Over the past few years, structures for lossless ion manipulations (SLIM) have used traveling waves (TWs) to move ions over long serpentine paths that can be further lengthened by routing the ions through multiple passages of the same path. Such SLIM "multipass" separations provide unprecedentedly high ion mobility resolving powers but are ultimately limited in their ion mobility range because of the range of mobilities spanned in a single pass; that is, higher mobility ions ultimately "overtake" and "lap" lower mobility ions that have experienced fewer passes, convoluting their arrival time distribution at the detector. To achieve ultrahigh resolution separations over broader mobility ranges, we have developed a new multilevel SLIM possessing multiple stacked serpentine paths. Ions are transferred between SLIM levels through apertures (or ion escalators) in the SLIM surfaces. The initial multilevel SLIM module incorporates four levels and three interlevel ion escalator passages, providing a total path length of 43.2 m. Using the full path length and helium buffer gas, high resolution separations were achieved for Agilent tuning mixture phosphazene ions over a broad mobility range (K₀ ≈ 3.0 to 1.2 cm²/(V*s)). High sensitivity was achieved using "in-SLIM" ion accumulation over an extended trapping region of the first SLIM level. High transmission efficiency of ions over a broad mobility range (e.g., K₀ ≈ 3.0 to 1.67 cm²/(V*s)) was achieved, with transmission efficiency rolling off for the lower mobility ions (e.g., K₀ ≈ 1.2 cm²/(V*s)). Resolving powers of up to ∼560 were achieved using all four ion levels to separate reverse peptides (SDGRG¹⁺ and GRGDS¹⁺). A complex mixture of phosphopeptides showed similar coverage could be achieved using one or all four SLIM levels, and doubly charged phosphosite isomers not significantly separated using one SLIM level were well resolved when four levels were used. The new multilevel SLIM technology thus enables wider mobility range ultrahigh-resolution ion mobility separations and expands on the ability of SLIM to obtain improved separations of complex mixtures with high sensitivity.

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