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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 N2 pressures and higher

T-Wave pulse amplitudes were required compared to those on

a standard Synapt instrument. Increasing the N2 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 N2 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 N2

drift tube at atmospheric pressure1. 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 N2 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 In

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 N2 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)nn+ has been demonstrated for various peptides in-

cluding Bradykinin2. 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)33+ species.

Figure 4 m/z vs arrival time and m/z 1061 mobility chroma-togram (a) Synapt at 0.5mb N2 and a 300m/s 9V wave (b) Synapt G2 at 2.5mb N2 and a 600m/s 40V wave.

B K_10.raw : 1

K G_B K_01. raw : 1(a)

(b)

4

B K_10.raw : 1

K G_B K_01. raw : 1(a)

(b)

4

(M+H)33+

(M+H)22+

(M+H)11+

(M+H)33+ (M+H)2

2+

(M+H)11+

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 N2 and a 300m/s 9V wave (b) Synapt G2 at 2.5mb N2 and a 800m/s 40V wave.

0 2 4 6 8 10 12

Arrival Time (ms)

Nor

mal

ised

Inte

nsity

(%)

0 2 4 6 8 10 12

Arrival Tim e (m s)

Nor

mal

ised

Inte

nsity

(%)

m/z 246 333 786 480 684 812 942 1056 1171 1285

0 2 4 6 8 10 12

Arrival Time (ms)

Nor

mal

ised

Inte

nsity

(%)

0 2 4 6 8 10 12

Arrival Tim e (m s)

Nor

mal

ised

Inte

nsity

(%)

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 2x10-2mb. 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 N2. The standard Synapt was operated

with ~0.5mb of N2 in the IMS cell and the Synapt G2 with

~2.5mb N2. 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 N2 and a 300m/s 6V wave (inset are the data for the peptides run individually) (b) Synapt G2 at 2.5mb N2 and a 1300m/s 40V wave.

0

20

40

60

80

100

1.5 2 2.5 3 3.5 4 4.5

Arrival Time (ms)

Rel

ativ

e In

tens

ity

(%

0

20

40

60

80

100

2 2.5 3 3.5 4 4.5 5

Arrival Time (ms)

Rel

ativ

e In

tens

ity (%

(GRGDS)2+

211.7 Ų(SDGRG)2+

222.7 Ų

(a)

(b)

1.5 2 2.5 3 3.5 4 4.5

0

20

40

60

80

100

1.5 2 2.5 3 3.5 4 4.5

Arrival Time (ms)

Rel

ativ

e In

tens

ity

(%

0

20

40

60

80

100

2 2.5 3 3.5 4 4.5 5

Arrival Time (ms)

Rel

ativ

e In

tens

ity (%

(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/z200 400 600 800 1000 1200

%

0

100

%

0

100KG_ADC_01 37 (0.806) TOF MS ES+

2.87e6430.9138226.9514

203.0322

362.9269

838.8392702.8635*566.8892

498.9016 634.8764

770.8513 906.8262 974.8135

1042.8010

1110.7886

1178.7756

KG_ADC_02 38 (0.689) TOF MS ES+ 1.89e6226.9522

203.0324

430.9147

362.9276

*566.8892

498.9020

702.8642

634.8767838.8397

906.8271 974.8143

1042.80201110.7887

1178.7767

Non-M obility

Mobility

m/z200 400 600 800 1000 1200

%

0

100

%

0

100KG_ADC_01 37 (0.806) TOF MS ES+

2.87e6430.9138226.9514

203.0322

362.9269

838.8392702.8635*566.8892

498.9016 634.8764

770.8513 906.8262 974.8135

1042.8010

1110.7886

1178.7756

KG_ADC_02 38 (0.689) TOF MS ES+ 1.89e6226.9522

203.0324

430.9147

362.9276

*566.8892

498.9020

702.8642

634.8767838.8397

906.8271 974.8143

1042.80201110.7887

1178.7767

Non-M obility

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 8 3 8 .8

KG_ADC_02.raw : 1

m /z 8 3 8 .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 634.8766ppm -1.37 -2.83 -0.22 0.03 -1.19 -0.28 0.07 -0.29ppm -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 1178.7760ppm -0.73 -0.17 0.41 -0.09 -0.21 -0.12 0.04 -0.32ppm 0.27 0.48 1 0.9 0.61 0.83 0.13 0.61