Mass Spectrometry Applied to Environmental Analysis

48 1 CHAPTER 12

Capillary Electrophoresis/Mass Spectrometry Applied to Environmental Analysis WILLIAM C. BRUMLEY and WITOLD WINNIK US EnvironmenfalProfecfionAgency, Nafional Exposure Research Laboratory, CharacferizafionResearch Division, P.O. Box 93478, Las Vegas. NV 89193-3478, USA

12.1. INTRODUCTION A major objective of the analytical chemistry research program at the EPA’s National Exposure Research Laboratory (Las Vegas) is to foster and advance the adoption of new environmental monitoring technologies, especially those suited to pollutants for which conventional analytical approaches fail. An analytical separations technique that has enormous, untapped potential for environmental analysis is capillary electrophoresis (CE), especially when coupled with the proven workhorse of mass spectrometry (MS) for identificatiodquantitation. Several excellent reviews and book chapters have been written on capillary electrophoresis/mass spectrometry (CE-MS) in the last 5 years [l-51. None of these, however, has focussed exclusively on environmental applications of CE-MS. They have instead emphasized the various interfaces and results published for applications involving biological or pharmaceutical molecules. Also, the electrophoretic aspect of these separations has received little coverage making the subject difficult for those new to CE as well as to CE-MS. There are, of course, several excellent books devoted to CE [&lo]. In this introduction of the chapter, we attempt to answer two preliminary questions, What is CE and why develop environmental applications for CE? We then survey the existing instrumental approaches and applications of CE-MS to environmental analysis and attempt to outline some future perspectives.

References pp. 523-527

482

Chapter I 2

12.1.1. Brief history CE owes its present popularity to the paper by Jorgenson and Lukacs [l I] on the fluorescence detection of labeled amino acids. The high efficiency observed in that separation, along with the highly sensitive detection system, helped to place modem CE on a firm foundation. The authors also offered a theoretical framework for understanding the high efficiency observed in CE. In little over a decade, more than ten instrument makers now offer integrated CE systems [12]. The foundations of CE were laid much earlier in the work of Hjerten [13], Virtanen [14] and Mikkers et al. [15]. A history of CE has been written by Vesterberg [16]. The biannual reviews of CE in Analytical Chemistry began in 1990 and have continued through 1994 [17-191. Excellent early reviews of the field include those of Jorgenson [20] and Ewing et al. [21].

12.1.2. Experimental formddetection methods Figure 12.1 illustrates some of the principles involved in both capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). MEKC (also called MECC) was introduced by Terabe et al. in 1984 [22,23]. Terabe et al. also introduced applications of cyclodextrins (CDs) and urea in MEKC [24] for improving separations involving hydrophobic molecules. The principle of CE involves a very simple experiment: the application of a high voltage difference between two buffer reservoirs connected by a fused silica capillary [6-lo]. Electrophoresis (i.e. the migration or mobility of ions in an electric field) accounts for the movement of ions of the appropriate charge towards the cathode or anode in narrow bands. Electrophoretic flow is shown in Fig. 12.1 by a smaller, dark arrow. In addition, an electroosmotic (EO) flow transports bulk liquid with buffer from one reservoir to

O v = Surfactant (negative charge)

+ =Analyte

= Electroosmotic Flow

= Electrophoresis

Fig. 12.1. Separation principle of CE and MEKC. Detector window is usually placed near the end of the capillary. Adapted from Ref. [18].

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the other depending on conditions. Usually, for bare silica, an excess of mobile positive charge exists in solution because of the ionization of silanol groups on the silica surface. This ionization causes the formation of a layer of mobile positive charge near the negatively charged silica surface. These resulting charge layers are very thin in CE, and in a very short distance (10-100 nm) become indistinguishable from the bulk electrolyte. The EO flow is illustrated by the large, gray arrow (Fig. 12.1). This flow is characterized by a flat, piston-like profile rather than the parabolic profile characteristic of pressure-driven systems. This flat-flow profile gives extremely narrow peaks and high efficiency in CE. The separation of neutral analytes under MEKC is based on their affinity for micelles (aggregates of surfactant molecules) that migrate under these conditions; these micelles can be viewed as forming a pseudostationary phase [25]. A comparison of CE with other separation techniques reveals several interesting characteristics (Table 12.1) [26].CE possesses the highest theoretical plate numbers and thus the highest efficiency when compared with the more common liquid and gas separation techniques. Solvent usage for CE is nearly one-thousandth that of HPLC. The mass detection limit of CE is about one-thousandth that for GC or HPLC. Recent efforts using laser-induced fluorescence (LIF) detection have reduced TABLE 12.1 COMPARISON OF CE, HPLC, cap-GC AND cap-LC Parameter

CE

HPLC

cap-GC

cap-LC

Solvent flow rate Injection volume Injection fouling Plates MW limit Detection limit Column price (US$) Column preequilibration Irreversible adsorption Versatility (need new column) Volatiles Semivolatites Polar, non-volatiles

0.5-10 pVmin

1 pl to 100 nl No 2.7 million 200000 kDa 1 ag to 1 ng 5-100 Yes (buffer)b

1-3 mVmin 1 pl-1 ml No 30-100 K 5000 kDa or GPCa 1 pg to 1 ng 300 Yes

1-3 mumin 1-loop1 Yes 100-200 K 1200 kDa 1 pg to 1 ng 300-600 No

I-1OpVmin 1-500 nl No 50-400 K 5000 kDa or GPC Ipgtolng 50-400 Yes

No (exceptions)c

Yes

Yes

Yes

High (no)d

Medium (Yes)

Medium (Yes)

Medium (Yes)

Rare Yes Yes

Rare Yes Yes

Yes Yes

Rare Yes Yes

~~

NOS

aGel permeation chromatography or size exclusion chromatography to 150 OOO 000 kDa. bBuffer pre-equilibration for 1-5 min. ‘Proteins can exhibit strong adsorption to uncoated capillaries. Versatility is usually achieved via the buffers and additives; some proprietary coatings available. Derivatization extends the range of applicability.

References pp. 523-527

Chapter I 2

484

TABLE 12.2 COMPARISON OF SEPARATION ABILITIES OF CE, GC, AND LC FOR SOME POTENTIAL ANALYTES Analytes

CE

Cap-GC

Cap-LC

HPLC

PNAs Phenols Anilines Benzidines Alkali metals Alkaline earths Lanthanides Transition metals Small organic acids Arom. acids Aliphatic amines Organometallic compounds Proteins, peptides PCBs Surfactants Pesticides Herbicides Polymers Drugs

+ (MEKC) + (CZEa, MEKC)

+

+ + +

+ + + +

+ (MEKC) + (MEKC)

+ (CZE) + (CZE)

+(most) + (most)

-

+ -

-

-

+ (chelates)

+ (chelates) + (ion-pairing) + + (ion-pairing)

+ (CZE)

-

+ (CZE)

i (some)

+

+ (CZE, MEKC) + (CZE) +

f (some)

f (some)

+ (as derivatives)

+ (CZE, CGEC)

-

+ (CGE, MEKC) + (MEKC) + (CZE, MEKC) + (CGE) + (CZE, MEKC)

f (some)

+ (MEKC)

+ +

f (some) f (some) f (some)

+ (ion-pairing)

f (some)

+ + + + + + +

+ (1C)b + (IC) + (ion-pairing) + (chelates) + (ion-pairing)

+

+ (ion-pairing) f (some)

+ (GPCd)

+ + + +

+ (GPC)

+

a Free zone electrophoresis. bIon chromatography. CCapillarygel electrophoresis. dGel permeation chromatography.

the detection limit to the 10 yoctomolar (10 X M) level (six molecules) [27]. The ruggedness and low cost of CE become apparent given the low cost of columns and the fast equilibration that takes place between post-run rinses and buffer conditioning of the capillary. CE possesses superior attributes of laboratory waste minimization. The wide range of analytes accessible to CE is readily apparent in the comparison of separation techniques for a range of analytes in Table 12.2 [26]. Additional experimental formats for CE include isoelectric focussing (IEF), isotachophoresis (ITP), and capillary gel electrophoresis (CGE). IEF uses a static pH gradient within the capillary so that all analytes migrate to their respective isoelectric points; compounds must then be eluted from the capillary or detected in situ at their respective zones. ITP involves the creation of sharp zone boundaries of analytes sandwiched between the zones (based on intrinsic mobilities) of leading and trailing electrolytes. The concentration of the analyte zone is electrically forced to become that of the leading electrolyte (often in practice a concentrating effect). This results in

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a varying electric field potential throughout the capillary (at constant current) such that all zones move at the same velocity. ITP is used for both separation and oncolumn concentration applications. CGE is often performed where a gel-filled capillary (polyacrylamide) is used to sieve proteins (SDS PAGE was introduced by Cohen and Karger [28]) or DNA segments according to molecular size. The useful range for CGE is from about 14 kDa to about 200 kDa [6]. Detection systems for CE consist of optical methods based on UV absorption [6], LIF 129,301, and indirect UV and LIF modes [31-341. Electrochemical detectors [35,36], radioisotope detection [37], and MS [38,39] are among the options commercially available or under development. Detection sensitivity for U V , the most widely available detector, is one of the major factors that has limited the development of 'environmental applications of CE. Although detection in CE is very mass sensitive (i.e. the absolute amount of substance on-column), concentration limits of detection are substantially higher because of the small injection volume typically used (ca. 1-10nl). While this is advantageous for microenvironment studies (e.g. a single cell), it is a serious limitation for environmental analysis. Various approaches including improved sample handling and enhanced detector sensitivity (e.g. using LIF) for overcoming the limitations of nanoliter injection volumes are discussed later in more detail in Section 12.2.6.

12.1.3. Why environmentalapplications? One of the broad areas of great potential for CE in environmental monitoring is outlined in the goals adopted by the US EPA and set forth in its Environmental Technology Initiative (ETI) formulated in 1993. Under ETI, environmental monitoring technologies are sought that offer continuous monitoring capabilities (i.e. prevent pollution in waste streams), are inexpensive, are exportable to foreign countries, are low generators of waste, minimize exposure of personnel to hazardous chemicals, and address a broad range of analytes. These widely divergent goals could be easily addressed by CE in its various formats. The capillary format of CE ensures little waste production. This format in turn lends itself to parallel (simultaneous) processing of samples (e.g. 24 at a time) via bundled capillaries [40,41]. CE is easily compatible with state-of-the-art laser detection systems such as LIF. The highly diverse separations chemistries now available and those yet to be developed offer a specificity and adaptability that is unparalleled by any other single technique. CE, because of its fast separations capability, has been suggested as the basis for chemical sensors [42,43]. The microminiaturization of CE is under development 1441. The fast separations capability and adaptability of CE make it of interest for both laboratory- and field-based methods of analysis. Indeed, the chemistry and manipulations developed for laboratory-based methodology will directly influence attempts References pp. 523-527

486

Chapter I 2

to reach goals such as those of the ETI by solving an immediate analytical problem. Head-to-head comparison of CE methods with those of capillary GC or HPLC are relevant to the assessment of the capabilities of CE. However, the limited range of analytes accessible to capillary GC and GUMS (conceptualized in Fig. 1 of Ref. [26]) only partially showcases the broad range of CE applications. Potential analytes for CE include ionic compounds such as metal ions, organometallics,anions, organic acids and bases, surfactants, and herbicides (see also Table 12.2). Analytical chemists have long recognized a major challenge in the frontier of nonvolatiles analysis. This has resulted, for instance, in the explosive development of new mass spectrometric techniques in biochemical analysis [45]. Non-volatiles of interest in the environment include ecological indicators of stress or health, biomarkers of exposure, and surfactant molecules whose metabolites act as hormone modulators that can affect wildlife and humans. A cyclical dilemma derives from the knowledge that many such compounds are not regulated, and, therefore, there is no need for their monitoring. Conversely, such compounds are not monitored solely because effective methods do not exist for their identification and quantitation. A partial resolution to this cycle is to develop research methods for these problematic compounds to assess their occurrence, action, and fate in the environment. Such data would ultimately serve as a basis for regulation and risk reduction.

12.2. CE MODES AND SEPARATIONS ILLUSTRATED 12.2.1. CZE An example of free zone electrophoresis is shown in Fig. 12.2 for the separation of propylamines and tetrabutylamine [46] using indirect UV detection. In this mode, a background electrolyte (imidazole 5 mM) which absorbs in the UV, is displaced by the analyte ions, resulting in a decrement in absorption; these operating conditions are similar to those used in CE-MS, where a relatively low concentration of buffer is used (e.g. 50 mM borate would be normal in typical CE-MS applications). The ions, as cations, reach the detector window (on-column detection is typical) at various migration times. From these times (column 2 of the inset in Fig. 12.2), the apparent mobilities &) may be calculated, and the effective mobilities 01,) are then obtained from the equations

where: peOfis the apparent mobility of the system peak which results from the displaced background ion moving with the velocity of the electroosmotic flow, and

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487

I

I

-4.oooo

0.00

1

2.00

4.00

1

1

1

1

8.00

1

1

1

1

8.00

1

1

1

10.00

Migration Time (min)

Fig. 12.2. Separation of aliphatic amines; buffer and conditions: imidazole (0.005 M) (pH 5.0); 57 cm X 0.075-mm i.d. capillary; 30 kV, detection 214 nm, indirect (50 cm to detector). Peak identities: MT = 1.44, ammonia; MT = 1.92, propylamine; MT = 2.14, dipropylamine;MT = 2.26, tripropylamine; MT = 2.43, tetrabutylamine (internal standard). Work performed in the authors’ laboratory.

where V is the applied voltage (V), 1 is the effective capillary length to the detector (cm), L is the total capillary length (cm) and t is the migration time (s). The mobilities calculated for the organic amines agree with predictions of the basic equation describing electrophoreticmobility:

where p, is the electrophoretic mobility, q is the ion charge, 7 is the solution viscosity and r is the ion radius. This equation predicts that small highly charged species exhibit high mobilities and that large, minimally charged species have low mobilities. This principle ignores the effects caused by solvation spheres of ions; the mobilities of such ions are often as shown in Fig. 12.3. correlated with the Stokes’ radius (i.e. as ll(rnl~)-O~~) References p p . 523-527

Chapter I 2 0.14

P

0.12

-

0.10

-

m

Ammonium Ion

0.08

0.06

0.04 0.02

0.00

Electrophoretic Mobility 10-4 crn2 V-1st Fig. 12.3. Correlation of l/Stokes’ radius or l / ( m / ~ ) O . ~for several amines. Adapted from Ref. [46].

12.2.2. Dispersion and efficiency

CE is normally dominated by dispersion, resulting from longitudinal diffusion. This term is described in the equation 0 = 2Dt

= 2D1WpeV

where D is the diffusion coefficient of the solute. Other contributions to dispersion include sample injection, wall interactions,joule heating, and electrodispersion forces. When efficiency depends primarily on the value of D,then the following equation can be derived from the familiar equations of chromatography for calculating the number of theoretical plates: N = peVl12DL = peEl12D N = 5.545(t/w,,2)

where t is the migration time and wlD is the peak width at half height.

CE-MS Applied to Environmental Analysis

489

An equation for the resolution of two species can then be written down and related to the familiar equation of chromatography: R = 2(t2 - tl)/(wl

+ w2)= (tz- tl)/4a

where t is the migration time, w is the baseline peak width, cr is the standard deviation in time and subscripts 1 and 2 are solutes such that R = 1/4N1/2@dif4p,,)

where Pdiff

=P2

-PI

and

This equation predicts that resolution of two species will be maximized when Oldiff) is large and @,, + peof)approaches zero. Obviously, considerations of practicality limit the time of runs to reasonable periods (e.g. c30 min).

12.2.3. Electrodispersion An important consideration for CE-MS separations is the contribution to zone broadening due to mismatch of mobilities between background electrolyte and analyte ion This problem becomes significant when the buffer or background electrolyte is not 100-1000 times more concentrated than the analyte. This mismatch results in peak fronting (or tailing) due to zone dispersion caused when the analyte mobility is greater than (or less than), the mobility of the background (leading) electrolyte and is described by the Kohlrausch regulating function [47]

where c, is the concentration of analyte, cIis the concentration of leading electrolyte, p xis the mobility of analyte, pcis the mobility of counter ion and p I is the mobility of

the leading electrolyte. This relationship controls the concentrating (or diluting) effects caused by injecting samples less (or more) concentrated than the running buffer.

References p p . 523-527

490

Chapter 12

0.015 m

E

i

0.010

Ir

0.000

I

0.0

5.0

10.0

15.0

20.0

Miaration Time (min) Fig. 12.4. Separation of seven sulfonyl urea herbicides; buffer and conditions: ammonium acetate (0.0375 M) (pH 5.0); 25% acetonitrile; 57 cm X 0.075-mm i.d. capillary; 30 kV; detection 214 nm (50 cm to detector). Peak identities in the order of increasing migration time: bensulfuron methyl, sulfometruon methyl, nicosulfuron (Accent), chlorimuron ethyl, thifensulfuron methyl (Harmony), metsulfuron methyl, and chlorsulfuron; internal standard, 3-nitrobenzene sulfonic acid. Work performed in the authors’ laboratory.

An example of these effects can be seen in Fig. 12.2 where the peak shapes of the amines become increasingly broadened as the migration time increases. Peak distortion can be minimized by matching background and analyte mobilities and by reducing the amount of analyte. When analyte concentration approaches MOO that of the background ion, peak distortions due to zone dispersion are minimal. Another example of this phenomena is an electropherogramof seven sulfonyl ureas and internal standard shown in Fig. 12.4. Peaks in this separation exhibit theoretical plate numbers between 200000 and 300000 and that of the internal standard is about 1000000. This efficiency may be contrasted with data shown later for a CE-MS determination of these compounds, where the background electrolyte concentration

49 1

CE-MS Applied to Environmental Analysis TABLE 12.3 PROPERTIES OF COMMON SURFACTANTS USED IN MEKC Surfactant

Acronym

Aggregation number

CMC/IO-3

Charge

Sodium docecyl sulfate Sodium tetradecyl sulfate Cholic acid Polyoxyethylene octyl phenol n-Dodecyl-B-D-maltoside Pol yoxyethylene(6)dodecanol Polyoxyethylene-(23)dodecanol Cetyltrimethyl-ammoniumbromide Dodecyltrimethyl-ammoniumbromide Cetyltrimethyl-ammoniumbromide

SDS STS NA Triton X-100 NA NA Brij 35 CTAB DTAB CTAB

62 138 2 4 140 NA 400 40 78 55 75

8.1 2.1 14 0.24 0.16 0.09 0.1 1.3 1.5 3.5

Anionic Anionic Anionic Non-ionic Non-ionic Non-ionic Non-ionic Cationic Cationic Cationic

NA, not available.

is 5 mM. In Fig. 12.4, the buffer (ammonium acetate 37.5 mM, 25% acetonitrile, pH 5.0) provides for an efficient separation. 12.2.4.MEKC Surfactant molecules form micelles in aqueous solution above a critical micellar concentration (CMC) that is characteristic for each surfactant type. The number of surfactant molecules per micelle is usually characteristic of a particular surfactant. For example, Table 12.3 lists some examples of CMC and molecules per micelle (aggregate number) for common cationic, neutral, and anionic surfactants; sodium and 62 molecules per micelle. It dodecylsulfate (SDS), has a CMC of 8.1 X should be pointed out that cationic surfactants can be used to reverse the direction of EO flow by forming a double layer at the silica wall, resulting in a net positive charge at the wall surface. The positive head of the surfactant aligns opposite the negatively charged wall while a second molecule’s hydrophobic tail associates with that of the first, allowing its positive head to point towards the bulk solution. Current research is directed, in part, to developing monomolecular micelles that have a CMC of 1 molecule. These micelles may be more compatible with CE-MS, since they are effective at the 5 mM concentration level. Another problem under study is improving the separation of highly hydrophobic molecules (e.g. polynuclear aromatic hydrocarbons) under MEKC conditions. With micelles present, the separation of neutral as well as charged analytes is possible. The affinity of an analyte for the micelle means that the analyte is imparted a mobility that falls in the range between that of the EO flow and that of the micelle. The capacity factor from chromatography is similar to that of MEKC but modified due to the finite migration time of the micelle as References pp. 523-527

492

Chapter 12

k' = (t, - to)/to( 1 - t,./tm)

where tr is the migration time of solute, to is the migration time of unretained solute moving at EO flow and tm is the migration time of micelle. The equation for resolution under MEKC is therefore modified as:

R = (N"V4)[(a - l ) h ] [ k ; / ( k ; + 1)]{[1 - (tdtm)]/[l- ( t d t , ) k ; ] } where a = k;/k,', k; is the capacity factor of analyte 1, k; is the capacity factor of

0.01w

8 C m

9

8

9

0.0050

0.0000

0.00

10.00

20.00

30.00

36.00

Migration Time (min) Fig. 12.5. Separation of synthetic dyes; buffer and conditions: borate (0.050 M); sodium cholate hydrate (0,100 M), 10% acetone (pH 8.35); 25 kV; 57 cm X 0.050-mm i.d.; 214 nm (50 cm to detector). Peak identities: MT = 13.28, 4-hydroxyphenyl acetic acid (internal standard); MT = 14.40, cresol red; MT = 17.96, orange 11; MT = 18.25, acid blue 40; MT = 18.48, acid orange 8; MT = 19.27, nuclear fast red; MT = 19.58, impurity; MT = 20.14, acid red 151; MT = 21.35, tropaeolin 0; MT = 25.53, anthraquinone-2,6-disulfonicacid, sodium salt (internal standard).Work performed in the authors' laboratory.

CE-MS Applied to Environmental Analysis

493

analyte 2 and N is the number of theoretical plates. Thus, resolution can be improved by optimizing the efficiency, selectivity, and the capacity factor. In practice, the capacity factor is varied by varying the concentration of micellar agent. The selectivity is altered by adding additional components such as organic solvents, cyclodextrins, and non-ionic surfactants. Resolution is also improved by extending the time window in which neutral analytes elute, i.e. between to and tm. This may be important in another sense for MEKC-MS by virtue of extending tm using organic solvents and partial filling of the capillary with micellar solution, Under such conditions, it might be possible to prevent micelles from ever exiting the capillary into the mass spectrometer. An example of the separation of synthetic dyes by MEKC is given in Fig. 12.5; in the reference capillary liquid chromatography (LC) and MEKC are compared [48]. The complementary nature of the two techniques was obvious from the altered elution order in comparing the two separations.

12.2.5. Electrokinetic chromatography (EKC) In this variation of MEKC and CE, a true stationary phase is employed as in reverse phase HPLC. The capacity factor and resolution equations are thus obtained by letting tm tend to infinity. EKC may offer the practical alternative to MEKC for MS applications. The efficiencies are high, and conditions worked out for monitoring applications using optical detection could be used directly in EKC-MS separations. An example of ongoing work is the separation of 16 polynuclear aromatic hydrocarbons (Fig. 12.6) [49].Although 3-pm particles of CI8 derivatized silica were used under isocratic conditions, gradient capability with such systems is currently feasible.

12.2.6. Sample handling Because extremely small volumes of solutions are injected in CE (nominally picoliters to tens of nanoliters), preconcentration,clean-up, and on-line sample concentration must be addressed in the context of the analysis. Chien and Burgi comprehensively reviewed injection techniques for CE [50]. By means of field amplification techniques, relatively large volumes can be injected without introducing intolerable peak broadening. In fact, 30-100% of the capillary volume can be used for injection [51,521, yielding injection volumes greater than 1 pl. A characteristic of environmental analysis, in contrast to biochemical analysis, is the large amount of sample that is usually available for analysis (e.g. lakes, rivers, estuaries, harbor sediment, industrial sludges, agricultural soil, smokestack fly ash, and industrial waste streams). In addition, for solid samples the requirements of homogeneity and representativeness usually demand samples of 1-100 g. Thus, techniques of analyte concentration by solvent evaporation, supercritical fluid extraction (SFE), solid phase extraction (SPE), and derivatization have been used. Nielen has References pp. 523-527

Chapter 12

12

13

384

15

1-

0

20

40

Retention Time (min) Fig. 12.6. Separation of 16 PNAs by EKC; buffer and conditions: CI8-derivatized 3-pm silica, 80% acetonitrile, borate buffer (0.004 M), pH 8.2, 33 X 0.075-mm i d . (LIF detection 257 nm excitation), 15 kV. Peak identities: 1, naphthalene; 2, acenaphylene; 3, acenaphthene; 4, fluorene; 5, phenanthrene; 6, anthracene; 7, fluoranthene; 8 pyrene; 9, benz[a]anthracene; 10, chrysene; 11, benzo[b]fluoranthene; 12, benzo[k]fluoranthene; 13, benzo[a]pyrene; 14, dibenz[a,h]anthracene; 15, benzo[g,h,i]perylene; 16, indeno[l,2,3-cd]pyrene. Adapted from Ref. [49].

selectively reviewed sample handling for CE from aqueous matrices [53]. Oncolumn preconcentration with adsorbents [54] and ITP [55] have also been used in sample preparation. Two examples of on-column concentration of ionic analytes are illustrative of the approaches. Nielen [53] has described the concentration of phenoxyacid herbicides in aqueous matrices using a field amplification approach. The capillary is largely filled with the sample, the polarity of the experiment reversed (cathode at injection end), and the voltage applied until the observed current reaches about 95% of its normal value. At this point the analytes are concentrated in a narrow band at the inlet end of the column, and the column has filled with running buffer. The polarity is then changed to the normal mode (anode at injection end), and the final separation M were achieved using UV detection in comeffected. Detection limits below bination with SPE and field-amplified injection techniques, representing a 100-fold

CE-MS Applied to Environmental Analysis

495

improvement. Kaniansky et al. [56] used ITP-CZE coupled columns with the ITP stage employing a leading electrolyte of potassium citrate/morpholinoethanesulfonic acid and a terminating electrolyte of tris(hydroxymethyl)aminomethane/citrate. Their system effected a 1000-fold concentration improvement resulting in the detection of paraquat and diquat at M in aqueous matrices. Liu et al. [57] have used a micellar agent to effect preconcentrations (factors of ca. 75) on-column for hydrophobic molecules, extending the on-column concentrating techniques to neutral molecules. Clean-up requirements for CE are generally similar to those appropriate for other forms of final separatiorddetermination consistent with the properties of the analytes and the detector (e.g. GC-MS, HPLC-UV, and TLC-VIS). A final solvent (with some electrolyte) miscible with aqueous buffers is usually essential for maintaining electrical continuity in the capillary. CE depends on harnessing electrical forces for the manipulation and separation of charged species. In this respect, it exhibits similarities to the field of mass spectrometry where ionization of molecules results in the ability to measure mass-to-charge ratios. Sample handling in CE can also take advantage of the manipulation of charged analytes and of neutral analyte’s imparted mobility (charge) via interactions under MEKC. Thus, preliminary concentration or separation can be effected based on ITP, IEP, CZE, or MEKC, before the final separation/detection using one of the forms of CE. These operations can be used in lieu of or in combination with techniques based on adsorption chromatographies. It is conceivable, therefore, that traditional clean-ups involving solvent exchanges with pH control can be replaced by sample handling in a CE format. Electrical manipulation and control of analyte and derivatizing agents in migrating zones enables the use of the capillary as a microreaction vessel as well as a separations tool. Handling of environmental samples for CE is treated more thoroughly in a recent review [58]. 12.2.7. Immunoaffinity and immunoassay in CE format

Schultz and Kennedy [59] pointed out two possible CE formats for immunoassay using LIF detection. An application of the general approach was published by Chen and Evangelista for drug analytes [60]. Such approaches should also be possible for environmental analytes. The use of affinity chromatography as a clean-up step prior to CE determination is also another interesting approach to sample analysis; with this approach, the analyte itself could be the affinity target, or, alternatively, a major interference could be the target. Alternatively, affinity complexes themselves have been directly detected using LIF with fast CE separation [61]. 12.2.8. Non-MS detection and sensitivity Absolute mass sensitivity of detection by optical spectroscopy (no preconcentration or field amplification) is about to M in a sample by UV or indirect UV References pp. 523-527

496

Chapter 12

20.0000

-

10.0000

0.0000

l.--&-4 0.00

10.00

20.00

Migration Time (min) Fig. 12.7. Separation of aliphatic and other amines with LIF detection; buffer and conditions: phosphate (0.039 M), SDS (0.070 M), urea (2.0 M), 23% methanol, pH 7.0, 47 cm X 0.050mm i.d. capillary, 30 kV, LIF detection (488 nm excitation and 520/20 nm emission, 40 cm to detector). Peak identities (as fluorescein isothiocyanate derivatives): MT = 9.77, 2-(2-aminoethyl)pyridine; MT = 1 1.80, 1,5diaminopentane; MT = 12.09, p-toluidine; MT = 12.68, butylamine; MT = 13.35, diethylamine; MT = 13.89, propylamine. Work performed in the authors' laboratory.

or indirect fluorescence detection. For a relative molecular mass of 100, this calculates to 1 pg to 100 fg on-column for a 10-nl injection. In the case of LIF, the detection limit may approach lo-" to 10-14M in a sample resulting in 10 ag to 10 zg injected on-column. Reaching these mass sensitivities presents one of the greatest challenges in the development of CE-MS. Detection of compounds derivatized with fluorescent agents is an important subcategory of this challenge. For example, the fluorescein-containing derivatives of amines and phenoxy acids whose separations are shown in Figs. 12.7 and 12.8 [26], respectively, can be detected at ppb to subppb levels. The derivatized molecules have relative molecular masses well beyond that of the usual background ions; this is an advantage. The production of artifacts due to derivatization of co-extractives and reaction by-products presents a challenge

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-m

ern 2-

' p ??

I 12.00

1

1

15.00

1

1

1

1

1

1

1

1

20.00

1

1

25.00

1

1

1

1

30.00

Migration Time (min) Fig. 12.8. Separation of phenoxyacid herbicides with LIF detection; buffer and conditions: phosphate (0.039 M), SDS (0.046 M), urea (1.670 M), (pH 7.0), 30 kV, 47 cm X 0.050-mm i.d., LIF detection (488nm excitation, 520120 emission, 40 cm to detector). Peak identities (as the 5-(aminoacetamido)fluorescein derivatives): MT = 14.89, 3,4-dichlorophenoxy acetic acid; MT = 16.84, 4-(4-chloro-otoly1oxy)butyric acid; MT = 17.25, 4-(2,4-dichlorophenoxy)butyric acid; MT = 17.73, 4-chloro-otolyloxy acetic acid; MT = 18.21, 2,4-dichlorophenoxy acetic acid; MT = 18.74, 2,3-dichlorophenoxy acetic acid; MT = 19.13, 2-(4-chloro-o-tolyloxy)propionicacid; MT = 19.51, 2-(2,4-dichlorophenoxy)propionic acid; MT = 25.19, 2,4,5-trichlorophenoxy acetic acid; MT = 28.06, 2-(2,4,5-trichIoro)propionic acid. Work performed in the authors' laboratory.

that could be forcefully addressed by mass spectrometry with its specificity based on mlz values and MS-MS selectivity. The situation is similar to strategies employed in enhancing electron capture response for GC-ECD and in using electron capture negative ion mass spectrometry to confirm identity and amount. All of these applications suggest a central cross-cutting role for CE in method development for environmental analytes. This role is depicted in Fig. 12.9. [%I. References pp. S23-S27

498

Chapter 12

Semivolatiles

I

Anions

Herbicides

Nonvolatiles

Biomarkers

Speciation

Preparative Applications

Preconcentration Micro Reactions

I

Micromanipulation

Fast Separating Sensor

Fig. 12.9. Central cross-cutting role of CE and M E K C in analytical methods development for environmental analytes.

12.3. INTERFACES AVAILABLE Three on-line techniques are currently in use for interfacing CE with mass spectrometry: continuous-flow fast atom bombardment (CF-FAB), electrospray (ES), and ion spray (IS). The first technique differs from the other two in the method of ion transfer from the liquid to gas phase. In the CF-FAB interface, a continuous flow of liquid, typically 5-15puVmin, is delivered through a 10-100-pm capillary to the tip of the CF-FAB probe, which is positioned in the high-vacuum ion source of a mass spectrometer. In the electrospray and ion spray interfaces, the capillary effluent is delivered to the nebulizer operating at nearly atmospheric pressure. The liquid is dispersed as uniform, small droplets (ca. 1-pm radius) by the action of a strong electric field on the liquid emerging from a narrow-bore capillary. There are two types of CE-FAB and CE-[ES] interfaces currently used, which are based on liquid junction and sheath flow designs. These designs differ by the manner in which the make-up liquid is introduced. Since typical flow rates from a CE capillary range from 1 to 100 nl/min, a make-up liquid is needed to support the CF-FAB and ES ionization processes in interfaces operating at 1-50 pl/min. Performance details for these different types of CE-MS interfaces are discussed in the following sections.

12.3.1. CE-CF-FAB-MS Fast atom bombardment mass spectrometry, first developed by Barber et al. [62],

CE-MS Applied to Environmental Analysis

499

has become a commonly used MS method for analysis of polar, thermally labile, or non-volatile compounds, including its interfacing with microbore LC [63]. Since both CE and FAB-MS are suitable for analysis of such compounds, interfacing of CE with FAB-MS has become an area of intensive research [38]. Generally, a CEMS unit comprises a modified CE system, a coupling device, a sample introduction interface, and a mass spectrometer. The continuous-flow fast atom bombardment technique is used in one of the most common interfaces coupling liquid streams with mass spectrometers [64,65] (Fig. 12.10). Most interfaces developed for CE-MS are modifications of the interfaces utilized for LC-MS [38]. The FAB-MS technique requires a viscous matrix. Typically, glycerol or nitrobenzylalcohol are used as the viscous matrix components. Generally, 5-25% glycerol in different solvents constitutes a good matrix for CF-FAB experiments. The liquid is delivered to the FAB interface in form of a make-up solution. In the sheath flow (or coaxial) CE-CF-FAB design, developed by Deterding, Moseley, Tomer, Fergenson, and DeWit [66-711, with further elaboration by Thibault et al. [72] and based on the design used for LC-MS coupling [73], the coaxial make-up liquid allows for independent optimization of the composition and the flow rates of the CE capillary effluent and the FAB matrix mixture. This arrangement also ensures the required electric continuity for CE is maintained via the make-up liquid. Since the CE capillary outlet is positioned in the high-vacuum ion source, the sheath flow design is restricted to relatively small-diameter CE capillaries (1015p m i.d.) to avoid an excessive vacuum-induced (or pressure-driven) flow. Such pressure-driven flow in the capillary would result in a reduced efficiency of the CE separation because it induces a parabolic instead of the preferred plug-type flow profile [38,64]. The small column inner diameter, however, significantly restricts sample loadability. Small sample injection volumes (low nanoliter range) result in poor concentration detection limits. This is a severe drawback in environmental applications, where analyte volumes are practically unlimited but analyte concentrations are usually very low. In a liquid-junction design, the analytical capillary column is aligned with a transfer column [75,76] (Fig. 12.10). The junction is immersed in the make-up solution. The narrow gap between these two columns (approximately 25 pm) allows the make-up liquid and the analytical capillary effluent to flow into the transfer column, which then delivers the mixture to the tip of the FAB probe. A variety of liquid-junction FAB interfaces have been reported along with the potential of tandem MS [77-1011. In an early application, the two capillaries were affixed to a glass plate [75]. In a later design, the width of the gap between the two capillaries was adjustable [ 1021. The liquid-junction CE-CF-FAB interface was found relatively easy to set up and handle [77], but extreme care was needed in maintaining good separation efficiency and avoiding loss of resolution caused by the interface. Suter and Caprioli have compared the liquid-junction and the sheath-flow inter References pp. 523-527

500

Chapter 12 CF-FA6 carrier solvent

/

/

/

\

\

10 prn ID \

I I I

I

0

0

+5kV

+25kV

CF-FAB carrier solvent

/

\

Fig. 12.10. Liquid junction design for CF-FAB-MS interface to CE. Adapted from Ref. [77].

faces for CE-CF-FAB [77]. The sheath-flow interface showed superior performance with respect to the number of theoretical plates and thus resolution, but it was more difficult to operate owing to arcing through the narrow-bore CE capillary walls and column plugging problems. In addition,' in the sheath-flow CF-FAB interface, the low column loadability reduces concentration detection limits. Lower efficiency of any design may be advantageous in some cases if the overall performance of the technique is scan-time limited. Since magnetic sector instruments commonly used

CE-MSApplied to Environmental Analysis

50 1

with the CF-FAB interfaces are characterized by inherently slow scan speeds, they require relatively broad peaks for optimal signal-to-noise ratios. Generally, at least three-to-five scans are required to ensure sufficient signal averaging. This problem can be circumvented by using a selective ion monitoring (SIM) or selective reaction monitoring (SIR) scan instead of the full-range scan. In this approach, however, the gain in sensitivity may be offset by loss of selectivity of the method because the full spectrum of the analyte is not provided. 12.3.2. CE-ES-MS and CE-ISMS designs In an electrospray interface, the analyte solution is electrostatically nebulized in a 3-5 kV electric field where it forms a fine mist of highly charged, nearly uniform (ca. 1-pm radius) droplets. Further evaporation of solvent from the droplets is facilitated by a countercurrent flow of heated nitrogen. When the droplet charge-to-size ratio approaches or exceeds the Rayleigh limit, the ionized analyte particles are transferred from solution to the gas phase. The Rayleigh limit is the condition in which the charge on the droplet is just sufficient to overcome the surface tension holding the droplet together. Although the details of the ionization process are not yet fully understood, it is postulated that analyte ions formed in solution are simply transferred into the gas phase during the evaporation process. The gas phase ions are introduced into the mass spectrometer (typically a quadrupole mass spectrometer) either through a small sampling orifice or a small-diameter capillary. Since the mass spectrometer separates ions according to their mass-to-charge ratio, even extremely large molecules (>lo0 kDa) can be analyzed when they are multiply charged using a regular quadrupole mass spectrometer (mass range 1000-4O00 m a ) . An ion spray interface is a modified electrospray design in which the liquid nebulization process is aided by pneumatic assistance of a coaxial gas stream [74]. The ion spray source, similar in design to the electrospray source, works at nearly atmospheric pressure. The pneumatic nebulization assistance of the ion spray allows for increased liquid flows (ca. 50 pl/min). The first electrospray-based CE-MS interface was described by Smith and coworkers [103]. In this first prototype, the electrospray needle was simply a 100-pm i.d. capillary column carefully positioned inside a stainless steel (300-pm i.d.) capillary whose terminus was kept at 3-5 kV potential relative to the counter-electrode. The steel capillary tube played the role of both a CE cathode and an electrospray electrode. A metallized CE capillary terminus utilized in the subsequent designs improved electrical contact between the capillary eluent and the cathode. Also, introduction of a make-up liquid through either liquid-junction or sheath flow systems allowed for a greater flexibility of the CE eluent flow rate and permitted use of smaller i.d. capillaries. Pleasance et al. [ 1041 described CE-IS-MS systems combining three adapted, commercially available capillary electrophoresis systems with an API 111triple quadReferences pp. 523-527

502

Chapter 12

rupole mass spectrometer equipped with an in-house built ion spray interface (Fig. 12.11). The interface possessed two different, interchangeable make-up liquid delivery systems: the sheath-flow and the liquid-junction designs. They were able to change from one configuration to the other in a few minutes, although obtaining the correct distance between the capillaries in the liquid junction system frequently required several attempts [104]. When the gap was too large, the analyte diffused into the fluid in the liquid-junction interface causing deterioration of the electrophoretic resolution. It was also important to obtain tight fits of the capillaries inside one another and relative to the probe tip of the ion spray. A diluted solution of volatile make-up buffers is commonly used. For example, formic acid (0.2%)was found to be the most suitable make-up buffer system for positive ion CE-IS-MS analyses of certain marine toxins and antibiotics. The sheath-flow design made a flow-injection analysis possible by introducing standard solutions through the make-up buffer capillary. This is an important feature of the coaxial interface since it allows for tuning the mass spectrometer specifically

Fig. 12.1 1. CE-ISMS interface with sheath flow and liquid junction designs. Adapted from Ref. [104].

CE-MS Applied to Environmental Analysis

503

to the diagnostic peaks of the compounds present in the analytical sample, and it also provides independent means of quantitation. Pleasance et al. concluded that the sheath-flow configuration was more robust and flexible while giving more reproducible results compared with the liquid-junction configuration [ 1041. Both CE-MS and CE-MS-MS modes of operation were shown to be feasible, although the mass analyzer was not always able to scan fast enough to obtain the adequate number of scans (e.g. 10 mass spectra) for a given CE peak. Even though the absolute detection limits of the CE-ES-MS technique are quite impressive (low picomole-femtomole level), the relatively high concentration detection limits (low ppm level). which are a consequence of the low sample injection volumes (typically 1-30 nl), remain a challenging problem. Achieving sufficiently low concentration detection limits is an important issue for CE-MS environmental applications. Isotachophoresis (ITP) has been found to be one of the most suitable preconcentration methods for capillary zone electrophoresis. On-line isotachophoretic analyte focusing has been demonstrated for the analysis of a mixture of anthracyclines [ 1051; this technique showed a 200-fold improvement in concentration detection limit with respect to CE-MS [105]. Smith and co-workers [ 1,74,111,112] suggested that, due to the competitive nature of ion transfer from droplets to the gas phase in the electrospray and ion spray interfaces, it is beneficial for method absolute sensitivity to minimize the buffer mass flow rate from the ES capillary. This goal can be achieved by using small i.d. capillaries and decreasing the concentration of the supporting electrolyte or the EO flow rate in the capillary. Diluting the CE electrolyte system, however, might not always be feasible since it could cause deterioration of the separation efficiency [74]. The efficiency of electrospray processes responsible for electrolyte transfer from the liquid phase to the gas phase depends, among other factors, on the mass flow of all electrolyte species delivered to the interface. It was found that if the mass flow of electrolyte into the electrospray interface exceeds the ion-transfer capabilities of the ES system, then the MS detector response to the increasing concentration of the analyte changes from a linear relationship to an undesirable flat response [113]. Because a mass spectrometer equipped with an electrospray interface behaves at the high ion concentration regime of operation, which is claimed to be typical for most CE conditions [ 1,74,112], it functions as a concentration-sensitivedetector (e.g. signal depends on the concentration of the sample in the carrier flow and not on the mass flow rate). It was emphasized by Smith and co-workers that the system response could then be improved by reduction of the CE column i.d. and thus mass flow rate into the capillary. A similar effect can be achieved by selectively decreasing the CE electric field and thus the electroosmotic flow for the time of the analyte band elution [1,112]. The E,O flow rate reduction is shown to increase the electrophoretic peak area with only a slight decrease in peak height, thus improving overall response of the system (i.e. the mass spectrometer can acquire more scans when the analyte is more slowly delivered to the mass analyzer). In contrast to Smith’s group, References pp. 523-527

504

Chapter 12

Niessen and co-workers [38] discussed the CE-MS response dependence on the concentration of charged species in the CE effluent by focussing their attention on the low concentration regime rather than on the ES saturation effects region. It was emphasized that, in the buffer-sustained spray, a decrease in the number of analyte molecules eluting from the capillary caused by using small i.d. capillaries (as advocated by Smith et. al. [1,74,112]) results in poor column loadability and therefore a poor response, as predicted for a mass flow-sensitive system [38]. In light of the work done by Smith, Niessen, and a recent article on the mechanism of electrospray mass spectrometry [113], it seems that the response, measured by the analyte ion intensity in the mass spectrum depends on the concentration of all electrolytes present in the solution. The response could improve or could worsen in response to a reduced flow rate of the CE effluent. For example, for a constant and M) concentration of the background ammonium and sodium ions in low methanol, the (M + H)+ ion intensity in an ES-LC-MS experiment increases proporM (Fig. 12.12) tionally to the analyte concentration within the range of to [ 1131. A further increase in the analyte concentration of the LC effluent results in a “saturation effect” and, ultimately, in a small decrease in signal intensity for analyte M [113]. In contrast, as evidenced by a series of ESconcentrations greater than 10-6

10-7

107

1 06

105

104

\

103

Fig. 12.12. Analyte response as a function of its concentration for constant background electrolyte concentration. Adapted from Ref. [113].

CE-MS Applied to Environmental Analysis

505

LC-MS experiments [ 1131 involving an analyte present at a constant concentration ( M) with varying different ammonium ion concentrations, the intensity of the MS analyte signal is suppressed by the presence of the ammonium buffer (Fig. 12.13) [113]. The latter effect is most significant for the relatively high buffer concentrations (1 O4 to 10-3 M) and less significant for the ammonium ion concentrations lower than Therefore, reduction of the mass flow rate from the CE capillary column and, as a consequence, lower concentrations of both the CE running buffer and the analyte delivered to the tip of the electrospray needle may have opposite effects on the analyte signal intensity. The effects depend on the concentrations of the ionic species in the CE effluent. For example, the detection limit is improved if the gain in the analyte signal due to the restriction of the buffer flow rate outweighs the loss of signal caused by the lower mass flow rate of the analyte (cf. Figs. 12.12 and 12.13). Signal intensity in the ES-MS system is also influenced by the rates of desolvation and transfer of ionic species from the liquid to the gas phase. Therefore, ion surface activities and solvation energies are important properties in estimating the practical

107

1 (b) NH: 106

=

105

Bu~N' CocH' HerH' Ni2t(terpy)2 CodH' MorH'

:

cs+ 104

I

I

I

I I 'I"

I

I

I I I Ill1

I

I

I

I I I l l

2

NH4CI (M) Fig. 12.13.Analyte response (at constant concentration) as a function of background electrolyte concentration. Adapted from Ref. [113].

References pp. 523-527

Chapter 12

506

TABLE 12.4 EXPERIMENTALLY DETERMINED RATIOS OF COEFFICIENTS k Ion

k

cs+

1 1.6 1.6 1.o 1.3 3 5 6 10 5

Li+ Na+

K+ NH4' MorH' CodH' HerH' CocH+ Ni+2(tPY)2

Ion

k 5

8 9 14 10 10

Coefficients relative to ks+ = 1. Valid for concentrations M. Abbreviations: Mor, morphine; Cod, codeine; Her, heroin; COC,cocaine; tpy, tripyridyl; Bu, n-butyl; Et, ethyl; Pr, n-propyl; Pen, npentyl; C7, n-heptyl; C11, n-undecyl.

value of a CE buffer for ES-MS. Generally, there is a positive correlation between high surface activities, low solvation energies and the transfer coefficient values (Table 12.4) [1131. These experimentally determined relative coefficients describe ion ability to migrate to the surface of the electrospray micro-droplets and then to desolvate to give gas-phase ions. This finding has a practical implication. It was suggested that ions characterized by high values of these coefficients should not be used as ES-MS buffers [ 1131. Consequently, since relatively high concentrations of surfactants must be present in the buffer system used in MEKC, it is difficult to interface this technique with a mass spectrometer due to analyte signal suppression by the surface active agents. 12.3.3. CE-ICP-MS A simple, direct CE-ICP-MS interface was described [114] by Olesik and coworkers. Fused silica capillaries (97-pm i.d., 40-50 cm long) were used in the study. The capillary terminus was painted with silver paint to ensure good electrical contact with the buffer and then tightly fitted into the center tube of the nebulizer. The end of the capillary was placed within 0.5 mm of the tip of the outer tube. A pneumatically induced pressure drop across the capillary gave a 2.0 pl/min flow in the CE capillary. This relatively high flow rate of the effluent caused a significant CE peak broadening and, consequently, loss of electrophoretic separation efficiency [ 1141. Detection limits reported for inorganic samples were generally in the low ppb range. The authors reported short analysis time (below 2 min) and good reproduci-

CE-MS Applied to Environmental Analysis

507

bility of the method (peak area and elution time reproducibility within 3% relative standard deviation). A similar interface was reported for coupling CE to the ICP/atomic emission detector (AED), which is use.d for capillary GC element-specific detection [115]. This configuration was used for detecting organometallic compounds of environmental interest. What makes this detector particularly interesting is its ability to act as a universal detector for CE, much as a flame ionization detector does in GC. Thus, signals specific to chlorine, carbon, nitrogen, or oxygen, for example, allow detection of compounds that do not absorb in the UV or visible regions and are not suitable for indirect modes of detection. 12.4. ENVIR0NMENTA:LAPPLICATIONS OF CE-MS

Capillary electrophoresis has become an important tool in biochemistry primarily because of its high efficiency and selectivity and very large molecular mass capabilities. Additionally, the small volume of analyte used in CE experiments has made possible the analysis of cellular contents [ 1 161. Besides the numerous papers describing CE and CE-MS applications in the biochemical area, a significant number of reports illustrate the importance of this technique in the field of small molecule and environmental analysis. Henion and co-workers reported application of a CE-IS-MS system equipped with a liquid junction interface for determining sulfonated azo dyes [82,117] (Figs. 12.14 and 12.15). The concentration detection limits ranged from high parts-per-billion to low parts-per-million, and the separation efficiencies varied from 50 000 to 300 000 theoretical plates. The liquid junction coupling did not contribute significantly to peak broadening, and the rnethod provided good reproducibility (3.5-1 2.8% relative standard deviation for manual injection, 2 4 % reductive standard deviation for automated injection). CE-ES tandem mass spectra of the deprotonated parent ion showed a diagnostic peak at m/z 80 corresponding to the sulfonate anion (Fig. 12.16). Three sulfonated azo dyes were detected at low parts-per-million levels in a spiked wastewater extract by MS-MS parent scans of this common fragment. A CE-ES-MS sheath flow design was used for analysis of a laser dye (Rhodamine 6G) [ 1181. Strong silica-wall-analyte interactions were found detrimental to the CE separation efficiency [ 1201. Use of the cationic surfactant, cetyltrimethylammonium chloride (CTAC) in the CE buffer avoided this problem. A reversed electroosmotic flow was observed, probably due to the formation of a cationic layer on the surface of the CE capillary [118]. Detection limit for Rhodamine 6G was ca. 100 ng/pl. Good selectivity and increased flow rates (and consequently shorter time of analysis) were observed. Reduced sensitivity was reported for this approach which was caused by the high surfactant mass flow from the CE capillary into the ES interface (refer to Table 12.4). Another drawback of the method was extensive contamination of the electrospray counter-electrodeby the eluting surfactant [ 1181. References pp. 523-527

508

loo

Chapter 12

1

A

S03H OH

Acid Blue 113

FW - 637

i

E

(M-2H) 285

-3NH2 OH

Acid Black 1 FW - 572

- I 1

Acid Red 14 FW - 458

~,1,,,1,,,1,,111,

m

240

280

320

260

400

440

480

Fig. 12.14.CE-IS mass spectra of six sulfonated azo dyes. Adapted from Ref. [117].

Pseudo-electrochromatography-negative-ion electrospray MS (PEC-ES-MS) of food colors was reported by Niessen and co-workers [120]. In this combination of capillary electrophoresis and electrochromatography with LC, the mobile phase was pressure-driven through a packed CE column and introduced into the sheath-flow ES interface. The pressure-induced parabolic instead of plug-like flow profile gave rise to lower efficiency of separation. Five food colors were used as model compounds to test this method. The performance of PEC-ES-MS was found comparable with that of micro-LC-MS. Brumley [121] described separation of eight aromatic sulfonic acids using a boric acid-borate running buffer at pH 8.3. Efficiency for the CE-MS separation of a leachate from the Stringfellow Superfund site using a sheath flow CF-FAB-MS system ranged from 20 000 to 50 000 theoretical plates and resulted in the detection of 4-chlorobenzene sulfonic acid (Fig. 12.17). To date, this represents the only Superfund sample that has been subjected to CE-MS determination. Limitations in sensitivity prevented the identification of additional components in the leachate. Performance of the sheath flow CE-ES-MS and nanoscale capillary liquid chromatography (nCLC-ES-MS) in their application to the separation of various sul-

CE-MS Applied to Environmen.ta1 Analysis

509

33

pmoles

v v

.c.'

c 40 0

270

fmoles -

20

0

2

4

1.3

pmoles

6

8

Minutes

10

12

14

15

COUNTS = 149088

Fig. 12.15. Reproducibility and ion current responses for various levels of acid blue 113; SIM of m/z 317, [M - 2H]-'. Adapted from Ref. [I 171.

fonamides was evaluated [122] (Fig. 12.18). It was concluded that these two methods were complementary. The degree of fragmentation of the sulfonamides was regulated by changing the skimmer voltage. High skimmer voltage favored fragmentation and formation of species with fewer charges [122]. Both nCLC and CE systems suffered from a small dynamic range of operation caused by the proximity of the upper limit of column loadability to the lower MS detection limit. Several sulfonamides were: detected by CE-UV-MS-MS and CE-SIM-MS techniques [123]. Capillary gel electrophoresis was first reported to be interfaced with an API triple quadrupole mass spectrometer by Garcia and Henion [ 1241. Despite the relatively high buffer concentrations required for achieving good CE resolution, the CE buffer did not elute into the ion spray chamber. In this preliminary work, mixtures containing aromatic sulfonates and amino acids were resolved. A liquid junction CE-CF-FAB-MS system was used to separate a mixture of eight sulfonylurea pesticides [1251 (Figs. 12.19-12.22). Selective ion monitoring (SIM) MS electropherograms showed very good efficiencies for most of the peaks. Fullscan collision-induced dissociation spectra for each component of the mixture were recorded. An effort was made to shorten the time of analysis by using shorter CE columns (length less than 50 cm). The need for improvement of the concentration detection limit of the method was emphasized [1251. References pp. 523-527

510

A

317

100

-

Chapter 12

50.350

80 *..

C

2 5

60

-C0

40

0

20 0 0

2

4

6

8

10

12

14

16

Minutes

100

1

B

20

COUNTS = 7101

297

2-

I-I

(M-2H)

31 7

C

a

Acid Blue 113 FW 637

156

-

60

50

18

80

120

160

200

MIZ

240

280

n 320 350

COUNTS = 317-1060

Fig. 12.16. CE-IS-MS-MS total ion electropherogram and CE-IS-MS-MS of [M- 2HI2- ions of acid blue 113; 10 pM injected; buffer 60:40 acetonitrile/20 mM ammonium acetate. Adapted from Ref. [82].

Quaternary ammonium salts were used in the initial studies of the CE-ES-MS interface conducted by Smith and co-workers [126-1281 (Figs. 12.23 and 12.24). The separation efficiencies reported for the SIM-MS electropherograms ranged from 160 000 theoretical plates for the tetrabutylammonium ion to 330 000 theoretical plates for the trimethylphenylammonium ion. Excellent absolute detection limits for these compounds spanned from low femtomolar to attomolar levels. The low abso-

CE-MS Applied to Environmental Analysis

1

51 1

4-chbrobenzenesulfonate

0.04

0.02

0.00

I

I

0.0

I

I

I

I

I

10.0

I

I

1

I

I

20.0 22.25

Time (min) Fig. 12.17. CZE of a leachate from Stringfellow Superfund site; buffer 50 mM sodium borate at pH 8.3; UV detection at 214 nm. Inset i.s the CE-MS ion electropherograrn corresponding to 4-chlorobenzene sufonate (dz191); sodium borate buffer, 10 mM (pH 8.3). Adapted from Ref. [121].

lute detection limits obtained in these analyses could be explained by the high values of the electrospray liquid-gas phase transfer coefficients (Table 12.4) that are typical of quaternary ammonium cations. Adducts of four polycyclic aromatic hydrocarbons to deoxyguanosine were studied using liquid-junction 133-CF-FAB-MS [81] (Fig. 12.25). A common loss of deoxyribose allowed for the application of the multiple reaction monitoring MS technique. Five organophosphonic acids, which are hydrolysis products of some warfare agents were detected and characterized by negative ion CE-IS-MS [129]. High ES nozzle-skimmer voltage differences gave rise to more abundant fragmentation of the [M - 11- ions. Thibault et al. [130] and Pleasance et al. [I311 studied the application of capillary electrophoresis mass spectrometry (sheath flow CE-IS-MS) to marine toxins [ 104, 1311 (Figs. 12.26-12.28). Analysis for saxitoxin and neosaxitoxin showed very good linearity within the concentration range 1.5-300puglml [ 1311. The concentration detection limit for these two compounds was ca. 5 pM. Good sensitivity (260 fmol) References pp. 523-527

512

/:I'

I

,,1~3

11"

268 (M+H)+

,

,

0 100

I

268 (M+H)+

B 50 113 0 100

1

113

~~1~

0 -

290 (M+Na)+

225810 SKIMMER +11 V

1

1

1

140910

1

50

C

,SHMMER+4V

290 (M+Na)+ 1

268 (M+H)+

C

87160

,,

1 1

156 I

Chapter 12

156

1 1

SKIMMER +18 V

290 (M+Na)+ 1

;(M+H)y

50

1

*,

I

57500 SKIMMER +25 V

290 (M+Na)+

92

'r

34620

'"]d;:i

SKIMMER +40 V

0

,

50

0 100

5

0

B ' 0 8 113

,,

,

36730

,

,,SKlMMER+55V

~

268

290

T""I

'-"p

36800 ,SKlMMER+75V

156

0 50

100

150

200

250

300

350

M/Z

Fig. 12.18. Effect of skimmer voltage on CE-ES-MS fragmentation of sulfisoxazole; needle voltage 3 kV; spray current 0.250PA; block temperature 260°C; spray temperature 55°C. Adapted from Ref. [122].

513

CE-MS Applied to Environmental Analysis

loo

I

1

jILi

1111

V

I

I

I

1.0 59

0.0 1

2.0 11

UV at 229 min

Time (min)

I

I

3.0 17

4.0 22

I

5.0 28

I

I

6.0 34

7.0 40

8.0 45

Time (min)/Scan Fig. 12.19. CE-MS total ion elcctropherogram of eight sulfonyl urea herbicides about 7 pmol each; 5 mM ammonium acetate/acetonitrile (75:25) (pH 5.0); 1000 Vlcm; 35 cm X 75-pm i.d. capillary. Inset is UV detection with same system minus interface and mass spectrometer. Adapted from Ref. [125].

100

A

3

B

5

l 3

I 1

0

2

4

6

Time (min)

I

8

1

0

0

0 5

15 25 35 Time (min)

45

Fig. 12.20. Comparison of CE separation of seven sulfonylurea herbicides (about 7 pmol each) using UV (A) and CE-MS detection (U); 5 mM ammonium acetatdacetonitrile (7525) (pH 5.0); 300 V/cm 100 cm x 75-pm i.d. capillary. SIM CE-MS required an additional 80 cm of capillary. Adapted from Ref. [125].

References pp. 523-527

514

Chapter 12

411

h

#! v

Nicosulfuron M,W, 410

c(M+NH4)+

50

428

0 300

#!

100-

400 (M+H)+ 358

500

mlz

600

700

B Chlorsulfuron M,W, 357

v

Q)

400

300

1

0

2

4

600

500

m/z

700

C

4 6 8 Time (min)

2

10

0

2

D

4

6

8

10

Time (min)

Fig. 12.21. CE-MS full scan mass spectra for two sulfonyl ureas and electropherograms comparing no pressure compensation with pressure compensation to speed elution. Adapted from Ref. [ 1251.

515

CE-MS Applied to Environmental Analysis

-

h1-

100

a,

> .-

5 -

25

a,

213

[r

0

-

50

150 I 100

200

250

300

a 100

(M+H)+

, I

350

50

450

100

150

200

350

300

250

400

450

167

7

m], ,l,/;.,h, /11

155(+2H)

9

100

400

7

175

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75

(M+H)+

a,

c

(M+H)+

a,

>

I

199

50

100

150

200

250

300

350

400

,

25

395

50

450

100

150

200

250

25

0.90

--8

1.84

2.77

3.71

4.74

Time (min) 100,

300

350

400

450

MI2

MI2

._

,

411 0

I

-ap

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1w Low-pressure

75

50

0

._

4d

$

25

0

mlz

0

2

4

6

8

10

12

14

15

Time (min)

Fig. 12.22. CE-MS-MS mass spectra of (a) selected herbicides obtained from the electropherogram given in (c); (b) Fargo soil extract spiked with bensulfuron methyl at 41 1 ngltL1; (c) overlay of TIC of eight sulfonylurea herbicides showing the use of low pressure compensation to speed elution of last two; 35 cm X 75 y m i.d. capillary at 800 Vlcm. Adapted from Ref. [125].

was reported for CE-CF-FAB-MS analysis of saxitoxin. The full-scan MS-MS spectrum of the protonated molecular ion of saxitoxin exhibited some structurally informative fragments. References pp. 523-527

516

Chapter 12

RIE CZE MS Quaternary Ammonium Salts

(CH,),N+

rn/z 74

(CH,),C,H,N+

m/z 136

v 0

(C,H,),N+

m/z 130

(C,H,),N+

m/z 242

10

20

30

40

Time (minutes) Fig. 12.23. CE-MS separation of quaternary ammonium salts; 2 m Adapted from Ref. [128].

X

100-pm i.d. capillary at 45 kV.

12.5. FUTURE PROSPECTS

The following discussion attempts to briefly mention some areas under development that may have an important impact on development of CE-MS or are important areas critical to advancing the art. 12.5.1. Quantitation

It is evident in the citations above that very little quantitative work has been performed with CE-MS techniques. One of the prerequisites in environmental analysis is the specific confirmation of identity of the analyte and an accurate, precise determination of its concentration in the sample. Mass spectrometry fulfills this role in

CE-MS Applied to Environmenr'al Analysis

I

517

CZE MS of Quaternary Ammonium Salts

2 rn x 100 prn Capillary

40 kV 1O-* Phosphate Buffer pH=4

Y

L M

I

0

10

20

30

40

Time (minutes) Fig. 12.24. CE-MS separation of quaternary ammonium salts; 2 m X 100-pm i d . capillary at 40 kV; 10 mM phosphate buffer at pH 4. Adapted from Ref. [127].

GC-MS analyses. It is expected that a significant percentage (e.g. 10-236) of monitoring determinations based on CE coupled with optical or other detectors will require some further validation by an independent technique. Performance data anticipated for CE-MS are expected to conform to data quality objectives achieved by GC-MS and LC-MS techniques. Some typical quantitations achieved thus far by LC-MS are illustrative of the successes and limitations of these newer ionization techniques and their performance with regard to matrix effects, levels of analytes, and co-elution of co-extractives. For example, Betowski and Jones [I32 3 reported achieving better than 10% relative standard deviation for standard compounds with an external standard method for LC-thermospray(TS)-MS of organophosphates. Levels quantitated ranged from subng or low ng to 500 ng on-column. Analysis of actual environmental samples actually showed better results by LC-MS compared by GC-MS for this class of compounds. Jones et al. [I33 3 addressed the difficulties of validating LC-MS methods with interlaboratory studies. Using either LC-PB-MS or LC-TS-MS for analytes consisting of carbamates, chlorinated herbicides, and benzidines, results showed great References pp. 523-527

Chapter 12

518

447

100

%Fs

100

1

m/z 489

acetylarninofluoreneadduct

- m/z 458

yoFs.

0.

methylanthracene adduct

.

: h *.

-.A -.

:

yoFs- arninobiphenyl adduct

D

B

100

yoFs

500 550

20

- m/z 489 -- > d z 373

40

60

MSIMS Data

80

100 20612

. acetylaminofluorene adduct

m/z 435 -- > d z 319 %Fs. aminobiphenyl adduct

34352

100 m/z 447 -- > d z 331 %Fs . arninofluorene adduct

22168

100

100

%Fs Scn

- m/z 458 -- > d z 342

. methylanthracene adduct 50

100

150

200

250

300

350

Fig. 12.25. (A) CE-MS separation of PNA adducts of deoyguanosine observed by constant neutral loss scans where [M + H]+loses 116 Da; [M + HI+ ions at pH 9.5, 0.01 M ammonium acetate; (B) multiple reaction monitoring of same at pH 3.7. Adapted from Ref. [81].

variation among laboratories. The percentage RSDs ranged from 4 to 99% with recoveries ranging from not detected to 290%. Stable isotope labeled analogs that coelute with the target analytes provide the best means for coping with enhancement or degradation in signal due to co-eluting components. Since these studies at their lowest levels (5 pglml) represent a likely starting point for CE-ES-MS, a great quantity of performance data is needed for assessing quantitation in real environmental samples. In the absence of suitably labeled analogs, careful selection of surrogates for recovery and for internal standards will be essential in assessing performance. Close correlation between CE/optical detection and CE-MS will be essential as well as careful spiking studies within identical matrices. A clear understanding of analyte responses and factors affecting them is also essential in operations characterizing samples for unidentified components.

519

CE-MS Applied to Environmental Analysis

0.016 1

t

-

(4

.012

h NEO

-

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.008

.004

GTX2

-

7

0, 0.0

100

-

1

2.0

6.0

AL 8.0

10.0

rnlz396

=- 50 cr" 25 s

4.0

h

v

75

(4

c.

Time (rnin) Fig. 12.26. CE-IS-MSof four marine toxins (paralytic shellfish poisons); 90 cm X 50;um i.d. capillary (UV at 200 nm), Trisma buffer (pH 7.2); 24.4 kV; with 0.2% formic acid at 8pYmin make-up flow; selected ion electropherograms as shown. Adapted from Ref. [131]. References pp. 523-527

Chapter 12 O.O1

-

0.012

-

GTX2

200nm NEO

GTX3

-

0.008

-

0.004

00.0

75 50 25 -

loo

loo

-

50

-

25

2.0

6.0

4.0

8.0

10.0

II MH+ B

t MH+

-

0.0

2.0

4.0

6.0

8.0

10.0

12.0

Time (min) Fig. 12.27. CE-IS-MSseparations comparing UV (200 nm), coaxial interface, and liquid-junction interface results. Adapted from Ref. [104].

12.5.2. Sensitivity

One of the outstanding issues in implementing CE-MS concerns achieving lower detection limits. Implementation of array detectors in high resolution instruments is one approach towards improving mass detection limits [ 1001. Other approaches include the use of ion trap MS [ 134,1351 and TOF instruments [ 136,1371 or the coupling of these with double focussing instruments or with each other in hybrid configurations [ 138,1391. Simple calculations indicate that picomolar sensitivity oncolumn involves 6 X 10'' molecules available for ionization before consideration of ionization efficiency and analyzer losses. Even if significant instrumental improvements reduced the detectable number to 6 X lo5 or further, the sensitivity realized would produce a detector subject to enormous background problems. Particularly

CE-MS Applied to Environmental Analysis

521

rnh 316

0.0

3.0

6.0

9.0

12.0

Time (min) Fig. 12.28. Sensitivity of CE-ISMS for consecutive injections of the indicated amounts of saxitoxin and neosaxitoxin; Trisma buffer (pH 7.2); 24.4 kV; with 0.2% formic acid at 8 pl/min make-up flow; selected ion electropherogramsas shown; capillary 90 cm X 50-pm i.d. Adapted from Ref. [104].

careful attention to solvent impurities, electrolyte purity, and laboratory contamina-, tion would be required. Therefore, sample handling, clean-up, and selectivity will continue to be prerequisites for environmental analysis. 12.5.3. MEKC-MS and EKC-MS

In light of the published experimental data, the development of MEKC-MS techReferences pp. 523-527

522

Chapter 12

niques depends on lowering the surfactant concentration or eliminating its elution from the column. Even the application of monomolecular micelles may still suffer from the increased ability of such molecules to dominate the ionization processes in ES . In the light of these limitations, EKC-MS provides a viable alternative to neutral molecule separations while MEKC-MS continues to be developed. EKC columns can be implemented in commercial CE instruments for screening purposes in conjunction with MEKC or as a primary technique. Similar columns can then be used in EKC-MS for confirmation of identity and quantitative validity. This approach should also work well with derivatized substrates for LIF detection. Such derivatives could have substantially higher masses than underivatized analytes, and this would result in a relative molecular mass beyond the high background region ( a d z 400). The increased organic content should increase the surface activity of the derivatized substance and improve ionization efficiency.

12.5.4. Preconcentration The application of off-column and on-column concentration techniques will continue to be important for CE-MS applications. A clearer understanding of ITP and electrolyte systems for the on-column concentrationklean-up of analytes will be an important development [ 1401. Systems involving micellar agents for the concentration of neutral analytes will be another useful development. The development of selective adsorbents for solid phase extraction appears to be needed. Nanoscale sample handling, microreaction vessels, and CE sample manipulations appear to be fruitful areas for investigation.

12.5.5. New developments Accurate mass measurements will continue to be an important tool in the identification of compounds in environmental matrices. Liquid introduction of compounds will greatly increase the universe of possible analytes that need to be considered. A high sensitivity, high-sampling speed technique called mass peak-profiling should help to assign accurate masses to analyte ions [138]. Applications of this approach to non-volatiles subjected to CE-MS and EKC-MS should expand our knowledge of the distribution and function of heretofore intractable substances. New improvements to ES and other interfaces are expected to make CE more compatible with MS [142-1521. The study of Wilm and Mann [147] presented a micro ES source that produced 200-nm droplets directly at a flow rate of 25 nVmin and an overall transmission efficiency of 8 X 10"' from solution to MS detector. Work in the area of capillary/capillary coupling and other approaches that make use of CE itself to improve sample introduction to MS is highly significant [ 1531.

CE-MS Applied to Environmenr+alAnalysis

523

12.6. NOTICE The US Environmental Protection Agency (EPA), through its Office of Research and Development, funded and performed the research described here. This work has been subjected to the Agency’s peer review and has been approved as an EPA publication. The US Government has the right to retain a non-exclusive, royalty-free license in and to any copyright covering this article. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

12.7. ACKNOWLEDGMENTS The author thanks: Dr. M. Jung (work performed while he held a National Research Council/NERL, CRD-LV Research Associateship ) for obtaining the separatiodderivatization of the arnines and phenoxyacid herbicides (Figs. 12.7 and 12.8); and Dr. Pete Matchett (enrollee of the Senior Environmental Employees Program, assisting the US EPA under a cooperative agreement with the National Association for Hispanic Elderly) for the separations of aliphatic amines and sulfonyl ureas (Figs. 12.2 and 12.4). Work was performed while W.W. was holding a National Research CouncilNERL, CRD-LV Research Associateship.

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