ANALYTICAL CHEMISTRY, VOL. 51, NO. 6, MAY 1979 (11) Y. Kat0 and T. Hashimgto, J. Polym. Sci., Part A - 2 , 12, 813 (1974). (12) D. Berek, D. BakoH, L. Soh&, and T. Bleha, J. Polym. Sci., Polym. Lett Ed., 12, 277 (1974). (13) D. Berek, D. BakoH, T. Bieha, and L. Soltes, Makromol. Chem., 178, 391 (1975). (14) T. Bieha, D. BakoS, and D. Berek, Polymer. 18, 897 (1977). (15) P. M. James and A. C. Ouano, J. Appl. Polym. Sci., 17, 1455 (1973). (16) J. N. Little, J. L. Waters, K. J. Bombaugh, and W. J. Pauplis, J. Polym. Sci.. Part A - 2 , 7, 1775 (1969). 117) . . J. N. UMe. J. L. Waters. K. J. h b a w -h . and W. J. PauDiis. J , Chromatwr. Sei., 9, 341 (1971). (18) D. Braun and G. Heufer, J. Polym. Sci., Pari 6.3, 495 (1965). (19) T. A. Maidacker and L. B. Rogers, Sep. Sci., 8, 747 (1971). (20) J. Y. Chuang and J. F. Johnson, Sep. Sci., 10, 161 (1975). 121) . . J. E. Hazell. L. A. Prince. and H. E. StaDelfeldt. J. Poivm. Sci.. Part C. 21, 43 (1967). (22) K. Heiising, J. Chromatogr., 36, 170 (1968). (23) E. G. Bartick and J. F. Johnson, Polymer, 17, 455 (1976). (24) D. BakoS, D. Berek, and T. Bleha, Eur. Polym. J., 12, 801 (1976). (25) L. W. Nichol, M. Janado, and D. J. Winzor, Biochem. J., 133, 15 (1973). (26) H. Vink, Eur. Polym. J., 9. 887 (1973). (27) P. A. Baghurst, L. W. Nichol, A. G. Ogston, and D. J. Winzor. Biochem. J., 147, 575 (1975). (28) M. Schweiger and G. Langhammer, Plaste Kautsch., 24, 101 (1977).
641
(29) B. G. Belenkii and L. Z. Vilenchik, "Chromatography of Polymers" (in Russian), Chimia Edition, Moscow, 1978. (30) M. Cantow, R. S. Porter, and J. F. Johnson, J. Polym. Sci., Part 8, 4, 707 (1966). (31) K. H. Aitgelt, Sep. Sci., 5, 777 (1970). (32) J. JanEa, J. Chromatogr., 134, 263 (1977). (33) J. JanEa and S.Pokorng, J. Chromatogr., 148, 31 (1978). (34) J. JanEa and S.Pokorng, J. Chromatogr., 156, 27 (1978). (35) J. JanEa, J. Chromatogr., in press. (36) J. JanEa and S.Pokornq, J. Chromatogr., in press. (37) P. J. Fiory, "Principles of Polymer Chemistry", Cornell University Press, Ithaca, N.Y., 1953. (38) J. C. Giddings, J. Gas Chromatogr., 1, 38 (1963). (39) C. Horvath and H. J. Lin, J. Chromatogr., 128, 401 (1976). (40) J. W. Daily and G. Bugliareilo, Ind. Eng. Chem., 51, 887 (1959) (41) J. H. Knox, Anal. Chem., 38, 253 (1966). (42) M. Kubh and S. Vozka, J. Polym. Sci., Part C , in press. (43) H. Morawetz, "Macromolecules in Solution", John Wiiey and Sons, Inc., New York, 1965.
RECEIVED for review October 23, 1978. Accepted January 5 , 1979.
Simultaneous Determination of Vitamin A Acetate, Vitamin D, and Vitamin E Acetate in Multivitamin Mineral Tablets by High Performance Liquid Chromatography with Coupled Columns Stephen A. Barnett" and Leroy W. Frick Mead Johnson & Company, 2404 Pennsylvania Avenue, Evansville, Indiana 4772 1
A reverse phase high performance liquid chromatographic method for the simultaneous determination of vltamin A acetate (retinol acetate), vitamin D, (ergocalciferol) and vitamin E acetate (d,/-a-tocopherol acetate) in multivitamin mineral tablets has been developed. The method requires dissolution of the sample in water-ethanol-pyridine solution (50:46:4), extraction of the vitamins into warm hexane, addition of cholesterol benzoate internal standard, and separation with a methanol-water gradient elution on coupled pBondapak Phenyl-pBondapak C,( columns. Detection of the vitamins and internal standard is monitored at 280 nm with separation accomplished in approximately 50 min. The assay is specific for each vltamln, and typical relative standard deviations for analysis of dosage forms are 0.059, 0.065, and 0.023 for vltamin A acetate, vitamin D,, and vitamin E acetate, respectively.
Compendia1 procedures for the analysis of vitamins A, D, and E lack specificity, are time-consuming, and are not amenable to simultaneous determination from a single sample preparation. In general, analysis of each of these vitamins by compendial methodology requires saponification, solvent extraction, and may require sample cleanup by open column adsorption chromatography. Quantitation is accomplished by spectrophotometric or colorimetric analysis of column eluent. Results using compendial methods are dependent upon the manipulative skills of the individual analyst, and generally lack precision within and between laboratories. Recent growth of reverse-phase high performance liquid chromatography has enhanced the analytical capability of the vitamin chemist. Procedures for the separation of mixed 0003-2700/79/0351-0641$01 .OO/O
vitamin standards (A, DP,E, and K) by high performance liquid chromatography are found throughout the literature ( 1 ) . Thompkins and Tscherne have described the determination of vitamin D, in gelatin-protected vitamin A and D, beadlets using adsorption chromatography after sample dissolution in dimethyl sulfoxide ( 2 ) . Osadca and Araujo have separated vitamin Dz from vitamin D3 on reverse phase packings in the presence of other vitamins in dosage forms ( 3 ) . Vitamins A, D, and E, were separated quantitatively by interfacing high performance liquid chromatography with continuous flow analysis ( 4 ) . T o date, the other forms of separation analysis, such as gas-liquid chromatography and thin-layer chromatography, do not possess the combination of sensitivity and flexibility required for simultaneous determination of these compounds. This paper describes a procedure for the simultaneous determination of vitamins A acetate, D,, and E acetate from a single sample extract using high performance reverse phase liquid chromatography and internal standard techniques. The procedure eliminates saponification, lengthy extractions, and sample cleanup, is specific for the compounds of interest in the presence of interference, and has very good internal precision compared to current compendial methods. This technique provides the high degree of resolution, reproducibility, and ease of quantitation required in the industrial quality assurance laboratory. Increased use of such new ill greatly reduce the imprecision and inaccuracies technology w associated with vitamin analysis.
EXPERIMENTAL Reagents and Solvents. Hexane, methanol, tetrahydrofuran, and pyridine were obtained from Burdick and Jackson, Muskegon, Mich. Retinol acetate d,l-a-tocopheryl acetate and cholesterol benzoate were obtained from Sigma Chemical Company, St. Louis,
0 1979 American
Chemical Society
642
ANALYTICAL CHEMISTRY, VOL. 51, NO. 6, MAY 1979
Mo. Ergocalciferol, was obtained from the United States Pharmacopeia at Rockville, Md. Millipore-filtered, doubledistilled water was used in the preparation of all aqueous solutions. A mixed vitamin standard solution containing vitamin A acetate, 0.88 mg/mL, vitamin D1,2.5 pg/mL, vitamin E acetate, 0.65 mg/mL and cholesterol benzoate, 5 mg/mL, was prepared in a solution of methano1:tetrahydrofuran (50:50). Instrumentation. The liquid chromatograph used was a Hewlett-Packard dual pump microprocessor controlled, Model 1084A, equipped with a Waters Associates, Model 440 UV Detector, operating at 280 and 254 nm. Analytical columns were Waters Associates pBondapak Phenyl, 10 pm, 3.9 mm X 30 cm, coupled with a pBondapak CI8, 10 pm, 3.9 mm x 30 cm. Separations were flow and solvent-programmed with an operating back pressure of approximately 32 MPa and were performed at ambient temperatures. Sample Preparation. An accurately weighed sample, estimated to contain 1 0 ~ 2 0 0 0 units 0 of vitamin A, 50(f1000 units of vitamin DZ,and 3545 units of vitamin E acetate was transferred to a 250-mL separatory funnel containing 25 mL of distilled water, 23 mL of absolute ethanol, 2 mL of pyridine, shaken vigorously for 2 min and placed in an air oven at 50 "C for 30 min. The sample was removed from the air oven, again shaken vigorously for 2 min. and extracted three times by gentle agitation with 50-mL portions of warm hexane (45-50 "C). The hexane extracts were collected and water-washed five times with 50-mL volumes of water, filtered through anhydrous sodium sulfate, and evaporated to dryness under reduced pressure with the aid of a warm (NMT 40 "C) water bath. A solution of methanol and tetrahydrofuran (62.5:37.5) was used to transfer the residue to a 10-mL volumetric flask containing 2.0 mL, of cholesterol benzoate internal standard solution (5 mg/mL) dissolved in tetrahydrofuran.
Chromatogram of mixed vitamin standard containing vitamin A acetate, 0.88 mg/mL (1); vitamin D,, 2.52 pg/mL (2); and vitamin E acetate, 0.66 mg/mL (3)on a single MBondapak C,, column separation was solvent programmed from 91.5 to 100% MeOH over 19 min. Retention times appear at apex of each peak Figure 1.
-
n
I I
m
h 131
L
z
14)
RESULTS AND DISCUSSION The compositions of multivitamin mineral preparations in tablet dosage form usually include raw material vitamins which are protected by a carbohydrate/protein matrix. Dissolution of this protective matrix with subsequent liberation of the vitamins is not generally a problem when compendia1 methods are employed, since the aggressive nature of the required saponification reaction facilitates removal of the vitamins. However, because of degradation during saponification and/or formation of reaction by-products which may interfere with spectrophotometric measurements, a simultaneous, quantitative extraction of t h e three vitamins is precluded. Several buffered enzyme and solvent systems were examined t o achieve simultaneous extraction of the vitamins from both raw material and dosage forms ( 5 ) . In general, a solution of water, ethanol, and pyridine (50:46:4) was found to be a n efficient means for dissolution of the vitamins (6). T h e suitability of several pure solvents and binary solvent systems was examined in order to achieve a twofold purpose; (a) to permit the simultaneous extraction of t h e dissolved vitamins and (b) to minimize extraction of interfering matrix components. Pure solvents ranging in polarity from petroleum ether through chloroform, as well as binary solutions of diethyl ether and hexane, were examined. A partition with warm hexane (45-50 "C) resulted in reproducible phase separations. T h e reverse phase mode was selected for chromatographic separation of the vitamins because of their relatively nonpolar nature (7). Reverse phase systems also offer stability of operation and reproducibility. Separation is based on the partition of nonpolar vitamin molecules between a polar mobile phase and a nonpolar stationary phase. T h e mixed vitamin standard solution was chromatographed under a variety of isocratic and gradient conditions with methanol: H 2 0 ,acetonitrile:H,O, solvent pairs on Waters CI8, phenyl, amino, and cyano columns. Isocratic separations were also investigated using the Rheodyne RP-18 5 p column. Flow programming options were examined. Analysis of the various chromatographic systems indicate that a methanol-water
Figure 2. Chromatogram of mixed vitamin and internal standard containing vitamin A acetate, 0.88 mg/mL (1);vitamin D, 2.52 Kg/mL (2); vitamin E acetate, 0.66 mg/mL (3);and cholesterol benzoate 1.0 mg/mL (4). This separation was performed using a single pBondapak C,, Column under conditions identical to Figure 1
gradient would provide favorable separation against time (8). The chromatogram shown in Figure 1 exemplifies the typical vitamin separation on the MBondapak C18 column with detection a t 280 nm. The selection of a suitable internal standard to be used with this extraction and separation system was simplified by experience gained in this laboratory in the analysis of vitamin D, in natural products and in infant formula preparations (9). Since degradation of the vitamins A, DZ,and E usually results in the formation of more polar fragments, the choice of an internal standard is limited to a nonpolar compound eluting a t the end of the chromatogram. The compound chosen for use as an internal standard must be stable in t h e solvent system selected and, if possible, be similar in molecular structure. Cholesterol benzoate satisfies these criteria. Figure 2 shows the separation of the vitamins from this internal standard. A multivitamin mineral tablet blank (containing all tablet components except vitamins A, D, and E) was formulated to quantitate recovery of t h e extraction procedure. A sample of this ground tablet blank was added to a 250-mL separatory funnel containing 25 mL of water. A volume of t h e mixed vitamin standard was evaporated to dryness and transferred as a spike to the funnel with 23 m L of ethanol and 2 mL of pyridine. Specificity of the extracting procedure was examined concurrently by identical treatment of a n unspiked tablet blank. The samples were dispersed in the water-ethanolpyridine solvent and treated as the sample preparation. Aliquots of the spiked and unspiked tablet blank extract were chromatographed against the mixed vitamin standard. No
ANALYTICAL CHEMISTRY, VOL. 51, NO. 6, MAY 1979
I
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. , , . .,
% MEOH
70
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77
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-FLOW PROGRAM
643
Flow ml!min
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1
1
1
I
5
10
15
20
25
30
35
40
45
50
55
60
20
I
TIME, MINUTES
Flgure 3. Profile of solvent and flow programs used with coupled pBondapak Phenyl-pBondapak C,, columns: insert illustrates elution of peaks of interest relative to profile. (1) Vitamin A acetate; (2) vitamin D,; (3)vitamin E acetate; (4) cholesterol benzoate
Table I. Analysis of Multkritamin Mineral Tablets for Vitamins A Acetate, D , , and E Acetate. Single MBondapak C, Column
Vitamin A Acetate
RsullL Vitamin D, YnitrlTablrt
Vitamin E Acetate UnitrlTablet
I Ln
12800
1630
36
13000
1800
34
n
12600
1860
35
(11
12200
1780
35
Mean
12800
1770
35
Theoormical
12000
532
33
interferences from the tablet blank were detected in the vitamin or internal standard retention windows. Extraction and recoveries of 9.597, and 99% for vitamins A acetate, DZ, E acetate, respectively, are typical. Tablets of finished dosage form (containing vitamins A, D, and E) were then analyzed using the internal standard technique. Results shown in Table I for vitamin A and vitamin E acetates agree with theoretical formulation; however, results for vitamin D2 are approximately 21/z times the theoretical concentration. Investigation of this disparity for vitamin D, results led to the analysis of samples of raw material vitamins A and D2 t h a t were used in the dosage formulation. Results for vitamin A were in agreement with theory but vitamin D2 results were approximately twice the theoretical concentration. Samples were subjected to analysis under the same chromatographic conditions but with detection a t 254 nm. Chromatograms obtained at 254 nm also displayed significant interference with the vitamin D2 peak. Procedures were reexamined to ensure that the substance apparently coeluting with the vitamin D2 was not an artifact of sample preparation. Several additional extracting solvents were examined. All efforts for both raw material and dosage forms resulted in the presence of an interfering peak in the vitamin Dz retention window. Chromatographic conditions were then modified to improve the resolution of the separation system. Improved separations of complex multicomponent samples can be achieved by changing parameters controlling k'. Values for k' can be changed during a chromatographic separation by modifying temperatures, mobile phase compositions, stationary phase compositions, and/or the ratio of mobile to stationary phase (IO). Separation of vitamin Dz from infant formula products in this laboratory suggests that a significant improvement in separation is achieved by solvent and flow programming using coupled columns. Under conditions of gradient elution, the changing distribution of solute between mobile and stationary phase is separated into fractions on the first column
dL Flgure 4. Chromatogram of mixed vitamin and internal standard solution, containing vitamin A acstate, 0.88 mg/mL (1);vitamin D,, 2.52 pg/mL (2); vitamin E acetate, 0.66 mg/mL (3);and cholesterol benzoate, 1.0 mg/mL (4). This separation was performed on coupled pBondapak Phenyl-pBondapak C,, columns under conditions described in Figure
3
(pBondapak Phenyl) with increased separation occurring on the less polar surface of the second column (pBondapak C18). A variety of flow and solvent programming conditions using methanol:H20 were examined for use with coupled columns. Chromatographic conditions selected for use with this coupled column system are summarized in Figure 3. A mixed vitamin standard solution containing internal standard was chromatographed under the conditions of solvent and flow programming on the coupled columns. Figure 4 provides an example of the separation of the mixed vitamin standards and internal standard. With detection a t 280 nm, precision of the chromatographic system is indicated by ten injections of the mixed standard. Relative standard deviations of 0.007, 0.041, and 0.010 for vitamins A acetate, D2, and E acetate, respectively, are typical. Reproducibility of the various retention times in the chromatographic system is *0.5%. Samples of the tablet blank were prepared and chromatographed. No interference from blank excipients or actives was observed in the vitamin or the internal standard retention windows. Both raw material and samples of finished dosage form were then reassayed. Results for vitamin A and D2from the raw material agree with theoretical concentrations. Figure 5 provides a comparison of chromatograms of the raw material on single and coupled columns. Table I1 summarizes
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 6, MAY 1979 Table 11. Analysis of Multivitamin Mineral Tablets for Vitamins A Acetate, D , , and E Acetate. Coupled Columns (pBondapak Phenyl-pBondapak C , 8 ) ReS"lt$
I I
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121
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1:
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SINGLE COLUMN SEPARATION
Vitamin D2 UnitrITablet
Vitamin E Acetate UnitrITablst
10800
550
34
11500
610
32
10700
630
32
11700
650
32
11500
670
33
11900
680
32
12200
700
34
12400
640
33
1
7
5
Vitamin A Acetate UnitrITablet
COUPLED COLUMN SEPARATION
Figure 5. Comparison of chromatograms of raw material vitamin A
12800
620
34
12500 __
-
650
33 -
Mean =
11800
640
33
RSD =
0.059
0 065
0.023
Theoretical
12800
532'
33
and D beadlets on single and coupled column systems. Vitamin A acetate (1); vitamin D, and interference (2);vitamin A acetate (3);vitamin D, (4); interference (5) extracted and, therefore, presents the greatest challenge to the extraction system. Experience to date with the analysis of multivitamin mineral tablets by this method indicates that difficulties with vitamin D2 analysis on single WBondapak CIScolumns originates in the raw material vitamin A and D. Attempts to identify this interference continue. Some lots of raw material contain no such interfering substance, thus allowing separation of finished product extracts on single columns in less than 20 min. However, the concentration of the interfering substance and/or substances does not appear to be predictable between sample lots of raw material. At this point, combinations of flow and solvent programming on coupled columns appear necessary for the degree of separation required in the quality assurance environment. Separation of this order has not been achieved to date under isocratic conditions, at constant flow, on single columns or with surface modifying reagents (i.e., hexadecyltrimethyl ammonium bromide). Until raw material variation is either eliminated or controlled or until the required improvement in separation on single columns can be realized,
the results for the three vitamins from dosage form and Figure 6 provides comparison of the chromatograms using single and coupled column systems. No significant reduction of the interfering peak was realized by detection a t 254 nm. Reconciliation of the differences noted between the relative standard deviation of the standard and samples can be addressed as follows. (1)The difference between relative standard deviation of standard and sample for vitamin E is insignificant. The relatively high concentration of vitamin E allows consistent data handling by the chromatographic system software. (2) T h e difference between relative standard deviation of standard and sample for vitamin D2 is directly related to the inability of the system software to reproducibly quantitate analytes of low concentration. (3) The difference between standard deviation of standard and sample for vitamin A is probably caused by lack of uniform extraction. Concentration is not a factor. Vitamin A is the most polar of the three vitamins simultaneously
N 131
7 7
z
SINGLE COLUMN SEPARATION
z
COUPLED COLUMN SEPARATION
Figure 6. Comparison of chromatograms of dosage form assayed for Vitamins A, D, and E on single and coupled columns. (1) Vitamin A acetate, (2)vitamin D, and interference, (3)vitamin E acetate, (4) cholesterol benzoate, (5) vitamin A acetate, (6) vitamin D, (7) interference, ( 8 ) vitamin E acetate, (9) cholesterol benzoate
ANALYTICAL CHEMISTRY, VOL. 51, NO. 6, M A Y 1979
t h e coupled column system is the method of choice.
ACKNOWLEDGMENT The authors are indebted to H. M. Baine, Jr. for assistance in the development and application of this method.
LITERATURE CITED (1) Williams, R . C.; Schmit, J. A,; Henry, R. A. J. Chromatogr., Sci. 1972, 10. 494-501. (2) Tompkins, D. F.; Tscherne, R . J. Anal. Chem. 1974, 46, 1602-4. (3) Osadca, M.; Araujo, M. J . ASSOC. off. Anal. Chem., 1977, 6 0 , 993-7. (4) MacDonald, J. C. Am. Lab. 1977, (8),69-76.
645
(5) Nunes, M.; Robinson, J. R., Mead Johnson Co., unpublished work, 1975. (6) Barnett. S. A.. Mead Johnson Co., unDublished work, 1976. i 7 j Schmit, J. A; Henry, R. A.; Williams, R. c',; Dickman. J. F. J . Chromatogr. Sci. 1971, 9. 645. (8) Snyder. L. R.; Kirkland, J. J. "Modern Liquid Chromatography", American Chemical Society: Washington, D.C.. 1973. (9) Frick, L. W., Mead Johnson Co., unpublished work, 1977. (10) Karger, 6.L.; Snyder, L. R.; Horath, C. "An Introduction to Separation Science", John Wiley and Sons: New York, 1973.
RECEIVED
for review August 24, 1978. Accepted January 29,
1979.
Universal Detector for Monitoring Organic Carbon in Liquid Chromatography Rolf Gloor' and Hans Leidner Federal Institute for Water Resources and Water Pollution Control, Ueberlandstr. 133, CH-8600 Dubendorf-Zurich, Switzerland
A detector based on the principle of measuring organic carbon as C 0 2 after oxidation has been developed. The construction and principle of the system are described. Sensitivity is discussed and compared to other LC detectors (UV, RI). The minlmum detectable concentration of organlc carbon is 3 X lod g/mL. Applications using Sephadex gel chromatography and reversed phase HPLC are shown. The detector Is restricted to nonorganic solvents.
T h e majority of detectors used in liquid chromatography (LC) today (UV-vis, fluorescence, electrochemical) are sensitive only for specific types of compounds, rather than being universal in their use (1-3). Among the universally used detectors, only the refraction index (RI) has been employed extensively, while others (transport-FID, photoionization, LC-MS) are still in an experimental stage, or their sensitivity is insufficient for many applications. In addition to its lack of sensitivity, RI yields both positive and negative signals which make its use very questionable for monitoring complex mixtures whose components may coelute. Therefore, there is still a need for a universal type detector comparable to the flame ionization detector (FID) in gas chromatography (GC). As part of a general effort in the biological department of our Institute to characterize organic water constituents, we have used gel permeation chromatography to gain information on the molecular weight distribution of organic matter in water samples of various types. Since dissolved organic carbon (DOC) is one of the key parameters in water analysis and gives t h e best information for comparison (4,we were searching for a detection system capable of monitoring the organic matter in the column effluent according to its carbon content. DOC is generally determined by oxidation of organic matter, followed by measurement of the COz produced with infrared absorbance or by FID (after reduction to methane). Axt ( 5 ) reported earlier on an analyzer capable of measuring organic carbon continuously in water samples by using the infrared technique. Based on this work, we developed a simple and inexpensive on-line detector for monitoring organic carbon in column effluents.
EXPERIMENTAL Construction of the Detection System. An illustration of the detection system is shown in Figure 1. The oxidation oven (1)(a 50 X 2.5 cm ceramic tube, 98% Alu203
standard unit, Koppers Dusseldorf, West Germany) is vertically
inserted into a furnace (2) (Heraeus, 6450 Hanau, West Germany). The oven is filled for 30 cm with ceramic splinters approximately 5 mm in length. A 5-cm-long copper tube (3), loosely filled in the lower half with platinum wool and copper pieces, is placed on top of the ceramic splinters (4). The copper tube has an outer diameter that fits snugly inside the ceramic tube. Copper forms an oxide surface in the high temperature of the oven and thus acts as a catalyst for oxidation of organic carbon. The copper tube also shields the outer ceramic tube from temperature shocks caused by the inlet flow (which enters as drops, as discussed below). Both ends of the ceramic tube, which extend 10 cm out of the oven on each side, are sealed with Teflon stoppers. If the oven is properly insulated at both ends, the temperature of the Teflon stoppers should not exceed 100 "C. The sample and carrier gas inlet is a '/4-in. stainless-steel tube with a 1/16-in.capillary tubing inserted into the top Teflon stopper. All connections (T-piece for carrier gas inlet, T-piece 1/16-in.for splitter (5)) are standard Swagelok units. The condenser (6) is a specially constructed intensive water condenser with a built-in syphon unit (7). A 20-mm syphon efficiently closes the system from the atmosphere. The infrared cell used is a Beckman 865 infrared analyzer (8) (Beckman, Fullerton, Calif.). Mode of Operation. The column effluent drops continuously into the hottest part of the oxidation oven (1) (800-900 "C) onto the platinum/copper oxide catalyst, which immediately oxidizes organic matter in the effluent to COP. A carrier air stream (C02-free)supplies the oxygen necessary for oxidation and carries the COz vapor mixture into the condenser (6). The condensed water separates from the gas phase and leaves via the syphon (7) which prevents any access of atmosphere to the system. About 30% of the carrier gas also leaves through the syphon, while the rest passes the infrared cell (8) through a side channel (9). The concentration of COPin the carrier gas is monitored on a strip chart recorder (11). The cell gas effluent reenters the system after having passed the COz filter (10). Fresh air, replacing the 30% lost through the syphon, is added by a needle valve (12) to the carrier stream. The respective amount of fresh air added is calculated from the two flowmeter readings (13, 13') and is adjusted to make up about 30% of the total carrier flow. This arrangement of the air circulation ensures a constant flow through the IR cell while the syphon acts as a pulse damper for pressure fluctuations caused by falling drops. It is not necessary to know the exact amount of carrier gas passing through the cell since the concentration of COz in the carrier stream is measured rather than the absolute amount.
RESULTS AND DISCUSSION Sensitivity. Figure 2 illustrates the excellent sensitivity
of the detection system. The response of the detector for three different compounds (fructose, propionic acid, phenol) was compared to t h a t of a UV absorbance detector (Varichrom,
0003-2700/79/0351-0645$01.00/0 @ 1979 American Chemical Society
