Fourier Transform Infrared Spectrometer
Thermo Scientific, iS10
by Anthony Butterfield
12/01/2009
This is an abbreviated operating procedure for the Fourier Transform Infrared Spectrometer
(FTIR) in our lab. Refer to the Thermo Scientific iS10 manual for more specific information.
1. The power to the FTIR should be on and should remain on even if the computer is turned off.
Do not ever turn the FTIR off. If it is for some reason powered down, turn on the FTIR
(there is a switch on the transformer)
2. When the FTIR is ready, the LED for the laser should be on.
3. If the computer is turned off, turn it on.
4. Open the OMNIC software.
5. If using the Attenuated Total Reflection stage (ATR), select “DuraScope Accessory, 1
Bounce” in the “Smart Accessory Change” window. If using transmission FTIR, select
the “Transmission” option.
6. Click the Experimental Setup button (“Expt Set”).
7. Select your experiment properties.
a. Typical number of scans = 32 (more will give more averaged, smoothed spectra,
and less will give more noisy spectra)
b. Typical resolution = 4 (more will leave out more spectra detail and less will give
more detail)
c. Typical data final format = Absorbance or Transmittence.
d. Typical time between backgrounds = 120 min.
8. Click OK to close the experiment properties window.
9. Choose an appropriate control sample.
10. Place the control sample on the Attenuated Total Reflection stage (ATR) or in/on another
appropriate sample holder.
11. If using the ATR with solids:
a. Place the sample over the window.
b. Turn Screw 1 (above the sample), raising or lowering the pin until it touches the
top of your sample. Do not tighten this screw; it is only meant to compensate for
samples of varying height and the load is measured at the base of Screw 2.
c. Tighten Screw 2 (at the corner of the sample) until the load gauge reaches 8. Do
not exceed a load of 9 and keep in mind the repeatability of your experiments may
be affected by the load.
12. Click the Collect Background button (“Col Bkg”) and then OK. If the Preview Data
option is selected in the Experiment Setup window, then you will see single scans of
spectra. To collect the data you have to select “Start Collection” on the top right of the
OMNIC window. Otherwise your background will be collected automatically. You can
watch the progress on the bottom left corner of the OMNIC window.
13. Once it is finished you may add the background to the window by clicking Yes.
14. Now you may put your sample of interest in or on your stage and click Collect Sample
(“Col Smp”).
15. Once the scan is finished click add to window to view the spectra.
16. Repeat steps 14-15 for as many samples you wish. If using ATR, be sure to clean the
stage between each sample. You may need to test a new background periodically and, if
you see significant peaks you will want to repeat the collection of a background.
17. To save samples you have several options:
a. To save singular spectra click the spectra in the display window (when selected it
will turn red). Then click “file” and “save as”. From here you can save it as a
OMNIC spectra (.spa), which will allow you to use the analytical tools in the
OMIC software later, or as a comma separated text file (.csv) to be read with
another program, such as Matlab or Excel.
b. You may also save a group of spectra as a group spectra file (.spg) by using ctrl-w
to select all spectra and then click “file” and “save group”.
18. You may be able to identify your substance by clicking “analyze” and “search”.
Figure 1: Air and Water Valves for the Bioreactor.
19. Turn on PC computer on the west side of the Biolab and sign in (user name: biolab, and
password: biolab). The Opto program is only usable if you log in, on the local machine,
using the biolab account user name: BIO_LAB COMPUTE.
20. Assemble the bioreactor, if it is not already assembled for the type of experiment you
plan to do. Make sure both the dissolved oxygen electrode and pH electrode are
immersed in a distilled water solution or the
aqueous solution you plan to use for fermentation.
I suggest you fill the reactor vessel up to the 1.5
liter mark. Fill the vessel using an open port in top
of the reactor and a funnel and empty the vessel
using the peristaltic pump provided.
21. Switch on the main power to the bioreactor located
on the Power Controller module (see Figure 2).
This turns on everything else (unless someone has
changed the switches on the different units),
including the primary control unit located on top of
the power control unit (see Figure 2).
22. The orange display on the primary control unit
should default to the setup screen on startup (Figure
3). If not, push the Startup Key. The bioreactor can
be setup to run in either a fermentation mode or a
cell culture mode. Make sure the unit you are using Figure 2: Bioreactor Modules.
(the control unit can control up to 4 reactors, we
only have one – Unit 1) is setup for
fermentation mode by pressing the USE key
using the Select Buttons on the setup screen.
Then set the unit to fermentation mode by
pressing the Select Button again on the next
screen. Also, you have the option to select
the language used for the controller. I
suggest you use English.
23. The Main Menu (showing the status of the
bioreactor) is obtained by pushing the Main
Menu Key depicted in Figure 3.
Figure 3: Bioreactor Primary Control Unit
24. Note that on startup (default) the O2 loop on
Control Panel.
the Main Menu is set to Manual mode, while
all the other loops are set to OFF. With our
setup this activates the gas coming from the gas mixer. Set the O2 loop to OFF to turn
off the gas coming from the gas mixer. You can set this back to MANUAL when you are
ready to use the gas mixer and have connected the line from the mixer to the rotameter
(see #10 below).
25. Fill the water jacket with tap water using the temperature control setting (Main Menu) on
the primary control unit by
(a) Setting the temperature control to AUTO
(b) Entering a temperature setpoint that is at least 5 C below the current bioreactor
temperature. The controller will respond to the call for cooling by opening the solenoid
valve, filling the water jacket. The water overflow will go into an overflow tube located
in the water jacket (in the back) and drain to the sink. Flush with at least 2 jacket volumes
of water. You can stop the water from flowing to the drain by setting the setpoint to the
desired bioreactor temperature (typically above the current temperature).
26. Test the Agitator Control by setting the agitator control to AUTO and setting a setpoint
between 0 and 1200 rpm. This is how you control the agitation speed if you are not using
the Gas Mix Controller or the dO2 controller.
27. Gas flow is controlled for fermentation two ways:
a. Manifold Control (Figure 1e) – make sure the connection from the outlet of the
manifold is connected to the inlet of the rotameter (using the quick disconnects).
Each gas is controlled by a separate needle valve located on the manifold. The
flow rate to the bioreactor is controlled by the rotameter located downstream from
the manifold. Typically, the needle valves will be in an ON/OFF situation, adjust
them only to give the maximum flow rate of 1200 CCM (cubic centimeters per
minute) on the rotameter. Use the rotameter to adjust the total volumetric flow to
the bioreactor. Make sure the pressure regulators on the cylinders that you are
using are set to approximately 10 psig.
b. Gas Mix Control – when using this control, the dO2 setting, make sure the output
connection from the gas mixer is connected to the inlet of the rotameter (using the
quick disconnects) and the valves on the individual gas cylinders are open (open
only those you will use). Make sure the pressure regulators on the cylinders are
set to approximately 10 psig.
28. You have four control choices to choose from in this mode: Agitation, Oxygen,
Agitation/Oxygen or None. Each of those is described in the manual, however the
Agitation/Oxygen control is the one used most frequently, since it controls the dissolved
oxygen level by both agitation and the mix of air and oxygen. However, if you want to
keep a constant agitation rate throughout your fermentation, you may want to use the
Oxygen control mode. In the dO2 details screen, press the setup button. From here you
can set the high and low limits for agitation and the gains for process control. Change the
P-Gain and I-Gain ONLY if you understand what you are doing. Now press the Cascade
selector button. From here you can choose one of the four options. Usually, you will
choose the Agitation/Oxygen option but the Oxygen option is also possible.
29. Dissolved Oxygen Probe Calibration – The dO2 sensor is a polarographic probe
manufactured by Ingold (Mettler-Toledo, InPro 6000 series O2 sensor). If the sensor has
been disconnected from the transmitter for longer than 5 minutes it has to be polarized
prior to calibration by connecting it to the transmitter or a polarization module for 6
hours. You should calibrate the dissolved oxygen probe before using it for your
fermentation. Normally, a single point calibration is sufficient. This is done by placing
the probe into a medium of known dissolved oxygen concentration, typically distilled
water at a constant temperature and pressure that is in equilibrium with a sparging gas
(typically air). Note the probe only measures % saturation. The actual dissolved oxygen
concentration has to be obtained from reference tables. Keep in mind that most reference
tables for dissolved oxygen assume atmospheric pressure at sea level. A correction must
be made to those values for pressure at our altitude.
30. To calibrate the dO2 probe choose the dO2 screen from the Main Menu. From here you
can choose the setpoint, choose the upper and lower limits for the display screen (Disp Hi
– Disp Lo), calibrate the probe (Calibrate) and set the process control parameters
(SETUP) such as the deadband, output multiplier, and the P and I gain. Set the control to
AUTO. Next, push the CALIBRATE button. Normally you do not have to set the zero,
but if you want to (dual point calibration), the easiest method is to momentarily
disconnect the O2 probe from the transmitter (no more than 5 minutes) and once the
reading is stable at zero, push the set zero selector button and enter zero. Reconnect the
O2 probe to the transmitter. The Set Span selector is used for the 100% calibration.
Once the probe reaches a steady value, indicating equilibrium with the medium and the
temperature is constant, press the Set Span button and enter 100, indicating the probe
(and medium) is 100% saturated with the sparging gas. Keep in mind that it usually takes
a long time for a solution to get completely 100% saturated.
31. pH Control – Choosing the pH selector button from the Main Menu, brings up the pH
control screen. From here you can choose the setpoint, choose the upper and lower limits
for the display screen (Disp Hi – Disp Lo), calibrate the probe (Calibrate) and set the
process control parameters (SETUP) such as the deadband, output multiplier, and the P
and I gain. Typically, you will use the default parameters for SETUP. You should,
however, calibrate your pH probe before you use it. This is explained in the bioreactor
manual, starting on page 63 (actually page 65 starts on calibration). Note that you have to
physically remove the probe from the bioreactor in order to do the calibration. Be sure to
rinse the probe with distilled water before you place it back into the bioreactor.
pH is controlled by two concentrated solutions of acid and base. Typically, you will use
a sulfuric acid solution and a sodium hydroxide solution, approximately 10% by weight.
DO NOT USE HYDROCLORIC ACID IN THE BIOREACTOR. HCl corrodes
stainless steel. The acid and base solutions are placed in their respective sterile bottles
and connected to the appropriate pumps using the existing plastic tubing. Make sure all
the lines to and from the pumps are filled before connecting the lines to the bioreactor.
Use the triport on top of the reactor for the acid, base and antifoam lines.
32. Once everything is setup, you can choose the setpoint for the pH and the controller will
control the pH in the bioreactor at that setpoint by turning on and off the acid and base
pumps. I suggest this is the last controller you setup.
33. Antifoam Control – the bioreactor is setup for antifoam control. Pumping a concentrated
antifoam solution into the vessel using one of the peristaltic pumps does this. There is a
foam sensor mounted in the headplate of the vessel that senses the presence of foam and
responds by turning on the antifoam pump. Make sure there is ample antifoam in the
addition bottle and that the tubing going to and coming from the pump is completely
filled and connected to the triport on the headplate.
34. Go back to the Main Menu and make sure the readings are reasonable for the variables
you selected. You are now ready to monitor the readings using the computer software.
COMPUTER SOFTWARE AND DATA ACQUISITION
The New Brunswick Scientific software, BIOCOMMAND LITE, is used for data acquisition
and for on-line plots of the various parameters. See the manual for complete details of the
software.
1.
2.
3.
4.
5.
Open BCLite from the Desktop.
In the first window select “Open an Existing Recipe”.
Select File name “Recipe Bio Bob.rcp”.
Go back to desktop
Open the shortcut to OptoDisR.
a. You should see the dO2, Agitation, Temperature, and pH logging.
6. Go back to BioCommand Lite.
7. Click “Start Batch”.
8. Select no, “do not save” changes to recipe.
9. Enter a novel Batch Display Name and then a Destination File Name.
10. After you click OK for the Destination File Name data will being recording.
11. To monitor/change set points go to Views, and double click “Batch Summary.1”. To
change a set point, double click on the line for that controlled (e.g. “Agit”)
12. To monitor trends and history go to Views. Double click on “Trend” under “New
Views” and click “Loops”. Then click “Set up Loop” and select the loop you wish to
monitor (e.g. dO2, pH, or so on). Use the arrow keys to add additional loops to the plot.
13. To add an alarm, go to the Alarm Tab, and choose the controller and set alarm bounds.
14. When finished gathering data, click “End Batch” on the BioCommand software, and click
“Exit Opto” in the OptoDisR software.
15. You can open OptoDisplay results in Excel.
a. Open Excel.
b. Go to file => open.
c. My Computer => C: Drive.
d. Open “Opto Data” folder.
e. Change Files of Type to “All Files”.
f. The logs are saved by date, so find the correct file.
g. When the text import wizard opens, click Next.
h. In Step 2, put a check in comma and hit next.
i. Now select “Finish”.
j. When it is open, columns G & H do not pertain to our reactor.
16. You can later read these files using separate software by New Brunswick, DBViewer32,
and then transfer the file to MS Excel.
a. Open the DBViewer32 program.
b. Select “Open” and find the file name you gave to your experiential run in Step 9.
MEASURING GLUCOSE CONCENTRATIONS
There are several ways to measure glucose concentrations during the fermentation. Two that
are available in our lab are the following:
(1) Sigma Chemical Company’s GLUCOSE TRINDER, [Procedure No. 315]. This kit is
used primary for the quantitative, enzymatic determination of glucose in serum or plasma
using a spectrophotometer at 505 nm and is linear up to 750 mg/dL. See the procedure
written by Sigma for detailed information.
(2) Molecular Probes’ AMPLEX RED GLUCOSE/GLUCOSE OXIDASE ASSAY KIT (A22189). This kit uses a fluorescence microplate reader and is also an enzymatic
determination, but uses excitation I the range of 530-560 nm and fluorescence emission
detection at approximately 590 nm. The reagents in this kit are very sensitive to light and
somewhat air sensitive. Thus, they must be use promptly. See the procedure and product
information sheets from the company for detailed information. This kit is used primarily
(3) HPLC. See the Projects Lab HPLC Page.
MEASURING YEAST CONCENTRATIONS
There are two ways to measure yeast concentrations during fermentation:
(1) Turbidimetric (Optical Density) Method – This is a very simple method that is based
strictly on how turbid the solution is, assuming that the yeast cells are the only
contribution to the turbidity of the solution. Turbidity is measured by absorbance in a
spectrophotometer set at a suggested wavelength of 660 nm. You must develop a
calibration curve (linear works best) for the yeast then compare the unknown sample with
this curve. You may use the Projects Lab’s UV-Vis or Plate Reader.
(2) ATP Assay Kit by BioTrace International, - This kit is based on the measurement of total
adenosine triphosphate as an indicator of the amount of yeast cells. Again, an enzymatic
reaction of d-Luciferin with ATP in the presence of Luciferase is used which produces
light and the light intensity is measured with a luminometer. There are several
information sheets on this method with procedures. Consult those documents.
(3) The Chemometec Cell Counter. See the Projects Lab Cell Counter Page.
SHUTDOWN AND CLEANUP PROCEDURES
1. Turn off the agitator, gas flow, and temperature control.
2. Empty the vessel using the peristaltic pump and then flush/agitate the reactor at least
twice with hot distilled water. Use the agitator to clean the vessel during each flush, but
make sure to turn off the agitator during filling and emptying. You may have to use a
dilute acid solution (sulfuric acid) or a mild detergent solution to get the vessel
completely clean.
3. MAKE SURE THE VESSEL IS FILLED WITH DISTILLED WATER (1.5 LITERS)
AT THE END OF FLUSHING AND CLEANING. The dO2 probe and the pH probe
need to be immersed in water when left overnight. For long-term storage, see the
bioreactor manual.
4. Turn off all the controls on the primary display unit
5. Turn off the main power switch.
6. Turn off the main water valves and the main air valves show, as well as all the valves on
the gas cylinders.
7. Sign off and shut down the computer.