INSTRUMENTATION
tral range. An internal reflectance standard permits immediate reference to confirm instrument setting and sta bility. The chromatogram adapter can also be supplied as an accessory to Carl Zeiss PMQ II users. The Photovolt Densitometer Model530 (Photovolt Corp., New York, N. Y.) operates on the principle of straight transmission measurement with a high degree of collimation-rejected scattered light. Λ variety of light sources can be used. For fluorescence measure ments two mercury vapor lamps are utilized. One affords a sharp 254-nm emission and the other a broader uv range centered at about 350 nm. A uvtransmitting glass filter is used in front of the mercury arc to eliminate the visi ble radiation when utilized as an ultra violet source. Most commonly used is a stabilized, small-filament tungsten lamp. This light source is essentially re-imaged on the thin-layer plate giv ing a small area of illumination. The upper collimating slit picks up only
the central transmitted beam and re jects the scattered light. The multiplier photometer permits high-resolution scanning with a 0.1-mm slit. The multiplier photometer has a large indicating meter calibrated in absorbance units. It has a four-decade switch in addition to a continuous sen sitivity control. The photomultiplier tube is contained in a detachable hous ing, and a choice of tubes is available which covers the visible and ultraviolet region of the spectrum. Automatic scanning requires the use of a motor drive for the stage and for the variable response recorder. The synchronous motor advances the plate 1 in./min. The photometer output is then fed into the recorder. The key feature of the recorder is its variable recording capability. Twelve responses are avail able. The recorder can be set so that either linear or nonlinear responses re sult, so that the logarithm (absorbance) or steeper curves may be plotted.
The authors are grateful for the as sistance and cooperation of the instru ment manufacturers mentioned in the article.
Conclusion
References
There is now available suitable in strumentation for the quantitative eval uation of thin-layer chromatograms. The sources of error involved in the spotting, layer thickness, development,
(1) M. S. J. Dallas, / . Chromatog., 33, 337 (1968). (2) E. J. Shellard and M. Z. Alam, ibid., p. 347. (3) K. Shibata, Methods Biochem. Anal., 7,77 (1959).
visualization procedures, and quality of the plates can be greater than that in herent in the use of any given instru ment. Since this article is not intended to be an exhaustive survey of densitom eters for thin-layer chromatography, the reader should refer to the Ameri can Chemical Society's 1969-1970 Lab oratory Guide to Instruments, Equip ment and Chemicals for further infor mation on other instruments. Readers are also referred to E. J. Shellard's "Quantitative Paper and Thin-Layer Chromatography," Academic Press, 1968, and G. Stahl's "Thin-Layer Chro matography," (second edition), Aca demic Press, 1967. Acknowledgment
COMMENTARY by Ralph H. Muller
ΠΡκιβ DISCUSSION of thin-layer chromatography is timely and useful. It is at once apparent that these elegant instruments are far more reliable and reproducible than the complex system (chromatogram) which they are called upon to measure. The timeliness is evident if we consider Stahl's state ment, "The golden era of paper chro matography began and by 1956 over ten thousand publications on the use of this 'universal' method had come out." In the case of TLC, by the end of 1965, over 4500 publications had appeared as well as a dozen monographs and re views in 11 languages. Paper and thin-layer chromatography have been applied to almost every con ceivable class of substance, but in quan92 A
·
titative evaluation a half dozen or more functional relationships have been pro posed and used, relating the optical measurement to concentration. It seems that further improvements must come from the chemical and manipula tive factors leading to more reproduci ble matrix systems. For the present, the instrumental resources are more than adequate. We continue to be confused by the term "quenching" as applied to that technique in which a chromatographic spot may, in one way or another, di minish the fluorescence of a substance incorporated in the thin layer or sprayed on the chromatogram. In most monographs or treatises, the term is used ambiguously. Obviously, there
ANALYTICAL CHEMISTRY, VOL. 42, NO. 3, MARCH 1970
are two cases involved. In true fluo rescence quenching, the "quencher" de activates the excited fluorescent sub stance and diminishes or obliterates its emission of radiation. In the other case, by absorbing the exciting uv radia tion, it acts in a manner not much more subtle than interposing a piece of card board or a thick sheet of glass. Either phenomenon can be useful in locating or measuring a spot, but the functional re lationship with concentration should be hyperbolic in the first case and logarith mic in the second. For true quenching, the simple explanation in terms of de activation by "collision of the second kind" should suffice. As first pointed out by Klein and Rossland, Franck, and others, this leads to the equation by
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Circle No. 132 on Readers' Service Card
Commentary
Stern-Volmer. The extinction is a hy perbolic function of quencher concen tration, and a simple algebraic manipu lation can furnish a linear plot. Chem ists may prefer chemical explanations, such as complex formation with the ap propriate equation, but both may be right. In any case, an exact formula tion can be obtained from good data with the advantage of knowing some thing about what is really going on. We think it might be useful to look into flying-spot scanners. At least 15 years ago, it was possible to buy scanners for a matter of a little over $100. These were designed for radio hams and TV hobbyists and consisted of a small cathode ray tube acting as a flying light source. The light was fo cused on a photographic negative (could be a chromatogram) and thence to a photomultiplier tube. The output could be displayed on a large oscillo scope or two-dimensionally on a TV screen. This would give an instan taneous and repetitive scan of the pat tern. By more recent techniques, such as CAT (Computer Averaged Tran sients), feeble signals can be "lifted" out of noise, with a signal-to-noise ratio of n/y/n, where η is the number of scans. It would seem that the advantage of high-speed scanning systems is not trivial because it could take advantage of running a large number of calibrat ing standards along with an unknown. Aside from the time required for spot ting a sample and standards, the time required for chromatogram develop ment is the same. Inherently, radioactive techniques provide an approach which is simpler to interpret than optical methods. One of the earliest applications in scanning labeled metabolites {β) gave improved precision and a saving of time of the order of 1800:1 compared with con ventional radioautographic methods. Neutron activation methods are ex traordinarily sensitive in many in stances but depend upon accessibility to large irradiation facilities. Much more attractive is characteristic X-ray ab sorption using isotope excited X-ray sources. Tiny but acceptable sources can be prepared for X-rays of all ele ments between Ζ equals 22-93 (1). They are also suitable for taking ad vantage of the absorption edge tech nique. The nuclear techniques have the advantage of digital readout and inte gration if scalers are used instead of count meters. References (1) Millier, R. H., "Radioisotopes in the Physical Sciences and Industry," Inter national Atomic Energy Agency, Vienna, 2, 65 (1965). (2) Millier, R. H., and Wise, Ε. Ν., ANAL. C H E M , 23, 207 (1951).
