Microscopic Examination of Modified Starches IUnL'AS J O H N S C H O C H and EILEEN C. MAYWALD M, Molfett
Research Laboratories, Corn Products Relining Co.. Argo,
Various microscopic techniques are useful for charaeteriaing commercial modified starohes. Granule aggregation can he detected by examination in water and glycerol media. The composition of starch blends can frequently he evaluated by granule counts in a haemacytometer. The species of a pregelatinized starch is determined by destroying the gelatinized material with ennyme and examining the ungelatinized residue. The KoEer miomsoope hot stage provides ;a simple and accurate measurement of gelatinization temperature, particularly useful for studying the effects of various adjuncts on starch gelatinization. Chemical modifieation of granular starohes progressively lowers the gelatinization temperature, and the extent and uniformity of derivatisation can be estimated by this means. Positively charged dyes-.#., methylene b l u e s t a i n anionic pmducts such as oxidized starches, phosphate esters, and carboxymethyl ethers. Cationio starches stain only with negatively charged dyes-eg., light green SF. Intensity and uniformity of staining provide evidence of hlended starches and nonuniform derivatives.
T.
H E microscope has been traditionally employed for identifying the species of an unknown staroh from the size and shape of its granules. Newer microexamination techniques have proved invaluable for the diagnosis of process engineering problems, for the analytical characterization of modi6ed starches, and as an important aid in general starch research. A brief inspection under the microscope frequently provides information which could not possibly be obtained by any other means. The present atudies employed a polarizing microscope fitted with Ahrens prisms and capable of magnifications of 100 t o 500 diameters. Individual objectives should be centerable and of the "quick-ohsnge" type. A highly useful accessory is the K 0 t h micro hot stage. point deterniina.. orieinallv . designed . for melting~. tions and microsublimations, hut ideally suited for determi ning gelatinisation temperatures of starches.
111.
Starches may sometimes be dried under too rigorous conditions of heat and moisture, producing a slight gelatinization of the surface of the granule; as a consequence, the granules Nil1 adhere together in clumps which do not break apart in glycerol medium. Figure 1 depicts a rice starch properly dried 60 that each granule is separate. I n contrast, Figure 2 shows a second rice starch in which the granules are dumped into large aggregates. This product was dried in a flash dryer, and the clumping nras provoked by excessive moisture of the starch feed to the dryer, together with too high an inlet air temperature. Flash drying is usually sat,isfactary for starch, hut operations should he checked occasionally with the microscope. Granule aggregation has several undesirable consequences. The bulk density of the dry starch is snbstmtially reduced-for example, the density of the aggregated rice starch in Figure 2 was 26 pounds per cubic foot compared with 33 pounds for the disaggregated sample in Figure 1. I n addition, aggregated starch is definitely inferior for such u ~ e s ad cosmetic dusting powder or a release agent for automobile tire molds. In most cases, these aggregates fall apart immediately and completely when the starch is suspended in water rather than in glyoerol. Any aggregation which persists in water medium may cause trouble in subsequent use, sometimes gelatinizing t o give micro lumps which do not break down on cooking. An instance is the clay coating of paper, where unmodified starch is pasted and converted with liquefying enzyme t o provide a carrier and adhesive for the clay. There have been isolated instances where granule aggregates have persisted through this process, subsequently giving specks on the highgloss paper and interfering with printing. Hence, examination of the starch in both glycerol and water media offers a useful test for quality control. Nonqueous mountants will sometimes reveal the presence of adjuncts. For example, a commercial mixture of corn dextrin and ca~ho~ymethylcellulose (for use M a rayon warp size) was readily identified when examined in glycerol medium under polariaed light, which showed the' birefringent fibrils of the cellulose derivative. Similarly, the presence of admixed harm
PRELIMINARY EXAMINATION
Initial inspection of a starch should he made in water suspension, never on the dry granules. A 0.2 to 0.3% suspension gives about the proper population of the field. The species of starch, judged by the size and shape of the granules and the position and fissuring of the hilum, can then be determined by comparison of the unknown against samples of known starches, preferably on the same slide. The procedure of alternate inspection of the unknown against the known frequently brings out minor differences which cannot be derived from comparison with a photomicrograph, although there are many good ones in the literature (6,8). A collection of permanent slides of the various starches mounted in Canada balsam or in one of the polymerizing synthetic resins is convenient for this purpose. The position of the hilum in the granule, whether centric or eccentric, is an important aid in identifying the starch species. With certain starches the hilum is difficult to disown; the eccentricity of the interference cram under polarized light then offers the best criterion of its location. The relative brightness of the polarbation cram is also frequently helpful for identification. The starch should likewise he examined in glycerol medium, primarily t o provide information on granule aggregation.
Figure 1.
382
Disaggregated rim glycerol
V O L U M E 28, NO. 3, M A R C H 1956
383 defined and msy be easily and accurately counted in the hemacytometer. GRANCLE SIZE DISTRIBUTION
During studies on size fractionation of starch granules, it wm necessary t o obtain granule size distribution curves for corn and sorghum starches. To do this, photomicrographs were taken of a number of representative starch samples in water medium, and these were enlarged to exactly 500 diameters. Granule diameters were then measured manually with a rule to the nearest millimeter-i.e., equivalent to 2 microns. It was found necessary to measure 600 to 1000 granules of each starch sample to obtain reproducible curves. Figure 3 shows the granule size distribution of these starches on a population basis. The “number average” granule diameters for commercid corn and sorghum starches were 9.2 and 15.0 microns, respectively, as calculated by the formula Number average diameter = Z (number %of each granule size X granule diameter) 100 Figure 2. Aggregated rice starch, mounted i n glyceml
or boric acid can be discerned by mounting in light mineral oil. Fissures and hilum damage are readily detected by mounting in clove ail, which has the same refractive index (1.530) as starch, thus making the hilum stand out in sharp contrast. GRANULE COUNTS
It is frequently necessary t o estimate the proportion of two different types of starch granules in a sample. This can be done with a blood-counting cell or hemacytometer, preferably of the “brigheline” type with Nenbauer double rulings. For the estimation of waxy starches, an aqueous suspension of the sample is stained lightly and uniformly with iodine and the redstaining waxy granules and the blue-staining normal granules are then counted. All waxy starches (except genetically pure preparations) contain a small percentage of blue-staining starch granules due to c r o pollination. ~ For quality control over waxy starch praduotiau, accurate counts on 500 to 600 granules can be made within 10 minutes by means of a hand tally. Precision is +1 part in 10, adequate for routine control. Commercial blends of waxy maiee and normal corn starch are now common and their identity and composition can also he readily established by this method. Mixtures of two different starch species are more difficult t o determine by count because of difference in granule size. I n such oases, the unknown sample must he compared against known synthetic mixtures. For example, mixtures of Idaho potato starch and corn starch, made up to contain 10, 25, 50, and 75% by weight of corn starch, were observed to contain 49, 70, 84, and 94%, respectively, by number count of corn starch granules. Granule counts may also be used to estimate minor proportions of partially swollen granules in process starch samples, Such gelatinization may be caused in an aqueous starch slurry by the incautious addition of agents such as alkali, or by local overheating, such as might be caused by contact with the wall of a steamjacketed kettle. The presence of partially swollen granules may cause such processing difficulties sa plugged filters, slow settling, and excessive solubles, sa well as indispersible grit in the finished dry starch. The literature makes frequent mention of Congo red as a preferential stain for partially swollen granules, However, diminution or absence of the characteristic interference cram when viewed under polarized light is s. much more positive and readily discernible criterion of swollen granules. I n most instances of this sort, the partially swollen granules are sharply
These two starches cannot be definitely distinguished by visual inspection, because this difference in average size cannot be readily detected by the eye. From a practical standpoint, siae distribution curves are significant only when converted to a weight percentage basis. Assuming that all granules have the same density, this calculation wsa made by multiplying the number percentage . of each .. 6rsnule size by the cube of its diameter. ~
71Veight
%= number % of granules of specified diameter X cube of dirtmeterX 100 . . .. Z (number % ’ , X cube a! diameter)
r
10.
8.
i,4 -/ ,
s
22-
4
6
8
10 12 14 16 18 20 Gronuie Diorneter, 8 ” microns
22
24
Figure 3. Granule size distribution of corn and sorghum starches, calculated on a population basis Arrows indicate respectise “number avevage granule diamqtun”
Figure 4 shows the granule size distribution of the same corn and sorghum starches calculated on a weight percentage basis. For example, a value of “8 weight % ’ of granules of 17-micron diameter would signify that 8% by weight of the total starch has a granule diameter between 16.5 and 17.5 microns. The “weight average granule diameters”-Le., the diameter of a granule of average w e i g h t f a r corn and sorghum starches are 14.1 and 17.4 microns, respectively, as calculated by the formula 2 (weight
% of each granule Siee X granule diameter) 100
Such data have utility in following the recovery of starch in tabling and centrifuging operations. As a further example, it might be desired to separate that fraction of corn starch having a granule diameter of 8 microns or less. This fraction consti-
ANALYTICAL CHEMISTRY
384
12-
Figure 5. Kofler microscope hot stage for determination of gelatinization temperatures 6
S
IO 12 14 i6 18 Granule Diameter. 8” micronr
20
22
24
Figure 4. Granule size distribution of corn and sorghum starches, oalculated on a weight percentage hasis Arrows indicate respective “weight average granule diameters”
tutes 45Y0 of the total number of granules, but it represents less than 8% by weight of the total starch. PREGELATINIZED STARCHES
A variety of pregelatinieed stmehes are manufactured by drying 8 precooked starch paste an drum dryers, on heated squeeze rolls, or in a spray dryer. These products are marketed for such purposes as paper heater size, foundry core hinders, and a8 a bodying agent for oil-well drilling muds. The method which is used for drying is usudiy determined by suspending a sample in glycerol and examining it under the microscope. If the particles appear as irregular chunks, the pasted starch N&S probably dried in a relatively thick layer on a drum dryer, then ground and screened. If the starch is in the form of thin irregular plates, like shattered window glass, drying was effected on the squeeze rolls. Spray drying gives hollow spheres enclosing an air cell. It i8 frequently necessary to identify the species of starch in such pregelatinized produots. Preliminary examination should be made in water suspension under the polarizing microscope. If the product has been insufficiently pasted, i t will show a multitude of ungelatiniaed granules with their characteristic polarization crosses. I n such instances, the species of starch e m be readily identified, An approximation of the proportion of ungelatinieed starch can be made by comparison against synthetic blends of granular and pregelatinized starches. Commercial products have been encountered in which as much as 20 to 30% of the starch remained ungelatinized and, hence, ineffective for the contemplated use. Usually the amount is less than 1%, and frequently it is almost vanishing. These latter products present difficulties in identification, since the few remaining granules are obscured by the mass of gelatinized flakes. I n these cases, 5 to 20 grams of the starch sample is stirred into 100 ml. of a filtered solution of a low-temperature amylase, such as Rhozyme S or Vanzyme. The ensyme solution must first be carefully filtered t,a remove any granular starch which may be used as a carrier for the enzyme. The mixture is maintained at 40‘ C. for 30 to 60 minutes to digest and dissolve the gelatinized starch flakes, leaving the ungelatinized granules intact. The solution is then centrifuged, the supernatant liquid is decanted, and the trace of insoluble residue examined under the polarizing microscope. No commercial pregelatinized starch has yet been encountered which was not identifiable by this means. Apparently, even thoroughly cooked products contain a few thousandths of a per cent of intact granules which somehow escape gelatinization. Because of its sensitivity, the method must be used with considerable care and discrimination. In the laboratory of a starch processing plant, there are always enough air-borne starch granules to represent a significant source
of contamination. If corn and potato starch granules are detected in a sample, some question may arise whether this represents a blend, or whether contamination has occurred during manufacture. Considerable difficulty is encountered with high-protein corn and wheat flours, where protein obscures the ungelatiniaed granules. I n this connection, when the honeycomb structures of cell walls are observed in B pregelatinized high-protein corn sample, an origin of dry-milled corn is presumed. This enzyme digestion technique sometimes has usefulness in identifying the starch species employed in surface-sizing a. fabric. A strip of the fabric is suspended in a warm emyme solution for 15 to 30 minutes. If the strip is then agitated, a perceptible amount of materid will slough off the surface. The strip is removed and thoroughly rinsed with a jet from the wash bottle. The combined enzyme solution and rinsings are centrifuged, and any slight trace of precipitated material is examined under the microscope. Persistent granules can frequently be identified, particularly if a thick-boiling starch has been employed in sizing. DETERMINAAON O F CELATINIZATION TEMPERATURE
The gelatinization temperature is best defined as the point at which the starch granules lose their polarization crosses when heated in a swelling medium. This loss of polarization immediately precedes swelling. Actually, all the granules in a given sample do not lose their polarization simultaneously, but usually over a range of some 8” to 10‘ C. Hence, a proper measurement of gelatinieation temperature should indioate both the initial and terminal paints. Determination of gelatinization temperature by the usual hot water bath technique is tedious and very frequently inaccurate. Even with slow heating in a triple bath, hot walls cause local gelatinization of the granules. However, it has been found that the Kofler electrically heated microscope stage (Arthur H. Thomas Co., Philadelphia) provides a quick, simple and accurate evaluation of gelatinization temperature. The Kofler stage (Figure 5 ) consists of a circular metal block heated by internal resistance coils. On this block is placed a glass slide carrying a droplet of starch suspension. , To ensure uniformity of temperature, a glass baffle is placed above the slide, and the whole assembly is covered with a close-fitting glass plate. A thermometer is inserted in the metal block in such a position that the stem can be kept under consttlnt observation. A small variable transformer provides accurate temperature control. The hot stage should be tested by determining the melting points of known substances. To determine the gelatinization range, the starch is slurried in water t o give a 0.1 to 0.2% suspension. A small drop of this suspension is then spotted on a microscope slide. The drop is surrounded by a continuous ring of high viscosity mineral oil and a cover glass dropped on in such a way that the %gueousdrop is completely enclosed by an oil barrier, with no air bubbles under the cover glass. The purpose of this oil barrier is twofold: (1) to prevent the escape of steam which fogs the glass baffle, and (2) to prevent air channels from penetrating under the cover glass and disturbing the field, The rate of temperature rise
385
V O L U M E 28, NO. 3, M A R C H 1 9 5 6
The gelatinization range becomes an important consideration when starch is used in other than simple water systems. For example, increasing concentrations of ammonium nitrate or urea progressively lower the gelatinization temperature, while ammonium sulfate markedly raises it. Corn starch, in water Same, in NHdNOa so!ution 10% 20% 30% 40% 50% 60%
70 % 80 %
Temperature,
OC
Figure G . Gelatinization curves for corn starch, for hydroxyethyl corn starch (0.12 degree of substitution), and for a 50-50 mixture of these two starches Latter sample showed no granules gelatinizing in range of 55' to 60° C .
n-hen approaching and during the gelatinization range should be about 2" C. per minute, corresponding to the 23-volt setting on the variable transformer. The field is continuously watched under normal lighting to determin ethe temperature at which the first three or four granules show a change in physical outline. Loss of polarization crosses in these granules can be immediately confirmed by introducing the Ahrens analyzer. This is the initial point in the gelatinization range. Heating is continued] and the field is now watched under polarized light. When the polarization crosses have disappeared in all but perhaps tn-o or three granules in the entire field, this temperature is recorded as the completion of the gelatinization range. Frequently, a very few granules will persist for an additional 5" C. or more, but it seems advisable to neglect these. Duplicate or triplicate runs should he made and the results averaged; in general, precision has been zk0.5O C. I t is often advantageous to include a midpoint in the gelatinization range-namely, the temperature at which approximately half the granules have lost their polarization crosses. In certain of the present studies, full gelatinization curves have been obtained by observing the temperatures at which the following percentages of granules have lost birefringence: initiation, 10, 25, 50, 75, 90%) completion. These points can be determined accurately by visual inspection during the gelatinization, and this technique gives much more reproducible curves than the r a t e r bath method, despite the fact that no exact count of gelatinized granules can be made. Typical sigmoid gelatinization curves are illustrated in Figure 6. The various species of starch have different gelatinization temperatures, n-hich are fairly uniform n-ithin any given variety Corn starch, various batches Waxy maize starch Sorghum starch, various hatches
fi2-07-70' C . 62-730 62-71.5' C . 6 2 . 5-68-7?O C 68-75' C .
c.
fiR-74"
c.
68-ir . 5 0 .c.
C. 6 8 . 5 - 7 5 . 5 0 c. 68.5-75'
Waxy sorghum starch Idaho potato starch Maine potato starch Dominican tapioca
67.5-70-740 c. 56-67' C. 59-62.5-67. C. 58.5-64.5-70O C .
These represent temperatures which cannot be approached too closely in the starch factory mithout risking partial gelatinization. The waxy starches have substantially the same gelatinization temperatures as their normal blue-staining counterparts. Hence, the presence of the linear starch fraction does not affect initiation of granule swelling, though it is recognized that waxy granules swell more rapidly and are much more fragile than the normal blue-staining starches,
Same, in urea soluhion 18% 20% 30% 40 %
62-13'
C.
62-75'
C.
54.5-70.5' C. 52-Gfi.5' C. 44.5-63" C. 42-61' C.
Room temp.-57' Room temp.-55' Room ternp.-53'
C. C. C.
51-64' C. 39-550
c.
Room temp.-47' C. Complete a t room temp.
Same. in (NHdzSO4 Rolution 10%
30%
Esterification or etherification of granular starch progressively lowers the gelatinization temperature as the degree of substitution (D.S.) is increased ( 3 ) . This is exemplified by the gelatinization ranges of several hydroxyethyl ethers and of the "Feculose" acetate esters (9) of corn starch. Feculose starch acetate, 0 . 0 4 D.S. 0 . 0 8 D S. 0 . 1 2 D.S. Hydroxyethyl corn starch, 0 04 D.S. 0 12 D.S. Unmodified corn starch
66-58-63' C. 48-52-56' C. 41-46-81' C. 57.5-67.5' C. 4 5 .
