Victor Pollak

Optical noise in photometric scanning of thin media chromatograms : II. Double-beam difference systems

Abstract In this the second part of a theoretical treatment of the quantitative analysis of thin media chromatograms the double-beam difference system of scanning has been investigated. This system is much more sensitive than any single-beam arrangement. The incorporation of a “flying-spot” system as opposed to fixed slits permits the quantitative analysis regardless of zone geometry. Perfect balance between both scanning beams and a high degree of stabilization of the light source are essential for good performance. The limits in sensitivity are obtained when the optical noise approaches the electrical noise.

Optical noise in photometric scanning of thin media chromatograms. I. General theory and its application to single-beam transmission measurements.

Abstract An attempt has been made to apply the concepts of “signal” and “noise” so important in communication theory to the analysis of the performance of photodensitometers used for the quantitative assessment of thin media chromatograms. Very low concentrations of separated substances are considered thus permitting the linearization of the relationships involved. It has been shown that for the best performance under these conditions a highly stabilized light source is essential and that the spectral width of the scanning beam should be the same, or somewhat less than that of the absorbing zone of interest. At low concentrations flying-spot scanning as opposed to fixed-slit scanning is of no real advantage. The double-beam system described in the following paper is vastly superior to any single-beam arrangement.

Fluorescence photometry of thin-layer chromatograms and electropherograms

Abstract The paper brings a theoretical analysis of the performance of photometric methods for the quantitative evaluation of thin-layer chromatograms using fluorescence. Fluorescence quenching is considered as an extension of straight photodensitometry into the UV without requiring UV secondary optics nor UV sensitive photodetectors. For quantitative analysis quenching is not recommended. The principal advantages of fluorescence photometry are good accuracy and high sensitivity without the need for costly and sophisticated instrumentation. These advantages are due mainly to a steady and in the ideal case altogether noise-free baseline and to the inherently linear relationship between photometric response and concentration. Intermediate integration on photographic film permits a further considerable increase in sensitivity. When evaluating the film recording, it may be necessary to compensate for the non-linearity of the gradation curve. When a zone is encountered, optical noise begins to appear. The signal-to-noise ratio is constant, independent of amplitude, as in double-beam densitometers. Its magnitude is also of the same order. A serious drawback of the fluorometer is the dependence of the readings upon the intensity of the exciting radiation. It appears feasible to apply double-beam scanning also to fluorescence measurements. The benefits to be expected are mainly increased accuracy and elimination of the influence of the intensity of illumination. The sensitivity may become limited by the lower light intensity available in the fluorescence transmission mode, which seems best suited for the double-beam approach, because of the relative prominence of electrical noise. In most cases, however, double-beam fluorometers should be able to produce better results than double-beam densitometers. Single-beam fluorescence methods have a performance which is much superior to single-beam densitometry but inferior to double-beam densitometry.

The quantitatives assessment of fluorescence on thin media chromatograms : A theoretical study

Abstract Owing to inherent wavelength conversion in fluorescence analysis, the measuring signal is much less affected by optical background noise than is the case in absorptiometric measurements by both transmittance or reflectance procedures. This explains the higher sensitivity and accuracy obtained in fluorescence analysis. A further advantage of fluorimetry is the much wider range of concentrations over which the response is nearly linear. Measurements from either side of the medium are shown to be nearly equivalent in this respect. More light is, however, available if the measurements are carried out from the illuminated side.

Comparison of optical transmittance and reflectance measurements on thin-media chromatograms

Abstract This paper compares the relative merits of transmittance and reflectance procedures in the photodensitometric analysis of substances separated on thin-media chromatograms. The considerations used are based upon the equations of Kubelka and Munk, which describe in simplified terms the optical transfer properties of turbid media. The parameters used in the comparison are sensitivity, linearity and signal amplitude. The theoretical values of these parameters were calculated using a computer and represented in graphical form. For improved linearity a logarithmic transform of the primary signal was satisfactory in the case of transmittance. For reflectance an inverse representation gave the best linearity. Both transforms promise improvement only if the primary signal-to-noise ratio is reasonably high. When very low concentrations are to be measured, the primary signal provides adequate linearity and no transform is necessary. At larger concentrations transmittance with logarithmic conversion and reflectance with inverse representation give nearly equal performance for most media. With media of very high optical density the light intensity available in the transmittance mode may be insufficient compared to the electrical noise of the photodetector and reflectance is then the mode of choice. Reflectance may also have a slight advantage over transmittance with regard to optical noise if specularly reflected light can be kept away from the photodetectors. Any linearising transform has to be carried out before the total amount of substance is determined by integration. For full exploitation of the benefits of these transforms a flying spot scanner is essential. A method is also described which permits the approximate determination of the coefficients of scattering and absorption of the medium from simple photodensitometric measurements. Knowledge of these parameters is required if more sophisticated methods of linearisation than logarithm forming or inversion are to be employed, e.g ., by using a computer. The conclusions obtained do not apply to methods based upon fluorescence.

The concentration profile of substances separated on thin-media chromatograms

Abstract The present paper brings a theoretical study to determine the concentration profile in a zone of separated substance in thin-layer chromatography. If the solution is applied to the origin as a narrow band uniform over the entire width of the substrate, the theoretical concentration profile of the zone is a Gaussian curve. If the width of the applied band cannot be neglected, the zone profile becomes the integral of a Gaussian curve. In the case of the Gaussian profile the amount of solute retained in the zone is proportional to the peak concentration value. Several reasons why concentration profiles observed in practice may deviate from the Gaussian distribution are discussed.

An experimental high-performance photodensitometer for quantitative chromatography : I. Design and construction

A new and experimental photodensitometer designed for quantitative chromatography is described. The principal features of the instrument were based upon the results of an extensive theoretical analysis and incorporate a mechanical arrangement for the production fo a flying spot and an optical path in which two beams of light are separated after interaction with the medium. The device is constructed so as to be suitable for operation in the three principal modes; in reflectance measurements only the ratio of the beam signals is formed, whilst in transmittance measurements the ratio is converted to logarithmic form, in the fluorescence mode only a single beam is used. The spectral range of the instrument extends from the red end of the visible spectrum to the medium ultraviolet, and quartz optics are utilized in most of the optic elements. A quartz halogen lamp and a xenon-mercury lamp may be used alternatively as the light source. Changeable interference filters are employed to determine the spectral position of the light beams and semiconductor photo-diodes with sensitivities extending into the ultraviolet are used as photo-detectors. In the determination of the sensitivity limits of the device the photo-diodes were replaced by photomultipliers and the apparatus was shown to fulfil most of the calculated thoeretical predictions.

An electrical model for the optical behaviour of thin-media chromatograms

Described is an electrical transmission line with resistive parameters as a model simulating the optical behaviour of chromatographic media as described by the Kubelka and Munk equation. The model is characterised by its attenuation constant γ, its characteristic impedance ξ0 and the reflection coefficient ϱ. The significance of the latter is derived from Ohms Law, in order to make the treatment understandable to persons not possessing a background in electrical engineering. A concentrated parameter multi-section representation of the transmission line model is described covering the whole range over which the Kubelka and Munk equations are valid. In order to reduce the complexity of the model, the number of sections may be reduced making the simulation valid only for a restricted range of optical parameters of the medium.

Semiconductor photodetectors and electrical noise in optical photodensitometric equipment for quantitative assessment of thin media chromatograms

Abstract This paper considers the electrical noise originating in photodetector devices and associated amplifier equipment of photodensitometric instruments intended for quantitative assessment of thin media chromatograms. From a comparison of optical and electrical noise values the minimum intensity of illuminating light is calculated, which is necessary to meet a prescribed performance standard. Photomultiplier tubes and solid-state PIN-diodes are compared and it is shown that it is feasible to use these devices for high grade photodensitometric work. Advantages accrue both in the electrical and mechanical design of the instrument, and there is a resultant decrease in the expected costs plus an easier mode of operation.

Linearity considerations in quantitative thin-layer chromatography

Abstract The relationship between the densitometric response of thin-layer chromatography (TLC) and the quantity of separated substance contained in a particular spot is discussed. Attention is focused upon remittance measurements. The relationship between the optical response signal and the concentration of the substance is highly non-linear. The quantity of substance contained in a particular spot is obtained by integration of the linearized spot signal over the area of the spot, an operation which requires a linear relation between optical signal and concentration. Transform equations which achieve approximate linearity over a wide range of concentrations are developed with modified solutions to the Kubelka and Munk equations as basis. The design possibilities for (flying) point scanners are briefly discussed. The need to obtain a baseline (surface) by algorithmic processing and the remaining plate noise are discussed as the ultimate limitation to the performance of the point scanning devices. Dual-wavelength scanning is able to reduce substantially both problems. The principles of quantitation are then discussed and the possibility of substituting the reading of peak values for integration. A comparative analysis of slit scanning versus point scanning is made.