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ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
Table 11. Limits of Detection of Isocyanates in Air limit of detection (approx.) sampling sample, time, ppm HDI, L s (approx.) mg/m3 TDI ppm MDI 0.1 0.2 0.5 1.0
20-25 45-50 120 140
0.02
0.003
0.002
0.01
0.002
0.001
0.004
0.0006
0.0004
0.002
0.0003
0.0002
by the conventional impinger method. Thin-layer chromatography was selected as the method for analysis, although high speed liquid chromatographic analysis would have been a suitable alternative. The samples were analyzed and the results obtained are shown in Table I. Column 2 shows the conditions selected to generate the required test atmospheres. T h e diluting gas stream was adjusted to 2 L/min. Columns 3-5 give the data for the new tube absorption method. Note that test volumes for the new method were in the range 0.2-1.0 L, whereas test volumes for the conventional impinger technique (column 6) were 100 L. Columns 7-10 complete the necessary information concerning the impinger technique experiments. T o determine how the new tube absorption technique compares with the conventional impinger procedure, the values in column 5 should be compared with those in column LO. The agreement is considered excellent, especially for a procedure based on thin-layer chromatography. The new technique should give values fully equivalent to those of the more conventional impinger method, but should allow satisfactory analysis of samples of 1 L or less. Also, a correspondingly shortened sampling time can be achieved, in the order of 2 min or less. T h e lower limits of detection for the method are based on the ability to detect a 2-ng sample on the TLC plate. Since the whole test volume is concentrated in one spot, the limits of detection which can be expected are described in Table 11. If desired, isocyanate concentrations expressed in mg/m3 can be converted to ppm values for those cases in which the
molecular weight can be defined. The following equations can be used: concentration in ppm = concentration in m g / m 3 x
24
-
mol wt mol wt H D I = 168.2; T D I = 174.2; M D I = 250.1 molecular volume at 20 " C = 24 With higher isocyanate concentrations, it may be necessary to either decrease the sampling volume or collect and dilute the sample from the tube, taking a suitable aliquot for spotting in order to have the proper size sample to be chromatographed. These variations and others which are in accord with good chromatographic technique should be applicable to this method. In conclusion, this technique should provide a new tool for workers in the medical and industrial hygiene fields who deal with short time period sampling of atmospheres containing isocyanates a t very low concentrations. I t is applicable to aliphatic, alicyclic, and aromatic isocyanates, and can be carried out with relatively inexpensive equipment. It is based on previously proven "nitro reagent" chemistry and can be adapted to other means of analysis such as high speed liquid chromatography.
LITERATURE CITED (1) "NIOSH Manual of Analytical Methods", 2nd ed.,Vol. 1; P&CAM No. 141 "2,4-Toluenediisocyanate (TDI) in Air"; U.S. Dept. of Health, Education and Welfare: Cincinnati, Ohio, 1977. (2) "NIOSH Manual of Analytical Methods", 2nd ed.,Vol. 1; P&CAM No. 142 " p ,p-Diphenylmethanediisocyanate(MDI) in Air"; U.S. Dept. of Health, Education and Welfare: Cincinnati, Ohio, 1977. (3) Keller, Jurgen; Dunlap, K. L.; Sandridge, R. L. Anal. Chem. 1974, 4 6 , 1845. (4) Dunlap, K . L.;Sandridge, R. L.; Keller, Jurgen Anal. Chem. 1976, 4 8 , 497.
(5) Neilson, Arthur: Booth, K. S. Am. Ind. Hyg. J . , 1975, 36, 169.
RECEIVED for review April 11, 1979. Accepted May 18, 1979. This publication describes work funded by the International Isocyanate Institute, Incorporated.
Determination of Sulfide in Pyritic Soils and Minerals with a Sulfide Ion Electrode Darwin L. Sorensen,' Walter A. Kneib,2 and Donald B. Porcella' Utah Water Research Laboratory, Utah State University, Logan, Utah 84322
The determination of sulfide in geological materials is often done by leaching naturally occurring sulfate from the sample, oxidizing the sulfide to sulfate, and measuring it gravimetrically. This method is time consuming and carries inaccuracies caused by coprecipitation interferences with gravimetric determination of sulfate. These problems have led authors such as Maxwell ( 1 ) and Jeffery ( 2 ) to recommend methods for sulfide determination which rely on the release of sulfide ion. Murthy et al. ( 3 , 4 )have developed a method for determining sulfide which generates hydrogen sulfide from mineral sulfides in the presence of hydriodic acid and mercury catalyst. The H2S is precipitated as CdS and analyzed by iodimetric titration. The titration procedure is time consuming and is not sensitive enough to measure very low levels of sulfide with good precision. Morie (5) measured H2S in Present address: Department of Microbiology, Colorado State University, Fort Collins, Colo. 80523. 2Presentaddress: 5114 Aspen Ave., NE,Albuquerque, N.M. 87110. 0003-2700/79/0351-1870$01.00/0
cigarette smoke by scrubbing the smoke through a sulfide antioxidant buffer and measuring the sulfide concentration in the buffer with a sulfide ion electrode. We developed a sensitive and precise method for soil and mineral sulfide by combining the sulfide ion electrode measurement, using Cd as a titrant, with a modification of the H2S generation/ precipitation technique.
EXPERIMENTAL Apparatus. The apparatus of Murthy e t al. (3)was modified to include a cold trap between the reaction flask and the first gas scrubbing bottle (Figure 1). This trap was maintained at -30 to -35 "C with a Thermoelectrics Unlimited, Inc. Stir Kool Model SK 12 cooling plate. The cold trap removed mercury vapor (mercury "poisons" the sulfide electrode) and other potential contaminants which are carried over from the reaction flask. Nitrogen was used as the carrier gas (hydrogen may be used) and a magnetic stirrer caused mixing in the reaction flask. A heating mantle held between 110 and 115 "C was used to warm the reaction flask. 1979 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
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Table I. Results of Analysis of Sulfide Materials
material analyzed
theoretical sulfide content, %
mean (X) sulfide found (%)
n
std dev
53.4 34.9 22.2 13.4
52.03 34.18 20.95 13.35
16
6.49 (12.5)a
5 2
___ 2.94 (14.0) ___
0.56 0.20 1.25 0.35 0.60 0.07
3 3 3 3 3 3
0.076 (13.6) 0.050 (25.0) 0.042 ( 3 . 4 ) 0.035 (10.0) 0.060 (10.0) 0.01 (14.2)
pyrite (FeS,) chalcopyrite (CuFeS,) cadmium sulfide (CdS) lead sulfide (PbS) mineral overburdens BG48 BL48 FG60 FL60 RG60 RL60 a
-_
__ -_
1
95% confidence limits, upper lower 55.49 -_. 24.60
48.57
0.75
0.37
0.32 1.35 0.44
0.08
___
%
___ ___
17.30
0.75
1.15 0.26 0.45
0.09
0.05
(Standard deviation as % of mean.) up to volume with deionized water. The concentration of sulfide ion in the diluted SAOB was then determined using the calibrated sulfide electrode, and the concentration was converted to percent sulfide in the sample. All samples were finely ground, and ranged from 10 to 200 mg depending on expected sulfide content. Compounds which readily dissolve in HI (CdS, PbS) were allowed to react for 30 min. More slowly dissolving minerals (FeS,, CuFeSJ and mineral overburden were allowed to react for 1 h.
-
c
STIRRER
ICE W A T E R T O COOL - H E A T SINK (0 I’C I
COOLING PLATE
Figure 1. Layout of reaction apparatus (SAOB is sulfide antioxidant
buffer)
All sulfide measurements were made with an Orion Research Model 67-16 silver/sulfide ion electrode and a Model 90-02 double junction reference electrode with saturated KC1 in 0.1 M KOH outer filling solution. Electrode potentials were measured with the Corning Model 130 millivolt meter. Reagents. The sulfide antioxidant buffer (SAOB) stock described by Orion Research ( 6 ) was used as the gas scrubbing solution in the apparatus. All standards and samples were read using the sulfide ion electrode in 50% v/v SAOB stock. Hydriodic acid about 57% (Fisher, “Certified” reagent, preserved) was used in the sulfide liberation reaction. Standard cadmium titrant was used to determine sulfide concentration in the concentrated stock sulfide standard prepared each day from cadmium sulfide. This titrant was about 0.1 M in Cd from 3CdS04.8H20. The exact cadmium concentration of this solution was determined by atomic absorption spectrophotometry against standards made from high purity cadmium metal. Cadmium was used as the titrant because CdS is less soluble than Pb (suggested by Orion (6)) and gave a sharper titration end point. Procedure. The sulfide ion electrode was calibrated as follows. Concentrated sulfide standard was prepared by passing 150-200 mg CdS (Fisher, “Certified” reagent) through the analytical process described below. Exactly 50 mL of the diluted (50%) SAOB from the gas scrubbing bottles were titrated using a 10-mL buret with the standard cadmium titrant. The silver/sulfide electrode was used as a potentiometric indicator. The end point was determined graphically, and the sulfide concentration calculated. Dilutions were made of this concentrated solution ( l : l O , 1:100, 1:250) to construct an electrode response standard curve (6). We calibrate the electrode daily. The procedure used by Murthy and Sharada ( 4 ) was followed for liberating H2S. A small pellet of mercury in the reaction flask is the catalyst and we found that 30 to 35 g of mercury gave best results. This mercury was reclaimed and used repeatedly. The HZS was absorbed into 50 mL of SAOB stock (25 mL in each gas scrubbing bottle). The SAOB containing the sulfide was transferred with rinsing to a 100-mL volumetric flask and made
RESULTS AND D I S C U S S I O N Statistical data from 42 analyses of sulfide minerals, compounds, and overburden materials are presented in Table I. In early trials with the technique, it became obvious that pyrite was the slowest dissolving common sulfide mineral and hence the most difficult to analyze accurately. Therefore, frequent analysis was made of this mineral to assure t h a t analytical conditions were adequate. Results of any analysis where gas leaks (H2Sodor) occurred or other problems arose, were discarded. (Caution. H2S is extremely toxic. Adequate ventilation must be provided in case of accident.) T h e expected relative standard deviation for this technique in our laboratory was about 1.3%. Increasing the number of replicate analyses of each sample and discarding obvious or statistically determined “outliers” (which was not done for the data in Table I) will improve the precision of the technique. At least triplicate determinations should be made for each unknown sample and the mean value and error reported. Sulfide concentrations in the 50% SAOB below 0.5 mg/L gave less reliable millivolt readings. This limits practical sensitivity of the method t o about 0.03% sulfide-sulfur in a 150-mg sample. The original work by Murthy e t al. (3) claimed the method was capable of determining sulfide in the presence of sulfate. We found t h a t under the conditions necessary to obtain quantitative recovery of sulfide from pyrite, we were also able t o recover significant amounts of the sulfur from sulfate salts as sulfide. Analysis of reagent grade FeS04.7H20 and CuS04.5H20 yielded 87 and BO%, respectively, of the sulfate-sulfur as sulfide. Apparently the reducing potential is high enough t o reduce sulfate to sulfide in the reaction flask. Because of this interference from sulfate, we leached the mineral mine overburden samples free of sulfate with triplicate washings of cold 2:3 (v/v) HCl in water followed by triplicate washings with distilled water before sulfide analysis. No correction in the percentage sulfide content has been made for the mass of soluble material lost from the overburden by leaching (Table I). This loss is assumed negligible. The data can be used with appropriate corrections in determining a lime requirement to control the soil p H of mine overburden. LITERATURE CITED (1) Maxwell, J. A. “Rock and Mineral Analysis”; Interscience Publishing: New York, 1968; pp 236-239.
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
(2) Jeffery, P. G. "Chemical Methods of Rock Analysis"; Pergamon Press: New York, 1970; pp 415-416. (3) Murthy, A. R. V.; Narayan, V. A.; Rao, M. R. A. Analyst(London) 1956, 81. 373-375. (4) Murthy, A. R. V.; Sharada, K. Anaksf (London) 1960, 8 5 , 288-300. ( 5 ) Morie, G. P. Tob. Sci. 1971, 107, 34.
(6) Orion Research Incorporated, "Determination of Total Sulfide Content in Water", Applications Bulletin No. 12, Orion Research Inc., Cambridge, Mass., 2 pp.
RECEIVED for review April 2, 1979. Accepted May 29, 1979.
High Performance Liquid Chromatographic Determination of Bovine Insulin Alan Dinner" and Leslie Lorenz Eli Lilly and Company, P.O. Box 678, Indianapolis, Indiana 46206
During the past four years, the use of reversed-phase high performance liquid chromatography (HPLC) for the purification and/or quantitation of biologically active molecules has mushroomed ( 1 , 2 ) . Reports of the application of this technique to the separation and analysis of underivatized peptides have recently started to appear in the literature. Since an early report by Burgus (3),the HPLC characteristics of several small (less than 10 amino acids) peptides have been discussed (4-8), and Vogelsang has reported (9) on the HPLC analysis of the polypeptide (15 amino acids) antibiotic, gramicidin. In more recent publications and symposia, the use of reversed-phase HPLC for the analysis of higher molecular weight peptides and proteins (10-12) has been discussed. Work in our laboratory required a rapid and accurate method for the measurement of the polypeptide bovine insulin (51 amino acids, mol wt 5734) in the presence of the byproducts most commonly encountered during its purification (see Table I). The development of the desired method would relieve some of the need for the slow and tedious classical insulin analyses involving DEAE-cellulose chromatography or polyacrylamide disc-gel electrophoresis (13). Alternatively, the method should be amenable to modification to allow collection of minor insulin components which are difficult to obtain in high purity by classical methods.
EXPERIMENTAL A Waters Associates model 6000A solvent delivery pump, an autoinjector with a Rheodyne sampling valve, and a Varian Associates Vari-Chrom absorbance detector are used in all determinations. The column is a Lichrosorb RP-8,lO km, 250 mm x 4.6 mm (id.) from E. Merck and Company. A temperature bath set at 30 "C which circulates water through a jacket surrounding the HPLC column is also employed. Chromatographic Conditions. Reagent grade ammonium sulfate was obtained from J. T. Baker Chemical Company, and the pH of the solution is adjusted to 3.5 with dilute sulfuric acid. Sterile water is used for the aqueous solution. The 24.5/75.5 acetonitrile (Burdick and Jackson, glass distilled) - 0.2 M pH 3.5 ammonium sulfate solution for isocratic elution is prepared by mixing appropriate volumes of each solvent after which the mixture is degassed under vacuum. The flow rate is 6.0 mL/min, the injection volume is controlled at 20 kL, and the detection is at 215 nm. All insulin samples used in this work are obtained from these laboratories. The analysis of data is performed on a digital data system using the approach of Savitzky and Golay (14) to determine appropriate peak parameters.
RESULTS AND DISCUSSION Reversed-phase HPLC proved to be a satisfactory method for the determination of bovine insulin. The higher molecular weight proinsulin-like impurities, porcine insulin dimer, and bovine and porcine proinsulin (the latter contains 84 amino acids with a molecular weight of 9082 (13a,b))were all retained on the RP-8 column under the conditions utilized to elute bovine insulin; a n increase in the amount of acetonitrile in 0003-_2700/79/035 l-lm;OO/O
Table I. Potential By-products in Bovine Insulin bovine monodesamido insulin porcine insulin porcine monoarginine insulin porcine monodesamido insulin bovine monoarginine and diarginine insulin bovine and porcine insulin dimer bovine and porcine proinsulin bovine and porcine proinsulin-like components Table 11. Elution Time of Insulin-Like Proteins component bovine diarginine insulin bovine monoarginine insulin bovine insulin bovine monodesamido insulin porcine insulin porcine monodesamido insulin porcine monoarginine insulin
elution time, s
k
357
13.9
441
17.4
590
23.6
770
31.1
910
36.9
1170 720
47.8
29.0
the mobile phase did result in the elution of these components. The insulin-like proteins, such as porcine insulin, porcine monodesamido insulin, bovine monodesamido insulin, and porcine monoarginine insulin, all separated from the bovine insulin under the isocratic conditions employed (see Table I1 and Figure 1). The response curve from bovine insulin was found to be linear from 2.5 to 12.5 mg/mL (50 b g to 250 pg of insulin on the column). A study to ascertain the precision associated with this method was performed on a set of 30 individually prepared samples from a common lot of insulin. The resulting relative standard deviation was 0.9970 for peak area and 1.22% for peak height. I t is apparent that seemingly minor structural changes among the insulin-like proteins result in significant differences in their retention characteristics. Although porcine insulin and porcine monodesamido insulin (13) differ only by the replacement of an aspartic acid in the latter for an asparagine in the former, these two 51 amino acid-containing proteins are resolved from one another. We have also observed that minor alterations in the mobile phase cause drastic changes in elution time. Whereas the proinsulin-like proteins do not elute within 30 min using 24.5% acetonitrile, the elution time drops to 1@15 min with 26-2770 acetonitrile. Finally, we have observed that insulins from other species, (porcine, rabbit, ovine, and human) although structurally similar (15) to one another, also have characteristic retention volumes under the conditions we have employed for the bovine insulin quantitation. We are in the process of correlating the structural features of the various insulins with their retention characteristics, and hope to report on this a t a later date. The different sensitivity of various insulin proteins to binding on the RP-8 column has enabled us to develop a rapid 63 1979 American Chemical Society
