Pressurejump relaxation technique with optical detection W. Knoche and G. Wiese Citation: Review of Scientific Instruments 47, 220 (1976); doi: 10.1063/1.1134584 View online: http://dx.doi.org/10.1063/1.1134584 View Table of Contents: http://scitation.aip.org/content/aip/journal/rsi/47/2?ver=pdfcov Published by the AIP Publishing Articles you may be interested in High pressure-jump apparatus for kinetic studies of protein folding reactions using the small-angle synchrotron xray scattering technique Rev. Sci. Instrum. 71, 3895 (2000); 10.1063/1.1290508 Pressurejump relaxation apparatus using bipolarpulse conductivity detection Rev. Sci. Instrum. 65, 199 (1994); 10.1063/1.1144777 A new highpressure cell for differential pressurejump experiments using optical detection Rev. Sci. Instrum. 60, 3685 (1989); 10.1063/1.1140475 High pressure pressurejump apparatus Rev. Sci. Instrum. 51, 252 (1980); 10.1063/1.1136168 Pressurejump apparatus suitable for repetitive experiments Rev. Sci. Instrum. 49, 1747 (1978); 10.1063/1.1135332
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Pressure-jump relaxation technique with optical detection W. Knoche and G. Wiese Max-Planck-Institut fiir Biophysikalische Chemie. Gottingen. West Germany
(Received 14 October 1975)
An autoclave is described which allows the measurement of chemical relaxation induced by a pressure jump and observed by the change in optical absorbance. The autoclave fits into the optical arrangement of conventional temperaturejump equipment. Relaxation times longer than 0.5 msec can be determined.
INTRODUCTION
During the last 20 years many chemical relaxation techniques have been developed which differ in the way they disturb a chemical equilibrium and in the method of observing concentration changes. 1 Two frequently used techniques are the temperature-jump method with Joule heating 2 •3and the pressure-jump method. 4.5 I n the former the progress of a reaction is usually measured by observing the optical absorption, whereas in the latter electrical conductivity is usually observed. In this contribution a pressure-jump apparatus using absorbance detection is described. This system has rarely been used until recently6.7; however, it may be preferred in many cases. The main advantage of optical detection is that it is not restricted to ionic reactions. The advantages of the pressure-jump method compared with the temperature-jump method (using the same detection system) are (a) Applicability to unstable solutions: Some solutions, especially those containing biochemical compounds, are so sensitive that they may be destroyed by the high electrical field and high current applied to the solutions during Joule heating. For instance. the study of solutions of ribosomes of E. coli using the temperClture-jump technique did not give reproducible results. On the other hand, reproducible results were obtained for more than 100 pressure-jump experiments using a single sample of solution in the cell we now describe. 8 (b) Measurements can be repeated at a faster rate: After a temperature-jump experiment the temperature of the solution has to attain its original value before a further experiment is performed; this usually takes about 5 min. A pressure-jump experiment can be repeated every 0.5 min. Such a high repetition rate is especially necessary when signal averaging is used to increase the signal-to-noise ratio. (c) Longer relaxation times can be observed. Temperature-jump relaxation measurements are restricted to times shorter than about 0.1 sec. After this time cooling of the solution strongly disturbs observation of the chemical reaction. There is no long time limit to the pressure-jump method. The drawbacks of the new apparatus compared with the other ones mentioned above are (a) relaxation 220
Rev. Sci. Instrum., Vol. 47, No.2, February 1976
times shorter than about 3 x 10- 4 sec cannot be observed, and (b) detection by optical absorption is much less sensitive than detection by electrical conductivity. APPARATUS A. Optical arrangement and data acquisition
Since the outer dimensions of this pressure-jump autoclave are exactly the same as those of a conventional temperature-jump cell,2.3 the optical arrangement of the temperature equipment is used without any modification. It has been described in the literature quoted. The output signal is fed to a storage oscilloscope and via an AID converter to a small computer which evaluates the relaxation times. For details of the data acquisition and processing see Refs. 9 and 10. B. Autoclave
The autoclave IS made of bronze. Two sectional views are shown in Fig. I. The sample volume is held in region I which has a capacity of about I m!. The windows 2 are made of quartz. The sample cell is made from black PVC 3 to avoid contamination and
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Scm Sectional views of the autoclave. I-Sample volume; 2-quartz windows; 3- PVC covering; 4-screw locking the sample volume; 5-high-pressure chamber; 6-pressure-transmitting plastic membrane; 7-bursting Q1embrane; 8-bayonet locking device for the exchange of the bursting membrane; 9-connection to manual high-pressure pump; IO-piezoelectric capacitor; II-temperaturemeasuring device; 12-pressure-reducing pump. FIG.
I.
Copyright
© 1976 American Institute of Physics
220
This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitationnew.aip.org/termsconditions. Downloaded to IP: 142.244.5.147 On: Thu, 27 Nov 2014 03:01:29
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creased slowly (compared to the relaxation time) until the bursting membrane ruptures, whereupon the pressure drops to ambient within about 10- 4 sec. The bursting pressure depends on the thickness and the type of material used for the membrane (0.07-0.12 mm, brass or aluminum) and is usually 50-200 atm. The fast pressure drop causes a piezoelectric capacitor 10 to generate a voltage pulse which triggers the oscilloscope and the data-capturing device. The temperature of the autoclave is determined with an accuracy of ±O.loC by measuring the resistance of an NTC resistor II. In the compartment above the rupture disk the pressure can be reduced to about 5 Torr by a small pump 12. This enables disturbances caused by acoustical noise to be avoided. To exchange the bursting membrane, only the bayonet socket 8 has to be loosened, and the metal band 7 has to be moved forward. Thus, the measurements can be repeated at a rate of 2/min.
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FIG. 2. Change in optical absorption vs time. a-O.l M FeCI 3; = 15°C; PH = 1.82; 2 msec/di.v; PH indicator added; observed at 510 nm. b-O.I M pyruvic ethylester; T = 23.5"C; PH = 2.85; 5 sec/div l l ; observed at 320 nm. c-Mixtures of30 Sand 50 S subunits of E. coli ribosomes in 11.7 mmol Mg"+. 10 mmol tris-HCl, 50 mmol KCI, I mmol EDTA, 0.1 mmol DTE; PH = 7.3; T= 22°C; A(260 nm)30S = 17.5; A(260 nmlsos = 35.1. Three experiments superimposed on the oscilloscope screen. 5 msec/div (lower trace); 50 msec/div (middle trace); 500 msec/div (upper trace); observed at 350 nm." T
Figure 2 shows oscillograms to demonstrate the range of applicability of the new apparatus. In Fig. 2(a), the rise time of the signal is given by the filter time constant of 0.2 msec. The short time limit of the apparatus is not determined by the bursting time of the membrane but by mechanical vibrations caused by the pressure jump; these strongly disturb the measurements for about 0.5 msec. These disturbances could be avoided by a more stable mounting of the autoclave which, however, was not possible when using the optical arrangement of our temperature-jump equipment.
reflection of light by the bronze. The cell is filled with solution when inverted, and it is closed by screw 4. The vessel is then positioned in the optical arrangement and the pressure chamber 5 is filled with water. The pressure is transmitted to the sample volume through a thin plastic membrane 6. The pressure chamber is covered by the bursting membrane 7, which is rigidly sealed when the bayonet socket 8 is positioned and turned through 90°. A high-pressure manual water pump is connected to the inlet 9 via a flexible tube. The pressure in the autoclave is in-
[ M. Eigen and L. De Maeyer, in Techniques of Chemistry, edited by G. G. Hammes (Wiley-Interscience, New York, 1973), Vol. 6, Part 2, p. 63. 2 G. G. Hammes, ibid, p. 147. 3 R. Riegler, C.-R. Rabl, and T. M. Jovin, Rev. Sci. Instrum. 45,580 (1974). 4 W. Knoche, Ref. I, p. 187. 5 W. Knoche and G. Wiese, Chern. Instrum. 5, 91 (1973-74). 6 D. E. Goldsack, R.E. Hurst, and J. Love, Anal. Biochem. 28, 273 (1969). 7 R. M. Clegg and E. L. Elson, Biopolymers 14, 883 (1975). " E. Schulz, R. Jaenicke, and W. Knoche, presented at the 5th International Biophysics Congress, Copenhagen, 1975. 9 M. Krizan and H. Strehlow, Chern. Instrum. 5,99 (1973-74). to H. Strehlow and J. Jen, Chern. Instrum. 3, 47 (1971). [[ H.-J. Buschmann, M.S. thesis, University Gottingen, 1974.
221
Pressure-Jump relaxation
Rev. Sci. Instrum., Vol. 47, No.2, February 1976
221
This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitationnew.aip.org/termsconditions. Downloaded to IP: 142.244.5.147 On: Thu, 27 Nov 2014 03:01:29
This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitationnew.aip.org/termsconditions. Downloaded to IP: 142.244.5.147 On: Thu, 27 Nov 2014 03:01:29
Pressure-jump relaxation technique with optical detection W. Knoche and G. Wiese Max-Planck-Institut fiir Biophysikalische Chemie. Gottingen. West Germany
(Received 14 October 1975)
An autoclave is described which allows the measurement of chemical relaxation induced by a pressure jump and observed by the change in optical absorbance. The autoclave fits into the optical arrangement of conventional temperaturejump equipment. Relaxation times longer than 0.5 msec can be determined.
INTRODUCTION
During the last 20 years many chemical relaxation techniques have been developed which differ in the way they disturb a chemical equilibrium and in the method of observing concentration changes. 1 Two frequently used techniques are the temperature-jump method with Joule heating 2 •3and the pressure-jump method. 4.5 I n the former the progress of a reaction is usually measured by observing the optical absorption, whereas in the latter electrical conductivity is usually observed. In this contribution a pressure-jump apparatus using absorbance detection is described. This system has rarely been used until recently6.7; however, it may be preferred in many cases. The main advantage of optical detection is that it is not restricted to ionic reactions. The advantages of the pressure-jump method compared with the temperature-jump method (using the same detection system) are (a) Applicability to unstable solutions: Some solutions, especially those containing biochemical compounds, are so sensitive that they may be destroyed by the high electrical field and high current applied to the solutions during Joule heating. For instance. the study of solutions of ribosomes of E. coli using the temperClture-jump technique did not give reproducible results. On the other hand, reproducible results were obtained for more than 100 pressure-jump experiments using a single sample of solution in the cell we now describe. 8 (b) Measurements can be repeated at a faster rate: After a temperature-jump experiment the temperature of the solution has to attain its original value before a further experiment is performed; this usually takes about 5 min. A pressure-jump experiment can be repeated every 0.5 min. Such a high repetition rate is especially necessary when signal averaging is used to increase the signal-to-noise ratio. (c) Longer relaxation times can be observed. Temperature-jump relaxation measurements are restricted to times shorter than about 0.1 sec. After this time cooling of the solution strongly disturbs observation of the chemical reaction. There is no long time limit to the pressure-jump method. The drawbacks of the new apparatus compared with the other ones mentioned above are (a) relaxation 220
Rev. Sci. Instrum., Vol. 47, No.2, February 1976
times shorter than about 3 x 10- 4 sec cannot be observed, and (b) detection by optical absorption is much less sensitive than detection by electrical conductivity. APPARATUS A. Optical arrangement and data acquisition
Since the outer dimensions of this pressure-jump autoclave are exactly the same as those of a conventional temperature-jump cell,2.3 the optical arrangement of the temperature equipment is used without any modification. It has been described in the literature quoted. The output signal is fed to a storage oscilloscope and via an AID converter to a small computer which evaluates the relaxation times. For details of the data acquisition and processing see Refs. 9 and 10. B. Autoclave
The autoclave IS made of bronze. Two sectional views are shown in Fig. I. The sample volume is held in region I which has a capacity of about I m!. The windows 2 are made of quartz. The sample cell is made from black PVC 3 to avoid contamination and
12 12
\ -.. 9 11
Scm Sectional views of the autoclave. I-Sample volume; 2-quartz windows; 3- PVC covering; 4-screw locking the sample volume; 5-high-pressure chamber; 6-pressure-transmitting plastic membrane; 7-bursting Q1embrane; 8-bayonet locking device for the exchange of the bursting membrane; 9-connection to manual high-pressure pump; IO-piezoelectric capacitor; II-temperaturemeasuring device; 12-pressure-reducing pump. FIG.
I.
Copyright
© 1976 American Institute of Physics
220
This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitationnew.aip.org/termsconditions. Downloaded to IP: 142.244.5.147 On: Thu, 27 Nov 2014 03:01:29
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APPLICATIONS
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creased slowly (compared to the relaxation time) until the bursting membrane ruptures, whereupon the pressure drops to ambient within about 10- 4 sec. The bursting pressure depends on the thickness and the type of material used for the membrane (0.07-0.12 mm, brass or aluminum) and is usually 50-200 atm. The fast pressure drop causes a piezoelectric capacitor 10 to generate a voltage pulse which triggers the oscilloscope and the data-capturing device. The temperature of the autoclave is determined with an accuracy of ±O.loC by measuring the resistance of an NTC resistor II. In the compartment above the rupture disk the pressure can be reduced to about 5 Torr by a small pump 12. This enables disturbances caused by acoustical noise to be avoided. To exchange the bursting membrane, only the bayonet socket 8 has to be loosened, and the metal band 7 has to be moved forward. Thus, the measurements can be repeated at a rate of 2/min.
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FIG. 2. Change in optical absorption vs time. a-O.l M FeCI 3; = 15°C; PH = 1.82; 2 msec/di.v; PH indicator added; observed at 510 nm. b-O.I M pyruvic ethylester; T = 23.5"C; PH = 2.85; 5 sec/div l l ; observed at 320 nm. c-Mixtures of30 Sand 50 S subunits of E. coli ribosomes in 11.7 mmol Mg"+. 10 mmol tris-HCl, 50 mmol KCI, I mmol EDTA, 0.1 mmol DTE; PH = 7.3; T= 22°C; A(260 nm)30S = 17.5; A(260 nmlsos = 35.1. Three experiments superimposed on the oscilloscope screen. 5 msec/div (lower trace); 50 msec/div (middle trace); 500 msec/div (upper trace); observed at 350 nm." T
Figure 2 shows oscillograms to demonstrate the range of applicability of the new apparatus. In Fig. 2(a), the rise time of the signal is given by the filter time constant of 0.2 msec. The short time limit of the apparatus is not determined by the bursting time of the membrane but by mechanical vibrations caused by the pressure jump; these strongly disturb the measurements for about 0.5 msec. These disturbances could be avoided by a more stable mounting of the autoclave which, however, was not possible when using the optical arrangement of our temperature-jump equipment.
reflection of light by the bronze. The cell is filled with solution when inverted, and it is closed by screw 4. The vessel is then positioned in the optical arrangement and the pressure chamber 5 is filled with water. The pressure is transmitted to the sample volume through a thin plastic membrane 6. The pressure chamber is covered by the bursting membrane 7, which is rigidly sealed when the bayonet socket 8 is positioned and turned through 90°. A high-pressure manual water pump is connected to the inlet 9 via a flexible tube. The pressure in the autoclave is in-
[ M. Eigen and L. De Maeyer, in Techniques of Chemistry, edited by G. G. Hammes (Wiley-Interscience, New York, 1973), Vol. 6, Part 2, p. 63. 2 G. G. Hammes, ibid, p. 147. 3 R. Riegler, C.-R. Rabl, and T. M. Jovin, Rev. Sci. Instrum. 45,580 (1974). 4 W. Knoche, Ref. I, p. 187. 5 W. Knoche and G. Wiese, Chern. Instrum. 5, 91 (1973-74). 6 D. E. Goldsack, R.E. Hurst, and J. Love, Anal. Biochem. 28, 273 (1969). 7 R. M. Clegg and E. L. Elson, Biopolymers 14, 883 (1975). " E. Schulz, R. Jaenicke, and W. Knoche, presented at the 5th International Biophysics Congress, Copenhagen, 1975. 9 M. Krizan and H. Strehlow, Chern. Instrum. 5,99 (1973-74). to H. Strehlow and J. Jen, Chern. Instrum. 3, 47 (1971). [[ H.-J. Buschmann, M.S. thesis, University Gottingen, 1974.
221
Pressure-Jump relaxation
Rev. Sci. Instrum., Vol. 47, No.2, February 1976
221
This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitationnew.aip.org/termsconditions. Downloaded to IP: 142.244.5.147 On: Thu, 27 Nov 2014 03:01:29
