Gradient controller

Implementation of SFC has initially been hampered by instrumental problems, such as back-pressure regulation, need for syringe pumps, consistent flow-rates, pressure and density gradient control, modifier gradient elution, small volume injection (nL), poor reproducibility of injection, and miniaturised detection. These difficulties, which limited sensitivity, precision or reproducibility in industrial applications, were eventually overcome. Because instrumentation for SFC is quite complex and expensive, the technique is still not widely accepted. At the present time few SFC instrument manufacturers are active. Berger and Wilson [239] have described packed SFC instrumentation equipped with FID, UV/VIS and NPD, which can also be employed for open-tubular SFC in a pressure-control mode. Column technology has been largely borrowed from GC (for the open-tubular format) or from HPLC (for the packed format). Open-tubular coated capillaries (50-100 irn i.d.), packed capillaries (100-500 p,m i.d.), and packed columns (1 -4.6 mm i.d.) have been used for SFC (Table 4.27). 

High Performance Liquid Chromatographic (HPLC) Analysis. A Waters HPLC system (two Waters 501 pumps, automated gradient controller, 712 WISP, and 745 Data module) with a Shimadzu RF-535 fluorescence detector or a Waters 484 UV detector, and a 0.5 pm filter and a Rainin 30 x 4.6 mm Spheri-5 RP-18 guard column followed by a Waters 30 x 3.9 cm (10 pm particle size) p-Bondapak C18 column was used. The mobile phase consisted of a 45% aqueous solution (composed of 0.25% triethylamine, 0.9% phosphoric acid, and 0.01% sodium octyl sulfate) and 55% methanol for prazosin analysis or 40% aqueous solution and 60% methanol for naltrexone. The flow rate was 1.0 mL/min. Prazosin was measured by a fluorescence detector at 384 nm after excitation at 340 nm (8) and in vitro release samples of naltrexone were analyzed by UV detection at 254 nm. 

Comparison of the boundaries of the observed flow patterns with the analytical criteria derived by Quandt showed that the bubble, dispersed, and annular flow patterns are subclasses of a pressure gradient-controlled flow. Similarly, flow patterns identified as slug, wave, stratified, and f ailing film are subclasses of a gravity-controlled situation. 

It is much more difficult to describe the relationship of the bulk field gradients, easily recognised in the flow of water in clouds and of oxygen in the ozone layer described in Section 3.4, to that of the gradients controlling the chemical flow in cell liquids. The effects of electric fields due to charge distribution in various parts of the cell is an obvious possibility. 

FPLC components (two P 500 pumps, V7 injection valve, gradient controller, UV-1 detector, Frac-100 fraction collector, 50 mL Superloop [Pharmacia], dualchannel chart recorder, or similar components). 

Instrumentation. HPLC separations were performed on a Waters Associates system consisting of two Model 590 pumps, a Model U6K injector, a Model 680 automated gradient controller, and both Model 440 fixed wavelength (254 nm) and Model 480 variable wavelength... 

An isocratic system is used with single solvents, a premixed solvent mixture, or step gradients. It has the advantage of needing only a single pump, no mixer, and no gradient controller. Because of this, isocratic systems are simpler and... 

HPLC system gradient controller, pump (one or two) optimized for low flow rates (frequently used flow rate below 1 mL/min), and a photodiode array detector or a variable wavelength detector (preferred wavelength 214 or 225 nm). An oven might be used for temperature control in the column and of the solvents delivered. An analogic recorder to directly follow the optical density of the hand-collected fractions. A fraction collector may be useful but not necessary. 

Figure 2. Flow Sheet of the HPLC Unit for Rapid Protein Analysis. A binary gradient system with gradient controller, Model 21500 pumps,Model 2152 controller, Pharmacia, Piscataway, NJ, U.S.A. (A), Variable wavelength UV-Visible detector, Model LC 95, Perkin Elmer, Norwalk, CT, U.S.A (B), Constant temperature circulating bath, Model DL-8 Haake Buchler, Saddlebrook, NJ, USA (C), Heat exchangers (D), Sampling valve,...

Automated gradient controller (Waters Model 680, MiUipore Corp., Milford, MA, USA)... 

Eluent delivery was provided by two model 510 high-pressure pumps coupled with automated gradient controller model 680 (Waters Chromatography Division, Millipore, MA, USA) and a model 7125 infection valve with a 20-(il loop from Rheodyne (Cotati, CA, USA) Stainless-steel columns (30 x 0.40 cm I.D.) packed from Tracer Analltica (Barcelona, Spain) with 10 un particle diameter Spherisorb ODS-2 (Merck, Darmstadt, FRG) were used. Four different LC mobile phase compositions were tested acetonitrile-water... 

---