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INTRODUCTION

Interest in ion mobility (IM) separation continues to grow and

it is now utilised over a broad range of application areas.

Over the last ten years or so there has been a notable

advance in ion mobility technology coupled with mass

spectrometry with significant increases in mobility resolution

and the prospect of additional instrument functionality [1-4].

One such system is a travelling wave (T-Wave) driven multi-

pass cyclic ion mobility (cIM) separator embedded in a Q-ToF

instrument [3]. Here we report on the preliminary design and

performance of a second-generation research platform with

enhancements to the ion optics, ToF mass analyser, detection

system and instrument control.

DESIGN AND PERFORMANCE OF A SECOND-GENERATION CYCLIC ION MOBILITY ENABLED Q-TOF Kevin Giles, Jakub Ujma, Jason Wildgoose, Martin R. Green, Keith Richardson, David Langridge, Nick Tomczyk Waters Corporation, Altrincham Road, Wilmslow SK9 4AX, UK.

Two 45mm long stacked ring ion guides (SRIGs), with axial

fields, are positioned either side of the array. These can be

used for ion transport or trapping. At the entry to the high-

pressure region there is a standard He cell. Either side of the

high-pressure region are 150mm long SRIG devices with

axial fields operating around 10-2

mb. The cIM was operated at

1.8mb of N2 and T-Waves in the range 35 to 43V at 375ms-1

.

Time-of-Flight Analyser

The new ToF analyser has an offset V/W geometry (Fig. 1),

providing 40/120cm longer flight path respectively than the

previous analyser. Further, to improve the ToF resolution vs

transmission characteristics, the standard transfer T-Wave

SRIG was replaced with a segmented quadrupole device

operating with an axial field (Fig. 2a). This quadrupole

provides optimal conditioning of the ion beam prior to entry

into the orthogonal acceleration region of the ToF. See poster

TP 391 for further description of the ToF and quadrupole

guide.

Acknowledgements

We are grateful to Bharat Chande, James Harrison, Darren Hewitt, John Iveson, John

Lewis, Witold Niklewski, Mark Pickering, Graham Scambler and Chris Wheeldon at

Waters Wilmslow for their involvement in the design and construction of the cyclic IM

research platform.

References

1. Koeniger et al, An IMS-IMS Analogue of MS-MS, Anal. Chem., 2006, 78, p4161

2. Merenbloom et al, High-Resolution Ion Cyclotron Mobility Spectrometry, Anal.

Chem., 2009, 81, p1482

3. Giles et al, Characterising a T-Wave Enabled Multi-pass Cyclic Ion Mobility

Separator, ASMS 2015 Proceedings, St Louis

4. Deng et al, Serpentine Ultralong Path with Extended Routing (SUPER) High

Resolution Traveling Wave Ion Mobility-MS using Structures for Lossless Ion

Manipulations, Anal. Chem., 2017, 89, p4628

5. Green et al, A Novel Wide Dynamic Range oa-ToF Detection System Based on

Dual 10-bit ADCs and FPGA Processing, ASMS 2016 Proceedings, San Antonio

CONCLUSION

A second-generation cyclic IM-enabled Q-Tof research platform has been built

Mobility resolution in excess of 500 (Ω/∆Ω) indicated for reverse sequence peptides SDGRG and GRGDS

Improved m/z and mobility transmission window using pre/post cyclic IM SRIGs with axial field

IMSn capability illustrated using the (M+6H)

6+ charge state of

bovine ubiquitin

m/z resolution of up to ~120,000 demonstrated with offset V/W geometry ToF analyser and dual gain ADC detection system.

OVERVIEW

Investigations into the design and characteris-

tics of a new Q-cyclic ion mobility-ToF platform based on Synapt G2-Si architecture

Mobility resolution over 500 (Ω/ΔΩ) indicated

m/z resolution up to 120,000

IMSn functionality

Increased detection system dynamic range

EXPERIMENTAL

The new platform is based on a Synapt G2-Si (ESI-Q-IM-ToF) instrument and is shown schematically in Figure 1. All samples were introduced by infusion through an ESI source.

Cyclic Ion Mobility

The new cIM arrangement is shown in Figure 2a. The cyclic device and array are the same as the original prototype instrument and provide a 100cm, single pass, mobility path length; however the new device is built from four quadrants (Fig. 2b), for ease of construction, compared to the single unit design of the first version.

RESULTS

The mobility separation characteristics of the new system

have been investigated by infusion of a mixture of the reverse

sequence peptides SDGRG and GRGDS. Figure 4a shows

the multipass separation of the (M+H)+ ions at m/z 491.2.

Figure 4b is a plot of resolution vs number of passes (n)

around the cyclic device, with the fitted curve (red)

highlighting the expected n dependence. For the data

points beyond n=18, the more mobile SDGRG ions have

caught up with the less mobile GRGDS ions in the cyclic

device and so the individual species were mobility selected

and allowed to cycle for more passes. The inset plot in Figure

4b is a composite plot of the arrival time distributions

(ATD) of the two individual species for 50 passes, from which

a resolution of ~550 is indicated.

TrapT-Wave

TransferQuad

SRIG SRIGArrayHeCell

SRIG SRIG

Cyclic IM

~1.8mb

(a)

(b)

Figure 2. (a) Mechanical drawing and outline of the cIM region. (b) Pho-

to of the cIM device and a quadrant section (inset).

Detection System

The standard Synapt detection system (8-bit, 3.0GHz

analogue-to-digital converter (ADC)) has been replaced with

a dual gain system [5], operating with two 10-bit, 3.6GHz

ADCs with independent analogue pre-amplifiers of different

gain. The combined output of the two ADCs is up-sampled to

7.2GHz and provides up to 6 decades of in-spectrum dynamic

range. This represents a 60-fold increase compared to the

standard acquisition system.

Instrument Control

The instrument control system utilised comprises a highly

configurable web-based graphical user interface for tuning, as

well as a Lua scripting environment, allowing design and

adjustment of custom acquisition modes. Each operating

mode of the cIM device is described by a short segment of

XML detailing the voltages to be applied and a duration. A full

cyclic sequence consists of a block of these XML segments

which can be embedded in a Lua script (Fig. 3).

Figure 3. XML code providing the IM sequence: inject(0)/separate(3)/

eject-detect(7). Times are specified in ms, other settings in volts.

The prototype cIM instrument utilised a long, high-pressure,

post-mobility T-Wave transfer device which required specific,

mobility range dependent, wave settings to maintain temporal

fidelity. The second generation system has a much shorter

SRIG with axial field. Figure 5 shows the comparative

transmission windows for the two systems. The high mobility

(and low m/z) cut off in Figure 5a is due to the fixed amplitude

T-Wave rejecting these ions.

The multifunction capability of the cIM is illustrated in Figure

6. Here the (M+6H)6+

charge state of bovine ubiquitin has

been m/z selected for mobility separation. Figure 6a shows

the 1 pass separation for the non-activated and pre-cIM

collisionally activated ions. Figure 6b shows the sequence of

1 pass separation with a 1ms segment of the separated ions

being ejected and stored in the pre-array SRIG, followed by

the 1 pass separation of these ions following re-injection to

the cIM. Figure 6c shows an expanded view the 1 pass

separation of the segment re-injected under both non–

activating and activating conditions. The activated species

(generated by collisional activation on re-entry to the array)

has an ATD close to that in Figure 6a but in this case

originates from a sub-section of precursor ions.

JU_20161202_alpha_MM _highmz_1spin.raw : 1

JU_20161114_beta_M M_1spin.raw : 1

100 500 1000 1500m/z

0

5

10

Dri

ft T

ime

(ms)

0

5

10

Dri

ft T

ime

(ms)

Inte

nsi

ty

0

1

Inte

nsi

ty

0

1

Figure 5. Plots of m/z and arrival time vs m/z for a tuning solution (a)

prototype cyclic instrument (b) latest generation cyclic platform.

(a)

(b)

0 40 80 120 160 200 240 280 320 360 400

Nor

mal

ised

Inte

nsity

Arrival Time (Bins)

0 20 40 60 80 100 120 140 160 180 200

Nor

mal

ised

Inte

nsity

Aligned Arrival Time (Bins)

0 20 40 60 80 100 120 140 160 180 200

Nor

mal

ised

Inte

nsity

Arrival Time (bins)

Ubiquitin 6+Not activatedActivated

1.0 ms Segment

Segment1 pass

(a)

(b)

(c)

Raw output

m/z1425 1426 1427 1428 1429 1430 1431 1432

%

0

100

Figure 6. Arrival Time plots for Ubiquitin (M+6H)6+

(a) single pass sepa-

ration (b) 1 ms segment selected from single pass ions followed by a sin-

gle pass on the ions from the segment (IMS2) (c) expanded view of se-

lected segment plot, with and without pre-separation activation.

0 10 20 30 40 50

0

100

200

300

400

500

600

Re

so

lutio

n

Number of Passes

Model Allometric1

Equation y = a*x^b

Plot Resolution

a 77.58079 ± 1.11603

b 0.50458 ± 0.0045

Reduced Chi-Sqr 26.51152

R-Square(COD) 0.99854

Adj. R-Square 0.99847

460 465 470 475 480 485 490 495 500

Time (ms)

SDGRG (1+) GRGDS (1+)

(a)

(b)

Figure 4. Mobility separation of singly charged SDGRG and GRGDS

ions (a) ATD vs number of passes, (b) resolution vs number of passes.

Inset are individual mobility spectra obtained for 50 passes.

m/z 491.2

SDGRG (1+)205.3 Ų

GRGDS (1+)208.5 Ų

ResolutionΩ/∆ Ω

71

104

149

219

317

Figure 1. A schematic diagram of the Q-cIM-ToF platform.

SourceQuadrupole

Cyclic IM

ToFAnalyser

Ion optics

a

b

c d

a - pusherb - V reflectronc - W reflectrond - detector

Figure 7. High resolution data. 2D plot of arrival time vs m/z for the

(M+6H)6+

charge state of bovine ubiquitin (collisionally activated).

30 35 40 45 50

Arrival Time (ms)

Ubiquitin 6+

(mw 8559.6)

2 Passes

m/z res

~120,000

Mobility res ~100

(for 1+ species)

JU_20170518_Ubi6_highres_2passe s_act_repeat.raw : 1

1427.5

1428

1428.5

1429

1429.5

m/z

The longer ToF analyser and dual gain ADC detection system

provide higher m/z resolution and higher dynamic range. Fig-

ure 7 is a plot showing data acquired in the ‘W’ mode of

ToF operation and 2 passes around the cyclic IM device. A

maximum m/z resolution of ~120,000 hass been obtained in

the ‘W’ mode and ~65,000 in the ‘V’ mode.