Sensitivity for Capillary Michael Albin, Paul D. Grossman, and Stephen E. Moring Applied Biosystems Division of The Perkin-Elmer Corporation 850 Lincoln Centre Drive Foster City, CA 94404
Capillary electrophoresis (CE) con tinues to grow rapidly as an analyti cal technique in a wide range of ap plication areas (1-5). Typically, CE separations are performed in fusedsilica capillaries with internal diam eters of 25-100 μηι. High heat dissi p a t i o n efficiencies i n t h e n a r r o w columns allow separations to be per formed at high field strengths ( 2 0 0 500 V/cm), resulting in short analy sis times and peak efficiencies of up to m i l l i o n s of t h e o r e t i c a l p l a t e s . Other attractive features of the tech nique include its compatibility with on-line detection a n d automated sample loading w i t h m i n i m a l (μι.) sample volume requirements. The evolution of automated instru ments has helped to spur the devel opment of a v a s t a r r a y of applica tions a n d s e p a r a t i o n media (6, 7). Separation modes such a s free solu tion electrophoresis, isoelectric fo cusing (IEF), micellar electrokinetic capillary chromatography (MECC), and sieving-based separations cou pled with rapid, automated method development have led to widespread acceptance. C u r r e n t research con tinues in a r e a s a s diverse a s DNA sequencing, the analysis of n a t u r a l products in foods, and clinical analy sis of serum samples. The narrow diameter separation columns that permit the advantages noted above are also responsible for t h e major l i m i t a t i o n of t h e t e c h nique. Although mass sensitivity is extremely high because of the very small detection volumes inherent in 0003 - 2700/93/0365 -489A/$04.00/0 © 1993 American Chemical Society
Enhancement Electrophoresis
CE analysis, the concentration sen sitivity—particularly in the case of UV absorbance detection—is gener ally on the order of 10- to 100-fold less than that of HPLC. In addition, the short optical p a t h l e n g t h (~ 50μιη capillary i.d.) typically results in c o n c e n t r a t i o n l i m i t s of d e t e c t i o n (LODs) on the order of 10" 6 M. Approaches to help a d d r e s s this problem can be divided into t h r e e categories: sample concentration strategies, alternative capillary ge ometries and improved optical d e sign, and alternative detection modes. I n this INSTRUMENTATION article we will discuss the applicabil ity, advantages and disadvantages, and availability of each approach. Given the prevalence of absorbance based detection, we will focus on en hancements for this detection mode.
Sample concentration strategies Methods t h a t enhance sensitivity by increasing the concentration of the analyte(s) include on-line concentra-
tive pressure, and vacuum. Hydrody namic injections provide a sample plug representative of analyte com position w i t h a n injection volume t h a t depends on the injection time, capillary dimensions, buffer viscos ity, a n d p r e s s u r e drop across t h e capillary. One drawback of hydrody namic injections, however, is t h a t significant band broadening can oc cur because of the parabolic profile t h a t is c h a r a c t e r i s t i c of p r e s s u r e driven flow (2). The total length of the sample plug is limited by the impact of the plug length on peak width. The relation ship between plug length / inj and the contribution to peak variance from injection plug σ ^ is given by
- 1% of the total sepa ration length.
INSTRUMENTATION tion using discontinuous buffer sys tems and pre-electrophoresis sample concentration techniques. A brief de scription of sample injection tech niques is given below, followed by il l u s t r a t i o n s of discontinuous buffer systems and pre-electrophoresis con centration schemes. S a m p l e i n j e c t i o n i n CE. Injec tion modes for CE are based on ei ther hydrodynamic or electrokinetic principles. In hydrodynamic injec tion, sample is introduced into t h e capillary by applying a pressure dif ferential 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 t h e capillary by applying a voltage across the capil lary while one end is immersed i n the sample solution and the other in buffer. Sample is drawn into the cap illary by a combination of electro phoresis and electroendosmotic flow. The amount of material injected is a function of the electrophoretic mobil ity of each solute, the electrical con ductivity of the sample buffer and the running buffer, and the electro endosmotic flow. Two important fea t u r e s m u s t be kept i n mind. First, because the amount of material i n jected is a function of several param eters that can be hard to control, it is
ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1993 · 489 A
INSTRUMENTATION difficult to get a high degree of r e producibility for s a m p l e injection over the course of analyses. Second, the amount of each sample compo nent loaded onto the capillary will vary as a function of the mobility of each sample species. S t a c k i n g . Sample stacking with d i s c o n t i n u o u s buffer s y s t e m s h a s been used extensively in many areas of electrophoresis (8-10). W h e n a sample is dissolved in a solvent with electrical conductivity lower t h a n t h a t of the electrophoresis r u n n i n g buffer, a concentration or stacking
(a)
(b)
(c)
6
8 10 Time (min)
c u r s . If i>eo > vel, a s i n m o s t countermigration separations (pH > 6), no injection takes place. The difficulties encountered in ap plying s t a c k i n g at high pH can be overcome by using polarity switching during electrokinetic injection. With a high veo and normal polarity, posi tive ions in t h e injection plug will stack at 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 s t a r t to migrate out. Termina tion of the injection before the posi tive ions migrate out of the capillary (normal p o l a r i t y t i m e exceeds r e verse polarity time) results in a sam ple plug containing both positive and negative ions in a narrow zone. The traces in Figure 1 show injec tions of sample peptides under three different injection conditions: (a) a 1-s vacuum injection, (b) a 5-s elec t r o k i n e t i c injection in w h i c h t h e sample is dissolved in the electro phoresis 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 μg/mL. The graph (Figure Id) illustrates t h e q u a n t i t a t i v e c h a n g e in t h e a m o u n t of m a t e r i a l injected when electrokinetic injection is used as a function of the ionic strength of the
phenomenon occurs upon electroki netic sample injection. The electric field strength in the low-conductiv ity sample m e d i u m is h i g h e r t h a n t h a t in the running buffer, and ions rapidly migrate to the interface be tween the lower and higher conduc tivity zones. Upon reaching the in t e r f a c e , t h e a n a l y t e s t h e n slow (stack), causing contraction of t h e sample zone. T h e u l t i m a t e e f f e c t i v e n e s s of stacking is limited by the trade-off b e t w e e n r e s o l u t i o n a n d injection plug length discussed above. Addi tionally, while the concentration of the sample zone increases as stack ing proceeds, t h e conductivity i n creases and the rate of sample con c e n t r a t i o n falls off asymptotically during the injection time. Thus, the / inj can become larger t h a n desired before a steady-state concentration can be reached in the sample zone. Finally, the process of electrokinetic injection, and therefore the effective n e s s of s t a c k i n g , d e p e n d s on t h e electroosmotic flow velocity veo. When veo is in the same direction as the analyte migration, the stack ing efficiency is decreased relative to the case in which f eo = 0. The larger the magnitude of νeo, the worse the stacking efficiency. When veo and the analyte migration vei are in opposite directions, with vel » veo, the stack ing efficiency is optimal. Note, how ever, t h a t this s i t u a t i o n rarely oc
12 (d)
(d) 5 Running buffer = 20 mM
(a)
LowpH
High pH Low pH
(b)
LowpH
High pH Low pH
4 3 1-s Vacuum injection
2 1
H
(+)
0 0
5 10 15 Sample buffer concentration (mM)
20
Figure 1. Comparison of sensitivities for different sample injection techniques using a 50^g/mL peptide mixture. (a) 1 -s vacuum injection; (b) 5-s, 5-kV injection with sample dissolved in 20 mM sodium phos phate, pH 2.50; (c) 5-s, 5-kV injection with sample dissolved in water; (d) peak area vs. sample buffer ionic strength with electrokinetic injection. Separation conditions: 20 mM sodium phosphate buffer, pH 2.5, 30 °C, detection at 200 nm.
Focusing interface
(c) (-)
(+) 0
2
4 6 Time (min)
8
Figure 2. Chemical enhancement by stacking. (a) Application of sample plug; (b) narrowing of sample zone; (c) dissipation of pH gradient and migration of concentrated zone to cathode; (d) separation of 10^g/mL peptide mixture in 10 mM sodium citrate, pH 2.5 (top) and in 10 mM NH4OH (bottom) with 30-s vacuum injection, 30-kV applied potential, and absorbance detection at 215 nm. (Adapted with permission from Reference 10.)
490 A · ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1993
(d)
(a)
A Β
Capillary column Buffer reservoir (b)
-V
Focusing interfaces
+V
Water
3x
A
B
(A+B)
Buffer reservoir (c)
+V
0
2
-V
4 6 8 Time (min)
10
12
Buffer reservoir
Figure 3. Chemical enhancement by field amplification. (a) Sample injection; (b) sample focusing; (c) sample separation; and (d) electropherograms showing improvement in sample stacking of a large injection volume. Top: 35-cm sample plug loaded and sample buffer removed; middle: conventional sample stacking using a 1 -cm sample plug; bottom: same as top but without removal of sample buffer. Sample is a mixture of (A) phenylhydantoin (PTH)-aspartic acid (40 μΜ) and (B) PTH-glutamic acid (34 μΜ each); column: 50-μππ i.d., 100-cm-long untreated fused-silica capillary; buffer: 100 mM 2-(N-morpholino)ethanesulfonic acid/100 mM histidine at pH 6.2. (Adapted from Reference 12.)
(a) f=0
(d)
(b) t=r
(c) f=2/'
6
10 14 Time (min)
18
Figure 4. Chemical enhancement by isotachophoresis (ITP). (a) Sample injection (L is the leading electrolyte and Τ is the terminating electrolyte); (b) voltage application; (c) equilibrium; (d) comparison of separations of fluorescein isothiocyanate-amino acids by ITP-CE (top) and CE (bottom). CE separation conditions: 5-s, 5-kV injection of 10-mmol mixture, 25-kV applied potential, and LIF detection at 488 nm; ITP-CE separation conditions: 25-μί mixture (ITP); 5-s, 5-kV CE injection, 25-kV applied potential (CE), 10-kV applied potential (ITP). (Adapted with permission from Reference 14b.)
sample buffer. The peak areas for a 1-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-s electrokinetic injection at 5 kV for a sample dis solved in the electrophoresis running buffer. However, the relative peak areas for the two injection methods are very different because of the de pendence of injection mass on analyte mobility. Additionally, the peaks for the electrokinetic injections have higher efficiencies; for the second peak, Ν = 247,000 for electrokinetic injection but Ν = 188,000 for vacuum injec tion. For the sample dissolved in wa ter, 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 at the expense of separation efficiency and resolution. Another way to achieve stacking is to use a pH gradient within the cap illary, as illustrated in Figure 2. In troduction of a sample plug in a buffer having a pH above the pi val ues of the analytes (a high pH me dia) surrounded by buffer at a low pH results in migration of the an ionic peptides toward the 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 gradi ent dissipates under the influence of the applied potential and the concen trated zone migrates toward the cathode. Aebersold and Morrison (,10) have reported detection limits of < 1 μg/mL (10-fold enhancement). Field amplification. Burgi and Chien (11) have demonstrated the ability to fill virtually the entire col umn length with sample and then fo cus prior to separation. This method can be thought of as sample stacking in reverse; instead of focusing the sample in the direction of electro phoresis, one focuses in the opposite direction. The principles involved for anionic species are illustrated in Figure 3. First, a large sample plug is hydrodynamically injected onto the col umn in a low-conductivity buffer. Second, the sample is focused at the cathodic end of the capillary at the sample buffer-running buffer inter face using a voltage polarity opposite to that employed for the electro phoresis. Third, when focusing is
ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1993 · 491 A
INSTRUMENTATION complete (as indicated by a change in c u r r e n t ) , t h e polarity is a g a i n r e versed and t h e s e p a r a t i o n is per formed. For a negatively charged sil ica surface, anions tend to stack at the back end of t h e s a m p l e buffer plug. Using a polarity opposite to the separation mode will result in driv ing out the sample buffer ahead of the negatively charged analytes. The use of this method (12, 13) re sults in significant i m p r o v e m e n t s (> 1 order of m a g n i t u d e ) over con ventional stacking methods (compare t h e m i d d l e a n d top e l e c t r o p h e r o g r a m s in Figure 3d). This method can be used to d e t e r m i n e positive ions by coating the capillary; how ever, it cannot be used to determine cations and anions simultaneously. I s o t a c h o p h o r e s i s (ITP). The principle of ITP (8, 14, 15) is illus trated in Figure 4. The capillary is first filled w i t h electrolyte whose mobility is greater t h a n t h a t of any ions in the sample of interest (lead ing electrolyte, L). Following sample injection, the sample end of the cap illary is placed in a second electro lyte whose mobility is less than that of the analytes of interest (terminat ing electrolyte, T) and voltage is ap plied. The resulting field is not ho mogeneous throughout the capillary (i.e., resistance varies along the cap illary length). Separation occurs be tween the boundaries based on the i n d i v i d u a l ion m o b i l i t i e s . As t h e components separate (time = f ), the field strength within the individual zones c h a n g e s . The h i g h - m o b i l i t y ions have a higher conductivity and slow down. At equilibrium, all bands migrate at the same velocity ("isotacho" time is 2 0 . An ion diffusing out of its zone speeds up or slows down, d e p e n d i n g on t h e velocity of t h e n e i g h b o r i n g z o n e it e n c o u n t e r s , thereby rejoining its focused zone. The technique can be used prior to CE by performing the concentration step in one column and t h e n using the appropriate plumbing to transfer the concentrated sample plug to the separation column. This technique can yield an increase in sensitivity of up to 3 orders of magnitude. Alter natively, judicious choice of buffers in a discontinuous system allows the use of this method with current CE systems (8). Figure 4d (14b) illus trates the results for a 25-μΙ^ injec tion onto an ITP system followed by electrokinetic injection onto a CE system (upper trace) in contrast to the direct CE analysis (lower trace). In this case, sensitivity enhancement on the order of 100 t i m e s was ob tained.
Chromatographic concentra tion. With proper selection of separa tion media and conditions, stacking, field amplification, and ITP can all be adapted for use with currently avail able instrumentation. Another method for on-line trace enrichment involves loading large volumes of ana lytes (to 100 μι, with micropumps) onto m i c r o c o l u m n s of c h r o m a t o graphic material followed by elution (with a p p r o p r i a t e solvents a n d / o r e l e c t r o o s m o t i c flow) o n t o a CE column. A device employing an LC pump to load material is shown in Figure 5a. An LOD of 50 nM (5 \iL sample loaded) was determined for the drug p a p a v e r i n e (16). High efficiencies m a y be m a i n t a i n e d when electrodesorption (backflushing with a p plied field) is employed (Figure 5b, trace C) as compared to electroendosmotic (forward flow) elution (Fig ure 5b, trace B). Thus a very large s e n s i t i v i t y e n h a n c e m e n t c a n be gained when large injection volumes are used. However, difficulties in the m a n u f a c t u r e of a n o n - l i n e system that is both reproducible and cost ef fective are likely to restrict this tech nique to applications involving sam ple cleanup or trace enrichment. Capillary geometry and improved detector cell design According to Beer's law, the optical absorbance of a sample is directly p r o p o r t i o n a l to t h e optical p a t h length through which the absorbance m e a s u r e m e n t is performed. There fore e x t e n s i o n of t h e optical p a t h should lead to increases in detection
(a)
(b) A 1
Stator
2 3 4 5 6
Rotor
1
Stator
B
C
10mAU lOOmAU 10mAU
0480
8
16 24 32 0 4 8 Time (min)
Figure 5. Chemical enhancement by on-line chromatographic concentration. (a) Schematic of concentrating device. Components: (1) capillary inlet/outlet; (2) 1.5 mm χ 0.3 mm PTFE tubing; (3) metal screen; (4) 1.5 mm χ 0.5 mm PEEK tubing; (5) 5-μιτι RoSil Ce packing material; (6) PTFE screen. (Adapted with permission from Reference 16.) (b) Separation of (A) 0.2 μ ι papaverine (0.1 mM) with direct injection; (B) 5 mL papaverine (0.1 mM) with precolumn on-line concentration during electrophoresis; and (C) 5 μί. papaverine (0.01 mM) after 45-s electrodesorption.
492 A · ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1993
noise levels and maintaining a sig nificant signal gain (Figure 6b). The results demonstrated a 14-fold sig n a l - t o - n o i s e r a t i o (S/N) improve ment over the unbent configuration. The extended optical pathlength in the Ζ-format contains a n i n h e r e n t downside. In any detector cell geom etry, a finite volume of fluid is in contact with the detector at any given time. T h u s the o u t p u t of the detector represents a signal t h a t is averaged over a finite detection vol ume. It has been shown (21) that the contribution to peak variance attrib utable to a finite detection volume, oJU, is given by
Set = ' i t ' 1 2
(2)
where / d e t is the length of the detec tion region. As this length increases, s e p a r a t i o n efficiency m a y be af fected. For a 0.05-mm cell, the plate height c o n t r i b u t i o n from t h e flow cell is 2.1 χ 10~ 8 m e t e r s per plate, which r e s u l t s in a l i m i t i n g p l a t e c o u n t of 4 8 , 0 0 0 , 0 0 0 . In r e a l i s t i c cases, this influence is negligible. However, when / d e t = 3 mm, the lim iting p l a t e count value of 133,000 plates per meter can have a severe impact on the overall performance of a separation. Figure 7 shows the sensitivity en hancement obtained for an optimized
(a)
(d) Incident ray 1
Sapphire ball lens
Ζ-cell format. The bottom trace in Figure 7a shows a 1-s vacuum injec tion on a 75-μπι i.d. capillary with transverse illumination of the capil lary; the top trace shows the same injection on a 75-μπι i.d. optimized Ζ-cell with a 3-mm p a t h l e n g t h . In this example, a 17-fold increase in sensitivity is evident for the Z-cell w i t h a n e q u i v a l e n t S/N (the LOD i m p r o v e s from 1.2 μ g / m L to 0.07 μg/mL in this example). The tradeoff for this sensitivity gain is a slight decrease in resolution (- 15%), pre sumably because of the increased de tector cell length. A l t e r n a t i v e s h a p e s . An alterna tive to bending a cylindrical capillary to gain a longer optical p a t h l e n g t h (22-24) is to perform electrophoresis in "flattened" channels (Figure 6c). A n u m b e r of workers have examined square and rectangular capillaries of varying dimensions and m a t e r i a l s . The a d v a n t a g e s provided by f l a t tened geometries is t h a t the narrow separation channel dimension can be m a i n t a i n e d for very efficient h e a t dissipation (25), whereas the longer dimension provides the longer opti cal path required for enhanced detec tion sensitivity. However, the fragil ity of t h e e l o n g a t e d , t h i n - w a l l e d structures currently available is of concern.
Figure 8a compares injections in a 50-μηι i.d. cylindrical capillary with a sample concentration of 50 μg/mL (top trace) and a 50 μπι χ 500 μπι i.d. r e c t a n g u l a r silica capillary, where the absorbance is measured across the longer axis and sample concen trations are 5 μg/mL (middle trace) and 0.5 μg/mL (bottom trace). For equivalent hydrodynamic injections
(a)
Incident ray 2 Protective coating *. Silver coating
Capillary d
Ί 4
I 5
1 1 1 6 7 8 Time (min)
Γ 9
(b)
Capillary ' Sample D,
(b) Quartz ball lens
D2
Detector
Photodiode 75-μηη i.d. Capillary (e)
(c)
Quartz window Ball lens Positioning screws •«
Clamping plate
6.5
Groove
Capillary
Figure 6. Schematics of optical cell designs for extended optical pathlength. (a) Standard on-column detection; (b) optimized Z-cell; (c) rectangular capillary; (d) multiretlection cell; and (e) end-column detection.
7.0 7.5 Time (min)
8.0
Figure 7. Comparison of separations of a 5 ^ g / m L peptide mixture obtained with different cell formats. (a) Top: 75-μπι i.d. optimized Z-cell with a 3-mm pathlength. Bottom: 75-μπι i.d. cylindrical capillary with transverse illumination, (b) Resolution comparison of 75-μπι cylindrical cell (top) and optimized Z-cell (bottom). Separation conditions as in Figure 1.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1993 · 493 A
INSTRUMENTATION and capillary lengths, the ratio of the rectangular to the cylindrical capil laries yields t h e following experi mental (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 capil lary is likely attributable to a greater degree of Joule h e a t i n g and an in crease in t h e injection plug. T h e former effect can be seen by noting the strong field dependence of t h e electrical conductivity of the buffer in t h e r e c t a n g u l a r capillary com pared with the field independence of the electrical conductivity in the cy lindrical case (Figure 8b, where Ke = Li/SV a n d L is t h e t o t a l c a p i l l a r y length, i is the current, S is the crosssectional area of the capillary, and V is the applied voltage). Efficient heat dissipation in the rectangular capil lary is a more serious problem be cause, for a given thickness or diam eter, the surface-to-volume ratio is twice as large as t h a t of a cylinder having an equivalent diameter. The expected 10-fold gain in sensitivity from the optical p a t h l e n g t h is not fully obtained because of the broad ening effects described above and be cause of t h e need to optimize both the orientation of the capillary and t h e o p t i c s for e f f i c i e n t e n e r g y t h r o u g h p u t (as in t h e case of t h e Z-cell). M u l t i r e f l e c t i o n f l o w cell. The optical p a t h l e n g t h of t h e capillary can be effectively multiplied by using mirrors to reflect the incident light inside the capillary prior to detec tion. The optical p a t h l e n g t h in creases while the narrow separation dimension is m a i n t a i n e d . U s i n g a silver-coated capillary, Wang et al. (26) demonstrated a 40-fold increase in sensitivity for a cell construction w i t h a 44-fold i n c r e a s e in p a t h length. A critical parameter in such a cell c o n s t r u c t i o n is t h e incident light angle θ (Figure 6d), which con trols the number of internal reflec tions and the pathlength per reflec tion. The number of reflections, and hence ultimate sensitivity, must be restricted to limit / d e t and thus mini mize the loss in efficiency caused by the flow cell size. The distance be tween incident light and detection (D2) should be < σ of the narrowest peak width. E n d - c o l u m n d e t e c t i o n . An al ternative to looking radially across t h e c a p i l l a r y is to look down t h e length of the capillary. In this format light is transmitted through the cap illary by total i n t e r n a l reflection.
The absorbance indicates the sum of the absorbance signals resulting from all a n a l y t e c o m p o n e n t s . As components elute from the column, the total absorbance decreases in a steplike manner. Xi and Yeung (27) described a means of interfacing con ventional light sources to capillaries with internal diameters ranging from 10 to 50 μπι (Figure 6e). For a 50-μπι i.d. capillary and 3-mm injec
ta)
6
8 10 Time (min)
12
(b) 0.04
Round Rectangular
0.03 0.02 0.01 0
100 200 300 400 Field strength (V/cm)
Figure 8. Comparison of separations achieved with alternatively shaped capillaries. (a) Top: separation of 50^g/mL peptide mixture with 50-μπι i.d. cylindrical capillary; middle: δ-μς/Γηί peptide mixture with 50 μιτι χ 500 μηη rectangular capillary; bottom: 0.5^g/mL peptide mixture with 50 μπι χ 500 μπι rectangular capillary. Separation conditions as in Figure f. (b) Ohm's law plot for 50-μπι cylindrical and 50 μπι χ 500 μπι rectangular capillary.
494 A · ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1993
tion plugs, a sevenfold increase in sensitivity was obtained. Alternative detection modes UV l a s e r s . The use of high-intensity light sources offers an alternative to extending the optical pathlength to obtain greater sensitivity in an ab sorbance d e t e c t i o n m e a s u r e m e n t . The availability of UV l a s e r s will h a v e a n i m p a c t on spectroscopic based i n s t r u m e n t a t i o n as costs de crease and as performance and opti cal characteristics improve. CE is no exception. A krypton fluoride laser a t 248 nm has been used in a thermooptical detector for P T H - a m i n o acid analyses c a r r i e d out with CE (28). A temperature rise in the sam ple zone, proportional to t h e l a s e r power and sample concentration, is m e a s u r e d as a change in the RI of the zone by a secondary laser probe beam. In another study with UV laser ex citation (29), the second harmonic of an argon ion laser (257 nm) was used to m e a s u r e underivatized aromatic amino acids. Vibrations in a capil lary under applied tension were gen e r a t e d by t h e r m a l h e a t i n g of t h e sample zones and were shown to be proportional to the analyte concen tration. Measurements of the emit ted acoustic wave provided submicromolar-level sensitivities. For n o n a b s o r b a n c e - b a s e d detec tion schemes, usually either the analyte(s) of i n t e r e s t c o n t a i n a chrom o p h o r e or f l u o r o p h o r e " l a b e l " compatible with the mode of detec tion or such a label can be introduced in a straightforward, reproducible manner (exclusive of indirect detec tion formats). In addition to greater sensitivity, a second detection mode can e n h a n c e detection selectivity. The advantages and disadvantages of alternative detection methods are discussed briefly below. F l u o r e s c e n c e d e t e c t i o n . Fluo rescence detection typically results in sensitivity gains of 1-3 orders of magnitude over those of absorbancebased detectors. The degree of en h a n c e m e n t d e p e n d s on t h e l i g h t source, its i n t e n s i t y and stability, the optical match of the excitation s o u r c e to t h e f l u o r o p h o r e ( s ) , t h e q u a n t u m efficiency of t h e l a b e l , background fluorescence, and the de tector configuration. After UV absor bance, fluorescence is the most fre quently used detection mode when high sensitivity and selectivity are r e q u i r e d . Although most a n a l y t e s are not intrinsically fluorescent, a large section of the chemical litera ture covers fluorescent labeling tech-
from light scattering at the capillary wall. Using a 25-mW argon ion laser, workers have obtained picomolar d e t e c t i o n l i m i t s for d e r i v a t i z e d amino acids and mass sensitivities of 1-2 zeptomoles = 1 0 - 2 1 moles = 600 molecules (33). In another case (34), attempts were made to perform separations by using an a r r a y of capillaries to increase system throughput. In lieu of the usual 90° orientation of excitation and emission beams, a confocal arrangement was used. Excitation (laser) and emission are accomplished through a single microscope objective such t h a t only t h e interior of the capillaries is probed. The advantage of this configuration is t h a t s c a t t e r i n g i s r e j e c t e d b y proper spacial filtering.
niques. Laser-based fluorescence is particularly well suited for CE because of the small illumination volumes and light intensities required. Optimization of t h e detector and the chemistry has resulted in impressive performance. Mass sensitivities i n the zeptomole r a n g e (10~ 2 1 moles or hundreds of molecules) have been obtained by using on-capillary laser-induced fluorescence (LIF). A n u m b e r of configurations a n d der i v a t i z a t i o n chemistries have been reported; a representative sample is given in Table I. I n one innovative scheme known as s h e a t h flow, t h e sample is detected after eluting from t h e end of the column i n a flowing s t r e a m of liquid, t h u s e l i m i n a t i n g t h e b a c k g r o u n d effects r e s u l t i n g
Table I. Fluorescence configurations and sensitivities Detection limits LabeT/Analyte
Excitation source
System components "
Argon ion laser PMT, lock-in GBQGAf 457 nm amplifier carbohydrate Fiber-optic collection
Concentration
Mass ,b
5» 10 moles
3 *10
a
Reference
M
30
FITC/ amino acids
Argon ion laser CCD 2 - 10 =" 488'514nm Axial moles illumination
1 * 10 " M
31
Native/ proteins
Argon ion laser PMT'optics 275.4 nm
1 «10 , n M 3*10 '"M*
32
FTH/ amino acids
Argon ion laser Sheath-flow 488 nm cuvette Microscope objective
1 - 10"M
33
2 - 10 " moles
"CBOCA: 3-(4-carboxybenzoyli-2-quinolinecarboxaldehyde: FITC- fluorescein sohiocyanate; FTH: Fluorescein thiohyoantoin; PMT- photonulbplier tube. CCD charge-coupled device. * Under sample stacking conditions
Table II. Sensitivity enhancement techniques: Sample concentration Enhancement
Applicability to commercial systems
10-100
Direct
Simple to implement; dependent on sample matrix ionic strength relative to separation buffer
Field amplification
50-1000
Direct
Cannot be applied to cations and anions simultaneously
ITP
100-1000
Chromatographic concentration
100-1000
Method Stacking
Indirect (with appropriate buffer choices/ method) No"
Comments
Need to develop separation methods for different analyte systems; no automated pre-electrophoresis system currently available Difficult to manufacture costeffective system
* Waters has indicated the availability of CE columns containing a section packed with chromatographic material for this purpose. However, no data have yet been published.
Fluorescence detection and other detection modes can be used i n 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, r e sulting in a decrease i n the signal. The advantage is t h a t 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, sensitivities do not compare with those that can be obtained with an equivalent direct detection mode. Indirect fluorescence detection can provide a degree of sensitivity enhancement over t h a t of conventional direct UV absorbance methods. The indirect methodology h a s also been used i n a n absorbance mode when analytes do not contain significant chromophores (36); however, sensit i v i t i e s a r e g e n e r a l l y poor ( 1 0 ~ 4 10" 5 M).
Electrochemical detection. Conductivity and amperometric detection offer significant sensitivity enhancement over that possible with absorbance techniques (37-40). The m e a s u r e m e n t i s i n d e p e n d e n t of p a t h l e n g t h , allowing very n a r r o w 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 ( 1 0 - 3 0 kV) from the electrochemical potentials (~ 1 V). In general, isolation is performed by introducing a break at the point of capillary and electrode coupling. Numerous detector configurations have been demonstrated in the laboratory, but i t h a s been difficult to reproduce them. Detection limits of 1 amol have been obtained for serotonin and catechol (39). These high sensitivities, coupled with extremely small sampling volumes, may make, cellular studies possible. Radiochemical d e t e c t i o n . Radiochemical detection has often been effective when extremely high sensitivity h a s been required, a n d i t is still widely used for HPLC. By using a variety of configurations (41, 42) researchers have obtained sensitivit i e s o n t h e o r d e r of 1 0 " 1 6 - 1 0 - 1 8 m o l e s of r a d i o a c t i v e l a b e l ( 1 0 ~ 9 10 -n
M
32p.labeled
b a g e s
a n d
99Tc
radiopharmaceuticals). Despite the a d v a n t a g e s of this technique (very
ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1993 · 495 A
INSTRUMENTATION high sensitivity a n d selectivity with few b a c k g r o u n d s i g n a l s a n d t h e fact t h a t labeled analytes behave chemi cally a s t h e u n l a b e l e d s p e c i e s ) , it is l e s s l i k e l y t o b e u s e d b e c a u s e of t h e h a n d l i n g a n d d i s p o s a l difficulties a s sociated with radioactive m a t e r i a l s .
Summary The relative gains achieved by u s i n g t h e different cell g e o m e t r i e s a n d t h e chemical m e a n s discussed above are s u m m a r i z e d in T a b l e s II a n d III. A d v a n t a g e s a n d d i s a d v a n t a g e s of t h e different m e t h o d s a r e i n d i c a t e d in t e r m s of a p p l i c a b i l i t y , effects o n t h e separation, and m a n u f a c t u r i n g con c e r n s . O v e r a l l d e t e c t i o n l i m i t s for t h e different m o d e s a r e s u m m a r i z e d in T a b l e IV. V e r y low c o n c e n t r a t i o n d e tection limits can be obtained, and we believe they will become more available to t h e commercial i n s t r u m e n t user as the application m a r k e t develops. (Already two commercial L I F systems are available, and cer t a i n l y t h e n u m b e r a n d v a r i e t y of d e t e c t i o n c o n f i g u r a t i o n s will e x p a n d . )
Future directions A n e x a m i n a t i o n of t h e l i t e r a t u r e r e veals t h a t the number and diversity of p u b l i c a t i o n s is g r o w i n g a t a n e a r -
e x p o n e n t i a l r a t e ( F i g u r e 9). A m o r e d e t a i l e d look a t t h e l i t e r a t u r e r e v e a l s some interesting perspectives. • T h e u s e of c o m m e r c i a l i n s t r u m e n t a t i o n is cited in 30.4% of all a r ticles. T h i s p e r c e n t a g e is g r o w i n g on an annual basis. • N i n e t e e n p e r c e n t of t h e p a p e r s d e s c r i b e t h e u s e of d e t e c t i o n m e t h o d s other t h a n absorbance on noncom m e r c i a l s y s t e m s ( f l u o r e s c e n c e , 12%; M S , 4 . 8 % ; e l e c t r o c h e m i s t r y , 1.6%; r a d i o a c t i v i t y , 0 . 3 % ; R a m a n , 0.2%). • Applications are well d i s t r i b uted among DNA, protein, and the small-molecule pharmaceutical and i n d u s t r i a l c h e m i c a l a r e a s (~ 6 0 % t o tal); the rest comprise general re v i e w s a n d d i s c u s s i o n s of n e w C E t e c h n o l o g y a s well a s o t h e r e s o t e r i c application areas. I t e m s t h a t s h o u l d be a d d r e s s e d in the future include sensitivity, t h r o u g h p u t , s a m p l e m a t r i x effects, capillary wall chemistry and repro d u c i b i l i t y , s y s t e m c o s t , a n d e a s e of u s e . S e v e r a l m e t h o d s c a n b e u s e d to c o m p e n s a t e for t h e s e n s i t i v i t y l i m i t a t i o n s of e x i s t i n g c o m m e r c i a l s y s tems, and new techniques and ad v a n c e m e n t s will c o n t i n u e to b e m a d e available. In addition, the methods a r e by n o m e a n s m u t u a l l y exclusive.
Table III. Sensitivity enhancement techniques: Capillary format Method Z-Cell
Z-Cell/optics
Enhancement8 Pathlength (i.d.) 3-5
10-20
3mm (75 μΓΠ)
3 mm (75 μηι)
Applicability to commercial systems* Direct
Direct
Rectangular
9.5
500 μιη (50 μπι)
~ Direct
Multireflection
40
3.3 mm (75 μηι)
No
End-column
UV laser
7
10-50
3 mm (50 μπι)
50 μπι (50 μιτι)
No
No
T h e c o u p l i n g of m e t h o d s (e.g., s a m ple s t a c k i n g a n d o p t i m i z e d Z-cells) can yield significant sensitivity en h a n c e m e n t s . U l t i m a t e l y t h e u s e r will h a v e to o p t i m i z e t h e s e p a r a t i o n con d i t i o n s w i t h i n t h e l i m i t a t i o n s of t h e sample matrices and sample workup m e t h o d s , a n d c o u p l e t h e s e to t h e h a r d w a r e a v a i l a b l e from t h e g r o w i n g field of m a n u f a c t u r e r s . As for a l t e r n a t i v e d e t e c t o r s , t h e t r e n d will al m o s t c e r t a i n l y be in t h e d i r e c t i o n that HPLC has taken: toward more modular, interconnecting systems. The authors would like to thank the numerous individuals at Applied Biosystems, users, and colleagues involved with CE for their comments and encouragement over the past several years.
References < 1 ) Li, S.F.Y. Capillary Electrophoresis: Prin ciples, Practice, and Applications; Elsevier: New York, 1992; pp. 1-586. (2) Grossman, P. D.; Colburn, J. C. Capil lary Electrophoresis: Theory and Practice; Academic Press: San Diego, 1992; pp. 1-352. (3) R u h r , W. G.; Monnig, C. A. Anal. Chem. 1992, 64, 389 R-407 R. (4) P e r r e t t , D.; Ross, G. Trends Anal. Chem. 1992, 11, 156-63. (5) Lauer, H. H.; Ooms, J. B. Anal. Chim. Acta 1991, 250, 45-60. (6) Vindevogel, J.; Sandra, P. Introduction
Table IV. Sensitivity of detection modes Detection mode
Comments Direct applicability of methods High noise levels Some resolution loss Commercially available Moderate cost 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 loadings Questionable manufacturability Questionable reproducible manufacturability Instruments require modifications Alignment requires modifications Need to use nonaqueous buffers Data as step functions Cost Specific wavelengths available Lifetime/reliability
" Enhancement relative to standard cylindrical capillary of identical i.d. "Some optical alignment or specific part may be necessary for individual instruments.
496 A · ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1993
Sensitivity (M)
Absorbance Direct Indirect Fluorescence Direct (lamp-based) Direct (laser-based) Indirect (laser-based) Electrochemical Conductivity Amperometric Radiochemical
10~5-10-6 10-4-10"5 10-7-10"8 10"9-10-12 10"6-10-7 1(T5-10~7 10-8-10"9
io- 9 -io- 11
500 400 300 200 100 0 1982
1985 1988 1991 Year
Figure 9. Growth of CE citations from 1981 through 1992.
to Micellar Electrokinetic Chromatography; (40) Gaitonde, C. D.; Pathak, P. V. Huthig: Heidelberg, 1992; pp. 1-231. /. Chromatogr. 1990, 514, 389-93. (7) Deyl, Z.; Struzinsky, R./. Chromatogr. (41) Altria, K. D.; Simpson, C. F.; Bharij, 1991, 569, 53-122. A. K.; Theobald, A. E. Electrophoresis 1990, 11, 732-34. (8) Schwer, C ; Lottspeich, F . / . Chromatogr. 1992, 623, 345-55. (42) Pentoney, S. L.; Zare, R. N.; Quint, (9) Gebauer, P.; Thormann, W.; Bocek, P. J. F. Anal. Chem. 1989, 61, 1642-47. /. Chromatogr. 1992, 608, 47-57. (10) Aebersold, R.; Morrison, H. D. /. Chromatogr. 1990, 516, 79-88. (11) Burgi, D. S.; Chien, R-L. Anal. Biochem. 1992, 202, 306-09. (12) Chien, R-L.; Burgi, D. S. Anal. Chem. 1992, 64, 489 A-496 A. (13) Chien, R-L.; Burgi, D. S./. Chromatogr. 1991, 559, 153-61. (14) a. Stegehius, D. S.; Tjaden, U. R.; van der Greef, J. /. Chromatogr. 1992, 591, 341-49. b. Stegehius, D. S.; Irth, H.; Tjaden, U. R.; van der Greef, J. / Chromatogr. 1992, 538, 393-402. (15) Foret, F.; Sustacek, V.; Bocek, P. /. Microcolumn Sep. 1990, 2, 229-33. Michael Albin received his B. S. degree in (16) Débets, A.J.J.; Mazereeuw, M.; chemistry from The Polytechnic Institute Voogt, W. H.; van Iperen, D. J.; Lingeof New York (1979) and his Ph.D. from man, H.; Hupe, K-P.; Brinkman, U.A.T. The Pennsylvania State University /. Chromatogr. 1992, 608, 151-58. (17) Moring, S. E.; Colburn, J. C; Gross(1984). Following postdoctoral studies on man, P. D.; Lauer, H. H. LC-GC 1990, 8, electron transfer mechanisms at Cal Tech, 34-39. he pursued an industrial career centered (18) Bruin, G.J.M.; Stegeman, G.; van on the interface between chemistry and in Asten, A. C ; Xu, X.; Kraak, J. C ; Poppe, H.J. Chromatogr. 1991, 559, 163- strumentation in a number of fields. He 81. has been involved in CE development for (19) Chervet, J. P.; van Soest, R.E.J.; three years. Ursem, M./. Chromatogr. 1991, 543, 439-49. (20) Tindall, G. W.; Perry, R. L.; Albin, M.; Moring, S. E., unpublished work. (21) Said, A. A. AIChE 1959, 5, 69. (22) Izumi, T; Nagahori, T.; Okuyama, T. /. High Résolut. Chromatogr. Chromatogr. Commun. 1991, 14, 351-57. (23) Tsuda, T.; Sweedler, J. V.; Zare, R. N. Anal. Chem. 1990, 62, 2149-52. (24) Chen, Y.; Zhu, A. Set. China Ser. Β 1992, 35B, 649-58. (25) Jansson, M.; Emmer, Α.; Roeraade, J. / High Résolut. Chromatogr. 1989, 12, Paul D. Grossman received his B.S. de 797-801. (26) Wang, T.; Aiken, J. H.; Huie, C. W.; gree and his Ph.D. in chemical engineer Hartwick, R. A. Anal. Chem. 1991, 63, ing from the University of California at 1372-76. Berkeley and his M.S. degree in chemical (27) Xi, X.; Yeung, E. S. Appl. Spectrosc. engineering from the University of Vir 1991, 45, 1199-1203. (28) Waldron, K. C; Dovichi, N. J. Anal. ginia. He is the co-editor of a recently pub Chem. 1992, 64, 1396-99. lished book on CE. His research interests (29) Odake, T.; Date, K.; Kitamori, T.; involve the application of novel electroSawada, T. Anal. Sci. 1991, 7, 507-08. phoretic techniques to DNA analysis. (30) Liu, J.; Shirota, O.; Wiesler, D.; Novotny, M. Proc. Natl. Acad. Sci. 1991, 88, 2302-06. (31) Sweedler, J. V.; Shear, J. B.; Fishman, Η. Α.; Zare, R. N.; Scheller, R. H. Anal. Chem. 1991, 63, 496-502. (32) Lee, T. T.; Yeung, E. S.J. Chromatogr. 1992, 595, 319-25. (33) Zhang, J. Z.; Chen, D. Y.; Wu, S.; Harke, H. R.; Dovichi, N. J. Clin. Chem. 1991, 37, 1492-96. (34) Huang, X. C ; Quesada, Μ. Α.; Mathies, R. A. Anal. Chem. 1992, 64, 967-72. (35) Yeung, E. S.; Ruhr, W. G. Anal. Stephen E. Moring is working on product Chem. 1991, 63, 275 A-282 A. research and development at the Applied (36) Pianetti, G. Α.; Taverna, M.; Baillet, Α.; Mahuzier, G.; Baylocq-Ferrier, D. Biosystems Division ofPerkin Elmer. He /. Chromatog. 1993, 630, 371-77. received his B.S. degree from the Univer (37) O'Shea, T. J.; Telting-Diaz, M. W.; sity of Maryland (1972) and his M.S. de Lunte, S. M.; Lunte, C. E.; Smyth, M. R. Electroanalysis 1992, 4, 463-68. gree in biochemistry from the University of (38) Yik, Y. F.; Li, S.F.Y. Trends Anal. Kansas (1977). He has been involved in Chem. 1992, 11, 325-32. CE development since 1988 and was re (39) Curry, P. D.; Engstrom-Silverman, C. E.; Ewing, A. G. Electroanalysis 1991, sponsible for the development ofUVand 3, 587-96. fluorescence detector technology.
Magnetic Resonance of Carbonaceous Solids F
ocusing on new magnetic resonance methods for characterizing solid carbonaceous fuels, this new volume covers 2-D methods, high-resolution ,3C and Ή NMR (CRAMPS) analyses, dynamic nuclear polarization tech niques, temperature-dependent relaxation measurements, chemical methods to improve quantitative reliability, ultrafast MAS methods, and new signal enhancement techniques. Also discussed are topics of current interest in the area of ESR, including spin-echo methods, 1-D and 2-D pulsed ESR techniques, relaxation time measurements, and ultrahigh frequency measurements. In addition, measurements are made on a set of Argonne Premium Coal samples to compare different magnetic resonance techniques on a homogenous set of carbonaceous fuel samples. Robert E. Botto, Argonne National Laboratory, Editor Yuzo Sanada, Hokkaido University, Editor Advances in Chemistry Series No. 229 666 pages (1993) Clothbound ISBN 0-8412-1866-8 $149.95 O R D E R F R O American Chemical Society Distribution Office Dept. 56 1155 Sixteenth Street. NW Washington. DC 20036 Or CALL TOLL FREE
M
800-227-5558 (In Washington, DC. 202-872-4363) and use your credit card!
ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1993 · 497 A
