APPLICATION OF ULTRAVIOLET RAYS IN CHROMATOGRAPHY*)

E. M. Brumberg

In 1903 the Russian botanist M. S. Tsvet1,2 described a remarkable method for separating substances similar in chemical nature, which he called chromatography. For a considerable time this method was undeservedly forgotten, and only in the thirties, after a number of interesting biological investigations had been carried out with its aid,3,4 did it again attract the attention of chemists. At present the method of chromatography is widely used in the most diverse branches of organic and inorganic chemistry, and the number of works performed by this method is steadily increasing. Chromatography is used both for analytical and research purposes and for obtaining preparations of pure substances.

At the beginning of 1949, in the Leningrad laboratory of S. I. Vavilov, at his initiative, the development was begun of certain new techniques of chromatography based on the use of rays of the ultraviolet region of the spectrum. S. I. Vavilov always showed great interest in the development of work on chromatography. In 1941, with the aim of popularizing Tsvet’s method, he commissioned a member of his laboratory, B. Ya. Sveshnikov, to write a review article on this question for the journal Priroda.5 In 1946 Sergei Ivanovich organized the first edition of the selected works of M. S. Tsvet.6 The work presented here on “ultraviolet chromatography” was carried out by us with the participation of the biochemistry laboratory of the X-Ray Institute and of several other biochemical laboratories, under the general and always attentive guidance of S. I. Vavilov.

Chromatographic analysis in its first variant, the principal features of which have been preserved to the present time, compa-

*) Revised text of a report read at the conference on chromatography at the Chemical Division of the Academy of Sciences of the USSR on November 23, 1950.

tively simple and does not require complicated apparatus for its implementation. A solution containing the mixture of substances under investigation is introduced into the upper part of a glass “column” filled with a powdered adsorbent (Fig. 1), after which a solvent is passed through the column under pressure.

Fig. 1. Adsorption column. Air is sucked out of the conical flask to accelerate the flow of the solvent.

The various substances contained in the solution introduced into the column, owing to differences in the magnitude of the adsorption forces binding them to the adsorbent, move along the column following the solvent

at different rates; as a result, a chromatogram is formed in the column, consisting of several sharply bounded zones, clearly visible to the eye in those cases when colored substances are being separated. The isolation of substances in pure form is carried out either by cutting the adsorbent column into parts after pushing it out of the glass tube, followed by washing the adsorbed substances out with a solvent into separate vessels, or by successively displacing the zones from the column by further passing solvent through it.

Fig. 2. Chromatogram on filter paper. After separation of amino acids, the chromatogram was developed with ninhydrin.

Recently, chiefly for analytical purposes, the separation of substances on strips of filter paper has often been used. A specimen of such a paper chromatogram is shown in Fig. 2. One of the advantages of paper chromatography is the possibility of carrying out analysis on very small quantities of substances, literally on drops. Its second advantage is the possibility of performing a simple qualitative analysis. On a paper strip, next to the drops of the mixture being analyzed, along a line perpendicular to the direction of motion of the solvent in the strip, drops of solutions of pure substances are applied, the presence of which is suspected in the mixture. Identical substances in the test drop and in the comparison drops, participating in the common process of movement with the solvent, move over the paper at exactly the same level; by examining the paper chromatogram thus obtained, one can immediately say not only how many components the mixture consisted of...

“mixture,” but also what substances were included in it. For this it is necessary only that all these substances be visible on the chromatogram*).

One of the main difficulties hindering the broad application of chromatography is the complexity of the methods for observing the position of separated substances in chromatograms or the passage of substances displaced by the solvent from the chromatographic column, in those frequently encountered cases when the substances under investigation are colorless**).

The methods used to detect zones formed in chromatograms by colorless compounds may be divided into chemical and physical methods. Chemical methods include the conversion of the analyzed compounds into colored ones before or after their development (chemical development of chromatograms). These methods are not always applicable, since it is by no means possible for every compound to find a convenient color reaction. Regeneration of the original substances from substances obtained after carrying out color reactions is usually very difficult and often altogether impossible, since most such reactions are irreversible. The attachment, before separation, of identical chromophoric groups to the substances being separated, imparting color to these substances, is inconvenient, since it usually greatly diminishes the difference in the adsorption capacities of the components, seriously complicating the application of the adsorption method to the given combination of substances.

Among physical methods one should include, first of all, methods based on determining the refractive index of solutions leaving the column ¹¹, ¹², and certain other techniques for studying chromatograms, which will be discussed in this article. Let us note only that refractometric methods, usually based on the use of interferometers of various systems, require the use of complex and bulky apparatus and are very tedious, since they amount to observations of small displacements of the system of interference fringes over the long period of time necessary for the separation of substances. In addition, refractive methods are difficult to apply for detecting substances on an adsorbent and, in particular, are unsuitable for all paper chromatography.

However, it is precisely the use of physical methods for observing the separation of substances in chromatograms that is of the greatest interest for this field, especially when the question is the preparation of pure substances, since chemical

*) In this article we cannot describe many other variants of the chromatographic method. Their description may be found in special reviews on this question ⁷, ⁸, ⁹, ¹⁰.

**) The first works using the chromatographic method were carried out in application to plant and animal pigments.

interference in the final phase of the process is capable of destroying one of the principal advantages of chromatography: the separation of substances by a physical method without chemical alteration of their structure.

Of substantial importance in chromatography, alongside the many and at times very ingenious methods of studying chromatograms that are currently employed, may be methods based on the use of ultraviolet rays.

Ultraviolet rays may be used here in two ways:

1) for exciting the intrinsic fluorescence of the substances being separated, or the fluorescence of products into which these substances are specially converted for their subsequent detection in chromatograms by a luminescent method, and

2) for detecting substances in chromatograms by their absorption of rays in the ultraviolet region of the spectrum.

THE USE OF THE FLUORESCENCE OF THE SEPARATED SUBSTANCES

The fluorescence of substances, both in paper chromatograms and in an adsorption column, is usually excited by long-wave ultraviolet rays with a wavelength of 365 mμ, isolated from the radiation of a mercury lamp by Wood’s black light filter. Any portable analytical lamp used in luminescence analysis, which makes it possible to direct ultraviolet rays onto the chromatogram, is suitable for this purpose. This simple and very sensitive method of examining chromatograms was already duly appreciated in the early works of Winterstein and Schön \(^{13}\) and of Karrer and Schöpp \(^{14}\), who used it in the study of carotenoids. Since then it has been successfully applied in chromatography.

The wavelength of the rays used to excite the fluorescence of chromatograms should, strictly speaking, be chosen in accordance with the absorption spectra of the fluorescent substances. This is especially important when working with very small concentrations of the substances under investigation, when their absorption of ultraviolet rays at the positions corresponding to spectral transmission bands is very slight. Thus, for example, when separating a mixture of certain yellow pigments it is considerably more advantageous, for exciting fluorescence, to use not long-wave ultraviolet rays but blue rays, observing the chromatogram through a yellow light filter that does not transmit blue rays. In other cases it is more advantageous to use shorter-wave rays, for example, with a wavelength of 313 mμ.

When exciting fluorescence by long-wave ultraviolet rays with a wavelength of 365 mμ, it is quite possible to use a column in a tube made of ordinary glass, which still transmits these rays sufficiently well. When working with more

with short-wave ultraviolet rays, the column tube should be made of special uviol glass or of quartz.

The fluorescent techniques of chromatography, like all methods of luminescence analysis, are distinguished by an extremely high sensitivity, determined in turn by that astonishing sensitivity with which the eye, adapted to darkness, notices a faint glow against a dark background. Thus, for example, in P. I. Plotnikov’s experiments on the separation of the fluorescent dyes 3-aminophthalimide and 3,6-diaminophthalimide, carried out by him in the laboratory of Academician S. I. Vavilov, the limiting concentration of these substances was only \(10^{-7}\ \text{g}/\text{cm}^{3}\). This sensitivity is approximately two orders of magnitude higher than the sensitivity of observation of the same dyes in visible light. It could be increased still further by increasing the brightness of the source of the exciting light. The limit to decreasing the concentrations of the substances under study is set, however, not so much by the decrease in the brightness of their fluorescence upon dilution of the solutions as by competition from the fluorescence of the adsorbent itself. Precisely for this reason, when dealing with very low concentrations of fluorescent substances, it is very important to use, for exciting their fluorescence, rays with wavelengths corresponding to the maxima in their excitation spectra and, as far as possible, to the minima in the excitation spectra of the adsorbent.

The fluorescent method may be used in the chromatographic separation of both organic and inorganic substances; in the latter case one usually has to employ luminescent development of the chromatogram.

The principal shortcoming of the fluorescent method of examining chromatograms, as indeed of many other techniques used for this purpose, is its insufficient universality, arising from the fact that there are not more, but considerably fewer, fluorescent substances and fluorescent reactions than colored ones.

USE OF THE ABILITY OF SEPARATED SUBSTANCES TO ABSORB ULTRAVIOLET RAYS

It is known that many substances, completely transparent and colorless in visible light, absorb rays in the ultraviolet region of the spectrum very strongly. It should be borne in mind that there are considerably more substances absorbing ultraviolet rays even in the limited region of the spectrum convenient for experimentation (from 400 to 250 m\(\mu\)) than there are colored substances or substances capable of luminescing. Among organic compounds, these include all compounds containing one or several benzene rings or chains of conjugated bonds—

substances*), especially if at the ends of these chains there are groups capable of carrying a charge \((\mathrm{NH_2}, \mathrm{OH}, \mathrm{NO_2}, \mathrm{CO}\), etc.), as well as many inorganic compounds and ions.

Among the first attempts to use the ultraviolet absorption of substances for chromatographic purposes may be counted the experiments of Brokman \(^{15}\), who carried out the separation of substances in an adsorption column on a luminescent adsorbent. When such a column is illuminated in the dark with ultraviolet rays, dark zones can be seen against the light background of the adsorbent luminescence at the locations of substances absorbing ultraviolet rays. This method will be discussed in more detail below.

The experiments described below were performed in the laboratory of Acad. S. I. Vavilov. As in the examples described earlier, the discussion will concern methods of viewing chromatograms in invisible rays independently of the processes by which these chromatograms were obtained.

If, by means of some visual instrument with a luminescent screen or an electron-optical converter, or by means of photography, one obtains in ultraviolet rays a visible image of the chromatogram, then in this image one can see the zones formed by substances absorbing ultraviolet rays just as we see them in the case of colored substances when viewing the chromatogram itself. This technique makes it possible, quite simply, to extend the usual variants of chromatography to many colorless substances, thereby substantially broadening the range of applicability of Tsvet’s chromatographic method.

Photographic Method

The photographic method was tested first. A chromatogram obtained on filter paper was photographed in ultraviolet rays with a photographic camera equipped with an achromatic photographic objective made of fused quartz and rock salt. The light source was a quartz mercury lamp, in front of which light filters were placed that isolated various regions of the ultraviolet part of the mercury spectrum. With an appropriate choice of light filters, depending on the absorption spectra of the substances under investigation, sharp photographs could be obtained of chromatograms that were completely invisible when viewed in ordinary visible light. This method is also applicable for recording chromatograms obtained in an adsorption column**),

* Such are, for example, many substances important for biochemistry—aromatic amino acids, purine and pyrimidine bases, many vitamins, and a number of others.

** On light filters for photographing in ultraviolet rays, see the article by I. I. Breĭdo and M. P. Bukhman \(^{17}\).

Visual Method

Subsequently, various visual techniques were developed for observing a chromatogram in ultraviolet rays, based on the use of fluorescent screens that convert invisible ultraviolet images into visible ones.

E. M. Brumberg and S. A. Gershgorin^16 constructed a special viewing tube*), the scheme of which is given in Fig. 3. The chromatogram is illuminated by the light of a mercury lamp passed through a light filter that absorbs visible light and transmits all the ultraviolet radiation of the lamp in the spectral interval from 400 to 250 mμ. The mirror-lens objective of the tube gives, on a fluorescent screen, an image of the chromatogram, viewed through a small microscope that replaces the tube eyepiece.

Fig. 3. Diagram of a viewing tube for observations in ultraviolet rays: 1 — objective mirror, 2 — fluorescent screen.

To obtain a sharp image of the chromatogram, light filters isolating rays of narrow portions of the spectrum should be placed in front of the tube or in front of the light source, just as was done when photographing chromatograms. However, the use of such light filters reduces the brightness of the image on the screen extremely strongly, since up to the present time there are still no satisfactory filters for the ultraviolet region of the spectrum. We achieved the same purpose of isolating different portions of the ultraviolet spectrum by using replaceable fluorescent screens made of fluorescent substances selectively excited by ultraviolet rays of separate limited portions of the spectrum. This method makes it possible, considerably better than the use of light filters, to utilize the radiation of the light source in the selected wavelength region**).

*) In this tube a mirror-lens objective with a lens compensator of the system of D. S. Volosov, D. Yu. Gal’pern, and Sh. Ya. Pechatnikova^18 was used.

**) Light filters used for photography in ultraviolet rays also strongly attenuate rays passed by them in the region of those wavelengths. In the case of photography this drawback is compensated for by increasing the exposure.

Observations with the aid of a viewing tube are applicable both for paper chromatography and for chromatographic analysis in a quartz adsorption column. The use, instead of the tube, of an ultraviolet microscope with small magnifications would make it possible to extend this technique, as well as the photographic method, to microchromatography, intended for chromatographic analysis of very small quantities of substances in capillary columns. Subsequently, a simpler method was also employed for examining chromatograms by means of fluorescent screens, suitable, however, only for work on filter paper. For this purpose we constructed a special apparatus—an ultrachemiscope[^19], the scheme of which is presented in Fig. 4, and its external appearance in Fig. 5. The source—

Fig. 4. Ultrachemiscope. 1—bactericidal lamp, 2—light filter, 3—filter paper, 4—screen.

of ultraviolet rays in the ultrachemiscope is a low-pressure mercury lamp in a uviol-glass envelope (bactericidal). A thin light filter of dark purple glass of the UFS-1 grade is mounted in the upper lid of the box; it absorbs the visible radiation of the lamp and transmits ultraviolet rays with wavelengths from 410 to 250 mμ. The spectral transmission curve of this light filter is presented in Fig. 6. The filter paper under investigation is placed on the light filter and is pressed tightly against it by a fluorescent screen; on the screen, at points located opposite the places of accumulation in the paper of substances that absorb ultraviolet rays, shadows appear. Thus, a shadow image of the chromatogram is developed.

The low-pressure mercury lamp is heated only very slightly during burning, which makes it possible to bring the chromatogram and the screen close to it, thereby substantially gaining in their illumination. The principal portion of the energy in the radiation of a low-pressure mercury lamp is concentrated in the resonance spectral line of mercury with wavelength 254 mμ, owing to which this lamp is a very good source of short-wave ultraviolet rays; however, under our conditions, when the lamp burns at several—

...at elevated pressure, owing to the absence of ventilation in the housing; its radiation contains sufficiently represented and longer-wavelength lines of the mercury spectrum.

Fig. 5. External appearance of the ultrachemiscope.

In this case as well, in order to obtain a sharp image of chromatograms it is necessary to isolate individual regions of the ultraviolet spectrum; such isolation is achieved, just as * also when working with a viewing tube, not by the use of light filters, which in the present case, because of the proximity of the screen to the paper and to the lamp, there is even nowhere to place, but by the use of various fluorescing

Fig. 6. Spectral transmission curves of the UFS-1 and UFS-3 light filters.

screens whose fluorescence is excited by limited portions of the spectrum.

Screens for such observations were prepared by depositing thin layers of powdered inorganic luminophores on glass. In operation the screen is turned with the fluorescing layer toward the source of ultraviolet rays—the ultrachromoscope. The excitation spectra of most luminophores suitable for our purpose are sharply bounded only on the long-wavelength side; the luminophore begins to be excited by rays of a definite wavelength and is excited by all rays of shorter wavelength. In practice, among the brightly luminous luminophores used in technology, one can find powders with any spectral position of the excitation boundary. The limitation of the excitation region of the screen on the short-wavelength side is produced by means of a light filter that absorbs the short-wavelength ultraviolet rays, beginning with rays of a definite wavelength. Such a light filter is usually a thin layer of varnish having the corresponding absorption boundary and applied to the layer of luminophore.

Here we shall have to digress somewhat and describe the method of color transformation, which makes it possible to obtain color images in the rays of the ultraviolet region of the spectrum. This method, originally developed by us for purposes of ultraviolet and infrared microscopy, consists in replacing, in an invisible image obtained in ultraviolet rays, ultraviolet rays of different wavelengths by visible rays of different colors.^21 The simplest way to do this is by means of luminescence. Let us imagine a three-layer fluorescent screen containing three layers of different luminophores deposited one on another, differing in their excitation and fluorescence spectra. The first layer, facing the source of ultraviolet rays, is excited only by rays with wavelengths of 270 mμ and shorter; the fluorescence of the second layer can be excited by rays with wavelengths of 320 mμ and shorter, but under our conditions it is excited only by rays of the limited wavelength interval 320–270 mμ, since rays with wavelengths shorter than 270 mμ are absorbed by the first layer, which with respect to the second layer plays the role of a light filter; the third layer consists of a fluorescing substance excited by rays of all wavelengths less than 400 mμ, but under our conditions, for reasons analogous to those indicated above, it is excited only by rays with wavelengths from 400 to 320 mμ. The fluorescence of the different layers differs in color; for example, the first layer gives blue fluorescence, the second—green, and the third—red.

The color of shadows or of the optical image projected onto such a screen depends on the spectral distribution of energy

in the ultraviolet rays incident on the screen and, consequently, on the absorption spectra of the substances through which these rays have passed on their way to the screen. Thus, in ultraviolet rays, color images of chromatograms are obtained, in which the zones formed by different substances differ in color just as, in the visible region of the spectrum, zones formed by colored pigments differ. The method of color transformation is also applicable in the photographic method of studying chromatograms[^20].

In many cases, for chromatographic purposes one may confine oneself to obtaining, by means of photography or single-layer fluorescent screens, gray or monochromatic images of chromatograms. Observations in rays with wavelengths of 270–250 mµ prove to be the most universal, since most substances have high absorption coefficients precisely in this extreme region of the mercury spectrum; here, too, many substances whose absorption begins in the longer-wavelength region of the spectrum absorb sufficiently strongly. Only in those cases where the spectral absorption curve of at least one of the substances under investigation has a minimum in the indicated range of wavelengths is it necessary, in addition to short-wave filters, to use other light filters for photography or to resort to screens with another spectral sensitivity. In these cases, when the substances under investigation have different absorption spectra, it is advantageous to use multilayer screens, which automatically make it possible, like our eye, not only to see distinctly in the chromatograms zones formed by substances with different spectral absorption curves, but also to recognize these substances by color. This is especially important when the zones in the chromatogram are insufficiently separated. With the color method of observation, even in the initial phase of separation of substances, colored rims are visible at the edges of the zone, indicating the presence of different substances in the zone, whereas the uneven distribution of brightness observed in this case in a gray image can easily be taken for an uneven distribution of the density of a single substance.

Ultrachemiscope, being a convenient source of short-wave ultraviolet rays, has also found application in other variants of ultraviolet chromatography described below; almost all of them are based on the use of this light source in combination with luminophores possessing selective excitation and luminescence spectra. One of the main merits of the low-pressure mercury lamp as applied to chromatographic problems is the relatively low brightness of its radiation in the visible region of the spectrum. Figure 7 shows the relative intensity of the spectral lines of a low-pressure mercury lamp

of the bactericidal type and of the high-pressure lamp lines usually used in luminescence analysis. The large content in the radiation of the high-pressure lamp of visible and long-wave ultraviolet rays makes it impossible to use it directly with the above-mentioned dark glass filter UVS-1. The blue and red rays of the light source partially transmitted by it and the bright fluorescence of the filter paper, excited by the intense long-wave radiation of this lamp, interfere with observation of the fluorescence of the screens. This also applies to all other visual methods of examining chromatograms by means of ultraviolet rays described below.

Fig. 7. Energy distribution in the spectra of low- and high-pressure mercury lamps. The intensity of the strongest line in both spectra is taken as 100%.

The ultrachemiscope also makes it possible to examine chromatograms formed by fluorescing substances in the light of their own fluorescence, without in this case using fluorescent screens*).

E. M. Brumberg, I. N. Berezhnaya, V. P. Dutkinskii, and S. E. Manoilov²¹ also described a number of methods for using ultraviolet rays in the chromatographic separation of substances in an adsorption column.

Above we indicated a method for observing chromatograms in a column on a fluorescing adsorbent¹⁵. We modified this method somewhat¹⁹. The separation of the substances was carried out not on a special fluorescing adsorbent, but on ordinary adsorbents, appli-

*) With the aid of an ultrachemiscope one can observe the bright luminescence of many minerals and some other inorganic substances, excited only by short-wave ultraviolet rays. A similar illuminator for luminescence analysis in short-wave ultraviolet rays, but with a less intense low-pressure lamp, was constructed earlier in the laboratory of S. I. Vavilov by Z. M. Sverdlov.

...used in chromatography—starch, silica gel, etc., to which small amounts of powdered phosphors were mixed. In this case, too, we made use of selectivity in the excitation spectra of phosphors, selecting powders excited by rays of the required wavelength. In individual cases a mixture of several phosphors, differing in excitation spectra and in the color of fluorescence, was introduced into the column. In such a column, colorless substances formed colored zones, as it were becoming colored for the time of their passage through the column. Such a division of functions between the adsorbent and the phosphor makes it possible to choose freely both the one and the other. The adsorbent and the phosphor are subject here only to the following additional requirements: the adsorbent must not absorb ultraviolet rays in that region of the spectrum which is used to excite the phosphor, and the phosphor must be a poor adsorbent. These requirements are satisfied by many adsorbents used in chromatography, and by many phosphors.

Observations were carried out in a quartz column illuminated by an ultrachemiscope.

At the same time an apparatus was constructed for working by the method of elution analysis. Substances separated into zones in the column, upon further passage of the solvent through the column, were successively carried out of the column by it and collected in vessels placed under the column. In the lower part of the column a small chamber was made with flat parallel quartz windows. A diagram of this apparatus is shown in Fig. 8. Behind the quartz chamber, close to it, there was a fluorescent screen coated with the corresponding phosphor. The rays of the ultrachemiscope, after passing through the quartz chamber, excited the luminescence of the screen. When substances absorbing ultraviolet rays passed through the chamber, a dark or colored shadow appeared on the fluorescent screen. At each appearance and disappearance of the shadow

Fig. 8. Diagram of an apparatus for chromatographic analysis under the control of a fluorescent screen. Pressure is supplied from above.

the vessels collecting the liquid displaced from the column were changed. Such an arrangement was implemented both for the case of forcing the solvent through under pressure from above (the scheme shown in Fig. 8), and for the case of drawing the solvent by suction from below the column (scheme of Fig. 9).

This method, in which the absorption of solutions that have already left the adsorbent is recorded, makes it possible to use any adsorbents, including black ones; such are, for example, almost all resins—cation exchangers used for retaining cations in ion-exchange methods of chromatography.

Fig. 9. Same as in Fig. 8. The pressure is produced by suction of air from below the column.

Photoelectric method

At the same time, an arrangement analogous to that described above was made, differing from it only in that the measurement of the intensity of the rays that had passed through the quartz chamber at the end of the column was recorded not visually, but with the aid of a photoelement (selenium), installed behind the fluorescing screen. In view of the fact that the substances investigated by us absorbed chiefly the ultraviolet rays of the short-wave part of the radiation of the mercury lamp, a screen coated with luminophore (willemite) was used, excited by rays with wavelengths shorter than 270 mµ and luminescing with green light. Between the photoelement and the screen was placed a green light filter, transmitting the light of the screen’s luminescence but retaining all the ultraviolet and visible radiation of the lamp transmitted by the dark light filter of the Ximiskop. Thus, by means of luminescence, only the short-wave rays exciting the glow of the screen acted on the photoelement. It would, of course, have been possible to use in the same scheme the direct action of ultraviolet rays on a corresponding photoelement intended for operation in the ultraviolet region of the spectrum. This would have required, however, the use of complex and expensive ultraviolet light filters.

The photoelectric method of recording the moment of emergence from the column of substances absorbing ultraviolet rays,

provides broad possibilities for further electrical automation of the process of separating substances, especially valuable when chromatography is used under production conditions.

Ultraviolet Colorimeters

Often, chromatographic separation of substances is immediately followed by determination of the concentrations of the solutions obtained. Such determinations are carried out by one of the known methods of quantitative analysis. In those cases where one has to deal with colorless substances that absorb ultraviolet rays, it is natural to use spectrophotometry or colorimetry in ultraviolet rays for the indicated purpose*).

The chief difficulty in the general case of applying ultraviolet spectrophotometry or colorimetry is the separation of the substances being determined from other substances that absorb ultraviolet rays. By applying adsorption methods in combination with chromatography, we have the favorable case in which these methods are applied to substances already separated by one of the most perfect methods.

In the laboratory of S. I. Vavilov, two models of a simple visual colorimeter for the ultraviolet region of the spectrum were developed, which can be successfully used in combination with ultraviolet chromatography for quantitative analysis of mixtures of substances that absorb ultraviolet rays.

The layout of one such colorimeter is shown in Fig. 10. It is an ordinary Duboscq-type chemical colorimeter, in which a number of modifications have been made. In front of the colorimeter there is an illuminator consisting of a metal housing with a mercury lamp. Two quartz lenses and two mirrors with an aluminum reflecting layer direct the ultraviolet rays into the colorimeter. In front of the colorimeter there is mounted a light filter that retains the visible rays. The bottoms of the cups for the solutions are made of quartz. Flat fluorescent screens are glued to the lower ends of the glass plungers immersed in the solutions. All the remaining optics of the colorimeter remain glass.

The comparison fields of the colorimeter are illuminated by the light of the fluorescence of the screens, excited by ultraviolet rays that have passed through the solutions poured into the cups. Determination of the ratio of the concentrations of substances in the test solution and in the solution

*) In certain cases, chiefly for inorganic substances, one may use the method of volumetric analysis in ultraviolet rays²².

9*

comparison is made according to the heights to which the vessels are raised in the position corresponding to equalization of the brightness of the fields.

In this case, similarly to what was done in the chromatography experiments, the isolation of individual portions of the ultraviolet spectrum corresponding to the regions of greatest absorption in the spectra of the substances under investigation is carried out not by light filters, but by the use of single-layer or multilayer fluorescent screens selectively excited by rays of narrow portions of the spectrum. This makes it possible to gain substantially in the brightness of the fields being compared, which is especially important when working with solutions that strongly absorb ultraviolet rays. Single-layer screens are made interchangeable, with different spectral sensitivity.

Fig. 10. Diagram of an ultraviolet colorimeter.
1—mercury lamp, 2 and 3—quartz lenses, 4–5—mirrors, 6—light filter, 7—fluorescent screen for observations in white light, 8 and 9—interchangeable fluorescent screens.

When the screens are removed, the instrument may be used for determining the concentration of solutions of fluorescent substances and as an ordinary colorimeter for measuring the concentrations of colored substances. In the latter of these cases, immediately after the ultraviolet light filter (along the path of the rays) there is installed a fluorescent screen coated with a mixture of phosphors emitting white light. This, as it were, converts the illuminator of the colorimeter into a luminescent daylight lamp, providing bright and uniform illumination of the comparison fields of the colorimeter with white light.

In another type of visual ultraviolet colorimeter, the vessels with the solutions have a constant length, and equalization of the brightness of the colorimeter fields illuminated by the fluorescence light of screens placed behind these vessels is carried out by means of a gray photometric wedge, which attenuates the light of one of the screens.

The methods of chromatography described above, based on the use of ultraviolet absorption by substances, were tested on material from several biochemical investigations.

S. E. Manoilov studied by this method nucleoproteins, nucleic acids, and certain amino acids (tyrosine and tryptophan) of hydrolysates of proteins from normal and tumor tissues.

Ya. A. Epshtein and M. P. Fomina applied ultrachemistry in the chromatographic study of certain purine and pyrimidine bases. By C. E. Bresler and E. I. Nidzyan^23, an ultraviolet technique in combination with the method of labeled atoms was used to prove the presence of an enzymatic reaction of phosphate transfer from adenosine triphosphoric acid to ribonucleic acid under the action of liver enzymes.

Despite the fact that there are considerably more substances absorbing ultraviolet rays than colored substances, chromatographic techniques based on the use of ultraviolet rays, like many other methods, are not universal. There remain many substances that do not possess significant absorption within the spectral region used by us, from 400 to 250 mμ. Advancement into the shorter-wavelength region of the spectrum is still hindered by the lack of convenient light sources and light filters suitable for this purpose; in addition, many solvents and adsorbents and, in particular, filter paper lose transparency in the short-wavelength region.

Some broadening of the applicability of ultraviolet chromatographic techniques can be achieved by using chemical reactions leading to the formation of substances that strongly absorb ultraviolet rays in a wavelength interval convenient for observation. In this case, just as in observations in visible light, such reactions may be applied both before and after the separation of substances. The use of reactions for the ultraviolet region has a number of advantages in comparison with ordinary color reactions. These advantages are determined by the fact that there are considerably more ultraviolet reactions than color reactions. Indeed, in most colorless compounds absorbing short-wavelength ultraviolet rays, it is considerably easier, by means of a chemical reaction, to cause a small displacement of the absorption band into the longer-wavelength region of the spectrum than to achieve the appearance of new absorption bands in the visible region, i.e., to convert a colorless substance into a dye; the latter is usually associated with a very substantial and difficultly reversible rearrangement of the molecule. An increase in absorption or a slight shift of the boundary of the absorption region, as a rule, is achieved by attaching to the molecule lighter chromophoric groups than in the case of shifting absorption into the visible region of the spectrum. This is especially important when the attachment of such groups is carried out before the chromatographic separation of substances, since the attachment of identical heavy groups to different substances usually strongly brings their adsorption properties closer together. This latter circumstance was illustrated in experiments on chromatographic separation in a paper chromatogram of aliphatic amino acids, carried out by us jointly with R. I. Plotnikov and

M. P. Fomina. Into these amino acids, which do not absorb ultraviolet rays (at least rays with wavelengths greater than 200 mµ), radicals \(C_6H_5CO\) and \(1,3,5\text{-}C_6H_3(NO_2)CO\) were introduced in different experiments by treating an alkaline solution of the amino acids respectively with benzoyl chloride and dinitrobenzoyl chloride. Amino acids colored in this way could be readily observed by means of an ultrachemiscope in paper chromatography. It turned out that the benzoyl derivatives of amino acids, although they had smaller coefficients and a shorter-wavelength absorption limit than the dinitrobenzoyl derivatives, were nevertheless separated on the chromatogram much more easily. Treatment with benzoyl chloride made it possible to detect about 21 amino acids on the chromatogram. After separation, the amino acids can be transferred into solution and obtained in pure form by boiling it. Such coloring of substances for the ultraviolet can also be applied to inorganic ions by converting them to the corresponding valence state or by obtaining absorbing complexes. Similar procedures are also applicable for the ultraviolet development of ready-made chromatograms. For example, divalent zinc can be detected very clearly on a chromatogram by converting it into the sulfide, which is completely transparent and therefore invisible on the chromatogram in visible light, but absorbs ultraviolet rays with wavelengths shorter than 330 mµ extremely strongly.

In conclusion, it is interesting to assess the prospects for the use of infrared rays in chromatography. Short-wave infrared rays with wavelengths less than 1.2 µ, in which a visible image of the chromatogram can still be obtained by means of photography or electron-optical converters, are of little interest for the present problem, since there are very few substances absorbing rays of all these wavelengths. The use of a longer-wavelength region of the spectrum appears very tempting; in this region many substances have strong absorption of vibrational and rotational origin. Here one could apply a method based on recording, with the aid of a suitable indicator (photo- or thermoelement), the moment at which a substance passes through the chamber at the end of the column. However, the application of this method is impeded by the lack of solvents sufficiently transparent to rays of the long-wave infrared region (unless \(CCl_4\) and certain other scarcely accessible solvents are counted). More promising, perhaps, is the use of infrared rays and gas chromatography, where the gas analyzer of M. L. Veingerov’s system may be successfully employed as an indicator.

The chromatographic procedures described in the present article, based on transferring observations into the invisible region of the spectrum, whose development was begun in the laboratory of S. I. Vavilov, may, of course, subsequently be modified and varied—

be used in various ways, depending on the nature of the object and on the methods used to obtain the chromatograms. The realization of the possibilities afforded by the use of the specific property of many substances to absorb preferentially ultraviolet rays of short wavelengths should aid chemists who apply and develop Tsvet’s chromatographic method in their important and interesting work.

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