Instrumentation

Modern chromatographic methods are instrumental techniques in which the optimal conditions for the separation are set and varied by electromechanical devices controlled by a computer external to the column or layer. Separations are largely automated with important features of the instrumentation being control of the flow and composition of the mobile phase, provision of an inlet system for sample introduction, column temperature control, online detection to monitor the separation, and display and archiving of the results. Instrument requirements differ significantly according to the needs of the method employed. Unattended operation is usually possible by automated sample storage or preparation devices for time-sequenced sample introduction.

Gas Chromatography

For GC a supply of gases in the form of pressurized cylinders is required for the carrier gas and perhaps also for the detector, for operating pneumatic valves, and for providing automatic cool-down by opening the oven door. To minimize contamination, high purity gases are used combined with additional purification devices. Each cylinder is fitted with a two-stage pressure regulator for coarse pressure and flow control. Fine tuning is achieved using metering valves or by electronic pressure control combining electromechanical devices with sensors to compensate automatically for changes in ambient conditions. The column oven is generally a forced air circulation thermostat heated resistively and capable of maintaining a constant temperature or of being programmed over time. The detector and sample inlet are generally thermostated separately in insulated metal blocks heated by cartridge heaters. The most common method of introducing samples into a GC inlet is by means of a microsyringe (pyrolysis, headspace and thermal desorption devices can be considered specialized sample inlets). For packed-column injection a small portion of (liquid) sample is introduced by microsyringe through a silicone septum into a glass liner or the front portion of the column, which is heated and continuously swept by carrier gas. The low sample capacity and carrier gas flow rates characteristic of narrow-bore open-tubular columns require more sophisticated sample-introduction techniques based on sample splitting or solvent elimination and refocusing mechanisms.

The principal methods of detection are varied, conveniently grouped under the headings of gas-phase ionization devices, bulk physical property detectors, optical detection and electrochemical devices. Further classification is possible based on the nature of the detector response - universal, selective or specific. The flame ionization detector and thermal conductivity detector are examples of (near) universal detectors; the flame photometric detector, thermionic ionization detector and atomic emission detector are element-selective detectors; and the photoioniz-ation detector and electron capture detector are structure-selective detectors. GC coupling to mass spectrometry and IR spectroscopy is straightforward and widely utilized for automated structure identification as well as detection. Detection in the gas phase is a favourable process and GC detectors are among the most sensitive and versatile by virtue of the range of mechanisms that can be exploited.

Liquid Chromatography

Modern LC employs columns with small particle sizes and high packing density requiring high pressures for operation at useful mobile-phase velocities. Syringe-type or single- or multiple-head reciprocating piston pumps are commonly used to provide the operating pressures needed in configurations that depend on the design of the solvent-delivery system. A single pump is sufficient for isocratic operation. A single pump and electronically operated proportioning valves can be used for continuous variation of the mobile-phase composition (gradient elution) or, alternatively, independent pumps in parallel (commonly two) are used to pump different solvents into a mixing chamber. Between the pump and sample inlet may be a series of devices (check valves, pulse dampers, mixing chambers, flow controllers, pressure transducers, etc.) that correct or monitor pump output to ensure that a homogeneous, pulseless liquid flow is delivered to the column at a known pressure and volumetric flow rate. These devices may be operated independently of the pump or in a feedback network that continuously updates the pump output. Mobile-phase components are stored in reservoir bottles with provision for solvent degassing, if this is required for normal pump and detector operation. Loop-injection valves situated close to the head of the column are universally used for sample introduction. This allows a known volume of sample to be withdrawn at ambient conditions, equivalent to the volume of the injection loop, and then inserted into the fully pressurized mobile-phase flow by a simple rotation of the valve to change the mobile-phase flow paths. Although most separations are performed at room temperature, either the column alone or the whole solvent-delivery system may be thermostated to a higher temperature when this is desirable or required for the separation. The separation is monitored continuously on the low pressure side of the column using several bulk physical property, photometric, or electrochemical detectors fitted with microvolume flow cells.

Common detection principles are UV absorbance, fluorescence, refractive index and amperometry. Coupling to MS and IR spectroscopy is becoming more common, as is online coupling to nuclear magnetic resonance (NMR) spectrometers. Detection is a more difficult aspect in the condensed phase and neither the variety nor operating characteristics of LC detectors compare favourably with GC detectors, although they allow a wide range of sample types to be analysed routinely. Special materials are used in the fabrication of biocompatible and corrosion-resistant instruments for the separation of biopolymers and for ion chromatography. Individual pumps can handle solvent delivery requirements over a decade range or so of flow rates. The diversity of column diameters used in modern LC for analysis and preparative-scale applications demands flow rates that vary from a few |L per minute to tens of litres per minute. Consequently, instruments are designed for efficient operation within a particular application range and are not universal with respect to column selection. Furthermore, analytical detectors tend to be designed with sensitivity as the main concern and preparative-scale detectors for capacity, such that the two are generally not interchangeable even when the same detection principle is employed. For preparative-scale work some form of automated sample fraction collection is necessary and economy of operation may dictate incorporation of an integrated mobile-phase recycle feature.

Supercritical Fluid Chromatography

Instrumentation for SFC is a hybrid of components used in GC and LC modified to meet the requirements of operation with a compressible fluid. The mobile phase is typically carbon dioxide (with or without modifier) contained in a pressurized cylinder and delivered to the pump in liquid form. Syringe pumps or cooled reciprocating piston pumps modified for pressure control are commonly used. A high precision pressure transducer is installed between the pump and sample inlet for programming the inlet pressure or fluid density during the course of a separation. Simultaneous measurement of the column temperature and pressure control allows constant density or density programming under computer control if the appropriate isotherms are known or can be approximated. Two pumps are generally used to generate mobile-phase composition gradients comprising liquid carbon dioxide and an organic solvent. Loop-injection valves similar to LC are the most convenient devices for sample introduction. The column oven is usually a forced air circulation thermostat similar to those used in GC. The full range of flame-based detectors used in GC can be used with only slight reoptimization as well as the main optical detectors used in LC, after modification for high pressure operation. A unique feature of the chromatograph is a restrictor required to maintain constant density along the column and to control the linear velocity of the fluid through the column. Orifice-type restrictors are usually placed between the column and detector for flame-based detectors and back-pressure regulators after the detector flow cell for optical detectors.

MEKC and CEC

MEKC and CEC employ the same instruments as used for capillary electrophoresis with the addition of overpressure capability for the buffer reservoirs when used for CEC. The separation capillary is terminated in two buffer reservoirs containing the high voltage electrodes that provide the electric field to generate the flow of mobile phase. The buffer reservoirs can be moved into place pneumatically and sequenced automatically to introduce a sample vial for sample introduction or a run buffer vial for separation. The column area is thermostated to maintain a constant temperature. A miniaturized optical detector positioned between the buffer reservoirs is commonly used for on-column detection. Some form of interlock mechanism is used to prevent operator exposure to the high voltages, up to 30-50 kV, typically used. A high level of automation is achieved under computer control and unattended operation is generally possible.

Planar Chromatography

The total automation of sample application, chromatogram development and in situ quantitation in planar chromatography has proved difficult. Instead the individual procedures are automated, requiring operator intervention to move the layer from one operation to the next. Samples are typically ap plied to the layer as spots or narrow bands using low volume dosimeters or spray-on techniques. Application volume, method, location and sample sequence are automated for unattended operation. The chromatogram is obtained by manual development in a number of development chambers of different design, or can be automated such that the conditioning of the layer, the selected solvents for the development, and the development length are preselected and controlled through the use of sensors. For multiple-development techniques the layer can be alternately developed, dried, new solvent introduced and the process repeated with changes in the development length and mobile-phase composition for any or all the programmed development steps. Apparatus for forced-flow development is also available and resembles a liquid chromatograph with the column replaced by the layer sandwiched between a rigid support and a polymeric membrane in an overpressure development chamber to allow external pressure to be used to create the desired mobile-phase velocity.

After development the chromatogram is recorded using scanning or video densitometry. The unique feature compared with detection in column chromatography is that the separation is recorded in space rather than time while in the presence of the stationary phase. The common forms of detection are optical methods based on UV-visible absorption and fluorescence. In mechanical scanning the layer is moved on a translation stage under a slit projecting the image of the monochromatic light source on the layer surface and the light reflected from the surface monitored continuously with a photodiode or similar device. Substances that absorb the light produce a proportional decrease in the intensity of the reflected light that can be related to the amount of sample present (for fluorescence there is a proportionate increase in the amount of light emitted at a wavelength that is longer than the absorbed wavelength). Electronic scanning is not as well developed but involves uniformly illuminating the whole layer and imaging the plate surface onto a video camera, or similar device, to capture and integrate the static image of the absorbing zones.

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