HPLC General Operating Procedure

1. Turn on the ELSD laser by pressing the button until the light turns green (Fig 1).
a. Allow the ELSD to warm up for 15 minutes
b. Check the solvent level of each bottle and replace/refill as needed (Fig 2).
Fig 1: Face of the ELSD

Press this Laser

button to turn
on
Gas pressure
button
Fig 2: Solvent bottles to pumps A and B.

2. If you need to change one or more solvents, 30 ml of solvent must be pumped

through the reservoirs
a. When changed, solvents must be miscible. If not, an intermediate solvent
should be used that is miscible with both old and new solvents.
b. Stop the flow in the Pump / Sampler / Timebase : HPLC screen (Fig 4) by
selecting Control > Stop from the top menu.
c. Remove cap with tubing and filters from existing solvent bottle.
d. Rinse tubing and filters with new solvent.
e. Affix cap with tubing and filters to new solvent bottle.
f. Open the purge valve on the right side of the pump. Make sure the waste
container is connected to the purge valve before opening (Fig 3).
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Fig 3: Pump, purge valve and waste container.

Pump

Purge valve

Waste container
connected to purge
valve

g. Restart the flow by selecting Control > Continue from the top menu.
h. Set up the gradient to pump 30 ml of new solvent through the reservoirs.
i. From the Pump / Sampler / Timebase : HPLC screen, select
Control > Flow > Flow Gradient (Fig 4) from the top menu.
i. Set up gradient parameters (Fig 5a) by inserting lines on the Flow
Gradient screen (Fig 5b).
j. Run the gradient by clicking the OK button on the Flow Gradient screen.
k. When the gradient is finished, stop the flow by selecting Control > Stop
from the top menu.
l. Close purge valve (finger tight). Overtightening may damage the purge
valve.
m. Restart the flow by selecting Control > Continue from the top menu.
Fig 5a: Example gradient parameters for changing pump C solvent reservoir. Solvent
reservoir A contains ACN.
Retention Time Flow Rate
%B
%C
%D
0.0
0.5
0
0
0
2.0
5
0
100
0
8.0
5
0
100
0
10.0
0.5
0
0
0

Fig 4: HPLC Pump/Sampler/Timebase Screen

Control >
Flow option

Fig 5b: Enter gradient parameters on the Flow Gradient screen.

3. Connect the column inline between the pump and the ELSD.
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a. Open the oven column compartment (Fig 6).

Fig 6: Oven column compartment

b. Ensure the metal or stainless steel tubing from the injection valve is
connected to the column inlet (left side) and that the metal or stainless
steel tubing from the column outlet (right side) is connected (Fig 7). The
tubing from the column outlet should first be connected to plastic tubing
and allowed to drain into a bottle before connecting directly to the ELSD
(Fig 8).
Fig 7: Column connections
Outlet:
Connection to
plastic tubing
or ELSD

Inlet:
Connection to
injection valve

Fig 8: Connection to plastic tubing and flask

Instead of
connecting to the
ELSD, the column
outlet will connect
to the plastic tubing

Column
outlet

c. Tighten column screw with two wrenches without overtightening (Fig 9).
Fig 9: Use both wrenches when tightening the column screw
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4. Flush the column.

a. Flush the column with 100% ACN for 5-10 column volumes. The column
should be stored in 100% ACN. If not, flush with the same solvent in
which the column was stored.
Note:
A 150 mm column will require flushing with 0.3 ml
A 250 mm column will require flushing with 2.5 ml

b. Once the column is flushed, manually set up the starting gradient.

c. Connect the column outlet to ELSD and immediately do step 5.
5. Turn on and adjust N2 pressure.
a. Open the main valve on the N2 tank (Fig 10).
b. Open the outlet valve on the regulator (small black knob).
Fig 10: N2 tank valves.
Check this pressure
gauge when
adjusting N2

Main valve

Outlet valve

Adjustment
knob

c. On the ELSD, press the Gas Pressure button (Fig 1) until the light turns
and stays green. Initial pressure should be about 3 bar before the button
is pressed.
d. Adjust N2 pressure using large black knob on the regulator.
e. Double check pressure on ELSD and fine tune using large black knob
Note:
For blanks, use 0.8 bar
For standards or samples, use 1.9 bar

6. Set up a run folder on computer

a. Open the Chromeleon browser
b. For each new experiment, create a new folder by copying and renaming
an existing folder.
c. Clear the folder contents.

Chromeleon Browser

Directory Folder

Run Folder

Run

7. Set up the run.

a. Create a new run by right clicking and selecting Insert Sample. Once a
run exists, use Append Sample.
b. The Name field should contain the name of the analyte/sample.
c. The Program field should contain the name of the gradient.
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d. The Status field should be set to Single.

e. Save the newly created sample by selecting File > Save from the menu at
the top of the screen, or click on the Save icon.
f. Ensure the manual injection valve is in the load (down) position
g. Prepare to run the batch by selecting Batch > Start from the top menu.
h. The run will highlight green.
Note:
The status of the run determines the current sample processing status.

Single indicates that a sample is unprocessed. If the manual injection valve is

switched to the load position while the sample is in the single status, the run will
automatically begin.
Running indicates the run is currently being processed.
Finished indicates that processing of a run is complete.
Interrupted indicates the run was manually terminated before processing was
completed.

8. Inject the sample.

a. Draw 2-10 l of sample into syringe (Fig 11).
Fig 11: Glass-barrel syringe used for injection

b. Insert the syringe into the injection valve and introduce the desired
volume into the 20 l injection loop.
Fig 12: Injection valve in load position

c. Inject the sample by switching the manual injection valve from the load
(Fig 12) to inject position (Fig 13). Sample will now be sent to the
column.

Fig 13: Injection valve in inject position

d. Check pump pressure, N2 pressure at the ELSD, and ELSD voltage output
to ensure correct values.
9. Viewing the chromatogram during data collection
a. Press the CTRL + Tab buttons simultaneously to switch the Pump /
Sampler / Timebase : HPLC screen (Fig 14).
b. If the batch is running properly, the Retention Time counter should
increment, the Pump should be in Connected mode and the grayed
gradient graph should progressively brighten as the retention time
increases.
Fig 14: Timebase screen

Retention time
counter

Connected
mode

10. Before starting a new batch, turn the pump switch to the load (down) position.
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General Shut down Procedure

1. Turn off the N2 by first closing the main valve on the tank (Fig 10). When both
gauges go to 0, turn the small black knob into the closed position. Do not open
the purge valve.
2. Turn off the ELSD by pressing the Laser button (Fig 1) until it turns red.
3. Disconnect the column from the ELSD and connect to the plastic tubing. The
plastic tubing should drain to a bottle (Fig 8).
4. Flush the column with ACN by running the following gradient:
Retention Time
0.0
5.0
10.0

Flow Rate
0.5
0.5
0.5

%B
50
0
0

%C
0
0
0

%D
0
0
0

5. Disconnect the column from the stainless steel tubing and replace with the small
black column plug. Tighten the plug until the column pressure starts to rise, then
immediately stop the flow through Chromeleon (Control > Stop). The flow must
be stopped quickly to avoid exceeding the pressure over 400 bar. Then tighten
the column plug all the way.
6. Dispose of the waste in the jar connected to the ELSD, purge container, and any
other waste containers.