JANUARY,1937
INDUSTRIAL AND ENGINEERING CHEMISTRY
bucket-type spinning machine. The rayon resembled viscose rayon. Although the dry strength was excellent, the wet strength was low. By forming a urea-formaldehyde resin in the rayon, it was possible to obtain a wet strength substantially equal to the dry strength. I n addition to developing a number of commercial possibilities in which the hydroxyethyl ether is used directly, some of its derivatives were prepared. It could be nitrated readily to yield an excellent lacquer base with low viscosity and excellent clarity and stability. When cellulose is acetylated, it is usually necessary to go through a second hydrolytic step to achieve acetone solubility. I n contrast, the hydroxyethyl dicellulose ether may be acetylated to yield in one step almost perfect acetone solubility. Films of the acetyl derivatives show good physical properties. As the degree of alkylation increases, the ether may be acetylated more easily. The stearate, xanthate, and benzoyl derivatives were made. These and the methylated ether were similar to the corresponding cellulose derivatives. Doubtless, many other derivatives can be prepared. The data presented represent a brief summary of the re-
117
search work of several years. Further details are given in patents (5).
Acknowledgment The authors wish to thank the C. F. Burgess Laboratories, Inc., and the Carbide and Carbon Chemicals Corporation for their courtesy in permitting the publication of this paper.
Literature Cited (1) Dreyfus, H., British Patent 166,767 (1920); U. 5. Patent 1,502,379 (1924). (2) Dreyfus, H., French Patent 462,274 (1912); Lilienfeld, L., U. 5. Patent 1,188,376 (1916). (3) Hubert, E., German Patent 363,192 (1920); J. SOC.Chem. Ind., 42, 348A (1923). (4) Pickering, S. U., J. Chem. Soc., 63, 890-999 (1893). (5) Schorcrer, A. W., U. S. Patents 1,863.208 (June 14, 1932): 1,914,172 (June 13, 1933); 1,941,276, 1,941,277, 1,941,278 (Dec. 26, 1933); Shoemaker, M. J., Ibid., 1,877,606 (Sept. 13, 1932) ; 1,898,601 (Feb. 21, 1933) ; 2,028,296 (Jan. 21, 1936) ; 2,029,131 (Jan. 28, 1936). (6) Suida, W., Monatsh., 26, 413-27 (Jan. 12, 1906); J. SOC.Chem. Ind., 24, 543 (1905). RECEIVED May 6, 1936.
Refractive Index of Egg Albumen Changes with Age, Season, and Development ALEXIS L. ROMANOFF AND ROYAL A. SULLIVAN Cornel1 University Agricultural Experiment Station, Ithaca, N. Y.
P
ROTEIN solutions, because of their chemical complexity and amphoteric nature, are usually difficult to characterize by ordinary chemical methods. Physical measurements, on the other hand, are particularly applicable because of the large molecular weights involved. It has often been shown that the refractive index of a protein solution is an indication of the amount of protein present. From the electromagnetic theory of light it is evident that the refractive power of protein solution must be due to the volume occupied by the protein molecules. Consequently, such factors as temperature, bound water, and degree of dispersion of the protein will influence the refractive index of its solution (6). In dealing with the albumen of birds' eggs, the problem is more complicated, not only because of the presence of several proteins, but also because four natural divisions or layers of albumen are clearly distinguishable in the fresh egg. These layers are differentiated morphologically during the process of egg formation. Each layer, presumably, is destined to carry on distinct biological functions a t least in the early course of embryonic development. Therefore, it may logically be assumed that each layer is of different chemical structure and consequently of different refractive properties. As a part of the investigation of various physico-chemical properties of albumen of birds' eggs, an extensive study of the refractive index was made. The object was to establish (1) the exact values of four layers of albumen of the fresh eggs of several representative species of birds, (2) the frequency distribution in a flock of hens, (3) the variation within a normal breeding and incubating season, (4) the constancy with individual birds, ( 5 ) the changes in the unfertilized
eggs with age under low and high temperatures, and (6) the changes in the fertilized incubated eggs.
Materials and Methods A large number (1177) of eggs of the following species was used : white Leghorn chickens (Gallus domesticus) , ring-necked pheasants (Phasianus torquatus), bobwhite quail (Colinus virginianus), Bourbon red turkeys (Meleagris gallopavo), and white Peking and white runner ducks ( A n a s domesticus). Usually all the eggs were tested within 12 hours after being laid. Prior to the analysis they were kept in the refrigerator at about 40" C. and 70 per cent relative humidity. In special tests of strictly fresh eggs, the eggs were used not later than half an hour after txey were laid. The various layers of egg albumen were separated and sampled. The egg was broken into two parts, and the contents drop ed gently into a large Petri dish. Then the layers were removezby means of pipets in the following order: (1) the outermost fluid layer with a pipet having a bore at the point 1 mm.; (2) the middle fluid layer with the same pipet, after making an incision in the sac of the dense albumen with scissors and permitting the fluid of this layer to run out; (3) the middle dense layer with a pipet having a bore at the point 2 mm.; and (4)the innermost chalaziferous layer together with t h e chalazas was removed with forceps.
The refractive index was read at 25" C. with a Zeiss refractometer, Abbe type.
Strata of Fresh Egg Albumen Table I shows that there is a noticeable difference in the refractive index of the different layers of albumen of the eggs just laid.' The value was lowest for the outer fluid 1 The proportional amounts by weight of each layer of chicken egg albumen, for example, was found to be 23.18, 57.29. 16.84,and 2.69 per cent of outer fluid, middle dense, middle fluid, and chalaziferous layer, including the chalazas, respectively.
INDUSTRIAL AND ENGINEERING CHEMISTRY
118
0.0001 to 0.0010. The curves for the different layers are nearly parallel. Therefore the same relation seems to exist between the different layers, regardless of their absolute values. Biologically the refractive index may be of some significance in the reproduction of birds. Among 775 incubated eggs, 29.1 per cent was infertile from hens laying eggs with a high refractive index of albumen and 41.1 per cent waa infertile from hens laying eggs with a low refractive index. The difference, 12.0 * 3.40, is statistically significant. On the other hand, the hatchability of fertile eggs was almost the same in both groups of hens.
1.362
-e
1.360
n
1.358
z
u
2
1.356
a z
1.354
2
I-
u
U
1.352
Variation within the Breeding Season
z
LL W
a
OUTER
t Naor
FLUID
M I D D L E DENSE MIDDLE FLUID
1.350
VARlAiiON
VOL. 29, NO. 1
[
CHALAZIFEROUS
1-
1.3491- 1.3501- 1.351I- 1.3521- 1.3531- 1.35411.3500 1.3510 1.3520 1.3530 1.3540 1.3550
HENS
4
6
22
31
27
21
15
REFRACTIVE INDEX OF OUTER FLUID LAYER
FIQURE1. FLOCK DISTRIBUTION OF 105 HENS, INDEXOF OUTER FLUID BASEDON REFRACTIVE LAYEROF ALBUMEN
-
x
W
0
I
7 6 -
For the period of 7 months from December to June, inclusive, there were used for the study of the refractive index of various layers of albumen, fifty chicken eggs on an average every month from the same group of hens. The observations (Figure 2) indicate that the r e f r a c t i v e I index of albumen is highest just a t the b e g i n n i n g of the natural breeding season-that is, from I February to March. The values of all four layers of a l b u m e n are lowest a t the closing of the breeding season in July.
layer; it increased with each consecutive layer -z 1.355 inward, irrespective of their apparent densities. W 2 4 The general relation for the refractive index of the Ivarious layers of albumen is the same for the eggs YE 3 of all species studied. kl 2 However, the separation of albumen by means E l of a screen into only two layers-the dense (or OUTER FLUID firm) and the liquid layer (when the latter in-%-XMIDDLE DENSE cludes both the outer fluid and the middle fluid MIDDLE FLUID portions)-as has been done by some workers 8 CC CHALAZIFEROUS (1, d ) , did not reveal any difference in the re7 fractive index between these two samples. To DEC. JAN FEB. MAR. APR. MAY JUNE explain such a phenomenon, the data on more FIQURE 2. SEASONAL CHANGES IN REFRACTIVE INDEXOF LAYERSOF EQQ ALBUMENFROM A than 500 fresh chicken eggs (not over 12 hours GROUPOF TWELVEHENS old) were compiled, using for comparison the refractive index of the dense portion and of the ?CI. I.z", fluid portion of albumen (outer fluid and middle fluid combined); on the average, the refractive index of both samples came to the same value of 1.3540. 1.360
-
Constancy with Individual Hens Observations showed that the refractive index of egg a l b u m e n is a constant characteristic of an i n d i v i d u a l hen.
,
I
I\
Frequency Distribution in the Flock of Hens To find the variability in the average refractive indices of the four layers of egg albumen of individual birds, 105 white Leghorn hens from two to three years old were used. Up to eight eggs from each hen were examined. Figure 1 illustrates the flock distribution based on the refractive index of the outer layer of albumen. In each of the six groups of hens the average value for the refractive index of the outer layer is arranged within the limits of b
TABLEI. REFRACTIVE INDEXOF VARIOUS LAYERSOF FRESH EGGALBUMEN Outer fluid 1.3529 1.3560 1.3668 1.3535
Refractive Index Middle Middle' nD dense fluid Chalaziferoua 1.3552 1,3582 1.3606 1.3567 1.3575 1.3588 1.3581 1.3590 1.3603 1.3561 1.3594 1.3628
1.3542 1.3565 1.3550
1,3557 1.3580 1.3566
c
Species Chicken Pheasant
%Duck: ;t&
Peking Runner Average
No. of Eggs 717 6 5 5
4 5
...
1.3569 1.3598 1.3585
1,3612 1.3630 1.3611
1.359
-
1.358
-
1.357
-
2 1.356
-
a
C c I
L I
X
z W
F
2
1.355
-
1.354
-
LT
-pD-
-
-X-X-
L353 DAYS
0
5
IO
OUTER F L U I D MIDDLE
DENSE
MIDDLE F L U I D CHALAZIFEROUS
15
20
PERIOD OF EXPOSURE TO 20°c.
FIGURE3. CHANGES WITH AGE IN REFRACTIVE INDEXOF LAYERS OF FRESH EGGALBUMEN FROM SEVENHENS
JANUARY, 1937
INDUSTRIAL AND ENGINEERING CHEMISTRY
The hen, which lays eggs with either a high or low refractive index of albumen, maintains that value throughout the breeding season. Furthermore, the seasonal changes were found to be much more pronounced with the birds possessing low refractive indices than in those with high indices (Table
119
age of about one week practically disappeared as a morphologically distinct layer.
Changes in Fertilized Incubated Eggs
During incubation there is rapid diaintegration in the strata of albumen of the developing egg. At the end of 24 11). hours the middle fluid layer almost completely I disappears. After 3 to 4 days of incubation, the TABLE11. SEASONAL CHANQES IN REFRACTIVE INDEXOF VARIOUS LAYERS middle dense layer d i s a p p e a r s . The inner O F ALBUMENOF FRESHCHICKEN EQGSFROM TWO GROUPSO F BIRDS' chalaziferous layer also disappears in the early EXHIBITING HIGHAND Low INDICES period of the development of the egg, except Refraotive Index, nDb >Outer Fluid-Middle Dense-Middle Fluid-Chalazifero& the which are found to be floating loosely Season High LOW High Low High Low High Low in the structureless mass of albumen resembling 1.3642 1.3555 December 1.3530 1.3497 1.3553 1.3498 1.3577 1.3523 the outer fluid layer. 1.3618 1.3603 January 1.3532 1.3513 1.3570 1.3649 1.3554 1.3532 This process of disintegration in the strata of 1.3619 1.3609 February 1.3541 1.3517 1.3581 1.3553 1.3556 1.3539 Maroh 1.3542 1.3526 1.3613 1.3887 1.3550 1.3544 1.3569 1.3558 albumen of fertilized incubated eggs is presum1.3619 1.3590 April 1.3533 1.3510 1.3549 1.3528 1.3568 1.3545 1.3604 1.3578 1.3551 1.3542 1.3542 1.3524 1.3527 1.3508 May ably due to a rapid dehydration, or loss of water, 1.3688 1.3584 June 1.3512 1.3503 1.3539 1.3515 1.3554 1.3531 a t the expense of external evaporation, on the Six in each group. b The figures are averages of about 25 eggs from each group of birds per month. one hand, and of transference of water to the yolk, on the other (10). Since the refractive index has a direct relation to the density or to the solid content of the colloidal solution, rapid change in the Changes in Unfertilized Eggs with Age refractive index of the disintegrated egg albumen would be The attempt was made to trace the precise changes in the expected in the course of embryonic development. refractive index of various layers of albumen of chicken eggs with age. The unfertilized eggs from the same group of 1.42 hens were tested either immediately after laying or after storing at 20" C. and 60 per cent relative humidity for various 1.4 I periods up to 21 days. The results of observations (Figure 3) e show that differences in the refractive properties of various " 1.40 strata of egg albumen rapidly diminish immediately after z u the egg is laid and, of the first three layers, almost comX 1.39 w pletely disappear within 3 to 5 days. n Presumably because of this rapid change in the distinct -zw 1.38 refractive properties of various strata of egg albumen with 2 age, the majority of workers failed to recognize such differIences. In the use of eggs not strictly fresh, it appears 2 1.37 possible to obtain the same values of the refractive index E! CHICKEN ; 1.36 for all three layers, and to interpret the data of further changes -0--0- PHEASANT as one linear curve of increase with age of the eggs (4). -4-4- W A I L 1.35 The eggs of five species of birds when incubated a t 37.5' C. TURKEY and 60 per cent relative humidity also show a gradual, almost DUCK b linear, increase in the refractive index of the outer fluid and 1.34 Ll I I I 1 1 I of the middle dense layer of albumen. The refractive inPERCENT0 IO 20 30 40 50 60 70 80 90 dex of the inner chalaziferous portion of albumen, particularly P E R I O D O F INCUBATION of the chalazas, was little changed (Figure 4); a slight drop FIQVRE 5. RELATIVE CHANQES IN REFRACoccurred a t one week of age, corresponding to that observed TIVE INDEXOF ALBUMEN FROM DEVBLOPING at 20" C. The refractive index of the middle fluid layer soon EQGS approached the value of the middle dense layer, which a t the Q
1
-
-
-X-X-
1
1
I
Each point represents from four to six observations.
-
1.365
..
1
OUTER
FLUID
M I D D L E DENSE
CHALAZIFEROUS
I
Observations on eggs of five species of birds show that the refractive index of the disintegrated or composite ('outer" layer of albumen increased rapidly with the incubation period (Figure 5). It reached the maximum value during the first third of the period, then remained almost constant during the second third, and finally had a sudden drop to a low value. Although the curves of these changes in the eggs of various species of birds vary slightly, they could not be interpreted definitely in terms of the specificity of various albumens. All the curves follow one general trend as just described and shown in Figure 5 . It is interesting to note that these changes coincide well with changes in the reciprocal of electrical conductivity (9).
21
Discussion
P
U
u
w X
-
1.360
-
W
2
iV
2 1.355
-01-0-
w
K
0
PHEASANT
-4-4- QUAIL
I' DAYS
CHICKEN
-i-X-
.-
7
14
21
0
7
14
TURKEY DUCK
21 0
7
14
PERIOD OF EXPOSURE TO 375OC.
FIQURE4. RELATIVECHANGESIN REFRACTIVE INDEXOF ALBUMENOF UNFERTILIZED INCUBATED EGGS Each point represents from three to five observations.
Reiss (6) showed that the index of refraction of a protein solution is directly proportional to its concentration. Consequently, the four layers of fresh egg albuqen are of different concentration, as has already been demonstrated experi-
120
INDUSTRIAL AND ENGINEERING CHEMISTRY
mentally (8). The writers’ recent observations indicate that the dry matter of the outer fluid, middle dense, middle fluid, and chalaziferous layers of albumen of fresh chicken eggs is, on an average, 10.70, 12.85, 13.72, and 15.82 per cent, respectively. Also the densities of these four representative layers of albumen are found to be 1.03149, 1.03457, 1.03690, and 1.04685. Thus the present data on the refractive indices of various strata of albumen agree very well with their dry matter and density-that is, with their concentration. There is considerable difference, as may be expected, between the refractive index of albumen of a developing egg and that of an unfertilized egg hydrolyzed and partially decomposed with age. During the process of embryonic development, the egg albumen, besides being hydrolyzed through the loss of water externally and through the transference into the yolk and to the developing embryo, presumably undergoes certain chemical transformations caused by the preferential absorption of certain proteins and protein-fraction amino acids. The change in the chemical composition unquestionably would influence the refractive index of egg albumen (3). However, the greatest change in the refractive index of albumen is due to changes in concentration. Consequently, there is a direct relation between the refractive index and the dry matter of the developing egg (7, IO). This relation is still better shown in eggs dehydrated with age (1). Therefore, the refractive index has a value in determining the age of the egg. Baird and Prentice (2) stated without presenting experimental evidence that the refractive index of albumen has a constant value with age-that is, about 1.360. Presumably, this value was obtained not only without a consideration of the stratified structure of albumen but from observation of quite old eggs. The present data on the refractive index of egg albumen within the breeding season may be of biological interest as well as of economic importance. Therefore, it is desirable to have repeated observations on the complete cycle of changes throughout the year on flocks of birds widely separated geographically. Such knowledge may throw more light on the causes of seasonal variability in the fertility and in the hatchability of eggs, and on seasonal and regional perishability of eggs used for human consumption.
Summary 1. Each of the four distinct layers of albumen in the fresh eggs of chicken, pheasant, quail, turkey, and duck
VOL. 29, NO. 1
shows specific refractive properties. The actual values of the refractive index increase with the successive layers, from the outermost to the innermost layer; on an average for 717 chicken eggs the values are 1.3529, 1.3552, 1.3582, and 1.3606. 2. The refractive index of albumen of fresh eggs varies seasonally. Its value is highest at the beginning of the natural breeding and incubating season from February to March. 3. The refractive index of albumen as observed in 105 hens is characteristic of the individual birds. Throughout the breeding season the same relation exists between the different layers of albumen, regardless of the absolute value. 4. The refractive inclex of albumen of unfertilized eggs increases linearly with age because of dehydration in all layers except the innermost (chalaziferous) layer, in which it drops, then reaches its original value, and thereafter remains constant. This change in the refractive index with the constant humidity of 60 per cent is more rapid a t a high (37.5’) than a t a low temperature (20’ C.), 5. During the incubation of fertilized eggs, there is rapid disintegration in the strata of albumen. The refractive index of this disintegrated albumen increases rapidly with the incubation period. It reaches the maximum value of about 1.40-1.42 during the first third of the period, then remains almost constant during the second third, and finally has a sudden drop to a low value. 6. Since the refractive index of albumen of birds’ eggs has a direct relation with the concentration and density, it is an approximate measure of the total solids of the various layers of albumen.
Literature Cited (1) Almquist, Lorenz, and Burmester, IND.ENG.CHP~M., Anal. Ed., 4,305-16 (1932). (2) Baird and Prentice, Amlust, 55,20-3 (1930). (3) Hand, J . Biol. Chem., 108,703-7 (1934). 14) Holst and Almquist, Hilgardia, 6,45-8 (1931). i 5 j Reiss, Arch. E&. Path., 51, 18 (1903). (6) Robertson, “Physical Chemistry of Proteins,” p. 363 (1918). (7) Romanoff, Cornel1 Univ. Agr. Expt. Sta., Memoir 132, 1-27 (1930). (8) Romanoff, Science, 70, 314 (1929). (9) Romanoff and Grover, J . Cell. Comp. Physiol., 7 , 425-31 (1936). (IO) Romanoff and Romanoff, A n d . Record, 55,271-8 (1933). RECJOIVED June 13, 1936
:a Tin
