Application Note: TRIO VS Dual Stream Staggered Injection

Description: This application is useful to increase sample throughput by taking advantage of front and/or back-cut time to load the next sample in the sequence early. It requires that the "period of interest" in the chromatography is significantly shorter than the overall chromatographic run-time. There are two columns that are running in "staggered" mode, where samples are pre-loaded onto the other channel while the previous channel is still in acquisition. There is no pre-column. The gradient programs regenerate the analytical columns in each stream.

Samples are processed in pairs, the application does NOT integrate with data systems, but contact closures are used to maintain synchronization with the detector.

Benefit: Sample injection occurs onto each column in a staggered fashion and is offset by the acquisition time. Optimum sample throughput is achieved by sample injection onto column 1 during the front cut and/or column equilibration of column 2. Increased throughput is the end result, with up to 100% increase in time saved

Equipment Requirements:

• HTS PAL.
• TrioVS Module
• TrioValve 1 - LCVlv1 = Injection valve for both columns.
• TrioValve 2 - LCVlv2 = Column selection valve.
• TrioValve 3 - LCVlv3 = Stream selection valve
• Two HPLC Pump systems

Required Special PAL Objects and Settings: Signal settings
START = Immediate INJECT = TTL In1 Ready from MS (Active State Low) (Set to "Immediate if signal is not available) injected = SW-Out1 Start Acquisition - to MS InjectdA = SW-Out2 Start gradient pump 1 InjectdB = TTL-Out1 Start gradient pump 2

Interface 2 must be used to communicate with the pumps and MS, this is because 3 signals are required as shown above.

MS acquisition method should be set up to acquire for only the period of interest. If no Ready is available from the MS, set the Sync Signal "Inject" to immediate and the injections can proceed based on the time parameters in the method.

Injection Valve Flush Time- The Injection Valve Flush Time variable is used to minimize these effects by ensuring that the sample will not be injected into a higher organic strength mobile phase than the starting gradient conditions. It also ensures that there will be enough time for the sample to exit the sample loop and make it into the LC

Software Requirements: Cycle Composer with Special Macro