Anal. Chem. 1986, 58,195-200
195
Immobilization as a Mechanism for Improving the Inherent Selectivity of Photometric Reagents M a u r i A. Ditder,* Henri Pierre-Jacques, a n d Sheryl A. Harrington Department of Chemistry, College of the Holy Cross, Worcester, Massachusetts 01610
It is shown that immobilization of metal chelating photometric reagents can have the effect of increasing their selectivity. The ligands studied, 3-( 2-pyridyl)-5,6-bls( 4-suifonatophenyi)-1,2,4-triazine and 2,4-bis(5,6-bis(4-suifonatophenyi)-l,2,4-trlazln-3-yl)pyridlne, become seiectlve for Cu' over Fez' upon immobiiiration. It is argued that the seiectivity results from the inability of the 3:l (ligand-to-metai) Fez+ complexes to form due to restrictions imposed by reagentsubstrate interactions. The Cu' complex is affected to a lesser extent due to the less demanding 2:l stoichiometry. Immobilized metal chelating reagents are widely used as preconcentrating agents in analytical detection schemes. Generally the analyte is eluted prior to the quantitation step. In some cases, though, elution can be deleted and the quantitation performed directly on the analyte-substrate system. There has been recent interest in the use of visible photometric and fluorometric methods for this purpose. One of the major reasons is that with the reagent immobilized on or attached to a fiber optic it is possible to devise a reusable optical probe ( I ) . Of course, direct analysis (no elution step) is essential. In these cases the immobilized reagent must serve both as a photometric and chelating agent. In related work it has been shown that immobilization of a fluorogenic-metal chelating reagent can be useful in eliminating potential interferences. The approach is to employ a reagent that complexes the analyte tightly and then isolate the substrate and thus the analyte by filtration. The system can be washed to eliminate some of the interferences prior to quantitation (2,3). While this approach was shown to be useful in eliminating interfering species that do not adsorb on or bind to the solid system it is not well-suited for dealing with interference from species that react with the photometric reagent. This report demonstrates that immobilization of photometric reagents can result in an increase in their inherent selectivity. Evidence is presented that suggests that this arises from the immobilization process restricting the formation of complexes with a high reagent-to-analyk ratio thereby making the reagent specific to complexes with low stoichiometric ratios. This has the potential to be generally applicable in a variety of photometric methods employing immobilized reagents. The systems described in this report are based on the analytically useful ligands 3-(2-pyridyl)-5,6-bis(4-sulfonatophenyl)-1,2,4-triazine and 2,4-bis(5,6-bis(4-sulfonatophenyl)-1,2,4-triazin-3-yl)pyridine. These will be referred to by the trivial name ferrozine and the abbreviation 2,4-BDTPS, respectively. The structures of these ligands are shown in Figure 1. Both are reported to be applicable to the quantitation of Cu+ and/or Fe2+. Bidentate ferroin ligands like these form 2:l and 3:l ligand-to-metal complexes with Cu+ and Fe2+,respectively (4-7). In aqueous solution the molar absorptivities at the appropriate visible X's maximum are greater for the Fe2+complexes than the corresponding Cu+ complexes. In this report we show that these reagents can be made selective for Cu+ over Fe2+by their immobilization on an anion-exchange resin.
The inability of the Fe2+complexes to form is consistent with evidence reported in an earlier study on the applicability of similar ligands to a preconcentration-elution scheme. It was found that for some ligands the tris complex with Fe2+ was greatly weakened by adsorption onto surfaces. For example, it was observed that if the Fe2+complexes of ferrozine or the related compound 3-(2-pyridyl)-5,6-diphenyl-1,2,4triazine were adsorbed on Norit the complexes would totally dissociate. That is, the ligands would adsorb on the Norit while the Fe2+was released to the solution (8). The ligands used here have previously been employed for the detection of copper in the presence of iron in a number of systems. In these earlier applications the interference due to spectral overlap was eliminated through one of the classical approaches of masking or multiwavelength techniques ( 4 , 5 , 9). In addition, there are ferroin ligands that are specific for copper in the presence of iron. Nevertheless, the procedure here is of interest because of the unique mechanism involved and because of its potential for general applicability to the increasing number of quantitation schemes relying on immobilized reagents.
EXPERIMENTAL SECTION Instrumentation and Equipment. All transmission and reflectance spectra were obtained with a Perkin-Elmer Model 559 UV-vis spectrophotometer. Scattering problems associated with transmission measurements of samples containing suspended particles of ion-exchange resin were diminished by using the integrating sphere available as an accessory to this spectrophotometer. Reflectance spectra were also obtained with the integrating sphere accessory. The reflectance spectra were converted from percent reflectance to the Kubelka-Munk function of absolute reflectance with the aid of a Perkin-Elmer Model 3600 data station. Reagents. The ligands, 3-(2-pyridyl-5,6-bis(4-sulfonatophenyl)-1,2,4-triazineand 2,4-bis(5,6-bis(sulfonatophenyl)-l,2,4triazin-3-y1)pyridine as their sodium salts, were from G. Frederick Smith Chemical Co. (catalog no. 569 and 664, respectively). Dowex 1X-8 200-400 M anion-exchange resin was used as the immobilization substrate. The resin was washed with 1.0 M NaCl prior to use. The buffer solutions were prepared from sodium acetate and acetic acid. Iron and copper solutions were prepared by appropriate dilution of stock solutions, which were generally prepared from chloride salts and acidified with HC1. For the chloride-free experiments a sulfate salt and H2S04were substituted. The Cu2+solution used in the quantitative uptake measurement was standardized by ion exchange for H+ and subsequent titration with NaOH. The hydroxylamine hydrochloride solution used in the volume dependence study (Figure 9) was Feand Cu-free by extraction by the GFS Chemical Co. General Procedures. Ligands were immobilized via 10-min equilibration with dilute aqueous solutions of ligand. For example, 100 mg of ferrozine in 1.00 L of water was equilibrated with 10.0 g of resin. Complete uptake of the ligand was verified by the absence of UV absorbance of the filtrate. For both immobilized and nonimmobilized metal-ligand complexes buffered solutions of Cu2+and Fe3+were reduced with hydroxylamine hydrochloride prior to reaction with the ligand. Buffering was with pH 4.3 acetate/acetic acid buffer. Immobilized systems were reacted by suspending (via continuous mixing) the resin-ligand in the buffered metal ion solution. Nonimmobilized systems were studied by transmission spectrometry in 1.0-cmquartz cells. Immobilized systems were studied
0003-2700/86/0358-0195$01.50/00 1985 American Chemical Society
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wso; & MFerrorine
so;
0
0
v'N 'Nd
so;
N
N-N
2,4-BDTPS
Figure 1. Structures of the ligands immobilized.
by either transmission or diffuse reflectance spectrometry. Transmission spectrometry was performed on dilute aqueous suspensions (25 mg of resin in 4.0 mL of solution) in 1.0-cm quartz cells. Reflectance spectrometry was performed on isolated resin in 0.2-cm quartz cells. The resin-ligand-metal systems were isolated by vacuum filtration. The spectra were taken within 30 min to avoid air oxidation. Reflectance spectra were initially referenced to ion-exchange resin. These were run without dilution. A spectrum of ion-exchange resin relative to BaSO., was used to convert the relative reflectance spectra of the samples to absolute reflectance spectra. The assumption that BaSOc has an absolute reflectance of unity is inherent in this process. A more detailed discussion of the use of reflectance spectrometry to characterize immobilized systems is available in a previous article (10). Specific Experimental Parameters. Unless indicated otherwise in the discussion or figure captions, experimental conditions M metal ion, 2 X mol of ligand/g were as follows: 5 X of resin, 1% (by mass) hydroxylamine hydrochloride, and 0.12 M total acetate and acetic acid from buffer (pH 4.3). For transmission studies 25 mg of resin was suspended in 4.0 mL of solution. For other systems 500 mg of resin was equilibrated with 50 mL of solution. The immobilized Cu+ systems were monitored at 490 nm and the Fez+systems at 560 nm.
RESULTS AND DISCUSSION In order to test the effect of immobilization on the selectivity of ferrozine, the formation of its copper and iron complexes on the surface of ion-exchange resin was monitored as a function of time. Representative results are shown in Figure 2. In this case the signal monitored is the absorbance of a suspension of resin in the reaction mixture. Similar results are obtained from the reflectance of filtered resin. It is apparent that the formation of the Cu+ complex is not substantially inhibited by immobilization of the ligand. Within 5-10 min color formation is essentially complete. However, the color formation due to Fez+is effectively suppressed, as even after 150 min it is still much weaker than that due to Cu+. This is particularly notable when compared to the characteristics of the totally solvated ligand where the signal from the Fez+complex is greater than that of the Cu+ complex. In solution, the molar absorptivities at the visible X's maximum are 25 400 at 560 nm for Fez+ (5) and 4320 at 470 nm for Cu+ (6). It is also important to note that in solution the color formation due to the Fez+ complex is described as almost instantaneous (11). Thus, the effect of immobilization is substantial. From the graph, the signal for Cu+ relative to Fez+ after 5 min is about 40/1 as opposed to the 0.17/1 ratio for nonimmobilized ligand. However, if equilibration continues for an extended period the selectivity of the system for Cu+ relative to Fez+diminishes as the colored Fez+ complex slowly forms. Thus, potential applications of this system would likely require that the signal
2k
I
7; Time
-
I
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I
Minutes
Figure 2. Absorbance from the reaction of Cu+ (0)or Fe2+ (0) with immobilized ferrozine. Absorbance was of 25 mg of resin (10 mg of M metal. ferrozinelg of resin) and 4 mL of 5 X
be measured after a relatively short reaction time. In order to optimize this selectivity and to consider its application to other systems, we have investigated its source. A reasonable explanation can be developed on the basis of differences in the stoichiometric ratios for the complexes. From a consideration of the systems it is reasonable to hypothesize that the alignment required for the 3:l bidentate ligand to metal stoichiometry exhibited by Fez+is impossible to achieve with the initial distribution of the ligand on the resin surface. One aspect to consider is simply the problem of bringing three bulky ligands together on a flat surface. In addition, the ligand alignment that optimizes ligand-resin interactions will certainly differ from that which optimizes ligand-metal interactions. Each ligand will bind to the resin through two ionic sites. In addition, further interactions between the T systems of the ligand and resin are likely (12). The multicentered nature of this binding to the substrate will inhibit the adoption of the three-dimensional orientation required for the octahedral geometry characteristic of complexes with three bidentate ligands. For the Cu+ complex the effects of the resin would not be expected to be as pronounced. Only two bulky ligands must be aligned on the solid instead of the three required by Fe2+. Furthermore, since it is not uncommon for tetrahedral systems to be distorted toward planarity an intimate ligand-resin interaction may not be as unacceptable for the metal complex. The loss of selectivity that occurs with time can be explained by a slow rearrangement of the ligands to allow Fe2+ligand binding a t the expense of ligand-resin interactions. There are at least three ways in which the reorientation-realignment might occur. One or perhaps two of the ligands could retain a strong (at least two sites/ligand) interaction with the surface while the other ligand(s) breaks free from the resin and occupies the remaining sites in the octahedral complex. Alternatively, all three ligands may retain an interaction with the resin through one instead of two of the ionic sites, thus increasing the freedom of the ligand and permitting appropriate interaction with the metal. A final possibility is that the ligands could migrate to selected sites on the surface where the resin's topography is sufficiently rough to allow the ligands to assume an orientation appropriate for the required ligand-metal geometry. Whichever of these is the more accurate picture, a realignment of the preferred ligand-resin environment must occur. It is also highly probable that some minor reorientation also occurs in the formation of the Cu+ complex. Even with the less stringent requirement many ligands may need slight readjustments to accommodate the Cu'. The important difference is in the extent of rearrangement required.
ANALYTICAL CHEMISTRY, VOL. 58, NO. 1, JANUARY 1986
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0
0.3-
,.-.
0
A
a_ U
0.20
o
A 0
O'lI
A
0.1-
o G
O I
60
0
0 al
0 55
C
0 I
0
0'5
Concentration of X - - M Effects of anion concentration and identity on signal from F$+: (0) c&i3o,,(0)cr, (A)BT, (0) NO3-. All solutions also contain 0.03 M CI- and 0.06 M C2H302-.
0
I
120 Time
I
- Minutes
240
Formation of Cu+ ( 0 )and Fez+(0)complexes with immobilized 2,4-BDTPS as a function of tlme. Fifty mllliliters of 2.5 X M metal Ion was equilibrated with 0.5 g of resin. Flgure 4.
Flgure 3.
In summary, we speculate that for Cu+ a detectable complex (distorted relative to the ideal geometry) forms in which the ligand-resin interaction is largely retained and the ligandmetal interaction accommodates the resulting alignment. However, the Fe2+complex with its higher stoichiometric ratio and thus more demanding ligand alignment cannot form without extensive ligand rearrangement. The necessity of the extensive rearrangement slows the formation of the Fe2+ complex and thereby provides the temporary selectivity. In order to support the above model, we have investigated the effects of changing the mobility of the ligands. The selectivity should be related to ligand mobility in that as we make the ligand more mobile the Fe2+complex should form faster. Mobility of the ligands can be controlled by varying the concentration or identity of nonreacting anions in the solution employed. That is, if a higher concentration of anion or anions with a stronger affinity for the resin are used the temporary displacement of one of the ligand binding sites should be more pronounced. Movement of the ligand may not require displacement of both binding sites as the ligand should be able to "walk" along the surface if the sites are displaced from the surface one at a time. In the original solution the major source of anions was the large excess of the reducing agent hydroxylamine hydrochloride. In order to study anion effect, this was decreased from 1 to 0.2% and anions from other salts were added. The signal from Fe2+after 30 min was monitored as a function of concentration of C1-, Br-, NO3-, (as Na salts), or C2H302-(from the buffer). The results are shown in Figure 3 as the Kubelka-Munk function of reflectance from the resin. This function renders the reflectance data proportional to concentration (13). For each anion it is apparent that as concentration increases, the rate of reaction of the Fe2+with the ligand increases. Furthermore, the effect of the ions increases in order of C2H30< < C1- < Br- < NO,. This order is identical with that of the affinity of these ions to a strongly basic ion-exchange resin. Both of these observations are consistent with our model of selectivity based on temporary misalignment of the ligands. That is, both conditions that should increase ligand mobility bring about an increase in the rate at which color development from Fe2+ occurs. An alternate approach for studying the effect of the mobility of the ligand is to use a similar ligand with more sulfonate functional groups. This was achieved with 2,4-BDTPS (see Figure l),which contains the same chelating functionality as ferrozine. Indeed, the only difference is the presence of a second 5,6-bis(4-phenylsulfonate)-1,2,4-triazine group. The two additional sulfonates will increase the number of sites of ionic binding to four. Also, the two additional phenyl rings
Flgure 5. Diffuse reflectance spectrum of Cu+ complex with immobilized ferrozine on anion exchange resin (curve a) and transmission spectrum of Cu+(ferrozine),in aqueous solution (curve b). Both systems contain 5 X M Cu+ and are buffered at pH 4.3 with acetic acidlacetab (0.07 F total acetate).
will increase the 7r interactions between the substrate and ligand. These additional sites of binding will result in a dramatically decreased mobility. Not only is the ligand less likely to become totally free in solution but the displacement of a single ionic site will not result in the "walking" movement speculated for ferrozine as the three remaining ionic sites will still inhibit movement. The properties of the immobilized tetrasulfonate ligand are as expected. Figure 4 illustrates that unlike the ferrozine system, a long-term selectivity for Cu+ over Fe2+is obtained. The reaction with Cu+ is essentially complete within 5 min; yet even after 240 min the signal from an equal amount and concentration of Fez+is scarcely detectable. Two comparisons are relevant here. In aqueous solution, the absorbance of the totally solvated Fe2+complex is more intense than that of the Cu+ complex (e = 32 200 at 565 nm vs. 9700 at 460 nm) (5). Therefore, as with ferrozine, the selectivity is attributable to the effects of immobilization. However, by using immobilized ferrozine and conditions identical with those for the 2,4BDTPS here, the selectivity of Cu+ over Fez+falls to 1.2/1 after 240 min. In comparison, the data in Figure 4 show a selectivity of 20/1 for Cu+ over Fe2+using 2,4-BDTPS with a 240-min reaction. Thus, the effect of the extra binding sites is, as predicted, a decrease in the extent to which the Fez+ complex interferes for longer reaction times. The reflectance spectra of the immobilized complexes provide further support of the proposed model. Curve a in Figure 5 shows the reflectance spectrum of the immobilized Cu+ complex with ferrozine. The transmission spectrum of the analogous totally solvated system is included for comparison as curve b. The spectra are presented in terms of the Kubelka-Munk function and absorbance, respectively, to make them amenable for comparison (13). There are significant differences apparent. For the immobilized complex
lg8
ANALYTICAL CHEMISTRY, VOL. 58, NO. 1, JANUARY 1980
-
e
e
1.0
e
g LL 0.5
c
o
0 0
0
I
25 450
600
750
-nm-
Figure 6. Diffuse reflectance spectrum of Fe2+(1.7 X
900
M) com-
plex with lmmoblllzed ferrozlne after 150 min (curve a) and transmisslon M) in aqueous solution (curve spectrum of Fe*+(ferrozine),(2 X b). Both systems are buffered at pH 4.3with acetic acid/acetate (0.07 F total acetate). the major spectral peak is shifted bathochromically by about 30 nm relative to the solvated system. Also, the major peak is split into two partially resolved peaks for the immobilized system. These differences are likely a manifestation of the strain placed on a ligand-metal system due to the ligand-resin interactions. The proposed distortion from tetrahedral toward planarity to accommodate the multisite ligand-resin interactions would lower the symmetry of the complex and lead to a splitting of the groups of degenerate d orbitals. Since the absorption band is due to a metal ligand charge transfer and the affected d orbitals are all occupied, the distortion could certainly result in a more complex spectrum. Since some of the filled d orbitals must be destabilized by distortion, the energy difference between the highest occupied metal orbital(s) and the unoccupied ligand orbital will decrease. Thus, the observed bathochromic shift in the portion of the spectrum available is consistent. The differences in environmental polarity of the systems as well as strain on the ligand itself may also contribute to the spectral shift. Similar differences are observed between the immobilized and solvated spectra of the Cu+-2,4-BDTPS complex. An alternate interpretation of the spectral changes is that the strain distorts the system to the point of lowering the ligand-Cu+ ratio to 1:l. Some ferroin-type ligands are reported to form this type of complex with Cu+ under appropriate conditions (5,14). Curve a in Figure 6 shows the reflectance spectrum of the Fez+-ferrozine complex. Once again the absorbance spectrum of the analogous solvated complex is included for comparison as curve b. For this system we have postulated that the complex has formed at the expense of the resin-ligand interactions. Without this there would be extreme distortion of the ideal octahedral geometry. The remarkable similarity between the spectra of the immobilized and free complexes gives no indication of undue strain on the immobilized complex. Furthermore, the similarity also provides evidence that the complex that forms on the resin is of the same ratio as the free complex. A study of the complexes of Fe2+with a similar ferroin ligand, 1,lO-phenanthroline, found that the spectral characteristics of 1:l and 2:l complexes were much different than those of the typical 3:l complex (15). While the above studies point toward a mechanism for selectivity based on a decreased availability of ligand it is also appropriate to consider a mechanism based on decreased availability of the metal. This could occur in several ways. The Fez+might bind directly to the resin and thus be masked or unavailable to the ligand. The loss of selectivity might arise from the slow conversion of a resin-metal complex to a ligand-metal complex. Since the resin functional groups are of positive charge, the Fez+ would have to bind as a complex anion. The major anions in solution (C1- and C2H302-)are
-
50
75
Time -Minutes
Flgure 7. Comparison of the rate of color formation of the Cu+ferrozlne system with CI- (0)and SO4*- ( 0 )as the counterion. M Cu+ and 0.5 g of resin were Twenty-five milliliters of 5 X equilibrated.
not good candidates for promoting this type of binding of Fez+. To verify this, a sample of resin without ligand was equilibrated with Fez+. Quantitation of the Fez+in solution after filtration verified that it was not bound or masked by the resin. Another mechanism by which the resin could make the Fez+ unavailable would be one of repulsion. Since the surface of the resin is positively charged, it might repel the positively charged Fez+. It could be argued that since Cu+ forms a stable complex chloro anion (C1- is present at 0.14 M from the hydroxylamine hydrochloride) it would be more suited to freely move to the surface. According to this mechanism Cl- would serve to catalyze the reaction of Cu+ but not Fez+ with the immobilized reagent. The function of the catalyst would be to carry the metal to the surface and then release it to the reagent. This mechanism does not seem to offer an explanation for the observed differences between ferrozine and 2,4-BDTPS with respect to the reaction of Fez+. That is, there would be no reason for 2,4-BDTPS (with its greater negative charge) to be less reactive than ferrozine if cation repulsion is the controlling factor. To further evaluate this mechanism, the reaction of Cu+ was followed in a low C1- environment. In this study chloride salts were replaced with SO:- salts and the resin was washed with NaZSO4prior to use. Figure 7 shows that the initial formation of the Cu+ complex is rapid even with minimal C1- present. Further, it appears that high concentrations of C1- serve to inhibit the complex reaction of Cu+. In the absence of C1- a stronger signal is ultimately obtained. This probably reflects a competition between C1and ferrozine for the Cu+. Although Cu+ uptake by the resin is quantitative, only a fraction is bound to the spectrophotometric reagent. In any event, it appears that C1- is not responsible for the unique reactivity of Cu+. Applicability. The experiments described above show that this type of procedure (e.g., immobilization of an analytical reagent) can be used to eliminate the signal from some potential interferents. However, interference can also occur through a blocking of the reaction sites. In this particular case before the system can be considered feasible, it needs to be shown that Fe2+does not interfere by masking or making the ligand unavailable to the Cu+. Since it is shown above that the 3:l Fez+ complex does not form or forms slowly, the concern here is with a stable, colorless complex of lower mole ratio that might form. If this occurs and Fez+ is present at high enough concentrations, it could affect the formation of the Cu+ complex by lowering the availability of reactive ligands. To investigate this possibility, we tested the uptake of Fez+by immobilized ligand. For a 5-min equilibration time, the Fez+uptake from a 5 X M solution by sufficient resin to provide a 15-fold molar excess of ligand was negligible. Thus, there is no evidence of extensive poisoning of the resin by Fez+.
ANALYTICAL CHEMISTRY, VOL. 58, NO. 1, JANUARY 1986
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1.o-
0.4-1
-a
c
,-.
LL
OL v
0'5j
LL
0
0
O
I
450
650
0
0
0
0
I
nm
a50
Figure 8. Experimental verification of the possibility of measuring Cu+ in the presence of Fe2+ with immobilized ferrozine. The spectrum of Cu+ complex alone (curve a), the mathematical combination of the spectra of the Fez+and Cu+ complexes (curve b), and the spectrum of a combination of Fe2+ and Cut reacted with immobilized ferrozlne (curve c) are shown. Equal concentrations and volumes of Cu+ and Fez+ were used. A 30-min reaction time was used.
Figure 8 shows the effect of Fez+on the reaction of an equal amount of Cu+. Here 1.25 X lo4 mol of Cu+ and/or Fez+in 25 mL has been equilibrated with 0.5 g of resin containing 10.0 mg of ferrozine/g of resin. Curve a represents the spectrum of Cu+ alone. Curve b represents the spectrum of Fez+ alone added to curve a, thereby giving a predicted spectrum of equal amounts of Cu+ and Fez+combined. Curve c is the actual spectrum of a mixture of Fez+and Cu+ and thus represents the experimental spectrum predicted by curve b. In this case the predicted interference error from Fez+ (curve b) in the determination of Cu+ is about 10%. The measured interference error (curve c) is even less than this. In either case this represents substantial improvement over the solution (nonimmobilized system) where the Fe2+ signal would be greater than that from the Cu'. The difference between the predicted Cu+ plus Fez+ spectrum and the experimental spectrum is likely due to a further slowing of the Fez+reaction in the presence of Cu+. Since Fez+ has a more demanding ligand alignment, its potential reaction sites arising from ligand mobility are also appropriate for Cu+ while the reverse is not necessarily true. Thus, initially, Cu+ further deactivates the resin toward Fez+. Applications of this technique will depend on a simple relationship between Cu+ and reflectance of the resin system. For reflectance spectrometry the most common approach to quantitation is through the Kubelka-Munk function of reflectance. Theoretically, the Kubelka-Munk function of the absolute reflectance of an infinitely thick sample should be linearly related to concentration. In order to obtain the absolute reflectance of a sample, relative reflectance measurements of sample vs. unreacted resin were corrected by multiplying by the reflectance of the unreacted resin relative to a BaSO., plate. Since the reflectance of BaS04 is close to total in the visible region, the relative readings are converted to the absolute scale. Using absolute values and prepared calibration curves, we found a linear relationship from the origin through 3.5 X lo4 mol (50 mL of solution) using 0.5 g of modified resin. Negative deviation from linearity is observed a t higher levels. Since the uptake of Cu+ is complete, the response should be dependent on moles instead of volume and concentration. However, with the uptake of Fez+being small, its reaction is not expected to be a function of the total moles but rather the concentration. Figure 9 shows the effect of diluting a sample of either Cu+ ( 0 )or Fe2+ (0) while the number of
moles is held constant. Other reagents involved are adjusted proportionally to hold their concentrations constant. As expected, the dilution has minimal effect on the Cu+ signal while the Fez+signal decreases as the volume of solution increases. This suggests two factors that may be of importance in adapting this type of procedure to a particular analysis. First, the sensitivity to Cu+ will increase if larger volumes of solution are used. The resin system serves as an effective preconcentrating agent in addition to improving selectivity. Second, the fact that only the Cu+ signal is unaffected by dilution indicates that sample dilution will improve selectivity. Of course, the necessity to add large amounts of reagent (buffer and hydroxylamine) to achieve the same concentration is a drawback to using large volumes. Unless ultrapure reagents are used, the resulting blank is proportionally increased. Several experiments described above with respect to investigations of the explanation of selectivity suggest means to further improve the selectivity. It was shown that the response of Fez+is diminished, particularly over a long range, if ligand mobility is decreased. In particular, lowering anion concentration dramatically slows the formation of the interfering Fe2+complex. However, in terms of applying this to real samples we have found that the lower anion concentration (achieved by using less hydroxylamine hydrochloride) also slows the formation of the copper complex. If hydroxylamine hydrochloride is lowered from 1to 0.2% (by mass), the F(R? value does not reach a constant level within an hour. Use of the tetrasulfonated 2,4-BDTPS virtually eliminated the long-term reaction of the Fez+complex. With this ligand the reaction of Cu' is essentially complete after 5-10 min, similar to the ferrozine (see Figure 4). The only drawback to this is that the sensitivity to Cu+ is diminished relative to the ferrozine system. This is contrary to the behavior of the solvated ligands where 2,4-BDTPS is more sensitive (e = 9700 vs. 4320). This probably reflects a less stable 2,4-BDTPS complex relative to ferrozine. The extra resin binding sites probably make this immobilized ligand less willing to accommodate the metal and shift the distribution of Cu+ toward the immobilized chloro complex anion. Registry No. 2,4-BDTPS, 98991-46-9;C2H3OZ-,71-50-1;Cl-, 16887-00-6;Br-, 24959-67-9; NO3-, 14797-55-8;Cu, 7440-50-8;Fe, 7439-89-6; ferrozine, 98991-45-8; Dowex lXB, 12627-85-9.
LITERATURE CITED (1) Seitz, W. R. Anal. Chem. 1984, 56, 16A. (2) Ditzler, M. A.; Doherty, G.; Sieber, S . ; Allston, R. Anal. Chlm. Acta 1982, 142, 305. (3) Ditzler, M. A. TrAC, Trends,Anal. Chem. (Pers. E d . ) 1983, 2, V I I I . (4) Schilt, A. A.; McBride, L. The Copper Reagents: Cuproine, Neocuprolne, and Bathocuproine", 2nd ed.; G. F. Smith Chemical Co.: Columbus, OH, 1972. (5) McBride, L. "The Iron Reagents", 3rd ed.; G. F. Smith Chemical Co.: Columbus, OH, 1980.
Anal. Chem. 1986, 58,200-202 Kundra, S. K.; Katyal, M.;Singh, R. P. Anal. Chem. 1974, 46, 1605. Schilt, A. A.; Chriswell, C. D.; Fang, T. A. Talanfa 1974, 21, 831. Hutchinson, D. J.; Schllt, A. A. Anal. Chlm. Acta 1983, 154, 159. Yee, H. Y.; Goodwin, J. F. Clin. Chem. (Winston-Salem, N . C . ) W74, 2 0 , 188. Ditzler, M. A.; Allston, R. A.; Casey, T. J.; Spellman, N. T.; Willls, K. A. Appl. Spectrosc. 1983, 37, 269. Jaselskis, B.; Nelapaty, J. Anal. Chem. 1972, 4 4 , 379. Ordemann, D. M.;Walton, H. F. Anal. Chem. 1976, 48, 1728. Kortum, G. "Reflectance Spectroscopy"; Sprlnger-Verlag: New York, 1969.
(14) Lundgren, J. L.; Schllt, A. A. Anal. Chem. 1977, 4 9 , 974. (15) Lee, T. S.; Kolthoff, I . M.;Leusslng, D. L. J . Am. Chem. SOC. 1948, 7 0 , 3596.
RECEIVED for review March 18, 1985. Resubmitted August 16, 1985. Accepted August 16, 1985. Financial support for this research through National Science Foundation Grants PRM-8117525 and CHE-820613 is acknowledged.
Critical Study of Chromatic Parameters in Color Break I ndicator Eva1uat ion J. Martinez Calatayud,* M. C. Pascual Marti, and P. Campins Falco Departamento de Quimica Analltica, Facultad de ciencias Qulmicas, Burjasot, Valencia, Spain
Some chromatic parameters are contrasted In order to arrive at the one most sultable for lndlcator evaluation. Several lndlcator transltlon curves are studied and slmulated transltion curves are performed. Total color dlfference, A€ * , was found to be the most adequate parameter for color break Indicator evaluation.
Titrations are, a t present, one of the most useful methods in analytical chemistry. Titrations require the use of one of several dyes in order to detect the end point; this detection is based on the transition color of the indicator near the equivalence point of the reaction between titrant and titrand. The number of recommended indicators for any single titration is always high (five or six as minimum). There are some published papers dealing with the objective selection of the most adequate indicator for a specific determination. The study requires an objective evaluation of color transition of the indicators (clarity and accuracy) by using tristimulus colorimetry. Different parameters have been used to evaluate the quality of indicators; Reilley proposed the grayness, length, and position of color transition curves ( I ) . Photometric titration curves were used by VytFas (2). Judson (3) deals with chromaticity difference values, AC, between adjacent points (also called chromatic separation, Au) and luminosity parameters. The papers by Bhuchar are concerned with the specific color discrimination (SCD) (4) and total color difference, LE* (also called chromatic difference) (5). Cacho in 1982 (6) introduced the optical concentration, J,for indicator evaluation. The standard deviation of color matching of MacAdam, A S (7,8), has been used in some few cases. The ideal parameter must have a uniform behavior for different shapes and situations of color transition curves and, of course, be in agreement with the visual color perception. This paper deals with a critical study of these parameters, in order to recommend the most suitable one for analytical chemistry studies. This attempt is based on simulated transition curves performed with sets of single or mixture dye solutions. The study has been completed and includes some actual titration curves previously performed (9-12). THEORY Chromaticity difference, AC, for CIE, L*a*b* is expressed as AC = ((Aa*)' (Ab*)')l/'
+
Total color difference is given as
AE* = ((AL*)2+ (Aa*)2 + (Ab*)2)1/2 Optical concentration, J , is
J = ( X + Y + Z ) / ( X o + Yo + 2 0 ) Yo = 100 20 = 118 for standard illuminant C. X, Y, and 2 are tristimulus values Xo = 98
of a solution obtained with absorbance data. Standard deviation of color matching, A S is given as
AS2 = gllAx2 + 2g,,AxAy
+ gZ2Ay2
where Ax and Ay are the changes in CIE 1931 coordinates and gll, 2g12, and g2, are the values of constants of MacAdam ellipses corresponding to color points. Specific color discrimination is given as
SCD = (l/)AS/ApH where pH can be replaced for the corresponding parameter v, E , etc.
EXPERIMENTAL SECTION Reagents and Apparatus. Many indicator transition curves have been tested, in this paper some of them have been chosen as representatives (different color area and shape of the curve): eriochrome black T, PAR, and arsenazo I11 for Pb(I1) titration; tiron for Fe(II1) titration; methyl orange-indigo carmine mixture for CO2- titration; and crystal violet in the nonaqueous titration. Some simulated titration curves are performed. Any point of such curves was obtained with the below cited procedure and corresponds to the color of a solution formed with different dyes. A set of such solutions, varying the dye ratios, gives one curve. The dye mixtures are naphthol green B-methyl red, indigo carmine-thymol blue, and indigo carmine-thymol blue-neutral red. Two transitions with a single dye are also performed: methyl red and naphthol green B. A Shimadzu UV-240 spectrophotometer with a 1cm path length cell and peristaltic pump, LKB, was used. Procedure. Color transition curves were obtained by recording absorbance and transmittance values in the 380-770 nm range, Y , 2) were calculated at 10-nm intervals. Tristimulus values (X, by using the CIE distribution function for standard illurninant C at 10-nm intervals. RESULTS AND DISCUSSION For the parameters studied, optical concentration depends on the chromatic system used; thus the CIE-L*a*b* 1976 system (13) was the choice for this study, because it seems to define, to date, the most perceptually uniform color space
0003-2700/86/0358-0200$01.50/00 1985 American Chemical Society
