User’s Guide
RM Synthesizer
Version 1.02
Copyright ©2010
Daedalus Innovations LLC
Aston, Pennsylvania
United States
TABLE OF CONTENTS
LIST OF FIGURES .................................................................................................... 2
WARNINGS / INFORMATION ................................................................................... 3
ITEMS INCLUDED WITH THE RM SYNTHESIZER .................................................. 4
ITEMS TO BE SUPPLIED BY THE END-USER ........................................................ 5
INSTRUMENT SITE CONSIDERATIONS ................................................................. 6
CONNECTION DETAILS ........................................................................................... 7
THEORY OF OPERATION ...................................................................................... 10
CONVENTIONS USED IN THIS MANUAL .............................................................. 14
VALVE DESCRIPTIONS ......................................................................................... 14
BACK PANEL CONNECTIONS ............................................................................... 16
FRONT PANEL CONTROL ..................................................................................... 17
PREPARING THE INSTRUMENT FOR USE .......................................................... 17
THE MIXING CHAMBER / ADDING REAGENTS .................................................... 19
SEALING THE MIXING CHAMBER ......................................................................... 21
PROTOCOL FOR MAKING SAMPLES ................................................................... 22
TRANSFERRING SAMPLES FROM THE MIXING CHAMBER TO THE NMR CELL
................................................................................................................................. 25
CLEANING THE MIXING CHAMBER ...................................................................... 26
RESETTING THE SYSTEM FOR THE NEXT SAMPLE .......................................... 28
TIPS ......................................................................................................................... 29
CALIBRATING THE SYSTEM ................................................................................. 31
REPLACING THE WINDOW SEALS ....................................................................... 32
SPECIFICATIONS ................................................................................................... 36
FURTHER INFORMATION ...................................................................................... 38
1
LIST OF FIGURES
Figure 1: Possible bench-top setup ............................................................................ 6
Figure 2: Possible fume hood setup ........................................................................... 7
Figure 3: Cleaning manifold assembly diagram ......................................................... 8
Figure 4: Piston position during operation ................................................................ 10
Figure 5: RM Synthesizer Operational Description .................................................. 12
Figure 6: RM Synthesizer Flow Diagram .................................................................. 13
Figure 7: Example valve actuation progression ........................................................ 14
Figure 8: Back panel port connections ..................................................................... 16
Figure 9: Front panel controls .................................................................................. 17
Figure 10: Mixing chamber components: Poston, reagent plate, and main seal
(MCCS). ................................................................................................................... 19
Figure 11: View showing internal placement of the mixing chamber components ... 20
Figure 12: Progression of steps for assembling and sealing the mixing chamber prior
to sample preparation ............................................................................................... 21
Figure 13: Increasing the sample chamber pressure using the piston ..................... 24
Figure 14: Progression of valve actuation for the sample transfer steps .................. 26
Figure 15: Removing the piston ............................................................................... 28
Figure 16: Replacing the mixing chamber windows ................................................. 33
Figure 17: Reassembly of the window plug .............................................................. 33
Figure 18: Reinserting the window plug ................................................................... 34
Figure 19: Face plate tightening pattern ................................................................... 34
Figure 20: Removing the light housing ..................................................................... 35
Figure 21: Internal control board schematic ............................................................. 37
2
WARNINGS / INFORMATION
When the system contains alkane gas the system should be left turned
on since an internal fan constantly circulates the air in the instrument
case to prevent possible build-up of flammable gases.
The maximum pressure rating of this device is 1 kbar (14,500 psi).
Use of the system above this pressure can result in component failure
and possible injury to the user.
The system has been leak tested to the maximum pressure, however
when using gases, especially expensive deuterated reagents it is
prudent to not leave gases loaded in the system for extended periods
of time. It is good practice to retract the gases from the RM
Synthesizer back into the syringe pump, and then push it back into the
gas cylinder.
The MCCS seal is single use only. Reuse of the seal or undertaking
multiple displacement cycles of the piston at high pressure can cause
the seal to fragment leading to the loss of pressure tolerance or
causing the piston to be jammed in the cap or mixing chamber.
Do not open the HP ALKANE TO BOOSTER valve on the RM
Synthesizer if the reservoir is not filled with liquid CO2. This could
displace the internal separator of the gas booster to an unknown
position. Thus it is very important that the separator be reset to the
position closest to the alkane inlet on the gas booster (see Figure 6)
for proper function and transfer of samples to the NMR tube under
pressure.
3
ITEMS INCLUDED WITH THE RM SYNTHESIZER
Below is the list of components that are supplied with the RM Synthesizer to
facilitate setup. This list includes part numbers for possible replacement sources
where appropriate.
Quantity Description Source
1
1
30 ft.
16
16
4
4
1
3 packs
1
3
1
12 ft
1
1
1
200
10
2
North America only
1
1
1
1
1
1
1
1
Elpac Power Systems 12V, 0.5A auto
ranging power supply
AC power cord
1/16” O.D. x 0.03” I.D. stainless steel
tubing (1 – 10 ft. coil, 1 – 20 ft. coil)
15,000 psi glands – ¼”-28 threads High Pressure Equipment
15,000 psi sleeves High Pressure Equipment
1L Pressure resistant bottles Ace Glass P/N 5557-20
PTFE 3-hole GL-45 caps VICI P/N JR-9000-0001
PEEK Cross w/fittings – ¼”-28 threads VICI P/N CXMPK
Cheminert glands and collars – ¼”-28
threads; 5/pack
PEEK plug – ¼”-28 threads VICI P/N CPPK
PEEK shut-off valves – ¼”-28 threads Cole-Parmer P/N 02014-
PEEK check valve – ¼”-28 threads Cole-Parmer P/N 013551/16” O.D. polyethylene tubing Various sources
5/16” x 1/4” wrench
3/4” wrench
3/16” hex driver
MCCS mixing chamber cap seals
(primary)
WS01 mixing chamber window seals
(spares)
Stir bar
Nitrogen gas regulator; 150 psi max. out
Tescom Extreme Pressure Regulator;
6000 psi max in; 2,500 psi max out
0-3000 psi brass gauge
0-3000 psi brass gauge
CGA 320 brass nipple
CGA 320 brass nut
HiP 15-21AF1NMB adapter
HiP 15-21AF1NMC adapter
High Pressure Equipment
Company P/N 15-9A1-030
Company P/N 15-2AM1
Company P/N 15-2A1
VICI P/N CFL-1N
00
26
Daedalus
Daedalus
4
ITEMS TO BE SUPPLIED BY THE END-USER
Some additional tools may be required to complete the setup of the instrument.
Metal tubing cutter: A 1/16” metal tubing cutter and deburring tool will be
necessary to cut sections of tubing for making connections between the syringe
pump and RM Synthesizer and connections to the gas cylinders.
Teflon tape: This may be required when making attaching gas cylinder regulators.
The gas cylinder requirements are outlined below.
N2 gas cylinder: Standard nitrogen gas cylinder. High purity is not necessary. For
customers outside of North America a suitable regulator capable of outlet pressures
of at least 120 psi – 150 psi (~8 bar - ~10 bar) is ideal.
CO2 gas cylinder: Proper function of the RM Synthesizer requires that CO2 be
delivered in liquid form. A standard CO2 gas cylinder will NOT work for this purpose.
Many companies can apply 2,000 psi helium head pressure to a cylinder to allow the
CO2 to exit as a liquid. This requires the tank be equipped with an eductor or siphon
tube so liquid CO2 will be drawn from the bottom of the tank. High purity is not
required. For customers outside North America a regulator capable of outlet
pressures of at least 1,800 psi (124 bar) is preferred. Lower outlet pressures are
possible, but it should not drop below the liquefaction pressure of CO2 (~850psi @
RT).
Alkane gas cylinder: Typically the propane and ethane gas cylinders used with the
RM Synthesizer do not have a regulator attached. The reason for this is the alkane
is generally loaded into the cylinder at or near the liquefaction pressure. So in most
cases the gas is withdrawn at maximum cylinder pressure hence no regulator is
needed. However, it is likely an adapter will be required mate the 1/16” tubing from
the RM Synthesizer to the cylinder outlet. Due to the variance in cylinder types
Daedalus does not attempt to supply suitable adapters. Companies such as High
Pressure Equipment Company (www.highpressure.com) can help identify a proper
adapter once the outlet specifications for the particular alkane tank are identified.
5
INSTRUMENT SITE CONSIDERATIONS
The operation of this instrument involves the use of high pressure
carbon dioxide and flammable gases. Quantities of these gases are
vented from the system after sample preparation. For the safety of the
user this instrument should be setup in an exhaust hood or other
suitable ventilated environment.
The setup instructions below assume that the syringe pump to be used with the RM
Synthesizer is the Daedalus Xtreme-10. The proposed arrangement of components
is merely a guide. Other configurations are possible, and may be necessary
depending on the space available. It is important to stress the necessity of using the
equipment in a ventilated space for the safety of the user.
Figure 1: Possible bench-top setup
The bench-top setup in Figure 1 assumes that the instrument in installed under an
appropriate exhaust hood. If that is not feasible, the instrument can be installed
inside a fume hood, however many fume hoods have a smaller depth than the length
of the Xtreme-10. This may require that the Xtreme-10 be placed towards the back
as shown in Figure 2. The Xtreme Controller would have to be placed outside the
hood, or mounted on top of the Xtreme Pump Box. Alternatively, the Xtreme-10
6
could be turned orthogonal to the placement in the figure with a portion protruding
outside the hood. If this configuration is selected, the Xtreme-10 should be placed
on the left side of the hood with the inlet / outlet side facing to the right hand side.
This places the internal tubing fully in the hood for optimal safety. The Xtreme
Controller could then be placed on top of the Xtreme Pump Box. Not shown are
nitrogen and carbon dioxide cylinders which would be outside the fume hood.
Figure 2: Possible fume hood setup
CONNECTION DETAILS
Once the instruments have been positioned the equipment should be connected
using the included 1/16” stainless steel tubing, the high pressure 15-AM1 glands,
and 15-2A1 sleeves. These steps should be followed when making a high pressure
connection:
i) Deburr the end of the tube section.
ii) Assemble the gland then sleeve onto the tube end.
iii) Insert the end of the tube into the fitting until it bottoms.
iv) Tighten the gland to 55 in·lb. A “bottoming out” or “dead stop” should be felt
when the connection is properly assembled.
Always use two wrenches when tightening fittings: one to tighten the
gland and one to prevent counter rotation of the fitting receptacle.
Failure to do so could break loose internal connections.
Make the following connections using high pressure tubing and fittings unless
otherwise specified:
7
1) Connect the OUTLET port of the Xtreme-10 to the HP ALKANE INLET port on
the back of the RM Synthesizer.
2) Connect the INLET port of the Xtreme-10 to the alkane gas cylinder.
3) Connect with high pressure the CO2 INLET port on the RM Synthesizer to the
CO2 gas cylinder regulator.
4) Connect the N2 INLET port on the RM Synthesizer to the N2 gas cylinder
regulator.
5) Connect a section of tubing to the WASTE port of the RM Synthesizer using a
high pressure fitting. Assemble the Cheminert CFL-1N gland and sleeve on the free
end. Feed this end through the small port of one of the three-port bottle caps until it
is at the bottom of the bottle. Tighten the Cheminert gland to secure the line. The
remaining ports of this bottle cap should be left open to allow gases to vent.
Figure 3: Cleaning manifold assembly diagram
8
6) Assemble the cleaning manifold according to the Figure 3 using the included parts
and connect to the RM Synthesizer as indicated.
7) The NMR cell is connected to the OUTLET/INLET valve on the RM Synthesizer
as indicated later in the instructions. This valve can also serve as the introduction
point for deuterated alkane in situations where the syringe pump is not fully loaded
with deuterated solvent.
(This section is intentionally blank)
9
THEORY OF OPERATION
The RM Synthesizer was designed to bring together all the necessary elements for
preparing reverse micelle samples in liquid alkanes at high pressures. One issue
with preparing samples in a vessel where the desired analysis cannot be performed
is the need to move the pressurized sample into a more appropriate container. In
the case of reverse micelle samples, the efficacy of the sample is dependent on the
pressure such that in moving the sample the pressure cannot dip below a certain
threshold without losing integrity of the construct.
There are a variety of methods that could be used. The first is to avoid the two
vessel system entirely; however, mixing in the high pressure NMR tube is not very
efficient and was discarded early in the development. Another method is to use a
high pressure gas to push that sample from one vessel into another. After
considering a variety of gases there are not many candidates that are immiscible
with liquid alkanes, readily obtainable, and do not require significant compression
cycles to bring the pressure of the gas up to the level required. Nitrogen is one of
the best candidates. However, for certain applications, the pressure required for the
transfer step increases the density of the nitrogen gas above that of the liquid alkane
such that the nitrogen is no longer pushing the sample; rather it replaces the sample
in the secondary chamber. The
method used by the RM Synthesizer is
to displace the sample, by way of a
piston, from the mixing chamber into
the NMR tube. The relative piston
position during preparation and
transfer is shown in Figure 4.
The force driving the piston
displacement is provided by the
expansion of a high pressure fluid
delivered by a secondary pressure
source. Again, there are a variety of
alternative methods that could be used to achieve this action. Rather than explain
why other methods are not used, this discussion will focus on the what RM
Synthesizer provides for this step. The liquid alkanes used in the preparation of
reverse micelles are highly compressible. Data from the website:
http://webbook.nist.gov/chemistry/ for liquid ethane shows that ethane undergoes a
decrease in volume of approximately 7% over the range of 4,000 – 7,000 psi. This
Figure 4: Piston position during operation
10
change in volume can be harnessed to rapidly move the piston when a reservoir at
high pressure is allowed to relax to a lower pressure. This process is rapid, and the
pressurizing fluid does not need to go through a transition from liquid to gas to
deliver the needed force.
This entire action could be provided solely by the syringe pump. However, the
syringe pump plays a role immediately after the actual transfer step by rapidly
returning the sample to the required encapsulation pressure. This will be discussed
later in the example protocols. The end result is the syringe pump has the role of
maintaining the reverse micelle sample pressure over being used to drive the piston
directly. In addition, and perhaps more importantly, more of the precious deuterated
alkanes would be wasted using it as the fluid for this step.
Instead the secondary pressure source is provided by an internal fluid reservoir or
gas booster. It turns out that carbon dioxide has nearly identical compressibility as
liquid ethane. Thus the reservoir can be filled with CO2 and this fluid pressurized to
the required level using liquid ethane delivered by the syringe pump. A separator
piston in the reservoir keeps the fluids apart. Once the transfer steps are performed,
the CO2 fluid can be used to push any deuterated alkane out of the reservoir
keeping the losses to an absolute minimum.
The piston displacement literally rams the sample into the waiting NMR tube.
Because it might be possible to deliver more pressure to the piston than the tube
can withstand the dimensions of the piston and length of the connection tube to the
NMR cell were selected such that the piston displaces less than the volume of the
NMR cell plus tubing. For most situations this is not strictly necessary. However, for
the small fraction of cases where this might be important, it is suggested this
approach be maintained.
Since the volume displaced is less than the volume of the NMR cell, additional
solvent molecules must be added to the mixing chamber fill this extra space. This is
again done by taking advantage of the compressibility of liquid ethane. The sample
in the mixing chamber is over pressurized just enough that when it is released, and
the piston pushes the sample into the NMR cell, the expanded fluid volume will fully
fill the volume of the NMR cell at the encapsulation pressure. The method for taking
all these details into account is described later in this manual when outlining
potential sample preparation protocols.
11
Figure 5: RM Synthesizer Operational Description
12
Figure 6: RM Synthesizer Flow Diagram
13
CONVENTIONS USED IN THIS MANUAL
When describing operations of this device the identification of the valves involved
will be made by listing the valve name that is stenciled on the instrument such as HP
ALKANE or PISTON BOOSTER valve. The same method will be used to identify
ports on the back of the instrument. Where it is prudent, the specific sequence of
valve actuation will be identified on the platform figure with indicators corresponding
to the step. Shown below is a typical example.
To transfer the sample from the mixing chamber to the sample tube perform the
following steps diagramed in Figure 7:
i) Open the OUTLET / INLET valve; wait for two seconds.
ii) Close the PISTON BOOSTER valve.
iii) Open the HP ALKANE valve to equalize the system pressure.
iii
ii
i
Figure 7: Example valve actuation
progression
VALVE DESCRIPTIONS
Refer to Figure 5 and Figure 6 for pictorial representations of the valve connections
and how they control fluid flow.
OUTLET / INLET: This is the connection point for the reverse micelle NMR cell. The
transfer line that comes with the NMR cell should be used for this connection. In
cases where the syringe pump will not be fully loaded with deuterated alkane, this
valve can be used as an inlet port for the initial fill with deuterated solvents. The
14
syringe pump would then use protonated solvents to pressurize the sample. Due to
the compressibility of ethane and propane, this will likely result is a sample that has
20-30% protonated solvent.
INLET TO BOOSTER: This connects the CO2 INLET port directly to the gas
CO
2
booster. This valve allows the gas booster to be refilled with high pressure CO
.
2
VENT PISTON: This releases the pressure on the piston to the WASTE port. This
valve should not be opened with the PISTON BOOSTER open as this will release all
gas in the gas booster reservoir.
HP ALKANE TO BOOSTER: This connects the HP ALKANE INLET port to the
opposite side of the separator in the gas booster. This valve is opened when
changing the internal pressure of the gas booster or when releasing pressurized
alkane back into the syringe pump.
HP ALKANE: this connects the HP ALKANE INLET to the mixing chamber. This is
opened when preparing the reverse micelle samples.
PISTON BOOSTER: Opening this valve delivers the pressurized CO2 from the gas
booster to the piston contained in the mixing chamber cap. This can be used both to
change the internal pressure of the mixing chamber without adding additional alkane
to the sample as well as more commonly for transferring the sample from the mixing
chamber into the NMR sample tube.
WASTE: Allows the contents of the mixing chamber to be flushed out to the
WASTE port. This is used for venting the chamber after the sample preparation as
well as for passing cleaning fluids out of the chamber.
FOR CLEANING: This connects the N2 INLET port directly to the mixing
N
2
chamber. This supplies high pressure nitrogen gas for pushing the cleaning fluids
out of the mixing chamber through the WASTE port. This valve should not be
opened when the chamber is at a pressure above the nitrogen gas cylinder output.
CLEANING MANIFOLD: This provides a path for the selected cleaning fluid into the
chamber. This valve should only be opened when the chamber pressure is at
atmospheric otherwise it will cause a backflow into the cleaning manifold bottles.
Mixing chamber water ports: There are a series of channels cut into the mixing
chamber forming a continuous loop. The ends of this loop terminate at the two
15
brass barbed fittings on the right side of the mixing chamber. These can be
connected to a water bath to provide thermal regulation of the sample.
BACK PANEL CONNECTIONS
Shown in Figure 8 are the ports used to connect the system to the external fluid
sources required for operation of the RM Synthesizer.
Figure 8: Back panel port connections
HP ALKANE INLET: Connects to the outlet of the Xtreme-10 Syringe Pump. The
tubing should be 1 kbar rated.
WASTE: T his port handles the outflow from the mixing chamber as well as the high
pressure gas from the piston. The tubing should be rated for high pressure, and the
end should be in a vessel that can tolerate organic solvents. It should be anchored
so it does not break loose when high pressure gas is vented.
CO2 INLET: This is the high pressure carbon dioxide inlet. Standard CO2 cylinders
are not able to deliver liquid CO
. Instead connect to a cylinder with an eductor tube
2
pressurized with helium to at least 1,800 psi for best performance.
N2 INLET: This port is for connecting a standard nitrogen cylinder with an output of
125-150 psi. This inlet links to the mixing chamber as well as an internal regulator
rated to a maximum of 250 psi which delivers 10 psi gas to the LP N
INLET.
2
LP N
INLET: The output from this port is 10 psi nitrogen gas. Connect this to the
2
cleaning manifold pressurizing port. It is this gas pressure that promotes fluid flow
out of the manifold bottles. There is no valve between the N
INLET and LP N2
2
16
INLET so always have it connected or plugged to prevent discharging the nitrogen
cylinder.
CLEANING MANIFOLD: The outlet of the PEEK cross from Figure 3 should be
connected to this port. Solvents from the cleaning manifold flow from this port to the
mixing chamber.
FRONT PANEL CONTROL
Figure 9: Front panel controls
POWER: Provides power for the lamp and mixing electronics. It also turns on a
venting fan inside the instrument. Even if the lamp and mixer are not required the
power should be turned on when in use to keep air circulating inside the instrument.
LAMP: This knob controls the intensity of the light source illuminating the mixing
chamber
MIX: This knob controls the sample stirring speed.
PREPARING THE INSTRUMENT FOR USE
The procedure described is the basic setup required for use of the instrument. It
assumes the Xtreme-10 Syringe Pump is being used as the pressure source.
(1) Fill the gas booster with 1,800 psi CO2 by opening the CO2 INLET TO
BOOSTER valve.
(2) Open the HP ALKANE TO BOOSTER valve while filling the gas booster to allow
the high pressure CO
pushing out any ethane present in the reservoir.
to push the internal separator all the way to one end thus
2
17
(3) Close the HP ALKANE TO BOOSTER and CO2 INLET TO BOOSTER valves.
The gas booster is now filled with CO2.
(4) Fill the syringe pump completely with liquid ethane. Follow the procedures in the
Xtreme manual for refilling the pump. To extract liquid from the ethane cylinder it
may be necessary to place the cylinder in warm water while filling the pump. It will
take time for the liquid to transfer. Depending on the temperature ethane liquefies
around 610 psi. See http://webbook.nist.gov/chemistry/ for detailed information
about the properties of liquid alkanes. Open the HP ALKANE TO BOOSTER to fill
this void as well.
(5) The nitrogen cylinder can be connected, but does not need to be open until the
cleanings steps. Set the cylinder pressure to between 125-150 psi.
At this point the system is ready for reverse micelle preparation work. The
remaining steps are provided as an example of how to load the syringe pump with
extra grams of ethane. This is optional, and does not need to be performed for
routine operation of the instrument. However, this operation might be useful if a
higher starting pressure is desired, or if the time between syringe pump recharges is
to be increased.
Only perform this procedure with the gas booster reservoir filled with
CO2 to act as a brake for the internal separator. If the reservoir is
empty and subsequently filled entirely with alkane the piston would be
moved to the fully displaced position. Later additions of liquid CO2
may not be sufficient to push the separator back into position and as a
(6) With the syringe pump fully filled with liquid ethane and the pump INLET valve
closed, set the pump to pressurize to 7,000 psi. With the HP ALKANE TO
BOOSTER valve already open this will pressurize the gas booster to 7,000 psi.
Once complete proceed to the next step.
(7) Close the HP ALKANE TO BOOSTER and OUTLET valve on the pump and
fully refill the pump again with liquid ethane. Be sure to Close the INLET valve on
the pump after the refill step.
result the transfer step may not work as expected because of
insufficient distance for the piston to travel.
18
(8) Open the HP ALKANE TO BOOSTER and pump OUTLET valves to
depressurize the gas booster. This will push out the ethane in the gas booster back
into the syringe pump. Dependent on the pump fill factor and the initial CO2 head
pressure, the pressure in the syringe pump will be around 1,500 psi. The system is
now filled with sufficient ethane to make fifteen typical ethane samples with
encapsulation pressures of 4,000 psi The CO2 in the gas booster will need to be
recharged after each sample. This will be addressed later when the protocol for
transferring the sample and resetting the system is outlined.
THE MIXING CHAMBER / ADDING REAGENTS
The internal volume of the mixing chamber is approximately 1.65 ml when fully
assembled. There are slight variations between cells so an exact measurement
should be obtained using the syringe pump (see CALIBRATING THE SYSTEM). Of
this volume approximately 1.1 ml can be displaced by the piston. The dead volume
is comprised of the empty volume of the connection tubing empty, the reagent plate
well, and spaces created from placing windows in the mixing chamber walls. This
large available sample volume is required because the NMR cell has an active
volume of approximately 1.2 ml. There are two observation windows in the walls of
the chamber. The lamp abuts the back facing window, while the front facing window
is for observation of the sample preparation process.
In addition to the main mixing chamber body and top cap there are three additional
components as shown in Figure 10. These are the piston, the reagent plate, and oring seal (MCCS).
Figure 10: Mixing chamber components:
Poston, reagent plate, and main seal (MCCS).
19
Figure 11 shows the assembly diagram of the cell and the relative position of the
components. The reagent plate serves two functions: The wall of the reagent plate
stops the descent of the piston and prevents the stir bar from being crushed.
Second, surfactants, especially liquid surfactants, can be weighed directly on this
plate prior to inserting into the mixing chamber. The reagent plate should be placed
at the bottom of the mixing chamber.
Make certain the WASTE valve is closed prior to adding reagents to
prevent the reagents from being forced out through this port in
subsequent steps.
Add the reagents in the following order (not all items are used with every sample):
(1) Add the protein / aqueous phase first
(2) Add any liquid co-surfactants such as hexanol
(3) Add the liquid surfactants
(4) Add the dry surfactants
(5) Add other additives such as pentane or carbon disulfide
(6) Always add the stir bar last
Figure 11: View showing internal placement of the mixing chamber components
20
SEALING THE MIXING CHAMBER
The high pressure seal is provided by the o-ring component P/N MCCS. This seal
provides both a static containment seal during the sample mixing as well as a
dynamic seal when the piston is driven down during the sample transfer step. This
combination of forces tends to degrade the seal such that it must be replaced after
every sample.
The following photos in Figure 12 show the steps for assembling the mixing chamber
for the high pressure work.
(1) Insert the o-ring seal (MCCS) into the large chamber O.D. Push it down as far as
it will go.
(2) Open the OUTLET / INLET valve, and push the piston to just slightly past the oring seal. Do not push it all way down as this will force the sample out the OUTLET /
INLET valve. The valve is opened to relieve pressure generated by pushing the
piston into place.
(3) Place the cap with the four screws into position. The small O.D. section of the
cap should fit into the mixing chamber main body large O.D. section.
Figure 12: Progression of steps for assembling and sealing the mixing chamber
prior to sample preparation
21
(4) Tighten the four screws in a star pattern using a 3/16” hex driver in the pattern
shown in the Figure 12. Once tightened, close the OUTLET / INLET valve.
At this time it is recommended that all valves be checked to make sure they are
closed, especially those that were actuated in the previous sample preparation
process.
PROTOCOL FOR MAKING SAMPLES
The encapsulation pressure for samples in liquid ethane varies according to the
surfactant, the water loading, and the co-surfactants / solvents added to the system.
Typically for samples in pure ethane in CTAB with modest water loadings the
encapsulation pressure is around 4,000 psi. For AOT this increases to around 8,000
psi without the addition of co-surfactants / solvents. Since there are so many
variables involved the best method for learning the art of preparing samples in
ethane is to practice. One way to do this is to prepare a series of ethane samples
using a protein-free water stock with bromophenol blue added. The visual indicator
will help the user learn the tricks and visual markers that are apparent as the
pressure on the sample is increased ultimately to the encapsulation point.
For a CTAB surfactant sample with 10% hexanol, a water loading of 15, in pure
ethane the steps are as follows:
(1) Add the reagents and seal the chamber.
(2) Open the HP ALKANE valve to introduce ethane into the chamber. Usually the
target pressure for this step is around 1,500 – 2,000 psi. Increase the pressure if it
is not already at this level. The OUTLET valve on the syringe pump should be open
and remain so throughout the sample preparation process.
(3) Set the stir bar speed to around 2/3rds. At this time the water and protein
solution will likely not be visually observable.
(4) Slowly step up the pressure to 3,000. Using small increments of 500 psi should
make apparent when the water begins to mix by a clouding of the sample. This is
the point where it is thought the CTAB solubilizes. The mixing should continue at
this pressure until this phenomenon is observed to assure the sample is in fact
mixing.
22
(5) Continue stepping up the pressure in small increments of 250 psi while
continuing to observe the mixing process as noted by the cloudy nature of the
sample.
(6) Around 3,500 – 4,000 psi the sample should become noticeably more clear. At
this point the experience of the user becomes important. Most samples will not
completely solubilize so some flocculence may be apparent in even a good sample.
Stop the stirring process and allow the insoluble particles to settle. If the previous
steps were followed, and a mixing transition was observed a transparent sample
usually indicates it is close or at the encapsulation point. The alternative is to
increase pressure some more, but doing will increase the total viscosity of the
sample and defeat the purpose of encapsulating proteins in ethane. However, it
may be necessary to do so to achieve stable samples so a judgment must be made.
A sample that is properly encapsulated will not become clearer with the addition of
more pressure.
Increasing the pressure in the chamber directly using ethane is not always the best
option since this would have negative consequences on the sample viscosity as well
as the pressure could not be subsequently reduced as this would draw sample out of
the chamber. However, the pressure on the piston can be used to increase the
chamber pressure without diluting the sample or making irreversible additions. This
procedure is optional, and is presented only as another method available for finding
the proper encapsulation pressure. To perform this piston pressurization routine
refer to Figure 13 and perform the following:
i) Close the HP ALKANE valve to isolate the mixing chamber.
ii) Open the HP ALKANE TO BOOSTER valve.
iii) Open the PISTON BOOSTER valve to expose the piston to the pressure
source.
iv) Using the syringe pump increase the pressure on the piston to a target
pressure above the internal chamber pressure.
v) Watch for additional clearing of the sample.
vi) After testing is complete, reduce the syringe pump pressure to below the
internal chamber pressure (if possible – see (ix))
vii) Close the HP ALKANE TO BOOSTER and PISTON BOOSTER valve.
viii) Resume preparing the sample with the newly obtained data.
ix) Optional: It may be necessary to release the piston pressure to reduce
the chamber pressure back to what it was originally. If necessary, be sure
23
all valves are closed, and open the VENT PISTON valves to release the
pressure. Close the valve after venting is complete.
ii,vii
i
ix
iii,vii
Figure 13: Increasing the sample
chamber pressure using the piston
(7) Once the sample has been encapsulated it is recommended the internal sample
pressure be increased by an additional 250 psi to account for potential pressure loss
from the reverse micelle NMR cell over time. This is the encapsulation pressure of
the sample and will be referenced later in the protocol.
(8) As discussed in the Theory of Operation section, the volume displaced by the
piston is less than the volume of the NMR tube. Therefore, the sample must be over
pressurized to compress more molecules of ethane into the chamber such that once
the transfer is initiated the sample will expand to fully fill the NMR tube. The over
pressurization required is dependent on the NMR cell style as described below:
Varian: Density
× 1.08 = Density
encap
Bruker: Density
× 1.03 = Density
encap
Using the density data ethane obtained from http://webbook.nist.gov/chemistry/ look
up the density for ethane at the encapsulation pressure, plug it into the formula, then
find the corresponding density for the over pressurization target. Over pressurize
the sample to the calculated target pressure then close the HP ALKANE valve. The
sample is ready to be transferred.
over
over
24
TRANSFERRING SAMPLES FROM THE MIXING CHAMBER TO THE
NMR CELL
With the sample now over pressurized to expand to fill the NMR cell internal volume,
what remains is to apply sufficient pressure to the piston such that the sample is
displaced from the mixing chamber into the NMR cell when the OUTLET / INLET
valve is opened. The following steps outline the procedure.
(1) Preset the NMR cell valve. The needle in the NMR cell valve displaces volume
during actuation. Thus the valve must be opened to the same position each time to
assure the internal volume matches the over pressurizing ratio from the previous
section. To set the position close the NMR cell valve until just snug, then open the
valve (counter clockwise) two full turns, then close (clockwise) one full turn. Be sure
to use the transfer tube provided as its internal volume is included in the over
pressurizing ratio. Connect this tubing between the NMR cell and the OUTLET /
INLET valve.
(2) Open the PISTON BOOSTER and HP ALKANE TO BOOSTER valves. Be
sure the HP ALKANE valve is closed before performing this operation
otherwise the sample will be pushed back into the syringe pump. This allows
the syringe pump to pressurize the piston. Increase the pressure on the piston to
2000 psi above the encapsulation pressure (this is not the current mixing chamber
pressure) or equal to the over pressurization pressure, whichever is higher. Close
the HP ALKANE TO BOOSTER valve after the target pressure is reached. The
piston is now set for transfer.
(3) Reset the syringe pump pressure to the encapsulation pressure of the sample.
This is not the over pressurization pressure.
(4) Perform the sample transfer by actuating valves according to the following
(Refer to Figure 14):
i) Open the OUTLET / INLET valve; wait for two seconds.
ii) Close the PISTON BOOSTER valve.
iii) Open the HP ALKANE valve to equalize the system pressure.
25
iii
ii
i
Figure 14: Progression of valve actuation
for the sample transfer steps
(5) After the system pressure equalizes to the encapsulation pressure, the NMR cell
valve can be closed. The OUTLET / INLET and HP ALKANE should remain open
during this step.
(6) After the NMR cell is sealed close the OUTLET / INLET and HP ALKANE
valves to prevent accidental expulsion of the pressurized fluid.
(7) Disconnect the transfer line from the NMR cell. It is now ready to be inserted
into the NMR spectrometer for sample assessment.
(8) Be sure to clean the mixing chamber after each sample.
CLEANING THE MIXING CHAMBER
The RM Synthesizer is shipped with three low pressure glass reservoirs that can be
filled with solvents for cleaning the mixing chamber and associated tubing after
sample preparation. Typically the solvents are water, ethanol, and dichloromethane.
The LP N
supply that plugs into the reservoirs to provide the fluid pushing force. A high
pressure nitrogen line feeds directly into the mixing chamber to push the cleaning
solvents out of the chamber. Be sure the WASTE port is connected to a suitable
waste container and waste gases are properly exhausted away from users.
The nitrogen gas cylinder should be opened and delivering at least 125 psi nitrogen
gas.
GAS port on the back of the instrument delivers a low pressure gas
2
26
(1) Open the VENT PISTON valve to release the high pressure CO2 gas to the
WASTE port. The remaining ethane in the mixing chamber will now push the piston
back to the retracted position.
(2) Open the WASTE valve to vent the sample to the WASTE port. Close the valve
after the sample is vented.
The order of cleaning solvents should be:
i) Dichloromethane
ii) Ethanol
iii) Water
iv) Ethanol
v) Dichloromethane.
Ethanol (ii) will dissolve most common surfactants and water (iii) will solubilize the
protein. Both reagents influence reverse micelle formation so these need to be
displaced with subsequent rinses (iv & v). Dichloromethane serves as the bridge
between ethanol and alkane solvents. It evaporates quickly when the cell is opened
to air however trace dichloromethane will not cause problems for reverse micelle
formation.
For each cleaning solvent:
(3) Open the CLEANING MANIFOLD valve, and open the appropriate solvent
PEEK shut-off valve in the manifold. Allow the mixing chamber to fill with the fluid.
At the same time the transfer tube to the NMR cell can be flushed by opening the
OUTLET / INLET valve for a short time. Be sure to catch the fluid in a suitable
receptacle. Stir the sample in the mixing chamber to promote solubilization.
(4) Close the CLEANING MANIFOLD valve and the PEEK shut-off valve.
(5) Open the N2 FOR CLEANING valve. This allows the high pressure nitrogen gas
into the mixing chamber.
(6) Open the WASTE valve to expel the cleaning solvent into the waste receptacle.
Also open the OUTLET / INLET valve to blow out the transfer tube.
(7) Close the N2 FOR CLEANING first then the WASTE valve.
27
(8) Go back to step (3) and repeat for each solvent in the list.
(9) After cleaning the chamber, the cell cap can be removed. A screw from the cap
can be threaded into the piston to remove it from the chamber (Figure 12). The seal
(MCCS) should be removed and discarded.
(10) Wipe out any additional trace material with a cotton swab. The mixing chamber
is ready for the next sample.
Figure 15: Removing the piston
RESETTING THE SYSTEM FOR THE NEXT SAMPLE
The following guidelines are for resetting the system; primarily resetting the internal
gas booster reservoir separator piston to the proper position for the next sample.
This method can be used to confirm that the separator piston has been moved to the
fully reset position.
At this point the reservoir pressure and syringe pump pressure are probably well
above the CO2 gas cylinder outlet pressure (1,800 psi). Before the reservoir can be
recharged the pressure in both the reservoir and syringe pump should be below this
level. All the system valves should be closed at this point.
(1) Open the HP ALKANE TO BOOSTER valve and the OUTLET valve on the
syringe pump. This will allow excess alkane to flow back into the syringe pump.
The reservoir pressure may be higher than the syringe pump so there may be a
jump in the pressure reported by the syringe pump.
28
(2) Change the setpoint on the syringe pump to 1,700 psi and start the pump
running. The high pressure CO2 fluid in the reservoir will now push the separator
piston back towards the fully reset position. Since some fluid was used during the
sample preparation process it should not be expected to complete the full
displacement.
(3) With the pressure in both the reservoir and syringe pump below 1,800 psi the
CO2 INLET TO BOOSTER valve can be opened to allow fluid from the gas cylinder
to recharge the booster. Once this valve is opened any excess alkane remaining will
be forced back into the syringe pump, and a temporary pressure increase should be
displayed by the syringe pump until it starts to readjust to the 1,700 psi setpoint.
This process can take a few moments since the pressure differential is small.
(4) Once the syringe pump is stable at 1,700 psi it can be assumed the separator
has been fully reset since the applied 1,800 psi CO2 pressure is no longer
influencing the syringe pump reading. Close the CO2 INLET TO BOOSTER the HP
ALKANE TO BOOSTER valves. The syringe pump OUTLET valve can be closed
as well.
(5) The system is now reset and ready for the next sample preparation.
The primary goal of this protocol is to assure the gas booster reservoir is fully
recharged with CO2. This method can be adapted to the user’s needs and time
constraints.
TIPS
The following list contains some tips for operation of the RM Synthesizer some of
which were contained in the text of this manual. It also contains tips that will
hopefully help in making successful reverse micelle samples in ethane. It is by no
means exhaustive.
After the first few uses of an ethane or propane cylinder, the outlet pressure
will likely be below the liquefaction pressure. Placing the tank in warm water
can help deliver liquid alkane from the cylinder.
Propane may not start to fill the mixing chamber until the pressure reaches
300 psi. The mixing chamber can be chilled to help condense propane, but it
is by no means necessary to make it work.
29
Propane samples can use the 1,800 psi CO2 fluid to actuate the piston. The
over pressurization method should still be followed, but it is unlikely the piston
will need more pressure than 1,800 psi.
Once encapsulation has been achieved it is good practice to go 250 psi
above that to overcome leakage from the NMR cell over extended periods of
time.
Insoluble material is common even in good samples. Stop the stir bar and
allow the insoluble material to settle to properly observe the sample.
The insoluble material should be allowed to settle prior to transferring the
sample to the NMR cell.
After the sample has been transferred, and the NMR cell closed, the cell
should be inverted several times to help solubilize any reagents that may
have fallen out of solution during the transfer.
The pressure in the NMR cell can be increased after it has been closed.
Reconnect the NMR cell to the OUTLET/INLET valve using the transfer line.
Open the OUTLET/INLET valve and pressurize the mixing chamber to the
encapsulation pressure or the new pressure desired. Open the NMR cell
valve and let the system equilibrate to the new pressure. Close the NMR cell
valve, and the OUTLET/INLET valve. Disconnect and check the sample at
the new pressure.
Use the piston to perform test increases in pressure in the mixing chamber.
This may be preferable to adding additional solvent and diluting the sample
unnecessarily.
No matter how good the seals, the system will leak over time. If the system
will not be used for several weeks it is good idea to retract any deuterated
solvents from the RM Synthesizer, into the syringe pump, and then push that
fluid back into the alkane cylinder.
Occasionally the stir bar becomes lodged in the surfactant slurry. It can be
dislodged using a strong magnet; either on the side of the mixing chamber or
balanced along the edge of the mixing chamber cap.
Typical CTAB samples encapsulate around 4,000 psi, AOT above 8,000 psi,
and a triple surfactant mix consisting of 70% C12E4, 25% AOT, 5% DTAB
might not encapsulate around 9,000 psi. Around 8,000 psi the viscosity of
ethane is equal to propane so the benefit of working in ethane is lost.
30
The encapsulation pressure is dependent on the dielectric constant of the
medium. The elevated pressure is required to raise the dielectric of ethane to
match the dielectric of the surfactant.
The encapsulation pressure can be lowered through the use of additives.
This serves to increase the dielectric constant of the bulk solvent. Examples
of additives are pentane, dichloromethane, and carbon disulfide.
Encapsulation conditions found in pentane in general are translatable to
ethane with minor changes. For example CTAB sample may require 1.5%
more hexanol in ethane than pentane.
Water loading is the ratio of molar water to molar surfactant in the reverse
micelle sample. It has a large impact on the size of the particle.
Past studies using reverse micelles have worked to keep the water loading
low; typically in the range of 10-15 to keep the particle as small as possible.
However, larger proteins likely require more water to maintain the proper
hydration shell. Thus water loadings of 20-25 should be considered for
proteins larger than 40 kDa. At this size the protein is dominating the size of
the particle so the excess water has less impact.
Ethane can support much higher water loadings than are common in pentane.
This will require slightly higher encapsulation pressures.
CALIBRATING THE SYSTEM
The mixing chamber and components are designed to provide an internal volume
near 1.65 ml. Of this volume only 1.1 ml can be displaced by the piston. The actual
volume of the mixing chamber and the amount displaced are important numbers.
The former being important for the initial reagent calculations, and the latter for
determining the over pressure requirements for the transfer step to the NMR cell.
These numbers can be determined by a simple protocol. Use protonated alkanes
for these measurements as it will be vented from the mixing chamber. The gas
booster reservoir should be charged with CO
(1) Setup the mixing chamber as would be done for a typical preparation process.
Include the reagent plate and stir bar.
(2) Open the HP ALKANE valve just enough to let in a little alkane gas then close
the valve. The syringe pump OUTLET valve should be open.
to the normal operating pressure.
2
31
(3) Open the WASTE valve to vent the alkane. This will purge the mixing chamber
of air.
(4) Change the setpoint on the syringe pump to 1,000 psi, and let it stabilize.
(5) Note the current volume on the syringe pump, and open the HP ALKANE valve
just enough to fill the chamber.
(6) After the syringe pump is again stable at the setpoint note the new volume. The
difference between the volume in step (5) and this new volume is the mixing
chamber volume.
(7) Open the CO2 INLET TO BOOSTER valve and the PISTON BOOSTER valve. It
is assumed the CO2 gas cylinder valve is open. This delivers 1,800 psi CO2 to the
mixing chamber piston. It will push the alkane out of the mixing chamber causing
the syringe pump to compensate to maintain the 1,000 psi setpoint.
(8) After the syringe pump has again stabilized note the new volume reading. The
difference between this volume, and the volume in step (6) is the volume the piston
can displace for the sample transfer step.
(9) Close all valves.
(10) Open the VENT PISTON valve to vent the CO2 gas behind the piston. After
venting is complete close the valve.
(11) Open the WASTE valve to vent the alkane from the mixing chamber. After
venting is complete close the valve.
(12) Repeat steps (5-11) several times to obtain an average reading for the mixing
chamber and piston displacement volumes.
REPLACING THE WINDOW SEALS
The performance of the seals around the mixing chamber sapphire windows can
degrade over time. Before changing the seals, be sure all other sources of leaking
are eliminated. Leaking window seals are evidenced by the external pressure
source noticeably not being able to maintain pressure. It can be further identified by
32
filling the chamber with ethanol, pressurizing with the piston, and monitoring for
leaks. Do not change the seals unless necessary. To remove:
Figure 16: Replacing the mixing chamber windows
(A) remove the four face plate screws. (B) Using a small L-shape hex wrench or
equivalent, push out the plug from the inside. (C) Using a forceps, remove the
sapphire window and seal. Discard the seal. (D) Remove debris from the window
/ seal surfaces.
Use a new window seal (WS01) and reassemble the plug by placing the window in
the recess of the plug. Then place the o-ring seal around the window as shown in
Figure 17.
Figure 17: Reassembly of the
window plug
33
Wet the seal with ethanol, and reinsert the assembled plug into the chamber wall
(Figure 18).
Figure 18: Reinserting the
window plug
Reattach the face plate using the four socket cap screws and incrementally tighten
the screws using the pattern shown in Figure 19. This will better distribute the force
on the window seal.
Figure 19: Face plate tightening
pattern
To replace the backside window, first remove the lamp housing by unscrewing the
power plug at the base and the two screws holding the housing to the mixing
chamber plate (Figure 20). Proceed as previously described.
34
Figure 20: Removing the light
housing
(This section is intentionally blank)
35
SPECIFICATIONS
Power requirements 100-120 VAC / 200-240 VAC, 50/60 Hz
Input current < 0.5A rms
Power output 12 VDC, 0.5 A maximum
Temperature range 10 °C to 70 °C
Weight 23 lbs (10.5 kg)
Dimensions 16.25” W x 13.25” D x 9” H (41.3 cm x 33.7 cm x 23 cm)
Pressure range 0-14,500 psi (1 kbar)
Wetted parts 316 stainless steel
Operating medium Alkanes, Carbon Dioxide, Water, Oils, Alcohols, Inert Gases.
Mixing chamber volume 1.65ml nominal
Piston displacement volume 1.1ml nominal
Pressure connections All ports are HiP AF1 (1/4”-28 UNF) for use with 1/16” tubing
This equipment has been tested and found to comply with the limits for a Class A
digital device, pursuant to part 15 of the FCC Rules. These limits are designed to
provide reasonable protection against harmful interference when the equipment is
operated in a commercial environment. This equipment generates, uses, and can
radiate radio frequency energy and, if not installed and used in accordance with the
instruction manual, may cause harmful interference to radio communications.
Operation of this equipment in a residential area is likely to cause harmful interference
in which case the user will be required to correct the interference at his own expense.
This Class A digital apparatus complies with Canadian ICES-003.
This system conforms to the European Community Council Directive 2004/108/EC for
electrical equipment for measurement, control and laboratory use. The standard used
for emissions requirements EN 61326-1:2006; Clause 7.2, and the immunity
requirements conformed to EN 61326-1:2006; Table 2.
This system conforms to the European Community Low Voltage Safety Directive
2006/95/EC. The standard used was EN 61010-1:2001 for electrical equipment for
measurement, control, and laboratory use, Part 1: General requirements.
Documentation can be provided by contacting in writing:
Daedalus Innovations LLC
200 Racoosin Drive, Suite 106
Aston, PA 19014
United States.
36
Figure 21: Internal control board schematic
37
FURTHER INFORMATION
This document may be updated periodically to reflect questions from users. Please
check back at www.daedalusinnovations.com in the support section for more recent
versions of this document.
Technical support can also be obtained by emailing questions to
support@daedalusinnovations.com, or contacting Daedalus directly at 610-358-
4728.
Other correspondence can be directed to:
Daedalus Innovations, LLC
200 Racoosin Drive, Suite 106
Aston, PA 19014
38
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