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INSTR UMENTATI0 N
Advisory Panel Jonathan W. Amy Glenn L. Boornan Robert L. Bowman
Jack W. Frazer G. Phillip Hicks Donald R. Johnson
Howard V. Malrnstadt Marvin Margoshes William F. Ulrich
Thin-Layer Densitometry
Morton S. Lefarl 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
THE CSE OF thin-layer chromat.ography (TLC) as a method of providing
mpitl separation of mixtures is wellknown and documented. Since there are only microgram amounts on the T L C plate, usually of the order of 0.001 to 0.1 mg, the quantitative analysis of sepnrated components has been difficult. The procedure of scraping nntl collecting the adsorbent folloir-ed by extraction with solvents is laborious antl difficult especially when several components of Ion- concentrat,ions are t o be qunntitnted. h h y investigators have reported that analytical blanks are generally high and not, reproducible on-ing to impurities in the adsorbent and to particles which remain in suspension in the eluat,e. Some substances siich as certnin steroids and amines may be decomposed during elution from the ahsorbent depending on the type of adsorbent and elution technique used. These p r o h l m s have been recognized by the scientific instrumentat,ion indnstry, and equipment is now available ~ h i c l ihas both the desired precision antl sensitivity. T h e purpose of this article i, to disciiss some of the aspects of ihin-layer densitometry. General Considerations
The rewlts of investigators vary greatly as to the relationship between absorbance and the Concentration of a material spotted on a TLC plate. (Readers cliould note that technically the term “absorbance” should be replaced hy the mathematical equivalent “optical denqity” for scattering samples, where the photocurrent depends on the ‘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 describing their TLC densitometers, v,-e have also done so in this article.) Several investigators have reported a direct relationship under suitable conditions 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 compound concentration (where the term “concentration” actually means concentration x volume or quantity of the analyte). Other authors have reported a linear relationship between the logarithm of the weight of material spotted and the square root of the integrated absorbance reading. There are also reports of a linear relationship between the absorbance and the logarithm of the concentration of compound or between 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 relationship 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. T h e behavior of a light beam when i t impinges upon a n absorbing material distributed on and in the surface of the gel is complex and not clearly understood. The optical phenomenon in a translucent material can be written as: There I,, I, It I,
= incident light = absorbed light
= transmitted light = reflected light
Shibata (3) reported a t least six other types of resultant light, which may also be applicable to TLC densitometry. One of the criteria which affect the precision in the densitometry of colored material.; 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 mu.;t be reproducible and show a definite relationship between the amount of light absorbed by the compound and the amount of material spotted. T h e design of a n 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 fluorescence, and fluorewmce quench, Visible Reflectance. For the detection of some compounds, the developed chromatogram can be visualized b y spraying with a reagent to develop a color Charring with acid and heat is also used extensively to make the compound visible. Howwer, with the current instruments available, this i,c perhaps the most difficult technique with which to obtain quantitative data. It is critical that the developing reagents are sprayed evenly 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 during the scanning period and that compounds of high concentration have reacted completely. This is critiral 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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We’re not lost In the crowd.
There are lots of UV flow monitors, but only ISCO:
. . . records directly
in three ranges of linear absorbance.
. . . has the least stray
light-low enough t o permit quantitative results in practically all applications.
. . . will automatically
operate a fraction collector so separate UV-absorbing peaks are deposited into separate tubes.
. will monitor two columns at one wavelength or one column at two wavelengths.
ISCO quantitative UV monitors operate at 254 and 280 m p with single and dual beam optical systems. They are sensitive, stable, and will operate in a cold room. They are priced no higher than conventional UV monitors. Request Brochure UA31 for complete details.
INSTRUMENTATION SPECIALTIES CO. 4100 SUPERIOR LINCOLN, NEBRASKA 6 8 5 0 4 PHONE (4021 4 3 4 4 2 3 1 CA8LE l S C O U 8 LINCOLN
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the oven temperature and time of reaction must be carefully and reproducibly controlled. Samples must be applied 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 background absorption. Ultraviolet Fluorescence. Those compounds that fluoresce visible radiation 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 contain samples to assess the amount of fluorescence “noise” present in the adsorbent layer. This may be caused by the use of solvents containing fluorescent impurities. Since some compounds show a deterioration of fluorescence with time, calibration curves should be prepared for each substance being analyzed. Fluorescence Quench. This is presently 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 t o those which exhibit fluorescence 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 instrument for a few minutes before starting the analysis to “dark adapt.” Quench systems usually show a nonlinear relationship between the integrated area under the scan curve and the sample concentration. A separate calibration curve must be prepared for each compound since different substances show different “extinction coefficients.” Transmission os. Reflectance. With transmission scanning, the background absorbance varies with layer thickness. The adsorbent thickness must, therefore, be the same a t 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 thickness 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. ,4 compact spot will, therefore, permit the scatter to become relatively 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. I n an investigation utilizing the Joyce Loebl Chromoscan densitometer, Dallas ( 1 ) reported that the thickness of the adsorbent layer is a critical factor in densitometry. Peak height, width, and area all depended on layer thickness. As the layer thickness decreased, the peak height and area increased Fvhen scanning with reflected light. With transmitted light, both peak height and area decreased as the layer thickness decreased. S o loss of peak resolution was observed as the layer thickness increased 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 standard deviation in the volume of different 2-pl capillary pipets was 2.2%. This error !vas greater than that due to the chromatography, the densitometry, and the method of determining the area under the curve combined. Other investigators 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 snme 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 .41am ( 2 ) reported that for two alkaloids, the mean square between readings on different plates is higher than the mean square between readings on the same plate. For these oxindole alkaloids, the average result obtained b y a number of readings from one plate was associated with a lower variation than the a\-erage result obtained b y a number of measurements from different plates. The average result obtained from a number of measurements from one plate may be higher or lower than the theoretical value.
High performance and low cost don't often come together. The Beckman GC-M Gas Chromatograph, however, gives you both, plus s i m p l e setup, ease of operation and rugged reliability. A c c o r d i n g t o your needs, t h e instrument is available with a dual flame ionization detector, a thermal conductivity detector, or one of each.Thus, with a single instrument you can perform analyses with both types of detectors. No t i m e i s lost shutting d o w n t o
1
change detectors o r readjust electronics. Other features of t h i s reliable, research instrument i n c l u d e : c o m p a c t size, independent column and detector temperature control, linear temperature programming with directly selected heating rates, dual-column compensation and high sensitivity.
tation field. And a Beckman representative is nearby, wherever you are. Beckman has a complete line of gas chromatographs to satisfy your GC requirements. Contact a Beckman sales representative, or write for Data File 305, Scientific Instruments Division, Beckman Instruments, Inc., 2500 Harbor Blvd. , Fu Ilerton, California 92634.
The GC-M is backed by the finest, most extensive applications and service groups in the instrumen-
Finally, a high performance GC you can afford.
Circle NO. 90 on Readers' Service Card
ANALYTICAL CHEMISTRY, VOL. 42, NO. 3, MARCH 1970
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Better results can be obtained by the coinLination of measured values from different' plates than by the combined values obtained from one plate. Mobile Phase. -4change in the st,ationary and mobile phase used for the separation affects the shape and size of the separated component. Different sy,steins offer different rates of movements depending on the adsorption isotherm and (or) the partition coeficicnts of the component with respect to the solvent system. The solvent system that permits a low R, usually gives a small, round compact spot. Systems that result in high R, of the compound usually offer larger, often elongated and sometimes laterally diffuse spots. The cliangea in size and shape are reflected in a diffcrent distribution and deposition of compound on the adsorbent. Of importance is that the same quantity of sulxtance may give a different, responsc v-ith regard to the densitometer rendings after separation utilizing diffcrent solvent systems, Instruments
The Nester-Faust Uniscan 900 (Sester-Faust Manufacturing Corp., Sewark, Del.) utilizes t,he illumiiiation and detection geometry used for many years in measuring colored solids and srirf;rces. The chromatogram is illuminated at a 45' angle to the surface of the plate by an incandescent soiirce and then scanned at, 90' to the surface. TKOmethods of operation are a\~nilnhlein the visible reflectance mode. The iiistr~unentcan be operated as a single-channel photometer where space is not available for use in scanning the plntc surface along with the compound to be analyzed. The differential scan mode may be used which provides for a reference pnth adjacent to the sepnrnted sample. For uv fluorescence (native fluorescence or quenching mode) a second optical head is supplied as well as a plug-in front panel section n-it11 the necessary controls for amplification and power for the source and pliotoinultigliers. A separntc module is used to afford regiilated high-voltage power for the low-pressure mercury source and the ~~liotoniiiltil~lier. The uv fluorescence head contnins a low-pressure mercury source and filters to provide maximum emission at 2537 A or 3654 A . Two slits, 1 min and 2 mm, are utilized in the instrument. The thin-layer plate is inserted in a carrier assembly. The appropriate detector head then moves over the plnte a t a rate of 8 in. in 2.5 Circle NO. 69 on Readers' Service Card
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ANALYTICAL CHEMISTRY, VOL. 42, NO. 3, MARCH 1970
min. The optional Summatic 1502 electronic digital integrator provides for automatic detection and digital printout of the area under the analog curve. -4plug-in log-to-linear converter is available which can convert the logarit,hmic function output to a function linear with concentration. Front panel adjustments a l l o ~the transfer function log-to-linear to be modified to accommodate wide deviations from Beer's laW. The Schoeffel hlodel SD-3000 (Schoeffel Instrument Corp., Westwood, N. J.) double-beam spectrodensitometer utilizes a 150-watt. Xenon or 200-watt Xenon-Mercury lamp as a light source. The radiated light, is focused onto the entrance slit of a monochromator by means of a quartz condenser and surface reflector. The monochromator has a linearly calibrated navelength selector dial, enabling the user to select monochromatic light in the range 200-iO0 nm. The light emitted from the monochromator exit slit is magnified by a quartz optic and a folded-beam mirror system, thus uniformly illuminating the sample and reference area on the TLC plate. The beams of light are scattered by the TLC plate and appear to the detectors as a pair of secondary light sources. Since the illuminated areas can be varied in width as well as in length, maximum resolution (narrow slit v-idth) or good background-noise integration (wide slit width) can be obtained. The scattered light (secondary light sources) can be detected either directly from underneath the plate or from above the plate by phototube interception of the scattered light at, 4 5 O t o the optical normal (reflection). If necessary, interference ivedge monochromators (continuously adjustable from 400-650 n m ) can be inserted for emission analysis. The sample and reference signals are balanced by means of R continuously variable gain balance adjustment on a plate location where reference and sample area have identical optical densities. Both signals are separately and continuously amplified and automatically computed into an optical density which equals log (reference signal)/ (sample signal). On special request, a linear ratio of (sample signal) / (reference signal) can also he provided to facilitate fluorescence and reflection measurements. Signals are recorded by a strip chart recorder and integrated by a disk integrator. The TLC stage drive is controllnble and synchronized in a number of ratios to the recorder speeds of the system. For obtaining maximum quantitation accuracy, signal interference, caused by sample spillover into reference areas, is prevenbed by scoring the adsorbent
A N e w s w o r t h y C o m p e n d i u m o f H o w EA1 H y b r i d , D i g i t a l a n d A n a l o g , s y s t e m s h e l p m a k e a c o m p l e x w o r l d r e l a t i v e l y s i m p l e r .
SO OTHERS MAYBREATHE EASIER
GCPEAKSAND THESOFTWARE DEMON
IMITATION POLLUTIONCAN BEASOLUTION
KINETIC DATA ~IUdINGFULLY SHAPED BY COMPUTER
Breathing is a pretty-much taken-for-granted activity. Until it stops. Or until you look into the efforts of researchers and physicians in that field. Measuring respiratory pressure and volumetric-flow rate is pretty straight-forwardly accomplished with conventional transducers. But there are other important parameters that are determined by these characteristics: total inspired volume and breath-by-breath volume. Then there's total work, compliance and respiratory resistance. All of these values used to have to be calculated by hand. Tedious and time consuming. More lately they were cranked into a digital computer. Less tedium, but still a delay. Now, thanks to EA1 analog computers, all calculations can be accomplished instantaneously and presented simultaneously with the original measurements on a strip chart. Besides the obvious value of showing a researcher what's happening when it's happening, our lower-cost machines also open new doors to monitoring patients. We've got some dramatic stories on how EA1 analog computers are helping in other areas of research into physiological dynamics. By writing to TIBio-medicalll, Dept. 206K, you'll get them by return mail. As with motherhood and the flag, consensus holds that computerized data reduction is with us to stay. But, in practice, it all can get a bit sticky,.Take data from an analytical instrument like a GC. A few giants in the industry continue to stumble over problems in GC like noise, signal processing, or really useful software. EA1 is still the pioneer here in its PACE I11 analytical data system. One seemingly small thing is a software technique f o r resolving complex GC peaks. It consistently and accurately apportions complex areas, rangi'ng from overlapping components to poorly resolved shoulder peaks. Part of the technique accommodates the usual "skewIt in component elution to give consistent improvement in accuracy of quantitative analysis. (Our research people gave a paper on it at the 158th National ACS meeting.) It's all part of the whole PACE 111 system--a turnkey data system for many analytical instruments--GC, mass spec, and the like. For a copy of the paper and a detailed booklet on PACE 111 write to Dept 206K. And if you're at the Pittsburgh Conference, plan to attend our seminars and see PACE I11 in operation. A topic certain to stir up the citizenry these days is pollution--any kind of pollution. Take a simple thing like free oxygen in water. Overload the water with oxygen-hungry chemicals--no oxygen. Or develop too many organisms--plant life prospers (called eutrophication) and no oxygen. Either way, no fish. And with no fish, you've upset the water ecology. Pragmatic scrutiny tells us we can't shut down our industries to bring back pristine, airy waters. Fortunately, we can imitate these conditions by computer simulation, and get a drip on the ameliorative aspects of a solution. Recently, EA1 provided the HEW with.a hybrid-computer simulation of the Delaware River Estuary. From this simulation engineers can tell where to best locate stand-by reservoirs, what flow rates to employ, and when to use them. We've written this one up. A request to "Delaware", Dept. 206K, will get you a copy, and get us both cracking on another solution. In olden times petrochemical-process design involved finding rate and equilibrium constants f o r several reactions required a trial-and-error method. Much trial. Much error. Most process designs involve the solution of ordinary differential equations --in a lumped-parameter system where changes are taking place in time but not space. With the use of analog computers, solutions poured forth. However, distributed parameter systems involve changes in time and space simultaneously--expressed by partial differential equations. Many approaches to PDE solution have evolved f o r digital computers. But such solutions consume more and more hardware, with ever-present error creeping back in as problem compqexity increases. Hybrid computers clear this difficulty up. Kinetic data are programmed into the analog portion, actual results go into digital computer memory. The analog makes a series of process condition runs, the digital stores the data, matches the results from the plant and computes least mean-square deviations. The "solution'l has been found when results of simulation most closely match actual conditions, and no further reductions can be made in mean square deviation values. Optimization is achieved--in time, money and results. After much struggle, EA1 is pleased to offer a software package in this arcane speciality--write to ItKinetict', Dept. 206K. Electronic Associates, Inc. West Long Branch, N.J. 07764.
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An e q u a l . o p p o r t u n i t y e m p i o y e i w l h unequaled e m p l o y m e i t o p p c r ! u n l t y
ANALYTICAL CHEMISTRY, VOL. 42, NO. 3, MARCH 1970
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Be No. 90,001 Over 260 catalog-type ad pages list all the alternatives. Which makes LAB GUIDE the easiest, most efficient way for you to shop for instruments. Complete facts about competing sources of supply are at your fingertips. Set up so they’re simple to find. With mail-in cards to get you more information about any advertised product in a hurry. But don’t take our word for it. Check the record. Last year alone, your colleagues used the LAB GUIDE to get product information from manufacturers 90,000 times. Which only goes to show it’s worth looking into LAB GUIDE. . . and being no. 90,001, The LAB GUIDE is issued annually, in July, to subscribers to the research journals of the American Chemical Society. Circle No. 25 pn Readers’ Service Card
If your plant could use analysis, make an appointment with ARL. ARL is the country's largest and oldest manufacturer of instruments for spectrochemical analysis, with a complete line of rugged, reliable, easy-to-use Quantometers - both optical emission and x-ray. ARL instruments routinely examine solids, liquids or gases quantitatively, in demanding production or process control applications. Precision and versatility are built in. These modular ARL analyzers provide effective quality control at low cost. They are compatible with a variety of EDP devices - integrally mounted or remote. An ARL District Sales Manager is the man who can determine the Quantometer best suited to your materials analysis needs. For more information just write o r call. Brochures on all instruments are yours for the asking.
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layer into 10-nm width strips. The zones are spotted alternately, providing a blank reference strip between samples. The Joyce Loebl Chromoscan densitometer (Technical Operations Inc., Burlington, Mass.) operates on the double beam principle. It is a nullpoint instrument in which a grey wedge is balanced against the reflection from or transmission through the sample. hIeasurements can be made by reflectance, transmission, fluorescence, or uv absorption through the use of an auxiliary high-resolution attachment. The light source is a quartz iodide tungsten lamp, with gelatin or interference filters. I n the uv, mercury and deuterium lamps can be utilized. The uv lamp can be placed alongside the visible soiirce in a dual lamphouse for rapid changing between sources. The parallel focused beam with a 90' incident angle can be adjusted to any shape within a 1-cm diameter circle. For reflection measurements, only light scattered at a 45' angle to the incident light beam passes to the photomultiplier. With the standard instrument plates, up to 25-mm widths can be scanned. For plates u p to 200 x 200 mm, an accessory is available. I n transmission measurements, a separate photomultiplier is positioned several centimeters behind the sample so that scattered light is not measured. One of the unique features of this instrument is the use of plastic cams so that a correction factor may be built into the analysis affording linearity of results. Different angles of straight
cams may be used for scale expansion when dealing with weak reflection or small extinction changes. A variable slit is available in the range of 0-1 mm in width and 0-3 m m in height, Filters are ut>ilizedto vary the spectral characteristics of t~he light. Sample movement. and the recording drum can be rigidly linked, which in turn may be coupled through a gear box to give different expansion ratios of recorder/ sample movement 5 9 : l . i i n automat>ic six-digit readout integrator is provided on the instrument. The Farrand VIS-UV Chromatogram Analyzer (Farrand Optical Co., Inc., Bronx, K.Y,) measures uv-absorption by either fluorescence quenching or by emission of native fluorescence. It also measures uv or visible absorption direct.ly with or without staining of the plate or the spotted compounds. It can perform these functioiis by either double-beam ratio or single-beam operation (Figure 1 ) . The arc from a xenon lamp is imaged in magnified form at the entrance of the exciter monochromator. By optical techniques and slit masks, a sliver of light, is produced which illuminates the surface of the plate. The sample carrier moves in such a manner that, the length of the light beam is a t right angles to the direction of scanning. I n this manner, the spot to be analyzed and the lane on each side are illuminated. The two reference signals are then averaged. The double-beam procedure lessens the effects of lamp iiitensity variations and plate-background gradients. This instrument utilizes two monochromators, one in the excitation portion and one in the analyzer leg with provisions for filters and polarizers. I n the reference leg, a filter is used routinely. The spectral range of
Figure 1. Schematic block diagram of Farrand chromatogram analyzer
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E l e c t r o n T e c h n o l o g y , subsidiary o f Esterline C o r p o r a t i o n , 6 2 6 S c h u y l e r Ave., Kearny, N.J. 07032' (201) 998-8100
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Cryogenic Chromatography b y and Effective With the 7620 GC's cryogenic module, sub-ambient oven temperature control is as easy and effective as high-temperature control has been for years. It's easy because the cryogenic module controls oven temperature continuously down to -70°C and holds it within 0.1"C, for isothermal or programmed operation. It's synchronized with programmer operation: a blinking light tells you when the oven has cooled to the starting temperature, whether cryogenic or near ambient. It operates either with economical liquid CO, down to -70°C or with compressed air in the trans-ambient range. And it's a standard option: you can order it installed on a new 7620 or install it yourself later, with relative ease, without modification to the programmer. The most important characteristic of the 7620's cryogenic module is its effectiveness. As evidence, we show the chromatogram of a complete separation of Argon from Oxygen in a sample of air, performed in 8 minutes on a 6-foot column (Molecular Sieve 5A) in a 7620 oven operated isothermally at -30°C. Any GC that can do this is clearly performing well in the sub-ambient region. Still more evidence of the 7620's precision at subambient temperatures is presented in Bulletin 7620, yours on request. Prices start at $5150 for a dual T C detector instrument; the cryogenic option adds $350. Hewlett-Packard, Route 41, Avondale, Pa. 19311. In Europe: 1217 Meyrin-Geneva, Switzerland.
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What will a 7700 do for YOU? 1. I t w i l l analyze the output of a variety of laboratory instruments. (Chromatographs, spectrometers, spectrophotometers, physical testing machines.)
2. I t will provide a more efficient data reduction system because data is collected off-line by high-performance recorders. (Each laboratory instrument can now have access to a computer. The amount of data collected i s limited only by the number of recorders.)
3. The 7700 can undertake routine analysis without computer interface. However, optimized data manipulation i s available by conversing with the computer. (Conversing i s via Teletype. You needn't be a computer expert to take advantage of all the 7700's capabilities.) 4. A separate software program is provided for each laboratory instrument. This means the entire computer capacity can be devoted to a particular analysis. (All equipment for data processing is contained in the 7700. There are no wires to any other equipment other than the Teletype.)
.....
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ANALYTICAL CHEMISTRY, VOL. 42, NO. 3, MARCH 1970
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* INSTRUMENTATION
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this instrument ranges from 200-785 nm. A built-in integrator/counter integrates the area under the curve by translating the pen position into a signal which is a function of scan time. After integration, the data are converted t o digital form for a digital counter. The Zeiss Chromatogram Spectrophotometer (Carl Zeiss, Inc., New Yorlr, N. Y.) is designed to determine quantitativc and qualitative reflectance, transmission-absorption, fluorescence, and fluorescence quenching. I t s major components consist of a dual light Soiirce (deuterium and tungsten), the l I 4 Q I11 prism monochromator, an optical imaging assembly, the chromntogram scanning tnble, and an electronic tlctectioii system. I n addition to the standard sources, a Yenon and mercury lamp can be furnished. The monochromator provides a spectral range of 200-2500 nm. Controls at the optical imaging assembly
permit precise focusing of the monochromator slit or prism image. Slit width is continuously variable from 0.01-2.0 m m and slit height may be adjusted from 2-14 mm. For ideal coverage of circular spots a continuously variable circular aperture ranging from 6-30 m m is provided. The instrument can be operated in two basic arrangements as shown in Figure 2. Arrangement M-PY facilitates monochromatic illumination normal to the sample surface while the detector is located a t 45' for determination of diffuse reflection or emission. Arrangement Pr-IIl positions the source a t 45O to the sample surface while diffuse reflection or emission is observed normal to the sample surface via the monochromator. I n the transmission mode the detector is placed underneath the sample. Intensities thus observed are indicated on linear and logarithmic scales of the photometer and can be documented on recorders with or without integrators. For spectrophotometric analysis of a specific spot, the chromatogram table is held stationary nhile the monochromator is automatically scanned through a desired spec-
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Figure 2. Simplified path of beam i n Zeiss chromatogram spectrophotometer Arrangement M - P r ( M = monochromator; Pr = sample): Radiation from light source first traverses the monochromator, then strikes sample. Arrangement Pr-M: Radiation first strikes sample then traverses the monochromator
1. Light source: in arrangement M-Pr, hydrogen lamp, incandescent lamp, or high pressure xenon lamp; i n arrangement Pr-M, Hg lamp, hydrogen lamp, or incandescent lamp i n special lamp unit 2. Monochromator
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ANALYTICAL CHEMISTRY, VOL
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3. Intermediate optical system (sim. plified) 4. Deviating mirror 5. Chromatogram 6. Intermediate optical system 7 . Filter 8. Detector
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Buy Heath 700 Series Spectroscopy Modules.. And Get Multi-Instrument Capability At One-Instrument Cost Choice of Readout. The modular versatility of the Heath 700 Series extends also to readout. The 701 and 703 Systems can b e supplied with the Photometric meter Readout, the Multispeed Chart Recorder o r Multispeed Log/Linear Current Recorder. O r combine readout modules to give simultaneous chart recording and direct meter readout. And the Photometric meter Readout can also be used a s a n interface for a digital voltmeter, such a s the Heath 805A. Pick the readout that suits your n e e d s . . . and your budget. The Heath 701 Spectrophotometer features 1900 to 7000 Angstrom range (which can be extended with PM interchange] . . . less than 0.05% stray l i g h t . . . & 1 A wavelength tracking a c c u r a c y . . . b e t t e r than 1 A r e s o l u t i o n . . . & 0.2 A wavelength reproducibility . . . electronic digital-controlled stepped scanning and many other precision features. 701 Systems range from $2245 (less readout) t o $2785 [with recording readout]. The Heath 703 AA-AE-AF System includes adaptability to all major types of total consumption and laminar flow burne r s . . . accurate, repeatably xy, yz burner positioning.. . a hollow cathode lamp turret that accepts four l a m p s . . . l a m p power supply stability that's better than 0.1% . . . h i g h intensity and multi-element lamp capability and built-in chopper. 703 Systems are available from $2736 (without readout] t o $3278. Investigate The Heath 700 Series N o w . . . and start conserving your budget while building the most versatile, widely applicable spectroscopy system on the market today. EU-701B, Single Beam System with Photometric Meter Readout ..................... .$2500.00* EU-703B, AA-AE-AF System, with Photometric Meter Readout ..................... .$2998.00*
Budget. Mention the word and even the most hardened manager c r i n g e s . . . h e knows what to expect well in advance of seeing the final figures. How d o you cope with expanding instrumentation requirements a n d a static or even reduced equipment budget? Do you delay needed purchases with the hope of "maybe next year"? Play the "numbers" game and borrow from one area to pay for another? Or could you find the kind of equipment that serves many needs today a n d can b e updated tomorrow without obsolescence? Read o n , . . Heath Can Help Solve Your Spectroscopy Budget Problems . . . Our 700 Series Spectroscopy Systems are unconventional in both design and price. But they give you precision Molecular Absorption and AA-AE-AF Spectrophotometric capability (and that's just the beginning) all for under $3900. So, if you're caught between a n increasing need for spectrophotometric laboratory equipment and a diminishing amount of money, the 700 Series may b e just what you're looking for. Modular Versatility. Because some functions in spectroscopy are common to many instruments, Heath h a s put these functions in separate modules that can b e interchanged a s needed. The Heath 701 high precision Spectrophotometer Single-Beam System, for example, consists of the Scanning Monochromator, UV-Visible Light Source Module, Sample Cell Module and Photomultiplier Module, locked together on the 701 Base. Cost? Only $2500 with photometric meter readout. To get complete atomic absorption, atomic emission, atomic fluorescence capability you don't have to buy another monochromator a n d another photomultiplier module . . . just add the AA-AE-AF Module and the 703 B a s e . . . only $1335 more. Modules to provide many other types of spectrophotometric measurements a t equally low cost are o n the way.
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ANALYTICAL CHEMISTRY, VOL. 42, NO. 3, MARCH 1970
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tral range. An internal reflectance standard permits immediate reference to confirm instrument setting and stability. The chromatogram adapter can also be supplied as an accessory t o Carl Zeiss P l I Q I1 users. The Photovolt Densitometer Xodel530 (Photovolt Corp., New York, N. Y.) operates on the principle of straight transmission measurement with a high degree of collimation-rejected scattered light. A variety of light sources can be used. For fluorescence measurements two mercury vapor lamps are utilized. One affords a sharp 254-nm emission and the other a broader u v range centered a t about 350 nm. -4uvtransmitting glass filter is used in front of the mercury arc to eliminate the visible radiation when utilized as an ultraviolet source. N o s t commonly used is a stabilized, small-filament tungsten lamp. This light source is essentially re-imaged on the thin-layer plate giving a small area of illumination. The upper collimating slit picks u p only
the central transmitted beam and rejects 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 sensitivity control. The photomultiplier tube is contained in a detachable housing, 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 available. The recorder can be set so that either linear or nonlinear responses result, so that the logarithm (absorbance) or steeper curves may be plotted.
visualization procedures, and quality of the plates can be greater than that inherent in the use of any given instrument. Since this article is not intended to be a n exhaustive survey of densitometers for thin-layer chromatography, the reader should refer to the American Chemical Society’s 1969-1970 Laboratory Guide to Instruments, Equipment and Chemicals for further information 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 Chromatography,” (second edition), Academic Press, 1967. Acknowledgment
The authors are grateful for the assistance and cooperation of the instrument manufacturers mentioned in the article.
Conclusion
References
There is now available suitable instrumentation for the quantitative evaluation of thin-layer chromatograms. The sources of error involved in the spotting, layer thickness, development,
(1) M. S. J. Dallas, J . Chromatog., 33, 337 (1968). (2) E. J. Shellard and M. Z. Alam, &id., p. 347. (3) K. Shibata, Methods Biochem. Anal., 7, 77 (1959).
C 0 MMENTARY by Ralph H. Muller
HIS DISCUSSION of thin-layer chroT m a t o ,wraphy is timely and useful. It is a t 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 statement, “The golden era of paper chromatography began and by 1956 over ten thousand publications on the use of this ‘universal’ method had come out.” I n the case of TLC, b y the end of 1965, over 4500 publications had appeared as well as a dozen monographs and reviews in 11 languages. Paper and thin-layer chromatography have been applied to almost every conceivable class of substance, but in quan-
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titative evaluation a half dozen or more functional relationships have been proposed and used, relating the optical measurement to concentration. I t seems that further improvements must come from the chemical and manipulative factors leading to more reproducible 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, diminish the fluorescence of a substance incorporated in the thin layer or sprayed on the chromatogram. I n most monographs or treatises, the term is used ambiguously. Obviously, there
ANALYTICAL CHEMISTRY, VOL. 42, NO. 3, MARCH 1970
are two cases involved. I n true fluorescence quenching, the “quencher” deactivates the excited fluorescent substance and diminishes or obliterates its emission of radiation. I n the other case, by absorbing the exciting uv radiation, it acts in a manner not much more subtle than interposing a piece of cardboard or a thick sheet of glass. Either phenomenon can be useful in locating or measuring a spot, but the functional relationship with concentration should be hyperbolic in the first case and logarithmic in the second. For true quenching, the simple explanation in terms of deactivation 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
