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12/2/11 R.C
Equipment Overview:
This document outlines the basic procedure for using the Sedex light
scattering detector and the PE series 200 HPLC system to detect sugars. To
use this system to detect and analyze other compounds consult the lab
manager.
This system is comprised of the following components:
Series 200 pump, UV/VIS detector, SEDEX Light Scattering Detector,
Vacuum Degasser, Column Heater, Phenomenex Luna 5 micron NH2
column.
The column is operated in reverse phase using a mixture (mobile phase) of
80% acetonitrile and 20% water. The UV/VIS detector is not normally used
to detect sugars.
Safety:
Acetonitrile is highly toxic. Gloves and safety glasses must be worn
while operating this equipment.
Prior to operating this equipment please read the MSDS for
acetonitrile located in this document.
Filling or emptying the mobile phase bottles must be done in the fume hood.
Pre-Operation Checklist
Safety Glasses and gloves must be worn when operating this equipment.
Before operating this equipment review all safety data specific to the chemicals you
will be using. M.S.D.S documents for acetonitrile and methanol are located in the back
of this book.
Prepare samples and reagents before powering up the HPLC.
All samples must be labeled with content, date and owner information.
Select the 25 micro liter HPLC injection syringe and 0.45 micron filter located in the
HPLC supplies container.
Check in with lab personnel to review operating procedure before operating this
equipment.
Make certain the mobile phase bottles (A = water), (B = ACN, acetonitrile)
are at least half full, if they are less than half full contact the lab manager.
Filter all samples through a 0.45 micron syringe filter prior to injecting the sample into
the HPLC column. Note, the injection loop volume = 20 micro liters, this is the
maximum injection volume.
Do not adjust the front panel controls on the SEDEX detector.
Do not modify this equipment without prior approval from laboratory staff.
Startup:
1. Turn on the computer and log on as posted on the computer monitor:
2. Apply power to the HPLC system in the order listed below:
A) Turn on power strip located just to the right of the feed bottles.
This applies power to the column heater and the vacuum degasser.
B) Turn on the series 200 UV/VIS detector; the switch is located on the back of
the unit above the power cord.
C) Turn on the series 200 pump; the switch is located on the right side of
the pump.
D) Turn on the Sedex 55 detector; the switch is located on the right rear of
the unit.
E)
Allow the column heater to run for 10 minutes before injecting samples.
F)
Make sure the mobile phase waste container is secured next to the SEDEX.
G) Verify that the lights on the dot Link box - located on top of the NCI 900 box
To the right of the series 200 pump/detector are displaying green LEDs for
the A, B and F locations. If this is not the case consult the lab manager.
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In the instruments window select SEDEX then click on the RUN icon located
in the lower center of the screen. Select CLEAR SETUP if it is not grayed
out, if it is, proceed to the next step and confirm the clear setup command. If
the setup is cleared proceed as follows. Wait until the Status window,
located on the upper left hand of the display, indicates the setup has been
cleared; the setup window should display SEDEX No Method.
B)
C)
After clearing the SETUP click OK to close the Setup Instrument Window.
Note the status window will momentarily display SEDEX Ready. If this
message is not displayed contact the lab manager.
D)
E)
defines the number of samples you can run before you must start the
software again. Do not change these values!
F)
Select the RAW DATA path folder and navigate to the location of your
RAW data folder the one you created earlier - for the Sedex data. Click
OK. This path should now be displayed in the raw file path dialog window.
G)
Select the RESULTS DATA path folder and navigate to the location of your
RESULTS data folder for the Sedex data. Click OK. This path should now
be displayed in the results file path dialog window.
H)
Click OK. You have just defined the locations for your RAW and
RESULTS data.
I)
Observe the Status window in the upper left had corner of the display, it
should display a setting up message then, after a slight delay SEDEX ready
will be displayed.
8.
9.
Filter all samples through a 0.45 micron syringe filter prior to injecting the sample into the
HPLC column. This will prevent clogging the column. If the column becomes clogged it is
unlikely that it can be cleared. A new column will be required at a cost in excess of $600.00.
Prior to injecting samples you must flush out the column by running a blank sample.
A blank sample should be run each day before starting your sample runs.
10.
Running a Blank:
Follow the sample injection procedure listed below but do not inject
anything into the sample loop.
When running a blank sample, allow the system to run for the full 20 minutes.
Running a blank will help limit the number of false peaks.
11.
Sample Filtration:
Using a Syringe filter sample the yeast material through a 0.45 micron syringe
filter into a 3 - 10 ml syringe. Then inject the filtered yeast solution into a clean, small
beaker.
You will fill the HPLC injection syringe from this beaker. This procedure insures that all
solution injected into the HPLC has been filtered.
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Verify that the injection valve on the series 200 pump is rotated counter
clockwise to the eight oclock position
C)
D)
E)
F)
G)
Insert the needle on the injection syringe into the injection port as
illustrated below.
H)
I)
Rotate the injection valve on the series 200 pump to the right until it stops
at approximately the 10 oclock position. Rotating the injection valve to
the right will start the run.
Wait 30 seconds to allow the sample to flow through the sample loop then turn
the injection valve back to the left until it stops at the 8 oclock position. You will
notice the preset flow rate displayed on the pump module reads 2.00 mL/min.
The run time is preset to 20 minutes. At the end of the 20 minute run the pump
will automatically reduce the flow rate from 2 ml/min to 0.5 ml/min.
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12.
13.
14.
15.
A)
Confirm that the injection valve is rotated all the way to the left.
B)
C)
D)
E)
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16.
B)
Verify that your sample has been properly filtered as outlined in step 11.
C)
Fill the injection syringe with the sample. Remember that 20 microliters is
the maximum injection volume.
D)
E)
F)
Repeat these steps for up to 30 injections. If you require more that 30 runs
in a single session you will be required to restart the software from step 4.
17.
Viewing Chromatograms:
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19.
Printing Chromatograms:
Documents print to the Starley HP-4000
Select the print function from the top menu. The data is sent to the printer located
in the computing lab adjacent to the senior lab.
Copy your data to a flash drive after each session.
20.
Shut Down:
A)
Close main cylinder valve on Nitrogen Tank
B)
C)
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D)
E)
F)
Turn off the power to the pump, SEDEX detector and UV/VIS detector.
G)
Turn off power strip located just to the right of the feed bottles.
This applies power to the column heater and the vacuum degasser.
H)
Cleanup work area, label and properly store samples and mobile phase.
I)
Troubleshooting Tips:
1. PE Series 200 status reads No Method, cannot start the pump.
Try selecting the PE Series 200 in the Instruments window then open the RUN
window, select Take Control. Wait for the seizing message to clear and then open the
Hands On dialog box and select start pump.
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