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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
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Table I. T h e Formulation of a Constant Molarity Buffera Acidic Buffers-Sodium Formate, Buffers A and B NaOH pellets 8g formic acid 7.5 mL 0.1 g EDTA, Na, boric acid 0.4 g phenol, liquefied 0.4 m L final volume t o 1 L with Millipore Milli-Q water for buffer B, adjust to p H 4.25 with formic acid for buffer A, adjust t o pH 3.25 with constant boiling HC1 Basic Buffers-Sodium Borate, Buffers C and D NaCl 10 g EDTA, Na, 0.1 g boric acid 2g phenol, liquefied 0.4 m L 900 m L H,O, Milli-Q titrate with 30% NaOH to pH 10.1 (buffer C) and bring u p t o 1 L titrate with 30% NaOH t o p H 1 2 . 4 (buffer D ) and bring u p t o 1 L Formic acid and constant boiling HC1 were distilled over ninhydrin. All buffers were filtered through 0.22p m Millipore membrane and stored under nitrogen. All reagents and solvents were of highest purity grade from either Pierce Chemicals or Fisher Scientific. a
aldehyde makes the OPA analyzer easier to construct and operate. On the other hand, fluorescamine does not yield a fluorescent adduct with ammonia and, hence, is suitable for the amino acid analysis of the hydrolysates of protein-containing polyacrylamide gel slices (Stein et al., 1974). Post-column chemistry is the major factor contributing to detection sensitivity; however, other parameters may also influence sensitivity, affecting the chromatographic speed and resolution. High pressure liquid chromatographic techniques have been applied in order to achieve rapid speed. Unfortunately, the construction and maintenance costs increase exponentially as the chromatographic pressure rises arithmetically. The approach to rapid chromatography has been to use fast solvent flow. High flow rate will not necessarily exert high column pressure if the shrinking and swelling of the chromatographic resin can be avoided. Shorter post-column reaction time is always desirable since long reaction time causes diffusion and sacrifices resolution. The introduction of microbore columns, small diameter tubing, and high capacity, uniformly sized microbead ion-exchange resin has also brought a new level of sophistication to amino acid analysis. Experimental Section The Preparation of the Constant Molarity Buffer System. The formulation of a constant molarity buffer system is shown in Table I. The buffer system consists of four buffers, buffer A, pH 3.25, buffer B, pH 4.25, buffer C, pH 10.1, and the regenerant, buffer D, pH 12.4. All solutions contain 0.2 M Na+. Buffers A and B have formate as the counterion and buffers C and D have borate as the counterion. The top panel of Table I shows the formulation of buffer B. Buffer A was made by adjusting buffer B to pH 3.25 with constant boiling hydrochloric acid. The formulation of buffer C is shown on the lower panel of Table I and the regenerant was prepared by adjusting buffer C to pH 12.4 with sodium hydroxide. All reagents and solvents used in this buffer system are of the highest purity grade possible. Impurity in the reagents used for buffer preparation will cause an unstable baseline, buffer change peaks, and severe baseline shift. Buffers were passed through a 0.2-ym membrane filter
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Figure 1. The construction diagram of a fluorometric amino acid analyzer. The nitrogen flow is shown by the dashed line. The solid line represents the buffers and reagents flow. See text for detail. Table 11. The Analyzer Parts and t h e Suppliers master regulator, t w o stage ( G l ) pressure reducing valve ( G 2 ) manifold and injector, motorized tee ( T ) and gauge Teflon tubings and f i t tin gs pump, reciprocating (P) fluorometer recorder, dual pen column and heater isolation valve, three-way and two-way ( I ) programmer resin
Matheson, E. Rutherford, NJ W. P. Nugent Co., Inc., So. Salem, NY Laboratory Data Control, Riviera Beach, F L Rainin Instrument Co., Inc., Brighton, MA Rainin or Dionex, Sunnyvale, CA or Omnifit' Inc., Cedarhurst, NY Milton Roy (LDC), or Beckman, Palo Alto, CA Gilson Medical Electronics, Inc., Middleton, WI Linear Instrument Corp., Irvine, CA Omnifit or Glenco Scientific, Inc., Houston, TX Angar Scientific Corp., E. Hanover, NJ Glenco or Xanadu, Springfield, NJ Dionex or Hamilton,b Reno, N V
' Flangeless Teflon tubing connection. through Aminco, Silver Spring, MD.
Also available
(Millipore) and stored in capped bottles. Construction of the Analyzer. The schematic design of the analyzer is shown in Figure 1. The suppliers and the parts used in this construction are listed in Table 11. The major items are the programmer ($2000), fluorometer ($1350),recorder ($700), and pump ($600). The motorized injector ($1200) is one of the essential parts in automation. However, the instrument can be operated without it. All components which come in contact with reagents are constructed of Teflon and glass. The Chromatographic Column and Buffer Delivery. The ion-exchange chromatography takes place in a microbore column packed with either Hamilton HCA 8.75 resin or Dionex DC-4A resin. No differences in peak resolution were noticed between these resins. Hamilton resin resulted in lower chromatographic pressure (250 psi at flow rate of 12 mL/h) than Dionex resin (350 psi). A column with an inner diameter of 3.0 mm, length of 25 cm, and pressure rating of greater than 500 psi was found to
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be satisfactory. It can be purchased from Glenco as a water-jacketed HPLC column with two adjustable plungers on both ends or obtained from Omnifit and wrapped with heating tape (Fisher Scientific) and heated through a variable autotransformer. The chromatographic temperature was set at 50 “C. The buffers (A to D) and the reagent (R2) were removed from the reservoirs by the constant displacement method using a Milton Roy minipump or Beckman Accupump. The reservoirs are laboratory glass bottles capped with silicone stoppers. Two lengths of Teflon tubing were “threaded” through the stopper, permitting the inflow of nitrogen and the outflow of buffer. Three three-way isolation valves, designated as I in Figure 1,were connected in series and used to control the selection of the desired elution buffer. The isolation valves were controlled and energized by a programmer which will be described below. The buffer flow rate was set at 12 mL/h by adjusting the stroke of pump P1. It is important that the check valve and the plunger seal inside the pump be corrosion-resistant and inert to the chromatographic reagents. All buffers and reagents are stored under nitrogen to minimize introduction of airborne contaminants. The dashed line in Figure 1 shows the nitrogen flow. The reagent, R1, was delivered by the constant pressure method using high nitrogen pressure controlled through the master regulator G1 (vide infra). G2 is a Monnier pressure reducing valve, usually set at 2 psi to protect the buffers and reagents from air contamination and oxidation. The Detection System. Our analyzer is capable of using either o-phthalaldehyde or fluorescamine as the detection agents. The design allows us to switch the use from one reagent to another very quickly. Essentially, the detection system consists of reagents R1 and R2, fluorometer, and recorder. Several coils were used for various purposes. These coils were made from various lengths of Teflon tubing with an outer diameter of 0.6 mm and an inner diameter of 0.3 mm (Dionex). Coil C, 300 cm in length, was used as a bubble suppressor. Coil D, 20 cm, was used as a restriction coil and coil E, 2500 cm, was used as a pulse dampening coil. Coils A and B were used as a delay coil and a reaction coil separately. Their length varies from one system to another. For the o-phthalaldehyde analyzer with proline detection system in which reagents R1 and R2 were filled with OPA and alkaline hypochlorite solution, respectively, coils A and B were made of 270 and 680 cm of tubing, respectively. For the fluorescamine analyzer, R1 and R2 were filled with Fluram (Trademark of Roche) and borate solution respectively, coils A and B were of 3 and 55 cm length, respectively. For the OPA analyzer without imino acid detection system in which the reagent, R2, the pump P2, coil E, and coil A can be removed, coil B was of 270 cm length. Two pens of the dual pen recorder (see Table I) were adjusted so that they operated at different sensitivity scales; normally one pen is set to have tenfold higher sensitivity than the other. The Reagents’ Preparation and Their Flow. OPA solution was prepared by adding o-phthalaldehyde solution (1.2 g in 15 mL of methanol), 6 mL of 30% Brij 35 (Pierce) and 2 mL of P-mercaptoethanol into 1 L of 1.0 M potassium borate buffer, pH 10.4. The OPA solution was stored in the dark under nitrogen. The fluorescamine solution was prepared by dissolving 30 mg of Fluram in 100 mL of Piersolve (Pierce, ethylene glycol monomethyl ether). Acetone cannot be used since nitrogen penetrates the solvent and forms an “aerosol” mixture. This causes a lot of fine bubbles and extremely noisy chromatograms
result. Acetone could be used as a solvent where the constant displacement delivery method is used. Alkaline hypochlorite solution was made by dissolving 0.4 mL of Clorox in 100 mL of 0.2 M potassium borate, pH 12.9. The flow rate of the reagent R2 was controlled by the minipump P2 and was set at 3.6 mL/h. The reagent R1 was placed in a pressurizable container (Glenco); its flow was regulated by the nitrogen pressure controlled by the pressure gauge, G1. The pressure delivery method is pulseless. The flow rate of OPA reagent used in this laboratory was 6 mL/h and the maximum delivery pressure was 30 psi. The flow rate of fluorescamine reagent was set at 5 mL/h and maximum delivery pressure was 28 psi. Automation. The programmer and the automatic sample injector are essential components in automation. A seven-function, ten-step Glenco chromatographic programmer was used. Three isolation valves (I), one two-way valve (V), two pumps (P1 and P2), and the valve drive unit are connected to each of the functions. The recorder shares the same functional command as the two-way valve. The fluorometer, column heater, and the 20-port rotary valve are plugged into the household ac outlet and left on all the time. The automatic injector can be made from a motorized twin-deck 20-port rotary valve attached with the valve drive unit. The desired volume of each sample loop can be made by cutting Teflon tubing of various lengths. The injector was interfaced with the analyzer through a manual four-way valve and allowed the loading of samples through the action of an intake syringe as shown in Figure 1. Quantitation. The amino acid composition can be determined by either the peak height or peak area. These methods have been discussed elsewhere (Lee et al., 1979). Results and Discussion The Constant Molarity Buffer. Due to the wide pH buffering capacity of sodium citrate, this trivalent salt has been used traditionally in analyzer buffers (Spackman et al., 1958). This conventional buffer system contains four solutions with increasing pH and ionic strength from buffer A to buffer C. The regenerant, buffer D, was made of 0.2 M sodium hydroxide solution. This buffer system gave buffer change peaks at A to B change and B to C change and caused baseline shift, especially in the basic region. It also induced resin shrinking and swelling and built up high column pressure requiring frequent column repacking. The use of the strong basic solution in the regeneration process required a much longer re-equilibration time. The constant molarity buffer produces no buffer change peak at the A to B change and no significant baseline shift throughout the entire chromatogram. Buffer A was used to elute the acidic amino acids up to cystine. Buffer B elutes valine to phenylalanine and buffer C is responsible for the separation of basic amino acids. The use of the constant molarity buffer results in less shrinking and swelling of the ion-exchange resin, lower chromatographic pressure, faster re-equilibration, and rapid chromatographic speed. It is important to use the highest purity grade chemicals and solvents for the preparation of buffers and to avoid contamination during manipulation. The source of contaminants is commonly from dirty glassware and water. Airborne contamination must also be avoided since the acidic composition of the initial buffers tends to extract ammonia and other basic materials from the atmosphere. Figures 2 and 3 show typical chromatograms of a standard mixture containing 200 pmol and 1 nmol, respectively, of each amino acid normally found in the
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50 60 70 TIME (MINUTES) Figure 2. The OPA analyzer chromatogram. A standard mixture containing 200 pmol of each amino acid was analyzed. The lower tracing shows the blank run, in which two peaks are seen. The first peak is from the buffer contaminants and the second peak is ammonia. 0
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protein hydrolysate. The formate-borate buffer system gave a small artifact peak at the emergence of buffer C and co-eluted with histidine. This is probably due to buffer contaminants in the acidic buffers which are concentrated by the resin and released by alkaline buffer. Fortunately, the size of this peak is quite consistent under controlled conditions allowing histidine to be determined by subtraction. It is recommended that if the sample quantity and time are not a major limitation it is probably more efficient to split the sample into two portions, analyzing the acidic and neutral amino acids in one run and the basic residues in another. If such a practice is used, the addition of internal standards is suggested. The Operation Conditions of an OPA Analyzer. The OPA analyzer is a very simple amino acid analyzer both in design and operation. I t can be used for amino acid analysis if the proline and hydroxyproline content of the protein hydrolysate do not require determination. The reagent bottle, R2, the pump, P2, the delay coil A, and the dampening device (coil E, see Figure 1)can be removed. The OPA flow rate (6 mL/h) was adjusted through the gauge, G1. Due to the mixed delivery methods used, the OPA flow rate has to be adjusted while the buffer is pumping. The flow meter can be made of a graduated pipet of any volume and the flow rate determined by introducing an air bubble and measuring the traveling time with a stopwatch. At the flow rate of 18 mL/h (12 mL/h buffer and 6 mL of OPA), 270 cm of coil B will provide a reaction time of 38 s, ample time for OPA reaction. An analyzer chromatogram of a standard amino acid mixture containing 200 pmol of each amino acid is shown in Figure 2. The peak produced by cystine is low. Ammonia forms a fluorescent adduct with OPA and hence can be detected. The shoulder peak implicates the presence of ammonia in our buffer preparation. Attempts to remove ammonia by passing the buffers through an ammonia trap (Dowex ion-exchange resin) have achieved limited success. The Fluorescarnine Analyzer. The necessity of using a fluorescamine analyzer arises when the protein hydrolysate contains a large amount of ammonia and/or when the cystine content needs to be precisely determined. Ammonia does not produce a fluorescent response with
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Figure 3. The fluorescamine analyzer chromatogram. A standard mixture containing 1 nmol of each amino acid was analyzed. The ammonia peak does not appear in the lower tracing of the blank control. See text for detailed discussion.
fluorescamine. Thus, the fluorescamine analyzer is especially suited for the composition analysis of a protein hydrolysate from a polyacrylamide gel slice, since polyacrylamide yields a huge amount of ammonia on hydrolysis which interferes with the analysis of the chromatographically neighboring peaks on both OPA and ninhydrin analyzers. Unlike OPA, fluorescamine produces a good fluorescent response with cystine. Figure 3 shows a fluorescent chromatogram of a standard mixture containing 1 nmol of each amino acid. The chromatographic conditions were the following. The reagent reservoir, R2, was filled with 1.0 M potassium borate buffer, pH 10.33, and delivered a t 3.6 mL/h. The fluorogenic reagent was placed in the reservoir, R1, and the flow rate was set a t 5 mL/h. The excitation and emission filters inside the cell compartment of the fluorometer were replaced with filters suitable for fluorescamine (Gilson Medical Electronics). The relatively small peaks for aspartate and glutamate seen in Figure 3 were expected since the optimum pH was not achieved (not attempted) in this particular chromatographic cycle. The flow rate of the reagent, R2, could be increased in order to achieve the optimum pH for maximum fluorescent response for these two amino acids. However, the faster flow rate will generate a larger pulse and a better dampening design is needed. An extra dampener made of a tee connected with a blocked, airfilled tubing is used, as shown in Figure 1. It is important to use anhydrous solvent for the preparation of the fluorescamine reagent. Prepurified nitrogen and other dry inert gas could be used for reagent delivery. The Secondary Amine Conversion. Neither OPA nor fluorescamine will react with imino acids. The detection of proline was accomplished by first converting it into a primary amine with an oxidant, then permitting reaction with the fluorogenic substance. Incorporation of the oxidant into the fluorometric analyzer has been reported (Felix and Terkelson, 1973; Bohlen and Mellet, 1979). The technique involves the injection of the oxidation agent into the effluent a t the time of emergence of proline. The method is useful when the proline is widely separated from
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detected and quantifiable although the peak size is smaller than for other amino acids. It is also noticed that the cystine peak increases significantly under this chromatographic condition and allows more precise quantitation of cystine. Other oxidants have also been attempted and more work must be done. Detailed information will be described elsewhere. In summary, we have described how to construct an inexpensive fluorometric amino acid analyzer. This analyzer can use either OPA or fluorescamine as the detection agent, and the interchange from one system to another is rather simple. The operation of such an analyzer is also discussed. The constant molarity buffer should reach wide acceptance and use by scientists. We hope that this communication will help to stimulate the analyzer industry to provide more analyzers for the scientific community of high quality at a low price. Acknowledgment We thank Drs. Stein and Laursen for helpful discussions and valuable suggestions. We also thank Ellen Pitler for the preparation of the manuscript. The technical assistance of D. Forde and M. Lee is acknowledged. This work was supported by the U S . Army Medical Research and Development Command under Research Contract No. DADA-17-69-C-9137.
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neighboring peaks (normally, glutamate and glycine). For those chromatographic cycles where proline elutes close to glutamate, especially in the fast chromatographic system (ours is less than 75 min including regeneration time), this method could result in inaccurate determination of imino acids and disturb the analysis of primary amino acids. To solve this problem, our approach was to deliver the oxidation agent continuously throughout the entire chromatographic cycle. One of the oxidants that we tried in an earlier stage of this project was dilute sodium hypochlorite solution, which was placed in the reagent reservoir R2. The chromatographic conditions are stated in the Experimental Section. Figure 4 shows a typical chromatogram of a sample of a standard amino acid mixture containing 2 nmol of each residue. The proline peak was
Literature Cited Benson. J. R.; Hare, P. E. Proc. Natl. Acad. Sci. U . S . A . 1975, 72, 619. Bohlen, P.; Mellet, M. Anal. Biochern. 1979, in press. Felix, A. M.; Terkelson, G. Arch. Biochern. 8iophys. 1973, 157, 177. Lee, H-M.; Forde, M. D.; Lee, M. C.; Bucher, D. J. Anal. Biochem. 1979, in press. Spackman, D. H.; Stein, W. H.; Moore, S. Anal. Chern. 1958, 30, 1190. Stein, S.; Bohlen, P.; Stone, J.; Dairman, W.; Udenfriend, S. Arch, Biochem. Biophys. 1973, 155, 202. Stein, S.;Chang. C. H.; Bohlen, P.; Imai, K.; Udenfriend, S. Anal. Biochem. 1974, 60, 272.
Received for review January 10, 1979 Accepted January 26, 1979
Preparation of I-Nitroanthraquinone Ch. Comninellis, E. Plattner, and Ph. Javet Institut de G6nie chimique, Ecole Polytechnique F6d6rale de Lausanne, Ecublens, 10 15 Lausanne, Switzerland
High purity 1-nitroanthraquinone is prepared in aqueous hydrogen fluoride taking advantage of the high solubility of its main impurity, 2-nitroanthraquinone, in the medium.
Introduction The nitration of a number of aromatic compounds in anhydrous hydrogen fluoride was first disclosed by Fredenstagen in 1930 and described with some details by Simons in 1941. In a Polish patent application (Galinowsky, 1962), the preparation of pure 1-nitroanthraquinone is claimed in a process working under pressure at 100 "C, and involving anhydrous HF, anthraquinone, and potassium nitrate. In the experimental conditions described, a mixture of isomers was obtained very similar in composition to the mixture obtained by the usual nitration in sulfuric acid. The separation of pure 1-nitroanthraquinone from such mixtures is technically very difficult and justifies further 0019-7890/79/1218-0126$01 .OO/O
investigations in this field (Coffey, 1953). The present work describes the conditions of nitration of anthraquinone in aqueous hydrogen fluoride, and the separation of pure 1-nitroanthraquinone based on its low solubility in aqueous H F (Comninellis, 1973). 0
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0 1979 American
Chemical Society
