LITERATURE CITED D. De Soete, R. Gijbels, and J. Hoste, "Neutron Activation Analysis" (Voi. 34 of "Chemical Analysis", Monographs on Analytical Chemistry and Its Applications), Wlley-lnterscience, London, 1972. J. Kuusi, Nod. Appl. Techno/., 8, 465 (1970). K. Mukai, K. Takano, and K. Takada, in "Nuclear Techniques in the Basic Metal Industries", iAEA, Helsinki, 1972, p 63. A. J. Lundan and 0. P. Mattila, in "Nuclear Techniques in the Basic Metal industries", iAEA, Helsinkl, 1972, p 3.
(5) S. S. Gurna and A. S. Bhatnagar, Indian J. Pure Appi. Phys., 11, 805 (1973). (6) L. Alaerts, J. P. Op De Beeck, and J. Hoste, Anal. Chim. Acta, 78, 329 (1975). (7) A. J. Cox, P. E. Francois, and R. P. Gatreil, int. J. Appl. Radiat. Isof., I S , 541 (1968).
RECEIVEDfor review April 26, 1976. Accepted J u n e 21, 1976.
Automated Composite Analysis of Major Sinter Components Om P. Bhargava* and W. Grant Hines Chemical and MetallurgicalLaboratories, The Steel Company of Canada, Limited, Wilcox Street, Hamilton, Ontario, Canada, LBN 3T1
Automated systems were developed for the composlte analysis of the major calcite sinter components: Al2O3 (0.4 to 1.5%); Slop (3 to 12%); CaO (5 to 14%); MgO (2 to 10%); and total iron (50 to 6 6 % ) with a standard deviation of 0.03,0.03,0.1, 0.1, and 0.2, respectively. The sinter sample is fused in a vitreous carbon crucible wlth a mixed flux of sodium peroxide and sodium carbonate yielding a complete solution suitable for the analysis of the above-mentloned components In a single solution. The vitreous carbon crucibles are expenslve but up to 18 fusions can be carried out per crucible. The simplificatlon of sample dissolution and ease of operation justify the cost. Fusing iron ore or sinter sample in a rirconlum crucible with sodium peroxide permits determination of the two key components-iron and sllica-on the AutoAnalyzer. The rlrconlum crucible is almost indestructible. Thls application could prove useful at mine sltes for monitoring and quality control during and after ore beneflcation.
Routine analysis of sinter-a recycled iron-bearing feed t o blast furnaces-is carried out currently by x-ray fluorescence on a VXQ-72000. T h e r e is no back-up unit a n d traditional methods of chemical analysis (including our scheme for rapid analysis (I))are slow a n d not available around the clock. T h e objective, therefore, was t o devise a n operationally simple alternative to cope with VXQ breakdown or provide a n economical alternative at lower workloads. AutoAnalyzer systems have been developed in our laboratory for determining acid soluble aluminum in steel, zinc, a n d galvanizing ( 2 ) , a n d silicon ( 3 ) in steel a n d other matrices. Satisfactory performance of these methods around t h e clock in the routine control laboratory for several years has soundly established this technique. AutoAnalyzer methods have also been developed for t h e successful determination of t h e major components of blast furnace slag, viz., CaO, Si02, MgO, a n d A1203 ( 4 ) .Owing t o t h e high concentration of iron in sinter as well as t h e appreciably different concentration range of t h e aforesaid components, t h e methods described there needed modification for application t o sinter.
EXPERIMENTAL Sinter Dissolution. Fusion in Platinum Crucible. Blast furnace slag responds to complete dissolution after sintering the sample with sodium peroxide at 380 "C in a platinum crucible for 20 min, in a muffle furnace. However this sintering technique was not successful for solubilizing the sinter, even after increasing the sintering time t o an hour. Grinding the sinter sample down to 250 mesh and increasing the ratio of peroxide to sample still left some residue unattacked.
Kilsby ( 5 )employed a 51 mixed flux (sodium carbonate-boric acid) for fusing the iron ore or sinters in a platinum crucible a t 900 "C for 20 min in a muffle furnace. This approach was tried but proved unsuccessful leaving some unattacked sample. Increasing the fusion temperature from 900 to 1100 "C and the fusion time from 20 to 30 min did not help. Additionally, the flux was increased and a blast Meker burner was used, but still there was no improvement in dissolution. Alternative approaches were then explored. Fusion i n Zirconium Crucible. In our scheme ( 3 )to determine silica in iron ore, sinter, and slag, it was demonstrated that iron ores and sinter are completely solubilized after fusion in a zirconium crucible. I t was thought worthwhile to investigate whether this approach is compatible with the analysis of the other components, viz., A1203, MgO, CaO, and iron. Determination of silica had already been established. Determination of iron as an o-phenanthroline complex did not pose any problem. After establishing the silica and iron systems on the AutoAnalyzer (fusion in zirconium crucible) efforts were directed towards investigating the use of this solution for determining magnesia. The solutions were run identically to the magnesium-Titan yellow system for blast furnace slags. However, the absorbance remained unchanged with increasing concentration of MgO in sinter samples. In our earlier study, we had established that iron interference is eliminated by incorporating the compensating solution. It became apparent that zirconium was the interfering species. This was confirmed by studying the effect of zirconium using synthetic solution. Zirconium greatly suppresses the absorbance of the magnesium-Titan yellow complex. Hence, an alternative solution was sought which was free from zirconium contamination. Fusion i n Iron Crucible. Fusion of sinter sample with sodium peroxide resulted in heavy erosion of the iron crucible contributing excessive and inconsistent amounts of iron to the solution. In spite of modifying the compensating solution to accommodate this increased and inconsistent excess of iron, the system did not work. Bisulfate Fusion i n Vycor Crucible. Fusion of sinter (0.100 g) in a Vycor (silica) crucible with potassium bisulfate (3 g KHSO4 fused powder) was then tried. The fused melt was leached with 50 ml of 3.6 N HC1 and filtered. The magnesium-Titan yellow complex was developed (manual operation) after establishing the optimum concentration of compensating solution used to mask the interfering elements. The system was then put on the AutoAnalyzer incorporating Titan yellow and sodium hydroxide into a single solution. This also eliminated the problem of occasional staining of the mixing coil. This system was suitable for determining MgO in the range 2 to 9%. The next sinter component attempted was alumina. Prior knowledge of zirconium interference in aluminum determination precluded fusion in such a crucible. The solution used for magnesia determination (bisulfate fusion in Vycor crucible) was then investigated. On dissolving the sinter bisulfate fusion melt in 3.6 N HCl, a slight white residue was noticed. The solution was filtered. Recovery of alumina was somewhat low. Spectrographic examination of the residue showed evidence of aluminum. It was therefore concluded that bisulfate fusion of sinter is not suitable for alumina determination. Again an alternative solution was needed. Fusion i n Niche1 Crucible. A method for determining alumina in sinter (6) etc. has been in use for 7 years and an automated version (AutoAnalyzer)for over 3 years. The sample is fused in a nickel cru-
ANALYTICAL CHEMISTRY, VOL. 48, NO. 12, OCTOBER 1976
1701
ER
Y ~
20-TURN
I
8-TURN
I -
I
0*42 .AIR 0.42 0*42 1.6%ASCORBIC ACID lxx) 1 BUFFER & CHROMAZUROL
I
'7--
WASTE
WASTE
COLORIMETER RECORDER 550nm 20mm FLOW CELL
'
*SOLVAFLEX TUBING PUMP
I
,P
COLORIMETER RECORDER 550 nm 20 mm FLOW CELL
Figure 1. Alumina determination
Figure 3. CaO determination
0.80 0*42 1 0 . 2 % Fe 1.6% NH4M07024 2 A% Na F 0.80
&+mM
20-TURN
1
I
I f-1
WASTE
WASTE
COLORIMETER RECORDER 660nm 20mm FLOW CELL Figure 2. Silica determination
cible with sodium peroxide and solubilized with dilute HCl. After dilution, the colorimetry is carried out by treating the solution (aliquot) with ascorbic acid to reduce the ferric iron. The color complex of aluminum with Chromazurol is then developed in a solution buffered with sodium acetate at pH 5.3. Satisfactory precision and accuracy were obtainedyor this component. The scheme is satisfactory for the simultaneous determination of silicon and iron fusing the sample in a zirconium crucible. However, separate fusions were required for magnesia (bisulfate in a Vycor crucible) and alumina (nickel crucible) to prevent the interference of zirconium in magnesia and alumina determinations and interference of nickel in iron, silica, and magnesia determinations. I t is most desirable to have a single solution (for the analysis of all the major components in the sinter) which is free from interference. Fusion in Vitreous Carbon Crucible. In the past, we had attempted to use the vitreous carbon crucible using both sintering at 380 OC with sodium peroxide as well as direct fusion on a Meker burner. Sintering was not adequate in decomposing the sinter while fusion eroded the crucible quite considerably. Sodium carbonate is incorporated in some fusion fluxes (using iron or nickel crucibles) to prevent a violent explosive reaction when decomposing some steelmaking reagents such as Calsibar and ferroalloys. It was thought worthwhile to incorporate sodium carbonate with sodium peroxide to fuse sinter samples in a vitreous carbon crucible. This modification completely solubilized the sinter in about 2-min fusion time. After acidifying the melt, a clear solution free from carbon specks and with no contaminants (such as zirconium, nickel, or iron) was obtained. Apparatus. 1)A Technicon AutoAnalyzer 11, comprising Sampler 11, Proportioning Pump 111,Colorimeter with a flow-through cell and interference filters (550 nm, 660 nm, and 520 nm) and two-pen chart recorder, was used in this study. 2) Vitreous carbon crucible, 20 ml. This crucible (Grade V25) "Le Carbonne-Lorraine" is available from Spectrex Ltd., 2245 St. Francois Rd., Dorval, P.Q. Reagents. Reagents are grouped according to the component being determined. Alumina. Ascorbic acid 1.6%w/v aqueous is used. Combined buffer-Chromazurol solution is prepared by dissolving 54 g of sodium acetate trihydrate in 700 ml of water and filtering. Chromazurol-reagent (0.08 g) is dissolved in 200 ml of denatured (1 1702
16-TURN
I
0.80
'*
1*40
AIR C O W SOL"
1 TITAN YELLOW-NaOH
a42
I
,r;v I
COLORIMETER RECORDER 550mn 20mm FLOW CELL Figure 4. Magnesia determination
t 1)alcohol, filtered, combined with the filtered buffer solution, and diluted to a liter with water. Silica. Iron 0.2% solution is made by dissolving high purity iron (1.00 g) in 80 ml of 8% v/v HzS04. After the reaction is completed, 10 ml of 3% w/v freshly prepared ammonium persulfate are added, the solution is boiled for 2 min, and diluted to 500 ml. Ammonium molybdate 1.6%w/v aqueous is used. Sodium fluoride solution, 2.4% w/v aqueous, is stored in a polyethylene bottle. Calcium Oxide. The wash solution is 0.024 N HCl. Triethanolamine 20% v/v is used. The buffer is prepared by dissolving 30 g of sodium hydroxide pellets in 600 ml of water, adding 10 g of Borax (NazB407.10HzO) and 5 g of potassium cyanide and diluting the solution to a liter with water. Glyoxal bis(hydroxanil),GBHA, is made by dissolving 0.10 g of the reagent in 300 ml of denatured alcohol and then adding 200 ml of water. After mixing, this solution is stored in an amber colored glass-stoppered bottle. Magnesia. The compensating solution is prepared as follows. Dissolve 7.0 g EGTA (Ethylenebis(oxyethylenenitro1o)tetraacetic acid) in 15 ml of 10% w/v sodium hydroxide. The pH is adjusted to 7 with 2 N HC1. Sodium fluoride, 3 g, is then added and the contents are diluted to 500 ml with water. This follows the addition of 2.5 g of hydroxylamine hydrochloride and 0.225 g of aluminum chloride hexahydrate. After mixing, 70 ml of triethanolamine are added and the volume is made up to a liter with water. This solution is stored in a polyethylene bottle. Titan yellow-sodium hydroxide solution is made as follows. Dissolve 60 g of sodium hydroxide in 600 ml of water and cool to room temperature. Into a 100-ml beaker, triturate 0.05 g of Titan yellow (also called Clayton yellow) in a minimum volume of water; then add 50 ml of water, mix to dissolve, and filter through a rapid filter into a 400-ml beaker. Wash the filter with about 200 ml of water. Add 100 ml of glycerol to the Titan yellow solution, mix, and add this solution to the sodium hydroxide solution, Dilute the combined solutions to 2 1. with water. Store in a polyethylene bottle. Iron. Hydroxylamine hydrochloride solution, 1%w/v aqueous, is made fresh each day. Sodium acetate trihydrate is 4% w/v.
ANALYTICAL CHEMISTRY, VOL. 48, NO. 12, OCTOBER 1976
~~
T a b l e 111. Calcium Oxide in Sinter Sample identification Jan. 20164 May 28/69 Dec. 11/57 March 18/69 April 25/71
Sample identification
Figure 5. Iron determination
Jan. 20164 Dec. 11/57 April 25/71 May 28/69 March 18/69
T a b l e I. Alumina i n S i n t e r % A1203
April 25/71 March 18/69 May 28/69 June 20164 Dec. 11/57 Nimba BCS Ore
7 7 7 7 7
4
s
R
Assigned or Cert.
0.027 0.022 0.035 0.029 0.026 0.026
0.51 0.68 0.73 0.91 1.12 1.05
0.45 0.60 0.70 0.91 1.04 1.08
Sample identification Dec. 11/57 March 18/69 May 28/69 April 25/71 Jan. 20164
April 25/71 May 28/69 March 18/69 Jan. 20164 Dec. 11/57
n
S
5
0.023 0.033 0.014 0.030 0.030
5 5 2 5
7 7 7 7 7
0.07 0.08 0.13 0.12 0.08
6.30 9.79 10.95 11.18 12.90
6.17 9.90 10.90 11.26 12.83
% Si02 f Assigned
4.25 5.06 5.20 6.65 11.43
n
S
16 16 16 16 16
0.08
0.15 0.05 0.08 0.12
% MgO i Assigned
2.60 4.42 5.71 7.04 8.06
2.6 4.3 5.7 7.4 8.1
T a b l e V. T o t a l I r o n in Sinter
T a b l e 11. Silica in Sinter Sample identification
s
T a b l e IV. Magnesia i n Sinter
COLORIMETER RECORDER 520 nm 20mm FLOW CELL
Sample identifications n
% CaO f Assigned
n
4.20 5.10 5.20 6.68 11.38
0-Phenanthroline solution is prepared by dissolving 1g of reagent in a liter of water containing 1ml of HCl. Wash solution (about 45% Fe) is prepared by treating an appropriate weight of iron ore or sinter sample (to provide the baseline) using the sample dissolution described below. Procedure. Sinter Dissolution. Sinter (0.1000g) is mixed with 0.5 g of sodium carbonate and 2 g of sodium peroxide in a vitreous carbon crucible and fused at low heat over a Meker burner for 2 min. The somewhat cooled melt is extracted with water followed by treatment with 25 ml of (3 + 2) HC1 and boiled for 2-3 min. The solution is then transferred to a 1-1. volumetric flask containing 40 ml of 8% v/v sulfuric acid and about 500 ml of water and the contents are diluted to volume. This solution is then ready for the determination of A1~03, SiOz, CaO, MgO, and total iron on the AutoAnalyzer. Internal reference sinter samples as well as NBS, BCS, and IS0 reference standard iron ores and sinters are dissolved identically along with the unknown samples and run on the AutoAnalyzer to establish calibrations. Alumina. The analytical system for determining alumina is assembled according to schematic flow diagram shown in Figure 1.The sample, air and ascorbic acid (1.6%)a t 0.42 ml/min, each are mixed and passed through an 8-turn mixing coil. Buffer solution containing Chromazurol(O.08g/lJ at 1.00 ml/min is added and passed through a 20-turn mixing coil. The absorbance of the Al-Chromazurol complex is recorded at 550 nm. Sample and wash/(water) cycles are 30 and 50 s, respectively. Silica. The analytical system for Si02 is assembled according to the schematic flow diagram shown in Figure 2. The sample, iron solution (0.2%) and air at 0.80,0.42, and 1.00 ml/min, respectively, pass through a 6-turn mixing coil followed by ammonium molybdate (1.6%) at 0.80 ml/min and mix through a 16-turn coil to form the silicomolybdate complex. Molybdenum blue is then formed by addition of NaF (2.4%)at 0.80 ml/min, and passage through a 16-turncoil to the col-
n
s
4 3 3 3 4
0.07 0.11 0.22 0.19 0.13
% Fe 2 Assigned
50.0 52.1 54.1 54.1 58.7
50.2 52.2 53.7 53.9 58.7
orimeter. Absorbance is measured at 660 nm. Sample and wash (8% HzS04) cycles are 30 and 45 s, respectively. Calcium Oxide. The analytical system for CaO is assembled as shown in Figure 3. The sample, air, and triethanolamine at 0.42,0.42, and 0.32 ml/min, respectively, pass through a 6-turn coil and meet buffer (NaOH 30 g/l., Borax 10 g/l., and KCN 5 g/L) at 0.32 ml/min. After passing through an 8-turn coil, the stream meets glyoxal bis(hydroxani1) at 0.70 ml/min and traverses a 28-turn coil. The absorbance of the complex is then recorded at 550 nm. The sample and wash (0.024 HC1) cycles are 60 and 90 s, respectively. Magnesia. The analytical system for MgO is assembled as shown in Figure 4. The sample, air, and compensating solution at 0.60,0.42, and 0.32 ml/min, respectively, pass through a 16-turn coil and meet a Titan yellow-NaOH-glycerol solution at 1.40 ml/min. After passing through a 20-turn coil, the absorbance is recorded at 550 nm. The sample and wash (8%HzS04) cycles are 50 and 60 s, respectively. Iron. The analytical system for the determination of iron is shown in Figure 5. The sample, air, and hydroxylamine (1%)at 0.16,1.00,and 1.6 ml/min, respectively, pass through a 16-turn mixing coil and meet sodium acetate at 0.42 ml/min. After traversing an 8-turn coil the stream meets o-phenanthroline and passes through a 16-turn coil to the colorimeter. The absorbance is measured at 520 nm. The sample and wash (45%Fe solution) cycles are 50 s each. The wash solution contains 45% Fe to establish the baseline and provide increased sensitivity in the range 50 to 66% Fe.
RESULTS AND D I S C U S S I O N As indicated earlier, t h e fusion of sinter samples in a vitreous carbon crucible with sodium peroxide incorporating sodium carbonate provided complete solution free f r o m contaminants (interfering elements) such as zirconium, nickel, or iron from the respective crucibles. It was possible t o analyze satisfactorily t h e components A1203, SiOz, CaO, MgO, and total iron in sinter in a single solution on t h e AutoAnalyzer using t h e necessary reagents and the appropriately devised analytical schematics. T h e results are recorded in Tables I through V for alumina, silica, calcium oxide, magnesia, and total iron, respectively. The tables also show the s t a n d a r d deviation and comparison with the assigned values.
ANALYTICAL CHEMISTRY, VOL. 48,
NO. 12, OCTOBER 1976
1703
ACKNOWLEDGMENT Acknowledgment is made to K. J. Alkerton and W. D. Lord for assistance in experimental work, and t o T h e Steel Company of Canada, Ltd., for permission to publish.
LITERATURE CITED Bhargava and W. G. Hines, Paper presented at the 50th Annual Conference of the Chemical Institute of Canada, Toronto, June 1967. (2) 0. P. Bhargava, G. F. Pitt, J. F. Donovan, and W. G. Hines, Technicon International Congress 1970, New York, N.Y.; published in the Congress Proceedings. (1) 0.P.
(3) 0. P. Bhargava, G. F. Pitt, and W. G. Hines, Talanta, 18,793 (1971). (4) 0. P. Bhargava, J. F. Donovan, and W. G. Hines, Paper presented at the 25th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 1974. (5) P. E. Kilsby, Technicon International Congress 1970, New York, N.Y.; published in the Congress Proceedings. (6) 0. P. Bhargava and W. G. Hines, Anal. Cbem., 40,413 (1968).
RECEIVEDfor review April 29, 1976. Accepted J u l y 1, 1976. P a p e r presented at t h e P i t t s b u r g h Conference on Analytical Chemistry and Spectroscopy, Cleveland, Ohio, March 1976.
Determination of Total Estrogens in Urine with 3-Methyl-2benzothiazolinone Hydrazone Hugh Y. Yee* and Bobette Jackson The Deparfnient of Pathology, Hutzel Hospital, 432 East Hancock, Detroit, Mich. 4820 1
An estrogen method has been developed that utilizes a colorimetric reaction consistlng of the oxidative coupling of 3methyl-2-benzothiazolinone hydrazone (MBTH) to the phenol portion of the steroid molecule. Colorlmetric measurements are made at 530 nm manually or with an AutoAnalyzer at an analysis rate of 50 samples per hour. Comparison with a fluorometric method gave a correlation coefficient of 0.96 and with a gas chromatographic one, a coefficient of 0.94, so that the proposed method may be suitable as an alternative method for the determination of total estrogens in pregnancy urine samples.
T h e importance of assaying urinary estrogen concentrations t o assess fetal growth and well being has been established and documented ( I ) . To date, most procedures have not combined simplicity a n d specificity. Perhaps, t h e most rapid m a n u a l method for use is one that uses Amberlite XAD-2 resin t o separate estrogens from urine, reaction with a Kober reagent, and a fluorometric measurement (2). A comparable colorimetric method has n o t been m a d e available, as relatively longer times are needed ( 3 ) . We have investigated a procedure to assay urinary estrogens that uses either ethyl ether-ethanol extraction ( 4 )or ammonium sulfate precipitation (5) of t h e steroids and reagents that a r e suitable for manual or automated quantification. Larger laboratories with a greater number of estrogen assays t o perform would t e n d to use an automated procedure, and m a n y excellent procedures are available (6-9). Most of t h e published procedures are carried oat at an analysis rate of 15 t o 40 samples per hour, a n d necessitate the use of a fluorometer. W e have succeeded in extending the manual method reported here t o a semiautomated estrogen method with an analysis rate of 50 samples per hour. T h e color reaction used in this procedure has been previously used for t h e determination of phenols in water supplies (IO, 11).
EXPERIMENTAL Apparatus. The manifold used for the automated procedure is shown in Figure 1. The AutoAnalyzer system (Technicon Corp., Tarrytown, N.Y.) consisted of sampler 11, pump 11, colorimeter, and recorder. Manual measurements were made with a Gilford Model 300-N spectrophotometer (Gilford Instruments Laboratory, Oberlin, Ohio). An International B-20 A high speed refrigerated centrifuge 1704
* ANALYTICAL CHEMISTRY. VOL.
(Damon/IEC Division, Needham Heights, Mass.) was used to centrifuge the ammonium sulfate precipitates. Spectra were obtained with a Coleman Model 124 spectrophotometer (Coleman Instruments, Inc., Maywood, Ill.) with Model 165 recorder. Fluorometric measurements were made with an Aminco Model SPf 125 spectrofluorometer (American Instruments Co., Silver Spring, Md.). A Varian Aerograph Model 1440 chromatograph was used for the assay of estrogens by gas chromatography. Reagents. Isolation, Hydrolysis, and Purification. Reagents used were 6 N H2SO4; 0.5 N HzS04 in ethanol; 0.5 N HzS04 in methanol; 1 N NaOH; 0.1 N NaOH; acetate buffer (2 molh.; pH 4.7); Glusulase, an enzyme mixture from Helix Pomatia containing approximately 200 000 unitdm1 of glucuronidase and 100 000 units/ml of sulfatase (Endo Laboratories, Garden City, N.Y.); 1 M K2C03; (NH&S04; ethyl ether; methanol (aldehyde free); ethanol. Manual Color Development. Use 0.2% w/v ceric ammonium sulfate (G. F. Smith, Columbus, Ohio) in 1.5%v/v HzS04, store in an amber colored bottle; 0.15% w/v aqueous solution of 3-methyl-2-benzothiazolinone hydrochloride (MBTH) (Aldrich Chemical Co., Milwaukee, Wis.), keep refrigerated when not in use and discard after 2 weeks; 0.3% w/v EDTA (disodium salt); estriol standards 20 and 40 mg/l. in 25% v/v methanol or 100%methanol. Automated Color Development. Wash water, add 0.1 ml Brij-35 (30%solution; Technicon Corp., Tarrytown, N.Y.) per liter; 0.2% w/v ceric ammonium sulfate in 2% v/v H2S04, store in an amber colored bottle; 0.05% w/v MBTH in aqueous solution, keep refrigerated when not in use and no more than a %week supply; 0.3%w/v EDTA (disodium salt), add 0.1 ml Brij-35per liter; stock estriol standard, 1mg/ml in ethanol; dilute with 25% v/v ethanol to obtain standards of 5,10, 20, 30, and 40 mg/l. Procedure. Isolation: ( a ) A m m o n i u m Sulfate Precipitation. In a 40-ml glass-stopper centrifuge tube containing 3.5 g (NH&S04, pipet 5 ml of urine from a 24-h collection,add 0.1 m16 N HzS04, and mix the contents. Warm the contents of the tube in a 55 "C water bath for several minutes. Stopper the tube and vortex vigorously until the ,salt dissolves. Transfer the contents of the centrifuge tube to aplastic centrifuge tube. Centrifuge the tube in a high speed centrifuge at 17 000 rpm at 0-5 "C for 30 min. Remove the tube from the centrifuge ana aspirate off the supernatant liquid, taking care not to remove any of the precipitate. Add 2 drops of 1N NaOH and 1ml of water. Vortex the contents. Add 3 ml of water and mix. Centrifuge for 5 min at 3000 rpm to pack any undissolved material. Transfer the purified urine extract to a clean 40-ml glass-stopper centrifuge tube. Hydrolysis. Add 1ml of acetate buffer and 0.5 ml of Glusulase. Mix and incubate the contents at 55 "C for 120 min. Purification: ( a ) A m m o n i u m Sulfate Isolation. Cool the tube containing the hydrolysate. Add 25 ml of ethyl ether and shake vigorously 3 times for 10-s intervals (release pressure after each 10-s shaking). After the layers have separated, aspirate off the lower phase. Wash the ether phase by shaking with 10 ml of 1M KzC03 and aspirating off the lower phase. Wash with 10 ml of water and aspirate off
48, NO. 12, OCTOBER 1976
