Reaction is sufficiently slow t o allow the use of many of the common organic liquid phases as well as Chromosorb P as solid support unless quantitative results are required. Figure 4 shows a chromatogram of AsF3 (b.p., 63” C.) eluted from a tricresyl phosphate-Chromosorb P column. Excellent elution characteristics are also obtained for AsF3 when the material is dissolved in carbon tetrachloride, chloroform, or methylene chloride and injected by syringe using standard techniques. Identification of the AsF3 peak was confirmed by standard qualitative procedures (6).
be determined quantitatively. The commonly used diatomaceous earth solid supports react with many metal fluorides and must be absent in such cases. For the strongly oxidizing metal fluorides it would appear that liquid phases and solid supports are restricted to the relatively inert polymers of perhalogenated hydrocarbons. ACKNOWLEDGMENT
The authors thank Franjo M. Zado for helpful suggestions during the course of this work. LITERATURE CITED
CONCLUSIONS
This work demonstrates that a number of transition metals may be eluted by gas chromatography in the form of the metal fluorides if proper precautions are taken to remove all moisture and other reactive materials from the system. MoFC and WFe may
(1) Dal Nogare, S., Juvet, R. S., “GasLiquid Chromatography,” p. 45, Interscience, New York, 1962. (2) Ibid., p. 15. (3) Ellis, J. F., Forrest, C. W., J. Inorg. Nucl. Chem. 16, 150 (1960). (4) Ellis, J. F., Forrest, C. W., Allen, P. L., Anal. Chim. Acta 22, 27 (1960). (5) Ellis, J. F., Ivesp, G., “Gas Chromatography, 1958, D. H. Desty, ed., p. 300, Butterworths, London, 1958.
( 6 ) Feigl, F., “Spot Tests,” Vol. I, p. 95, Elsevier, New York, 1954. ( 7 ) Ibid., p. 113. ( 8 ) Ibid., p. 115. (9) Gudzinowicz, B. J., Smith, V. R., ANAL.CHEM.35, 465 (1963). (10) Gutzeit, G., Ramirez, E. J. (to
General American Corp.), U. S. Patent
Transportation (Nov.
2,658,842
10, 1953). (11) Hamlin, A. G., Iveson, G., Phillips, T. R., ANAL.CHEM.35, 2037 (1963). (12) Iveson, G., Hamlin, ,,A. G., “Gas Chromatography, 1960, R. P. W. Scott, ed., p. 333, Butterworths, London, 1960. (13) Lysyj, I., Newton, P. R., ANAL. CHEM.35, 90 (1963). (14) Phillips, T. R., Owens, D. R., “Gas Chromatography, 1960,” R. P. W. Scott, ed., p. 308, Butterworths, London, 1960. (15) Swinehaft, C. F., private communication; in “Fluorine Chemistry,” Joseph Simons ed., Vo1. I, p. 195, Academic Press, d e w York, 1950. (16) Shrewsberry, R. C., Musulin, B., Science 145, 1452 (1964).
RECEIVEDfor review June 24, 1965. Accepted October 11, 1965. Research supported by the National Science Foundation (Grant NSF-GP-2616).
Bioa na Iytica I Tec hniques A Report of the 18th Annual Analytical Chemistry Summer Symposium W . B. MASON Department of Biochemistry, The University of Rochester, Rochester 20, N. Y. Two hundred and fifteen registrants at the 18th Annual Summer Symposium on Analytical Chemistry spent an educational three days at the University of Wisconsin, Madison, on June 81 lth. The theme was bioanalytical techniques, and 14 technical papers were presented covering developments in histochemistry, enzymatic analysis, amino acid chromatography, electrophoresis, thin layer chromatography, procedures utilizing radioactive reagents, gas chromatography, and automation. There was also a special symposium by undergraduates describing student research projects.
STRUCTURAL AND CHEMICAL ORGANIZATION OF BIOLOGICAL MATERIALS
T
was opened with an overview of the organization present in biological materials, presented by W. B. Mason, School of Medicine and Dentistry, University of Rochester, who also served as program chairman. As better visualization has become possible, the concept of a cell has HE SYMPOSIUM
changed from “a large sack of ill defined materials” to a highly organized entity possessing many specialized substructures. It was emphasized that all biological materials are characterized by structural and chemical organization which is highly complex. This was-illustrated by reference to muscle and muscle contraction, to energy metabolism and adenosine triphosphate (ATP) formation, and to protein synthesis. SCOPE AND TECHNIQUES OF ENZYMATIC ANALYSIS
Michael Vanko, Albany Medical College, chose the enzyme lactic dehydrogenase (LDH) as a focal point in discussing the determination of clinically important enzymes. LDH catalyzes the reversible reaction between pyruvic acid and reduced nicotinamide-adenine dinucleotide (NADH) to form lactic acid and NAD. NADH and NAD have different absorbances a t 340 mp, and this provides a convenient basis for following the reaction rate. One enzyme may be used in the determination of another, as in the assay of
glutamic-pyruvic-transaminase (GPT) . This enzyme catalyzes the reaction between Iralanine and a-ketoglutaric acid to give Irglutamic acid and pyruvic acid. By including purified LDH and NADH in the assay system, newly formed pyruvic acid is converted to lactic acid with the consumption of an equal molar quantity of NADH. Thus the rate of change in absorbance a t 340 mp provides a direct measure of G T P activity. I n a more complex system, two enzymes are used in measuring a third. This was illustrated by the determination of creatine phosphokinase (CPK). In this case, the reaction catalyzed by CPK is coupled with reactions utilizing pyruvic kinase and LDH. Under appropriate conditions the rate of change in absorbance a t 340 mp is proportional to CPK activity. In closing, Dr. Vanko noted that purified enzymes are becoming increasingly available on a commercial basis and that detailed procedures now exist in which they are employed for determining many compounds of biological interest. VOL 37, NO. 13, DECEMBER 1965
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USE OF ENZYME HISTOCHEMISTRYIN CLINICAL RESEARCH
Enzyme histochemistry employs chemical reactions to produce insoluble chromogenic precipitates at the sites of enzyme activity. Some applications of these techniques in clinical investigclr tions were described by Eric A. Schenk, School of Medicine and Dentistry, University of Rochester. Generally speaking, enzyme changes are apparent much earlier than morphological changes, or those which produce alterations in histochemical staining. This was illustrated by studies on LDH distribution in heart muscle folloxing clinical or experimental myocardial infarcts. In the area of damage there is also a disruption of energy metabolism with an increase of lipid. This can be demonstrated with fatsoluble dyes. Another illustration was taken from studies on sprue, a malabsorption disorder. Investigations have shown structural and enzyme changes in the intestinal mucosa from patients having both forms (tropical and nontropical) of the disease. The significance of these changes is not yet clear. The major limitations in enzyme histochemistry stem from low specificity of the chromogenic reactions and poorly understood reaction kinetics. The techniques are not quantitative and interpretations must be subjective. Dr. Schenk noted that histochemistry can proceed only as fast as allowed by the sister sciences of chemistry and biochemistry, and expressed the hope that chemists present in the audience would provide inspiration and guidance for new histochemical developments. QUANTITATIVE HISTOCHEMICAL TECHNIQUES
Most histochemical work has been by staining techniques, which is understandable because morphologists are familiar with stains. The aim of histochemistry is to correlate structure and function, and David Glick, Stanford University School of Medicine, emphasized that in order to understand life processes we must know how much as well as where. Only in relatively recent years have analytical techniques become sufficiently micro to permit quantitative study of morphologically defined biological structures. The use of micro burets and a concomitant reduction of sample size and reaction volume, for example, permitted the titrimetric determination of urease activity in sections of gastric mucosa measuring only a few microns in thickness. Microcolorhetry was illustrated by the determination of ascorbic acid in tissue sections. Fluorometry is especially well suited to quantitative histochemical work 1756
ANALYTICAL CHEMISTRY
because of its inherent high sensitivity. Many enzymes can be measured by use of appropriate reactions for coupling the enzyme with NAD or NADP, both of which are strongly fluorescent under appropriate conditions. By using turbidity as an index of microbial growth and working with 0.01-pl. drops, microbiological growth assay techniques have been applied in the range 0-10 X gram biotin. This represents an improvement in sensitivity of about 106 over standard growth assay procedures. Elemental analysis of tissue sections can be performed by x-ray absorption. By using this technique to obtain the absolute weight of individual cells, the increase or decrease after histochemical reactions can also be quantitated. Simultaneous determination of many metals in a small segment of tissue section can be achieved by coupling a laser beam and an emission spectrograph. Since the silver content of biological material is very low, silver can be used as an internal standard by placing the tissue section on photographic emulsion and focusing the laser beam through a microscope. Upon discharge of the laser, the tissue is vaporized into the electrodes of the spectrograph and the vapor plume triggers the electrode discharge. This technique has been used to study the distribution of calcium and magnesium in sections of gastric mucosa and in the skin of live animals. MICROELECTROPHORESIS OF NUCLEOTIDES FROM INDIVIDUALLY ISOLATED CELLS
An interest in the chemical basis of memory stimulated the work which was described by M. L. Moss, Institute for Muscle Disease, New York City, on the microelectrophoresis of acid-soluble nucleotides in brain tissue. Electrophoresis is carried out on a single rayon monofilament, which is equivalent to a 10,000:1 reduction of a typical paper strip. Ultraviolet photomicrographic techniques were used to locate and estimate the separated nucleoside phosphates. The lower limits of sensitivity are estimated to be about 10-6 or lO-7pM which should allow the technique to be useful with samples consisting of only a few individual nerve cells. MICROCOLUMN CHROMATOGRAPHY OF AMINO ACIDS AND SMALL PEPTIDES
Steps which have been effective in reducing sample requirements for column chromatography of amino acids were described by P. B. Hamilton, Alfred I. duPont Institute, Wilmington. The objectives of this work were to increase the resolving power and to achieve a sensitivity adequate for quantitative determinations using microgram amounts of sample.
Resin composition, particle size, and degree of cross-linkage are critical factors. Best results have been obtained with spherical particles of sulfonated polystyrene-divinyl benzene polymers, nominally 8.5% cross-linked, and measuring 17-18 microns in diameter. Column dimensions are also critical, increased sensitivity being obtained with smaller diameters. Resolution improves with increasing column length, but the pressure required for adequate flow soon becomes very great. A practical compromise is a column measuring 0.636 X 125 cm. and an operating pressure of approximately 450 p.s.i. Applications were described showing the determination of free amino acids in 0.1 ml. of blood plasma, 1.0 ml. of spinal fluid, 50-250 pl. of urine and the amino acids in the hydrolysate from 25 pg. of protein. Further increases in sensitivity have been achieved through use of capillary columns and a photometer based on the DuPont Model 400 process stream analyser. With this equipment it has been possible to determine the free amino acids in 10 pl. of serum or the hydrolysate from 1-5 pg. of protein. The practical limit of sensitivity may be set by the ability to exclude contamination. This was illustrated by finding amino acids in meteorites which could be attributed to thumb prints on the glassware used. Dr. Hamilton speculated that much of the present work on amino acids in meteorites may not be meaningful. The technique for microcolumn chromatography of amino acids and small peptides is now a t a point where it can be applied to the solution of many problems. STARCH GEL ELECTROPHORESIS
The need to experiment with buffers used in starch gel electrophoresis was emphasized by 0. Smithies, University of Wisconsin. I n the separation of human hemoglobins, for example, poor results were obtained with buffers composed of borate or TRIS. By combining the two into a single buffer, however, good separations were obtained. “Don’t settle for the first buffer you find, try several. If you don’t like what you find, invent a new one.” Relative molecular sizes can be estimated from the relative mobility in gels having different starch concentrations. This was illustrated by studies on partially polymerized albumin. I n the case of cattelo (cross between cow and buffalo) hemoglobin, this same technique showed that differences between three types of hemoglobin were largely due to differences in molecular charge. I n some instances, it is desirable to work with dissociated fragments rather
than the parent protein. Starch gels are useful in characterizing such fragments, as was illustrated by studies on the inheritance of haptoglobin subtypes. Although still imperfectly understood, it is clear that starch gel electrophoresis is an extremely useful technique. ACRYLAMIDE GEL ELECTROPHORESIS
Samuel Raymond, Hospital of the University of Pennsylvania, focused attention on the chemistry of acrylamide gel and on techniques for circumventing technical difficulties associated with its use. A neutral gel is easily and reproducibly prepared from buffered solutions of the monomer. This gel is inert toward proteins, enzymes, and histochemical stains and does not exhibit electroendosmosis. By copolymerization with appropriate substituted acrylamides, it is possible to form gels having ion exchange properties. Flat slabs are the preferred form for electrophoresis because this is the best geometrical shape for heat dissipation, and because a large number of samples can be handled simultaneously. The latter is important in comparing the mobility of different specimens, since acrylamide gels have poor dimension stability. Flat slabs have high load capacity and also permit two-dimensional electrophoresis for increased resolution, molecular weight estimations, and determination of isoelectric points. Continuous circulation of buffer between the two electrode compartments is recommended to minimize pH changes secondary to electrolysis; the changes will be equal and opposite at the two electrodes. More uniform and reproducible results are obtained when a 40- to 60-minute period of electrophoresis precedes application of the sample. Migration is linear with time after adequate electrophoretic equilibration between buffer and gel. Separated components may be eluted through the slab by the use of a special electroconvection cell, or histochemical techniques may be used for comparisons on the same slab. A number of proteins which appear to be homogeneous on paper electrophoresis were used to illustrate the improved resolution which is obtained with acrylamide. THIN LAYER CHROMATOGRAPHY
Using the fractionation of extracts from plant and animal tissues as examples, the fundamentals of thin layer chromatography were reviewed by Harald H. 0. Schmid, Hormel Institute, University of Minnesota. The principles are similar to those in conventional chromatography and most of
the same solvents and substrates can be used. Advantages of thin layer chromatography lie in its simplicity, the speed and efficiency of separations, its capacity relative to paper, and the high sensitivity which can be achieved. Rp values have little meaning in thin layer chromatography and migration should be measured relative to reference compounds. Physical or biological properties should be used to confirm the identity of separated components. Special reference was made to a micro technique for characterizing liquids which is based upon the critical solution temperature, and the pattern of changes observed at the interface with known liquids as this temperature is reached. The correlation between molecular weight and critical solution temperature is quite different for hydrocarbons, fatty acids, triglycerides, and cholesterol esters. Molecular weight can be estimated for a member of any class by knowing the critical solution temperature. A newly introduced technique which involves a layer having continuously variable composition along one axis was described. A useful application consists of applying a sample along the variable composition axis and chromatographing a t right angles. In this manner it is easy to determine the best proportion between two substrates for a given separation. Chromatography in the direction of varying composition should permit separations which cannot be achieved on uniform layers, but as yet there have been no practical applications.
DOUBLE LABELING TECHNIQUES IN ANALYSIS OF BIOLOGICAL MATERIAL
The relative merits of introducing the loss label before or after the derivatizing label were examined in detail by N. D. Lee, Bio-Science Laboratories, Los Angeles, in a discussion of double labeling techniques. The latter sequence has been more commonly used and was illustrated by the determination of aldosterone in urine. The former sequence requires compounds having very high specific activity and has only recently become feasible. It was illustrated by the determination of testosterone in plasma. In both determinations many separation steps are necessary before the final radioactivity measurements can be made. Isotope derivative dilution methods were originally developed for analysis of amino acid mixturesand protein hydrolysates but have been used increasingly in recent years for analysis of blood, urine, and various biological preparations where it is necessary to determine very
small amounts of materials occurring in complex mixtures. Important applications have been for steroid hormones such as cortisol, corticosterone, progesterone, and various estrogens, as well a-s aldosterone and testosterone. An important factor in the development of double labeling techniques has been the commercial availability of pure reagents having high specific activity for use as loss label and as derivatizing agents.
IODINE-131 IMMUNOCHEMICAL ASSAY OF PEPTIDE HORMONES
Specificity and sensitivity are limiting factors in almost all analytical problems encountered with biological materials. There are no gross chemical differences between peptide hormones and other proteins. The concentration of these hormones in plasma rarely exceeds 10-lOM at rest, or 10-8M under physiological or pathological stimulation. By contrast the concentration of other plasma proteins is about 10-3M. By far the most successful method, and in many cases the only method, for determining these hormones in plasma has been radioimmunoassay. This technique was devised in 1960 by Berson and Yalow for the assay of plasma insulin, and has since been applied successfully to the measurement of glucagon, growth hormone, parathyroid hormone, adrenocortical stimulating hormone (ACTH), and thyroid stimulating hormone (TSH) . Studies involving determination of growth hormone were used by Jesse Roth, National Institute of Arthritis and Metabolic Diseases, Bethesda, Md., to illustrate practical problems encountered with the iodine-131 immunochemical assay technique. Antibodies to the hormone are obtained from animals following repeated injections of the hormone. Purified hormone, labeled with 1131, binds to the specific antibody. Free and antibodybound h o r m 0 n e - 1 ~are ~ ~ separated by paper electrophoresis or chromatography. At a given concentration of antibody and of h ~ r m o n e - I ~the ~ l , ratio of bound-to-free labeled hormone ( B / F ) is decreased as the concentration of unlabeled hormone is increased. Thus the concentration of hormone in plasma is determined by comparing the B/F obtained in a solution containing the plasma to the B/F of a series of solutions containing known concentrations of hormone. I n addition to the assay of hormones in plasma, the method has been applied successfully to rapid assay of hormone concentrations in protein fractions during purification procedures, to detection of small concentrations of circulating antibody (especially nonprecipitating \101. 37, NO. 13, DECEMBER 1965
1757
antibodies) and to quantitative evaluation of antigen-antibody interactions. In closing, Dr. Roth called attention to the need for pure hormones for use in radioimmunoassay procedures. Chemists present in the audience were urged to work on preparation and puriiication of these materials.
GAS CHROMATOGRAPHIC TECHNIQUES FOR BIOLOGICALLY IMPORTANT STEROIDS
Procedures for urinary estrogens and pregnanediol were selected by H. H. Wotiz, Boston University School of Medicine, to illustrate some applications of gas chromatography in analysis of biologically important steroids. When the urinary levels are high, as in pregnancy, estrone and estriol are easily identified and estimated using a procedure which involves only extraction, washing, acetylation, and chromatography, all of which can be completed in less than 2 hours. Preliminary purification by thin layer chromatography on silica gel is required when less than 1-pg. amounts of steroid are to be measured. In addition it is sometimes advantageous to concentrate the steroids on alumina to avoid overloading the thin-layer plates with extraneous materials present in crude extracts. This is the case with estradiol. Pitfalls which must be circumvented include uneven heating, dead spaces, destruction on column walls or substrate, losses through adsorption, and solvent impurities. All of these problems become progressively worse as one attempts to measure increasingly smaller amounts. If the full sensitivity of electron capture detectors can be utilized, it is possible that gas chromatography may ultimately become more useful than the
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ANALYTICAL CHEMISTRY
double labeling technique for determining steroid hormones.
DIGITAL READOUT TECHNIQUES IN ANALYTICAL CHEMISTRY
Conventional analytical instrumentation employs readout devices which give an arbitrary number that is not in itself useful. Subsequently, this arbitrary reading must be transformed into a useful form, usually the concentration of the substance in the original sample. Elementary processes are ordinarily involved in this conversion, ranging from simple linear interpolation to translating the data to logarithms by means of a table, then performing elementary arithmetic on the logarithms. Even so, these mathematical transformations often require more expenditure of time than taking the original readings. Further, they are drudgery and are exceedingly wasteful of a highly trained worker’s time. They are also a common laboratory bottleneck. This is particularly true where large numbers of analyses are performed repetitively on samples having a standard matrix and the variation between samples is relatively narrow, as is often the case in biological and clinical work. E. A. Boling, Boston Veterans Administration Hospital, and Tufts University and Boston University Schools of Medicine, described several special purpose laboratory instruments in which data reduction is incorporated into the instrument itself. These included an internal standard flame photometer for sodium and potassium, an analog device for use with nonlinear curves (spectrophotometer or absorption flame photometer) and a scanner for electrophoresis strips. It seems fair to predict that similar
developments will soon eliminate the mathematical drudgery that has accompanied many procedures. MULTIPLE AUTOMATIC ANALYSIS
Fundamental principles of the commercially available instrument known as the Technicon Auto Analyzer were reviewed by Harry Hochstrasser, Veterans Administration Hospital, Cleveland. This device employs continuously following streams of sample and reagents which are automatically proportioned, mixed, filtered, dialysed, or heated, as necessary, to produce a final stream which may be fed to a colorimeter, flame photometer, or fluorometer. Most determinations performed in clinical chemistry laboratories have been adopted for use with the AutoAnalyzer. This automatic method has been extended by the development of systems in which multiple determinations are made on a single sample. An example is the simultaneous determination of chloride, bicarbonate, sodium, and potassium in blood serum. This effects a saving in space, time, and sample; however, the recorder peaks must be reduced to useful form. More recently, further refinement has permitted the synchronous determination of eight or more constituents from a single sample. -411 results for one sample appear on a single sheet of calibrated paper. This graphical readout minimizes opportunities for errors in data reduction and transcription. Systems have also been devised for 12 and 18 synchronous determinations. Techniques for multiple automatic analysis will extend the physician’s power of observation by providing broad chemical profiles, and will facilitate screening of large population groups for unrecognized and untreated diseases.
