Anal. Chem. IQQ3,85, 3454-3459
3454
Optical Improvements of a Z-Shaped Cell for High-Sensitivity UV Absorbance Detection in Capillary Electrophoresis Stephen E. Moring' and Richard T. Reel Applied Biosystems, Division of Perkin Elmer, 850 Lincoln Centre Drive, Foster City, California 94404
Remco E. J. van Soest LC Packings, Baarsjesweg 154, 1057 HM Amsterdam, The Netherlands
Absorbance detection in capillary electrophoresis (CE)offers excellent mass sensitivity but relatively poor concentration sensitivity. This dichotomy arises from the short optical path lengths found in current detector designs. Minimum detectable concentrations range from lod to lo-' M. The theoretical and physical design aspects affecting signal, noise, and optical transmission of various commercial (CE)detector cells are discussed. A new Z-shaped cell is described which uses a quartz ball lens to optimize light throughput in a 3-mm capillary section. The optical enhancement of the cell provides more than 1 order of magnitude improvement in the signal-to-noise ratio (MDCto 10-8 M) over that of a conventional cell design with sapphire ball optics. The effect of the increase in cell path length and volume is evaluated for electrophoretic efficiency and resolution. For peak efficiencies up to 200 000 plates, extention of overall path length from 0.075 to 3 mm results in less than 14% loss in efficiency. The new cell design also provides an improvement of linear dynamic range from -3.5 orders of magnitude with a conventional cell to more than 4 orders of magnitude.
INTRODUCTION Capillary electrophoresis (CE) is an important alternative and complement to gas chromatography (GC) and highperformance liquid chromatography (HPLC). Numerous reviews have noted the need for enhanced detection for the technique to gain greater acceptance." Commercial instrumentation currently provides detection options that include only UV/visible absorbance and fluorescence. Despite the high sensitivity offered by fluorescence detection (minimum detectable concentration 1o-S M with conventional light sources and to M with a laser source), the technique requires cumbersome chemical derivatization strategies that are impractical for routine analysis of a broad range of analytes. Furthermore, derivatization of these analytes at low concentrations results in poor yields and multiple impurities. UV absorbance typically yields minimum detectable concentrations (MDC) in the lW-lV M range and,
* To whom all correspondence should be addressed.
(1) Goodall, D.M.;Lloyd, D.K.; Willi-, S.J. LC-GC 1990,8,788799. (2) Kuhr, W . G . Anal. Chem. 1990,62,403R-414R. (3)Novotony, M.V.;Kobb, K.A.; Liu, J. Electrophoresis 1990,11, 736-749. (4)Kuhr,W.G.; Monnig, C. A. Anal. Chem. 1992,64,389R-407R. 00092700/93/0365-3454$04.00/0
because of its broad applicability, remains the most popular detection mode. A major challenge facing absorbance detection in a capillary format is improvement of the optical interface. Bruin et al.6 compared various interface designs and evaluated their performance with respect to sensitivity, noise, and linearity. This evaluation, as well as work carried out by Chervet et al.,S showed that an extended optical path, in which the capillary is bent to a Z-shaped configuration, can result in a significant gain in signal. However,the increase in sensitivity with this design was limited by the relatively high background noise caused by low light transmission through the bent section of the capillary. We have evaluated the theoretical and physical aspects of light transmission through various capillary cell geometries. The optical characteristics of a new Zshaped cell design are compared to commercially available detector cells. Signal gain, noise, limits of detection, dynamicrange, and the effects of cell volume on electrophoretic efficiency and resolution are demonstrated and discussed.
THEORY In CE the detector cell is defined by the section of illuminated capillary seen by a detector element (photodiode, etc.). The factors affecting sensitivity include analyte concentration, extinction coefficient,detector cell light path, area of illumination, and light energy throughput of the optical cell. The latter two affect only the background noise levels. The most critical factor in designing an optical interface for absorbance detection is the ability to focus light within the central bore (lumen) of the capillary. A simple design which has been used in commercial CE detectors consists of slit optics for transverse illumination of a section of the capillary (Figure 1A). The effective light path (A) is defined by integration of ray path lengths across the inner bore of the capillary between the slit boundaries, where the average path length for a capillary with a given inner radius (r)6is simplified to A = rrI2
(1)
An increase in the area of illumination longitudinally along the capillary does not result in an increase in absorbance. It does, however, improve light energy transmission, decrease detector noise, and, therefore, enhance the signal-to-noise ratio. The optical path length can be extended significantly, if focusing optics are used so that light is channeled to approximatea radial path (AT) in the transverse plane (Figure (5) Bruin, G. J. M.; Stegeman, G.; van Asten, A. C.; Xu,X.,Kraak,J. C.;Poppe, H.J. Chromotogr. 1991,559,162-181. (6)Chervet, J.; van Scat, R.; Umm, M.; Salunann,J. J. Chromotogr.
1991,543,439-449.
0 1983 Amerlcan Chemlcal Soclety
ANALYTICAL CHEMISTRY, VOL. 65, NO. 23, DECEMBER 1, 1993 3455 A
I I I I
A
Flgura 1. On-columndetection schemes: (A) transvem lnumlnatbn of capillary section with SIR optics; (6) Illumination at lerge solid angle with sapphire ball lens optics.
lB), where
-
2r (2) For this purpose, a ball lens made of sapphire has been used (refractiveindexof 1.91with66% transmittanceat 200n1n).~ In the longitudinal plane, the light path (AL) is a function of the light angle (8) incident with the capillary and can be described by the expression A,.
AL = 2r/cos(arcsin(nJ% sin 8 ) )
(3)
where n, and nb are the refractive indexes of the capillary wall and the buffer in the capillary lumen, respectively. With a solid angle of -30°, the cell path length is increased hy a average of 10% in the longitudinal plane. The optical light path of a CE cell can be extended further by bendingaportionof the capillary intoa Z-shape,providing an increased path length. In this cell design, light enters the bend of the capillary and reflects through the lumen by total internal reflection. A ray trace analysis of the refraction of a parallel beam of light in the plane of the capillary bend (Figure 2A) illustrates the limits of useful incident light for sample illumination in that plane. To maximize the light traversing the lumen, the incident beam must be offset from the capillary axis. In addition, the incident beam should he limited to the boundary of light that enters the bend and passes through only the capillary lumen. These boundries are defined by the outer edge of the bend to the limit where the refracted light no longer passes through the lumen and is trapped within the outer wall of the capillary. An estimationof the average path length of light traversing the capillary lumen in the planes parallel and perpendicular to the capillary bend can he determined by analysis of light rays at angles between the limits defined by the rays that escape the outer wall and those that become trapped within the wall of the capillary. Calculation of these boundaries by ray trace analysis at a wavelength of 200 nm (Figure 2B) shows that these limits occur between 40' and 60D,respectively, for a 75 pm i.d. by 280 pm 0.d. fused-silica capillary. The path length for a given ray passing through the &shaped cell can he calculated from the equation A = N,L,
/
Focal
Point
2. Ray trace analysis of light at capillary bend wkh Z-shaped optical cell gometry: (A) axle1 lilumlnatlon with collimated IMght (6) angular llmlts of light transmission Inside channel. Rays e (escaping)
!+ne
(4)
where NR is the number of times that a given ray passes through the lumen (capillary inner diameter) and 4 is the
andd(trapped)arenotusehrlanddonotpasstomedetectw. Effective rays, band c. range between 40' and 59'. (C) Focus of lerge solid engia of light with an axial offset.
length of a given ray for each pass through the lumen:
L, = di/cos(arcsin(nJ% sin 8 ) )
(5)
and
NR = L/(d,tan(arcsin(n,n, sin 8)) + (do- di) tan 8) (6) where di and do are the capillary inner and outer diameters, respectively, L is the length of the detector cell, and 8 is the angle of the ray in the wall of the capillary. Combining eqs 4-6, the average path length of rays passing through the bend can he approximated by the following expression:
(dJd,
- 1)tan 01 cos(arcsin(n,&
sin 0))) (7)
where 0. and are the light boundary angles within the walls of the capillary. The light rays within the defined boundaries can be extended backwards in two perpendieular planes out through the bend in the capillary. The resulting bundle of light is characterized as an elipsoid cone of light. Light entering the capillary bend with an equivalent solid angle would be expected topass predominantlythrough the capillary lumen. With the light source aligned parallel to the central axis of the optical segment capillary, only a very small fraction of rays would be expected to pass around the lumen and pass out theotherendofthecbend. Furthermore,whenafocusing lens is placed in front of the capillary bend with the correct offset as shown in Figure 2C, the light path length and the total light - transmission through . the capillary lumen can be maximized. Comnarison of the sensitivitv of different cell desiensmust include the effeds of capilla& diameter on the v&me of sample injected. The volume of hydrodynamic injection differsfor the50- and75-q4.d. capillarieafor agivenpressure and injection time. The injection volume is determined by the Poiseuille equation:
(7)Mo~,S.E.;Colbum,J.C.;Gmssman,P.D.;Lauer,H.H.LC-CC where IrJo,8,34-46.
V, = hPmd't/128~Lt
(8) AP is the injection pressure differential across the
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 23. DECEMBER 1, 1993
Table I. Calculation of Effective Path bngthB for 3-mm Optical Cell with Various Capillary DimensionB capillary dimens (pm) effectivepath semitivity gain i.d. 0.d. length (EPLY (mm) actual' theoretical'
Y Photodiode
\
Flpure 3. optlcal deslgns fw cells used in sensnivny measurements: (A) optlcal cell wim transverse Illumination of capillary using a 2-mm sapphlreballlensanda0.5mma~ure:(8)Zcellwimaxlallllumlnatbn of capillary using a 4 m m qcarlz ball lens and 1.5-mm aperture.
capillary, Lt is the capillary length, d is the capillary internal diameter, n is the viscosity of the run buffer, and t is the injection time. For the same capillary length, injection time, and pressure, the injection volume for a 75-pm4.d. capillary is 5 times larger than the 50 pm i.d. capillary. The sample plug (zone) length for a given capillary is determined by factoring out the cross-sectional area from eq 8:
L, = APd't/32rlLt (9) The injection plug length is a major factor which contributes to the peak efficiency obtained in an electrophoretic separation. Equivalent sample zone lengths provide the same number of theoretical plates for analytes with the same migration time and where Joule heating and diffusion caused by temperature differences arenegligible. The ratioofsample plug lengths for the same capillary length, injection time, and pressure for a 75- and a 50-pm4.d. capillary is 2.25.
EXPERIMENTAL SECTION Evaluation of various detector cell designs was carried out on the Applied BiosystemsModel 270A-HT capillary electrophoresis system (Foster City, CA). Comparison of detection parameters was performed on the Applied Biosystems sapphire optical cell and amodified LC Packings (Amsterdam,The Netherlands) CE Zell. The sapphire cell consists of a two-piece assembly with a 2-mm sapphire hall lens (Swiss Jewel Co. Philadelphia, PA) and a 0.5-mm aperture (Figure 3A). The modified cell consisted of a capillary housing, a 75 pm i.d. X 280 prn 0.d. capillary bent into a Z-shape with a 3-mm optical path, and an optical fixture containing a 4-mm quartz ball lens (SwissJewel Co.) and a 1.5mm aperture (Figure3B). The sapphire cell was tested with two capillary sizes: 50 pm id. X 360 pm o.d., and 75 pm i.d. X 280 pm 0.d. (PolymicroTechnologies,Turdon, AZ). A 1-em portion of the polyimide coating of various capillaries was removed for light transmission. The capillary used in the %shaped optical cell had the coating removed prior to bending, before being permanently mounted into the cell assembly. Performance Evaluation Methods. Sensitivity and the limits of detection were determined by direct absorbance measurements carried out by contiguous flushing of serial dilutions of dimethyl sulfoxide in deionized water through the capillary. Measurements wererecordedonaKipp& ZonenMcdel BD 41 chart recorder (Delft, The Netherlands) with the Model 270A-HT detector wavelength at 200 nm and a rise time of 0.5
50 50 75 75 100
100
280
360 280
360 280 360
0.94
0.75 1.33
12.4
1.01
9.9
1.67 1.37
3.1
18.8 15.1 17.7 14.3 16.1 13.1
Numerical inmation of eq 6 performed hy MathCad, version 3.1 (Mathsoft,Inc., Cambridge, MA). *Direct absorbance measurement @A/& in AU/ %) of DMSO in water at 200 nm. < Ratio of inner dimension, EPL/capillary i.d. 8. Buffer salts and organic solventa were obtained from MaUiuckrodt, Inc. (Paris, KY)or Sigma Chemical Co. (St. Louis, MO). Light throughput of the cells was monitored by measurement of the detector photodiode voltage. Photodiode voltages were converted to current by using an exponential function in order to correlate the photodiode output with light transmission. Comparisons of the electrophoretic performance of the % shaped and sapphire optical cells were made by the analysis of a set of pyrrolidinone analogs and a mixture of test peptides. Samples containing three acidic pyrrolidinone analogs were provided by American Cyanamid Co (Princeton, NJ). The pyrrolidinone sample mixture was solubilized in an aqueous solution containing 10% acetone at 3.0 pgimL per component. Ahighionicstrength buffer containing2OmMscdiumphosphate at pH 6.5,90 mM hexanesulfonic acid, and 10 mM tetramethylammonium phosphate was used to assess the effect of high running current on electrophoretic resolution. Analyses were performed at 15 kV (208 Vicm) and 45 "C with a detection wavelength of 242 nm. A peptide performance evaluation standard was used for efficiency comparison. The standard consisted of three basic peptides, RKRSRKE,dynorphin3-13,anddynorphin1-13 (AB1 performance evaluation standard, Foster City, CA) at a concentration of 50 pgImL each. The samples were made up in a run buffer (20 mM sodium phosphate, pH 2.5) and a low ionic strength buffer (1 mM sodium phosphate. pH 2.5). The electrophoresiswascarriedoutat20kV (278V/cm)at30OCwith a detector wavelength of 200 nm. The determination of detector cell dynamic range was carried out with the use of DMSO, uracil, and fluoresceinobtained from Sigma Chemicals. The electrophoretic conditions for the detenninationofdynamicrangeincludedtheuseofa20mMscdium borate buffer, pH 9.0, and electrophoresis at 20 kV (278 V/cm) at 30 "C with a detector wavelength of 200 nm. All CE experiments were performed with capillaries of a total length of 72 cm and a length to the detector cell of 50 em. AU injections were made with vacuum of 16.8 kPa.
RESULTS AND DISCUSSION Optimization of Optical Parameters. The optimal capillary dimensions, relative aperture, focus, and offset of the image of the light source were carefully evaluated in designing the optics of the high-sensitivity %cell. Various capillary sizes (inner andouter diameter aspect)wereassmed to determine the maximum effective path length for light reflected through a 3-mm optical segment of the Z-cell. The path lengths for 5W100pm capillaries with outer diametera ranging from 280 to 360 pm were calculated using eq 6. The results are presented in Table I. The largest effective light path occurs with capillaries with the smallest outer/inner diameter ratio. These calculations were verified by sensitivity measurementafor 2-cells constructed withcapillariesofthree different sizes. The theoretical sensitivity gain shows the potential enhancements over the conventional capillary (sapphire) cell design with the same internal diameter. The calculations suggest that a cell with a 50 pm i.d. hy 280 pm
ANALYTICAL CHEMISTRY, VOL. 65, NO. 23, DECEMBER 1, 1883
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3457
3 mm 2-Cell weh
ball lens
100
//
4 70 urn offset 50 pm offset
A
3 mm (LCP CE-Zell)
75 pm sapphire cell
n
50 pm sapphire cell 0.000
0
200
100
300
Lens Angle of Rotation (")
0.002
0.004
0.006
0.008
0.010
Concentration of DMSO (K)
Flgurr 5. Comparison of detector cell geometry and senslthrky by
direct absorbance measurement. Offset
Aperture
Image
Capillary
axis
Flguro 4. Effect of offset of aperture image In Z-cell on senslthrlty and ilgM transmission (endon vlew of capillary bend). Absorbance of 0.005% DMSO at 200 nm.
0.d. capillary may provide significant sensitivity gain without large losses in electrophoretic resolution and efficiency. However, comparison of the absolute sensitivity of the 50pm4.d. Zcell with that of the 75-pm4.d. Z-cell were disappointing. Even though capillarieswith inner diameters larger than 75 pm provide a longer effective light path, their use generally results in losses in resolution and efficiency due to excessive Joule heating. Therefore, a 75 pm i.d. by 280 0.d. capillary format was chosen as the best compromise in terms of sensitivity and resolution. Ray trace analysis of the light angles reflected through the lumen of the capillary indicates that the selection of a relative aperture (f-number) of -0.7 maximizes light transmission and effective path length. To obtain the optimal focus, a 4-mm quartz ball lens was positioned at a distance of 0.25 mm from the capillary (lens surface to the outer capillary surface perpendicular to the optical axis). The alignment of the ball lens with the Z-cell was found to be an important factor in the optimization of sensitivity. The effect of a 50- and 70-pm offset of the lens to the optical axis as shown in Figure 2C was determined by rotating the optical fiiture relative to the bend and the axis of the capillary. Figure 4 shows the absorbanceand transmission as a function of the offset and offset angle of the lens. Both these results and measurements by ray trace analysis demonstrate an optimal offset of 70 pm from the center of the capillary. Sensitivity Measurements. The sapphire and Z-shaped optical cells were tested using direct absorbance and electrophoretic methods. Figure 5 shows the relative sensitivity of different capillary diameters and cell geometries. Table I1 shows the important figures of merit for the direct absorbance measurements. These data demonstrate the typical sensitivities for 10 individual Z-shaped optical cells. The sensitivity measurements (aa/ac) were made by comparing absorbance values for continuous plugs of dimethyl sulfoxide at three different concentrations. The signal-tonoise ratios (WN) were inconsistent compared to aA/ac. In this case the S/N is more a function of the changes in noise level caused by differences in the level of light throughput for the various detector cell designs. The sensitivity gain observed with the unmodified Z-cell over the sapphire cell with the
Table 11. Comparison of Detector Cell Design and Sensitivity by Direct Absorbance Measurement of DMSO in Water at 200 nm aAlac photodiode noise cell geometry (Aut%) output (nA) (mAU) S/NO 3-mm Z-cell with ball lens 3-mmLC packingsCE-zell 75-pm sapphire cell 50-pm sapphire cell
12.0 7.5 0.93 0.29
2.10 0.077 0.675 0.633
0.02 0.18 0.06 0.08
500 41.6 10.8 3.75
OAt O.OOOl%.
same capillary dimensionswas &fold, whereas the Z-cell with 4-mm quartz lens provided a 12.9-fold increase. A comparison of the detector noise measurements of the three cell design variations reveals that the ball lens reduces the noise level by 8-10-fold. The gain in sensitivity of the Z-cell plus the ball lens over that of the sapphire cell can be attributed predominantly to an increase in light path length of the cell. The difference in the sensitivity between the Z-cell with and without the lens is a function of both the effective path length and the light energy throughput. The high noise level without the ball lens is a result of insufficient light transmission. Evaluation of Resolution, Efficiency, and Sensitivity in CE. The Z-shaped optical cell was evaluated for electrophoretic performance with regard to resolution and efficiency. In Figure 6, the detector responses for three acidic pyrrolidinone analogs (injected with equivalentsampleplug lengths) are compared for the two detector cell geometries under conditions of significnatJoule heating. The effect of capillary diameter and cell path length on sensitivity, efficiency, and resolution are further demonstrated in Table 111. Ae expeded, peak efficiency and resolution decrease by a factor of 20% and 6 7% ,respectively, with the increase in capillary diameter. The effect of the increase in cell path length on these measurements is substantially less (5.0% and 276, respectively). The increase in sensitivityobserved with the Z-shaped optical cell (75-pm4.d. capillary) versus the sapphire optical cell (50-pm4.d. capillary) is -60-fold. Considering the higher mass injected in a 75- versus a 50-pm capillary (a factor of 2 greater),this gain represents a 30-fold increase. Under the conditions used above, little loss in resolution and efficiency with the Z-shaped cell was observed. Under conditions where the analysis time is faster (e.g., shorter capillaries and high electric field strengths), or where peak sharpening occurs (e.g., electrophoretic stacking in low ionic strength sample solutions), loss of peak efficiency is more dramatic. Electrophoresis of the peptide performance evaluation standard in a low ionic strength buffer and under nonstacking versus stacking conditions shows the extent of efficiency loss associated with the change in capillary dimension and an increase of cell path length (Table IV). The path length of the 3-mm Z-shaped cell essentially defines the
3458
ANALYTICAL CHEMISTRY, VOL. 65, NO. 23, DECEMBER 1, 1993 SWOW
-
50 km 1.d.
Sapphire Cell
II)
Q)
75 km 1.d.
-m
Sapphire Cell
c
L
400000-
a n 300000
-
.-u 2
200000-
0 0)
z 100ow-
0 4 0
1000
2000
Analysis Time (sec.)
Flgure7. Calculation of theoretical efficiency as a functionof analysis time for 50- and 75-pm4.d. capillaries and different detector cell path d l s at 30 O C ; electric field 417 V/cm). lengths (diffusion 3 X Efficiency curves for caplllaries with different Injection volumes (times): sapphire optlcal cell, (A) 50-pm4.d. caplllary, lnjectlon 4.6 nL (1.O 8); (6) 50-pm1.d. capillary, Injection 11.5 nL (2.5 8); (C) 75-pm4.d. capillary, Injection 11.5 nL (0.5 8). (D) 3-mm 2-shaped cell with ball lens and 7Cpm-1.d. capillary, injection 11.5 nL (0.5 s).
3 mm Z-Cell with ball lens
I
I
I
I
6
8
10
12
Tlme
-
(mln.)
Flguro 8. Electrophoretic comparison of detector response with sapphire cell and 2-shaped optical cell. See Experimental Section for electrophoretic condltlons.
Table 111. Comparison of Analytical Figures of Merit for Sapphire and 3-mm Z-Shaped Cells with Pyrrolidinone Analogs capillarycell
sapphirecell(50pm) sapphirecell(7Spm) 3-mm %cell with ball lens (75 pm)
inj lug w i h cunent Na area MDCc (mm) (MA) R a (plates) (rcVs) (ng/mL) 4.6 5.2 5.2
40 107 101
1.43 91432 4248 1.35 72943 12117 1.33 69 309 172 258
1980 450 32
Resolution calculationsfrom peaks 2 and 3. b Theoretical plate calculationsfrom peak 2. Minimum detectable concentrationbased on S/N = 3. Table IV. Comparison of Measured Efficiency for a Peptide Test Standard with Different Capillary Cell Geometries total theoretical dates
50-am 75-rm 75-pm 3-mm sample buffer conditions sapphire cellas sapphire cellbvc Zcelll1ensb.C stacking nonstacking
275 700 180 OOO
139 500 91 100
of the 50-pm4.d. capillary). Under the same conditions, the same peak is 150 times larger than the 50-pm segment of illumination in the sapphire cell. In the latter case, peak distortion caused by “optical dilution” is insignificant. However, as can be seenwith the 2-shaped cell, the lose in efficiency under stacking conditions k apparent, although small ( 14% ). Under nonstackingconditions, the difference in the two cells is insignificant. For peak width that are less than approximately 3 times the detector cell path length, one begins to observe peak distortion or broadening due to the increased cell length. In a given electrophoretic system under the influence of a constant electrical field and temperature, and where no analyte/wall interaction exists, the total efficiency is the result of the contribution of dispersive forces caused by diffusion (u&, capillary diameter (Joule heating, UA?), injection plus length (uw2) and detector cell length (up)
120 800 89 500
E 1.0-8 (2.3 mm) vacuum injection. 0.5-8 (2.6 mm) vacuum injection. See ExperimentalSection for electrophoreticconditions.
volume of the cell. Under stacking conditions, the width at base (ca. 8 mm for 0.5-8 vacuum injection and a migration time of 6 min) is more than twice as wide as the path length of the 3-mm, Z-shaped optical cell. Optical distortion of the peak in this case is significant, albeit small. The cell volume of the sapphire optical cell is equivalent to the volume of the capillary illuminated by the light source (50-60-pmsegment
N = L2/uT2 and the total dispersion (u$)
(10)
Figure 7 illustrates the theoretical efficiency using eqs 10and 118 for a hypothetical analyte over a broad range of electrophoretic mobilities for constant field strength, temperature, and diffusion rate for a typical low ionic strength buffer. A true comparison of efficiency for the 50- and 75-pm4.d. capillaries must take into consideration the differences in injection time for equivalent mass injections (Figures 7B,C). From the theory as well as actual measurement, the lose in resolution (R N NV2) caused by the use of an extended cell optical path is minimal compared to the effect of using a larger inner diameter capillary tube. The loss in resolution in this case can be justified by a substantialgain in sensitivity. Figure 8 illustrates a practical example of the sensitivity enhancement for the free solution electrophoresisof a typical protein digest at a picomole per microliter concentration. The greatest loss in peak efficiency in this model is the result of the increase in capillary diameter and injection plug length. The limits of theoretical efficiency with the 3-mm Z-shaped cell, however, are defiied by the smallest peak width detectable by the cell. Because this width can never ‘appear” (8) G~txwnaqP.D. In CapilkvyElectrophLx TheoryandF’ractice; Grossman, P. D., Colburn, J. C., E&.; Academic Press: San Diego, CA, 1992; pp 2E-43.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 23, DECEMBER 1, 1993
34SQ
A
50 Fm Sapphlre Cell
000
75pm Sapphlre Cell
1.
A
h
with lens sapphire cell sapphire cell
&Cell with Ball Lens
Concentration
of
DMSO (pglrnl)
B
5
g
1000
. m 5 * Y 0
7 8
-1 10
I 12
I
14
I
16
Tlmo
1 18
I
20
I 22
I 24
I
(mln.)
F@uo 8. Comparlson of detector cell pertormence for the analysis of a tryptic d l g d of &lactogbbuHn (10 pmoVpL). CondMons: 20 mM sodium phosphate buffer, pH 2.5,20 kV at 30 OC, and detection at 200 nm. VacuumInJectlons were adjusted for mass equhralent badng.
to the detector to be less than 3 mm, the maximum efficiency for a 500-mm capillary (N= 5002/(32/12))is -330 OOO plates. Where separations require very high resolution, as in the case with the application of capillary gel electrophoresis (peak efficienciesare typically on the order of 1million plates), the loss of efficiency may preclude the use of the Z-cell with capillary lengths under 1m. Dynamic Range. The dynamic range of the sapphire and ball lens-modified Zshaped optical cells was evaluated using direct absorbance measurements at 200 nm. Figure 9A demonstrates linearity over 3.5 and 4.0 orders of magnitude for these cells, respectively. The minimum detectable concentration (MDC at S/N = 3) for DMSO under these conditions was 1.2 &nL (50-pm id.) and 650 ng/mL (75-pm i.d.) for the sapphire optical cells compared to 40 ng/mL for the modified Zcell. The upper end of the linear range (regressioncoefficient 0.999) was 5 mg/mL with the sapphire cell (dynamic range 7.7 X 103) and 500 pg/mL with the modified Zcell (dynamic range 1.25 X 104) for the same capillary inner diameter. The detector response saturated for both cells at 1.0 AU. This phenomenon is presumably the result of leakage of light around the capillary lumen. In the case of the sapphire cell, a small fraction of light is scattered at the inner capillary wall and passes to the photodiode without entering the capillary lumen. With the modified Zcell, a larger fraction of light p-s around the outside of the capillary and through the hole in the cell mounting plate holding the capillary. In one example, the fraction of light bypassing the Zcell lumen was measured at -3% of the photodiode current. However, despite the difference in the cells, there is an overall increase in the linear range because of an extension of the lower end of the range with the modified Zcell. Under electrophoreticconditions,the Zshaped optical cell extended the lower end of the linear dynamic range over 1order of magnitude. The lower limit detection for uracil
Fluorescein to1
A
area
A width
1:
.l,014 .01
26
Uracil 0 area width
100
...
"7
.1
.
~
1
... ....
1
"7
lo
100
.-
...-.1
1000
Concentration (pglrnl) Q. Comparlson of dynamic range for dlfferent detector cell types: dkect absorbance measvement (A) and electrophomtk conditions for modlfled Z-cell (B) (seeExpertmentalSection for detal). -0
and fluorescein was 15 and 10 ng/mL, respectively. Figure 9B illustrates the dynamic range of the Zcell, the upper limit detector response, and the onset of capillaryoverloading. Peak distortion due to conductivity perturbation occurred when an excess of 4 ng (20-nL injection) was injected, resulting in a dramatic increase in peak width.
CONCLUSION The ball-lens-modified Z-shaped cell design provides a significantbreakthroughfor absorbance detedion in capillary electrophoresis. A sensitivity gain of over 10-fold is realized with a limit of detection on the order of 1V M and an expansion of linear dynamic range of 4 orders of magnitude. The sensitivitygain is realized without significant loss of peak efficiency and resolution in free-solution CE due to the increase in cell volume. The modified Zshaped optical cell also improves quantitative precision because noise levels are significantly reduced, allowing for more accurate peak integration and lower limits of quantitation.
ACKNOWLEDGMENT The authors are grateful to MichaelAlbin, Paul Grwman, and John Wiktorowicz for their comments and suggestions in the preparation of the manuscript, and Max Safarpour of American Cyanamid for his donation of test samples.
RECEIVED for review May
4, 1993. Accepted August 20,
1993." 0
Abstract published in Adoance ACS Abstracts, October 1, 1993.
