Conductive Housing(HV Contact)

Separation Capillary

Electrospray Tip

Protective retractable cover

Static Conductive Liquid Inlet

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Transient Isotachophoresis (t-ITP)- The BGE is a well-known terminating electrolyte (CH3COOH)- Sample diluted in ammonium acetate (ammonium as LI)

+ -LIS4S3S2S1

Transient formation of a conductivity gradientAll boundaries are stabilizedLarge sample zones can be injected

What are the Accessible Flow Rates?

The union of capillary electrophoresis (CE) with electrospray ionization – mass spectrometry has great potential as the fundamental properties of each technique are ideally complementary to each other. While CE separations are usually performed at very low flow rates, the ESI process is known to provide enhanced performance with increased sensitivity and decreased ion suppression at these low flow rates. To take advantage of these fundamental properties, we have designed a robust sheathless interface which through a porous region at the tip allows for the generation of stable electrospray ionization with flow rates ranging from below 10 nL/min to >340 nL/min. This flow range enables the use of CE-MS in either the mass or concentration-sensitive regions of the ESI process. Sheathless coupling further conserves the resolution gained by capillary electrophoresis, byeliminating the analyte dispersion that results from stepping up the low flow generated by electoendosmosis to a substantially higherpressure driven make-up flow utilized in sheathflow approaches. In this presentation we assess the potential of this platform for the analysis of peptide samples of increasing complexity. Particularattention has been dedicated to parameters such as sensitivity and peak capacity. To increase the mass loading abilities of the platform, various online preconcentration methodologies have been integrated, providing concentration sensitivity down to the low picomolar level while achieving peak capacities above 300.

Abstract

Sheathless Interfacing of CE and ESI-MS through a Porous Tip

Implementation

Experimental Conditions for CE-ESI-MSPA 800 plus Capillary Electrophoresis system30 mm ID bare fused, positively or neutrally coated capillaries (85<L<100 cm)Inlet OD: 150 μm, outlet OD ~40 μm 10% acetic acid (pH=2.2) or 0.1% formic acid as background electrolytesElectric field ranging from 300 to 350 V/cmGenerated current between 1 and 5 mASample introduction: hydrodynamic or electrokinetic injection

Bruker UHR-QTOF MaXis Distance between porous tip and MS from 3 to 8 mmESI voltage between 750 - 1750 VWide scanning range

Stable spray between 4 nL/min and 330 nL/min Two different behaviors observed• “Concentration sensitive:” flow rate >30 nL/min• “Mass sensitive:” flow rate <30 nL/min

Infusion of an Angiotensin 1 solution (2 μM in 10% acetic acid)

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Base peak electropherogram

Bare fused silica capillary, 10% acetic acid BGE

6 nL injected (1% of the total capillary volume)

6 fmol of BSA tryptic digest injected (1 μM)

Depending on the peptide: 2 nM < LOD < 10 nM (12 - 60 amol injected)

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CZE Separation

CE is a miniaturized technique

Separation capillary – open tube of Vtot <1 μL

Classical Capillary Zone Electrophoresis

Sample volume ≤1% Vtot

Purpose of integrating preconcentration techniques

Increase mass loading without decreasing peak efficiency

Increase of Mass Loading

Conductivity

Electric Field

Depending on the peptide: 70 pM < LOD < 210 pM (14 - 42 amol injected)

EIEs of 3 peptides obtained by analyzing a BSA tryptic digest. Injection volume = 200 nL (~30% Vtot , 250 amol injected). BSA tryptic digest concentration = 1.25 10-9 mol.L-1

t-ITP-CZE SeparationESI voltageStainless steel cylinder filled

with BGEBGE

Porous section

Inlet for filling the stainless steel cylinder

with BGE

ESI voltageStainless steel cylinder filled

with BGEBGE

Porous section

Inlet for filling the stainless steel cylinder

with BGE

Increase of Peak Capacity in Bare Fused Silica Capillaries

Use of vacuum in a bare fused silica capillary(Tryptic digest of BSA at 20 nM)

Experimental Conditions: Vacuum applied at the inlet was varied as follow. (A) 0 psi. (B) 0.5 psi. (C) 1 psi. (D) 1.5 psi. (E) 2 psi. Injection volume = 200 nL (~30% Vtot)

Vacuum at the inlet can be used to tune peak capacity without affecting the achievable efficiency

Use of Neutral Capillaries

Sample: E. coli tryptic digest at 0.5 mg/mL in 50 mM LE

42 nL, 6.2% Vtot, 21 ng loaded,

+ 34.5 mbar at the inlet (4.2 nL/min following Poiseuille)

15 peptides arbitrarily chosen migrating between 26 min and 79 minAverage peak width at half peak height of 11 seconds, 60 min separation window

Peak Capacity > 320

ConclusionThe intrinsic properties of the interface allows the operation of the system either in the concentration-sensitive or mass-sensitive range of the ESI process, resulting in the achievement of exquisite sensitivities. Preconcentration technique such as t-ITP can easily be integrated to improve the mass loading of the platform. Sample plugs as long as one third of the separation capillary (>200 nL) can be used without significantly affecting the achievable resolution. Depending on whether t-ITP is used or not, the platform provides concentration limits of detection in the low nanomolar to subnanomolar range, respectively. Finally, it was demonstrated that the intrinsic properties of the interface even permit the use of non-charged neutral capillary coatings that exhibit only minute EOF. With this approach we demonstrated that a very high peak capacity can be reached together with a very high sensitivity, ideal for the analysis of very complex samples only available in minute amounts

Enhancing Sensitivity and Peak Capacity with Sheathless CE-ESI-MSJean-Marc Busnel1, Bart Schoenmaker2, Rawi Ramautar2, Alegria Carrasco-Pancorbo2, Chitra Ratnayake1, Jerald S. Feitelson1

Jeff D. Chapman1, André M. Deelder2, Oleg A. Mayboroda2

1-Beckman Coulter, Inc., Brea, CA 92822, USA

2-Biomolecular Mass Spectrometry Unit, Leiden University Medical Center, The Netherlands