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Original Operating Manual HPLC 1100
Original Operating Manual HPLC 1100
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.
3. Check the waste bottle, if the waste bottle nearly full, change a new waste bottle.
4. Continue follow article 2.
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6. If you choose detector different from the last user, the window of Configuration will occur following the picture below.
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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.
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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.
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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.
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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.)
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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 l) 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.
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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.
1. Click 1 time then choose Set up DAD signal the window of DAD signal will occur (see picture below).
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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.
1. Click 1 time then choose Set up FLD signal and the window of
FLD Signal will occur. (see picture below).
1. Click 1 time then choose Set up RID signal the window of RID Signal
will occur.
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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.
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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.E..
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 F 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.
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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.
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.
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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.
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 E
Or click button at the window of Instrument1 (Online) Method & Run Control.
Or click from the window of Sample info.
6. Instrument will ready to run in progress and show status .(Blue color)
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- If you want to set command macro, you will click Post-sequence Cmd / Macro then chooses type of
command, there are 4 types
STANDBY: when finished sequence the instrument go to standby mode.
LAMPALL OFF: when finished sequence, only lamp of detector turn off.
PUMPALL OFF: when finished sequence, only pump turn off.
macro KSHUTDOWN.MACL,go : when finished sequence , every modules of HPLC shutdown.
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- 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@
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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 L).
6. Sample Info for Vial: fill comment detail of sample or condition of HPLC or blank.
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
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- 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.
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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)
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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).
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.
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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.
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.
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then the window of Specify Report will occur. (see picture below)
1. At Menu bar choose Report > Specify report or click icon then the window of Specify Report will occur.
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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.
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.
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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.
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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 and choose Set up Pump then change to 100% Water with
flow rate 0.5 ml/min then follow article 1.
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
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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.
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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 m 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
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5. Pipette sample into the syringe and filter sample by pressing plunger for fitting sample through membrane filter into
collected vial follow the picture.
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5
7 4
3
2
1
3. Mobile phase through membrane filter into collect flask and switch on pump.
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