Using the unique x2 dual-gradient technology, 2D-LC methods can be performed on a single system. Use the switching valves inside the NCS-3500 column compartment for on-line 2D-LC or combine the x2 dual-gradient technology with the micro fractionation option to perform off-line 2D-LC. The UltiMate^® 3000 Proteomics MDLC offers a wide flow rate range and high flow rate flexibility, allowing the system to perform an extensive range of applications, as shown in the figure below:

UltiMate 3000 Proteomics MDLC system configurations, applications, and column technology.

These 2-D solutions use the UltiMate 3000 Proteomics MDLC system to address challenging proteomics applications.

• Combination of two orthogonal chromatographic techniques, for example, IEX and RP-HPLC or high and low pH RP-HPLC
• Separation of complex samples
• High peak capacities
• Easy system control and visualization of 2D-LC data with Chromeleon software
• Simple method development and optimization
• High method flexibility

The UltiMate 3000 Proteomics MDLC system provides:

• Unique x2 dual-gradient systems that enables multidimensional separation on one system
• The advanced UltiMate 3000 well plate sampler for both injection and fractionation, providing fully automated off-line 2D-LC and protein pre-fractionation
• Nanoflow capabilities for the final analytical stage
• On-line fraction collection on MALDI targets with the Probot^™ Microfraction Collector/Spotter
• Complete range of column types for your separation needs
• Fractionation control and visualization of 2-D retention map using Chromeleon^® Chromatography Data System
• DCMS^Link™ for single-point control of UltiMate 3000 through Xcalibur^™ (Thermo Fisher), Analyst^® (AB/Sciex) and HyStar^™ (Bruker Daltonics) for easy ESI coupling
• Easy automation of separation processes, leading to higher sample throughput, more reliable results, and improved laboratory productivity

2D-LC Salt Steps

The 2D-LC salt step solution is the easy-to-use, flexible solution for moderately complex proteomics analyses. It allows 2-D separations by using the autosampler and salt injections to transfer fractions from the first to the second dimension.

The 2D-LC salt step LCi solution is based on injections of salt plugs onto an ion-exchange column. Typically, a protein digest is injected onto a strong cation-exchange column for separation in the first dimension. Peptides are eluted and separated as fractions by injecting salt plugs with increasing salt concentrations. Each fraction is subsequently separated on a reversed-phase column in the second, orthogonal dimension. The total number of salt plugs used depends on the sample complexity. This method can be fully automated. 2D-LC salt step kits are available for nano, capillary, and monolithic configurations. The same fluidic configuration also supports Proteomics Preconcentration.

Flow schematic for 2D-LC salt steps.

2D-LC Off-Line

The off-line 2D-LC solution supports the separation of highly complex proteomic samples. The advantages of off-line MDLC techniques over on-line approaches include high flexibility in column dimensions and mobile-phase compositions, as well as the ability to re-analyze sample fractions. The micro fraction collection option of the WPS-3000PL Nano/Cap Pulled Loop autosampler supports fully automated off-line 2D-LC, a unique solution for the analysis of complex proteomics samples.

The typical workflow for automated off-line MDLC involves sample injection and separation in the first dimension, collection of eluting peaks by the WPS autosampler, re-injection, and subsequent separation in the second dimension. The analytes are finally detected by UV and MS detection. Off-Line 2D-LC kits are available for peptides (SCX-RP) and proteins (WAX-RP).

Flow schematic for off-line 2D-LC.