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
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( G L Y & ~ ~ G L Y - A A ) ~
1 f
(n-x)GLY
NoOMe
f
(x)GLY-AA
1
2
3
rIcc/nn
4
5
Figure 1. Schematic illustration of a resin probe experiment is shown
Figure 2. Percent coupling is plotted a s a function of the DCC to amino acid ratio
The letter. n. indicates the equivalents of glycine initiilly present on the resin.
A single stock Boc-Gly solution was used for all sample points, and the concentration of Boc.Gly in each reaction mixture was constant. Activated amino acids were mixed with DCC solutions to generate the active intermedii ate. Following preincubation for 0 (0). two hours (D), or four hours (A),each solution was incubated with a resin probe. The amount of glycine remaining on the resin reflects the coupling power of each DCC test solution
Following the coupling reaction, dipeptide has formed ( x equivalents), leaving n - x equivalents of unreacted amino acid. The amino acid and dipeptides are removed from the resin by saponification with sodium methoxide reagent and the amino acid content is determined
resins were mixed and the isolated peptides exhibited a constant 3H/14C ratio. The results were taken as evidence for a common (anhydride) intermediate during the DCC reaction. The authors then suggested that the DCC/amino acid ratio should be 0.5 and that excess DCC is superfluous or even deleterious during peptide synthesis. The conclusion of an experiment based on a single four-hour determination may be subject to serious error in view of the fact that DCC activated amino acids are known to couple with resin bound amines rapidly, providing high yields (90-99?6) within five minutes (3, 10, 1 1 ) . The rate of coupling of many anhydrides may be even greater (12).The suggestion that a 0.5/1 level of DCC to amino acid should be used during activation, has not been experimentally demonstrated. We, too, have been concerned with the stoichiometry of the DCC coupling reactions and have carried out experiments in which the DCC to amino acid ratios have been greater than 0.5 without encountering problems. Similarly, other laboratories have employed equivalent amounts of DCC and amino acids (13, 14) during activation. We now report the results of resin probe experiments which were carried out to define the limits of DCC stoichiometry during peptide synthesis. The effect of elevated levels of DCC on coupling and inactivation processes are described.
EXPERIMENTAL
Procedure. Initially, a stock solution was prepared containing Boc-Gly (0.012M) in methylene chloride. DCC solutions were prepared in methylene chloride a t the following concentrations: O.O58M, 0.047M, 0.023M, O.O18M, 0.012A4, and 0.006M.These DCC concentrations were chosen to provide a series of 5, 4, 2, 1.5, 1.0 and 0.5-fold excess, respectively (relative to the BmGly content) when equal volumes of Boc-Gly and DCC solutions were mixed. Samples of deprotected glycine resin (25 mg each; 0.934 pMlmg) were weighed into eighteen reaction vials, At zero time, BoeGly stock solution (20 ml) was mixed with each DCC solution (20 ml each). A sample of the activated amino acid (4 ml) was added to a corresponding resin probe in the reaction vial. Following preincubation of the activated amino acid for two or four hours a t each DCC concentration, a sample of each solution was added to the corresponding resin probe. Each resin was reacted for 15 minutes, and the reaction was stopped by filtration [sintered glass funnel (2 ml)]. Following washes with methylene chloride (4 ml), ethanol (4 ml), and methylene chloride (4ml), the resins were dried in vacuo and 10 mg of each was weighed into individual vials and saponified with sodium methoxide reagent (2 ml) a t 55 "C for 15 minutes ( 4 ) . The saponification mixture was filtered through a disposable pipet containing a plug of glass wool. The resulting solution was diluted with 0.2M citrate buffer (pH 2.2), and was analyzed for glycine content with a Beckman Model 120C amino acid analyzer. Analysis of the samples was expedited by eluting with pH 4.25 buffer, permitting the determination of the glycine content in 35 minutes. Percent coupling was determined relative to the glycine content of the unreacted saponified resin and was plotted as a function of the DCC to amino acid ratio (Figure 2).
Materials. Methylene chloride was obtained from VMR Scientific Company, and was used without further purification. Dicyclohexylcarbodiimide was purchased from Aldrich Chemical Company and tert-butyloxycarbonylglycine (Bot-Gly) was purchased from Schwarzhlann. Chloromethylated resin (Bio-Beads SX-2) was obtained from Bio-Rad Laboratories. Glycine resin was prepared by the method of Monahan and Gilon (15)(0.98 mequiv glycine/gram). The procedure was modified by extending the reaction time to 3 hours a t 80 "C. Glycine content was determined by amino acid analysis of the deprotected resin following saponification. The saponification reagent ( 4 ) contained sodium methoxide (0.3M) in ethanol. General Procedure for Resin Probe Experiments. The rationale of a typical resin probe experiment is shown in Figure 1. A resin sample containing an esterified amino acid (e.g., glycine) is prepared, and the amine protecting group is removed. Individual samples of resin (e.g., 25 mg) are incubated with the test solution(s) in order to probe the reaction parameter of interest (in this case, the relationship between coupling yield and equivalents of DCC). Following the desired time interval, the reaction is terminated by filtration, and the resin is washed free of materials not covalently bound to the resin. A sample of the dried resin (about 10 mg) is then saponified, and the glycine content is determined by quantitative'amino acid analysis. The change in glycine content relative to an unreacted resin reflects the coupling efficiency of the test solution and the influence of specific conditions in an experiment.
Using resin probes, it has been possible to evaluate the influence of DCC excess on the coupling reaction and on the availability of activated amino acids for coupling. In Figure 2, the percent coupling is plotted as a function of the DCC to amino acid ratio. The results (curve 0) indicate that the coupling reaction is relatively insensitive to DCC concentration during the coupling intervals (15 minutes) employed in this study. Little decrease in coupling levels was observed as the ratio of DCC to amino acid increases from 1 to 5 equivalents. We have consistently observed a maximum incorporation at a DCC to amino acid ratio of 1.5, and have run resin probe experiments at this level. Anhydride formation might be expected a t 0.5 equivalent of DCC to amino acid, whereas acyl isourea derivatives should be the active intermediate at 5 equivalents of DCC. High ratios of DCC might be necessary in cases where anhydride formation is undesirable. When activation is immediately followed by reaction with the resin probe, no significant decrease in coupling has been observed, even a t elevated DCC levels (2/1-5/1). The life time (availability) of the active intermediate is sensitive to elevated DCC concentration. Preincubation of
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RESULTS AND DISCUSSION
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
activated amino acid solutions for two or four hours prior to addition to the resin probe resulted in relatively small changes in the 0.5-2 range (DCC/amino acid), whereas substantial loss of acylating ability was observed a t high equivalents of DCC (e.g., 5/1) after four hours. The loss of activated amino acid a t high DCC levels during long preincubation times is of theoretical interest. However, operationally, the coupling reaction and acylating power of the solution appear to be independent of DCC to amino acid ratio during the initial phase of the reaction. Resin probe experiments, described in this report and previously, must be evaluated relative to the specified set of reaction conditions. If different conditions are employed (e.g., solvents, nucleophiles, concentrations, temperatures, etc.), it is prudent to evaluate the effects of the modification by carrying out new resin probe experiments rather than extrapolating from previous data. In addition, i t is desirable to maintain the relative coupling levels in the range of 40-80% in order to provide a system that is sensitive to change (i.e., increased coupling or inactivation). Often maximum sensitivity is obtained when the amount of activated amino acid present is equivalent to the free amine content of the resin. Dorman (7) has suggested an alternative to our resin probe method based on the quantitative isolation of solution components. Routine isolation of solution components can be difficult as the composition of the reaction mixture and/or the solvent is changed and different solvents are used. In contrast, resin probes provide ease of execution,
rapid separation and isolation of reactants and products, rapid quantitative analysis, and the general applicability for probing and optimizing reaction conditions. We feel that a comparison under a variety of experimental conditions will favor Resin Probe Analysis.
LITERATURE CITED (1) A. M. Tometsko, Biochem. Bbphys. Res. Commun.,50,886 (1973). (2) D. F. DeTar and L. Silverstein, J. Am. Chem. SOC.,88, 1020 (1966). (3) A. M. Tometsko, M. Schreiner. and J. Comstock, Anal. Biochem., 67, 182 (1975). (4) A. M. Tometsko and E. Vogelstein, Anal. Bbchem.. 84, 438 (1975). (5) J. Rebek and D. Feitler, J. Am. Chem. Soc., 95,4052 (1973). (6) J. Rebek and D. Feitler, J. Am. Chem. Soc., 96, 1606 (1974). (7) L. Dorman, Bbchem. Biophys. Res. Common.,60, 318 (1974). (8) J. C. Sheehan and G. P. Hess, J. Am. Chem. SOC.,77, 1067 (1955). (9) D. F. DeTar, R. Silverstein. and F. F. Rogers, Jr., J. Am. Chem. Soc.. 88, 1024 (1966). (10) L. Corley, D. H. Sachs, and C. B. Anfinsen, Biochem. Biophys. Res. Common.,47, 1353 (1972). (11) K. Esko, S. Karlsson, and J. Porath, Acta Chem. Scad., 22, 3342 (1968). (12) M. A. Tilak and C. S. Hollinder, TetrahedronLet?., 1297 (1968). (13) R. B. Merrifield, A. R. Mitchell, and J. E. Clarke. J. Org. Chem., 39, 660 /197A\ I . _ . .,.
(14) J. M. Stewart and J. D. Young, “Solid Phase Peptide Synthesis”, Freeman, San Francisco, Calif., 1969, p 24. (15) W. M. Monahan and C. Gilon, Biopolymers, 12, 2513 (1973).
RECEIVEDfor review May 5, 1975. Accepted July 21, 1975. This research was supported in part by the Monroe County Cancer and Leukemia Association, Inc, the Genesee Valley Heart Association, and by a Public Health Service Contract (Number N01-CP-45611) from the National Cancer Institute.
Determination of Gasoline Octane Numbers from Chemical Composition Mark E. Myers, Jr., Janis Stollsteimer, and Andrew M. Wims Analytical Chemistry Department, Research Laboratories, General Motors Corporation, Warren, Mich. 48090
The research and motor octane numbers (RON and MON, respectively) of a gasoline are measures of its quality of performance as a fuel. Octane number (rating) is affected by the isoparaffin, aromatic, lead (tetraethyl lead, TEL, and tetramethyl lead, TML), sulfur, and olefin contents of gasolines. The octane number is conventionally determined on a test engine by comparing the test gasoline with standard mixtures of 2,2,4-trimethylpentane (isooctane) and n-heptane (I). The octane number of a gasoline may range from that equivalent to isooctane (octane number of 100) to that of n-heptane (octane number of 0). This engine testing of fuels is somewhat expensive and time consuming. The purpose of this investigation was to develop a correlation between octane number and readily measurable characteristics of a gasoline (determined by conventional chemical instrumentation) using linear regression analysis. Such a method is of particular value when only a limited amount of gasoline is available. For engine testing, a 4000ml sample is normally used, but only 100 ml is required by the method to be described.
VARIABLES AFFECTING OCTANE NUMBER Isoparaffins. It has been known for many years that a close correlation exists between the amount of branching in the structure of a paraffinic hydrocarbon and its octane
number. The role of branching is implied by the choice of the two pure hydrocarbons which define the extremes of the octane scale, and is further confirmed if one looks a t the experimental octane numbers of other pure hydrocarbons (2). A measure of the amount of branching is the “isoparaffin index”, which is the measured ratio of CH3:CHz in the paraffins. For 2,2,4-trimethylpentane, this ratio is 5.0 and for n-heptane it is 0.4. The CH3:CHz ratio for an unknown paraffin or for a mixture of paraffins can be determined experimentally by nuclear magnetic resonance (NMR) spectrometry. A typical NMR spectrum of a gasoline is shown in Figure 1. Also shown are the six principal spectral regions for gasolines and the types of protons they represent. The locations of these regions are indicated as parts per million (ppm) chemical shifts from the resonance of tetramethylsilane (TMS), a material added as an internal reference. The two regions of primary interest here are those due to methylene protons and methyl protons in paraffins. The integrals of these regions are referred to as E and F , respectively; F / 3 is proportional to the total number of methyl groups and E/2 is proportional to the total number of methylene groups. T h a t is, F / 3 = k (total number of CHs groups)
(1)
E / 2 = k (total number of CH2 groups)
(2)
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