1

WAT ERS P RODUCTSACQUITY UPLC® H-Class

MassLynx™ Software

OpenLynx™ Open Access Software

K EY WORDSOALogin, Open Access, Automated

Method Switching, Method Filtering

Automated Method Switching and System Optimization in a Walkup EnvironmentDarcy ShaveWaters Corporation, Milford, MA, U.S.

INT RODUCT IONWhen initially working with newly synthesized compounds, it is often necessary to

run a variety of LC methods to determine the best one for future analysis. Methods

can vary in mode (analytical or preparative), column size, run time, gradient

conditions, solvents, modifiers, MS ionization mode, and other variables. The most

cumbersome part of method scouting is ensuring the system is in the appropriate

state before proceeding with the next injection.

Waters developed new software that compares the current and the upcoming

methods and notes any changes between the two. The appropriate action to

ensure quality performance is then initiated automatically. This could include

purging solvent lines, flushing and washing columns, and equilibrating the system.

Implementation increased user confidence in the fidelity of the system and

decreased system administrator set up time.

RESULTS AND DISCUSSIONThe walk-up system was set up with a large number of methods, due to the flexibility

of the ACQUITY UPLC H-Class System with its Solvent Select Valve, Column

Manager, and PDA Detector. The solvent select valve (SSV) gave the choice of six

modifiers, with either acetonitrile or methanol as the organic solvent. The column

manager allowed the use of four columns, of varying chemistries. The combination

of just these two parameters led to the creation of 48 possible methods. Allowing

users to choose from three gradient profiles increased the number of available

methods to 144.

144 methods – simplifying the choiceThe diversity of compounds being synthesized in medicinal chemistry laboratories

requires access to many different methods for analysis. This diversity can make it

difficult for a chemist to choose the best method for their compound. This can mean

delays in the development of the compound.

AP PLICAT ION BENEFITSIn this work we demonstrate use of system

automation software that was developed to

facilitate the transition from one injection to

another. By reducing the need for pre-, post-, and

switch methods, the system setup is simplified.

Confidence in system performance increases as

users know it will be in an optimal state when their

sample is run. Additional benefits include:

n Create and use targeted methods

vs. generic ones

n Reduces administrator setup

n Ensures system is at optimal

conditions before injection

n Increases column lifetimes

2 Automated Method Switching and System Optimization in a Walkup Environment

To make it easier for chemists to choose the most appropriate method, the method

filtering capabilities of OpenLynx OALogin were used (Figure 1). This software

simplifies the choice of methods and allows the administrator to create many specific

methods, instead of a few generic ones. This filter allows users to choose methods

based on the properties of their compounds by answering a few simple questions.

Figure 1. Method Selection window showing method description.

The naming convention for methods is simplified to aid in development. Methods

are named using an A_B_C_D pattern, where A is the column (1 to 4), B the organic

phase (1 or 2), C the modifier (1 to 6), and D the gradient profile (1 to 3). A more

detailed description of each method is included to aid in the choice.

EX PERIMENTAL

LC conditionsLC system: Waters ACQUITY UPLC

H-Class System

Column 1: ACQUITY UPLC BEH C18

1.7 µm, 2.1 x 50 mm

Column 2: ACQUITY UPLC BEH C8

1.7 µm, 2.1 x 50 mm

Column 3: ACQUITY UPLC BEH Phenyl

1.7 µm, 2.1 x 50 mm

Column 4: ACQUITY UPLC HSS C18

1.7 µm, 2.1 x 50 mm

Column temp.: 45 °C

Flow rate: 0.6 mL/min

Mobile phase B: Acetonitrile

Mobile phase C: Methanol

Mobile phase D1: Acetic acid

Mobile phase D2: Formic acid

Mobile phase D3: Trifluoroacetic acid

Mobile phase D4: Ammonium acetate

Mobile phase D5: Ammonium bicarbonate

Mobile phase D6: Ammonium formate

Gradient Profile 1: 5-95% organic/70 seconds

Gradient Profile 2: 5-50% organic/70 seconds

Gradient Profile 3: 50-95% organic/70 seconds

PDA conditionsRange: 210 to 400 nm

Sampling Rate: 20 points sec-1

Data managementMassLynx Software with OpenLynx Open Access

Application Manager

3 Automated Method Switching and System Optimization in a Walkup Environment

Automated method switchingGiven the wide variety of methods possible, it was previously necessary for the system administrator to define

pre-, post-, and switch methods for each inlet method available. System Automation Software reduces this need by

allowing the administrator to define rules for managing the transitions between methods. System Automation will

look at any method changes between injections and perform the administrator-defined actions (Figure 2).

There are three changes between injections that will trigger the System Automation software. These include changes

to the modifier, the column, or the gradient conditions. A visual representation of the system and the various flow

paths is presented in the ACQUITY UPLC Console to assist in the setup of the automation software (Figure 3). This

representation also shows real-time information about the system, including flow rates and valve positions.

Figure 2. ACQUITY UPLC System Automation flowchart.

Figure 3. Representation of ACQUITY UPLC System with real-time flow information.

4 Automated Method Switching and System Optimization in a Walkup Environment

Figure 4. Configuring the system equilabration settings.

A change to the modifier will cause the software to run the “flush solvent selections” command (Figure 4). This

primes the tubing from the new solvent position upon change of the selection valve. With the start of the next

injection, the correct modifier will be ready and none of the previous modifier will enter the system. The amount of

modifier used to flush the valve can be configured as either a volume or a time (at the default flow rate of 4 mL/min).

The “equilibrate system” command sustains initial conditions of a method when mobile phase has changed. The

equilibration time is dependent upon either a designated time or column volume and system volume (Figure 5).

Equilibrating the new column prepares it for the next injection.

Figure 5. Configuring the solvent selection flush settings.

5 Automated Method Switching and System Optimization in a Walkup Environment

Because the System Automation Software performs the equilibration of the column based upon the initial

conditions of the upcoming method, the design of the gradient profile was changed. No longer is the step

back to initial conditions needed at the end of the gradient. The equilibration does not need to be built into

the method; it is handled by the software. The profiles for the three gradients used are seen in Figure 6.

Figure 6. Profiles for gradients 1 (5-95%B), 2 (5-50%B), and 3 (50-95%B).

6 Automated Method Switching and System Optimization in a Walkup Environment

The column volume is defined in the Column Library (Figure 8). The Column Library is also where the administrator

defines the conditions for rinsing the column or storing it.

Rinsing the column applies conditions to resume the use of a column after switching it back online or when using a

column after a minimum period of dormancy, preparing it for use but not equilibrating it.

Storing the column applies conditions to a column to prepare it for storage, either when switching offline or after a

minimum period of dormancy.

For either situation, you can define the percentage of each solvent (A to D) to use, along with a flow rate and either

a time or a column volume (Figure 9). Storage of a column is recommended when it will not be used for a period of

time to avoid adsorbent degradation or buffer precipitation.

Figure 7. Column Library details.

Figure 8. Rinse Column and Store Column settings in Column Library details.

7 Automated Method Switching and System Optimization in a Walkup Environment

If you are switching columns often, storing the old column and rinsing the new column upon every switch can be time

consuming. By using the Startup, Recovery, and Shutdown options (Figure 10), it is possible to mitigate these delays.

Startup gives the option to rinse the new column only if a specified amount of time has passed. It also allows you to

tell the system to equilibrate the new column and run a blank injection, if you wish. Shutdown allows you to specify

an amount of time a column must be idle for, before it is put in storage conditions. This dialog box also allows you

to specify steps to be taken when the system is recovered from an error state. These steps can include rinsing and

equilibration the column as well as running a blank injection, before resuming a queue.

A Solvent Library (Figure 10) is also available, with features much like the Column Library. In the Solvent Library, the

administrator associates each mobile phase line with a defined solvent. The administrator can also define aliases for

each solvent, such as MeOH for methanol. This helps to keep solvents correctly configured when automation settings

are exported to other systems. There is also the option to validate the solvents. This ensures that the names (and

aliases) specified in the Solvent Library match the names in the inlet editor. This is helpful when transferring methods

between systems, where the solvents may not be configured in the same way.

Figure 9. Startup, Recovery, and Shutdown dialog box.

Figure 10. Solvent Library.

8 Automated Method Switching and System Optimization in a Walkup Environment

Once the system is running, a journal (Figure 11) is kept of all the actions performed by the System Automation

Software. The journal notes when methods are downloaded, what actions the method change dictates, and when those

actions are performed. This enables the user to quickly know what is happening and if any problems have arisen.

Setup of the system automation software only needs to be performed once. The settings can then be exported in

an .html format for importation on other systems (Figure 12). This ensures uniformity across all instruments.

Figure 11. System Automation journal.

Figure 12. Exporting system automation settings.

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

Waters, ACQUITY UPLC, and UPLC are registered trademarks of Waters Corporation. MassLynx, OpenLynx, eCord, and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2010 Waters Corporation. Produced in the U.S.A.October 2010 720003779en AG-PDF

CONCLUSIONSn Method Filtering allows users to quickly and easily find an appropriate method

from a long list of available methods.

n System Automation Software enables automatic switching between methods,

removing the need to create switch methods.

n Automation can be optimized for time or for volume.

n The administrator is enabled to create more simple gradients as the gradient

does not need system re-equilibration steps to be built in.

n When used with a Column Manager, the software automatically stores columns

going off-line and conditions columns coming on-line.

n The Column Library keeps track of the columns being used on the system.

Columns with eCord™ Technology automatically populate the Column Library,

while those without can be manually entered.

n The Solvent Library ensures that the correct solvents are being used by

matching the setup in the library with the setup in the Inlet Editor.