automated pressure and temperature control apparatus for x...

Automated pressure and temperature control apparatus for x-ray powder diffraction studies

P. T. C. So, S. M. Gruner, and, E. Shyamsunder@ Department of Physics, Joseph Henry Laboratory, Princeton University, P. 0. Box 708, Princeton, New Jersey 08544

(Received 3 September 1991; accepted for publication 8 November 1991)

A system for performing x-ray diffraction on biological samples as a function of pressure and temperature is described. It is capable of operating in a pressure range of 1 bar-3 kbar (0.1-300 MPa) and in a temperature range of - 30 to 80 “C. The system incorporates microprocessor-based pressure and temperature controllers which provide automated control with excellent stability characteristics: Fluctuations in pressure and temperature can be maintained within f 1 bar (0.1 MPa) and AO.05 “C!, respectively. Use of the apparatus is illustrated by application to a pressure-induced phase transition in a lipid-water liquid crystal.

1. INTRODUCTION

An understanding of the workings of biological mole- cules frequently involves study of their structure and function of both temperature and pressure. Although x-ray diffraction is one of the most important tools for the determination of biomolecular structure, few laboratories use x-ray apparatus which can operate over both a wide range of temperature and pressure. In this report, we de- scribe a relatively inexpensive apparatus which can be used for diffraction studies of small quantities of biological materials over temperature and pressures which are frequently of interest.

Biological systems are known to be affected by the application of relatively modest pressures.lp3 Although the mechanism by which modest pressure couples to biological function is not understood, it is assumed that the coupling involves some degree of structural change in either the proteins or biomembranes of the system. Even though extensive fluorescence’ and vibrational spectroscopic” studies have been performed on proteins over the 1 bar-3 kbar (0.1-3 MPa) range where effects on organisms are known to occur, only a few high-pressure x-rayk7 and neutron diffraction*1g studies have been performed to probe the as- sociated structural alterations. Our main interest here is in devising an apparatus which may be used to perform small- angle x-ray scattering (SAXS) studies on biomembrane materials. The motivation for studying biomembranes is that the factors controlling the mesomorphic phase behav- ior observed in biomembrane lipid-water dispersions have been suggested to also modulate the operation of proteins imbedded in the lipid bilayers of biomembranes.10**2

II. SPECIFICATIONS

The starting point for the design of any high-pressure apparatus is the determination of the temperature and pressure ranges over which it must operate. Once these ranges are known, appropriate high-pressure construction techniques’3 may be applied. It is then necessary to esti-

‘)To whom correspondence should be addressed.

mate the precision with which the temperature and pressure must be controlled.

( 1) Range of variables desired: Mesomorphii: phase transitions in biological lipid-water liquid crystals typically occur in a temperature range of - 40 to 90 “C (Refs. lO- 12) and in the pressure range of 1 bar (O.l~MPa) to about 3 kbar (300 MPa). At the higher pressures or lower tem- peratures, the liquid-crystalline phases transform to gel phases.“6 Furthermore, many physiologically interesting effects occur below 3 kbar (300 MPa). For example, re- versal of anesthesia typically occurs at room temperature at about 0.3 kbar (30 MPa). l4 The phase transition behav- ior of lipids has been implicated in the mechanism of anesthesia, although the issue remains controversial. l4 Our investigations of the connection between lipid phase transitions and anesthesia require pressure control that is vari- able within the 3.0-kbar range (300 MPa).”

(2) Degree of control required: Many of the interesting biological lipid-water systems form lattices- with unit cell repeat spacings on the order of 50 A. Careful measure- ment of the lattice is fundamental to understanding the system. For example, Gruner16 has proposed a theory of lipid-protein interactions in which living cells homeostati- tally regulate the spontaneous radius of curvature, Roe, of lipid monolayers to a value in the range of lO-lo2 A. A determination of this important parameter involves mea- surement of the lattice size of nonlamellar phases to an accuracy of 1% or about 0. l-l A. Since the unit cell spacing in the nonlamellarphases varies with pressure and temperature at about 1 A/100 bar (1 A/10 MPa) and 0.5 A/l “C, respectively,4s6 it is desirable that the control accuracy of pressure and temperature be better than 10 bar ( 1, MPa) and 0.2 “C, respectively. The requirements of stability of temperature and pressure for lyotropic (i.e., mul- ticomponent) lipid-water systems during mesomorphic phase transitions imposes a less stringent constraint than for comparable studies on single-component conventional liquid-crystal systems, because the weakly first-order transitions are always accompanied by coexistence of the two phases over a temperature range of 2-10 K or a pressure range of 50-100 bar (5-10 MPa).

.

1763 Rev. Sci. Instrum. 63 (2), February 1992 0634.6746/92/021763-08$02.00 @ 1992 American Institute of Physics 1763

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(3) X-ray flux requirements: Lipid-water mixtures have scattering powers - 10 - 3-10 - 5, requiring the use of relatively large samples. Typical experiments use - 5 mg of sample material with a total volume of about -5 ~1. The x-ray cell design has to balance the competing requirements of strength to withstand the pressures and transparency to x rays so that pattern collection times are not inordinately long.

(4) Additional constraints: It is also desirable that the high-pressure cell and the pressurizing medium do not react with the biological samples. The pressure should be transmitted hydrostatically to ensure pressure uniformity. Finally, experiments conducted at fixed water concentrations are desirable. The pressure should be transmitted without altering the water content of the sample, so that direct pressurization via a gas or water phase is not desirable.

Ill. APPARATUS

A. Basic idea

Pressure control: Since biological samples are typically composed of relatively incompressible materials such as water, one can effectively regulate sample pressure by minute adjustment of the sample volume. A variety of techniques have been employed to exploit this idea.17 The system described here uses computer-controlled translation of a piston to achieve pressure variation and control.

Temperature control: Thermoelectric modules (Melcor Materials Products Corp., Trenton, NJ) are used to control sample temperature. The thermoelectric modules can either heat or cool the sample stage by varying the magnitude and direction of the current applied through the thermoelectric modules. A temperature-controlled x-ray diffraction stage based on this approach has been described by Mudd et aZ.‘*

B. Mechanical

Figure 1 shows an overall schematic side view of the x-ray apparatus. The x-ray sample chamber consists of an evacuated aluminum chamber with vacuum-tight feed- throughs for electrical and high-pressure connections. A pair of slits is mounted inside the evacuated chamber for collimation. The main beam is blocked with a lead beam stop which is mounted inside the vacuum chamber, but which can be moved by externally accessible micrometer carriers. The diffraction pattern is recorded with a two- dimensional x-ray CCD detector described in Templer et ai.”

Beryllium cell: Beryllium is the material of choice for high-pressure x-ray study because of its relative high x-ray transparency and its high tensile strength. Transparency to x rays requires the use of cells that have thin walls; on the other hand, the ability to withstand high pressure requires the use of thick-walled cells. The absorption of x rays is an exponential function of the material thickness. Figure 2 shows a plot of the intensity of x rays transmitted through beryllium, which has about 1% Be0 as the dominant impurity (Brush-Wellman Inc., Elmore, OH). The tensile

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FIG. 1. Side view of the high-pressure x-ray scattering experimental ap paratus. CuKa x rays are produced by an x-ray generator and focused via Franks camera optics (Charles Supper Company, Natick, MA). The x rays then pass through a thin mylar windows (M) into an aluminum sample chamber housing (Y) which is evacuated to reduce air absorption of the x rays. The x-ray beam is collimated to a cross-sectional area of 0.5 X 2 mm by two orthogonal pairs of tantalum slits (C) and then passes into the Be sample cell. The sample cell is pressurized via &in.- (1.59- mm-) o.d. high-pressure tubing (T) (HIP products, Erie, PA) connected to the pressure generator via a vacuum feedthrough. Diffracted x rays bypass a beam stop (B) and exit the vacuum chamber via another mylar window (M). The diffraction is recorded on a CCD-based x-ray detector (see Ref. 19) (D). The sample (S) is placed in a &in.- (3.17%mm-)

diam, $in.- (1.905-cm-) deep hate in a &in.- (0.635cm-) diam, l-in.- (2.54-cm-) long beryllium rod (Be). Tolerances on critical dimensions were specified at 0.001 in. (0.0254 mm). The rod sits in the steel jacket (N). Entrance and exit cones in the jacket allow a maximum scattering angle of 60”. The push piece (P) is forced by the I$-11 screw piece (A) to compress a Teflon packing containing antiextrusion brass rings (Pa). The steel jacket is encased in a temperature-controlled brass jacket (not shown) which is heated and cooled by thermoelectric modules. Sample temperature is sensed by a platinum resistance thermometer (R). The entire assembly rests on an insulating G10 base (G), which is mounted on a stepper motor-controlled XZ two-axis translation stage that allows for remote alignment of the sample in the beam.

strength of an unsupported Be cylinder for a given ratio w = Router/&w of outer and inner radii is given by Dawson.13 The inner radius is fixed by the constraint of having to use a minimum size sample volume for the weakly diffracting x-ray samples. Our cell was designed to accept standard quartz x-ray capillaries ( 1.5 mm diam). A plot of the tensile strength as a function of the outer radius is shown in Fig. 2. One can see that a compromise exists between the two design criteria at a diameter of about 0.5 cm, i.e., further increases in diameter do not improve the tensile strength significantly, whereas the transmitted intensity is reduced by a factor of about 2. We note that the calculation shown in the figure is for unsupported cylinders of Be. Careful design of a supporting jacket can extend the

1764 Rev. Sci. Instrum., Vol. 63, No. 2, February 1992 X-ray powder diffraction 1764


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FIO. 2. (a) Be cell design considerations: Shown in this figure are the two conflicting design considerat ions in a high-pressure x-ray cell design. The line (solid symbols, right ordinate) shows the intensity of transmitted x rays as a function of thickness (outside diameter) of the Be cell. Absorp- tion is governed by the composit ion of the material used, which contains about 1 % Be0 as the dominant impurity. Traces of Be0 improve the strength of the cell. Also shown in open symbols (left ordinate) is the tensile strength P/P, of an unsuppor ted Be cylinder given by P/P, 2.E (e? - I)/v%? (seeRef. 13), wherew = R2/R, is theratiooftheouter radius R2 to the inner radius R,. For Be, the maximum tensile strength PO = 40 kpsi ( -2.7 x lo8 N m ..’ 2, ( Brush-Wellman Inc., Elmore, OH).

upper limit to beyond this value imposed by the tensile strength of Be, as discussed by Dawson.13 The Be cell used has been safely pressurized up to pressures of 3 kbar (300 MPa).

The use of beryllium has its disadvantages. Since beryllium oxide dust is extremely toxic, more complicated handling and maintenance procedures have to be prac- ticed. Indeed, this last consideration led us to a design that minimizes the machining of the beryllium. Consequently, we decided against using a design in which the top of the beryllium cell is shaped like a mushroom plug. Although the mushroom plug design is elegant and can form better seals, our design uses a straight cylindrical rod of beryllium and a more elaborate seal. The beryllium cell sits inside a steel holder (Fig. 1). The steel is a high nickel content maraging steel (Vasco-Max C-300, from Teledyne Vasco, Latrobe, PA) which can be heat treated in air to achieve tensile strengths of 300 kpsi ( -2X 10’ N m - *). The beryllium cell in Fig. 1 is similar in construction to the one reported by Utoh and Takemura.s The sample is placed in a &in.- (0.635cm-) diam l-in.- (2.54-cm-) long beryllium rod in which a &in;- (0.318-cm-) diam $-in.- ( 1.905cm-) long concentric hole has been drilled. The beryllium rod is in turn placed in a tight-fitting hole inside the steel holder. The high-pressure seal is formed at the lip formed between the holder and the beryllium rod using Teflon packing with brass antiextrusion rings. The large screw piece (part A in Fig. 1) forces the push piece to compress the packing. The packing is compressed until the pressure inside the packing

1765 Rev. Sci. Instrum., Vol. 63, No. 2, February 1992

is just over 3.0 kbar (300 MPa). In order to get reproduc- ible compressions of the packing, a torque wrench was used to tighten the screw to 20 ft Ibs (27 N m). The pressure tubing is hard soldered to the other end of the push piece, and is passed out of the vacuum chamber via a vacuum feedthrough.

The steel jacket is placed in a demountable brass jacket (not shown). Two thermoelectric modules are sandwiched between the jacket and a brass heat sink cooled with circulating ethanol-water solution. The use of ethanol-water allows the cooling solution temperature to be below 0 “C. Thermal contact between the brass and the thermoelectric modules is established via a thin film of Thermal Joint Compound (type 120, Wakefield Engineering, Wakefield, MA). The thermoelectric modules may also be soldered with indium solder to the polished copper plates. We have found that, while this technique forms an excellent thermal contact, it makes it difficult to replace the thermoelectric modules (which occasionally fail). The thermal joint compound does not seriously compromise the thermal contact and allows quick replacement. The brass jacket and the steel jacket are placed on a thermally insulating G-10 mount in the vacuum chamber. The entire assembly is placed on a remote-controlled two-axis translation stage (not shown) to facilitate alignment of the sample cell with the x-ray beam. The temperature is measured by a platinum resistance thermometer embedded in the steel con- tainer.

Low-pressure studies: At pressures of about 0.5 kbar (50 MPa), we have found that plastic cells made of Delrin instead of Be are satisfactory. The use of Delrin cells was first reported by Heald and Simmons.*’ We have found a dead-ended $-in.- (0.635-cm-) diam Delrin cylinder coupled directly by means of commercially available stainless- steel Swagelok fittings (Penn Valve and Fitting Co., Marl- ton, NJ) to be an inexpensive and quick alternative to Be cells. No supporting steel jacket is needed, resulting in a much faster temperature response. Delrin also has the ad- vantage that it is chemically inert and does not react with biological materials. Delrin does, however, limit the highest pressure that can be measured with the system, to about -0.5 kbar (50 MPa) at room temperature.

Pressure generation and control: A commercially available pressure generator (HIP Model No. 37-5.75-60, Erie, PA), which uses a manual hand-crank to translate a piston, was modified as follows: The maximum torque required at the highest pressure was measured to be 64 ft lbs (87 N m). The original handle of the generator was re- moved and a large gear wheel was mounted in its place. The gear wheel was coupled via a 1O:l gear system to a reversible dc motor which can provide a torque of 10 ft Ibs ( 14 N m) (True-Torq Model EB-PSC, Blanchester, OH). Limit switches were placed to restrict the travel of the piston. The volume of the system ( - 50 ml) was such that entire pressure range of interest from 1 bar to 3 kbar (0. l- 300 MPa) could be covered by translating the piston by 5 cm.

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Smallpressure ranges: It was found that an inexpensive means of precisely controlling small ranges of pressure

X-ray powder diffraction 1765


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FIG. 3. Block diagram of the pressure controller. Pressure is controlled by a single board computer (SBS-2300). The controller senses the analog output of a pressure transducer and compares it to the set pressure. It then turns on the motor in an appropriate direction until the set pressure is reached. For coarse control to within f 5 bar (0.5 MPa), the pressure generator was directly driven by reversible de motor. For applications involving fine control to within f I bar (0.1 MPa), the system could also control a stepper motor connected to a high-pressure needle valve. The pressure controller can be operated either locally or by a remote computer via a RS-232 line, or via digital I/O lines. A vacuum fluorescent display connected to the SBS-2300 shows the sample pressure.

could be accomplished by connecting a dead-ended high- pressure needle valve to a line connecting the high-pressure pump and specimen chamber. As the valve is opened or closed, the displacement of the needle valve results in a pressure change. In our experiments, the needle valve was driven by a stepper motor, which was in turn controlled by the single board computer controller so as to provide automated control.

Pressure transmission: It is frequently necessary to perform experiments at fixed water concentrations to deter- mine the structure of the lipid-water phases. In addition, contact between the sample and pressurizing fluid should be avoided because many samples react with the pressurizing fluid. A mixture of 50% ethanol and water is used as the pressurizing medium in order to allow experiments down to - 30 “C. The sample was placed in a conventional thin-walled 1.5-mm-diam quartz capillary and sealed with a small plug of mercury of approximately 1-2 ~1 volume. The capillary was then placed inside the beryllium cell. The mercury acted like a frictionless piston to transfer hydrostatic pressure, and also did not interact with the sample. The same method might also work with protein crystals in solutions of mother liquor. Mineral oils have been used to coat the protein crystal,7 but oils are known to react with lipids. Because the hydrostatic pressure is the same on both inside and the outside of the quartz capillary, even the very thin-walled capillaries survive extensive pressure and temperature cycling.

It is worth remarking that the requirement of relatively large sample volumes and of good pressure control at low pressures precludes the use of diamond anvil cells in our experiments.


C. Electrical

The central feature in the electrical designs of both the pressure and temperature controllers is a single board computer (SK) (SBS-2300, Octagon System Corporation, Westminster, CO) which can be coupled to additional elec- tronics to monitor and control either pressure or temperature. This board features a microprocessor, two channels of g-bit digital-to-analog converter (D/A), eight input channels of 12-bit analog-to-digital converter (A/D), dig ital I/Q lines, and a programmable EPROM. The control algorithm is implemented in BASIC code that can be burned into the EPROM. Control parameters can be easily modified or optimized by changing the software codes without any hardware alterations. In addition to the digital I/O lines that can be used for parallel I/O, a serial RS-232 port allows the controllers to conveniently communicate with a remote IBM AT computer which oversees the whole data acquisition system and which is used to develop the control algorithm.

D. Pressure control

A block diagram of the pressure controller is shown in Fig. 3. The pressure at the sample is monitored by a pi- ezoresistive transducer (Sensotec Inc., Columbus, OH) which provides an output voltage that is linearly propor- tional to the applied pressure. The amplifier circuit provides an output signal with a sensitivity of 10 mV/bar ( 10 mV/O*l MPa). To reduce noise, the signal is digitized 20 times via the SBC’s AD converter and the resulting values are averaged. The pressure so obtained is then compared with the set pressure. If the detected time-averaged differ- ence exceeds 1 bar (0.1 MPa), the computer turns the



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FIG. 4. (a) Block diagram of the temperature controller. The temperature of the brass sample cell holder is sensed by a platinum resistance thermometer (RTD). A microprocessor-based temperature controller senses the output of the thermometer and compares it with the set temperature. A current of up to 6 A can be passed through the thermoelectric modules in either direction to heat or cool the sample stage. T’he temperature controller can be operated either locally or by a remote computer via a RS-232 line or via digital I/O lines. (b), (c) Details of the sensing and control circuitry: The use of hybrid chips such as the Analog devices 2B31J simpli- fies the design. The RTD has a resistance of 100 a at 0 “C.

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stepper motor in the appropriate direction to either raise or lower the pressure. Control of the stepper motor is accomplished via two digital I/O lines which activate the motor and set its direction of rotation. By changing the direction

expansion, thereby increasing or decreasing the pressure.

E. Temperature control

of rotation, the piston of the pressure c&t& valve could Figure 4 shows the block diagram of the temperature be advanced to compress the liquid, or reversed to allow controller. A platinum resistance thermometer (F3 102,



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FIG. 5. (a) The pressure response as a function of time was measured as the set pressure was changed to 2 kbar (200 MPa) from an initial set pressure of 100 bar ( 10 MPa). The curvature in the ramp is due to the nonlinear compressibility of water at high pressures. The system’s slew rate was determined to be about 5 bar/s (0.5 MPa/s), and is higher at the higher-pressure range due to decreased compressibility of water. (b) The stability of pressure response as a function of time at 2 kbar (200 MPa) at 20 ‘C is shown at a finer scale. The pressure equil ibrated accurately to within *5 bar (0.5 MPa) of the set pressure.

Omega Engineering, Inc., Stamford, CT) in a bridge circuit is used as the temperature sensor yielding a sensitivity of 0.1 mVPC!. The output is then digitized by the A/D on the SBC board and compared to the set point. The computer compares the specimen temperature against the desired temperature and generates a correction signal. This digital signal is translated to a correction voltage via an &bit D/A, which in turn is amplified to drive current through thermoelectric modules via a power transistor Darlington pair. The thermoelectric modules (Melcor Ma- terials Products Corp., Trenton, NJ) are connected in se- ries pairs across a 20-V source to the Darlington pair [Fig. 4(b)]. Each module can handle up to 6 A of current and can either heat or cool depending on the direction of the current flow through the module. The direction of the current flow is determined from the SBC correction signal and is used to control a current reversing relay, which directs the current from the Darlington pair either one way or another through the thermoelectric modules. The relay is switched only during changes of sign of the correction signal (i.e., at zero thermoelectric current) to avoid relay contact arcing.

Discussion of corm-oiling algorithms: All controls are exercised digitally. The feedback system’s gain can be modified dynamically to allow minimization of overshoot and oscillations.

The control program consists of three phases with suc- cessively smaller gain. During the initial phase, the system operates at the maximum gain in order to quickly approach the desired set temperature. As the actual temperature of the system becomes sufficiently close to the set temperature, the system proceeds to the next phase. By determining the heating or cooling rate of the thermal load, the gain of the system can be smoothly decreased as the set temperature is approached. This minimizes overshoot. When the set temperature is reached, the system enters the final phase with the smallest gain so that the system will not oscillate around the equilibrium point. Fur- ther, note that the system’s D/A and A/D have rather


limited resolution: one bit of D/A and A/D roughly cor- responds to 0.1 and 0.04 “C, respectively. To achieve the desired resolution, the output digital D/A signal is cycled over a few bits sufficiently rapidly compared to the thermal response time of the system so that the t ime-averaged output may correspond to a fraction of a bit. Similarly, the input A/D signal is time averaged to minimize system noise.

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FIG. 6. Stability of pressure response as a function of time at 300 bar (30 MPa) was measured while the sample temperature was increased from 20 to 70 ‘C for the Delrin cell. The solid line with circles shows the pressure response of the system with the pressure controller on and the line with squares shows the pressure response of the system with the pressure controller off. The insert shows the temperature response of the system as a function of time while the pressure response was being measured. Note that the pressure of the system drifted by over 10 bar ( 1 MPa) without the pressure controller. On the other hand, with the pressure controller connected to the high-pressure needle valve, the system’s maximum pressure fluctuation was less than 1 bar (0.1 MPa) in the first 2 min after the onset of the perturbation and was damped down to f 1 bar (0.1 MPa) afterwards.



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FIG. 7. (a) The temperature response as a function of time was measured as the set temperature was changed. The system’s slew rate was determined to be about 1 “C/min inside the operating range. (b) The stability of temperature as a function of time at 20 “C at 1 bar (0.1 MPa) was measured. At both temperature ranges, the temperature equilibrated accurately to within *0.05 ‘C of the set temperature. Initial oscillation was damped down within 2 mm after the set temperature was reached. The subsequent fluctuation is less than 0.05 “C.

IV. PERFORMANCE

A. Pressure control

The performance of the pressure controller was monitored by recording the output of the pressure sensor as a function of time. Figure 5(a) shows the response of the pressure controller as a function of time as the set pressure is changed by 2 kbar (200 MPa). The system was determined to have a slew rate of 5 bar/s (0.5 MPai’s). Figure 5(b) shows the stability of the pressure controller as a function of time. The system accurately equilibrates at the set pressure to within 5 bar (0.5 MPa). The most important cause of the 5-bar (0.5-MPa) fluctuation seen in Fig. 5 (b) is the backlash in the screw in the pressure generator. Using a small high-pressure valve controlled by a stepper motor for fine control of pressure, we have found that it is

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possible to control the pressure to within f 1 bar (0.1 MPa). The stability of pressure was also measured by set- ting the pressure at 300 bar (30 MPa), and changing the temperature of the sample from 20 to 70 “C. Without the pressure controller, the pressure in the specimen chamber would have changed by over 10 bar (1 MPa) due to the thermal expansion of water; however, with the pressure controller, this pressure change was eliminated. The initial oscillation due to the perturbation was quickly damped down in 2 min (Fig. 6).

B. Limitations of the pressure cell

The restriction of using a cell that involves minimum machining of the Be cell lead us to a more elaborate seal design. Repeated disassembly of the cell during sample re-

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FIG. 8. Traces through x-ray powder diffraction patterns of a water dispersion of dioleoylphosphatidylethanolamine were taken at (a) 10 “C, 1 bar (0.1 MPa) and (b) lo%, 300 bar (30 MPa). Exposure times were 1000 s. S is the magnitude of the scattering vector. The lipid was observed to undergo a phase transition from a hexagonally packed tubular H, phase to a lame&r L, phase as pressure was increased isothermally from 1 to 300 bar (0.1-30 MPa). The position of the beam stop is at S=O. The tic marks show seven orders of diffraction from a hexagonal lattice with lattice spacing 7.81 *to.05 nm in (a) and four lamellar orders with lattice spacing 5.30 =t 0.05 nm in (b) . The quality of diffraction was sufficiently good to allow a low-resolution electron density reconstruction of the inverted hexagonal phase which is shown in Fig. l(b) of Narayan et al. (see Ref. 21).



placement appears to result in a small distortion of the Be cell at the point at which the seal is made, where the antiextrusion rings swage into the cell wall. Although the swaging has so far not affected the sealing properties, it is not desirable. The use of steel as jacket material instead of beryllium copper also leads to long temperature equilibra- tion times. Although this is not a severe restriction in an x-ray experiment, where the low flux of laboratory x-ray sources leads to long data collection times, the use of a Be-Cu jacket is recommended, provided a machine shop that can handle the hazards of machining Be-Cu is available. Although the hazards of machining BeCu are small compared to the hazards of machining pure Be, appropriate precautions should be taken.22

C. Temperature control

The temperature response was monitored by putting a thermocouple in the sample chamber. Surprisingly, the Delrin pressure ceil has excellent thermal characteristics. Figure 7(a) shows the temperature recorded by a thermocouple at the position of the sample in the high-pressure steel cell as a function of time after the set temperature was changed. The relatively slow response of the sample temperature is because of the large thermal mass of the high- pressure cell, resulting in a slew rate of 1 “C/min. In com- parison, small Cu sample holders used for l-bar (0. l- MPa) studies have a slew rate that is close to 1 “C/s. The temperature stability is shown in Fig. 7(b). It can be seen that fluctuations in the temperature of the sample are less than 50 mK.

D. Limitations of temperature control

The resolution of our present system is limited by the 8-bit D/A. For controllers that need to achieve greater accuracies, a D/A with more bits would be desirable.

It is found that the performance of the temperature controller is relatively poor if the set temperature is within f 5 “C of the circulating bath temperature. Fluctuation in this range can exceed *OS “C!. However, one can easily circumvent this difficulty by choosing a bath temperature well outside the experimentally interesting temperature range.

Perhaps the biggest drawback is the rather large thermal mass, due to the steel jacket of the Be cell, which severely slows the slew rate for temperature variations. We are experimenting with more compact cell designs to improve the slew rate.23 The use of a Be-Cu jacket instead of a steel jacket should improve the system performance as well.

E. X-ray diffraction experimental performance

Small-angle x-ray scattering experiments utilizing this pressure and temperature-controlled system were carried out on various biological specimens. Figure 8 presents the powder diffraction patterns of an aqueous dispersion of dioleoylphosphatidylethanolamine at 10 “C, 1 bar (0. I MPa) and at 10 “C, 300 bar (30 MPa). The lipid is observed to undergo a phase transition from a hexagonally

packed tubular phase (HI,) to a lamellar phase (L,) as the system pressure is isothermally increased. The number of orders observed in the pattern was sufficient to allow an electron density reconstruction of the HI, phase.

Correctionsfor Be cell absorption: The cylindrical sym- metry of the Be ceil should ensure that the absorption due to the Be cell should be independent of angle, provided the cell is correctly centered with respect to the incoming beam. We checked for this first by performing an electron density reconstruction at 1 bar (0.1 MPa) through the Be cell, and then comparing the results to another reconstruction performed using a thin quartz capillary (Charles Sup- per, Co., Natick, MA., wall thickness 10 pm). Although data collection times for recording equivalent diffraction patterns with the Be cell were about three times longer, the reconstructions were identical to within our signal-to- noise.

ACKNOWLEDGMENTS

This work was supported by the Office of Naval Re- search (contract No. N00014-90-J- 1702)) the Department of Energy (Grant No. DE-FG0287ER60522), and by the National Institute of Health (Grant No. GM32614). Peter So acknowledges support from the Princeton Materials In- stitute.

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1770 Rev. Sci. Instrum., Vol. 63, No. 2, February 1992 X-ray powder dijfraction 1770


automated pressure and temperature control apparatus for x...

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