1

Quick Manual to the Waters UPLC System

1. Application of Chiral HPLC Columns

Warning! The HPLC columns are destroyed if the pressure overrides 2000 psi. The
highest pressure applied for routine analysis is 1400 psi. Before starting any
operation check that the high pressure limit is set to 1400 psi.

1.1 Prime pumps and wash needle

Prime the pumps from the Binary Solvent Manager under the Control menu. Prime and
wash the needle before starting the run. This option can be found in the Sample Manager
under the control menu.

1.2. Starting the analysis

Open the Empower Pro and click on the Run Sample button. Specify the project and a
new window (Run Sample) appears. Click on the Acquity Sample Manager symbol. In
this new window, all system parameters are visualized (this window is recommended to
be open under the entire analysis). The most important information involves the pressure
profile of the pumps (pumps A and B). The window also shows Maximum pressure,
the Minimum pressure and their difference Delta. The Delta value should be less
than 10 psi for a certain solvent composition and flow rate. Be observant of any
inconsistencies in the pressure and pump plots.

Go back to the Run Sample window and switch on the lamp. This can be done in the
Acquity PDA detector panel. When you click on the Lamp button a question appears.
Choose Yes to switch on the lamp.
 2

1.3 Changing the eluent composition and the flow-rate

The Run sample window shows the eluent composition. Eluent A is usually
isopropanol, while eluent B is usually hexane (at least for straight phase chiral HPLC
analysis). Click on any of the components and a new window appears. Specify the
composition of eluents A and B directly in % units. Usually the highest amount of
isopropanol is 50% and the lowest amount is 0.2%. The maximum flow-rate is 1.4
ml/min and the lowest is 0.2 ml/min for routine analysis.

Always change the eluent gradually. For example, changing 1% isopropanol/99% hexane
to 30% isopropanol/70% hexane can be done in three steps: 10% isopropanol/90%
hexane, wait 5 min; 20% isopropanol/80% hexane, wait 5 min and then 30%
isopropanol/70% hexane. Apply the same caution when changing the flow rate. Notice
that the viscosity of isopropanol is higher than that of hexane so increasing the amount of
isopropanol leads to increased pressure with a constant flow-rate. When you increase the
amount of isopropanol monitor the change of pressure, it cannot exceed 1400 psi!

1.4 Monitoring the base line

After adjusting the eluent and the flow-rate you need to monitor the base line. Choose
one of the existing instrument methods in the Instrument methods panel. For example
take johannaluke, which can be used for the IC/OD-H columns (excellent for
separation of esters, alcohols and sulfonylimines) Click on the Edit button of the
Instrument methods panel. A new window opens. Click on the BSM (Binary solvent
manager) button. Give the same flow-rate and eluent composition that you have adjusted
above or define a gradient system. Choose the column by setting the switches according
to the column position you intend to use. Save this method in the main menu and quit.
You will return to the Run sample window. Click the monitor button. This starts the
monitoring program. The Run button lights up on the menu panel.

Check the base line and check the actual pressure, eluent composition and the flow-rate.
The delta value has to be less than 10 psi after 5 min monitoring. If you are satisfied
 3

with the base line, you have to stop the run by clicking the red-sphere button in the
main-menu line.

1.5 Sample preparation

Dissolve your silica chromatography (or crystallization) purified sample in a mixture of
hexane and isopropanol. Use the same composition as in the eluent that you intend to use.
Avoid using isopropanol, if you have ester groups in your analyte. You may improve the
solubility by adding small amounts (less then 50%) of CHCl3 to the hexane solution. Fill
the sample vial to the upper mark (about 1 ml) and place the sample in the sample tray.
Write up the tray position.

1.6 Injection of a sample

After you have stopped running the instrument method monitoring, check the injection
panel. In the Method Set exactly the same method has to appear as in the Instrument
methods. For example you may use johannaluke method. Change the sample tray
position (plate/well) and file name. Then inject your sample by clicking the injection
button (left side).

After the injection check the actual pressure, eluent composition and the flow-rate. If the
eluent composition or the flow-rate suddenly changes, then you do not use the same
Instrument method and Method Set. Wait until the analyte peak appears in the
chromatogram, and stop the run only after this. Check that Instrument method and
Method Set agrees and then start the next injection.

1.7 Review of the spectrum

You have the possibility to review the spectrum under (or after) the analysis. Right-click
in the spectrum window and choose Review. It may take up to 30 seconds to get the
review, wait patiently.

1.7.1 Extracting the UV spectrum. When the Review window appears, click the 3D-
chanels button in the bottom of the screen. Mark the entry in the appearing Table. Then
 4

the UV spectral distribution appears in one of the windows. Right click in the sample
spectrum and choose Extract chromatogram @ XX nm. Then a cursor appears at the
right side, which shows the actual wavelength of the analysis. You may draw the cursor
to the required wavelength. In the spectrum window, the actual spectrum recorded at the
adjusted wavelength will appear.

1.7.2 Overlaying the spectra of the enantiomers. Right-click the apex of the peak from
your sample and choose the Extract spectrum at XX min menu. A new window appears
with the UV spectrum of this peak. Note that the enantiomers of a certain compound give
exactly the same UV spectrum. This can be used for identification of the enantiomers.
Right-click on one of the two UV spectrums (which are believed to belong to the
enantiomers of the same substance). Choose the Properties menu. A new window
appears. Choose the overlay menu and click the Overlay in a single plot button, then
choose the Scaling menu and choose the Normalization X and Normalization Y.
This gives you the normalized overlays of the two UV spectra, which have to be
completely identical for the enantiomers.

1.7.3 Getting integrals. On the main menu choose the Process button and click the
Integrate, then choose the Peaks button at the bottom of the window. This gives the
integrals of all detected peaks in a table form. Choose the rows of the appearing Table
reporting uninteresting integrals (solvent peaks, impurities, etc) and let the rows of the
desired peaks be unmarked. Right-click and choose remove. After the retention time
column, a number (about 10) of unimportant columns appear. Mark them with the cursor,
right-click and choose hide columns. After the areas and area% columns again a
number of superfluous columns appear. Mark them and hide them. Then you need to
print the table of integrals by right-click and Print, the spectrum (right-click and
Print) and the overlayed UV spectra (right-click and Print). Do not forget to write up
the solvent composition, the flow-rate, the filename and the date of the analysis.
 5

1.8 Going to standby mode.

Choose the Run Samples window. Decrease the flow rate to 0.01 ml/min and the eluent
composition to 1% isopropanol/99% hexane. Follow the change of the pressure and wait
until Delta is under 10 psi. Then, switch off the UV-lamp on the Lamp panel. The
UV lamp has a finite lifetime and therefore it always has to be switched off after the
analysis. You also have to check that the green light in the upper module of the
instrument over Lamp is not on.