Technology
Chromatography aids biological research Among new developments are techniques to measure fatty acids, ions in aqueous environments, and serum proteins
ίΗ
ίΐίιϋ
Recent advances in chromatography are finding wide applicability in bio chemical, biological, and medical re search. Among new developments are techniques for measuring fatty acids, ions in aqueous environments, and serum proteins. The techniques were among those described at a symposium on analysis of biologically important compounds, sponsored by the Division of Analytical Chemistry. One of the new techniques, devel oped by a team at State University of New York, Buffalo, makes ultravioletabsorbing derivatives of fatty acids. SUNY's Dr. H. DuPont Durst notes that recent studies have shown that all types of fatty acids, of both low and high molecular weight, are more im portant in human systems than had been previously realized. Fatty acid mixtures are easily resolved by chro matography. But since most fatty acids don't absorb UV radiation—at least not in the wave-length range of most commercial UV detectors—detecting quantities in the neighborhood of 1 ng can be difficult. It is essential, Durst says, to tag the acids with a strongly UV-absorbing species. There have been several ways, he explains, but they all have short comings. They require expensive or toxic reagents, the UV absorption isn't high enough, or they produce by-prod ucts that interfere with separation. The SUNY approach is to form phenacyl esters, using crown ethers as catalysts. Fatty acids are neutralized with potassium hydroxide in methanol, then alkylated in the presence of the ether. Durst points out that crown ethers have great ability to complex metal salts, especially those of potassi um. And by causing solvation of the cation, they aid dissolution of such salts in nonpolar aprotic solvents. Under those conditions, the nucleo-
philic properties of the carboxylate anion are enhanced; 92 to 98% yields of alkylated material are not uncommon. Only a small amount of the crown ether is needed to catalyze the reac tion, and there are no by-products to interfere with separation. Durst explains that a, p-dibromoacetophenone was chosen as the alkylating agent because it is inexpensive, gives highly crystalline products, and yields esters with high molar absorptivity at 254 nm. But the method also can be used to prepare other useful derivatives such as benzyl, p-nitrobenzyl, or p-chlorophenacyl esters. Also, a variety of solvents, including benzene, acrylonitrile, cyclohexane, methylene chlo ride, or carbon tetrachloride, may be used with no decrease in yield or prod uct purity. Dr. Eli Grushka, head of the Buffalo group, notes that the technique already is being used in analyzing the urine of retarded children. The method is being evaluated for use with dibasic acids and other biologically active com pounds with acidic functionality. Demand is growing, says Hamish Small of Dow Chemical Co., for auto mated or semiautomated analyses of ions in aqueous environments, includ ing body fluids such as blood and urine. Ion exchange resins can provide excellent separations of ionic species, Small points out. But, he adds, in au tomated analyses, a chromatographic separation is useful only when it can be coupled to a suitable detector. In the case of ions, it would be very desirable to use conductometric detection, since conductance has a simple dependence on species concentration. In practice, however, the conductivity of the species of interest is generally "swamped" by that of the much more abundant eluting electrolyte. According to Small, he and asso ciates William C. Bauman and Timo thy S. Stevens have solved that partic ular problem. After ionic species are separated by conventional elution chromatography, the eluent passes through a second column containing a combination of resins that "strip out" the eluent, leaving a background of deionized water. Small outlines a typical cation de termination: A sample containing lithi um, sodium, and potassium ions is in jected into a separating column con taining a cation exchange resin. The ions, resolved in the separating bed, exit at various times from the bottom of the column, in a background of hy
drochloric acid eluent. The effluent then passes into a stripper column, which contains a strong base resin in the O H - form. There, the hydrochloric acid is removed by the strong base resin and the alkali metal chlorides are converted to their hydroxides. These
Dionex showed its ion anaiyzer at ex position at ACS meeting in Chicago pass unretarded through the stripper column to the conductivity cell, where they are monitored and quantified. Elution time is typically three min utes per ion, Small says. Anions can be similarly analyzed, using sodium hy droxide as the eluent, an anion ex change resin in the separating column, and a strong acid cation exchange resin in the H+ form as stripper. The Dow chemist notes that a Cali fornia firm, Dionex Inc., has been li censed to develop and market commer cial products based on the new tech nology. Progress in developing a centrifugal elution chromatograph for assay of serum proteins was revealed by Dr. Charles D. Scott of Oak Ridge Nation al Laboratory. The multicolumn inSept. 8, 1975 C&EN
21
strument has as many as eight chro matographic channels in a centrifugal rotor. Cuvette windows at the end of each chromatographic column allow photometric monitoring of each column eluate as it passes through a stationary photometric detector. Preliminary re sults indicate, Scott says, that several serum proteins can be determined in dividually from a single serum sample by affinity chromatography. An anti body to each of the serum proteins is bound to the stationary phase of each of the columns. Several other developments de scribed at the symposium deal with specific applications and techniques. For example, Dr. Charles W. Gehrke of the University of Missouri, Columbia, detailed the use of chromatographic techniques to monitor "biological markers" in cancer. According to Gehrke and his coworkers, body fluid levels of polyamines, nucleosides, organ ic acids, and many other compounds can provide useful indications of the course of disease and the efficacy of treatment. Dr. Phyllis R. Brown of the Universi ty of Rhode Island is carrying out work aimed at developing methods for ana lyzing phosphorus compounds by highpressure liquid chromatography. And Dr. Peter T. Kissinger of Purdue Uni versity has a new method for measur ing ascorbic acid in body fluids, using liquid chromatography with electro chemical detection. The method also may be used to measure ascorbic acid concentration in foods and pharmaceu ticals. •
Identifying spilled oil may be difficult
ΓΗ CAC7.0 The consequences of oil spills, particu larly crude oil spills in international waters, have led to something approach ing a clamor for a method of identi fying the source of the crude oil. The assumption is that, if the source can be identified, the carrier, who is usually considered to be the spiller, also can be determined and punitive action taken against him. This approach presumes that chemi cal analysis of spills can be performed quickly, reliably, and at reasonable cost. It further presumes that crude oils from various fields have unique characteristics which can be deter mined by analysis. But it may not be that easy, according to Harold J. Cole man, of the Energy Research & Devel opment Administration's Bartlesville Energy Research Center. He has just finished a study of the methods avail able for analysis of spills. With the benefit of 50 years of re 22
C&EN Sept. 8, 1975
search on petroleum by what was then the Bureau of Mines' Bartlesville labo ratories to back him up, Coleman con cludes that although it might be possi ble to establish the source of a given spill, it would be exceedingly difficult. The biggest impediment isn't the ana lytical methods available, but the lack of necessary data on the world's oils. From the analyst's viewpoint, Cole man told the Division of Petroleum Chemistry, a major problem is the "weathering" of a spill. The more vola tile components are quickly lost, and there are often subtle changes that take place in the remainder before the analyst is brought into the picture. Both effects render the spill different in composition from that at the time of the spill. Even so, there is still a rea sonable expectation that some of the unique characteristics are not changed and are detectable, but with great ef fort. Among the analytical methods used in spill identification are gas-liquid chromatography, atomic absorption spectrophotometry, infrared spectros copy, mass spectrometry, and ultravio let spectrophotometry. In the case of GLC, the major problem is interpreta tion of results. Often recommended for oil charac terization is the vanadium and nickel contents of crude, both of which vary widely. Even when weathering has been prolonged, it is believed that the vanadium-to-nickel ratio remains con stant. The preferred analytical method for vanadium and nickel is atomic ab sorption spectrophotometry. Infrared spectroscopy has great ap peal because it is easy and cheap to perform. Unfortunately, Coleman says, the results are often of limited value, because of variability in duplicate runs. The method is regarded as valu able as a confirmatory technique, and is of greatest use if weathering is slight. Mass spectrometry is severely limit ed by the cost of equipment, and the necessity for highly trained personnel. Where available, however, the method can yield useful characteristics for oil spills research. The only factor interfering with the use of sulfur and nitrogen contents is disagreement on the quantitative ef fects of weathering on the samples. As in the case of vanadium and nickel, the sulfur-to-nitrogen ratio appears to be stable, since weathering affects both about the same way. Sulfur and nitro gen analyses are considered to be among the more reliable tests applied to crude oil spills. Other techniques are of value as con firmatory tests, but Coleman believes his study indicates that the most use ful are GLC, vanadium-nickel analysis by atomic absorption, and sulfur-nitro gen determinations. They all are sim ple, reliable, rapid, and inexpensive. However, the lack of data on crude oils from various fields around the world renders even the best analyses impo
tent if there is nothing to compare the analyses with. Coleman believes that international cooperation will eventually relieve the problems and that spill analysis will become more useful in determining the source of a crude and the identity of the spillers. However, at present, he says, spill analysis is not capable of routinely identifying either. •
Cyclohexanones new route to catechols
1H
JEUUL J
Catechols find numerous uses in pro duction of plastics, antiseptics, photo graphic chemicals, dyes, and other products. Their production has cen tered on reactions of alkylcyclohexanediones, which result in high yields of alkylcatechols. However, despite the relatively easy aromatization of diones, the synthesis of diones themselves is difficult and expensive. Aiming at decreasing reactant costs, Mahmoud S. Kablaoui of Texaco's re search and technical department has developed a synthesis for alkylcatechols using less expensive, commercially available intermediates—specifically, cyclohexanones. Cyclohexanones can be used to produce catechols with nearly the same procedure that had been pre viously used with diones. Alkylcyclohexanones can be reacted with sulfuric acid in an acetic anhy dride solvent to produce an intermedi ate that is then hydrolyzed to a mix ture of o-alkylphenols and alkylcat echols. Distillation of the mixture, Kablaoui told the Division of Petrole um Chemistry, separates the catechols frqm the alkylphenols in about a 30%/70% split. Although the primary work was done with acetic anhydride solvents, Ka blaoui says that the desired reactions also can be carried out in hydrocarbon solvents to which some acetic anhydride has been added. The overall yield with hydrocarbon solvents is 5 to 10% less. The use of sulfuric acid is necessary, and its concentration has a marked ef fect on the aromatization step. The progress of the reaction is measured by evolution of sulfur dioxide, which comes off as a by-product. Two moles of sulfuric acid per mole of cyclohexanone seems to give the best yield. Higher concentrations of sulfuric acid favor the formation of alkylphenols. The mechanism for the reaction pro posed by Kablaoui involves o-acetylation of the enol form followed either by two hydride abstractions to give o-cresol or by two hydride abstractions and acetoxylation to give 3-methylcatechol and sulfur dioxide. •
