High pressure optical cell for biochemical studies Richard Kenneth Williams Citation: Review of Scientific Instruments 46, 250 (1975); doi: 10.1063/1.1134181 View online: http://dx.doi.org/10.1063/1.1134181 View Table of Contents: http://scitation.aip.org/content/aip/journal/rsi/46/3?ver=pdfcov Published by the AIP Publishing Articles you may be interested in All quartz optical cell of constant diameter for use in high pressure studies Rev. Sci. Instrum. 47, 770 (1976); 10.1063/1.1134736 High pressure cells for the study of optical properties of liquid systems Rev. Sci. Instrum. 45, 1427 (1974); 10.1063/1.1686520 High Pressure Optical Cell for the Study of Biochemical Reactions Rev. Sci. Instrum. 44, 239 (1973); 10.1063/1.1686098 A High Pressure Optical Cell for Study of Biochemical Solutions Rev. Sci. Instrum. 43, 738 (1972); 10.1063/1.1685744 Teflon and Sapphire Cell for Optical Absorption Studies under High Pressure Rev. Sci. Instrum. 32, 752 (1961); 10.1063/1.1717497
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: 129.120.242.61 On: Tue, 02 Dec 2014 16:27:33
High pressure optical cell for biochemical studies Richard Kenneth Williams Department of Chemistry. The University of Guelph. Guelph. Ontario. Canada (Received 29 July 1974; and in final form. 17 September 1974)
The adaptation of a commercially available high pressure optical cell to monitor changes in absorbance in aqueous solutions by use of a fused quartz cuvette and a high pressure seal, inert to n -hexane, is described. Measurements of percent transmittance to an accuracy of ±O.2% have been obtained. Contact of the sample solution with the metal surface of the optical cell is avoided, and the cell is, therefore, suitable for studies of biochemical reactions.
INTRODUCTION Recent studies by means of optical cells on the effect of pressure on enzyme unfolding,l and on model compounds for hydrophobic bonding,2 have provided information on the nature of the stability of proteins, and on the nature of the hydrophobic bond. The techniques used in the first study involved exposure of enzyme to the metal walls of the optical cell, while in the latter a specially built optical cell and inner fused quartz cuvette were used. The importance of isolating biochemical solutions from possible metal ion contamination renders the first cell of limited use, while the difficulties of designing and using a high pressure optical cell which reduces such contamination is likely to deter potential users. The present study describes a solution to these difficulties based on the use of a commercially available high pressure optical cell and fused quartz cuvette.
it was preferred to use an indium metal gasket seal with a Viton O-ring as elastic support to it, in the manner of H. L. Klimann. 2 In this design the indium metal being the inner seal can be assumed completely inert to hexane while the Viton O-ring provides an elastic seal at low pressure. In order to obtain a seal with indium metal it is necessary to press the metal into place. This was achieved by use of a specially designed rig of tool steel inserted in the optical cell sample compartment. The rig is shown in Fig. 3. It consists of a central cylinder slipping over the sapphire window retainers, and two gaskets cut to fit to 0.025 mm clearance on both sides. The material used in making the
25'4mm
EXPERIMENTAL The Cuvette The fused quartz cuvette was supplied by Hellma Canada Ltd. (528 Huron St., Toronto, Canada). It was based on a previous design,2,3 and is shown in Fig. 1. A 2 mm port allowed filling of the cuvette through a polyethylene tube, and the cuvette was sealed with a polyethylene sheet and Teflon cap. The Teflon cap had a pinhole vent which allowed transmission of pressure to the sample solution, the polyethylene sheet acting as a flexible membrane. The length of the cuvette, 6 mm, allowed it to fit between the windows of the optical cell which had a separation of approximately 7 mm.
(a)
FIG. 1. (a) Collimating lens. (focal length 4.45 em) (b) Cuvette with Teflon cap and polyethylene sheet membrane-end view cross section. (c) Cuvette -side view cross section.
(b) 2mm 4-+
The Optical Cell This was a 6900 bar high pressure cell supplied by American Instrument Co., Silver Spring, MD as shown in Fig. 2. The windows were of uv-transparent sapphire.
24 mm
The High Pressure Seal It was found that "Viton" (polyfluorocarbon) O-rings were completely resistant to n-hexane under pressure and did not leach impurities into the solvent at pressures up to 1380 bar. However, to avoid possible criticism of later work 250
Rev. Sci. Instrum., Vol. 46, No.3, March 1975
(cl
Copyright @ 1975 by the American Institute of Physics
250
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: 129.120.242.61 On: Tue, 02 Dec 2014 16:27:33
251
251
R. K. Williams: Optical cell
silver nitrate column based on silica gel, in a ratio of 10 g AgN0 3 to 90 g silica gel (20-100 mesh).
19·4 em
FIG. 2. The high pressure optical cell (6800 bar). (a)-O-ring seal; (b)-window retainer (304 stainless steel); {c)-sapphire window (10 mm thickness); (d)-seal support ring (416 St. St.) ; {e)-window support plug (440-C, St.St.); (f)screw plug (416 St.St.); (g)-body of optical cell (403 St.St.).
abc
d
e f 9
cylinder and gaskets was tool steel (sps 245), while the indium metal was supplied as 99% indium wire, grade No.4, by the Indium Corporation of America (1676 Lincoln Ave., Utica, NY). On assembly of the optical cell the two indium metal gaskets were squeezed into the available space by the action of tightening the window support screw plugs. A high pressure seal has been described by A. Sawaoka and N. Kawai,4 using a lead gasket with rubber O-ring. Both materials, however, are more sensitive to corrosion and leaching of impurities than those described herein. Thermostating The optical cell was thermostated by means of a brass cylindrical water jacket fitted to its outer diameter to a separation of 0.025 mm. Water from a thermostat bath, controlled to ±0.05°C, was circulated through the water jacket. Thermal equilibrium of the sample was obtained in less than one minute as measured by means of a thermocouple.
Separator The optical cell pressurizing fluid, n-hexane, was separated from the oil used in the pressure pump by a brass syringe of 100 ml capacity (Fig. 4). The syringe inner plunger was fitted with a Teflon jacket into which was cut a groove for a Viton O-ring. This allowed a flexible seal. The syringe was wrapped in a polyethylene bag filled with water to prevent contact of oil with hexane. The syringe was placed in a standard 12 cm o.d. pressure vessel, to which it was attached by a stainless steel needle by means of a Viton O-ring in the manner of Koskikallio, Osborne, and Whalley.5.6 The syringe needle was soldered to the inside wall of a pressure tube attached to a pressure vessel, and the pressure tube was closed by a pressure valve. This arrangement allowed the syringe to be filled with hexane by applying suction to the oil in the pressure vessel. 5 Spectrometer and Optical Assembly A Beckmann D.U. spectrometer was used for all measurements, the high pressure optical cell being placed between the monochromator and detector. A fused quartz cylindrical plano-convex lens of focal length 4.45 cm [Fig. 1 (a) ] was used to collimate light from the monochromator before it passed through the optical cell. The collimating lens was obtained from Applied Physics Specialties (27 Prince Andrew Place, Don Mills, Ontario, Canada). Between readings the sensitivity of the spectrometer was adjusted by reflecting its light beam around the spectrometer by four front surfaced aluminum mirrors, two of which were
Pressure Valves High pressure valves in contact with n-hexane, the pressurizing fluid, were lubricated with a mixture of uvtransparent paraffin oil (Nujol) and graphite. The paraffin oil was purified by successive washings with concentrating sulphuric acid followed by column chromatography on a
--8
~-d
~. ~"---""el~--b
d b
--a
FIG. 4. Pressure fluid separator. (a)-syringe needle; (b)-Viton polyfluorocarbon O-ring; (c)-brass syringe barrel; (d)-Teflon jacket of plunger; (e)-brass plunger.
o
-+~--c
e
28'6mm
FIG. 3. Seal ring spacer assembly. {a)-spacer cylinder; {b)-window retainers; (c)-sapphire window; (d)-spacer gasket ring; (e)-indium metal gasket; (f)-Viton polyfluorocarbon O-ring; (g)-seal support ring; (h)-window support plug. Rev. Sci. Instrum., Vol. 46, No.3, March 1975 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: 129.120.242.61 On: Tue, 02 Dec 2014 16:27:33
252
R. K. Williams: Optical cell
mounted on optical benches and could be moved into and out of the light path. The optical benches (supplied by Ealing Scientific Co., 719 Lajoie Ave., Dorval 760, Quebec, Canada) were thermostated by a water jacket. RESULTS
The high pressure optical cell described above has been used in a study of the association of ribonuclease and cytidine-3'-phosphate as a supplement to a previous study of the effect of pressure on the activity of ribonuclease. 7
252
Measurement of percent transmittance was obtained to an accuracy of ±0.2%. 1J. F. Brandts, R. J. Oliveira, and C. Westort, Biochemistry 9, 1038 (1970). 2H. L. Klimann, Ph.D. dissertation (Princeton University, Princeton, NJ,1969). 3R. K. Williams, Rev. Sci. Instrum. 44, 239 (1973). 'A. Sawaoka and N. Kawai, J. Phys. Soc. Jap. 15, 133 (1968). 5J. Koskikallio and E. Whalley, Trans. Faraday Soc. 55, 809 (1959). 6A. R. Osborne and E. Whalley, Can. J. Chern. 39, 1094 (1961). 'R. K. Williams and C. Shen, Arch. Biochem. Biophys. 152, 606 (1972).
Rev. Sci. Instrum., Vol. 46, No.3, March 1975 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: 129.120.242.61 On: Tue, 02 Dec 2014 16:27:33
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: 129.120.242.61 On: Tue, 02 Dec 2014 16:27:33
High pressure optical cell for biochemical studies Richard Kenneth Williams Department of Chemistry. The University of Guelph. Guelph. Ontario. Canada (Received 29 July 1974; and in final form. 17 September 1974)
The adaptation of a commercially available high pressure optical cell to monitor changes in absorbance in aqueous solutions by use of a fused quartz cuvette and a high pressure seal, inert to n -hexane, is described. Measurements of percent transmittance to an accuracy of ±O.2% have been obtained. Contact of the sample solution with the metal surface of the optical cell is avoided, and the cell is, therefore, suitable for studies of biochemical reactions.
INTRODUCTION Recent studies by means of optical cells on the effect of pressure on enzyme unfolding,l and on model compounds for hydrophobic bonding,2 have provided information on the nature of the stability of proteins, and on the nature of the hydrophobic bond. The techniques used in the first study involved exposure of enzyme to the metal walls of the optical cell, while in the latter a specially built optical cell and inner fused quartz cuvette were used. The importance of isolating biochemical solutions from possible metal ion contamination renders the first cell of limited use, while the difficulties of designing and using a high pressure optical cell which reduces such contamination is likely to deter potential users. The present study describes a solution to these difficulties based on the use of a commercially available high pressure optical cell and fused quartz cuvette.
it was preferred to use an indium metal gasket seal with a Viton O-ring as elastic support to it, in the manner of H. L. Klimann. 2 In this design the indium metal being the inner seal can be assumed completely inert to hexane while the Viton O-ring provides an elastic seal at low pressure. In order to obtain a seal with indium metal it is necessary to press the metal into place. This was achieved by use of a specially designed rig of tool steel inserted in the optical cell sample compartment. The rig is shown in Fig. 3. It consists of a central cylinder slipping over the sapphire window retainers, and two gaskets cut to fit to 0.025 mm clearance on both sides. The material used in making the
25'4mm
EXPERIMENTAL The Cuvette The fused quartz cuvette was supplied by Hellma Canada Ltd. (528 Huron St., Toronto, Canada). It was based on a previous design,2,3 and is shown in Fig. 1. A 2 mm port allowed filling of the cuvette through a polyethylene tube, and the cuvette was sealed with a polyethylene sheet and Teflon cap. The Teflon cap had a pinhole vent which allowed transmission of pressure to the sample solution, the polyethylene sheet acting as a flexible membrane. The length of the cuvette, 6 mm, allowed it to fit between the windows of the optical cell which had a separation of approximately 7 mm.
(a)
FIG. 1. (a) Collimating lens. (focal length 4.45 em) (b) Cuvette with Teflon cap and polyethylene sheet membrane-end view cross section. (c) Cuvette -side view cross section.
(b) 2mm 4-+
The Optical Cell This was a 6900 bar high pressure cell supplied by American Instrument Co., Silver Spring, MD as shown in Fig. 2. The windows were of uv-transparent sapphire.
24 mm
The High Pressure Seal It was found that "Viton" (polyfluorocarbon) O-rings were completely resistant to n-hexane under pressure and did not leach impurities into the solvent at pressures up to 1380 bar. However, to avoid possible criticism of later work 250
Rev. Sci. Instrum., Vol. 46, No.3, March 1975
(cl
Copyright @ 1975 by the American Institute of Physics
250
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: 129.120.242.61 On: Tue, 02 Dec 2014 16:27:33
251
251
R. K. Williams: Optical cell
silver nitrate column based on silica gel, in a ratio of 10 g AgN0 3 to 90 g silica gel (20-100 mesh).
19·4 em
FIG. 2. The high pressure optical cell (6800 bar). (a)-O-ring seal; (b)-window retainer (304 stainless steel); {c)-sapphire window (10 mm thickness); (d)-seal support ring (416 St. St.) ; {e)-window support plug (440-C, St.St.); (f)screw plug (416 St.St.); (g)-body of optical cell (403 St.St.).
abc
d
e f 9
cylinder and gaskets was tool steel (sps 245), while the indium metal was supplied as 99% indium wire, grade No.4, by the Indium Corporation of America (1676 Lincoln Ave., Utica, NY). On assembly of the optical cell the two indium metal gaskets were squeezed into the available space by the action of tightening the window support screw plugs. A high pressure seal has been described by A. Sawaoka and N. Kawai,4 using a lead gasket with rubber O-ring. Both materials, however, are more sensitive to corrosion and leaching of impurities than those described herein. Thermostating The optical cell was thermostated by means of a brass cylindrical water jacket fitted to its outer diameter to a separation of 0.025 mm. Water from a thermostat bath, controlled to ±0.05°C, was circulated through the water jacket. Thermal equilibrium of the sample was obtained in less than one minute as measured by means of a thermocouple.
Separator The optical cell pressurizing fluid, n-hexane, was separated from the oil used in the pressure pump by a brass syringe of 100 ml capacity (Fig. 4). The syringe inner plunger was fitted with a Teflon jacket into which was cut a groove for a Viton O-ring. This allowed a flexible seal. The syringe was wrapped in a polyethylene bag filled with water to prevent contact of oil with hexane. The syringe was placed in a standard 12 cm o.d. pressure vessel, to which it was attached by a stainless steel needle by means of a Viton O-ring in the manner of Koskikallio, Osborne, and Whalley.5.6 The syringe needle was soldered to the inside wall of a pressure tube attached to a pressure vessel, and the pressure tube was closed by a pressure valve. This arrangement allowed the syringe to be filled with hexane by applying suction to the oil in the pressure vessel. 5 Spectrometer and Optical Assembly A Beckmann D.U. spectrometer was used for all measurements, the high pressure optical cell being placed between the monochromator and detector. A fused quartz cylindrical plano-convex lens of focal length 4.45 cm [Fig. 1 (a) ] was used to collimate light from the monochromator before it passed through the optical cell. The collimating lens was obtained from Applied Physics Specialties (27 Prince Andrew Place, Don Mills, Ontario, Canada). Between readings the sensitivity of the spectrometer was adjusted by reflecting its light beam around the spectrometer by four front surfaced aluminum mirrors, two of which were
Pressure Valves High pressure valves in contact with n-hexane, the pressurizing fluid, were lubricated with a mixture of uvtransparent paraffin oil (Nujol) and graphite. The paraffin oil was purified by successive washings with concentrating sulphuric acid followed by column chromatography on a
--8
~-d
~. ~"---""el~--b
d b
--a
FIG. 4. Pressure fluid separator. (a)-syringe needle; (b)-Viton polyfluorocarbon O-ring; (c)-brass syringe barrel; (d)-Teflon jacket of plunger; (e)-brass plunger.
o
-+~--c
e
28'6mm
FIG. 3. Seal ring spacer assembly. {a)-spacer cylinder; {b)-window retainers; (c)-sapphire window; (d)-spacer gasket ring; (e)-indium metal gasket; (f)-Viton polyfluorocarbon O-ring; (g)-seal support ring; (h)-window support plug. Rev. Sci. Instrum., Vol. 46, No.3, March 1975 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: 129.120.242.61 On: Tue, 02 Dec 2014 16:27:33
252
R. K. Williams: Optical cell
mounted on optical benches and could be moved into and out of the light path. The optical benches (supplied by Ealing Scientific Co., 719 Lajoie Ave., Dorval 760, Quebec, Canada) were thermostated by a water jacket. RESULTS
The high pressure optical cell described above has been used in a study of the association of ribonuclease and cytidine-3'-phosphate as a supplement to a previous study of the effect of pressure on the activity of ribonuclease. 7
252
Measurement of percent transmittance was obtained to an accuracy of ±0.2%. 1J. F. Brandts, R. J. Oliveira, and C. Westort, Biochemistry 9, 1038 (1970). 2H. L. Klimann, Ph.D. dissertation (Princeton University, Princeton, NJ,1969). 3R. K. Williams, Rev. Sci. Instrum. 44, 239 (1973). 'A. Sawaoka and N. Kawai, J. Phys. Soc. Jap. 15, 133 (1968). 5J. Koskikallio and E. Whalley, Trans. Faraday Soc. 55, 809 (1959). 6A. R. Osborne and E. Whalley, Can. J. Chern. 39, 1094 (1961). 'R. K. Williams and C. Shen, Arch. Biochem. Biophys. 152, 606 (1972).
Rev. Sci. Instrum., Vol. 46, No.3, March 1975 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: 129.120.242.61 On: Tue, 02 Dec 2014 16:27:33
