Fluorometric and Spectrophotometric Determination of Aluminum in Industrial Water CHARLES A. NOLL and LOUIS J. STEFANELLI Befz laboratories, Inc., Philadelphia 24, Pa.
b A method for determining micro amounts of aluminum in industrial waters is described. The aluminum is complexed with fluoride to prevent its reaction with 8-quinolinol while interfering ions are removed by exposing them to a weak cationic ion exchange resin and an 8-quinolinol-chloroform extraction. The aluminum is then made reactive to the 8-quinolinol and its chloroform-extracted 8-quinolinate is measured either fluorometrically or spectrophotometrically. The range of the test is from 0.00 to 0.50 p.p.m. as AI with a standard deviation of *0.02.
ion might be complexed with fluoride ion to keep it in solution while the other ions were removed seemed promising. By the careful control of pH, the aluminum fluoride complex remained intact when exposed to a weak cationic ion exchange resin and an acidic 8-quinolinol-chloroform extraction which was used to remove residual amounts of ions not retained by the resin. The aluminum in the complex was then made reactive to the 8-quinolinol reagent by using a large amount (10 ml.) of the ammoniumacetate buffer. EXPERIMENTAL
N
o ALUMINUM methods described in
the literature are applicable for determining small concentrations of aluminum in industrial waters quickly and accurately. Cooling waters, for example, which are treated with sodium dichromate, zinc, and complex phosphates to prevent the corrosion of the equipment, present a particularly difficult environment in which to determine micro amounts of aluminum. Gentry and Sherrington (5) measured absorptiometricdly the chloroform extract of 8hydroxyquinoline in the presence of many diverse ions; however, the fiuoride anion, which is not at all uncommon in industrial water seriously interfered with this method. Merritt and Walker (8) reported that 8-hydroxyquinaldine would not precipitate the aluminum but would precipitate many other ions. Although Hynek and Wrangell (7) used this principle in conjunction with other techniques to remove diverse ions, the authors observed that the residual amounts of the ions not precipitated by this reagent alone caused the results to vary. Claassen, Bastings, and Visser (8) extracted known quantities of aluminum from complexone-cyanide solution containing diverse amounts of interfering elements. This method was not satisfactory for routine control of industrial water treatment processes because of the length of time required for each determination. Freund and Miner (4) found that by complexing aluminum with fluoride and then by exposing the solution to an ion exchange resin, a separation of aluminum and zirconium could be effected. The concept that aluminum 1914
0
ANALYTICAL CHEMISTRY
Apparatus. All fluorometric measurements were made with a Turner fluorometer using 7-60 (360 mp.) primary filter and 2A (415 mp.), 2 X D secondary filters. All spectrophotometric measurements were made on a Bausch & Lomb Spectronic 20 colorimeter using their 0.5-inch 0.d. test tube a t 380 mp. Reagents. Aluminum Stock Solution. Dissolve 1.759 grams of aluminum potassium sulfate [A1K(So4)21 2 H 2 0 ] in distilled water containing 50 ml. of 5N hydrochloric acid and dilute to 1liter (1). Buffer Solution. Add 238 ml. of ammonium hydroxide (NHdOH, sp. gr. 0.90) to 500 ml. of distilled water. acetic acid Slowly add 111 ml. of glacial and dilute to 1 liter. 8-Quinolinol Solution. 2% Dissolve 2 grim, of 8-hydroxyquinoline (m.p. 74-6' C.) in 6 ml. of glacial acetic acid and dilute to 100 ml. with distilled water. Ion Exchange Resin. IRC-50 R.G. (20 to 50 mesh.) (Mfg. by Rohm & Haas Co.) Sodium Sulfate, c.P., Anhydrous. Sodium Fluoride Solution. Dissolve 2.2105 grams of C.P. NaF reagent in distilled water and make up to 1 liter. Solution, 1 ml. = 1 mg. as F-. Store in polyethylene container. Size of Sample. Use 100-ml. samples for waters which are untreated and uncycled-Le., wells, rivers, etc. Twentyfive-milliliter samples are taken for waters which are treated and cycled such as cooling waters which contain chromate ion. The specific conductance of the samples should not exceed 2500 pmhos. Procedure for Total Aluminum. To n well shaken appropriate size sample in a borosilicate glass beaker, 5 ml. of (concd.) HCI was added and then
Table 1.
Ten Determinations at Each AI Concentration
Method Fluorometric
Mean,
p.p.m. 0.10 0.40 Spectrophotometric 0.10
0.40
Standard deviation *0.004 f0.016 *O. 003 k0.018
evaporated to approximately 1 ml. After the addition of 5 ml. of (coned.) HNOs acid, the evaporation was continued to dryness in a steam bath t o avoid baking the residue. The residue was dissolved by wetting it with 2 ml. of 0.1N HCI, heating to just boiling, dislodging the residue by means of a plastic policeman if necessary, and finally adding 80 ml. of distilled water. After pipetting 4 ml. of the N a F reagent, the thoroughly mixed solution was transferred to a shaking jar which contained 20 ml. of moist IRC-50 ion exchange resin beads (resin from which water had been decanted prior to use). One-half milliliter of the buffer reagent was added by means of a pipet and the sample was shaken on a shaking machine (approx. 12 strokes per 5 seconds) for 20 minutes ( k 3 minutes). Then the sample was decanted into a separatory funnel. Two milliliters of 8-quinolinol solution was pipetted into it and was allowed to remain in contact with it from 2 to 5 minutes. (The authors used an average time of 3 minutes). Two chloroform extractions followed, 25 ml. and 10 ml., respectively. The shaking time for each extraction was 1 minute and each solvent layer was discarded. Ten milliliters of the buffer reagent was added and the above procedure, starting with the addition of 2 ml. of 8-quinolinol reagent, was repeated with two exceptions. The contact time was 2 minutes and the extractions were not discarded. These chloroform extractions were filtered through a No. 5 Whatman filter paper (9 cm.), full of the anhydrous NazS04,into a 50-ml. volumetric flask. The extracts were then made up to volume with chloroform and tnensured both fluoronietrically and spectrophotometrically on the previously described instruments. Procedure for Soluble Aluminum. Use a clear sample and follow the procedure for total aluminum. Calibration. Standard curves using concentrations of 0.00, 0.10, 0.30,
Table II.
centrations in parts per million. Chloroform was used as a reference blank on both instruments. Maximum absorbance was found t o be at 380 mp. for the spectrophotometer. (See Discussion).
Freedom from Ionic Interferenc,e
M sximum amount eniployed without interference, p.p.m. SpectroFlJorophotometric metric
RESULTS
I
Ion Iron as Fe+3 Copper as Cui2 Chromium as Cr + 3 Zinc as Zn+2 Manganese as
5.0 1. 0 10.0 5.0
10.0
Mn+2
0.3
0.3
poa-3
30.0
30.0
30.0 5.0 IC'OO. 0
lCOO.O
30.0 5.0 1000 .O 500.0
100.0
100.0
60.0 20.0
60.0 20.0 100.0
Polyphosphate as Orthophosphate as Boron as B+3 Chloride as C1Sulfate as S@4-2 Kitrate as KO3Chromate as Cr04-2
Fluoride as FSilica as Si02 Table Ill.
100.0
Standard deviations were calculated on the basis of 10 determinations for two concentrations of aluminum in distilled water. See Table I. Standard deviations were also calculated for the same aluminum concentrations recovered in the presence of the individual ions listed in Table 11. The 'F' test was applied to these data and no practical interference was caused by the concentrations of ions listed in Table I. The values as shown by the data in Table I indicate the maximum concentrations of the possible interferences investigated. The data as shown in Tables 111 and IV indicate values for aluminum concentrations recovered in a solution of the mixed ions. The concentrations of the ions were chosen on the basis of compatibility-Le., ability to remain in solution-and averages as near as possible t o actual concentrations existing in industrial waters. Finally, data was obtained on industrial waters submitted by 10 companies. The analyses of the samples are shown by the data in Table V. Standard amounts of A l + a ion were added t o the samples and their recoveries are shown by the data in Table VI.
3.0 0.5 5.0
Precision and Accuracy of Method Effect of impurities"
Aluminum, p.p.m.b Added Found
Method Fluorometric Spectrophotometric
(1.40 I .60 0.40 1.60
0.40 1.40 0.41 1.60
a Ions present, p.p.m.: copper as Cut2, 1.0; manganese as PAn+Z 0.1; polyphosphate as 5.0; mthophosphate as Pod-3, 5.0; fluoride as F-, 5.0; iron as Fe+3, 1.0; Mg+* as CaCOa, 20.0; Ca+2as CaC03, 80.0. Aluminum added to 25-ml. volumes.
DISCUSSION
Using a Bausch & Lomb Spectronic 20, maximum absorbance was found t o be at 380 mp. Because of the 20-m,~ bandwidth of this instrument, optimum wavelength should be determined on each spectrophotometer. B y means of a Beckman DU spectrophotometer Hynek and Wrangell (7) observed the maximum absorbance for the 8-quinolinol-chloroform extraction t o be at
and 0.50 p.p.in. of a uminum as Al+3 based on 100-ml. volumes were developed by plotting average fluorescence readings obtained on the fluorometer and average absorbance readings on the spectrophotometer us. aluminum con-
.
Company Sample Ca as CaCOa, p.p.ni.
Mg ae CaCO3, p.p.m. Phenolphthalein alkalinity as CaCO3, p.p.m. Methyl orange alkalinity as CaCO3, p.p.m. Sulfate as S04-2, p.p.m. Chloride as C1, p.p.m. Silica aa SiOp, p.p.m. Polyphosphate as P04-*,3p.p.m. Orthophosphate aa PO4- , p.p.m. Specific conductance at 18' C., pmhos Chromate as CrOd-*, p.p.m. Chromium as Cr+3, p.p.m. Total iron as Fe+3, p.p.m. PI1 Zinc as Z I I + ~ p.p.m. ,
Table V. A
Cooling tower
Table IV.
Cooling water
Cooling tower
Aluminum, p.p.m.b Added Found
Method Fluorometric Spectrophotometric a
0.40
0.44
1.60 0.40 1.60
1.42 0.48 1.68
Ions present, p.p.m.: copper as C U + ~ ,
0.10; manganese as Mn+2, 0.10; boron as B 4-3, 5.00; iron as Fe +3, 0.10; chloride
as C1-~,1000.00;sulfateas S0~-2,1000.00; nitrate as NOS-', 20.00; Mg+2as CaCOa, 40.00; Caf2as CaC03, 160.00; chromium
as Cr+a, 20.00. Aluminum added to 25-ml. volumes.
389 mp. while Wiberley and Bassett found the maximum t o be 390 mp. (IO). The values used t o plot the standard curves were in accord with Beer's Law for the concentrations quoted under the heading Calibration. The average number of dial readings for fluorescence in increments of 0.10 p.p.m. of aluminum was 13 ( A 1 dial division), while the average number of absorbance units was 0.042 (& 0.004 units). The p H range which is essential to the successful use of this method is practically selfcontrolling. It was found by experimentation that if the specific conductance of the sample (18' C.) was no greater than 2500 pmhos. and 0.50 ml. of the buffer reagent was used t o activate the IRC-50 beads, the resulting p H value would vary between 4.4 and 4.9. Two milliliters of the acidic 8-quinolinol reagent lowers the p H approximately 0.4 units. Quinolinol and most quinolinates are not all extractable at a p H value lower than 4.0 (9); consequently, a p H lower than 4.4 after exposure t o the beads will result in an incomplete extraction and a low aluminum value. Adjusting a p H lower than 4.4t o a higher value caused
Analyses of Industrial Waters Used in Table VI B C D E F
Cooling tower
Precision and Accuracy of Method Effect of impurities"
Cooling tower-
Raw water
G Boiler feed
H I J Shower Untreat. water well River
408 172
296 112
528 216
724 160
1 0
30 7
1 0
36 6
126 84
4
0
0
0
0
0
0
6
0
0
0
S
10 490 66 46
92
3.54 __ -
3ti 9 4 2 0 5.4
160 85 36 5.2
570 126 53 3.1 3.4 1050 8.9.5 0.09 0.10 6.4 1.25
...
...
875 7.3 3.15 0.20
5.5 1.02
12 890 68 43 5.6 2.8
1250 12.7 0.36 0.50 5.9 0.97
Si0
102 102 1.3
1.7
1350 36.7 0.36 0.05 7.1 0.15
29 122 54 34.4 4.1
1000 18.5 1.35 1.8
7.4 0 .0
12 43 24 8.7
... ...
170
...
142
...
100
... ... ...
YO0
...
... .
.
I
80
...
... ...
7 2
...
...
1
.
500
... ... ...
7 6
...
VOL. 35, NO. 12, NOVEMBER 1963
6
6 2 7 4.0 3.3
... ...
40
...
0.45 5.5
...
1915
results to vary. Therefore, it was necesesry to start, from the beginning with an aliquot of the original sample. Excesses of 8-quinolinol reagent caused no interference by fluorescing as wa9 found by Collat and Rogers (3) but rather aided in the reproducibility of fluorescence as stated by Goon et al. (6). The constant volume of 8-quinolinol used in the procedure eliminates any interference with respect to the absorbance measurements. Gentry and Sherrington (5) proved that large amounts of Ac-, C1-, NO8-, and SO,-* ions caused no interference with their method; however, if they are present in sufficient amounts to cause the specific conductance to exceed the limit of 2500 pmhos. by any significant amount, a variation in results was noted by the authors with respect to the authors’ method. The ionic strength of the solution due to the large excesses of ions no doubt causes the exchange process to continue beyond the controlling effect of the small amount of buffer present. When this occurs more hydrogen ion is released resulting in a pH value too low for the complete extraction of the 8-quinolinates. Regeneration of the Resin. One hundred per cent regeneration of the IRC-50 resin can be easily accomplished by passing 10 bed volumes of 4% HCI acid through a resin bed con-
Table VI.
Aluminum Recoveries from Industrial Waters
Fluorometric Company - A B
A1 found in samde, - . p.p.m. 0.00 0.00 0.00 0.00 0.00 0.05 0.14
c
n
E F G H I J
0.05
0.00 0.15
Al added, p.p.m. 0.60 0.60 0.60
0.60 0.60 0.20
0.20 0.20 0.40 0.20
Total AI found, p.p.m. 0.62 0.62 0.57 0.60
0.52 0.26 0.35 0.25 0.35 0.35
tained in a column a t a rate of 1 gallon per hour and then by backwashing the resin free of chlorides. ACKNOWLEDGMENT
The authors thank W. L. Nieland, E. C. Feddern, and L. J. LaSalvia for their technical assistance in the development of this procedure. The authors also thank the Rohm st Haas Co. for supplying the IRC-50 resin and the information regarding its use. LITERATURE CITED
(1) American Society for Test,iFg and
Materials, Philadelphia, Pa.,
Manual
on Industrial Water and Industrial Waste Water,” 2nd ed., p. 268, 1959.
Spectrophotometric - __ Total A1 in samDle, found. - . Al added. p.p.m. p.p.m. p.p.m. 0.00 0.60 0.60 0.00 0.60 0.62 0.06 0.60 0.62 0.60 0.60 0.65 0.00 0.60 0.52 0.06 0.20 0.25 0.12 0.20 0.32 0.08 0.20 0.23 0.00 0.40 0.37 0.13 0.20 0.35
AI found
(2) Claassen, A., Bastings, L., Visser, J., Anal. Chim. Acta 10, 373 (1954).
(3) Collat, J. W., Rogers, L. B., ANAL. CHEM.27,961 (1955). (4) Freund, H., Miner, F. J., Ibid., 25,564 (1953). ( 5 ) Gentry, C. H. R., Sherrington, L. G., Analyst 71,432 (1946). (6) Goon, E., Petley, J. E., McMullen, W. H.. Wiberlev. ” . S. E., ANAL. CHEW 25,608 (1953). (7) Hynek, R. J., Wrangell, L. J., Ibid., 28,1520-7 (1956). (5) Merritt, L. L., Jr., Walker, J. K., ANAL. CHEM.16,387 (1944). (9) Morrison, G. H., Freieer, H., “Solvent Extraction in Analytical Chemistry,” pp. 10, 164, Wiley, New York, 1957. (10) Wiberley, S. E., Bassett, L. G., ANAL.CHEM.21, 609 (‘1949). RECEIVEDfor review May 6, 1963. Accepted August 21,1963.
Identification of the Methyl Esters of the Stable Krebs Cycle Acids by Gas Liquid Chromatography H. H.
LUKE, T. E. FREEMAN, and L. B. KlER
Crops Research Division, U. S. Department of Agriculture, University of Florida Agricultural Experiment Station and fhe College o f Pharmacy, Gainesville, Fla.
b A gas liquid chromatographic procedure for the determination of the stable acids of the Krebs cycle involves preparation of methyl esters of the acids b y their reaction with diazomethane. Subsequent GLC analysis of the esters employed columns of polar (polyester LAC 728) and nonpolar (D-C Silicone Grease) liquid phases on Chromosorb W. Using these two columns, methyl esters of succinic, fumaric, malic, citric, maleic, isocitric, and cis- and frans-aconitic acids were separated and identified. The two unsaturated acids (fumaric and cis-aconitic), upon esterification, exhibited varying degrees of cis-trans isomerization. In both cases these cis and trans isomers could b e separated on one of the columns used. The effect of volatility and steric hindrance on the separation of these isomers is noted. 1916
ANALYTICAL CHEMISTRY
T
(nonketo) Krebs cycle acids are short (four to six carbon atoms), straight-chain dicarboxylic or tricarboxylic molecules. With the exception of fumaric and cis-aconitic, these are saturated acids. Although Spencer (10) recently reported the determination of a-ketoglutarate by GLC, the keto Krebs cycle acids are generally unstable and difficult to isolate from biological material. They also form reaction products during esterification which cause difficulty during GLC analysis. Therefore, the keto Krebs cycle acids were not included in this study. Krebs cycle acids are of considerable biological significance in that they form a vital part of carbohydrate metabolism and ATP synthesis. Thus, the effect of various stresses on biological material could be more thoroughly examined if a rapid, precise microtechnique such as gas liquid chromatogHE STABLE
raphy (GLC) were available. Although GLC analysis has been extensively applied to fatty acids, and to some extent to other organic acids (4), little has appeared in the literature concerning its use to identify the esters of the stable Krebs cycle acids. Mirocha and DeVay (8) determined the ethyl esters of fumaric, succinic, and malic acids; Esposito and Swam (4) separated the methyl esters of succinic and fumaric acids; and Ackman, Rannerman, and Vandenheuvel (1) determined methyl succinate. Nothing, however, was found in the literature concerning the GLC analysis of the esters of isocitrate and cis-aconitate. In fact, previous reports of the analysis of this group of organic acids have been incidental to the problem a t hand. Therefore, the current study was initiated to develop a GLC method of determining the six stable Krebs cycle acids.
