Instrumentation

Of the techniques described above, dye affinity chromatography and high performance dye affinity chromatography are the main ones employing sophisticated instrumentation. Closed columns (i.e. columns with adapters at both ends) packed with affinity adsorbents can be run and studied with the highest possible precision on automated and computerized chromatography instruments. Such instruments are, for example, the FPLC® System of Pharmacia Biotechnology, and the Waters 650E Advanced

Protein Purification System. Of course, the systems can be used also with nonaffinity chromatography materials. The FPLC® system (pressure limit 4 MPa/580 psi, flow rate range 0.02-8.3 mL min-1) is composed, typically, of a chromatography controller, which offers the facility for connecting to a computer, and controls the rest of the system components: two dual-syringe high precision pumps; a mixing device; an injection motor valve; a sampling loop device to introduce, without dilution, volumes of up to 50 mL; an absorbance detector system with a recorder to monitor column effluents; and a fraction collector with a solenoid valve. Additional motor valves can be used for selecting different liquid phases and columns. The same company also manufactures the BioPilot® system, made especially for exploiting large column beds and processing large sample volumes; it has the similar basic philosophy as the FPLC®. The system offers a flow rate range 0.5-100 mL min- at pressures up to 2 MPa/290 psi, and sample volumes up to 150 mL introduced via a loop device. A money-saving alternative to FPLC® offered by Pharmacia Biotechnology, is the GradiFrac™ which incorporates, in a one piece instrument, a fraction collector and a gradient maker. It requires additionally a peristaltic pump, an absorbance monitor, an injection valve, a mixing device, a solenoid valve to form the gradient, and a fraction collector with a solenoid valve. The Waters system is also a nonmetallic liquid chromatograph, operating at flow rate ranges of 0.1-45 mL min- or 0.5-80 mL min-1, depending on the pump heads used, having a pressure limit of 750 psi. It is composed of a chromatography and gradient controller which offers the facility for connecting to a computer, a high precision two-head pump, a solenoid four-solvent delivery valve for solvent selection and mixing, a sample injection valve, and an absorbance detector to monitor column effluents. In contrast to the FPLC®, in the 650E system the liquid phase gradient is made at low pressure and prior to entering the pump.

Dye affinity adsorbents based on conventional supports result in low back pressures, when packed in short, wide columns to minimize particle deformation, can be run using peristaltic or other appropriate types of pumps. In contrast, adsorbents composed of small size particles suitable for HPAC (e.g. 5-10 |im) usually need to be run on high pressure instruments, such as those employed in HPLC applications. In this latter case, it is most advisable that titanium pump heads and tubings be used to prevent metal corrosion. However, small size particles with narrow size distribution (e.g. Monobeads®) can be run on medium pressure instruments (e.g. FPLC® and Waters 650E).

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Solar Panel Basics

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