INSTRUMENTAL CHEMICAL ANALYSIS AT THE CONNECTICUT AGRICULTURAL EXPERIMENT STATION On the occasion of the 255th Meeting of the NEACT The means of excitation may be an electric arc or spark a t the Connecticut Agricultural Experiment Station, or a flame; a t the Experiment ,Station an arc and one of the high lights of the day was an inspection tour sometimes a spark are used. Spectrographs are of the laboratory facilities. Of particular interest to divided into two general groups on the basis of the the chemistry teachers were the analytical laboratories means used to separate the emitted light: the prismatic and the instruments in use for analytical purposes. and the grating instruments. The A. R. L. Dietert Just prior to the tour, Dr. Harry J. Fisher, Chemist in instrument a t the Experiment Station is of the grating Charge, Department of Analytical Chemistry, de- type; it contains a concave piece of metal ruled with scribed many of the pieces of equipment that would be 15,000 lines to the inch. This grating separates the seen in use. The following is an abstract of the manu- emitted light, after it has passed through a slit, into a script from 11-hichhe spoke. pattern determined by the wave lengths present. The The Rejractometer. This is the simplest instmment positions of the lines on the film,therefore, show what used in the laboratory outside of the balance. When a elements were present in the sample, and the intensiray of light strikes a transparent object a t an angle, it ties of the characteristic lines indicate how much was is bent in passing through the object a t an angle that present. The spectrograph can rapidly provide proof depends on the relative densities of the object and the of the presence of most elements and highly accurate air. By measuring the amount of this bending or re- quantitative determinations of elements in mixtures fraction the density of the object can be determined. can be made under certain conditions. The spectroRefractometers are prismatic instruments for measnr- graph can be used to detect and determine any metallic ing the angle of refraction of light in passing through element and a few nonmetallic elements, and does not liquids. By such measurements the concentration of distinguish between compounds of the same element. an aqueous solution of one compound may be deterAt the Experiment Station thousand of analyses of mined, for instance, or one vegetable oil may be dis- plant materials and biological tissues have been made. tinguished from another. The Experiment Station has Accurate quantitative determinations of Ca, Mg, K, three different refractometers+n AbbB, a Zeiss butter Na, P, Fe, Al, Mn, Cu, Zn, and B are made sirnulrefractometer, and an immersion or dipping refractom- taneously on pasture grasses, tobacco, and similar maeter. The first two instruments are used chiefly for terials, and toxicological specimens are tested for Pb, identifying oils, determining the proportions of two TI, Zn, As, Sb, Hg, Ag, and other elements. The savoils in a mixture, and for determining the moisture con- ing in time over chemical methods is enormous. tent of simps; among the uses of the immersion reVisible and Ultraviolet Absorption Spectrophotometers. fractometer (which dips into a solution in a beaker) The spectrograph measures emitted light, and deterare the determinations of alcohol and of sugar in bever- mines elements only; other instruments measure absorbed light, and may be used to determine compounds ages. The Emission Spectrograph. When materials are as well as elements. Energy in the form of light may heated to a su5ciently high temperature, some of the be absorbed by molecules in various ways, involving energy absorbed is re-emitted as light. Some of this electronic, vibrational, and rotational motions. Comlight is in the visible spectrum but much of it is in the plicated molecules undergo very complex vibrations and ultraviolet. The light emitted is not in the form of a each of these is affected by light of certain specific wave continuous spectrum but is a series of distinct limes. lengths. Visibly colored materials are colored because they This series of limes is a unique characteristic of the element, and is given by no other element. The spectro- absorb light of some wave lengths from incident white graph consists of a means of exciting (energiaing) ele- light and let light of the other wave lengths pass ments, an optical train to separate the light emitted through; in the same way, if some material is exposed according to wave lengths, and a means of recording to a source of ultraviolet light yielding a continuous and measuring the lines of different wave lengths. spectrum it will absorb certain wave lengths, more or
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less characteristic of the material. Consequently, measurement of the decrease in transmission of light a t those wave lengths will afford a means of both detecting and determining that material. The spectrophotometer used a t the Experiment Station is the Beckman. It is a prismatic instrnment with quartz optics that provides a choice of two sources of light, a tungsten bulb for the visible and a hydrogen lamp for the ultraviolet. At each wave length the amount of light transmitted by the sample is measured electrically by a photoelectric cell to give the absorption curve. The spectrophotometer is used for a wide variety of determinations; two examples are the determination of sulfa drugs in poultry feeds and the determination of vitamin A in fish oils. In the first example, the sulfa drug is diazotized and coupled with an amine and the intensity of color of the resulting dye solution is measured. In the second case, the oil is saponified, the unsaponifiable matter is separated, dissolved in alcohol, and the absorption in the ultraviolet is determined a t a specific wave length (325 m*). Both of these examples illustrate the fact that usually in analysis, using the visible-ultraviolet spectrophotometer, the material being analyzed is not examined directly but first is subjected to various chemical manipulations to produce a solution suitable for the quantitative estimation of a component. Some materials can be analyzed by direct absorption measurements: For instance, the concentration of a solution of potassium chromate or potassium permanganate can be determined directly by measuring its absorption a t the right wave length. Infrared Spectrophotometers. The principle of construction and use of infrared spectrophotometers is identical with that of the visible type of instrument, but since glass and quartz are very nearly opaque to infrared light, crystallme salts, most of which are water soluble, are used instead. Sodium chloride crystals are transparent to infrared light of wave lengths up to about 15 microns, and potassium bromide, up to 25 microns; for longer wave lengths a mixed thallium bromide-iodide crystal must be used. Prismatic instruments of satisfactory optics would have been very difficult to construct were it not for the fact that very large artificial crystals of sodium chloride and other salts are now grown on a commercial scale. It is obvious that aqueous solutions cannot be examined in cells the windows of which are plates of a soluble salt; moreover, water itself bas such a complicated absorption pattern that it masks the absorption of the solute. Infrared readings on solids are, therefore, made either on their solutions in organic solvents such as carbon disulfide or carbon tetrachloride that possess little absorption a t the points being measured, or else they are made using ground suspension ("mulls") in Nujol. Liquids and gases may be measured directly. To provide a continuous spectrum of infrared light a heated, silver-coated Carborundum rod k n o m as a "Globar." or an oxide rod known as a "Nernst dower!' is used; 'and the transmitted light is measured Gther by
a sensitive thermocouple or a thermopile. Plotting the many rea&mgs necessary to obtain an absorption curve of a compound in the infrared is very laborious, and for this reason most of the modern instruments are of the recording type that automatically plot the curve on a sheet of paper on a rotating drum. Some instruments of the double-beam type draw curves directly in terms of percentage transmission. The Perkin-Elmer instrument used a t the Experiment Station is a recording instrument, but the drawn cunves must be recalculated to yield percentage transmission curves. The great advantages of absorption measurements in the infrared are three: (1) Every organic compound yields an absorption pattern that as a whole is characteristic of that compound only; (2) groups within compounds, such as carbonyl, carhoxyl, hydroxyl, amino, etc., show more or less specific absorption a t certain regions, and an infrared curve will therefore indicate whether an unknown compound is an amine or a ketone or an acid (or a ketone alcohol), or other structural types; (3) if two different compounds are present the absorption curve of the mixture is strictly (or nearly) the additive resultant of the curves of the individual compounds, and therefore both compounds can be identified and their relative amounts can be determined. Mixtures of organic compounds that are so similar in structure as to defy analysis by ordinary chemical means may be analyzed by means of the infrared spectrophotometer. One example of this is the mixture of hexachlorocyclohexanes used as an insecticide under the name of "benzene hexachloride." "Benzene hexachloride" contains a t least five different geometrical isomers whose ordinary chemical properties are almost identical; one of these, the so-called gamma isomer, is, however, much more effective as an insecticide than any of the others, and i t is, therefore, commercially important to know the percentage of the gamma isomer in various "benzene hexachlorides." This can easily be done by infrared absorption measurements, because each isomer shows absorption a t certain points that is characteristic of that isomer only. Fluorometers. Absorption spectra result from electrons being forced from their normal levels in the atom to higher levels; the return of the electron to its normal level may result in emission spectra or in the radiation of the absorbed energy in the form of heat (infrared) or other radiation. When the light produced is within the visible region of the spectrum, we have the phenomenon commonly known as Jluorescence. Fluorescent substances on exposure to ultraviolet light emit visible light-that is, they appear to glow. This property is of considerable value from the analytical standpoint just because the number of strongly fluorescent compounds is limited; if a compound is fluorescent, the measurement of the fluorescence of a solution containing it offers a sensitive means of determining its concentration. In principle, a fluorometer consists of a device for
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illuminating a solution with a source of ultraviolet light and for measuring, usually a t right angles to the direction of illumination, the light re-emitted by the solution. The instruments on the market measure the intensity of the fluorescence with photoelectric cells; such instruments employ glass filters to eliminate any effect on the photoelectric cell of stray light from the ultraviolet source. The fluorometer used a t the Experiment Station is made by the Coleman Instrument Company. I t finds its greatest use in the determination of two vitaminsthiamine and riboflavin; riboflavin is naturally fluorescent, and thiamine on oxidation yields a fluorescent compound known as "thiochrome." Quinine can equally well be determined fluorometrically. Polarimeters. Organic compounds having four different groups attached to the same carbon atom are "optically active." Such compounds exist in two isomeric forms that are mirror images of each other. When a beam of plane-polarized light-light vibrating in one plane only-is passed through a solution of one of these isomers, the beam is rotated about its axis. One isomer will rotate the beam in a clockwise direction and the other will rotate it counterclockwise. A polarimeter consists of a device for producing plane-polarized light, a tube to hold solutions through which the polarized light passes, and a scale for measuring the angle through which the light has been turned in passing through the tube. The polarimeter in use a t the Experiment Station was made by 0. C. Rudolph. I t is accurate to 0.002' of arc. The most important use to which it is put is the determination of concentrations of sugars. Natural sugars are optically active, and comparatively few other water-soluble compounds occurring in plant products are. The rotations of different sugars change a t different rates with changes of temperature, so to determine one sugar in the presence of another, the rotations of a solution are measured a t both 20" and 87'C. This principle is made use of to
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detect the adulteration of honey with glucose. X - R a y Diff~actionApparatus. X-rays are produced by the bombardment of a metal target by electrons. These electrons are generated in a vacuum tube, and the wave length of the X-rays formed depend on the element of which the target (usually molybdenum or copper) is composed. That is, X-rays are emitted from the tube not as a continuous spectrum but as discrete lines of wave lengths characteristic of the target element. X-ray spectrophotometers, that is, instruments %or measuring the absorption of X-rays by substances, have analytical uses. They are not common, however, because the information they supply can be obtained more easily with other instruments. The instrumkut that is finding considerable use as an analytical tool is not properly a spectrometer-that is, it does not measure an X-ray spectrum-but is an X-ray diffraction apparatus. It depends on the principle that when a beam of X-rays strikes a crystal the various monomolecular layers within the crystal act essentially like lines of a diffraction grating to reflect-diffract-the beam a t an angle that is a function of the interatomic distances in the crystal. Various devices are used for recordmg this diffraction. The Soils Department of the Experiment Station uses a Norelco instrument which is equipped with a Geiger-Muller counter t o measure the intensity of the diffracted beam a t various angles and automatically records the "crystal pattern." Since X-ray diffraction analysis is crystal measurement, only crystalline materials can be analyzed. The Norelco instrument, thus, determines compounds, not elements, and therefore readily distinguishes, for instance, a mixture of tricalcium phosphate and strontium sulfate from a mixture of calcium sulfate and tristrontium phosphate. The Soils Laboratory uses the instrument to identify and determine components of soils in connection with The Soil Survey.
