Protocol: HPLC (amino acids)

Transcription

1 Page 1 of 6 1. Chemicals Composition M MW gram volume (ml) product code 0-pthaldialdehyde C 8H 6O Sigma P0657 Perchloric acid HClO Dilute 70% stock solution 1:1 with demi to obtain 35% PCA Baker mercaptopropionic acid HSCH 2CH 2CO 2H Sigma M5801 Boric acid (B14) H 3BO Sigma B7901 Sodium hydroxide (N26) NaOH Sigma Sodium azide (@N07) NaN Sigma Sodium tetraborate decahydrate (N50) Na 2B 4O H 2O Sigma S9640 DL-Norvaline C 5H 11NO Sigma N7502 Acetonitrile CH 3CN Methanol CH 3OH Sigma Disodium hydrogen phosphate (N25) Na 2 HPO Security Guard Cartridges kit ZORBAX Eclipse plus C18, Solvent saver plus 3.0x150 mm 3.5 micron Phenomenex, KJO4282 VWR (Agilent) Crimp top vial, clear VWR, Vial snap ring, 1.5 ml VWR, Snap cap ND11, Silicone withe/ptfe red VWR, 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 Na 2 HPO 4 and 3.81 gram of Na 2B 4O H 2O 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

2 Page 2 of 6 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 ). 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 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 minutes to 0,3 ml/min and after 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 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 pretreatment for the blanc at the end of the sequence.

3 Page 3 of 6 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 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 Degasser DGU-14A

4 Page 4 of 6 Pump A LC-20AD L Pump B LC-20AD L Autosampler SIL-10ADvp C Oven CTO-10ASvp Fluorescene detector RF-10AXL System controller SCL-10Avp C Configuration of Shimadzu HPLC system: System Controller Det A (SCL-10Avp) Sound alarm error Enable status log External start All runs Pressure units MPa Auto injector online Fraction collector Injection trigger communication RS232C, COM1 Autosampler (SIL-10ADvp) 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 Number of valve rotations Pump A (LC-20AD) Total delivered volume (L&R) 60 liter Pump B (LC-20AD) Total delivered volume (L&R) 60 liter Fluorescence detector Acquisition source Digital (RF-10AXL) Y-axis unit Volts X-axis multiplier 1e-006 Base period 100 msec Detector port A 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) Pumps (LC-20AD) Mode Binary gradient T flow 0.64 ml/min B conc 2% B curve 0 P. max 25 Mpa P. min 0 Mpa Lamp on

5 Page 5 of 6 Fluorescence detector Ex. wavelength 350 nm (RF-10AXL) 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 Sampling frequency Hz Run time 30 min Acquisition delay 0 min System Controller Trigger type External (SCL-10Avp) Power on Status log Display pump A Display pump B Oven (CTO-10ASvp) Temperature 40 C Temperature max. 85 C Time program # Time (min) Module Event Value SCL-10Avp Start Pumps B. conc Pumps B. conc Pumps B. conc Pumps B. conc Pumps B. conc Pumps B. conc 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. ly the differences from amino acids method are mentioned below: Filename: Shutdown.met (ClassVP methods Shutdown.met) Pumps Fluorescence detector Oven mode Binary gradient T flow 0.05 ml/min B conc 50% Lamp on no Acquisition channel on yes Sampling frequency Hz Run time 1 min Temperature 0 Temperature max. 85 C Autosampler pre-treatment files OPA derivation pre-treatment of the samples

6 Page 6 of 6 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 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 Wait time 1 minute