V O L U M E 23, NO. 2, F E B R U A R Y 1 9 5 1 The pressure resulting from the digestion of the 5- t o IO-mg. samples specified should amount to only 8 few atmospheres. That the heavy-walled tubes used are amply strong t o withstand the pressure is demonstrated by the fact that not a single tube failed a t any time during this investigation. I n order t o test the tubes further, 50-mg. samples of dextrose and 33-mg. samples of corn oil were digested a t temperatures up t o 525" C. for several hours. Thin is 55" C. above the temperature specified for the method and 15" C . above the strain point of the glass used. Samples of this size a t the higher temperature generate very much more pressure than do the 5- t o IO-mg. samples a t the temperature used in the method. Even under these extreme condit.ions, there was still no failure of the tubes. As 8. safety precaution, commercid Carius miorotubes should not be used for this procodurc and t.he tubes should come t o room tcmperature before they are removed from the shield and opened. If desired, the digestion box can bo opened behind a safety glass shield, and the pressum within the tubes can be released before they are removed from the hox by applying a small, sharp flame to the tips of the tubes until they open as a result of the slight internal pressure. The results obtained nith this met.hod show excellent precision
365 and accuracy, and bot.h umking and elapsed times are very favorable when compared with the long digestion procedures often recommended for hctcrocyclic or refmotor? nit.rogcn compounds. LITERATURE CITED
(1) Belohdr, R., and Godbert, A. L., "Semi-Micro Quantitative Organio Analyais." P. 91. London, Longmans, Green and Co.. 1945. ( 2 ) Clark. E. P.. 1.Assoe. Oi% Am. Chemists, 24, 641 (1941). (3) Drevon, B., and Roussin. J . pham. chim., (9) 1, 24 (1940). (4) Friedrieh, A,. Kiihaas. E., and Schniirch. R., Z. physiol. Cham.. 216,68 (1933). (5) Gunning, J. W., 2. anal. Chem., 28, 188 (1889). (6) Levi, T. G., and Gimignani, L., Cess. chirn. ital.. 59, 757 (1929). (7) .~ Ogg, C. L.. and Willits. C. 0.. J. Assoc. Ofic.Am. ChsmbFta.. 33.. 100 (1950). (8) Seoor, G. E.. Long, M. C., Kilpatriek, M. D.. and White, L. M.. Ibid.,33. 872 (1950). (9) White, L. M., and Seoor, G. E.. ANAL.CEIBM.,22, 1047 (1950). (10) White. L. M., nndSecor, G. E., IN". ENG.CHEM..ANAL ED..18, 457 (1946). (11) White, L. M., Seeor, G. E., m d Long, M. D. C., J. Assoc. OBc. Ag7. Chcmists,31. GSi (194X).
Microscopic Fusion Analysis of Sterols VICTOR GILPINL, Yale University, New Haven, Conn.
I-
move8 rapidly into melt. During the coolA recently d e v e l o p e d ing period, the crystallization velocity abmethod of miemsoopio has been shown in the identification of ruptly decremes and parallel or curving identification has been incompounds by means of microscopic fusion blsdos and rods appesr; meanwhile, the erysvestigated by which mPthods. Although all such methods depend tal front changes from smooth to serrated. minute quantities nf on the same phenomena, two differing techOn further cooling, the crystallization velocity sterols can he easily a n d niqucs have been developed. For convenincreases slightly. Transverse shrinkagr rapidly recognized. Aliencc, these may be called the. Kofler method, cracks appear soon after complete solidifics, t h o u g h there are limitain which a hot stage is used (3), and the Mction. tions to the method, it Crone method, in which a hot stage is not Partially Remelting Solidified Melt ("Meltshould pmve a valuahle u a d ( 1 , 4). The latter method has the adback"). Bladelike crystals grow (Figure 1). a i d i n the field of sterol vantaees of m a t e r soeed and simnlieitv. . - AlSolidified Preaaration. The microcrvPtalanalysis. mo& all the data on compounds studied by line solid often shows a herringbone pattern t,he McCrone technique appear as a part of (Figure I). Bladelike crystals show nearly x large cryst$lagraphic program (6). It is parallel extinction, negative elongat,ion, low birefringence. thv aim of the present study t o explore the possibilities of the and newly eentored O.A. or off-oenter Bz. (optic sign positive, McCrone analysis as an independent means of identification of 2V large). n h t e d compounds. The sterols are excellent materials for this m r k , because they melt a t moderate temperatures, usually without decomposition. Y RECENT years, considerable interest
~~
To describe interference figures where no ,optic axis is visible the following terms have been employed: Indefinite-for relatively clear figures, from single crystals, who? orientation cannpt he definitely specified; one brush-for relativcly clear figurcs in which one isogyre sweeps the field on rotation the stage; diffuso-for a. conoscopie view consisting of supenmposed figures, the result of several crystals in the field a t once. Not all observations yield useful data for all compounds-for example, heating some compounds gives rise to no sublimation, decomposition, or mesomorphs ("liquid crystals"); in such cases, observations on heating the solid are omitted. Again, if the erystdlilliaation velocity of the solidifying melt change with temperature is not reported, it is t o be inferred that such change is slight. Omission of the optic sign and estimate of 2V from interference figure descriptions implies that the figure8.a~not sharp enough for reliable determination of these properties. All conoscopic observations were made with a 0.85 numerical aperture dry objective; all photographs and observations (except refmct,ive index) were made with crossed Nieals. CHOLESTEROL
Cooling and 'Solidification of Melt. The melt su~ercoals slightly. A microcrystalline mass solidifies spontaneously and L Present address. Department of Chemistry. Michigan State College. East Lansing. Mieh.
Figure 1. Change i n Crystal Habit duringSolidification of Cholesterol Mixed Fusion of Compound with Thymol. Iilrtdev and needles st boundary show poor profile angles; one refractive index equals melt, and one refract.ivc index is greater than melt,. After a few minut,es, nuclei of a new phase, possibly an addition compound, appear at the boundary. The bladelike crystals of thi8
366
ANALYTICAL CHEMISTRY
phase show very low birefringence, positive elongation, and both refractive indices slightly greater than melt. CHOLESTERYL ACETATE
Cooling and Solidification of Melt. There is no supercooling. A fine-grained, uniform, gray mesomorph grows a t moderate crystallization velocity. Pressure applied to the cover glass causes the mesomorph to turn a vivid rust color. This, and other mesomorph colors, can usudly be seen equally well with the naked eye hy reflected light. From several nuclei of the solid, circular spherites with a smooth circumference grow a t medium erystallillization velocity. As the temperature falls, the crystallieittion velocity slows t o nearly aero, and, at the same time, the number of solid nuclei increases tremendously (Figure 2). Solidified Preparation. The radiating hairlike crystals in the spherites show fairly low birefringence, parallel extinction, negative elongation, and a diffuse interference figure.
Figure 4.
Solidified Cholesteryl Hcnroate
birefringence and relatively low crystallizntion velooity (Figure 3); (b) larger areas of narrow blades of the stable polymorph, showing relatively higher crystallisatiou velocity and birefringence than (a) (Figure 3). Strings of small air huhhles are often ovident in (b). The transformation velocity of solid polymorphs is nil a t room temperature, but the transformation known as boundary migration (4) gradually changes t,he appe&rancoof the stable polymorph.
Figure 2. Sphorites of Crystalline Cholesteryl Acetate WIOLESTERYL PROPIONATE
Heating the Solid. The solid melts t o a bright blue mesomorph showing white streaks. Further heating gives tho isotropic liquid. Figure 5 . Supercooled Mesomorph of Cholesteryl o-lodobenzoate Showing Air Bubbles Solidified Preparation. Itadial needles in the unstablc spherites show parallel extinction, negative elongation, and R diffuse interference figure. Crystals a i the stable polymorph show nearly parallel extinction, negative elongation, and a diffuse interference figure. Mixed Fusion with Thymol. From the stable polymorph, stubby, slightly curved teeth grow into the melt. One refrsctive index equals melt; the other is slightly greater than melt. CAOLESTERYL HEYZO.&TE
Figure 3.
Solidified Cholesteryl Propionate
Round spherifea-unstable polymorph
Cooling and Solidification of Melt. There is no supercooling. A fine-grained, gray-white mesomorph moves with high crystallization velocity. Pressure applied t o the cover glass causes the mesomorph t o turn bright blue, which changes first to bright green and later to rust color as the preparation cools. Two types of solid soon appear from numerou nuclei: (a) fine needles, in spherites of an unstable polymorph, showing very low
Heating the Solid. It melts to a bluish mesomorph ivith scattered white streaks. Further heating gives the isotropic liquid. Cooling and Solidification of Melt. There is no supercooling. A fine-grained, white mesomorph movcs a t medium crystallieation velocity acros8 the preparation. Pre.sure on the cover slip causes the mesomorph to become a gray color that gradually changes to bright blue. The solid appppcars .w fairly large bright areas. The serrated crystal front moves at, a medium crystsllieatiou velocity which increases on cooling. Just above room temperature, the solidification is rapidly completed by the appearance of many rectsngulsr-shaped nuclei, which grow rapidly. Some crystals develop fine shrinkage cracks.
V O L U M E 23, NO. 2, F E B R U A R Y 1 9 5 1
367
Solidified Preparation. Elongated crystals show close to 25' extinction, and medium to high birefringence (Figure 4). Almost all crystals show a centered indefinite interference figure. Mixed Fusion with Thymol. Spikes and dendrites grow into the melt. These show approximately parallel extinction, negative elongation, poor profile angles, and low contrast with theliquid. CHOLESTERYL o-IODORENWATE
Heating the Solid. It melts to a grayish mesomorph showing white st.resmers. Further heating givcs the isotropic liquid.
Figure 6.
Solidified 7-Ketocholesteryl Acetate
Cooling and Solidification of Melt. A fine-grained, vhite mesomorph moves slonly across the preparation. The mesomorph supercools to room tempcmture, and does not solidify when sectled wit.h solid. When the cover slip is moved the appearance of the preparation changes to gray with white streamers. The n-hite streamers, especially noticeable near air bubbles (Figure 5 ) , show parallel extinction, positive elongation, and an indefinite, centered interference figure.
whitish border forms at the interfaco. The contrast between d i d and melt is very low. 7-KETOCHOLESTERYL BENZOATE
Heating and Solid. The solid melts bo a hluish mesomorph showing white streamers. Further heating gives the isotropic liquid. Cooling and Solidification of Melt. The melt suporcools slightly. A fine-grained, white mesomorph slowly covers the preparation. The crystalline solid, with a fairly smooth front, moves with a medium crystallisstion velocity which increases slightly on cooling. Just shove room temperature, several c o m e spherules complete the solidification. Meltback. Broad blades of very low birefringence me formed, interspersed with a few highly birefringent needles (Figure i ) . Prominent transverse shrinkage cracks appear soon after solidification. Solidified Preparation. Highly bircfringent needles show parallel extinction and negative elongat,ion. The interference figure varies from indefinite, ccntered, t o one brush. Low hirefringent areas all show oenterrd Bz, (optic sign negative, 2E = ' 39 ', no dispersion). Mixed Fusion with Thymol. Sharply pointed, highly birofringent needles and stubbier, low birefringent blades grow a t the melt boundary. The needles show 40" to 50" profile angles, the blades about 60' to io". Keedles have one refractive index slightly less than melt, one refractive index greater t,han melt, and a diffuse interference figure. Blades show both refractive indices slightly less thsn melt, and a centered Bz, figure. Both habits exhibit parallel extinction and negative elongation. 7(#)-HYDROXYCHOLESTERYL RENZO.%TE
Cooling and Solidification of Melt. to an isotropic glass.
The pwparation solidifies
7-KETOCHOLESTERYL ACETATE
Coaling and Solidification of Melt. The' melt supercools slightly. .4 smooth continuous crystal front grows with medium crystallization velocity which inoreases on cooling. Spheritea oft,en form (Figure 6 ) .
Figure 8. Growing Crystal Front of 7(0)-Hydroxycholeste~?lBenzoate
Meltback. Seeding with tho d i d and careful reheating just helow the melting point results in slow growth of sheaves of needles and narrow blades (Figurc 8). Transverse shrinkage cracks appear on cooling. Solidified Preparation. Needles show pasallel extinction, negative elongation, one refractive index equal to melt, and one refractive index slightly greater ihnn mclt. eholestergl Benzoate, Showing Transverse Shrinkage Cracks
,
Solidified Preparation. Fine hairlike crystals show extremely low birefringence; the polarization colors are nearly always dark gray, The extinction is oblique a t about 45'. Mixed Fusion with Thymol. A smooth gray front with a
I-CHOLESTERYL METHYL ETHER
Cooling and Solidification of Melt. The melt supercuds lu a viscous liquid. On seeding with solid, fen-shaped sheaves of needles and laths grow very slowly. The needles gradually codesce to a fine-grained, smooth front. Solidified Preparation. The original needles show parallel extinction, negative elongation, and both refractive indices
ANALYTICAL CHEMISTRY
368 g r a t e r than melt. I n later stages of Solidification the solid exhibits a peculiar chracteristic herringbone appearance (Figure 9). OICHOLESTERYL ETRER
Heating the Solid. The solid melts t o a milky mesomorph, then almost immediately t o the isotropic liquid.
Figure 11. Round Spherites of Unstable Form of Cholestane Being Transformed on C o n t a c t with Stable Form
b. Growing Crystal Front of Cholesteryl Methyl E t h e r
..
. - ..
CooImg and solidification of Melt. There is no supercooling. A relatively coarse-gained and highly birefringent mesomorph forms rapidly. Solid farms quickly, and the fairly smooth front moves with medium crystallization velocity which increases rapidly on coaling. A microcrystalline mass often completes the solidification. Complex shrinkage cracks appear.
light and dark rings. When the two polymorphs meet, there is R very slow tmndarmation (Figure 11). Solidified Preparation. Smallcr nuclei of the stahle form show roughly uniform extinction, parallel to the long axis of the nucleus. All v i e w show low contrast with the melt, and a nearly centered indefinite int.erferencefigure. Some large spherites have narrowbladed cryst,als with 10' extinction. The unstable form shows all refractive indices slightly greater lhan melt: the fast ray is paxtllel to the sphorite radius.
Figure 12. Solidified Cholestane-3-one Showing Low Birefringent Unstable Form and High Birefringent S t a b l e Form
Solidified Preparation. Fairly large areas of roughly uniform calor show a ropy or rippled appearance when rotated to extinction (Figure 10). The interferonce figure is usually one brush, or an off-center hisectrix 12V large). A few small scattered arem show uniform parallel extinction and negative elongation. Many sharply pointed needles grow in the boundary, all showing parallel extinction, negative elongation, and a nearly centered indefinite interference figure. One refractive index is slightly greater than melt and the other equals melt. CHOLESTANE
Cooling and Solidification of Melt. The melt supercools considerably. Numerous nuclei of the stable solid appear, and grow with a berely perceptible crystallization velooity into oval or lens-shaped areas of narrow, bladelike crystals. In the melt, other nuclei of an unstable solid form grow, with a much slower crystallization velocity, into round spherites showing concentric
Mixed Fusion with Thymol. Thymol markedly increases the crystallization velocity of the stable form; fairly rapid salidifica tion takes place a t the houndmy between the two liquids. Very thin platelike crystals of the stahle form grow into the melt. These all show 90' profiles. CHOLESTANEJ-ONE
Cooling and Solidification of Melt. The melt supercools slightly. A smooth, fine-grained solid front of extremely low birefringence moves with a medium crystallization velocity which increases slightly on cooling. Rapid chilling of the melt leads to formation of spherites of radiating hairlike crystals. Meltback. In the melt and in the solid highly birefringent nuclei of a more stable polymorph appear (Figure 12). The crystalliaation velocity of the unstable form is muoh greater than that of the stable form. The transformation velocity slows to zero a t room temperature. Solidified Preparation. Hairlike crystals of the low birefringent unstable form have parallel extinct,ion and negative
V O L U M E 23, NO. 2, F E B R U A R Y 1 9 5 1 elongation. Much of the stable polymorph is microcrystdline. A few larger crystals show 5' extinction, positive elongation, and a diffuse interference figure. Mixed Fusion with Thymol. The smooth front a t the boundary of the thymol and the unstable form is invisible except between crossed Nicols. A few tiny rods grow from the stable form. These show 19' to 25" extinction, one refractive index equals melt, and one refractive index is greater than melt. The rods rapidly dissolve in the thymol.
369
Iem thm melt and one refractive index considerably loss than melt. 2-BKOMOCHOLESTANE3-ONE
Heating the Solid. The solid decomposes slightly if no1 heated csrefully. Cooling and Solidification of Melt. The melt supercools considerably. The melt solidifies fairly rapidly into a multitude of tiny nuolei.
3(8),5(a),6(8)-CHOLESTANETKlOL
Cooling and Solidification of Melt. The melt supercools slightly. A jagged solid front of lathlike crystals moves with fairly high crystallization velocity which increases on cooling. Shrinkage cracks, usually transverse, appear in the solid
/I
Figure 15. Growing Crystal Front of
Solidified Preparation. Most, of t,he solid, consisting of broad crystals of low birefringenw, shows pnrallel extinot,ion, positive elongation, and varying 0.A . vieas. Dispersion is negligible and the O.A.P. is normal to the leugth of the crystal. A few narrow laths of higher hirefringonce show parallel extinction, negative elongation, and ii one hrush or centered indefinite int,erference figure).
Melthack. A wotusion of flne needles mows with medium crystallization velocity. The needles gradually coalesce to form a smooth solid front. Just above room temperature many small round spherites appear ahead of this front, and coniplete the solidification (Figure 14). Transverse shrinkage cracks appear in the original needles. Solidified Preparation. All the noedlcs, fine and coarse, show parallel extinction, negative elongation, and a diffuse interference figure. Mixed Fusion with Thymol. A profusion of needles grows into the thymol, showing parallel extinction, negative elongation, H diffuse interference figure, and either 62" or 124" profiles. One refractive index is slightly less than melt, and one refractive index is greater than melt. ALCHOLESTENE
Figure
14. 2-Bromocholestane-3-one Showing Two Stages of Solidifioation
Mixed Fusion with Thymol. Parallel needles and laths grow into the melt (Figure 13). All show parallel extinction, and poorly defined profile angles. Some of the crystals, showing negative elongation (as above), have one refractive index very s!iihWy less than melt and one refractive index greater than melt. The other crystals, showing lower birefringence and positive eloncation (as above) have one refractive index very slightly
Cooling and Solidification of Meit. The molt supercools to i t viscous liquid a t room temperature. O n ' seeding, bladelike crystals gradually unite to form a sawtooth front (Figuro 1.5) which moves with a very low orystallimtion velocity. Solidified Preparation. Of several habits noticeable at the crystal front, the following we most common: ( a ) 90" profiles showing a centered Bz,; O.A.P. parallel to one of the profile edges; ( b ) 73" profiles, the interference figure showing an O.$. a t the edge of the field; optic sign negative, estimated 2V equals 80' (from curvature), slight dispersion 7 greater than v; ( c ) 40' profiles, with an interference figure showing off:center Bz.. The extinction is close to parallel, but difficult to measure, as the sides of most crystals are not paralld. Mixed Fusion with Thymol. The larger crystal8 split up at. the thymol boundary into long spikes with poor profile angles; most of these show prtrdllel extinction and a centered Bz., hut B few show a centered O.A. The crystdlieation velocity increases noticeably in the presence of thymol. AcCHOLESTENE3-ONE
Cooling and Solidification of Meit. The melt supercools to a viscous liquid. After 5 to 10 minutes, numerous scattered nuclei form, and grow exceedingly slowly into round spherites (Figure 16).
, 370
ANALYTICAL CHEMISTRY
Solidified Preparation. Very fine to coarse needles radiate from a oommon oenter. All needles show pardlel extinction, negative elongation, one refraotive index slightly less than melt, and one refractive index slightly greater than melt. Some parts of the spherites have nearly uniform orientatioir and a lower birefringence; these areas show a slightly off-center Bza (optic sign positive 2E = N O ) , with the O . A . P . parallel to the spherite radius. Dispersion is slight, r greeter than 0. More highlv birefringent parts of the spherites show n diffuse interference figure.
melt, and one refractive index is considerably greater than melt. All crystals show a diffuse interference figure. CHOLESTERYL PTOLUENESULFONATE
Heating the Solid. The solid melts to a clear liquid if heated carefully. Excessive heading produces n dark red decomposition product
2,4-CHOLESTADIENE
Cooling and Solidification of Melt. The melt supercools to a viscous liquid and must be soeded. A fan of blunt rods spreads a u t from the seed;. the orystals gat smaller and become interlaced to form a smooth front that moves a t a very loa. erystalliaation velocity.
Figure 17. 2,4-Cholestadiene Growing i n t o Thymol
Figure 16. Spherites of ALCholestene3-one G m w i n g i n Melt
Solidified Preparation. Lathlike crystals show psrallel extinction, and negative elongation. Mixed Fusion with Thymol. Laths grow into the melt (Figure 17). Well-defined profile anglos soon round off. The most frequently occurring habits are as follows: (a) square profiles, showing parallel extinction and centered indefinite interference figure; ( b ) 47" or 94" profiles showing parallel extinction, and off-center O.A. figure; ( c ) 54O or 108' shom.ing bluish dispersion colors, centered O.A., optic sign positive, 2V estimated from curliature equals SO", moderate dispersion r greater than 8 . All views show refractive index greater than melt.
Cooling and Solidification of Melt. The melt supercools to an isotropic glass. Meltback. Rapid rehesting to just helow the melting point causes complete solidification as % microcrystalline white solid. Cautious heating, horever, produces many fine-grained spherites of very low birefringence that show ooncentrio light and dark circles. Many scattered nuclei of a highly birefringent polymorph also sppesr (Figure 19). These grow slowly a t the expense of the dark form, a t higher tcmperatures. Both the crystallization velocity and transiormation velocity we zero a t room tempcrnture.
8-CHOLESTANYL BENZOATE
Heating the Solid. The solid melts to a metallic gray mesomorph. Further heating gives the isotropic liquid. Cooling and Solidification of Melt. Thcre is no supercooling. A white mesomorph appears; portions of this show relatively high polmisation colors and extinction. The solid grows a8 clusters of long, slightly curving blades. The jagged front moves with medium crystallization velocity which increases on cooling. Just above room temperature numerow nuclei appear and complete the solidification. During cooling of the solid, the phenomenon of boundary migration is evident (Figure 18). The rate of this transformation slows to nearly zero a t room temperature. Solidified Preparation. Blades show approximately parallel extinction, negative elongation, and a diffuse interference figure. Mixed Fusion with Thymol. Short, slightly curving, sharply pointed spikes grow,at the boundary. These show varying degrees of oblique extinction, one refractive index nearly equals
Figure 18. Solidified 6-Cholestanyl Benzoate Showing Boundary Migration Solidified Preparation. Keedles of the unstable, dark form show parallel extinction, negative elongation, and a diffuse interference figure. The nuclei of the stable polymorph are usually microcrystalline; a few relatively elongated crystals show parallel extinction and negative elongation. CHALINASTEROL
Cooling and Solidification of Melt. The melt solidifies to a glass. Meltback. On careful reheating, many white nuclei appear. With continued heating below the melting point, many of these
371
V O L U M E 23, NO. 2, F E B R U A R Y 1 9 5 1 enlarge into ragged spherites of fibrous,. irregular needles and narrow blades (Figure 20). The crystallization velocity is zero a t room temperature. Solidified Preparation. Crystals show varying extinction, from prtrsllel to slightly oblique, negative elongation, one refractive index equal to melt, and one refractive index slightly greater than melt.
purple color as numerous solid nuclei appear. S o continuous crystal front forms. Most solid nuclei remain very small; a few grow into relatively large, leaflike crystals with irregular outlines. Distinctive curving shrinkage cracks appear (Figure 221.
CHALINASTEKYL ACETATE
Cooling and Solidification of Melt. The melt supercools slightly. Numerous spherites of coarse, radiating needles appear during the whole solidification. Many of these coalesce into a fairlv smooth ci-~stalfront, which moves a t a moderate crystalliaation velocity. Meltback. Blades of varying . . width mow u-ith an irregular front. A s solidification proceeds, the blades break up into meshed needlcfi (Figure 21), and the front becomes smooth. Solidified Preparation. I'eedles in spherules have fairly low birefringenoe, parallel extinction, and some show positive, some negative elongation. Tho interference figure is diffuse. Those blades from the meltback t,hat show uniform extinction USUEIIY have parallel extinction, positive elongation, O . A . at the edge of the field, optic sign positive, 2V large, and O.A.P. a t right angles to the length of the blade. A f m blades exhibit negative elongation, and a ecnrered Bz,. Many interference figures &re diffuse owing to overlapping blades. Mixed Fusion with Thymol. Thin short blades with irregular profiles and very low birofringence grow into the melt. One refractive indox eiluitls m .It, and one rcfrxctivc index is slightly greater than melt,.
Figure 21. Solidified Meltback of Chalinasteryl Acetate
Meltback. Broad blades grow into the mesomorph. A strongly serrated front shows acute profile angles. After growing a short distance, the blades appear to curve sideways, and the rest of the melt than solidifies in nuclei as sbovc. Cracks appear in the blades.
CH.&LINASTERYL BENZOATE
Cooling and Solidification of Melt. A he-grained white inemmorph spreads over the incll. This gradually turns a
Figure 22. Shrinkage Cracks in Solidified Chalinasteryl Benzoate Figure 19. Light and Dark Polymorphs of Cholesteryl p-Toluenesulfonate
Solidified Preparation. Largo nuclai do not show uniform extinction. They often show O.A. a t edge of field, hut other indefinite interference figures are frequently visible. Blades from the meltback show approximately parallel extinct,ion, varying Bz. or O.A. conoseopic viows, O.A.P. normal to long axis of the blade, optic sign pasitivo, and 271 large. Mixed Fusion with Thymol. Overlapping thin leaves, with a smooth profile, farm a t thc boundary. There may also bc groups of short, curving needle% CAALINASTERYL PNENYLURETHANE
Cooling and Solidification of Melt. The melt solidifies to a glass. Meltback. Before the melting point is reached, the preparation solidifies as a white, microcrystalline mass. When this solid is partially remelted, clear blade or wedge-shaped crystals grow a short distance into t,he melt before the crystdlizstion velocity slows to zero as the temperature drops (Figure 23).
ANALYTICAL CHEMISTRY
372 Solidified Preparation. Extinction is not uniform in the large crystals. Most of them, however, show an O.A. just outside the fiold, with a one brush interference figure. One refractive index is greater than glass, and one refractive index is nearly equal to glass. PORIFFJZASTEROL
Cooling and Solidification of Melt. The melt supercools slightly. Many round spherules form, and coalesce to form a fairly smooth front which move8 with a fairly slow crystallization velocitv. A feu- snherules comdete the solidification. RonIC brottd areas develop complex shrinkage cracks (FigureI 24).
into the melt, but the profile angles are usually poorly defined. .411 needles show both refractive indices are greater than melt, P~RIFFJZASTERYL ACLTATE
Cooling and Solidification of Melt. There is nu supercooling. A dightly serrated front of blades plus a fen needles grons with moderate orystillization velocity which increases slightly on cooling. A few nuclei usually nippear in the melt to complete the solidification. A icu, l.rninsvcwe shrinkage cracks appear in the broader erysials.
Figure 25. Solidified Poriferasteryl Acetate Figure 23. Chalinasteryl P h e n y l u r e t h a n e Growing in Melt ~ ~ l t b ~~~l~~ ~ k the . melt,ing point many nuclei a mo~e stable polymorph appear. These nuclei show very low hirefringence (Figure 24), and gron- rapidly, on remelting, only the stable form grows into the melt, as a profusion of rough]y parellel needles, whose medium crystallieation velocity increases on cooling. Transverse shrinkage crack's appear in the dark needles. The transformation velocity is zero st room t,emperature.
Meltback. Imgor crystals form, but the behavior is no diff,srent. Solidified Preparation. Needles show extinction vmsina from 0 t,o 20" (Figure 25). Most needles exhibit pardlei exdinction and negative elongation, with a n O.A. just outside the field of view and O.A.P. a t right angles to the long needle axis. Most, blades arc not single crystals, and show a diffuse interference figure. -4 few blades show nearly centered O.A., optic sign positive, and 2v Mixed Fusion with Thymol. Thin blades and platw of low birefringence, and thick needles of higher birefringence, show a variety of angles, of whioh square and 105' profiles are mast common. Needles have one refractive index slightly greater than melt and one refmotive index greater than melt. Pltttes and blades h a w bath refractive indices slightly greater than melt. PORIFERASTANOL
Cooling and Solidification of Melt. The melt supercools slightly. A slightly serrated front of needles and laths moves u,ith a moderate orystallization velocity which increases on cooling. Coarsc tramverse or fine longitudinal shrinkaggo cracks appear in some park of thr solid.
asterol Growing into Light Unstahle Form
Solidified Preparation. The dark stable needles show parallel ext,inction, positive elongation, and a nearly centered, indefinite interference figure. Fine needles in the spherules of the unstable form show parallel extinction, negative elongation, and indefinite interference figure. Broader areas of the unstable form usually show a centered (indefinite) interference figure. Mired Fusion with Thymol. The dark, stable needles grow
F i g u r e 26.
Poriferastanol Growing into Thymol
373
V O L U M E 23, NO. 2, F E B R U A R Y 1951 Solidified Preparation. Most of the solid consists of laths of very low birefringence showing slightly oblique extinction (ahout 5'), positive elongation, with variable O.A. views, optic sign positive, 2E large, and O.A.P. at right angles t o the long neodlo axis. A few needles shorn- higher birefringence, slightly oblique extinction, and negaiive elongation.
more highly birefringent needles show hoth refractive indices greater than melt. STIGMASTEROL
Cooling and Solidification of Melt. There is usually no supercooling. Parallel narrow laths, of low birefringence, grow with fairly f m t crystallization velocity. The crystal front is not continuous, hut consists of noncont,iguous laths. The space between the laths freezes 8 s an irregular matrix of randomly oriented small cryst&. Prominent transverse shrinkage cracks appear (Figure 28). OcesJionally there is some supercooling, and spherules appear; in addition, bright patches of an unstable, more highly birefringent polymorph are seen, hut very little of this survives to room temperature where bhc transformation velocity is relatively slow.
Solidified Poriferastanyl
Figure 27.
loetate
4 finely serrated front guu's Mixed Fusion with Thymol. . into the thymol. .After a short time the solid-melt boundary is obscured by the solidifierttion of many sheaves of tiny needles, which show verv low birefringence and ~ m d l e lextinct,ion (Figure 21 PORIFERASTAI YYL ACE7PATE
- .. ...
.
.-
cooling and Soliditication of Melt. There 1s no supercooling. :\ ~ B - Y Sof payish nuclei forms and coalesces to give a serrat,cd front composed of sheaves of laths and needles; the medium icrysblliaation velocity increases slightly on oooling. Four-sided nuclei form cont,inually in the melt during cooling. Fine longit,ndinal or coarse transverse cracks appear in mmc crystals.
Solidified Preparation. Must laths of the stable form have parallel extinction and positive elongation. The interference figure shows O.A. views of several orientations (optic sign positive, 2V large) and O.A.P. a t right angles to t h e long crystal axis. A few laths show parallel extinction, negative elongation, and indefinite interference figures. Mixed Fusion with Thymol. Lrtths grow into the melt showing parallel extinction, hoth refractive indices greater than melt, and (a) 73" or 146' profile angles, negative elongation, and indefinite interference figures, and ( b ) square ends, positive elongation, and indefinite interference figures. There are also man? crystals with poor profiles. STIGMASTERYL ACETATE
Figure 28.
L a t h s of Stigmasterol w i t h Shrinkage Cracks
Solidified Preparation. Tho first. solid to form is a heterogenemass (Figure 2;). The later crystals may he either laths of low birefringence, with parallel or slightly ohlique extindim, positive elongation, and off-eenter Bz, figure (optic sign positive, 2V large) needlen and laths of higher birefringence, showing parallel extinction and negative elongation, Mixed Fnsion with Thymol. Overlapping laths and needles, with acute profiles, grow a short distance into the melt. The
OUR
01
Cooling and Solidification of Melt. The melt superoools slightly. A few nuclei form and coalesce to give a continuous, slightly serrated front, moving with medium orystallisation velocity. A fcw fine transverse cracks appear. Solidified Preparation. Solid consists of blades and needles intempemsod with finegrained matorial of random orientation. The more frequently occurring blades show very low birefringenec, parallel extinction, positive elongation, slightly off canter O . A . , opt,ic sign positive, 2V ahout 75" (from curvature), and O.A.P. a t right angles to t,hc long cryat,al axis. Most of the more highly birefringent needles have pnrsllol extinction, negative elongation, and an interference figum which is usually indefinite, hut o e & x m l l y may he O.A. at edge of field, and O.A.P. a t right angles to tho long needle &.xis. A few of the highly birefringent needles show 30' extinction. Mined Fusion with Thymol. A profusion of overlapping thin blades and needles grow into the melt (Figure 29). Most of these exhibit parallel extinction and positive elongation. Profile angles of 105' are often visible.
374
ANALYTICAL CHEMISTRY CAMPESTEROL
Cooling and Solidification of Melt. The melt supercools slightly. A few nuclei coalesoe to farm a smooth, fine-grained front, which moves with moderate crystallization velocity. On cooling further, the crystallization velocity decreases, the fine crystals grow into long, narrow laths of nearly uniform width; simultaneously the crystal front changes from smooth to slightly serrated. Transverse shrinkage cracks appear.
Figure 30.
b y means of a mixed fusion (4), it is readily seen that they form solid solutions, and are probably isomorphous. DISCUSSION
This work clearly reveds both the strength and weakness of fusion analysis 818 B means of identification. The appearance of some fusions-for example, cholestan-is so remarkable that subsequent recognition is inst,antancous, even if the observer is ignorant of optical crystallography. On the other hand, for compounds which decompose markedly a t the melting point,, the method is nearly uselem; this will also be true if the compound does not crystitllise-clionarterol, for example. Conipounds which behave like elionnstcrol may sometimes be induoed to orystallize by prolonged heating just below the melting point. Such treatment, however, requires apparatus not used in the present study. Fortunately, the formation of glasses which will not crystallize is rather rare. Isomorphism, as illustrated between the acetates of chalinsstcrol and clionasterol, may also cause difficulties. This again is & relatively rare occurrence; moreover, the fusion technique would identify the unknown as one of a very few possibilitics.
Carnpesterol Growing into Thymol
Solidified Preparation. Laths of very low birefringence show a. oontered O.A., optic sign positive, 2V large, and 0 . A . P normal to the long axis of the rod. Laths of highor bircfringence have parallel extinotion, and either (a) negative elongation, with O.A. a t edge of field, and a one brush intcrfcrcnee figurc, or ( b ) positive elongation, andvarying off-center O.A. views. Mixed Fusion with Thymol. Needles :and narrow l@hs grow (Figure 30). The profile angles are acutc, but are generally poorly defined in crystals broad enough to p c d t measurement:. One refractive index is Slightly- gmitrr than melt, and one refractive indcx is greater than melt,. CAMI'ESTERYL ACETATE
Cooling and Solidification of Melt. Thorc is mine supercooling. A few roughly sphcrulitic nuclei of radiating blades and needles unite to form 8. slightly serrabed front which move8 slowly. Occasional rough spherulcs form all during cooling of the molt. Complex fine shrinkagc clacks usually apposr in the broader orystals. Melthack. Interlaecd blades and iicedles grow with a slightly serrated front which gradually becomes nearly smooth. Solidified Preparation. Blades show scveral views (Figure 31), of which the following axe the most common: ( a ) p a r d e l extinction, positive elongation, O . A . a t edge of field (optic sign positive, 2V large), O.A.P. normal to long crystal axis, and ( b ) parallel extinotion, ncgative elongation, intcrfercnee figure indefinite (probably % slightly off-center &,). Mixed Fusion with Thymol. A mass of thin overlapping short blades grow into the thymol. CLIONASTEROL
Cooling and Solidification of Melt. A fine-grained, gray, barely visible mesomorph spread8 over the melt. The preparation solidifies to s glass, and i t is impossible t o cause crystallization by ordinary methods. CLIONASTERYL ACETATE
Behavior an fusion is identical with that shown by chalinasteryl acetate. If the two compounds me compared with one another
Tho use of eompilcd fusion data to identify an unknown presents small problom. In laboratories habitually concerned with relatively few compounds, there are no difficulties. The fusions of a few known compounds are easily memorized, and unknowns can then be recognirled in a few seconds. If the unknown can be any one of hundreds, this method becomes impossible. Punched cards srcm i o offer the simplest way out of this diffioulty. A labarstory could card index the gradually amumulating fusion data ( 5 ) in any one of number of ways. The card for each coinpound should preferably have 8 photograph of the fusion attached. Identifioation of an unknown from information available in this farm should be relatively rapid. A recent puhlication (3)has shown how similar data, for crystal optics, may be conveniently indrxed on such cards, ACKNOWLEDGMENT
The author wishes t,o thank W. Borgmann for supplying the pure sterols used, and for his interest in this work. LITERATURE CITED
Goetu-Luthy. N., J . Chem. Education. 26, 159 (1949). Kirkpatrick, A. F., ANAL.CHEM.,20,847 (1948). (3) Xofler, L., and Kofier, A,, "Mihomethoden sur Kenneeichnung organisoher Stoffe und Stoffepernische." Innabruok, Universi-
(1) (2)
tiitsverlag Wagner, 1948.
MoCrone. W. C., ANAL.CHEM.,21,436 (1949). (5) McCrone. W. C., et el., 16% monthly '.'Crystdlopraphic Data." (4)
RECSWEDMarch 15, 1950.
