matic cycle for help in trouble-shooting possible failures. APPLICATIONS
The automated apparatus described here has been successfully utilized for the synthesis of several peptides, among which were bradykinin (15),several analogs of this nonapeptide plasma kinin (It?), angiotensinylbradykinin (121, a decapeptide from tobacco mosaic virus protein (19) and insulin (8). There was, in each case, substantial saving of time and effort in the synthesis of these peptides and the overall yields were better than those usually achieved by conventional techniques. These advantages should become even more important as the synthesis of longer peptides is under taken. Since the various peptides which have been prepared have contained most of the common naturally occurring amino acids, it is believed that the method will have rather wide applicability to problems of peptide synthesis. However, several problems concerned with the chemistry of peptide synthesis by the solid phase method remain, and certain amino acids and combinations of amino
acids still present difficulties. These questions are under active investigation. It has already been suggested (12) that the principles of solid phase synthesis should be applicable to the sgnthesis of other polymers of defined structure. The flexibility which has been incorporated into the design of this instrument is expected to facilitate its application to the automated synthesis of such polymers in addition to the specific application described here for the synthesis of peptides. LITERATURE CITED
(1) Anderson, G. W., McGregor, A. C., J . Am. Chem. SOC.79, 611
(3) Bodanszky, IT.,Sheehar Ind. 1964, p. 1423. (4) Carpino, L. A., J . Am. Chem. SOC. 79, 98 (1957). (5) Gish, D. T., Katsoyannis, P. G., Hess, G. P., Stedman, R. J., Ibid., 78, 5934 (1956). (6) Jones, F. D., “Ingenious RIechanisms
for Designers and Inventors,” Vol. 1, p. 69, Industrial Press, New York,
1930. (7) Kashelikar, D. V., Ressler, C., J . Am. Chem. Xoc. 86,2467 (1964).
(8) McKay, F. C., Albertson, K. F., Ibid., 79, 4686 (1957). 191 Marshall. G. R..blerrifield.’ R. B.. Biochemistiy 4 , 2394 (1965). (10) Merrifield, R. B., Biochemistry 3, 1385 (1964). (11) AIerrifield, R. B., Endeavour 24, 3 (1965). (12) Merrifield, R. B., in “Hypotensive Peptides,” E. G. Erdos, N . Back, and ~
Sicuteri, eds., Springer Verlag, New York, 1966. (13) Merrifield, R.E., J . Am. Chem. SOC. 85, 2149 (1963). (14) blerrifield, R. B., Science 150, 178
(1965\.
\ - - - - ,
(15) Merrifield, R. B., Stewart, J. M., Nature 207, 522 (1965). (16) Paul, R., Kende, D. S., J . Am. Chem. SOC.86, 741 (1964). (17) Schwyzer, R., Sieber, P., Kappeler, H., Helu. Chim. Acta 42, 2622 (1939). (18) Stewart, J. M.,Woolley, D. W., in “Hypotensive Peptides,” E. G. Erdos, N. Back, and F. Sicuteri, eds., Springer Verlag, Kew York, 1966. (19) Stewart, J. M.,Young, 5. D., Benjamini, E., Shimizu, XI., Leung, C. Y., Federation Proc. 25, 653 (1966). (20) Thomas, A. B., Rochow, E. G., J . Am. Chem. SOC.79, 1843 (1957).
RECEIVED for review September 6, 1966. Accepted October 10, 1966. Work was supported in part by Grant A 1260 from the U. S. Public Health Service.
Automated Measurement of Powder Diffraction Patterns LUDB K. FREVEL Chemical Physics Research laboratory, The Dow Chemical Co., Midland, Mich.
b A novel system is described which converts digitized film data from a precision microphotometer into a printout of interplanar spacings and corresponding peak intensifies. Useful information is also presented on sample preparation, film handling, calibration procedures, and data processing. A computer program written in Algol 60 for the Burroughs B 5500 pertains to the Guinier powder method utilizing crystal-monochromatized CuKal radiation. Optional punched-card output of the diffraction data permits ready application to automated searching procedures of powder patterns.
W
the extensive compilation of reliable quantitative powder diffraction standards issued by the National Bureau of Standards (NBS) (IO, 11) and with the demonstrated practicality of computerized matching of powder patterns (S), it becomes expedient to consider digitized output of diffraction data in the form of punched cards or tape so as t o speed up the phase identification process and a t the same time introduce a quantitative aspect to powder intensity data. Photographic registration of powder patterns 1914
ITH
ANALYTICAL CHEMISTRY
furnishes convenient storable records; and this advantage, among others, accounts for the fact that in many analytical laboratories most of the qualitative phase-identification is done with films. For this reason, automating the measurement of films took priority over designing a digitized ouput for recording diff ractometers. In 1964 a fully digitized precision microphotometer became commercially available (8) which was suitable for automated measurement of powder patterns taken with crystal-monochromatized x-rays. The equipment consists of three parts: the 24-304 Jarrell-Ash microphotometer, the IBM 526 card punch, and the Bristol Dynamaster potentiometer recorder. The precision microphotometer is equipped with a movable stage, a plate-holder with transverse motion, an optical system with projecting line images, and a precision slit with controls changing length, width, and transmission density. The stage is driven by a precision ball screw which can be operated manually or by a 12-speed reversible drive. For reproducible mounting of films, a special adaptor is mounted on the plate-holder of the stage. Two matched phototubes
in a compensated circuit form the basic electronic system which, via a ratio recorder, provides a trace of the diffraction pattern. The microphotometer drive-screw position is encoded with a 10e-bit shaft-position encoder in the nonambiguous Wang code. Encoding of the transmittance reading is achieved through a 103-bit encoder mounted directly on the recorder slide-wire drive. The outputs of the two encoders are transferred into a series of logic circuits and fed to the IBhI 526 card punch. For reproducible positioning of the commonly used 48-mm.-wide film, the adaptor shown in Figure 1 can be readily made from a clear 4- x 10-inch spectrometric plate. Four brass guides, a brass stop, and four brass positioners are securely fastened to the plate with epoxy resin. The slotted brass cover holds the film flat on the glass plate. Two circular holes are provided t o give ready optical access to the clear unexposed edges of the film so that the span control can be adjusted for a loo’% transmittance reading. Small translations of the entire adaptor are achieved with a positioning screw secured to the 4-inch plate-holder. For routine work, the slit width is 25 microns; the slit
height, 2000 microns; the wedge position is 6; and the scanning rate is 5 mm./minute. To test the full capabilities of digitized film data, a computer program for the Guinier method utili~ingCuKai radiation ( 7 ) was written (for Dow use only) to convert the card output for such films from the above microphotometer into a print-out of interplanar spacings and peak intensities. For greatest reproducibility of relative intensities it is recommended that powder samples be dusted onto a 7.5-micron A1 foil (G.E. A9946A) coated with a very thin layer of petrolatum. Excess powder is removed gently by tapping the sample holder and by delicately stroking the powder surface with a piece of cleansing tissue. The randomly oriented layer of powder, usually less than 10 microns thick, is oscillated during a 2- to &hour exposure. If double emulsion film is used, then the side toward the camera wall should be masked during developing t o avoid pseudo-doublets or broadened diff raction lines. Care should be taken not to wrinkle the A1 foil in the sample holder. By observing these precautions, one obtains clear, well focused patterns from randomly oriented crystallites with negligible absorption. This reduction of background intensity in powder patterns (over conventional patterns taken with p-filtered x-rays) permits the successful automatic photometering of films and warrants the writing of a computer program to process the digitized data. DESCRIPTION OF PROGRAM
The word format for the automatically punched cards is as follows: columns 1 through 6 contain the translation data, s,, in microns; columns 7 through 9 contain the transmittance data, T,, ranging from 0.000 to 0.999; column 10 specifies the wedge position. Three manually punched cards and a flag card are added to the card output; namely, a title card ahead of the card output, the flag card behind the output, a background card listing seven selected values of the background transmittance, and an output-option card (print-out, punched card output, or both). Any number of such decks can be stacked and computer-processed at one time. The program to handle the cards accomplishes six functions: identification of the 4 1 reference lines, calibration of the effective camera-diameter, calculation of interplanar spacings, calculation of peak intensities, elimination of A1 reference lines, and tabulation of the results. For the asymmetrical 45' subtractive position of the Guinier camera, the five reference spacings from the A1 foil (2.3379 A., 2.0247 A,, 1.4317 A,,1.22094A,, 1.16896 A.)are identified in the following manner. By virtue of the high degree of preferred orientation of the A1 foil, the fifth 8 1 spacing (4222) = 1.16896 12.) has a very high intensity and serves as an unambiguous reference line which is initially positioned within
-
bass positioner l
A
l
9
glass plate
E
spring clamp
n
1
f
brass stop
I
6
0
Ye+
-
slotted Teflon block
screw
P
slotted brass cover
0
Q
L Figure 1.
Film adaptor and slotted brass cover
&20 microns at the 100000-micron mark of the precision screw. The fourth A1 spacing [d(311) = 1.22094 A.] is then identified by finding the translation, s,. of a subsequently larger spacing satisfying the inequality : 8415 (1 - 6) 5 Isy
- 1000001 5 8415 (1
+ 6)
(1)
where 6 is a tolerance factor (0.0036) to allow for film shrinkage and where 8415 niicrons is the separation between the fifth and fourth A1 reference lines calculated from Equation 2 for an average effective camera diameter of 2r, = 114611 microns, and h = 1.5405
A. 8415
=
2r, {sin-l
where s(222), s(311), s(200), and s(1ll) are the translation values for the fifth, fourth, second, and first A1 reference lines. Calculation of an interplanar spacing corresponding t o a given measured sv is carried out by first finding its nearest A1 reference line-e.g., if sv satisfies Criterion 4 8,
?
s(11l)
2
then the first A1 reference line is taken as closest to s,. The Bragg angle difference, Ae,, (in radians) between the line corresponding t o sy and the first -41 reference line is given by Equation 5: s,
[M] h
+ 4200)
As, =
-
- s(1ll)
(5)
2r
The sought interplanar spacing, d,, is then computed from Equation 6: \
If two or more lines fulfill Criterion 1, the program selects the line matching 8415 microns most closely. With the fourth reference line identified, the program proceeds to identify the'third reference line> and in like manner the remaining two A1 reference lines. Next, the effective camera diameter, 2y, is ccmputed from tjvo pairs of widely separated -41 reference spacings and averaged according to Equation 3: 2r
=
4
4222) - s ( l l l ) / 0.767299 , ~ ( 3 1 1) S(lll)l 0.69387 14222) - s(200)1 0.658247
['
+ + +
/~(311)- ~(20O)lI (3) 0.584825
d, =
A
2 sin[e(111)
+ Ae,]
-
0.77025 sin(0.335732 A&)
+
(6)
Computation of the peak intensity for d, involves the cons,ersion of transmittance T , to x-ray intensity 6, and the appropriate correction for backFrom an accurate ground intensity scale prepared with crystalmonochromatized CuKal radiation, a smooth plot was obtained for ~,(T)'vs. T = 1 - T,. This empirical curve can be fitted adequately by Expression 7 aura 6,(T)
=
c,log,
(,?)' I--\
u=o
COsna
(7)
where co is an arbitrary scale factor and the parameter au and exponent nu have the values shown in Table I. SatisVOL. 38, NO. 13, DECEMBER 1966 e
1915
C
factory agreement between observed intensity and &(7) is documented in Table 11. For a particular sy, the appropriate background transmittance, Tv,b, is obtained by linear interpolation between two bracketing T-values of the background. These two bracketing 2’”s are selected from the seven entries punched on the background card. The peak intensity I , corresponding to d, is then computed from Equation 8:
1, = where
8v(T)
- fiv(7b)
(8)
= 1 - Tv,b
rb
.IO
1.632
a
.20
.3a
E
.4c
L 4
Elimination of the aluminum pattern is accomplished by subtracting the calculated intensities for the five A1 lines from their respective observed peak intensities. For the highly oriented A1 foil, the observed relative peak intensities are given in Table 111.
t
s
ln
.50
z a
I-
-60
*?O
.8C
Table 1.
Parameters for gV(7) .go
1 1
0.15 0.23 0.27 0.40 0.70 0.020 0.0165
Table II. T
n 0.105 0.194 0.266 0.336 0,391 0,440 0.533 0.606 0.657 0.700 0.736
Comparison of Gob0
o
5
I .oo
Figure 2.
Gobs
with zYV(7) &(7)
D- . on __
0,767 0.794 0.818 0.839 0.887 0.869 0.879 0.889 0.899 0.903
120 130 140 150 160 170 180
5.03 10.07 14.79 20.05 24.75 29.45 40.18 50.99 60.41 70.07 79.84 89,92 100.40 111.49 123.05 134.80 143.89 152.43 162.05 170.70 177.85
1.000
.,.
m
10
15 20 25 30 40 50 60 70
80 90
100 110
Scale factor co is 43.6. Intensity equal to 1 is barely visible on film.
Table 111. hlck
111 200 220 311 222
1916
(9)
dobs
ANALYTICAL CHEMISTRY
precision have also improved with automation. For a direct comparison of the two procedures, the same film used for Case 2 of a recent publication (3) was measured automatically and the digitized data processed by the described d,I-program. The data of Table IV correspond to the first two columns of the previously published Table I1 ( 3 ) . Spacings for v = 2, 4, 8, 10, 12, 16, 19, and 21 belong to the sphalerite phase
- I,,&kk)
is positive, then this difference is entered as the intensity of a superposed line with an interplanar spacing equal to d(hk8). The final step ol‘ the program is the tabulation of the processed data. h general format of the print-out is reproduced in Table IV. The time to process the digitized data of four patterns totaling 165 lines amounted to 8 seconds of machine time on the Burroughs B5500 computer and 25 seconds I / O time.
1 0 0.29 0.34 0.13 0.38 1.00
Because the very intense 222 reflection occurs in a region of the film where generally only weak reflections are observed, its intensity remains essentially unaffected by any fortuitous superposition. Therefore, it is a good approximation to calculate the intensities of the A1 lines from Equation 9:
If I,b,(hk8)
Relative Peak Intensities for AI-Foil d(hk8), I(hkk)
A. 2,3379 2,0247 1.43170 1.22094 1,16896
Slow scan o f t w o overlapping reflections (0.25 mm./minute)
UTILITY OF PROGRAM
Several examples will be cited to illustrate the advantages as well as limitations of automated measurement of high grade powder patterns. Previous to our acquisition of the fully digitized microphotometer, films were measured twice on a precision comparator to reduce human errors of transcription. Intensities were estimated subsequently with a comparison scale. The combined operation took three to four times as long as the automated procedure. Reproducibility and
Table
IV. d,l-Measurement
(Micro-
photometer)
+
Sample: p-ZnS (sphalerite) AgClo.ezBro~as Reference Std.: A1 4.0494 A,, A = 1.5405 A. Camera diam.: 115.025 zt 0.008 mm. U
dv, A.
Iv
3.2552 66.7 2 3.1247 347.3 3 2.8203 304.6 4 2.7055 71.5 7 1.9944 113.5 8 1.9136 237.7 9 1.7001 11.1 10 1,6316 112.1 11 1,6258 17.8 12 1.5617 8.9 14 1.4096 10.0 15 1,3741 3.1 16 1.3527 11.9 17 1.2939 2.5 18 1.2606 18.3 19 1.2415 37.9 21 1.2100 4.7 23 1,1510 11.4 Unlisted spacings 5, 6, 13, 20, and 22 pertain t o A1 standard. 1
with a cube edge of 5.4113 rt 0.0006 A., somewhat larger than a = 5.4060 A. (10) for pure p-ZnS. In view of the improved accuracy, this increase of the unit cell is considered real and is attributed to solid solution. X-ray fluorescence analysis of the sphalerite sample revealed -1 to 2 wt. yo Cd and 0.3 wt. yo Fe. If CdS is in solid solution with p-ZnS, its mole fraction x can be calculated from Equation 10:
d 3 ___ (a, - a m ) = 4
2
(TCd+Z
- TZ,+$) 0.23 x
=
Table
V.
Dow d, A.
d, A.
6.7 4.88 4.43
20
3.39 3.17
100
2.93
30
10
15 87
(10)
where a, = cell edge of sphalerite, aZng = cell edge of pure p-ZnS, TCdl.2 and rz,+z are the respective ionic radii for Cd+2 and Zn+2 (9). A cell edge expansion of 0.0053 A., therefore, corresponds to 1 mole % CdS, in agreement with the elemental analysis for Cd. The second phase, AgC10.62Br0.38, was prepared to have a lattice constant of 5.639 A., almost identical with that of KaC1. For the completely resolved reflections corresponding to Y = 1, 3,7,9,14, 17, 18, and 23, the cube edge calculates to be 5.6391 f 0.0011 A,, in better agreement with 5.639 A. than the previously reported value of 5.6367 f 0.0019 A. for AgCla.e,zBro.ss. Although the scanning of a film proceeds automatically, the diffractionist still must inspect the trace of the diffraction pattern to detect unsymmetrical peaks resulting from overlapping reflections and to reject any spurious peaks due to scratches or defects on the film. Figure 2 shows a slow scan (0.25 mm./minute) of the overlapping lines 10 and 11. By resolving the reflections as indicated by the dotted lines, it is possible to estimate the position and transmittance of the shoulder (line 11) and insert a manually punched card in the output deck. Line 15 is spurious and arises from a slight defect in the 2-year-old film. Normally, such a spurious peak is readily recognized on the trace and its card is rejected from the output deck. Spurious peaks can also be picked up if the per cent noise sensitivity switch is set too low and consequently statistical fluctuations of the background transmittance are registered. I n general, the utility and reliability of phase identification by powder diffraction will be decidedly improved by greater absolute accuracy in the measurement of interplanar spacings greater than 3.5 A. For many ASTM powder patterns, these very spacings are in error by as much as 1-294 and are not amenable to careful comparison between standard and matched phase. Moreover, with complex mixtures the extent of searching for potential matches is greatly increased by inferior standard patterns. The analysis of a light gray
Comparison of PbHAsOc Powder Patterns
ASTM 1-0635
2.56
10
2.42
10
2.20
'
20
6.76 4.831 4,404 3.927 3,376 3.145
hkC
21 19 22 5 100
84
2,905 2,767 2,6697 2,6010 2,5530 2,4486
30
2,4162 2.3931 2,2745
16
2,2506
5 15 9 2 6
:Eo;{ 2,0993
5
5 6 14 4 8 1
6.7508 4,8328 4,4050 3.9296
010 001 110
011
{;:;z
Ti1
020
3.1457 2,9190 2.9065 2.7673 2,6696 2,6012 2,5526 2.4479 2.4273 2,4164 2,3932 2,2750 2,2557 2.2503 2,2025 2,1853 2,0985 2,0604 2.0576 2.0400 1.9648 1.9523 1,9506 1,9492 1.9061 1,9014 1,8739 1,8625 1,8196 1.7932 {I.7793 t1.7777 1.7191 1,7018
111
120 200 021 210 201 i21 121 211 002 201 012 211 030 220
iiz
130
221 112 031 2,0399 2 022 1.9654 11 221 1.95 20 102 1,9509 14 131 122 131 1,9022 6 212 2 1.8739 310 1,8633 4 122 1.8197 3 311 12 1.7935 230 1.78 20 15 202 1.7782 212 231 222 1.68 15 12 1,6882 040 311 1.6874 320 1,6806 032 1,6468 4 1.6473 231 1,6394 521 1,6290 3 1.6294 140 1,6208 i32 1 1,6116 003 041 1.5933 222 1,5729 5 1.5738 013 1,5669 132 1,5584 113 11 10 1.5498 1.55 141 321 1,5485 512 1,5442 141 1.5246 1,5247 4 113 1,4793 1.4780 6 1.48 10 232 1.4739 203 1.4688 1 1,4692 330 1.4683 240 1,4595 023 1,4541 3 400 i23 1,4395 1,4402 2 322 2 1.4349 213 331 1.4336 301 1.4293 410 1,4207 1,4158 441 312 1,4146 1,4147 1 31 1 1,3983 232 3 1.3954 042 5 1.39 2 1.3838 123 1.3830 Lattice constants for PbHAs04: a = 5,8389 f 0.0011 A., b = 6.7508 =t0.0012 A., c = 4.8543 i 0.0016 A., p = 95' 24' f 0.5' at 23' C. Space group Pb/a. 2.0601
1
1
c : 68; {::;6
{; :E:
i{: :E {; :%E
VOL. 38, NO. 13, DECEMBER 1966
0
1917
Table VI.
d v , A.
V
IV
10 11 92
7,0672 6.7682 5,2143 4,8239 4.4789 4,4233 3.9290 3,8126 3,6709 3.6272 3.5466 3.3801
57.0 37.5 145.8 25.2 73.6 24.9 7.1 302.6 3.2 3.6 4.7 162.5
13 14 15 16 17 18
3,2689 3,2126 3,1530 3,0781 2.9817 2,9221
6.7
19 20 21 22 23 24 25 26 27 28 29 30 32 33 34 35 36 37 38 39 40 41 43 44
2,8557 2,8125 2,7709 2,6964 2.6783 2.6464 2.6095 2’ 5775 2,5563 2.4558 2.4121 2,4055 2,2544 2,2379 2.2118 2,1830 2,1337 2.1019 2,0650 2.0575 2.0433 2,0282 2.0002 1.9634
45
1,9505
1
2 3 4 5 6 7 8 9
1.9439
46
131.8 118.7 70.7 336.2 30.8
dNB6,
A.
KH&304 I’NBB doalo, A.
Modified PbHAsQl Ioala
dcslcr
A*
U-3.6 Modified KzPb(SeQ4)z Residuum &lo
I C
7.050 5.21
3.810
3.078 2,980
190
293
82 255
5,222
3 815 ~
3.081. 2,984
151.7
5.5
6.7706
31
4.8235
28
4.4222 3.9285
33 7
81.3 267.8
;1 E)
149
3,1518
125
x: :xZ)
45
2,7710
7
2,6812
7
2.697
91
2.6976
86.1
2.610
18
2,6112
11.1
2,6067
9
6.5 0.3
2,5565 2.4536 2.4118 2,4017 2.2569
21 6 24 12 7
2 2111 2,1824
22 13
2.1051 2.0654 2.0578 2,0442
9 3
1,9643 1,9499
16 21
2.412 2.397
12
