Adsorption of Riboflavin by Lactose Influence of Concentration ABRAHAM LEVITON pare lactose containing as Bureau of Dairy Industry, U. S. Department high as 300 micrograms concentrates, lactose adof Agriculture, Washington, D. C. riboflavin per gram. Much sorbs riboflavin selectively. The relation between dehigher concentrations may be realized, but the rate of crystallization under gree of adsorption and initial riboflavin concentration under conditions of complete crystalthe conditions required for the preparation of the more concentrated adsorbates becomes exceedlization is linear. A minimum concentration of ingly slow. Concomitantly, the lactose crystals riboflavin exists below which no adsorption occurs. Under conditions of incomplete crystallization change from a characteristic tomahawk to a thin platy form. Riboflavin exerts a definite retarding the value of the riboflavin concentration below which no adsorption occurs is displaced toward action on the rate of crystallization of lactose, lower and lower values as the degree of superan effect which becomes more and more prosaturation with respect to lactose is lowered, nounced as the riboflavin concentration is inand reaches a final minimum value of 2.5 microcreased, and as the degree of supersaturation grams per ml. It has been found practical to prewith respect to lactose is decreased. In crystallizing from whey
in these experiments, had a pH of 4.5 and a solids concentration of 30.5 per cent, and contained lactose in slight excess of the quantity (17.4 grams per 100 ml. water) required to effect saturation at 5" C. Adsorbates containing natural riboflavin were prepared along approximately the same lines as indicated above. Following the recovery of alcohol, however, and the concentration of the mother liquor, the addition of carbon was omitted. The concentrate, after storage at 2-5" C. for one day, was filtered and stirred at 7' for a week. The adsorbate which crystallized to the extent of 12 grams per 100 grams of concentrate was washed with 70 per cent alcohol. Analysis showed 170 micrograms riboflavin per ram, representing approximately 85 per cent recovery of ribogavin. In one experiment in which 5 parts of the concentrate were diluted with 1 part by weight of water, the adsorbate which was recovered contained 230 micrograms riboflavin per gram, representing a yield of 75 per cent. The method outlined was applied on a small pilot plant scale to the preparation of adsorbates. The concentrated li uor from which the adsorbate crystallizes is obtained as a by-pro&ct in an extraction process developed in this laboratory for the preparation of soluble proteins and lactose. Directions for the recovery of this concentrated liquor have been published (3). METHODS.Experiments were conducted on measured portions of the stock solution fortified with measured uantities of synthetic riboflavin and lactose. These fortified soqutions were seeded with 0.05 per cent milk su ar. Sealed 22 X 175 mm. test tubes containing the solutions o f varying riboflavin and sugar content were rotated in a thermostat maintained a t 5.0 * 0.1" C. Tubes were removed periodically, their contents filtered rapidly (within 15 seconds), and the lactose residues washed well, but without elution, with 70 per cent alcohol saturated with lactose. Riboflavin determinations were conducted fluorimetrically and colorimetrically. Preliminary experiments had established the good agreement between results obtained with colorimetric, microbiological, and fluorimetric methods. It was found sufficientl accurate in all colorimetric and fluorimetric methods to consdeer riboflavin as the only pigment associated with lactose. AbsorDtion curves were obtained bv means of a sensitive Dhotoelect& spectrophotometer employing a Hilger monochrdmator with 0.0025-inch slits.
I
N THE crystallization of lactose from concentrated whey, a portion of the coloring matter of whey is adsorbed.
This property of lactose is undesirable in certain respects since it necessitates additional processing in the man'ufacture of lactose; but when properly understood and utilized, it becomes of practical significance. The importance of the adsorption of riboflavin by lactose as a basis for the preparation of riboflavin adsorbates has pagsed unnoticed, notwithstanding the fact that the general subject of adsorption on crystals during their growth has been amply investigated. The phenomenon relating t o the adsorption by lactose was extensively investigated, and a8 a result the preparation of potent and pure adsorbates has been placed upon a practical basis. A large number of factors influence the degree and rate of adsorption. Of these, variations in riboflavin concentration and in the degree of supersaturation with respect t o lactose are most significant. These variables will be considered here to the exclusion of others, such as temperature, nature of solvent, nature of adsorbent, hydrogen-ion concentration, and stirring, which require further study. PREPARATION OF STOCK
SOLUTIONS
AND ADSORBATES
Experiments were conducted largely on whey concentrates of known riboflavin and lactose concentration. These concentrates were obtained from spray-processed Cheddar cheese whey powder (2). Briefly, 1 part of powder was treated with 17.5 parts of 70 per cent ethyl alcohol by weight. After several minutes the undissolved proteins were removed by filtration, and after 24 hours lactose which had crystallized was recovered from the acidified filtrate. Following neutralization of the mother liquor, the alcohol was recovered and the residual liquor Concentrated t o contain a proximately 40 per cent solids. Decolorizing carbon was addec! and the mixture was agitated one week at 2' to 5' C. and then htered. This riboflavin-free filtrate, the stock solution 589
590
Vol. 35, No. 5
INDUSTRIAL AND ENGINEERING CHEMISTRY RELATIVE ABSORPTION OF ADSORBATE SOLUTIONS
Figure 1 is a plot of the relative absorption of solutions of two adsorbates, one containing natural and the other synthetic ribofla.i.in. The relative absorption of the concentrate from which the natural riboflavin adsorbate was derived is also plotted over a limited range of wave length. The ordinates are expressed in terms of absorption of natural riboflavin adsorbate: the absorption a t 445 millimicrons is taken as 100 per cent. Solutions of the adsorbates were prepared to contain a p proximately 8 micrograms riboflavin per ml. simply by dissolving the required quantities of the adsorbates. Figure 1 shows that the location of the maxima a t 370 and 445 millimicrons and thc shape of the curves representing the absorption of the adsorbates are in agreement with each other and in agreement (not illustrated) with corresponding features of the absorption curves of crystalline riboflavin. The highly selective nature of the adsorption is demonstrated by the decrease in relative absorption of the concentrate with increases in was’e length in the region 400-425 millimicrons compared with corresponding increases in the absorption of the adsorbates. This selectivity is also shown by the good agreement between results of microbiological and of colorimetric and fluorimetric methods in nhich no attempt was made to exclude absorption of radiation by any interferinn pigments which might have been present. RELATION BETWEEN ADSORPTION AKD I x l r I A L RIBOFLAVIN CONCENTRATION
Figure 2 shows the relation between the degree of adsorption and the initial concentration of riboflavin. The concentration of riboflavin is expressed in micrograms per ml. of the original concentrate. Criticism may be directed against the use of initial rather than equilibrium concentrations as abscissa, and against the use of solutions instead of pure solvents as standards of refercnce; but inasmuch as the data are plotted to emphasize the practical rather than any theoretical aspect of the problem, the treatment is suitable. In these experiments crystallization was allowed to proceed to completion.
5
301
P
- -ADSORBATE (NATURAL FLAVIN)
o
-ADSORBATE(SYNTHETICFLAVIN? 0 -RIEOFL4VIN COKCENTilATE
350
375
403
WAW
425
4x7
475
500
LENGTH IN MILLIMICRONS
Figure 1. Relative Absorption of Solutions Containing Natural Riboflavin Adsorbate, Synthetic Riboflavin Adsorbate, and Whey Solids from Which the Natural Riboflavin Adsorbate Was Derived
The relation between degree of adsorption (micrograms riboflavin adsorbed per ’gram lactose) and initial concentration of riboflavin is linear. The lines corresponding to various levels of supersaturation with respect to lactose converge a t a point representing a concentration of 2.5 micrograms riboflavin per ml. This concentration, then, is the minimum concentration belorn7 which no adsorchion occurs. I n all experiments bud one, lactose crystallization reached completion under the conditions of agitation employed within 3 to 5 weeks. Crystallization, however, from the concentrate containing 42 micrograms of riboflavin and 0.084 gram of added lactose per ml. had hardly begun even after 5 weeks. Riboflavin exerts a definite retarding action on the rate of crystallization of lactose, an effect dGch becomes more and more pronounced as the riboflavin concentration is increased and as the degree of supersaturation with respect to lactose is decreased. This retarding action occurs concomitantly with a change in habit of the crystals from their characteristic tomahawk to a thin platy form (Figure 3). The linear relation and the existence of a minimum concentration below which adsorption does not Whey Is Concentrated as in the Commercial Process for the Manufacture occur are not features peculiar t o of Milk Sugar; Slightly Modified, This Process Permits Recovery of the system under study. Analogous Riboflavin Adsorbate without Using Solvents
INDUSTRIAL AND ENGINEERING CHEMISTRY
May, 1943
591
relations in the crystallization of inorganic salts from solutions of dyestuffs were reported by Rigterink and France (4). Table I gives additional data on the variation of percentage adsorption with variations in the quantity of sugar recovered under conditions of complete crystallization. The data in the fourth column indicate that, for practical purposes, the percentage adsorption may be considered independent of the quantity of sugar recovered under conditions of complete crystallization although small systematic variations are present which point to the existence of a maximum adsorption value. The percentage adsorption is proportional to the difference between the initial riboflavin concentration and the minimum concentration below which adsomtion does not occur: consequently, it' increases with increasing Adsorbate, Consisting of Riboflavin and Milk Sugar, Is Recovered by a Chemical Centrifuge riboflavin concentration. The value of the minimum calculated from the difference between the initial concentration of riboflavin and the quantity is well adapted for practical purposes and establishes a basis adsorbed per ml. of concentrate agrees with the value obtained for the controlled preparation of adsorbates from whey. It by the extrapolation of the curves of Figure 1. The table and graphs show that the degree of adsorption, a, is equal to should be borne in mind, however, that this formula applies the difference between the initial and minimum riboflavin only under conditions in which lactose crystallization' is concentration, c-2.5, divided by the quantity of lactose, s, complete. separating per ml. of concentrate. This empirical formula, a=- c
RELATION BETWEEN ADSORPTION AND CRYSTALLIZATION
- 2.5
When crystallization is incomplete (Figure 4),the quantity of riboflavin adsorbed is influenced by variations in the percentage of lactose which has separated. These curves were obtained a t 5" C. for five series of solutions, the initial riboflavin concentrations of which ranged from 5.25 to 42.0 micrograms per ml. At low riboflavin levels (but exceeding 2.5 micrograms per ml., the lowest level below which adsorption does not occur), no significant adsorption took place until an appreciable percentage of sugar had crystallized. The slopes of the curves, excepting the slope representing crystallization from the most potent concentrate, indicate that the degree of adsorption increases a t first with increase in the percentage crystallization, reaches a maximum, and then decreases. From a study of these curves and of the data already discussed, it is clear that, as the degree of supersaturation with respect to lactose is decreased, the value of the riboflavin concentration below which no adsorption occurs during the early stages of crystallization is displaced toward lower and lower values and reaches a final minimum value of 2.5 micrograms per ml. From a practical standpoint these curves indicate the extent t o which and the manner in which crystallization should be carried out to obtain potent adsorbates consistent with good yields.
S
3 - 0.249 4- 0268 RIBOFLAVIN CONCENTRATION MICROGRAMS PER ML.
Figure 2.
Relation between the Degree Initial Riboflavin Concentration
of Adsorption and
Relation is valid under conditions of complete lactose crystallization for concentzatcs containing 0.084-0.268 gram exce8s lactose per ml. at 5 O C.
APPLICATION TO RIBOFLAVIN RECOVERY
Applying the data to the preparation of adsorbates from whey, lactose can be produced containing between 200 and 300 micrograms riboflavin per gram, representing 75-80 per cent recovery of riboflavin. Greater concentrations may be realized, but the rate of crystallization of lactose under the conditions required for the preparation of more concentrated products becomes exceedingly slow. The data are capable of application not only to those con-
592
INDUSTRIAL AND ENGINEERING CHEMISTRY
Figure 3.
Vol. 35, No. 5
Photomicrographs of Lactose Crystals (X150)
Above. Shortened tomahawk form crystallizing at 5' C. from concentrate containing 46 micrograms riboflavin and 0.21 gram excess lactose per gram concentrate. Below. Shortened t0mahaw.k form derived from concentrate containing 16.0 micrograms riboflavin and 0.145 gram excess lactose per gram concentrate.
centrates which form the subject of this paper but also to any number of related concentrates. These embrace concentrates obtained from whey in the commericial production of milk sugar and in the production of fermented products (alcohol and lactic acid, for example). They may be used either separately or t o supplement one another, or they may be combined ITith concentrates derived from sources other than milk. Obviously, certain details of processing must be worked out in order to establish optimum conditions, and in this respect each particular concentrate presents its own problem. LIMITATIONS OF ~ I E T H OEmpirical D. formula 1 defines the conditions which must be met in order to obtain potent adsorbates consistent with good yields. I n the application of this equation to whey, a material containing both riboflavin and lactose, it may readily be seen that the potency of
Aboae. Thin platy form derived from a concentrate containing 46 micrograms of riboflavin and 0.15 gram of excess lactose pcr gram. Below;. Thin platy form derived from a concentrate containing 16.0 micrograms of riboflavin and 0.095 gram of excess lactose per gram.
the adsorbate is limited by the pre-existing concentrations of riboflavin and lactose in whey. The yield, Y , is given by the following formula deriyed from formula I: y = -c
- 2.5 C
To obtain a satisfactory yield, it is necessary to concentrate the whey to a considerable degree. A liquor containing lactose in a high degree of supersaturation is subsequently obtained which, if crystallization were permitted to proceed to completion, would result in the recovery of weak adsorbates albeit in good yields. The graphical relations in Figure 4 indicate how t o recover concentrated adsorbates consistent with good yields. At each riboflavin level corresponding to a definite degree of
INDUSTRIAL AND ENGINEERING CHEMISTRY
May, 1943
TABLEI. RELATION BETWEEN RIBOFLAVIN RECOVERY AND LACTOSE RECOVERED UNDER CONDITIONS OF COMPLETE CRYSTALLIZATION Riboflavin Concn. c , Microgram/ M1. 2.10
0.2 0.2 0.2
Riboflavin Reoovery, Per Cent 0.20 0.20 0.25 0.25
Residual Riboflavin EO, Micrograms/ MI. 2.1 2.1 2.1 2.1
0.084 0.191 0.249 0.268
36.2 16.0 11.0 9.7
58.0 58.2 52.2 49.5
2.2 2.2 2.6 2.6
10.5
0.084 0.191 0.249 0.268
94.0 43.0 33.0 30.0
75.2 78.2 78.2 76.6
2.6 2.3 2.3 2.5
21 .o
0.084 0.191 0.249 0.268
218.0 97.0 75.0 67.5
87.3 88.2 88.9 86.2
2.6 2.5 2.3 2.9
31.5
O.OS4 0.191 0.249 0.268
338.0 151 .O 117.0 105.0
90.2 91.6 92.4 89.3
3.0 2.6 2.4 3.3
42.0
0.084 0.191 0.249 0.268
5.25
.
Lactose Recovered a, Gram/Ml. 0.084 0.191. 0.249 0.268
Riboflavin Adsorbed, Micrograms,’ Gram Lactose
0.5
’
3:o 2.2 2.8 Av. 2.49 0.25 Av. co derived from Figure 1 2.50 20i:o 160.0 146.0
9i:i 95.0 93.1
supersaturation, adsorption on the growing crystals is insignificant a t the onset of crystallization and increases sharply only after the degree of supersaturation has reached sufficiently low levels. The values of these low levels are indicated for concentrates containing 5.25 and 10.5 micrograms riboflavin per ml., but unfortunately data are lacking for their evaluaOion a t higher concentrations. The qualitative application of these data to the preparation of adsorbates indicates the desirability of conducting the crystallization operation in two steps; in the first only insignificant quantities and in the second the greater portion of riboflavin would be adsorbed. I n the crystallization of lactose from ethyl and methyl alcohols, a considerable percentage of milk sugar can be recovered in the first stage, and consequently satisfactory conditions exist for the recovery of concentrated adsorbates in good yields in the second stage (3). A difficulty encountered in the preparation of concentrates exceeding 200 micrograms per gram is the diminished rate of crystallization under the conditions required for their preparation. To overcome this difficulty it is essential to increase simultaneously the rate of diffusion of lactose and riboflavin or to provide increased adsorbing surface, Encouraging possibilities are the use of increased stirring rates and copious seeding material. A factor of some significance is pH. Preliminary experiments indicate that maximum adsorption is attained a t a pH of 6.7, corresponding to 75 per cent crystallization. This may mean that adsorption is more rapid at pH 6.7, but it does not necessarily mean that both adsorption and crystallization are more rapid. Concerning the change in habit of the lactose crystal from its characteristic tomahawk t o a thin platy form, experiments show that there is no definite relation between the degree of adsorption and crystal habit. Although adsorbates containing in excess of 300 micrograms riboflavin per gram tend to crystallize as plates, there is overlapping; and tomahawk-shaped crystals containing more than 300 micrograms per gram and platy crystals containing less are recoverable. Figure 3 emphasizes the wide variety of conditions under which the platy form is obtained. This form was described by Herrington ( I ) , who observed it as a transitional form in
593
the development of the normal crystal. The platelike crystals in Figure 3 are products of exceedingly slow crystal growth and do not represent a transitional form. CONCLUSION
A word may be said about the practical significance of the product. At present there is a shortage of riboflavin in a form suitable for human consumption. Because of this shortage public hearings before the Federal Security Administrator on the subject of mandatory riboflavip requirements in the definitions and standards for enriched flour and allied products have been postponed although all other provisions of the definitions and standards of identity are already effective. This shortage will probably be met, in part a t least, by the expanded production of both the synthetic vitamin and various types of concentrates. To what extent the adsorbate described in this paper can compete with these products depends largely on the utilization of the other ingredients of whey, either in their natural or in converted form. The cost of production incidental to the manufacture of the adsorbate would then constitute but a small proportion of the total cost of production.
40QrRIBOFLAVIN MICROGRAMS
LACTOSE CRYSTALLIZED IN PER CENT
Figure 4. Relation between Quantity of Riboflavin Adsorbed per MI. of Concentrate and Percentage Crystallization of Lactose Lactose recovered at 100 per cent erystallization, 0.25 g r a m per ml.
Concerning the purity of the product, the adsorption curves indicate that the adsorbate is interchangeable with admixtures of milk sugar and crystalline synthetic riboflavin of like potency. Pharmaceutical preparations of riboflavin in tablet form usually consist of synthetic riboflavin admixed with the vehicle or diluent milk sugar; consequently the product derived from whey may be utilized directly, or if lacking in potency it may be supplemented with the synthetic vitamin to meet pharmaceutical requirements. LITERATURE
CITED
(1) Herrington, B. L., J. Dairy Soi., 17,633-43 (1934). (2) Leviton, A,, U. 9. Patent 2,116,931(1937); Leviton, A,, and Leighton, A,, IND. ENQ.C H ~ M30, . , 1305-11 (1938). (3) Leviton, A., U. S. Patent application 390,941 (April 29, 1941). (4) Rigt.erink, M.D.,and France, W. G., J. Phys. Chem., 42, 1079 (1938).
