Anal. Chem. 1996, 68, 3045-3049
High-Pressure Stopped-Flow Spectrometer for Kinetic Studies of Fast Reactions by Absorbance and Fluorescence Detection Pascal Bugnon, Ga´bor Laurenczy, Yves Ducommun, Pierre-Yves Sauvageat, and Andre´ E. Merbach*
Institut de Chimie Mine´ rale et Analytique, Universite´ de Lausanne, BCH, CH-1015 Lausanne, Switzerland Roger Ith and Rene´ Tschanz Atelier Me´ canique, Section de Chimie, Universite´ de Lausanne, BCH, CH-1015 Lausanne, Switzerland Michael Doludda, Rolf Bergbauer, and Ernst Grell Max-Planck-Institut fu¨ r Biophysik, Kennedyallee 70, D-60596 Frankfurt/a.M., Germany
The development of a stopped-flow instrument that operates over a temperature range of -40 to +100 °C and up to 200 MPa is described. The system has been designed so that measurements can be performed in absorbance and fluorescence modes simultaneously, without dismantling the unit. It can easily be combined with an optical system of a conventional ambient pressure setup by using light guides. Optimum optical performance and a wide operating wavelength range (220-850 nm) are achieved as the light is not passing through the pressurizing fluid. A special design for the pistons has been developed; thus, the apparatus has proven to be leak-free, even under extreme conditions (high pressure, low temperature, various solvents). The dead time of the system is found to be less than 2 ms at 298 K and is pressure independent up to 200 MPa. We examined the kinetics for the formation of the Mg2+-8-hydroxyquinoline chelate in aqueous solutions at pH 8.0 in order to develop a convenient alternative test method for high-pressure stopped-flow spectrometers with absorption and fluorescence detection. High-pressure techniques have been recognized to be very important for the elucidation of chemical reaction mechanisms. Relaxation techniques such as T-jump, P-jump, or NMR, and flow techniques such as stopped-flow, have been used with success at high pressures.1-5 The usual high-pressure stopped-flow (HPSF) apparatus were usually restricted to an absorbance detection.6-10 Balny et al.11,12 reported a high-pressure stopped-flow instrument (1) van Eldik, R., Ed. Inorganic High Pressure Chemistry; Elsevier: Amsterdam, 1986. (2) van Eldik, R.; Merbach, A. E. Comments Inorg. Chem. 1992, 12, 341-378. (3) Winter, R., Jonas, J., Eds. High Pressure Chemistry, Biochemistry and Materials Science; Kluwer Academic Publishers: Dordrecht, 1993. (4) Hubbard, C. D.; van Eldik, R. Instrum. Sci. Technol. 1995, 23, 1-41. (5) Powell, D. H.; Merbach, A. E. In Encylopedia of Nuclear Magnetic Resonance; Grant, D. M., Harris, R. K., Eds.; Wiley: Chichester, 1996; pp 2664-2672. (6) Heremans, K.; Snauwaert, J.; Rijkenberg, J. Proceedings of the 6th AIRAPT High Pressure Conference, Boulder, CO, 1977; pp 646-650. (7) Sasaki, M.; Amita, F.; Osugi, J. Rev. Sci. Instrum. 1979, 50, 1104-1107. (8) van Eldik, R.; Palmer, D. A.; Schmidt, R.; Kelm, H. Inorg. Chim. Acta 1981, 50, 131-135. (9) Ishihara, K.; Funahashi, S.; Tanaka, M. Rev. Sci. Instrum. 1982, 53, 12311234. S0003-2700(96)00382-4 CCC: $12.00
© 1996 American Chemical Society
being capable of kinetic measurements at low temperature with fluorescence detection. However, recent advances, especially in the domain of biochemistry,13 necessitated the design and construction of a high-pressure stopped-flow fluorometer with better performances (e.g., dead time, wavelength range). We present a description of the third generation of our highpressure stopped-flow10,14 instrument, capable of measuring rates of reactions in absorbance or in fluorescence, or both. To test this new apparatus, we measured at variable pressures the reaction of complex formation between 8-hydroxyquinoline and magnesium(II) in aqueous solution, at 278 K and pH 8.0, with both fluorescence and the absorbance detection. DESCRIPTION OF THE HPSF APPARATUS The high-pressure stopped-flow apparatus may be divided into the following parts: the autoclave, including the static and dynamic components of the stopped-flow circuit (see Figure 1); the drive mechanism; and the high-pressure control unit. The autoclave consists of a hollow cylinder made of stainless steel (AISI 630), with inner and outer diameters of 5 and 11 cm, respectively. The autoclave has an integrated cooling system in the form of a double helix, which has been machined into the surface of the bomb to ensure good temperature control. This allows a minimal temperature gradient throughout the autoclave. With the thermostating system taken into account, the effective outer diameter of the bomb, which is supporting the pressure, is 10 cm, not 11 cm. The outer surface of the bomb is covered by a stainless steel sleeve (AISI 310), which is supported by the lower plate. The autoclave is sealed at the top with a stainless steel disk (AISI 630). A Berylco (Berylco 25, NGK Berylco) screw cap secures the disk in place, with six Berylco screws used to compress the disk against a Viton seal of 90 shore. The thrust rod, which is made of hardened steel, passes through the disk. The sealing between the movable thrust rod and the disk is (10) Nichols, P. J.; Ducommun, Y.; Merbach, A. E. Inorg. Chem. 1983, 22, 39933995. (11) Balny, C.; Saldana, J. L.; Dahan, N. Anal. Biochem. 1984, 139, 178-189. (12) Balny, C.; Saldana, J. L.; Dahan, N. Anal. Biochem. 1987, 163, 309-389. (13) Doludda, M.; Lewitzki, E.; Ruf. H.; Grell, E. In The Sodium Pump; Bamberg, E., Schoner, W., Eds.; Steinkopff: Darmstadt, 1994; p 629. (14) Ducommun, Y.; Nichols, P. J.; Merbach, A. E. Inorg. Chem. 1989, 28, 26432647.
Analytical Chemistry, Vol. 68, No. 17, September 1, 1996 3045
Figure 2. Windows mounts and cell.
Figure 1. Schematic of the high-pressure stopped-flow unit: vertical section of the high-pressure autoclave.
ensured by a Turcite seal, which is held between two bronze bearings and compressed by a nut. Three threaded ports machined into the drum of the cylinder, 4.9 cm from the bottom of the autoclave, pass through the wall and end at the observation cell. The static component of the stopped-flow unit consists of two cylinders made of Vespel (polyimide polymer, Dupont de Nemours). Included in this section are the mixer mount (Durham 4 jet tangential mixer) and a capillary hole which leads to the observation cell. The observation cell has dimensions of 2 mm × 2 mm × 10 mm, giving path lengths of 10 mm for absorbance measurements and 2 mm for fluorescence measurements. The cell is built into the Vespel block, with the window mounts for the entering light, the absorbance detector, and the fluorescence detector enclosing three sides (Figure 2). The window mounts for the entering light and for the absorbance detection consist of a Berylco holder with a sapphire window (10 mm diameter, 5 mm thickness, resistance > 800 MPa, Djeva), held in position with a silicon toric seal. The internal diameters of these two mounts differ so as to accept a fiber guide (minimal diameter, 5 mm) or the photomultiplier (diameter, 16.8 mm). The window mount for the fluorescence detection consists of a Berylco holder with a sapphire window (8 mm diameter, 16 mm thickness, resistance > 800 MPa, Djeva), which is supported by the Vespel block and a Teflon seal. A temperature sensor (platinum resistance, Pt 100) is positioned close to the cell; however, it remains at ambient pressure. The sensor is linked to a digital thermometer (Technoterm 7600/700) through a port in the lower plate. The dynamic component of the stopped-flow unit includes two reagent syringes and a collection syringe and is fixed to the static component by four screws (Figure 1). Three silicon toric O-rings ensure the seal. The three syringes are screwed to a cylindrical 3046 Analytical Chemistry, Vol. 68, No. 17, September 1, 1996
Figure 3. Reagent syringe detail.
Vespel base. The syringes’ barrels and the piston caps are made of poly(chlorotrifluoroethylene) (PCTFE) (Figure 3). The piston has an internal plunger, which can be adjusted with a tension screw to allow for the compression of an O-ring, which in turn alters the effective diameter of the piston head. This ensures a good seal between the inner wall of the syringe barrel and the piston head. A spiral thrust spring keeps the head at the desired diameter so as to avoid the effect of material contraction under high pressure and/or low temperature. The plunger depressor allows the thrust spring to be relaxed, thus contracting the piston head, which facilitates the dismantling of the syringes. During a typical injection the thrust rod pushes the anvil (made of Anticorodal), which then pushes the pistons. The syringe block can be replaced with a flushing adaptor to enable flushing of the nonremovable cell block and flow circuit. The thrust rod is driven by a pneumatic press (Festo DC) with a translatory jack (140 mm diameter, 70 mm stroke). The pneumatic press is mounted on a track, conveniently enabling it to be moved aside during loading of the pressure vessel (Figure
for the insertion of a flexible light guide to direct the emitted light initially through a monochromator before detection.
Figure 4. Lateral view of the high-pressure stopped-flow instrument, including the fluorescence photomultiplier. After loading of the pressure autoclave, the pneumatic press is moved from position A to A′.
4). This modularity of the system enhances its ease of use. The operator is protected from possible injury during the operation of the pneumatic press through a combination of a metal cage with a perspex door, which isolates the drive assembly. The drive assembly can only be operated when the pneumatic press is over the autoclave and the door is closed. Several antireverse valves regulate the inflow and outflow of compressed air in the press. The driving length of the thrust rod is selected by adjusting a micrometer screw on the press jack (stroke adjustment). Just before stopping, the pressing action pivots a small mirror, which diverts the beam from an emitting LED to an optical captor, thus inducing the trigger. The pressure for the system is generated by a pressure intensifier. The hydraulic fluid is connected to the bottom of the autoclave by a capillary tube (Novaswiss, inner diameter 0.5 mm). The pressure intensifier consists of a liquid tank, five gates, and two pumps (Novaswiss, 400 MPa), linked by stainless steel capillary tubes. A pneumatic pump is used for large variations of pressure, while a manual pump is used to more accurately adjust the pressure to the desired value. A piezoelectric sensor (Burster 8219R-4000) is use to measure the pressure. The hydraulic fluid is generally distilled water, which is less compressible than n-hexane or n-pentane. To enhance sensitivity and improve accuracy, the absorbance photomultiplier is mounted directly at the detection window, and the light source is connected to the excitation window via a flexible light guide. For fluorescence measurements, a quartz cylinder (diameter 6 mm, length 90 mm) is inserted into the window mount to guide the light through an integrated cutoff filter prior to the photomultiplier. The design of the fluorescence mount also allows
CHARACTERISTICS AND PERFORMANCE OF THE HPSF APPARATUS The main characteristics of this apparatus are described as follows: the stopped-flow measurements can be performed at pressures up to 200 MPa, in a temperature range between -40 and +100 °C. The sample circuit is chemically inert (limited in very basic media by the Vespel properties), and there are no metallic parts in contact with the sample solutions. The volume of the cell is 40 µL, and the dead volume is 65 µL. A 250-300 µL volume is used for each injection, so the sample handling system can be loaded with sufficient reagent for more than 20 experimental runs without dismantling the high-pressure unit, which involves depressurization and removal of the stopped-flow unit from the container. Optimum optical performance is achieved as a result of the use of sapphire windows, which are in direct contact with both the experimental solution and the optical system, without passing through the pressurizing fluid, as is often the case with other instruments.11,15 In consequence, we have access to a wide operating wavelength range (220-850 nm). It is possible to measure simultaneously in the absorbance mode and in the fluorescence mode. Furthermore, the high-pressure stopped-flow system can easily be combined with the optical system of a conventional ambient pressure setup by using inexpensive light guides. To test the efficiency of the mixer, we measured the very fast reaction of phenolphthalein protonation. Upon mixing phenolphthalein dissolved in 5 × 10-3 M NaOH with 10-2 M HCl, no residual absorption due to ionized indicator was detected at 552 nm (maximum absorbance coefficient of the protonated species) and at the highest sensitivity of the instrument. We examined the efficiency of our apparatus by determining the dead time of the instrument, i.e., the time needed for the reaction mixture to flow from the mixing chamber to the observation cell. The dead time, td, measured in the absorbance mode using the reaction of reduction of 2,6-dichlorophenolindophenol with L-ascorbic acid,16 was
