Fluorescence > Components > High-Pressure Cell System

High-Pressure Cell System

High-Pressure Cell System   
The ISS High Pressure Cell System (HP Cell) has been designed for Absorption, Fluorescence, Raman spectroscopy and polarimetry studies of solid and liquid samples. The cell, built in a stainless-steel alloy, reaches pressures up to 400 MPa (4,000 bar) using a hydrostatic pump. The HP Cell can be mounted directly into ISS spectrofluorometers and several commercial instruments; also, it can be used as a stand-alone unit.

The HP Cell comes either in a 3- or 4-windows version; the windows are located at 90 degrees from each other. Quartz and sapphire windows feature a 10mm aperture; MgF2 windows (used for polarimetry studies) feature a 3mm aperture.

The high aperture 10mm-diameter windows allow for using as an excitation light source either a laser, a light emitting diode (LED) or a collimated beam from a xenon arc lamp. The unusually high numerical aperture (NA = 0.18) for a cell makes it unique for high sensitivity fluorescence studies.

Electrical Feedthrough
A plug is available for the HP Cell whenever a voltage needs to be applied to a sample. The plug is mounted on top of the HP Cell; the pressure is applied by connecting the flexible tubing from the pump horizontally onto the plug. Pressures up to 300 MPa can be reached with the plug.

HP Cell with the plug for electrical feedthrough. The pressure is applied through the tubing connected horizontally to the plug. The voltage is applied on the electrodes.

Windows
Optical windows are made out of quartz [SiO2], sapphire [single-crystal aluminum oxide Al2O3] or MgF2. The windows are mounted onto polished plugs and held in place by precise mechanical holders.

Material Max pressure (MPa) Aperture (mm) Birifringence(Δn) Transmission range (nm)
Quartz [SiO2] 300 10 +0.009 180 - 2,000
Sapphire [Al2O3] 400 10 -0.008 250 - 5,000
MgF2 250 5 +0.006 150 - 6,500

The choice of the windows material is dictated by the specific application and measurement to be acquired. Quartz windows, constructed of UV grade fused silica, withstand pressures up to 300 MPa. Fused silica is a polycrystalline, isotropic material with no crystal orientation; these windows are suitable for polarization measurements. Sapphire windows withstand pressures up to 400 MPa. Yet, sapphire is a synthetic, hexagonal single crystal anisotropic material, which displays substantially different optical characteristics when measured along different axes; sapphire if birefringent, that is a light beam propagating along the optical axis experiences two different values of the index of refraction. These windows are less suitable for polarization measurements. MgF2 windows feature the lowest birefringence; moreover, their transmission extends to 150 nm making them suitable for circular dichroism studies.

Temperature Control
The HP Cell body includes a built-in path for circulating the temperature control liquid from an external bath circulator directly attached to it. As the cell is made of a stainless-steel alloy with excellent thermal conductivity, temperature stabilization is rapid. The HP Cell is designed for operating in the temperature range from -20°C to +60°C.

Cell

Pressure
  • up to 300 MPa (3,000 bar) with quartz windows
  • up to 400 MPa (4,000 bar) with sapphire windows
  • up to 250 MPa (2,500 bar) with MgF2 windows
Temperature
  • -40°C to +80°C
  • Controlled through built-in liquid circulation circuit
  • Thermal Conductivity
    • 2 W cm-1 K-1 at 20°C
    • 16 W cm-1 K-1 at 200°C
Windows
  • 10 mm clear aperture
  • I-Seals for easy use and replacement
  • Quartz
    • 19 mm diameter x 8.5 mm thick
  • Sapphire
    • 19 mm diameter x 6.4 mm thick
  • MgF2
    • 19 mm diameter x 8.5 mm thick
Internal Cell Chamber
  • Diameter: 22mm
  • Height: 50.3mm
Dimensions
  • 146mm (W) x 118mm (L)
  • 39 mm from base to window centers

Pressure Generator

Gauge
  • Up to 600 MPa
Reservoir Volume
  • 116 mL
Generator Volume
  • 10 mL (manual or automatic pump)
Tubing
  • 1/8" diameter
  • Flexible
  • Up to 415 MPa
Thermocouple
  • Either Ni-Cr-Ni or Fe-Const

Sample Holder Volumes

Empty Cell, 3 Quartz Windows
  • 15.0 mL
Empty Cell, 4 Sapphire Windows
  • 24.5 mL
Holder fitted with 11 mm bottle and cap
  • 11.0 mL
  • Max allowed height of bottle with cap: 40.0 mm
Round cuvette, 11 mm outer diameter
  • 0.75 mL
Round cuvette, 9 mm outer diameter
  • 0.80 mL
Square cell, 6x6 mm
  • 0.30 mL

Hydrostatic Liquid

Pressure Media
  • Spectroscopic-Grade Water
  • Low Fluorescence Background Ethanol

Absorption and Emission Spectra

The effect of pressure on the emission spectrum of Prodan in Dipalmitoyl-phosphatidylcholine (DPPC) multilamellar vesicles at 20°C in tris buffer (1o mM, pH=8.0) containing 0.8M ethanol. Excitation wavelength was 359 nm.
(Courtesy of Prof. Parkson L.-G. Chong, Temple University, Philadelphia, PA, USA)

Lifetime Measurements

Effect of pressure on fluorescence lifetimes of staphylococcal nuclease. Frequency response profiles at atmospheric and high pressure using the HPCell. Excitation wavelength was 295 nm and emission was monitored at 350 nm.
(Courtesy of Prof. Catherine A. Royer, Centre de Biochimie Structurale, Université Montpellier I, Montpellier, France)

High pressure (2 Kbar) data: blue (phase) and red (modulation) lines
Atmospheric pressure data: green (phase) and purple (modulation) lines

Kinetic Studies

Along with researchers of Temple University (Philadelphia, PA) ISS developed a special cuvette for kinetics studies. The solutions in the cuvette are separated by a membrane that disrupts upon the application of a low voltage signal, which is transported into the special cuvette by wires embedded into the top plug.

ISS provides the suitable baseplates for mounting HPCell into several spectrofluorometers and spectrophotometers.

Spectrofluorometer mounting kits are available for:

ISS PC1
K2
ChronosBH
ChronosDFD
AVIV AFTF105
Edinburgh Instruments FS900
Hitachi F4500
Horiba Fluorolog 2, 3
Fluoromax 2, 3
Jasco FP-8300
SLM 4800, 8000, 8100, 48000
Varian Cary Eclipse

Spectrophotometer mounting kits are available for:

Shimadzu UV2501PC, UV1650PC
Varian Cary 14 UV/VIS, Cary 50, 100, 300

Spectropolarimeter mounting kits are available for:

Jasco J-710, J-715, J-810, J-815

If the high-pressure cell cannot be mounted into an instrument, fiber-optic bundles are utilized for connection. Please contact ISS for details on the fiber optics kit.

High-Pressure Cell in a PC1 Spectrofluorometer.

High-Pressure Cell in a Chronos Lifetime Spectrometer.

High-Pressure Cell in a Jasco FP-8300.

Selected References for the Use of HPCell in Various Research Areas

Comparing Fast Pressure Jump and Temperature Jump Protein Folding Experiments and Simulations.
Wirth, A.J., Liu, Y., Prigozhin, M.B., Schulten, K., Gruebele, M.
J Am Chem Soc. 2015 Jun 10;137(22):7152-7159. doi: 10.1021/jacs.5b02474. Epub 2015 Jun 2.
Pressure modulation of Ras-membrane interactions and intervesicle transfer.
Kapoor, S., Werkmüller, A., Goody, R.S., Waldmann, H., Winter, R.
J Am Chem Soc. 2013 Apr 24;135(16):6149-56. doi: 10.1021/ja312671j. Epub 2013 Apr 16.
Circular Dichroism and Site-directed Spin Labeling Reveal Structural and Dynamical Features of High-pressure States of Myoglobin.
Lerch, M.T., Horwitz, J., McCoy, J., Hubbell, W.L.
Proc Natl Acad Sci U S A., 2013, 110(49), E4714-22.
Misplaced Helix Slows Down Ultrafast Pressure-Jump Protein Folding
Prigozhin, M.B., Liu, Y., Wirth, A.J., Kapoor, S., Winter, R., Schulten, K., Gruebele, M.
PNAS, 2013, 110(20), 8087-8092.
Dissecting the structure, thermodynamic stability, and aggregation properties of the A25T transthyretin (A25T-TTR) variant involved in leptomeningeal amyloidosis: identifying protein partners that co-aggregate during A25T-TTR fibrillogenesis in cerebrospinal fluid.
Azevedo, E.P., Pereira, H.M., Garratt, R.C., Kelly, J.W., Foguel, D., Palhano, F.L.
Biochemistry. 2011 Dec 27;50(51):11070-83. doi: 10.1021/bi201365r. Epub 2011 Nov 30.
Refolding of endostatin from inclusion bodies using high hydrostatic pressure.
Chura-Chambi, R.M., Genova, L.A., Affonso, R., Morganti, L.
Anal Biochem. 2008 Aug 1;379(1):32-9. doi: 10.1016/j.ab.2008.04.024. Epub 2008 Apr 23.
V(i)-value analysis: a pressure-based method for mapping the folding transition state ensemble of proteins.
Mitra, L., Hata, K., Kono, R., Maeno, A., Isom, D., Rouget, J.B., Winter, R., Akasaka, K., García-Moreno, B., Royer, C.A.
J Am Chem Soc. 2007 Nov 21;129(46):14108-9. Epub 2007 Oct 26.
pH dependence of the dissociation of multimeric hemoglobin probed by high hydrostatic pressure.
Bispo, J.A., Santos, J.L., Landini, G.F., Goncalves, J.M., Bonafe, C.F.
Biophys Chem. 2007 Feb;125(2-3):341-9. Epub 2006 Sep 27.
Proton dependence of tobacco mosaic virus dissociation by pressure.
Santos, J.L., Bispo, J.A., Landini, G.F., Bonafe, C.F.
Biophys Chem. 2004 Sep 1;111(1):53-61.
Pressure and Temperature Dependence of the Dilute Solution Segmental Dynamics of Anthracene-Labeled Polyisoprene
Punchard, B.J., Adolf, D.B.
Macromolecules, 2002, 35, 3281-3287.
Pressure and Temperature Dependence of the Melt Segmental Dynamics of cis-1,4-polyisoprene Via Time Resolved Optical Spectroscopy
Punchard, B.J., Adolf, D.B.
J. of Chem. Phys., 2002, 117, 7774-7780.
Time-resolved Optical Spectroscopy Study of the Local Dynamics of cis-1,4 and vinyl-1,2-polybutadiene in Dilute Solution at High Pressure
Punchard, B.J., Kirpatch, A., Adolf, D.B.
Polymer, 2002, 43, 6287-6293.
Unusual Properties of Highly Charged Buffers: Large Ionization Volumes and Low Barrier Hydrogen Bonds
Hess, R.A., Reinhardt, L.A.
J. Am. Chem. Soc., 1999, 121, 9867-9870.

Accessories

Manual Pressure Generator

Hydrostatic pressure is generated through a manual pump connected to the HP Cell by a flexible tubing. The pump includes a reservoir for storing the liquid (water or ethanol), and valves for allowing the pressurizing liquid to circulate thorough the circuitry. A pressure gauge indicates the value of the pressure. All of the components are conveniently mounted on a single baseplate which can be located next to the HP Cell.

Automated Pressure Generator

ISS and Pressure Biosciences (PBI) have partnered to offer the Automated Pressure Generator HUB440, a compact, portable, bench-top, ultra-high pressure generator that uses compressed air-driven pressure intensifier to generate fluid pressure up to 500 MPa with an input compressed air pressure of just 900 KPa. The HUB440 can be operated via a simple front panel, or controlled using the integrated Data Acquisition and Control Module for dynamic pressure control and connectivity to external devices.

Cylindrical Cuvettes

The cuvette is placed into a holder, which is positioned into the HP Cell. A Teflon stopper on the cuvette separates the internal solution from the pressurizing liquid; the stopper acts as a piston and allows for the transmission of pressure to the solution contained in the cuvette.

Square Cuvettes

The cuvette is placed into a holder, which is positioned into the HP Cell. A Teflon stopper on the cuvette separates the internal solution from the pressurizing liquid; the stopper acts as a piston and allows for the transmission of pressure to the solution contained in the cuvette.