Anal. Chem. 1987, 59, 2767-2773
2767
Sequential Determination of Both Acids and Bases by Optosensing Flow Injection Analysis Using a Single-Line Manifold Bruce A. Woods, Jaromir Ruzicka, and Gary D. Christian* Center for Process Analytical Chemistry, Department of Chemistry, BG-IO,University of Washington, Seattle, Washington 98195
Optosensing flow lnjectlon analysis (FIA) Is a technlque that utllzes Immoblllted selective Indicators for determining a 88 iected specles. The lndkator color change Is monltored spectrophotometrically. Thls technlque Is lnvestlgated as a method for performing rapld sequential acid and base FIA tltrations. Dllute add, base, and buffer are lnvestlgated as carrlers, to determlne dllute aclds and bases over a wlde concentration range. W h a buffer as carrler, dilute aclds and bases can be determined In the same slnglallne system at the rate of approxlmately 120 samples per how uskrg a 30-pL Injected sample volume. Peak height and peak wldth measurements are both used to extend the working range of the system for aclds and bases In the range of 0.001 to 0.11 M. Standard devlatlons range from 1% to 8%. Some problems with indicator sorptlon encountered when uslng soluble indlcator and buffer carrlers are dlscussed.
The rapid determination of acids and bases, separately or simultaneously, is of great practical importance in chemical, pharmaceutical,nuclear energy, and other industries. Classical batch titrimetry, even when automated and computerized, remains a laboratory technique, cumbersome and too complex to be confortably used in a process environment. Flow injection analysis (FIA), being versatile and fast, would allow monitoring of chemical processes in real time. Yet it is essential to exploit its potential in full in order to develop systems which, due to their simplicity and ruggedness, will become useful tools for process control. It will be shown in this work how advanced concepts of FIA-i.e., gradient titrations ( I ) , single point titrations (2),fast singleline titrations ( 3 ) ,optosensing (4), and matched indicator-buffer carriers (5,6)-are combined to develop a system which allows fast sequential determinations of both strong acids and bases over a wide concentration range using the same carrier-indicator/detector system. FIA titrations using a single-line manifold with a gradient chamber ( I ) are actually used in process control (7).These are novel and faster than classical batch titrations, but they have the drawback of being mechanically complex and use a large volume mixing chamber plus stirrer. The result is large titrant consumption and limited sensitivity due to large dispersion in the mixing chamber, which also requires dilute titrant (NaOH) that is unstable a t such levels. A soluble acidobasic indicator is added to the titrant carrier and the change of this indicator is monitored with a spectrophotometer. The equation for these types of titrations is (1,3, 8) t = (Vm/Q) In 10 log Cos - (V,/Q) In 10 log CNaOH + (VJQ) In 10 log (Sv/Vm) where t is the peak width in seconds, V , is the volume of the mixing chamber in milliliters, Q is the carrier flow rate in milliliters/minute, Cos is the original concentration of the 0003-2700/87/0359-2767$01.50/0
sample, CNaoH is the concentration of the basic carrier in this case, and Svis the voIume of injected sample in milliliters. This equation contains all the relationships that can affect the speed of a titration and the detection limit, i.e., volume of the mixing chamber, carrier flow rate, concentration of the carrier, and sample volume. Astrom (2),in single-point-basedtitrations, measured peak height rather than peak width as used by Ruzicka et al. (I). For that purpose, he used a mixture of linear buffers, with the aim of obtaining a linear relationship between peak height and acid concentration. The linear buffer was used to maintain a constant buffering capacity carrier to ensure a rapid response from the glass electrode used as a detector in this two-line system. Single-line titrations, employed by Ramsing ( 3 ) ,allowed titration cycles to be shortened and sample and reagent consumption to be reduced from milliliters to microliters. Yet, since only peak width was measured, the method had a limited range and dilute titrants with acidobasic indicators had to be employed. FIA analysis of concentrated acids and bases, as done by Ishibashi (5,6),relies on the use of a buffered carrier stream containing a dissolved indicator, the pKI, of which closely matches the pK and pH of the buffer. This yields, within a limited range, a linear relationship between peak height and concentration of analyte and allows the simultaneous determination of acids and bases using the same carrier stream, since, when measuring the absorbance of the indicator at a pH nearly equal to the pKI, value, acids and bases yield opposite deflections, measured as positive and negative peaks. In this work they use a two-line manifold for acids and a complicated three-line manifold for bases and utilize either spectrophotometric or glass electrode detection methods. Finally, FIA optosensing (9)relies on the color change observed by means of fiber optics at the surface of a solid phase placed into a carrier stream. It has been shown that acidobasic indicators covalently bound to cellulose are wellsuited for highly reproducible pH measurements, even in solutions of low buffering capacity and of low ionic strength (10).Since these nonbleeding indicators are commercially available in a wide range of pKI, values and colors, it was decided to use them for the present study.
THEORY The present method, in contrast to all previous FIA approaches using either peak height or peak width measurement, aims to extend the measuring range by selecting the point of readout rather than by changing the experimental parameters (such as sample volume, carrier stream concentration, flow rate, or volume of flow channel). The key to understanding how this is achieved is the concept of controlled dispersion, according to which, at limited dispersion, the carrier stream is not mixed with the injected sample zone components-and therefore no chemical reaction will occur. At medium dispersion, the carrier stream and sample are mixed in moderate 0 1987 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 59. NO. 23,DECEMBER 1. 1987
S -7
w
F m 1. Generalized schematic of the singkAine tiition system. SP is the syrlnge pump, S is the sample to b e injected by a vabe, and
FC is the flow cell (see Figure 3 for details). The source light and detector D are communicating through fiber-optic cables. 3-D is a geometrically disoriented reactor. The components shown in the broken line areas are integrat.33 in a micrwonduii the SIZe of a credit card.
proportions, and a t large dispersion, the sample material is extensively diluted by the carrier stream. For convenience, the extent of dispersion has been described by the sample dispersion coefficient, Ds = CoS/Cswhere Cosis the sample concentration prior to injection and Cs is the concentration of sample material in that element of fluid from which the readout (e.& peak height) is taken. When a chemical reaction is to take place between an injected sample solution and a reagent contained in a carrier stream, as desired in the present case, a mixing must take place, yielding a distribution of reactant concentrations which depends on the dispersion within the analyzer channel. In analogy to the sample dispersion coefficient, Ds = CoS/Cs, also the reagent dispersion coefficient may he defined as: DR = CoR/CR (1) where CORis the original reagent concentration (as pumped into the carrier channel), while CRis the reagent concentration in that element of fluid which yields the analytical readout. Since the true concentrations of sample, (2‘s. and reagent, Cb, materials in said element of fluid are a result of a mutual dispersion and the ensuing chemical reaction, the concentrations defined above, Le., Cs and CR (which would be the result of the mutual dispersion alone), are higher than the actual concentrations at the point of detection because they are not corrected for the mutual equivalent amounts consumed. For the present purpose, however, the differences in C values are neglected for the sake of brevity. For the same m u , the species are considered to react rapidly and in molar ratios of 1:l. Hussein and Christian ( I I ) have described the correction for the reagent consumed in a single-line system when the reagent spectrum also overlaps that of the measured products of multicomponent analytes. In a singleline system (Figure l),in which the carrier stream contains a reagent (Codinto which a sample (Cos)is injected, the concentrations Csand CR&9 obtained hy mutual dispersion of sample zone and carrier stream, are mirror images of one another. Illustrated in Figure 2 are the time or scan profiles of the sample (S) and reagent (R)materials, as they would appear in the absence of chemical reactions, a t time t a t the together with the respective Ds and DR detector position (D), values. Obviously, when the sample concentration is at its highest (Cs(mm,),the reagent concentration is a t its lowest (CR(-)), and therefore, whenever Ds approaches unity, then the reagent concentration approaches zero. Consequently, a species to he measured cannot he formed in the center of the sampIe zone whenever DS= 1,because ea DR- a,the reagent concentration approaches zero. It can he further shown that 1/Ds + ~ / D =R 1 (2) and therefore
DR= D s / ( D s - 1)
(3)
Figure 2. (a) Single-line FIA system. (b) Curves showing the interreiated mixing and displacement of the carrier reagent and sample in
a single line flow injection analysis system. C, is the concentration of the dispersed Sample and CRis the concentration of the dispersed reagent. The CuNes are for the case in which COR and C o swere injected at equivalent COncentratlonsand this equivalence in the detector is obtained where CR = C,.
It follows then that in a single-line FIA system, the sample and reagent concentration lines cross at D = 2 (where D R = Os),for the special case in which Cos= C O R , that is, when the concentration of sample is equivalent to the concentration of pumped carrier solution (Figure 2). If, however, the starting concentrations Cosand C O R are different, then since
Cs = C o S / D s
(4)
i t follows that for the degree of dispersion required for the concentrations to be equivalent (substituting eq 1 and 4 into eq 5, and making Cs = CR),the relationship
Cos= CoR(Ds- 1)
(5)
holds. If a sufficient excess of reagent is to he maintained even in the center of the sample zone (say at least a 5-fold stoichiometric excess), if peak height is to be measured, then for the case when the original sample and reagent concentrations are equal, a medium dispersion at Cmas(Ds = 6 and Cmaxis the concentration at the peak maximum) must be arranged hy the usual means (i.e., selecting channel geometry and/or injected sample volume). On the other hand, for FIA titrations where peak width is to he measured, an equivalence is sought between the reagent (titrant) and analyte (sample), and an element of dispersed sample zone must be located where Cs = C,. Since for the equivalence condition, Cos = CoR(Ds- l), then if reagent concentration COR is kept constant while the concentration of the injected sample is increased, then Ds must increase in order for the equivalence to be maintained. This will be seen as an increase of peak width as monitored at a set distance from the base line. To summarize, the Concentration gradients of sample and reagents in the single-line system are mirror images of one another, and if the Ds value approaches unity, a lack of reagent in the core of the sample zone will occur. This results in a “hole” in the center of the FIA peak where there is a substoichiometric amount or even a total lack of reagent for reaction with the analyte in the sample plug. Two equivalence points will be observed whenever the condition of eq 5 is fulfilled. The distance or time, teq,between these points will be in proportion to the concentration of the analyte. Thus, with Ds, D,, and COR values remaining constant during the experiment, the peak will he widest for the highest Csvalue, and with decrease of analyte concentration, t,, will decrease
ANALYTICAL CHEMISTRY. VOL. 59, NO. 23, DECEMBER 1, 1987
to zero at that Coswhen the condition of eq 5 is fulfilled at the peak maximum. From then on, further decrease of Cos will result in decreasing peak height until base line and the limit of detection me reached. Measurement of peak width and of peak height allows a wide range of sample concentrations to be accommodated hy the same FIA system using the same reagent stream. It has been shown (3) that if the flow geometry, flow rate, and channel dimensions are properly selected, the dispersion pattern of the material will conform with the tanks-in-series model with N value8 of 1-2, while Ds of 2 to 4 will he reached, allowing the condition of eq 5 to be fulfilled at the center of the injected zone (peak maximum) at a realistic C" concentration. Consequently, the asymmetric concentration gradient will yield a calibration curve which for peak width t , = k log Cos (6) and for peak height H
A = k H = k'Cos
(7)
where A is the absorbance of the indicator used, measured at an appropriate wavelength. In order to obtain a linear relationship hetween A and H,the carrier stream and indicator have to he selected in such a way that p H = pKbd = pKI. as closely as possible. Both strong acids and strong bases will yield responses, negative or positive, at low concentrations ohserved as peak heights and for high concentrations as peak widths, providing an additional advantage over previously discussed systems. The last factor to he considered is the form of the indicator used. Previously, indicator dissolved in the carrier stream has been used ( I , 3-6). This did not present any problem for peak width measurements, but for peak height measurements a two-line FIA manifold had to be used to ensure that each element of dispersed sample zone was supplied and mixed with the same quantity of indicator. However, in a single-line manifold according to Figure 1,no indicator will be present in the center of the sample zone at Ds = 1,and the indicator concentration would vary along with De Therefore, two options had to be considered and tested in the present work, i.e., (1)use of an indicator immohilized in the flow cell, and (2) an indicator dissolved in the carrier stream. While execution of the first option requires a highly stable indicator covalently bound to a suitable substrate to he available, the second option requires a correction to be made of the indicator dispersion, due to the formation of a concentration gradient between the carrier stream and the injection sample zone.
EXPERIMENTAL SECTION Construction of t h e ManiFolds. The manifold was constructed from a 45 x 70 x 10 mm block of black poly(viny1 chloride) (PVC) and WBS equipped with a homemade Teflon rotary injection valve. Plumhing of the manifold was done with Teflon tubing (0.5 mm i.d. X 1.5 mm 0.d).The length of tubing hetween the injection valve and detector was 15 cm and had an internal volume of approximately 30 rL. The sample loop was made of Teflon tubing and had a volume of approximately 30 rL. The reflectanceflow cell (details are shown in Figure 3A) was constructed with two Crofon 1110 fiber-optic cables (Du Pont). These fiber-optic cables are a poly(methy1 methacrylate) fiber and have 16 strands in each cable. The fiber-optic cables were glued into the PVC block to he equivalent to a bifurcated fiber optic bundle. When the epoxy had set, the fiber was trimmed and sanded with 600-gritsandpaper and then polished with 5+m polishing paper to provide a smooth surface that was flush with the surface of the block. Some thin pieces of dental "raincoat" rubber were then cut and glued around the fiber-optic bundle to provide a small flow cell. A 3-mm circle of the immobilized indicator waa punched out of the plastic dip strip and the indicator pad separated from its plastic backing. T h e immobilized indicator
2769
A ~~
h i t e Reflector
Out
D
- S
outleL Detector 3. Details o f h cwrstrudm ofhRow ceb used hmls wak: (A) reflectance cell: (E) absorbance cell.
pad was then placed in the small hole over the face of the fiher-optic bundle. A white block of PVC was then fastened with screws to seal the flow-through cell and also to act as a diffuse white reflector behind the indicator pad. The absorbance cell (details are shown in Figure 3B) was constructed hy drilling a 3-mm hole through a black PVC block. Fiber-optic cables of Crofon 1110 were glued into smaller black PVC blocks and polished as described previously. These blocks were then fastened to the main PVC block with screws to seal the flow cell. T h e inlet and outlet Teflon tubings were glued into place in a standard Z-cell configuration. The dimensions of the connecting tubing hetween the injection valve and the deteetor cell were chosen to optimize fast titrations. Ramsing (3) has reported that for optimal fast titrations the dispersion should be in the range, D = 2 4 . Smaller reactor or mixing volumed lead to a lower dispersion value than larger reactor volumed. Dispersions can also be controlled hy lmoaing the tubing hetween the injector and flow cell so that it has a three-dimensional configuration (12). The knotted configuration lowers dispersion hy reducing the axial dispersion that occurs in a straight-line system by increasing the radial dispersion, leading to plug flow which is similar to that created hy a like mechanism in a single head string reactor (13). Reagents Used. AU solutionswere prepared from analytical grade reagents and deionized water. Dilute acids and bases were prepared by diluting commercially available 1.ooO N HCI (VWR Scientific) and 1.0 N NaOH (Banco). The Concentrationsof stoek solutions were checked by titrating against tris(hydroxymethy1)aminomethane(Aldrich, Gold Label) and potassium hydrogen phthalate, respectively,by the method of Gran (14). All buffers used were prepared by modifying recipes of Perrin and Dempsey (15).Buffers were prepared hy dissolvingthe acid's salt in approximately 900 mL of water and then adjusting the pH to approximately the desired value with acid and diluting to 1L. The final pH was determined with a glass electrode on a small aliquot of the buffer. Buffers containing methyl red indicator were prepared in the following manner. A 0.002 M stock solution of methyl red indicator (J. T. Baker) was prepared in methanol (Burdick and Jackson). Five hundred microliters of the methyl red indicator stock was added to 100 mI,of methanol, then approximately 800 mL of water and the acid's salt were added. T h e pH of the buffer was adjusted to approximately the desired pH and then diluted to 1liter. The final pH was determined as descrihed previously. The immobilized indicator used was a Colorphast indicator (Merck catalog no. 9582) for the pH range of 4 to 7. T h e indicator changes from its yellow acidic form to a blue alkaline form. The wavelength used to monitor the color change, 640 nm, and pK, of 5.28, had been previously determined (IO). Equipment Used. A Model 390 buret pump (Fisher Scientific) was used in this study. The buret pump provides a more precise and pulse-freeflow than can he provided by a peristaltic pump.
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ANALYTICAL CHEMISTRY, VOL. 59,NO. 23, DECEMBER 1, 1987
Table I. Summary of Titration Systems carrier 0.1 M NaOH 0.1 M HC1
0.11 M acetate, pH 5.7
0.5 M acetate, pH 5.7
sample rangea
peak height or width*
std dev'
std devd
fig no. and curve
0.02-0.05 M HCl 0.03-0.1 M HCl 0.02-0.11 M NaOH 0.02-1.1 M NaOH
height width height width height width height width height width height width
2.3% 0.03 M HC1 5.2% 0.03 M HCl 2.6% 0.03 M NaOH 7.8% 0.03 M NaOH 2.0% 0.005 M NaOH 1.1%0.005 M NaOH 2.8% 0.005 M HC1 4.2% 0.005 M HC1 1.6% 0.01 M NaOH 4.1% 0.01 M NaOH 2.2% 0.01 M HCl 2.2% 0.01 M HCl
5.1%
SB, 0 7B, 0 SA, 7A, SA, 0 7A, 0 SB, 0 7B, 0 SA, w 7A, w SB, w 7B, w
e 0.005-0.1 M NaOH
0.005-0.02 M HCl 0.005-0.1 M HC1 0.001-0.01 M NaOH 0.01-1.0 M NaOH 0.005-0.1 M HCl 0.01-0.1 M HCl
5.8%
13% 10% 0.3% 13% 7.6% 1.1%
4.6% 12% 10%
+ +
Sample range is the measurable concentration range obtained in these experiments. *Indicateswhether peak height or width wa8 used for the measurement of the peaks. Standard deviations are based on either peak height or width measurementsfor 10 replicate injections at the listed concentration. For peak width standard deviations are for variation in the timed width of the peak. Standard deviations based on calculated concentrations. 'Too few points on calibration curve to report linear working range. A carrier flow rate of 0.5 mL/min was used throughout this work. A small 20-W quartz projector lamp was used as a light source and was controlled with a homemade voltage regulator. A Bausch and Lomb Mini-Spec 20 spectrophotometer was used as the detector. The regular lamp had been removed from the Mini-Spec 20 and the fiber-optic cable inserted in its place by using a homemade adapter. A logarithmic converter was used between the Mini-Spec 20 and the recorder (Houston Instruments Microscribe 4500), so that all peaks would be linear with absorbance readings from the spectrophotometer. For the reflectance measurements, all values are reported as A,, which is the apparent absorbance reading on the spectrophotometer for the signal reflected from the pad with a diffuse white reflector behind the pad. A Corning Model 155 pH meter with Corning X-EL semimicro glass electrode was used to check the pH of all buffers prepared in this work. The pH meter was calibrated with two buffers of pH 4.00 and 7.00.
RESULTS AND DISCUSSION The results for the various systems investigated in this work are summarized in Table I. The sample range reported in the table represents the measurable concentration range determined. The measured peak widths are determined a t an arbitrary height on the FIA peak and the width is measured between the leading and trailing ends of the peak at the chosen point on the peak. The selected point used for width measurements must then be used for all standards and samples. The height chosen will obviously affect the measurement range (see Figures 4 and 5 below). For this study, we generally selected a height of about 50% of the peak height for the most concentrated sample. In FIA titrations, ideally a region will be picked where Cs = CR. Standard deviations are reported based on peak height measurements and for peak widths on a time basis and on a concentration basis. Use of Dilute Base as Carrier. Titrations of dilute acids using a dilute NaOH carrier and an immobilized indicator were first conducted to compare results with those of previous workers. Titration curves for this are presented in Figure 4, where we are monitoring the disappearance of the blue base color of the immobilized indicator. Typical peak widths are 2-4 s for dilute HC1 in the concentration range of 0.02-0.1 M. A goal was to determine if it is possible to perform FIA measurements based on using peak height measurements, followed by peak width measurements after reaching a saturation point in peak height. As the HCl concentration increased, the calibration curve had a short linear segment for peak height measurements before it started to reach a saturation point; this was due in part to the indicator S-shaped titration curve approaching a plateau at the higher concentrations. A peak was not observed for a 0.01 M HC1 standard. The peak for 0.02 M HC1 was below the level we had chosen
0.9
L
0.3
1 TIME
Flgure 4. Titration of dilute acid using the optosensing system. The carrier Is 0.11 M NaOH at a flow rate of 0.5 mLlmIn. A Merck 9582 pH indicator pad was used with monitoring of the color change of the pad at 640 nm. Injected samples (30pL each) of HCI are as follows: (0) 0.10 M, (0) 0.075 M, (0) 0.05 M, (A)0.03 M, (-) 0.02M. 0.6 -
'
/A
1
6sec
1111 \
TIME
Flgure 5. Titration of dilute base using the optosensing system. The carrier is 0.1 M HCI. Other conditions are the same as in Figure 4 except the sample concentrations of NaOH are the following: (0)1.1 M,(O)O.11M,(~)0.077M,(A)0.055M,(O)0.033M,(-)0.022 M.
for peak width measurements. The relative standard deviation of peak width measurements could probably be reduced if an electronic peak width measuring device were employed. Use of Dilute Acid as Carrier. Titrations of dilute bases using a dilute acid carrier are shown in Figure 5. Experimental conditions are the same as for the previous experi-
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 23, DECEMBER 1, 1987
-
0.7
A
1000
3 min
I 0 100
*r
I( k
M NaW
0010
0 001
1
465
765
111
I
i
0'
7
129 Peak Width
B
165
1875
,
216
(SK)
0.100
TIME
Flgure 6. Titration of dilute bases and acids with the optosensing system. The carrier is 0.1 M acetate, pH 5.7. The other conditions are the same as in Figure 4. The NaOH samples are as follows: (a) 0.077 M, (b) 0.055 M, (c) 0.033, (d) 0.022 M, (e) 0.011 M, (f) 0.005 M, (9) 0.001 M. The HCI samples were as follows: (h) 0.075 M, (i) 0.05 M, (j)0.03 M, (k) 0.02 M, (I)0.01 M, (m) 0.005 M.
ments, except that dilute acid is used as the carrier stream and dilute bases are injected as standards. Peaks were not observed for NaOH concentrations below 0.02 M. The results indicate that one could use peak height data for determining dilute base concentrations and then use peak width measurements for higher concentrations of bases. Caution must be exercised when using immobilized indicators, to ascertain that peak height measurements are within the linear range of the indicator. At the extreme ends of the range of the indicator, areas of extreme nonlinearity may be encountered due to the indicator's S-shaped titration curve. Use of Dilute Buffer as Carrier. In order to perform titrations of both acids and bases using the same carrier with an immobilized indicator, we used dilute buffers as carriers. Previous workers have reported performing either acid or base titrations using soluble indicators, but they have not reported the ability to titrate both acids and bases using the same system. The results of this experiment are shown in Figure 6. To be comparable with the above studies with dilute base and dilute acid carriers, the buffer was dilute (0.1 M) acetate. When dilute base is used as samples and examining peak height changes, there is a large change only between 0.001and 0.005 M NaOH, and this apparently represent9 a limited range for a precise correlation between peak height and dilute base concentration. For samples with NaOH concentrations between 0.005 and 0.1 M, peak widths were between 5 and 25 s. The peak for 0.001 M NaOH was below the level used for peak width measurements. A peak was measured for 1.0 M NaOH but it was 45 s wide, which was a long time for our desired objective, and the calibration curve slope shifted between 0.1 and 1.0 M NaOH. When a 0.5 M acetate buffer was used as carrier solution, the width for a 1.0 M NaOH sample was reduced to approximately 11 s, which was acceptable for our objectives. When either base, acid, or buffer carrier is used, a notable difference was observed in the detection limits with the different carriers (see Figures 7 and 8). When dilute acid or base was used as carrier, the immobilized indicator is in either its extremely acidic or basic form. To measure a change in color, the sample plug must undergo only limited dispersion, so as not to be too diluted or neutralized by the carrier, as would happen if there was a high degree of mixing (a case similar to titrations occurring in a gradient chamber with high dispersion and suppression of the sample peak due to mixing and neutralization). The sample plug must then flush the carrier out of the interstices of the cellulose pad on which the indicator is immobilized. And the indicator must change enough
M HCl 0.010
0001
3
45
57
69
81
9 15
99
Peak Width (set)
Flgure 7. Calibration curves for 30-pL injected samples showing peak width versus log concentration when using an immobilized indicator in a singlaline manifold. (A) NaOH samples injected using the following carriers: (e)0.1 M HCI, (0)0.1 M acetate, pH 5.7, and (H) 0.5 M acetate, pH 5.7. (8)HCI samples injected using the following carriers: (0) 0.1 1 M NaOH, (0)0.1 M acetate, pH 5.7, and (H) 0.5 M acetate, pH 5.7.
A
Perk Ht (rnrn)
-20
1
0.00
0.02
0.04
0.06 M NaOH
0.08
0.10
I 0.12
B
Peak Ht
(rnrnl
0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10 MHU
Flgure 8. Calibration curves for 30-pL injected samples showing concentration versus peak height when using an immobilized indicator in a single-line manifold. (A) NaOH samples injected using the following carriers: (e)0.1 M HCI, (0)0.1 M acetate, pH 5.7, and (H) 0.5 M acetate, pH 5.7. (B) HCI samples injected using the following carriers: (0) 0.11 M NaOH, (0)0.1 M acetate, pH 5.7, and (). 0.5 M acetate, pH 5.7.
in color to be measurable by the spectrophotometer. Dilute samples cannot accomplish all of these, especially the requirement of a measurable change in the color of the immo-
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 23, DECEMBER 1, 1987
bilized indicator when the indicator is in one of its extreme colored forms. An example of this would be for the dilute NaOH and HC1 carriers (0.1 M) that were employed; for concentrations of acid or base samples below approximately 0.02 M, no peaks were observed (Figures 4 and 5). Below this concentration the sample cannot flush out the carrier from the interstices of the pad and still cause a color change in the indicator from either its extremely acidic or basic color form. When a buffer is used as a carrier, we choose a buffer pH that is in the middle of the pH range (and range of color change) covered by the indicator. This results in a measurable color change, even when dilute samples are injected. This is because we are now operating in the middle of the zone of color change where even small changes in pH result in a color change. We are no longer operating at the extreme limits of the indicator as in the previous acid and base carrier cases. Using a dilute buffer would also result in less suppression of the sample signal, as occurs when using either strong acid or base as a carrier. In the above systems, peak width measurementscan be used to determine acid or base concentrations when either acid, base, or buffer are used as carriers. If the peak falls below the level used for peak width measurements,then peak heights can be used for the determination of low concentrations of acids or bases. As the peak size increases, one can make a transition from using peak heights for low concentration samples to peak width measurements for higher concentration samples. This allows one to expand the range of concentrations analyzed by this technique. But as shown in Table I, the largest linear working range is still obtained by using peak width measurements, and peak height measurements have only a limited working range. When the standard deviations are examined for both peak height and width measurements, it would appear that all the carrier systems studied would offer similar precision. But when these deviations are used to calculate precisions based on concentration, the buffered carrier systems appear to offer better precision. And it appears that the more dilute buffer offers slightly better precision than the higher concentration one. But if a more dilute buffer is used, then the linear working range is decreased for peak height and one must use peak width measurements sooner. The dilute buffer cannot be used at higher sample concentrations where the more concentrated buffer can be employed. When a buffered carrier is used and low sample concentrations are injected, it appears that the carrier is diluted because we see negative peaks (see Figure 8A). Due to the signal readout following the S-shaped titration curve of the indicator for peak height measurements,this would be an ideal application for a more elaborate data acquisition system employing polynomial fit calibration curves. In the present system we are limited to fitting the calibration curve with a number of short linear segments. Rhee and Dasgupta (16,17) report a magnification of the error in calculated concentrations when using peak width measurements. This is due to the logarithmic dependence of the width on the concentration. This was seen as the deviation in concentration being a factor of 2 to 12 times larger than the deviation in peak width times. We see a similar effect in this work but only by a factor of approximately 0.3-5. Other workers (2,3)reported only variations based upon peak width or height measurements, but evaluation of their data shows similar trends in precision of concentration values based on peak width measurements.
Use of Soluble Indicator and Dilute Buffer as Carrier. A system was investigated that used soluble methyl red indicator and dilute acetate buffer as carrier. We were investigating the potential for performing FIA titrations under
6 sec P
TIME
Figure 9. Use of soluble indicator and dilute buffer. The carrier is 0.01 M acetate, pH 5.0, with 500 pL of methyl red stock solution added per liter of carrier. The injected sample volume is 30 pL and monitoring is at 520 nm. (A) Hole that results for FIA titrations performed under limited dispersion condltions. Dashed line shows expected shape of peak. (B) Peak showing the effects of indicator sorption-desorption on the “hole” during titrations. Conditions are the same as for (A).
limited dispersion conditions. Under limited dispersion, the result would be incomplete mixing of the sample and reagent carrier stream, resulting in a “hole” in the middle of the peak. This “hole” could then possibly be used for background subtraction when performing FIA titrations on complex industrial matrices. Our initial work was to see if FIA titrations could be performed under limited dispersion conditions and what conditions would be required to obtain a “hole” in the FIA peak. We were able to obtain some data but were plagued by problems of indicator sorption-desorption to the tubing used for constructing our system. Other workers developing FIA titration systems have not reported problems with indicator sorption-desorption. Ruzicka and Hansen (8) used alcohol in their FIA titration system when employing bromthymol blue indicator. The alcohol was added to prevent sorption of the acidic form (yellow color) of bromthymol blue indicator to the poly(viny1chloride) tubing (PVC) used for construction of their systems (18). Adding up to 10% alcohol to the methyl red and buffer solutions did not decrease the sorption-desorption problem. We tried tubing made of PVC, Microline, and Teflon to avoid this problem. Satisfactory results, even with the addition of ethanol, were obtained only with manifolds constructed of Teflon tubing. PVC tubing suffered the most from indicator sorption effects, turning orange after just a few days of use. Microline was not as bad as PVC but sorption was still a problem. Some of the results obtained showing the desired “hole” and the problem encountered with the sorption-desorption of the indicator are shown in Figure 9.
When using methyl red indicator in an acetate buffer, we obtained a calibration curve for dilute HCl concentrations of 0.02-0.075 M, with a correlation coefficient of 0.99 for peak width measurements. These peak widths were measured
Anal. Chem. 1907, 59, 2773-2776
across the “hole”, i.e., from the leading to trailing edge of the double peak. For HC1 samples below 0.02 M, the peaks were below the starting base line. This is probably due to a leveling of the acid sample plug by the buffer and dilution of the buffer by the sample plug.
CONCLUSIONS Selecting the readout (peak height or width) is a powerful tool for increasing the range and extending the detection limits of any FIA method. Titrations are a special case in that an equivalence point is sought for peak width evaluation. When acid or base concentration is determined as in the present work, selection of readout is a choice between titrimetric assay and assay based on pH measurement. Technically, the present work resulted in the development of a sturdy, simple system which allows monitoring at a high sample frequency of 100-120 samples per hour in real time (with a delay of less than 30 s, which is the time between sample injection and availability of the readout), low sample consumption with 30 FL of sample injected (less than 0.5 mL required totally), and low reagent consumption (less than 0.5 mL) per assay. Additionally, both acids and bases can be determined with this system. The successful application of the optosensing technique to the determination of the pH of “acid rain” is the topic of a paper that is currently in preparation.
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The use of FIA as a method to evaluate materials for the construction of chemical systems has also been demonstrated.
LITERATURE CITED (1) Ruzlcka, J.: Hansen, E. H.: Mosbak, H. Anal. Chlm. Acta 1977, 92, 235-249. (2) Astrom, 0. Anal. Chlm. Acta 1979, 105, 67-75. (3) Rarnsing, A. U.; Ruzlcka, J.; Hansen, E. H. Anal. Chlm. Acta 1981, 129, 1-17. (4) Ruzicka, J.; Hansen, E. H. Anal. Chlm. Acta 198$, 173, 3-21. (5) Ishibashi. N.; Imato, T. Frezenlus’ Z . Anal. Chem. 1986, 323, 244-248. (6) Imato, I.; Ishibashi, N. Anal. Scl. 1985, 1 , 481-482. (7) Wolcott, D. K.; Hunt, D. 0. Presented at the 11th FACSS Meetlng, Philadelphia, PA, 1984; paper 353. (8) Rwlcka, J.; Hansen, E. H. f l o w In/ectlon Analysis; Wiiey: New York, 1981; pp 138-141. (9) Ruzicka, J.: Hansen, E. H. Anal. Chlm. Acta 1985, 173, 3-21. (10) Woods, E. A.; Ruzlcka, J.; Christlan, 0. D.; Charison, R. J. Anal. Chem. 1986, 58, 2496-2502. (11) Haj-Hussein, A. T.: Chrlstian, G. D. Mlcrochem. J. 1986, 34, 67-75. (12) Ruzlcka, J.; Hansen, E. H. Anal. Chlm. Acta 1984, 161, 1-25. (13) Reijn, J. M.; van der Linden, W. E., Poppe, H. Anal. Chlm. Acta 1981, 123, 229-237. (14) Rosetti, F. J. C.; Rosetti, H. J. Chem. Educ. 1965, 42(7), 375-378. (15) Perrin, D. D.; Dempsey, E. Buffers for pH and Metal Ion Control; Chapman and Hail: London, 1974. (16) Rhee, J. S.; Dasgupta, P. K. Mkrochlm. Acta 1985, I I I , 107-122. (17) Rhee, J. S.; Dasgupta, P. K. Mlkrochlm. Acta 1985 IIZ, 49-64. (18) Ruzicka. J., private communication, Seattle, WA, July 1988.
RECEIVED for review March, 6, 1987. Accepted August 14, 1987.
Determination of Ingredients of Antipyretic Analgesic Preparations by Micellar Electrokinetic Capillary Chromatography Shigeru Fujiwara Pharmaceuticals Research Center, Kanebo, Ltd., Miyakojima-ku, Osaka 534, J a p a n
Susumu Honda* Faculty of Pharmaceutical Sciences, Kinki University, Kowakae, Higashi-Osaka 577, J a p a n
The principal ingredients of antipyretic analgesic preparations were determined simultaneously by micellar capillary eiectrokinetlc chromatography wlth sodium dodecyl sulfate as the anionic surfactant. All of these compounds mlgrated to the cathode and were well-resolved within ca. 20 min between the aqueous and micellar phases, wlth number of theoretical plates values ranglng from 70000 to 130000. On-column detection at 214 nm wlth ethyl p-aminobenzoate as the internal standard allowed accurate and reproducible determlnation of these compounds. Application to a commerclai antlpyretlc analgesic tablet demonstrated the usefulness of this method.
Electrophoresis in an open capillary tube (capillary zone electrophoresis, CZE), has brought forth many advantages regarding the separation of ionic substances. Electroosmotic flow having a flat profile perpendicular to the capillary axis carries ions rapidly with high column efficiency (1-5). Oncolumn detection allows simultaneous microanalysis of the 0003-2700/87/0359-2773$01.50/0
component ions of a sample with the number of theoretical plates (NTP) values reaching several hundred thousands or more. Recently, Terabe et al. (6, 7) introduced the micellar solubilization technique to CZE and extended its applicability to nonionic substances. Introduction of a sample composed of lipophilic components to a fused silica capillary tube filled with a buffer solution containing an ionic surfactant such as sodium dodecyl sulfate (SDS),followed by application of a high voltage between both ends of the tube, causes distribution of the components between the aqueous and micellar phases, which migrate toward the cathode. Since the distribution is kinetic, dependent on the concentration of the surfactant, good separation can be obtained for multiple components by changing the concentration of the surfactant. This new analytical tool of micellar electrokinetic capillary chromatography (MECC) has been successfully applied to the separation of the phenolic compounds (7), phenylthiohydantoin derivatives of amino acids (8))and purines (9). In this paper, we have described the application of this method to the analysis of the ingredients of antipyretic analgesic preparations, with an emphasis on quantitative aspects. 0 1987 American Chemical Society
