(25) (26) (27) (28) (29) (30) (31) (32) (33) (34)
L. E. Thomas, Arch. Biochem., 5, 175 (1944). J. Giral, An. lnst. Invest. Clent. Univ. NuevoLBon, 1, 115 (1944). S. Udenfriend and J. R. Cooper, J. Bial. Chem., 106, 227 (1952). D. B. McCornick, Anal. Siochem., 37, 215 (1970). K. Uehara, J. Biochem. (Tokyo),68, 119 (1970). R. Hakanson, Anal. Blochem., 51, 523 (1973). J. A. Ambrose, J. Clin. Chem., 20, 505 (1974). W. K. Wong, M. E. Flynn, and T. Inouye, Clln. Chem., I O , 1098 (1984). F. M. Schemjakin, 2.Anorg. Allgem. Chem., 217, 272 (1934). F. R. Duke and G. F. Smith, Ind. fng. Chem., Anal. Ed., 12, 201 (1940); 17, 572 (1945).
(35) (36) (37) (38)
V. W. Reid and R. K. Truelove, Analyst, 77, 325 (1952). V. W. Reid and D. G. Salmon, Analyst, 80, 704 (1955). V. Kratochvil and SobBslavsky, Chem. PrCm., 6, 515 (1956). C. H. W. Hirs, W. H. Stein, and S. Moore, J. Bid. Chem., 211, 941 (1954).
RECEIVED for review June 20, 1975. Accepted August 4, 1975. This work was by grants from the NIH
(GM-15431, GM-21949).
Removal of Trace Elemental Impurities from Polyethylene by Nitric Acid R. W. Karin,’ J. A. Buono,2 and J. L. Fasching3 Department of Chemistry, University of Rhode Island, Kingston, R.I. 0288 7
The importance of trace-element techniques can best be emphasized through the increasing list of applications and developing instrumentation ( I , 2 ) . With this added sophistication, it becomes mandatory to document and reduce all levels of contamination as much as possible (3). Not only do reagents contribute, but all materials that come into contact directly or indirectly with standards and samples must be considered possible sources of contamination. The contamination problem can be best controlled by the use of inert apparatus whenever possible ( 4 ) . Because of inertness and relative cleanliness, polyethylene, Teflon, and high-purity synthetic quartz have been selected as the best container materials ( I , 3). When involved with large sample populations, polyethylene is principally used instead of Teflon and quartz because of economic considerations. A large number of scientists have employed a wide range of techniques for preparing materials for use in trace analysis ( I , 3-11). From these studies, only a few documented their cleaning procedures with limited results. We report in this article some additional studies on the subject. In this work, the use of nitric acid for leaching polyethylene as a cleaning step in preparation for trace element analysis was investigated. Reagent grade (concentrated) and 8N nitric acid were used to evaluate the usefulness of leaching trace impurities from polyethylene. The leaching process was studied for a period of four days to determine the optimum cleaning period.
topes being determined, the polyethylene pieces were irradiated upon completion of each 24-hour leaching period to provide the same type of time-related information. The samples used for longlived isotope determinations were irradiated, leached, and then counted a t the end of each 24-hour cleaning cycle. Liquid standards were heat sealed in pre-cleaned polyethylene tubing and were prepared under controlled conditions using a laminar-flow clean-bench. Three standards were used to avoid chemical incompatibility and spectral interferences. One of these contained elements having isotopes with short half-lives and the remaining two were used for elements with isotopes having long halflives. A 0.65% Co-A1 wire was used to monitor the neutron flux distribution during long irradiations. Activation Analysis. All irradiations were carried out a t the Rhode Island Nuclear Science Center. The nuclear reactor is a swimming pool reactor with a 2-megawatt power rating. Pneumatic irradiation tubes were used to provide rapid handling of samples. The reactor neutron flux was 4 X 10l2n/cm2-sec and the thermal-to-fast neutron-flux ratio is 40 in the pneumatic tube location. Ten-minute irradiations were used for short half-life isotope determinations and activation of the long half-live isotopes was performed with a 7-hour irradiation. These two types of irradiations could provide information for 13 elements (Table I). Short-lived isotopes were counted following a decay period of 3 minutes. Samples were counted first and standards were counted immediately upon completion of the first count. Both sample and
Table I. Isotope Information Used in Activation Analysis Short-lived isotopes Isotope
EXPERIMENTAL Sample Preparation. Polyethylene samples were prepared using two different techniques. In one series of experiments, tops from polyethylene snap-top containers were carefully removed without the use of metal tools. They were placed into two large tanks containing 8N and concentrated nitric acid. Samples were systematically leached up to four days. Five pieces were removed from each tank of acid a t the end of a 24-hour cleaning cycle and rinsed with distilled, deionized, and filtered water. Samples were air-dried in a laminar-flow clean-bench to prevent particulate contamination. Heat sealed polyethylene bags were used to contain samples for irradiation. Prior to use, the bags were cleaned by leaching for several days in 8 N nitric acid. In the second series of experiments, pieces of polyethylene tubing were first irradiated and then systematically leached. In the case of the short-lived iso-
56Mn 66cu 24Na 2V 391 28Al
2296
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
Gamma-ray, keV
846.7 1039.0 1368.6 1434.2 1642.0 1778.9
Long-lived isotopes
Standard one
98Au lz2Sb lZ4Sb
64.7 h r 64.3 day 60.3 day 40.2 hr
411.8 564 .O 602.7 1596.6
27.8 day 35.3 h r 35.3 h r 83.9 day 5.3 y r 5.3 y r
320.1 554.3 776.5 889.3 1173.1 1332.5
Standard two
51~r Present address, New England Nuclear Corporation, Atomlight Place, North Billerica, Mass. 01862. Present address, AID Division of Fisher Scientific, 590 Lincoln Street, Waltham, Mass. 02154. To whom correspondence concerning this article should be addressed.
Half-life
2.6 h r 5.1 min 15.0 h r 3.8 min 37.0 min 2.2 min
82Br “Br
%c 6OCO 6OCO
Table 11. Ratios of Unleached Samples with Leached Samples Day
8 N Kitric acid 0
1
2
3
4
1.000 1.ooo 1,000 1.ooo 1.ooo
0.77 0.15 0.95 i 0.05 0.29 i 0.04 0.95 0.10 0.79 i 0.21
0.83 0.06 0.93 0.10 0.28 i 0.03 1.08 0.44 0.72 0.18
*
0.66 0.19 0.97 f 0.08 0.30 0.02 1.69 1.91 0.89 0.08
0.74 0.43 1.OO 0.07 2.81 i 0.93 0.60 0.41 0.87 0.41
1.ooo 1.ooo 1.ooo 1.ooo 1.000
0.81 0.35 1.00 0.09 0.30 i 0.06 1.51 0.20 1.09 ~t0.34
0.65 1: 0.22 1.16 0.48 0.29 i 0.02 1.17 i 0.30 0.75 0.22
0.76 0.15 0.94 0.03 1.15 i 1.71 1.09 f 0.21 0.85 rt 0.09
Isotope
56Mn 24Na 5%
38c1 28Al
*
*
* * * *
* *
1 6 N S i t r i c acid
56Mn “Na 52V
3 8 ~ 1 28Al
*
-
1 -0.00
2.00
1.00
v
*
I
*
I
-0.00
2. 00
1. 00
3.00
DRYS
Figure 1. Leach ratio data summary for aluminum, sodium, and vanadium
!. 0 0
*
0.80 0.10 1.03 i 0.08 3.21 + 0.32 1.36 f 0.43 1.08 0.12
52
3.00
DRYS
-0.00
* *
2.00
3.00
Figure 3. Leach ratio data summary for chromium and manganese
I
Figure 4. Leach ratio data summary for bromine, antimony, and gold
DRYS
Figure 2. Leach ratio data summary for copper and chlorine
RESULTS A N D DISCUSSION
standard were counted for 200 seconds of clock time. Data were collected using an Ortec 40 cm3 Ge(Li) coaxial detector with a resolution of 2.3 keV for the 1332-keV gamma ray of 66Co. This was coupled to a Nuclear Data 2200 4096 multichannel analyzer with computer compatible magnetic tape output (AMPEX TM-7) for spectrum analysis. A computer program (12) was used to process the resultant Ge(Li) spectra. Long-lived isotopes were counted for 8000 seconds of clock time following a decay period of 7 days to allow relatively short-lived isotopes responsible for high background radiation levels sufficient time to be reduced to an insignificant level.
Results for the first series of experiments conducted substantiated the use of nitric acid as a leaching agent for the removal of trace contamination. The values with standard deviations reported in Table I1 not only indicate the overall effectiveness of the technique, but also confirm the random character of impurities found. The values are leach ratios calculated by dividing the results (average of five samples each) for days 1 through 4 by the value for the unleached sample. A value less than one would therefore indicate contamination removal, and a continuous downward slope
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
2297
Table 111. Concentration of Thirteen Elements in Polyethylene and Detection Limits
1. 0 0
-0.00
2 . DO
Isotope
Concentration
Detection limita
5 i ~ r lg8Au
60.
ng/g
14.
2 . 1 ng/g
0.2 7.0 0.6 0.2
ng/g ng/g 82Br 186. ng/g ng/g lzzSb 7.5 ng/g ng/g “sc 2.4 ng/g ng/g COCO 13.8 ng/g 3.0 ng/g 140~a 4.8 ng/g 0.4 ng/g 56Mn 0.51 d g 0.003 pg/g 66cu 0.25 pg/g 0.09 pg/g 24Na 17.1 pg/g 0.20 Y d g 52 v 0.84 pg/g 0.001 pg/g 3 8 ~ 1 3.1 pg/g 0.14 pg/g 28A1 9.0 Pg/g 0.02 pg/g a Detection limits were calculated by assuming a detectable
3.00
DRYS
Leach ratio data summary for cobalt, scandium, and lan-
peak area of three times the standard deviation of the background area.
would substantiate a steady reduction of trace elements. The standard deviations calculated confirm the nonhomogeneous profile of trace contamination found in the polyethylene samples. The change in concentration of acid from 16N to 8N did not appear to affect the results obtained. The authors theorize that the contamination variation was introduced by handling during manufacture, by the type of material used for formulation, and by any subsequent processing. These differences in manufacturing explain the change in location of the trace impurities from within the polymer matrix to just below the surface or on the surface. Consequently, large deviations occur from sample to sample and from day to day. The second set of experiments was designed from an entirely different approach. The samples used for the short half-life isotope determinations were re-irradiated after each additional 24-hour leaching cycle. There was no evidence of radiation damage during the multiple short irradiations. In this manner, trace metal contamination was determined without being affected by any random character attributed to a changing sample population. Figures 1 through 5 indicate the actual relative contamination removal. The long-lived isotopes evaluated were the result of
a simple 7-hour irradiation followed by sample counts taken after each 24-hour leach cycle. All values indicated that the leach ratios stopped declining by the end of the third day. Because of the basic inherent inhomogeneity of the trace element contamination, values reported in Table I11 and Figure 6 represent results that can be considered typical. The contamination may be found well within the matrix of the polymer, just below the surface, or absorbed onto the surface of the polymer and/or in different chemical states. Based on this conclusion, the values reported for aluminum indicated trace element contamination that is not only nonleachable, but possibly located well below the surface of the polyethylene or in a chemical state that is not soluble in “ 0 3 . Results such as those obtained for copper indicate that this metal is being leached out from just below the surface. This suggests that contamination that is leachable is due to its location near the surface of the polymer and/or the chemical state. Values that level off after one day of leaching are characteristic of soluble trace element contamination that would be found adsorbed onto the surface instead of absorbed into the matrix. This would also indicate possible contamination due t o handling. The importance of these data is further emphasized
Figure 5.
thanum
i MA 2 4
AC 11
V 52
t L [
I
YN 5 6 CL 3:
,13 TAGWORD =
3814
CHRNNEL NUMBER
Figure 6.
Gamma-ray spectrum of leached polyethylene sample
2298
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
Table IV. Experimental Leaching D a t a for Polyethylene Blood, literature Element
Amount leached
Removal, 94
N u m inu m Antimony Bromine Chlorine Chromium Cobalt Copper Gold Lanthanum Manganese Scandium Sodium Vanadium
0 .oo 2.55 ng/g 38.0 ng/g 2.64 d g 27.0 ng/g 2.59 ng/g 0.16 pg/g 1.08 ng/g 4.06 ng /g 0.05 pg/g 1.15 ng/g 7.50 d g 0.76 i i d g
0 34 20 86 45 19 65 53 100 88 47 44 90
a
valuea
0.32 4.7 46.0 2900. 26.0 0.33
d g ng/g ng/g d g ng/g ng/g 1.07 Pg/g 0.04 ng/g
...
0.03 bg/g 75.0 ng/g 199. ng/g 0.02 ng/g
All values from Bowen ( I , 3 ) unless otherwise noted.
ing, handling, and type of production process all contribute to the changing trace element profiles obtained. The results indicate the usefulness of using 8N nitric acid and a 3-day leach period for optimum trace element contamination removal. The inhomogeneity of the contamination in a chemical and/or physical sense has perhaps been the basis for the lack of documentation in the literature. It is this same inconsistency that requires a minimum 3-day cleaning period to optimize for the least amount of contamination. ACKNOWLEDGMENT We are grateful to the nuclear reactor staff a t the Rhode Island Nuclear Science Center and the University of Rhode Island Computer Center for the facilities and assistance provided for these analyses. We also wish to express our thanks for the technical assistance supplied by Paul B. Monaghan, Charles M. Grill, and Cathy Montibello. LITERATURE CITED
when a comparison is made between the absolute amount removed by this cleaning procedure and the type and quantities of trace elements being determined. In the case of trace element analysis of whole blood, it can be seen from Table IV that amounts of trace elements removed can be as much as or more than the literature valves reported. This can create very serious problems if not minimized by the proper cleaning procedure. The percent removal indicates what portion of the contamination detected is removed. These values will not remain constant because of the random character of the trace element contamination and will be different for each sample. I t is important to note that ratios obtained resulted in a leveling off of leach ratio values by the end of the third day and, therefore, all sample containers should be leached for a t least three days to optimize for the least trace-element contamination possible. The concentrations and detection limits shown in Table I11 likewise indicate typical values for polyethylene; however, the random nature of the contamination changes these values for each sample determined. The manufactur-
J. W. Mitchell. Anal. Chem., 45, 492A (1973). D. S.Ahearn and C. A. McMenaury, Amer. Lab., 3(6), 63-67 (1971). E. C. Kuehner. R. Alvarey, P. J. Pauisen, and T.J. Murphy, Anal. Chem.. 44, 2050 (1972). R . E. Thiers in "Methods of Biochemical Analysis", D. Glick, Ed., Voi. 5, Interscience. New York, N.Y., 1957, pp 274-309. E. C. Kuehnr and D. H. Freeman in "Purification of Inorganic and Organic Materials", M. Zief. Ed., Marcel Dekker, New York, N.Y., 1969, pp 297-306. T. Ruzicka and T. Stary, "Substoichiometry in Radiochemical Analysis", Pergamon Press, New York, N.Y., 1966, pp 54-58. V. C. Smith in "Uitrapurity," M. Zief and R. Speights, Ed.. Marcel Dekker, New York, N.Y.. 1972, pp 173-191. D. E. Robertson, Anal. Chem., 40, 1067 (1968). W. A. Haller, R. H. Fiiby, and L. A. Rancitelli, Nucl. Appl, 6, 365 (1969). D. E. Robertson in "Ultrapurity," M. Zief and R. Speights, Ed., Marcel Dekker, New York, N.Y., 1972. W. H. Zoller and G. E. Gordon, Anal. Chem., 42, 257 (1970). J. L. Fasching, J. P. Maney, and P. K. Hopke, private communication, 1974. H. G. M. Bowen, "Trace Elements in Biochemistry", Academic Press, New York. N.Y., 1966.
RECEIVEDfor review April 25, 1975. Accepted July 25, 1975. This research was supported by the Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health, Grant No. 1R 0 1 HD 06675.
Evaluation of Carbodiimide Stoichiometry by Resin Probe Analysis Andrew M. Tometsko and Jeanne Comstock Department of Biochemistry, University of Rochester Medical Center, Rochester,
Resin Probe Analysis is an analytical technique which facilitates the determination of the effective limits of a set of reaction conditions by expediting the isolation and analysis of one or more resin bound reactants or products. In introducing the general method of resin probe analysis ( I ) , a reaction mixture was chosen which would provide a significant change in activated amino acid as a function of time, namely, the triethylamine (TEA) inactivation of dicyclohexylcarbodiimide (DCC) activated carboxylic acids ( 2 ) . Additional resin probe experiments ( 3 ) have analyzed the influence of coupling time on reaction yield, the importance of amino acid structure on inactivation, and the limits of TEA concentration compatible with coupling. The method has also been employed in the development of a quantitative method for analyzing resin bound amines ( 4 ) .
N.Y. 14642
Our reservation concerning recent reports (5, 6) and discussion (7) of the influence of DCC concentration on amino acid coupling reactions prompted a series of resin probe experiments designed to define the limits of DCC stoichiometry. Although dicyclohexylcarbodiimide ( 8 ) has been widely employed in the synthesis of peptides as a carboxyl activating agent, the reaction mechanism and stoichiometry have not been adequately resolved. DeTar and Silverstein (9) have indicated that anhydride intermediates are formed through the reaction of the acyl isourea derivative with available carboxyls. More recently, Rebeck and Feitler (6) have carried out an experiment in which 14C carbobenzoxyglycine anhydride and DCC activated 3H carbobenzoxyglycine were reacted with resin samples for four hours. The
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
2299
