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Anal. Chem. i986, 58,256-258
Work is currently in progress to improve the response time of the system on a hardware level.
LITERATURE CITED (1)
Kuwana, T.; Darlington, R. K.; Leedy, D. W. Anal. Chem. 1984, 36, 2023.
(2)
Kuwana, T.; Winograd, N. In "Electroanaiytlcal Chemistry"; Bard, A. Marcel Dekker: New York, 1974; Vol. 7, p 1. Muller, R. H., Ed. "Advances in Electrochemlstry and Electrochemical Englneering"; Wiley-Intersclence: New York, 1973; Vol. 8. Kuwana. T.: Heineman. W. R. Acc. Chem. Res. 1976. 9 .~ . 241. Mlles, R. S I A , Surf. Interface Anal. 1983, 5, 43. Robinson, J. In "Electrochemistry-Speciallst Periodical Reports"; Pletcher, D., Ed.; The Royal Society of Chemistry, Burlington House: D.. Ed.;
(3)
1
London, 1984; Vol. 9. Skully, J. P.; McCreery, R. L. Anal. Chem. 1980, 52, 1885. Brewster, J. D.; Anderson, J. L. Anal. Chem. 1982, 5 4 , 2560. Bewick, A.; Kunlmatsu, K.; Pons, B. S.; Russell, J. W. J . Nectroanal.
Chem. 1984,
160, 47.
Blubaugh, E. A.; Doane, L. M. Anal. Chem. 1982, 54, 329. Pyun, C.-H.; Park, S.-M. Anal. Instrum. NY 1984, 13, 156. Amgerstein-Kolowska,H.; Conway, B. E.; Sharp, W. B. A. J . Electroanal. Chem. 1973, 43, 9. Gilroy, D.; Conway, B. E. J .
fhys. Chem. 1985, 69, 1259. Brelter, M. M. Electrochim. Acta 1963, 8, 447. Vijh, A. K.; Conway, B. E., Chem. Rev. 1987, 67, 623. Bewlck, A.; Russel, J. W. J . Nectroanal. Chem. 1982, 732, 329. Loo, B. H.; Furtak, T. W. Electrochim. Acta 1980, 25, 505. Dohrmann, J. K.;Sander, U.; Strehbiow, H. -H. 2.Phys. Chem. (Munich) 1983, 134, 41. Bewlck, A.; Kunimatsu, K.; Pons, B. S. Electrochim. Acta 1980, 25, 465.
McIntyre, J.
D. E.; Kolb, D.
M. Symp. Faraday Soc.
1970, 4 , 99.
(21) Wawzonek, S.; Berkey, R.; Blaha, E. W.; Runner, M. E. J . Electrochem. Soc. 1958, 103, 456. (22) Kolthoff, I. M.; Reddy, T. B. J . Electrochem. SOC.1981, 108, 980. (23) Turner, W. R.; Elvlng, P. J. J . Electrochem. Soc. 1985, 712, 1215. (24) Jones, R.; Spotswood, T. M. Aust. J . Chem. 1982, 15, 492. (25) Eggins, B. R.; Chambers, J. Q. J . Electrochem. SOC.1970, 117, 186. (26) Koshy, V. J.; Swayambunathan, V.; Periasamy, N. J . Electrochem. Sot. 1980, 127, 2761. (27) Tripathi, G. N. R. J . Chem. fhys. 1981, 74, 6044. (28) Trlpathy, G. N. R.; Schuler, R. H. J . Chem. fhys. 1982, 76, 2139. (29) Tripathy, G. N. R.; Schuler, R. H. J . fhys. Chem. 1983, 87, 3101. (30) Fukuzumi, S.-I.;Ono, Y.; Keii. T. Bull. Chem. Soc. Jpn. 1973, 4 6 , 3353. (31) Fujihara, M.; Hayano, S. Bull. Chem. Soc. Jpn. 1972, 45, 644. (32) Pyun, C.-H.; Park, S.-M. J . Electrochem. Soc. 1985, 732, 2426. (33) Kosower, E. M.; Cotter, J. L. J . Am. Chem. Soc. 1984, 86, 5524. (34) WMters, L. J.; Borror, A. L.; Smlth, N. Tetrahedron Lett. 1987, 24, 2313. (35) Tunuli, M. S.; Fendler, J. H. J . Am. Chem. Soc. 1981, 703, 2507. (36) Ebbesen, T. W.; Ferraudl, G. J . fhys. Chem. 1983, 87, 3717. (37) Bock, C. R.; Meyer, T. J.; Whltten, D. J. J. Am. Chem. Soc. 1974, 96, 4710. (38) Watanabe, T.; Honda, K. J . fhys. Chem. 1982, 86, 2617. (39) Blubaugh, E. A.; Doane, L. M. Anal. Chem. 1982, 54, 329. (40) Mohammad, M.; Igbal, R.; Kahn, A. Y.; Bhatti, M.; Zahir, K.; Jahan, R. J . fhys. Chem. 1981, 85, 2616. (41) Winograd, N.; Kuwana, T. J . Am. Chem. Soc. 1970, 92, 224. (42) Winograd, N.; Kuwana, T. J . Am. Chem. SOC.1971, 93, 4343. (43) Flnklea, H. 0.; Bogges, R. K.; Trogdon, J. W.; Schultz, F. A. Anal. Chem. 1983, 55, 1177.
RECEIVED for review March 13, 1985. Resubmitted June 24, 1985. Accepted August 19, 1985.
Poly(ethy1ene glycol) Molecular Weight by Laser Nephelometry Scott C. Cole,* Ellie Nielsen, and W. P. Olson Hyland Therapeutics Division, Travenol Laboratories, Inc., 4501 Colorado Boulevard, Los Angeles, California 90039
M. J. Groves' and J. Brent Bassett Searle Research and Development, 4901 Searle Parkway, Skokie, Illinois 60077 Poly(ethy1ene glycol) (PEG) of 3-4 kdalton is used widely in the preparation of human clotting factors intended for intravenous therapy (1-3). Although intravenous PEG 4000 is completely excreted in man, PEGS of higher molecular weight (MW) may not be (4), and might accumulate in, for example, hemophiliacs. A PEG of low MW is more selective in the precipitation of proteins than is a PEG of high MW, but a greater mass of low MW PEG is required to achieve the desired precipitation (5). Therefore, PEG MW relates to excretion and to the reproducibility of the fractionation process. As a matter of good manufacturing practice, a pharmaceutical manufacturer wants to confirm PEG MW to within 10% of the value claimed by the PEG manufacturer. Two methods for the estimation of PEG MW are listed in USP XXI: viscometry and determination of hydroxyl groups by phthalylation (6). Viscometry is completely nonspecific; therefore many laboratories use the phthalylation method developed by Elving and Warshowsky (7). That method is specific for hydroxyl groups only and is subject to considerable error if the pyridine is not dry (8). A simpler nephelometric test appears to be specific for PEG and is a quantitative analysis (9). We have modified that test so as to estimate PEG MW. The test involves the formation of particles when Ba2+ Present address: Department of Pharmaceutics, College of Pharmacy, University of Illinois at Chicago, Room 252, 833 S. Wood St., Chicago, IL 60680.
and iodine are added to PEG.
EXPERIMENTAL SECTION Apparatus. All nephelometry was done with the laser nephelometer "PDQ" (Hyland Diagnostics, Northbrook, IL). The " P D Q contains a He/Ne light source (632.8 nm) and a photomultiplier mounted at an angle of 31O to the incident light. Relative light-scattering (RLS) values to the nearest tenth are presented by a digital display. The RLS values are on a scale of zero to 200, and by convention the values are expressed as a percentage, (9). Particle diameters were estimated with a Brookhaven photon correlation spectrometer (Brookhaven Instruments, Ronkonkona, NY) with a He/Ne laser light source. The spectrometer was calibrated with latex beads of 100,360,and 500 nm diameter (Dow Diagnostics, Indianapolis, IN) and of 270 nm (National Bureau of Standards, Gaithersburge, MD). Reagents. Chromatographically purified PEG samples were provided by the Glycol Department of Dow Chemical (Midland, MI) and ranged in MW from about 200 to 8500. These were used as standards. Other PEG samples were from Dow Chemical and Union Carbide (Tarrytown, NY) and were commercial grade material. Barium chloride was reagent grade and the 200 mM solutions were made fresh daily as, on standing, they became turbid. The iodine solution (50 mM as monatomic I, whether I, or I-) was prepared according to USP XXI (10). Perchloric acid (reagent grade) was made up as a 500 mM solution. Water was distilled water made at our plant or demineralized, filtered water; we found no difference between these in the tests. Assay. PEG standards or sample(s) (50 f 0.5 mg) were weighed and each was dissolved in 100 mL of distilled or de-
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Chemical Society
ANALYTICAL CHEMISTRY, VOL.
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58,NO. 1, JANUARY 1986
Table 11. Estimated Molecular Weights, by Nephelometry, of Mixtures of PEG Standards
r
MW theoryb interpolated”
sample composition” 6
100
-
MW
(2/3) 1450 + (1/3) 4500 (1/3) 1450 + (2/3) 4500 (2/3) 4500 + (1/3) 8000 (1/3) 4500 + (2/3) 8000
9
x
0
0
3
257
9
6
MW x 10-3 (Circles)Relative light-scattering of PEG/Ba/I particles as a function of PEG MW, for PEG MW 1.3-9 kdalton, experimental points. (Triangles)Calculated points using least-squares fit. (Inset)Relative light-scattering of PEG/Ba/I particles as a function of PEG MW, for PEG MW 0.2-9 kdalton.
2467 3483 5667 6833
1702 2117 4236 6809
“All samples prepared as described in text. Fractions in parentheses indicate dry-weight proportions of the standards in a given sample. Whole numbers are Mw of selected Dow standards. Theoretical estimation based on mixture composition. e Experimental estimation based on interpolations from the RLS curve of the Dow standards.
Flgure 1.
Table I. Precision in the Estimation of Molecular Weight of Poly(ethy1ene glycol) by Nephelometry” sample TB840205AlH (Dow)
MW RLS method USPb USPc RLSd ne sf RSDB 3141
3354
TB830830AlH (Dow) 3240 B-207 (Union Carbide) 3053
3405 3124
B-251 (Union Carbide) 3473
NDh
3094 3359 3080 3422 3242 3270 2933 3186
20 10 20 4 20 20 12 4
94.5 133 155 219 132 92 91 43
3.05 4.0 5.0 6.4 4.1 2.8 3.1 1.3
Samples are commercial material. Mw estimated with the USP phthalic anhydride method, submitted and certified by the m anufacturer. ‘Mw estimated with the USP phthalic anhydride method, by a reference laboratory. Relative light-scattering(nephelometric) method (this paper). e Number of sample preparations. ’Standard deviation. #Relative standard deviation (coefficient of variation). Not determined. mineralized, filtered water in a volumetric flask. For the standard curve, PEGs of 1386, 3166, 4456, and 8458 MW were used. Samples and standards were stable for 3 months at 5-8 OC. One milliliter of each sample or standard was placed in a test tube, 5 mL of 500 mM perchloric acid was added, and the mixture was vortexed. To 0.1 mL of each, 4.9 mL of water was added and mixed. Duplicate 2-mL samples were placed in nephelometer tubes and, to each, 0.5 mL of 200 mM BaClz and then 0.25 mL of 50 mM iodine were added; the tubes were vortexed at each addition. Twenty minutes after mixing, nephelometer readings of duplicate samples were averaged.
RESULTS Nephelometric Data. Relative light-scattering as a function of PEG MW is shown in Figure 1. The general shape of the curve (inset) was similar to that of Lee and Groves for latex spheres and lipid emulsions (11). The ascending side of the curve, prepared with PEGs of 0.6 kdalton or less, could not be confused with the descending side (PEG MW > 1.386 kdalton) as PEG 600 has the appearance and consistency of a very viscous gel and the lower PEGs are liquids. In contrast, PEG 1386 has the appearance of a wet powder, and those of higher MW look like dry flakes or granules. Our interest was in verifying the MW of PEG powders claimed by the manufacturer to be in the range of 3-4 kdalton. Standard curves in the range of 1.3-9 kdalton took the form of Y = when Y = RLS, X = PEG MW, and a and b were constants. For a typical curve, a = 12500, b = 0.582, and the correlation coefficient (r) for four points was 0.995. Computations were
0
MW
Figure 2.
6
3
9
x
PEG/Ba/I particle diameter as a function of PEG
MW.
done on an HP-85 or a TI-59. The precision and accuracy of the nephelometric test can be appreciated from Table I. The relative standard deviation was no more than 6.4%, which we found acceptable. If we assumed that the certified PEG MW values provided by the manufacturers were true values, the RLS method for the estimation of PEG MW showed percentage accuracies ranging from 1.49 to 8.26%, which also was acceptable. When several PEG powders were mixed in various proportions by weight, the MW estimated by the RLS method was significantly different from either of the PEGs and from a theoretical weighted average (Table 11). Interpolated MW values were estimated form a curve prepared with RLS data of the Dow standards, using the calculated curve MW = 4.35 (RLS)-0.6s, Particle Size Data. We first attempted to measure the size of the PEG/Ba/I particles by scanning electron microscopy, but the particles disappeared under vacuum. However, the data of the photon correlation spectrometer (Figure 2) indicated that PEG/Ba/I particles increased in diameter with increasing PEG MW. The linear approximation, Y = aX + b, was obtained where Y = PEG/Ba/I particle diameter in nm, X = PEG MW, a = 0.134, and b = 153. For seven points, r = 0.967.
DISCUSSION Our data indicate that PEG MW, in the range of 3-4 kdalton, can be estimated from the RLS data with good precision and accuracy. Above 4 or 5 kdalton, we suspect that the RLS assay is not useful; the curve is sufficiently flat that considerable error is likely. On the other hand, we speculate
258
Anal. Chem. 1986, 58,258-261
that the method will work well with PEGs of less than 3 kdalton but have not developed data to prove this. The preparative biochemist will have interest in the 3-4 kdalton PEGs. The industrial pharmacist who formulates parenteral and oral drugs will have interest in PEG 400, which is useful as a cosolvent for water-insoluble drugs (12). The particle size data from the photon correlation spectrometer suggest another method for estimating PEG MW from PEG/Ba,/I particles. Because a test based on these particles is both a qualitative and a quantitative analysis, our tests-whether done with the nephelometer or the photon correlation spectrometer-are more useful than the viscometric or phthalylation methods detailed in USP XXI. The RLS method, like the viscometric and phthalylation methods, is an averaging technique and does not provide data on PEG MW distribution. The technique cannot differentiate between a conventional normal distribution and bimodal distribution as might occur if two very different PEGs were mixed. The use of chromatographically purified PEG samples as standards suggests that an HPLC method could also be used for estimated MW of PEG. This was tried previously in our laboratory using a pStyrage1 column. In our hands, this method gave poor results, and HPLC as an analytical technique for PEG was abandoned (13). Our data support the observation of Lee and Groves (11) that maximal RLS obtains as particle diameter approaches the wavelength of the incident light. However, we caution the analyst that the significance of RLS curves, such as our Figure 1,can be determined only if one maintains constant particle number or particle size. We also caution that analyst against the use of a UV-vis spectrophotometer in place of a
nephelometer measuring scattered light at an angle from the incident light. The two systems give very different results (9).
ACKNOWLEDGMENT We thank Dow Chemical for the gift of chromatographically purified PEGs. Victor Khachatourians performed the RLS analyses and Rick Srigley and Steven Kuwahara reviewed the manuscript; all are from Hyland Therapeutics. Registry NO. PEG (SRU), 25322-68-3; barium chloride, 10361-37-2; iodine, 7553-56-2,
LITERATURE CITED (1) Chandra, S.;Wickerhauser, M. Thromb. Res. 1978, 72, 571-582. (2) Johnson, A. J.; MacDonald, V. E.; Brind, J. Vox Sang. IS’lS, 3 6 , 72-76. (3) Wickerhauser, M.; Williams, C.; Mercer, J. E. Vox Sang. 1979, 3 6 , 281-292. (4) Shaffer, G. B.;Critchfield, F. H. J . Am. Pharm. Assoc., Sci. Ed. 1947, 3 6 , 152-157. (5) Ingham, K. C. Methods Enzymol. 1984, 704, 351-355. (6) “The United States Pharmacopeia”; 21st rev.; United State Pharmacopeial Convention, Inc.; Rockville, MD, 1985;pp 1589-1590. (7) Elving, P. J.; Warshowsky, B. Anal. Chem. 1947, 79, 1006-1010. (8) Siggla, S.;Hanna, J. G. “Quantitative Organic Analysls via Functional Groups”; 4th ed.; Wiley: New York, 1979;pp 22-28. (9) Cole, S.C.; Christensen, G. A.; Olson, W. P. Anal. Biochem. 1983, 134 368-373. (10) “The United States Pharmacopeia”; 21st rev.; United States Pharmacopeiai Convention, Inc.; Rockvllls, MD, 1985;pp 543-544. (11) Lee, G.; Groves, M. J. Powder Techno/. 1981, 28 (I),49-54. (12) Groves, M. J.; Bassett, B.; Sheth, V. J . Pharm. Pharmacol. 1984, 3 9 ,
799-803. (13) Luckhurst, D., personal communication, 1985, Hyland Laboratorles, Los Angeles, CA.
RECEIVED for review Februrary 25, 1985. Resubmitted September 9, 1985. Accepted September 9, 1985.
Flash Thermal Desorption as an Alternatlve to Solvent Extraction for the Determination of C8-C35 Hydrocarbons in Oil Shales Phillip T. Crisp* and John Ellis Department of Chemistry, University of Wollongong, Wollongong, N.S. W. 2500, Australia Jan W.de Leeuw and Pieter A. Schenck Delft University of Technology, Department of Chemistry and Chemical Engineering, Organic Geochemistry Unit, de Vries van Heystplantsoen 2, 2628 RZ Delft, T h e Netherlands The small quantities of volatile hydrocarbons that occur in oil shales ( ~ 1 % of total organic matter) may be derived either from hydrocarbons originally present in sedimentary organic matter or from low-temperature pyrolysis reactions occurring over millions of years. Volatile hydrocarbons provide, therefore, a characteristic “fingerprint” of the organic matter in the oil shale, which may be used to demonstrate the nature and extent of deposits and which may correlate with certain retorting characteristics. Measurement of volatile hydrocarbons has been limited due to the time required for solvent extraction of rock samples and for the workup of the resulting extracts. An alternative to solvent extraction is the release of adsorbed hydrocarbons in oil shales by gentle heating. This is done in the commercial Rock-Eval instrument ( I ) , which measures the total amount of hydrocarbons desorbed from a crushed rock sample in a furnace at 250-300 “C before determining the pyrolysis yield at 300-550 “C. The Rock-Eva1 instrument was designed for the investigation of petroleum source rocks and has recently been applied to the evaluation of Canadian oil shale deposits ( 2 , 3 )and to the oil shales of the Toolebuc Formation in Queensland ( 4 ) . Ex-
tensions of the Rock-Eva1 method allow the thermally desorbed hydrocarbons to be trapped either cryogenically (5) or on an adsorbent (6)and analyzed by gas chromatography. Previous work on the comparison of compounds solvent-extracted from shale samples and tho,se thermally desorbed by the use of a small furnace (5) found significant differences with compounds eluting after CI5. Significantly less material >CI6 was found in the thermal desorbate and this was attributed to the catalytic cracking of long-chain compounds by minerals in the shale matrix (7) during thermal desorption. In the present study, the residence time of volatile constituents in the heated mineral matrix has been reduced by carrying out desorption in a Curie-point pyrolysis unit. Under these conditions, the compounds detected are remarkably similar to those obtained by solvent extraction and analysis time is greatly reduced. Since the organic matter found in oil shales is entirely comparable to that in petroleum source rocks (8) and volatile hydrocarbons provide a valuable maturation indicator (9),exploration companies may also wish to apply the Curie-point technique to the geochemical investigation of petroleum deposits.
0003-2700/86/0358-0258$01.60/0 0 1985 American Chemlcal Soclety
