Anal. Chem. 1087, 59, 1452-1457
1452
flection. Few solid samples are rugged enough to remain undamaged by tightly focused laser pulses. Many samples are damaged by exposure to tightly focused CW radiation. Hadamard encoding may extend the range of microscopy with other generally useful laser spectroscopies such as Raman spectroscopy. Experiments toward this goal are in progress.
LITERATURE CITED
2 W
ZUQ
2750
2SM
2-
MM
5lM
asw
MM
SIONAL INTENSIW (AQC u u n h )
I# 17 I#
-
” ,, 7 /
B
m n
zsm
ass
2150
USQ
SIWAL INXEYIIW (ADC u u n h )
Flgure 6. (A) Distribution of data shown in Figure 3A; (B)Simulatlon of the distribution of the data taken from a sample approximating the configuration of the sample of the Figure 3A.
The major advantage of Hadamard encoding is that it allows acquisition of moderate to high spatial resolution spectroscopic information from an unfocused laser. The need to distribute laser power is not limited to transverse photothermal de-
(1) Morris, M. D.; Peck, K. Anal. Chem. 1988, 5 8 , 811A-822A. (2) Low, M. J. D.; Morterra. C. Appl. Spectrosc. 1984, 38, 807-812. (3) Coilette, T. W.; Parekh, N. J.; Griffin, J. H.; Carreira, L. A,; Rogers, L. B. Appl. Spectrosc. 1988, 40, 164-169. (4) Peck, K.; Fotiou, F. K.; Morris, M. D. Anal. Chem. 1985, 5 7 , 1359- 1362. ( 5 ) Wetsei, G. C.; McDonald, F. A. Appl. fhys. Left. 1982, 47, 926-928. (6) Lepoutre, F.; Fournier, D.; Boccara, A. C. J. Appl. fhys. 1985, 5 7 , 1009- 1015. (7) Benchikh, 0.; Fournier, D.; Boccara, A. C.; Teixeira, J. J . Phys. 1985, 4 , 727-731. (8) Jackson, W. B.; Amer, N. M.; Boccara, A. C.; Fournier, D. Appl. Opt. 1981, 20, 1333-1343. (9) Murphy, J. C.; Aamodt, L. C. J . Appl. fhys. 1980, 57, 4580-4588. (10) Coufal, H.; Moiler, U.; Schneider, S. Appl. Opt. 1982, 27. 116-120. (11) Coufal, H.; Moiler, U.; Schneider, S. Appl. Opt. 1982, 27, 2339-2343. (12) Fournier, D.; Lepoutre, F.; Boccara, A. C. J . fhys. Colloq. 1983, C6, 479-482. (13) Fotiou, F. K.; Morris, M. D. Appl. Spectrosc. 1988, 40, 704-706. (14) Fotiou, F. K.; Morris, M. D. Anal. Chem. 1987, 59, 185-189. (15) Harwit, M.; Sloane, N. J. A. Hadamard Transform Optics; Academic: New York, 1980. (16) Habibi, A,; Robinson, G. S. Computer 1974, 7 , 22-34. (17) Pratt, W. K.; Kane, J.; Andrews, H. C. R o c . I€€€ 1989, 5 7 , 58-68. (18) Pratt, W. K. IEEE Trans. Comrnun. Techno/. 1971, 79, 980-992. (19) Crowther, W. R.; Rader, C. M. Proc. I€€€, 1986, 5 4 , 1594-1595. (20) Goodman, J. W. Introduction to Fourler Optics; McGraw-Hili: New York, 1968. (21) Levi, L. Applied Optlcs; Wiley: New York, 1968. (22) Goodman, J. W. Stastistical Optics; Wiiey: New York, 1985; pp 36 1-688.
RECEIVED for review September 19, 1986. Resubmitted January 12,1987. Accepted February 11,1987. This work was supported in part by Grant GM37006 from the Public Health Service and in part by Grant CHE-8317861 from the National Science Foundation.
Peroxyoxalate Chemiluminescence Detection with Capillary Liquid Chromatography Andrew J. Weber and Mary Lynn Grayeski*
Department of Chemistry, Seton Hall University, South Orange, New Jersey 07079
Peroxyoxalatechemlhmlnescence Is evaluated In a detectkn mode for packed captllary llquid chromatography. Relatively large volume flow cells (>1 ML) based on a sheathlng flow of chemlkmkrescent reagents around the column eftluent are evaluated In terms of sensltlvlty and band broadenlng. Because the postcolumn reagent flow ls large In proportlon to the total flow, the effluent condltlons have relatlvely llttle effect on the chemiluminescentslgnal over a wide range of organlc/aqueous solvent compos#lons. Detedon lhrlto In the femtomole range are posslble for certaln fluorophors.
The advantages of capillary liquid chromatography, including the potential for greater resolving power as a result 0003-2700/87/0359-1452$01.50/0
of increased column lengths and reduced solvent consumption, have recently been described in the literature (1-5). However, utilization of such columns requires the use of extremely small injection volumes and reduced detector cell volumes to reduce the contribution of extracolumn dead volumes. Fluorescence and UV detectors using submicroliter cell volumes (6-8) or on column detection (8-10) have been reported. Electrochemical techniques with reduced cell volumes have also been applied (11,12).However, these schemes require extremely small detection volumes with potential loss of sensitivity. Peroxyoxalate chemiluminescence (CL)has been demonstrated to be a sensitive and selective technique for the detection of suitable fluorophors in conventional (13-15) and microbore high-performance liquid chromatography (HPLC) (16). The increased sensitivity of CL over conventional @ 1987 American Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 59, NO. 10, MAY 15, 1987
1453
fluorescence detection can be explained in terms of the chemical excitation of the fluorophor. The postcolumn reaction of an oxalate ester, typically bis(2,4,6-trichlorophenyl) oxalate or bis(2,4-dinitrophenyl) oxalate, with hydrogen peroxide, in the presence of a base, produces the proposed high-energy intermediate 1,2-dioxetanedione. This intermediate forms an encounter complex with the fluorophor in which an electron is transferred from the ground-state fluorophor to the intermediate. Dissociation of the complex, followed by a charge annihilation process results in population of the first excited singlet state of the fluorophor. The emission of light is the result of the subsequent decay of the excited fluorophor to the ground state as in conventional photolytic excitation. One advantage of using chemical excitation is increased sensistivity, which is obtainable due to the reduced background since extraneous radiation associated with photolytic excitation, i.e. Rayleigh and Raman scattering, and source instabilities are eliminated (I7). With chemical excitation, sensitivity can be further increased by using larger volume cells, without increasing peak width (16, 18). This report demonstrates the applicability of CL detection to packed capillary liquid chromatography. The effect of flow cell volume on signal intensity and peak width as a measure of extracolumn band broadening has been investigated. The effect of the nature of the reagents and flow rates will be described, and the instrumental contribution to the noise will be discussed.
EXPERIMENTAL SECTION Chemicals. All solvents were HPLC grade from Fisher Scientific (Fair Lawn, NJ). Perylene and 2-aminoanthracene were purchased from Aldrich Chemical Co. (Milwakee, WI) and were used without further purification. Stock solutions were prepared at 1.0 mM in 4/1 methanol/ethyl acetate and at 0.8 mM in methanol, respectively. Two stock solutions of 7-(diethylamino)-4-methylcoumarin (DEAMC), obtained from Eastman Kodak (Rochester, NY) and used as received, were prepared in methanol at 29 mM and 6.1 mM. The chromatographic eluent was a 4/1 mixture of methanol and citrate buffer. Sodium citrate, tribasic, (Sigma Chemical Co., St. Louis, MO) was prepared at 1.7 g/L dissolved in water and adjusted to pH 7.8 with 50% HC1 prior to the addition of methanol. The mobile phase was delivered at 5 pL/min. All solutions were filtered through 0.45-pm filters prior to use (Millipore Corp., Milford, MA). Bis(2,4,64richlorophenyl) oxalate (TCPO) and bis(2,4-dinitrophenyl) oxalate (DNPO) were donated by A. Mohan and recrystallized from ethyl acetate. Stock solutions (5 mM) were prepared daily, as needed, in ethyl acetate. Hydrogen peroxide solutions were prepared by appropriate dilution of 70% aqueous stock (Du Pont, Wilmington, DE) to yield 1.2 M working solutions in either methanol or 9/1 methanol/5.9 mM citrate buffer, pH 7.8. HPLC and Detection System. The HPLC system consisted of a Gilson Model 302 pump (Villers-le-Bell,France) equipped with a 5.S liquid end capable of flow rates from 5 ML/min to 5 mL/min. Pump pulsations were reduced by using a Gilson Model 802 manometric module. Sample introduction was performed by using a Valco (Houston, TX) Model C-14-W injection valve with a fixed 60-nL injection volume. Columns were connected to the valve by using a Vespel ferrule (Alltech Associates, State College, PA) and a Valco compression nut, tightened to withstand pressures of 4500 psi. The detector was a Kratos (Ramsey, NJ) Model 950 filter fluorometer. The low-volume flow cells connected to the capillary column were modifications of the previously described microbore cell (16) (Figure la, MV1, and b, MV2). The cell core was made from a 20 WL“Microcap” (Drummond Scientific, Brommall, PA). Solutions of ester and hydrogen peroxide were delivered postcolumn in 1:1flow-rate ratio from 10-mL ground-glass syringes by a Sage (White Plains, NY) Model 351 syringe pump. The reagents were combined by using a standard low-pressure chromatography Kel-F tee, the outlet of which was delivered to the detector mixing tee. The column effluent was delivered to flow-cell
-
//I I
Ira
7
6
”
rtl lh
u YW 2
I
Flgws 1. (a) Capillary flow cell MV1: (1) effluent entry tube; (2)
0.25-mm4.d. X 3-cm PTFE tube; (3) Waters Associates mixing tee: (4) 0.25-mmi.d. X k m PTFE tube, postcolumn reagents inlet; (5)Row cell connecting tube: (6) ’/,&. Swagelok ZDV union; (7) blank off plate to position fitting and seal out light: (8)flow cell core; (9) FS 950 cell holder: (10) waste line (Teflon tube); (11) cell outlet. (b) Caplllary flow cell MV2: (1) packed caplllary column; (2) 0.25-mm4.d. X 5 c m PTFE tube; (3)1/,6-ln. Swagelok ZDV union cut in half to reduce volume; (4) glass wool plug; (5)effluent entry tube: (6) Parker-Hannafln mixing tee: (7) 0.25-mm4.d. X 4-cm PTFE tube, postcolumn reagents inlet; (8) blank off plate to position fitting and seal out light: (9) flow cell body; (10) FS 950 flow cell holder; (11) waste line; (12) cell outlet. Not to scale.
1454
ANALYTICAL CHEMISTRY, VOL. 59, NO. 10, MAY 15, 1987 a
1
2
3
4
b
5
Figure 2. Capillary column connector: (1) packed capillary column: (2) 0.25-mm4.d. X 4-cm PTFE tube; (3)1/,6-in. Swaglok ZDV union; (4) quartz wool plug: (5) effluent entry tube leading to effluent entry tube
pictured in Figure la. MV1 by a 200-pm i.d. X 100-mm length of fused silica tubing (effluent entry tube). The capillary connection consisted of a piece of PTFE tubing (0.25-mm i.d. X 40 mm) into which a small quartz wool plug (0.8-mm length) had been inserted (Figure 2). The packed capillary column (construction described below) and effluent entry tube were inserted into opposite ends of the PTFE tube and pressed tightly against the quartz wool plug. The quartz wool plug served as a frit to retain the packing. This connection was subsequently placed in a l/&n. Swagelok male union to maintain the connection, of which the column side was secured with a Vespel ferrule to facilitate removal. Geometric cell volumes were calculated by measuring the distance between the outlet of the effluent entry tube and the cell outlet. Changes in cell volume could be accomplished by either inserting or withdrawing the effluent entry tube. To prevent leaks of the postcolumn reagents from the mixing tee, the effluent entry tube was passed through a short segment of PTFE, which was connected to the mixing tee in a conventional manner. To further reduce the extracolumn volume, the postcolumn connections were modified (Figure lb, MV2). A Parker-Hannafin mixing tee replaced the detector mixing tee, the flow cell connecting tube, and the ZDV coupler. The flow cell proper was connected directly to this mixing tee. The column side of the capillary connector was secured in a modified ZDV coupler, while the effluent entry tube was maintained in the mixing tee as detailed in Figure lb. Cell volumes were determined as before and changed by changing the length of the effluent entry tube. The length of tubing used with this arrangement was 40 mm. The detector signals were taken from the 10 mV output to a Fisher 5000 Recordall strip chart recorder. The packed capillary columns were constructed by using 200-pm4.d. deactivated fused silica tubing in 1-m lengths obtained from SGE, Inc. (Austin, TX). The columns were packed by using a Haskell Model DHI-300 pneumatic intensifier pump (Burbank, CA) as follows. To an appropriate amount of 5-6-pm Zorbax C-8 (Du Pont Instruments, Wilmington, DE) a sufficient quantity of acetonitrile was added to produce a slurry ratio (microliters of slurry solvent/milligrams packing) between 3.3 and 3.5. This mixture was swirled until homogeneous, and then placed in an ultrasonic bath for 5 min. The empty columns were connected below the slurry reservoir, constructed from a 15-cm X 4.6-mm4.d. conventional LC column blank, by using Vespel ferrules. The packing was retained during the packing procedure by connecting the column outlet to a 1/16-in.Swagelok female union into which a 2-pm frit had previously been inserted. The slurry was added to the reservoir and the empty volume carefully filled with methanol. The system was then sealed. With methanol as a packing solvent, a pressure of 6000psi was applied and maintained until 5 mL of solvent had been collected. The system was shut off and allowed to depressurize completely before the column was removed. Lengths of 20-30 cm were cut from the 1-m packed capillary and used as chromatographic columns. Static Experiments. A Turner Design Model 20e photometer was used to evaluate the effect of the organic content of the mobile phase on the chemiluminescent signal. The photometer was equipped with an injector capable of injecting either 50 or 100 pL into the sample vessel and initiating the measurement of light. Integrals of the area under the intensity-time decay curves were obtained from the internal integration routine of the photometer. Integration was performed for 30 s, during which more than 80% of the total light was emitted for the conditions listed below. All experiments were performed with a solvent system equivalent to a mobile phase to postcolumn ratio of 1:lO. In an 8-mm-0.d. X 5-cm sample vessel, 100 p L of a solution containing 5 mM TCPO and 40 nM analyte, 2-aminoanthracene, was added to 20 uL of
L
m
r--r-l 0
4
8
0
4
8
TIME (min) Flgm 3. Comparison of chemiluminescent slgnal for injection of 60-nL of 29 mM D E A N solution on column obtained by using (a) TCPO and (b) DNPO as postcolumn reagents.
the appropriate mobile phase. The sample vessel was then placed in the sample chamber and was injected with 100 p L of 1 M hydrogen peroxide. Integration was initiated with the final injection.
RESULTS AND DISCUSSION Reagents and Buffer. An important consideration in adapting peroxyoxalate CL detection to HPLC is the solvent composition of the mobile phase in proportion to the postcolumn CL reagents so that the solvents needed for separation are compatible with those needed for efficient CL. In conventional reverse-phase HPLC/CL applications, the chromatographic eluent typically constitutes up to 40% of the total flow (13-15, 17-19) and the influence of the aqueous content of the mobile phase must be considered in the selection of suitable postcolumn solvents because the commonly used oxalates TCPO and DNPO are most stable and efficient in organic solvents, which are usually immiscible with the aqueous mobile phase, thereby requiring the use of a cosolvent to improve their water miscibility. Although the contribution of the chromatographic eluent in capillary LC/CL applications is expected to be low, as in microbore LC/CL (16), these effects cannot be entirely neglected. The initial solvent and pH conditions were chosen based on previous HPLC/CL work. Ethyl acetate was selected as the solvent for the oxalate esters TCPO and DNPO, hydrogen peroxide was dissolved in methanol as the cosoivent, and the mobile phase was buffered at pH 7.8 to catalyze the chemiluminescent reaction. Although previous reports have used ester to peroxide ratios of 1:2 (13, 15,17),results in our laboratory obtained by using simplex optimization procedures indicate maximum signal intensity at ratios of 1:l. Therefore, the postcolumn reagents, TCPO and hydrogen peroxide, were delivered at a combined flow rate of 300 pL/min in a 1:l ratio into a 2.2-pL flow cell with holder MV1 (Figure la). The initial 60-nL injections of 29 mM solution of DEAMC did not yield any detectable signal because the contribution of the mobile-phase buffer was too small relative to the postcolumn reagents to catalyze the reaction. T o increase the buffer concentration, the hydrogen peroxide was prepared in a 9/1 methanol/5.9 mM citrate buffer (pH 7.8) making the final solvent composition 49.2% ethyl acetate, 45.0% methanol, 5.8% buffer (approximately 6 times more than originally used). Subsequent injections of DEAMC in this system resulted in a signal-to-noise ratio of 110 (Figure 3a).
ANALYTICAL CHEMISTRY, VOL. 59, NO. 10, MAY 15, 1987
1455
Table I. Influence of Flow-Cell Volume on Signal Intensity and Peak Widtha
V,,,
pL
N
relhtens, AU
CV
Wlp, s
CV
6
0.051 0.149 0.184
30.3 12.8 5.6
38.4 32.2 28.8
18.7 5.7 4.6
0.47 1.25
16
2.18
6
a See Experimental Section for chromatographic conditions: postcolumn flow rate, 50 pL/min; TCPO, 5.4 mM; H2O2,1.2 M in 9/1 CH30H/5.9 mM citrate buffer (pH 7.8); test solute, DEAMC 85 ng on column (6.1 mM); PMT, 0.75 kV; time constant, 6 s. Cell holder MV1.
This modification in experimental design with respect to buffer addition suggests that because the proportion of mobile-phase contribution to total flow rate is so small (less than 2% in this example), the effect of the mobile-phase composition has little effect on the chemiluminescent signal. If this is the case, one of the problems associated with adapting CL detection to conventional LC may be eliminated. In the earlier reports of these applications, mobile-phase conditions were modified so that they were compatible with optimum postcolumn chemiluminescent reaction conditions. With capillary LC, it may be possible to optimize separation and postcolumn conditions independently because of the low mobile-phase contribution to the total system flow. To investigate this effect, static experiments evaluating the effect of different organic/aqueous ratios on the chemiluminescent signal were conducted after postcolumn flow rate studies were evaluated. According to results obtained by Rauhut and co-workers (20) and in our laboratory (21),TCPO and DNPO produced high CL efficiencies when compared to other aryl oxalates. Therefore, DNPO was compared to TCPO in the capillary LC postcolumn detection system. The use of DNPO resulted in peak heights 75% as high as the signal intensity generated by TCPO and a 3.3-fold decrease in signal-to-noise ratio was observed (Figure 3b). The difference in signals for TCPO and DNPO can be explained in terms of kinetics and flow rates. Because the ester and peroxide react prior to entering the flow cell, decomposition and the occurrence of dark side reactions tend to occur to a greater extent with faster reactions as is the case with DNPO. Therefore at flow rates slow enough for the signal to be observed in the flow cell under the given conditions, too much decomposition of the DNPO occurs prior to the flow cell resulting in reduced signal. Increasing the flow rate to compensate for the increased reaction rate results in removal of the analyte from the flow cell at a much faster rate, thereby offsetting the desired effect. These losses are not as severe for TCPO since it is a much slower reacting oxalate than DNPO (21). Based on these results, TCPO was utilized in the investigation of flow-cell volume and the evaluation of postcolumn band broadening. Flow Cell. A . Effect on Signal Intensity. The cell volume is a particular concern in capillary chromatographic techniques because of the very low volume of the eluted peaks. The use of conventional flow cells for absorbance measurements may result in loss of chromatographic resolution and reduced sensitivity as a consequence of the logarithmic dilution of the analyte in the flow cell. As previously described in the application of CL detection in microbore LC (16),this technique is attractive for capillary LC because it requires the addition of make-up flow. However, in contrast to the microbore application previously reported, the design of the flow cell differs in that the chemiluminescent reagents act as a sheathing flow, thereby reducing the dispersion in the relatively large volume cells (22,23). To investigate the effect of cell volume on signal intensity (Table I), three cell volumes were obtained by either inserting
0
200
400
POST C O L U M N FLOW RATE
I
0
(uL/min)
Figure 4. Effect of postcolumn Row rate on chemiluminescent Intensity: 0, 0.47 FL cell volume; 0 , 2.18 I.LL cell volume. or withdrawing the effluent entry tube in the flow-cell proper as described in the Experimental Section. A considerable gain in intensity is obtained by increasing the cell volume. This increased intensity is attributed to the longer residence times afforded by the larger volume cell, resulting in the measurement of a larger fraction of photons emitted during the course of the reaction. Since in the capillary detection system, the solute, and the chemiluminescent reagents are mixed in the flow cell, a second advantage provided by the increased volume cell is the improved diffusional mixing as illustrated by the reduction in the coefficient of variation (CV), increasing the intensity and improving the signal-to-noise ratio (Table I). Postcolumn Flow Rates. The effect of the postcolumn flow rate on signal intensity was examined by using cell volumes of 0.47 and 2.18 pL. The signal decreased with increasing flow rate (Figure 4). This effect may be attributed to the removal of the solute from the cell at a much faster rate (shorter residence time). Postcolumn flow rates lower than 50 pL/min could not be readily achieved due to equipment limitations.
Effect of Percent Organic Solvent on Chemiluminescent Signal. With a postcolumn flow rate of 50 pL the contribution of the mobile phase to the solvent mixture in the detector cell is relatively small (less than 10%). To determine the effect of variations in the mobile phase on the chemiluminescent signal, static experiments were conducted in which solvent systems were evaluated in the same proportions as would be found in the detector cell (see Experimental Section). On the basis of a mobile phase/postcolumn solvent mixture of 1/10, various compositions from 20 and 80% of acetonitrile or methanol with water or buffer were used to generate a chemiluminescent signal (Table 11). (100% organic was also evaluated but mixing in the static system was incomplete for this system because of the way the reagents were added. These values were therefore not included in the analysis.) At the 95% confidence level statistical analysis of the results show no significant difference in chemiluminescent intensity for percentage or nature (acetonitrile vs. methanol) of organic content. It should be noted that the presence of the buffer also did not affect the signal. Variations in pH were not evaluated
ANALYTICAL CHEMISTRY, VOL. 59, NO. 10, MAY 15, 1987
1456
reported range of 2-5 ( 2 4 , s ) . For a 60-nL injection volume, &NJ was calculated to be 1.8 X pL2 for the worst case ( K = 2). Thus, it can be assumed that the contribution of the injector is less than 10% of the total extracolumn variance and can therefore be neglected. The experimentally determined extracolumn variance, &C,E, can therefore be considered to be equal to the sum of the individual contributions of the effluent entry tube, a2CON, and the Cell c12DET,
Table 11. Effect of Variations in Organic Solvent on
Chemiluminescent Signal relative chemiluminescence intensity
methanol/
acetonitrile/
methanol/
% organic”
buffer
buffer
water
80
156 118 133
50
122 119 113
122 139 127 114 130 129 123 132 131
126 129 142 134
139 124
20
125
O ~ E C , E= a
135 128 121 128 131
because pH of the mobile phase can be readily changed by using a higher buffer concentration in the postcolumn reagents to control the final pH. These results suggest that it should be possible to use a variety of mobile-phase compositions in the 20-8070 range of acetonitrile or methanol to optimize the separation while operating the postcolumn conditions under favorable chemiluminescent conditions. B. Effect of Flow Cell on Band Broadening. The total variance for a chromatographic peak can be treated as the additive sum of the variance contributions of each of the individual components present in the system. Therefore, the total variance, a2TOT, is expressed by =
a21NJ
+ a2C0L + a2CON + a 2 D E T
(1)
where the contributions are due to the injector, a21NJ, the column, a2COL, the connectors, $CON, and the detector, ‘J2Dm. Equation 1 can be rewritten as a2TOT
=
a2COL -k a 2 E C
(2)
where a2EC represents the extracolumn band variance. Large postcolumn volumes may contribute substantially to the extracolumn variance, adversely affecting chromatographic performance. Therefore, the extracolumn variance, c?~C, was evaluated for the packed capillary CL detection system as described below. A decrease in peak width is observed for an increase in cell volume (Table I), which can be attributed to improved mixing of the reagents and analyte in the larger flow cell. To evaluate the band broadening of extracolumn contributions due to the postcolumn reactor, the column was removed and the injector was connected directly to the flow cell by a 140 X 0.2-mm-i.d. effluent entry tube. The effluent entry tube length of 14 cm was necessary because of equipment constraints. The theoretical contribution of the injector volume to the total variance is ~
N
= J VINJ/K
~
(4)
Theoretically, the volumetric band variance due to the effluent entry tube is given by (24) a2cON,T = a2r6Lu/24 D M (5)
‘The % organic refers to the organic content of the portion that corresponds to the mobile-phase composition, which is 10% of the final volume used for the chemiluminescent measurement.
a2TOT
2 + C~~ D E T ~
(3)
where K is an experimentally determined constant with a
where r is the tube radius, L, the length, and u, the linear velocity. By assuming a diffusion coefficient in the mobile phase, D,, of 1 X cm/s for the test solute, the theoretical variances for the 14- and 4-cm tubes are calculated to be 0.155 and 0.044 pL2,respectively, at a linear velocity of 0.27 cm/s. Assuming the variance in the flow cell is due to laminar flow (25))the theoretical contribution to the band dispersion can be calculated as 2
a DET.T
= ~DET/K
(6)
where the constant K can be assumed to be equal to 12 for a plug flow profile in a cylindrical cell geometry (24).This theoretical detector variance based on actual cell size can be compared to a calculated detector variance, a2Dm,C, found by subtracting the contribution of the connecting tube, (TPCON,T, from the experimentally determined extracolumn variance u2EC,E (Table 111) (from eq 4). Initially, a 3.44-pL cell was constructed by using the cell holder shown in Figure la. A 1.0 mM solution of perylene was used to obtain the experimental extracolumn band variance, a 2 E c g . The column was then connected and the total variance, a2TOT, was measured. In this manner the column variance can be obtained and used in the evaluation of the detector band broadening for the modified cell holder (MV2) (Figure lb). With the modified holder, the extracolumn volume due to the effluent entry tube is reduced by 72%. Peak variances for the two cell holders were compared (Table 111),showing that reduction of the extracolumn volume results in a 40% decrease in the experimental variance (0.673 pL2 vs. 0.398 pL2), accounting for 22% of the total variance, (r2TOT, when using the modified holder. In spite of the relatively large volume flow cell, calculated band broadening is less than theoretical for two reasons: (1) an “effective cell volume” in the range of 2.1-2.5 pL obtained as a result of the “wall-less” flow cell created by sheath flow (22, 23) and (2) the CL reaction kinetics, which also provide for a smaller cell volume, as described previously for the microbore CL detection system (16). The nature of the CL measurement is such that a signal is observed only when the analyte is in contact with “fresh” CL reagents and a signal is not observed if the analyte contacts “spent”reagents, thus effectivelyreducing cell volume in areas where spent reagents are located. The variation in cell volume
Table 111. Evaluation of Postcolumn Detection System Band Variance’ effluent entry
tube length, cm 14
4
U’Tot,
&‘
2.056 1.781
~ * E C , E , wL‘
0.673 0.398
‘J’CON,T,
rL2
0.155 0.044
o’Det,C,*
fiL2
0.518 0.354
‘J2Det,T,
rL2
0.986 0.986
‘See Experimental Section for chromatographic conditions: flow cell volume, 3.44 rL; postcolumn flow rate, 50 pL/min; TCPO, 5.4 mM; H202, 1.2 M in 9/1 methanol/5.9 mM citrate buffer; PMT, 0.75 kV;time constant, 6 9.; test solute, perylene 1.0 mM. = &C,E ‘J’IYlN
T.
ANALYTICAL CHEMISTRY, VOL. 59, NO. 10, MAY 15, 1987
1457
flow cell design, the sheathing flow produced by the chemiluminescent reagents serves to reduce the extracolumn band broadening by creating, in effect, a flow cell within a flow cell. Reduced band width is aLs0 obtained as a result of the reaction kinetics. A potential advantage of this detection scheme in capillary liquid chromatography is that the detection system may be optimized separately from the chromatographic system if the mobile-phase flow rate is low relative to the postcolumn flow rate because of the low contribution of the mobile phase to the final reaction conditions in the flow cell. I
0
i
4
8
1 TIME ( m i d
2
Figure 5. Separation of 1.5 ng of 2-aminoanthracene and 3 ng of perylene on packed capillary column. Postcolumn reaction conditions were as follows: 5.4 mM TCPO 1.2 mM hydrogen peroxide in 911 methanoV5.9 mM clrate buffer, pH 7.8; flow rate, 50 pL/min; cell volume, 3.44 pL; PMT, 0.8 kV; time constant, 6 s.
between the two cell holders may be attributed to the difficulty in reproducibly positioning the effluent entry tube in the flow-cell core. Elimination of this tube should result in further reductions in the extracolumn variance. This may be accomplished by insertion of a PTFE frit into the column to retain the packing (24) permitting the separation column to be fed directly into the flow-cell core. Detection Limits. On the basis of the previous studies, a cell volume of 3.44 pL was used for the determination of detection limits for a mixture of 2-aminoanthracene and perylene separated by capillary LC (Figure 5.) Samples in the range of 0.34-1.5 ng of 2-aminoanthracene and 1.5-3.0 ng of perylene were injected and from these responses detection limits for these compounds based on a signal-to-noise ratio of 4 were determined to be 450 and 600 fmol, on column, respectively. Increased sensitivity may be obtained by the use of a detector with better optics as previously reported (16) and by the use of pulseless pumps to deliver the chemiluminescent reagents, permitting the operation of the PMT at higher voltages. The pulsed power of the Sage Model 351 syringe pump was apparently translated through the drive gears resulting in base line disturbances preventing a reduction in detection limits. Pressurized gas may be used as an alternative approach to reagent delivery to alleviate this problem.
CONCLUSION Chemiluminescence offers several advantages as an alternative detection scheme in capillary liquid chromatography. The main advantage is the use of large volume flow cells (>1 pL), which result in enhanced sensitivity. As a result of the
ACKNOWLEDGMENT The authors thank A. Mohan for the oxalate esters, C. Mannon and R. Weinberger of Kratos Analytical Instruments for equipment, and R. Hartwick for help with column packing. LITERATURE CITED (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25)
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RECEIVED for review March 14,1986. Resubmitted December 16, 1986. Accepted February 11,1987. This work was supported in part by a grant from Research Corporation. This work was presented in part at the 1986 Pittsburgh Conference, Abstract No. 63 (Atlantic City, NJ).
