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 ag- ganic materials, solutions of soaps and detergents are able
gregated structures called micelles. Individual molecules to dissolve materials that remain insoluble in pure water.
of such materials possess hydrophobic and hydrophilic seg- Increasingly, micellar solutions in various solvents have
ments (Fig. 1).At a sufiicient concentration in aqueous so- become the focus of research due to their potential a s con-
lution, aggregation between hydrophobic segments is fa- trollable reaction and biomimetic media ( I ) . Biochemists
vored because it excludes water. Micelle formation thus also have 10% been interested in the micelles formed by
has the effect of creating nonpolar regions in a total struc- natural compounds such a s lipids, as well a s in the pmper-
ture stable in polar aqueous solutions. B~~~~~~the nonpo- ties of detergents used in extractive and preparative
lar regions of micelles are able to soluhilize nonpolar or- schemes (2, 3). Despite this, few undergraduate physical
chemistry experiments deal with
these systems.
Hydraphilic
The development of a variety of lu-
minescent techniques during t h e
O\ 8 p a s t decade offers a convenient
s means of introducing measurements
'O '0- on aqueous micellar systems into the
Hydrophobic
~ a * teaching laboratory (4). Three types
of experiments involving the lumi-
nescent characterization of aqueous
solutions of sodium dodecyl sulfate
0
(SDS) will be described below: deter-
mination of critical micelle concen-
tration (CMC), effects of sodium
chloride concentration on the CMC,
and finally determination of micellar
aggregation number (Nag,). Sodium
. 0
dodecyl sulfate was chosen because of
its ease of usage, wide applications,
\
o-j-'d, and well-characterized properties.
Luminescent Determination of
Sodium Dodecyl Sulfate Solution
Properties

Critical Micelle Concentration (CMC)

The concentration a t which a mate-
rial forms a micelle is known as the
critical micelle concentration (CMC).
A simple method for the determina-
tion 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 lim-
ited fluorescence in aqueous solution
b u t increased solubility a n d en-
Micelle
Presented at the American Chemical So-
ciety Meeting, New York, August 1991.
'To whom reprint requests should be ad-
Figure 1. Schematic structures for sodium dodecyl sulfate molecules and micelles. dressed,

A8 Journal of Chemical Education

 Nile Red
Figure 2. Fluorescent probes useful in critical micelle wncentration
(CMC)determination.
hanced fluorescence in nonaqueous solutions. The fluores-
cence 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 in-
crease in fluorescence at the CMC, because the formation
of micelles permits better solubilization and enhanced flu-
orescence of the probe. This type of fluorescence response
is shown in Figure 3, which demonstrates how the fluores-
cence 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
& A -
e s the svstems beine measured. The introduc-
tion and equilibration of probes among various solution en-
vironments 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, reproduci-
ble determinations of CMC values for SDS could be
achieved using diphenylhexatriene (DPH) and t h e
phenoxazone Nile Red as probes.
Experimental Details
All data shown were obtained with a Perkin-Elmer LS-
50 fluorimeter and commercially available reagents and
fluorescent probes. Best results were obtained by fluores-
cence labelling of stock solutions a t high detergent concen-
tration 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 6 8 10 12 14 16 18
SDS CONCENTRATION (mM)
Figure 3. Fluorescent intensity of Nile Red at different sodium
dodecyl sulfate concentrations. At the CMC(about8.0mM), the fluo-
rescence intensity shows a diswntinuity.
the stock detergent solution was used for the final dilu-
tions. The specific procedure used in generating Figure 3
was as follows. Initially, deionized water and 50 mM solu-
tions 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 (Al-
drich, Kodak, Molecular Probes) solution. This gave a total
probe concentration of about 3 x 10" M. Fluorescent label-
ling was allowed to proceed in the dark overnight. Label-
ling appears to be improved by addition of methanolic Nile
Red after partial solubilization of the SDS. Prior to mea-
surement, 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 solu-
tions usually could be minimized by dropwise addition of
water using a Pasteur pipet or squeeze bottle, or by appli-
cation 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
Variation of Sodium Dodecyl Sulfate Critical Micelle
Concentration with Sodium Chloride Concentration
width of 20 nm,using an excitation wavelength of 550 nm
and a 10-nmslit width. The intensitv at 630 nm was then
recorded and plotted a s a function O ~ S D Sconcentration. A
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 de-
tergent 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 discon-
tinuity, 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 litera-
ture 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.
-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
Biophysical Chemistry
0 REF [B]
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 ac-
curate 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 label-
ling 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 under-
graduate 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 determina-
tion should be possible even on filter fluorimeter instru-
mentation.
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 varia-
&on of ~ D CMC
S with 'added wdium chloride'concentra-
tion was measured usine Nile Red as described above. He-
sults for four salt concekrations are shown in the table.
Sodium dodecyl sulfate aggregates a t increasingly lower
concentrations as salt concentration is increased, consis-
tent 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
ionic detergents of 1:l e l e c t r o l ~ t y p ethe
, expression
log, (CMCM= -Kgloglo(CdW)+ AGmiJ2.3 RT (1)
results, where CMC equals the critical micelle concentra-
tion, Chis the counterion concentration, W is the number of
moles of waterfliter at tem~eratureT. R is the eas con-
stant, K, is a parameter representmg the electrical energy
-
associated wlth micelllzatlon, and activity coefficients
 Biophysical Chemistry

5 10 15 20 25 30 35 40 45 50
SDS CONCENTRATION (mM)

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 inter-
cept, 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, result-
*
ing in a final value of AGmi,of -37.5 1.1kJ1mole at 298 K.
his compares reasonably well with the reported litera-
ture 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 repre-
sented in Figure 5 by the solid circles and squares, respec-
tively. 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 mra-
surements for Nile Red (not shown) suggest that the error
(Continued on next page)
Volume 71 Number 1 January 1994 All
The Modern Student laboratory
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 mi-
celle. Analogous to a molecular weight determination for
polymers, the N., to be discussed represents an "aver-
a 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 lumi-
nescent measurements have been applied to the determi-
nation of this parameter (4). Steady-state measurements
usually rely on the decrease or "quenching" of lumines-
cence of a probe associated with the micelle through the
presence of a second species, the "quencher". How lumines-
cence 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) chlor-
ide hexahydrate luminescence quenched by 9-
methylanthracene. Given several assumptions about
pmbe-quencher interactions and behavior (4,111, a math-
ematical expression relating the ratio of luminescence in-
tensities to the total detergent concentration, free deter-
gent concentration and quencher concentration can be
derived. Initially, the expression
(IIZ,) = exp (-[QI/[Ml) (2)
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
[MI = ([DET]- [FREEI)I&, (3)
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 con-
centration [QI. The feasibility of this measurement for un-
dergraduates 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 tris-
t2.2'-bi~vridvl1rutheniumdI1 chloride hexahvdrate (Al-
drich) 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 (Al-
drich) solution was added to one flask and 50 pL of metha-
nol to the other. Emission spectra were recorded between
A12 Journal of Chemical Education
Biophysical Chemistry
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 so-
lutions 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
of 142 + 4 Llmole, giving an N,of 67 2. Combining the
slope and intercept (-1.06 + 0.11) gives a free detergent
concentration of 7.5 + 0.8 mM. These values compare rea-
sonably 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 sub-
stitutes the CMC value for IFREEI rn t h e ~exDrrssmns
r 141.
While such an assumption would permit a one-step deter-
mination 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 appli-
cable 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
tween the [FREEI and CMC values for SDS, since counter-
" be- .
ion effects can lower CMC values from those in Dure aaue-
ous solution.
Conclusions
Three experiments involving the physical characteriza-
tion of aqueous sodium dodecyl sulfate solutions using lu-
minescence techniques have been described and tested.
These experiments i r e well within the capabilities of un-
dergraduates and involve readily available reagents and
instrumentation. Because of their relative simplicity, rele-
vance to current research, and broad possibilities for ex-
tension, it is hoped that these types of experiments will be
of interest to instructors interested in modernizing or im-
proving the physical or biophysical chemistry curriculum.
Acknowledgment
The authors would like to express their thanks for the
generous support offered by the Physical Chemistry Proj-
ect of the Mid-Atlantic Consortium of the Pew Science Pm-
gram, Merck and Company, and Bucknell University in
completing this work.
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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.
lo. Mukejee, P A d u Colloid Interfoe.Sci. 1961,1,241: cited in Referenee 9.
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