1968
ANALYTICAL CHEMISTRY, VOL. 50, NO. 14, DECEMBER 1978
Fundamental Splitting Conditions for Pyrogram Measurements with Glass Capillary Gas Chromatography Yoshihiro Sugimura and Shin Tsuge" Department of Synthetic Chemistry, faculty of Engineering, Nagoya University, Nagoya 464, Japan
Fundamental splitting conditions for pyrolysis-gas chromatography (PGC) with high-resolution glass capillary columns were studied using a furnace-type pyrolyzer. Polystyrene was used as a standard sample to give fairly simple pyrograms with peaks of wide range of volatility. The effects of the splitting ratio, the temperature of the splitter, the flow rate of carrier gas, the sample size, and the packing before the splitter on the reproducibility and the quantitativeness of the resulting pyrograms were examined and compared with those by a packed column. As an application, typical high-resolution pyrograms of polyethylene were also demonstrated.
Since the thermal degradation of high-polymers usually yields very complex products, there has been an increasing interest in the application of high-resolution glass capillary columns to PGC. In the practical case, however, there exist some serious difficulties in the sampling technique, Le., the introduction of the pyrolysis products onto the capillary column, and the column maintenance for long term use. Splitters are used in the usual operation of the capillary columns since the sample capacity of the columns ordinarily is in the nanogram range for each component. In the splitting mode operation, however, the sample composition actually entering the capillary column sometimes differs from the original one depending on the volatility of each component, sample size, and the splitting conditions such as the splitting ratio, the flow rate of carrier gas in the splitter, the dead volume, and the temperature of the splitter. This situation of the sampling usually causes less quantitative and poorer reproducible results, especially for complex mixtures with a wide range of volatility. To overcome this problem, modification of the splitters and various splitless sampling techniques have been studied (1-3). Schomburg et al. ( I ) reported a critical review about the operation of glass capillary columns, which pointed out some important factors which affected the performance of the conventional splitters using artificial mixtures containing homologous n-alkanes. fatty acid esters, and alcohols. On the other hand, Simon et al. ( 4 ) , Meuzelaar et al. (t5)and Leeuw et al. (6) utilized a Curie-point pyrolyzer with small dead volume for the direct splitless sampling in PGC. In usual PGC, however, very slow linear velocity of the carrier gas in the pyrolyzer, which is encountered necessarily in the splitless mode operation, sometimes causes undesirable secondary reactions. Therefore, the splitless sampling cannot always be properly adopted in PGC. Another problem in the use of capillary columns is the column contamination which results in a loss of resolution. This is especially serious in PGC since tarry components are formed more or less in the thermal degradation of highpolymers. In this work, the fundamental splitting conditions for PGC with glass capillary columns were studied using a furnace type pyrolyzer. Monodispersion polystyrene was used as a standard sample which yields a fairly simple pyrogram with peaks of wide range of volatility. The reproducibility and the 0003-2700/78/0350-1968S01.00/0
quantitativeness of the resulting pyrograms were compared with those observed with a packed column without splitting. As an application, typical high-resolution pyrograms of polyethylene were also presented.
EXPERIMENTAL Samples. Monodispersion polystyrene (PSt) of MW = 1800000 supplied from Waters Associates was used as a testing standard sample which yields fairly simple pyrograms with peaks of wide range of volatility; monomer (MW = 104), dimer (MW = 208) and trimer (MW = 312). As a typical polyolefine sample, high-density polyethyelene (PE) was also used. Apparatus. A vertical microfurnance-type pyrolyzer (7). Yanagimoto GP-1018 (Figure 1) was directly attached to a gas chromatograph, Shimadzu 7-AG. The flow diagram of the pyrolysis-gas chromatographic system is shown in Figure 2, where a coiled glass capillary column with 11 cni of diameter can be directly mounted on the inlet and outlet glass connection tubes with the same radius of curvature as that of the capillary column. In order to prevent the memory effect of less volatile components, a small portion of carrier gas was also fed from the side branches at the inlet system. However, the original inlet glass connection including the splitter was exposed to the column oven temperature. Therefore, as shown in Figure 3, the whole inlet tube was so modified as to be heated up to any desired temperatures between the oven temperature and 400 "C. Occasionally, supporting materials were charged in the dead space of the inlet tube. Pyrolysis-Gas Chromatographic Conditions. Glass capillary columns were drawn and coiled with a machine, Shimadzu GDM-1.Two capillary columns (o.d., 0.9 mm X i d . , 0.3 mm X 30 m long, and X 50 m long) suspension-coated with OV-101were prepared in basically the same way described by McKeag et al. (8). The 30-m long column (column A) was used for the separation of the degradation products from PSt, and the 50-m long column (columnB) for PE. Column temperature for PSt was programmed from 50 to 250 "C at a rate of 8 OC/min, and that for PE from 40 to 250 "C at a rate of 2 OC/min, respectively. In ordinary PGC, nitrogen was used as carrier gas at a flow rate of 0.8 mL/min at the capillary column after splitting by 25:1, 50:1, or 100:l. Scavenger gas of 50 mL/min (N2)was added at the outlet tube just before the FID. The FID was operated at 30 mL/min of hydrogen and 0.4 L/min of air. For comparison, a parked column (column C) (i.d., 3 mm X 1 m long) packed with 5% of OV-101 on Diasolid H (80-100 mesh) was also used in a temperature programming mode from 50 to 300 "C at a rate of 10 "C/min. The injection block of the gas chromatograph was maintained at 300 "C. Sample size ranging from 30 to 200 pg was pyrolyzed at 490 "C for PSt and 650 "C for PE under a flow of nitrogen carrier gas. The peak area of the resulting pyrograms was integrated by an integrator. Shimadzu, Chromatopack E-1A.
RESULTS AND DISCUSSION Typical pyrograms of P S t a t 490 "C are shown in Figure 4, (a)-(c): (a) is obtained by column C (packed column), (b) by column A with the heated inlet tube, and (c) by column A without heating of the inlet tube. The main peaks of all these pyrograms are the monomer (MSV = 1041, the dimer (MW = 208), and the trimer (MW = 312). Naturally, the apparent column efficiency for the capillary column is significantly improved, compared with the packed column. The trimer peak on pyrogram (c), however, has a remarkable C 1978 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 50, NO. 14, DECEMBER 1978
1969
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Figure 3. Modified glass inlet tube with splitter. A: inlet glass tube, 6: power supply for heater, C: heater, D: thermocouple, E: heat insulation, F: split vent, G: glass capillary column
Table I. Reproducibility Data of PSt Pyrograms with Packed Column std. dev." rel. std. deg. prod. rel. % (u) dev., ?La monomer 77.0 0.4 dimer 8.7 0.4 trimer 14.3 0.5 The statistical calculation was made for six runs.
Figure 1. Vertical microfurnace-type pyrolyzer. A: push button, B: sample holder chuck, C: carrier gas inlet, D: sample holder, E: quartz tube, F: heat insulation, G: power supply, H: injection port of GC, I: sample holder hook
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leading, whereas that on pyrogram (b) has a sharp and symmetric shape where the inlet tube including the splitter (Figure 3) is maintained at a temperature close to the
repeated
maximum column temperature (250 ' C ) . These differences have close relations to the reproducibility and the quantitativeness of the pyrograms. Tables I and I1 summerize the repetitive data of the P S t pyrograms obtained by the packed column and the capillary column in three different splitting ratios (25:1, 50:1, and 1OO:l): (A) empty inlet tube without heating; (B) empty inlet tube with heating a t 250 "C, and the packed inlet tube with 5% of OV-101 on Diasolid H (80-100 mesh); without heating ( C ) ; wrapped with asbestos tape to maintain the temperature (D); and with heating a t 250 " C (E),respectively. For each case, the weighed sample between 30 and 200 pg was pyrolyzed a t 490 "C, and all the data of 6 repetitive runs were taken into the statistical calculation. When the splitting inlet tube is empty and its temperature is exposed to that of the column oven, a homogeneous state of the degradation mixtures uith a wide range of volatility could not be achieved at the splitter. Actually, the peak shape of the trimer on Figure 4(c) mentioned above, suggests that a t least that component does not completely reach the state of vapor a t the initial stage of splitting. This leading phenomenon has a tendency to become conspicuous a t larger sample size. Generally, the statistical data imply that higher molecular weight fractions are more liable to be split off and have lower reproducibility. This tendency becomes more pronounced a t lower splitting ratios. On the other hand, when the empty inlet tube is heated up to 250 "C, both the reproducibility and the quantitativeness of the data become almost comparable to those obtained by the packed column, regardless of the Splitting ratio. Such conditions are quite satisfactory for PSt since it decomposes almost entirely into the fragments appearing on the p5Togram and the formation of tarry components is negligible. In ordinary polymers, hobever, a significant amount of the tarry components has to be expected to form, which often causes
G G Figure 2. Schematic flow diagram for pyrolysis-gas chromatograph with glass capillary column. A: pyrolyzer, 8: carrier gas inlet (N2), C: scavenger gas inlet (N2),D: hydrogen inlet, E: air inlet, F: resistance tube, G: vent, H: glass inlet tube with splitter, I:glass capillary column, J: glass outlet tube, K: FID, L: column oven
0.5 5.1 3.8
1970
ANALYTICAL CHEMISTRY, VOL. 50, NO. 14, DECEMBER 1978 X
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Flgure 4. Typical pyrograrns of polystyrene at 490 O C . (a) by a packed column (column C) without splitting, (b) by a glass capillary column (column A) with heated empty inlet tube at 250 O C using split ratio of 100:1, (c) by column A with empty inlet tube at column temperature (initial 50 " C ) using split ratio of 100: 1
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4'Figure 5. Typical pyrograrns of polystyrene at 490 O C using packed inlet tube with 5 % OV-101 and column A. (a) without heating the inlet tube using split ratio of 100:1, (b) with heating the inlet tube at 250 O C using split ratio of 100:1, (c) without heating the inlet tube using split ratio of 25:1, (d) with heating the inlet tube at 250 O C using split ratio of 2 5 1
ANALYTICAL CHEMISTRY, VOL. 50, NO. 14, DECEMBER 1978
1971
Table 11. Reproducibility Data of P W Pyrograms with Capillary Column Using Various Splitting Conditions packed inlet tubea empty inlet tubea without heating heated at 250 " C without heating wrapped with heated at 250 "C state of inlet tube (A) (B) (C) asbestos ( D ) (E) splitting ratio rel. a re]. u rel. a rel. a rel. u (flow rate) deg. prod. rel. % u (%) rel. % u (%) rel. % a (%) rel. % a (%) rel. % u (%) 25:l monomer 89.2 2.4 2.6 77.6 0.4 0.5 87.7 2.9 3.3 79.0 0.3 0.4 78.8 0.6 0.8 8.6 0.4 4.3 8.4 0.5 6.5 (20 mL/min) dimer 8.4 0.5 6.0 3.8 0.8 20.5 4.2 0.5 11.4 trimer 6.5 2.0 30.8 14.0 0.8 5.6 8.5 2.6 30.8 12.6 0.5 3.7 12.6 0.9 6.8 50:l monomer 85.5 2.1 1.7 78.4 0.2 0 . 3 77.9 0.4 0.5 2.4 77.7 0.5 0.6 84.1 1.4 8.4 0.5 6.4 6.9 0.7 9.8 (40 mL/min) dimer 7.9 0.5 5.7 5.6 0.6 11.0 8.1 0.5 5.6 trimer 7.7 1.5 19.3 14.5 0.4 2.8 10.2 1 . 4 14.0 13.5 0.5 3.4 13.6 0 . 3 2.5 2.1 77.9 0.2 0.2 77.1 0.3 0.4 1OO:l monomer 81.9 0.9 1.1 77.8 0.5 0.7 80.3 1.7 8.3 0.4 4.8 (80 mL/min) dimer 7.6 0.5 8 . 1 0.5 5.8 6.2 8.2 0.2 2.4 6.3 0.6 9.0 trimer 10.4 1.3 12.7 14.0 0 . 3 2.1 13.4 2.2 10.1 13.9 0.5 3.2 14.6 0.4 2.7 a The statistical calculation was made for six repeated runs. u = standard deviation, rel. u = relative standard deviation.
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Figure 6. High resolution pyrograms of polyethylene at 650 "C using a glass capillary column (column B). (a) without hydrogenation using split ratio of 50:1, (b) with hydrogenation of the degradation products using Pt catalyst at split ratio of 50:l. C,:n-C,alkane, C,=:n-C, (Y olefin. and
C,==:n-C, a,odiolefin serious column contamination and loss of column efficiency (9).
Therefore, in order to protect the capillary column, supporting materials coated with the same liquid phase as in the capillary column (OV-101) were charged in the inlet tube just before the splitting point (Figure 3). When the packed inlet tube is in the column oven without any additional heating, the leading of the trimer peak almost disappears and the quantitativeness of the data is a little bit improved. The poor reproducibility, however, is almost comparable to the cases of the empty inlet tube without heating (A). On the other hand, if the packed inlet tube is wrapped with asbestos tape, or heated up to 250 "C, both the reproducibility and the quantitativeness approach those of the packed column. However. as shown in Figure 5 . (a) and (b), the trimer peak given by the simply wrapped inlet tube has still very little leading; whereas when it is heated up to 250 "C, the leading becomes negligibly small. Similarly, the peak shape of the higher fragments also becomes slightly broader a t lower splitting ratios, compared with Figure 5, (c) and (d). With
the empty heated inlet tube, any peak broadening was not observed regardless of the splitting ratio and the sample size up to 200 pg of PSt, whereas appreciable tailing of the trimer peak was still observed using the heated packed inlet tube at a lower splitting ratio (125). Summerizing these results: ( I ) when the formation of any significant tarry component is not anticipated, the simple heating of the splitter is very effective both for the quantitativeness and the reproducibility; otherwise (2) the heated packing insertion just before the splitting point is quite satisfactory not only for column protection from contamination but also for quantitative measurement of the pyrograms. In the latter case, however, higher splitting ratio (higher flow rate of carrier gas a t the splitter) and smaller sample size are preferred. Practically, these packing materials in the inlet tube should be renewed after appropriate runs, depending on the operational conditions. When some specific reactions for the degradation products are necessary, these packings can be replaced with special catalysts or reagents. As an application of this technique, the pyrograms of PE
1972
ANALYTICAL CHEMISTRY, VOL. 50, NO. 14, DECEMBER 1978
are shown in Figure 6, (a) and (b), which were obtained by column B before and after hydrogenation reaction followed by thermal decomposition of about 200 pg of P E at 650 "C. In the hydrogenation mode, the carrier gas was changed to hydrogen and a catalyst tube (id., 3 mm X 15 cm) packed with 10% Pt coated on Diasolid L (80-100mesh) was inserted between the pyrolyzer and the inlet tube. Both the catalyst and the inlet tube were maintained a t 200 'C. Further work on the high-resolution pyrograms of various polyolefines is currently in progress.
(2) K . Grob and G. Grob, J . Chromatogr. Sci., 7, 584 (1969). (3) K. Grob and G. Grob, J . Chromatogr. Sci., 7, 587 (1969). (4) J. P. Schrnid, P . P. Schrnid, and W. Simon, Chromatographia, 9 , 597 ( 1 976). (5) H. L. C. Meuzelaar, H. G. Ficke, and H. C den Harink, J . Chromatogr. Sci., 13. 12 (1975). (6) J. W. de Leeuw, W. L. Maters, D. v. d. Meent, and J. J. Boon, Anal. Chem., 49, 1881 (1977). (7) S. Tsuge and T. Takeuchi. Anal. Chem., 49, 348 (1977). (8) R. G. McKeag and F. W. Hougen, J . Chromatogr., 136, 308 (1977). (9) A. Mitchell and M. Needlernan, Anal. Chem., 50, 668 (1978).
LITERATURE CITED
RECEIVED for review July 25, 1978. Accepted September 12,
(1) G Schornburg,R Dielman, H Husmann, and F Weeke, J Chromatogr , 122, 55 (1976)
1978.
Stability Constants of Calcium and Lanthanide Ions with Murexide K. S. Balaji, S. Dinesh Kumar, and P. Gupta-Bhaya" Department of Chemistry and The Biosystems Laboratories, Indian Institute of Technology, Kanpur 2080 16, U.P., India
pH titration less suitable as compared to spectrophotometric methods. However, for these determinations, one requires accurate values of stability constants at well defined pH for metal-murexide equilibria. It is well known that the positions of equilibria are pH-dependent (3). In this paper, we report values of stability constants for murexide complexes of Ca2+,Gd3+,Eu3+,Tb3+,and La3+ a t well defined pH, maintained by buffer solutions (pH < 6, acetate; pH > 6, phosphate). A method has been developed to take into account the binding of metal ions to buffer ions (acetate and phosphate). This binding reduces the concentration of free metal ion. The values so determined differ significantly from older values reported in literature, where metal ion-buffer ion binding was ignored.
The stability constants of the metallochromic indicator murexide (Ammonium Purpurate) with Caz+, Eu3+, Gd3+, La3+, and Tb3+ have been determined at several well defined pHs maintained by buffer solutions. The binding of buffer ions to metal ions has been taken into account in the analysis of the data. I n the case of Caz+, two different methods have been used to determine the stability constants and the results agree very well. The stability constant values are significantly different from the values published in literature, because in the earlier determinations the binding of buffer ions to metal ions was neglected. As expected, the discrepancy between the older values and ours is larger under conditions where the binding of buffer ions to metal ions should be stronger. For the trivalent lanthanide ions, our values differ from the older values by several orders of magnitude.
EXPERIMENTAL All reagents used were of AnalaR grade. La, Gd, and Tb were purchased as their trichlorides (99.99% pure) from Indian Rare Earths Corporation, and were used as such. Eu203(99.970pure) was purchased from Sigma Chemical Company and was converted to its perchlorate by repeated treatment with perchloric acid. Water used was doubly distilled and deionized. Spectrophotometric determinations were made with a precalibrated Beckman DU spectrophotometer. Murexide solutions were prepared in the respective buffers. Their concentrations were determined spectrophotometrically at 506 nm ( t = 1.26 X IO4 mol-' cm-' L (3),this value was determined in our laboratory). The purity of the murexide used was greater than 99.9% according to microanalytical data obtained in our laboratory. Metal solutions were standardized volumetrically against standard EDTA ( 4 ) . In all measurements an appropriate quantity of NaC104or KC1 was added to maintain the total ionic strength at the desired values. All measurements were made at the temperature specified. The titrations were carried out in the following way. Method A. Metal solution was added in small steps using a microliter pipet to a buffered solution of murexide. After each addition, the absorbance at 470 nm (480 nm for Eu3+)was noted with murexide solution in the reference cell. The choice of wavelength is dictated by the maximum in the difference spectrum. Method B. Murexide solution was added in small steps, using a microliter pipet, t o a buffered solution of metal ion. After each addition. the absorbance at 470 nm (480 nm for Eu3+)was noted
The equilibrium between metal ions and the metallochromic indicator murexide (Ammonium Purpurate) has been used to determine the stability constants of metal-ligand equilibria by monitoring spectrophotometrically the displacement of metal-murexide equilibrium due to metal-ligand binding. Some of the most important metal-ligand equilibria involve biological macromolecules as ligands. Calcium ion is one of the most important metal ions in biochemistry (1). Trivalent lanthanide ions are useful spectroscopic probes for calcium binding sites ( 2 ) . This makes the stability constants for binding of calcium and trivalent lanthanide ions to biological macromolecules important. A detailed analysis of titration data on binding of metal ions t o biomolecules provides us with equilibrium constants of individual sites and free energy of coupling between mutually interacting binding sites in a macromolecule. T o carry out such an analysis, one needs the concentrations of free metal ions as a function of the total concentration of metal ions. The titration has to be carried out under well defined conditions of temperature, ionic strength, and, in particular, pH because macromolecular structure and binding are strongly pH sensitive. This requirement makes alternative methods like 0003-2700/78/0350-1972$01.00/0
C
1978 American Chemical Society
