Download Chapter 2: Methods for High Pressure

C HAPTER T WO
C ELLS FOR H IGH P RESSURE
C RYSTALLOGRAPHY
Overview
A high pressure crystallographic cell must accommodate large x-ray
scattering angles, be agile enough to rotate through a wide range of
orientations, be precise enough to keep the crystal orientation fixed, and
be robust enough to hold several thousand atmospheres of pressure. Three
techniques described in this chapter are:
•
pressurizing using a beryllium cell
•
pressurizing using a diamond anvil cell
And in an alternate approach:
•
cryogenic cooling under pressure
The last technique involves cryogenic cooling under pressure with the goal
of freezing-in pressure induced deformations, allowing the high pressure
state to be studied without needing x-ray compatible pressure cells on the
beamline.
This chapter describes basic high pressure techniques, reviews each
method, and highlights merits and drawbacks of each. Details about
sample preparation and data acquisition are in Chapter 3.
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© C o p yr i g h t b y P a u l Ke n j i U r a ya m a , 2 0 0 1 . Al l r i g h t s r e s e r v e d .
Basic Pressure Techniques
To ensure safety, materials, seals, and protocols should be
developed with the pressure range in mind. Liquids have a lower
compressibility than gas, so represent a substantial safety factor. A liquid
pressurizing medium should be used whenever possible. Sample volume
should also be kept as small possible, keeping the stored energy and load
(= pressure × surface area of sample cell) minimized. Spain & Paauwe’s
High Pressure Technology (1977) is an essential reference for high
pressure safety and design.
Materials and Seals
Stainless steel provided adequate material strength and chemical
resistance for most applications in this thesis. High pressure steel had the
advantage of being more easily machined before hardening by heat
treatment. However, commercially available stainless steel parts, such as
pressure couples (High Pressure Equipment Company, Erie, PA) and
syringe tubing (Small Parts, Miami Lakes, FL), made extensive work with
high pressure steel unnecessary.
The main high pressure seals used were O-ring, cone, and
Bridgeman type seals. O-ring seals require a groove which allows the Oring to deform without extruding. The seal works so long as the O-ring
remains confined, and more complex designs include anti-extrusion rings
or angular grooves. The O-ring is often an elastomer, like rubber, Viton ® ,
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pressure seal
co llar
Figure 2-1. The Cone Seal
From Spain & Paauwe (1977). A collar, used in conjunction
with a gland, screws into a socket, forcing the cones to mate
to form a seal. (a) A properly seated seal. Note the annular
seal formed by the slightly mismatched cone angles. (b) When
the seal is over tightened, the cone becomes deformed and
does not reliably seal. (c) If the collar is too close to the cone,
the seal cannot be tightened enough and the seal is not made.
and Buna-N (nitrile); or soft metal, like copper or indium. Elastomeric Oring seals typically operate to about 500 bar.
Cone seals are robust, versatile, and easy to assemble and
disassemble. It is the seal of choice up to 7 kbar. The cone angle of the
female piece is slightly larger than the cone angle of the male piece,
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forming an annular line seal when the two pieces mate. Care must be taken
not to over tighten. Figure 2-1 compares a properly seated cone seal with
ones that are improperly seated.
A Bridgeman seal relies on a differential area across a packing
material to provide a sealing pressure. One way to achieve a differential
area is by using an unsupported area, as in Figure 2-2. The area exposed
to high pressure P a p p l i e d is A. The unsupported area of the plug is a.
Under static conditions, the force applied to A is equal to the force applied
Pi = Papplied
Figure 2-2. The Bridgeman Seal
From Spain & Paauwe (1977). (a) Parts of the Bridgeman
seal. (b) Applied pressure deforms the seal. The unsupported
diameter d is less than diameter D on which pressure is
applied, thus under static conditions, the pressure in the
sealing material must be greater than the applied pressure.
Since the pressure in the sealing material is greater than the
applied pressure in the medium, the medium cannot escape.
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to the seal area (A – a). The pressure in the seal is
 A 
Pseal = Papplied 
 > Papplied .
 A−a 
(2.1)
Thus the pressure in the sealing material is greater than the applied
pressure. The Bridgeman seal is extremely robust, but awkward for
frequent assembly and disassembly. A well designed seal can go up to 50
kbar.
Pressure Measurement
A Sensotec (Columbus, OH) Model UHP pressure transducer
measured pressure with an accuracy of 0.5% and a maximum pressure of
100 kpsi (6.90 kbar). The detecting element was a strain dependent
resistor. A Sensotec Model 450D reader sensed the amplified transducer
output, with a factory set shunt resistor providing a calibration reference
for adjusting the reader gain.
The transducer had a ten-minute decay time for an “overshoot”
hysteresis which is about 3% of the amplitude of the pressure jump. For
example, when going from 1 bar to 2000 bar, the reading decayed to about
1950 bar over a 10 minute period. This behavior was reproducible and the
final value held steady, so it was not due to a pressure leak. Because
sample equilibration times were at least one hour, this transducer
characteristic did not affect our results. A transducer with a faster
response time would be needed for pressure-jump kinetic experiments.
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Generator, Valves, and Fittings
The pressure generator, valves, and fittings were from High Pressure
Equipment Company (Erie, PA). The generator was a cylindrical pressure
bomb with a small diameter (~cm) central bore through which a piston
hydrostatically compressed the pressurizing medium. The piston was hand
tightened. Model 50-6-15 has a stroke capacity of 11 ml and a maximum
pressure rating of 30 kpsi (2.07 kbar). Model 37-5.75-60 has a stroke
capacity of 10 ml and a maximum pressure rating of 60 kpsi (4.14 kbar).
The packing seal should be replaced periodically (about every 2
years), and the generator should be pressure cycled before use to re-pack
the seal.
Valves were rated for the appropriate pressure (30 kpsi or 60 kpsi).
Since valves used cone seals, they should not be over tightened.
Fittings used cone seals unless specifically noted. Tubing sizes
were either 1/4 or 1/8 inch. Small inner diameter, flexible stainless steel
tubing hard soldered into commercial cone seals provided flexible joints.
Setup and Operation
The high pressure setup (Figure 2-3) was designed for convenient
pump filling and loading. Filling the generator involved opening the fillvalve V2 and closing the output-valve V1, then turning back the generator
piston. Applying pressure to the sample involved closing the fill-valve
V2, opening the output-valve V1, and turning the piston in.
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sample
vessel
flexible
pressure
tubing
pressure generator
V1
reservoir for
pressurizing
medium
V2
pressure sensor
sample
vessel
valves
generator
Figure 2-3. High Pressure Setup
Schematic and picture. In the picture, the generator is
manually operated (handle spokes on left). The water
reservoir is on the right. The sample is in the middle.
The pressure sensor is not shown.
The fluid in the pump was de-ionized water. Since corrosion was a
problem within the generator, future experiments should use an inert oil
such as Fluorinert™ (a fully fluorinated, chemically inert hydrocarbon by
3M™) within the generator.
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Fluids with lower compressibility transmit pressure more efficiently.
Though mercury had a lower compressibility that water, it was not used
due to its toxicity.
Pressure Cells Used in High Pressure Protein
Crystallography
Pressure cells for high pressure protein crystallography are: 1) a
beryllium pressure cell, and 2) the diamond anvil cell (DAC). The DAC
was not used for this thesis. It is described briefly for completeness. This
section also describes the pressure cell used during high pressure cooling.
Beryllium Pressure Cell
Kundrot & Richards (1986) used a crystallographic beryllium cell to
solve the structure of lysozyme at 1 kbar. Tilton (1988) designed a
beryllium cell for gas pressures up to 200 bar to look at the binding of
nitrogen to myoglobin. Both cells were cylindrical beryllium rods with an
axial bore for housing the crystal. Because the cells were made entirely of
beryllium, there were no blind spots created by x-ray opaque supports.
Beryllium Toxicity
Beryllium toxicity (Daugherty, 1992) should be considered when
working with beryllium. Large inhaled exposures of the metal, its
sulfates, fluorides, chlorides, or oxides may lead to acute berylliosis,
indicated by the inflammation of nasal passages (rhinitis), pharynx
(pharyngitis), and/or trachea and bronchi (tracheobronchitis).
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