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SOP: HPLC-chiral-1.2
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1 Responsibilities.
First person responsible: Marille Delville.
Second person responsible: Helene I.V. Amatdjais-Groenen.
2 Definitions.
Read the information on the Instrumentation page of the website of the Bio-organic Chemistry department:
http://www.ru.nl/bio-orgchem/instrumentation/chromatography/hplc/
3 Starting the chiral HPLC and logging into LC Real Time Analysis.
1.
Check solvent amounts of isopropanol and heptane.Make sure the solvent level is above the MINIMUM
level. Otherwise, fill to MAXIMUM level using new bottle HPLC grade heptane or IPA p.a. grade solvent.
For heptane ask Marille Delville.
2.
Switch computer & monitor on; start the program LabSolutions LCsolution (screen on picture 1 appears).
3.
4.
5.
A screen like picture 3 will appear and you will hear a beep as a confirmation of the communication
between the computer and the HPLC.
6.
Copy and paste a general method you want to use from the methods folder to your own folder. Now it is
possible to change this method.
7.
Organic Chemistry
SOP: HPLC-chiral-1.2
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Run time is
40 minutes
Picture 4: Total Run time.
Detector A
Fill in wavelength(s).
Check if lamp is ON.
Controller
Auto sampler
Check values (picture 6).
Auto purge
Check if degasser is selected
In this example:
B = 10%
Organic Chemistry
SOP: HPLC-chiral-1.2
Page 3 of 5
Increase flow to 1 mL/min in steps of 0.2 mL/min (tab Pump in the Instruments
Parameters View).
2.
Click on
the DOWNLOAD button.
All the parameters are transferred to the HPLC-instrument.
3.
Check the actual parameters at the display on the right side of the screen (picture 7):
Flow, B. Conc. (solvent composition), Pump Pressure, Oven Temperature.
4.
5.
Sample treatment
Step 1. Dissolve 1 mg sample into the eluent (heptane, IPA or mixture)
Step 2. Filter the solution over a membrane filter into a clean screw-cap sample vial.
Use a screw-cap with a red septum (red side pointing to the solvent) to close the vial.
Picture 7:
Actual parameters.
2.
Press Data Acquisition icon on the navigation panel at the left (blue) side of the screen (picture
8)
3.
Press Single Start button on the navigation panel. A new window (picture 9) will appear.
Sample Name
Sample ID
4.
Make sure all the data in step 4 are correct and push the OK button. This will start the measurement.
Picture 8:
Navigation
panel.
Picture 9: Single Run parameters.
*C:\Labsolutions\Data\Yourfolder\Methods\Methodname.lcm
Organic Chemistry
SOP: HPLC-chiral-1.2
5.
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2.
3.
(Batch Start icon) to start the batch (green arrow and box, picture 10).
7 Shutdown procedure.
Manual shut-down procedure:
1.
Decrease the flow in steps of 0.2 mL/min to 0.00 mL/min in the actual parameters screen (picture 7).
2.
Set the UV-detector in tab Detector A of the Instrument Parameter View to Off.
3.
2.
Organic Chemistry
SOP: HPLC-chiral-1.2
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3.
4.
5.
Fill in your Batch file and your last sample must be the shut-down sample (picture 13).
6.
7.
8.
9.