curacies and poor resolution of visible polarimeters, the optical rotatory dispersion curves are of much greater significance in categorizing the rare earth complexes than are the molecular rotations at a single wavelength. DISCUSSION
The completely modified Model 141 polarimeter has many attractive features. The Bausch & Lomb monochromator containing a double grating modified Czerny-Turner mounting provides twice the dispersion of a single grating monochromator and the increased spreading of the spectrum permits the use of wider slits for a given band-pass than those used in a single grating monochromator ( 4 ) . The extremely low stray light of the double grating B&L monochromator allows the entire spectrum to be obtained without the necessity of additional operations on the optical system and subsequent changes in sensitivity. The second major advantage is the high intensity xenon lamp source which can be used over the entire wavelength range (650-240 nm), thus eliminating the necessity of changing lamps to cover the spectral region ( 4 ) . (4) “Perkin-Elmer Polarimeters 1 4 1 ~ 1 4 1 M ~ 1 4 1 M C Perkin-Elmer .” Corporation, Norwalk, Conn., Bulletin 541/8.70.
This modification allows handling samples of high absorbance throughout the entire spectrum. The addition of the B & L wavelength drive, the P-E transmitting potentiometer, and the Coleman recorder permits the scanning and recording of spectra such as the plain positive O R D of sucrose, Figure 4 (scan speed of 25 nm/min), the plain negative ORD, or the highly complex spectrum of the Nd-D(-)PDTA complex, Figure 6 (scan speed of 5 nm/min). The multi-millivolt Coleman recorder allows the changing of the optical rotation range while the spectrum is being scanned. The scan time of the O R D spectrum for the NdD(-)PDTA complex, Figure 6, was about 1 5 minutes including both the base line and the actual ORD spectrum. The scan time for the sucrose ORD spectrum, Figure 4, was about 40 minutes including the base line. Thus, effectively, the time required to run an ORD spectrum is considerably shortened with these modifications. RECEIVED for review June 19, 1972. Accepted November 17, 1972. This work was presented in part by the authors at the 23rd Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 9, 1972. This work was supported by a CSU research grant.
Improved Experimenta I Technique for Reverse Isotope Dilution Method W. H. Graham and W. E. Bornak‘ Rohm and Huus Company, Bristol Research Laboratories, Box 219, Bristol, Pa. 19007 THE CARRIER-ADDITION or reverse isotope dilution (RID) method is a useful technique for both qualitative and quantitative identification of radioactive compounds in complex mixtures. The method is particularly useful in pesticide residue analytical work because very low levels of multiple degradation products can be determined quantitatively from a single radioactive precursor, the parent pesticide ( I ) . I n practice, the weighed, unlabeled compound is equilibrated with the labeled counterpart of known specific activity. The diluted radioactive material is then isolated, purified, and radioassayed. Ordinarily, nicely crystalline materials are used and purification involves multiple recrystallization steps. The criterion of purity is a constant specific activity after consecutive recrystallizations. The traditional R I D expcrimental method then involves solution, crystallization, filtration, drying to constant weight, and radioassay of the crystals. The process is repeated until consecutive radioassays of specific activity are identical. We describe here the principles involved, the apparatus and procedures used, and the results obtained in trials of a modified R I D technique. In the modified R I D method, the test 3f constant specific radioactivity is determined differently. [nstead of isolating the crystalline material after each rexystallization, it is necessary tc radioassay only the mother liquor. This follows from the fact that at equilibrium at To whom reprint requests should be sent. :1) V. F. Raaen, C. A. Ropp, and H. Raaen, “Carbon-14,’’ McCraw-Hill Co., New York, N.Y.. 1968. has an excellent discussion of isotope dilution methods.
Table I. Solubility of ETU on Repeated Recrystallizations
Recrystallization No. Soluble dpm of total activity
1
2
3
4
Wash
1353 12.3
1471 13.4
1440
1258 11.5
813 7.4
13.1
constant temperature and with a constant volume of solvent, an equal amount of solute will be dissolved independent of the amount of solid or crystalline phase present. Therefore, two consecutive recrystallizations of a chenlically pure but radiolabeled material will contain the same amount of radicactivity in the same volume of mother liquor at constant temperature. At that point, the crystalline material may be isolated and radioassayed to determine its specific activity and complete the analysis. EXPERIMENTAL
The modified RID method was carried out in either small pressure vials or in a specially constructed filter apparatus. The pressure vials used were the conical bottom type with inert Teflon (Du Pont)-coated septa, commonly used in silylation reactions. We used 1-ml and 5-ml Kontes (Kontes Glass Co., Vineland, N.J.) Microflex tubes. Separation of solvent from crystals was accomplished by decanting with a hypodermic syringe with a 25-gauge needle. It was necessary that the needle opening be a small bore to prevent entry of crystalline material. Proper crystal growth technique produced large enough crystals so this presented no problem with the compounds studied. ANALYTICAL CHEMISTRY, VOL. 45, NO. 3, M A R C H 1973
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Table 11. Analytical Results
Example No. 1
2 3 4 5
Cold carrier ETW ETU
EUc EU JBd
Initial Initial weight, charge, dpm mg 55.3 1440 46.9 5742 201.8 412 184.2 916 46.2 105'6 68.8 528
Dpm in mother liquor for: recrystallization No. 1 2 3 4 5 6 460 125 66 56 52 57 5 NSb 5048 634 87 20 284 35 15 6 9 6 745 43 18 21 17 20 990 53 10 9 8 417 64 15 11 17
6 EDAe Ethylenethiourea. NS indicates not significant above background count rate. c Ethyleneurea. d JaffCs base : 3 42-imidazolin-2-yl)-2-imidazolidinethione, e Ethylenediamine.
First, it was necessary to determine a suitable solvent system for the cold carrier being used. Ideally, the solvent should dissolve the carrier completely a t high temperatures and only slightly a t low temperatures. The steam bath and ice bath, 100 "C and 0 "C, provided our operating range. However, if necessary, an even wider range could be employed. The pressure vials permitted the use of solvents above their boiling points. Also, since there was no loss of solvent, mixed solvents retained a fixed composition. In a typical determination, cold carrier was weighed into the tared vial. Then a n aliquot of the radioactive solution of interest was added t o the vial. The volume of solution added varied from 1-100 ,ul and radioactivity levels between 400 and 11,000 dpm. The radioactive solution should, of course, be compatible with the solvent system chosen. However, if this is not possible, addition of the solution to cold carrier followed by evaporation before addition of the recrystallization solvent is recommended. After addition of a measured volume of solvent, the vial was sealed by means of the screw-cap and septum and heated until the crystals were dissolved. The crystallization was allowed to take place slowly a s the vial and contents cooled for a few minutes in air, and afterwards the vial was stored for several minutes in a n ice bath until temperature equilibration was reached. This method usually resulted in larger crystals than resulted from sudden cooling in the cold bath. The vial cap was then ordinarily removed (it could be left on) and the solvent withdrawn with the syringe. The excess solvent was shaken t o the bottom of the vial and decanted again. The needle was emptied into a counting vial and washed twice with fresh solvent into the counting vial. All the recovered solvent was counted as one sample in a liquid scintillation spectrometer. The whole crystallization was then repeated by adding the same volume of recrystallizing solvent. Ordinarily four to six recrystallizations were necessary to reach a constant specific activity. At that point, the final crystals, still in the original vial, were dried in a desiccator and weighed, then radioassayed. A series of six recrystallizations on four samples or twentyfour recrystallizations were carried out in less than one day. The filter apparatus consisted of a glass cylinder with a fritted filter disk in the middle and pressure screw-caps a t each end. These were constructed by special order from Kontes. Crystallization was carried out in one end of the apparatus and filtration accomplished by inverting and blowing the mother liquor directly into a counting vial. The crystals were assayed as before after sufficient crystallizations were carried out. The weight of crystals remaining after the final crystallization was recorded so that the radioactivity accounted for 624
ANALYTICAL CHEMISTRY, VOL. 45, NO. 3, MARCH 1973
Final wt. of
crystals, mg 34.5 37.0 44.2 83.5 22.5 35.2
Crystal radioassay, dpm/g 16860
NS 543 1063 533 908
of initial activity in crystals 65 0 27 21 2 12
accountability of radioactivity 97 101 92 103 102 103
could be balanced against that in the initial charge. Ordinarily the accountability of total radioactivity was near 100%. RESULTS AND DISCUSSION
The method was initially tested by spiking 24 mg of cold, recrystallized ethylene thiourea (ETU) with 1 p1 (10,963dpm) of a standard 14C-ETU solution and recrystallizing four times from 0.6 ml of methanol followed by a wash with ethanol. The data (Table I) indicate that within 1-2% the 14CC-ETU dissolved and assayed was the same each time; as expected, the wash nkmber was lower because equilibrium did not take place. The final crop of crystals was 9.8 mg. Of this 9.3 mg radioassayed at 476,120 dpm/g. This corresponds to 11,430 dpm per 24.0 mg starting weight or 104% of the initial activity present as ETU. Summing up the soluble dpm in each recrystallization step and the activity in the final crop of crystals, 11,001 dpm or 100 of the radioactivity was accountable. The results using several cold carriers are summarized in Table 11. Examples 2 and 6 are from a photolysis study with I4C-ETU exposed to a combination of lights simulating the ultraviolet portion of sunlight. The other examples are analyses of tomato foliage extracts from studies in which 14C-ETU was applied topically to the plants. As can be seen, the test for constant radioactivity in the mother liquor was met before crystal radioassay was carried out. The accountability, as above, is the sum of activity in the mother liquors plus the activity in the final crop of crystals. The advantages of the method are speed and simplicity; the small amount of cold carrier and radioactive material needed; ease of handling sensitive carrier compounds; and increased boiling range of solvent systems, including mixed solvents, available through use of pressure vials. The chief advantage of the method is its speed. This is particularly true if the compound being assayed is air-sensitive; the vial need not be opened if the syringe needle is inserted through the cap septum. Although a conventional RID recrystallization could be carried out with comparably small amounts of material using special and somewhat expensive microequipment, it would be much more laborious. The economy of the method is obvious considering that the cost of some carriers or their synthetic precursors may amount to $1&$100/gram. RECEIVED for review September 12, 1972. Accepted NO. vember 10, 1972.
