SPECTROPHOTOMETRIC AND FLUOROMETRIC MEASUREMENT OF CHANGES IN LIPIDES H. J. DUTTON
AND B. G . EDWARDS
Changes that occur in the lipide fraction of dehydrated eggs during storage have been studied by spectrophotometric and fluorometric techniques. The reaction of lipide amines with aldehydes, the destruction of carotenoids, and a process that has been interpreted as polymerization of the unsaturated fatty acids were found to proceed faster at 98’ than at 70° F.; little change was detected at L5O F. The fluorescing substance that develops in the fat of dehydrated egg0 during storage has been tentatively identified as the reaction product of lipide amines with aldehydes.
DEHYDRATED EGG(1 gram)
I
EXTRACTION OF LIPIDES (ether) I ’-t
I
I
(residue)
(extract) DILUTETO 26 ML. II
AI (4 n
to
1
ALIQUOT (IO ml.)
IOT
dild. nl.)
I
SAPONIFICATION (3 ml. alc. KOH, 40° C., 15 min.)
I
PARTITION 5 ml. HIO)
(5 ml. ether I
+ I
I
\
(hyperphase) (hypophase) WASH (3ml. 0.1% NanCOs) I
r ( 2
--
7 -
DILD.TO 25 ML. WITH ETHER
Figure 1. Flow Sheet of Analytical Procedure
SAPONIFIABLE FRACTION
I
DIL. ~ 0 ’ 2ML. 5 WITH DISTD.HIO
I
AND PARTITION
(10-ml. aliquot &SO4
+ 1.6ml. 7.3 N
+ 3 ml. ether) I
WASH
-
(3 ml. ether)
DILD.TO 10 ML. WITH ETHER UNS
during storage arise presumably from three chemical changes: destruction of naturally occurring carotenoid pigments, production of yellow-to-brown materials from unsaturated fatty acid groups, and development of brown products resulting, probably, from the interaction of amine and aldehyde groups of lipides (6). While carotenoid destruction results in loss of color, the other two changes tend to darken the color of the dried egg product. The decomposition of carotenoids in egg powders may be expected to be primarily oxidative, involving atmospheric oxygen either directly (7, IS),or indirectly through enzymic or chemical intermediates (1, 91). The alteration of unsaturated fatty acid groups in this product may also involve oxidative reactions. Although complex and partly obscure, the latter reaction8 are believed to result in the formation of peroxides and aldehydes and to yield brown polymers (4, 14). However, polymerization and browning of unsaturated fatty acids may not involve oxidation but may occur as a result of a Diels-Alder type of reaction (16). Attempts to follow the course of oxidation of the fats of dehydrated egg during storage by means of peroxide determinations have demonstrated little change; in fact, initial peroxide values have been found to decrease during storage (8). These data have led to the impression that the fat of dehydrated egg is quite stable. The present report, however, describes several changes found to occur in egg lipidea during storage. The “amine-aldehyde” interaction in egg lipides (6)is apparently a condensation process analogous t o the glucose-protein reaction of the protein fraction (9,16, 19,SO). The brown substances produced, which will be referred to subsequently as lipide amine-aldehyde reaction products, absorb light throughout the
I
ACIDIFICATION
AL LIPIDE
HANGES in color of the lipide fraction of dehydrated egg
I
1 -
I
c
eon
FI BLE
ACID
ETHER
DILD.TO 10 ML. WITH HtO
USE OF DIETHYLETHER. Despite the fact that other solvents have been reported to extract the fat more completely (IS)), diethyl ether was chosen becauve of it5 low boiling point and the resultant minimizing of deleterious effects upon the color of the residue. LENGTHOF EXTRACTION. Extraction time was 4 hours, since longer periods produced no signifiwnt increases in the amounts of either fat or carotenoid pigment extracted. PURITY OF SOLYENTS.This is a critical factor in spectrophotcmetric and fluorometric procedures. However, satisfactory results were obtained by using, without redistillation, the highest purity grades of anhydrous ether and ethanol. SAPONIFICATION MIXTURE. The use of a completely reproducible saponification mixture was found desirable for the preparation of fractions whose absorbing components are cleanly separated. Such a mixture, modified from Olcott and Mattill (l7),was made as follows: 28 grams of KOH were dissoIved in 40 ml. of distilled water with thorough mixing in a stoppered flask. The mixture while hot WBB added to 90 ml. of 95% alcohol. (This was prepared from absolute alcohol of the best grade, since commercial 95% alcohol after treatment with alkali was not satisfactory for spectrophotometric purposes.) The alcoholic alkali was well mixed in a stoppered flask. T o compensate for unavoidable inaccuracies in weighing the KOH, three 3-ml. aliquots of the solution were titrated with standard acid. On the basis of the titrations, the alcoholic alkali was diluted to a concentration of 2.92 normal: 3 ml. of the 2.92 N alkali were used for saponification.
ACID AQUEOUS 1123
1124
3o 40
I.(\
RED 9 MONTHS AT 98'F.
20
\
Vol. 37, No. 11
INDUSTRIAL AND ENGINEERING CHEMISTRY
Total Lipides Unsaponifiable Acid Ether Acid Aqueous
*-.-.---I--.--
interpreted in conjunction with chemical reactions. Experience has shown that either spectrophotometric measurements or chemical methods alone may lead t o erroneous conclusions. One gram of dehydrated egg powder was continuously extracted for 4 hours in a micro-Soxhlet apparatus with anhydrous diethyl ether from a freshly opened bottle. From the extract, diluted to 25 ml. with the solvent, two aliquots were removed: (a) 4 ml. were diluted to 10 ml. with. the anhydrous ether, to give the sample designated as total lipide which thus had a dilution eaual to that of the Samples to be subsequently described; ( b ) 10 ml. were 7 15'F. transferred to a 65Q---+ ml. separatory funnel, freed fromether by evaporation 4 saponified, and fractionated ac3 cording t o flow sheet of Figure 1. Absorption measurements were made with a Beckman M o d d DU spectrophotometer upon t h e total lipide and unsaponifiable fractions and on the acid-ether and acidz a q u e o u s soluble 2 10 ACID-ETHER (b) components of the Isaponifiable fracn s U tion prepared ax^ in0 dicated in Figure 1. v) To facilitate com18 parisons between samples, t h e ab16 sorptions were cal14 culated by t h e equation: 12
r
_------
STORED 9 MONTHS AT 70°F.
40
3G
ck
STORED 9 MONTHS AT 15°F.
t
2
log Io -
IO
I, = __
B
70-F.
6
where c = concentration, grams of dehydrated egg extracted per 100 ml. of solution Is, I , = i n c i d e n t and transmitted light intensity, respective1 d = o ticarpath lengt!, cm.
4
WAVE LENGTH ( m y )
Figure 2. Absorption Spectra of Total Lipide Extracts and of Absorbing Components of Dehydrated Eggs Stored for 9 Months at Three Temperatures
I
2C
1
18 16
visible and ultraviolet regions. This absorption diminishes with increasing wave length and shows a slight maximum a t 270 mp. I n addition to these absorption properties, solutions of the brown product show marked fluorescence. The reported correlation of fluorescence, both in the salt-soluble potassium chloride protein fraction and in the whole egg powder, with loss of palatability (9, 18) led to this investigation of fluorescence as well as of spectral absorption properties of the stored egg. Spectrophotometric and fluorometric studies were carried out upon commercially spray-dried egg powders. Samples prepared at Lynden, Wash., by the Washington Cooperative Egg and Poultry Association for a previous investigation (11) were available for the present study. The egg powders had been stored in paper-lined barrels at 98', 70", and 15' F. Samples were collected at intervals of 1, 3, 6, and 9 months from the approximate centers of the barrels. I n the absence of other controls, the powder stored at 15' F. for 1 month was used. During the intervals between sampling and analysis the materials were kept at -30' F. ANALYTICAL METHODS
Completeness of chemical separation and washing wm checked spectrophotometrically, and spectrophotometric data were
I-
z
14 12
y
IO
TOTAL LIPIDES
k: w
4
=
2
u
$w
8 9 2
8
r t
7
cd
In early work to check the chemical fractionations, complete absorption spectra were measured over the range 250-550 mfi.
ACID-AQUEOUS
6
d 5 4
3
70'F.
2 I
I
3
6
STORAGE TIME (MONTHS)
Figure 3. Change of Fluorescence and Absorptionof Dehydrated Eggs with Duration of Storage in the Total Lipide Extract and in Several Fractions
November, 1945
After the chemical method was established, it s d c e d for analytical purposes to measure optical density a t a few specified wave lengths. Complcte absorption data are given, however, for the final (9-month storage) samples (Figure 2). Fluorometric measurements were made on the total lipide extract and the three fractions (diluted from 3 t o 10 ml.) by means of a Coleman electronic photofluorometer with filters B1 and PCI (excitation, 365 mp, mercury). The fluorescence standard was a solution of quinine sulfate dissolved in 0.1 N sulfuric acid a t a concentration of 0.4 microgram per ml. Blank readings for the solvents, either ether or water, were subtracted from the observed readings. A function designated as the relative fluorescence coefficient, was calculated by the equation:
+,
where Z,/Zq,,
11%
INDUSTRIAL AND ENGINEERING CHEMISTRY
crease appeared to proceed a t a nearly constant rate, which waa greater a t higher temperature; little or no change was found at 15" F. The course of the lipide amine-aldehyde reaction with time and temperature is illustrated in Figure 3c. The zero time intercept of 3.8 X 10-8 probably represents absorption due t o substances other than the lipide amine-aldehyde reaction products, which were present in the acid-aqueous fraction, Evidence on this point has been presented (6),and further evidence is suggested in the legend for Figure 4. At the highest temperature (98" F.) the initial rate of reaction was high but decreased with time. At the intermediate temperature (70" F.) the reaction proceeded more ~ 1 0and ~ a1 t a~ nearly constant rate. No reaction was observed a t 15" F.
= ratio of fluorescence intensities of sample (cor-
rected for solvent fluorescence) to that of standard quinine sulfate solution c = concentration, Y m s of egg powder extracted per liter of so ution ABSORPTION SPECTRA
The absorption spectra of the total lipide extracts of dehydrated egg stored for 9 months at 98", 70", and 15" F. are plotted in Figure 2. Absorption spectra of significant fractions derived from the total extract are also shown. With increasing temperature of storage, the absorption of the total extract decreases in the visible region of the spectrum and increases in the ultraviolet. Apparently the lowering of absorption in the visible region is caused by destruction of the carotenoid pigments (unsaponifiable fraction, maxima 425,445, and 475 mp), and the rise in the ultraviolet is accounted for by a rise in the absorption of the acid-ether and acid-aqueous fractions. The increased absorption of the acid-aqueous fraction is apparently caused by the reaction of lipide amines with aldehydes (6). Two hypothe ses are suggested to explain the intensified absorption of the acid ether fraction-an increase in amount of conjugation of fatty acid double bonds and the development of polymerization products from the unsaturated fatty acids. Some conjugation has been found in freshly dehydrated eggs as indicated by maxima at 268 and 280 mp (triene conjugation) and at 305 and 315 mr (tetraene conjugation). Conjugation is also evident in the sample stored at 15" F. (Figure 2c). The absorption coefficients at these maxima are larger on the material stored at the higher temperatures, and the increase might be explained as formation of conjugated products. However, the increases could result from the development of generally absorbing polymerization products. In fact, analysis of the data by the method of Brice and Swain (6)does not reveal any increase in Conjugation. It appears probable, therefore, that polymerization accounts for the observed rise. Figure 3 shows the rates at which changes take place in the absorption and fluorescence coefficients of the total lipide extract and component fractions during storage. The points plotted represent averages of two to four separate determinations. Results were producible t o *5%. The breaks in many of the curves after 6 months of storage were unexpected. However, anomalies of this sort had been observed previously (8,19). The rate of destruction of carotenoids (Figure 3a) aa revealed by absorption measurements upon the unsaponifiable fraction (A = 445 mp) rises both with temperature and duration of storage. The Ioss of 27% of carotenoids during 6 months of storage a t 98' F. is comparable to the 29% loss during 6 months of etorage a t room temperature reported by the Purdue University Agricultural Experiment Station (IO). Figure 3b shows the increase during storage in the absorption at wave length 270 mp of the acid-ether fraction, which is tentativeiy interpreted aa being due t o polymerization. This in-
u,
I
2c
s - OOI
I 2
,I 4
I 6
] 8
R E L A W E FLUORESCENCE COEFFICIENT
Figure 4. Relation of Absor tions to Relative Fluorescence Lefficients of Acid-Aqueous Fraction The mro fluorescence intercept of the abmorption for the acid-aqueous fraction ham a value of 3.1 X 10-8. Thio value is
equivalent to that for the ~ero-timemtora e mample (Figure Sc) and, therefore, probt%lly reprementm the aboo tion due to mubmtancw other than x e lipide amine-aldehyde reaction product pmaent in the acid-aqueoum fraetion.
FLUORESCENCE
Comparison of the fluorescence values of the total lipide extract from samples stored 9 months a t 98' and 15" F. showed that the extract of the high-temperature sample fluoresced ten times as strongly as that of the low-temperature sample. The effect of storage upon the fluorescence coefficients of the total lipide extract was then determined (Figure 3 4 . The rate of development of fluorescence for the egg powder stored a t 98" F. waa initially high but decreased, at 70" i t was approximately constant, and at 15"it was zero. An attempt was made to learn something of the chemical ncc ture of the compound that gives rise to fluorescence in the total lipide extract by memuring the changes in fluorescence of the chemically separated, unsaponifiable, acid-ether and acidaqueous fractions. From a consideration of the chemical separations indicated in Figure 1 and the fluorescence results in Figure 3, d and e, it is evident that the fluorescing material passed from the total lipide to the acid aqueous phase upon ether partition of the acidified saponifiable fraction. No fluorescing material could be demonstrated in the unsaponifiable and acid-ether fractions. Their relative fluorescence coefficients did not increase significantly but varied at random between 0.47 and 1.8 (data not shown). It is therefore apparent that the fluorescing materials of the total lipide extracts are released from their lipide combinations in the stored egg powder by the saponification process and are isolated in the acid-aqueous fraction (compare reference 8). Since the curves describing development of fluorescence in the total lipide extract (Figure 3d) and acid aqueous fraction (Figure 3e) appear to parallel the curves for absorption at 270 mp (Fig-
1126
INDUSTRIAL A N D E N G I N E E R I N G CHEMISTRY
ure 3c) in the acid-aqueous fraction, the identity of the fluorescing and absorbing substances is indicated. This conclusion is supported by the approximately linear relation that exists in the acid-aqueous fraction between absorptions and relative fluorescence coefficients (Figure 4). Thus the fluorescing material appears to be the same as the substance responsible for the absorption in the acid aqueous fraction. This substance is believed to be the fluorescent lipide amine-aldehyde reaction product (6). Evidence is now at hand to indicate that oxidative changes take place in the fat and fat-soluble components of egg powders stored in the presence of air, as follows: Loss of carotenoid pigments as reported here, as well as of vitamin A (II), during storage indicate oxidative reactions; the formation of lipide aminealdehyde reaction products likewise implies oxidative change, since aldehydes have been shown to develop during the oxidative breakdown of fats. The development of lipide amine-aldehyde reaction products in egg fat tends to give the extracted lipides a brown color as does also the polymerization of the unsaturated fatty acids. Formation of these brown lipide substances is one factor in the color change of dehydrated egg in high-temperature storage. An.other factor is the coincident loss of carotenoid pigment. Aside ,from the importance of the lipide amine-aldehyde reaction to .color, there is evidence that the change in concentration of this -substance is correlated with changes that occur in the palatability .of both high-moisture ( > 5 % ) and low-moisture (c17°C) eggs (unpublished results).
Vol. 31, No. I1
staff of this laboratory who are investigating the causes of deterioration of dehydrated eggs. LlTERATURE CITED
(1) Balls, A. K.,Axelrod, B., and Kies, M. W., J . Biol. Chem., 149. 491 (1943). (2) Balls, A.K.,and Swenson, T. L.. Food Reseamh, 1, 319 (1936). (3) Bate-Smith, E. C., Brooks, J., and Hawthorne, J. R., J . SOC. Chem. I d . ,62,97(1943). (4) Brauer, R. W., and Steadman, L. T., J . Am. Chem. Soc., 66,563 (1944). Brice, B. A., and Swain, Margaret L., J . O p t . Sac. Am., 34,772 (1944). Edwards, B. G . , and Dutton, H. J., IND.ENQ.CHEM.,37,1121 (1945). Eschor, H. H., Hela. Chim. Acta, 15,1421 (1932).
Fevold, H. L., private communication. Fryd, C. F. M., and Hanson, 9. W. F., J . SOC.Chem. Ind., 63,3 (1944).
Hauge, S. M., Zscheile: F. P., Carrick, C. W., and Bohren, B. B., IND. ENQ.CHEM., 36, 1065 (1944). Klose. A. A., Jones, G : I., and Fevold, H. L.,Zbid., 35, 1203 (1943).
Kuhn, R.,and Btockman, H., Ber., 65B,894 (1932). MacLean, H., and MacLean, I. S., “Lecithins and Allied Substances”, New York, Longmans, Green and Co., 1927. Mattill, H. A., Oil & Soap, 18,73 (1941). Norris, F. A., Rusoff, I. I., Miller, E. S.,and Burr, 0.O., J . Bid. ChsPn., 147,273 (1943). Olcott, H. S., and Dutton, H. J., IND. ENQ.CEEM.,37, 1119 (1945).
Olcott, H.S.,and Mattill, H. A., J . Am. C h m . Soc., 58, 1628 (1936).
Pearce, J. A., and Thistle, M. W., Can. 6. Rescarth, 20, 276 (1942).
ACKNOWLEDGMENT
The authors acknowledge the advice and encouragement of G. H. Kunsman, H. D. Lightbody, and other members of the
Stewart, G.F.,Best, L. R.. and Lowe, B., Boo.Inst. Food Tech.. 1943,77. Stewart, G . F., and Kline, R. W., Zbid., 1941, 48. Sumner, J. B., and Dounce, A. L., Enzwnadogda,7,130(1939).
Hygroscopicity of Softened Glue Composition WILLIAM C. GRIFFIN Atlas Powder C o m p a n y , W i l m i n g t o n , D e l .
A
LTHOUGH it is widely recognized that the functioning of
industrial glue compositions is closely related to their water contents, no data are found in the literature concerning the hygroscopicity of glue compositions containing the usual softeners. As part of a study of the evaluation of sorbitol as a plasticizer for glue, hygroscopicities of a typical hide glue softener with sorbitol, glycerol, and a mixture of sorbitol and glycerol were investigated along with the hygroscopicities of the individual components. In use, a glue composition is seldom a t a given set of conditions long enough for equilibrium to be reached. Equilibrium values, however, represent the point toward which the composition will always be changing, and a knowledge of how the equilibrium moisture content varies with humidity is of value in calculating the maximum changes expected within established humidity limits. A second attribute of hygroscopicity which was not investigated in the present study is the rate a t which moisture is exchanged between the composition and the air under nonequilibrium conditions. This rate of moist’;ie exchange is a complex function depending upon such variables as film thickness, velocity of water diffusion within the composition, rate of air circulation, distance of the composition from equilibrium mois-
ture content, etc., and i t was not considered feasible to undertake such an investigation a t the present time. Briefly, the method used for the glue cornpositions was to eaet thin films on glass and to expose them to circulated air of a chosen constant humidity and temperature until they attained constant weight. From known initial compositions and final weights, the equilibrium moisture contents were calculated. MATERIALS AND APPARATUS
The ingredients employed in compounding the experimental softened glue samples follow. Their moisture contents were determined by titration with Karl Fischer reagent (1): GLUE. A balanced grade of hide glue containing approximately 16% water (analyzed !5,3%), 410 to 415 grams gel strength (Bloom), 135 millipoise viscosity, ground to 24-26 mesh, pH 5.9 in 20% solution. GLYCEROL.Dynamite grade, 0.5% water. SORBITOL.Commercial solution (Arlex), 16.0% water. The differences introduced by using this product in place of ure sorbitol have been found to be within the experimental error orthe method employed. The test samples were conditioned in a laboratory model Carrier processing cabinet in constantly recirculated air of con-
