Fluoroscope and Geiger Counter for Measurin Ultraviolet Absorption of Chromatograms T. D. PRICE and P. B. HUDSON Departments of Biochemistry and Urology and Institute of Cancer Rere Columbia University College of Physicians and Surgeons, New York 32,,
In search for new criteria for radiochemioal purity of labeled nucleotides, it was necessary to determine aocurately distribution of the compounds on paper ehromatograms. Measurement of light absorption in the 240- to 290-mp region of the far ultraviolet was a n obvious approach, hut the techniques of photoelectric densitometry could not he applied in this spectral region without improved means to confine sensitivity of a lamp-detector combination to light of wave length absorbable by the compounds under investigation. Materials erpeoted to have appropriate wavelength-dependent sensitivity to ultraviolet radiation were tested as deteotors. The halophosphate fluoroscope and the stainless steel cathode Geiger tube were found sensitive to radiation in the far ultraviolet and unresponsive to the light of longer wave length emitted by all ultraviolet lamps. Employed a s described, they are effeotive detectors of two new categories of simple and precise densitometric instruments. Sensitivity of the counting photometer would appear to meet any requirement of chromatographic densitometry, and that of the fluoroscopic photometer can be increased enormously by modsoations indicated. The instruments are easily aoolied for measuring - ahsorption of chromatograms hecause of insensitivity to mom illumination. The prevalence of ultra" iolet -mnnO nnmnnllnr(r Epnaratprl hu s n ~ vc absorptiG- _._._Iz) -,nr_r-_ -hmmatography and performanee oharacteristies of the instruments suggest analytioal applications of wider
.
.-
__...
PHa
Fluoroscopio Boreens were preparea uy aeposmng caic iini eniorofluorophosphste, a common phosphor of fluorescenL .amps, an one side of heat-softened sheets of polyethylene. Blue, violet and ultraviolet light from the mercury vapor lamp are removed by an optical filter such as Argus LY, Wratten K,, or Corning 3486 to permit exclusive excitation of a phototube by yellow nud red fluorescent light from the fluoroscope, The last of these filters is to be preferred, although the first was used in the studies reported; the first two filters exhibit significant yellow fluorescent emission to long-wave-length ultraviolet light. The fluoroscope and optical filter were employed in the opening of a Photovolt search unit, Type E, which was used with the Photovolt electronic photometer, Model 501-A. A Mineralight lamp (8) served as source of high intensity ultraviolet radiation of short wave length. The complete instrum fluoroscopic photometry is shown in F
__
__I_ I1 7
___I I -
of many organic compoundsseparated hy chromatographic and electro~horetk techniques. Widely employed for locating invisible spits and lines (1, 14-16', 60, 2S), the property has hitherto proved amenable to precisian measurement on paper only with use of an ultraviolet monochromator (5,8, 24, 65, 67). An inexpensive unit applicable for meitsurements on preparative chromatograms of large area and on two-dimensional obromatogrs,ms was required for studies on purine and pyrimidine derive tives. Densitometric methods of the type useful for compounds with absorption in the visible spectrum (S, 4, 7, 1s) were tried and found inadequate, mainly hecause of t h e unavailahility of suitahle hand filters effective in the ultraviole t spectrum. Investigations were therefore directed to clevelopment of detectors unresponsive to light beyond the ahr;orbing range; the specific requirement being an instrumental reisponse determined exclusively by the intensity of light in the 240- to ZYU-mp region of the far ultraviolet. Detectors found satisfactory are an ultraviolet-sensitive fluoroscope used with B conventional photometer responding to the visible fluorescent light (Figure I), and an ultraviolet-sensitive Geiger-Mailer tube used with a count ratemeter (Figure 2): Wben a low pressure mercury lamp emitting largely a t 254 mp (8, 18) i8 employed as the light source, responses of the fluoroscope and Geiger tube are determined quantitatively by the intensity of incident light in the wavelength ranges 240 to 313 mp and 240 to 280 mp, respectively.
Figure 1. Instrumental Assembly for Fluoroscopic Photometry
Figure 2.
Photon Count Ratemeter
~~~
The detector used in the photon counting assembly (Figure 2) is constructed by simple modifications of a Tracerlah TGC-2 Geiger tube to provide for continuous flow of counting gas, and to allow entry of the short-wavelength light. A small tubulature was blown in the stainless steel-coated glass wall of the tube
from the Plicose Mfe. Co.. New York. N. Y . . Trinsmittance to 254 mp ultraviolet Gdiation is 94%. ' A tank of helium-butane (98.7 t o 1.3, Matheson Co., Inc., East Rutherford, N. J.) W&E connected t o the tubulature, and a pinhole provided a t the p e 1122
1128
ANALYTICAL CHEMISTRY
riph,ery of the end window for escape of counting gas. This modified Geiger-Muller tube was inserted in a probe unit of the type commonly used for nuclear radiation detectors, and connected to a conventional count ratemeter. The Tracerlab SU-3B laboratory monitor was employed without modification in the studies reported. Some of the ratemeters require adjustment of the power supply for an output of 1450 to 1650 volts needed in operation of the photon counting tubes. Addition of a logarithmic scale to the indicating panel of the ratemeter, as in Figure 2, facilitates photometric application. For vertically adjustable support of the Geiger tube, the detector arm of a transmittance-density unit, Photovolt Model 52 C, was employed. A 4-watt General Electric germicidal lamp mounted directly under the opening in the platform of the density unit served as light source. The light aperture was defined either by the opening in a disk inserted a t the platform level, or by the opening in a polyethylene-coated lead mask used on top of the chromatogram. KO light filter was employed in the present applications of the basic photon count ratemeter. PROCEDURES
Standards with known amount of nucleotide per unit area of paper were prepared in the following manner. Twenty-microliter aliquots of standard solutions in 2% aqueous ammonium bicarbonate were placed on Whatman S o . 50 chromatographic paper with a micropipet held in a vertical position. The tip was lowered t o contact with the paper for reproducible delivery. Resulting spots are slightly elliptical but adequately constant in area. Evaluation of the technique on 16 spots prepared with a solution of guanylic acid revealed the area t o be 2.48 f 0.044 sq. cm. as measured with a planimeter, and 2.52 =t0.040 sq. cm. when computed as circular areas after averaging measured lengths of major and minor ases for the diameter. Distribution of nucleotides through the spots was evaluated by ultraviolet absorption and found to be symmetrical with respect to the center, where the concentration is generally 3 to 5% higher than a t the periphery. For convenience, measurements on spots from applied solutions were routinely made at the centers, the resulting small systematic error being of no practical significance. Reproducibility of the spot application technique as regards ultraviolet absorption a t the center of spots is indicated by the precision data of Table I, which are typical. Guanylic acid spots were converted to the acidic fluorescent form (Table I j by exposing each side of the paper to dry hydrogen chloride gas for 1 minute. The gas was generated by adding about 0.5 ml. of concentrated sulfuric acid to excess anhydrous calcium chloride. Chromatograms for measurements of both radioactivity and light absorption were developed on Whatman No. 50 paper from straight lines of sample applied a t the base line. Ultraviolet absorption of purine and pyrimidine derivatives on paper was found to vary with ionic charge as in aqueous solution, and this variable was normalized by exposing developed chromatograms rto an atmosphere of ammonia, carbon dioxide, and water produced by spontaneous decomposition and volatilization of satu-
Table I. Concn. of S o h . Applied, Mg./MI. _. 0.100
0.500
1 .oo 2.50 6.00
Form n n a n a
n a
n a n
R
7.50
IO.00
Fluoroscope placements and absorbance readings were made in triplicate; the readings rarely varied by more than 0.02 unit, and average values found on chromatograms were recorded directly on linear coordinates as in Figure 5, curve 2. Application of the Geiger tube for measurement of ultraviolet absorption was as detecting component of the photon count ratemeter shown in Figure 2. For routine photometric application the area of paper measured was defined by a light aperture in the opening of the platform supporting the chromatogram. Flat placement of paper over the aperture was maintained with a transparent architect's triangle or a lead mask with large opening. The Geiger tube detector, because of its extreme sensitivity to short-wave-length ultraviolet radiation and complete lack of response to room illumination, was employed with the detector arm elevated 4 to 8 cm. above the paper, as shown in Figure 2. The instrument was prepared for use by sweeping the tube with counting gas for 5 to 10 minutes until a constant counting rate resulted with the light beam intercepted by an undeveloped portion of the paper. Vertical adjustment of the detector by means of a wedge a t the base of the arm provided a rough means of establishing a standard counting rate on the untreated paper, and rotation of the detector provided the fine adjustment. The latter was possible by virtue of diminished photoemissive sensitivity of the side of the tube where stainless steel had been oxidized in the course of glassblowing to provide a side arm. Maximum counting rates on three sensitivity scales of the ratemeter employed are 20,000, 2000, and 200 counts (photons) per minute. If, as was generally done, the instrument is adjusted so that 10, transmittance of the paper, provides 20,000 counts
Dependence of Instrumental Response on Concentration of Guanylic Acid in Neutral and Acidic Forms
a
0,250
rated aqueous ammonium bicarbonate (pH 7.6). The air-dried chromatograms were then examined visually over an ultraviolet lamp, using an ultraviolet fluoroscope with small hole through the center to facilitate outlining of the absorbing areas with pencil. Lanes of transit to be measured were paralleled by straight lines graduated a t 0.5-cm. intervals. Yeast nucleic acid (Schwarz) was purified by extraction and filtration with hot aqueous 10% lithium chloride, precipitation with 2 volumes of ethyl alcohol, drying, re-extraction with 2% aqueous ammonium bicarbonate, and a final precipitation with 2 volumes of ethyl alcohol. The dried precipitate was hydrolyzed to ribonucleotides with 0.35 lithium hydroxide at 37' C. for 17 hours, and the hydrolyzate neutralized with pxcess solid ammonium bicarbonate. For quantitative analysis, triplicate 10-pl. s ots of hydrolyzate and duplicate 10-~1.spots of standard nucfeotide mixtures were applied at the base line on Whatman No. 50 paper. Three papers \+-ere developed with separate standard chromatographic solvents (20, 22, 28), each designed to provide a t least one nucleotide in a state of unequivocal purity. Fluoroscopic photometry measurements were made with the apparatus (Figure 1j described. The spot, or the area of a chromatogram to be measured, was approximately centered on the Mineralight lamp, and the exact area delineated by the opening in a polyethylene-coated lead mask. Absorbance measurements were made by placing the fluoroscope-phototube unit on the mask in a position centered by means of guide lines inscribed on the mask.
n a n a
Conventional Photometer Absorbance Deviation A B C D Av. Av. % 0 . 1 5 0.025 16. 0,095 0.015 1 6 .
0.18 0.12 0 . 1 7 0.14 0.12 0.08 0.10 0.08 0 . 2 3 0.25 0.27 0 . 2 7 0.15 0.18 0.16 0.18 0.43 0.37 0.42 0.44 0.27 0.28 0.22 0.24 0.53 0.53 0 . 5 3 0 . 5 4 0.30 0.26 0 . 2 8 0.28 0.72 0 . 6 5 0.84 0 . 6 8 0.34 0.33 0 . 3 1 0 . 3 1 0.73 0.73 0.72 0 73 0 3 R O.3 8 0 . 3 7 0 : 3 9 0.75 0.76 0.73 0.73 0.42 0 . 4 2 0.43 0 . 4 1
0.25 0.17 0.42 0.25 0.53 0.28 0.67 0.32 0.73 0.38 0.74 0.42
0.78 0.74 0 . 8 0 0 . 7 9 0.50 0.46 0 . 5 1 0.52
0.78 0.50
~~
0.015 ,5.8 0,013 7 . 5 0,025 8 . 0 0,022 8 . 9 0,003 0 . 7 0.01 3.6 0,027 4 1 0,013 3 . 9 0,004 0 . 5 0,007 1 . 9 0.017 2 3 0,005 1 . 2 0.019 2 4 0,019 3 . 8
Fluoroscopic Photometer Absorbance Deviation A B C D Av. Av. % 0 . 2 3 0.22 0.20 0.24 0.15 0.16 0.16 0 . 1 7 0 . 3 3 0.37 0.35 0.37 0.26 0 . 3 1 0.29 0.32 0 . 5 8 0.52 0 . 5 6 0 . 5 8 0 . 5 6 0.54 0.49 0 . 4 9 0.80 0.80 0.80 0.79 0.72 0.70 0.74 0 . 7 2 1.00 0.99 0.98 0.99 0.94 0.95 0.94 0.95 1.08 1.07 1.06 1.07 1.051.071.061.07 1 . 0 9 1 . 1 0 1 . 0 8 1.08 1.11 1.12 1 . 1 0 1 . 1 1 1.12 1.10 1.14 1.12 1 . 1 4 1.18 1.14 1.14
0,013 5 . 9 0,005 3 . 1 0.35 0,015 4 . 2 0.29 0.02 6 . 8 0.56 0.02 3 . 6 0 . 5 2 0.03 5 . 8 0.80 0.004 0 . 5 0 0.72 0.01 1.4 0.99 0,005 0 . 5 1 0 . 9 5 0,005 0.53 1.07 0 . 0 5 0 . 4 7 1.06 0 , 0 0 7 0 . 6 6 1.09 0.007 0.64 1.11 0,005 0.45 1.12 0,007 0.63 1.14 0.004 0.33 0.22 0.16
A
Photon Count Ratemeter Absorbance Deviation B C D Av. Av. ?t
0.16 0 . 1 5 0.16 0.18 0.17 0.15 0 . 1 5 0 . 1 6 0.30 0 . 3 3 0 . 3 3 0 . 3 4 0.28 0.32 0 . 3 3 0 . 3 5 0 . 6 0 0.52 0.59 0.59 0.57 0.49 0 . 5 2 0.54 1.00 1 . 0 1 1 . 0 2 0 . 9 9 0 . 8 2 0 . 8 2 0.87 0.85
0.16 0.009 5 . 6 0.16 0.007 4 . 8 0.32 0.013 3 8 0 . 3 2 0.020 6 2 0 . 5 8 0,027 4 . 8 0 . 5 3 0.025 4 . 7 1 . 0 0 1.010 1 . 0 0.84 0.020 2 . 4
1.60 1.60 1 . 5 8 1.62 1.37 1.37 1.37 1 . 4 4 2.28 2.26 2.26 2 . 2 8 2,022.022.062.10 2.65 2 . 6 6 2 . 6 2 2 . 6 3 2.44 2.50 2.43 2.40 3 . 1 0 2.82 3 . 0 7 3.02 2.82 2.52 2 . 8 8 2.88
1.60 1.39 2.27 2.05 2.64 2.44 3.00 2.78
0.010 0.026 0.010 0.03
0.63 1.9 0.44 1.5 0.015 0 . 5 7 0,029 1.2 0.069 2 . 3 0.13 4 . 6
V O L U M E 2 6 , NO. 7, J U L Y 1 9 5 4
1129
per minute, then absorbance values corresponding to full scale pointer deflections are 0.0, 1.0, and 2.0, respectively. Zero deflection corresponds to infinite absorbance on all scales. With special techniques employing standard absorbers, absorbance values of a t least 5.00 can be easily measured with no diminution of precision. Maximum instrumental stability results if the detecting system is maintained in full operation during the period of use. For scanning purposes, a chromatogram may be drawn rapidly across the beam, indication of absorbing areas being provided by an aural feature as well as the indicating pointer. For precision measurements, a chromatogram is advanced along the lane of transit and readings are recorded on graph paper at each graduation, and a t intermediate points of maximum and minimum absorbance.
resulted from covering the end window with a 0.1-mm. sheet of cellulose acetate (18) characterized by 0, 15, and 60% transmittance at 280, 290, and 300 mp, respectively. In contrast to the fluoroscope, response of this detector is determined preferentially by intensity of the lines of shorter wave length emitted by the mercury lamps, and the tota! photoemissively effective radiation
Concenfrofion o f Solufion Applied, I
A and C 0 and D
2
3
m g, / m l . 4
5
- Adenylic A c l d -
Uridylic Acid
/’
PROPERTIES AND PERFORMANCE
The unique property common to both detectors is dependence of their response upon wave length of incident light. This is shown by the spectral sensitivity curves, Figure 3. Comparison of curves F and A reveals that excitation of the halophosphate fluoroscope by a low pressure mercury lamp occurs principally in a spectral region where absorption by nucleotides is very high. B quantitatively significant fraction of the response, however, is due to light in the wave-length range 280 to 313 mp, a region where absorption by nucleotides is small or negligible. The former property is responsible for high sensitivity of the detecting system in measuring nucleotides a t low concentration, and the latter is a t leaPt partially responsible for the saturation effect shown in Figure 4 and Table I for samples of high concentration. The empirical spectral sensitivity curves of the stainless steel cathode Geiger tube, C-1 and C-2, are properly evaluated with cognizance that peripheral location of the photoemissive cathode results in unusually high sensitivity to scattered light when the tube is used in a position coaxial to the monochromatic beam. This placement was employed for the measurements plotted as curve C-1, and a relatively low discharge rate persisting above 280 mp was found to be caused by scattered radiation of shorter wave length. Thus, a t any wave-length setting of the monochromator, a background counting rate of 25 counts per minute
Concenfrafion in paper,
210
220
230
240
Wovelength,
Figure 3.
250
260
270
280
290
c m.2
A , E . Effect of absorption on discharge of GeigerMiiller tube. I a n d Io are counts per minute due t o short-wave-length ultraviolet photons transmitted through the paper with and without nucleotide, respectively. C, D. Effect of absorption of incident ultraviolet on fluorescent emission b y halophosphate fluoroscope. Log Io/I values are absorbance readings on logarithmic scale of electronic densitometer used with phototube assembly designed t o confine response t o fluorescent light.
14,000
200
r/
Figure 4. Absorption of Ultraviolet Light by Two Ribonucleotides as Standard Deposits in Whatman No. 50 Paper
300
m &.
Spectral Sensitivity of Stainless Steel Cathode G-;\.I Counter and Halophosphate Fluoroscope
Showing limitation of response to light of wave length absorbable b y nucleotides A . Absorption curve of typical nucleotide. C-1, C-2. Intensity of Geiger tube discharge due t o monochromatic light emitted by hydrogen discharge lamp and Mineralight mercury lamp, respectively. I n (7-1, t h e response above 280 m r i s due t o scattered radiation of lower wave length. F . Intensity of fluorescent light from calcium halophosphate fluoroscope in response t o monochromatic ultraviolet light from Mineralight lamp. Beckman quartz prism monochromator with fixed slit width of 0.5 mm. was used for C-1, and Bausch a n d Lomb grating monochromator was employed with slit width settings a t 0.1 and 1.0 mm. for C-2 and F, respectively.
from the lamps is in a range absorbable by nucleotides. These considerations provide an explanation for the slightly inferior sensitivity of the GeigerMuller tube as compared with the fluoroscope for measuring low concentrations of neutral nucleotides, and a greater sensitivity for the measurements a t high concentration (Figure 4 and Table I). Absolute sensitivity of the detectors per se has not been critically evaluated, but information a t hand indicates that quantum efficiency of the halophosphate fluoroscope is greater than that of the stainless steel cathode. Efficiency for conversion of ultraviolet to visible light is generally around 20% for the phosphors of fluorescent lamps, and a major fraction of the visible light from halophosphate is in the usable spectral region, 500 to 700 mp (fa,18). Factors responsible for low sensitivity of the present fluoroscopic photometer include the small fraction of fluorescent light intercepted by the secondary detector, a phototube, and the limited sensitivity of the latter. Preliminary experiments in measurement of fluoroscopic light with an RCB 931 A photomultiplier connected to a count ratemeter provide a thousandfold increase in sensitivity. The latter modification is essentially a scintillation counter with wave-length-dependent sensitivity for ultraviolet radiation. Sensitivity of the fluoroscopic photometer and the photon count ratemeter as units (Figures 1 and 2) has been compared to that ~
1130
ANALYTICAL CHEMISTRY
of the Photovolt 501-A photometer used with the Type B ultraviolet-sensitive photoelectric tube. The latter combination has been referred to, for convenience, as a conventional ultra, violet photometer. Responses to a standard 254mp light source are : 5uoroscopic photometer : conventional photometer : photon count rate meter = 1:15:300. The extraordinary sensitivity of the latter inRtrument is conferred by the metering system, whereby each photon effective in releasing an electron causes a pointer deflection of 0.4 mm. on the most sensitive scale. Other cathodes, platinum, lead, and aluminum, have been tested, and found to provide a sensitivity even higher than the steel cathode. Stability of both instruments during use is comparable to that of conventional photometers, and no variation in performance has been detected during 6 months of use. Change of long-wave limit and photosensitivity of Geiger tubes reported by earlier workers (19, 21) has not occurred on application of the present tube in the manner described. Precision is also similar to that of conventional photometers, and reproducibility of instrumental response has not proved to be a significant source of error in measurements on chromatograms. Dependence of instrumental response on absorption of ultraviolet light by nucleotides on paper is shown by the typical standard curves, Figure 4. At concentrations up to 4 to 87 per sq. cm. the specific absorption is seen to be very high, nearly constant, and, proportional to extinction coefficients of the individual compounds in neutral aqueous solution. With further increase in concentration of absorber, instrumental response is determined to a continuously increasing extent by the light of wave length least efficiently absorbed. As with conventional photoelectric colorimeters and densitometers, a characteristic departure from linearity in the absorbance-concentration curves is therefore expected and quantitative determination is made by reference to the empirical curves. Data of the type used for plotting the standard curves are presented in Table I, and they serve to high light certain performance characteristics. Values in the A, B, C, and D columns are instrumental readings on 2.50 sq. cm. spots from 20 p1. aliquots of sodium guanylate. The search unit of the above-mentioned conventional ultraviolet photometer contained a 3-mm. ultraviolet filter of Corning 9863 glass designed to remove most of the visible light. All values have been included, although a few are rejectable on a statistical basis. Guanylic acid was selected for the comparison to illustrate noninterference of fluorescent light in absorption measurements with the fluoroscope and photon counter; this nucleotide exhibits a rather intense fluorescence in the acid form. The fluorescence on paper is detectable visually and instrumentally through band filters with peak transmittance a t 470 and 420 mp, and may extend into the near ultraviolet. The effect of this light on the conventional ultraviolet-sensitive photoelectric tube leads to apparent absorbance values which are in error on the low side, but the significant changes in absorbance found with the present instruments after acid treatment of guanylic acid are correlatable with known shifts in the absorption spectrum on acidification of aqueous guanylate solutions. The extent of unreliability indicated by the deviations shown in Table I is typical. Precision of instrumental response and absorbance by paper are regarded as minor contributors to random variation. Major contributors appear to be variable spot application technique and variable light scattering by the paper. A systematic investigation of the latter and its effect on response of detectors with varying geometry would be a valuable contribution to quantitative paper chromatography. APPLICATIONS '
The radiochemical application which motivated development of the present instruments, and for which they have been used routinely, is illustrated by typical graphic data shown in Figure 5.
Comparison of curve 1 or 2 with curve 3 reveals an example of radiochemical contamination narrowly averted. While the slow uridylic acid, presumably associated with a trace of magnesium or manganese (N), is well separated from polynucleotides a t the base line, the proximity of a 660-count-per-minute orthophosphate-phosphorus-32 peak a t D could, if unrecognized, result in serious contamination on cutting out and eluting the uridylic acid for determination of specific activity by customary procedures. To determine the specific activity and provide a rigorous criterion of radiochemical purity, the radioactivity of successive areas along the lane of transit of a labeled compound may be compared quantitatively Fith the chemical quantity of compound in the identical areas. Techniques for measuring comparable radioactivity and weight distributions of compounds on chromatograms will be presented in a later report.
v
i
03
IO
Distance from Baseline.
20
25
c m.
Figure 5 . Distribution of Ultraviolet-Absorbing and Radioactive Compounds on a Chromatogram 1. Intensity of ultraviolet light transmitted through 1 X 2 mm. areas of paper a t indicated points along lane of transit na measured with Geiger counter 2. Absorption over successive 0.7 X 2.7 om. areas centered a t graduations by lead mask and measured with fluoroscopic photometer 3. Intensity of Pas beta-radiation from identical masked area8 of paper used for measurements plotted in 2. Cridylic aoid-Pa2 from perfused human seminiferous tubules has traveled as two components B and C. Separated radioactive compounds are shown a t A and D,while ultraviolet absorbing impurities are concentrated a t A and E. Absorption at F is due t o materials from paper, and occurs in lanes with no sample.
Applications of a more general nature may be classified as those for which the present simple units are suitable, additional applications made possible by using a monochromatic light source, and applications of other wave-length-specific detectors operating on the same principles as the halophosphate fluoroscope and the steel cathode Geiger tube. Auxiliary to any of these applications in paper chromatography, appropriate ultraviolet fluoroscopes for visualization are very helpful; they confer to observations in the ultraviolet spectrum a facility comparable to that provided by color in compounds absorbing light of the visible spectrum. Haines and Drake ( 1 5 ) first applied the fluoroscopic principle for 3-keto, A'-unsaturated steroids using a phosphorcoated glass plate. Apparently these fluoroscopes are not easily adapted to the marking of chromatograms, as the absorbing areas are outlined by tracing from an ultraviolet photograph (14, 16). An automatic chromatographic stage and a continuous recorder are further accessories which should facilitate measurement for most of the applications. Because the present models of fluoroscopic and Geiger tube
V O L U M E 26, NO. 7, J U L Y 1 9 5 4
1131
Table 11. Photon Count Ratemeter Applied to Quantitative Estimation of Nucleotides on Chromatograms rolution A. 30.0 mg. each of adenylic acid, guanylic acid, cytidylic acid, and uridylic acid per 10 ml.: aisomer of guanylic acid and approximately 1 t o 1 mixtures of a and b isoiners of other nucleotides used. Solution T. Hydrolyzate from 180 nig. of yeast nucleic acid in 10 ml. Solution B. 80.0 m g . of each nucleotide per 10 ml.; isomeric composition a s above.
Nucleotide Adenylic acid Guanylic acid
Developing Solvent Isobutyrate ( 2 0 ) .Immoniurn sulfate (22)
Solution -4
T
B A
Y
B
Maximum Absorbance Log Io/I Av. dev. 1.18 1.58 1,78 0.87 0.73 1.37
detectors are designed for measuring absorption in the 240- to 200-mp region of the far ultraviolet, they are applicable to organic compounds n-ith conjugated double bonds, including most colored compounds. T o date they have been applied for distribution curves of purine and pyrimidine derivatives, adrenal hormones, proteins, porphyrins, and B number of dyed compounds on chromstograms. Both instruments have been used for rapid determination of ultraviolet-absorbing compounds in solution, the measurements being done on spots applied to chromatographic paper in the manner described above. Thus, the ultraviolet absorption of 100 spots corresponding to 100 effluent fractions from an ion exchange separation of nucleotides can be measured in ahout 20 minutes. Concentration of an identified absorbing compound in the Polution applied is determined by reference to 5tandard curves (Table I and Figure 4). The phot,on count rstemeter has been applied to quantitative analysis of purified ?-rust nucleic acid after hydrolysis and separation of the nucleotides by paper chromatography. Results of measurements on developed chromatograms and the calculated relative concenti,ations are shown in Table 11. Although maximum absorbance vdues are seen to be highly reproducible for identical samples, :ireas under the curves provide a more generally applicable basis for quantitative determination (3-7). This is especially true for the ribonucleotides, as the unknown and standards may contain the u and b isomers in different ratios. The ribonucleotide composition of yeast nucleic acid, as computed from areas under the ~011:il absorbance curves, is in close agreement with that reported by Deutsch et al. (10). The use of an ultraviolet monochromator as a source of incident light for fluoroscopic and photon counting detectors offers several advantages, which are analogous to those provided by spectrophotometry as compared to photoelectric colorimetry in the visible spectrum. The methodology for accurate spectrophotometric measurement of ultraTiolet absorption as applied to chromatograms in the form of paper strips has already reached an advmced stage, as s h o m b>-reports of Brown and Marsh (6) and Brown ( 5 ) . In developing similar procedures for measurement of ultraviolet absorbance at :my area of a large chromatogram, use .of detectors insensitive to visible light has been found to confer a simplicity and versatility highly desirable for some purposes. Thus, absolute insensitivity of the stainless steel cathode Geiger t u h e to visible light has permitted installation of a small incantlrscent lamp inside the monochromator to provide bright visual illuminstion of the slit image on the precise area of a paper being riir:isurcttl for absorption a t 254 mp. The tn-o detrctorE described in the present communication are representative memhrrs of two distinct categories of materials. fluorescent and photoemissive, with the common property of markedly wave-length-dependent sensitivity to ultraviolet light.
0.05 0.08 0.03 0.05 0.01 0.02
Zonal Area 4 v . dev. 8.50 0.32 12.0 0.72 16.8 0.49 7.0 0.21 9.9 0.62 13.1 0.30
Sq. cm.
Concentration, pX/I\11. Known From areas 8.64
Ratio ,to Adenyllc
.M/ M
12.3
1.00
12.2
0.99
17.28 8.27
16.54
Although the relation between intensities of incident ultraviolet and emitted fluorescent light has previously been used for absorption measurements ( 9 ) , wavelength specificity of fluorescent responses has apparently not been applied to limit spectral sensitivity of a lamp-detector combination. Fluorescent components are obtainable for fluoroscopes effective in many broad Trave-length bands of the ultraviolet spectrum ( 2 , 12, 18). Most of the fluorescent papers and plastics contain organic dyes characterized by excitation spectra mavima in the near ultraviolet, 300 to 380 mp. The inorganic phosphors of fluorescent illuminating lamps are usually not excited by such radiation, and respond principally or exclusively in the far ultraviolet. Fluorescent responses to radiation in the extreme ultraviolet, of wave length as low as 23 mp, have been reported ( 2 ) for common materials potentially suitable as fluoroscopic phosphors. Lowwave limits of wave-length bands in the ultraviolet spectrum may be established with sharp cutoff filters, materials for whirh are readily available down to 200 mp (18). The photon counting method employed for measuring intensity of ultraviolet radiation is dependent upon rarely used conditions wherein photoemission from a metal cathode occurs in a high potential electrical field across a gas medium capable of supporting and quenching a Geiger cascade. Studies on similar systems for evaluating very feeble radiations in the ultraviolet or visible spectra, 90 to 750 mp, have been reported by Locher (19), and lllandeville and Scherb ( 2 1 ) have summarized the findings of related investigations. Photoemission of an electron occurs only if the wave length of incident radiation is below a certain maximum value (long-wave limit, Amax ) which corresponds to the minimum energy (work function, h v O )which will suffice to detach an electron. For a metallic cathode, the long-wave limit is generally predictable from known properties of the elementary components, iron, nickel, and chromium in this case. Substitution of work function values of these elements in the Einstein photoelectric equation (11) provides a computed long-wave limit of 280 mp, which coincides with the present observations. Such rigorous limitation on the maximum wave length of light to which photoemissive elements are sensitive (11, 17, 18) clearly provides a generally applicable basis for establishing the longwave limit of lamp-filter-detector combinations to measure absorption in selected bands of the ultraviolet spectrum. The remarkable metering sensitivity conferred by applying a photoemissive metal as the cathode of a Geiger tube permits choice of detectors on the basis of selectivity and stability of response, and with little regard for the quantum yield of electrons. ACKNOWLEDGMENT
The authors are deeply appreciative of the support of the present work by the U. S. Atomic Energy Commission, Division
1132
ANALYTICAL CHEMISTRY
of Biology and Medicine. They also wish to thank David Rittenberg of this university and B. A. Silard of the Photovolt Corp. for valuable suggestions contributing to development of the instruments. Calcium halophosphate phosphor was kindly donated by Robert Gleason of the Sylvania Electric Co., New York,
N. Y. LITERATURE CITED
Balston, J. S . , and Talbot, B. E., “A Guide to Filter Paper and Cellulose Powder Chromatography,” London, H. Reeve Angel & Co., Ltd., 1952. Beese, N. C., J . Opt. SOC.A m e r . , 29, 278 (1939). Block, R. J., Science, 108,608 (1948). Block, R. J., Le Strange, R., and Zweig. G., “Paper Chromatography,’’ Xew York, iicademic Press, 1952. Brown, J. A , ANAL.CHEM., 25, 774 (1953). Brown. J. A,, and Marsh, M. M., Ibid., 24, 1952 (1952). Bull, H. B., Hahn, J. Ti-., and Baptist. V. H.. J . Am. C h e m . Soc.. , 71. - ,650 - - -(1949). . Carter, C. E., I b i d . , 72,1466 (1950). Danckwortt, P. W.. “Luminessens-hnalyse im Filtrierten Ultravioletten Licht,” Leipsig, dkademische Verlagsgesellschaft, 1940; Ann Arbor, llich., Ed%-ardsBrothers, 1944. Deutsch, A., Zuckerman, R., and Dunn, 31. S., ANAL. CHEM., 24,1769 (1952). Dushman, S., “Fundamentals of dtomic Physics,” New York, McGraw-Hill Book Co., 1951. ~~
Forsythe, W. E., and Adarns. E. Q.. ”Fluorescent and Other Gaseous Discharge Lamps,” Sew York, Murray Hill Books, 1948. Fosdick, L. S., and Blackwell, R. Q . , Science, 109, 314 (1914). Haines, W. J., in “Recent Progress in Hormone Research,” Vol. VII, p. 255, Sew York, Academic Press, 1952. Haines. W. J., and Drake, S . h.,Federation Proc., 9,180 (1950). Holiday, E. R., and Johnson, E. =1.. Nature. 163. 216 (1949). International Critical Tables, Vol. TI, p. 68, New York, McGrawHill Book Co., 1929. Koller, L. R.. “Ultraviolet Radiation,” Sew York, John Wiley Br Sons, 1952. Locher, G. L., P h y s . Reo., 42, 525 (1932). Xagasanik, B., Vischer. S . , Doniger, R., Elson, D., and Chargaff, E., J . B i d . Chem., 186, 37 (1950). Nandeville, C. E., and Scherh, 31. V., Nucleonics, 7, 5, 34 (1950). Markham, R., and Smith, J. D., Biochem. J . , 49,401 (1951). hlarkham, R., and Smith, 6 . D., S a t w e . 163, 250 (1949). . 24, 1024 (1952). Paladini, A. C., and Leloir, L. F., A N . ~ LCHEhf., Parke, T. V., and Davis, W.IT‘., Ibid., 24, 2019 (1952). Smith, K. C., and Allen, F. W., Federation Proc., 12,269 (1953). Tennent. D. M., Whitla, J. B., and Florey, K., QKAL. CHEM.,23, 1748 (1951). Wyatt. G. R., Biochem. J., 48, 584 (1951). RECEIVEDfor review October 5 , 1953. Accepted April 2 2 , 1954.
Detection of Glycosides and Other Carbohydrate Compounds On Paper Chromatograms J. A. ClFONELLl and FRED SMITH Division o f Agricultural Biochemistry, University of Minnesota, St. Paul, M i n n .
A method is described for the detection of nonreducinp glycosides. The chromatogram is first sprayed with a weak periodate solution whereby the a-glycol grouping is split and, at the same time, the periodate ion is reduced to iodate. Except for those small areas of the paper which contain the glycosides, the periodate solution remains largely unaffected. Hence upon spraying with benzidine, the glycosides are located by the appearance of white spots on a blue background, the latter arising from the oxidizing action of periodate on benzidine. The method is suitable for the detection of other carbohydrate compounds capable of being oxidized by periodate. Variation of the benzidine test enables certain carbohydrate compounds to be differentiated.
T
HE location of minute amounts of reducing sugars on paper
chromatograms usually presents little or no difficulty and this is also true of reducing methylated sugars (2, 7 , fa). Nonreducing oligosaccharides, such as sucrose and alkyl glycofuranosides, may also be detected fairly easily by means of any one of a number of spray reagents that contain an amine and a n acid capable of effecting fission of the glycosidic group (8, 3, 7 ) . Alkyl glycopyranosides, being relatively stable to acids, cannot be detected in this way and in this work the ammoniacal silver nitrate reagent (6, 8) has been only of limited use and furthermore the test is time-consuming. Because the reducing group in alkyl pyranosides is effectively masked, use has been made of the fact that when the periodate ion cleaves the a-glycol structure, it is itself reduced to iodate (9). A test with benzidine for the removal of periodate, rather than one for the formation of a dialdehyde ( 4 ) from the carbohydrate compound, has provided a awe, simple, and rapid procedure for locating glycopyranosides
(IO). I n order to accomplish this, the paper chromatogram. itre $prayed with a dilute solution of periodate followed b y a benndine solution. The glycosides, which react vrith periodates, are located b y the appearance of colorless spots on a blue background, which arises from the action of the periodate on benzidine. The periodate benzidine reagent is fairly sensitive (2 y of glucose, fructose, or ethylene glycol may be detected) but does not appear to be so sensitive for aldoses and ketoses as the Tollens xmmoniacal silver nitrate reagent (12)and those containing aromatic amines, such as aniline (ff), p-anisidine ( 7 ) , and X,Sdimethyl-p-aminoaniline ( 2 ) . Its value is in the detection of nonreducing carbohydrate compounds containing two or more hvdroxyl groups such as glycosides, alcohols, acids ( I ) , lactones ( 1), rrrtain partially methylated sugars, nonreducing oligosaccharidrs and polysaccharides. EXPERIMEYTAL
Reagents. A saturated aqueous solution of potassium metaperiodate. BENZIDINE REAGENT A. Mix 1 volume of 0.1M benzidine in ethyl alcohol with 1 volume of 0.8N hydrochloric acid. BENZIDINE RE.4GENT B. Mix 10 volumes of 0.1M benzidine in 50 (volume) yoaqueous alcohol with 2 volumes of acetone and 1 volume of 0.2.V hydrochloric acid. IRRIGATING SOLVENTS.Mixtures of tert-amyl alcohol, npropyl alcohol, and water were employed. The Rj values in Table I were obtained with these solvents in the volume ratio of 4 to 1 to 1 5 , respectively; the values in parentheses were for the solvent ratio of 4 to 1 t o 1. For comparison, Rf values of 0.23, 0.21, 0.30, and 0.31 were obtained for D-glucose, D-galactose, ~r arabinose, and n-fructose, respectively, for a 5 t o 1 t o 1 mixture of these three solvents. Application of Periodate Reagent. The chromatogram is dried in air in the usual way and sprayed with the metaperiodate solu-
