TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS Figure 1 A schematic diagram of the Synapt G2 HDMS instru- ment INTRODUCTION One of the more recent developments in ion mobility (IM) separation technology has been the travelling wave-based approach used in a commercially available hybrid quadrupole/ IM/oa-ToF instrument (Synapt HDMS, Waters Corp.). Whilst this technology has provided greater access to the potential of ion mobility spectrometry in combination with mass spectrometry for many analysts, the mobility resolution afforded by the travelling wave separator is relatively low in comparison with current, albeit significantly larger, instruments in some research laboratories. What is presented here is a novel approach towards increasing the mobility resolving power of the travelling wave device together with a description of an enhanced detection system for acquiring mobility data. Kevin Giles , Tony Gilbert, Martin Green and Garry Scott Waters MS Technologies Centre, Manchester, UK The trap T-Wave accumulates ions whilst the previous mobility separation occurs, then the ions are released in a packet into the IMS T-Wave where mobility separation is performed. The transfer T-Wave delivers the mobility separated ions to the oa- ToF mass analyser. Ion arrival time (or drift time) distributions are recorded by synchronising the oa-ToF mass spectral acquisitions with the gated release of ions from the trap T-Wave device. The T-Wave mobility separator uses a repeating train of DC pulses to propel ions through the gas- filled cell in a mobility dependent manner. To provide increased mobility resolution, higher N 2 pressures and higher T-Wave pulse amplitudes were required compared to those on a standard Synapt instrument. Increasing the N 2 pressure alone in the IMS cell is problematic since driving the ions into this device then requires higher voltages which can lead to unacceptable losses through scattering and/or fragmentation. The solution to this was to employ a high pressure Helium- filled cell on the front of the IMS cell to provide a low loss interface to the high N 2 pressure region, see Figure 2. References (1) C Wu, W.F. Siems, J. Klasmeier and H.H. Hill, Anal. Chem. 72 (2000) 391 (2) AE Counterman , SJ Valentine, CA Srebalus, SC Henderson, CS Hoaglund, DE Clemmer, J.Am. Soc. Mass Spectrom., 9 (1998) 743 RESULTS AND DISCUSSION Inverse Sequence Peptides The inverse sequence peptides GRGDS and SDGRG (mw 490) have been studied previously and the doubly charged molecular ion species of each at m/z 246 were determined to have a difference in collision cross section (Ω) of 5.2% in an N 2 drift tube at atmospheric pressure 1 . In Figure 3, the mobility separation attained on the standard IMS cell and the new high pressure cell are shown for a mixture of the two peptides. CONCLUSION A new high resolution Travelling wave IMS cell operating at N 2 pressures of ~2.5mb with a high pressure Helium- filled entry cell has been described Mobility resolution increases of 3-4X are achieved over the standard IMS cell in a Synapt instrument Near baseline mobility separation achieved on doubly charged isomeric peptide doublet with 5% difference in collision cross-section indicating Ω/∆Ω of ~45 Use of a new ADC-based detection system illustrating high dynamic range in mobility mode of acquisition Glu-Fibrinopeptide B Fragmentation The doubly charged precursor ion of glu-fibrinopeptide b at m/z 785.6 was selected using the quadrupole mass filter and fragmented in the trap T-Wave collision cell. The fragments were then separated by mobility in the IMS T-wave cell. Figure 5 shows the resulting total ion current arrival time distribution and those of selected fragments from the standard mobility cell and the new mobility cell. Again, highlighting the increased mobility resolution. ENHANCEMENTS TO THE ION MOBILITY PERFORMANCE OF A TRAVELLING WAVE SEPARATION DEVICE OVERVIEW Mobility resolution improvements on a travelling wave separator and an en- hanced detection system Studies undertaken on a Synapt G2 HDMS instrument Mobility resolution increase of 3-4x and increased dynamic range with an ADC- based detection system Figure 2 A schematic diagram of the new T-Wave IMS cell with the Helium-filled entry cell Helium Nitrogen Helium Out Nitrogen Out Ions Out Ions Helium Cell T-Wave IMS Cell Stacked Ring Ion Guide Helium Nitrogen Helium Out Nitrogen Out Ions Out Ions In Helium Cell T-Wave IMS Cell Stacked Ring Ion Guide Using the helium cell, working pressure of up to 2.5mb of N 2 in the IMS were possible, compared to the 0.5mb typically used on a standard Synapt IMS cell. The increase in mobility resolution poses significant issues for the TDC-based detection system used on a standard Synapt, reducing the available dynamic range for both intensity and mass accuracy. Whilst moving to an ADC-based detection system was expected to alleviate these issues, no commercially available systems were capable of dealing with the demands of recording IMS arrival time data. Consequently a completely new ADC system was designed and built in- house. The ADC system comprises an 8-bit ADC sampling at 3Ghz feeding to a field programmable gate array for signal processing and subsequently to a block of memory for accumulating the 200 sequential mass spectra that form the mobility arrival time spectrum. Summing of the mobility data is performed on the ADC card and ultimately transferred to the acquisition PC after each ‘scan’ period. With the new, higher pressure mobility cell, the mobility reso- lution (Ω/∆Ω) has increased to ~45 from the resolution of ~11 for the standard mobility cell. Bradykinin Multimers The formation of multiply charged multimeric species of the form (M+H) n n+ has been demonstrated for various peptides in- cluding Bradykinin 2 . The first isotopes of these species have the same m/z value but can be separated by their differences in mobility. Figure 4 shows the mobility separation of the nominal m/z 1061 ion species of Bradykinin using the standard mobility cell and the new, high pressure cell. With the new, higher pressure mobility cell, the mobility reso- lution (Ω/∆Ω) has increased by approximately a factor of three over the standard mobility cell. In addition, these data illustrate that even at five times the pressure of the standard cell, the new design with the helium cell is able to efficiently transmit the relatively labile (M+H) 3 3+ species. Figure 4 m/z vs arrival time and m/z 1061 mobility chroma- togram (a) Synapt at 0.5mb N 2 and a 300m/s 9V wave (b) Synapt G2 at 2.5mb N 2 and a 600m/s 40V wave. (a) (b) 4 (a) (b) 4 (M+H) 3 3+ (M+H) 2 2+ (M+H) 1 1+ (M+H) 3 3+ (M+H) 2 2+ (M+H) 1 1+ Figure 5 Mobility separation of the fragment ions of the dou- bly charged m/z 785.6 ion of glu-fibrinopeptide b (a) Synapt at 0.5mb N 2 and a 300m/s 9V wave (b) Synapt G2 at 2.5mb N 2 and a 800m/s 40V wave. 0 Normalised Intensity (%) 0 2 4 6 8 10 12 Arrival Time (ms) Normalised Intensity (%) 0 Normalised Intensity (%) 0 2 4 6 8 10 12 Arrival Time (ms) Normalised Intensity (%) m/z 246 333 786 480 684 812 942 1056 1171 1285 TIC TIC (a) (b) ADC-based Detection System A solution of sodium formate was infused into the electrospray source of the Synapt G2. Acquisitions were made in both non- mobility and mobility mode. The mass spectra in Figure 6 are summed mass spectra over 30 scans with a single point internal lock mass correction using the m/z 566.8892 ion. The m/z differences between the calculated and measured m/z values are presented in Table 1. As can be seen there is excellent agreement with the calculated values for both data sets. Figure 7 shows the mobility data for the sodium formate sample. The mobility arrival time peaks for single m/z species are approximately 3 ToF pushes (mass spectra) wide and the ion concentration in these peaks is around 66X (200/3) that of the non-mobility mode. From the acquired data, the m/z 430.9 ion flux in non-mobility mode is calculated to be around 0.2 ions per push (3000 counts/s), consequently, in mobility mode this increases to 13 ions per push in the mobility peak. METHODS Instrumentation The instrument used in these studies was a Synapt G2 HDMS instrument (Waters Corp.), shown in Figure 1, which has a hybrid quadrupole/IM/oa-ToF geometry. Ions are generated using an electrospray ionisation source and pass through a quadrupole mass filter to the IM section of the instrument which contains three travelling wave (T-Wave) ion guides. Experimental All samples were infused into the electrospray source at 5μL/ min. The source conditions were tuned for each sample to give best signal. The trap and transfer T-Wave cells were operated with Argon at a nominal pressure of 2x10 -2 mb. The helium cell performance was optimised with a resultant gas flow of 200mL/ min (pressure not measured directly), approximately twice the main IMS cell flow of N 2 . The standard Synapt was operated with ~0.5mb of N 2 in the IMS cell and the Synapt G2 with ~2.5mb N 2 . T-Wave settings are listed for each experiment. Figure 3 The arrival time distributions of the inverse pep- tides at m/z 246. (a) Synapt at 0.5mb N 2 and a 300m/s 6V wave (inset are the data for the peptides run individually) (b) Synapt G2 at 2.5mb N 2 and a 1300m/s 40V wave. 0 20 40 60 80 100 1.5 2 2.5 3 3.5 4 4.5 Relative Intensity (% 0 20 40 60 80 100 2 2.5 3 3.5 4 4.5 5 Arrival Time (ms) Relative Intensity (% (GRGDS) 2+ 211.7 Ų (SDGRG) 2+ 222.7 Ų (a) (b) 1.5 2 2.5 3 3.5 4 4.5 FWHM ~0.09ms m/z 200 400 600 800 1000 1200 % 0 100 % 0 100 430.9138 226.9514 203.0322 362.9269 838.8392 702.8635 * 566.8892 498.9016 634.8764 770.8513 906.8262 974.8135 1042.8010 1110.7886 1178.7756 226.9522 203.0324 430.9147 362.9276 * 566.8892 498.9020 702.8642 634.8767 838.8397 906.8271 974.8143 1042.8020 1110.7887 1178.7767 Non-Mobility Mobility m/z 200 400 600 800 1000 1200 % 0 100 % 0 100 TOF MS ES+ 2.87e6 430.9138 226.9514 203.0322 362.9269 838.8392 702.8635 * 566.8892 498.9016 634.8764 770.8513 906.8262 974.8135 1042.8010 1110.7886 1178.7756 TOF MS ES+ 1.89e6 226.9522 203.0324 430.9147 362.9276 * 566.8892 498.9020 702.8642 634.8767 838.8397 906.8271 974.8143 1042.8020 1110.7887 1178.7767 Non-Mobility Mobility Figure 6 Summed mass spectra for sodium formate (a) with- out mobility separation (b) with mobility separation (a) (b) Figure 7 Mobility chromatogram and m/z vs arrival time for sodium formate KG_ADC_02.raw : 1 m /z 838.8 KG_ADC_02.raw : 1 m /z 838.8 Table 1 Mass measurement accuracy for sodium formate: non- mobility data and mobility data m/z 158.9646 226.9520 294.9395 362.9269 430.9143 498.9017 566.8892 634.8766 ppm -1.37 -2.83 -0.22 0.03 -1.19 -0.28 0.07 -0.29 ppm -0.11 0.7 -1.24 1.96 0.9 0.53 0.07 0.18 m/z 702.8640 770.8514 838.8389 906.8263 974.8137 1042.8011 1110.7886 1178.7760 ppm -0.73 -0.17 0.41 -0.09 -0.21 -0.12 0.04 -0.32 ppm 0.27 0.48 1 0.9 0.61 0.83 0.13 0.61