Agilent Technologies 1200 Series HPLC

Standard Operating Procedure

1. Sample preparation prior to analysis

I. Powering on and preparing the instrument
II. Creating a method
III. Creating a sequence
IV. Data retrieval and analysis

1. Sample preparation
• Ensure samples are free of particulate matter. A 0.45 µm disposable filter is recommended to
filter samples.
• Load samples into labeled HPLC vials (VWR cat # 82030-974 may be used). Each vial
holds a maximum of 1.5 mL but do not need to be completely filled; because the autosampler
needle collects sample from the bottom of the vial, only ≈0.5 mL is necessary. For smaller
samples, a glass insert may be used (Figure 1). Glass inserts serve to lower the internal
volume of the vial are simply placed inside the HPLC vial and loaded with sample. The cap
is then screwed onto the vial as normal.

Figure 1. An HPLC vial loaded with sample (right) and an optional 400 µL glass insert (left).

• Place sample vials in the autosampler tray, making note of the position of each sample in the
tray (you will need this information later).

I. Powering on and preparing the instrument

• Turn on the computer (the password and username can be found underneath the keyboard)
and give the old computer ample time to “warm up” (5-10 minutes is recommended).
• Turn on the HPLC by turning on the power strip. The instrument will proceed through its
normal startup procedure. You should see an info bubble in the bottom right corner of the
computer telling you “1200 is connected.” Wait for the startup procedure to finish; all lights
 should be green (or off, in the case of the autosampler module). Orange or red lights indicate
a problem.

Figure 2. The HPLC as it appears during normal use.

• Open the program “HPLC Online” otherwise known as ChemStation. The program should
start up and open after initializing. If the instrument modules are not recognized, repeat
previous steps.
 Figure 3. Chem station after it has been opened. Note the entire top panel, including the image
of the tray, are all intractable; clicking on images opens a menu with relevant controls.

• To enable instrument control, make sure “Method and Run Control” in the lower left corner
of the program is highlighted.
• Ensure solvent bottles are filled with solvent. If running low, fill bottles with HPLC grade
solvent. Then adjust the solvent bottle fill level in the program by clicking on the picture of
the solvent bottles and selecting “solvent bottles filling,” inputting your new volumes.
NOTE: the program will automatically shut off if the solvent bottle fill drops to 0, regardless
of whether or not solvent is actually in the bottle. If you do not remember to adjust solvent
bottle filling, your run will shut off!
 Figure 4. Solvent bottles filling menu.

• Ensure that the waste jug underneath the HPLC is not full, or near full, or it will overflow
during the run. Dispose of waste as necessary.
• Install your column, making sure a secure connection is established by tightening screw-
plugs to finger-tight. NOTE: when putting away columns, ensure they are stored in the box
with endcaps in place. Columns should not be allowed to dry out.
• Loosen the “to waste” knob (Figure 2) so that flow (and any initial bubbles) will be directed
to the waste jug.
• If you already have a method created, load that method (Method→Load Method…, then
select your method from the list). If not, navigate to and complete section II: creating a
method. Method must be created and loaded to proceed with instrument operation.
• In the instrument control panel in ChemStation, hit the “on” button. The instrument will
begin to pump solvent. At this point, check your column/system for leaks. NOTE: if the
instrument detects a leak via dripping solvent, it will automatically turn red and shut off (and
won’t tell you why).
• Depending on your column, it is recommended to flush with organic solvent first until
column appears clean (no peaks eluting). Quickly change solvents by clicking on the arrows
above the solvent bottles (Figure 3) and selecting “Set up Pump…” Once clean, set the
solvent levels to match your initial chromatographic conditions to equilibrate the column.
Column should be equilibrated until pressure and UV-vis baseline has stabilized.
• You are now ready to create a sequence to run or, alternatively, perform single runs. Select
between these options by clicking the image of three vials (vs. the single vial) in the upper
left-hand corner of the chemstation program (Figure 3). A sequence may be started by
pushing the “start” button.
II. Creating a method
• Choose a method on which to base your own. Then save it as your own by going to
File→Save As→Method… whereupon you will be prompted to select a name for your new
method. NOTE: it is useful to save your file name ending in a number (ex. Name_1) so that
new versions may be saved when changes are made.
• Now edit your method by selecting Method→Edit Entire Method… and select the desired
method sections to edit (typically all checkboxes are checked). Select “OK.”
• You will now be able to fill in your method information which should include your analyte,
solvents, detection wavelengths, injection volume, flow rate, column type, chromatographic
conditions, and total runtime. This information may be edited later. Select “OK.”
• Set up Pump: on this screen you will set your flow rate, stop time, chromatographic
conditions, and pressure limits. Initially, ensure your solvents are labeled correctly and the
letters A, B, C, and D match the actual solvent bottles. For an isocratic run, only use of the
“solvents” section is required. If a gradient run is required, use “timetable;“ create a
timetable using the “insert” and “append” buttons to modify your chromatographic run.
Make sure to include ample time in your run for your column to equilibrate back to initial
conditions. NOTE: information in the “timetable” section will always override information
in the “solvents” section. Select “OK.”

Figure 5. Set up pump menu with a gradient elution scheme.

• Set up Injector: here you will select your injection type and volume. Standard injection of 10
µL is a recommended starting point. Select “OK.”
 Figure 6. Injector setup.

• DAD Signals: here you will select what wavelengths to monitor throughout your run. Ensure
the “store” box is checked so that data is saved! If you require detection in the UV region,
make sure the required lamps Vis and UV are selected; if the UV region is not needed,
unselect this box to preserve the deuterium lamp. Also ensure that your reference
wavelength is a clean area in your spectrum- this wavelength should have 0 absorbance.
Make sure in the “time” section that “stoptime” is set to “as Pump.” All other conditions
may be changed as desired. Select “OK.”

Figure 6. What a functional DAD signals menu looks like.

• Column thermostat method: here you can select the temperature at which the column will be
maintained. If temperature is to change throughout a run, push the “more” button and input
the timetable desired using the insert and append buttons. Select “OK.”
 Figure 7. Column thermostat setup.

• Signal Details: Each signal that is to be stored must be added to the method by selecting it
from the drop-down menu and then pushing the “add to method” button. In the example
shown, three signals have been added and are therefore stored simultaneously. Select “OK.”

Figure 8. Signal details setup.

• Edit Integration Events: make sure “manual events” box is checked, then select “OK.”
• Specify Report: here you will adjust what displays on the screen, and the default settings are
generally acceptable. Select “OK.”
• Instrument Curves: if you wish to store data pertaining to solvent percentage, flow, or
temperature, check the appropriate boxes and this data will display in your report.
• Run Time Checklist: the boxes next to data acquisition, standard data analysis, and save
method with data should be checked. Select “OK.” Your method is now ready to use.
III. Creating a sequence
• Access the sequence table by clicking on the image of the sample tray and selecting
“sequence table.” Alternatively this may be accessed via Sequence→Sequence Table...

Figure 8. An example of what a sequence table should look like.

• Here you will program your sequence. Under “vial,” input the number only (do not type
“vial”) that corresponds to the location of the vial in the sample tray. Chose a unique name
for your sample, then click the “method name” box and select your desired method from the
drop-down list. Then continue to fill in injections/vial (typically 1), sample type (typically
Sample), and injection volume (which should ideally match your method). Use the “insert”
and “append line” buttons to add samples to the sequence list.
• When sequence is created as desired, select “OK.”
• SAVE YOUR SEQUENCE! Select Sequence→Save Sequence As… or File→Save
As→Sequence… You will then be prompted to enter a name for your sequence. NOTE: this
name is how you will locate your data later so it is important to remember the name you
select.
IV. . Data Retrieval and Analysis

Figure 9. The data analysis screen in chemstation.

• In the lower left-hand panel of chemstation select “Data Analysis”

• Select the name of your sequence (or, alternatively, your single run) from the list to the left
• Now you may select the individual sample you wish to analyze from the list displayed at the
top of the screen. To view all spectra collected for this sample, show “all loaded signals” as
in the example shown. Alternatively, if only one signal is desired, select it instead; this is
recommended for integration.
• The “integration” and “spectrum” buttons below your sample list will be most frequently
used. To view a complete UV-vis spectrum of any of your chromatographic peaks (as long as
you opted to save this data in your method) click the “spectrum” button and click the
appropriate “select spectrum…” button that appears in the toolbar below.
• To integrate your peaks, select the “integration” button (shown in Figure 9). Select the
desired signal you wish to monitor from the drop-down menu as described before.
Automatic integration results may now be read or, if desired, manual integration may be
preformed by clicking the “Manual Integration” button (highlighted in Figure 9) and then
drawing your own baseline under the peak(s) of interest.
• Record your integration results. This is your data! Congratulations.
• When finished with HPLC, shut down the system to conserve energy. This is done by
turning off the power strip behind the instrument (note: do not touch the buttons on the
instrument) and turning off the computer.