SUPREX

Another HX-based method, stability of unpurified proteins from rates of hydrogen exchange (SUPREX), was developed by the Fitzgerald and coworkers [38]. It is analogous to chemical dena-turation methods employing CD or fluorescence, which were previously known. 

Similar to PLIMSTEX, SUPREX data are first plotted as AD (deuterated-undeuterated) of an apo/holo state protein as a function of denaturant concentration and then fitted using nonlinear least-squares analysis. The fitted curve affords a transition midpoint (CsJ ggx a denaturant concentration at this point) that can be used to calculate AGp the free ena-gy of folding, and an w-value (defined as the sharpness of the transition in the fitted curve (5AGj)/(5[denaturant]) (Eq. 11.22)  

In Equation 11.22, is the equilibrium constant between the exchange incompetent and competent states that is related to protein folding dynamics (Eq. 11.23) (see Section 1.3). R is 

---

Figure 8.22 Schematic diagram of the Suprex MPS/225 integrated aupercritical fluid extractor, cryogenically focused interface and supercritical fluid chromatogra d>. The bold lines represent the direction of fluid flow in the load and inject positions.

Reproduced with permission from Suprex Corporation). 

The Model 50 Supercritical Fluid Microextractor from the Suprex Co. (Pittsburgh, PA) was adapted for this design. A schematic of our multi-vessel extractor design can be found in Figures 1 and 2, for six and twelve multi-vessel systems, respectively. The following is a detailed description of the main components. The design, explained here for the extraction from six and twelve vessels is in principle applicable to any number of vessels, provided that other components of the system are scaled up. 

Modifier Pump. The first feature in our adapted design is the introduction of a liquid pump via an instrument controlled VALCO (Model E04, Valeo Instruments, Houston, TX), four position selection valve. We have used an LKB Model 2150, dual piston pump for pumping modifier and entrainer fluids (LKB-Produkter AB, Bromma, Sweden). However, any suitable liquid pump could be substituted. Only pure fluids such as carbon dioxide have been introduced with the Suprex system syringe pump. With the addition of this second pump to deliver liquids, modifier is introduced directly into the extraction vessel. A wide range of alternative fluids and fluid mixtures can be rapidly selected with this dual pumping option. The criteria for selection of a modifier pump include the ability of the pump heads to withstand pressures in the range of 100 to 300 atm and interfacing capabilities, i.e. the ability to be turned on and off by the Suprex contact closure controls. 

Autofill Valve. The VALCO autofill valve and actuator is an option available on the Suprex Model 50 extractor that presented problems during routine operation even without the multi-vessel adaptations. With the multi-vessel option, rapid refill of the syringe pump is a necessity for routine unattended operation. This is accomplished via a VALCO 1/16 five port valve (Model E04) mounted on an electric actuator. The valve has a pressure rating of 11,000 PSI. In the tank mode, the autofill valve directs flow from the mobile phase tank to the pump. In the column mode, flow is directed from the pump to the column. 

With this system, two extract delivery lines are required, one for each of the vessels as well as two delivery nozzles. The advantages of this mechanism are its simplicity, ease of operation, and its capacity. The mechanism is also compatible with the method-chaining program in the SUPREX. Since the extraction vessels are treated as pairs,when the program asks for a count number on the columns, an entry of six, which is the maximum allowable, will result in twelve extractions, i.e. 2N. An entry of four will result in eight extractions, etc. However, because at each actuation of the solenoids, a pair of columns is selected, this mechanism does not allow room for independent method use between the extraction vessel pairs. 

System Control The control unit operates all the functions of the extractor. It is composed of a CRT, keyboard, contact closure outputs and inputs and nine control cards each with special functions. This unit requires almost no modification since its functions are highly specialized and the circuits are delicately assembled. The most important function to the user is its ability to interface with other instruments. This is done via the contact closures. Of primary use in our design is the contact closure which controls the event end output pulse. In the Suprex Model 50, this is contact closure B. This event end contact closure is the main interface in our design to other in-... 

Figure 1. Schematic representation of two-vessel suprex extraction system.

We acknowledge the assistance of Nikhil S. Dodhiwala who performed most of the SFE work, of Janet Benedicto and Lisa Balch, who performed all gas chromatographic and gas chromatographic/mass spectrometric analyses reported in this manuscript, and of Karin Bauer of Midwest Research Institute, who provided input on the statistical analysis of the preliminary results from our method optimization study. We would also like to acknowledge the assistance of Ashok Shah and Carl Stadler of Suprex Corporation who helped with the design of the dual-extraction setup on the Suprex SE-50 system, and of Hewlett-Packard Company who made available to us a Hewlett Packard extractor. 

Joseph M. Levy, Eugene Storozynsky, and Mehdi Ashraf-Khorassani Suprex Corporation, 125 William Pitt Way, Pittsburgh, PA 15238... 

State University of New York at Buffalo, 1,48,73,92 Suprex Corporation, 336 U.S. Environmental Protection Agency, 179 University of North Dakota, 165 University of Notre Dame, 106 University of Nottingham, 121 University of Texas, 31 Virginia Polytechnic Institute and State University, 206... 

Figure 11.5. SFC separation of an ethoxylated amine using pressure programming. Courtesy of Suprex.

In addition to the previous three commercial extractors, some authors have developed custom models [78,79], adapted in most cases from a supercritical fluid extractor [25,58,80]. For example, Heemken et al. altered a Suprex SF extractor for use in ASE [81] they disconnected the syringe pump from the COj cylinder and filled it with a suitable ASE solvent. The restrictor was replaced with a stainless steel capillary tube leading into the trapping vial and an additional nitrogen pipe was installed at the inlet valve of the extraction vessel for purging after extraction. In the extraction of PAHs from soil [79], a custom extractor and commercially available equipment provided equivalent results on the other hand, in the extraction of benzene and toluene from soil [78], the former provided even better results than the latter. 

Applied Separations 930 Hamilton St. Allentown, PA 18011 Isco 4700 Superior, Lincoln, NE 68505 Durability, Inc. 1872 Pratt Dr., Suite 1630, Blacksburg, VA 24060-6344 (Isco-Suprex) Thar Designs, Inc. 730 William Pitt Way, Pittsburgh, PA 15238... 

Suprex, Alumex, Supreme, Rogers - kaolins fi om two different locations in SC and GA R.T. Vanderbilt Company, Inc., Norwalk, CT, USA... 

---