Molecular Cell Physiology José Kiewiet 11/02/2011
1. Chemicals
Composition M MW gram volume (ml) product code
0-pthaldialdehyde C8H6O2 134.13 Sigma P0657
Perchloric acid HClO4 3.5 100.46 Dilute 70% stock solution
1:1 with demi to obtain 35% PCA
Baker 6022
3-mercaptopropionic acid HSCH2CH2CO2H 106.14 Sigma M5801
Boric acid (B14) H3BO3 61.83 Sigma B7901
Sodium hydroxide (N26) NaOH 40.00 Sigma 480878
Sodium azide (@N07) NaN3 65.01 Sigma 438456
Sodium tetraborate decahydrate (N50) Na2B4O7. 10 H2O 381.37 Sigma S9640 DL-Norvaline C5H11NO2 117.15 Sigma N7502 Acetonitrile CH3CN 41.05 Methanol CH3OH 32.04 Sigma 34860 Disodium hydrogen phosphate (N25) Na2HPO4 141.96
Security Guard Cartridges kit Phenomenex, KJO4282 ZORBAX Eclipse plus C18, Solvent saver plus 3.0x150 mm 3.5 micron VWR (Agilent)
959963-302
Crimp top vial, clear VWR, 548-1134
Vial snap ring, 1.5 ml VWR, 548-0011
Snap cap ND11, Silicone withe/PTFE red VWR, 548-0016
Preparation 0.1 M Borate buffer
1. Dissolve 1.2 gram boric acid in 100 ml of demi
2. Dissolve 0.8 gram sodium hydroxide in 100 ml of demi
3. Mix both solutions under stirring and adjust the pH to 10.2 with 5 M HCL
Preparation OPA reagent
1. Dissolve 25 mg of OPA in 500 µl methanol by pipetting 2. Add 4.5 ml of 0.1 M Borate buffer
3. Add 21 µl 3-Mercaptopropionic acid (work in fume hood) 4. Mix the solution well and filter it through a 0.25 µm PES filter
Preparation eluent A (1 liter)
1. Dissolve 1.42 gram of Na2HPO4 and 3.81 gram of Na2B4O7. 10 H2O in 950 ml of demi
2. Add 325 mg of sodium azide
3. Adjust the pH to 8.2 and fill up to a volume of 1 litre
Preparation eluent B (1 liter)
1. Mix the following liquids together (work in fume hood): 450 ml methanol
450 ml acetonnitrile 100 ml milliQ
Molecular Cell Physiology José Kiewiet 11/02/2011
2. Sample preparation
Sample preparation
1. Filter supernatant/sample through Millipore HPLC filters (Millex-HV 0.45µm PVDF 4mm non-ster 100/Pk, cat.No. SLHVR04NL).
2. Add to a 1.5 ml glass vial:
MilliQ 875 µl
0.1 M Borate buffer 25 µl 1 mM (internal standard, e.g. Norvaline) 25 µl
Sample/standard 25 µl +
Total 950 µl
3. Close the vials with caps (Omnilabo cat.no. 261763). 4. Remove air bubbles.
5. Put the follow into the HPLC rack #7 the right order: blanc, standards/samples, after every 8-10 standards/samples a vial with 10% MeOH (for rinsing the column between samples) and end with a blanc (for the shutdown method).
3. Procedure
Start up
1. Check if there is enough eluent A and B.
2. Make fresh OPA and put it in a reagent vial in position 1. Put in position 2 a reagent vial with 10% MeOH.
3. When the machine is running on 30% MeOH switch the tubes to eluent A and B and purge the system (open the pump valves and press “purge” button on the pump). 4. Close the pump valves when the purging is finished.
5. Open classVP software and select “instrument 1”.
6. Open the wanted method (e.g. AminoAcid_001.met): File Method Open
7. Start the machine by selecting: Method Instrument setup. Change the flow rate to 0.1 ml/min and press download (now the machine start pumping, heating etc.). Check if the pumps are working, make sure that the button “act” (main controller) and the buttons “pump” (pumps) are selected.
8. Increase the flow rate after 30-60 minutes to 0,3 ml/min and after 60-90 minutes to 0,64 ml/min (watch the pressure).
9. Press the “zero” button on the fluorescence detector.
Run procedure
1. Make a new sequence file by selecting: File Sequence New
2. Fill in the sample name under “Sample ID” and “Datafile”, end with (001) (e.g. 101202 JK (001))
3. Under “Data Path” select the appropriate folder to store the acquired data. 4. Under “Method” select the wanted method (e.g. AminoAcid_001.met).
5. Under “Autosampler” select the wanted pretreatment file (e.g. Pretreat_OPA.ape). 6. Fill in the start and end vial of your samples starting with zero as loaded in the rack #7. 7. Injection volume should be 5 µl and press the button OK.
8. Change in the sequence: (1) the methods for the 10% MeOH vials (MeOH_Rinse.met) and for the blanc at the end of the sequence (Shutdown.met). (2) The pretreatment files under “autosampler” for the 10% MeOH vials (Pretreat_MeOH.ape) and no
Molecular Cell Physiology José Kiewiet 11/02/2011
9. Change the export folder by selecting: Method Advanced Path for export files Change the path to your appropriate folder in the export folder. Select wanted export parameters.
10. Save the sequence file: File Sequence Save as
11. Start the sequence run by selecting: Control Sequence Run. Select the right sequence file and press start.
12. When asked to save changed method, press NO.
Shutdown procedure
In case the sequence file is not including a shutdown method:
1. Select shutdown method: File Method Open (e.g. filename: shutdown.met). 2. Select: Method Intsrument Setup and press the download button. The display on the
HPLC pump should now show a flow rate of 0.025 ml/min. In case the machine will not be used for a long time:
3. Switch from eluent A and B to 30% MeOH and run the shutdown method.
4. HPLC setup
Shimadzu HPLC system consists of:
Module Type Serial number Version
Software ClassVP v6.2
Molecular Cell Physiology José Kiewiet 11/02/2011
Pump A LC-20AD L20104473038 1.07
Pump B LC-20AD L20104473039 1.07
Autosampler SIL-10ADvp C21053970957 5.32
Oven CTO-10ASvp
Fluorescene detector RF-10AXL
System controller SCL-10Avp C21014071741 5.42
Configuration of Shimadzu HPLC system:
Det A On
Sound alarm error On Enable status log On External start All runs
Pressure units MPa
Auto injector online On Fraction collector On Injection trigger On System Controller (SCL-10Avp) communication RS232C, COM1 Sample rack 7 Rinsing volume 100 µl Needle stroke 52 mm
Rinsing speed 35 µl/sec Sampling speed 15 µl/sec
MTP tray single
Cooler temp 4 °C
Purge time 25 min
Rinse mode No rinsing
Rinse dip time 0 sec Number of injection 5000 Autosampler (SIL-10ADvp)
Number of valve rotations 20000 Pump A (LC-20AD) Total delivered volume (L&R) 60 liter Pump B (LC-20AD) Total delivered volume (L&R) 60 liter Acquisition source Digital
Y-axis unit Volts
X-axis multiplier 1e-006
Base period 100 msec
Detector port A
Fluorescence detector (RF-10AXL)
Xe operating time 500hr
5. Methods description
Amino acids method
This method describes all the settings necessary for performing the amino acids analyses. The settings for the components can be accessed within the ClassVP software in the menu item “instrument setup”.
Filename: AminoAcid_001.met (ClassVP methods AminoAcid_001.met)
Mode Binary gradient
T flow 0.64 ml/min B conc 2% B curve 0 P. max 25 Mpa P. min 0 Mpa Pumps (LC-20AD) Lamp on Yes
Molecular Cell Physiology José Kiewiet 11/02/2011 Ex. wavelength 350 nm Em. wavelength 450 nm Sensitivity gain X4 Sensitivity Low Sensitivity 3 Spectrum type Em Start em 400nm Stop em 500nm
Scan speed Fast
Acquisition channel on Yes Sampling frequency 3.3333 Hz
Run time 30 min
Fluorescence detector (RF-10AXL)
Acquisition delay 0 min Trigger type External System Controller
(SCL-10Avp) Power on Yes
Display pump A Yes Status log
Display pump B Yes
Temperature 40 °C
Oven (CTO-10ASvp)
Temperature max. 85 °C
Time program
# Time (min) Module Event Value
1 0.01 SCL-10Avp Start 0 2 0.50 Pumps B. conc 2 3 30.00 Pumps B. conc 57 4 30.10 Pumps B. conc 95 5 33.00 Pumps B. conc 95 6 33.10 Pumps B. conc 2 7 38.00 Pumps B. conc 2 8 38.10 SCL-10Avp Stop 0 Shutdown method
A sequence run is always ended with one water sample. This is for initiating the shutdown method. This method has the purpose to lower the flow rate, shutting down the oven and to switch of the lamp of the fluorescence detector. This is the standby mode and saves eluent, increases the lifespan of the lamp and of the column. Only the differences from amino acids method are mentioned below:
Filename: Shutdown.met (ClassVP methods Shutdown.met)
mode Binary gradient
T flow 0.05 ml/min
Pumps
B conc 50%
Lamp on no
Acquisition channel on yes Sampling frequency 3.3333 Hz Fluorescence detector
Run time 1 min
Temperature 0
Oven
Temperature max. 85 °C
Autosampler pre-treatment files
Molecular Cell Physiology José Kiewiet 11/02/2011
Filename: Pre-treatment_AA.ape (ClassVP methods Pre-treatment_AA.ape)
Mode Reagent
Source vial Reagent vial 1
Volume 50 µl
Mixing count 1
Mixing volume 100 µl
Bubbling Yes
Wait time 1 minute
Rinsing pre-treatment for the needle
Filename: MeOH_Rinse.ape (ClassVP methods MeOH_Rinse.ape)
Mode Reagent
Source vial Reagent vial 2
Volume 50 µl
Mixing count 1
Mixing volume 100 µl
Bubbling Yes
