INSTRUMENTATION
Advisory Panel Jonathan W. Amy Glenn L. Booman Robert L. Bowman
Jack W. Frazer G. Phillip Hicks Donald R. Johnson
Howard V. Malmstadt Marvin Margoshes William F. Ulrich
Thin-Layer Densitometry
Morton S. Lefar1 and Arnold D. Lewis Department of Analytical/Physical Chemistry, Warner-Lambert Research Institute, Morris Plains, N. J. 07950
Precise and sensitive instrumentation for the quantitative evaluation of thin-layer chromatograms is now available
' T ' H E USE OP thin-layer chromatography (TLC) as a method of providing rapid separation of mixtures is wellknown and documented. Since there are only microgram amounts on the TLC plate, usually of the order of 0.001 to 0.1 mg, the quantitative analy sis of separated components has been difficult. The procedure of scraping and collecting the adsorbent followed by extraction with solvents is laborious and difficult especially when several components of low concentrations are to be quantitated. Many investigators have reported that analytical blanks are generally high and not reproducible owing to impurities in the adsorbent and to particles which remain in suspen sion in the eluate. Some substances such as certain steroids and amines may be decomposed during elution from the absorbent depending on the type of adsorbent and elution technique used. These problems have been recognized by the scientific instrumentation in dustry, and equipment is now available which has both the desired precision and sensitivity. The purpose of this article is to discuss some of the aspects of thin-layer densitometry. General Considerations
The results of investigators vary greatly as to the relationship between absorbance and the concentration of a material spotted on a T L C plate. (Readers should note that technically the term "absorbance" should be re placed by the mathematical equivalent "optical density" for scattering samples, where the photocurrent depends on the 1 Present address, Rhodia Inc., P.O. Box 111, New Brunswick, N. J. 08903
solid angle collected by the photometer. However, since the term "absorbance" is used by most manufacturers in de scribing their TLC densitometers, we have also done so in this article.) Sev eral investigators have reported a di rect relationship under suitable condi tions between the integrated area under a densitometric peak, the absorbance value of the spot center, or the optical density of the entire spot and the com pound concentration (where the term "concentration" actually means concen tration χ volume or quantity of the analyte). Other authors have reported a linear relationship between the loga rithm of the weight of material spotted and the square root of the integrated absorbance reading. There are also re ports of a linear relationship between the absorbance and the logarithm of the concentration of compound or be tween the curve area and the square root of the quantity of the substance applied. If the Beer-Lambert law were obeyed, one could predict a linear rela tionship between the logarithm of the absorbance area and the logarithm of the concentration of the compound spotted. Unfortunately, this most often is not the case because of the translucence of the plate, the scattering of light, and other unknown factors. The behavior of a light beam when it impinges upon an absorbing material distributed on and in the surface of the gel is complex and not clearly understood. The opti cal phenomenon in a translucent ma terial can be written as :
/„ = /„ + /t + h where I0 Ia It Ir
= incident light = absorbed light = transmitted light = reflected light
Shibata (3) reported at least six other types of resultant light, which may also be applicable to TLC densitometry. One of the criteria which affect the pre cision in the densitometry of colored materials is the use of reagents which will react with the compounds to be quantitated to afford a stable colored spot without diffusion of color to the surrounding areas. The color intensity must be reproducible and show a defi nite relationship between the amount of light absorbed by the compound and the amount of material spotted. The design of an instrument which meets these criteria is, therefore, not a small task. Methods. There are primarily three modes of optical scanning of thin-layer plates. They are visible reflectance (or transmission), ultraviolet-excited fluo rescence, and fluorescence quench. Visible Reflectance. For the detec tion of some compounds, the developed chromatogram can be visualized by spraying with a reagent to develop a color. Charring with acid and heat is also used extensively to make the com pound visible. However, with the cur rent instruments available, this is per haps the most difficult technique with which to obtain quantitative data. I t is critical that the developing reagents are sprayed e\renly onto the plate. If this is not done (in practice difficult to perform), an uneven baseline will be obtained when the spot is scanned with a densitometer. The reagent must also be chosen and applied in a manner so that the spot intensity is constant dur ing the scanning period and that com pounds of high concentration have re acted completely. This is critical in the preparation of a calibration curve and in obtaining analytical precision. If the charring technique is used, then
ANALYTICAL CHEMISTRY, VOL. 42, NO. 3, MARCH 1970
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INSTRUMENTATION
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the oven temperature and time of re action must be carefully and reproducibly controlled. Samples must be ap plied so that a reference lane is adjacent to each row of spots. If the plate has been uniformly charred or sprayed, it will permit the "cancelling out" of back ground absorption. Ultraviolet Fluorescence. Those compounds that fluoresce visible radia tion when excited with ultraviolet light can give good quantitative results since the area under the scan curve is usually linear with concentration over a wide range. Using this technique, nanogram amounts of compounds may be detected provided the plates are of good quality. The baseline is generally straight and not greatly affected by the thickness of the adsorbent. It is important to prescan developed plates which do not con tain samples to assess the amount of fluorescence "noise" present in the ad sorbent layer. This may be caused by the use of solvents containing fluores cent impurities. Since some compounds show a deterioration of fluorescence with time, calibration curves should be prepared for each substance being ana lyzed. Fluorescence Quench. This is pres ently one of the most popular methods for visualization and quantitation of materials since neither a color-forming reagent nor charring is necessary. The number of compounds which can quench fluorescence is quite large when compared to those which exhibit fluo rescence upon ultraviolet excitation. The most useful fluorescent phosphors are those which radiate 5220 A energy when excited with 2537 A ultraviolet light. Fluorescent plates should be excited first with uv light in the in strument for a few minutes before starting the analysis to "dark adapt." Quench systems usually show a non linear relationship between the inte grated area under the scan curve and the sample concentration. A separate calibration curve must be prepared for each compound since different sub stances show different "extinction co efficients." Transmission vs. Reflectance. With transmission scanning, the background absorbance varies with layer thickness. The adsorbent thickness must, there fore, be the same at all parts of the plate. The compound being analyzed should absorb the same amount of light regardless of the thickness of the layer. Most commercially coated plates give little problem with variations in thick ness when the instrument used was operated so that the area adjacent to
ANALYTICAL CHEMISTRY, VOL. 42, NO. 3, MARCH 1970
the spot being analyzed is used as a reference. Since the scanning beam must pass through a small amount of adsorbent layer before it can contact the spot, the amount of scattered light will vary. A compact spot will, there fore, permit the scatter to become rela tively unimportant. The manner in which the compound is distributed on the adsorbent layer becomes of greater significance when using a reflectance method since this takes place mainly at the surface of the plate and detector response may decrease with increasing concentration of compound. Layer Thickness. In an investiga tion utilizing the Joyce Loebl Chromoscan densitometer, Dallas (1) reported that the thickness of the adsorbent layer is a critical factor in densitom etry. Peak height, width, and area all depended on layer thickness. As the layer thickness decreased, the peak height and area increased when scan ning with reflected light. With trans mitted light, both peak height and area decreased as the layer thickness de creased. No loss of peak resolution was observed as the layer thickness in creased with transmitted light, but an appreciable loss of resolution occurred with reflected light. Precision in Spotting. Although Dallas found a slightly higher standard deviation for scanning by transmitted light, consideration of the variance due to the method of evaluation and to the densitometry indicated that the stan dard deviation in the volume of differ ent 2-μ.Ι capillary pipets was 2.2%. This error was greater than that due to the chromatography, the densitometry, and the method of determining the area under the curve combined. Other in vestigators have also reported that the application of compounds to a TLC plate is a major source of error. Calculating Amount of Material. If there is a considerable variation in the area measured under the curve for the identical amount of material on the same plate and from different plates, it is desirable to use a calibration curve for each compound to determine the regression line equation. This can then be utilized for the calculation of the amount of material actually present. Shellard and Alam (2) reported that for two alkaloids, the mean square be tween readings on different plates is higher than the mean square between readings on the same plate. For these oxindole alkaloids, the average result obtained by a number of readings from one plate was associated with a lower variation than the average result ob tained by a number of measurements from different plates. The average re sult obtained from a number of mea surements from one plate may be higher or lower than the theoretical value.
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INSTRUMENTATION
Better results can be obtained by the combination of measured values from different plates than by the combined values obtained from one plate. Mobile Phase. A change in the sta tionary and mobile phase used for the separation affects the shape and size of the separated component. Different systems offer different rates of move ments depending on the adsorption iso therm and (or) the partition coeffi cients of the component with respect to the solvent system. The solvent sys tem that permits a low ~Rf usually gives a small, round compact spot. Systems that result in high Hf of the compound usually offer larger, often elongated and sometimes laterally diffuse spots. The changes in size and shape are reflected in a different distribution and deposi tion of compound on the adsorbent. Of importance is that the same quantity of substance may give a different re sponse with regard to the densitometer readings after separation utilizing dif ferent solvent systems. Instruments
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