Software interface

The previous prototype instrumentation required not only the use of Masslynx for LC/MS control, but a significant amount of custom electronics and a custom software interface as well. As these custom components began to fail, it created significant problems

in the operation of the system. At one point there were three operational hydraulic amplifier prototypes, which has since been reduced to one, and the functionality of that system is only possible because of a workaround. Thus, when designing the present system, it was desired to use the built-in functionality of the control software provided by Waters Corporation with a nanoAcquity UPLC/MS system. Little was changed with regard to normal operation within Masslynx 4.1, the software that is primarily responsible for the operation of the mass

spectrometer. The largest change was to how one creates a sample list.

A screen capture of the sample list from Masslynx can been seen in Figure 4-11. Normally, each row corresponds to a single analysis. Because of the need to store a gradient prior to trapping the sample, multiple rows are needed for a single analysis. Methods

pertaining to each step were created to facilitate ease of use. The first line needed is a

gradient loading method. At the end of the gradient loading, valves states are reconfigured to allow for trapping and the mobile phase conditions are changed to be amenable to this mode of operation. The second line required for operation is for sample injection and trapping. There are a significant number of timing events associated with this method. Initial settings entered into the events tab must be set to allow trapping operation. This should be a

continuation of the valve states used at the end of the gradient loading method with mobile phase conditions amenable to sample trapping (0.5% acetonitrile). Trapping continues for an amount of time defined by the user and typically a volume three times the sample loop volume is used. Once this is complete, valve states are changed to allow for high pressure operation. When this occurs, an additional signal is sent to the mass spectrometer to begin data collection and the -903 pump is activated by applying pressure via the pneumatic solenoid valve. For short analysis times, the end of this method could be configured to turn

off the -903 pump, depressurize the system, and return the valve states to the “gradient loading” configuration to allow for the next run to proceed. Typically, more data analysis time is required and therefore “linker” methods are employed.

The needs for the “linker” methods results from a limitation of 32-bit operating systems as mentioned in Chapter 2. With these high-resolution separations, data collection rates were at times exceeding 2GB/hr per function (one for low energy collisions, one for high energy). Thus, in less than two hours, each function would reach the maximum 4GB limit imposed by the 32-bit system. While the mass spectrometer continues to perform scans and the XUPLC system continues to elute analytes, no data is saved. In order to overcome this limitation, multiple runs were linked together. There are a few disadvantages to this type of operation, one of which will be discussed in more detail in section 4.3.3. The most obvious disadvantage is that approximately 15 seconds of data are lost for each additional linked run. This is certainly not a large amount of data, but it would be ideal to not lose any data.

Unfortunately upgrading to a 64-bit operating system is not sufficient as Masslynx was written in a 32-bit environment and significant software changes would be required to facilitate the use of the 64-bit operating system.

While Masslynx is the program that is used to control the mass spectrometer and provides access to modify both LC and MS conditions, the nanoAcquity console and nanoAcquity inlet method editor are the two programs that are actually running the nanoAcquity. Masslynx provides a convenient link to these programs and streamlines the user experience by requiring only one application to be physically opened by the user. The nanoAcquity console is critically important to the operation of this system. There has been much discussion regarding valve state changes with little mention of how this actually occurs.

During setup, column flushing, and other non-routine activities, the valve states can be directly controlled by accessing the “rear panel” from the troubleshooting pull-down menu within the nanoAcquity console. This is highlighted with a red oval in Figure 4-12. In order to have enough valve states to properly control all aspects of the system, switches on both the sample manager module as well as the nanoAcquity binary solvent manager were needed. These can be toggled by clicking on the icon for each of the swtiches changing between a red, open switch, and a green, closed switch. Conveniently enough for operation, “red”

corresponds to a closed Valco on/off valve, while “green” indicates that the pneumatic solenoid is energized to allow gas flow to the on/off valve, opening it (Figure 4-13). Fortunately, changing these switches can be automated. This is accomplished by accessing the inlet method editor. Once in this part of the program, one can edit the timing events associated with both the pump and the autosampler by navigating to the “Events” tab and inputting the appropriate values (Figure 4-14). In order to assist in the creation of methods, a spreadsheet was created where inputting the gradient loading time will output all the values needed across all the tables for all windows.