V O L U M E 2 8 , NO. 1 2 , D E C E M B E R 1 9 5 6 Ibid., 28, 328 (1956). Ells, 5’. R., J . O p t . SOC.Amer. 31, 534 (1941). Ells, V. R., Xarshall, C. E., PTOC.Soil Sci. SOC.Am. 4, 131
(1939). Feldman, C., ANAL.CHEX 21, 1041 (1949). Fox, C. L., Jr., Freeman, E. B., Lasker, S. E., Ani. Soc. Testing Materials, Tech. Pub. 116, 13 (1951). Fred, ll.,Xachtrieb, 3’. A . , Tomkins, F. S.,J . Opt. SOC.Amer. 37, 279 (1947). Gilbert, P. T., Jr., Ind. Laboratories 3, 41, (1952). Gilbert, P. T., J r . , Hawes, R. C., Beckman, -1.O., ANAL. CHEX.22, 7 7 2 (1950). Gilliland, J. L., =Im. Soc. Testing Materials, Tech. Pub. 116, 33 (1951). Griggs, A I . .1.,Johnstin, R., Elledge, B. E., ISD. ENG.CHEM., AXAL.ED. 13, 99 (1941). Heyes, J., Angew. Chem. 50, 871 (1937). Heyes, J., others, 2 . physik. Chem. A174, 291 (1935). Honma, 31., Smith, C. L., ANAL.CHEM.26, 458 (1954). King, W ,H., Jr., Priestley, W., Jr., .im. Soc. Testing Materials, Tech. Pub. 116, 97 (1951). Kingsley, G. R., Schaffert, R. R., ANAL.CHEM.25, 1738 (1953). Kuemmel, D. I?., Karl, H. L., Ibid., 26, 386 (1954). Laidler, K. J.,Shuler, K. E., Chem. Rem. 48, 154 (1951). Lohse, H . W.,Can. J . Research 12, 519 (1935). Luke, C. L., Campbell, 11. E., -4x.4~.CHEM.26, 1778 (1954). Lundegardh, H., “Quantitative Spektralanalyse der Elemente,” Vol. I and 11. Gustav Fischer, Jena, 1929, 1954. AIcClelland, J. A. C . , Whalley, H. K., J . SOC.Chem. Ind. 60, 288 (1941). llalmstadt, H. V., Schols, R. G., AXAL.CHEM.27, 881 (1955). llargoshes, M., Vallee, B. L., Ibid., 28, 180 (1956).
1847 (40) Nargoshes, AI., Vallee, B. L., “Methods of Biochemical Analysis,’’ David Glick, ed., Vol. 111. Interscience, S e w York,
1956. (41) Mavrodineanu, R., A p p l . Spectroscopy 10, No. 2 , 50; Y o . 3. 137 (1956). (42) Mavrodineanu, R., Boiteux, H., “Analyse Spectrale Quantitative par la Flamme,” Masson et Cie, Paris, 1984. (43) Meloche, V. W., Am. Soc. Testing Materials, Tech. Pub. 116, 3 (1951). (44) XIelochi, V. W., Ramsay, J. B., Mack, D. J., Philip, T. T. AXAL.CHEM.26, 1387 (1954). (45) llitchell, R. L., Robertson, I. M , , J . SOC.Chem. Ind. 55, 269T (1936). (46) Nosher, R. E., Bird, E. J., Boyle, A. J., AN.AL.CHEV.22, 715 (1950).
(47) Rlosher, R. E., Boyle, A. J., Bird, E. J., Jacobson, 9. D., Batchelor, T. h l . , Iseri, L. T., Myers, G. B., Am. J . Clin. Pathol. 19, 461 (1949). (48) Pagliassotti, J. P., Porsche, F. W., Ibid., 23, 198 (1951). (49) Porter, P., Wyld, G., Ibid., 27, 733 (1955). (50) Rothermel, D. L., Am. SOC. Testing llaterials, Tech. P u t . 116, 117 (1951). (51) Schrenk, W.G., Glendening, B. L., ; ~ N A L .CHEM.27, 1031 (1955). (52) Schuhknecht, W., Angew. Chem. 50, 299 (1937). (53) Sevringhaus, J. W.,Ferrabee, H. W., J . B i d . Chem. 187, 621 ( 1950). (54) Sloviter, H. A , , Sitkin, A , , J . O p t . SOC.Amer. 34, 400 (1934). Tennent, C. B., Ax.4~.CHEM.28, 858 (1956). (55) Standen, G. W., (56) Todd, H. E., Tramutt, H. >I., I b i d . , 26, 1137 (1954). (57) Vallee, B. L., llargoshes, AI., I b i d . , 28, 175 (1956). (58) West, P. W., Folse, P., Montgomery, D. I h i d . . 22, 667, (1950). RECEIVED for review July 9, 1956. Accepted September 4, 1956
Ninth Annoal Summer Symposinm-Rapid Methods of Analysis
Instrumental Methods of Gamma-Ray Spectrometry R. E. CONNALLY General Electric Co., HanFord Atomic Products Operation, Richland, Wash.
The gamma-ray spectrometer and its application to the rapid analysis of mixtures of gamma-emitting radioisotopes are discussed. The parameters of importance in the selection of the gamma-sensing unit are shown. The pulse height analysis systems commercially available for use in gamma-ray spectrometry are evaluated regarding their use as both qualitative and quantitative analytical instruments. The precision to be expected with each type of system is also discussed.
T
HE gamma scintillation spectrometer has found wide ac-
ceptance in the field of radiochemistry in the last 3 years. I t s major advantages in the analysis of gamma-emitting radioisotopes are the speed, specificity, and simplicity of the method
(5). DISCUSSION
T h e gamma scintillation spectrometer is used to analyze the gamma spectra of radioisotopes in a manner similar to the analysis of the emission spectra of elements with the emission spectrograph. T h e sensing unit required for the detection of the emitted gamma radiation is the thalium-activated sodium iodide crystal. h typical gamma spectrometer sensing element is shown in Figure 1. T h e gamma photons from the sample, penetrating the crystal, have a high probability of interacting with the crystal. This interaction results in the conversion of a single gamma photon, via a recoil electron, to a shower of light photons which are detected by one of several types of multiplier phototubes (such as the Duniont 6292 shoffn).
T h e current output from the multiplier phototube anode is directly proportional to the energy lost by the incident gamma photon. These current pulses are fed into a n amplifier of sufficient gain to produce voltage output pulses in the amplitude, range of 0 to 100 volts. T h e oscilloscope photograph in Figure 2,b, is a time exposure of the amplifier out.put pulses resulting from multiple cesium-barium-137 gamma interactions with the scintillation sensing element. One method of analysis of the pulse spectra is by use of a single-channel pulse-height analyzer which plots the curve shown in Figure 2,a. T h e analyzer slowly scans the pulse distribution over a 0- to 100-volt range with a n acceptance slit a few volts in width. Only those pulses falling within the acceptance slit are passed on to a rate meter and plotted. For example, when the slit is a t 75 volts, there are few pulses of this amplitude in the pulse spectrum, as shown on the photograph in Figure 2,b; thus, only a few pulses pass through the acceptance slit to the rate meter and recorder. 8 s the slit sweeps on t o the 65-volt region, there are a large number of pulses which are of proper amplitude to pass the accept,ance slit and be recorded. As the acceptance slit completes its scan down to zero energy, the complete pulse height distribut’ion is plotted. This same differential curve has been obtained by vertically scanning the face of the oscilloscope with a recording densitometer ( 7 ) . T h e curve of pulse height distribution, known as the differential scan, of a monoenergetic gamma emitter can be divided into two characteristic curves (Figure 2,a). T h e sharp symmetrical peak is the result of total absorption of the gamma energy by the X a I ( T1) crystal, and is normally referred to as the full-energ:. peak. T h e continuous curve below t’he full-energy peak is due to Compton interaction, wherein the gamma photon loses only
ANALYTICAL CHEMISTRY
1848 part of its energy to the crystd. The loeat.ion of the full-energy peak on the pulse amplitude or gammsrenergy axis is proportional to the gamma energy of the incident photon, and is the basis for the qualitative application of the gamma scintillation spectrometer. The area under the full-energv peak is related to the number of gamma photons interacting with the crystal, and is the basis for the quantitative application of the gamma scintillation spectrometer. The Compton continuum serves no useful purpose in full-energy peak analysis, and must be corrected for when a mixture of gamma emitters is analyzed.
RECOIL ELECTRON LIGHT PHOTON
ELECTRON PbTH FOCUSlNB SHIELD
Figure 1. Diagram of g a m m a detector
Selection of Sensing Unit. As the area under the full-energy peak is the s m w e of quantitative information and the Compton continuum is only a background which requires correction, i t is desirable to increase the ratio of full-energy events to Compton events. This can be accomplished by increasing the size of t h e crystal, which increases the probability t h a t a Compton-scattered gamma photon will lose ill1 of its energy to the crystal by multiple interactions. Until recently the crystal l a / , inches in diameter and 2 inches thick w i t s the maximum practical size, because multiplier phototubes of larger diameter were not available. Today there are 3- and Qnth multiplier phototubes available for this application. The 3 by 3-inch NaI(T1) crystal operated with B 3-inch or &inch multiplier phototube is becoming a popular choice for the sensing element. With larger crystals the background increases a t a greater rate than the signal. However, in most applications the improvement in full-energy peak to Compton ratio is worth the increase in background. Far quantitative applications i t is desirable to have a calibration curve relating full-energy peak area. to gamma. activity. Such a curve can be obtained by using isotopes of known decay scheme. Each isotope is initially calibrated by absolute beta counting or other radiochemical methods, rtnd then used to establish a point on the gamma. detector efficiency curve. However, the number of possible calibration points is quite limited. Litzer and coworkers hrrve recently applied a more general technique wherein any gamma source emitting one or two major gamma rays can be used for calibration (9). R, the ratio of mea. under the full-energy peak to the area under the total spectrum, is determined in a n experiment in which scattering is negligible. et, the probability of interaction by the incident gamma photons for the geometry used in the experiment, is calculated. From these data the intrinsic peak efficiency is obtained as shown in Figure 3. T h e calibration curve does not include the geometry factor (solid angle), which must be taken into consideration when relating full-energy peak area to gramma. activity. Berger and Doggett have calculated R, which they call photofraction (3). However their calculated values are somewhat higher than experimentally determined values. The use of 3- rtnd 4 i n e h crystals with re-entrant well can increase the counting signal to background ratio by a factor of 3 or 4. The presence of a well in the larger crystals has the effect of
Y
(n
6
10.1
COUNTS
PER
Figure 2. 0.
SECOND
PER
SLIT
WIDTH
Ib.1
TIME
IN
MICROSECONDS
Two methods of pulse spectrum display for cesium-barium-137
(Lelt) differential
b. (Right) oheilloseope p ~ t i e r n
V O L U M E 2 8 , NO. 12, D E C E M B E R 1 9 5 6
1849
slightly decreasing resolution. The 4J/4-inoh orystal (Figure 3), in add;ltion to having a well, was operated in anticoincidence with a guard crystal surrounding the detector crystal. Such a n anticoincidence system substantially reduces the Compton continuum and background oounting rst.e ( I ) . Preparationof Sample. For qualitative applications the reproducibility of the Compton continuum region is of negligible importance, and thus the size, phase, and iocation of the sample
Figure 4..
1.0
8
2
Gamma-ray tl'ometer
3
4
ergy-(M.ev.) energy for sodium iodide orystals r f l (9)
x
6
spee-
8
IO
ANALYTICAL CHEMISTRY
1850
following set of rules (10) to be used in designing a low-Conipton system: 1. Maximize distance between crystal and shield. ' 2. Maximize Z (atomic number) of all scatterers. Photoelectric cross Eection varies as 2*. Compton cross section varies as Z. 3. hIaximize scattering angle betn-een source, scatterer, and crystal.
where E1
E
Figure 5.
= =
energy of scattered photon in m.e.v. energy of incident photon in m.e.v.
4. Remove all unnecessary material from between source and crystal. Beta absorber, when required, should be of low Z material. il typical solution to the low-Compton shielding problem results in a crystal, phototube, and light shield assembly of minimum mass, low 2 material. T h e shielding is usually 4 inches of lead or mercury located 5 inches or more from the crystal and enclosing the crystal and phototube on the sides and bottom. For low background work the shielding extends up and around the sample (Figure 5 ) . T h e gamma photons emitted from the sample generate characteristic x-rays in the high Z shield, resulting in a spurious x-ray peak in the gamma energy scan. This x-ray peak can be reduced by a factor of a t least 100 by the use of a graded inner shield consisting of ' / l a inch of cadmium or tin lined with 15 mils of copper ( 2 ) . Qualitative Analysis. T h e various gamma emitters are identified from the gamma scan by the location of their full-energy peaks on the energy axis, according to the curves shown in Figure 6. Where two full-energy peaks do not differ by sufficient magnitude to be resolved, or where a full-energy peak is hardly detect-
Typical low-Compton gamma detector system L
T[ SI
!I 9-11
-
RU I03 Cr -51
A R E A = 1.07 p x h
Counts Per Minute Per Acceptance Slit Width
x 10-3
Figure 6.
Typical three-component gamma spectrum
V O L U M E 28, NO. 12, D E C E M B E R 1 9 5 6
1851
able above t1:c c’unii~to~i c.ontinuum of t,he more energetic gamma version of the same instrument is the single-channel stepping type, photons, preliminary clieniical separations n-ill be required. wherein the entire spectrum is analyzed in 1- or 2-volt steps. For low activity samples, more precise d a t a can be obtained in a Quantitative Analysis. T h e net area under each photopeak is directly proportional to the absolute gamma emission rate of the given s m e p time. However, the analysis of d a t a is usually corresponding isotope. This is usually determined by taking the more complex t h a n with the continuous scanning type. MELTIPEAK ANALYZERS.A useful multichannel analyzer for product of peak height, width a t half maximum, and the normalizing factor, 1.07-based on the assumpt,iori that the fullthe analytical laboratory is the multipeak analyzer. With this energy peak is Gaussian ( 6 ) . instrument the boundary of each channel can be adjusted t o enFor the analysis of mixed gamma-emitting radioisotopes, howcompass the full-energy peaks of the isotopes of interest (Figure ever, the characteristic lower energy curve of the most energetic 8). T h e total counts in each channel are stored on separate full-energy peak must be drawn in from previously recorded scalers and the Compton cont’inuumcan be s u b t r a c k d by use of a standard curves detailing t,he Compton continuum region us. Compton continuum table and a n adding machine; graphical principal full-energy peak area, as shoTT-n in Figure 6. By subsolution is not necessary. The d a t a from such a system are adapttraction, the net height of the second most energetic gamma able to analog computation methods, in which the readout would photon can be established and it.s 1%-idthmeasured. T h e Compbe directly in units of full-energy peak counting rates corrected ton continuum of the second full-energy peak is then drawn in for Compton continuum interference. and the area of t,he third peak can be evaluated, etc. ( I n a few T h e multipeak analyzer is most effective in the routine analysis cases a radioisotope may have a minor full-energy peak more of five or less known gamma peaks. When unknown gamma energetic than its associated principal peak. This situation is emitters must be searched for and ident,ified, as in activation normally solved by solution of simultaneous equations or by analysis (Is),the five-peak analyzer is of little added value over successive approximation. ) the single-channel analyzer. I n the analysis of gamma photons in the energy range from ~ICLTICHANNEL A S ~ L Y Z E R ST. h e mult.ichanne1 analyzers 32 to 100 k.e.v., there appears to be a n escape peak 28 k.e.v. differ from the multipeak analyzers in that all channels below the full-energy peak. This is due to photoelect,ric int.erare adjacent, but can be moved as a group to any region actions wherein the resulting iodine K x-ray escapes from the in the spectrum. Instrument,s with 20 channels are availcrystal. Above 1.5 m.e.v. the effect of pair production becomes abie, but must be moved five or six times to cover a 0.1- to apparent in the gamma scan. I n addition to the full-energy 1-m.e.v. gamma energy spectrum r i t h good resolution. To be peak, there are peaks in the scan a t full-energy minus 0.51 m.e.v. used in qualitative analysis, 100 to 120 points must be plotted and full-energy minus 1.02 m.e.v. A41soa small peak occurs a t out. in analog form for final analysis. Although it is more com0.51 m.e.v. due to detect,ion of an annihilation photon resulting plex, the 20-channel analyzer has a real advantage over the singlefrom pair production in the shielding material ( 2 ) . channel type for short half life, low counting rate, and particle It follows that the precision of measurement of each peak accelerator applications, as it can accumulate information 20 becomes less as successive Compton continuum corrections have times as fast. to be made. I n pract’ice the five-component mixture is about the T h e multichannel analyzers of the 100- and 256-channel types maximum which can be analyzed without, the assistance of radioall have provision for automatic analog readout. T h e resulting active decay or chemical separation. curve can be solved graphically to obtain qualitative dat,a. Finally, the full-energy peak yield curve, sample geometry, decay scheme data, half life, and atomic weight may be applied to carry the results to a weight basis. However, in many applications, such as in measuring the yield of a separation step, the gamma scan d a t a can be retained on a total full-energy peak REG U L AT E 0 count basis. P U L S E GENERATOR -1unique met,hod of evaluating the full-energy peak wit.hout reference to Compton cont.inuum is by graphically determining the point a t n-hich the characteristic width rho occurs, and by GAMMA LINEAR integrating the peak area above this point with a planimeter (11). c This method is advantageous where very large Compton correcDETECTOR AMPLIFIER tions must be made, but, it must be limited to situations where the gross Compton cont,inuum region is rea-onably flat. T h e method requires recalibration if system resolut,ion should ehange. SINGLE-CHANNEL Pulse Analysis Systems. SISGLE-CHISSEL .ISALYZERS. The DISCRIMINATOR P U L S E HEIGHT typical block diagram of a single-channel gamma s;wtrometer DRIVE ANALYZER is shown in Figure 7 . T h e gamma detector, utilizing a 10-stage multiplier phototube, is vapahle of driving commercially available linear amplifiers via pulse cables 10 to 250 feet in length. This makes it possible to operate the gamma detector at remote SCALER D I SC R I M I NA T 0 R lorations where nercssary ( 4 ) . T h e regulated high voltage supply DRIVE is usually a 1-ma., 500- to 1500-volt model Tvith 0.01 % line regiilation and 0.1 % stability per 21 hours. LINEAR T h e amplifier drives the pulse height analyzer, which has a COUNTING discriminat,or level that can be adjusted continuously or stcpwise in order to cover the pulse height spectrum of intere.:t. T h e counting rate per acceptance slit viidth can be rend on the scaler or recorded on the count rate meter-recorder system. K h c n STRIP C H A R T the discriminator level is driven continuously, the recording tern usually consists of a linear count. rate meter driving a strip RECORDER chart recorder. T h e resulting curve is generally plotted automatically over a period of from 15 minutes to 2l/? hours, dcpendFigure 7 . Block diagram of single-channel gamma ing on sample counting rate and required precision. Another scintillation spectrometer
+ c
‘1
--I
I
4
I
~
~
I
ANALYTICAL CHEMISTRY
1852 Here again, the major advantage over the single-channel instrument is speed of analysis-that is, 1 to 5 minutes counting time compared to 30 to 150 minutes. Precision. The precision of a gamma spectrometer s l stem ie less than might be calculated from the counting statistics alone. A drift in high voltage, multiplier phototube gain, amplifier gain, and channel level will all have their degrading effect on precision of multiple determinations over an 8-hour period. I n general, the stability of a 0.1 % per 24-hour high voltage supply IS in the same order as that of the well-designed amplifier analyzer system, and the gain of the multiplier phototube ( 2 2 ) . The per cent standard deviation to be expected from the overall system for multiple determinations of the major constituent is shown in Table I. The precision for minor constituents n ill vary from about 5% to undetectable, depending upon the number of successive Compton corrections and the relative magnitude of the constituents. The accuracy is seldom greater than within 5%, which is the accuracy to which most calibration curves of full-energy peak 2s. gamma energy can be determined.
spectrometer. And finally, decay scheme studies and other nuclear data determinations have been facilitated by use of the gamma-ray spectrometer. SUSl3IARI
I n the analysis of radioisot,ope gamma spectra the sodium iodide cryst,al, activated TTith thaliuni, 113s gained prominence. The trend has been tonard increasing crystal dimensions in order to yield more nearly pure line pulse spectra. This improvement is further increased by removing the shielding material from t.he immediate vicinitj- of the crj 1. Radioisotope mixtures containing as many as five gamma-emitting isotopes can be determined qualitativelj-, and in many cases, quanthatively, The major constituent of a radioisotope mixture can readily be determined to a precision within 5y0 (standard deviation), and in special cases the precision has been improved to vithin 1%. The pulse analysis systems available for use in gamma-ray spectrometry vary from simple single-rhannel instruments to more comples 256-chnnnel tems, designed for high data accumula-
4PPLICATIO3S
Among the applications, the following can be mentioned. The determination of trace elements in semiconductor materials has been facilitated (fs),and the analysis of mixed fission products has been simplified (6). Selected points in a spent nuclear fuel solvent extraction plant have been monitored for specific isotopes ( 4 ) . A spectrometric method has been developed a- a rapid means of determining neptunium-239, molybdenum-99, and cerium-141 in gross fission products ( 1 1 ) . Radioisotopes shipped from Oak Ridge are checked for purity by use of a scintillation spectrometer (8). Localization of iodine-131 is greatly facilitated bj- elimination of the Compton scattering n i t h a scintillation
Table I. Average Precision Obtainable with Several Types of Gamma Spectrometer Pulse .Analysis Systems
Tj-pe of System Single channel, continuous sweep t j p e with count rate meter Single channel. step scanning n-itli scaler Multipeak with scalers ,\Iultichannrl
Counts Per Minute Per Acceptanc Sllt Width
x
10-3
Gamma
Figure 8.
Energy
-
( M.e.v.)
Typical three-peak analysis
Precision Reported for Major Constituents, 'Literature 7c Reference 5 2 1 1
(6) (11)
(12)
1853
V O L U M E 28, NO. 12, D E C E M B E R 1 9 5 6 (6) Crouthamel, C. E., Johnson, C. E., U.
tion rates. T h e application of the method continues to increase as complete gamma spectrometer systems are becoming commercially available and specific applications appear more frequently in the literature.
(7) (8)
(9)
(10) LITERATURE CITED
(11) (1) AAIbert,€1. D., Rcc. Sci. Instr. 24, 1096-101 (1953). (2) Bell, P. R., "Beta and Gamma Ray Spectroscopy," li. Sieghahn. ed.. Interscience. S e w York. 1955. (3) Berger, .\I. J., Doggett, J., Ret. Sci. Iiiistr. 27, 269-io (1956). (4) Connally, R. E., I . R . E . Trans. NS-3, T o . 2 . 28-31 (1956). (5) Connally, 11, E., Leboeuf, .\I. B., .kS.kL. CHEM. 25, 1095-100 (1953).
(12) (13)
S.Atomic Energy ('ommission, ANL-4924 (1952). Hine, G. J., .VucZeonics 1 1 , S o . 10, 68-9 (1953). Kahn, B.. Lyon, W. S., Ibid., 1 1 , No. 11, 61-3 (1953). Lazar, S . H., Davis, R. C., Bell, P. R., Ibid., 14, No. 4, 52-3 (1956). Lefevre, H. IT., private communication, General Electric Co., Richland, Wash. McIsaac, L-. D., V. S.Naval Radiological Defense Laboratory, TR-72 (1956). Miller, D . G., U. 8. Atomic Energy Commission, HW-39969 (1956). Morrison, G. H., Cosgrove, J. F., -&x.IL. CHEU. 27, 810-13 (1955).
R E C E I V E for D re\,iew July 10, 1956.
.Iccepted Septeinber 2 2 , 1930.
Ninth Annual Sommer Symposium-Analytical Problems in Biological Systems
Paper Chromatography in Steroid Determination LESTER M. REINEKE Research Laboratories, The Upjohn Co., Kalamazoo,
Mich.
Paper chromatograph? has been applied to the identification and quantitatite determination of steroids obtained from microbiological and chemical transformations. 4 clear indication of the probable structure is obtained b? ultraliolet absorption, chemical tests, and mobilit? (as compared to various known steroids) in a variet? of sollent s?stems of both the Bush and Zaffaroni t?pes. 4 quantitatile procedure using light absorption at 224 and 242 mp has been deleloped for progesterone and lla-hjdrox?progesterone. This procedure eliminates the necessit? for running blanks and steroid standards with each determination.
T
HE technique of paper chromatograph? is discussed here,
as applied to steroidal transformations induced microbiologicalli or chemically. The compounds considered are more polar than the sterols. I n these laboratories, paper chromatography is used only for identification or determination of purity, but the infoimation is also helpful in proof of structure. \T-hen Zaffaroni, Burton, and Keutman (31) reported the formamide-benzene and propylene glycol-toluene systems, the first practical method of applying paper chromatography to steroids brcnme available. [ I n this discussion, any solvent system (1-4, 1 1 , 16-17, 21, 23-25, 27, 28, 30) using a paper impregnated n i t h a high boiling polar solvent as the stationary phase and a nonpolar solvent saturated n i t h the stationarv phase as the mobile phase is referred to as the Zaffaroni type.] Later, Bush (6)used a series of s~ stems in n hich the stationary phase, water and methanol, is preferentially adsorbed onto the paper from the vapor of both phases during equilibration. The mobile phase, consisting of various nonpolar solvents, is used for development. With the Bush systems, a slightly elevated temperature is usually desirable. [Any solvent s>stem of similar nature (6, 8,14, 18, 19) is referred to here as of the Bush type.] Various systems for paper chromatography of steroids, other than sterols (5, 9, 13, 22, 26, 29), have been published but, in general, they are one of the above two types COMPARISOY OF DEVELOPRlEhT S Y S T E M S
1Iuch larger quantities of steroid can be applied to the Zaffaroni type of chromatogram than can satisfactorily be applied to the Bush type. The development time of the former is generally longer, but in the Znffaroni type of system resolution of steroids having ultraviolet absorption can be conveniently followed by
removing the strip from the chamber for ult'raviolet' scanning. The sheet may then be dipped t'hrough the mobile phase and the development continued. This is not possible with the BiiPh type of system. I n order to speed up and sharpen the resolution, it is a common practice, first reported by Zaffaroni (28), to reduce the amount of stationary phase by dipping the filter paper through the stationary phase diluted with a volatile solvent such as methanol, The diluent is then evaporated from the paper chromatogram before the development is started. T n o modifications of Zaffaroni's procedures (28, 31) have been made. (1) S o wick is used to saturat'e the battery jar; the nonpolar solvents are sufficiently volatile to saturate the chamber rapidly. (2) Channeled sheets are not used, because they present greater manipulation problems, particularly when a sheet may be removed from the bath several times for observation. The slight sideways diffusion of the steroidal material is useful in the separation of a small quantity of material from a larger amount of a closely moving material. If confined to the narrotv channel, the "edge effect" can obliterate the space between compounds of similar mobility. The mobilities on these systems are expressed as a ratio to a standard steroid, Rs, because the solvent front has often moved off the chromatogram before sufficient' resolution has been ob-
Table I.
LiteraSystem Symbol Cll CNF PT PTF K-1
ture
Reference
Zaffaroni-Type Solvent Systems0
Dried at 370 C., Min.
Phase Mobile Stationary (16) Methylcyclohexane Carbitol b .. hlethylcyclohexane Carhitol" 10 (3;) Toluene Propylene glycol , . (28) Toluened Propvlene glycolc 10 (1I ) CyclohexanePropklene glycolc 10 benzene (1 : 1) FBF (28) Benzene FormamideC 15 .. Skellysolve B e CFS Carbitol15 formamidef a Best grade of solvents commercially available is used. Only formamide is redistilled. Room temperature, 2 5 O ?C 2O C. * Diethylene glycol monoethyl ether. Diluted with methanol (1 : 1). Methanol evaporates during indicated drying time. d For materials such as 3-ketohisnor-4-cholenic acid, 27, glacial acetic acid can he added to reduce streakiness. e Essentially a normal hexane, b.p. 60-70' C,. f Two parts of Carbitol-formamide (1:l) dlluted with 1 part of methanol.
'
