A Sample Preparation Technique for the Application of Phase Contrast Microscopy to Polystyrene-Type Polymers Pascal A. Troylor, Special Services Laboratory, The Dow Chemical Co., Midland, Mich.
plastics represent RuBBsR-reinforced an .important part of the present plastics market. The performance of such modified polymers depends a great deal upon the nature and uniformity of their rubber dispersions. Therefore the development, production, and application of these rnateria.ls depend on the characterization of thc dispersions (1, 2 ) . The technique most useful for the resolution of dispersed rubber particles is that of phase contrast microscopy. Useful information is ohtaincd when a slice as thin as 2 microns is examined. This papcr deals with the prcparation of such slices, APPARATUS AND MATERIALS
MICROTOME.A sliding mierotomc is recommended for polystyrene-type polymers. The Spencer Model 860 sliding microtome gives good service with these materials. The knife-edge should bc very sharp and have a minimum of serrations (S). ROLL-OUTBATH. A Petri dish of glycerol is maintained at 110' C. on a small hot plate for the roll-out bath. MOUNTING LIQUID. The mounting liquid is prepared by dissolving Matheson Coleman and Bell reagent grade mercuric potassium iodide in glycerol and adjusting the refractive index to 1.59 a t 25' C. This solution should he filtered or decanted after settling to remove any dispersed solids which do not dissolve. MICRO~COPE. A Bausch & Lomh Model TBR dark phase contrast microscope using achromatic optics furnishes excellent contrast for polystyrene-type materials. PROCEDURE
Secure a piece of the plastic sample between wood blocks in the object clamp
of the microtome. With a razor blade trim a 0.5- to 5-mm. square slicing area. Tilt the microtome knife 30" and set it at a 45" slicing angle. Lock the automatic feed at 2 microns. Cut slowly and without hesitation, once the blade touches the object, until the slice is almost through. At this point push the blade hack to a safe distance and carefully unroll the curled slice with a finger. This reduces the tightness of the slice curl. Now cut the rolled slice free from the block, pick it up with a soft bristle brush, and flip it into the hot glycerol bath. If the slice roll out is not immediate upon contact with the hot glycerol, introduce a new slice. Generally, roll out is not delayed. Take up the rolled-out slice on a microscone slide. Wine the bottom of the slide'clean with a wet towel and airdry. Then with a razor edge carefully trim as much glycerol away from the slice as possible. Add a drop of glycerol -K2Hg14 (n*J 1.59) as a mounting medium. Tease the slice about in the drop for good mixing and then place a cover glass over the specimen. A thin cross section of plastic is now ready to be examined with the phase contrast microscope. DISCUSSiON
Proper preparation of a polystyrenetype polymer requires that the specimen be thin enough, lie flat, and be free of glaring interference from the surface. Generally a preparation is desired where the continuous phase component of the slice is completely masked out, and only the dispersed material is imaged by the microscope. The first consideration is met by using a sliding microtome and reducing the slicing area. It has been found in our laboratory that sections ranging in area from 4 to 25 sq. mm. are adequate
4 Figure 1. A phasecontrast photomicrograph revealing rubber dispersion in a rubber-reinforced polystyrene plastic MagniRcotion 4VOX Scale. 4.9 mm. = 10 microns
for full representation of most rubher dispersions. By keeping the area of slicing n-ithin this range good reproducibility of slice thickness is possible at a 2-micron feed setting. In many materials where rubber particle size and distribution are the only concerns, a slice of 4 sq. mm. is sufficient for good representation. For polystyrene thc roll-out bath is very effective when it is maintained at 110' C. In the case of materials which have higher heat distortion temperatures, it may he necessary to raise the bath temperature. When a slice of polymer is subjected to a bath temperature only high enough to give a flat slice, the polymer m.ill experience a slight annealing action. This aids glycerol wetting and brings no detectable physical damage to the specimen unless i t is highly oriented. In this case, most of the orientation will be lost. Polymeric objects which have heen injection-molded often solidify with high orientation. For such materials, if orientation is not important, it may be desirable to anneal in an oven a t 125' C. before slicing. This treatment will produce specimens which Kill flatten out at the normal roll-out temperature. If an oriented material is not annealed before slicing, a specimen will often take on the appearmce of a fried bacon strip and yield no flat areas unless the temperature, of the rollout bath is raised 20' or 30'. To presenie the polymer orientation, the sample preparation must be made without the roll-out bath. A slicing block about 1 inch long and I/p inch wide can he used. One can then ohtain a specimen long enough for each end to be captured by tweezers and held until it can be carefully placed in glycerol mounting liquid spread out on a microscope slide. A cover glass is quickly placed over the preparation before the slice frees itself from the glycerol and rolls up again. A specimen prepared in this way nil1 be poorly wetted by the glycerol mounting material, and microscope resolution will be low. To remedy this, repeated vacuum oven treatments of the mount will prove helpful, particularly if the temperature is maintained below the s o f e ening point of the plastic. The third consideration in sample preparation involves the type of mounting liquid. It should be inert to the specimen but capable of wetting the surface. Also, the suspended ruhher phase is best resolved by using a liquid with the same refractive index VOL 33, NO. 11, OCTOBER 1961
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as the polystyrene. This eliminates all interference caused by the rough cut surface of tlie polymer. When glycerol is used in the roll-out bath, a glycerol-potassium mercuric iodide solution (n% 1.59) meets all requirements and is a good mounting medium.
K i t h all three considerations met, a sharp knife, and a little experience, a cross section can be prepared on many impact-grade polymers in as little as 5 minutes. d typical photomicrograph produced by this technique is shown in Figure 1.
LITERATURE CITED
(1)
Claver, G . C., Merz, E. H., Ofic. Dig.
Federation Paint a n d Varnish Prodicction C h b s 28, 858-68 (1056). ( 2 ) Llcrz, E. H., Claver, C:. C., Barr, J . Polymer Sei. 27, 325-41 (1956). ( 3 ) Richard$, 0 . IT.. "Effective r s e and Proprr Carr of the Microtome," Aiiiierican Optical eo.: Buffalo 15, S . T., 1949.
Rapid Detection of Traces of Peroxide in Ethers Patrick R. Dugan, Mircobiological and Biochemical Center, Syracuse University Research Corp., Syracuse, N. Y
Thc reagent is an aromatic amine. and therefore n ill form Schiff bases with aldehydes: if they are present in tlie ethers. EXPERIMENTAL Solutions containing from 0.1 to 1% of a variety of aldehj des n ere prepard Reagents. . ~ ~ , ~ ~ - T ) i m c t h ! - l - ~ - p l i ~bj~ n ~di-solving lin absolute CH,OH: enedianiinc sulfate n a s prepared h>2 ml. of each n a ' reacted nith 2 ml.-sf dissolving 0.3 gram in 10 nil. of reth(3 rc,agent. distilled watw in a 100-nil. volumetric flask, arid brought t,o tlic mark with RESULTS A N D DISCUSSION frtsh C.P. absolutcx nictlianol. Procedure. T K O millilitcrs of tlic h d w p rcd-blue reaction product' was reagcnt \vas n i i r d ivith from 2 to !5 obscricd whcn the reagcnt iyas mixcd nil. of tctrahydmfuran ( T H F ) in a with T H F ; h o w v e r . no color changc test tulw and allocvetl t'o stnnti for 5 del-eloprd \\-hen t'he T H F had first niinutcs in rubtlucd light a t roo111 h c n distillrd over sodium, a proccw t ein p era t1.1 re. A c. o nip arisoi i t ul)r n-(Ire uscd for the r e m o d of pcroxides (and preparcti in thi. sun(' nianiicr by adding 2 nil. of tt.ti,ahydi,ofurati n-hicli also aldchytics) . had hccn clistill(~t1 o v r r d i u m in Srvrral grades of diethyl ether w u e p l a c ~of the 2 nil. of st,ot-kT H F . examined. In all cases in which tlic TTVO millilitcrs of tlic rcngcmt n-cw rrducing agcnt, sodium dicthyldithioalso added to 2 ml. of mvh of scvcral carhamate, had been addcd to the ether, samplcs of dicthyl et'liw, isopropyl no color change could bc drtccted. ether, and diosanc whirh had bem Honevcr, the typical colortd coniplcs stored in thc laboratory stock room. rcw1tc.d whrn ethcr n h i c h had not 1m.n stabilized n-ith sodium diethyldithiooarbamatc was used. Table I. Comparison of Absorbance Values of Lauroyl Peroxide to Benzoyl 'l'he color complex \vas formed n-hcn Peroxide a t 570 Mp with Respect to Active Oxygen Content 2 nil. of dioxanr n-hich had hren stored 1,aaroylfor scvcral months in a partially filled Peroxide Benzoyl bottlc was employcd; but no color Concn.,a pg./hIl. Lauroyl Peroxide Benzoyl Peroxide Ratio drvrloped when 2 nil. from a freshlj10 0.051 (89% T ) 0 . 1 0 2 ( i 9 % 2') 0.50 opcncd lmttle was uscd in the reaction. 20 0 . 1 0 2 ( i 9 7 0 2') 0.195 (645; 2') 0.52 30 0.155 (70% T ) 0.285 (52% 2') 0.54 If the absorbance (or %I") of the 40 0.211 (6170 2') 0.446 (3670 7') 0.48 colored solutions as previously rcport'cd Av. 0 . 5 1 is compared to the ratio of the active Active oxygen content, yo 3.8 6.8 ;zv. 0 . 5 6 osygcn content of benzoyl and lauroyl 0 hlicrograms per ml. of 2 nil. added to reaction t h e . peroxide, the absorbance values of the colored complex from both peroxide reactions appear nearly equal (see Table II. Absorption of Reaction Product of Aldehydes with Reagent 1 at Selected Table I). W a v e Lengths vs. Reagent Blank It is therefore suggested that the % Concentration of Aldehyde in Absolute CHdOHa amount of active oxygen can be de0 1 0 3 0 5 0 7 1 0 termined on the basis of this reaction 545 5iO 590 535 570 590 545 570 590 545 570 590 E i m O and that the amount of active oxygen Aldehyde mp mp mp nl9 in9 mp mp mp mp mp mp mp mp mpmM in ethers can possibly be quantitated Formaldewithout knowing the specific chemical hyde S o effect KO effect K-Propionstructure of the peroxide. aldehyde 100 108 97 65 TG 65 50 61 50 32 44 33 . . . . . . The method is a rapid qualitative K-T'alerindication of peroxide content in ethers aldehyde 113 118 110 . . . . . . , . . 98 106 100 . . . . . . 80 92 85 and possibly other solvents, and may A'-Hep< aldehyde 112 119 108 104 112 104 96 106 96 86 99 89 77 90 80 be an aid in controlling accidents Benzaldecaused by peroxide-induced explosions. hyde 65 94 99 Comparison of O/oT values a t 570 Glucose S o effect No effect mp (Table 11) shows that the aliphatic a i'alues at 545, 5i0, and 590 m M represent minima, maxima, minima, respectively, of aldehydes tested decolorized the reagent absorption peak a t which peroxide color is determined. somewhat when added in concentrations for detecting microgram quantities of benzoyl and lauroyl peroxides [AiV.AL. CHEM. 33, 696 (1961)] was developed to detmninc, the prtsence of these perosides as they migrated from polymers. 1rliic.11 had been formed using peroxidc ental)-sts in polymcrization, into various solvents. The publication referretl Fpccifically to cictection in benzene and mineral oil. I t has since been dctcrminctl in this laboratory that thc reaction is suitable for dctecting tracm of pcroxidc in diethyl ether, isopropyl cther. diosanc! and tetrahydrofuraii. Because the method is so rapid and simple, and because peroxide formation in ethers in general can be hazardous: particularly when ethers are evaporated in an anhydrous condition (Soller, C. B.,"Chemistry of Organic Compounds," p. 139, Saunders Publishing Co., Xew York, 1950), it should be a useful tool in the hands of invcstigators
A
METHOD
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ANALYTICAL CHEMISTRY
working with ethcrs as w l l as tho-e concernd with quality control.
