WO2015197072A1

WO2015197072A1 - Electrical contacts for ce-ms interfaces based on generating cracks/fractures along the capillary

Info

Publication number: WO2015197072A1
Application number: PCT/DK2015/050178
Authority: WO
Inventors: Nickolaj PETERSEN
Original assignee: Københavns Universitet
Priority date: 2014-06-23
Filing date: 2015-06-22
Publication date: 2015-12-30

Abstract

There is provided an electrical contact fora CE-MS interface. The contact is obtained by establishinga fractureor crack less than 2 micrometer in dimension(width)close to the spray tip. A reservoir is provided in liquid contact with the crack/fracture and filled with a reservoir liquid, and an electrode is placed in contact with the reservoir liquid. Several cracks/fractures can be created with their respective reservoirs. Beside providing the electrical contactfor the electrophoretic separation and/or electrospray ionization,the crack/fracturecan be used for electrokinetically introducing reagents or analytes into the capillary, for example, for providing deuterium for hydrogen-deuterium exchange (HDX) experiments.

Description

Electrical contacts for CE-MS interfaces based on generating cracks/fractures along the capillary

FIELD OF THE INVENTION

The present invention relates to electrical contacts for CE-MS interfaces. Specifically, the invention is based on the generation of cracks/fractures along the capillary of a CE-MS system. Fractures that are having dimensions of less than 2 μηι in width, have unique properties on the electrokinetic transport, thus generating field free pumping in the capillary towards the ESI tip.

BACKGROUND OF THE INVENTION

Interfacing capillary electrophoresis (CE) with mass spectrometry (MS) was first demonstrated back in 1987[1]. Since then, several different designs of CE-MS interfaces have been proposed. However, still today CE-MS is not used as a routine technique in laboratories worldwide, mainly due to the stability issues of the electrical contact required for the CE separation and the stability of the electrospray ionization (ESI). The CE-MS interfaces are difficult to make reproducible or they require complicated fabricating procedures, thereby increasing the cost of analysis.

One of the main difficulties of constructing an ideal interface is the requirement of a stable electrical contact close to or at the terminus of the capillary. The electrical contact is needed for both an electrophoretic separation (typically operated with currents in the range of 1-300 μΑ) and for providing high voltage across the ESI tip for electrospray ionization. The electrical contact should preferably not induce any band broadening of the separated analytes as due to, for example, dead volumes in junctions at the spray tip.

Traditional electrical contacts at the outlet of the CE-MS capillary and for the ESI are made by large (typically 10-30 μηι) junctions close to the end of the separation capillary used in liquid junction interfaces [2] or in the split flow interfaces [3]. The junction filled with buffer provides the required electric contact to an external reservoir containing the electrode. When applying the separation voltage, the separated analytes inside the capillary as well as the bulk flow of buffer inside the capillary are migrating into the reservoir of the side channel of a traditional dimension, where the electrical contact is provided, instead of towards the ESI spray tip. This is because the electro kinetic transport of the analytes, as well as the bulk flow (electroosmotic flow), follows the applied electrical field. The operation of CE-MS devices having electrical contacts based on gaps of 10-30 μηι, therefore, requires the application of an external pressure drop across part of the capillary, to pump at least a fraction of the separated analytes towards the ESI spray tip. The amount of sample migrating into the side channel (reservoir containing the electrode) can be minimized by either applying an external pressure across the junction reservoir and the ESI tip (Liquid junction interfaces [2]) or by applying a co-pressure in the separation capillary as used for split flow interfaces [3]. Appling the external pressure, however, induces band broadening, thereby minimizing the separation efficiency. Also, the influence of the pressure on the separation performance is highly dependent on the size of the junction, making it difficult to obtain repeatable migration times and intensities from device to device. One way to minimize the sample loss into the side channel without applying external pressure is by reversing the electroosmotic flow (EOF) in the side channel by coating procedures [4]. This approach requires coating procedures and the coating may degrade over time. Also, the electrical contact has been achieved by etching porous junctions in the separation capillary. The porous junctions of 1 mm to several cm are made by etching the fused silica capillary in hydrofluoric acid to a thickness of typically 30-50 μηι, allowing electric conductance across the thin porous glass wall (U.S. Pat. No. 7,544,932, Janini et al. Jan 2009). Different variations of porous junctions for improving the mechanical stability are disclosed in the patents U.S. Pat. No. 8,198,586 B2, Moini, June 12 2012, and U.S. Pat. No 8.449,746 B2, Jarrel, May 28 2013. Disadvantage: The fabrication of the porous junctions requires the etching to be monitored and stopped at the right depth. Also, the mechanical stability is not good. The porous junctions have limited conductivities and may not allow high current separations. Coated capillaries may further disrupt the electric contact of the porous junction. Another way to minimize sample and liquid loss into the side channel/junction can be obtained by applying an ion permeable polymer or conductive gel at the intersection. The gel or polymer is able to conduct the current and provide resistance to the bulk liquid flow, as described in U.S. Pat. No. 5, 169,510, Lunte et al. Dec 8, 1992 and EP 0576361 A2, Nakano Vinegar Co., Ltd, 29 Sep 1993.

Hence, there is a need for improved electrical contacts for CE/ESI systems that can be fabricated in a simple way, allowing high current separations and with minimal induced band broadening, having no requirement for surface modification or application of external pressure.

SUMMARY OF THE INVENTION

The present inventor has found a simple way of providing electric contact for CE-ESI- MS based on forming a crack/fracture along the capillary, whereby achieving a surprising effect of field-free pumping. Furthermore, the inventor has found that a single fracture/crack even with submicron width is able to conduct currents op to at least 300 μΑ, and the crack/fracture does not disturb the separation due to the perfect alignment of the capillary before and after the crack. The submicron dimensions of the fracture/crack generate field-free pumping towards the MS due to surface conductivity effects, that has been investigated in detail by the inventor using micro- and nanofabricated devices [5]. The same pumping effects that can be obtained on advanced microfabricated devices when combining micro- and nanochannels, can easily be achieved in conventional CE capillaries by applying the electric potential across a fracture having openings less than 2 μηι in width in conjunction to the standard separation capillaries typically having ID of 20-100 μηι. This unique property is simply related to the increased surface-to-volume ratio of the fracture compared to that of the separation capillary.

Prior art US5169510 discloses an electrical contact for a CE connected to a MS based on a fracture in the capillary, but in this document, the fracture is covered by a flexible, polymeric ion-permeable membrane. The capillary is scored, up to 5 cm, from the detection end of the tube, which is covered by a Nafion sleeve. The capillary is then broken at the scored region, creating a pair of two closely adjacent aligned tube sections in order to create a perfectly aligned joint. In their approach, the Nafion membrane works as an ion permeable membrane that provides the electric contact, but will not allowing bulk liquid to pass. In the patent US5169510, there is no disclosure of a crack / fracture of less than 2 micrometer, and there is no indication that a field-free pumping can be generated towards the MS without the Nafion membrane. Page 2 line 40-43 in the patent states that "An important feature of the invention resides in the fact that the joint cover is formed of flexible polymeric material which is ion-permeable and of low electrical resistance".

Another patent EP0576361 describes forming a fracture using e.g. a capillary cleaving tool for providing electric contact and covering the fracture with polyacrylamide gel containing an electrolytic buffer solution. Meanwhile, there is no disclosure of a crack / fracture of less than 2 micrometer, and there is no indication that a field-ree pumping can be generated without the polyacrylamide gel. In this patent, the polyacrylamide gel contains an electrolytic buffer solution that is able to provide the electric contact while preventing bulk liquid flow out of the fracture.

The inventor has also envisaged that the crack/fracture can be used for electrokinetically introducing sample or reagents into the separation capillary allowing e.g. on-column hydrogen deuterium exchange (HDX) and 2D separations in a simple way.

Accordingly, in one aspect the present invention provides a CE / ESI capillary assembly comprising: i) a CE / ESI capillary having a crack or fracture at the spray end of the capillary; ii) a reservoir provided in liquid contact with the crack or fracture and filled with a reservoir liquid; and iii) an electrode in contact with the reservoir liquid, wherein the width of the crack or fracture is less than 2 μηι.

In another important aspect, the present invention provides a method for preparing an electrical contact according to the first aspect of the invention. The method comprises the steps of:

Optionally, removing an external coating of the capillary by any known method, for example, by burning, etching or cutting away the coating, as the standard coatings (for example, polyimide) are not chemically and mechanically stable enough.

· Optionally, applying a heat shrinking tubing, or other coating to improve the mechanical stability of the capillary; Optionally, coating the internal surface of the capillary for separation improvement etc.

Optionally, fixing the capillary to a substrate in order to keep the parts of the capillary aligned after generating a crack/fracture.

· Generating a crack or /fracture one or several places along the separation capillary, wherein the width of the crack or fracture is less than 2 μηι.

Attaching one or more reservoirs in liquid connection with each one of the cracks or /fractures.

Filling the reservoirs with reservoir liquids, liquids can be different from reservoir to reservoir; and

Providing an electrode in contact with the reservoir liquids.

In a different aspect of the invention, several cracks/fractures can be created several places along the capillary.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a CE-MS interface, where the electric contact is applied through a fracture/crack located in the buffer reservoir.

Figure 2 shows a photo of a typical CE-MS device where the electric contact is applied through a fracture/crack located in the buffer reservoir. Figure 3 shows a close up picture of a CE-MS device seen from below.

Figure 4 shows a device for on-column hydrogen deuterium exchange, HDX, coupled with CE-MS or ESI-MS Figure 5 shows CE-MS separations using a device pictured in figures 1-3, without any applied co-pressure or pressure applied at the buffer reservoir.

Figure 6 shows CE-MS separations using a device pictured in figures 1-3. DETAILED DESCRIPTION OF THE INVENTION

A new stable electrical contact at the CE capillary outlet / ESI electrode is achieved by making one or several cracks/fractures across the capillary close to the ESI spray tip. Such a crack/fracture is functioning as a side channel that provides an electrical contact from the liquid inside the capillary to a liquid solution placed in a reservoir attached on top of the crack/fracture.

The fracture can be made in a reproducible manner by using, for example, an optical fiber cleaver. Optical fiber cleavers are designed for cleaving (breaking) optical fibers at a perfect 90° angle by applying a small stress to the optical fiber (our capillary for electrophoretic separation), followed by slightly touching the outer surface of the capillary by a cutting stone. When using such a tool for cleaving capillaries having an external coating or tubing, a perfect fracture is generated across the whole capillary, while the external coating / tubing is holding the two parts of the capillary together in their initial position resulting in perfect alignment and minimal band broadening of separated analytes. In the cleaving process the tool also cuts a opening in any applied external coating or tubing thereby providing the required electric contact through even non conducting tubing or coatings.

The mechanical stability of the device is improved by applying the external polymer coatings or tubings on the section where the fracture/crack is generated, and/or by mounting the capillary on a substrate before or after the crack/fracture has been made.

Other methods for making such fractures in a repeatable manner can be, for example, applying thermal or mechanical stress, or applying high electrical field strength locally across the capillary. A crack/fracture with a dimension of less than 2 microns possesses a high surface-to - volume ratio due to its relatively large surface area compared to its small volume. This high surface-to-volume ratio leads to a large excess of counter ions from the ionized surface (electrical double layer). These ions increases the conductivity of the liquid present in the crack/fracture [5] compared to the conductivity of the same liquid present in the CE capillary. For common CE separation buffers, this increase in conductivity is very significant in channels/cracks/fractures with one of the dimensions less than 2 μηι, due to the high surface-to-volume ratios of these channels. This increase in conductivity modifies the electro kinetic transport and leads to field free pumping [5] in the section after the fracture thereby pumping the analytes towards the ESI-MS.

Theory of the EOF pumping:

Normally, decreasing the cross sectional area of a part of the separation channel, in this case a side channel close to the ESI tip, will proportionally increase the electric resistance. This will increase the local electric fieldstrength in the narrow channel and leave the bulk electrokinetic flow independent on the changes in the channel dimensions. The whole flow is therefore still expected to migrate into the side channel instead of towards the ESI spray tip. In other patents where the electric contact also is based through a fracture in the capillary like US5169510, EP0576361 the flow is prevented by covering the fracture with an ion permeable membrane or conductive polyacrylamide gel. However, when decreasing the channel depth (here width of the fracture) to less than 2 μηι, then, the conducting properties of the ions located in the electrical double layer at the surfaces start to modify the apparent buffer conductivity [5]. Therefore, the fieldstrength no longer increases to the same proportion as the channel depth decreases. Having a side channel with increased surface-to-volume ratio will then generate a simple EOF pump, delivering the analytes towards the spraytip / ESI-MS (see Figure 1).

Referring to Figure 2 there is shown a photo of a typical CE-MS device where the electric contact is applied through a fracture/crack located in the buffer reservoir close to the ESI spray tip. Figure 3 shows a close up picture of a CE-MS device seen from below.

The pumping is achieved without the need of surface modifications of any channel segments or by covering the fracture with an ion permeable membrane [6] or conducting gel [7], but requires that the fracture be less than 2 μηι in width. The higher the surface to volume ratio of the fractures the more efficient the field free pumping.

The end of the capillary used for the ESI tip can be sharpened either by pulling using a torch or plasma [8] or by etching with hydrofluoric acid, either before or after the fracture has been prepared. The sharper tip will improve the spray stability as well as increase the sampling into the MS, since the plume is smaller and may be placed closer [9]. Referring to Figure 4 there is shown a device for on-column hydrogen deuterium exchange, HDX, coupled with CE-MS or ESI-MS.

Referring to Figure 5 there is shown a typical CE-MS separation using a device pictured in Figures 1-3, without any applied co-pressure or pressure applied at the buffer reservoir.. The CE capillary was 90 cm having a 50 μηι ID and as buffer 125 mM ammonium formate pH 4.5 was used. The applied potential was 30 kV. The sample contained 5 μg/mL of each Pethidine (m/z: 248u), nortriptyline (m/z: 264u), methadone (m/z: 310u), haloperidol (m/z: 376u), loperamide (m/z 478u). If the same analysis as shown in Figure 5 had been performed on a similar device but where the generated fracture had been slightly expanded to generate a gap of only a few μηι then no analytes could be detected with the ESI-MS due to lack of field free pumping and co-pressure was required.

Referring to Figure 6 there is shown a typical CE-MS separation of a BSA tryptic digested sample (3 μΜ) after desalting by a C-18 trap column. The CE capillary was 65 cm having a ID of 50 μηι and coated with poly(oligo(ethylene glycol)methacrylate) to minimize the integrations of the peptides with the inner surface of the capillary. A co-pressure of 50 mbar was applied since the coating also suppressed the elctroosmotic flow. Injection: 50mbar, 10s, Buffer: 0.1 % Formic acid, Voltage: 30 kV. The separation in Figure 6 where approximately 15 nL of the BSA tryptic digested sample (3 μΜ) was analyzed shows the high sensitivity of the interface. Furthermore, the separation shows that even coated capillaries could be applied however with the need of a slight co-pressure. The devices of the present invention, where the electric contact is achieved by fracture/cracks, are simple, robust and fast to prepare. The capillary before and after the crack is perfectly aligned and may be held in place by the external coating/tubing or by fixing the capillary to a substrate, for example, by gluing, before the crack/fracture is generated. Minimal band broadening is induced due to the perfect alignment of the capillary before and after the fracture. The fracture/crack is highly conductive due to the ions present in the electrical double layer, and a single fracture can conduct stable currents of at least up to 300 μΑ, which is the maximum current used for conventional instrumentation.

The fracture can provide stable electric contact for several hours required for electrophoretic separations.

The fracture/crack may also be used for operations with a co-pressure as for the split-flow interfaces, solving the problems of constructing these devices in a fast and repeatable manner. The fracture/crack is having a high fluidic resistance (backpressure) leading to none or minimal bulk flow into the reservoir.

The fracture/crack can be prepared close to the spray tip, only a few mm from the end, providing minimal band broadening. The spray tip can be drawn or etched before or after the fracture/crack has been made. The crack/fracture can be used for coupling other low flowing techniques to ESI-MS detection, since the electric contact also can be used alone for the ESI Voltage. In this way, spray tips can be pulled from glass capillaries and the electric contact for ESI can easily be established without the need for metal coatings.

The crack/fracture can even be used with coated capillaries for improving the separation performance of proteins since the crack can be created after the internal coating has been applied.

The devices may be mass-produced in a repeatable manner. No dead volume is introduced due to the perfect alignment of the channel before and after the crack. Redox reactions occur only in the reservoir, and bubble formation and pH changes do not affect separation.

In the main aspect of the invention, one of the dimensions of the crack/fracture should be less than 2 μηι. Providing the electric contact trough one or more narrow cracks/fractures having dimensions less than 2 μηι will allow the separated analytes to be pumped towards the ESI spray tip by field free pumping [5], since the conductivity of the solution in the fracture/crack is increased compared to the conductivity of the same solution when present in the separation capillary.

In another aspect of the invention, the tip is provided for ESI interface by any known method like pulling or etching, either before or after a fracture/crack is created.

The fracture/cracks are having a high fluidic resistance (backpressure) leading to none or minimal bulk flow out of the crack/fracture if applying external pressure during the separation.

For CE-MS the fracture for the ESI voltage can be prepared close to the spray tip only a few mm from the end providing minimal band broadening.

One of the main applications for the novel electrical contact disclosed in this application, is also for introducing deuterium into a capillary for on-column hydrogen deuterium exchange (HDX) of proteins, peptides and other large organic molecules in the capillary, prior to MS detection. Introducing deuterium this way allow fast diffusive mixing and easy control of the time of the exchange. Another important application is electrokinetically introducing a sample or a reagent into a separation capillary from an outside reservoir through the crack and into the capillary, which can be very useful in automated 2D separations.

Olivares, J. A., et al., Online Mass-Spectrometric Detection For Capillary Zone Electrophoresis. Analytical Chemistry, 1987. 59(8): p. 1230-1232.

Lee, E.D., et al., Liquid Junction Coupling for Capillary Zone Electrophoresis Ion Spray Mass-Spectrometry. Biomedical and Environmental Mass Spectrometry, 1989. 18(9): p. 844-850.

Moini, M., Design and performance of a universal sheathless capillary electrophoresis to mass spectrometry interface using a split-flow technique. Analytical Chemistry, 2001. 73(14): p. 3497-3501.

Mellors, J.S., et al., Fully integrated glass microfluidic device for performing high-efficiency capillary electrophoresis and electrospray ionization mass spectrometry. Analytical Chemistry, 2008. 80(18): p. 6881-6887.

Petersen, N.J., et al. Study of interface conductivity and its possible applications, in Micro Total Analysis Systems 2004. 2004. Malmo, Sweden: The Royal society of Chemistry.

Lunte, S.M., C.E. Lunte, and T.J. O'Shea, Ion-permeable polymer joint for use in capillary electrophoresis, US516951 O A, Dec 8, 1992.

Fujimoto, C.G.T., Eelectrophoretic electrode, method of/and system for capillary electrophoresis using the electrophoretic electrode and fraction collector assembled into the capillary electrophoresis system, EP0576361 A2, 29 Dec 1993.

Ek, P. and J. Roeraade, New Method for Fabrication of Fused Silica Emitters with Submicrometer Orifices for Nanoelectrospray Mass Spectrometry. Analytical Chemistry, 201 1. 83(20): p. 7771-7777.

Wilm, M.S. and M. Mann, Electrospray and Taylor-Cone Theory, Doles Beam of Macromolecules at Last. International Journal of Mass Spectrometry, 1994. 136(2-3): p. 167-180.

Claims

1. An electrical contact for CE and ESI comprising:

• a coated or non-coated CE / ESI capillary having a crack or fracture at the spray end of the capillary;

• a reservoir provided in liquid contact with the crack or fracture and filled with a reservoir liquid; and

• an electrode in contact with the reservoir liquid,

characterized in that the width of the crack or fracture is less than 2 μηι.

2. A CE-MS interface comprising one or more electrical contacts as disclosed in claim 1 , operated with or without an external pressure, where a sharp spray tip is provided at the spray end of the capillary by any known method either before or after generating the crack/fracture.

3. A method for preparing an electrical contact according to any one of the claims 1 , comprising the steps of:

• optionally, removing an external coating of the capillary by any known method, for example, by burning, etching or cutting away the coating;

· optionally, applying a heat shrinking tubing or other coating to improve the mechanical stability of the capillary;

• optionally, coating the internal surface of the capillary for separation improvement etc;

• optionally, fixing the capillary to a substrate in order to keep the parts of the capillary aligned after generating a crack/fracture;

• generating a crack or fracture one or several places along the separation capillary, wherein the width of the crack or fracture is less than 2 μηι;

• attaching one or more reservoirs in liquid connection with each one of the cracks or fractures;

· filling the reservoirs with reservoir liquids; and

• providing an electrode in contact with the reservoir liquids.

4. A method according to claim 3, wherein the crack or fracture is generated by using an optical fiber cleaver, or other methods for making such fractures in a repeatable manner, for example, by thermal effects, mechanical stress or applying high electrical field strength locally across the capillary. A method according to claim 3, where in the crack or fracture is generated through the whole cross-section of the capillary.

5. A method according to anyone of the claims 3, wherein the heat shrinking tubing is Teflon tubing.

6. A method according to anyone of the claims 1-5, wherein crack or fractures are generated several places along the separation capillary.

7. The use of the fracture/crack as disclosed in claim 1 , fabricated by any of the methods disclosed in claims 3-5, for introducing deuterium into a capillary for on-column hydrogen deuterium exchange (HDX) of proteins, peptides and other large organic molecules in the capillary.

8. The use of the fracture/crack as disclosed in claim 1 , fabricated by any of the methods disclosed in claims 3-5, for electrokinetically introducing a sample or a reagent into a separation capillary from the reservoir through the crack/fracture and into the capillary.