Biophysical Chemistry
Luminescent Characterization of Sodium Dodecyl Sulfate Micellar Solution Properties Keith Goodling, Kim Johnson, Lee Lefkowitz, and Brian Wesley ~ i l l i a m s ' Bucknell University, Lewisburg, PA 17837 Soaps and detergents are useful because they form aggregated structures called micelles. Individual molecules of such materials possess hydrophobic and hydrophilic segments (Fig. 1).At a sufiicient concentration in aqueous solution, aggregation between hydrophobic segments is favored because it excludes water. Micelle formation thus has the effect of creating nonpolar regions in a total structure stable in polar aqueous solutions. B~~~~~~the nonpolar regions of micelles are able to soluhilize nonpolar or-
Hydraphilic
O\
s Hydrophobic
0
. 0
\
o-j-'d,
'O
8 '0-
~ a *
ganic materials, solutions of soaps and detergents are able to dissolve materials that remain insoluble in pure water. Increasingly, micellar solutions in various solvents have become the focus of research due to their potential a s controllable reaction and biomimetic media ( I ) . Biochemists also have 10% been interested in the micelles formed by natural compounds such a s lipids, as well a s in the pmperties of detergents used in extractive and preparative schemes (2, 3). Despite this, few undergraduate physical chemistry experiments deal with these systems. The development of a variety of luminescent techniques during t h e p a s t decade offers a convenient means of introducing measurements on aqueous micellar systems into the teaching laboratory (4). Three types of experiments involving the luminescent characterization of aqueous solutions of sodium dodecyl sulfate (SDS) will be described below: determination of critical micelle concentration (CMC), effects of sodium chloride concentration on the CMC, and finally determination of micellar aggregation number (Nag,). Sodium dodecyl sulfate was chosen because of its ease of usage, wide applications, and well-characterized properties. Luminescent Determination of Sodium Dodecyl Sulfate Solution Properties Critical Micelle Concentration (CMC) The concentration a t which a material forms a micelle is known as the critical micelle concentration (CMC). A simple method for the determination of CMC in aqueous solutions lies in exploiting the solvent-dependent fluorescence associated with many fluorescent molecules (5). Molecules like those shown i n Figure 2 demonstrate poor solubility and limited fluorescence in aqueous solution b u t increased solubility a n d en-
Micelle
Figure 1. Schematic structures for sodium dodecyl sulfate molecules and micelles. A8
Journal of Chemical Education
Presented at the American Chemical Society Meeting, New York, August 1991. 'To whom reprint requests should be addressed,
Nile Red Figure 2. Fluorescent probes useful in critical micelle wncentration (CMC)determination. hanced fluorescence in nonaqueous solutions. The fluorescence response of these molecules therefore serves as a probe of their surroundings in solution, and the fluorescent molecules themselves are referred to as "probes". As the CMC is approached, due to micelle formation there is an increase in the number of nonpolar regions in solution. Aqueous detergent solutions containing these fluorescent probes would, therefore, be expected to show a rapid increase in fluorescence at the CMC, because the formation of micelles permits better solubilization and enhanced fluorescence of the probe. This type of fluorescence response is shown in Figure 3, which demonstrates how the fluorescence intensity of the probe Nile Red varies with aqueous SDS concentration. This use of suitable fluorescent probes is not without problems. High probe concentrations may change the ~ r o ~ e r t i of e s the svstems beine measured. The introduction and equilibration of probes among various solution environments robe "labellincr")is a dvnamic Drocess. Also. because these probes are solugle in water, they usu: ally must be added to detergent solutions in some quantity of organic solvent, which can perturb the CMC value from that in pure aqueous solution. In light of these difficulties, a variety of labelling and measurement techniques were explored in order to determine whether simple, reproducible determinations of CMC values for SDS could be achieved using diphenylhexatriene (DPH) and t h e phenoxazone Nile Red as probes. &
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Experimental Details
All data shown were obtained with a Perkin-Elmer LS50 fluorimeter and commercially available reagents and fluorescent probes. Best results were obtained by fluorescence labelling of stock solutions a t high detergent concentration in large volumes followed by dilution to the desired detergent concentrations. In order to maintain constant probe concentration, deionized water labelled similarly to
2
4
8 10 12 14 SDS CONCENTRATION (mM)
6
16
18
Figure 3. Fluorescent intensity of Nile Red at different sodium dodecyl sulfate concentrations. At the CMC(about8.0mM), the fluorescence intensity shows a diswntinuity.
the stock detergent solution was used for the final dilutions. The specific procedure used in generating Figure 3 was as follows. Initially, deionized water and 50 mM solutions of SDS (Sigma) containing the Nile Red fluorescent probe were prepared in separate 100-mLvolumetric flasks by addition of 200 pL of 15 mM methanolic Nile Red (Aldrich, Kodak, Molecular Probes) solution. This gave a total probe concentration of about 3 x 10" M. Fluorescent labelling was allowed to proceed in the dark overnight. Labelling appears to be improved by addition of methanolic Nile Red after partial solubilization of the SDS. Prior to measurement, the 50 mM SDS stock solution was then diluted to the desired SDS concentrations in 5-mL volumetric flasks using the labelled deionized water. Final dilutions were mixed gently and allowed to stand in the dark for 30 min-lh before measurement. Foaming of the SDS solutions usually could be minimized by dropwise addition of water using a Pasteur pipet or squeeze bottle, or by application of a gentle nitrogen gas or compressed air stream to the neck of the volumetric flask. Emission spectra for each solution were taken between 590 nm and 690 nm at a slit (Continued on neztpage) Volume 71 Number 1 January 1994 A9
The Modern Student laboratory Biophysical Chemistry Variation of Sodium Dodecyl Sulfate Critical Micelle Concentration with Sodium Chloride Concentration
0 REF [B]
width of 20 nm,using an excitation wavelength of 550 nm and a 10-nmslit width. The intensitv at 630 nm was then concentration. A recorded and plotted a s a function O ~ S D S consistent manual rather than automatid photomultiplier eain settine was used throuehout a eiven CMC determina;ion. The p&edure used foi DPH ( ~ o l e c u l aProbes) r was similar to that for Nile Red. Labelling of concentrated detergent solutions was conducted from dry tetrahydrofuran solutions, and a period of 30 min-1 h appears sufficient for both concentrated and dilute detergent solutions. The emission intensity was recorded a t 430 nm using a 390 nm red pass filter, with excitation a t 360 nm. Results
Figure 3 qualitatively resembles a titration curve, and continuous fluorescence intensity increases were observed before and after the discontinuity expected a t the CMC. The CMC value was taken as the mid-point of the discontinuity, determined using the linear extrapolations shown. For reagent grade SDS used without further purification, this procedure consistently gave CMC values for DPH and Nile Red in the vicinity of 8.0 mM, consistent with literature values (5-8).The uncertainty reported is based on an estimated 5-7% relative uncertainty in the value taken as the mid-point of the discontinuity.
A REF [7]
o REF [S]
Figure 5. Comparison of the data in Figure 4 with literature values for SDS CMCat various salt concentrations.All dataare at 298 K, except forthose of ref. (5) for which no temperature was specified. Reproducibility with these probes depends mainly on accurate dilutions and the amount of time over which the dilute solutions are allowed to undergo labelling prior to measurement. Longer labelling times improve results. Concentrated labelled SDS solutions appear to be stable for several days, and organlc probe solutions used for labelling appear stable for several weeks if pn~tectedfrom light. (For DPH. stock solutions in tetrahvdrofuran also should be kept on ice or frozen when not inuse.) If labelled stock detergent solutions were prepared in advance, the undergraduate authors testing these procedures usually were able to generate a 10-12 point intensitylconcentration plot within a 3-4 h period. Because high spectral resolution is not required with these probes, it is likely CMC determination should be possible even on filter fluorimeter instrumentation. Counterion Effects on Critical Micelle Concentration The availability of a simple method for evaluation of the CMC permits student investigation of how this is affected hv chanees in solution orooerties. To test this. the variaS with 'added wdium chloride'concentra&on of ~ D CMC tion was measured usine Nile Red as described above. Hesults for four salt concekrations are shown in the table. Sodium dodecyl sulfate aggregates a t increasingly lower concentrations as salt concentration is increased, consistent with the idea that micelles form in order to minimize the exposure of nonpolar awl chains to the ionic solution. Approximate solution theory permits an estimate of the free energy of micellnation iA G,,,) from such data 19,. For , expression ionic detergents of 1:l e l e c t r o l ~ t y p ethe log, (CMCM= -Kgloglo(CdW)+ AGmiJ2.3 RT
-4.0
-3.5
-3.0
-2.5
-2.0
LOG([Nal/W)
Figure 4. A log4og plot of [CMC]MI versus total [NatW from the table. The intercept of the linear fit selves as an estimate of the free energy of micellization. A10
Journal of Chemical Education
(1)
results, where CMC equals the critical micelle concentration, Chis the counterion concentration, W is the number of moles of waterfliter at tem~eratureT. R is the eas constant, K, is a parameter representmg the electrical energy associated wlth micelllzatlon, and activity coefficients
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Biophysical Chemistry
5
10
15
20 25 30 35 40 SDS CONCENTRATION (mM)
45
50
Figure 6. Plot of eq ( 4 ) for ~ u ( b p ~ )quenching ,+~ by 9 methvlanthracene in SDS micelles. The aaoreoation number and free detergent concentration can be determkdirom the slope and intercept of the linear fit.
have been neglected. A plot of loglo(CMC/W)versus loglo(CdW)should be linear with AGmiJ2.3RT as the intercept, and a plot for the data in table is shown in Figure 4. Here, the sodium ion concentration Cb was taken as the sum of the detergent CMC and the externally added salt concentration, a s the detergent itself releases sodium counterions in solution. The linear fit shown in Figure 4 has slope -0.692 + 0.057 and intercept -6.56 + 0.19, resulting in a final value of AGmi,of -37.5 1.1kJ1mole at 298 K. his compares reasonably well with the reported literature value of 4 2 . 4 kJImole a t 294 K (10). Measurement accuracy was assessed by comparing the results in the table with literature values determined using a variety of methods (5-8).Literature results for DPH ( 5 ) and the present results for Nile Red are represented in Figure 5 by the solid circles and squares, respectively. At low salt concentrations, these two fluorescent probes appear to give CMC values about 10-15% lower than those determined bv other methods. However, it is not uncommon for diffcl.int measurement techniques t o rive different results for C.MC valuri; fi,. Rrdicatr mrasurements for Nile Red (not shown) suggest that the error
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(Continued on next page)
Volume 71
Number 1 January 1994
All
The Modern Student laboratory Biophysical Chemistry estimates for precision given in the table are substantially correct. As for earlier CMC measurements in the absence of salt, the amount of time allowed for equilibration of the dilute solutions was found to be important, especially for dilutions at or near the CMC. Micellar Aggregation Number (Nagg) The micellar aggregation number (N.,) represents the number of detergent molecules or monomers found in a micelle. Analogous to a molecular weight determination for polymers, the N., to be discussed represents an "avera g e d value for a collection of micelles, which individually are dynamic structures capable of exchanging monomers among themselves. Currently, both steady-state and time-dependent luminescent measurements have been applied to the determination of this parameter (4). Steady-state measurements usually rely on the decrease or "quenching" of luminescence of a probe associated with the micelle through the presence of a second species, the "quencher". How luminescence quenching varies with either detergent or quencher concentration is then determined. Several pmbe-quencher combinations have been investigated, but a useful system was developed by Turm and Yetka (11)in a study of SDS micelles. This uses tris-(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate luminescence quenched by 9methylanthracene. Given several assumptions about pmbe-quencher interactions and behavior (4,111, a mathematical expression relating the ratio of luminescence intensities to the total detergent concentration, free detergent concentration and quencher concentration can be derived. Initially, the expression (2) (IIZ,) = exp (-[QI/[Ml) results, relating the ratio of luminescence intensities in the absence (1,) and presence ( I ) of quencher to the quencher concentration [QI and the micelle concentration [MI. The micelle concentration is given by (3) [MI = ([DET]- [FREEI)I&, where [DETI is the total detergent concentration, [FREEI is the concentration of detergent monomers not associated with micelles, and N,, the "average" number of detergent monomers associated with each micelle. Substitution of eq 3 into eq 2 and rearrangement results in (In (I,ln)-' = [DETll([QlN), - [FREEVKQIN), (4) and the aggregation number N, can be determined from the slope and intercept of a linear fit of (In (1oA))F' versus total detergent concentration a t a constant quencher concentration [QI. The feasibility of this measurement for undergraduates was tested by reexamination of the Turm and Yetka measurements for SDS. Experimental Driections
Stock 50 m M SDS solutions containing 7.2 x lo4 M trist2.2'-bi~vridvl1rutheniumdI1 chloride hexahvdrate (Aldrich) were hiluted to various concentrations-in parallel 10-mL volumetric flasks. At each concentration, 50 pL of a stock 1.05 x lo4 M methanolic 9-methylanthracene (Aldrich) solution was added to one flask and 50 pL of methanol to the other. Emission spectra were recorded between A12
Journal of Chemical Education
550 nm and 630 nm at a n 8.0-nm slit widthusina a 530-nm red pass filter, with excitation a t 450 nm and 8.0-nm slit width. The intensities of the quenched and nnauenched solutions at 630 nm were recorded. Results
A plot of eq 4 using the intensity ratios for several pooled runs is shown in Figure 6. The linear fit shown has a slope 67 2. Combining the of 142 + 4 Llmole, giving an N,of slope and intercept (-1.06 + 0.11) gives a free detergent concentration of 7.5 + 0.8 mM. These values compare reasonably well with the original Turro and Yetka values (N,, = 60 2, [FREEI = 7.5 mM) and the values cited in this reference (N.,= 62,631. The value of [FREE1mieht reasonablv be ex~ectedto be close to that of thc. CMC 1; fact, at 1raa;onr rrfennce subr 141. stitutes the CMC value for IFREEI rn t h e ~exDrrssmns While such an assumption would permit a one-step determination of CMC and N,, for SDS, it appears this method leads to underestimates of CMC values. It also should be noted that this probe-quencber combination is only applicable to anionic detergents. Neutral and cationic systems cannot be measured with this probe-quencher combination because it relies upon the association of the metal cation with the negatively charged micelle. This association mieht be the fundamental cause of the discre~ancv " between the [FREEI and CMC values for SDS, since counterion effects can lower CMC values from those in Dure aaueous solution.
.
Conclusions Three experiments involving the physical characterization of aqueous sodium dodecyl sulfate solutions using luminescence techniques have been described and tested. These experiments i r e well within the capabilities of undergraduates and involve readily available reagents and instrumentation. Because of their relative simplicity, relevance to current research, and broad possibilities for extension, it is hoped that these types of experiments will be of interest to instructors interested in modernizing or improving the physical or biophysical chemistry curriculum. Acknowledgment The authors would like to express their thanks for the generous support offered by the Physical Chemistry Project of the Mid-Atlantic Consortium of the Pew Science Pmgram, Merck and Company, and Bucknell University in completing this work. Literature Cited 1. Fendler, J. Membrane Mimetic ChPmutry;W h y : New Yar*,1982. 2. Tanford, Charles. The Hydmphobic Eflmf. 2nd ed.;Wiley: New York, 1988. 3. Neugebauer, J . A Guide lo the P G r l l p s and Usps 0fDe1egente in Biology and Biochemistry: Calbioehem Corporation; San Diego, 1990. 4. Gtieser,F.;Drummond,C. J.J Phys. Chem. 1988,92,55805593. 5. Chattaoadvav. A.: Landon. E A m l BlD~hem.ISM. 139.408412.
8. Sch0e.H. J. phye CLA.1966, GO.
29662913.
9. Rasen, M.;Sur/edonia ondinterfmioiPhpmmna, 2nd ed.: Wiley: NewYork, 1989:
Chapter 3.
Mukejee, P A d u Colloid Interfoe.Sci. 1961,1,241: cited in Referenee 9. 11. lbm,N.J.:Yetka,A. J A m . Cham. Soe. 1978,100,59514952.
lo.
