SOLUBILIZATION OF DYES IN MINERAL OIL AND ITS APPLICATION TO A MODEL BIOLOGICAL CELL MEMBRANE

Sydney Ross

Department of Chemistry, Rensselaer Polytechnic Institute, Troy, New York Received October 19, 1951

INTRODUCTION

Substances that are normally insoluble may be brought into solution in water in the presence of small concentrations of soaps or colloidal electrolytes. The same effect also takes place in nonaqueous solvents, where dilute solutions of oil-soluble surface-active' agents can solubilize substances normally insoluble in oil. The term solubilization was intro- duced by McBain to distinguish this effect from the related but different processes of hydrotopy, formation of ordinary colloidal sols, colloidal suspension, emulsification, blending, and cosolvency. Solubilization de- pends on the spontaneous formation by association of a colloidal mieelle in or upon which the molecules of the solubilized material are incorporated. Many studies have been made of solubilization, and a comprehensive review was published recently by Klevens (2), in which 258 separate references are cited. Of this work only a small fraction treats of solubili- zation in nonaqueous solvents. The topi c has, however, an interesting application in biology that has not previously been realized, namely, in the permeability of cells.

The lipoid theory of cell permeability postulates an organic solvent whose dissolving power, compared to that of water, parallels the permeat- ing power of the solute. No single, generally appropriate, pure solvent could be found, however, whose dissolving properties were such that for a large number of organic nonelectrolytes there would be a correspondence between 'their solubility in the solvent and their ability to permeate cell membranes. The best solution so far discovered comes from an exhaustive series of experiments done by Nirenstein (3), who correlated the vital staining of Paramecium by dyes with thesolubility of the dyes in a mixture of olive oil, oleic acid, and diamylamine. This model cell solution simulates the cell membrane rather than the complete cell, and therefore only sub- stances taken up by the model cell have a chance to go into the inner part of the real biological cell. Any such mixture, referred to as a "model biolo- gical cell," would have to meet the conditions that on extraction with aqueous dye solutions, the oil system would take up the same type and amount of dye as the living system would, and it would reject those dyes

497

498 SYDNEY ROSS

that are rejected by the living system. Nirenstein's "model biological cell" consists of 18 parts olive oil, 6 parts oleic acid, and 1 part diamylamine. The dyes that are extracted from aqueous solutions by this oil mixture correspond remarkably to those that are able to stain Paramecium.

As well as going far toward elucidating the mechanism of cell perme- ability, Nirenstein's results provide the possibility of a system in vitro that would come close to duplicating some of the behavior of a living organism. It still suffers from the disadvantages of uncertain composition, as olive oil is by no means a standard material, and even oleic acid is difficult to obtain pure. It therefore seems important to set on foot experiments that migh~ lead to the discovery of another model biological cell that would be free of these defects.

It is immediately apparent that oleic acid and diamylamine are able to combine, and that one of the constituents of Nirenstein's model cell is the amine salt of oleic acid. This substance belongs to the general class of surface-active agents, and the close relation of Nirenstein's results to the study of solubilization in oils becomes clear. This paper presents a series of qualitative and quantitative experiments that might be the basis for a desired continuation of Nirenstein's work.

EXPERIMENTAL

I

To eliminate the uncertainty of the composition of olive oil, these experiments have all been done on a highly refined white mineral oil, Drakeol No. 10, of the Pennsylvania Refining Co. The replacement of the olive oil denies us the presence of other substances, present in the oil, that are also effective solubilizing agents. It was found immediately that olive oil by itself was already a better solubilizing s,olvent than white mineral oil. A series of experiments were done with 35 different dyes, taken from Nirenstein's list, which were examined both for solubility in mineral oil alone and in mineral oil containing 1% di-n-butylamine oleate. Sixteen dyes were found to be solubilized by the addition of the amine oleate. A second series of tests was made using Nirenstein's technique, namely, dissoving the dye in water and then extracting it with a 1% solution of the amine oleate in mineral oil. The results did not correspond exactly with those of the previous test : a smaller number showing signs of solubilization. The presence of water, of course, complicates the system, as water may itself be solubilized in the oil, as well as the dye. These experiments showed, however, that the presence of even a small quantity of an amine oleate is enough to cause solubilization of many dyes, normally insoluble in oil, and that therefore the results obtained by Nirenstein can be grouped as examples of the phenomenon that is described by the title of this paper.

S O L U B I L I Z A T I O N OF DYES 499

II

McBain, Merrill, and Vinograd (5) have reported over 200 qualitative observations on the solubilization of dyes in nonaqueous solvents. Only a few of these were made with mineral oil as the solvent, and accordingly several similar experiments were made, using mineral oil, to find systems that would be suitable for further study. As a result of this search eight commercial surface-active agents were selected (Table I), for comparison

TABLE I List of Agents used for Dye Solubilization in Oil

No. Trade name

1 Span 20 2 Span 85 3 Glyceryl oleate 4 G-954 5 G-2000 6 Tween 81 7 Arlacel B 8 Amine soap

Major chemical constituent

Sorbitan monolaurate Sorbitan trioleate

Ma,nnitan monooleate Mannitan monopalmitate Polyoxyethylene sorbitan monooleate Mannitan monooleate Di-n-butylamine oleate

of their action on a series of water-soluble dyes. The dyes chosen are those federally certified for use in foods, drugs, and cosmetics, manufactured by the National Aniline Co. and supplied through its generosity. These dyes are described in the Merck Index, 5th edition, p. 593 (1940). As solubiliz- ing agents, the chemicals found to have the best effect are those generally used as oil-soluble emulsifying agents, more particularly the nonionic sub- stances, as exemplified by the fat ty esters of the sugar alcohols. Materials of this sort are made and sold under the trade names Span and Arlacel by Atlas Powder Co., and are essentially partial esters of the common fat ty acids (laurie, palmitic, stearic, and oleic) and hexitol anhydrides (hexitans and hexides), derived from sorbitol and mannitol. The agents known by the trade name Tween are derived from the sorbitan esters by adding polyoxyethylene chains to the non-esterified hydroxyls, thus changing the hydrophile-lipophile balance toward a more water-soluble product. The hydrophilic character of these surface-active agents is supplied by free hydroxyl and oxyethylene groups, and the lipophilie portion comes from the hydrocarbon chain of the fa t ty acid used. All of them are actually com- plex mixtures of several compounds. Neither these materials nor the dyes were purified beyond the stage supplied by the manufacturer, as the present problem is more concerned with the existence and relative magni- tude of the effect, than with an investigation of the effect of variations in the molecular structure of the agent. Nevertheless, because of the varia- tion in hydrophile-lipophile balance in these materials, some information on this point was obtained.

The amine soap was made by adding exactly equivalent amounts of y

5 0 0 S Y D N E Y ROSS

di-n-butylamine ( E a s t m a n Organic Chemicals, P1260) to oleic acid, whose equivalent weight had been obtained by titration.

Each of these agents is soluble in mineral oil at room temperature. Solutions of each, 1.00% by weight, were made in white mineral oil tha t had been dried by the addition of metallic sodium. To small portions of each of these solutions a pinch of each solid crystalline dye was added, and the mixture was stored in a constant temperature cabinet at 25°C. for 2 months, with occasional shaking during the first week or two. At the end of 2 months a deep color was present in each sample and the excess solid dye had settled to the bo t tom of the tube.

TABLE II Solubilization F. D. and C. Dyes by 1% Solutions of Various Agents in Refined Mineral Oil

Values are in micromoles of dye/liter of solution

F. D. and C. Dye

Blue No. 1 Blue No. 2 Green No. 1 Green No. 2 Green No. 3 Orange No. 1 Red No. 1 Red No. 2 Red No. 3 Red No. 4 Yellow No. 1 Yellow No. 5 Yellow No. 6

Solubilizing agents of Table I

ka

640 610 620 630 625 475 500 515 525 505 430 425 480

21.3 13.3 23.8 94.5

223 135 24.5 60.9 57.8 44.8 56.7

131 • 33.5

l

2

1.3 3.2 3.5 0

I 0.7 1.1 3.5 1.5

10.6 6.3 2.7 0 0.4

3 4

35.8 8~4

277 234 128 160

0 24.5 2.5 55.5 0.4 304 ' 7.7 61.5 4.2 57.0 5.5 78.8 0.1 19.7

118 2.6

53.4 274

3.3 11.9

5 •

0.9 0 2.0 0.9 2.2 2.3 2.4 0 0.3 7.1 0.8 2.9 0

6 7

0.3 9.7 0.4 0.4 0.1 143 0.5 60.1 0.2 0.2 0 17.5 1.6 3.5 0 0 0.1 0 1.6 2.1 1.7 0.3 1.0 0 0.6 0

Lambda (k) = wavelength in millimicrons of minimum transmission.

3.2 1.1 2.9 1.5 3.0 0 7.1 0 0

20.8 0 0 0

The frequencies of min imum transmission were determined by measur- ing, with a Beckman model B spectrophotometer, the transmission of an aqueous solution containing about 10 -4 mole/l, of each dye to be tested. These wavelengths are reported in col. 2, Table I I ; they were later used in the standardization and analysis of each dye. M a n y dyes are known to have a "spectral shift," i.e. a change in the wavelength of minimum transmission, with changes of concentration or on solubilization with certain agents. In none of these solubilized dye systems~ however; does a spectral shift occur.

The standardization curve for each dye was obtained from measure- ments of a series of aqueous solutions of known concentration. The use of aqueous solutions gets around practical difficulties tha t might have

SOLUBILIZATION OF DYES 501

occurred had solubilized dye-in-oil solutions been used, but invites the serious criticism that the transmission of a solubilized system may not correspond to that of an equal concentration of dye in water, even after a correction is made for the difference in the transmission of the solvents. The complete (visible range) transmission spectrum for F. D. and C. Blue No 1 dissovled in water was measured and compared to that of the same dye solubilized in oil with Span 20 and again with G954. No difference could be observed. For the other dyes the absence of any spectral shift, either in aqueous solution or in the solubilized system, was taken as an indication of the same congruity, and the congruity itself interpreted as a justification for the use of aqueous standards for the analysis of solubilized dyes in oils.

In Table II are reported the final results, corrected where necessary for the slight solubility of the dye in oil when no agent is present, so as to give solely the solubilizing effect of the added agent.

Some interesting conclusions can be drawn from these results. The dyes have, between themselves, greater variance in molecular structure than have the first seven of the eight solubilizing agents between themselves. Nevertheless, greater differences are to be observed by reading the columns of Table II horizontally than by reading vertically; the agents are less selective in their behavior than are the dyes; speaking generally and loosely, a good agent will solubilize each and every dye, disregarding (within rather wide limits) the details of its molecular structure; but change the structure of the agent (within rather narrow limits) and its solubilizing ability is greatly lessened, again for all dyes. Whatever may be its cause, this effect is encouraging in the search for a model biological cell, where a single solution must be capable of solubilizing many different compounds. There is a parallel between this behavior and the behavior of biological systems.

Although the data are not sufficiently extensive to give more than an indication, the hydrophile-lipophile balance of the agents appears to be important. The more hydrophilic Tween 81 (agent 6) and the amine soap (agent 8) are notably less effective solubilizers. At the other extreme the lipophilic Span 85 (agent 2) is also less effective. 1 The importance of this balance is no more than has been found to be generally true in most applications of surface-active agents, and it is a useful guide in selecting the suitable agent for a given application. A numerical index of the hydrophile-lipophile balance has been proposed in the publications of the Atlas Powder Company (1), but the method by which it is determined has not been published.

Another well-known characteristic of surface-active agents is indicated

1 Span 85 is believed to be more lipophilie than the other agents on the author i ty of the manufacturer (!) '

502 SYDNEY ROSS

in these results, and by a comparison of the effect of the amine soap with the mixture composed by Nirenstein, namely, the synergetic effect: the enchancement of an effect, beyond the ability of any single component, when a mixure is used. All the agents used here, with the exception of the amine soap, are mixtures; agents 4 and 7 are mixtures with the same principal constituen~ , yet differing in their effects. This too must be taken into account in devising any other model biological cell.

I¢

8.0

.3

E 60

z R I-- ~ 5.0 I- Z

Z 0

w ~ 3.C1 o

/ o

®

/ / /o

/

0 .02 .04 06 08 I0 .12 .14 .16 .18

% ARLAGEL "B"

Fro. 1. Solubilization of methylene blue in mineral oil by Arlacel B. Equilibrium concentration of dye solubilized in mg./l, v s . concentration of Arlaccl B in solution.

III

It now seemed of interest to select one of the agents and study the effect of varying its concentration. Agent No. 7 (Arlacel B) was selected, and its effect on three dyes, methylene blue (Merck Co. product, recrystallized from absolute alcohol), F. D. and C. Green No. 1 (Guinea Green), and F. D, and C. Orange No 1, was evaluated.

SOLUBILIZATION OF DYES 503

It was found that reproducible results could not be obtained unless care was taken to remove moisture from the system. Each solution of the agent in oil was treated with Drierite (CaS04) for 24 hr. in a rotating sample holder at 30°C. The Drierite was removed by centrifugation and a portion of the oil solution was retained as a blank for comparison with the solubilized dye system.

9C

Z o b -

e t

z 50 I=.1

Z 0

4 0 I.g

I 0 0

10

O

0 .04 .08 .12 .16 .20 .24

% ARLACEL "B"

FIG. 2. Solubilization of Guinea Green (F. D. and C. Green No. 1 Dye) in mineral oil by Arlacel B. Equilibrium concentration of dye solubilized in mg./l, v s . concentration' of Arlacel B in solution.

The dyes were solubilized in screw-cap test-tubes by adding excess solid dye to the dried oil solution of the agent, and placing the tubes in a rotating sample holder inside an air bath at 30°C. for a week. Excess solid was removed by centrifugation, and the color intensity of the system was measured with a Lumetron colorimeter, equipped with appropriate filters and a line-voltage stabilizer. The standardization curve was ob-

504 SYDNEY ROSS

rained from aqueous solutions, as before. Methylene blue is known to have a spectral shift as the concentration is increased (7), but it does not become significant even for the most concentrated of the solutions measured in this work (3 X 10 -~ mole/l). The transmission for the visible range of t h e spectrum is the same for the dye dissolved in water as it is for the solu- bilized dye; this holds for the three dyes.

9 . 0

8 . 0

7.O - I / o* E

6.0 Z _0 I,-

5.0 I--

/ W .

Z 0 0 .

W

~ 5 . C - -

,i / / -

. OI .02 .05 .04 .05 .06 .07

% ARLAC EL"B =

FIG. 3. Solubilization of F. D. and C. Orange No. 1 Dye in mineral oil by Arlacel B. Equilibrium concentration of dye solubilized in rag./1, v s . concentration of Arlacel B. in solution.

The results of this portion of the work are reported in Figs. 1, 2, and 3 showing the amount of dye put into solution in a liter of oil at different concentrations of agent. Each figure shows the same general pattern : slight solubilization at low concentrations and then at a critical concentration the beginning of a rapid rise in the amount of dye solubilized. For aqueous solutions of surface-active agents, the critical concentration at which solubilization begins to increase is interpreted as the stage at which significant amounts of the solute are spontaneously associating to form

SOLUBILIZATION OF DYES 5 0 5

large aggregates or colloidal micelles. The existence of a so clearly defined, or critical, eoncentration for mieelle formation (sometimes referred to as CMC--critieal mieelle concentration) is demonstrated in aqueous solu- tions of dodeeyl sulfonic acid by the change of other physicochemieal properties with concentration: such properties as equivalent eonduetivi-

=

d i l l

0

J rr"

E

) , . r'-,

E

t000

I I

800

700

/ I

\

40C

300

200

.GE No,

GUINEA /

1

/ METHYLENE BLUE /// ,Z

/ff44' I ~ ~'-4t--___ v ]

0 .02 .04 .06 .08 O]O QI2 0.14

T~ARL ACE L "B"

Fro. 4. An alternative method of plotting the solubilization data of the previous figures, to show the similarity of the effect of the solubilizing agent on each dye. T h e equilibrium amount of dye solubilized per rag. of agent v s . concentration of the agent in s61ution.

ties, transport numbers, and diffusion coefficients (6). Association colloids occur in nonaqueous media almost as much as in water, and it is therefore not surprising to find a critical micelle concentration in these oil solutions, just as in aqueous solutions of soaps and other association colloids. That the critical mieelle concentration is a function of the agent and is independ- ent of the dye being solubilized is demonstrated in Fig. 4, where the data

506 SYDNEY ROSS

are plotted in the form mg. dye/mg. Arlacel B vs. per cent Arlacel B. The curves for the three different dyes have the same form, and all have a minimum at the same concentration of agent (about 0.05% by weight), which may be taken as the CMC of Arlacel B in mineral oil.

By an application of the mass law to the equilibrium nA ~ A~, where A represents the formula of the solubilizing agent, and by assuming that the concentration of solubilized dye is a direct measure of the concentra- tion of the micelle, Am, it can be shown that the function illustrated by Fig. 4 cannot have either maxima or minima: Only if two or more micelles are simultaneously present can minima and maxima appear in this func- tion. In the equilibrium nA ~-A,~ there must therefore be at least two different values of n present. The energy differences between different micellar forms is so small that it is probable that a series of micellar sizes exists. I t is certainly correct to conclude that, in coincidence with the findings of McBain and Huff (4) for aqueous solutions of some colloidal electrolytes , it may also be said of these oil solutions that no one formula- tion of a micelle is valid over the whole range of concentration. Vold (8) has recently demonstrated the possibility of there being a distribution of micellar sizes in aqueous solutions of all colloidal electrolytes, even for those where the data have previously been sufficiently described by a monodisperse micellar system. The present results would encourage a similar interpretation for nonaqueous solutions of association colloids.

ACKNOWLEDGMENTS

The author acknowledges the students who have helped with the experimental por- tions of this work: with Part I, R. Minnieh and G. Forbes; with Part II, D. S. Beard, T. Donnelly, and R. Lacoste; with Part III, B. A. Becker, F. A. Phillips, Jr., and R. L. Zimmerman.

SUMM&RY'

1: The model biological cell of Nirenstein is shown to operate by solubilization of dyes in oil.

2. The solubilization in oil of the oil-insoluble F. D. and C. dyes is measured for different solubilizing agents at 1% concentration.

3. The solubilizing ability of each agent depends on its physicochemi- cal behavior in solution and, within wide limits, does not depend on the structure of the dye.

4. The effect was observed of a single agent, at different concentrations, on the solubilization of three different dyes.

5. The change of solubilizing power with change in concentration is of the same general form for one agent with different dyes.

6. In these oil solutions of association colloids, there is an equilibrium between different kinds of miceiles present in the same solution, changing with concentration and other factors.

SOLUBILIZATION OF DYES 507

REFERENCES

1. INDUSTRIAL CHEMICALS ~ DEPARTMENT, ATLAS POWDER CO., Atlas Surface Active Agents. Wilmington, Del., 1948.

2. KLEVENS, H. B., Chem. Revs. 47, 1-74 (1950). 3. NIRENSTEIN, E., Arch. ges. Physiol. (Pfii~gers) 179, 233 (1920). 4. McBAIN, 3. W., AND HUFF, H. M., J. Colloid Sci. 4, 383 (1949). 5. McBAIN, J. W., MERRILL, R. C., JR., A~D VINOGRAD, J. R., J. Am. Chem. Soc. 62,

2880 (1940). 6. MoBAIN, M. E. L., J. Phys. Chem. 47, 196 (1943). 7. MICFIAELIS, L., AND GRXNIOK, S., J. Am. Chem. Soc. 67, 1212 (1945) ; VICKERSTAFF, W.,

A~D LEMIN, D. R., Nature 157, 373 (1946). 8. VOLD, M. J., J. Colloid Sci. 5, 506 (1950).