Simple Photoelectric Microdensitometer 31. SPIEGEL-IDOLF AND R. H. PECKH.AII1. Temple University School of IIIedicine, Philadelphia, Penna.
scope equipment, n-hile one of the lenses is used instead of the Abbi: condenser and the other lens is set in place of the revolving nosepiece. The lenses are focused by help of the microscope’s rack and pinion and adjustment of the substage. In order to measure the light transmission of the plates by small steps interchangeable diaphragms of 1, 0.5, and 0.25 sq. mm., respectively, are placed in the plane of the stage which supports the film under investigation. The microscope is equipped with a compound revolving mechanical stage with 70-mm. transverse and 50-mm. back\Tard and forward range of rectilinear motion and with verniers reading displacement in either direction to 0.1 mm. The current output of the photoelectric cell is measured TTith a 440 Weston microammeter which permits direct readings in microamperes in steps of 0.1 for 15 microamperes. A single double-throw switch short-circuits the instrument when not in use.
A simple photoelectric microdensitonieter is described consisting mainly of light source, microscope stand, and photoelectric cell. With this arrangement it is possible to give graphical descriptions of x-ray diffraction patterns.
S
TUDIES (4) of the x-ray diffraction patterns of amino acids and proteins subjected to various alterations have made i t necessary to describe the corresponding changes in the x-ray diffraction patterns in a reliable m-ay. It seems desirable to determine the diameter of the diffraction rings with a higher degree of accuracy than is yielded by repeated direct measurements ( I ) , and a t the same time to evaluate the varying relative intensities of these rings. Furthermore, i t seems advisable to exclude personal equations by using a n objective method. Not all the available apparatus meet these requirement>. I n some cases the costs are prohibitive ( 2 ) . The arrangement of apparatus shown in Figure 1 has proved satisfactory for the authors’ special purposes.
A disadvantage of this arrangement consists in the fact that variations in the light source may influence the results. since measurements of the diffraction patterns are made consecutively and not simultaneously. This danger is lessened by using a lowvoltage bulb fed from a secondary electric circuit, and can be entirely eliminated by replacing the average transformer by one of constant output. I n the authors’ special case they have checked their results by reduplication in a satisfactory way. The greatest variations for any measurements were less than 2 per cent, which according to Weigert (6) is the limit for photometric determinations of this type. Kevertheless the authors now use a voltage stabilizer. ADVATTAGES OF METHOD. The image is focused in the plane of the cell to cover the whole cell evenly and thus integrate the light from the chosen area over the entire cell. Because of the small output used in these determinations the readings stay within the linear range of the cell. The amount of current varies between 1 and 14 microamperes. The current density can be controlled to give a full-scale reading for calibration on a rugged and standard ammeter by control of the primary light source. The photoelectric microdensitometer is made up of standard and simple optical parts, mounted on the microscope stage only for convenience and using electrical parts that are standard and can be used for other apparatus or borrowed from other setups for this purpose. The cost of the apparatus, including the shopwork but excluding the microscope stage, does not exceed $156 (including voltage stabilizer). The accurately working mechanical stage, n-hich is an essential part of the photoelectric microdensitometer, enables one to make readings at exact settings. By a procedure described below, the center coordinates of a n x-ray diffraction pattern and any of its diameters can be computed within 2 per cent. By making a large number of readings across the film under observation the construction of a relative light transmission graph is made possible. Such graphs have until nom been obtained only by very expensive self-recording instruments (Moll).
Apparatus A Bausch & Lomb adjustable lamp equipped with a 6-volt, lOS-watt ribbon filament bulb is used as a light source. With the help of a plane mirror and two additional condenser lenses the whole surface of a Type 2 Weston photronic cell is equally illuminated. A large monocular Zeiss microscope stand is used to support these parts, the mirror belonging to the usual micro-
Procedure
-
I FIGCRE 1.
I
.%SSEMBLY OF -4PPAR.ITUS FOR PHOTOELECTRIC h’1ICRODENBITOMETER
182
The determinations of the center of the diffraction pattern and of the diameter of an individual ring are made in the following way *
MARCH 15, 1940
ANALYTICAL EDITION 41m , 36.0 35.0 34.0 33.5 33.0 32.5 32.0 31.5
183 Mm. 9.0 8.5
Jfzcroa mperes 4.0 3.7 3.5 3.4 3.4 3.4 3.6 3.8
MWicroamperes
4.2 3.9 3.6 3.4 3.4 3.35 3.4 3.4
8,. 0
.1
7.0 6 , .I
E;
2
5 0 4 5
The readings on the horizontal scale were: Urn.
.
.lf m
.lizcroamperes
36.3 37.0 37.3 38.7
3.8 3.6 3.5 3.4
M t c i oamperes
38. .i 39 0 39.5 40.0
3 35 3.33 3 . .5 3.6
The minima of transmissions in these three sets of readings give the following three pairs of coordinates of three points located on the diffraction ring: Horizontal Scale
Tertical Scale
i:
i 33.0 6.5 1'3.4
24.6 24.6 38.7
FIGURE 2.
PRIXTOF X-RAYDIFFRACTIOS PATTERS O F dl-P-PHESYLAL.iNINE
Wheri these values are substituted in the general equation given above, three independent equations are obtained for D, E, and F which can therefore be computed.
POSITIT-E
(33.012 + 2 4 . 6 0 + 33.OE + F = 0 ++ 1694.16 1089 ++ 22 44..F6D0 ++ 33 33 .. OO EE ++ FF == 00 124.6)2 + i G . 5 1 2 + 2 4 . 6 0 i- 6 . 5 E + F = 0 605.16 + 4 9 . 2 5 + 2 4 . 6 0 - 6 . 5 E + F = 0 647.41 2 4 . 6 0 -- 6 . 5 E + F = 0 1 3 8 . 7 ) ? + 119.412 + 3 8 . 7 0 19.4E + F = 0 1497.69 + 376.36 + 3 8 . 7 0 f 1 9 . 4 E + F = 0 1874.05 + 3 8 . 7 0 t 1 9 . 4 E t F = 0 1694.16 + 2 4 . 6 0 + 33.OE + F = 0 647.41 + 2 4 . 6 0 + 6 . 5 8 + F = 0 1046.73 + 0.0 t 26.5E = 0 124.612 G05,lB
T h e center found by c o m p u t a t i o n h a s been marked with a n h i t e cross. T h e scale un left shows units of 4 mm. Letters on right refer t o points indicated in Figure 3 .
t
The film is approximately centered by help of the mechanical stage. The horizontal scale is held constant and the vertical scale is moved until the ring being measured is close to the aperture. Successive measurements of the transmission are made for each 0.5 mm. across the ring. The minimum transmission determines the coordinates of one point on the ring. The horizontal scale is still kept constant and the vertical scale is moved to the opposite side of the ring. The minimal point of the transmission across this part of the ring determines the coordinates of a second point on the ring. The midpoint of the two readings on the vertical scale determines the center of this approximately diametral chord; the vertical scale is left in this position and the horizontal scale is moved until the ring is near the aperture. The minimal transmissions xi11 now determine the coordinate of a third point on the ring. The coordinates of these three points are subst,ituted for X and Y , respectively, in the general equation of a circle: X2
+ Y2 + D X + EY + F
=
+ 19.4E + F + + 33.OE + F = 00 179.89 + 1 4 . 1 0 - 1 3 . 6 E 0 179.89 + 14 1 0 + 1 3 . 6 X 3 9 . 5 = 0 1874.05 -I- 38 7 0 1694.16 24.60
=
=
1694.16
0
-
-716.59 14.1
24.6 X 5 0 . 8 F = 859.84
-
-50.82
- 33.0
X 39.:
+F
= 0
D, E, and F are computed by the simultaneous solutions for all three points determined above. The coordinates of the center will be expressed by y 1 / 2 D and -'/?,E, and the diameterzof the ring ~ 1 1 equal d D 2 E 2 4F. EXAMPLE. An x-ray diffraction pattern of genuine serum albumin which consists chiefly of two diffuse rings was tentatively centered at 19.4 of the vertical and 24.6 of the horizontal scales. The transmissions !on the vertical scaleinear the ring to be determined were:
+ +
FIGURE3.
DENSITOMETRIC
STCDY OF ORIGINALPLATE SHOWN IN FIGURE 2.
Ordinates represent relative transmission of darkened portions of plate for 0.25 sq. mm. areas along a .radius, as compared t o transmission of unaffected center of plate.
0
2
4
6
8
10
12
14 16 MILLIMETERS
18 FROM
20 22 CENTER
24
VOL. 12. KO. 3
INDUSTRIlL AND ESGINEEIIIKG CHEMISTRI-
184
According to the above-mentioned relations betn-een D,E, and F , the coordinates of the center and the diameter of the ring, these latter values are computed from these figures. The coordinates of the center are 19.73 and 25.4, and the diameter of the ring is 26.47 mm. A less accurate but more convenient nietliod is the geometrical determination of the center and the diameter found by plotting the three points n-ith a large scale on coordinate paper. By using millimeter coordinate paper and a 10-time magnification, the results are 19.75 and 25.4 for the center coordinates and 26.3 mm. for the diameter of the ring. For the construction of relative light transmission graphs the horizontal and the vertical scales are now set for the coordinates of the center and the transmission of the center area is read on the scale of the ammeter. The center area has been protected by lead against the x-ray beam. Successive readings are made along the vertical or horizontal radii. The ratio of the ammeter readings for any point as compared to the center \vi11 yield the relative transmission of this point. By subtracting the stage scale readings for a given point from the respective center coordinates, the radius of the point, is determined. By plotting the relative transmissions against the distances of points from the center a curve is obtained, which graphically represents the brightness of the diffraction rings. The maxima and minima of this curve will indicate the diameters of the diffraction rings of the plate. In event of diffraction patterns which are not circular the same fundamental method is used, but more points must be used according to the pattern involved.
Data A graphical description of a n x-ray diffraction pattern is given in Figures 2 and 3. The technique used in making
the x-ray diffraction pattern has been described beforc (4). The dl-P-phenSlalanine is a preparation from the Eastman Kodak Company viliic*hhas been used before in iiltraspectrographic work (3).
Acknowledgment These investigations have been made possible through x grant of the Yational Research Council, Committee on Radiation, to one of the authors (Sp.-d.).
Literature Cited (1) K a t s , J. R., “Die Roentgenspektrographie als Untersuchungsmethode bei hochmolekularen Suhstanzen, hei Kolloiden und hei tierischen und pflanslichen Gem-eben”, in Abderhalden’s “Handbuch der hiologischen Arbeitsmethoden”, -4ht. 11, Physikalische Methoden, Teil 3, Heft 6, p. 222, Berlin, Urban & Schxaraenberg, 1934. ( 2 ) Sisson, IT. A . , and Clark, G. L., ISD. ENG.C H m r . , Anal. Ed., 5, 296 (1933). (3) Spiegel-ddolf, RI., Biochern. J., 31,1303 (19373. (4) Spiegel-Adolf, M., and Henny, G. C., J . Am. Chena. .z’oc., 61, 2175 (1939). ( 5 ) TTeigert, F., “Optische Methoden der Chemie”, Leipsig, Akrtrlemische Verlagsgesellschaft, 1927. FROM t h e Department of Colloid Chemistry, D. J. M c C a r t h y Foundation. and t h e Department of Ophthalmology, Temple Cniversity School of Medicine, Philadelphia, Penna.
Sealable Absorption Microtube ARTHUR N. PRATER, California Institute of Technology, Pasadena, Calif.
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I N C E the development of combustion microtechnique by Pregl, numerous investigators have proposed modifications of the absorption tubes. Most of these tubes have been designed to seal the absorbing agents from the air at all times except during the actual combustion. Through the use of a closed-type absorption apparatus, diffusion from the tube is eliminated, allowing the comout in oxygen alone. While the Pregl tube (3) is unquestionably the simplest and easiest to manipulate, the Tube - 9 , 5 mm, combustion must be finished I n n e r Tube - 8 . 0 m m . o , o . O v e r e l l lenqth 14C m , with air and temperature changes must be avoided when transferring between the combustion train and the balance room. These objectionable features are entirely overcome by the absorption tubes proposed by Friedrichs (2) and modified by Abrahamczik ( 1 ) . As reported b y Friedrichs, however, the tubes are of such small capacity that too frequent filling is necessary. The modification proposed by Abrahamczik has several distinct drawbacks: It is expensive to construct, very fragile, extremely difficult to fill, and difficult to wipe without accidentally dislodging the sleeve and wiping away some of the lubricant. Modified Friedrichs absorption tubes possessing none of these disadvantages have been used successfully in this laboratory for the past year. These tubes are constructed Closed
from thin-walled hydrometer tubing and when full weigh less than 15 grams, although they contain sufficient absorbent for twenty-five analyses. On standing overnight these tubes. \Then closed, increase in weight only about one quarter as much as do the regular Pregl tubes with the protecting rubber stoppers. The illustration gives the design and dimensions. I n use, all the absorbent is placed in the inner sleeve with the top and bottom protected b y cotton plugs. The lower portion of the ground joint is lubricated with a good grade of stopcock grease, care being taken to prevent grease from touching the portion of the ground surface above the protecting groove. Table I gives typical results with tubes of this design.
TABLE I. TYPICAL RESULTS~
-
Theory
Found
%
%
Adipic acid
C, 4 9 . 3 1 H, 6 . 9 0
C, 4 9 . 2 4 , 4 9 . 3 1 , 4 9 . 1 9 H, 6.97, 6.90, 6.77
Acetyl salicylic acid
C , 59.98 11, 4 . 4 8
C, 59.85 H, 4 . 6 1
C, 7 1 . 0 7 H. 6.72 a Microanalyses b y G. h. Swinehart.
C, 70.96 H, 6 . 6 7
Acetanilide
I n addition to their use in micromethods, these tubes have proved to be admirably suited for semimicroprocedures. I n this case the tubes are preferably made somewhat larger, the inner and outer t’ubes being 10 mm. in outside diameter and 12 mm. in inside diameter, respectively. Both the micro- and semimicrotubes may be purchased from the Greiner Glassblowing Laboratory, 255 West, Santa Barbara Ave., Los dngeles, Calif.
Literature Cited (I) Abrahamcsik, E., .lfikrochemie, 22, 227 (1937). (2) Friedrichs, A , , I b i d . , 19, 23 (1935). (3) Pregl and Roth, “Die quantitative organische Mikroanalyse”, 34, Berlin, Julius Springer. 1935.
kl.
