Checked your spectrophotometer lately? D o you know the exact temperature of your spectrophotometer cuvette in terms of absolute accuracy? Here's an easy way to find out. The YSI Model 45CU cuvette thermometer is a laboratory temperature standard specifically designed to check cuvette temperature. The instrument has four high accuracy ranges that let you measure 25°, 30°, 32° and 37°C with a readability, reproducibility and precision of ± . 0 1 °C and an absolute accuracy of ± . 0 5 ° C traceable to NBS. There is also a survey range of 20° to 40°C providing continuous coverage of the four high accuracy ranges. This portable, battery powered temperature standard has many additional clinical laboratory applications, including calibration and accuracy verification of temperature control systems, laboratory thermometers and other temperature related equipment. If knowledge of true temperature is critical to your measurements, check with the YSI Model 45CU cuvette thermometer.
SCIENTIFIC DIVISION YELLOW SPRINGS INSTRUMENT CO., INC. YELLOW SPRINGS, OHIO 45387
available for the detection of almost any group of compounds (18,19). Spot Characterization (Identification). Characterization of the spot is an important aspect of T L C . Separated compounds may be characterized by Rf values (useful in certain solvent systems and adsorbents); nuclear properties of 14 C and 3 H; chemical formation of colored or fluorescent compounds (spots may be sprayed in situ with a reagent, giving a colored or fluorescent spot characteristic of the material being analyzed); biological aspects [growth stimulation or inhibition of microorganisms and mammalian cell lines (20)]; UV or fluorescent measurements (spots are scanned in situ to give characteristic spectra; in certain cases where spots are nonfluorescent, spots are sprayed with a fluorescent reagent and then scanned); and elution and subsequent analysis by spectral methods such as U V - V I S , fluorescence, infrared, mass spectrometry, and atomic absorption (12, 21). Elution of the spots may be achieved by scraping the spots and extracting the compound from the adsorbent or by in situ elution of the spots without disturbing the adsorbent. T h e spots of interest can be scraped off with an adsorbent scraper or a knife. T h e adsorbent and the sample are then transferred to a small flask, and the sample is extracted with an appropriate solvent. A simpler approach is to use a spot collector, such as the Brinkmann spot collector which is equipped with a rounded glass tip to loosen the layer without scratching the surface of the glass plate. As the particles are loosened, they are immediately sucked into the collector and deposited on the filter disc. Less than a minute is required for the complete operation. T h e compound is subsequently extracted by sucking an appropriate solvent into the tip of the collector and through the sorbent so t h a t the resulting eluent accumulates in an evaporating flask while the sorbent remains on the filter disc. For maximum recovery, very polar solvents should be used, e.g., methanol, water, or acetone. With the use of these solvents, however, small particles (and silicic acid methylesters when using methanol) may pass through the filter regardless of the type of filter employed. By evaporating the polar solvent and dissolving the residue in a less polar solvent such as benzene, the precipitated contaminating material can be separated from the eluent by an additional filtration. A vacuum head can be useful for concentration eluent and, generally, obtaining smaller volumes. Both the vacuum head and the spot collector are equipped with a small hole for suction
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ANALYTICAL CHEMISTRY, VOL. 49, NO. 1, JANUARY
1977
control with a finger. If prolonged operating is required, the opening can be closed either by sliding the vacuum housing over the hole or by placing a piece of filter paper on top of it. Extraneous silica gel can be removed by applying vacuum to the tip and drawing solvent through the collector in the opposite direction. Disadvantages of this method include the use of a large amount of solvent compared with the compound, losses due to the powdery nature of the adsorbent, more time and care required, adsorbent particles getting into the solution, not all of the compound being extracted in certain cases (benzo[a]pyrene) (22), and the special care required when working with toxic or carcinogenic compounds. An automatic T L C zone scraper and sample collector available from Analabs is suitable for radiological studies and scintillation counting and can handle 24 samples simultaneously. Manual scraping is clearly not the ideal method for eluting spots from the plate for ancillary analyses. T h e ideal method would minimize sample losses due to scraping, require less care, time, and a minimum a m o u n t of solvent, and elute more than one sample simultaneously and quantitatively. T h e Eluchrom Automatic Elution System by Camag is used in our laboratory. This unit is capable of eluting six spots simultaneously and quantitatively and requires less than 0.2 ml of solvent without destroying the surface of the T L C plate (23, 24). T h e principle by which the unit works is simple and may be summarized as follows: T h e adsorbent is removed from around the sample zones (spots) by a special milling device. Elution heads are placed over each separate zone. T h e solvent up to 5 ml is then pumped through the elution head and passes through the adsorbent layer at a predetermined rate of flow (a minimum of 0.1 ml/min). T h e resulting solution contains ~ 9 9 % of the sample. Since a precise amount of solvent is used, the solution is ready for quantitative analysis. T h e resulting solution is adsorbent-free and ready for analysis by spectroscopy. (For a detailed discussion, see ref. 25.) Spots also can be lifted from the plate with a strimpix (by Applied Science) which is applied to the plate to bind the adsorbent layer into a continuous flexible film t h a t may be removed intact from glass plate. It can also be used to check, by UV or fluorescence (26), if the sample was completely removed. Documentation T L C laboratory results must be documented for reference purposes. Normally, the method of keeping a
