Operating Manual. High Performance Liquid Chromatograph. Scientific Equipment Center, Prince Of Songkla University

Operating Manual High Performance Liquid Chromatograph Agilent 1100 ; VWD, DAD, FLD and RID Scientific Equipment Center, Prince Of Songkla University

Operating Manual High Performance Liquid Chromatograph Agilent 1100 ; VWD, DAD, FLD and RID Editor : Songsuda Promthong Scientific Equipment Center Prince of Songkla University Hatyai, Songkhla.

Contents List Page 1. Preparation and Setting of HPLC.. 1 2. Power on and Login ChemStation software.. 2 3. The Priming of HPLC system. 3 4. Installation of the Column. 5 5. Setting Control value of Pump. 6 1. Isocratic Elution. 6 2. Gradient Elution 6 6. Setting Control value of Injection. 7 7. Setting Control value of Column temperature. 7 8. Setting Control value of Detector... 8 1. Setting Control value of VWD. 8 2. Setting Control value of DAD.. 8 3. Setting Control value of FLD.. 9 4. Setting Control value of RID. 9 9. Setting Signal from Online plot.. 11 10. Save as method.... 12 11. Download the saved Method.... 12 12. Sample analysis.. 13 1. Single Run.... 13 2. Sequence Run.. 14 13. To manage the Data file. 18 1. Download wanted chromatogram to analysis... 18 2. Integration data file... 19 3. Create Calibration curve... 22 4. Change unit of Concentration... 24 5. To Calculation of sample... 24 6. Adjust scale of Report.. 25 7. Result and Print.... 26 8. Batch... 26 14. Shut down HPLC... 28 15. Appendix.. 30 1. Sample filtration..... 30 2. Mobile Phase filtration.. 32

HPLC : Agilent 1100 1. Preparation and Setting of HPLC 1. Place mobile phase (HPLC grade solvents) filtered with membrane on solvent cabinet and put solvent line into mobile phase bottle then seal it. (see the mobile phase filtration and sample filtration at Appendix) 2. Connect the capillary tube line to used detector that following the direction below. 2.1 Open the cover of detector by pressing button and put it out. 2.2 Connect the capillary tube line that connected with the column thermostat to used detector at inlet port then screw it tightly for preventing leak. 2.3 Connect the capillary tube line into waste bottle at outlet port then screw it tightly for preventing leak. 2.4 Close the cover of detector. 3. Check the waste bottle, if the waste bottle nearly full, change a new waste bottle. 4. Continue follow article 2. 1

2. Power on and Login ChemStation software : follow the direction below. Power on and login Chemstation software must power on computer, HPLC instrument and Chemstation software, respectively. 1. Power on computer and (for HPLC1 select Customer) type password, then wait until show desktop and CAG Bootup sever occur. 2. Press switch on button of HPLC instrument on the left below of all modules such as Degasser, Quaternary pump, Auto sampler, Column Thermostat, DAD and used Detector. 3. Wait until initialization of HPLC instrument is complete, see the light on the right of Autosampler turn off (about 1 minute). 4. Login ChemStation Software by double click icon Instrument 1 Online. 5. If you choose detector similar to the last user, the window Instrument 1 (online) Method & Run Control will occur following the picture below. 6. If you choose detector different from the last user, the window of Configuration will occur following the picture below. 2

6.1 Check modules on the right in the consist of Quaternary pump, Auto sampler, Column Thermostat and Detector match with using (they are green color). 6.2 If modules are not complete or not correct, choose modules on the left from the by clicking chosen modules (green color) then click for automatic moving modules to the right in the, and if the consist of unused modules (gray color or green color), click unused module then click for automatic moving unused module to the left in the. Click when modules that you want to use are correct. 6.3 The window Instrument 1 (online) Method & Run Control will occur the same article 5. 7. Continue follow article 3. 3. The Priming of HPLC system. : follow the direction below. 1. At pump module open purge valve by turning a quarter anticlockwise and the flow channel to waste. (see picture below). 2. Click the solvent bottle then select Solvent bottle filling. The window of Solvent bottle filling will occur (see picture below). 3

2.1 Fill volume value of mobile phase at current volume in (A, B, C or D) Actual Volume. 2.2 Fill volume value of total bottle in (A, B, C or D) Total Volume. 2.3 Click on Prevent analysis if level falls below and fill minimum volume value. 2.4 Click on Turn pump off if running out of solvent. 2.5 Click to exit window Solvent bottle filling. 3. Click button at window of Instrument 1 (online) Method & Run Control to power on system of modules. 4. Priming system by at (picture) click 1 time then choose Set up pump. The window of Set up pump will occur following picture below. 2 4

4.1 Part of Solvent, fill the name of mobile phase by matching with bottle and line (A, B, C and D). 4.2 Purge bubble only used line, For example If you want to use line A, you type 0% behind of line B, C and D or click button 1 time for turn off it and line A is automatic 100%. If you want to use line B,C or D and the behind are, click button 1 time for turn on them and type 100 % behind of line B, C or D, line A is automatic 0 %. 4.3 Part of Control, at adjust flow rate to 0.5 ml/min then click. 4.4 Repeat 4 and 4.3 to adjust flow rate by adding flow 0.5 ml/min per time until flow rate is3 ml/min. Purge until that no more bubble in used line. 4.5 Repeat 4 for showing the window of Set up pump. Adjust composition of mobile phase at used line. For example, line A 60% and line B 40% then click and purge continue for 5-10 minutes or until sure that no more bubble. 4.6 Repeat 4 for showing the window of Set up pump. Adjust flow rate by reducing flow 0.5 ml/min per time until flow rate is 0.5 ml/min. 5. At pump module close purge valve by turning a clockwise and the flow channel to HPLC system (see picture below). 6 Disconnect capillary tube line both of side (from autosampler and to detector) that connected with column thermostat. 7 Continue follow article 4. 4. Installation of the Column. : follow the direction below. 1. Remove the cover of column thermostat by pressing button then push it out (see picture below). 2. Connect capillary tube line to column at inlet (line from autosampler) and connect capillary tube line to column at outlet that connect to used detector (see picture below). in out to 3. Place column at column thermostat and lock column with column clip then close the cover of column thermostat. 4. Continue follow article 5. 5

5. Setting Control value of Pump. : follow the direction below. 1. Isocratic Elution. (composition of mobile phase constant) 1. Click at then choose Set up pump for showing window of Set up pump. 2. Adjust flow rate by adding of flow 0.1-0.2 ml/min per time until used flow rate. For adjusting flow rate each time must be wait pressure steady then adjust the next flow rate. 3. Setting value of parameter in the window of Set up pump. Pressure Limit: Max (maximum pressure) Recommend 400 bar or depend on certification of column. Min (minimum pressure) setting value is 0 bar Stop Time: Time for running each inject. (Setting 0.0-99999 minutes or no limit depend on your work). Post time: Delay time before the next inject. (If you don t want to set, you choose off). 4. Continue follow article 6. 2. Gradient Elution (Mobile Phase composition is changed during the separation). 1. Follow article 1-3 at choice Isocratic Elution 2. At the window of Set up pump part of Timetable to setting the gradient program 2.1 Click at 1 time for add 1 line; add cover line for gradient (see picture below.) 6

2.2 Setting value of parameter in the Timetable. - At channel Time : set time. - At channel %B, %C, %D : set composition of Mobile Phase. - At channel Flow : set Flow rate. - At channel Max. Press. : set Max Pressure of the column. 2.3 Click at for exit the window of Set up pump. 3. Continue follow article 6. 6. Setting Control value of Injection. : follow the direction below. 1. Set injection volume by clicking 1 time then choose Set up injector and the window of Set up injector will occur (see picture below). 2. Choose injection method and set appropriate volume of injection (unit µl) in column of Injection Volume. The injection method have 3 choices. Standard Injection: for simple injection. Injecttion with Needle Wash: to needle wash after inject sample and you must specify position of wash vial. Use Injector Program: for Sample Pretreatment. 3. When you process follow article 6.2 then click to exit the window of Set up injector. 4. Continue follow article 7. 7. Setting Control value of Column temperature. : follow the direction below. 1. Set column temperature by clicking 1 time then choose Set up Column Thermostat and the window of Column thermostat Method will occur. 7

2. Set used column temperature by choose and at channel fill temperature value. If don t use column temperature, set temp = 25 C. 3. No change in other values then click to exit the window of Column thermostat Method. 4. Continue follow article 8. 8. Setting Control value of Detector. : follow the direction below. 8.1 Setting Control value of VWD 1. Click 1 time then choose Set up VWD signal the window of VWD Signal will occur. 2. At part of Signal > wavelength : Setting your Wavelength ( 190-650 nm ). 3. No changes in other values then click to exit the window of VWD signal. 4. Continue follow article 9. 8.2 Setting Control value of DAD 1. Click 1 time then choose Set up DAD signal the window of DAD signal will occur (see picture below). 8

2. Setting the value of wanted wavelength (from 190-850 nm and can select 5 wavelength for your sample (A,B,C,D,E)) 2.1 Part of Signal : click behind A or B or C or D or E for record your wanted wavelength. 2.2 Setting the value of used wavelength at Sample, Bw and Reference, Bw and if don t use Reference, Bw, click 1 time for off the Reference, Bw. 2.3 For store spectrum part of Spectrum > Store click and choose All in peak 2.4 Part of Spectrum > Range : setting value 190 to 850 nm. 3. No changes in other values then click to exit the window of DAD signal. 4. Continue follow article 9. 8.3 Setting Control value of FLD 1. Click 1 time then choose Set up FLD signal and the window of FLD Signal will occur. (see picture below). 2. Part of Signal: setting the value of Excitation and Emission. 3. No changes in other values then click to exit the window of FLD signal. 4. Continue follow article 9. 8.4 Setting Control value of RID 1. Click 1 time then choose Set up RID signal the window of RID Signal will occur. 9

2. Choose Polarity to. 3. No changes in other values then click to exit the window of RID Signal. 4. Click 1 time then choose Control the window of RID Control will occur. 5. Click on Purge reference cell and set time at at least 30-50 minutes to purge reference cell. 6. No changes in other values then click to exit the window of RID Control. 7. Continue follow article 9. 10

9. Setting of Signal from Online plot 1. At the window of Online plot click. then the window of Edit signal plot will occur. (see picture below) 2. Choose signal on the left from Available Signals by clicking chosen signal then click for automatic moving signal to the right in the Selected Signals. 3. And if the Selected Signals compose unused signal, click unused signal then click for automatic moving unused signal to the left in the Available Signals. 4. At the Selected Signals, highlight wanted signal by clicking signal then set value... Setting of x-axis range. Click at draw zero line. Setting of y-axis range. Setting of offset. If you want to auto adjust y-axis, click at auto y adjust. 5. Click. 6. Click to exit the window of Edit signal plot. 7. At the window of Online Plot, click at wanted signal occur scale of wanted signal. 8. Continue follow article 10. 11

10. Save as method: There are 2 ways. : follow the direction below. 1. At Menu bar choose File > Save as > method..(name of saved method is.m) 2. At Menu bar choose Method > Save method as ( name of saved method is.m) The window of Instrument1 (online): Method & Run Control will show the status of instrument. Instrument is not ready show status. (red color). Instrument is ready show status. (green color) Before beginning the analysis, the column must be equilibrated at least 30 minutes 1 hour, parameters setting are complete, baseline of detector is stable and Instrument is ready show status. 3. Continue follow article 12 (skip article 11). 11. Download the saved Method: There are 3 ways. : follow the direction below. 1. At Menu bar choose File > Load > Method then choose the saved method that you wanted. 2. At Menu bar choose Method > Load method then choose the saved method that you wanted. 3. Click icon then choose the saved method that you wanted. 4. When download saved method then process continue follow article 3-10 5. Then continue follow article 12. 12

12. Sample analysis: There are 2 ways. 12.1 Single Run. : follow the direction below. 1. At Menu bar choose Run Control > Sample info then the window of Sample Info will occur. 2. Fill data and detail in the window of Sample info Operator name Data file: There are 2 ways.. - Prefix/Counter: fill alphabet or number in channel of Prefix and fill number in channel of Counter by character sum of Prefix and Counter not exceeding 8 characters and automatic run data file for the next injection. (This way is recommended) - Manual: fill the name of data file by character not exceeding 8 characters all injection. Subdirectory: To create the subdirectory (Folder) for recording the data file. Location: Set position of sample vial. Sample name: Fill sample name. Sample Amount: Set zero 0. ISTD Amount: Set zero 0. Multiplier: Set = 1 13

Dilution: if sample isn t diluted, set = 1 or if sample is diluted, set value of dilution sample. Comment: fill comment detail of sample or condition of HPLC or blank. 3. Click to exit the window of Sample Info. 4. Click at. 5. Place sample vial on auto sampler at the setting position then command to run by clicking at menu bar then choose RunControl > RunMethod Or click button Or click at the window of Instrument1 (Online) Method & Run Control. from the window of Sample info. 6. Instrument will ready to run in progress and show status.(blue color) 7. Wait until the finished acquisition and status change to. 8. The next injection, repeat the sub article from1-7 of the article 12.1. 9. Continue follow article 13. 12.2 Sequence Run. : follow the direction below. 1. At Menu bar choose Sequence > New Sequence 2. At Menu bar choose Sequence > Sequence Parameters then the window of Sequence Parameters will occur. 14

3. Fill data and detail in the window of Sequence Parameters Operator name Data file: There are 2 ways - Prefix/Counter: fill alphabet or number in channel of Prefix and fill number in channel of Counter by character sum of Prefix and Counter not exceeding 8 character and automatic run data file for the next injection. (This way is recommended) - Auto Subdirectory: To create the subdirectory (Folder) for recording the data file. Part of methods to run: Choose According to Runtime Checklist. Wait time: Set zero 0. Shutdown: Select Command macro when the sequence run finished. - If you want to set command macro, you will click Post-sequence Cmd / Macro then chooses type of command, there are 4 types 1 STANDBY: when finished sequence the instrument go to standby mode. 2 LAMPALL OFF: when finished sequence, only lamp of detector turn off. 3 PUMPALL OFF: when finished sequence, only pump turn off. 4 macro SHUTDOWN.MAC,go : when finished sequence, every modules of HPLC shutdown. 15

- If you don t want to use command macro, you will click 1 time at Post-sequence Cmd / Macro to remove from Post-sequence Cmd / Macro. Sequence comment fills comment detail of sample or condition of HPLC or blank. 4. Click to exit the window of Sequence Parameters 5. At Menu bar choose Sequence > Sequence Table then the window of Sequence Table will occur. 6. Fill detail in the window of Sequence Table 1. Click to add new line to set the sequence of injection. 2. At Location: set position of vial. 3. At Sample Name: type sample name. 4. At Method: choose use method for injection. 5. At Inj/Location: set number for repeated sample injection. 6. At Sample Type: choose type of sample that compose3 types.. - Sample - Control Sample - Calibration (for standard) : if you choose calibration, you must set Cal Level: set level of calibration. Update RF: when recalibration, set Response Factor by choose No update or Replace or Average or Bracket. Update RT: when recalibration, set Retention time by choose No update or Replace or Average or Bracket. 16

7. Move cursor to the right for setting other data (see picture). 1. Sample Amount :Set blank (if not value you can blank ) 2. ISTD Amount : Set blank (if not value you can blank ) 3. Multiplier : Set blank (if not value you can blank ) 4. Dilution factor: if sample is not diluted you can blank or if sample is diluted, set value of dilution sample. 5. Inj Volume: set volume for injection (unit µl). 6. Sample Info for Vial: fill comment detail of sample or condition of HPLC or blank. 10. Click to exit the window of Sequence Table. 11. Save Sequence as : there are 2 ways - At Menu bar choose File > Save as > Sequence.. (name of saved Sequence is_.s) - or At Menu bar choose Sequence > Save Sequence As ( name of saved Sequence is _.S) 12. Click icon. 13. Command to Run by at Menu bar choose Run Control > Run Sequence Or click button at the window of Instrument1 (online) Method & Run Control 14. When start sequence run the method was loaded instrument will show status and when instrument will ready to run in progress, show status. 15. Until sequence run finished, status bar change from to or change from to when set command macro in sequence Parameter. 16. Continue follow article 13. 17

13. To manage the Data file (window of Data Analysis) 13.1 Download wanted chromatogram to analysis. : follow the direction below. 1. Go to the window of Data analysis: there are 3 ways - At desktop, double click icon Instrument 1 offline (recommanded) - Or at the window of Instrument 1 (online) Method & Run Control choose Data Analysis show the picture below. - or at the window of Instrument 1 (online) Method & Run Control, at Menu bar choose View > Data Analysis.. All 3 ways the window of Data Analysis will occur. (see picture below). 2. Load Data file by clicking at Menu bar choose File > Load Signal or click icon. then the window of Load Signal will occur 18

3. Double click Data from Folder then choose your subdirectory by double click it and all data files will show at the channel File name then click wanted file and click the data file will occur. (see picture below) 4. If you want to compare chromatogram, at menu bar choose File > Overlay Signal or click icon then click wanted file to overlay and click the overlay files will occur. (see picture below) 5. Continue follow article 13.2 13.2 Integration data file. : follow the direction below. 1. Load chromatogram by processing follow article 13.1. 2. There are 3 ways of Integration.. 2.1 Way 1: Auto Integration by - At Menu bar choose Integration > Auto Integrate or click icon. 19

2.2 Way 2: Integration Event by 1. At Menu bar choose Integration > Integration Events or click icon. then the window of Integration Events will occur (see picture). 2. At Integration Event : set value of initial event that you need. Slope Sensitivity mean slop at base peak Peak Width mean width at half of peak Area Reject mean peak area Height Reject mean peak height Shouldermean shoulder of peak 20

3. After setting value of initial event that you need, click icon to command software use setting value. 4. At Integration Event compose other integrated variables to use such as Baseline at Valley, Area sum etc.(see picture) by clicking icon to Insert line to other integrated variables, and click to Delete line of other integrated variables. 5. Click to exit the window of Integration Event (If you want to save, click YES and if not, click NO). Or click to auto save and exit the window of Integration Event. Note: Both of integration, auto integration and integration event, when you load other chromatogram that command software automatic to use setting value from the way that you choose to integration. 2.3 Way 3 : Manual Integration by A B C D E Where A : Manual set to draw baseline of peak. B : Manual set to draw baseline of negative peak. C : Manual set to Tangent skim peak D : Manual set to divide two peaks. E : To cancel peak. 1. Choose wanted icon A or B or C or D or E. 2. Place cursor to set initial value by hold clicking right mouse and draw to end point of peak. 3. Other peak, repeat choice 1-2. 4. Double click to show all integrated peak. 21

: Manual integration can not save value to all chromatograms. 3. Continue follow article 13.3. (If you don t create calibration curve, skip 13.3and 13.4, continue follow article 13.5). 13.3 Create Calibration curve: There are 2 ways 13.3.1 One point Calibration curve. : follow the direction below. 1. Load chromatogram of standard level 1. 2. At menu bar choose Calibration > New Calibration Table then the window of Calibrate: Instrument will occur. (see picture) 3. At Calibrate: Instrument choose Automatic Setup level Default Amount: fill concentration of standard. (or blank). 4. Click to Calibration Table 5. Fill name of compound at correlated with (, Retention Time) 6. If you don t fill concentration value in Calibrate window, you will fill at channel 7. Choose at channel value of Response Factor will occur at channel. 8. If RT is RT of Internal Standard, you will choose at channel and choose at channel the level of ISTD will occur at. 9. Repeat article 5 8 all compound and click 10. Save as the calibration curve by clicking At Menu bar choose File > Save as > Method.. 22

13.3.2 Many level of Calibration curve. : follow the direction below. 1. Process follows one point Calibration curve article 1-10. 2. Load second concentration by at Menu bar choose > load signal of second standard concentration. 3. At Menu bar choose Calibration > Add level then the window of Calibrate for level 2 will occur. 4. Fill second concentration at Default Amount or at Amt the window of Calibration Table all compounds then click and the Calibration Table window will occur. 5. Process follow article 2-4 for other level concentrations. Note: To average peak area of the all repeat standard concentration by Load Data file, At Menu bar choose File > load signal of wanted repeat standard concentration. At Menu bar choose Calibration > Recalibrate.. the window of Recalibrate will occur (see picture below) Choose Mode is Average for average value (choose Replace for replace value) then choose Level of concentration then click. Repeat until do all concentrations of standard. 23

6. Click to exit the window of Calibration Table. 7. Save as calibration curve, At Menu bar choose File > Save as > Method.. 8. Continue follow article 13.4. (If you don t change unit of concentration, skip13.4 and continue follow article 13.5). 13.4 Change unit of Concentration. : follow the direction below. 1 At Menu bar choose Calibration > Calibration setting 2 The window of Calibration setting will occur (see picture below) 3 Fill unit of concentration at channel Amount Unit. 4 Click to exit the window of Calibration setting 5 Continue follow article 13.5 13.5 To Calculation of sample. : follow the direction below. 1. Load Data file of wanted sample by clicking at Menu bar choose File > load signal of wanted sample to calculation. 2. At Menu bar choose Report > Specify report or click 24

then the window of Specify Report will occur. (see picture below) 3. At Destination,choose Screen 4. At Quantitative Results select type to Calculate by click will show types of Quantitative Results then choose for calculation - for Qualitative choose Percent - for Quantitative choose ESTD or ISTD or Normalization or %ESTD or %ISTD. 5. Click 6. Continue follow article 13.6 13.6 Adjust scale of Report. : follow the direction below. 1. At Menu bar choose Report > Specify report or click icon then the window of Specify Report will occur. 25

2. Click at then the window of Signal Options will occur (see picture below). 3. At Ranges choose Use Ranges then setting scale value at channel Min Value and Max Value of Time Range and Response Range. 4. Click to exit the window of Signal Options and click to exit the window of Specify report. 5. Continue follow article 13.7 13.7 Result and Print. : follow the direction below. 1. Click to show the window of result. 2. Click in the window of result or click from the window of Data Analysis. 3. Continue follow article 13.8. (If you don t create batch, skip 13.8 and continue follow article 14). 13.8 Batch. : follow the direction below. Menu Batch to calculation of batch results quantitative samples. Before using of Bath menu, you have to command sequence run, integrate all data files and create calibration curve. 1. At Menu bar choose Batch > Load Batch 2. The window of Load Batch: Instrument will occur then choose Batch name that automatic saved when you save as sequence and batch name is the same of sequence. 26

3. The window of Set up Batch will occur. (see picture) 4. At Method to Process Batch Data check name of method is used method if it is not used method click at to choose used method. 5. Select Data file that you wanted to batch Quantification by Chick in in front of wanted Data file then click OK. 6. ChemStation will load chromatogram of data file that you select. 7. Start calculation by clicking. 8. Then ChemStation start batch quantified until finished all selected data files. 9. Show batch results after quantified by click at. 10. Report of Batch will occur (see picture). 27

11. Print batch results by click 12. Click to exit the window of Batch. 13. Continue follow article 14. 14. Shutdown HPLC. : follow the direction below. 1. After finish run, A column can often be restored by pumping a optimize solvent through (depend on column). Solvent such as Acetonitrile, Methanol etc. To adjust of optimize solvent by click at choose Set up Pump then change to optimize solvent to 100% with flow rate 0.5-1 ml/min and wait until the column is stable with solvent (15-30 minutes). Note: If you use buffer, you must be left buffer by flushing column with water at low flow rate (0.5 ml/min) about 30 minutes to 1 hour or overnight. Click flow rate 0.5 ml/min then follow article 1. and choose Set up Pump then change to 100% Water with 2. Disconnect column, At pump module open purge valve by turning a quarter anticlockwise (see picture) then disconnect capillary tube line from both sides of column and connect it to column thermostat then in out end cap the column. 28

3. Storage system of HPLC with Isopropanol by clicking then choose Set up Pump and change to 100% Isopropanol with flow rate 0.5-1 ml/min and close purge valve of pump module by turning a clockwise and wait until HPLC system saturated with Isopropanol (15-30 minutes). 4. Click then choose Set up Pump to reduce flow rate to 0.0 ml/min by reducing 0.1 ml/min per time or at pump module turn on purge valve by turning a quarter anticlockwise then reduce flow rate to 0.0 ml/min by reducing 0.5 ml/min per time. 5. Power off system by clicking (see picture below) and click YES or OK. 6. Exit software chemstation by clicking at Menu bar then choose File > Exit and click YES or OK. 7. Switch OFF HPLC modules by pressing button of HPLC instrument on the left below of all modules such as Degasser, Quaternary pump, Auto sampler, Column Thermostat, DAD and used Detector. 8. Close window of CAG Bootup server. 9. Shut down Computer and switch off monitor. 10. Fill information in log book completely. 29

Scientific Equipment Center, Prince of Songkla University Appendix 1. Sample filtration To remove particulate matter which may damaging the instrument or column, sample must be filtrate through membrane filter or syringe membrane filter size 0.2 or 0.45 µm before injection of sample. : follow the direction below. 1. Accessories for filtration (see picture) or 2. To assemble the accessories follow the picture. 1 2 3 4 5 6 3. Screw tightly follow the picture. 4. Connect the syringe follow the picture. 30

or connect syringe membrane filter to syringe follow the picture. 5. Pipette sample into the syringe and filter sample by pressing plunger for fitting sample through membrane filter into collected vial follow the picture. 31

2. Mobile Phase filtration All Mobile Phase used on the HPLC must be HPLC grade quality. Mobile Phase must be filtered with 0.2 or 0.45 µm membrane filter to remove particulate matter which may damage the instrument or column before use. : follow the direction below. 1. Accessories for filtration (see picture) 2. To assemble the accessories and connect to pump follow the picture. 6 7 5 4 3 1 2 3. Mobile phase through membrane filter into collect flask and switch on pump. 4. Transfer filtrated mobile phase to the bottle. 5. Incase of HPLC have no Degasser, degas mobile phase to sonicate with Ultrasonic bath or Purge with Helium gas for 30 minutes before use 32