Ind. Eng. Chem. Prod. Res. Dev., Vol. 18, No. 2, 1979 117
Oxidative Coupling of Toluene to Stilbene Stephen S. Hupp and Harold E. Swift‘ Gulf Research & Development Company, Pittsburgh, Pennsylvania 15230
I n recent years, the conversion of toluene to stilbene has received considerable attention as the first step in a toluene to styrene process. One way this reaction has been achieved with high selectivity is by reacting toluene with lead or bismuth oxide. This paper reports on studies directed toward developing a supported form of lead oxide which exhibits high activity and selectivity, can be readily regenerated, and has good physical strength. Considerable success was achieved in developing a supported lead oxide having a low attrition index; however, such an oxidant had a low oxygen-carrying capacity, which limited its activity. Attempts to increase oxygen-carrying capacity resulted in losses in activity and/or physical strength.
Introduction Background. The oxidative coupling of toluene to bibenzyl and stilbene has been known for many years. Recently, there has been renewed interest in this reaction as a first step in the production of styrene from toluene and ethylene (Knox et al., 1976; Stanford Research Institute, Process Economics Review, 1977). The ideal reaction to product stilbene from toluene would be the direct dehydrocoupling reaction (eq 1).
However, this reaction is thermodynamically unfavorable and must be combined with a highly exothermic reaction to remove hydrogen and provide a driving force for the overall reaction. Virtually all reports of carrying out the coupling reaction have shown the use of oxidants containing oxygen, sulfur, or a halogen (Benson and Hardesty, 1968; Buyalos et al., 1970; Garst and Henry, 1971; Hargis and Young, 1969) to remove the hydrogen as water, hydrogen sulfide, or a hydrogen halide. Of these, the removal of hydrogen as water would seem the obvious first choice because of the relative ease of handling, recovery, and disposal, as well as the large driving force provided by the free energy of formation of water. The overall reaction (eq 2) is thermodynamically fa-
*aCH3 +
silver, thallium-silver (Weterings, 19761, and antimonylead-bismuth (Li, 1978). Of these, the most active and selective are the oxides of bismuth and lead. Toluene is not unique in undergoing the oxidative dehydrodimerization reaction. Other compounds have been oxidatively coupled in an analogous manner using similar oxidants, and the literature regarding such reactions is generally pertinent to the coupling of toluene. Examples are the coupling of propylene to give 1,5-hexadiene and benzene (Swift et al., 1971) and reaction of isobutylene to give 2,5-dimethylhexadiene-1,5 and p-xylene (Bozik and Swift, 1971). Reactions and Products. In the oxidative coupling of toluene to stilbene, toluene is first dehydrocoupled to bibenzyl (eq 3) which is subsequently dehydrogenated to
-
‘ 2 m c + PbO H 3
cis and trans-stilbene (eq 4). The more stable trans isomer
@cH2-cHzB --+
PbO
02
+
vorable, but generally proves to be inefficient, even in catalytic systems, because of the preponderance of nonselective free-radical reactions, leading to complete combustion of the hydrocarbons and the formation of oxygenated products. More selective reactions have been conducted using oxidants such as metal or nonmetal oxides as stoichiometric reactants, providing lattice oxygen which is depleted during reaction. The reaction of toluene with litharge (PbO) to form stilbene was reported as early as 1873 (Behr and Van Dorp, 1873). Many oxygen compounds have been reported since, including oxides of bismuth, cadmium, thallium, antimony, and arsenic (Hargis and Young, 1969) as well as mixed oxides such as bismuth-tin (Liu et al., 1977), bismuth0019-7890/79/1218-0117$01.00/0
H20
+ Pb (4)
predominates by a ratio of about 10 to 1. The ratio of stilbene to bibenzyl generally correlates rather well with the level of toluene conversion. Those conditions which favor higher conversion of toluene also favor the further reaction of bibenzyl to stilbene. Substantial amounts of benzene are usually produced, presumably by the oxidative dealkylation of toluene (eq 5).
The other major reaction which competes with silbene formation is the combustion of hydrocarbons to carbon dioxide and water. 0 1979 American Chemical Society
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Ind. Eng. Chem. Prod. Res. Dev., Vol. 18, No. 2, 1979
Reactants
Figure 1. Two-zone fluid bed reactor. Toluene coupling reaction and regeneration of oxidant are carried out in the same reactor. Oxidant circulates from the top reaction zone where it reacts with toluene to the bottom regeneration zone where it is restored by burning in air.
In addition to these major reactions, there are a number of nonselective reactions which form small amounts of products, including ethylbenzene, xylenes, phenol, benzaldehyde, naphthalene, biphenyl, diphenylmethane, diphenylacetylene, benzophenone, benzoin, phenanthrene, and anthracene. The economical use of stoichiometric oxidants in the toluene coupling reaction requires either that the materials be very inexpensive and may be discarded or that they may be restored easily for reuse. Fortunately, lead oxide can be regenerated by burning in air at or near the reaction temperature. Thus, it is used as a catalyst and completes the reaction and reoxidation cycle unchanged, though the reaction and regeneration are not conducted simultaneously. Such an operation, however, requires proper engineering in order to accommodate the need to remove the catalyst periodically from the reaction stream as it becomes depleted, reoxidize the catalyst, and resume the reaction. Reactor Systems. The reactor concepts evaluated during the course of this research are of two general types: fixed bed cyclic and moving bed continuous. In the fixed bed system, the toluene coupling reaction and catalyst reoxidation are carried out alternately in the same reactor. Two or three reactors with staggered cycles would be required to maintain continuous operation. This system was judged to be too costly for large-scale commercial operation because of the construction materials required for reactors which would be subjected to oxidizing and reducing conditions and the large expensive valves which would be necessary for short cycle operation. Conducting the reaction continuously with a single reactor requires that a portion of the catalyst be constantly removed, regenerated, and returned to the reaction stream. Fluid bed catalysts are best suited for this moving bed operation. The reaction and regeneration can be carried out in the same vessel if the toluene and regeneration air are introduced at different places in the reactor (see Figure 1). The turbulent fluidization circulates the catalyst from the top reaction zone where it is depleted, to the regeneration zone where it is restored, and back to the top to take part again in the reaction. Oxygen is consumed from the air introduced into the bottom leaving only nitrogen as an inert diluent as it contacts the toluene in the reaction zone. This reaction configuration was used by Monsanto investigators (Knox et al., 1976) and works well on the laboratory scale, but engineering problems involved in
+
Figure 2. Circulating fluid bed reactor. Reaction of the oxidant with toluene and regeneration with air are carried out in separate reactors. The oxidant is circulated continuously between the coupling reaction and the regenerator. N 2 Toluene
Pressure Con tro I Va Ive Products
Bayonet Filter
S t e a h Generator
Figure 3. Diagram of laboratory-scale fluid bed reactor. The toluene inlet is adjustable to introduce toluene at any level in the bed so the unit may be used as a single-zone or two-zone reactor.
scale-up and control of circulation make this a dubious choice for large-scale operations. The alternative mode of operation is to regenerate the catalyst in a separate vessel and circulate the catalyst continuously between the reactor and regenerator (see Figure 2). This design is similar to existing catalytic cracking operations and was judged to be the method which could most likely be used successfully in a commercial operation. This conceptual design for toluene coupling also prescribes certain essential characteristics of the oxidant. It was our goal to develop a fluid bed catalyst which has the following characteristics: (1)high activity and selectivity to minimize toluene recycle and minimize toluene loss to undesired products; (2) high oxygen-carrying capacity; the oxidant must carry as much usable oxygen as possible in order to reduce the energy required for circulation of solids; (3) high reoxidation rate; regeneration of the oxidant should be fast in order to minimize oxidant inventory and regenerator size; (4)high physical strength; the oxidant not only must be resistant to attrition but must be stable to the structural changes of repeated oxidation and reduction. Despite the decision to pursue toluene coupling in a circulating system, the design, construction, and operation of a small-scale unit would have been complex and expensive. The cost of such a unit was not justifiable at the catalyst research stage. It was decided to approximate the results which would be obtained in a circulating fluid bed unit by conducting the reaction in a single fluid bed reactor in cyclic operation. The length of the reaction cycle will be roughly equivalent to the average residence time of the
Ind. Eng. Chern. Prod. Res. Dev., Vol. 18, No. 2, 1979
118
Table I. Lead Oxide o n Various Carborundum Fluid Bed Carriers 96% A1,0,, 1.3 SiO,
100%A1,0,
catalys ta conversion selectivity stilbene bi benzyl benzene + CO, other products CO, (combustion) wt catalyst charged, g wt percent fines percent total Pb in fines attrition ratioC
96% A1,0,, 2.3% SiO,
10% 15% 15% PbOon PbOon 15% PbOon 15% SAHT P b O o n SAEHSSAEHS- P b O o n 33b SAEHS-5 99-16 SAEHS-3 Std
15% PbOon SAHT 96-13
15% PbOon SAHT 96-16 14.9
32.0
26.2
32.0
36.9
29.0
25.3
59.3 5.8 22.2 6.2 6.5 517 7.0% 14% 2.0
58.3 10.0 19.9 7.6 4.2 660 3.5% 12% 3.4
63.8 7.1 18.3 7.5 3.3
60.0 5.0 23.2 7.8 4.1 665 5.1%
63.0 7.5 20.2 7.0 2.3 661 4.9% 12% 2.4
58.1 8.6 22.2 7.4 3.9 759 0.6% 1.7% 2.8
IO5 1.0% 1.7% 1.7
39.8 24.5 20.9 9.4 5.5 1012 2.0% 7.2% 3.6
96% A1,0,, 3.3% SiO, 15% 15% PbOon PbOon SAEHS-2 SAEHS-1 20.8
16.9
63.2 10.9 15.7 7.2 3.0 559 0.9% 1.6% 1.8
60.6 16.2 12.9 8.1 2.3 636 0.6% 0.7% 1.2
Designaa Catalysts are arranged by increasing silica content and increasing severity of heat treatment of the support. tions are those given in Carborundum’s catalog. Attrition ratio is percent total Pb in fines divided by weight percent fines. This ratio gives a measure of the selective loss of Pb from the catalyst.
catalyst in the coupling reactor of a circulating system. Experimental Section All of the fluid bed reactions were carried out in reactor units similar to that shown in Figure 3. The reaction zone measured 2 in. in diameter by 18 in. long and was heated by two Calrod heaters attached to the outside wall and controlled by ECS proportional controllers. Above the reaction zone was a catalyst disengagement zone 6 in. in diameter by 7 in. long. This was maintained at 400 “C with an attached Calrod heated and ECS controller. Nitrogen, air, and steam entered the bottom of the reactor through a “D” porosity stainless steel fritted disk which supported the catalyst and dispersed the incoming stream to give uniform fluidization. Toluene entered the reactor through a Il4-in dipleg which could be adjusted to introduce the toluene a t any level within the catalyst bed. For cyclic operation, the toluene inlet was adjusted to 1 in. above the diffuser frit. The effluent stream exited at the top of the reactor through a “D” porosity stainless steel bayonet filter. Liquid products were collected in a wet ice trap and dry iceacetone trap and were analyzed by gas chromatography using 6 ft X in. stainless steel columns packed with 10% Silar 1OC Chromosorb Q. Carbon dioxide was collected on Ascarite and weighed by difference. The catalyst was regenerated with air until GC analysis of the effluent showed less than 0.5 mol 70 carbon dioxide and an oxygen content corresponding to that being introduced. The catalysts (or oxidants) were prepared by incipient wetness impregnation of aqueous Pb(NO3I2 on a low surface area catalyst carrier obtained from the Carborundum Co. The supports were used as received from the manufacturer except that they were screened to size (50-140 mesh) before impregnation. Catalysts were oven dried a t 120 “C for 8 h and calcined a t 700 “C for 8 h. Results a n d Discussion Active Phase. The metal oxide chosen as the active component for toluene coupling will largely determine the catalyst’s activity, selectivity, oxygen-carrying capacity, and reoxidation rate. Lead and bismuth oxides have been shown to be active for toluene coupling with good selectivity, but the reaction rate is quite slow. Even at toluene rates less than one-tenth gram per gram of catalyst per hour, only about one-third of the toluene is converted to coupled products. In addition, the oxygen-carrying capacity of these heavy metals is quite low. A catalyst
containing 15% lead oxide, for example, carries only 10.8 mg of oxygen per gram. A number of metals were added to PbO in an attempt to increase its performance. Some resulted in increased toluene conversion; however, in all cases there was an adverse effect on selectivity. S u p p o r t Material. Unsupported bismuth oxide was studied for the oxidative dehydrodimerization propylene and was found to be unsuitable for long-term use, even in a fixed bed reactor (Swift and Bozik, 1971). The best results with propylene were achieved using bismuth oxide impregnated on supports with surface areas less than 1 m2/g. Higher surface area supports gave poor selectivity. Similarly, low surface area supports were required for toluene coupling. Screening experiments using bismuth oxide supported on various alumina carriers showed selectivity to coupled products decreases rapidly with increasing surface area above about 0.1 m2/g. The requirement for an extremely low surface area support together with the need for a high loading of active metal oxide presents a problem in fluid bed operation. There is virtually no internal surface where the metal oxide can reside. Microscopic observation of the fluid bed catalysts showed that lead oxide is deposited as small crystals on the outer surface of the spherical support particles. This leaves the soft metal oxide very vulnerable to attrition. In a seven-day run with a catalyst of 15% PbO supported on Carborundum SAEHS-33 alumina, one-half of the lead was lost from the catalyst as fines which collected on the walls of the reactor disengagement zone. A solution to this selective attrition problem was sought by preparing catalysts in which the active metal oxides were prepared in mixtures with support materials rather than impregnated on them. These catalysts gave disappointing results in fixed bed screening tests. Some success was achieved by modification of the fluid bed carriers provided by Carborundum. A series of two-day tests was conducted using catalysts of lead oxide impregnated on a variety of Carborundum supports (Table I). These supports differed most notably in the silica content of the aluminas and in the severity of heat treatment to which the materials were subjected. In each run, the fines at the top of the reactor and the catalyst remaining in the reaction zone were collected separately and the lead content of each fraction was determined by X-ray fluorescence. Invariably, the catalyst fines were found to contain a higher percentage of lead
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Ind. Eng. Chem. Prod. Res. Dev., Vol. 18,
No. 2, 1979
Table 11. Lead Loading o n a Silica-Containing Support" 15% PbO
20% PbO
30% PbO
conversion 21.1 30.3 27.1 selectivity stilbene 56.6 55.4 56.0 13.4 bibenzyl 14.0 8.8 benzene t CO, 15.9 22.5 15.5 other products 8.2 7.0 10.8 CO, (combustion) 5.3 6.3 4.4 ' Catalyst support: carborundum SAEHS-1; 96% A1,0,, 3.3%SO,. Conditions: temperature, 600 "C; atmospheric pressure; toluene WHSV = 0.6; steam/toluene = 7 mol; 10-min reaction cycles. Table 111. Extended Run with PbO on Silica-Containing Support" _
_
~
~
day no. of samples conversion selectivity stilbene bibenzyl benzene + CO, other products CO, (combustion)
day no. of samples conversion selectivity stilbene bi benzyl benzene + CO, other products CO, (combustion)
1 1 28.2
2 3 25.5
3 2 25.3
4 3 26.2
5 3 27.0
57.7 9.2 20.2 8.6 4.3
58.5 57.9 58.7 57.7 10.3 9.4 9.0 10.0 19.9 20.5 21.0 20.4 7.2 8.3 7.2 7.9 4.1 3.9 4.2 4.0 a Catalyst: 15% PbO on Carborundum SAEHS-5; 100% Al,O,. Conditions: temperature, 600 C; atmospheric pressure; toluene WHSV = 0.060; steam/toluene = 6.7 mol; 10-min reaction cycles. Table V. Lead Oxide on Silica"
_ ~_ _ _ _ ~
3 3 31.3
Table IV. Extended Run wtih PbO o n Pure Alumina Support"
1 3 35.5
2 3 31.5
4 3 28.6
5 2 25.5
68.7 7.5 13.0 8.3 2.6
64.2 62.7 64.2 63.3 8.1 7.7 9.0 9.8 16.1 17.5 16.4 16.7 7.7 7.4 6.7 6.9 4.0 4.6 3.6 3.3
a Catalyst: 20% PbO on Carborundum SAEHS-1; 96% Al,O,, 3.3% SiO,. Conditions: temperature, 600 " C ; atmospheric pressure; toluene WHSV = 0.053; steam/ toluene = 7.1 mol; 10-min reaction cycles.
than did the catalyst from the bed, indicating that a disproportionate amount of lead was being attrited from the catalysts. Table I gives a relative measure of the lead loss in the "attrition ratio" which is derived by dividing the percentage of the lead found in the fines by the percentage of total catalyst collected as fines. The ideal catalyst would show an attrition ratio of 1,indicating that the active component was lost in proportion with the rest of the catalyst. It was found, as expected, that overall catalyst attrition decreased with increasing severity of heat treatment for a given composition of the support. It was not fully expected, however, that those supports which gave the least overall attrition would also lose the least lead. This, in fact, appears to be happenstance, since there is no consistent trend among the other catalysts. Two supports showed particular promise, one of pure alumina which has undergone severe heat treatment (SAHT 99-16), and the other a silica-containing support (SAEHS-1). A catalyst made from the first exhibited high activity and attrition resistance whereas one made from the latter exhibited superior attrition and lead retention properties. It is tempting to draw correlations of activity or selectivity with silica content or heat treatment from the results in Table I; however, these might be misleading since both activity and selectivity were found to vary with the amount of lead on the supports. The silica-containing support SAEHS-1, for instance, gave a maximum in activity and minimum in selectivity with around 20% lead oxide as shown by the results in Table 11. The benefit of the superior attrition properties of this silica-containing support is tarnished considerably by losses in activity of the catalysts with age. An extended run with a catalyst containing 20% PbO showed steadily declining activity (Table 111) while a catalyst supported on pure alumina showed virtually no change in five days (Table IV). Comparison of X-ray diffraction patterns of a fresh catalyst and used catalyst revealed that a large part of the lead had reacted with the support to form a mixed oxide
conversion selectivity stilbene bibenzyl benzene t CO, other products CO, (combustion)
5.8 8.2 18.3 55.2 3.8 14.5
' Catalyst: 15% PbO o n S O , ; Davison Grade 59. Conditions: temperature, 600 'C; atmospheric pressure; toluene WHSV = 0.072; steam/toluene = 5.5; 10-min cycles. Table VI. Lead Loading o n Pure Alumina Support" 15% PbO conversion selectivity stilbene bibenzyl benzene + CO, other products CO, (combustion) wt percent fines
20% PbO
25% PbO
32.0
31.4
28.1
63.8 7.1 18.3 7.5 3.3 1.0%
58.7 6.5 19.7 10.7 4.4 4.7%
58.9 59.6 8.8 10.9 19.3 18.5 8.5 7.2 4.4 3.7 9.2% 12.4%
30% PbO 25.1
Catalyst support: Carborundum SAHT 99-16; 100% N , O , . Conditions: temperature, 600 C; atmospheric pressure; toluene WHSV = 0.06; steam/toluene = 6 mol; 10-min reaction cycles.
compound identified as Pb8A12Si4019.This stoichiometry is such that in a catalyst of 20% PbO on this support (96% A1203,3.3% SiOJ, there is enough silica to react with 98% of the lead. The reaction of lead with the support is also evident based on microscopic observation. Lead oxide on pure alumina shows yellow crystals jutting out from the surface, whereas the silica-containing support shows an even, brown coating on the surface. A catalyst of 15% PbO on silica showed outstanding attrition resistance in a 2-day test, but the activity for toluene coupling was very low as shown by the results in Table V. A study with the pure alumina support SAHT 99-16 (Table VI) showed a dramatic increase in attrition with increased loadings of PbO, and the greater amount of the fines is unquestionably lead lost from the support. The limit of lead oxide which can be carried on this support is about 15 wt 70.Again, a variation in activity and selectivity is seen with increasing amounts of lead. With this support, higher activity and selectivity appear to be favored by the lower lead loading. Reaction Parameters. Lead catalysts have been studied in considerable detail to determine the effects of the reaction parameters. Results in Table VI1 show that with increasing temperature lead oxide gives higher conversion and deteriorating product distribution as increasing amounts of toluene go to benzene and combustion.
Ind. Eng. Chem. Prod. Res. Dev., Vol. 18, No. 2, 1979
121
Table VII. Parameter Study with a Lead Oxide Catalyst in the Fluid Bed Reactoru temperature pressure toluene WHSV steam/toluene, mol cycle time, min no. of samples conversion selectivity stilbene bibenzyl benzene + CO, other products CO, (combustion) oxygen consumption
575°C atm 0.061 6.7 10 5 14.2
600°C
625°C
10 10 28.2
10 3 40.0
71.2 13.2 10.6 4.1 0.9 3%
68.0 9.1 13.2 7.8 1.9 7%
56.7 5.4 21.3 9.4 7.2 18%
600°C atm 0.058 3.8 10 20 3 2 38.2 35.6 58.4 5.0 22.1 6.9 7.6 17%
53.5 4.5 26.3 6.1 9.6 35%
30 2 28.8
600 " C atm 0.24 2.9 5 2 20.5
10 3 19.9
20 2 15.1
51.1 5.4 29.5 5.0 9.0 41%
60.1 12.1 16.9 5.5 4.2 14%
56.4 13.5 16.5 7.2 6.4 31%
52.3 14.3 22.9 4.8 5.7 47%
Catalyst: 15% PbO o n Carborundum SAHT 99-16 (100% A1,0,).
Coupled Products
30
8
Benzene
8
v) 01
Combustion
1 01 0
A
&
.",AP A
AA
A A A A A I
10
I
I
20
I
A I
1
30
,
40
I
I
10
-AA
50
% Oxygen Depletion o f Catalyst
Figure 4. Selectivity to various products as a function of the percent oxygen depleted from lead oxide in catalyst--15% PbO on SAHT 99-16.
With increasing reaction time, lead catalysts give decreasing toluene conversion and declining selectivity to coupled products. In fact, this decreasing efficiency with oxygen depletion from the lead catalysts seems to dominate other reaction parameters in determining reaction selectivity. Figure 4 shows a plot of product selectivities vs. oxygen depletion for more than 25 runs at widely differing conditions of temperature, pressure, space velocity, steam-to-toluene ratio, and reaction time. Regardless of reaction conditions, the product distribution is mainly determined by the state of the catalyst. Similar plots of oxygen depletion vs. oxygen efficiency (Figure 5) show the same effect exaggerated by the large amount of oxygen required for the nonselective reactions. These plots are instructive in pointing out how very inefficient the lead catalyst becomes as oxygen is consumed. If one-third of the oxygen is reacted from the lead oxide, only about one-fifth of that oxygen is used to make desired products, while one-half of the oxygen goes to combustion. About
0
50
IO0 0
% Oxygen Depletion of Catalyst
Figure 5. Selectivity of the toluene coupling reaction (based on oxygen reacted from the catalyst) vs. the extent of reduction of lead oxide in catalyst--15% PbO on SAHT 99-16.
one-fourth of the oxygen is used to produce benzene, regardless of the state of the catalyst. Conclusions Of the four catalyst requirements presented in the Introduction, the only one which is easily met is that the catalysts can be quickly and easily regenerated at reaction temperature, provided they have not been excessively depleted. Each of the other requirements might be achieved separately, but to attain them all at once has proved difficult. Any catalyst (oxidant) which could be provided at present would involve a trade-off in the desired characteristics. In general, the conditions which favor high toluene conversion give poor product distributions. Conversely, excellent selectivity can be obtained at lower temperatures, but toluene conversion is very low. In certain cases, both high conversion and selectivity can be obtained but in very
122
Ind. Eng. Chem. Prod. Res. Dev., Vol. 18, No. 2, 1979
short reaction cycles with utilization of only a small fraction of the lattice oxygen. Attempts to increase the oxygen-carrying capacity have resulted in losses in activity and/or physical strength. Attrition resistance of the catalysts has been greatly improved by modifying the support, but the necessity for very low surface area and high loading of active component leaves a large amount of material on the outer surface where it is vulnerable to abrasion and would inevitably be lost in long-term operation. A promising develapment in this area is the identification of an active compound in which lead oxide is combined with the support material to provide a very attrition-resistant catalyst. There remain problems with the activity of this material which would have to be identified and solved. Acknowledgment The authors are grateful to Mr. Walter Chemerys of the Carborundum Company, who graciously supplied catalyst
supports, and to Gulf Research & Development Company for permitting this work to be published. Literature Cited Benson, H. L., Jr., Hardesty, D. E. (to Shell Oil), US. Patent 3 409 680 (Nov 5, 1968). Behr and Van Dorp, Chem. Ber., 6, 753 (1873). Bozik, J. E.. Swift, H. E.. J. Catal.. 22. 427 (1971). Buyalos, E. J., Green, P. A.. Scheirer, D. E. (to Allied Chemlcal), US. Patent 3 494 956 (Feb 10, 1970). Garst, R. H., Henry, J. P. (to Union Carbide), US. Patent 3557234 (Jan 19, 1971). Hargis, C. W., Young, H. S.(to Eastman Kodak), US. Patent 3 476 747 (Nov 4. 1969). Knox, W. d., Montgomery, P. D., Moore, R. N. (to Monsanto Chemical), U S . Patent 3 965 206 (June 22, 1976). Li, T. P. (to Monsanto Chemical), U S . Patent 4091 044 (May 23, 1978). Liu, K. H. D., Kawai, T., Yamazaki, Y.. Sekwu Gakkai Shi, 20, 249 (1977). Stanford Research Institute Process Economics Reviews, "Styrene Economics", Report No. PEP 77-1-2 (July 1977). Swift, H. E., Bozik, J. E., J . Catal., 21, 212 (1971). Weterings, C.A. M. (to Stamicarbon B. V.), U S . Patent 3 963 793 (June 15, 1976).
Received f o r review November 28, 1978 Accepted January 24, 1979
GENERAL ARTICLES A Highly Sensitive and Inexpensive Amino Acid Analyzer How-Ming Lee,' Doris J. Bucher, and Robert C. Seid, Jr.' Department of Microbiology, Mount Sinai School of Medicine of The City University of New York, New York, New York 10029
The construction and the operation of a highly sensitive microbore amino acid analyzer is described. The analyzer can use either o-phthalaldehyde or fluorescamine as the fluorogenic reagent for detection. It is based on a single-column chromatographic separation method and has several desirable features: simple construction, low cost, easy maintenance, low column chromatographic pressure, full automation, and high sensitivity. The preparation of a constant molarity buffer system and its application to the fluorometric microbore analyzer is also described. This buffer system has some advantages over t h e use of the traditional buffer with a gradient in ionic strength and pH. It exerts lower column pressure, achieves faster chromatographic speed, and needs shorter regeneration and equilibration time. Most importantly, this buffer produces a stable baseline and does not exhibit buffer change peaks near the methionine position. The detection of the imino acids was achieved by continuously pumping an oxidant to the column effluent throughout the entire chromatographic cycle.
Introduction There are about 20 companies currently manufacturing amino acid analyzers. The prices range from about $30 000 to above $80000. Although the amino acid analyzer was introduced over 20 years ago, the demand for this instrument is still increasing due to its wide applications in biochemistry, clinical chemistry, and other fields. Most of these commercial analyzers use ninhydrin colorimetric detection which has a sensitivity limit of about 1nanomole. Unless very sophisticated instrumentation is applied, the colorimetric analyzer can barely reach detection in the picomole range. Biological materials of interest frequently exist and are available to the scientist in only minute quantities. A highly sensitive amino acid analyzer at a 'Department of Bacterial Diseases, Walter Reed Army Institute of Research, Washington, D.C. 20012. 0019-7890/79/1218-0122$01.OO/O
moderate cost is highly desired. In theory, fluorometry is far more sensitive than colorimetry. Absorptiometric measurements made with a spectrophotometer or colorimeter can at best detect colored materials in concentrations of 0.1 ppm. By comparison, fluorometric measurements can detect concentrations of fluorescent materials as low as a few parts per trillion, thus exhibiting as much as a thousand times greater sensitivity. Two fluorogenic reagents, o-phthalaldehyde and fluorescamine, are now in general use, and fluorometric detection systems have been added to various amino acid analyzers (Lee et al., 1979; Stein et al., 1973). These two fluorogens have different properties and specific advantages. o-Phthalaldehyde is stable in aqueous solvent and was reported to be at least 10 times more sensitive than fluorescamine in amino acid detection (Benson and Hare, 1975). The aqueous solubility and stability of o-phthal0 1979 American Chemical Society
