Anal. Chem. 1983, 55,1115-1117 Biennial Lignito Symposium, San Antonio, TX, June 15-17, 1981; pp 627-667. (4) Vogel, A. “Textbook of Practical Organic Chemistry”, 4th ed.; Longmans: New York, 1978; p 883. (5) Gordos, J.; Schaenblin, J.; Spring, P. J. Chromafogr. 1977, 143, 171-181. (6) Rucker, G.; Natarajan, P. N.; Fell, A. F. Arch. Pharm. (Weinhelm, Ger.) 1971, 304, 883-893. (7) Corral, R. A.; Orazi, 0.0.; Duffield, A. M.; DJerassi, C. Org. Mass Spectrum. 19’71, 5, 551-563. (8) van den Dool, H.; Kratz, P. D. J . Chromafogr. 1983, 7 1 , 463-471. (9) Bucherer, H. T.;Steinelr, W. J. Prakf. Chem. 1923, 140, 291-316.
1115
(10) Ware, E. Chem. Rev. 1950, 46, 403-470. (11) Olson, J. K.; Schobert, H. H. Prepr. Pap.-Am. Chem. Soc., Div. Fuel Chem. 1979, No. 24, 2 (12) Sax, N. I . “Cancer Causlng Chemicals”; Van Nostrand-Reinhold: New York, 1981; p 377.
RECEIVED for review November 19,1982. Accepted February 18,1983. Reference to specific brand names and models is done to facilitate understanding and neither constitutes nor implies endorsement by the Department of Energy.
Crit icaI 1\11ic elIe Concent rat ion Determination of Nonionic Detergents with Cooimassie Brilliant Blue G-250 Ken S. Rosenthal” arid Frank Koussaie Department of Microbiology/Immunology, Northeastern Ohio Universities College of Medicine, Rootstown, Ohio 44272
A spectrophotometric assay for crltical mHcelle concentratlon determination of nonionic detergent solutions Is described. The assay utlilzes the same dye, Coomagsle Brililant Blue 0, and conditions as for the direct protein-dye blndlng assay.
was mixed briefly and an absorption spectrum was obtained against a water blank. Alternatively, only absorptions at 470 nm (A470) and 620 nm (A6ao) were obtained. The A,,, and A620 were plotted independently against detergent concentration. The CMC can readily be determined from this plot.
The use of nonionic detergents has become important to the study of membrane protein structure and function. These detergents differ in their chemical structure and biological interaction with proteins (I, 2). The extent of protein-detergent interaction can often be correlated with the hydrophile-lipophile balance (HLB) (3) or critical micelle concentration (CMC) of the detergent ( 4 ) . The HLB for most detergents can be obtained from the literature (5),but the CMC usually must be determined empirically (6). CMC determinations are usually made on the basis of a sharp change in the colligative properties, surface tension, solubilization of II hydrophobic dye, e.g., Orange OT, or conductivity of a detergent, solution with respect to detergent concentration (6). In this paper, we describe an extrapolative assay for CMC determination based on the change in absorption spectrum of Coomassie Brilliant Blue G (CBBG). The CBBG absorption spectrum changes as a function of the HLB of a series of detergents or the micelle concentration of a detergent solution. The assay utilizes the same conditions and reagents established for a widely used method for protein determination (7,8). This provides a rapid, simple assay for detergent characteristics in solution.
Coomassie Brilliant Blue G-250 (CBBG) has two major absorption peaks with maxima a t 655 nm and 465 nm when prepared as the protein-dye binding assay reagent. Upon binding to protein, a change in the absorption spectrum for CBBG occurs. The peak a t 655 nm shifts to 595 nm with a decrease in the peak at 465 nm. Protein determinations can thus be made by measuring the ASg5of the resulting blue solution. Upon mixing of the dye with organic solvents, such as 2-propanol or chloroform, a single absorption peak with maximum at 615 nm is observed (Figure 1). Low concentrations of Triton X-100, up to 0.008% (approximately 0.13 mM) caused no change in the absorption spectrum of the CBBG reagent. However, increasing the concentration beyond 0.010% (approximately 0.15 mM) caused a concomitant increase in the A620 with a decrease in &?@ The resultant spectra, at detergent concentrations greater than 0.02 % , resemble the spectrum of CBBG in the organic solvent (Figure 2). Similar concentration-dependent changes in CBBG spectra were observed for other nonionic detergents. These results indicate that a t high concentrations of nonionic detergent, which correspond to a high detergent micelle concentration with respect to monomer concentration, the dye is completely sequestered within a hydrophobic environment, the micelle interior. This spectral change was not observed for ionic detergents such as sodium dodecyl sulfate or sodium deoxycholate. For these ionic detergents increase in absorption of the CBBG with increasing detergent concentration occurs without a change in the absorption spectrum suggesting that the interaction between CBBG and these detergents differs from that with nonionic detergents. Correlation with HLB. Since incorporation of CBBG into a hydrophobic environment alters the spectrum of the dye, the relationship between hydrophobicity of a series of Triton X and Triton N detergents, differing in their HLB number (IO),and the A620 and A470 of CBBG was investigated. As can be seen in Figure 3, there is an inverse nonlinear relationship between the HLB and the absorption of the detergent-CBBG solution. Determination of CMC. Since there is a correlation between the HLB of a detergent and its CMC, and for the Triton
RESULTS
EXPERIMENTAL SECTION Reagents. Coomassie Brilliant Blue G-250 (CBBG), sodium dodecyl sulfate, and bovine serum albumin (BSA) were obtained from Sigma. The Triton detergents were kindly provided by Jan R. Gelfand, Rohm and Ham, Inc. The other detergents Emulgen 911 and Emulgen 913 (Kao-Atlas) and Lubrol PX and NP 40 (Shell) were obtained from John Chiang, Northeastern Ohio Universities College of Medicine (9). All other reagents were standard reagent grade. Preparation of Solutions. The CBBG reagent was prepared as described by Bradford (7) or alternatively, as obtained from Bio-Rad (8). Final concentration of CBBG in the reagent was 0.01% (w/v) and in the assay was 40 ppm (w/v). The detergents were made up as 10% (w/w) stock solutions in deionized water. CMC Assay. The assay procedure is as described by Bradford (7)for protein concentration determination. Briefly, varying concentrations of detergent solutions were added in a volume of 0.2 mL to 4.8 mL of the CBBG reagent. Final detergent concentration was based on the total 5 mL volume. The solution
0 1983 American Chemical Society
1116
ANALYTICAL CHEMISTRY, VOL. 55, NO. 7, JUNE 1983
I
\
1.11 W
0
m
a
0
(0
a .7 m .5 I
I
12
,
14
18
16
HLB
Flgure 3. Change in absorptivity of CBBG reagent with respect to HLB of TrRon X detergent. aqueous solutions of the Triton X 114, X 100, X 165, and X 365 (0.1 mL) were mixed with the CBBG reagent (4.9 mL) to a final concentration of 0.04% (wlw) and absorbance at 620 nm (A 820) and 470 nm (A 470) measured. 350
480
650
650
14 -
750
WAVELENGTH ( n m )
13. -
Flgure The absorption spectra of the CBBG reagent (4.- mL) with 0.2 mL of distilled water (-), with 0.2 mL of 2-propanol (- - -), and with 0.15 mL of 1% (w/w) Triton X 100, final concentratlon 0.03% Trlton X 100 (-.-).
12 I I
I O
-
3 -
2 I
1 0
/
/
1
01 TRITON X- 100
#
1 1 02
/
CONCENTRATION
1
8
1
03
i%)
Flgure 4. Crltical micelle concentration determination uslng the CBBG reagent. Varying concentratlons of Triton X 100 were prepared in the CBBG reagent. The A 620 and A 470 were plotted vs. concentration. The CMC was then determlned as the intersection of the lines corresponding to the A 620 or A 470 values above and below the CMC.
Table I. Detergent Properties (Homogeneous Solutions)
\ 350
450
550
650
750
Flgure 2. The absorption spectra of the CBBG reagent with concentrations of Triton X 100 below 0.006% (w/w) (-) and above 0.03% (w/w) (- -), the critlcal micelle concentration of the detergent.
a
CMC a (expt 1)
detergent
HLB
Triton N 60 Triton X 114 Triton X 100 Triton X 1 6 5 Triton X 305 Emulgen 911 Emulgen 913 Lubrol Px NP 40
10.9
0.0065
12.4
13.5
0.009 0.010
15.8
0.027
17.3
Detergent percent by weight.
CMCa (reported) 0.009b 0.010b
0.067 0.0025 0.004
0.008
0.0085
Reference 10.
-
X and Triton N detergents the HLB is directly proportional to the CMC (4), the change in absorption maxima with concentration of detergent was analyzed. A sharp break in the concentration-dependent absorbance of the detergent-CBBG solution was observed a t critical micelle concentrations of detergent. Initially, with increasing concentration of detergent no change in the absorption spectrum of the mixture was observed. However, a blue shift in the spectrum and a concomitant increase in ABZoand decrease in A470 was observed when micelles were formed. Increasing the detergent con-
centration and hence micelle formation, resulted in a further increase in and decrease in AdTO.This change was linear with increasing detergent concentration until the dye concentration became a limiting factor. Determination of the CMC for other detergents or mixtures of detergents, for which no CMC values have been reported, can be made simply from plots of the AeZ0and A470 with respect to concentration, as shown in Figure 4. The CMC values for the detergents (Table I) and detergent mixtures (Table 11) were obtained as described above. Critical micelle concentrations for Triton X 100 and Triton X 114, determined by the CBBG method, agree with literature values for the same (Table I). This
Anal. Chem. 1983, 55, 1117-1121
Table 11. CMC! Deterimination of Detergent Mixtures CMC, ratioa approx % detergent mixture by % molar ratio 0.009 Triton X 114 0.009 Triton X 114/Triton X 305 3:l 9: 1 0.012 1:l 3: 1 Triton X 114/?'riton I< 305 0.012 Triton X 114/Triton X 305 1:3 1:1 0.060 Triton X 114/Triton .X 305 1:19 3:19 0.067 Triton X 305 a Detergent solutions were prepared by mixing % wlw) ic ted solutions of both detergents in the proportions ir ._ Molar determinations and then assayed as described. based on an average molecular weight for Triton X 114 of 536d and for Triton X. 305 at 1526d. suggests that ths assay conditions do not significantly perturb the biophysical propeirties of the detergent solution.
IDISCUSSION The change in absorption spectrum upon transfer of CBHG from a hydrophilic to a hydrophobic environment allows measurement of detergent micelle formation. This change in spectrum, with a rel.ative increase in ABZoand decrease in A470upon CBBG interaction with organic solvents or micelles, differs from that observed for CBBG bound to protein or in ionic detergent solutialns. The CMC is readily determined as t,he concentration of detergent which initiates a change in the absorption spectrum a t 620 nm and 470 nm. Subsequent addition of detergent increases only the micelle, not the monomer concentration of the detergent (6). This is corroborated by the linearity of change in AG2,,and A470with detergent concentration above the CMC. The CBBG assay provides a rapid means of CMC determination for nonionic detergents. Unlike many other assays
1117
for CMC, the reagents and instruments required for the CBBG assay are readily available in most labs, the analysis does not require separation of two phase systems (as for a hydrophobic dye such as Orange OT (6)),and the change in CBBG absorption with micelle concentration can be followed at two wavelengths, facilitating the extrapolation. Determination of the detergent concentration of a solution above the CMC can also be obtained by using this assay. Nonionic detergents are increasingly being used in membrane research and such a routine colorimetric assay for CMC should be useful in determining an important physical chemical parameter for aqueous solutions of one or mixtures of multiple nonionic detergents. Registry No. CBBG, 6104-58-1; Triton N, 9016-45-9; Triton X 114,9036-19-5;Triton X 100,9002-93-1;Lubrol Px, 9002-92-0.
LITERATURE CITED (1) Gennis, R. B.; Jonas, A. Annu. Rev. Biophys. Bioeng. 1977, 6 , 195-238. (2) Gennls, R. B.; Strominger, J. L. J . Bo/. Chem. 1976, 251, 1277- 1282. (3) Grlfflth, W. C. J . Soc. Cosmet. Chem. 1949, I , 311. (4) Egan, R. W.; Jones, M. A.; Lehninger, A. L. J . Biol. Chem. 1978, 251, 4442-4447. (5) "McCutcheon's Detergents and Emulsifiers", North American edition; MC Publishing Co.: Glen Rock, NJ, 1980. (6) Jain, M. K.; Wagner, R. C. "Introduction to Biological Membranes"; Wiley: New York, 1980;pp 66-70. (7) Bradford, M. Anal Biochem. 1978, 72, 248-254. (8) Bio-Rad, Price List G, 1981. (9) Chiang, J. Program of Molecular Pathology, Northeastern Ohio Universities College of Medlcine, Rootstown, OH 44272. (IO) "Rohm and Haas Surfactants and Dispersants-Handbook of Physical Properties"; Rohm and Haas: Phlladelphia, PA, 1978.
RECEIVED for review November 8, 1982. Accepted March, 10, 1983. This investigation was supported by PHS Grant No. CA 28342 awarded by the National Cancer Institute, DHHS and JFRA 36 from the American Cancer Society to K.S.R.
Search System for Infrared and Mass Spectra by Factor Analysis and Eigenvector Projection S. S. Williams, R. B. Lam,' and T. L,, Isenhour* Department of Chemistty, University of North Carolina, Chapel Hiil, North Carolina 275 14
A factor analysis technique has been used to compress a library of combined infrared and mass spectra. With this method, a 75 % reduction in library sire has been achieved with little loss in compound dlscriminetion. A search system using the compressed iilbrary Is described. Characteristics of the searches arc! demonstrated with intralibrary and GCIR/ GCMS data.
The need for efficient search-identification methods in infrared spectroimetry and mass spectrometry has become increasingly important. This is primarily a result of increased growth of spectral libraries and the rapid maturation of gas chromatography/mass spectrometry (GC/MS) and gas chromatographylinfrared spectrometry (GC/IR). To date, Current address: Foxboro Analytical, 140 Waters St., Norwalk,
CT 06856.
many automated search algorithms have been developed which are capable of rapidly identifying compounds from infrared and mass spectra with a high degree of accuracy (1, 2 ) . Recently several workers have reported the combined technique of gas chromatography/infrared spectrometry/mass spectrometry (GC/IR/MS) ( 3 , 4 ) . The method exploits the complementary nature of infrared and mass spectra, allowing more confidence in identifying individual components of mixtures (5). In GC/IR/MS work published to date, all searches applied to GC/IR/MS data have used separate IR and MS search algorithms. This approach to searching has the drawback that each search must individually generate results while ignoring a substantial portion of the available data. In addition, it is possible in some cases that the individual searches will not yield the same results. Finally, with infrared spectral libraries rapidly growing in size, and mass spectral libraries already commonly over 25 000 compounds, the time required to carry out two separate searches and rationalize the results becomes unacceptable.
0003-2700/83/0355-1117$01.50/00 1983 American Chemlcal Soclety
