Chronicle

Conference on Molecular Spectral Analysis

On October 24–27, 1949, a city conference on molecular spectral analysis was held at the Leningrad State Order of Lenin University named after A. A. Zhdanov. More than 300 specialists—physicists, chemists, biologists, physicians, and students interested in questions of molecular analysis—filled the large physics auditorium of the Physics Institute of Leningrad University.

The organization of the conference on molecular spectral analysis should be recognized as highly timely. At a time when emission (essentially, atomic) analysis had already gained wide popularity and found numerous applications in industry and metallurgy, the possibilities of molecular spectral analysis were being used to a far lesser extent. Yet these possibilities are unusually deep and varied. In many cases molecular spectral analysis makes it possible, in the shortest intervals of time, to carry out qualitative and quantitative analysis of the most complex substances, using for this purpose negligible amounts of the substance, which moreover remains completely unchanged after the analysis. The aim of the conference was to unite workers of industrial enterprises, research institutes, and higher educational institutions. Production workers were to see what powerful means of investigation molecular spectroscopy could place in their hands, while scientific workers, in this joint work, were to clearly sense the needs and requirements of practice. The director of the Scientific-Research Institute of Physics of Leningrad University, Corresponding Member of the Academy of Sciences of the USSR, Prof. S. E. Frish, spoke about the cooperation of science and technology, about further technical progress, about the paths of bold innovation and the creative revolutionary daring of workers in industry and science, and about the aims and tasks of the conference when opening the conference.

The opening report, on the features and possibilities of modern molecular spectral analysis, was delivered by Prof. V. M. Chulanovskii (Leningrad University). The speaker emphasized first of all the great selectivity and sensitivity of the methods of molecular spectral analysis. The applications of absorption methods of analysis in the infrared region of the spectrum are especially varied. Owing to the fact that different groups of atoms in molecules give characteristic absorption bands in the infrared spectrum, the range of substances accessible to analysis—not only qualitative but also quantitative—is extremely broad.

CHRONICLE

An important advantage of infrared analysis is that, in this case, small quanta act on the molecule and do not cause photochemical changes in it.

Absorption in the visible and ultraviolet regions, associated with a change in the state of the electron shell of the molecule, can be used in solving a narrower range of problems; compounds with multiple bonds, especially substances with conjugated bonds, poly- and heterocyclic compounds, complex molecules of the dye type, porphyrins, etc., usually give extremely characteristic absorption spectra in this region. The possibilities of analysis by ultraviolet absorption spectra are somewhat broadened when, by suitable chemical treatment of the substance, separated double bonds are converted into conjugated positions.

The emission methods of molecular spectral analysis include the method of combination scattering and the method of fluorescent spectral analysis. The first of these has great selectivity, the second high absolute sensitivity.

However, infrared analysis still remains the most effective, and every effort must be made to provide scientific and industrial laboratories with the apparatus needed for introducing this technique.

The vibrational spectra of complex molecules were the subject of reports by B. I. Stepanov and M. V. Vol’kenshtein. In the first report, the role of theory in the development of molecular-spectral analysis was vividly emphasized. In turn, the study of the dependence of the vibrational spectrum of a molecule on its structure can contribute much to the development of theoretical concepts. Particularly valuable results can be obtained from comparing the infrared spectrum of a molecule with its combination-scattering spectrum. Sometimes the mere count of the number of frequencies observed in the spectrum already gives a sufficient idea of the configuration of the molecule: thus, for example, the molecule CO₂ gives two bands in the infrared spectrum and one band in the combination-scattering spectrum, which indicates the linearity of the molecule. In the case of water, the presence of three bands indicates its triangular configuration; four bands in the case of the ammonia molecule show that it is pyramidal, etc. Analysis of vibrational spectra also provides valuable information on rotational isomerism.

The characteristic frequencies of individual bonds, groups of bonds, and entire complexes are the most important analytical feature. At present, for structural analysis it is most essential to study deviations from characteristicity, since this provides information on the presence of these or other groups in the molecule and makes it possible to determine subtle details in its structure.

The report by M. V. Vol’kenshtein, being a direct continuation of the preceding report, was devoted to the significance of the intensities and polarization of vibrational spectra for structural analysis of molecules.

The speaker attaches especially great significance to intensities and believes that we are entering an “epoch of intensities.” The basis for using intensities and polarizations for analytical purposes is the theory developed by the speaker jointly with M. A. El’yashevich.

Characteristic intensities play a particularly important role. The study of anomalous intensities in the combination-scattering spectra of compounds with conjugated bonds, with aromatic rings, etc., is also of great interest, since it makes it possible to approach the solution of many difficult problems of organic chemistry.

Intensities in vibrational spectra vividly express the polar properties of chemical bonds, which also has substantial significance for molecular spectral analysis.

A number of subsequent reports illustrated how the study of infrared absorption spectra can lead to practically important results. In studying the structure of such complex substances as cellulose, in investigating the processes of transition from a glassy to an elastic state in polymers, adsorption processes, etc., changes in the infrared spectrum in the region of frequencies corresponding to “free” and “bound” hydroxyls are of particular interest. In the work of V. V. Nikitin (Institute of High-Molecular Compounds, Academy of Sciences of the USSR), a study of the absorption spectra of natural and mercerized fibers in the region of 1–2.5 μ showed that intermolecular interaction in them is effected by means of hydrogen-bond forces. A study of nitrated fibrous samples showed that, from the intensity of the band of free hydroxyls, one can judge the degree of nitration of cellulose. Interpretation of the absorption spectra of cellulose and its derivatives in the region of 1–9 μ made it possible to establish characteristic frequencies of vibrations of the groups O—H, C—H, C—O, CH₃ and NO₂, knowing which made it possible to analyze unknown cellulose materials and to follow the changes that occur during their chemical transformations. The application of the method of infrared absorption spectra to the study of the dissolution of incompletely substituted cellulose ethers made it possible to resolve the question of the nature of the interaction between particles of the solute and the solvent.

In the work of S. N. Zhurkov and B. Ya. Levin (Leningrad Physico-Technical Institute, Academy of Sciences of the USSR), a study of the absorption spectra of supercooled substances in the region of 1.5 μ at different temperatures was undertaken in order to clarify the mechanism of transition into the glassy state. The intensity of the band corresponding to free hydroxyls (near 1.44 μ) decreased with lowering temperature, while the absorption of bound OH groups (~1.55 μ) increased. The investigation led to the conclusion that hydrogen bonds arise between the molecules of the glass-forming substance.

In the work of E. F. Gross and Ya. I. Ryskin, the processes of transition of polyvinyl alcohol from a glassy to an elastic state were studied in the region around 3 μ. Changes in the position and intensity of the OH and CH bands showed that, when the polymer is softened, hydrogen bonds are broken.

Absorption in the region of 7.3 μ, as is known, is due to CH₃ groups, and the intensity of this band is proportional to the number of CH₃ groups. Knowing the molecular weight and determining spectroscopically the number of CH₃ groups, one can judge the degree of branching of paraffin hydrocarbons. Studies of this kind are extremely valuable for investigating the composition of petroleum, bitumens, and ceresins. The report of Z. E. Glebovskaya and A. A. Zakharov (VNIGRI) was devoted to these questions. The speakers used an apparatus assembled by themselves; optical amplification was employed, improved by A. A. Zakharov, who developed a circuit for connecting a galvanometer with negative feedback, eliminating mechanical interference.

Studies of absorption spectra in the near infrared region, or in the overtone region (0.7–2 μ), can in many cases prove extremely useful. The methodology used in this region is simpler (the possibility of using glass optics, photoresistors or photocells, working in thicker layers, etc.), and the overlap of bands here is less than in the region of fundamental frequencies.

The recording glass monochromator of high dispersion, developed and built in the laboratory of Acad. A. N. Terenin, makes it possible to obtain rapidly spectrograms of the substances under investigation in this region. This instrument should find wide application in our research and factory laboratories for the express qualitative analysis of organic compounds. In N. G. Yaroslavsky’s report at the present conference, the application of this method to the study of the adsorption of various molecules by porous media was described. The investigation carried out made it possible to establish directly the presence of active hydroxyl groups in the structure of the surface glass and to detect the existence of three states of water in microporous glass: associated, adsorbed, and valence-bound states, to which separate absorption bands in the spectrum correspond: 1.4 μ, 1.40 μ, and 1.365 μ. A spectral investigation of the adsorption of various molecules (aniline, pyridine, phenol, benzene, toluene) made it possible to make certain assumptions concerning the mechanism of interaction of these molecules with the surface of microporous glass during adsorption. With the aid of the same instrument, N. G. Yaroslavsky and A. N. Aleksandrov investigated the absorption spectra of acridine and several of its derivatives. Special attention was directed to elucidating the structure of 9-aminoacridine, since some of its derivatives exhibit increased biological activity (bactericidal properties). Spectral investigations showed that the groups NH₂ and NH in isolated molecules manifest themselves differently in the infrared spectrum. Studying the infrared spectra of 9-aminoacridine, the authors came to the conclusion that it has not the usual amino structure, but a tautomeric acridine-imine structure, which, as is believed, is responsible for the increased biological activity of this compound.

A report by M. G. Batishcheva and A. N. Mironova (LSU, VNIIZh) was devoted to the absorption spectra of fatty acids and their esters in the infrared region.

This work was undertaken in order to determine the possibility of qualitative and quantitative analysis of mixtures of high-molecular-weight fatty acids in the region of the valence vibrations of the CH and OH groups (2–4 μ). Spectra were obtained for a whole series of fatty acids, their esters, alcohols, etc. It was found that the intensity of the absorption band near 3000 cm⁻¹ (the CH₂ group at an unsaturated carbon atom) depends on the number of double bonds. This band can be used for the quantitative analysis of the unsaturated component in a mixture with saturated ones, and also for determining the total unsaturation of any complex mixture.

Similar methods were also applied to investigations of the processes of hydrogenation and oxidation of fats and oils. Thus the work mentioned leads to rapid and reliable methods for controlling production processes.

No less important for practice are the results given by the method of combination scattering. In the report by Ya. E. Shmulyakovsky (NII Khimgaz), an account was given of various applications of the method of combination scattering to the analysis of petroleum products. This method is a very effective means for solving problems of qualitative, quantitative, and structural analysis of hydrocarbons and their mixtures.

Special attention was paid to the little-studied class of unsaturated compounds of the type 2,2,3-trimethylpentene. Applying the characteristic features of branched hydrocarbons established by B. I. Stepanov, it was possible to decipher the composition of complex technical products. With the aid of the method of combination scattering

It was possible to explain the high values of the physicochemical constants of certain fractions by the presence of naphthenes in them, and also to solve several other practical questions.

In the report by E. F. Gross, K. V. Nelson, and Ya. M. Slobodina (Leningrad Physicotechnical Institute), the results were communicated of work undertaken to elucidate the mechanism of the polymerization process of isobutylene. For this purpose the method of combination scattering was applied, by means of which an analysis was made of the assumed structures, on the basis of chemical data, of isomers of polyisobutylene.

The method of combination scattering also gives very valuable results in studying the structure of complex organic substances. The report by G. V. Pigulevsky (Leningrad State University), “A Comparison of Natural Fatty Acids and Acids Obtained under Laboratory Conditions,” was devoted to this question. The speaker gave a number of examples of the successful study, by means of this method, of certain essential oils and fatty acids. This method also makes it possible to clarify the mechanism of chemical reactions, thereby facilitating the work of the chemist.

M. V. Chulanovsky reported on the possibilities of analyzing liquid multicomponent mixtures with the aid of a differential absorptiometer with filters. The report analyzed the possibility of applying integral absorption coefficients, by means of which the determination of the desired concentrations is reduced to solving a system of linear equations of the same type as in the case of monochromatic beams. A convenient scheme of a differential absorptiometer was proposed, which apparently in many cases can be used for solving a number of practical problems.

K. S. Popov, L. A. Graurman, and L. G. Karantsevich (All-Union Scientific Research Institute of Fats) showed how effective the methods of molecular spectroscopy can be in the chemistry of fats. Despite the difficulties of applying ultraviolet spectroscopy to substances absorbing in the far ultraviolet, such as, for example, fats that are triglycerides of fatty acids, in these investigations it proved possible to solve a whole series of practically important questions. With the aid of alkaline isomerization of fatty acids, it became possible to convert isolated double bonds into a conjugated position, which made it possible to obtain characteristic spectra for a number of fatty acids lying in the accessible region of the ultraviolet. This technique was applied to the determination of linoleic acid in vegetable oils, β-eleostearic acid in tung oil, etc.

Ultraviolet absorption spectrophotometry is also successfully being applied to the solution of many problems in pharmaceutical chemistry. Thus, for example, in the report by M. N. Fishcher and M. K. Bunyaeva, experience was presented in the spectrographic characterization of domestic penicillin preparations. The spectrographic method offered great possibilities for carrying out rapid and accurate routine control in the production of medicinal penicillin preparations.

In the report by O. N. Setkina (Technological Institute), various examples were given of the application of absorption photographic spectrophotometry to the analysis of complex organic substances, to the determination of the structure of complex molecules, and to the observation of chemical reactions and control of the kinetics of production processes.

The last two sessions were devoted to applications of molecular spectral analysis in biology and medicine.

In his report on ultraviolet microscopy, E. M. Brumberg showed the advantages of this new method. The high-aperture ultraviolet microscope with a mirror achromatic objective constructed by him made it possible to obtain microphotograms in the ultra-

violet light. Many biological objects that are completely colorless in visible light possess very intense absorption bands in the ultraviolet, so that in microphotographs obtained in ultraviolet rays a sharp contrast is often revealed. By varying the region of the spectrum used for photography, one can bring out now one, now another detail of the object. Especially ingenious is the method of “three-color photography” in the ultraviolet, invented by E. M. Brumberg and at present called the method of color transformation. Its essence is as follows: the object is photographed in three regions of the ultraviolet (for example, 400, 300, and 250 mμ); three photographs are obtained: in the first, the objects whose absorption bands lie near 400 mμ appear most contrasty; in the second, those that most strongly absorb the second region, and so on. Then all three photographs are covered with three different light filters (for example, green, red, blue), and by means of a three-color projector all these three color images are projected onto a screen. As a result we obtain a color image in which all the details having different absorption spectra in the ultraviolet will appear colored in different colors. Many photographs were demonstrated to the audience, showing the applicability of this visual method in biology, metallography, mineralogy, and histochemistry. A special fluorescent screen was also developed, responding to different regions of the ultraviolet with different colors of fluorescence. With the aid of this screen the microscopic image can be examined directly or photographed on ordinary color film.

The method of color transformation requires the use of filters to isolate particular regions of the ultraviolet. Brumberg proposed new types of filters that make this possible.

Brumberg’s ultraviolet microscope with a spectrographic attachment makes it possible to study the absorption spectra of micro-objects and therefore should become widely used in histochemistry. Especially valuable is the fact that this method makes it possible to study living cells, since no fixation of the preparation is required here.

In the presentations a unanimous opinion was expressed concerning the exceptional value and power of the methods of ultraviolet microscopy and concerning the urgent necessity for the broadest possible dissemination of ultraviolet microscopes.

In the report by Prof. L. F. Larionov (X-ray, Radiological, and Cancer Institute of the USSR Ministry of Health), it was shown how valuable the application of the newest physical methods is for solving the most vital questions of medicine and biology, for example, the problem of malignant tumors.

Using, for example, the strong fluorescence of carcinogenic substances of the dibenzanthracene or benzpyrene type, one can introduce them into a living animal and study the paths and behavior or observe the products of their transformation. From the examples cited in the report, the extraordinary sensitivity of the fluorescent method was clearly evident.

With the aid of ultraviolet spectroscopy, nuclear proteins—nucleoproteids—in normal and tumor cells were studied. With the aid of E. M. Brumberg’s microscope, the question of the distribution of nucleoproteids in the nucleus of a living cell was studied. The absorption of the nuclei of normal and tumor cells was also compared. It turned out that the cells of the peripheral layers of a growing tumor are richest in nucleoproteids. It was found that under the action of β- and γ-rays the absorption of tumor-cell nuclei increases; the absorption maximum of the nucleoproteids isolated from them “moves” from 254 mμ, and a spectrum of free protein appears with a maximum at 280 mμ. Especially sensitive to the action

β- and γ-rays proved to be the cells of the peripheral parts of a growing tumor. The speaker suggested that in this case cleavage of the nucleoproteid occurs. Spectrophotometrically it was shown that some therapeutic preparations cause changes in cells similar to those observed under the action of γ-rays.

Nuclear proteins were discussed in greater detail in the report by S. E. Manoilov. Nucleoproteids were isolated from tumor and normal tissues and their absorption spectrum was photographed. The absorption maximum of the true nucleoprotein lies in the region of 2540 Å, and not in the region of 2600 Å, characteristic of free nucleic acid. This shift gave the speaker grounds to suggest that in the nucleoproteid the nucleic acid is firmly bound to the protein. Subsequently it proved possible to carry out the enzymatic synthesis of compounds of amino acids with nucleic acids, i.e., to obtain a synthetic nucleoproteid, which was also confirmed spectroscopically.

The report by A. A. Ferkhmin was a fine illustration of the applicability of the method of microscopic ultraviolet spectrophotometry in histochemistry.

At the concluding session a report by M. V. Savostyanova was heard on certain methodological questions of molecular spectral analysis. The report raised very important sources of systematic errors in spectrophotometric methodology and indicated ways to eliminate them.

The report by engineer S. A. Khrzhanovsky was devoted to a review of domestic apparatus for molecular spectroscopy. The speaker acquainted the audience with a whole series of the latest models of spectral instruments allowing various studies to be carried out in the infrared, visible, and ultraviolet regions of the spectrum. Various auxiliary devices for the same purposes were also demonstrated. Throughout the conference there was also an exhibition of spectral apparatus, vividly illustrating S. A. Khrzhanovsky’s report with a whole series of the newest models of domestic instruments.

The conference showed how rich and broad the possibilities of molecular spectral analysis are. High polymers, various petroleum products, cellulose and penicillin, liquid fuels and glasses, fatty acids and cells of malignant tumors—these are the varied examples of the application of molecular analysis. From this list it is already clear that the development of methods of molecular spectroscopy and their introduction into the practice of factory and scientific laboratories constitute a most important task.

A. Ilyina