the radioactivity detector. In addition, a further nine less common detectors are also described.
The book starts with a short history of capillary columns and then discusses the essential apparatus for their use including, gas supplies, injection systems and column ovens. The preparation of capillary columns is then described including dynamic coating, static coating and capillary column connecting.techniques. Detector requirements for capillary columns are considered and the two commonly used detectors, the flame ionization detector and the nitrogen phosphorus detector discussed in detail. Capillary column theory is presented and such important parameters as optimum velocity and the column variance per unit length considered in detail and equations given. Practical aspects of the technique are described including back flushing techniques and heart cutting together with the apparatus necessary for these procedures. A number of applications of capillary columns are given, including the use chiral stationary phases, drug analysis and the use of high temperature stationary phases such as carborane.
The basic liquid chromatograph is described, including mobile phases supply systems, high pressure and low pressure gradient programmers, the various forms of pumps (piston and diaphragm pumps, syringe and rapid refill pumps), valves (sample and switching) and column ovens. The design and function of eight of the more common HPLC detectors are described, including the UV detector (fixed and variable wavelength), the fluorescence detector and the refractive index detector. The various stationary phases used in LC are considered and include the preparation of irregular and spherical silica gel, the description of the different types of bonded phase and their preparation (by reaction in solvent and by the fluidized bed method). The properties of the mobile phase are outlined and their interaction with silica gel and the different types of bonded stationary phases are also discussed. The characteristics of the special stationary phases, macrocyclic glycopeptides, cyclodextrin and other chiral stationary phases are included
Liquid Chromatography Detectors
Detector specifications are first considered that include sensitivity, noise, response, dynamic range and linearity together with sensor dispersion and dispersion in connecting tubes. The effect of Newtonian flow on detector dispersion is considered together with the time constant of the sensor and associated electronics. A wide range of different detectors are described including six based on refractive index measurement, five detectors based on UV absorption and three detectors based on fluorescence detection. There are four transport detectors described and four based on light scattering measurements. A description of the dielectric constant detector and the electrical conductivity is included together with a number of electrochemical detectors including the multi-electrode array detector.
Plate Theory and Extensions
The plate theory is developed and equations for the retention volume of a solute, the capacity ratio of a solute, the separation ratio of two solutes and the conditions for chromatographic separation derived. The different volumes that make up the dead volume are discussed and experimental methods for measuring the different dead volume components given. The Gaussian form of the elution equation is derived and methods of measuring the retention volume of closely eluting peaks discussed. The concept of column efficiency is introduced and a method for measuring column efficiency described. The points of inflection of a peak are defined together with effective plate number and the resolving power of a column. The concept of the summation of variances is introduced and used to calculate the maximum volume of sample that can be placed on a column. The technique of vacancy chromatography is considered and an equation for the peak capacity of a column developed. Finally the temperature changes that take place on the passage of a solute through a theoretical plate are examined in detail both theoretically and experimentally.
The Mechanism of Chromatographic
Solutes are retained in a chromatographic column because the solute molecules interact more strongly with the molecules of the stationary phase than those of the mobile phase. The different types of molecular interaction are described which include dispersive interactions, polar interactions (which include both dipole-dipole interactions and dipole-induced dipole interactions) and ionic interactions. Molecular interactions with mixed phases are also discussed and it is shown that interaction occurs as though each component of the phase is a separate phase and its contribution is proportional to its concentration in the mixed phase. Retention by surface adsorption is also discussed and the theory of monolayer and bi-layer adsorption developed. The sorption and displacement adsorption processes are considered including retention by exclusion. The preparation of silica gel is described and the preparation of silica gel mixtures having different exclusion properties outlined. Chiral interactions and chiral phases are also discussed.
The Thermodynamics of Chromatography
The basic thermodynamic equations pertinent to chromatography are introduced and the method of distribution analysis using the standard energy of distribution discussed. Thermodynamic analysis is demonstrated by analyzing the energy difference between the dispersive interactions of the methyl and methylene groups with an alkane stationary phase. Using the distribution data for the substituted methanes the energies involved in the dispersive interactions of carbon, hydrogen chlorine and bromine with an alkane stationary phase are also examined. Other types of molecular interaction are considered and the thermodynamic explanation of complex formation also examined. Thermodynamic argument is also used to identify the optimum operating conditions for chiral separations and the effect of solvent composition enantiomeric separations.
Dispersion in Chromatography
The principal of the summation of variances is first discussed and the alternative axis of the chromatogram introduced, namely curves relating sample concentration to either time, volume flow of mobile phase through the column or distance moved along the column. The Random Walk model for obtaining an expression for the variance of a dispersion process is explained and used to derive expressions for multipath dispersion, longitudinal diffusion and dispersion due to the resistance to mass transfer in the two phases. The effect of the compressibility of the mobile phase in a gas chromatography column on the variance equation is theoretically examined and the Van Deemmter equation discussed. The alternative dispersion equations developed by Giddings, Huber, Knox, Horvath for packed columns and by Golay for capillary columns are described and discussed.. Strong experimental evidence is given to indicate that the Van Deemter equation best explains the variance in a packed column over practical range of operating variable used in both gas and liquid chromatography.
Extra Column Dispersion
Dispersion of an eluted solute in the chromatographic apparatus other than the column can be extremely important and in the worst case seriously impair the performance of the column. This book examines quantitatively and qualitatively the dispersion that can take place in sample valves, connecting conduits, unions, frits and in the sensing volume of the detector. The dispersion that takes place in the column is first considered and from that the limiting value of the extra column dispersion can be determined and an expression giving this limiting dispersion is included. Low dispersion tubing is discussed and the special case of low dispersion serpentine tubing considered in detail. The design of low dispersion gradient elution apparatus is also described and the special case of microbore columns examined.