STANDARD OPERATING PROCEDURE (SOP)
Agilent 1200 HPLC System for Sugar Analysis
Purchased May 2010
Location: 2115, Pulp and Paper Labs
Contact: Dr. Sunkyu Park (sunkyu_park@ncsu.edu), 919-515-0473
To schedule training contact: Hui Chen (hchen14@ncsu.edu), 919-809-4751
Technical service: 1-800-227-9770
Sales Rep.: Tom Breeden (919) 466-2032
1. Preparation
Equipment: the following colors are used to indicate the status of the module or instrument:
Color
Status
Red
Error
Yellow
Not ready
Purple
Pre-run, post-run
Blue
Run
Light blue
Injecting
Green
Idle
Dark gray
Offline
Light gray
Standby (for example, lamps off)
Calibration standards: We are making up stock solutions of the following sugars:
Glucose, xylose, galactose, and arabinose with mannose together
An excel program (Linda’s HPLC standard sugar solution makeup) has the makeup and
calculates the amounts needed to make up 6 calibration standards. The concentrations are kept
constant.
Place the 2 ml vials in the holder as follows: slot (1) is always Milli-Q water, followed by the
standards in slots (2) – (7).
Instrument 1 online:
Turn on the five sections of the equipment, click on Instrument 1 online to set up for analysis.
Instrument 1 offline is used when samples are being run and you want to check or export
finished work.
Under Method and Run Control:
At this time, the mobile phase used is Milli-Q water (no need for degassing). Check the level of
the brown bottle before starting any sequences. Fill if necessary and reset the volume control
by the following means:
Click on instrument go to more pump then bottle filling.
Actual volume reset to 1.0 liter (if you fill up the bottle) and click on OK.
The following two statements should also be checked:
a. Prevent analysis if level falls below 0.100.
b. Turn pump off if running out of solvent.
Baseline:
On instrument control diagram: hit on to start the ovens, pumps and lamps. Wait until all
sections are ready (diagram color from yellow to green). Observe the baseline in Online Plot
(View-> Online Signals-> Signal Window 2); it should be stabilized before you start running
samples.
2. Sample Requirement
pH requirement for HPLC samples: 5 – 7.
For samples with pH< 5, neutralization is necessary.
a. Aliquot sample (5 – 10 mL) in a small flask or beaker.
b. Neutralize to pH 5 – 6 using calcium carbonate (ACS grade).
c. After reaching pH 5 – 6, allow sample to settle and decant off the clear liquid. The pH of
the liquid after settling will be approximately 7.
d. Prepare the sample vial by passing the decanted liquid through a 0.2 µm filter.
Caution: When calcium carbonate is used excessively, solid precipitation might occur even
after filtering, and could damage the column. Use calcium carbonate as little as possible, and
wait a sufficient time for settlement after neutralization.
3. Acquisition Standards
Sugars analysis
A standard method and sequence for sugar analysis will be developed and named Sugars1. This
will be a set flow rate, analyzing time for each sample, injection volume, injection depth and
oven temps.
If there is a parameter you want to change: use the “save as” icon first to rename the method
and then make your changes.
Establishing a method or editing a method:
1. Go to method.
2. Click on desired method then OK.
3. Back to method in the menu.
4. Click on: Edit Entire Method.
There are four sections
a. Method info;
b. Instrument Acquisition which will let you change: flow rate, stop time, injector info,
and oven temps;
c. Data analysis;
d. Run time checklist.
5. File save as >>>>whatever you want to name the new method.
Setting up a Sequence
a. Go to Sequence Template.
b. Under the Sequence menu, find Sequence Parameters…, input your name for operator
and in subdirectory tell it where the date is to be saved. It should contain the following:
path C:\chem32\1\data\your folder name. Select Auto for the data file naming system.
c. For Part of methods to run, select According to RuntimeChecklist.
d. Select the Post-Sequence Cmd/Macro checkbox, and then from the drop box, select
STANDBY. Enter an nRdy Timeout of 30 minutes. OK the dialog box. If you want to turn
off the pump and oven after signal acquisition, select macro “SHUTDOWN.MAC”, go.
e. Under the Sequence menu, find Sequence Output…, select the Print Sequence
Summary Report option along with the Report to Printer and Print individual reports
for each run as well, options.
f. Specify the destination for individual reports as the File, as specified here, not as
specified with each method. Select the File Settings as you need, then OK the dialog box.
In this case, you don’t need to specify report for each sample analysis.
Adding a sequence and start up to run samples:
1. Click on: Sequence.
2. Go to Sequence Table to input sample identification, vial position, sample name,
method name that you want to use for sugar analysis, Injection times per Vial (shown as
Inj/Vial), and usually please set it as 1.
3. Use Append Line to add new samples, also use Insert, Cut, Copy and Paste as you need.
4. After finishing setting up your sequence, click on OK to save your sequence.
5. Click on Run Sequence or Icon Start on the control board to start running samples.
4. Data Analysis
Evaluating the data:
1. Click on your data file:
2. Click on a specific sample. If it’s a calibration standard, it will show the four peaks plus a
lot of junk peaks if you do not specify data purifying standards in your method.
3. To clean up the unnecessary peaks, highlight the icon that has a peak with an X on it.
4. Drag and box the junk peaks to remove, (If you remove a good peak, use the green
arrow by a peak icon to undo).
5. Save icon is at the very right.
Exporting the data:
1. Double click the sample you want to export.
2. Click Report -> Specify Report to edit report calculation and print style.
3. Under destination: unclick screen, click file, uncheck all but xls, and then click OK.
4. Go Report -> Print Report to save the report in your targeting folder.
5. C:\chem32\1\data\your folder name to save your report into your hard disc.
5. Shut-down Process
1. Click on the icon off in the lower right corner of the diagram board to shut down the
ovens, pumps and lamps.
2. Turn off the five sections of the equipment.