Combined electrophoresis-electrospray interface and method

A system and method for analyzing molecular constituents of a composition sample includes: forming a solution of the sample, separating the solution by capillary electrophoresis into an eluent of constituents longitudinally separated according to their relative electrophoretic mobilities, electrospraying the eluent to form a charged spray in which the molecular constituents have a temporal distribution; and detecting or collecting the separated constituents in accordance with the temporal distribution in the spray. A first high-voltage (e.g., 5-100 KVDC) is applied to the solution. The spray is charged by applying a second high voltage (e.g., .+-.2-8 KVDC) between the eluent at the capillary exit and a cathode spaced in front of the exit. A complete electrical circuit is formed by a conductor which directly contacts the eluent at the capillary exit, or by conduction through a sheath electrode discharged in an annular sheath flow about the capillary exit.

RELATED APPLICATION DATA 
This application is related to copending, commonly-assigned U.S. patent 
application Ser. No. 07/034,875, filed Apr. 6, 1987, by R. D. Smith and J. 
Olivares entitled, "Combined Electrophoretic-Separation and Electrospray 
Method and System." 
BACKGROUND OF THE INVENTION 
This invention relates to a method and apparatus for analyzing chemical 
compositions and more particularly to a method and system for combining 
free zone electrophoretic separation of a mixture sample with 
electrospraying to interface with on-line detection or off-line collection 
apparatus. The invention finds especially advantageous application in 
combining capillary zone electrophoresis and mass spectrometry (CZE-MS). 
Numerous systems employed in the separation and analysis of analytes are 
known in the prior art. However, these prior art systems are not 
necessarily broadly applicable to the separation and/or analysis of 
analytes which comprise complex materials, or high molecular weight, 
nonvolatile, and highly polar compounds. 
One known method for separation of analyte mixtures, free zone 
electrophoresis in small diameter capillaries or capillary zone 
electrophoresis (CZE), is used for a wide variety of analyses including 
high resolution separations of amino acids, peptides, proteins and complex 
salt mixtures. CZE employs a capillary with an electric field gradient to 
separate the analyte constituents, particularly ions, by difference in 
electrophoretic mobilities in addition to electroosmotic flow in the 
capillary. The electroosmotic flow results when an electrical double layer 
of ions forms at the capillary surface and an electrical field is imposed 
lengthwise along the capillary. The field causes the ions to migrate 
towards the oppositely charged electrode at rates determined by the 
electrophoretic mobility of each analyte. In the resulting bulk 
electroosmotic flow, positively charged ions, neutral species, and 
negatively charged ions elute at different time intervals. The extent and 
speed of this separation are determined by differences in the 
electrophoretic mobilities of the analytes, the length of the capillary, 
the bulk electroosmotic flow and by the strength of electric field. 
FIG. 1 is a schematic illustration of the customary arrangement of a CZE 
system. In this arrangement, a complete high voltage electrical circuit 
must be formed between opposite ends of a fused silica capillary A filled 
with a buffer solution and extending through a flourescence detector B. 
This is accomplished by immersing both ends of the capillary in beakers C, 
D of the buffered solutions at each end of the system. 
CZE detection is currently limited to analysis by ultraviolet or 
fluorescent detection techniques, so as not to degrade the quality of the 
separation. Such detection techniques have been adequate for species that 
fluoresce, absorb, or are amenable to derivatization with fluorescing or 
absorbing chromophores. These detectors also impose cell volume and sample 
size limitations that preclude high separation efficiencies concurrent 
with high sensitivities. Structural information necessary for the correct 
identification of unknown analytes and their constituents cannot be 
obtained using these detectors due to the small sample volume and the 
limited spectroscopic data inherent in UV and fluorescence detection 
techniques. These limitations constitute a major drawback in the use of 
CZE for the separation and identification of complex mixtures since many 
compounds cannot be detected, and, if detectable, cannot be unambiguously 
identified. A detailed discussion of CZE can be found in an article by 
Jorgenson, et al., in the publication "Science" (1983), Vol. 222, 
beginning at page 266. 
A well-known analytical technique which combines a separation technique 
with an analytical detection device is gas chromatography-mass 
spectrometry (GC-MS). In this method, GC can provide separations of 
sufficiently volatile compounds which are then ionized and analyzed by 
mass spectrometry. GC-MS has become established as the definitive 
analytical technique for amenable compounds, i.e., compounds having 
sufficient volatility for GC separation and ionization by conventional gas 
phase electron impact or chemical ionization methods used in mass 
spectrometry. 
Such an established capability of broad application is not known to exist 
for nonvolatile compounds and mixtures. Systems for combining liquid 
chromatography with mass-spectroscopy are described in U.S. Pat. No. 
4,209,696 and in European Patent Application No. 84302751.7, which are 
incorporated herein by reference. In these systems, carrier liquid from a 
liquid chromatograph is electrosprayed and then analyzed by mass 
spectrometry. To work, electrospray requires an ionic strength of less 
than about 10.sup.-2 molar. Various other attempts to combine liquid 
chromatography with mass spectroscopy are described in "Microcolumn High 
Performance Liquid Chromatography," P. Kucera, Ed., J. Chromatography 
Library, Vol. 28, Chap. 8, pp. 260-300 (1984) and in "Small Bore Liquid 
Chromatography Columns: Their Properties and Uses," R. P. W. Scott, ed., 
Vol. 72, pp. 104-114 (1984). Unfortunately, these systems and ther LC-MS 
approaches suffer significant limitations due to their inability to 
effectively separate complex mixtures, their limited separation 
efficiency, and the time required for analysis or separation. Combined 
liquid chromatography-mass spectroscopy does not provide high resolution 
separations. In liquid chromatography, the maximum number of theoretical 
plates is limited to about 10,000 for reasonable separation times (under 
about one hour). In contrast, CZE has been shown to be able to provide 
over one million theoretical plates in the same time. 
Accordingly, a need remains for a method of separation that has the 
high-resolution separation of efficiencies of CZE and, additionally, an 
ability to analyze a wide range of nonvolatile compounds. 
SUMMARY OF THE INVENTION 
This invention relates to a system and method for interfacing the free zone 
electrophoretic separation of a sample and electrospray, respectively, so 
that the molecular constituents of the electrosprayed eluent produced have 
a temporal distribution and can be concentrated by evaporation of the 
solvent. The electrosprayed eluent can be subsequently analytically 
detected on-line using mass spectrometry, or other analysis methods, or 
can be collected off-line for analysis or other applications requiring 
highly-purified samples. 
A system and method for analyzing molecular constituents of a sample 
includes: forming a solution of the sample, separating the solution by 
capillary electrophoresis into an eluent of constituents longitudinally 
separated according to their relative electrophoretic mobilities, 
electrospraying the eluent to form a charged spray in which the molecular 
constituents have a temporal distribution; and detecting or collecting the 
separated constituents in accordance with the temporal distribution in the 
spray. 
A first high-voltage (e.g., 5-100 KVDC) is initially applied to the 
solution to separate its constituents. The separated eluent is 
electrosprayed and the spray is charged by applying a second high voltage 
(e.g., +/-2-8 KVDC) between the eluent at the capillary exit and a counter 
electrode spaced in front of the exit. A complete electrical circuit is 
formed by a conductor which directly contacts the eluent at the capillary 
exit. 
Capillary electrophoresis includes variations such as electrokinetic 
chromatography or isotachophoresis. Electrospraying includes processes 
which involve electric fields, and may include concurrent utilization of 
nebulizing gases or heating methods. 
The sample can include complex, high-molecular-weight, nonvolatile and 
highly-polar compounds. Ordinarily, the solution includes a buffering 
agent. Detection can be by apparatus that does not depend on UV or 
fluorescence of the constituents and that is capable of identifying and 
quantifying, or providing universal detection of, the constituents. 
The interface includes means for applying a first high voltage potential 
between the source of sample solution and the capillary outlet, to effect 
electrophoretic separation in the sample, and means for applying a second 
high voltage potential between the capillary outlet and the collector or 
detector, to electrospray and ionize the separated sample as it is 
discharged. In one embodiment, the capillary outlet end can be metallized 
to conductively couple the eluent to the second high voltage source. In a 
second embodiment, an annular sheath flow of a sheath flow liquid is 
discharged simultaneously around the sample flow from the capillary 
outlet. The latter interface permits greater and more uniform ion current, 
not limited by capillary flow rates and composition. It also simplifies 
interface design and fabrication, allows making electrical contact at the 
capillary end without gas generation problems, loss of separation 
efficiency due to dead space, and electrospray instability. 
The invention finds particular advantage in interfacing capillary-zone 
electrophoresis and mass spectrometry (CZE-MS). In one embodiment of this 
application, the CZE cathode serves as an electrospray needle for spraying 
a separated sample into a mass-spectrometer. The analyte eluent at the 
capillary outlet is biased relative to the mass spectrometer at a voltage 
potential sufficient to produce the electrospray, which is then sampled by 
the mass spectrometer. Electrospraying is carried out at near-atmospheric 
pressure. Accordingly, the mass spectrometer preferably includes a 
differentially pumped input chamber. The interface can further include an 
ion lens to aid ion transmission into the detector. It can also include 
means for desolvating or vaporizing the ionized spray to form an ion vapor 
phase stream into the mass spectrometer. 
The foregoing and other objects, features and advantages of the invention 
will become more readily apparent from the following detailed description 
which proceeds with reference to the drawings.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT 
Description of CZE-Electrospray Interface 
FIG. 2 shows an apparatus for combined CZE-electrospray-mass spectrometer 
(CZE-MS) in accordance with a preferred embodiment of the present 
invention. FIG. 2A is an electrical circuit diagram of a generalization of 
the system of FIG. 2. 
Referring first to FIG. 2A, a CZE-electrospray interface according to the 
invention generally comprises a capillary-zone electrophoresis (CZE) 
subsystem 10, an electrospray interface 12, a detection or collection 
device 14, and a high voltage electrical circuit 16. The CZE subsystem 10 
and electrospray interface 12 form integral parts of the electrical 
circuit 16. Specifically, the CZE subsystem forms a part of a subcircuit 
16A which includes a first high voltage supply 18 and capillary 20 having 
an outlet nozzle 22. The electrospray interface forms subcircuit 16B, 
which includes a second high voltage supply 24 and a counter-electrode 26 
in the detector/collector 14. The two subcircuits are electrically 
interconnected at nozzle 22 and node 28, for example, ground. Optionally, 
a third power supply (not shown) can be used to bias the counterelectrode 
relative to node 28. 
Referring next to FIG. 2, the CZE subsystem 10 includes an electrically 
insulated sampling box 30, provided to isolate the first high voltage 
system 16A from the outside environment. For example, a Lucite or 
Plexiglas box can be employed for this purpose. From a safety standpoint, 
this portion of the system is isolated because of the dangers to the user 
from this high voltage application. 
Within box 30 are a sample injection reservoir 32 and a buffer reservoir 33 
which contain the analyte sample and the CZE buffer solution in separate 
containers. High voltage system 16A includes a first high voltage power 
supply 18 and an electrode or microsampling arm 34 extending into the 
reservoir 33. An analyte sample solution 35 is formed in reservoir 32 by 
adding a suitable chemical solvent to a sample of the material to be 
analyzed. A buffering agent 36 is provided in reservoir 33. Typically, the 
reservoir to which either solution is added comprises a standard 
micro-beaker or other liquid container made of glass or the like. 
A capillary 20 is also disposed within the sampling box 30. The capillary 
20 may have a bend with a vertical inlet section 37 (depending upon sample 
introduction method) and horizontally-disposed outlet section 38, and 
includes respective inlet and outlet ends 39 and 40. The capillary inlet 
end 39 extends into sample solution reservoir 32 during injection of the 
sample solution 35 and into the buffer solution 36 in reservoir 33 during 
separation. Outlet end 40 is electrically connected, as hereinafter 
described, to form a closed electrical circuit for the first high voltage 
system 16A. 
Capillary 20 can be fabricated in the form of any capillary structure 
capable of effecting the capillary zone electrophoretic process. 
Particularly, however, nonconductive materials such as glass, fused 
silica, TEFLON.RTM. and the like are preferred materials of construction 
of such capillary. Preferably, the capillary has a length of 20 to 500 
centimeters and has an inside diameter which ordinarily ranges from about 
25 uM up to about 250 uM, although a wider range of dimensions is 
feasible. 
Capillary tube 20 has joined to its outlet end 40 a second high voltage 
input system 16B, including a second high voltage power supply 24. This 
system 16B is grounded or biased at a selected voltage above ground to 
complete a first closed circuit for high voltage supply 18, as shown in 
FIG. 2A. The electrical connection at the capillary outlet end serves as 
both the electrode for the CZE step and also as the spray needle for the 
electrospray step. More specifically, the system 16B forms a completed 
circuit with high voltage supply 24 through a physical connection with a 
high voltage line 42. Thus, high voltage power supply 24 is grounded at 
one end 44 and is connected at its other end through high voltage line 42 
to outlet end 40. This electrical connection also forms circuit 16B, which 
enables the analyte to be electrosprayed upon application of a second high 
voltage from voltage supply 24 through line 42. 
A large voltage drop is applied from the inlet end 39 to the outlet end 40 
of the capillary to enable electrophoretic separation of the analyte 
solution 36. The high voltage also causes a bulk electroosmotic flow of 
buffer towards the capillary outlet 40. The high voltage is applied from 
power supply 18 through microsampling arm 34 into the reservoir 33. The 
voltage drop along the capillary is the difference between the voltage 
from supply 18 and the voltage from supply 24. The voltage drop draws the 
buffer solution 36 into capillary 20. It also causes solution 36 to be 
electrophoretically separated into its individual molecular constituents 
as they pass at differing levels of electrophoretic mobility from inlet 
end 39 to outlet end 40. The amount of voltage provided from power supply 
18 into the sample analyte solution 36 ranges typically from about 5 
kilovolts DC up to about 100 kilovolts DC. If ions of both positive and 
negative electrophoretic mobility are to be analyzed, the electroosmotic 
flow must be sufficiently large to offset the electrophoretic motion in 
the opposite direction, so that all analytes of interest move towards the 
capillary exit. It should be noted that nonconducting capillaries can form 
an electrical double layer with electroosmotic flow in a direction and 
rate that depends on the surface or any surface treatment of the 
capillary. In such a situation the polarity of the voltages required for 
CZE separation may be reversed. 
The basis of the invention includes forming a completed electrical contact 
at or near the capillary exit 40 without immersing it in a beaker of 
buffer solution. Referring to FIG. 3, a schematic illustration of the 
capillary zone electrophoresis electrode which serves as the electrospray 
needle, a novel CZE-electrospray interface (ESI) system 12 is provided. 
The outlet end 40 of capillary 20 has a conductive stainless steel 
capillary sheath 46 located concentrically thereabout. The sheath 46 
comprises respective inner section 47 and outer section 48 joined one to 
the other. Sheath 46 is attached to capillary 20 by an adhesive such as an 
epoxy resin or the like. The sheath 46 is physically connected to high 
voltage power supply 24 by means of a copper conductive wire 42 (see FIGS. 
2 and 2A). A conductive metal-plated end section 50 is plated 
concentrically about the exit of the outlet end 40, including the exit 
portions of respective sheath sections 47 and 48, to form conductive tip 
60 contacting the electrophoretically separated solution 36. 
In one form of the invention, a metal coating is sputtered onto the 
respective exit portion of sheath 46 and outlet end 40 so that electrical 
contact is directly made with the eluent at tip 60 as soon as it emanates 
from the exit of nonconductive fused silica capillary 20. Typically, a 
metal such as gold, silver, or platinum is employed for this purpose. 
Preferably, the conductive tip is formed so that the dead volume after 
completion of the electrical circuits is minimized and there is virtually 
no flow turbulence within capillary 20 and, therefore, no substantial 
contribution to band broadening (or loss of separation) of the analyte 
sample. The ability to minimize flow turbulence, and thereby maintain 
continuous flow of the eluent 36 in capillary 20, is dependent upon 
capillary diameter and length. For example, the effective dead volume for 
a 100 um. i.d. capillary 1M in length should be not more than about 10 nL, 
and preferably less than about 1 nL. 
The electrical contact can be formed in other ways, which include (1) 
joining a metal capillary to the nonconductive CZE capillary; or (2) 
electrical contact through a small conductive capillary segment near the 
capillary exit. The latter can be done in numerous ways, but approaches 
that minimize the dead volume after the electrical contact are necessary 
so as to avoid loss of separation efficiency. 
High voltage system 16B creates an electrical potential between the 
capillary tip 60 and eluent 36 and the collection or detection apparatus, 
such as the counter electrode 26 of the mass spectrometer shown in FIGS. 2 
and 2A. The purpose is to produce an electric field resulting in the 
desired electrospray process. 
Depending upon whether positively or negatively charged constituents are to 
be desirably produced by subsequent electrospraying, either a positive or 
a negative (+/-) voltage is applied to the capillary end 40 relative to 
the counter electrode (sampling orifice) 26. Voltages of about +/-2,000 to 
8,000 volts DC can generally be used, with a voltage of about +/-3,000 to 
4,000 volts DC being preferred depending upon the distance to the counter 
electrode. The resultant electric field causes the eluent 36 to be 
discharged (electrosprayed) in airspace 62 from the conductive tip 60 of 
capillary tube 20. This produces a fine spray 64 of electrically charged 
droplets including gaseous ions, solvent and solvent-carrying analyte 
material, having a charge polarity determined by the field. 
These electrospray droplets are attracted towards counterelectrode 26, 
which has sampling provisions, i.e., an orifice 63, for on-line detection 
or off-line collection device, by the electric field created by high 
voltage system B. FIG. 7 shows a photograph of a fully developed 
electrospray flowing from capillary outlet 40. 
Electrospray Analysis 
Analysis of the electrosprayed eluent 64 can be conducted employing any 
on-line detection or off-line collection equipment capable of analyzing 
the molecular or atomic constituents of the eluent. Alternative analysis 
techniques are further described hereinafter. Preferably, molecular 
analysis by on-line detection techniques, and more preferably, by mass 
spectrometry, is employed as next described. 
In on-line detection gaseous phase analysis, hereinafter described, a 
counter-current flow of hot gas 68 is typically used to assist solvent 
vaporization of the spray 64 of charged droplets. Thus, vapor is removed 
from electrospray source region 62, which is at approximately atmospheric 
pressure. The resultant droplets have nearly uniform size, similar charge, 
and produce gaseous molecular ions. 
As depicted in FIG. 2, a ring member 66 is employed to heat the gas in the 
airspace which in turn heats the exiting electrosprayed analyte eluent 64. 
Generally, gas temperatures of from about 50.degree. C. up to about 
120.degree. C. can be employed for this purpose but a wider range of 
temperatures would be usable depending on flow rate. 
The countercurrent gas flow 68 of inert or reactive gases can be employed, 
alone or in combination with the previously-described thermal heating for 
desolvating the spray droplets. Typical inert gases include nitrogen, 
helium and the like, and typical reactive gases include ammonia, oxygen 
and the like. Countercurrent gas flows are directed through chamber 69 so 
as to impinge the electrosprayed analyte eluent 64 within the airspace 62. 
Typical gas flow rates of from about 0.1 liter per minute, up to about 20 
liters per minute, can be employed for this purpose. 
Operation of CZE-Electrospray Interface 
The method and system of this invention is broadly applicable to the 
analysis of any material soluble in water or polar solvents, particular 
ionized or partially ionized species. Compounds amenable to this process 
include normally neutral compounds on which a charge can be induced by 
manipulation of buffer solution composition and neutral compounds 
separated by buffer solutions containing micellar phases or microemulsions 
by partitioning between the bulk liquid and micelle phases. This includes 
materials separable by electrokinetic chromatography, as well as those 
separable by CZE and capillary isotachophoresis. In general, complete 
mixtures of positive, negative and neutral constituents in solution are 
amenable to separation and analysis by the subject invention. This method 
and system is more particularly applicable to the separation of organic, 
inorganic, and bio-organic molecules soluble in aqueous solutions. 
Nonaqueous solvents may also be used. Some organic solvents, especially 
those with some ionic characteristics, or those that can be seeded or 
mixed with ionic components, are also applicable. With respect to the 
solvent portion of the analyte sample solution 36, any solvent is suitable 
for use herein as long as it exhibits at least a minimum conductivity. The 
solution 36 preferably has a minimum surface tension, if used with gas 
phase ion detection methods, in order to permit maximum desolvation on 
subsequent electrospraying. Thus, compounds ranging from aqueous to 
organic solvents to mixtures of solvent components may be employed if a 
certain minimum ionic strength of the analyte solutions formed are 
achieved. Aqueous sample solutions preferably include a buffering agent. 
These solutions are preferably provided at concentrations below about 
0.01M. 
Buffer materials are also required for most CZE media. The buffer and 
solvent mixtures are chosen according to the sample employed in the 
electrophoretic process relative to the buffer selected. The buffer 
portion of solution 36 provides a number of important properties. First, 
the buffer imparts ionic strength for enhancing conductivity and 
minimizing field effects which distort separation of the individual 
constituents. It also provides a stable pH medium in which the solution is 
stabilized and effective constituent separations can be performed at 
different electrophoretic mobility levels. A solution is formed with a 
sufficient level of conductivity that subsequent electrospraying can be 
effectively performed. Buffer concentrations preferably ranging from about 
10.sup.-6 to about 10.sup.-2 molar are particularly useful in this 
invention. Typical compounds employed as buffers include ionic salts such 
as ammonium salts, inorganic salts such as sodium and potassium chloride, 
and organic salts such as potassium phthalate. 
Regarding electrical currents present in the system, it should be noted 
that a high voltage-low current relationship is typically maintained in 
the system. Currents which will facilitate the system and method of this 
invention and which can provide maximum separation of the analyte 
constituents are employed. Although the current is dependent upon such 
variables as the ionic strength of the solution, the capillary column 
length and inside diameter of the capillary, current is preferably 
maintained at or below the 100 uA level. The current is typically directly 
proportional to voltage and the maximum voltage is usually selected so 
that heating of the buffer solution in the capillary is minimized, since 
heating results in convective flow which degrades separation efficiency. 
In order to analyze the molecular constituents according to the method and 
system of the present invention, sample solution 36 is electrophoretically 
separated into its molecular constituents. The use of electrophoresis 
according to the teachings of the subject invention facilitates high 
efficiency separation or analysis of complex materials. First, voltage is 
briefly applied to the analyte sample solution 36 and migration of a small 
amount of the sample solution into a capillary 20 is achieved due 
primarily to electroosmotic flow. The buffer solution reservoir 33 is then 
introduced into the sampling box 30, the capillary is removed from the 
sample reservoir and introduced into the buffer reservoir, high voltage is 
applied thereto, and electrophoresis proceeds. 
Electroosmosis is caused by the migration of ions, from the diffusive layer 
of the electrical double layer at the capillary surface, under the 
influence of an electrical field imposed tangentially to the surface. The 
ions present in the analyte will then migrate towards the oppositely 
charged electrode carrying the capillary contents with them. The 
electroosmotic flow is sufficiently fast that positively charged ions, 
neutral molecular compounds, and negatively charged ions elute in short 
times, typically about 5-30 minutes for a 1M capillary. In a positive 
voltage gradient, positive ions will have the largest net mobilities and 
will elute first since they are repelled by the high voltage anode, 
resulting in positive electrophoretic mobilities, and also will be carried 
by the electroosmotic bulk flow of the solvent. Negative ions having the 
largest negative electrophoretic mobilities will elute last. Negative ions 
with very high electrophoretic mobilities may never elute from the column 
if the electroosmotic flow is not sufficiently fast, but usually 
conditions can be varied so that the electroosmotic mobility is always 
larger than the analyte's electrophoretic mobility. 
Therefore, the migration time through the capillary column 20 is for the 
most part determined by a combination of the capillary length, the 
molecule's electrophoretic mobility in the electric field, the electric 
field strength, and the electroosmotic flow of the supporting buffer 
solution. The various constituents forming analyte sample 36 have 
different relative electrophoretic mobilities. These differences in 
electrophoretic mobility produce a dissimilar rate of migration of the 
molecular constituents from the inlet 39 to the outlet 40 of capillary 20. 
This results in an effective, high efficiency separation of these 
different molecular constituents with respect to time so that the identity 
and quantity of each constituent can be individually and analytically 
determined or collected. 
In defining the optimum conditions for electrophoretic flow of analyte 
eluent 36 from inlet 39 to outlet 40, the following are some of the 
preferred conditions: a minimum metal surface contact or other electrical 
contact between the analyte flowing in the capillary to complete an 
electrical circuit near the point of electrospray formation, a 
substantially constant voltage drop from inlet 39 to outlet 40, and a 
continuous inner flow surface, having minimum discontinuous surface areas 
and substantially no dead volume is present, so that electroosmotic flow 
of the analyte eluent 36 in the capillary is created with a minimum 
introduction of any turbulent effects. 
The electrospray of the subject invention can be used in both the positive 
and negative ionization modes, although a small addition of oxygen or 
other electron scavenger to the bath gas is useful for negative ion 
production to avoid electrical breakdown. 
For mass spectrometric analysis, this atmospheric pressure ion source is 
then typically followed by a molecular beam sampling apparatus consisting 
of a nozzle-skimmer arrangement with an RF only quadrupole field or ion 
lens system for ion focusing and a quadrupole mass spectrometer for mass 
analysis and detection. Other mass-spectrometer inlet designs are 
feasible. For example, nonconductive capillaries can be used as disclosed 
in Whitehouse, C. M., et al., "Electrospray Interface for Liquid 
Chromatographs and Mass Spectrometers," Analytical Chemistry, Vol. 57, pp. 
675-679 (1985). 
In the preferred form of this invention, the electrosprayed droplets are 
allowed to continually divide and evaporate at near atmospheric pressure 
to form gaseous ions of analyte constituents employing electrospray 
techniques similar to those described in U.S. Pat. No. 4,209,696 and EPA 
No. 84302751.7. The solution flow in the capillary results preferably from 
electroosmotic flow rather than any pressure drop, so that separation 
efficiency is not degraded. Thus, spray 64 is formed without substantial 
distortion of the electropherogram thereby permitting analysis by numerous 
analytical detectors. 
The electrospray process utilized for mass spectrometric detection is 
similar to that developed by previous workers. As the droplets are formed 
by the electrospray process, desolvation of the solvent from the droplets 
begins to occur, and the analyte constituent passes from the liquid phase 
into the gaseous phase, the gaseous phase including gaseous ions of the 
analyte constituents. As the droplets move away from outlet 40, they 
continually decrease in size and their mass-to-charge ratios continually 
shrinks until an ionic vapor phase stream is formed which is capable of 
detection by mass spectrometry. Desolvation of the solvent from its 
association with the droplets can be facilitated thermally and/or by 
countercurrent gas flow. Electrospraying includes processes which involve 
electric fields, and may include concurrent utilization of nebulizing 
gases or heating methods. 
In any case, the desolvated vapor phase ions produced, along with the 
remaining portion of the analyte present within airspace 62, are conveyed 
to a mass spectrometer for analyzing the identity and quantity of the 
individual constituents contained in the analyte sample. 
Improvements In Mass Spectrometry For CZE-Mass Spectrometry System 
Certain features which improve or facilitate analysis using mass 
spectrometry have also been uncovered with respect to the analysis of 
electrosprayed eluent 64. These include, for example, the use of an RF 
only lens in the first vacuum region of the mass spectrometer. These 
lenses are known to provide nearly 100 percent containment of ions in 
triple, quadrupole mass spectrometers, where the lens is operated in an 
intermediate vacuum of about 5.times.10.sup.-4 torr, which is similar to 
the pressures used in the first vacuum region of the CZE-MS system 
interface. This RF only lens also acts as a high pass mass filter allowing 
only ions above a preselected mass of interest to pass into the mass 
analyzer. This cleansing effect (since high ion currents are to be 
expected from the buffer employed at low masses) provides spectra which 
are potentially free of space charge effects created when high ion 
currents containing ions of no interest to the analyst enter the normal 
quadrupole mass analyzer and are to be rejected during mass analysis. 
Quadrupole devices are mass analyzers in the form of mass-to-charge 
separators. 
A well-coupled RF-RF/DC pair of quadrupole lenses is also employed to 
minimize the fringe field effects observed when the lens combination is 
DC-RF/DC. This serves to maximize ion transmission into the mass analyzer. 
Finally, the use of quartz inlet capillaries to transmit ions from the 
atmospheric pressure electrospray ionization source is a feasible 
alternative to the nozzle-skimmer introduction method which allows the 
direct injection of transmitted ions into the RF only quadrupole. 
Alternative Analytical Applications 
The electrospray interface also provides a basis for combining CZE 
separations with other on-line analysis techniques. In these methods the 
electrospray is sampled so that either the small liquid droplets or gas 
phase ions are introduced into an analytical or detection device. Thus, 
this invention includes the combination of free zone electrophoresis (and 
variations which include electrokinetic chromatography and 
isotachophoresis) separation methods, using electrospray, with other 
detection methods which include: 
1. flame ionization detection; 
2. elemental analysis by inductively-coupled plasma or microwave plasma 
atomic emission for elemental analysis; 
3. ion mobility detection; 
4. photo ionization detection; 
5. element-specific ionization detection; 
6. electron capture detection; 
7. surface-sensitive analytical methods; 
8. infrared analysis of electrosprayed deposits. 
The common feature of all the above analysis methods is that a gaseous or 
aerosol sample is required. The electrospray process produces such a gas 
or aerosol which may be interfaced to these detection devices. Each 
analysis method requires somewhat different methods for sampling the 
electrospray. However, the methods are such that someone reasonably 
skilled in the above techniques, given the information disclosed herein, 
could successfully combine CZE with the selected analytical method. It 
should be noted that some methods will present difficulties due to limited 
sensitivities, and thus may impose some limitations upon the practice of 
CZE (such as the use of a larger than optimum sample that may degrade 
separation efficiency) or the analytical detection method. 
Off-line collection or analysis methods are also feasible using the 
electrospray. In these methods the electrospray is collected on a solid or 
liquid surface. The surface can be moved so that the temporal distribution 
of separated analytes is deposited on the surface as a spatial 
distribution. The separated sample collected on the surface can be 
utilized for other off-line analysis methods or other purposes where only 
a small sample is required. The spatially distributed material can also be 
analyzed by analytical methods which are compatible with solid samples on 
surfaces. These analytical methods include: 
1. mass spectrometry using a moving ribbon or belt with ionization methods 
which include ion or atom bombardment; 
2. infrared analysis of surfaces; 
3. any surface-sensitive analytical method. 
EXAMPLE 1 
Using the CZE-mass spectrometer system specifically depicted in FIG. 2, the 
identity and quantity of an analyte sample was determined. 
CZE was carried out using a 0-60 kV dc power supply, Glassman High Voltage 
Inc. (Whitehouse Station, N.J.) Model LG60P2.5. The high voltage electrode 
and capillary end (anode) and solution vials were contained in an 
insulating sampling box with a remote controlled sampling arm and 
injection timer to facilitate the interchange and injection of solutions. 
Fused silica capillaries, 100 um i.d. and 100 cm long, from Polymicro 
Technologies, Inc. (Phoenix, AZ), were used in all experiments without 
further treatment. The cathode (low voltage end) of the fused silica 
capillary was terminated in a stainless steel capillary sheath, 300 um 
i.d. and 450 um o.d. (see FIG. 3). The sheath potential was controlled 
with a 0 to 5 kV dc power supply and functions as both the CZE cathode and 
electrospray needle (see FIG. 2A). 
Electrospray ionization was carried out at atmospheric pressure in a 2.54 
cm long by 2.29 cm i.d. stainless steel cylinder. The cylinder terminated 
in an electrically biased (190 V dc) focusing ring 44 with a 0.475 cm 
aperture. The ion sampling orifice (or nozzle) 63 had a 0.5 mm i.d. 
orifice, was made from copper, which was in contact with a copper cylinder 
at ground potential. This cylinder surrounded the electrospray assembly 
and was heated to 60.degree. C. by a system of cartridge heaters (not 
shown). The electrospray needle, focusing ring 66, and ion sampling nozzle 
63 were disposed concentric with the mass analyzer. These components could 
be positioned independently relative to the fixed skimmer 70 (with the aid 
of linear motion drives), even while high voltage is on, in order to 
maximize ion formation and transmission. A flow of N.sub.2 forming a gas 
curtain, at a flow rate of 2.5 L/min, is fed between the focusing ring 66 
and the nozzle 63 and directed so as to flow counter to the electrospray 
to aid in the desolvation process. 
The vacuum system consisted of a three stage differentially pumped chamber, 
although many different arrangements are feasible. The first stage allows 
for a supersonic beam expansion through the ion sampling nozzle 63. This 
region is pumped to 0.85 Torr by a 150 L/s roots blower. A portion of the 
supersonic beam is sampled by a 1.2 mm i.d. beam skimmer, Beam Dynamics, 
Inc. (Minneapolis, MN), Model 1. The second differentially pumped stage 
houses a 22 cm long, 0.95 cm diameter quadrupole filter 72. This 
quadrupole is operated in the RF only mode with a -1.8 V dc rod bias and 
acts as an ion lens which facilitates ion transmission to the analysis 
quadrupole. The pressure in this region is maintained at 10.sup.-4 to 
10.sup.-5 Torr with a 1500 L/s turbomolecular pump. Another version of 
this instrument substituted an integral cyro-pump which provided a pumping 
speed of approximately 50,000 L/S and allowed larger orifice and skimmer 
diameters. An electrically isolated stainless steel plate (-28 V dc), with 
a 0.635 cm i.d. orifice, allows the mass spectrometer chamber to be 
maintained at 2.times.10.sup.-6 Torr using a 550 L/s turbomolecular pump. 
The 2000 amu range quadrupole mass filter, Extrel Co. (Pittsburgh, Pa.), 
Model CQPS1HV, and a channeltron electron multiplier 74, Detector 
Technologies, Inc. (Brookfield, Mass.), Model 203, operated in the analog 
mode. Data acquisition and mass scanning was performed with a Teknivent 
Corp. (St. Louis, Mo.) Model 1050 interface-IBM PC/XT based system. 
Additional operational parameters were as follows: applied voltage of 
40,000 V dc, electrospray voltage of 3,000 V dc, focus ring voltage of 190 
V dc, N.sub.2 flow rate of 2.5 L/min, source temperature of 60.degree. C., 
RF only quadrupole dc bias of -1.8 V dc, and an ion entrance aperture of 
-28 V dc. 
Injection of samples onto the CZE capillary was performed using the 
previously-described electromigration technique of Jorgenson et al. In 
electromigration, the anode end 39 of the column is introduced into the 
analyte solution, the injection voltage is turned on for a predetermined 
amount of time, the voltage is turned off and the buffer replaced; the CZE 
applied voltage (V.sub.app =40,000 V dc) and electrospray (V.sub.ESI 
voltage=3000 V dc) are then turned on and the separation is allowed to 
continue. (The CZE voltage V.sub.CZE) here refers to the voltage drop 
across the CZE column which has been modified from the traditional sense 
because the cathode is maintained at the electrospray voltage; thus 
V.sub.CZE =V.sub.app -V.sub.ESI.) 
A (50-50) water-methanol with 10.sup.-4 M KCl was used as the separation 
and electrospray medium. It was observed that water-methanol provides a 
considerable electroosmotic mobility (3.6.times.10.sup.-4 cm.sup.2 /V s) 
with the fused silica capillary. Thus, positively ionized compounds elute 
in less than 12.5 minutes from a 100 cm long column (with V.sub.CZE 
=37,000 V). 
EXAMPLE 1: RESULTS AND DISCUSSION 
Five ammonium salts were tested: tetramethyl ammonium bromide, tetraethyl 
ammonium perchlorate, tetrapropyl ammonium hydroxide, tetrabutyl ammonium 
hydroxide, and trimethyl phenyl ammonium iodide. These quaternary ammonium 
salts all give good electrospray signals with the dominant peak in the 
mass spectrum being the quaternary ammonium cation. 
FIG. 4 shows the electrospray ionization mass spectrum for the five 
components injected at 10.sup.-5 M concentration by continuous 
electromigration without CZE separation. The dominant peaks are due to the 
quaternary ammonium cations of: tetramethyl ammonium bromide (m/z-74); 
tetraethyl ammonium perchlorate (m/z-130); trimethyl phenyl ammonium 
iodide (m/z=136); tetrapropyl ammonium hydroxide (m/z=186); tetrabutyl 
ammonium hydroxide (m/z=242); and a background peak due to 
Na-MeOH+(m/z=55). 
The first CZE-MS separation of such a mixture, taken under multiple ion 
monitoring of the corresponding quaternary ammonium cation peaks, is shown 
in FIG. 5. FIG. 5 is an electropherogram of five quaternary ammonium 
salts, at 10.sup.-6 M (14-17 femtomole injection) concentration, obtained 
by CZE-MS: (A) tetramethyl ammonium bromide; (B) trimethyl phenyl ammonium 
iodide; (C) tetraethyl ammonium perchlorate; (D) tetrapropyl ammonium 
hydroxide; (E) tetrabutyl ammonium hydroxide. The amounts injected for the 
quaternary ammonium salts, 14-17 femtomoles, gave single ion 
electropherograms with good peak shapes and signal/noise ratios. 
FIG. 6 is an electropherogram of five quaternary ammonium salts, at 
10.sup.-7 M (0.7-0.9 femtomole injection) concentration, obtained by 
CZE-MS: tetramethyl ammonium bromide (m/z-74); tetraethyl ammonium 
perchlorate (m/z-130); trimethyl phenyl ammonium iodide (m/z 136); 
tetrapropyl ammonium hydroxide (m/x=186); tetrabutyl ammonium hydroxide 
(m/z-242). FIG. 6 shows the same separation obtained for a 0.7-0.9 
femtomole injection, obtained by decreasing V.sub.i to 0,000 V, and C to 
10.sup.-7 M. 
Though the separation efficiencies in FIG. 5 vary from 26,000 and 100,000 
theoretical plates, they are increased to between 35,000 and 140,000 
theoretical plates in FIG. 6. Such increases in efficiency with decrease 
in sample concentration and size suggest further improvement can be 
obtained with higher buffer ionic strength and either smaller diameter or 
longer capillaries. 
As described earlier, the cathode need not be in a buffer reservoir, but 
only biased negative with respect to the anode. Thus, a metallized segment 
of capillary tubing or other electrical contact with the buffer provides 
the essential control of the electric field. This approach (necessary for 
mass spectrometric interfacing) does not alter the electroosmotic flow, if 
a pressure drop along the length of the capillary is avoided, at least to 
an extent that is detectable with fluorescence detection just prior to the 
electrospray. The success of this approach is further supported by the 
high efficiency separations presented. On the basis of these results, 
electrospray ionization appears to provide an ideal interface for the 
combination of a highly efficient separation technique, CZE, with the 
sensitive and highly specific detector provided in the mass spectrometer. 
Complex mixtures of compounds pose a problem even to the trained mass 
spectroscopist because the identity and the quantity of each constituent 
is not readily ascertainable. 
Using the system and method of the present invention, a series of 
electropherograms of the abovedescribed five quaternary ammonium salts 
were produced (see FIG. 8). These electropherograms are obtained by 
tracing the ion current for a particular ion of mass m/z throughout the 
separation process (over time). Single mass spectra (see FIG. 9) for each 
of the components to be determined can then be obtained for a particular 
time in the separation process. These mass spectra are used to identify 
the molecular weight of the particular component, while the area under the 
electropherogram peak is used for quantification. 
ALTERNATIVE EMBODIMENT WITH SHEATH ELECTRODE FLOW 
The CZE-MS interface described previously above has a number of attractive 
features, including the fact that the effective detection volume is 
negligible and does not appear to contribute to CZE bandspread. In 
addition, the electrospray is created directly at the terminus of the CZE 
capillary, avoiding any post-column region which would contribute to 
extra-column bandspread (due to laminar flow) or analyte adsorption The 
previous example of operation of the electrospray interface did not 
require any additional gas flow for nebulization purposes, as in the "ion 
spray" configuration of Henion and coworkers described for LC-MS, and the 
attendant problems associated with the larger droplet size distribution. 
These problems include the need to sample the electrospray "off-axis", and 
reduced sensitivity since analyte in larger droplets will not be 
efficiently ionized. (Bruins, A. P.; Covey, T. R.; Henion, J. D., "Anal. 
Chem." 1987, 59, 2642-2646.) 
The foregoing example of CZE-MS interface does, however, impose significant 
limitations upon the CZE separation conditions. One restriction is related 
to the minimum flow rate required for generation of a stable electrospray. 
The electroosmotic flow rates, in fused silica capillaries relevant to 
CZE, range from zero to as much as several uL/min, depending upon 
capillary surface treatment, diameter field gradient, and buffer 
composition (i.e., ionic strength and pH). Stable electrosprays are 
increasingly difficult to maintain at flow rates under 0.5 uL/min, and can 
be perturbed by stray electric fields, mechanical perturbations (e.g., 
vibration), or minute variations in flow, causing increasingly severe 
oscillations in performance as flow rate decreases. A second restriction 
is related to buffer composition. For example, aqueous solutions or 
buffers with ionic strengths above about 10.sup.-2 M cannot be effectively 
electrosprayed. These restrictions are not as severe as might be assumed 
since mixtures of alcohols and water can be easily electrosprayed, but can 
limit application to certain analytes as well as the range of useful CZE 
conditions. In addition, buffer chemistry can strongly affect the ESI 
process; certain components may suppress or enhance ionization efficiency, 
but not necessarily have a desirable effect upon the separation process. A 
final limitation of our original interface is the necessity of metal 
deposition at the capillary terminus which provided the electrical contact 
used to define both the CZE and ESI field gradients. The capillary 
preparation process is time consuming due to the several steps required 
for metal deposition and the contact with a surrounding stainless steel 
sheath used to impart mechanical stability. In addition, the deposited 
metal slowly erodes and requires replacement after several days of 
operation. 
We have, therefore, developed an improved electrospray ionization interface 
(ESI) 110, 110A (FIGS. 10-14) and 110B (FIG. 19) for CZE-MS and other 
applications which removes the limitations of the above-described design. 
The object of the improved design is to retain the attractive features of 
the foregoing design (i.e., avoidance of extra-column volumes, a 
well-behaved electrospray process, etc.) but not to impose any additional 
limitations upon operation. The improvement replaces the metal contact at 
the CZE outlet 40 with a thin sheath of flowing electrically-conductive 
liquid. The net result of this change is that the desired features of our 
original interface are retained while circumventing previous limitations 
on CZE flow rate and buffer composition. In addition, these changes 
provide a qualitative improvement in ESI stability, a design which does 
not require special treatment of the CZE capillary, and the capability for 
easy replacement of the CZE capillary. These and other advantages 
discussed in EXAMPLE 2 below suggest the improved ESI interface would 
allow much wider application of the CZE-MS method and the basis for 
extension to other capillary electrophoresis techniques. 
EXAMPLE 2 
CZE-MS Instrumentation 
The system arrangement and instrumentation used for CZE-MS in this example 
is largely the same as that described above for EXAMPLE 1 and like 
reference numerals are used in FIGS. 10-13 to indicate similar components. 
CZE was conducted in a fused silica capillary 20 with electroosmotic 
sample introduced at a rate of 0.1-1 microliters/min at the high voltage 
(10 to 50 kV) electrode. The high-voltage region containing the buffer and 
sample containers 32, 33 is electrically isolated in an interlocked 
Plexiglas box 30. Untreated 100 um i.d. fused silica capillaries were used 
for all studies. 
As demonstrated previously, operation of both the CZE and ESI requires an 
uninterrupted electrical contact for the electroosmotically eluting liquid 
at or near the capillary terminus. In this example, the electrical contact 
for the buffer at the low voltage (detection) end 40 of the capillary 20 
was made by a sheath flow of liquid 116, generally methanol, propanol, 
acetonitrile or similar, easily electrosprayed substances, as described in 
the next section. This electrical contact also serves to define the ESI 
voltage and was typically in the range of 3 to 5 kV. The ESI focusing 
electrode 26 was typically at +300 V (for positive ion operation). A 
nozzle-skimmer bias of 80 to 150 V was found to give optimum performance. 
The skimmer 70 was at ground potential for EXAMPLE 1. A precise pulse-free 
liquid flow for the sheath electrode flow 116 was provided by a small 
syringe pump 74, Sage Instruments (Cambridge, MA) Model 341B. 
An auxiliary flow of gas 136 was also used on occasion and was introduced 
at a flow rate of 0.1 to 1 L/min as a sheath around the capillary terminus 
(ES1 electrode). The purposes of this gas flow were to (1) add oxygen to 
suppress discharges in the negative ion mode of operation and (2) provide 
cooling for the sheath liquid flow at high CZE currents. A secondary 
benefit of this flow was the suppression of gravitationally induced 
instabilities in the electrospray for buffers which had a tendency to be 
marginal (i.e., generally too high an ionic strength for the sheath 
electrode liquid flow rate being used). 
Ions created by the ESI process were sampled through a 1 mm orifice 
(nozzle) 63 into region 200 maintained at a presure of 1-10 Torr by a 
single stage roots blower pumping at 150 liters/second. The ions entering 
this region were sampled through a 2 mm diameter orifice of skimmer 70 
located 0.5 cm behind the nozzle orifice 63. Ions passing through the 
skimmer 70 enter a radio frequency (rf) only focusing quadrupole 72. The 
region 201 containing the quadropole 72 is maintained at a pressure of 
approximately 10.sup.-5 Torr by differential pumping with a 
specially-designed Leybold Hereaus cryopump, consisting of a standard 
compressor and coldhead with a custom cylindrical second stage baffle 
cooled to approximately 14K, which encloses the quadrupole and provides an 
effective pumping speed for N.sub.2 of &gt;30,000 L/s. The analyzer 
quadrupole chamber was pumped at 500 L/s with a turbomolecular pump. A 
single ion lens with an 0.64-cm aperture separates the ion focusing and 
analysis quadrupole chambers. The pressures in the focusing and analysis 
chamber were about 1.times.10.sup.-6 and 2.times.10.sup.-7 Torr, 
respectively. The counter current flow 68 of N.sub.2 (at -70.degree. C.) 
for desolvation of the electrospray was in the range of 3 to 6 L/min. The 
mass spectrometer (Extrel Co., Pittsburgh, PA) had a range of m/z 2000. 
ESI Interface Design and Construction 
FIGS. 12 and 13 show a schematic illustration of a preferred version of the 
sheath electrode ESI interface 110 developed in this work. The ESI probe 
body 114 is machined from polycarbonate with a central axial channel 115 
and mounted in an approximately region 199 on custom holders that are 
movable on a small optical bench rail (not shown). The CZE capillary 20 
and a flow of 1-4 microliters/min of sheath liquid 116 are enclosed in a 
central 1/16 in o.d. Teflon.RTM. tube 126 through a Teflon.RTM. tee (not 
shown) outside the probe body. A polycarbonate tip holder 118 carries a 
conductive electrospray electrode 120 fabricated from a 26 gauge (0.25 mm 
ID, 0.46 mm o.d.) 3.3 cm long stainless steel (SS) tube soldered 
concentrically into a 1.9 cm long 21 gauge (0.51 mm ID, 0.81 mm o.d.) SS 
tube 122. Tube 122 is fitted into an axial bore 123 in the rear of the tip 
holder and extends rearward into the end of Teflon.RTM. tube 126. The ESI 
end 121 of the SS electrode is machined to about a 45.degree. taper and 
then electropolished. As the tip holder 118 is screwed axially into the 
probe body 114, the SS electrode 120 slides over the protruding fused 
silica CZE capillary 20 forming an annular passage and outlet 125 around 
the end portion of capillary 20, forming an annular passage and outlet 
125, around the end portion of capillary 20, making electrical contact 
with a spring-loaded, clip-type high voltage connector 124 coupled to 
conductor 42 and thereby being maintained at ESI voltage of +/-4-7 kv, and 
snugly fitting into the central Teflon.RTM. tube 126 connected to the 
Teflon.RTM. tee. The axial position of the CZE terminus 40 of capillary 20 
relative to the SS electrode 120 is easily adjusted bY sliding the 
capillary in the aforementioned Teflon.RTM. tee. 
An auxiliary sheath gas flow capability is provided to prevent any 
deleterious effects due to heating at either the sheath electrode (due to 
high CZE currents) or the counter current flow of heated nitrogen, 
although under ordinary operating conditions this provision is not 
required. The central axial channel 115 contains six 1/16 in. o.d. 
Teflon.RTM. tubes 128 (two shown) that carry nitrogen or oxygen (about 0.1 
to 1 L/min.) for the probe gas sheath and the central tube 126 that 
contains the CZE fused silica capillary 20 and the sheath electrode liquid 
116 flowing at 1-10 microliters/min. Two 1/16 in i.d. holes 130 are 
drilled into the back half of the tip holder 118 and connect into a single 
coaxial channel 132 which surrounds the ESI source at the front half of 
the tip holder. These passages serve to direct approximately one half of 
the gas delivered by the six Teflon.RTM. tubes 128 forward through the tip 
holder and over the ESI tip. The rest of the gas flows backward through 
channel 115 within the probe body 114 in the spaces between the tubes 126, 
128. An additional electrode of 0.5 cm long 11 gauge (2.4 mm i.d., 3.2 mm 
o.d.) SS tubing 134, is mounted over the ESI tip and in the central 
channel 132 of the tip holder. It directs the coaxialgas flow 136 over the 
tip and is held at the ESI potential by a second clip-type spring 
connector (not shown) touching tube 134. 
A second alternative embodiment 110A is shown in FIGS. 10 and 11. The 
capillary 20 passes through a Lucite.RTM. plastic tee 140 mounted on a 
copper plate 142. A short stainless steel tube 144 having an inside 
diameter 144d of 0.300 mm passes through plate 142 and inside one leg of 
tee 140 concentrically to surround the capillary outlet end portion. The 
steel tube 144 is sized relative to capillary 20 having an outside 
diameter 20d of 0.250 mm so as to provide an annular passage 145 for the 
flow of sheath fluid 116 at 1-10 microliters/min around the capillary 
outlet 40. Such flow is introduced via a second fused silica capillary 146 
146 connected to the remaining leg of the tee by a short copper tube 148. 
The steel and copper tubes, the copper plate and the buffer liquid 
introduced via the second capillary are maintained at the ESI voltage. 
EXAMPLE 2: RESULTS AND DISCUSSION 
ESI Interface Operation 
The key improvement provided by the interface shown in FIGS. 10-13 is a 
much broader range of CZE operating conditions which may be adapted to 
mass spectrometric detection. For example, the interface allows operation 
at CZE flow rates previously too small for stable ESI performance (&lt;0.5 
uL/min). An ideal electrospray emanates from the fused silica capillary 20 
when the electrical contact is made via the conducting liquid 116. In such 
a mode of operation, ion production is stable and large liquid droplets 
are not produced as when a nebulizing gas is used. A similar electrospray 
can also be produced at higher flow rates from a metal capillary, or using 
the metal-coated fused silica capillaries described previously for CZE-MS 
(see FIG. 7). At lower flow rates, however, the electrospray enters an 
unstable mode in which much larger droplets and liquid streams are 
sporadically produced. Such unstable operation is also characterized by an 
electrospray emanating from various sites on the metal surface, or moving 
from one site to another, and resulting in large variations in measured 
ion currents. Metal surfaces also appear to make a corona discharge more 
likely, which generally causes loss of analyte signal. Where there are no 
metal surfaces, the electrospray 64 emanates from the apex of a small 
liquid cone 138 (FIG. 13), having a volume generally in the range of 5 to 
10 nL. This type of electrospray was considered the ideal situation, which 
we have successfully duplicated with the new interface. 
FIG. 13 shows a detailed diagram of the capillary terminus (see also FIG. 
11 for end view). The low voltage end 40 of the CZE capillary 20, towards 
which buffer flows due to electroosmosis, shares the electrical contact 
with that necessary for electrospray ionization. The voltage at the 
stainless steel capillary 120 (+3 to 6 kv for positive ion production) 
thus defines the CZE and ESI field gradients. The actual CZE electrical 
contact is effectively made with the thin sheath of liquid 116 which flows 
over the fused silica capillary. The CZE capillary 20 need extend axially 
only a short distance beyond the metal capillary 120 (&gt;0.2 mm) to provide 
good performance. The voltage drop across the sheath electrode appears 
smaller than predicted on the basis of bulk conductivity of the sheath 
liquid 116 and, under typical conditions, both ESI and CZE performance are 
consistent with the expected electric field gradients. Thus, the CZE 
effluent avoids contact with any metal surfaces and is isolated from loss 
by electrochemical reactions. Interestingly, if the CZE capillary is 
retracted into the stainless steel capillary, analyte signals are lost 
even though a visibly unperturbed electrospray is still produced. 
Presumably, analyte ions are lost due to an electrochemical process at the 
stainless steel capillary. 
The sheath electrode liquid 116 can be the same as the CZE buffer, but it 
is often advantageous to use another liquid to improve electrospray 
performance. Since aqueous buffers could not be electrosprayed, our 
initial studies with CZE-MS used methanol/water buffers (50/50, V%/V%) 
with a small amount of added electrolyte (e.g., kI). By using either 
methanol or propanol as the sheath electrode liquid, However, aqueous CZE 
buffers can be used having up to 0.05M ionic strength. Similarly, the 
previous limit of .ltorsim.0.01M ionic strength in methanol/water buffers 
can be circumvented. Typical sheath electrode flow rates are 5 to 10 
uL/min, but the range of 2 to 30 uL/min is practical. Since typical CZE 
flow rates for 100 um i.d. capillaries are in the range of 0.2 to 0.5 
uL/min, we anticipate that even greater ionic strength buffers (about 
0.1M) can be addressed with smaller diameter capillaries due to the large 
effective dilution by the sheath liquid. At higher CZE currents (&gt;50 uA), 
addition of a small amount of electrolyte to the sheath liquid was found 
to be useful to prevent excessive voltage drop and heating, and a 
resulting disruption of the electrical contact. Although methanol and 
isopropanol are not highly conductive liquids, their conductivity appears 
sufficient given the short distance from the stainless steel capillary to 
the site of electrospray emission (0.3 to 0.4 mm) for normal CZE currents 
(&lt;30 uA). Analyte signals are relatively insensitive to the flow rate of 
the sheath electrode liquid. 
The sheath electrode liquid can also be used to modify the electrospray 
process by either manipulation of the liquid phase chemistry related to 
ion desorption or, potentially, post-column derivatization to yield an 
analyte providing distinctive mass spectral information or more efficient 
ionization. Our results indicate mixing in the electrospray cone 138 (FIG. 
13) is extensive since ESI performance can be dramatically improved with 
CZE buffers which could not be otherwise addressed (i.e., aqueous 
solutions). We have also used the sheath electrode liquid 116 to introduce 
components into the electrospray (such as ammonium acetate, 
trifluoroacetic acid, etc.), and such methods offer significant potential 
for affecting ESI efficiencies. 
The present interface essentially removes any CZE buffer composition and 
flow rate limitations for ESI. The use of smaller diameter capillaries and 
CZE media having high viscosities, including gels, is now feasible. In 
addition, other forms of capillary electrophoresis (isotachophoresis and 
isoelectric focusing), as well as capillary electrokinetic chromatography, 
should be more readily coupled with mass spectrometry. 
ESI Spectra 
The CZE-MS interface allows previously intractable CZE buffers to be 
effectively electrosprayed. FIG. 14 gives ESI mass spectra for a CZE 
buffer containing 0.05 sodium dodecylsulfate (SDS) in water obtained using 
isopropanol at about 5 uL/min flow rate as the sheath electrode liquid. 
The critical micelle concentration (CMC) for this anionic surfactant is 
about 8.times.10.sup.-4 M; thus most of the SDS exists as micelles in the 
CZE buffer. Negative ion spectra were obtained using the additional sheath 
gas flow of oxygen to scavenge electrons and suppress discharges similar 
to the approach first described by Fenn and coworkers (Whitehouse, C. M.; 
Dreyer, R. N.; Yamashita, M.; Fenn, J. B., "Anal. Chem." 1985, 57, 
675-679). The negative ESI spectrum shown in FIG. 14A shows only the 
dodecylsulfate anion at m/z 265. A small amount of singly charged dimer 
was also observed under some conditions at high SDS concentrations. A 
similar mass spectrum is obtained for much more dilute 10.sup.-5 
solutions. The positive ESI spectrum is shown in FIG. 14B. An intense 
molecular ion due to sodium attachment, is observed, and sensitivity is 
similar to the negative ion mode. Other ions in the spectrum can be 
attributed to the sodium cation and impurities. These results are 
especially encouraging since it appears possible that capillary 
electrokinetic chromatography, which requires concentrations of generally 
nonvolatile surfactants in excess of the CMC (Terabe, S.; Otsuke, K.; 
Ando, T. "Anal. Chem." 1985, 57, 834-841), can be interfaced with mass 
spectrometry to provide variations in separation selectivity and extension 
to neutral compounds. 
Two additional observations can be made regarding the ESI mass spectra. 
First, while fragmentation is generally not observed for the positive ion 
mode, the negative ion spectra sometimes show useful fragment peaks. For 
example, the spectrum of diphenylacetic acid (FIG. 15) shows major signals 
at m/z 59 and 167, as well as the molecular ion (due to H+ loss). Second, 
the mass spectra shown in FIGS. 14 and 15 are in sharp contrast to those 
from other desorption ionization methods (i.e., thermospray, particle 
bombardment, and laser desorption), which give large background signals at 
m/z&lt;150. This may be related to the relative simplicity of the ESI process 
compared to other desorption ionization methods, and the fact that the 
method does not involve either bulk or local heating of the sample. 
CZE-MS Separation 
The CZE-MS interface using a sheath electrode flow was evaluated using 
mixtures studied previously of quaternary ammonium salts in water/methanol 
buffers. Essentially identical performance was obtained yielding 
1-3.times.10.sup.5 theoretical plates and detection limits in the 10 to 
100 attomole range. In contrast with our earlier interface, similar 
separations could also be obtained with aqueous buffers. 
THE CZE-MS total ion electropherogram for a mixture of quaternary 
phosphonium salts is shown in FIG. 16. Electroosmosis was used to inject a 
sample plug containing approximately 1 pmole/component, using methods 
described previously. The separation was conducted in a relatively short 
60 cm capillary, using an 11 kV CZE voltage drop. Buffer conductivities 
were generally chosen to be on the order of 10.sup.3 umh/cm. The 
separation in FIG. 16 was obtained using a 0.05M potassium hydrogen 
phtphale aqueous buffer adjusted ph 4.8 by titration with NaOH and 
contained a 10.sup.-3 M KCl. (It should be noted that inferior separations 
exhibiting extensive tailing were obtained at higher pH). The sheath 
electrode liquid was isopropanol containing 10.sup.-4 M ammonium acetate. 
Although 40,000 to 80,000 theoretical plates are obtained in the 
separation of the individual components, as shown for the single ion 
electropherograms in FIG. 17, the four component mixture shows only two 
peaks in FIG. 16. It is not surprising that the vinyltriphenyl and 
ethyltriphenyl phosphonium ions elute simultaneously due to the expected 
similarities in their electrophoretic mobilities; it is somewhat less 
expected that the tetrabutyl and tetraphenyl phosphonium ions also 
coelute. We anticipate that separations obtained using smaller i.d. or 
longer capillaries or with alternative buffer systems would provide 
improved electrophoretic resolution of these components. 
The single ion electropherograms for a four component mixture of 
sympathomimetic and relate amines are shown in FIG. 18. In each case, the 
protonated molecular ion dominated to ass spectrum. The separation was 
obtained with an 11 kV CZE voltage in a 60 cm.times.100 um i.d. capillary 
using a buffer similar to that above adjusted to ph 3.8 with HC1 and 
containing 10.sup.-4 M KCl. Although dopamine and 5-hydroxytrypamine are 
not electrophoretically resolved, the norepinephrine and epinephine are 
separated from the other components. Although no attempt has been made to 
optimize the CZE separations, such results are particularly encouraging 
due to the biological significance of these compounds. 
The CZE-MS interface described here provides the basis for much broader 
application. The liquid sheath electrode allows the electrospray 
ionization interface to be operated for almost any buffer system of 
interest for CZE. This includes aqueous and high ionic strength buffers 
which could not otherwise be electrosprayed. In addition, the interface 
provides simplicity of operation and day to day reproducibility not 
obtained previously. The CZE capillary can be easily and rapidly replaced 
and no special treatment of preparation is required. The electrospray 
interface does not affect CZE efficiency and avoids a pressure drop across 
the capillary, problems which may make other approaches problematic. The 
sheath flow also provides a convenient method to introduce reagents for 
mass spectrometer calibration, manipulation of the ESI process, or 
post-column derivatization. If higher sheath flow rates are desired, a 
nebulizing gas can be introduced to assist the electrospray process 
(Bruins, A. P.; Covey, T. R.; Henion, J. D., "Anal. Chem." 1987, 59, 
2642-2646). 
The CZE-MS approach offers a combination of separation efficiencies and 
detection limits making it uniquely suited for many biological samples. 
For example, it is possible to directly couple a micropipette to CZE, 
providing a basis for direct sampling of single cells and a rapid mass 
spectrometric characterization of cell contents. A range of both organic 
and inorganic analysis conventionally addressed by even LC-MS, ion 
chromatography, and ICP-MS may benefit from CZE-MS when higher resolution 
separations are required. Separations requiring less than a few minutes 
are possible by using short capillaries, particularly where sensitive 
detection methods allow the sample volume to be minimized. Other 
variations of capillary electrophoresis, including isoelectric focusing, 
capillary electrokinetic chromatography and isotachophoresis (Everaerts, 
F. M.; Beckers, J. L.; Verheggen, Th. P. E. M. "Isotachophoresis," J. 
Chromatogr. Library, Vol 6, Elsevier Science Publishers, Amsterdam, 1976), 
are amenable to mass spectrometric detection using the new interface. 
The sheath flow electrospray interface also makes it possible to interface 
to other forms of analysis. FIG. 19 shows a third alternative embodiment 
110B adapted for CZE-electrospray interfacing to plasmas, e.g., for 
elemental emission, atomic absorption or other known forms of 
spectroscopy. This interface uses two Teflon.RTM. fittings 150, 152. 
Capillary 20 enters T-fitting 152 through a Teflon.RTM. tube 154 and plug 
156 and exits through opposed tube 162. Sheath liquid is introduced to 
T-fitting 152 via tube 160 and flows outside the capillary 20 through tube 
162 into a stainless steel tube 120 surrounding capillary 20 in four-way 
fitting 150. The ESI voltage conductor 42 enters fitting 150 via 
Teflon.RTM. tube 164 and plug 166, and connects to steel tube 120. Sheath 
gas 136 is introduced to fitting 150 via Teflon.RTM. tube 168 and flows 
outside steel tube 120 toward capillary exit 140. The fourth leg of 
fitting 150, containing concentric outlets for the CZE eluent, sheath 
electrode liquid flow and sheath gas flow, is connected to an inlet end of 
a cylindrical glass spray chamber 170. Spaced axially from these outlets, 
spray chamber 170 has a stainless steel collar 172 which connects the 
entire interface to plasma analytic apparatus (not shown) and which is 
connected into the second high voltage circuit 16B to serve as the 
electrospray target. Operation of interface 110B is generally as described 
above for embodiments 110 and 110A. The main difference is the lack of a 
countercurrent flow to desolvate the electrospray droplets. Also, the 
sheath gas, typically helium or argon, is necessary to help sustain a 
plasma. This sheath gas flow is also larger than the auxiliary flows 
mentioned above, e.g., 0.1 to 2 L/min. 
Having illustrated and described the principles of our invention in a 
preferred embodiment thereof, it should be readily apparent to those 
skilled in the art that the invention can be modified in arrangement and 
detail without departing from such principles. The extension of CZE, 
electrokinetic chromatography or isotachophoresis interfaced using the 
described electrospray process to other analytical or detection devices as 
well as off-line sample collection is also part of this invention. 
Modifications of the electrospray process which involve additional liquid 
or gas streams, or additional provisions for heating or focusing of the 
electrospray are also part of this invention. We claim all modifications 
coming within the spirit and scope of the accompanying claims.