Determination of Trace Amounts of Chromium(ll1) Using Chemiluminescence Analysis W . Rudolf Seitz Southeast Water Laboratory, Encironmental Protection Agency, Athens, Ga. 30601 Wallace W . S u y d a m and David M. Hercules Department of Chemistry, Unicersity of Geargia, Athens, Ga. 30601 Trace Cr(lll) is determined by measuring Cr(lll)-catalyzed light emission from luminol oxidation by peroxide. Light emission catalyzed by other metals is quenched by adding EDTA to form complexes that are not active as catalysts. The Cr(ll1)-EDTA complex is kinetically slow to form. The detection limit for Cr(lll) is5 x 10-l0M (CO. 0.025 ppb). The minimum detectable quantity is 25 picograms. Response is linear up to 10-6M. The method was successfully applied to several natural water samples. No sample pretreatmentwas required. Analysis time is less than 30 minutes.
THEDEVELOPMENT OF CHEMILUMINESCENCE methods opens the possibility of rapid, highly sensitive analyses using inexpensive instrumentation. This capability is valuable when large numbers of samples must be analyzed or measurements a t a variety of locations must be made, as in monitoring trace metal concentrations in natural waters. The intrinsic sensitivity of the luminol system t o small metal concentrations makes it very attractive for trace analysis. Metal ions catalyze the oxidation of luminol (5-amino-2,3dihydrophthalazine-l,4-dione) by hydrogen peroxide in basic aqueous solutions. This extensively studied reaction is one of the most efficient chemiluminescent reactions known (1-3). In the presence of excess reagents, the intensity of light emission is proportional t o metal catalyst concentration, a property that can be made the basis for trace metal analysis. Babko and coworkers (4-7) have reported methods for cobalt, copper, and iron, all based on catalysis of the luminol reaction. By simply mixing the reactants while exposing the container t o a photographic film and measuring exposure as a function of concentration, they found detection limits of 1, 3, and 10 ppb for cobalt, copper, and iron, respectively. Recently Santini and Pardue (8) have constructed a more sophisticated apparatus for using the luminol reaction analytically, but they have not reported any analytical applications. Chemiluminescence methods are capable of resolving different chemical forms of a metal a t the trace level. For example, Cr(II1) catalyzes the reaction efficiently while Cr(V1) does not catalyze the reaction a t all. Also, for several metals, organic complexes d o not catalyze light emission while the (1) H. 0. Albrecht,Z. Phys. Chem., 136,321 (1928). (2) E. H. White, “A Symposium on Light and Life,” W. D. McElroy and B. Glass, Ed., The John Hopkins Press, Baltimore, Md., 1961, p 183. (3) F. McCapra, Quart. Rec.,20,485 (1966). (4) A . K. Babko and N. M. Lukovskaya, Zli. Anal. Khim.,17, 50 (1962). ( 5 ) A. K . Babko and L. I . Dubovenko, 2.Anal. Clieni., 200, 428 (1964). (6) A . K. Babko and N. M. Lukovskaya, Zuood. Lab., 29, 404 (1963). (7) A. K. Babko and I. E. Kalinichenko, Ukr. K h m . Zh., 31, 1316 ( 1965). (8) R. E. Santini and H. L. Pardue, ANAL.CHEW,42, 706 (1970).
“free” ion does (9). This effect has been used t o measure concentrations of organics by observing the extent to which copper-catalyzed luminescence is quenched in their presence (IO,11). A practical difficulty in using the luminol reaction for trace analysis is that several metals catalyze light emission making it necessary t o find some means of measuring one metal ion in the presence of others. This paper reports a method for specifically measuring trace concentrations of free Cr( 111) in the presence of other metals ions. Chromium catalysis of the luminol reaction has been reported as a n interference in analytical methods for other elements (12, 13) but no methods for Cr(II1) have been published. Specificity for Cr(II1) is achieved by adding EDTA and taking advantage of the fact that the Cr(EDTA) complex is kinetically slow to form (14). EDTA complexes metal ions that would otherwise interfere and makes them unavailable for catalysis. The method is sensitive to better than lOP9Mand is fast and specific. Natural samples may be analyzed with no pretreatment other than EDTA addition. Since the biological effects of chromium differ for Cr(II1) and Cr(VI), this method should be particularly helpful in setting water quality criteria for chromium. EXPERIiMENTAL
Apparatus. For convenience and to develop continuous analysis, the catalysis of luminol oxidation was carried out in the flow system diagrammed in Figure 1. The reactants are continuously mixed in a cell positioned directly in front of a photomultiplier tube which measures light emission. Luminol is dissolved in a 0.1M KOH-H3B03 buffer, which controls the pH a t which the reaction occurs. Luminol-buffer is mixed with hydrogen peroxide directly in the flow system; this avoids solution deterioration, which occurs on standing. The background solution is similar to the sample in composition except that it contains no trace metal catalyst. When the background solution flows through the cell, one observes the level of light emission characteristic of the absence of trace metal catalysts. For Cr(II1) analysis. the background solution is 2 X 10-2M EDTA. Slugs of sample are inserted into the background flow line using the sample injection valve. A Harvard Model 975 infusion pump drives the reactants through the cell. The syringes are 50-ml disposable plastic (9) A. K. Babko and N. M.Lukovskaya, Ukr. Kliini. Zh., 34,1279 (1968). (10) A. A. Ponomarenko and B. I. Popov. Zh. Anal. Khrm.. 19, 1397 (1964). (11) A. A. Ponomarenko and L. M. Amelina, Zh. Obshch. Khim., 35,2252 (1965). (12) I. E. Kalinichenko, Ukr. Khirn. Zh., 35,755 (1969). (13) I. E. Kalinichenko and 0. M. Grischchenko, ibid., 36, 610 (1970). (14) R. E. Hamm, J. Amer. Cliem. SOC..75,5670 (1953). ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
0
957
TO WMTE
I-
~ H - H ~ M(J / RECORDER
.
AMPLIFIER
,
CELL
Ill 6!
i
TEFLON TUBINQ
LIGHT EMISSION
n
n
Y’
I
I
I NITROGEN
Figure 1. Diagram of flow system for chemiluminescence determinationof trace metals
TIME (seconds)
Figure 3. Principle of reaction cell operation t, = Residence time in cell L(t) = Light intensity at a given time, t, after mixing
F9rnrn-l
@ END VIEW
Figure 2. DetaiIed view of reaction cell
syringes. Plastic is preferred since glass is capable of adsorbing and desorbing trace metals, thereby affecting light intensities; also plastic syringes with a rubber tipped plunger flow more smoothly than glass syringes. Three syringes can be mounted on one pump even though the mounting is designed for two. The lis-in. diameter thin-walled Teflon (DuPont) tubing is connected to the syringes by a short piece of l/B-in. i.d. Tygon tubing. The flows from the Hz02and luminol-buffer syringes are joined by a glass “Y.” A Chromatronix SV-8031 sample injection valve is used to insert slugs of sample into the background flow line. In one position, the background solution passes through a bypass loop, and sample can be drawn into the sample loop using a syringe. In the other position, the sample loop is connected to the flow line. In operation, air bubbles are left on either side of the sample to prevent mixing between sample and background solution. While the volume of the sample loop used for these studies was 3 ml, it could be reduced to 1 ml without seriously impairing the quality of the data, if smaller samples were necessary. With minor modifications, even smaller samples could be used. Figure 2 shows a detailed drawing of the reaction cell, The Teflon tubing of the flow system fits over 3-mm 0.d. glass nipples. Short sections of 1/3z-in.i.d. Teflon tubing are fitted inside the glass nipple and extend into the cell, ensuring that the reagents initially mix toward the center of the cell and reducing the possibility of solution back-up into the tubing. Stirring is provided by bubbling nitrogen through a 1/64-in, i d . Teflon tube fitted inside the 1/32-in.i.d. Teflon tubing, 958
*
ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
The small diameter produces smaller bubbles which stir more efficiently than large bubbles. The high pressure drop through narrow tubing reduces fluctuations in gas flow rate that result from fluctuations in the liquid pressure in the cell. Visual observation of mixing between colored and clear solutions indicated that this method of stirring is quite efficient. Gas flow to the cell is monitored with a flowmeter. The flow rate used in analyses was generally 35-40 ml/min. The chemiluminescence cell operates as an integrating device. In Figure 3 a hypothetical stopped-flow kinetic curve of light emission as a function of time after mixing is plotted for the luminol reaction. Light emission catalyzed by a particular increment of Cr(II1) solution entering the reaction cell in the continuous flow system will follow these stoppedflow kinetics while it remains in the cell. After achieving a steady state, there will be increments of Cr(II1) solution at all positions along the stopped-flow kinetics curve, and the observed light emission will be the sum of the light emissions catalyzed by each increment of Cr(II1) solution. If we assume that all Cr(II1) ions stay in the cell for an equal length of time, then at steady state the light emission will be the integral of the stopped-flow kinetic curve from the time of mixing (t = 0) for as long as the Cr(II1) ions remain in the cell. This time period has been designated as t,, the residence time in the cell, equal to the cell volume divided by the flow rate. The conditions used for the work reported here were cell volume 1.2 ml and flow rate 13.2 ml/minute (4.4 ml/minute per syringe). This gives a t , value of 5.5 seconds. Data for Cr(II1) using this apparatus are shown in Figure 4. As a slug of Cr(II1) sample goes through the cell, light emission reaches a constant value as steady state conditions are achieved. When the sample slug ends and background solution starts through the cell again, light emission quickly returns to the background level. The low frequency noise during Cr(II1)-catalyzed light emission is attributed to variations in flow rate and mixing rate. Its magnitude is about 5-10 of the peak height at all Cr(II1) levels. Light intensities were measured by an 1P22 photomultiplier operated at 900 volts using a Princeton Applied Research (PAR) Model 280 high voltage power supply. The photomultiplier tube was housed in a PAR Model 180 housing, and the anode current was measured with a PAR Model 270 DC photometer and recorded on a Hewlett-Packard Moseley 7100B strip chart recorder. The total cost of this equipment is less than $4000. The PAR instrumentation and the Hewlett-Packard Moseley re-
6 I
I
\
l 0
20
>
*
40
60
80
100
120
140
MINUTES
i
Figure 4. Light emission peaks catalyzed by Cr(II1) samples passing through the reaction cell
Figure 5. Change in chemiluminescence intensity with time due to Cr(II1)-EDTA complex formation
Conditions: 1WM H202, 10-3M luminol, 10-1M KOH-H3B03 buffer, cell pH -10.5; 10-4M EDTA added to Cr(II1) in deionized water
Conditions: 107M H202,10-3M luminol, 10-IM KOH-H3B03 buffer, cell pH 10.3, 2 X 10-2M EDTA in background solution and sample bottle, 10-6M Cr(III), room temperature
Peak 6 Peak 4 Peak 2
= 6.0 X = 4.0 X = 2.0 X
10-8M Cr(III) 10-8M Cr(II1) 10-8M Cr(II1)
1
LOG PEAK HEIGHT (ARBITRARY UNITS)
3.0
H
5 mins
corder are of higher quality than is required for this work so an apparatus with equivalent capability for Cr(II1) analysis can readily be constructed for less than $3000. Reagents. Luminol from Eastman Organic Chemicals was converted to the sodium salt by recrystallization from base and was purified by recrystallization from water. The purified sodium luminol was dissolved in the 0.1M H3B03-KOHbuffer, which was used to control the pH in the reaction cell. The concentration was maintained constant while the amount of KOH was varied to achieve the desired pH. Hydrogen peroxide solutions were prepared by diluting 3 H202. Both the HaB03-KOHbuffer and the HzOz solutions were made lW3M in EDTA, to prevent trace metal impurities in these reagents from catalyzing light emission. The EDTA in the background and the sample will also do this, but it is more effective to complex the trace metal impurities before they enter the cell. All reagents were prepared using water from a Continental Water Conditioning Company deionization system. Procedure. A sample volume of 500 ml was used in this study. Such a large sample is convenient because the amount of sample consumed for one measurement (2.5-3.0 ml) is small relative to the total sample volume: Analysis can be done by the method of standard additions without having to correct for volume changes. Smaller sample volumes can be used. To prepare samples, 50 ml of 0.2M EDTA stock solutions are added to 450 ml of sample. A 500-ml plastic bottle was used volumetrically by marking the 450-cc and 500-cc levels. One per cent precision ( 5 ml) is attainable with this procedure. No sample pretreatment, not even filtration, is necessary even when running samples of river water that have a high concentration of suspended solids. Standard additions were made with a 50- or 100-p1 Grunbaum pipet. A 0.100M standard solution of Cr(II1) was
, -9
./ -8
,
,
-7
-6
,
LOG Cr (111) CONC.
Figure 6. Plot of log peak height cs. log Cr(II1) concentration Conditions: 10-2M H202, 10-3M luminol, 10+M KOHH3BOabuffer, 2 X 10-2M EDTA in sample and background, cell pH -10.3, 10-3M EDTA in H202 and luminol solutions
prepared from Cr(N03), and a 10-3Mstandard was prepared by dilution. RESULTS AND DISCUSSION
Specific chemiluminescence analysis for free Cr(II1) (not organically complexed) is achieved by adding EDTA to the Cr(II1) sample. The Cr(II1)-EDTA complex is thermodynamically stable but kinetically slow to form. All other metals that catalyze the luminol reaction rapidly form EDTA complexes and, as the EDTA complex, have a greatly reduced catalytic effect. Once formed, the Cr(II1)-EDTA complex does not catalyze the luminol reaction. This was demonstrated by heating Cr(II1) in the presence of excess EDTA to form the complex and making standard additions to a deionized water sample. No light emission was observed. T o determine Cr(II1) by chemiluminescence, the kinetics of Cr(II1)-EDTA formation must be known to be able to select operating conditions so that analysis can be completed without significant complex formation. Hamm’s (14) studies of the Cr(II1)-EDTA system have shown: ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
959
CHROMIUM PEAK I HEIGHT
-2
-3 LOG
I
H 2 0 2 CONCENTRATION
Figure 7. Variation in chemiluminescence intensity with H 2 0 2concentration Conditions: 10-3M luminol, 10-lM KOH&Boa, 10-6M Cr(III), 2 X 10-M EDTA in sample and background, reaction cell pH 10.3
PEAK HEIGHT (ARBITRARY
UNITS) 40
20 L
l -4
I
-3
-2
LOG LUMINOL CONCENTRATION
Figure 8. Chemiluminescence intensity cs. luminol concentration Conditions: W Z MH202, 10-lM KOH-H3B03 buffer, lO-'M Cr(III), 2 X 10-2M EDTA in sample and background, 10-3M EDTA in luminol and H 2 0 2solutions, cell pH 10.3
The rate of Cr(II1)-EDTA formation is independent of EDTA concentration. This makes it possible t o vary EDTA concentration with no significant effect on the analysis. The rate of Cr(II1)-EDTA formation increases as the pH is increased from 1 t o 6. ' In this study, EDTA was added to Cr(II1) samples as the disodium salt, thus adjusting the pH to 4'4' The rate Of Cr(lll)PEDTA formation may be slowed by lowering pH. The rate of Cr(II1)-EDTA formation increases exponentially with temperature. Hamm also reported that a t higher Cr(II1) concentrations the rate of Cr(II1)-EDTA formation is proportional to the concentration of Cr(II1). However, a t the Cr(II1) concentration levels used in this study, the rate of Cr(II1)-EDTA formation was not first-order with respect to Cr(I1I) concentration. Figure 5 shows the decay in the amount of light emission catalyzed by 1OV6MCr(II1) in the presence of 2 X 10-2M EDTA as a function of time. In analysis, EDTA was always added t o the sample less than a minute before starting the determination. Response to Cr(II1) Concentration. Figure 6 shows a plot of Cr(II1) peak height as a function of concentration from 2 x lO-9M Cr(II1) t o 5 X 10+M Cr(II1). This calibration was prepared by making standard additions of Cr(II1) to deionized water. Above 1 X 10-6M Cr(II1) where the linearity of light emission us. Cr(II1) concentration falls off, peaks characteristically rise to a high initial value and then quickly decay to a constant value. Apparently Cr(OH)B (Ksp = is precipitating from solution a t high Cr(II1) 960
ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
96
130
I04
112
IO8
116
I20
I24
PH
Figure 9. Effect of pH on chemiluminescence intensity Conditions: 10-2.M luminol, 10-3MH202,10-1M KOH-H3B03.pHwas varied by adding different concentrationsof KOH
concentrations. The initial spike is believed to correspond to supersaturation of Cr(II1) in the reaction cell while the subsequent decay is due to precipitation causing some of the Cr(II1) t o be unavailable for catalysis. This effect places an upper limit on the concentration of Cr(II1) which can be measured by this technique. The lower limit is imposed by low level spontaneous light emission from luminol in the presence of base and peroxide. Under the conditions used in this study, the detection limit is 5 X 10-'OM Cr(III), where the detection limit is defined as the concentration producing a signal twice the noise level. The EDTA added to the H202 and luminol-buffer solutions complexes any Cr(II1) in the reagents so there is no significant reagent blank. Effect of HzOLConcentration. Figure 7 shows the Cr(II1)catalyzed chemiluminescence as a function of H202 concentraction in the presence of 10P3Mluminol. Below 5 X 10P3M H 2 0 2 ,light emission is proportional to peroxide concentration. Further increases in peroxide concentration d o not appreciably increase light emission. This effect has been used to analyze for peroxide (4). A H202concentration of 10-2M was selected for analysis. Effect of Luminol Concentration. Figure 8 shows Cr(1II)catalyzed luminescence as a function of luminol concentration in the presence of 10-*MHz02. Two effects are discernible. At low concentrations, the amount of luminol can be the limiting factor determining the intensity of light emission. At high concentrations adding more luminol reduces light emission, This may be due t o luminol forming a compiex with Cr(II1) ions, thereby reducing the availability of Cr(II1) for catalysis. An initial crude optimization of reagent concentrations indicated that 10-3Mluminol would be a good concentration for analysis so this level was used for most of the data reported here. Only later did we realize that additional sensitivity could be obtained using 4 X lO-4M luminol as shown in Figure 8. Effect of pH on Light Emission. Figure 9 shows the intensity of Cr(II1)-catalyzed light emission as a function of reaction cell pH. Although the maximum efficiency occurs at a p H around 10.9, the most convenient p H t o work at is that of the pK for the fourth proton of EDTA, 10.3. At this pH, HEDTA-3 and EDTA-4 are present in equal concentrations and have a buffering effect in addition to the buffering from the boric acid-KOH. The variation in light emission with p H has two important analytical consequences. First, if unknowns are t o be compared with standard solutions, the cell pH must be the same
Table I.
CHROMIUM HEIGHT
PEAK
i
LL 0 1
I
2
4
6
8
IO
FLOW R A T E (rnls/rnin/syringe)
Effect of Complexing Agents on Cr(II1) Catalysis of Luminol Oxidation Peak height, Catalyst relative units Uncomplexed Cr(II1) 100 Cr(II1)-glycine 8 Cr(II1)-citrate 0 Cr(II1)-tartrate 3 Cr(III)-2,4-pentanedione 5 Conditions: 10-2M H202,10-M luminol, 10-lM KOH-H3B03 buffer, 2 X 10-2MEDTAin sample and background, cell pH -10.5, 10-7Mconcentrationof catalyst, 10-3MEDTA in HzO?and luminol.
Figure 10. Chemiluminescence intensity as a function of flow rate Conditions: 10*MH~02,10-3Mluminol 10-1M KOH-HBB03buffer, 2 X 20-M EDTA in sample and background, cell pH 10.3, 10-3M EDTA in luminol and HzOz
for both. Second, if the equilibrium pH in the reaction cell differs between the sample and the background solution, the light emission catalyzed by a Cr(II1) sample will vary with time as the reaction cell pH moves toward the new equilibrium value. Thus, buffer systems should show a high buffer capacity. Effect of Flow Rate. Increasing the flow rate through the cell has two effects. The amount of catalyst entering the cell per unit time is proportional to flow rate. Thus, if the light emitting reaction goes to completion before the catalyst leaves the cell, one would expect the intensity of emitted light to be proportional to flow rate. Increasing the flow rate also reduces the residence time in the cell for the catalyst. If the light-emitting reaction does not go to completion within the cell, then reducing the residence time results in less of the light emission occurring in the cell and more light being lost in the exit tube from the cell. Figure 10 shows Cr(II1) catalyzed light emission as a function of flow rate. At low flow rates, the increase in peak height is almost proportional to the flow rate, while at higher flow rates the effect of shortened residence time in the cell becomes more important. These data imply that an increment of Cr(II1) sample will have lost virtually all of its catalytic activity after 10 seconds in the cell (10 seconds is the residence time for a flow rate of 2.25 ml/minute per syringe). This is probably because all the Cr(II1) will have been oxidized to Cr(V1) by the peroxide; Cr(V1) does not catalyze luminol oxidation. The chemical species oxidizing the luminol to produce light is very likely an intermediate formed in the oxidation of Cr(II1) to Cr(V1) by peroxide. The flow rate used for analysis was 4.41 ml/min per syringe. Although faster flow rates give somewhat greater sensitivity, the syringes empty very fast at these flow rates and the operator has less time to make standard additions. Effect of Complexation. It has been stated above that the Cr(II1jEDTA complex does not catalyze luminescence. Several other complexes of Cr(II1) were tested for catalytic activity. Solutions of 10-3MCr(III) in the presence of 10-2M ligand were prepared. Because kinetic inertness is a general property of Cr(II1) complexes, each solution was heated to make certain the complex would form. Table I lists the complexes that were tested and the intensity of light emission catalyzed by each. It is not certain whether the residual cata-
Table 11. Cations Tested for Interferences
Cation Co(II) Co(I1) CO(I1) Fe(111) Fe(II1) Fe(I1I) Fe(I1) Fe(I1) Sn(IVp Sn(1Vp Sn(IV)a Mg(I1) Ca(I1) Zn(I1) Ba(I1) Sr(I1) V(V)
Pb(I1) Mn(I1)
Al(II1) Ni(I1) '%(I) Cu(I1)
Cation concentration, (Cr(II1) concn 10-7M)
Light from Cr(II1) cation (relative units) (Cr only = 100)
+
10-1
3 5 5
x 10-7 x 10-7 x 10-0 10-5
x 10-5 2 x 10-5 4 x 10-5 2 x 10-5 6 x 10-5 io x 10-5
1 5
10-4 10-4 10-4
10-4 10-4 10-4 10-4 10-4
10-4 lo-'
10-4 10-4 10-4
125 175 219 124 159
176 143 186 92 65 42 LOO LOO LOO 100 100
100 100 100
100 100 100 100 100 100
Cd(I1) 10-4 Conditions: IO-ZM H202,lO-3M luminol, 10-IM KOH-H3BO3 buffer, 2 x lO-*MEDTA in sample and background, 10-?M EDTA in luminol and buffer. cell pH 10 2. 5 For Sn(1V).the Cr(II1)concentration was 6 X 10-'M.
lytic effect is due to uncomplexed Cr(II1) in the Cr(II1)--ligand standard or to the activity of the Cr(II1) complex itself. Cr(II1) organic complexes may fail to catalyze light emission because the complexation stabilizes Cr(II1) with respect to Cr(V1) so that H 2 0 2will no longer oxidize the Cr(II1) at the pH in the reaction cell. If the active species that oxidizes luminol is in fact generated in the oxidation of Cr(II1) to Cr(VI), this would explain why complexation inhibits catalysis. Interferences. Table I1 lists the cations which were tested for interferences. Of these ions, only Sn(IV), Co(II), Fe(II), and Fe(II1) interfere. Sn(1V) reduces luminescence presumably by oxidizing Cr(II1) to Cr(V1). This is not a true interference since this oxidation would have already taken place in naturally occurring samples. The Co(II), Fe(II), and Fe(II1) interferences can be compensated for by running a blank. This is done by heating the sample loop of the injection valve for two minutes in ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
961
Table 111. Chromium(II1) Analysis of Natural Water Samples
Sample
(MI
Oconee River
2 . 5 X 10-8 3 x 10-8 4 x 10-8 2.0 x 10-8 2.2 x 10-8 2.0 x 10-8 1 . 8 x 10-7 1.9 x 10-7 2.0 x 10-7 0.8 x 10-7 1 . 0 x 10-7 1 . 1 x 10-7
Lake Lanier Tap water Tara Pond
Cr(II1) concentration found Average
Equivalent Cr(II1) concn of blank, ( M ) 2
x
10-7
4
x
10-8
3
x
10-8
2
x
10-7
Table IV. Increase in Sample Peak and Corresponding Blank with Time for Tara Pond Sample Sample peak, Blank peak, arbitrary arbitrary Time, minutes units units Difference 0 18 42 59 80
75 91 89 96 96
46 64 59 70 68
29 21 30 26 28 Conditions: 10-2M H202, 10-3M luminol, 2 X 10-2M EDTA in sample and background, cell pH -9, 10-3M EDTA in H202
and luminol. water at 80 to 90 OC followed by cooling to room temperature for 6-8 minutes. This procedure causes all the Cr(II1) in the sample loop to form the Cr(II1)-EDTA complex which does not catalyze the reaction. The light emission catalyzed by Fe(II), Fe(III), and Co(I1) is unchanged by heating and cooling the sample loop and may be subtracted from the total. The possibility of running a zero-chromium blank greatly enhances the capability of the method. The Co(II), Fe(II), and Fe(II1) interferences prevent analysis only when they are present in sufficient excess to make the blank values large relative to the Cr(II1)-catalyzed luminescence. No anion effects of luminescence were observed at the following levels: Cl--O.lM, S042--0.01M, N03--0.01M, PO42--10- 4M, Br=l O+M, COS2--10- 4M, and F2--10-SM. In some samples other chemiluminescing systems may interfere. For example, HzOz is known to react with NaOCl producing an emission peaking at 635 nm (15). If this interference is present, it can be eliminated by appropriate filtering of the light from the cell. Analysis of Natural Waters. The method was applied to some natural water samples from the Athens, Ga., area. Table I11 lists the samples and the concentration of Cr(II1) found. Tara Pond is a small pond receiving the sewage of a nearby mobile home park. Both Tara Pond and the Oconee River are high in particulate matter. Tara Pond is high in organic matter. Each sample was analyzed in triplicate. N o sample pretreatment was used. EDTA was added just prior to analysis. The blank was run after standard additions of Cr(I1I) were made. Figure 11 shows a typical recorder tracing for analysis of Tara Pond water. In all four samples, there was a sig(15) H. H. Seliger, Anal. Biorhem., 1,60(1960). 962
ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
Figure 11. Typical recorder tracing for the analysis of Tara Pond water 10-M H202, 10-3M luminol, 2 X 10-M EDTA in sample and background, 10-3M EDTA in H202 and luminol-buffer, 10-lM KOH-HsB08, pH 9
nificant blank. The relative magnitudes of the blank and sample was one of the factors affecting precision. Certain difficulties were encountered with field samples that did not occur with prepared samples. The concentration of organic matter in Tara Pond water was so high that after 3 or 4 analyses, it coated the Teflon sample loop and rendered it wettable by water. When this occurs, the air bubbles are no longer effective in separating sample from background and the peak shape is distorted. The sample loop can be replaced in 5 to 10 minutes, and old sample loops can be cleaned in hot nitric acid. In natural samples, the top of the blank peak was not flat. Instead it rose to an initially high value and then decayed (as seen in Figure l l ) , causing difficulty in accurate measurement of peak heights. This effect was not due to Cr(II1) because the shape of the peak was the same for blank, sample, and sample plus standard additions of Cr(II1). In cases where this effect is severe (e.g., Tara Pond) it can be reduced by running the reaction at lower pH’s. Tara Pond was analyzed for Cr(II1) at a pH of 9. This reduces sensitivity but the intrinsic sensitivity of the method is still sufficient to measure natural Cr(II1) levels. The third problem with natural water samples was that the sample and blank peaks tend to increase with time. This can be attributed to an increase in Fe(II1)-EDTA by the slow reaction of hydrous ferric oxides (colloids) to lose Fe(II1) to the EDTA. Table IV lists the sample and blank peaks as a function of time for a sample of Tara Pond water. The possibility that the heat treatment of the sample loop might accelerate this reaction and thereby give wrong values was considered. If this is occurring, one would expect the difference between sample and blank to vary with the length of time for which the sample loop is heated. This was checked for Tara Pond water, and it was found that increasing
the heating time from 2 to 10 minutes did not affect the difference between sample and blank. Because the method described here measures only free Cr(III), it cannot be directly applied to samples where the value for total Cr is desired. The method also cannot be directly compared with existing methods for Cr because none of them measures only free Cr(II1).
RECEIVED for review October 20, 1971. Accepted January 11, 1972. This work was supported in part through funds provided the University of Georgia by PHS, N I H Research Grant No. G M 17913-01 from the National Institute of General Medical Sciences. Use of trade names does not imply endorsement by the Environmental Protection Agency or the Southeast Water Laboratory.
Influence of Solvent Matrix upon Phosphorescence Signals R. J. Lukasiewicz, J. J. Mousa, and J. D. Winefordner’ Department of Chemistry, University of Florida, Gainesville, Fla. 32601 The influence of methanol-water mixtures and of sodium chloride, sodium bromide, and sodium iodide aqueous solutions of several organic molecules at 77 O K upon phosphorescence signals is studied. Only a few per cent by weight of methanol in water, sodium iodide in a methanol-water mixture, and sodium chloride, sodium bromide, or sodium iodide in water resulted in phosphorescence signals at 77 O K several orders of magnitude greater than the phosphorescence signal for the same analyte in pure water solution. The salt solutions at 77 O K resulted in even larger phosphorescence signals than the methanolic solutions due to the additional effect of the heavy atom (iodide being the most effective of the three halides). The optimum aqueous solvent for routine analytical phosphorimetric measurements appears to be a 5-30% aqueous solution of sodium iodide. All studies are carried out with an open rotating quartz capillary cell.
APPLICATIONS OF MOLECULAR PHOSPHORESCENCE in chemical analysis have been limited since phosphorimetry was first reported as an analytical technique about fifteen years ago. Although phosphorimetry has been shown to be an extremely sensitive analytical method (Z-3), of considerable value in trace analysis of molecules which can achieve appreciable excited triplet state populations relative to the excited singlet states, technological problems in sample handling and choice of solvent have been the main obstacles to its wide-spread use. In the past, phosphorimetry has been limited almost exclusively to nonaqueous solvents and solvent mixtures which formed clear, rigid glasses at low temperatures. This requirement is indeed very troublesome because many factors both physical and chemical can cause serious cracking of the glasses and lead to poor analytical results. However, initial studies on the analytical utility of intensely cracked and snowed matrices in phosphorimetry were encouraging ( 4 ) . While a small degree of cracking is difficult 1
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(1) M. Zander, “The Application of Phosphorescence to the Analysis of Organic Compounds,” Academic Press, New York, N.Y., 1968. (2) J. D. Winefordner, P. A. St. John, and W. J. McCarthy, “Fluorescence Assay in Biology and Medicine,” Volume 11, S. Udenfriend, Ed., Academic Press, New York, N.Y., 1969. (3) W. J. McCarthy and J. D. Winefordner, “Fluorescence Theory, Instrumentation and Practice,” G. G. Guilbault, Ed., Marcel Dekker, New York, N.Y., 1967. (4) R. Zweidinger and J. D. Winefordner, ANAL.CHEM.,42, 639 (1970).
to control and leads to poor results, intense cracking and snowing of the matrix can be achieved for some solvents and excellent analytical results can be obtained ( 4 , 5 ) . Snowed and crystalline matrices often undergo considerable changes in structure on cooling, which cause severe stress on sample containers often shattering them completely. Use of a new quartz capillary cell for phosphorimetry in predominately aqueous, snowed matrices has been previously described (5). Aqueous solutions of phosphorescent molecules form on freezing, a translucent severely cracked glass, which gives greatly reduced phosphorescence signal intensity. However, addition of small amounts of lower chained alcohols to aqueous solutions produces a snowed matrix which gives excellent phosphorescence signals ( 5 ) . Similar results have been reported in hydrocarbon solvents (6, 7). The physical and chemical nature of the matrix and its effect on phosphorescence signals are not completely understood. Although appreciable molecular aggregate formation could occur in aqueous solutions and result in reduction of the phosphorescence signal oia triplet-triplet annihilation (8), molecular aggregation is apparently minimized by addition of alcoholic solvents, and the solutions studied are too dilute for appreciable triplet-triplet annihilation. Therefore, the mechanism accounting for the effect of the matrix upon the phosphorescence signals seems to rely upon diffuse reflection of incident exciting light within the crystalline matrix ( 9 ) resulting in multiple scattering at the boundaries of the individual particles ; multiple scattering enhances the probability of an absorptive process and thus increases the intensity of phosphorescence signal emitted. We wish to report on recent studies involving the influence of the matrix upon phosphorescence signals inasmuch as the matrix affects analytical phosphorimetry, and to discuss the development of new analytically useful aqueous solvents for phosphorimetry. Previous studies (5) have demonstrated the utility of mixed alcohol-water solvents in phosphorimetry. A more detailed study of phosphorescence signals as a function of solvent composition has revealed a close correlation between the physical and chemical structure of the (5) R. J. Lukasiewicz, P. Rozynes, L. B. Sanders, and J. D. Winefordner, ANAL.CHEM., 44, 237 (1972). (6) H. H. Richtol and F. H. Klappmeier, J. Amer. Chem. Soc., 86, 1255 (1964). (7) R. A. Keller and D. E. Breen, J . Chem. Phys., 43, 2562 (1965). (8) H. Sternlicht, G. C. Nieman, and G. W. Robinson, J . Chem. Phys., 38, 1326 (1963). (9) W. W. Wendlandt and J. G. Hecht, “ReflectanceSpectroscopy,” Interscience Publishers, New York, N.Y., 1966, p 46. ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
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