Instructions for operating the Hitachi 7000 series HPLC Apparatus

Instrument start-up

1. First check that the mobile phase and wash supply bottles are full enough so that you
won’t run out during your session. The wash solution should, ideally, be the same as
you intend to use as your initial mobile phase in your method, but normally using
methanol is sufficient.
2. Using the power buttons located on the front of each component, turn on the D-7000
interface unit, the L-7100 low pressure gradient pump, the L-7420 UV/VIS variable
wavelength detector, the L-7300 column oven, and the L-7200 autosampler unit.
3. Turn on the on-line degasser. This automatically degasses the
mobile phase constituents. Without degassing, you run the risk of bubbles forming in
the pump and more usually in the detector just when you are getting a signal.
4. Turn on the computer. Log on, or have the instructor log on for you.
5. Enter the D-7000 HSM instrument software by double clicking on the appropriate
icon.
6. Using the application icon (file drawer) select the appropriate application. Your
instructor may have assigned an application name for your group. If this is the case,
use that application only, and do not enter into other groups’ applications. Basically,
this is akin to your own file into which you will place your own method and store
your own data.
7. Click on the “i” icon then click on the initialize button. This will download information
from the software to the hardware and confirm that all of the appropriate components
are in place. Once initialization is complete (this takes awhile), you should see, in
gray print, all of the relevant components of the apparatus in the appropriate boxes.
Click ok.
8. Use the keypad on the front of the L-7100 pump to set the mobile phase to 100% A
(this will pump pure methanol through the system) and to set the minimum pressure
setting for the pump to 250 psi. This is accomplished by pressing the "manual set"
key then entering in the appropriate value for each parameter (followed by pushing
the enter key). The pump determines the correct A% by using the values you put in
for the other mobile phases. Thus, for 100% A, you will enter in 0% for the other
mobile phase components. Only adjust the mobile phase proportions and minimum
pump pressure; leave the other default values alone (accept them by pushing the enter
key).
9. Using the pump icon in the software, turn on the pump. Pumping pure methanol
through the column both before and after you do your experiments helps ensure
column integrity and prolongs column life. Pump methanol through the system at
least 10 minutes before you do your experiments.

Method Development
You will need to devise an appropriate method for the separation and quantification of
your analytes. The method you choose depends on what you need to accomplish, so the
following steps only point to the sorts of options you have available.

1. Click on the method setup icon. Choose the “basic” method to help you get started.
The icons at the top of the window can be clicked individually to allow you to control
the various components of the HPLC.
2. Click on the pump icon. Here you will see the mobile phase mix and the times at which
the various mixes will be used. If you use a constant mix, it is called an isocratic
method. If you vary the mix as you elute the analyte, it is called a gradient method. If
you want to alter the method, click the appropriate box and change the values as you
see fit.
3. Click on the Autosampler icon. This sets up the method the autosampler will use to
inject your sample into the mobile phase stream and load it onto the column. We have
found that using the cut method, with 30 microliters of waste both before and after the
sample, works well. Currently the syringe wash is set to just one wash, but if you
notice carryover of sample from run to run, this could be increased. Leave the syringe
speeds alone--they are set to appropriate values.
4. Click on the column oven icon. Set the temperature to some value above room
temperature.
5. Click on the detector icon (channel one). Here you can set the wavelength you would
like to use to detect your analyte and how long you would like to collect data. If you
have more than one analyte, and you know the best wavelengths to use for each of
them (and their retention times), you can have the detector move to the new
wavelength at selected times. How could you determine the best wavelength for
detection of your analyte?
6. Click on the report format icon. Here you can select which information will be printed
when you print out your chromatogram.
7. Finally, after you have adjusted the method to suit your needs, go up to the file menu
and use “Save As” to save the method under a name of your choice. This will be
available for you, in your application file, for later use.

Sample Table Creation

The sample table is used to tell the autosampler where to find the sample (or samples),
how much to withdraw, and what method to use. The method listed in the sample line
must be available in your application and certain parameters, such as information
regarding the autosampler, must correlate between the sample table and the method
chosen. Error messages allow you to correct these things before using a sample table.

1. Click on the sample table icon. Select “test” as a template for your sample. Click in the
boxes where you would like to type in corrected information, such as a description of
the sample and which method you would like to use. Just clicking in the method box
will generate a list of available methods. Generally, you should use a sample volume
at 5 to 20 microliters, since this amount will be sandwiched between the 30
 microliters selected in the cut method of injection in the method setup. Once you have
adjusted the sample table appropriately, save it using “Save As”, giving it an
appropriate name. This also will remain available to you whenever you open your
application file.

Collecting a chromatogram

The software will control all the components of the HPLC system, collect and store the
chromatogram, and allow you to analyze the data. The data will be stored in your
individual application.

1. Click on the acquire data icon (the eye).The software will ask you for the appropriate
sample table to use in your run. Select the one you want. The software will then
download the appropriate instrumental parameters and show you the current signal
output from the detector. As the mobile phase changes from pure methanol to
whatever you have stipulated as the first mobile phase of your method, the baseline of
the detector output will show substantial wander. You must wait until the baseline is
stable before beginning your run. This can take a long time—perhaps up to an hour!
While you are waiting, prepare your sample.
2. Sample preparation. You should use a sample which is dilute enough so you could
expect Beer’s Law to hold. If possible the solvent used should be close in
composition to the initial mobile phase. If it is quite different, then the initial part of
your chromatogram will appear quite bumpy. This is ok as long as your method
allows the baseline to settle down before your analyte(s) elute.
a) Filter your sample with a syringe and a 0.2 µm syringe filter into the special vial
for the L-7200 Autosampler. The septum for the vial should have a blue side and
a white side. The white side should be facing up, and the cap screwed down on
top of the septum (which has been pre-scored).
b) Label the vial, then place it in the autosampler tray in the number 1 position (back
and to the left). This assumes that you have listed that position in the sample
table.
c) Re-position the autosampler tray, making sure that it rests evenly on the support
bars and is pushed all the way back. If the tray is not positioned correctly you
will at best inject air into the column, potentially damaging it and certainly
causing long delay, and at worst you will damage the autosampler.
d) Once you have a stable baseline and your sample is in place, use the wash button
on the front of the autosampler to wash the syringe and injection path. Repeat two
more times.
3. You are finally ready to begin collecting data.Click the “start” button. The software
will take the necessary steps to load the sample onto the column and begin collecting
data.

Taking care of the data

1. You can stop your chromatographic run at anytime by clicking the “Cancel Run”
button. Otherwise the data collection will stop after the allotted time has passed. The
 detector trace will still be visible, though, so if you haven’t seen your analyte yet you
can continue to monitor the trace to get some idea of the retention time under your
conditions.
2. Once the data collection is over, you can analyze the data using the software. Click on
the reprocess data icon and select the number of the chromatogram you wish to work
with. Normally this will be the last one in your list; the one you just completed.
3. Click the display button at the bottom of the screen to bring the chromatogram onto the
screen. At this point you can, if you choose, re-draw the baselines below the peaks to
best reflect what you perceive to constitute the peak. (this will yield a corrected peak
area--important for quantitative work) If you do this, be sure to select the change
baseline feature under the process data menu selection at the top of the page. You can
also change the scale limits using this window.
4. Once you are satisfied with the way the chromatogram appears, click the recalculate
button. Then click the “modify report” button, and you should see what your printed
report will look like. If everything looks ok, select print from the file menu at the top
of the page.

Instrument Shutdown

1. Use the keypad on the pump to set the mobile phase to 100% A (methanol) again. Let
methanol pump for 10-15 minutes while you clean up your laboratory area.
2. Stop the pump by clicking on the pump icon along the left hand side of the screen, then
exit the D-7000 HSM software. Click the “Start” on the Windows NT desktop, then
opt to shutdown the computer. Windows NT will write unsaved data to disk, then
inform you that you may turn off the computer. It does not turn off the computer
automatically—you have to do it.
3. Turn off the in-line degasser and the components of the HPLC system.
4. Refill the mobile phase bottles with the appropriate solvents and refill the wash bottle
with methanol. This ensures the temperature of the solvents will reach a constant
value before the next time the system is used. If the solvent temperature is constantly
changing, severe drift will be seen in the detector output baseline.

Be sure to use only HPLC grade, ultra-pure solvents, and be especially sure that the
glassware used to transfer these solvents is perfectly clean.