Semipermeable films useful as dialysis membranes are prepared from starch-based composites. The films are shaped by conventional methods from a plasticized matrix comprising highly gelatinized starch and ethylene acrylic acid copolymer neutralized with a strong alkali. These films are remarkably transparent and resistant to degradation in the presence of aqueous solutions.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
While cellulose-based films have achieved prominence as semipermeable 
membranes, starch-based films have not been available that would withstand 
prolonged exposure to water. Starch and cellulose are high polymers 
composed of D-glucose units. Their molecules differ only in weight and in 
the manner in which the glucose units are joined together. Cellulose is a 
linear polysaccharide consisting of 6,000 to 8,000 1,4-linked 
.beta.-D-glucose units. Because of this 1,4-.beta.-linkage, these chain 
molecules can align themselves alongside each other to form linear 
crystals or microfibrils. These structural properties contribute to strong 
hydrogen bonding, film-forming capabilities, and high resistance to 
gelatinization in water. In contrast, most common starches contain 17-27% 
linear polysaccharide (amylose) consisting of 400 to 1,000 1,4-linked 
.alpha.-D-glucose units and the remaining composition is a branched 
molecule (amylopectin) having 10,000 to 40,000 1,4- and 1,6-linked 
.alpha.-D-glucose linkage. Because of the 1,4-.alpha.-linkage, the amylose 
molecules assume a spiral or helical shape having six glucose units per 
spiral. Starch readily disperses in hot water to form starch-pastes 
possessing unique viscosity characteristics and film-forming behavior. 
However, such films are very brittle upon drying and are very sensitive to 
water. This invention relates to certain starch-based formulations which 
yield durable semipermeable films having potential applications as 
dialyzing membranes. 
2. Description of the Prior Art 
In U.S. Pat. No. 4,337,181, Otey et al. teach the preparation of flexible, 
starch-based films which are water resistant, yet biodegradable. The films 
are formed by shaping a composite of a water-dispersible ethylene acrylic 
acid (EAA) copolymer and a starchy material by the methods of extrusion 
blowing, simple extrusion, or molding. The resultant products have 
disclosed utilities as agricultural mulch, as well as disposable packaging 
and bagging materials. In this procedure, gelatinized starch and EAA are 
combined in a plasticized matrix, and then the acidic portion of the EAA 
is at least partially neutralized with ammonia or an amine. It is by 
virtue of the neutralization and adjustment of the moisture content that 
the matrix can be extrusion blown. Films prepared by this process have no 
observed permeability properties. 
SUMMARY OF THE INVENTION 
It has now been surprisingly discovered that when starch-EAA formulations 
such as those of Otey et al., supra, are neutralized with a strong alkali 
as opposed to ammonia or an amine, it is possible to prepare films having 
semipermeable characteristics. These results are accomplished by 
converting to a plasticized matrix a mixture of highly gelatinized starch, 
EAA, and sufficient strong alkali to neutralize substantially all of the 
EAA. The matrix is thereafter shaped into a film by any method known in 
the art. 
In accordance with this discovery, it is an object of the invention to 
prepare starch-containing semipermeable films useful as dialysis 
membranes. 
It is also an object of the invention to prepare highly transparent, 
starch-based films which are resistant to degradation under conditions of 
dialysis. 
It is a further object of the invention to tailor the permeability of the 
films of the invention to predetermined molecular sizes by controlling the 
formulation constituents. 
Other objects and advantages of the invention will become readily apparent 
from the ensuing disclosure. 
DETAILED DESCRIPTION OF THE INVENTION 
"Films," such as those made in accordance with the invention, are defined 
by the polymer industry (Encyclopedia of Polymer Science and Technology, 
John Wiley & Sons, Inc., 1967, Vol. 6, page 764) as "shaped plastics that 
are comparatively thin in relation to their breadth and width and have a 
maximum thickness of 0.010 in." Self-supporting films are those capable of 
supporting their own weight. "Uniform films," as used in this application, 
refer to those which are virtually free of breaks, tears, holes, bubbles, 
and striations. 
"Semipermeable membranes" are those films through which certain substances 
pass while others are retained. Typically, it is at the molecular level 
that such membranes are semipermeable. 
"Composite" is defined herein in accordance with The American Heritage 
Dictionary of the English Language, New College Edition, published by 
Houghton Mifflin Company, page 27, to mean "a complex material . . . in 
which two of more distinct, structurally complementary substances, 
especially . . . polymers, combine to produce some structural or 
functional properties not present in any individual component." 
The starch-based films of the invention are prepared from any unmodified 
starch from cereal grains or root crops such as corn, wheat, rice, potato, 
and tapioca, from the amylose and amylopectin components of starch, from 
modified starch products such as partially depolymerized starches and 
derivatized starches, and also from starch graft copolymers. The term 
"starchy materials" as used in the specification and in the claims is 
defined herein to include all starches, starch flours, starch components, 
and other starch products as described above. The term "starch-based" in 
reference to the products of the invention is used in the broad sense of 
containing a starchy material. 
In the preparation of the instant starch-based films, the starchy materials 
must be highly gelatinized by the time of the shaping steps. By "highly" 
gelatinized, it is meant that all or substantially all of the starch 
granules are sufficiently swollen and disrupted that they form a smooth 
viscous dispersion in the water. Gelatinization is effected by any known 
procedure such as heating in the presence of water or an aqueous solution 
at temperatures of about 60.degree. C. The presence of strong alkali is 
known to facilitate this process. The gelatinization may be carried out 
either before or after admixing the starchy material with the EAA as 
discussed further below. 
The EAA copolymer must have sufficient carboxyl functionality so as to be 
compatible with the starch for purposes of preparing the disclosed films. 
It is believed that the pendant carboxyl groups supplied by the acylic 
acid component associate with the hydroxyl groups of the starch, thereby 
contributing to the compatibility and composite formation of the starch 
and the EAA. These carboxyl groups coincidentally contribute to the water 
dispersibility of the copolymer. We have found as a rule of thumb that if 
the EAA is water dispersible, it will also be sufficiently compatible with 
the starch. 
The preferred EAA is a product prepared by copolymerizing a mixture 
comprising about 20% acrylic acid and 80% ethylene, by weight. However, it 
is to be understood that EAA copolymers having somewhat different 
proportions of polymerized acrylic acid and ethylene would also yield 
acceptable starch-based films provided that they contain a sufficient 
number of carboxyl groups to be water dispersible. 
The strong alkali of choice for use in the invention is sodium hydroxide, 
though potassium hydroxide would also be effective. The amount added to 
the film compositions may be varied over a wide range so long as enough is 
initially present to equal at least one equivalent per equivalent of acid 
in the EAA. Normally, the level of alkali addition will be about 2 to 8 
weight percent based on the dry weight of the starch-EAA formulation. The 
alkali cation is believed to form a salt with the acid as evidenced by the 
weight change observed upon soaking and redrying the film. Addition of an 
amount of alkali in excess of that required for neutralization insures 
that the starch attains an adequate degree of gelatinization. 
On a dry weight basis, the starch:EAA ratio must be at least 20:80, and 
should not exceed about 60:40. The range is preferably from about 30:70 to 
about 50:50. Below ratios of 20:80, the permeability of the film to even 
the smallest of molecules becomes insignificant. At this level, we have 
found that only trace amounts of urea (MW 60) will pass through. As the 
starch:EAA ratio approaches 60:40, the degradation and tear resistances 
drop considerably, the film becomes translucent, and the other physical 
properties become fair to poor. Within the designated ratios, the 
proportions may be varied to tailor the films' permeability to the desired 
end-use. Permeability may also be controlled by selection of the starchy 
material type. 
If the starch has been pregelatinized, its moisture content at the time of 
addition to the starting mixture is not particularly critical provided 
that enough moisture is available in the system to permit dispersing the 
EAA. If the added starch is granular, sufficient moisture must be provided 
to allow partial or complete gelatinization. Either way, during the 
initial mixing of the formulation components, at least 10-20% water based 
on total solids should be present. Excess moisture is then removed from 
the composition by evaporation during the subsequent processing 
operations. 
In the preferred embodiment of the invention, the starch is gelatinized in 
the presence of the strong alkali during the mixing operation. The mixture 
should be heated sufficiently to simultaneously gelatinize the starch and 
melt the EAA, resulting in the formation of a plasticized matrix. With the 
aforementioned EAA copolymer comprising 20% acrylic and 80% ethylene, this 
can be accomplished at temperatures as low as 60.degree. C., though the 
operation is more readily conducted at 95.degree.-130.degree. C. 
Shaping of the matrix into the semipermeable films is most expeditiously 
achieved by means of extrusion blowing. The term "extrusion blowing" is 
well known in the art and distinguishes from simple extrusion in that it 
relates to shaping a tubular extrudate, or "bubble" into its form by 
internal and external cooling streams of air, the internal stream causing 
expansion of the bubble to several times the size of the die opening. 
Films prepared by this technique are commonly referred to as "blown 
films." By continuous feeding of the plasticized formulations of this 
invention into the blowing apparatus, uniform, continuous blown films can 
be readily obtained. 
The moisture content of the film formulation just prior to and after 
blowing must be maintained within the range of about 2 to 10% (w/w) and 
preferably between 5 and 8%. Compositions with moisture contents outside 
of this range do not produce a uniform, continuous film. 
A second stage of mixing at temperatures of 125.degree.-145.degree. C. is 
suitable for adjusting the moisture content to the appropriate level. 
Since the formulations are readily blown at these temperatures, further 
temperature adjustment is unnecessary. Of course, the gelatinization, 
mixing, moisture reduction, and film blowing could all be conducted in one 
continuous operation using commercial equpiment with heating, mixing, 
venting, and extrusion blowing capability. 
It is envisioned that the films could also be formed by other known methods 
to include simple extrusion, milling, and casting provided that the 
plasticized matrix is prepared accordingly. Typically, the formulations 
and preparatory steps for simple extrusion would be substantially the same 
as those set forth above. In casting, the EAA, starch, and alkali are 
dispersed in water in an amount of about 5-15 times the weight of the 
starch. Treatment in a high-shear blender accelerates dispersion. When 
heated, the resultant suspension is converted to a thin plasticized matrix 
which is readily cast and dried in a suitable manner. For purposes of 
milling, it is advisable to hold the moisture content to the minimal level 
required for gelatinization. The formulation of ingredients is passed 
through a conventional mill such as a rubber mill, and the resultant 
plasticized matrix is rolled into a thin sheet or film. 
The product as produced by any of the aforementioned procedures is a 
flexible composite of the gelatinized starch and the EAA salt. Without 
desiring to be bound to any particular theory, it is believed that the EAA 
salt associates with the gelatinized starch molecules and holds them in 
the same expanded flexible state in which they exist in the heated matrix. 
The instant films are transparent. 
As indicated by the relative diffusion rates of diverse molecules, these 
films have semipermeable properties useful in a variety of applications as 
known in the art. The absence of these properties in films produced by the 
process of Otey et al., supra, establishes the criticality of 
incorporating strong alkali into the formulation. 
The films are prepared for use as dialyzing membranes by conditioning them 
in water or other suitable aqueous solution. During soaking, they imbibe a 
considerable amount of water, causing expansion and a leaching out of any 
excess alkali. When mounted in test dialysis cells in the presence of 
water, the expanded membranes tend to remain stable for several weeks with 
no apparent distortion or change in appearance. 
Additives may be incorporated into the composites to alter their properties 
during preparation or in use. It was mentioned above that these films 
could be tailored for specific permeability characteristics by controlling 
the relative proportions of starch and EAA, as well as by the particular 
choice of starchy material. It is also possible to enhance permeability by 
incorporation of water-soluble additives which are leached out during the 
conditioning step. Many conventional plasticizers and other extractable 
materials as determined by the skilled artisan are suitable for this 
purpose. However, the principle by which their extraction from the 
membrane alters the permeability characteristics is not currently 
understood. 
The following examples further illustrate the invention but should not be 
construed as limiting the invention which is defined by the claims. 
All percents herein disclosed are "by weight" unless otherwise specified.

EXAMPLES 1-3 
A. Film Preparation. A mixture of air-dried corn starch (11% moisture) and 
sodium hydroxide dissolved in an amount of water to provide 50% solids in 
the complete formulation were blended for 2-5 min. at 
95.degree.-100.degree. C. in a steam-heated Readco mixer (type: 1-qt. lab 
made by Read Standard Div., Capitol Products Corp.) to gelatinize the 
starch. EAA pellets (type: 2375.33 manufactured by Dow Chemical Co.) were 
added while heating and mixing were continued. After the mixture was 
stirred and heated for a total of about 45 min., the resulting plasticized 
matrix was extrusion processed with an extrusion head attached to a 
Brabender Plasti-Corder (type: PL-V300, manufactured by C. W. Brabender 
Instruments, Inc., South Hackensack, N.J.). The screw of the extruder was 
3/4-in. in diameter, 9 in. long, and had a compression ratio of 2:1. The 
die consisted of 24 circular holes of 1/32-in. diameter. This extrusion 
process was repeated usually one or two more times until the moisture 
content of the exudate was between about 5 to 10%. This exudate was then 
blown into a film by passing it through the same extruder except that the 
die was replaced with a heated 1/2-in. blown film die. The barrel and die 
temperature ranged from 105.degree.-110.degree. C. As the level of starch 
was increased, extrusion blowing became increasingly more difficult until 
at about 60% the limits of the equipment were attained. 
B. Semipermeability Analysis. Rotating dialysis cells were constructed from 
"Plexiglas" as follows: Each half of the cell was prepared by cutting a 
round 9.4-cm. diameter hole from a 0.6-cm. thick "Plexiglas" acrylic disc 
(13 cm. diameter), and this disc was then laminated to the same diameter 
"Plexiglas" disc that was 1.3-cm. thick. A "Plexiglas" rod (3.5 cm. 
diameter and 3 cm. long) was glued to the outside corner of each half for 
attaching the motor chuck. Two filling holes were drilled 180.degree. C. 
apart on the edge of each compartment and provided with threaded plugs 
fitted with O-rings. Four-inch O-rings were fitted into grooves machined 
in the face of each compartment to seal the membranes clamped between the 
two halves. 
The maximum available volume in each compartment was 46 cm..sup.3 and the 
area of exposed film, measured across contact with the large O-ring (100 
mm. diameter) was 78.5 cm..sup.2 The entire measured area of the membrane 
was used to calculate observed permeabilities (P.sub.o). Even though the 
cell is only partly filled, the diffusion process apparently continues due 
to a thin film of solution being carried over the arc above the solution 
during rotation of the cell. 
Thickness of air-dried film specimens was measured at nine locations and 
reported as average thickness. The films were then soaked in water for 
several hours, and clamped, while wet, between the two cell halves. The 
cell was then attached to a motor chuck and the complete assembly 
positioned so that the axis of cell rotation was horizontal. A weighed 
syringe was used to introduce about 34 cm..sup.3 water into one side (B) 
and the same volume of solution into the other side (A). The filling holes 
were immediately plugged and the motor started. Since volumes A and B were 
essentially equal, P.sub.o was calculated from the following equation: 
EQU P.sub.o =V ln (.DELTA.C.sub.o /.DELTA.C)/2At 
where .DELTA.C.sub.o /.DELTA.C is the ratio of the concentration 
differences initially and at time t, A is the membrane area (78.5 
cm..sup.2) through which transport took place, and V is the volume of 
liquids (about 34 cm..sup.3) in each side of the cell. Initial 
concentration, C.sub.o, of each solute was 1.5% (w/w). 
Diffusion rates of solutes through the membranes were followed by 
colorimetric analyses for the individual runs of urea and sugars. Sodium 
chloride concentrations were measured by atomic absorption analysis of 
sodium on a Varian Techtron AA120 spectrophotometer. The diffusivity of 
each of the tested solutes was individually determined. The results are 
reported in Table I, below. 
EXAMPLE 4 
The procedure of Examples 1-3 was repeated except that the corn starch was 
gelatinized in aqueous NaOH at 90.degree. C., freeze dried, and then 
blended with the EAA and additional water to provide a formulation having 
50% solids. The diffusivity of the sugars and alanine were simultaneously 
determined by introducing a mixed solution thereof [each at 1.5% (w/w)] to 
side A of the test cell. Solutions withdrawn from the cells at the 
conclusion of each run were analyzed by HPLC. The results are reported in 
Table I. 
EXAMPLES 5-6 
The procedure of Examples 1-3 was repeated except that water-soluble 
additives (20% ethylene glycol, Example 5; 2% glycerol and 6% glycol 
glucoside, Example 6) were blended into the mixture at the time of 
formulation, and in Example 5 the amount of water added with the NaOH 
provided a solids content of 77%. The diffusivity of the sugars and 
alanine were simultaneously determined as in Example 4. The results are 
reported in Table I. 
EXAMPLES 7-10 
The procedure of Example 1 was repeated except hydroxyethyl starch (a 
modified corn starch sold under the trade name "Amaizo 742D," American 
Maize Products Co.) was substituted for the unmodified corn starch. In 
Examples 8-10, various levels of sucrose were blended into the mixtures at 
the time of formulation. Diffusivity of the sugars and alanine were 
simultaneously determined as in Example 4. The results are reported in 
Table I. 
EXAMPLES 11-16 
The procedure of Example 1 was repeated except that starch graft copolymers 
were substituted for the unmodified corn starch. The copolymers were 
prepared by grafting either methyl methacrylate, methyl acrylate, or 
acrylonitrile onto corn starch by the procedure reported in Polym. Lett. 
6: 599-602 (1968). Diffusivity of the sugars and alanine were 
simultaneously determined as in Example 4. The results are reported in 
Table I. 
EXAMPLES 17-19 
For purposes of comparison, the procedure of Example 1 was repeated except 
ammonia as ammonium hydroxide was substituted for the NaOH in the 
specified amounts. 
TABLE I 
__________________________________________________________________________ 
Film 
Ex- 
Starchy thick- 
P.sub.o .times. 10.sup.6 
(cm./sec.) 
am- 
material, EAA, NaOH, 
ness, Glu- 
Fruc- 
Su- 
Raf- 
Ala- 
ple 
%.sup.a %.sup.a 
Additives, %.sup.a 
p.p.h..sup.b 
mils 
NaCl 
Urea 
cose 
tose 
crose 
finose 
nine 
__________________________________________________________________________ 
1 starch, 20 80 . . . 5 1.6 . . . 
0.01 
. . . 
. . . 
. . 
. . 
. . . 
2 starch, 60 40 . . . 7.5 
2.5 31.6 
52.8 
6.3 
10 2.9 
2.5 . . . 
3 starch, 40 60 . . . 5 1.9 7.2 12 1.5 
1.1 0.4 
0.08 
. . . 
4 starch, (freeze 
40 60 . . . 5 1.5 5.1 17.5 
0.9 
1.2 0.6 
0.1 2.4 
dried) 
5 starch, 30 50 ethylene glycol, 
20 
5 1.7 11.8 
33.6 
2.0 
2.3 0.9 
0.2 4.0 
6 starch, 40 52 glycerol, 
2 
5 2.3 . . . 
21.6 
4.1 
. . . 
2.0 
1.1 . . . 
glycol glucoside, 
6 
7 hydroxyethyl, 
40 60 . . . 5 1.2 4.6 20 1.0 
1.1 0.4 
0.1 2.2 
8 hydroxyethyl, 
40 50 sucrose, 10 
5 1.9 8.7 37 0.8 
1.0 0.4 
0.1 1.9 
9 hydroxyethyl, 
40 40 sucrose, 20 
5 2.2 20.4 
66 4.8 
5.5 2.6 
1.2 4.3 
10 hydroxyethyl, 
40 30 sucrose, 30 
5 1.9 34.7 
112 5.1 
5.7 2.6 
1.3 6.5 
11 starch/methyl 
40/10 
50 . . . 5 1.8 9.7 25.4 
1.3 
1.7 0.8 
0.4 2.7 
methacrylate, 
12 starch/methyl 
40/20 
40 . . . 5 3.1 5.1 15 0.3 
0.4 0.3 
0 1.1 
methacrylate, 
13 starch/methyl 
40/10 
50 . . . 5 2.5 20.8 
88 3.7 
4.2 1.7 
0.6 6.1 
acrylate, 
14 starch/methyl 
40/20 
40 . . . 5 1.9 11.1 
34.7 
4.4 
5.2 2.7 
1.4 6.8 
acrylate, 
15 starch/acryloni- 
40/10 
50 . . . 5 1.3 11.1 
30 1.5 
1.8 0.7 
0.3 3.2 
trile, 
16 starch/acryloni- 
40/20 
40 . . . 5 2.2 11.2 
26.0 
0.8 
1.0 0.7 
0.3 2.2 
trile, 
17 starch, 20 80 . . . .sup. 0.sup.c 
0.8 . . . 
0 0 0 0 0 0 
18 starch, 40 60 . . . .sup. 0.sup.d 
1.0 . . . 
0 0 0 0 0 0 
19 hydroxyethyl, 
40 60 . . . .sup. 0.sup.d 
1.9 0 0 . . . 
. . . 
. . 
. . 
. . 
__________________________________________________________________________ 
. 
.sup.a All percents are expressed on a dry weight basis. 
.sup.b P.p.h. is parts per hundred of starchy material plus EAA plus othe 
additives, on a dry weight basis. 
.sup.c Formulation was neutralized with 4.3 p.p.h. NH.sub.3. 
.sup.d Formulation was neutralized with 3.2 p.p.h. NH.sub.3. 
With respect to the NaOH incorporated at 5 p.p.h. in the preceding 
examples, the molar amount of NH.sub.3 was 100% more for Example 17, and 
50% more for Examples 18 and 19. In Example 19, the starchy material was 
the hydroxyethyl starch used in Examples 7-10. Diffusivity determination 
was conducted as in Example 4, except for Examples 17 and 18 in which the 
concentration of each solute tested was 2% (w/w). 
EXAMPLE 20 
The procedure of Examples 1-3 was repeated wherein the formulation on a dry 
basis was 40% starch, 20% water-soluble sucrose, 40% EAA, and 5 p.p.h. 
NaOH, and the diffusivity of 7.65% ethanol was determined and analyzed by 
gas chromatography. Diffusivity results were: sodium chloride, P.sub.o 
=14.4.times.10.sup.-6 cm./sec.; urea, P.sub.o =26.7.times.10.sup.-6 
cm./sec.; and ethanol, P.sub.o =17.9.times.10.sup.-6 cm./sec. 
EXAMPLE 21 
The procedure of Examples 1-3 was repeated wherein the formulation on a dry 
basis was 40% starch, 15% urea, 45% EAA, and 5 p.p.h. NaOH. The resulting 
film was especially pliable, uniform, and transparent. Diffusivity results 
were: sodium chloride, P.sub.o =13.5.times.10.sup.-6 and urea, P.sub.o 
=24.times.10.sup.-6 cm./sec. 
EXAMPLE 22 
The films prepared in accordance with Examples 2, 3, and 6 were subjected 
to wet and dry tensile measurements using an Instron. Thirty 0.635 
cm..times.10 cm. strips were cut from each film. Ten were used for dry 
tensile and elongation measurements and the other 20 were soaked 4 hr. in 
water, after which excess water was removed by blotting lightly with paper 
towels. Ten of the soaked strips were used for wet tensile and elongation 
measurements. The effects of soaking were determined from the remaining 10 
strips. All of the films expanded and imbibed a considerable amount of 
water as indicated by the weight increase. After the wet samples were 
taken to dryness, they were reweighed. Weight losses indicate that the 
sodium hydroxide, in excess of that needed to neutralize the 20% acrylic 
acid in the EAA, and the glycerol and glycol glucoside (Example 6) are 
extracted within 4 hr. The results are reported in Table II, below. 
It is understood that the foregoing detailed description is given merely by 
way of illustration and that modification and variations may be made 
therein without departing from the spirit and scope of the invention. 
TABLE II 
__________________________________________________________________________ 
Tensile 
Formulation.sup.a strength, 
Elongation, 
Effect of water soaking.sup.c 
Starch, EAA, 
NaOH, 
kg./cm..sup.2.spsp.b 
%.sup.c 
Length 
Weight 
Weight 
Film % % p.p.h. 
Dry 
Wet 
Dry 
Wet 
increase, % 
increase, % 
loss, % 
__________________________________________________________________________ 
Example 2 
60 40 7.5 255 
24.4 
7.6 
27.5 
12.4 58.9 7.5 
Example 3 
40 60 5 238 
44.9 
50.4 
48.3 
9.5 40.4 1.8 
Example 6.sup.d 
40 52 5 198 
44.5 
35.1 
45.0 
8.4 33.8 8.7 
__________________________________________________________________________ 
.sup.a Based on dry weight exclusive of water. NaOH given in parts per 10 
parts formulation. 
.sup.b Values are average of 10 specimens. Wet strengths based on dry 
crosssectional areas; all samples broke at jaws. 
.sup.c Values are average of ten specimens. 
.sup.d Contained 2% glycerol and 6% glycol glucoside.