~
f i r CupiCCuy Michael Albin, Paul D. Grossman, and Stephen E. Moring Applied Biosystems Division of The Pehin-Elmer Corporation 850 Lincoln Centre Drive Foster City, CA 94404 Capillary electrophoresis (CE) continues to grow rapidly as an analytical technique in a wide range of application areas (1-5). Typically, CE separations are performed in fusedsilica capillaries with internal diameters of 25-100 p.High heat dissipation efficiencies in the narrow columns allow separations to be performed a t high field strengths (200500 Vlcm), resulting in short analysis times and peak efficiencies of up to millions of theoretical plates. Other attractive features of the technique include its compatibility with on-line detection and automated sample loading with minimal (pL) sample volume requirements. The evolution of automated instruments has helped to spur the development of a vast array of applications and separation media (6,7). Separation modes such as free solution electrophoresis, isoelectric focusing (IEF), micellar electrokinetic capillary chromatography (MECC), and sieving-based separations coupled with rapid, automated method development have led to widespread acceptance. Current research continues in areas as diverse as DNA sequencing, the analysis of natural products in foods, and clinical analysis of serum samples. The narrow diameter separation columns that permit the advantages noted above are also responsible for the major limitation of the technique. Although mass sensitivity is extremely high because of the very small detection volumes inherent in 0003-2700/93/0365-489A/$04.00/0 0 1993 American
Chemical Society
CE analysis, the Concentration sensitivity-particularly in the case of UV absorbance detection-is generally on the order of 10- to 100-fold less than that of HPLC. In addition, the short optical pathlength (- 50pm capillary id.) typically results in concentration limits of detection (LODs) on the order of lo-' M. Approaches to help address this problem can be divided into three categories: sample concentration strategies, alternative capillary geometries and improved optical design, a n d a l t e r n a t i v e detection modes. In this INSTRUMENTATION article we will discuss the applicability, advantages and disadvantages, and availability of each approach. Given the prevalence of absorbaucebased detection, we will focus on enhancements for this detection mode.
tive pressure, and vacuum. Hydrodynamic injections provide a sample plug representative of analyte composition with a n injection volume that depends on the injection time, capillary dimensions, buffer viscosity, and pressure drop across the capillary. One drawback of hydrodynamic injections, however, is that significant band broadening can occur because of the parabolic profile t h a t is characteristic of pressuredriven flow (2). The total length of the sample plug is limited by the impact of the plug length on peak width. The relationship between plug length lid and the contribution to peak variance from injection plug a& is given by
Sample concentration strategies Methods that enhance sensitivity by increasing the concentration of the analyte(s1 include on-line concentra-
Therefore, if lid becomes too large, a& can negatively influence separation efficiency. This typically occurs when l,, is z 1% of the total separation length.
tion using discontinuous buffer systems and pre-electrophoresis sample concentration techniques. A brief description of sample injection techniques is given below, followed by illustrations of discontinuous buffer systems and pre-electrophoresis concentration schemes. Sample injection in CE. Injection modes for CE are based on either hydrodynamic or electrokinetic principles. In hydrodynamic injection, sample is introduced into the capillary by applying a pressure differential across the capillary while one end of the tube is immersed in the sample solution. Hydrodynamic techniques include gravimetric, posi-
In electrokinetic injection, sample is introduced into the capillary by applying a voltage across the capillary while one end is immersed in the sample solution and the other in buffer. Sample is drawn into the capillary by a combination of electrophoresis and electroendosmotic flow. The amount of material injected is a function of the electrophoretic mobility of each solute, the electrical conductivity of the sample buffer and the running buffer, and the electroendosmotic flow. Two important features must be kept in mind. First, because the amount of material injected is a function of several parameters that can be hard to control, it is
-
ANALYTICAL CHEMISTRY, VOL. 65, NO. 10. MAY 15,1993
-
489 A
INS7RLJMENTAIIoN difficult to get a high degree of reproducibility for sample injection over the course of analyses. Second, the amount of each sample component loaded onto the capillary will vary as a function of the mobility of each sample species. Stacking. Sample stacking with discontinuous buffer systems has heen used extensively in many areas of electrophoresis (8-10).When a sample is dissolved in a solvent with electrical conductivity lower than that of the electrophoresis running buffer, a concentration or stacking
phenomenon occurs upon electrokinetic sample injection. The electric field strength in the low-conductivity sample medium is higher than that in the running buffer, and ions rapidly migrate t o the interface between the lower and higher conductivity zones. Upon reaching the interface, t h e a n a l y t e s t h e n slow (stack), causing contraction of the sample zone. The ultimate effectiveness of stacking is limited by the trade-off between resolution and injection plug length discussed above. Additionally, while the concentration of the sample zone increases as stacking proceeds, the conductivity increases and the rate of sample concentration falls off asymptotically during the injection time. Thus, the l i can ~ become larger than desired before a steady-state concentration can be reached in the sample zone. Finally, the process of electrokinetic injection, and therefore the effectiveness of stacking, depends on the electroosmotic flow velocity ueo. When v,,. is in the same direction as the analyte migration, the stacking efficiency is decreased relative to the case in which ue0= 0. The larger the magnitude of ueo, the worse the stacking efficiency. When u.. and the analyte migration u., are in opposite directions, with ,.u >>,,u the stacking efficiency is optimal. Note,however, that this situation rarely oc-
c u r s . If u., > v.,, a s i n most countermigration separations (pH > 61, no injection takes place. The difficulties encountered in applying stacking a t high pH can be overcome by using polarity switching during electrokinetic injection. With a high ,u and normal po1arity;positive ions in the injection plug will stack a t the sample-buffer interface, whereas negative ions will tend to migrate out of the injection end of the capillary. Reversing the polarity will cause anions to migrate into the capillary and stack as the positive ions start to migrate out. Termination of the injection before the positive ions migrate out of the capillary (normal polarity time exceeds reverse polarity time) results in a sample plug containing both positive and negative ions in a narrow zone. The traces in Figure 1 show injections of sample peptides under three different injection conditions: (a) a 1 - s vacuum injection, (b) a 5-s electrokinetic injection in which the sample is dissolved in the electrophoresis running buffer, and (c) a 5-s electrokinetic injection in which the analytes are dissolved in water. In all cases the sample concentration is 50 pg/mL. The graph (Figure Id) illustrates the quantitative change in t h e amount of material injected when electrokinetic injection is used as a function of the ionic strength of the
rigure 1. Comparison of sensitivities for different sample injection techniques using a 50-pglmL peptide mixture.
0
2
4
6
lime (min)
8
(a) 1-8 m u m inienion:[b) 5-6.5-kV i n w o n
wilh sampk dissolvBd in 20 mM sodium phas-
male, pH 2.50; (c) 5-s, 5-kV injeaion with sample digpolved in water; [dl peak area vs. mpbuffer ionic slrem wilh dmmkinelic inpdon. Separationconditions:x ) mM sodium phmwphae buffer. pH 2.5.30 “C. defeclion 81 2M) nm.
Figure 2. Chemical enhancement by stacking. (a) npplicalion of sample plug; (b) n a m i n g of sample wne:[c) IC) digsipation of pH gradiem and migration of comenlrald M W to a h d e ; (d) separation of lO-p#mL peplide mimre in 10 mM sodium dtrale. pH 2.5 [top) and in 10 mM “,OH (bouom) with 30-9m u m inwion. 30-kV applied pnenlial. and absolbBnm detenion a1215 nm. (Adapted with permission hom Reference 10.)
490 A * ANALMICALCHEMISTRY, VOL. 65, NO. 10, MAY 15,1993
(a)
- +
-
-+
ilumn
Buffer reservoir
+ o+ - + --+ '
-
Buffer reservoir
(a) Sample InjMion; (b) sample focusinp; (c) sample Mparelim: and (d) eklatmphemprams showing impovemem in sample stackmp d a law inpction wiume. Top: 35-msample plug loaded am sample m e r amoved: middle: comemiona sample stacking using a 1-m sample plug. m o m : same as mp M vilhoul amova4 d sample buifer. Sample Is a mixture d (A) phsnylhydanmin (PTH)-aspamca d 140 W) and (8)PTH-glutamic add (34 pM eadl):coiumn: Sollm 1.d. l00-m-bnp unreafed 1usBo.sIca capilary; buffer: 100 mM 2.(N.morpholino)sthanssu~n1c aadll00 mM hiaihne a1 pH 6.2. (Mapted horn Refefenca 12.)
Figure 4. Chemical enhancement by isotachophoresis (ITP), (a) Sample injection (L is the W i n g elmmtyle and T is the leminaling eleclrolyte); (b) w@ application; (c) equilibrium: (d) comparison of oeparalionsd lluorescdn isothiayanate-aminoadds by ITP-CE (lop) and CE (bottom). CE sparation conblions: 5-s.5-kV injenion of 10-mmol mixture. 25-hV applied potential. and LIF d M h m a1 488 nm; ITP-CE separationconditions: 25pL mixture (ITP); 5.5, 5-kV CE injection, 25-kV applied potentia4 (CE). IO-kV DM pnenlial IiTP). (Maned with mmi88ion from Referem I&.)
sample buffer. The peak areas for a I-s vacuum injection (3.5 nL for a 5-in. vacuum injection and a 72-cm capillary) are approximately the same as that of a 5-8 electrokinetic injection a t 5 kV for a sample dissolved in the electrophoresis running buffer. However, the relative peak areas for the two injection methods are very different because of the dependence of injection mass on analyte mobility. Additionally, the peaks for the electrokinetic injections have higher efficiencies; for the second peak, N = 247,000 for electrokinetic injection but N = 188,000 for vacuum injection. For the sample dissolved in water, the peak areas have increased fivefold over sample dissolved in the running buffer because the analytes have greater velocities in the lower ionic strength media. Increasing the time of the electrokinetic injection results in a further increase in peak area, ultimately a t the expense of separation efficiency and resolution. Another way to acbieve stacking is to use a pH gradient within the capillary, as illustrated in Figure 2. Introduction of a sample plug in a buffer having a pH above the PI values of the analytes (a high pH media) surrounded by buffer a t a low pH results in migration of the anionic peptides toward t h e anode when the electric field is applied. When the analytes enter the acidic region, the charge reverses and the migration direction is reversed. This results in a narrowing of the sample zone. In the final step, the pH gradient dissipates under the influence of the applied potential and the concentrated zone migrates toward the cathode. Aebersold and Morrison (10)have reported detection limits of e 1pg/mL (10-fold enhancement). Field amplification. Burgi and Chien (11) have demonstrated the ability to fill virtually the entire column length with sample and then focus prior to separation. This method can be thought of as sample stacking in reverse; instead of focusing the sample in the direction of electrophoresis, one focuses in the opposite direction. The principles involved for anionic species are illustrated in Figure 3. First, a large sample plug is hydrodynamicallyinjected onto the column in a low-conductivity buffer. Second, the sample is focused at the cathodic end of the capillary at the sample buffer-running buffer interface using a voltage polarity opposite to t h a t employed for the electrophoresis. Third, when focusing is
ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15.1993 * 491 A
~
INSTRUMENTATION complete (as indicated by a change in current), the polarity is again reversed and the separation is performed. For a negatively charged silica surface, anions tend to stack at the back end of the sample buffer plug. Using a polarity opposite to the separation mode will result in driving out the sample buffer ahead of the negatively charged analytes. The use of this method (12,13)results in significant improvements (> 1 order of magnitude) over conventional stacking methods (compare the middle and top electropherograms in Figure 3d). This method can be used to determine positive ions by coating the capillary; however, it cannot be used to determine cations and anions simultaneously. Isotachophoresis (ITP). The principle of ITP (8, 14, 15) is illustrated in Figure 4. The capillary is first filled with electrolyte whose mobility is greater than that of any ions in the sample of interest (leading electrolyte, L). Following sample injection, the sample end of the capillary is placed in a second electrolyte whose mobility is less than that of the analytes of interest (terminating electrolyte, T) and voltage is applied. The resulting field is not homogeneous throughout the capillary (i.e., resistance varies along the capillary length). Separation occurs between the boundaries based on the individual ion mobilities. As the components separate (time = Y), the field strength within the individual zones changes. The high-mobility ions have a higher conductivity and slow down. At equilibrium, all bands migrate at the same velocity Yisotacho” time is 21). An ion diffusing out of its zone speeds up or slows down, depending on t h e velocity of t h e neighboring zone i t encounters, thereby rejoining ita focused zone. The technique can be used prior to CE by performing the concentration step in one column and then using the appropriate plumbing to transfer the concentrated sample plug to the separation column. This technique can yield an increase in sensitivity of up t o 3 orders of magnitude. Alternativelv. iudicious choice of buffers in a di&oktinuous system allows the use of this method with current CE systems (8).Figure 4d (146) illustrates the results for a 25-pL injection onto an ITP system followed by electrokinetic injection onto a CE system (upper trace) in contrast t o the direct CE analysis (lower trace). In this case, sensitivity enhancement on the order of 100 times was obtained. 492 A
C h r o m a t o g r a p h i c concentration. With pmper selection of separation media and conditions, stacking, field amplification, and ITP can all be adapted for use with currently available i n s t r u m e n t a t i o n . Another method for on-line trace enrichment involves loading large volumes of analytes (to 100 pL with micropumps) onto microcolumns of chromatographic material followed by elution (with appropriate solvents and/or electroosmotic flow) onto a CE column. A device employing an LC pump to load material is shown in Figure 5a. An LOD of 50 nM (5 pL sample loaded) was determined for the drug papaverine (16). High efficiencies may be maintained when electrodesorption (backflushing with applied field) is employed (Figure 5b, trace C) as compared to electroendosmotic (forward flow) elution (Figure 5b, trace B). Thus a very large sensitivity enhancement can be gained when large injection volumes are used. However, difficulties in the manufacture of a n on-line system that is both reproducible and cost effective are likely to restrict this technique to applications involving sample cleanup or trace enrichment. Capillary geometry and improved detector cell design According t o Beer’s law, the optical absorbance of a sample is directly proportional to t h e optical pathlength through which the absorbance measurement is performed. Therefore extension of the optical path should lead to increases in detection
sensitivity. However, simply increasing the inner diameter of the capillary is not always an attractive alternative because increased Joule heating can result, leading to a loss of resolution from increased peak widths. Ways of increasing the performance of an absorbance detector by increasing the effective optical pathlength are described below. All flow cell designs, including the standard cylindrical capillary, require an effective means of coupling the excitation light into and through the capillary pathlength. A ball lens made from sapphire or quartz, placed close to the capillary (Figure 6a), is the most effective alternative (17). This wniiguration provides radial illumination of the center of the capillary, optimizing light throughput and minimizing stray light. Poppe et al. (18)have systematically investigatedflowcelldesignandcapillarydiameters. Alternatives are described below. Bent capillaries. The short optical pathlengths in microcapillaries can be extended by bending t h e capillary (2-cell) and illuminating through the bend. Chervet et al. (19) have manufactured 2-cells for CE that provide a 3-mm optical pathlength. Although the pathlength was increased by 40-fold over a 75-pm id. Cylindrical capillary, only about a fivefold sensitivity enhancement occurred because of increased background noise levels from poor efficiency in light throughput at the 3-mm bend. The original design was improved (20)by coupling proper optics to the 2-cell, thereby reducing
B
A
$0 mAU
3
C
00 mAUI10 mAU
I
Il;\l m-rn
0480
I
8 162432 0 4 8
Time (min)
Figure concentration. - 5. Chemical enhancement bv on-line chromatographic . . (a) Schematic of concentratingdevice. Components:( 1 ) capillary inletloutlet; (2) 1.5 mm Y 0.3 mm PTFE tubing: (3) metal screen: (4) 1.5 mm x 0.5 mm PEEK tubing: (5) 5-1” RoSil C, packing material: (6) PTFE screen. (Adapted With permision from Ref~rence16.) (b) Separation of (A) 0.2 pL papaverine (0.1 mM) with direct injection: (8)5 mL papaverine (0.1 mM) with precolumnon-line Concentration during electrdphorssis:and (C) 5 pL papaverine (0.01 mM) after 45-s electmdssorplion.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15,1993
Figure 8a wmpares injections in a 50-pm i.d. cylindrical capillary with a sample concentration of 50 pg/mL (top trace) and a 50 pn x 500 pm i.d. rectangular silica capillary, where the absorbance is measured across the longer axis and sample concentrations are 5 pgImL (middle trace) and 0.5 pg/mL (bottom trace). For equivalent hydrodynamic injections
2-cell format. The bottom trace in Figure 7a shows a 1-s vacuum injection on a 75-pm i.d. capillary with transverse illumination of the capillary; the top trace shows the same injection on a 7 5 p m i.d. optimized Z-cell with a 3-mm pathlength. In this example, a 17-fold increase in sensitivity is evident for the Z-cell with a n equivalent SIN (the LOD improves from 1.2 pgImL to 0.07 pgImL in this example). The tradeoff for this sensitivity gain is a slight decrease in resolution (- 15961, presumably because of the increased detedor cell length. Alternative shapes. An alternative to bending a cylindrical capillary to gain a longer optical pathlength (22-24)is to perform electrophoresis where Idee is the length of the detecin “flattened” channels (Figure 64. A tion region. As this length increases, number of workers have examined separation efficiency may be afsquare and rectangular capillaries of fected. For a 0.05-mm cell, the plate varying dimensions and materials. height contribution from the flow The advantages provided by f l a t cell is 2.1 x lo-* meters per plate, tened geometries is that the narrow which results in a limiting plate separation channel dimension can be count of 48,000,000. In realistic maintained for very efficient heat cases, this influence is negligible. However, when Idet = 3 mm, the lim- dissipation (25),whereas the longer dimension provides the longer optiiting plate count value of 133,000 cal path required for enhanced detecplates per meter can have a severe tion sensitivity. However, the fragilimpact on the overall performance of ity of t h e elongated, thin-walled a separation. structures currently available is of Figure 7 shows the sensitivity enconcern. hancement obtained for an optimized
noise levels and maintaining a significant signal gain (Figure 6b). The results demonstrated a 14-fold signal-to-noise ratio (S/N) improvement over the unbent configuration. The extended optical pathlength in the Z-format contains a n inherent downside. In any detector cell geometry, a finite volume of fluid is in contact with t h e detector a t any given time. Thus the output of the detector represents a signal that is averaged over a finite detection volume. It has been shown (21)that the contribution to peak variance attributable t o a finite detection volume, &, is given by 0$=I &/ 12 (2)
j ~
I ! 1
Flgure 6. Schematics of optical cell designs for extended optical pathlength. (a) Standard on-column detection: (b) optimized 2-cell: (c) rectangular capiilary: (d) multireflection u and (e) end-column detection.
.
Figure 7. Comparison of separations of a 5-pg/mL peptide mixture obtained with differentcell formats. (a) Top: 75-1” i.d. oplimked 2-cell with a 3-mm pathlength. Bottom: 75-pn i.d. cylindrical capillary with transverse illumination. (b) Resolution cwnparison of 75-pm cyind-I cell (lop) and optimized2-cell (bonorn). Separationconditions as in Figure 1.
ANALYTICAL CHEMISTRY,VOL. 65, NO. 10, MAY 15,1993 * 493 A
INS7RUMENTA7ION and capillary lengths, the ratio of the rectangular to the cylindrical capillaries yields the following experimental (and calculated) parameters: injection volume: 35 (38) and flow rate: 2.9 (3.0). The observed increase in peak width in the rectangular Capillary as compared with the cylindrical capillary is likely attributable to a greater degree of Joule heating and an increase in t h e injection plug. The former effect can be seen by noting the strong field dependence of the electrical conductivity of the buffer in the rectangular capillary compared with the field independence of the eleetrical conductivity in the cylindrical case (Figure ab, where K, = LiISV and L i s t h e total capillary length, i is the current, S is the m s s sectional area of the capillary, and V is the applied voltage). Efficient heat dissipation in the rectangular capillary is a more serious problem because, for a given thickness or diameter, the surface-to-volume ratio is twice as large as that of a cylinder having a n equivalent diameter. The expected 10-fold gain in sensitivity from the optical pathlength is not fully obtained because of the broadening effeets described above and because of the need t o optimize both the orientation of the capillary and t h e optics for efficient energy throughput (as in t h e case of t h e 2-cell). Multireflection flow cell. The optical pathlength of the capillary can be effectively multiplied by using mirrors to reflect the incident light inside the capillary prior to detection. The optical pathlength i n creases while the narrow separation dimension is maintained. Using a silver-coated capillary, Wang e t al. (26)demonstrated a 40-fold increase in sensitivity for a cell construction with a 44-fold increase in pathlength. A critical parameter in such a cell construction is the incident light angle I3 (Figure 6d), which controls the number of internal reflections and the pathlength per reflection. The number of reflections, and hence ultimate sensitivity, must be restricted to limit l,, and thus minimize the loss in efficiency caused by the flow cell size. The distance between incident light and detection (D,) should be 5 a of the narrowest peak width. End-column detection. An alternative to looking radially across the capillary is to look down t h e length of the capillary. In this format light is transmitted through the capillary by total internal reflection.
The absorbance indicates the sum of the absorbance signals resulting from all analyte components. As components elute from the column, the total absorbance decreases in a steplike manner. Xi and Yeung (27) described a means of interfacing conventional light sources to capillaries with internal diameters ranging from 10 to 50 bm (Figure 6e). For a 50-wm i.d. capillary and 3-mm injec-
I
I
I
I
6
8
10
12
Time (rnin)
Figure 8. Comparison of separations achieved with alternatively shawd capillaries. (a) Top separation of 50-wmL peptide minure wth 50-lrm n.d. cylindrica capllary: middle: 5-&YmL pepbde minure win 50 lrm x 5M) pm rectangu ar cap Ilary; bonom: 0.5-WmL peptide mlnure vlth 50 pm x 500 pm rectangular cspfllary Separation conditions as in FiQure1 Ib) Ohm’s law dot for 50-pm cylindrical and 50 prn x 500 Gm rectangular cap llary.
494 A * ANALYTICAL CHEMISTRY, VOL. 65, NO. I O , MAY 15,1993
tion plugs, a sevenfold increase in sensitivity was obtained. Alternative detection modes W lasers. The use of high-intensity light sources offers an alternative to extending the optical pathlength to obtain greater sensitivity in an absorbance detection measurement. The availability of W lasers will have a n impact on spectroscopicbased instrumentation as costs decrease and as performance and optical characteristics improve. CE is no exception. A krypton fluoride laser at 248 nm has been used in a thermooptical detector for PTH-amino acid analyses carried out with CE (28).A temperature rise in the sample zone, proportional to the laser power and sample concentration, is measured as a change in the RI of the zone by a secondary laser probe beam. In another study with W laser excitation (29).the second harmonic of an argon ion laser (257 nm)was used to measure underivatized aromatic amino acids. Vibrations in a capillary under applied tension were generated by thermal heating of t h e sample zones and were shown to be proportional to the analyte concentration. Measurements of the emitted acoustic wave provided submicromolar-level sensitivities. For nonabsorbance-based detection schemes, usually either the analyte(s) of interest contain a chromophore or fluorophore “label” compatible with the mode of detection or such a label can be introduced in a straightforward, reproducible manner (exclusive of indirect detection formats). In addition to greater sensitivity, a second detection mode can enhance detection selectivity. The advantages and disadvantages of alternative detection methods are diseussed briefly below. Fluorescence detection. Fluorescence detection typically results in sensitivity gains of 1-3 orders of magnitude over those of absorbancebased detectors. The degree of enhancement depends on t h e light source, its intensity and stability, the optical match of the excitation source to t h e fluorophore(s), t h e quantum efficiency of t h e label, background fluorescence, and the deteetor configuration. After W ahsorbance, fluorescence is the most frequently used detection mode when high sensitivity and selectivity are required. Although most analytes are not intrinsically fluorescent, a large section of the chemical literature covers fluorescent labeling tech-
niques. Laser-based fluorescence is particularly well suited for CE because of the small illumination volumes and light intensities required. Optimization of the detector and the chemistry has resulted in impressive performance. Mass sensitivities in the zeptomole range moles or hundreds of molecules) have been obtained by using on-capillary laser-induced fluorescence (LIF). A number of configurations and derivatization chemistries have been reported; a representative sample is given in Table I. In one innovative scheme known as sheath flow. the sample is detected after eluting'from the end of the column in a flowing stream of liquid, thus eliminating the background effects resulting
from light scattering a t the capillary wall. Using a 25-mW argon ion laser, workers have obtained pimmolar detection limits for derivatized amino acids and mass sensitivities of 1-2 zeptomoles = 10P1 moles = 600 molecules (33). In another case (34), attempts were made to perform separations by using an array of capillaries to increase system throughput. In lieu of the usual 90" orientation of excitation and emission beams, a confocal arrangement was used. Excitation (laserjand emission are accomplished through a single microscoDe obiective such that onlv the interior i f the capillaries is pmbed. The advantage of this configuration is t h a t scattering is rejected by proper spacial filtering.
Dsto&en WmiW
L.m*imya
Exdmlon .oUW
m
wnpwmtr.
conoMlmtlm RdMIIw
Mesa
CBWN ion laser PMT, lock-in 5 x IO-'' carbohme "%nm amptitier mol= Fiber-optic collection FiTC/ arninoacids A%
Native/ proteins FTtu
amino acids
ion laser CCD 514nm Axial
illumination Argon ion laser PMTIoptica
3 x IW'M
30
1 x lo-'- M
33
I
275.4 nm
nm 9% ,ion 1-r
lo-" Sheath-flow 2x CUVelW MleS Microscope q&e
*CBOCA: 3-(4-carboxvbenrovl)-2-quinolineoarboxslh~de;FITC: nuorescein isothiocyanait FTH: FluMesceinthiohydanmin; PMT: pholomultpller tube: CCD: charge-WpW d e v i . 'Under s a m p stadclng conditi.
Table II. Sensitivity enhancementtechniques: Sarnpl
50-1000
Direct Indirect
(with appropriate
buffer choices/ method) No'
Simple to implement: dependent on sample matrix ionic strength relative to separation buffer Cannot be applied to cations and anions simultaneously Need to develo separation methods fordidrent analyte systems;no automated pre-electrophoresis system currently available Dinicult to manulacture cos1 effective system
*Wales has m,cztedthe avsimLM ty of CE coldmns comaining a w o n pacled with CMOrnalO graphic rnatenal lor Inis p~rposeHowever. no data nave yet been PLM snw
Fluorescence detection and other detection modes can be used in an indirect format (35)in which a fluorescent additive is combined with the separation media to provide a fluorescent background signal. Analytes will displace a quantity of the fluorescent material in proportion to their individual concentrations, resulting in a decrease in the signal. The advantage is that the analytes need not possess a signal characteristic of the detector employed (e. g., the analyte need not fluoresce or require derivatization). However, because of the high fluorescence background, sensiti&ies do not compare with those that can be obtained with an equivalent direct detection mode. Indirect fluoreseenee detection can provide a degree of sensitivity enhancement over that of conventional direct W absorbance methods. The indirect methodology has also been used in a n absorbance mode when analytes do not contain significant chromophores (36);however, sensitivities are generally poor
lo-' M).
Electrochemical detection. Conductivity and amperometric detection offer significant sensitivity enhancement over that possible with absorbance techniques (37-40). The measurement i s independent of pathlength, allowing very narrow capillaries to be used, and the components required to fabricate a n electrochemical cell are inexpensive relative to laser-based detection schemes. However, electrochemical detection is complicated by the need to isolate the high voltage employed in the separations (10-30 kV) from t h e electrochemical potentials (- 1V). In general. isolation is performed by introducing a break a t the point of capillary and electrode coupling. Numerous detector configurations have been demonstrated in the laboratory, but it has been difficult to reproduce them. Detection limits of 1 am01 have been obtained for serotonin and catechol (39).These high sensitivities, coupled with extremely small sampling volumes, may make, cellular studies possible. Radiochemicaldetection.Radiochemical detection has o h heen effective when extremely high sensitivity has been required, and it is still widely used for HPLC. By using a variety of configurations (41, 42) researchers have obtained sensitivities on the order of 10-'6-10-18 moles of radioactive label (lo-'lo-" M SaP-labeledbases and '% radiopharmaceuticals). Despite the advantages of this technique (very
ANALYTICAL CHEMISTRY, VOL. 65, NO. IO, MAY 15,1993
495 A
INS7RUMEN7A7ION high sensitivity and selectivity with few background signals and the fact that labeled analytes behave chemically as the unlabeled species), it is less likely to be used because of the handling and disposal difficulties associated with radioactive materials.
Summary The relative gains achieved by using the different cell geometries and the chemical means discussed above are summarized in Tables I1 and 111. Advantages and disadvantages of the different methods are indicated in terms of applicability, effects on the separation, and manufacturing concerns. Overall detection limits for the different modes are summarized in Table IV.Very low wncentration detection limits can be obtained, and we believe they will become more available to the commercial instrument user as the application market develops. (Already two commercial LIF systems are available, and certainly the number and variety of detection eonfirations will expand.) Future directions An examination of the literature reveals that the number and diversity of publications is growing at a near-
exponential rate (Figure 9). A more detailed look at the literature reveals some interesting perspectives. The use of commercial instrumentation is cited in 30.4% of all articles. This percentage is growing on an annual basis. Nineteen percent of the papers describe the use of detection methods other than absorbance on noncommercial systems (fluorescence, 12%; MS, 4.8%;electrochemistry, 1.6%; radioactivity, 0.3%Raman, 0.2%). Applications are well distributed among DNA, protein, and the small-molecule pharmaceutical and industrial chemical areas (- 60%total); the rest comprise general reviews and discussions of new CE technology as well as other esoteric application areas. Items that should be addressed in t h e f u t u r e include sensitivity, throughput, sample matrix effects, capillary wall chemistry and reproducibility, system cost, and ease of use. Several methods can be used to compensate for the sensitivity limitations of existing commercial systems, and new techniques and advancements will continue to be made available. In addition, the methods are by no means mutually exclusive.
The coupling of methods (e.g., sample stacking and optimized Z-cells) can yield significant sensitivity enhancements. Ultimately the user will have to optimize the separation wnditions within the limitations of the sample matrices and sample workup methods, and couple these to the hardware available from the growing field of manufacturers. As for alternative detectors, the trend will almost certainly be in the direction that HPLC has taken: toward more modular, interconnecting systems. The authors would like to thank the numemus individuals at Applied Bimystems, useera, and colleagues involved with CE for their comments and encouragement over the past several yeam.
References (1) Li, S.F.Y., Capillary Elecfn@~oresis:plinciples, Prachce, and Applicahons; Elsevier: New York, 1992gp 1-586. (2) Grossman, P. , Colburn, J. C. Capillary Electrophoresis: Theoly and Practice; Academic Press: San Diego, 1992 pp.
i i g , C. A. Anal.
64, 389 R-407 R.
D.; Ross, G. Trends Anal.
11,156-63. €Ooms, I.; J. B. Anal. Chini. 50.45-60.
I, J.; Sandra, P. Introduction
Table IV. Sensitivity detection modes
Table ill. Sensltlvity enhancement techniques: Capillary format lbthod
Enhancermnt* Pathlength (1.d.)
3-5
3mm (75 W)
Appllcablll to
commerc%
Cornmenla
Direct
Direct applicability of methods Hih noise levels Some resolution loss Commerciallv available Moderate coit Direct applicability of methods More than an order of magnitude gain Minimal resolution loss Commercially available Moderate cost Some care in methods: buffer choice. flow rates High sample loading Questionable
syaems"
Direct
- Direct
lsOrbanCe
I
Direct
10-5-10-6
j
Indirect
1~4-10-5
I
Fluorescence Direct (lamp-based) IO-'-l(Ts Direct (laser-based) lW*-lO-'* Indirect (laser-based) l(T*-lO-' Electrochemical conductivity
10-5-1 0-7
Amperometric Ftadiochemical
IO-6-io-9
modifications Alignment requires modifications Need to use nonaqueous buffers Data as step function ,
No
cost
Specific wavelengths availaMe Lifetime/reliability
..
, _ ~ . Growth of CE citations from 1981 through 1992.
I
496 A * ANALYTICAL CHEMISTRY, VOL. 65. NO. 10, MAY 15,1993
io Micellar Electrokinetic Ckromaiograpky;
Huthi : Heidelberg, 1992;pp. 1-231. (7)Deyf, Z.;Struzinsky, R. J. Ckromatogr. 1991.569.53-122.
.
.
matog, 1992,623, 345'-55. (9)Gebauer, P.; Thormann, W.; Bocek, P. J. Chromaiog. 1992,608,47-57. (10) Aebersold, R.; Morrison, H. D. J. Chromaiogr. 1990,516, 79-88. (11) Burgi, D. S.; Chien, R-L. Anal. Biochem. 1992,M2,306-09. (12) Chien. R-L.: Burei. D. S. AnaL Ckem.
I. Microcolumn SeP 1990.'2, 229-33
34-39. (18)Bruin, G.J.M.; Stegeman, G.; van Asten, A . C.; Xu, X.; Kraak, J. C.; Poppe, H.I. Chromaiogr. 1991,559,16381. (19)Chervet, J. P.;van Soest, R.E.J.; Ursem. M. 1. Ckromatoer. 1991. 543.
Michael AIbin received his B. S. degree in chemistvfrom The Polytechnic Institute ofNew York (1979) and his Ph.D. from T h e Pennsylvania State University (1984). Following postdoctoral studies on electron transfer mechanisms at Cal Tech, he pumred an industrial career centered on the interface between chemktv and instrumentation in a number offields. He has been involved in CE development for three yean
RGnance of Carbonaceous Solids F
(25)Jansson, M.; Emmer, A.; Roeraade, J. J. High Resolui. Chromaiogr. 1989,12,
-"-.
707-IL"l .I.
(26) Wang, T.; Aiken, J. H.; Huie, C. W.; Hartwick, R. A. Anal. Chem. 1991,63, 1372-76. (27) Xi, X.; Yeung, E. S. Appl. Specirosc. 1991,45,1199-1203. (28) Waldron, K. C.; Doviehi, N. J. Anal. Chem. 1992,64,1396-99. (29) Odake, T.; Date, K.; Kitsrnori, T.; Sawada, T. Anal. Sci. 1991, 7, 507-08. (30)Liu, J.; Shirota, 0.; Wieslw, D.; Novatny, M. F'roc. Nail. Acad. Scr. 1991,88, 2302-06. (31) Sweedler, J. V.; Shear, J. B.; Fisbman, H. A,; Zare, R. N.; Seheller, R. H. Anal. Ckem. 1991,63,496-502. (32)Lee, T. T.; Yeung, E. S.J. Ckmmatogr. 1992,595,319-25. (33) Zhang, J. Z.; Chen, D. Y: Wu, S.; Harke, H. R.; Doviehi, N. J. Ciin. Chem. 1991.37. 1492-96. (34) H u m X. C.; Q u e s a d a , M. A,; Mathies, I? A. Anal. Ckem. 1992, 64,
Paul D. Grossman received his B.S. degree and his Ph.D. in chemical engineeringfrom the University of California at Berkelq and his M.S. degree in chemical engineeringfrom the University of Virginia. He is the co-editor of a recentlypub lished book on CE. His research interests involve the application of novel electrophoretic techniques to DNA analysis.
Robert € Bolto. Argonne National Laborator). Eartor Yuzo Sanada. Hokkaido University, Editor
4dvances in Chemistry Series No. 229 566 pages (1993)Clothbound ISBN 0-8412-1866-8
.I.
Qfi7-7')
(35) Yeung, E. 5.;Kuhr, W. G. h a l . Ckem. 1991,63, 275 A-282 A. (36)Pianetti, G. A.; Taverna, M.;Baillet, A.; Mahuzier, G.; Baylocq-Ferrier, D. J. Chromatog. 1993, 630, 371-77. (37)OShea, T.J.; Telting-Diaz, M.W.; Lunte, S. M.; Lunte, C. E.; Smyth, M. R. Electroanalysis 1992,4,463-68. (38)Yik, Y. F.; Li, S.F.Y. Trends Anal. Chem. 1992,11,325-32. (39)Curry, P.D.;En strom-Silverman, C. E.; Ewing, A. G. &eciroanalysis 1991, 3.587-96.
ocusing on new magnetic resonance methods for characterizing solid :arbonaceous fuels, this new volume :overs 2-D methods. high-resolution 'T md 'H NMR (CRAMPS) analyses. lynamic nuclear polarization techiiques. temperature-dependent .elaxation measurements. chemical nethods t o improve quantitative 'eliability. ultrafast MAS methods. and iew signal enhancement techniques. Also discussed are topics o f current nterest in the area of ESR. including spin-echo methods. 1-D and 2-D pulsed 2%techniques relaxation time neasurements.and ultrahigh frequency neasurements. In addition. measurements are made )n a set of Argonne Premium Coal jamples t o compare different magnetic .esonance techniques on a homogenous j e t of carbonaceous fuel samples.
$149.99
Stephen E. Moring is working on product research and development at the Applied Biosystems Division of Perkin Elmer. He received his B.S. degreefrom the University ofMav1and (1972) and his M.S. degree in biochemistvfrom the UniverSiryof Kansas (1977). He has been involved in CE development since 1988 and was responsible for the development of W a n d fluorescence detector technology.
W&hinoton EC 20036 Or CALL TOLLFREE
800-227-5558
(In Washington. OC.202-872-4363) and use your credit card!
ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15,1993
497 A
