ANALYTICAL CHEMISTRY, VOL. 50, NO. 12, OCTOBER 1978
dichlorobenzene, and trichloroethylene were measured in such a way. Similar detection limits of approximately 1 gmol L-' were achieved (12). Linear alkylbenzenesulfonates, which are widely used detergents, are also amenable to HPLC analysis by direct aqueous injection on a reversed-phase system. In addition, polycyclic aromatic hydrocarbons can be simultaneously enriched and separated by the approach described in this paper. For the latter two compound classes, it is recommendable or even necessary to apply a solvent gradient for elution. T h e detection limit for polycyclic aromatic hydrocarbons can be improved through the very sensitive detection by UV fluorescence. Determinations of polycyclic aromatic hydrocarbons in natural waters, however, are hindered by interferences from other absorbing or fluorescing compounds. If one wants to analyze larger volumes of water (up to several liters) or heavily contaminated waters (e.g., sewage effluents), we suggest the use of a precolumn for the preconcentration step. In this way, the detection limit can be lowered and the analytical column is protected from plugging by small particles and from detrimental effects caused by other, primarily polar, constituents which are not retained by the precolumn. In cases where one single chemical has to be determined in otherwise clean water samples, the long-term stability of the column is sufficiently good to render this method useful for routine investigations. We have analyzed more than 400 groundwater samples for tetrachloroethylene without observing any deterioration of the chromatographic system. The major advantage of this direct HPLC technique is certainly its quickness and ease of operation. The limits are set by the necessity of light absorbance or fluorescence of the chemical to be determined. Furthermore, the limited separation efficiency of HPLC systems prevents the analyses of complex mixtures as they are often encountered in environmental samples. We feel that similar methods using direct aqueous injection HPLC can be developed for many other organic water pollutants. Because of the large range of possible injection
1639
volumes, it is feasible to achieve sufficiently low detection limits. In this respect, the direct H P L C method compares favorably to techniques applying direct aqueous injection gas chromatography or gas chromatography-mass spectrometry (13). Only with precolumn concentration lower detection limits could be achieved (14). In the case of tetrachloroethylene, the achievable detection limit is ten times less than the maximum allowable concentration of tetrachloroethylene in treated effluents as it is set in Switzerland (0.7 wmol L-l). Natural waters, particularly groundwaters, can contain elevated levels of tetrachloroethylene as shown in a survey of an aquifer underneath an industrial section of Zurich (3). In this study, the tetrachloroethylene concentrations were reported to exceed a t 1 2 of 18 locations the detection limit of the method presented in this paper.
ACKNOWLEDGMENT T h e authors thank R. P. Schwarzenbach and S. G. Wakeham for their suggestions on the manuscript. LITERATURE CITED (1) L. H. Keith, Ed., "Identification and Analysis of Organic Pollutants in Water", Ann Arbor Science Publishers, Ann Arbor, Mich., 1976. (2) W. Glger, Mar. Chem., 5, 429 (1977). (3) W. Giger, E. Molnar, and S . Wakeham, in "Aquatic PollutantsTransformations and Biological Effects", 0. Hutzinger, I. H. von Lelyveld, and B. C. J. Zoeteman, Ed., Pergamon Press, Oxford, 1978, p 101. (4) J. J. Rook, Environ. Sci. Techno/., 11, 478 (1977). (5) W. Giger, M. Reinhard, C. Schaffner. and F. Zurcher, Ref. 1, p 433. (6) F. Zurcher and W. Giger, Vom Wasser, 47, 37 (1976). (7) W. Giger and E. Molnar, Bull. Eviron. Contam. Toxicoi., 19, 475 (1978). (8) P. Schauwecker. R. W. Frei, and F. Erni, J . Chromatcgr., 136, 63 (1977). (9) K . Krumrnen and R. W. Frel, J . Chromatogr., 132, 429 (1977). (IO) J. N. Little and G. J. Fallick, J . Chromatogr., 112, 389 (1975). (11)J. J. Kirkiand, Chromatographia, 8, 661 (1975). (12) C. Matter, Ph.D. Thesis, ETH Zurich, in preparation. (13) L. E. Harris, W. L. Budde, and J. W. Eichelberger, Anal. Chem., 46,1912
(1974). (14) T. Fuji, J . Chromatogr., 139, 297 (1977).
RECEIVED for review June 8, 1978. Accepted July 24, 1978. This work was supported in part by the Swiss Department of Commerce (Commission of the European Communities, Project COST 64 b) and the Swiss National Research Council.
Response Correction of Differential Refractometer for Polyethylene Glycols in Size Exclusion Chromatography Sadao Mori Department of Industrial Chemistry, Faculty of Engineering, Mie University, Tsu, Mie 514, Japan
Refractlve Indices of ollgoethylene glycols at 25 and 45 O C and those of polyethylene glycols (PEG) at 25 O C were measured and the response correctlon factors for refractlve lndlces In tetrahydrofuran and In chloroform were calculated. Ollgoethylene glycols from dlmer to decamer were obtalned by preparatlve slze excluslon chromatography. The method to estlmate refractlve Index of PEG has been proposed. The dlfference In response between uncorrected molecular welght averages and those corrected was small for PEG of narrow molecular welght dlstrlbutlons such as PEG 200 and 300. The response correctlon was Important for samples havlng broad molecular welght dlstrlbutlons.
T h e differential refractometer is the most widely used 0003-2700/78/0350-1639$01 .OO/O
detector for size exclusion chromatography (SEC) or gel permeation chromatography (GPC). This device continuously detects the difference in refractive index (RI) between a reference mobile phase (the solvent) and the mobile phase containing the sample (the solute) as it elutes from the column. The difference in RI monitored is directly proportional to the concentration of the solute in the sample. Since the RI response generally varies for different compounds, the differential refractometer must be calibrated for quantitative analysis with each compound to be measured. The relationship between molecular weight (MW) and RI of a polymer is generally assumed to be constant for the calculation of MW averages from the SEC chromatogram. While this assumption is true in general for polymers with MW above 10 000, there are changes in the RI-MW relationship below MW 1OOOO. The small changes in the constant would lead to errors in the
e 1978 American Chemical Society
1640
ANALYTICAL CHEMISTRY, VOL. 50, NO. 12, OCTOBER 1978
molecular weight averages calculated from SEC data. T h e effect of M W on the RI difference of polymers and oligomers has received little attention. A few examples for changes in the constant have been reported by some workers for polystyrene ( I ) , polyethylene glycol (2-4),oligoethylene glycol (5,6),and p-oligophenylenes (7). Barrall, Cantow, and Johnson ( I ) have found a small systematic MW-RI dependence for polystyrene u p to 300 000 and higher and reported the effect on this MW-RI dependence on SEC molecular weight measurements. T h e data for polyethylene glycol showed a 1% change in the refractive index difference from 1000 t o 10000 M W (2) and significant molecular weight dependence vanished around 4000 MW (3). T h e data for molten polyethylene glycol showed that the MW-RI relationship became essentially constant a t 20000 MW ( 4 ) . The refractive index falls linearly with the reciprocal of the molecular weight by about 1% from heptamer to dimer a t 20 "C ( 5 ) . T h e refractive index for p-oligophenylene became constant by MW 1000 ( 7 ) . Measurement of t h e refractive indices of oligoethylene glycols (OEG) from diethylene glycol to decaethylene glycol and polyethylene glycols (PEG) from PEG 200 to PEG 20000 have been carried out in this study and the correction factors for the refractive index response have been calculated. These factors have then been applied to the correction of the peak intensities of size exclusion chromatograms of several P E G and molecular weight averages calculated from the corrected chromatograms. T h e effect of the MW-RI dependence on molecular weight calculations is also discussed.
EXPERIMENTAL Apparatus. Two sets of liquid chromatographs were used. One was a JASCO (Nihonbunko Co., Hachioji, Tokyo, Japan) TRIROTAR high performance liquid chromatograph equiped with a variable loop sample injection port, a differential refractometer Model R401 (Waters Associates), and two stainless-steel columns 500 mm X 8 mm i.d. packed with Shodex A802 GPC gel provided by courtesy of Showa Denko Co., Ltd. through Hikari Kogyo Co., Ltd., Tokyo 104, Japan. The gel is polystyrene-divinyl benzene copolymer and has the exclusion limit of 8000 by polystyrene molecular weight according to the manufacturer's literature. The other set was a high-speed preparative liquid chromatograph Model LC-08 (Japan Analytical Industry Co., Ltd., Mizuho, Nishitama, Tokyo 190-12, Japan) equiped with a differential refractometer Model RI-3 and two preparative GPC columns 600 mm X 20 mm i.d. packed with ,JAIGEL 2H (corresponding to Shodex H202). An Atago new Abbe-type refractometer was used for the measurement of the refractive index of oligoethylene glycols and liquid polyethylene glycols. The refractometer was thermostated at 25 and 45 "C by circulating water from a constant temperature circulating bath. Samples. A series of PEGS having number-average molecular weights from 200 to 20000 were obtained from Sanyo Chemical Co., and diethylene glycol, triethylene glycol, and tetraethylene glycol were reagent grade chemicals of Tokyo Kasei Co. Preparative SEC. Chloroform solutions (3%)of PEG 200 and PEG 300 were prepared and injected into a Model LC-08 liquid chromatograph. A sample injection volume was 3 mL. Chloroform was used as the mobile phase. Oligoethylene glycols from diethylene glycol ( m = 2) to decaethylene glycol ( m = 10) ( m represents the monomer repeating unit, e.g., the degree of polymerization) were fractionated with a recycling operation. The recycling was repeated a maximum of three times. The fractionation procedure was carried out several times to obtain the necessary amounts of OEG. The purity of the fractions was confirmed by analytical SEC and observing their single peaks. These fractions were freed of chloroform at ahout 50 "C under reduced pressure and dried at room temperature in vacuo for 6 h, and then stored over silica gel in a desiccator. Measurement of RI. Refractive indices of PEG 200,300,400, and 600 (purified by preparative operation); OEG from m = 2 to m = 10; tetrahydrofuran (THF);and chloroform were measured
at 25 and 45 "C with an Abbe refractometer. Chloroform included 170ethanol and THF 0.026% BHT (2,6-di-tert-butyl-p-cresol or butylated hydroxytoluene). Refractive indices of PEG samples having MW over loo0 which are solid at 25 OC, were measured by the following method. A PEG sample of about 60 mg was weighed accurately and dissolved in 10 mL of THF at 25 "C. The solution was again weighed accurately. The unit of concentration was gram of sample per gram of solvent. The solvent had previously been deaerated under reduced pressure. The differential refractive index of the PEG solution vs. the solvent (THF including 0.025% BHT) was measured a t 25 "C by a differential refractometer Model R401 thermostated at 25 "C. The signals from the refractometer were recorded on a chart recorder. THF was in the reference side of the refractometer. First, THF was injected into the sample side and the baseline was settled on the chart. Then, the sample solution was injected into the sample side. This procedure was repeated five times. The difference in response of the sample solution vs. THF, Ans, was measured. The difference in response of the PEG 200 solution vs. THF, AnR, was measured as a reference. The PEG solution was measured daily. The RI of PEG samples at 25 "C, which are solid at this temperature, were calculated from Equation 1.
&/G
ns = ( n - ~~ T H F ) + A~R/ACR
~ T H F
(1)
where n, = RI of a PEG sample at 25 "C; n~ = RI of the PEG 200 sample at 25 "C, 1.4585; nTHF= RI of THF at 25 "C, 1.4044; I n s = the difference of response of the PEG sample solution vs. THF at 25 "C; AnR = the difference of response of the PEG 200 solution vs. THF at 25 "C; and SC,, ACR = concentrations of the PEG sample and PEG 200. Analytical SEC. Molecular weight averages of a series of PEG samples were determined using a TRIROTAR liquid chromatograph. THF was used as the mobile phase and flow rate was 1.0 mL/min. A 0.25-mL loop was used to inject the 0.570 sample solutions. A calibration curve was constructed using PEG samples of known molecular weights. About equivalent weights of PEG 200, 400, 600, and 1000 were blended and a 170 solution was prepared. SEC of the blend was performed in the similar manner. SEC using a Model LC-08 liquid chromatograph was also carried out. The mobile phase was chloroform and flow rate was 3 mL/min. The injection volume was 1.5 mL for 1% solution of PEG samples and 2% solution of the blend.
RESULTS AND DISCUSSION Refractive Index and Response Correction Factor. The refractive indices a t 25 and 45 "C of OEG from m = 2 t o m = 10 are shown in Table I together with those of OEG from m = 11 to m = 15. The RI values of the latter five OEG were obtained by plotting R I vs. m of OEG up to m = 10 and extrapolating the curve. The response correction factors for RI of OEG in THF and in chloroform shown in the table were calculated by t h e following equation
where nrn=lo, nrn.mer, and nsolvent represent refractive indices for decamer, monomer, and the eluent. Decamer was used as a reference. T h e response of each OEG on the chromatogram can be corrected by multiplying t h e peak intensity by t h e corresponding correction factor. There is an appreciable variation of refractive index with temperature, but the variation of the response correction factor with temperature is small (compare the values a t 25 and 45 "C in Table I). It would be possible to use the correction factor a t 25 or 45 "C for the response correction at different temperature, with an allowable error. The difference in refractive index between OEG and the solvent (THF and chloroform) increased with increasing temperature, e.g., RI values at 45 "C for decaethylene glycol was 110% (in THF) and 132% (in
ANALYTICAL CHEMISTRY, VOL. 50, NO. 12, OCTOBER 1978
1641
Table I. Refractive Indices and Response Correction Factors of Oligoethylene Glycols
25 " C
45°C
response correction factor" 25 " C 45 " C in THF in chloroform in THF in chloroform
1.4455 1.4529 1.4563 1.4589 1.4597 1.4610 1.4619 1.4623 1.4630 1.4636 1.4640 1.4645 1.4650 1.4655
1.4396 1.4460 1.4490 1.4520 1.4533 1.4545 1.4556 1.4564 1.4570 1.4575 1.4580 1.4585 1.4590 1.4595
1.426 1.208 1.129 1.075 1.060 1.035 1.019 1.012 1.000 0.990 0.983 0.975 0.967 0.959
RI m
2 3 4 5 6 7 8
9 10 11 12 13
14 15
6.147 1.935 1.472 1.244 1.188 1.106 1.055 1.035 1.000 0.972 0.954 0.933 0.913 0.893
1.378 1.219 1.144 1.086 1.062 1.041 1.023 1.009 1.000 0.992 0.984 0.977 0.969 0.962
2.891 1.705 1.430 1.231 1.162 1.104 1.055 1.023 1.000 0.982 0.964 0.947 0.930 0.914
a Calculated from Equation 2. n Z s o C of THF = 1.4044, n4joC of THF = 1.3936. n 2 s 0 cof chloroform = 1.4421, n4s"c of chloroform = 1.4304.
Table 111. Refractive Indices and Response Correction Factors for Polyethylene Glycols
Table 11. Molecular Weights Obtained from Observed Refractive Indices and z- Average Molecular Weights for PEG 200, 300, and 400 MW observed RI PEG200 PEG 300 PEG400
25°C 45°C 25°C 45°C 25°C 45°C
Mz
MI
1.4585 1.4515 1.4620 1.4550 1.4631 1.4570
251 242 379 348 462 467
MW
247
468
chloroform) of those corresponding values at 25 "C. Therefore, it is important to use the differential refractometer a t the calibration temperature for quantitative analysis in HPLC. The average m number corresponding to the RI of PEG 200, 300, and 400 was obtained from the plot of R I vs. m of OEG and the refractive indices of PEG 200,300, and 400. Molecular weight, M I , corresponding to the average m number can be calculated as shown in Table 11. The values of M I are close t o z-average molecular weights of P E G 200, 300, and 400, respectively. It can be said that the OEG mixture such as PEG 200 and 300 has t h e refractive index corresponding to that which OEG equal tc, z-average molecular weight of the mixture has. Polyethylene glycols having MW of lo00 and above are solid a t 25 "C. Therefore, the measurement of their refractive indices a t this temperature is impossible and meaningless. However, P E G possesses some value of refractive index in solution and the refractive index of t h e P E G solution is different from that of pure solvent. T h e refractive index obtained from Equation 1 under the appropriate assumption does not refer to P E G at solid state, b u t corresponds to the refractive index t h a t P E G would possess in solution. Refractive indices of P E G a t each molecular weight were obtained by plotting z-average molecular weights of PEG samples and refractive indices at 25 "C which were calculated from Equation 1. The results are shown in Table 111. The response correction factors for refractive indices of P E G in THF and in chloroform were calculated by Response Correction Factor =
n M = 2 0 000 - nso1vent
n~
106 ( m= 150 ( m = 194 ( m = 238 ( m = 282 ( m = 326 ( m = 370 ( m = 414 ( m = 458 ( m = 500 550 600 650 700 7 50 800 850 900 950
378
- nsolvent
(3)
where n~ is the refractive index for PEG of molecular weight M . P E G 20000 was used as a reference and the correction factor is unity. T h e correction factors for OEG were also
RI, 25 " C 2) 3)
4) 5) 6) 7) 8)
9) 10)
1000 1100
1200 1300 1400 1500 1700 2000 2500 3000 3500 4000 5000 6000 7000 8000 20000 a
1.4455 1.4529 1.4563 1.4589 1.4597 1.4610 1.4619 1.4623 1.4630 1.4640 1.4653 1.4660 1.4664 1.4668 1.4670 1.4674 1.4676 1.4678 1.4680 1.4682 1.4686 1.4689 1.4692 1.4694 1.4696 1.4700 1.4704 1.4708 1.4710 1.4713 1.4715 1.4718 1.4720 1.4721 1.4722 1.4724
response correction factor' in chloroform in THF 1.655 1.402 1.310 1.248 1.230 1.201 1.183
1.174 1.160 1.141 1.117 1.104 1.096 1.090 1.086 1.079 1.076 1.073 1.069 1.066 1.059 1.054 1.049 1.046 1.043 1.037 1.030 1.024 1.021 1.016 1.013 1.009 1.006
1.004 1.003 1.000
8.912 2.802 2.134 1.804 1.722 1.603 1.530 1.500 1.450 1.384 1.306 1.268 1.247 1.227 1.217 1.198 1.188 1.179 1.170 1.161 1.143 1.131 1.118 1.110
1.102 1.086 1.071
1.056 1.048 1.038 1.031 1.020 1.013 1.010
1.007 1.000
Calculated from Equation 3.
calculated using P E G 20 000 as reference and are shown in Table 111. I n a dilute solution, the refractive index difference, An, between a solution of concentration C and the solvent is expressed as
An =
nmlution
-
nsolvent
= KC
(4)
1642
ANALYTICAL CHEMISTRY, VOL. 50, NO. 12, OCTOBER 1978 Table IV. Accuracy and Precision of Calculated Refractive Indices from Equation 1 for PEG 300,400, and 600 RI calculated RI from Equation 1 PEG 300
1.4620
average accuracy av. dev.
1.4616 1.4622 1.4627 1.4622 i0.0002 0.0004
1.4631
average accuracy av. dev.
1.4632 1.4636 1.4638 1.4635 t 0.0004 0.0002
1.4660
average accuracy av. dev.
1.4670 1.4655 1.4663 1.4663 t 0.0003 0.0005
PEG 400
I
, 24
l
, 26
l
l
I 3G
2%
ZLLII3 4 ‘ 3 , L I ’ E
l 32
~
~
PEG 600
(mu)
Figure 1. Chromatogram of PEG 200 in THF. TRIROTAR high performance liquid chromatograph and Shodex A802 GPC columns R I X 8. The numbers on the chromatogram refer to the number for t h e monomer unit of oligoethylene glycol
observed
Table V. Effectiveness of Response Correction in the Calculation of Molecular Weight Averages for Narrow hlW Distribution PEG MW average uncorrected corrected
PEG 200 (in THF)
E, = 230
gn= 213
-
M,
=
226
fin= 208
d-= 1.09 d = 1.08 A?, = 241 Ill, = 226 &Gn= 225 G, = 208 d = 1.07 d = 1.09 PEG 300 G, = 323 = 320 &f, = 298 = 293 (in THF) d = 1.08 d = 1.09 = 333 ATa, = 3 2 2 PEG 300 (in chloroform) = 311 = 297 d._ = 1.07 d = 1.08 PEG 400 M u = 414 % , = 410 A@n = 390 M , = 386 (in THF) d = 1.06 d = 1.06 = 611 = 607 PEG 600 (in THF) M, = 583 IC,, = 580 I I 1 I 1 1 -1-1 d = 1.05 d = 1.05 160 171 180 193 201 2.b PEG 1000 G, = 1002 G, = 990 L -13u 3LL I (in chloroform) p, = 967 = 962 Figure 2. Chromatogram of PEG 200 in chloroform LC-08 preparative d = 1.04 d = 1.03 liquid chromatograph and JAIGEL 2H. RI X 8 (-) Observed chroE, = 2.01 x i o ’ GI‘,= 2.00 x 10’ PEG 2000 Response corrected chromatogram The numbers on matogram (in chloroform) &f, = 1.95 x l o 3 A?, = 1.94 x l o 3 the chromatogram refer to the number for t h e monomer unit of oligoethylene glycol d = 1.03 d = 1.03 E, = 3.43 x 103 E, = 3.42 x 10’ PEG 3400 where K is constant. T h e value An is proportional to the E, = 3.27 x 10’ En= 3.26 x i o 3 (in THF) response of the differential refractometer. This equation is d = 1.05 d = 1.05 customarily assumed to be valid at concentrations below 30 mg/mL (8). All measurements in this study were made a t function, Equation 1. The validity of Equation 1 is shown concentrations of about 6 mg/mL. Similarly, the next in Table IV where refractive indices for PEG 300, 400, and equation is applicable to a dilute solution 600, calculated from Equation 1,were compared with those measured by using an Abbe-type refractometer. The results (5) nso1ute - nsolvent = K ’ h were in good agreement within experimental error. Effectiveness of Response Correction in the Calcuwhere K’is constant. T h e assumption that K’in Equation lation of MW. Size exclusion chromatograms of PEG 200 5 is independent of molecular weight leads t o t h e empirical
PEG 200 (in chloroform
.c,
E,,
M,
‘e,, u,
L L J
3-
(e-)
’L
A@,
ANALYTICAL CHEMISTRY, VOL. 50, NO. 12, OCTOBER 1978
1643
-
5-
in T H F and in chloroform are represented in Figure 1 and Figure 2, respectively. Separation of triethylene glycol ( m = 3) and tetraethylene glycol ( m = 4) was insufficient in the T H F mobile phase. The peak of diethylene glycol in the chloroform mobile phase was extremely smaller than that in the T H F mobile phase. The peak intensity was corrected as shown by the dotted chromatogram in Figure 2. Molecular weight averages for P E G 200 and 300 were calculated by dividing each chromatogram every 0.1 mL in the T H F mobile phase and 0.5 mL in the chloroform mobile phase. The response of each peak height was corrected using the correction factor in Table I. Corrected and uncorrected molecular weight averages are listed in Table V. The difference in both values was small in the T H F mobile phase, that in the chloroform mobile phase large, resulting from the larger difference in refractive indices of the solute and the solvent. Uncorrected molecular weights in both solvents were
The precision for the molecular weight averages in Tables V and VI was determined to be within 1.5% as a relative deviation by replicate injections of the same sample. The response correction factor of OEG and PEG in other solvents can be calculated from Equation 2 or 3.
LITERATURE CITED (1) E. M. Barrall 11, M. J. R. Cantow, and J. F. Johnson, J . Appl. Polym. Sci., 12, 1373 (1968). (2) P. Rernpp, J . Chim. Phys., 54, 421 (1957). (3) W. Heller and T. L. Pugh, J . Polym. Sci., 47, 203 (1960). (4) J. D. Ingham and D. D. Lawson, J . Polym. Sci., Part A , 3, 2707 (1965). (5) W. Heitz, B. Boerner and H. Ullner, Makromoi. Chem., 121, 102 (1969). (6) A. Weissler, J. W. Fitzgerald, and J. Resnick, J . Appi. Phys., 18, 434 (1947). (7) I. Ziegler, L. Freund, H. Benoit, and W. Kern, Mkromol. Chem., 37, 217 (1960). (8) M. B. Huglin, J . Appl. Polym. Sci., 9, 4003 (1965)
RECEIVED for review April 6, 1978. Accepted June 30, 1978.
