Haleem J. Issaq and Edward W. Barr NCI Frederick Cancer Research Center Frederick, Md. 21701
Instrumentation
Recent Developments in Thin-Layer Chromatography Thin-layer chromatography (TLC) was first introduced by Izmailov and Shraiber (/) in 1938 as spot chromatography. Eleven years later Meinhardt and Hall (2) used adsorbentcoated glass plates for the fractionation of inorganic ions by a process called surface chromatography. T h e y divided the chromatographic process into four main parts: application of sample, development with solvent or reagent, immobilization of adsorbate, and visualization. In 1951 Kirchner et al. (3) used glass strips coated with adsorbents for the separation of terpenes. However, T L C did not become popular until 1956 when Stahl (4) built a convenient spreader for the preparation of the plates. [A more detailed history of T L C is provided by Pelick et al. (5).] T L C has developed into one of the more powerful research tools and is in routine use in many fields for separation, analysis, and quantitation. It is used in biochemistry for separation and screening of complex products such as amino acids, purines and pyrimidines, nucleotides and nucleosides, and toxic and carcinogenic compounds and their metabolites; in clinical chemistry as a diagnostic tool in the analysis of urine and blood; in pharmaceutical laboratories for the analysis of drugs, antibiotics, vitamins, and other products; in toxicology for such compounds as insecticides and pesticides; in the cosmetic industry for the analysis of dyes and perfumes; in the study of natural products such as lipids, steroids, and plant extracts; in forensic labs for the identification of samples for crime suspects; in organic synthesis to follow a reaction and test the purity of a product; and in inorganic and metallorganic studies. T L C is also useful for identification and confirmation purposes. T h e success of T L C is attributed to its sensitivity, selectivity, ease of operation, and low cost. This article will discuss recent developments in T L C such as new adsorbents and instrumentation. T h e advantages and limitations of T L C with respect to paper
chromatography (PC), high-pressure liquid chromatography (HPLC), and gas-liquid chromatography (GLC) will also be discussed. T h i s is not a review of T L C literature since this has been done in the past (6), nor will we dis^ cuss the application of T L C to the different fields. A review of the T L C bibliography (7) will suffice.
Adsorbents In their first report, Izmailov and Shraiber (1) suggested t h a t chalk, talc, magnesium oxide, lime, aluminum oxide, or other similar substances be used as adsorbents on microscopic slides. They also reported t h a t the most suitable thickness for the adsorbent layer is 2 mm; a thicker layer cracks upon drying, and a thinner one does not cover the plate uniformly. Today one can readily obtain plates precoated with silica gel, alumina, cellulose, polyamide, reversed phases, charcoal, graphite, magnesium oxide, and magnesium hydroxide (0.102-mm thickness). T h e coating may be hard or soft, with or without a binder or fluorescent indicator, polar or nonpolar, neutral, acidic or basic, and impregnated with silver nitrate or any other complexing agent. Some of these coats are permanent which permits them to be washed after development and used repeatedly. T h e best adsorbent to use for any specific separation is determined by several factors: chemical composition of the surface, water content, surface area, and geometrical arrangement of surface atoms or groups. Furthermore, chemical and physical properties of the sample are also important. A detailed discussion of adsorbents is provided by Snyder (8). T h e adsorbents generally best suited for use in the separation of different classes of compounds are listed in Table I. T h e adsorbents are also divided according to the type of chromatographic process involved: partition or adsorption (e.g., silica gel, cellulose, Kieselghur); ion exchange [cellulose phosphate, polyethylene amine (PËI),
NEW required reading from Waters — the Liquid Chromatography People New Analysis of Pharmaceutical Products This new 20-pg. refer ence describes rapid and economical assay and quantitation m e t h o d s for a w i d e range of drug products. Described are LC sep arations of cough prep arations, antibiotics, vitamins, and tran quilizers, as well as various specialized LC techniques. CIRCLE 234 O N READER SERVICE CARD
Paired-Ion Chromatography This new 16-pg. bro chure d e s c r i b e s the technique of Paired-Ion Chromatography, an al t e r n a t i v e t o i o n ex change. PIC™ allows simultaneous analysis of acids, bases, and neutral c o m p o u n d s . CIRCLE 235 O N READER SERVICE CARD
New Detectors Brochure Considerations for the choice of a LC detec tion system for specific applications are dis cussed in detail. The c a p a b i l i t i e s of t h e Model 440 UV/Visible Absorbance Detector and 400 Series Differ ential Refractometers are discussed in relation to specific applica tions. CIRCLE 236 O N READER SERVICE CARD
free from WATERS ASSOCIATES 137 Maple Street, Milford, Ma 01757 Telephone (61 7) 478-2000 The Liquid Chromatography
People
Table 1. Adsorbents and Their Applications Adsorbent
Cellu lose Florlsil Ion ex change Alumina Silica gel
Polyamide MgO Mg(OH) 2
Class of samples
Hydrophilic compounds Basic compounds Nucleotides Nonpolar or weakly polar compounds Amines, bases, antibiotics, and many other compounds (most universal adsorbent) Quinones, alkaloids, and natural products Planar and polycyclic aro matic hydrocarbons Planar and polycyclic aro matic hydrocarbons
cellulose]; reversed phase partition (silicone); polyamides (polyacrylonitrile); and Dextran gels (Sephadex G-25) (9).
Solvents Solvents used in developing T L C plates may be selected from the elutropic series depending on t h e type of sample to be analyzed. A solvent may be polar or nonpolar, one component, or a mixture of two or more. For ex ample, a mixture of six solvents was used for the separation of nucleic acid bases (10). T h e solvent m a y be used pure or with the addition of a base or an acid such as ammonia, which was added to prevent streaking of the methylated nucleic acid bases (11). In using two or more solvents in a mix ture for plate development, demixing might occur, i.e., a change in solvent composition along the adsorbent bed (8). When acetylaminofluorene was spotted on silica gel and developed in ethylene chloride:ethyl acetate 9:1, two solvent fronts were formed; t h e secondary solvent front, rather t h a n the primary front, moved the spots (12). Demixing is prominent when short developments are employed. For a detailed discussion of the role of the solvent, see ref. 13.
Coaters T h e production of a uniform coat of adsorbent is important not only for re producible RF values but for accurate quantitative work. Today there are various types of coaters, manual or au tomatic, which can be selected to meet research and quantity requirements. Precoated plates, commercially avail able for almost all types of adsorbents, are suitable for labs which use few plates daily. Where research dictates the use of mixed adsorbents on the same plate, a manual applicator will suffice. An automatic applicator is rel atively expensive. Other consider
84 A · ANALYTICAL CHEMISTRY, VOL. 49, NO. 1, JANUARY 1977
ations are the price of glass plates and the adsorbent a n d time necessary t o prepare the plates. T h e layer thickness may vary from 120 to 1000 Mm. T h e most popular layer thicknesses are 250 yum for ana lytical plates and 500 μιη for prepara tive work. T h e use of binders such as starch, Gypsum, or carboxymethyl cellulose causes t h e layers to adhere to t h e plate surface. T h e effect of binders on development was discussed earlier (14).
Spotting T h e sample is spotted on the plate with a micropipet, microsyringe, or capillary tube. These devices are espe cially useful in quantitative work in t h a t they dispense from 1 t o 25 μΐ of sample with a precision of ± 1 % or less. It is possible to connect the Hamiltontype microsyringe t o a repeating dis penser which discharges 1/50 of the syringe capacity a t each push of a but ton, minimizing the spot area. This is important when reproducible quanti tative work is sought. T h e above-mentioned spotting de vices are useful for microanalytical work. However, preparative T L C re quires t h a t a larger sample be used. T h e devices used for this purpose are called applicators or streakers, the lat ter of which will be used in this paper. T h e y range in price from $5 t o over $2000, depending on the type of work and the accuracy required, and are ei ther manual, semiautomatic, or fully automatic. T h e sample is applied as a line (not as a spot because of over loading) and should be uniform, nar row, reproducible, and, when neces sary, quantitative. When certain plates are used, such as Quanta, uni formity and thickness of the line are not prerequisites. Manual Streakers. This pipet has a capillary at both ends and a sample reservoir bulb in the middle with a ca pability of 2 ml. Solution flow is con trolled by t h e operator through a mouthpiece attached to flexible tub ing. Such devices require some skill in uniform blowing and, are not recom mended for use with toxic or carcino genic compounds. Semiautomatic Streaker. T h e plunger of a microsyringe (50-500-μ1 capacity) is depressed at a uniform rate while the syringe is moving man ually on a slanted dosage bar (Applied Science Streaker or Camag's Chromatpcharger) or by battery (Desaga's Microdoser). Use of this type of streaker requires t h a t t h e plate remain station ary while the syringe is moved. Fully Automatic Streaker. T h i s method requires that the plate be moved while the syringe is stationary. T h e plate is moved by a motor while
THE LC COLUMN
Jan./Feb. Packed with news from Du Pont Instruments.
Du Pont SEC proves five times faster than GPC system.
Du Pont announces two key improvements in Zorbax-ODS
The c h r o m a t o g r a m s below compare the separation of copolymers contain ing polystyrene at molecular weights of 100,000 and 200,000. One D u Pont size exclusion chromatography (SEC) c o l u m n was able to complete the sepa ration in 7 minutes while six gel perme ation chromatography (GPC) columns took 37 minutes. What's more, coupled with Du Ponts exclusive flow feedback controller, precision was found to be 5 times better with the D u Pont system at the molecular weight of 200,000. Du Pont SEC overcomes many of the limitations of GPC. In addition to reduc ing analysis time and providing high precision in many applications, SEC requires less solvent and is compatible with water, alcohol, T H F and all c o m monly used organics. Circle the reader service card for more information.
Development
Improved Zorbax"-ODS c h r o m a tographic packing gives the c h r o m a tographer two key capabilities: • Retentivity for many polar as well as non-polar compounds. • High efficiency for needed separation power. For example, Figure 2 shows the highly efficient separation of typical i n g r e dients found in commercial soft drinks. Note that, although these c o m p o u n d s are water s o l u b l e , they are readily retained on Zorbax-ODS with a mobile phase c o n t a i n i n g as m u c h as 30% organic modifier. This high retentive capacity permits the selective reduction in retention for caffeine due to its high solubility in T H F while retention and separation are maintained for the other ingredients. If you haven't tried improved ZorbaxODS, you owe it to yourself to test its performance. The material is available from Du Pont in prepacked, pretested columns. Circle the reader service card for more information, or write Du Pont Instruments, R o o m 2 5 4 7 0 , W i l m i n g ton, D E 19898.
Du Pont Instruments CIRCLE 51 O N READER SERVICE CARD 86 A ·
the sample is delivered. Instruments such as the Camag Linomat II, the Desaga Autoliner, and the ICN T L C Applicator are designed for laborato ries t h a t perform a substantial num ber of chromatographic separations on both paper and T L C plates. These units have various controls t h a t aid the operator in selecting and adjusting conditions to suit requirements such as length of sample application line (20-370 mm), width of sample line (0.5-5 mm), speed of sample dis charge, volume of sample to be ap plied (2-5000 μΐ), and interval be tween sweeps (5-360 s) depending on volatility of the solvent. Some of these streakers have an electrostatic dis pensing system t h a t eliminates drop formation during or after sample ap plication. Others have a jet spray gun and no syringes are used, which elimi nates undesirable drop formation. These streakers provide quantitative sample application. T h e glass reser voirs used have a capacity of 0.1-5 ml. Narrow width sample streakers pro duce lines which give larger separa tions than spots and improve the ac curacy in quantitative in situ scanning TLC, especially when the width is nar-
ANALYTICAL CHEMISTRY, VOL. 4 9 , NO. 1 , JANUARY
1977
T h e plate is developed after the sample has been spotted and dried in either a stream of air or an inert atmo sphere, depending on the reactivity of tbe sample. Development refers to the separation of the mixture on the plate by allowing the liquid phase to move u p the plate (adsorbent). T h e plate, depending on the mixture to be sepa rated, may be developed in a saturat ed or unsaturated atmosphere; the plate also may be activated (i.e., heat ed) before use to drive out the humidi ty. T h e first developing device was a test tube (3); the latest is a pro grammed multiple development (PMD) unit (75). Any size container, depending on the size of the plate, may be used for plate development. T h e container may be a closed jar or an especially manu factured cylindrical or rectangular tank. T h e tank most widely used for 8 X 8 plates measures 4 X 12 X 9 in. For 2-in. plates a cylindrical or rectangular tank may be used. T h e tanks and their covers are normally made of glass. Plates are normally developed in the tank without flushing with nitro gen. However, plates can be developed in nitrogen atmosphere by flushing the tank through a two-way valve and then allowing the tank's atmosphere to be saturated by the solvent vapor before development. T h e plate can also be developed in a refrigerated tank. Development under nitrogen and refrigeration is recommended for
easily oxidized and heat sensitive samples. T a n k s used for plate development include sandwich chambers (require 15 ml of solvent), hanging chambers, and N-chambers (faster equilibration time and less solvent required than rectangular tanks). A twin trough development chamber recently introduced by Camag has the following advantages: 20 ml of solvent are used: two solvent systems may be used, one to equilibrate the chamber and the other to develop the plate; and relative humidity can be controlled by using a sulfuric acid-water mixture in one trough and developing solvent in the other. P r o g r a m m e d Multiple D e v e l o p ment ( P M D ) . Two compounds separated by development over a certain distance may be expected to double in separation if development is continued over twice the distance. Theoretically, however, this is inefficient since the diffusion of the compounds increases with the distance traveled. Also, since the time of development is proportional to the square of the distance traveled, doubling the distance traveled quadruples the developing time. One possible solution is to make a series of shorter developments rather t h a n one long development. This is called multiple development (MD). M D can be made in the same direction with either a different solvent or with the same solvent. M D can also be made in a different direction (usually at a right angle) to the initial development by using a different solvent. P M D is the automatic use of M D in the same direction with the same solvent. There are several advantages to P M D : since it is automatic, operation time is minimized; since development is carried out repeatedly over the area of plate close to the solvent, both diffusion and development time are kept to a minimum; and the spot is reconcentrated once as the solvent front advances (common to both P M D and MD) and again as the solvent front recedes during the drying cycle (specific to P M D ) . Reconcentration occurs because the solvent still flows in an upward direction while the solvent front is receding. This reconcentration of the spot has two important results. Plate efficiency is improved. Reconcentrating the spot minimizes the spot width and area, thus making smaller amounts of compound detectable. Sensitivity, also affected by reconcentration, is proportional to amount of spot/area of spot. P M D requires caution in two areas. First, P M D generally employs a weaker solvent t h a n T L C so t h a t a trial and efror period may be necessary before the two are correlated (Figure 1). Sec-
Figure 1. Comparison of development of aflatoxins B,, B 2 , G,, and G2 in ether and in acetone:chloroform:water (12:88:1.5) by conventional TLC and PMD
Figure 2. Development of 1-hydroxy-2-acetylaminofluorene and 7-hydroxy-2acetylaminofluorene in benzene, methanol, ethyl acetate, chloroform, and acetone by use of the Vario-KS-Chamber
ond, any solvent demixing problem will be accentuated with P M D since the solvent front passes over the sample not once but several times. S i m u l t a n e o u s Plate D e v e l o p ment. It was mentioned earlier t h a t more than one plate can be developed in the same tank by use of the same solvent. It is also advantageous to be able to develop the same plate in more than one solvent system or at different conditions simultaneously. T h e advantages of such a system would be selection of the most suitable solvent system and selection of the most suitable development conditions. This may be achieved by the Camag Vario-KS-Chamber (16) unit, which has three glass conditioning trays (5, 10, and 25 compartments), two stainless steel slides, two glass solvent troughs, and a temperature control unit. It is very versatile, allowing the researcher to study the effect of relative humidity and saturation on T L C separations, select the most suitable solvent (up to five solvents can be used) (Figure 2), develop 10 samples (up to five different solvents on one 20 X 20 cm plate), and develop continuously at a predetermined temperature, which is most advantageous when reaction chromatography is used.
88 A · ANALYTICAL CHEMISTRY, VOL. 49, NO. 1, JANUARY 1977
Detection
Location of Spots on Plates. Colored and fluorescent spots on plates can be easily located by white and UV light, respectively. Spots of inorganic ions can be detected by "ripening", a process developed by Meinhardt and Hall (2). T h e major points of this process include applying gaseous reagents to the plate (after developing, the plate is placed in a tank containing gaseous reagents such as ammonia, sulfuric acid, iodine, or bromine); applying colorimetric reagents directly to the adsorbed species (nickel, for example, is readily located by pressing a filter paper saturated with dimethyl glyoxime solution against the surface of the plate); including a complexing agent in the adsorbent composition (this gives a colored complex with metal ions); and incorporating sulfur in the adsorbent surface (the dried chromatogram is exposed to infrared radiation or heated in an oven which reveals those metals t h a t formed colored compounds). Fluorescence quenching of fluorescent plates is another fast technique for detecting spots. Indirect detection methods utilizing combinations of the above techniques may also be used (17). Hundreds of reagents are
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
CIRCLE 238 ON READER SERVICE CARD 90 A ·
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
My TLC Plates? Quantum LQD Timesavers, of course.
p e r m a n e n t record is by sketching the chromatogram in the notebook and recording the RF values and the solvent and adsorbent used. Two modern methods of recordkeeping use photography (27) and the photocopier. T h e photocopier copies chromatograms t h a t absorb UV radiation at 366 nm but is not suitable for compounds which can only be detected under short UV (254 nm). T h e T L C plate and photocopy paper are placed on the machine and exposed. All visible chromatograms, whether charred or sprayed with a color developer, can be copied. An exception is when the spray reagent (indicator) absorbs at 366 nm. Copies are made only in blue. Compounds detected under short UV (254 nm) can be made copyable by t r e a t m e n t with iodine vapor or charring. Photocopying is as fast as photography b u t considerably cheaper, costing less than 10 cents/copy, which is less than Polaroid's M P 4 system unit cost. Also, expertise is not necessary. T h e copying sheets are 20 X 20 cm. Of course, a Xerox copy of plates is also possible for visible compounds. Quantitative TLC (22)
Equal or better resolution in 1/10 the time Unique preadsorbent sample dispensing area saves people-time where it matters: in sample prep aration and application As much as 90%. in fact. As the solvent migrates through the preadsorbent area, it collects the material to be separated at the solvent front and applies it to the silica gel as a concentrated band. The result: sample application and preparation need not be precise, but the resolution is still equal to or better than can be achieved with conventional plates. The LQD Timesaver can handle crude, dilute, organic or aqueous spotting solutions, and comes in several configurations, with or without separated channels. Save important people-time in your laboratory. Writeforcomplete information on LQD Timesavers and other TLC supplies.
quantum Industries 341 Kaplan Dr., Fairfield, NJ 07006 (201)227-4880
Quantitation of in situ spots on T L C plates is an established technique. T h e methods used are visual and spectrometric. T h e visual technique is carried out by spotting the unknown sample either alongside a standard or between two standards and then comparing the intensity of the spots by fluorescence, fluorescence quenching, or color intensity. T h e error in this method is ±15-50%. T h e spectrometric technique may be divided into three different modes. Reflectance or Transmittance: Colorless compounds are made visible by charring or by treating with a reagent to produce color. Fluorescence: Certain compounds, such as aflatoxins, fluoresce under UV radiation. T h e area under the peak is proportional to concentration [caution here: some compounds deteriorate under UV radiation (28)]. Compounds which do not possess natural fluorescence may be made to fluoresce by spraying with a reagent. Fluorescence Quenching: Spots appear as dark circles on the fluorescent plate. This method usually gives a nonlinear relationship between integrated area and sample concentration. Fluorescence is more sensitive than absorption; also, the measured signal is a linear function of the concentration. Errors in spectrometric measurements are between 5-10%. T h e sources of error are sample application, variation in spot size, uniformity of layer thickness, and variation from plate to plate. Errors can also be attributed to
CIRCLE 175 O N READER SERVICE CARD
92 A · ANALYTICAL CHEMISTRY, VOL. 49, NO. 1, JANUARY 1977
Now there's a reliable high performance recorder at a reasonable price... thenewVarian Model 9176 It's a best buy. Varian Model 9176 Strip Chart Recorder gives you the reliability, accuracy, precision and repeatability of recorders that cost much more. Model 9176 is designed to provide exceptional performance with gas and liquid chromatographs and with most other laboratory analysis instruments, including spectrophotometers, densitometers, pH meters, titrators, and EPR and NMR instruments. Choose Your Recorder. Single and dual pen models are offered with a choice of plug-in span and chart speed modules plus many performance options. Result: you can tailor a recorder that provides the best price/performance to meet your exact requirements. Model 9176 Features: New long-writing pens (4,500 linear feet) for unattended operation. New guarded differential input circuitry to eliminate noise. New crystal-clock controlled chart speeds. Fast, ^500 millisec full-scale response. And solid-state electronics, lead-screw pen drive, and proven chart drive motor for high reliability. For details circle Reader Service No. 226. To have a Varian representative call you, circle Reader Service No. 227. Varian Instrument
Division 611 Hansen Way Box D-070 Palo Alto, CA 94303
varian
The sure-fire way to beat the heat safely! A silicone rubbe' Hand prottvtur Ό' Ί . Ι Ι Μ IH; 'vt .itvutorv ware A i l &*l-Art
i^feyr local t a b Svppiy
aVa/er
Bel-Art Products Pequ.i • •
-, \
•• , e y O 7 4 4 0
Your favorite il ι • j | « r ί\ our favorite i i i s l o n i f r CIRCLE 23 ON READER SERVICE CARD
"UNBREAKABLE" Polymer Body Combination pH/Reference Electrodes Sealed reference neverneeds refilling Full 0 to 14 pH range Fast response (95% in less than 1 second) Complete with 30" lead and your choice US Standard/ Beck man or BNC connector Standard Size: (0.48" dia. χ 6.0" length)
Order M o d e l S200C
High-Performance TLC (HPTLC) and Micro TLC
Only $36.00 "Dependable Electrodes"
SensoreX ^
9713 Bolsa Avenue » Westminster. California 9 2 6 8 3 · (714) 554-7090
CIRCLE 192 O N READER SERVICE CARD 94 A · ANALYTICAL CHEMISTRY, VOL. 49, NO. 1, JANUARY
1977
the method used for measuring the area under the peak, triangulation, cutting, and weighing or integration. Errors can be minimized by using uni form layers on t h e same plate. Our ex perience indicates t h a t commercially precoated plates are reproducible to within ± 5 % (12). T h e area under the peak can be measured manually or automatically. Manual methods, such as triangula tion, cutting and weighing, or by using a planimeter, are relatively slow and subject to h u m a n error. T h e automat ic method eliminates these disadvan tages by electronically integrating the area under the peak. T h e source of error is the scanning speed of the T L C plate, combined with the counting/ second. Our experience shows t h a t a 2.1% error is introduced if t h e scan ning is fast (cm/min) and the counting is slow, whereas a 0.7% error is intro duced if t h e scan is slow and the counting is fast (29). In our laboratory, the SD 3000 and M P F - 3 densitometers with scanning attachments are connected to a Hew lett-Packard Lab Data System Model 3354A which types the integrated area, percentage of each spot and the total area, and the RF value. For a de tailed discussion of instruments useful for T L C , see ref. 30. References 31 and 32 are also useful sources of infor mation. Quantitation through elution, which involves several steps (as mentioned earlier), is of limited value in routine analysis. Limiting factors include in completeness of elution and the possi bility t h a t interfering materials will be carried over into the elute. Gravimet ric determinations are often very pre cise, but the amounts required for an analysis are very high. Spectrophotometric determinations of eluted frac tions are feasible only when the com pounds are colored or fluorescent in solution. T h e Eluchrom system de scribed earlier is a good method of quantitation. Another method, only applicable in the case of labeled mate rial, involves the use of a radioactive scanner for in situ quantitation. As mentioned earlier, scraping of zones often results in personal errors. These could be minimized with the "zonal scraper", suggested earlier for the analysis of radiolabeled materials, which might also be used to advantage for quantitative evaluation of T L C of nonlabeled substances (33).
j
H P T L C is a new technique for con trol of all phases (stationary, mobile, and vapor) (34). T h e sample is either applied to the dry phase or injected into the flowing system. H P T L C (which we have not examined) is sup-
posed to offer increased resolution and reproducible RF values up to ±0.01 RF units. Micro H P T L C (35) utilizes silica gel with a pore size of 60 Â to form a layer 12 /an thick. UV and visible absorbing samples were analyzed in the 100-pg to 100-ng range, while fluorescent substances were determined quantitatively between 10 pg and 100 ng. General Comparison of TLC with Other Chromatographic Techniques Although GLC and H P L C are faster, more accurate, and afford superior resolution, T L C offers some advantages when compared to the above techniques. T L C could handle up to 15 different samples simultaneously (36) on a 20 X 20 cm plate, whereas GLC and H P L C are limited to one sample at a time. T L C like H P L C could handle all types of compounds (except gaseous), but GLC is limited to compounds t h a t have appreciable vapor pressure or could be derivatized to products t h a t have an appreciable vapor pressure without thermal decomposition. T L C offers the analyst the advantages of in situ spectroscopy and bioautography (12, 20, 21). Furthermore, classification of a large number of compounds could be achieved faster and easier by T L C than by GLC or H P L C , Different ad-
sorbents and various solvent systems can be used for t h a t purpose (20). Generally, presample cleanup is not a must for T L C , while GLC and H P L C require reasonably clean samples for column injection. T h e Eluchrom makes simple, fast, and quantitative elution of samples from the plate and subsequent analysis by spectroscopic methods. Recently, T L C - A A S (21) and T L C / I R (12) have been achieved by use of the Eluchrom for sample elution. T h e coupling of the Eluchrom to mass spectrometry is under study and will be reported later. Overall, T L C is cheaper t h a n the other chromatographic techniques. Also, the use of the Eluchrom and other modern instrumentation makes T L C a very competitive and useful analytical tool. Acknowledgment T h e authors thank the commercial companies for supplying literature and information about their equipment. We also would like to caution the reader t h a t the instruments mentioned in this article may not be the only ones available, nor do we consider them the best on the market; they are, however, the instruments we are most familiar with. We thank George M. Janini for helpful comments and discussion.
Reliable. Fast and Easy. MCI automatic analyzer. Incorporates coulometry principle applied to Karl Fischer titration. Operation is full-automatic. Measuring time is shortened to 1/2—1/3 of conventional KF method. Accuracy is within 5//g for lO^g—1 mg H2O and within 0.5% for 1—10 mg H2O. Suitable for wide-range applications, including measurement of ultra-trace water content in liquids, solids and gases. Digital display in μ%. Range: 10/zg—lOmg H2O. An optional water vaporizer may be connected to the instrument for speedy and accurate measurement of water content in plastics, grain, etc. Printer (optional)
CA-02 Moisture Meter with Printer
References (1) N. A. Izmailov and M. S. Shraiber, Formatsiya, 3,1 (1938). (2) J. E. Meinhardt and N. F. Hall, Anal. Chem., 21,184(1949). (3) J. G. Kirchner, J. M. Miller, and G. J. Keller, ibid., 23, 420 (1951). (4) E. Stahl, Pharmazie, 11,633(1956). (5) N. Pelick, H. R. Bolliger, arid H. Man gold, "Advances in Chromatography", J. C. Giddings and R. A. Keller, Eds., Vol III, ρ 92, Dekker, New York, N.Y., 1962. (6) G. Zweig and J. Sherma, Anal. Chem., 48,66R(1976). (7) Camag Bibliography Service, Dieter Janchen, Ed., Camag, New Berlin, Wis. (8) L. R. Snyder, "Principles of Adsorp tion Chromatography", Dekker, New York, N.Y., 1968. (9) E. Stahl, in "Chromatography", E. Heftman, Ed., 3rd éd., ρ 172, Van Nostrand-Reinhold, New York, N.Y., 1975. (10) T. W. Mumms, K. C. Podratz, and P. A. Katzman, J. Chromatogr., 76, 401 (1973). (11) H. J. Issaq and E. W. Barr, ibid., in press. (12) H. J. Issaq, unpublished results. (13) L. R. Snyder, "Principles of Adsorp tion Chromatography", pp 185-240, Dekker, New York, N.Y., 1968. (14) J. G. Kirchner, "TLC", Interscience, New York, N.Y., 1967. (15) J. A. Perry, Anal. Chem., 47 (1), 65A (1975). (16) F. Geiss and H. Schlitt, Chromatographia, 1,393(1968). (17) H. J. Issaq and E. W. Barr, J. Chro matogr., in press. (18) E. Stahl, "Thin Layer Chromatogra phy", Springer-Verlag, New York, N.Y., 1969. (19) G. Zweig and J. Sherma, Eds., "Hand book of Chromatography", Vol II, CRC Press, Cleveland, Ohio, 1972. (20) H. J. Issaq, E. W. Barr, T. Wei, C. Meyer, and A. Aszalos, J. Chromatogr., in press. (21) H. J. Issaq, Anal. Chem., submitted for publication (1976). (22) J. C. Touchstone, Ed., "Quantitative Thin Layer Chromatography", ρ 31, Wiley-Interscience, New York, N.Y., 1973. (23) H. J. Issaq and E. W. Barr, J. Chro matogr. Sel., 13, 597 (1975). (24) H. J. Issaq and E. W. Barr, unpub lished results. (25) R. K. Viteck, C. J. Seul, M. Baier, and E. Lau, Am. Lab. (Feb. 1974). (26) "Applied Science", Gas Chromatogr. Newsl., 16 (3) (1975). (27) D. E. Heinz and R. K. Viteck, J. Chromatogr. Sci., 13, 570 (1975). (28) H. J. Issaq, E. W. Barr, and W. L. Zielinski, Jr., J. Chromatogr., in press. (29) H. J. Issaq, E. W. Barr, and R. Young, unpublished results. (30) M. S. LeFar and A. D. Lewis, Anal. Chem., 42(3), 79A (1970). (31) "Guide to Scientific Instruments", Science, 190 (4216A) (1975). (32) "1976-77 Lab Guide", Anal. Chem., 48(10) (1976). (33) M. K. Moryald and M. D. Mukhrejce, J. Chromatogr. Sci., 13, 399 (1975). (34) R. Kaiser, Z. Anal. Chem., in press (1976). (35) J. Ripphahn and H. Halpaap, J. Chromatogr., 112, 81C (1975). (36) E. Stahl, in "Chromatography", E. Heftman, Ed., 3rd éd., ρ 421, Van Nostrand-Reinhold, New York, N.Y., 1975.
MITSUBISHI CHEMICAL INDUSTRIES LIMITED Instruments Dept., Mitsubishi Bldg., 5-2, Marunouchi 2-chome, C Tokyo, 100 Japan Telex: J 2 4 9 0 Cable Address: KASEICO TOKYO CIRCLE 142 ON READER SERVICE CARD
96 A · ANALYTICAL CHEMISTRY, VOL. 49, NO. 1, JANUARY 1977
Research sponsored by the National Cancer In stitute under Contract No. N01-CO-25423 with Litton Bionetics, Inc.
