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Anal. Chem. 1986,58,3207-3215 8
7.2 6.4
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20
40
60
100 120 140 Concentration Ipprn)
80
160
180
200
220
FIgure 8. Calibration curves for maltose oligomers.
to the determination of trace concentrations of oligomers in resin leachates.
ACKNOWLEDGMENT We thank H. D. Woodcock for machining the electrodes and cells and V. A. Stenger for suggestions on reagent chemical testing.
LITERATURE CITED (1) Rocklln, R. D.; Pohl, C. A. J . Li9. Chromatogr. 1083, 6, 1577-1590. (2) Fleischman, M.; Korinek, K.; Pletcher, D. J . €kcfroanal. Chem. 1971, 3 1 , 39-49. (3) Fleischman, M.; Korinek, K.; Pletcher, D. J. Chem. SOC., Perkln Trans. 2 1072, 1396. (4) Lu, P. W. T.; Srinivasan, S. J. Electrochem. SOC. 1078, 125, 1416. (5) Van Effen, R. M.; Evans, D. H. J. Electroanal. Chem. 1078, 703, 383-397. (6) Robertson, P. M. J . Electroanal. Chem. 1980, 7 1 7 . 97. (7) Vertes, G.; Horanyi, G. J. Electroanal. Chem. 1074, 52, 47. (8) Schick, K. G.; Magerearu, V. G.; Huber, C. 0. Clin. Chem. (WinstonSalem, N.C .) 1078, 24, 448-450. (9) Morrison, T. N.; Schick, K. G.; Huber, C. 0. Anal. Chlm. Acta 1080, 120, 75-80. (10) Yuan, C. J.; Huber, C. 0. Anal. Chem. 1085, 5 7 , 180-184. (11) Hui, B. S.; Huber, C. 0. Anal. Chim. Acta 1082, 134, 211-218. (12) Schick, K. G.; Huber, C. 0. U S . Patent 4183791. (13) Kafil, J. B.; Huber, C. 0. Anal. Chim. Acta 1085, 775, 275-280. (14) Buchberger, W.; Winsauer, K.; Breitwieser, Ch. Fresenius' 2.Anal. Chem. 1083, 315, 518-520. (15) Hanekamp, H. B.; Bos, P.; Frel, R. W. TrAC 1082, 7, 135-140. (16) Bockris, J. 0. M.; Reddy, A. K. N. Modern Nectrochemistfy; Plenum: New York, 1970; Voi. 2, Chapter 8. (17) Reagent Chemicals, 6th ed.; American Chemical Society Specifications, ACS Publications: Washington, DC, 1981.
RECEIVED for review May 19,1986. Accepted August 28,1986.
Properties and Applications of the Concentration Gradient Sensor to Detection of Flowing Samples Janusz Pawliszyn
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300
A concentratlon gradlent sensor based on light beam deflection measurement (Schlieren optics) has been used to analyze samples InJectedInto flowing streams. This detector In Its nonselective mode of operation responds llnearly to an Increase In sample concentration. Its linear dynamic range extends from about 0.1 M to 5 X lod M sucrose (n = 10-s/lOd R I units) which Is the concentration detectlon ilmR for a slngie injection experlment. This iimlt can be decreased by an order of magnitude via muitlpie Injectlons and a lock-In detectlon technlque. The concentration gradlent sensor had a detectlon volume In the nanollter range, whlch resulted In the detectlon of a few picrograms of sucrose in a single injection experhnent. The concentration gradient sensor Is able to effectively distinguish between broad and sharp peaks. The enhancement In sensitivity of this technlque can be accomplished by expandlng the effluent from the capillary coiumn Into the larger diameter sample cell. Broadening of the peak can be prevented by the sheath flow method. A slmple optical arrangement In such a sensor allows for a convenlent and inexpensive design. Application of this technique to flow InJectkn and chromatographic eluant analysis is demonstrated and dlscussed.
Recent developmental trends in flow injection analysis and liquid chromatography have led to more efficient, small-dim e t e r chromatographic columns ( I , 2) and miniaturization of flow systems (3). These advancements stimulate search
for low-volume, highly sensitive detection methods ( 4 ) . One way this task can be accomplished is by scaling down known techniques; however, this is not always successful. Another approach is to develop new methods specifically suited for small-volume systems. Recently such a small-volume detection technique based on observations of concentration gradients has been proposed (5). The gradients are generated during the injection of the sample into the solvent. The magnitude of these gradients depends on many factors including environmental conditions and experimental design. The ability to maintain a concentration gradient during transport of the sample toward the detector depends on the internal diameter of the tubes used. For smaller tube diameters less loss in the gradient magnitude occurs (6). Therefore, the method of detection described here is specifically designed for the microflow systems. The concentration gradient of the sample in the solvent produced during injection creates a refractive index gradient. The magnitude of this gradient can be measured by allowing a nonadsorbed light beam (probe beam) to pass through the detector volume. The physical reason for light deflection when passing through this gradient lies in the relationship between the refractive index and light propagation velocity. Different parts of the light advance to a different degree with time, which generates the phase shift. This results in a tilted light beam path illustrated in Figure l a (Schlieren Optics (7)). The light path through the concentration gradient can be calculated by using the Fermat principle (the light path through the medium is such that the time necessary for its traversal is minimum). The relationship between the angle of deflec-
0003-2700/86/0358-3207$01.50/0 0 1986 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986 t
tCd t
a.
-r/
I
'\
I
_ _ -_--
u
L- .
b.
-J
Figure 1. Principle of the concentration gradient detector operation: (a) tilting of the light waveform propagating through the medium with inhomogenous refractive index and (b) propagation of the light beam
through the detector cell without the solute present (solid lines) and through the concentration gradient produced by the eluting peak (dashed lines). tion, 0, and the refractive index gradient normal to the light propagation and path length, d , can be written as (8)
d dn tan 0 = sinh - - = n dx
n dr where n is the refractive index of the medium. For most of the practical cases (d and 0 are small) we can approximate:
d dn
$ = --
(1) n dx Figure 1 b illustrates the detection principle behind this method. The solute plug or peak that is passing through the cell generates the concentration gradient due to the nonuniform distribution of this solute in the detector. This gradient forms the corresponding refractive index gradient dn/dx = (dn/dc)(dc/dx), which then tilts the propagating light beam. The information produced during this measurement relates to the universal property of the solute-refractive index n. Consequently, the concentration gradient produced by any solute that has a different n than the solvent will be detected by this method. One of the advantages of this method compared t o other nonselective techniques is that it can simultaneously provide selective spectroscopic information about the sample when an additional excitation beam is used (5,9). This paper describes simple designs of the concentration gradient sensor specifically suited for flowing sample analysis. The properties of this technique are investigated experimentally. Based on its nonselective mode of operation, potential applications of such a sensor to the detection of flowing samples are discussed.
EXPERIMENTAL SECTION In most of the experiments distilled water was used as a solvent. The solvent was degassed by filtering through 0.2-pm pore size cellulose nitrate membranes (Whatman Limited, Maidstone, England). A similar procedure was followed for the sample solution. Special care was taken to thermally equilibrate both
sample and solvent to room temperature before their use. Most experiments used sucrose as a solute (ACS Analytical Reagent Grade, Mallinckrodt, Inc., St. Louis, MO). Solutions in the O.l-lOW3 M range were prepared by dissolving appropriate amounts of sucrose in 500 mL of distilled water. Lower concentrations were obtained by the dilution of 100 mL of the M sucrose solution in appropriate amounts of distilled water. The concentrations of diluted solutions were monitored by the refractive index detector (Model R400 Waters Associates, Milford, MA). A detection limit established for the refractive index detector was 2X M (10). The dynamic range and detection limit for the concentration gradient sensor were determined first by decreasing the concentration of sucrose and then by increasing the concentration starting with distilled water as a sample. Figure 2 shows the experimental arrangement used in some of the investigations. In most cases a piece of square capillary tube (Wale Apparatus Co., Hellertown, PA) was used as a detector cell. The inside diameters of these cells were 1,0.3, and 0.2 mm. Light-emitting diodes (LEDs) coupled to multimode optical fiber (50 pm, graded index) (IRE 160 FB, Laser Diode Laboratories, Inc., New Brunswick, NJ) were used as probe beam sources. The end of the fiber was cut using a fiber breaker (Model F-BK1, Newport Corp., Fountain Valley, CA). The probe beam was oriented perpendicular to the capillary wall. In most of the experiments a PIN-SPOT/BD silicon detector together with a Model 301 DIV signal-conditioning amplifier (United Detector Technology, Hawthorne, CA) was used as the probe beam position sensor. The detector was equipped with a RT-830 color glass filter (Hoya Optics, Inc., Frement, CA) or an 830-nm interference filter (Ealing, South Natick, MA). The light source, detector cell, and position sensor were placed on the optical vibration-isolated table (Micro-g, Technical Manufacturing Corp., Peabody, MA). The sample was introduced in the vicinity of the probe beam by a small inside diameter (250 pm, 50 pm, 10 pm) fused silica capillary (Spectran Corp., Sturbridge, MA). The solvent was introduced to the detector cell by using microtubing made of Teflon fluorocarbon resin (Cole-Parmer, Wellesley, MA). The injection of the sample was performed by use of an ac 10-W switching valve (Valco Instruments Co., Inc., Houston, TX) equipped with a high-speed switching accessory kit and digital valve interface (DVI) (11). Small volumes of samples were injected by using "moving injection technique" (12). To ensure repeatability of the injections, the valve was operated using a sweep generator (Model 184, Wavetek, Inc., San Diego, CA) in its triggered mode. Flow of the solvent and the sample was generated by use of two syringe pumps (Model 249-2, Sage Instruments, Inc., White Plains, NY)equipped with syringes of various volumes (Hamilton, Reno, NV) and low-volume filters (ZUFI, Valco Instruments Co.). The fused silica capillary was fitted into the valve by use of a syringe adapter (VISF, Valco) and sealed with a piece of microtubing made of Teflon. This experimental setup was used in two major types of experiments. In one, the sample was injected at the beginning of the fused silica capillary and then was carried to the detector cell by the solvent flow. A t the same time the sheath flow of the solvent was generated in the Teflon tube. The sample was then allowed to expand in the detector cell. In the other experiment the fused silica capillary was filled with the sample solution. The solvent flow was halted, and the sample was then injected into the detector cell directly from the fused silica capillary and then carried to the probe beam by the solvent flow originating from the Teflon tube. In the chromatographic experiments, Waters liquid chromatograph equipped with a refractive index detector and a P-Bondapack CIBcolumn was used. Water was used as a mobile phase with a flow rate of 1 mL/min. About 0.1% of the effluent was split using a T connection and directed toward the concentration gradient sensor. This allowed detection by using the sensor and a commercially available detector simultaneously. A lock-in amplifier (5208, Princeton Applied Research, Princeton, NJ) equipped with a low-band adaptor was used to detect modulated signals produced by the multiple injections. The modulation frequency varied from 0.5 to 3 Hz. The signal was recorded on a Sargent-Welch recorder (Model XKR, Skokie, IL). Figure 3 shows the design of the concentration gradient sensor using fiber optic technology. The probe beam was produced by
ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
SAMPLE
Figure 2. Schematic of the experimental configuration. a light-emitting diode coupled onto a 50-wm optical fiber (Model 160 IRE, Laser Diode, Inc.). Similarly the silicon detector (FO02-400, United Detector Technology) was coupled with a 200-wm core optical fiber. Both optical fibers were fitted into opposite sides of a cross connection (ZXlC, Valco Instruments Co., Inc.) with the help of two pieces of tubing and ferrules. The flow of the sample and solvent occurred through the other two sides of the cross connector. The electronic circuit used is shown in Figure 3b. The simulated chromatogram was generated on the IBMcompatible computer (Leading Edge) using ASYST scientific software (Macmillan Software, New York, NY). The chromatogram was calculated by adding the first derivatives of the Gaussians (5) (Figure loa). Then the Gaussian white noise was added to this chromatogram (Figure lob), and the f i t and second integral of the resulted function were calculated (Figure 10c,d). The programs to generate the noise and integrals are standard features of the ASYST.
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This period allows extra broadening of the negative slope of the peak, which results in the concentration gradient loss. An additional loss can be created by extra-column effects related to the injection device and detector flow cell. Also, the parabolic concentration profile (13) across the diameter of the tube might affect the performance of the sensor, since measurement is of the average concentration gradient which exists in the detection volume. This effect should depend on the diameter of the capillaries used to transport the sample and on the flow rates. However, no relationship was observed between the ratio of the peak heights corresponding to positive and negative parts of the signal and these parameters for linear flow rates below 2 cm/s and capillary diameters below 250 wm. In fact, this value corresponds closely to the ratio of the appropriate peak widths as follows:
RESULTS AND DISCUSSION Figure 4a presents the usual response of the concentration gradient sensor to the peak generated during injection of the sample into the flowing stream of the solvent. Figure 4b shows ita numerical integral. During transport of the sample through the tube, the concentration profile undergoes diffusive and kinetic broadening, and in the ideal case it resembles the Gaussian curve. Detection of the solute by the concentration gradient sensor should result in the Gaussian derivative (see Figure l b in ref 5). However, as Figure 4a shows the signal observed is not symmetric. One of the reasons for this observation can be related to the time lag between detection of the positive and negative slopes of the concentration profile.
where h, w ,and u represent height, width, and standard deviation of the positive (+) or negative (-) part of the derivative. This could be expected since the magnitude of the signal in the concentration gradient method is related to ( l / ~( 5)) .~ Consequently, any broadening of the peak can be easily detected by this method in comparison to the magnitude of concentration techniques where the peak magnitude is proportional to l / u . For example, it is difficult to distinguish the numerical integral (Figure Ib) of the deflection signal from the "ideal" Gaussian. On the other hand, the ratio h+/h-in Figure 4a can effectively describe the extent of peak broadening. It should be pointed out that the total width of the
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1966
a
b
Flgure 3. Example of the concentration gradient detector design using LED and silicon detectors coupled to optical fibers ffed into a chromatographic cross connection: (a) geometrical configuration and (b) electronic circuit.
signal in the gradient technique is about 6 standard deviations while in the concentration method it is about 4 (see Figure 1 in ref 5). This fact can be easily noticed on Figure 4. The relative signs of the first and the second peak in the concentration gradient signal are related to the orientation of the position detector as well as the relative magnitude of the refractive indexes of the solvent and the solute. For example, methanol solution injected to water produces an inverted derivative compared to sucrose used as a solute. The quadratic relationship between the amplitude of the concentration gradient signal and 1/0 indicates that sensitivity of the sensor can be significantly increased by optimizing the solvent flow or by using shorter connections between the injector and detector. To illustrate this relationship, a sample was injected into various lengths of fused silica tubing (Table I). In these experiments the standard deviations, u+ and uof the peak were calculated as a distance between positive and megative optima from the t , in the vertical direction. The
Table I. Observed h +, h -,u+, and u- Values for Detection of Peaks Generated during 0.5-pL Injection of lo-* M Sucrose Solution into Various Lengths of the 250-pm-i.d. Capillary“
L,cm 400 200 100 60
U&d,
20 14 10 8
s
h+,mm h-, mm 6 12 29 45
4 8
20 31
(h+)il [ ( u + ) l k u+, s
27 18 12 10
0-,
s
30 20 15 13
@+)I
1 2.0 4.8 7.5
(u+)i]
1 2.3 5.1 7.3
“See Figure 4; detector cell diameter, 1 mm; no sheath flow (u.Iu.. = 0):solvent flow rate. 50 rtL/min. agreement between the theoretical relationship ( A a l / d and the experimental data is excellent. Figure 5 clearly indicates that by decreasing the peak width by a factor close to 3, the signal-to-noise ratio is improved by about an order of m a g nitude.
ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
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n a
b b
Figure 5. Concentration gradient signal corresponding to injection of 0.5 pL of lo-’ M sucrose into (a) 4 m and (b) 0.6 m of 0.25-mm4.d. capillary (see Table I): no sheath flow (u,/u, = 0);capillary flow rate, 50 WLlmin; detector cell i.d., 1 mm.
Figure 4. (a) Signal produced by concentration gradient detector after injection of 50 nL of M sucrose solution into 2 m of a 50-pm-i.d. capillary: sheath flow, u,Iu, = 0.5. (b)The signal’s numerical Integral: t , = 12.6 min; u+ = 18 s; a- = 21 s; h+/h.. = 1.4; detector cell diameter, 300 pm; flow rate, 0.4 pL/mln.
It can be noted, however, that the theoretical standard deviation calculated for the same experimental conditions using the Golay equation (6) and the expression N = ( t , / u ) 2 is about two-thirds of the corresponding reported value. In these experiments the expansion of the effluent was from a 250-pm4.d. capillary into a 1-mm-squaredetector cell. Clearly, significant peak broadening occurs during this process. This conclusion was confirmed by “on-column” detection (the probe beam was passing directly through the capillary). Theoretical standard deviations within an experimental error were obtained for this experimental arrangement. Expansion of the effluent, however, leads to significant enhancement in sensitivity. An improvement factor of (dD/dcI3is expected, where dDis the diameter of the detector cell and dc the diameter of the capillary, due to concentration gradient increase and longer probe beam path length (5). Using a 250-pm capillary and 1-mm detector cell, one can expect a 64 times increase in sensitivity. Only about half of this enhancement was observed experimentally. The difference is due to the peak broadening effect during which the significant loss of the concentration gradient occurred (sensitivity is proportional to l/a2). In order to prevent this destructive process, the sheath flow
technique was used. This method was applied extensively to narrow the flowing streams without disturbing their flow pattern (14-16). Recently this method was used in one of the designs of the fluorescence detector for capillary chromatography (17). In the experiments reported here instead of narrowing the flowing stream coming from the capillary column the effluent is expanded into the larger cell. The sheath flow that surrounds the capillary effluent prevents mixing during stream expansion. The design of the sheath flow expansion cell is shown in Figure 2. The solvent that generates the sheath is supplied directly into the Teflon tubing, while the sample is injected into the small inside diameter capillary using a valve and is then carried into the detector by the solvent flow. When the ratio of the linear sheath, us,and capillary, uc,flows is 1, no expansion or narrowing of the capillary stream occurs. Excellent preservation of the concentration gradients eluting from the capillary was observed for these flow conditions. With decrease of the u,/uc value, expansion of the effluent occurs. It was found that by using ratios down to 0.3, no significant peak broadening was observed (within 10% of theoretical value) for 250-pm-i.d. capillaries. In this case, smaller effluent expansion occurred (dD/dc= 2.5) compared to dD/dc = 4 for the us/uc= 0. Therefore, the sensitivity of the concentration gradient measurement is still larger for the no sheath flow (us = 0), even with the peak broadening effect, compared to u,/uc = 0.3. This is due to the triple-power relationship between
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sensitivity enhancement and the ratio of detector and capillary diameters. The situation changes somewhat when the diameter of the capillary becomes smaller and d,/dc becomes larger. For example, expanding the effluent from a 50-pm capillary into a 300-pm cell broadens the peak about 2 times. The sheath flow method does not work as effectively for this capillary as for a 250-pm-i.d. capillary due to the relatively thick capillary walls (17). Separation of capillary and sheath flows introduces mixing in the capillary flow for small u,/u, ratios. In order to prevent peak broadening, uJu, must be close to 0.5 ( d D / d c 2 ) . To improve this situation, sharpening of the capillary end is required. A detailed description of the sheath flow expansion method will be provided elsewhere (18). Table I and Figure 5 clearly indicate that the concentration gradient method can be used effectively to distinguish between broad and narrow peaks. This can be a very useful property, since slow refractive index variation due to gradient elution or temperature change might exist in real flowing systems such as chromatography or electrophoresis. Figure 5a shows the recorder trace obtained for a low-volume refractive index detection, while Figure 5b shows the simultaneous, samevolume concentration gradient signal produced during injection of the sample into the waterlethano1 solvent gradient. The experiment was performed by passing the probe beam through the 300-pm-i.d. square capillary core a t a 30” angle with respect to the wall’s plane (19). The deflection parallel to the flow direction provided information about the concentration gradient (refractive index gradient), while the deflection in the perpendicular plane corresponded to the magnitude of the sample concentration (refractive index). The position sensor employed allowed simultaneous measurement of the light deflection in both directions by recoding the change in the position and the sum outputs at the same time. Figure 6 clearly indicates that the concentration gradient sensor does not “see” base-line drift related to changing solvent composition but exhibits high sensitivity toward sharp peaks. Also, it can be noted that the concentration signal and its derivative clearly correspond to each other. Table I and Figure 5 indicate that a decrease in the length of the fused silica capillary will produce higher sensitivities and lower detection limits. Clearly, an ultimate detection limit must exist. The most favorable situation is when the probe beam diameter, w , is approximately equal to 2u of the injected or expanded peak. In that case almost full concentration change occurs within w. Simplifying the calculation, we can assume a triangular peak as follows: dnldx = A n / w , where An = n (sample) - n (solvent). Therefore, by use of eq 1 we can write 8 = ( d / n )dnldx = n d j w n and nmin.= wnO,,,,/d. It should be remembered that w is not only the width of the probe beam but that it also corresponds to 2u, and in any practical system 2u is a t best close to the diameter of the column. For the purpose of this calculation we can assume that n = 1,Omin, = rad (20,21),and d = w. Consequently, the smallest refractive index difference between solvent and the sample that can be detected by this method is about lo-’. In this simple calculation two important assumptions were made. Firstly, the solute peak or sample plug corresponds to twice the detector diameter. Although it is possible to inject such “sharp” plugs directly into the detector cell (see below), the refocusing of the dispersed peak to that level might be an impossible task. In these cases, the sensitivity of this method will be lower than predicted theoretically. Therefore, the concentration gradient detection scheme will more likely find an application in small-diameter flow systems due to low dispersion during the transport of the sample. Secondly, in our calculations the diameter of the detector, d , was made similar to the probe beam width, w. In most of the cases,
-
b
Figure 6. Response of the refractive index detector (a) and samevolume concentration gradient detector (b) for injection of 50 nL of sucrose into 2 m of 50-pm-i.d. capillary: no sheath flow (u,/u, = 0); detector cell i.d., 300 pm.
however, the beam width is substantially smaller than the cell dimension. For example, in most of our experiments the cell inside diameter was 300 pm while the probe beam diameter was about 100 pm. This fact should improve the sensitivity by a factor close to 3 when assuming the Gaussian rather than triangular peaks shapes. The refractive index of a sample-solvent mixture can be calculated by adding the specific refraction r = ( n - l)/clo of the components, where n is a refractive index and do is density (22). If we assume that the densities of the solvent and the solute are similar, then the change of the refractive index of the mobile phase, n, due to solute present can be expressed as
where M , is a molecular weight of the solute, c concentration of the solute, n, and n2 refractive index of the solute and
ANALYTICAL CHEMISTRY, VOL. 58,
NO. 14, DECEMBER 1986 3213
13
30
r---
r-
r r
Figure 7. Series of 8 nL of lo-' M sucrose solution injections into 300-pm4.d. detector cell: solvent flow rate, 0.4 pL/min.
mobile phase, respectively, and m~ the mass of 1L of solution. In most cases we can assume M,= 100 g/mol and nl - n2 = 0.1 (23). Therefore, the minimum detected concentration of the sample can be approximated to be about lo4 M. It should be emphasized that this concentration detection limit corresponds to the ideal situation when a very narrow peak is produced in the detector cell (d = 2a). For broader peaks the loss of sensitivity will be proportional to a' increase. For example, for peaks having a width of about 20d a detection M is expected. limit of only In order to investigate practical detection limits close to theoretical predictions, direct injection of the sample from a microcapillary close to the probe beam was applied. The experimental design was similar to the sheath flow experiment (Figure 2). The 50-pm capillary was filled with sample solution while the solvent was supplied by the Teflon tube. Small volumes of sample were injected into the detector cell from the capillary placed about 1 mm below the probe beam, while the solvent carried it into the detector volume. Figure 7 presents the series of 8-nL injections of lo4 M sucrose solution. The relative standard deviation of peak magnitude in such a flow injection system was below 1%. A 5 X lo4 M concentration of sucrose produced a signal that was 3 times that of the noise. This value is considered as a detection limit for single injection experiments. This results in nmin,= 5 x lo4, which is about an order of magnitude better than for most refractive index detectors (IO). This limit can be decreased even further when lock-in detection is used. With a 3-Hz injection frequency and 3-s time constant, this concentration detection limit was close to 6 X lo-' M. For the slow solvent flows (
