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Anal. Chem. 1985, 57,2183-2190
Characterization and Evaluation of the Use of Nonionic Polyoxyethylene(23)dodecanol Micellar Mobile Phases in Reversed-Phase High-Performance Liquid Chromatography Michael F. Borgerding' and Willie L. Hinze* Department of Chemistry, Wake Forest University, P.O.Box 7486, Winston-Salem, North Carolina 27109 Aqueous solutions of a nonlonlc surfactant, poiyoxyethyiene(23)dodecanoi [BrlJ-35], are characterized and evaluated as reversed-phase liquid chromatographic (RPLC) mobile phases. Base line separatlon of the ASTM recommended RPLC test mix on a Radial-PAK C-18 column demonstrates the viability of Brij-35 as a HPLC mobile phase. The ArmstrongNome theory was applied and found to adequately describe the partitioning behavior of solutes eluted with Brij-35. The pertlnent chromatographic parameters were determined and contrasted to those obtained with a conventional 30:70 aceton1triie:water mobile phase. The effect of ethanol addltlon on retentlon and efflclency was also assessed. The abllHy lo extract tobacco samples with this micellar system and perform a chromatographic separation of the extract demonstrate the practical potential of nonionic micellar liquld chromatography.
The use of aqueous micellar solutions, Le., solutions containing surfactant a t a concentration above the critical micelle concentration (CMC), has been recently proposed as a selective alternative for traditional mobile phases in reversed-phase high-performance liquid chromatography, RP-HPLC (1-21). This technique has been termed micellar or pseudophase liquid chromatography (MLC or PLC) (2). As with traditional RPLC, solute retention in MLC is primarily controlled by manipulation of the mobile phase composition. In addition to bulk surfactant concentration, a number of solute-solvent interactions unique to MLC can be adjusted in order to obtain the desired resolution of sample components. Such separations can be judiciously controlled by variation of the charge-type, hydrophobicity, and/or concentration of the micelle-forming surfactant employed (1-7, 15). Reports concerning the theory of PLC (2-9) as well as the efficiency (10,11),routine separations (1-3,5,6,12,14,16), enhanced detection (18,19),and the unique chromatographic advantages (17,20)obtainable promulgate the recent literature signaling that the MLC technique is definitely coming of age (21). Surprisingly, all work with MLC to date appears to have involved only charged, ionic micellar mobile phase systems composed of either anionic sodium dodecyl sulfate (NaLS) (1-7, 9-11, 13-15, 17-20) or the cationic surfactants hexadecyltrimethylammonium bromide or chloride (CTAB or CTAC) and dodecyltrimethylammonium bromide (DTAE!) (6, 8,ll-13,15,16). The use of uncharged, nonionic surfactants seems to have been largely neglected. Indeed, examples of the use of such surfactants in the field of chromatography in general appear to be sparse. The effects of aqueous-organic eluents (typically 50:50 v/v watermethanol mixture) containing various Tweens (polyoxyethylene sorbitan esters) as well as mixtures of a specific Tween and anionic NaLS or cationic CTAB on the chromatographic properties of silica gel have been reported (22-24). However, the conditions Present address: R. J. Reynolds Tobacco Co., Bowman-Gray Technical Center, Winston-Salem,NC 27102.
employed were apparently such to preclude micelle or mixed micelle formation. Additionally, the nonionic surfactants, Tween-80, Triton X-100 [l-(l,l-dimethyl-3,3-dimethylbutane)-4-polyethyleneoxy(9.5)benzene], and Neodol91-6 [a mixed alkyl-ethoxylated alcohol, C+1,(OCH2CH2),0H], have been added as one of the mobile phase components in the liquid chromatographic separation of proteins, including membrane proteins on a variety of columns (25-29). The presence of micelles was only demonstrated in the separation involving Neodol91-6. In this system, the chromatographic behavior was rationalized in terms of favorable van der Waals attraction of the proteins by the nonionic micelle and the reduced surface tension provided by the aqueous micellar mobile phase (29). The presence of micelles or their role in the separation process, if present, seems unclear in the other reported systems (25-28). In one instance, the surfactant was merely added to enhance the mobile phase flow rate (25). In view of this general lack of information, the present study was undertaken in order to determine the feasibility, effectiveness, advantages, and limitations of using nonionic micellar mobile phases in MLC. Specifically, the effects of micellar polyoxyethylene(23)dodecanol (Brij-35) mobile phases upon the chromatographic behavior of solutes in a test mix on a C-18 reversed-phase column were assessed. Brij-35 was chosen for this initial study over other nonionic surfactants (such as Tritons, Spans, Igepals, or Tweens) on the basis of its commercial availability, high purity, low cost, low toxicity, high cloud temperature, and low background absorbance compared to the other types of surfactants mentioned. The effect of surfactant concentration upon the elution volumes of the test solutes was determined and discussed in terms of the Armstrong-Nome partition theory (4,21). The effects of surfactant adsorption on the stationary phase were examined. The retention parameters, efficiency, sensitivity, and precision obtained for the micellar mobile phase were compared and contrasted to those obtained using a traditional 3070 (v/v) acetonitri1e:water hydroorganic mobile phase. The results clearly demonstrate the viability of nonionic micellar mobile phases in RP-HPLC.
EXPERIMENTAL SECTION Apparatus. The HPLC system was constructed of Waters (Waters Associates, Milford, MA) components and included dual M6000A pumps, Model 441 fixed wavelength UV detector, Waters Intelligent Sample Processor (WISP),Model 720 system controller, and an RCM-100 radial compression module. The column was a Waters Radial-PAK cartridge (10 cm X 5 mm) packed with C-18, non-end-capped 10-pm particles. Data acquisition and integration were performed with Computer Automated Laboratory Systems (CALS) software (Computer Inquiry Systems, Inc., Waldwick, NJ) and HP-1000 hardware (Hewlett-Packard, Avondale, PA). The data acquisition rate was 15 Hz and all data were acquired at 254 nm, unless otherwise specified. Reagents. Brij-35 [polyoxyethylene(23)dodecanol]was obtained from Fisher Scientific Co. (Raleigh, NC) as an aqueous 30% solution and was used as received. The standard six-component ASTM test mix composed of the solutes benzyl alcohol, acetophenone, methyl benzoate, benzaldehyde, benzene, and dimethyl terephthalate was employed in this work (30). Uracil
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Table I. Summary of Micellar Parameters for Brij-35 under Various Conditions
exptl conditions
104[CMC],"M
aggregation no.b
water alone 95:05 (v/v) water ethanol 9O:lO (v/v) water:ethanol 85:15 (v/v) water:ethanol 80.5:19.5 (v/v) water:ethanol
0.90 f 0.10 (0.6-1.0) 1.25 f 0.25 (0.99) 1.50 k 0.15 (1.24) 1.94 f 0.12 (1.65) 2.72 f 0.18 (2.40)
40 32 29 23 12
" CMC values as determined experimentally in this work. Values in parentheses are taken from the literature, ref 38 and 39. bThe aggregation number refers to the number of monomer Brij-35 molecules per micellar aggregate. The values given were taken from the literature, ref 38 and 39. was added as the void volume marker. These solutes were used as obtained without further purification. The acetonitrile used was Burdick and Jackson Distilled-in-Glass while the absolute ethanol used was U.S. Industrial Chemical Company's USP grade. HPLC water was distilled and deionized with a Barnstead NANOpure system. All water was filtered with a 0.2-pm filter. Procedures. Multipartition Theory Experiments. Micelle mobile phases were prepared by making the appropriate dilution of 30% Brij-35 solution with either water or a 15:85 (v/v) ethano1:water mixture. The final concentration of Brij-35 ranged between 6.0 and 60.0 mg/mL with both solvent systems. All micellar mobile phases were used without further filtering or degassing. A stock solution of test solutes was prepared in a 3070 (v/v) acetonitri1e:water mixture and working solutions were subsequently prepared by dilution with the appropriate mobile phase. The working concentrations were uracil (0.20 mg/mL), benzyl alcohol (1.5 mg/mL), benzaldehyde (0.02 mg/mL), ace-
Armstrong and Nome (4). Chromatographic Behavior. The chromatographic behavior of the micellar mobile phases was characterized in terms of the standard chromatographic parameters: k ', the capacity factor; t , solute retention time; N , the number of theoretical plates; and HETP, height equivalent to a theoretical plate. These parameters were calculated in the usual fashion ( I I , 3 0 - 3 4 ) using the computer acquired data. Namely, the capacity factor was determined by using the formula k' = (V,- Vo)/V, where V, is the elution volume of the solute and V,,is the column void volume; the HETP was determined using the relationship H = L / N , where L is column length in centimeters and N is the number of theoretical plates. The plate count was estimated by using the formula N = 4.00 (tR/WJ2, where t R is the retention time of the test solute (benzene) and W,is the peak width at 60.7% peak height. The efficiency data were provided automatically by the auxiliary computer program SCEFF (skew and column efficiency program) (35). Micellar and Solution Parameters. The CMC of Brij-35 (Table I) in distilled water and in aqueous-ethanol mixtures was de-
termined using the surface tension method (36). A Fisher Model 20 tensiometer was used for this purpose. The partial molar volume of Brij-35 (Table 11) was determined by the procedure of Mukerjee (37). Density measurements were made with a Mettler/Par DMA-46 density meter. The cloud point temperatures were determined for 1.0 and 6.0% surfactant solutions by two successive measurements of the temperature for onset of turbidity on heating and clearing on cooling. Viscosity measurements on indicated solutions were made with a Rion Viscotester VT-03 (Extech, Boston, MA). Ultraviolet-visible absorption spectra and absorbances were acquired with a Hewlett-Packard 8450A UV-visible spectrophotometer. Absorbance measurements were obtained between 200 nm and 800 nm, and solvent contributions were automatically subtracted from all spectra. Adsorption Studies. The amount of Brij-35 adsorbed onto the stationary phase as a function of surfactant concentration was measured by frontal chromatography experiments. For these studies, the WISP (automatic sampler) was removed from the chromatographic system and the pump was connected directly to the column to reduce system void volume. A Waters Lambda-Max Model 480 LC spectrophotometer was used to monitor the Brij-35 absorbance at 200 nm. Each surfactant solution was primed to the M6000A pump solvent selection valve prior to the experiment and all experiments were performed at 2.0 mL/min flow rate. To monitor the adsorption at a particular surfactant concentration, the solvent selection valve was switched to the mobile phase of interest simultaneously with the onset of data acquisition. The system holdup volume (system volume from solvent selection valve to detector, including the column) was determined in a similar fashion with sodium iodide as an indicator. Determination of Selected Aldehydes in Smoking Tobacco. Two commercially available smoking tobaccos were extracted by shaking for 30 min on a wrist-action shaker with an aqueous 30% Brij-35 solution. The sample was then injected into the liquid chromatograph without further preparation and separation of the components performed with a 6% Brij-35 mobile phase. Solute peak identification was made by comparison of retention times with known standard retention times.
RESULTS AND DISCUSSION Characterization of Brij-35 Micelles. Knowledge of the physical properties of Brij-35 is necessary to fully understand its behavior as a reversed-phase liquid chromatographic mobile phase. Brij-35, also referred to as polyoxyethylene(23)1auryl ether, is a nonionic surfactant, polymeric in nature, with an average molecular formula best described as CH3(CH2)110(CH2CH20)BH.The Brij-35 monomers cooperatively associate in aqueous media to form aggregates termed normal micelles. In water alone, the rod-shaped ( r = 2 nm, 1 = 18 nm) micellar aggregate of Brij-35 is composed of about 40 molecules (38). The Brij-35 micellar parameters (CMC and aggregation number) used for various calculations are summarized in Table I. The partial specific volume calculated in the usual fashion (37) was 0.9033 mL/g (=kO.Ool%) and 0.9132 mL/g (f0.009%) for Brij-35 in water and 85:15 (v/v) water:ethanol, respectively. The ultraviolet-visible absorption spectrum of aqueous solutions of Brij-35 exhibited an end absorption with slight
Table 11. Solute-Micelle Partition Coefficients for Normal Brij-35 Micellar Systems in Water Alone and in a 85:15 (v/v) Water:Ethanol Mixture"
test solute
slope
in water aloneb intercept R2 Pmd
P,;
slope
in 85:15 (v/v) waterethanol' intercept R2 Pmd 8.38 13.21 15.37 36.96
Pwme
193 0.2636 2.018 401 0.997 10.03 0.1752 1.588 benzyl alcohol 304 0.0854 1.030 16.10 644 0.0467 1.000 0.679 benzaldehyde 354 0.0706 0.991 815 1.000 20.38 0.0340 0.626 acetophenone 850 0.0237 0.800 1376 0.999 34.40 0.0167 0.519 methyl benzoate benzene 0.529 0.0118 1,000 49.63 1985 f f f f f dimethyl terepthalate 0.473 0.0065 1.000 80.56 3222 0.755 0.0157 0.999 52.66 1211 "Determined by treatment of the data according to the Armstrong and Nome equation ( 4 ) . bTo calculate the Pwmvalues in water alone, an aggregation number of 40 and a partial specific volume of 0.9033 mL/g for Brij-35 were used. 'To calculate the P,, values in aqueous 15% ethanol, an aggregation number of 23 and a partial specific volume of 0.9132 for Brij-35 were used. dThese P,, values are given per surfactant monomer. e These P,, values are given on a per micelle basis. f Not determined. 0.997 0.998 0.998 1.000
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2
7
1
A
I1 n
7
5
I II Minuter
Flgure 1. Chromatogram showing the separation of the ASTM test
mix with the 6.0% Brij-35 micellar mobile phase. The peaks represent (from left to right) uracil, benzyl alcohol, benzaldehyde, acetophenone, benzene, methyl benzoate, and dimethyl terephthalate. Conditions were as follows: 10 cm C-18 Radial-PAK cartridge column; flow rate, 1.0 mL/min: detection UV at 254 nm.
3.0
Chromatographic Separations and MLC Retention Mechanism. The viability of Brij-35 as a RP mobile phase is demonstrated by the base line separation of the seven solutes in the ASTM test mix (Figure l). Although the test mixture contains a range of solute functionality, a 6.0% aqueous Brij-35 mobile phase has sufficient solvent strength to elute all compounds within about 30 min. For comparison purposes, Figure 2A shows the chromatogram for the separation of these solutes using the traditional 3070 (v/v) acetonitri1e:water mixture as mobile phase. As can be seen, the separations are comparable except that the elution order of benzene and methyl benzoate observed with the micellar Brij-35 mobile phase is the reverse of that observed with the traditional hydroorganic one. Such alterations in selectivity have also been observed for ionic micellar mobile phases (15, 18, 21). A 6.0% Brij-35 mobile phase is approximately 500 times the CMC, therefore approximately 99.8% of the surfactant is present as micelles. The unique presence of micelles distinguishes the Brij-35 surfactant mobile phase from typical hydroorganic RP-HPLC mobile phases. While strictly a single phase in the thermodynamic sense, the micellar mobile phase
Q.a
:2.0
15.0
1E.E
21.2
24.0
15.2
ia.0
2:.a
24.0
15.0
18.0
21.0
24.0
Minutes
B i
shoulders centered at -225 and 250 nm. At the indicated wavelengths (in nm), Brij-35's molar absorptivity values (in M-l cm-l) are: 280 nm (2.0 M-l cm-'), 270 (2.8), 254 (2.9), 230 (6.2) 220 (9.5), and 205 (16.6). In contrast, the molar absorptivities of the nonionic surfactants Triton X-100, Tween-20, Tween-80, and Igepal CO-530 were larger by factors ranging from lo2to lo3 at these wavelengths! The fact that these nonionic surfactants absorb at many of the commonly used HPLC detector wavelengths restricts their use to isocratic elution as the base line rise which would occur with gradient elution is potentially unacceptable. However, if gradients are desired, one can compensate for the base line rise by the use of a computer-assisteddetector and an appropriate subtraction algorithm. Brij-35 offers some advantage in this regard as it does not possess a strong chromophore and its absorption, as mentioned above, is minimal. Lastly, it should be noted that, as with all nonionic surfactants, aqueous solutions of Brij-35 micelles have a cloud point temperature a t which phase separation occurs. All chromatographic work should be conducted below this cloud temperature in order to avoid clogging and possibly ruining the column. The cloud point for aqueous Brij-35 solutions (in the 1-6% concentration range) is approximately 100 "C. In contrast, the cloud temperature of Triton X-100 is only 64 "C.
6.0
i
3.0
3
6.0
9.0
12.0
Minutes 2
1 1
3.0
C
6.0
9.0
12.0
Minutes
Flgure 2. Chromatograms showing the separation of the recommended ASTM test mix solutes: (1) uracil, (2) benzyl alcohol, (3) benzaldehyde, (4) acetophenone, (5)methyl benzoate, (6) benzene, and (7) dimethyl terephthalate using isocratic elution with a 30:70 (v/v) acetonitr1le:water mobile phase, flow rate, 1.0 mL/min on a (A) new C-18 column, (B) Brij-35 exposed C-18 column, and (C) Brij-35 exposed C-18 column that had been "stripped" by elution with an aqueous 30% acetonitrile solution for 24 h.
contains two distinct environments (frequently called pseudophases), the micelles and the bulk liquid, respectively. A unique retention mechanism is therefore possible with MLC because partitioning occurs not only between the bulk liquid mobile and stationary phases but within the mobile phase from bulk liquid to micelle. The multipartitioning possible
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10'
05
7
0
01
02 03 04 Conc of BRIJ-35 in Micelles (g/ml)
c5
06
Figure 3. Dependence of V J ( V , - V,) on concentration of Brij-35 surfactant in the micelle (in g/mL) for the ASTM test mix solutes: (0) dimethyl terephthalate, (A)methyl benzoate, (0) benzene, (+) acetophenone, (0)benzaldehyde, and (X) benzyl alcohol, when eluted with an aqueous Brij-35 mobile phase.
with micellar mobile phases has been treated theoretically (4, 7,9,21). According to this model, the two partition coefficients which predominately affect RP-HPLC retention are P,, and P,, the stationary phase to bulk water and bulk water to micelle solute partion coefficients, respectively. The multipartition theory has been verified previously by application to cationic and anionic micellar mobile phases (4, 7,9,11-14,21). In this study, the theory was applied to the micellar Brij-35 mobile phase to ascertain if the nonionic surfactant exhibited analogous behavior. From the retention data for each test solute, the ratio V,/(V, - V,) was calculated. The stationary phase volume, V,, is the empty column volume minus the measured void volume (V,), and V , is the solute retention volume (4, 12, 14, 15). The expected linear relationship between the concentration of surfactant in the micelles and V 8 /V ( , - V,) is shown to be valid for the aqueous nonionic Brij-35 mobile phase (Figure 3). From such plots of V,/(V, - Vo)vs. [Brij-35], the slope and intercept were determined using linear regression analysis. The ratio of the slope to intercept is equal to [P,, - l ] a from which P,, can be calculated (4). Table II summarizes the data. Partition coefficients, P,, for distribution of the solutes between the micelle and bulk mobile phase varied from 401 for benzyl alcohol, the least retained solute, to 3222 for dimethyl terephthalate, the most retained test solute when eluting with Brij-35 in water alone. The capacity factors were 2.92 and 23.3, respectively, for these two solutes when eluted with a 6.0% Brij-35 aqueous mobile phase. The partition coefficients for these solutes, P,, seem to follow that predicted in terms of their relative hydrophobicity. If the multipartition theory is applied to Brij-35 in a 15:85 ethano1:water mixture as mobile phase (Table TI), 193 and 1211 are the Pw,values calculated for benzyl alcohol and dimethyl terephthalate, respectively. The shift to lower P,, values supports the multipartition theory, because the diminished polarity of the 15:85 (v/v) ethanokwater bulk liquid would be expected to shift the P,, equilibrium away from the micelle. Consequently, the theory developed for describing the dependence of the capacity factors on ionic surfactant micelle concentration appears to hold for the nonionic micellar mobile phase as well. While the mobile phase interaction in the retention mechanism is analogous for nonionic Brij-35 and ionic surfactant mobile phases, there appear to be some differences in the stationary phase interaction, particularly adsorption of the surfactant onto the reversed-phase packing material. There are no studies of nonionic surfactant adsorption from aqueous solution onto reversed-phase LC packing materials
reported in the literature. However, the adsorption of polyoxyethylene ether surfactants on other polar and apolar surfaces has been reported (40-45, 50). Although nonionic surfactant adsorption depends upon such factors as the surfactant characteristics (i.e., number of oxyethylene units, distribution of alkyl chain lengths, etc.), adsorption surface, and additives present in the system, their adsorption in general can be described by Langmuir-type adsorption isotherms. The plateau region of the isotherm generally commences at or near the CMC, with several instances of adsorption slightly above the CMC being reported (40-45, 50). Adsorption of polyoxyethylene ethers onto apolar surfaces may occur via hydrophobic bonding interactions with the surface. Such interactions may also produce multilayer adsorption on silica, with the primary and secondary monolayers being bound together in this fashion (40, 41, 44, 45, 50). Although there are some discrepancies, it has also been reported that ionic surfactant additivies in the mobile phase strongly adsorb to and coat (2-18 (and C-8) bonded stationary phases (8, 17, 46-49). Ionic surfactants generally follow Langmuir adsorption isotherms and there is apparently no additional adsorption once the surfactant concentration in the mobile phase is above the CMC value (8,17,20, 44-46). In fact, if the multipartition theory of MLC is to be strictly valid for a particular micellar mobile phase, this is one of the conditions that must be met (4, 21). Our results indicate that Brij-35 also coats the C-18 stationary phase. T o study this phenomenon, surfactant breakthrough patterns were obtained on a new column as well as a column that had been previously exposed to 6.0% Brij-35. In each case, the column was exhaustively washed with methanol (Le., a minimum of 1500 column volumes) and water prior to successive elution with aqueous solutions containing Brij-35 below and above the critical micelle concentration. Elution of 2.5 X 10" M (0.25 X CMC value), 5.0 X M (0.50 X CMC value), 1.0 X M (CMC value), and 2.0 X lo-* M (2.00 X CMC value) Brij-35 solutions for a minimum of 30 min each resulted in relatively little adsorption onto either the previously exposed or unexposed columns. Significant adsorption was detected however with both columns when using solutions of higher concentration. Figure 4A shows the breakthrough curves obtained on the column which had been previously exposed to Brij-35, when the Brij-35 concentration in the mobile phase was 2 X lo4 M, 2.0%, 4.0%, 6.O%,8.0%, and 10.0%. An elution front occurred with each surfactant concentration near the system holdup volume (Le., system dead volume plus column void volume) of 4.4 mL. More significantly, the 2.0% Brij-35 curve exhibits several additional fronts before reaching a plateau at approximately 22.0 mL. This indicates that the nonionic Brij-35 is interacting with and coating the C-18 stationary phase. In addition to the adsorption demonstrated by the multiple fronts in the 2.0% Brij-35 elution curve, there is evidence of additional adsorption in the higher concentration breakthrough curves. As can be seen in Figure 4A, the breakthroughs occurred at approximately the system holdup volume for all concentrations examined. However, an expanded absorbance scale presentation of the 4.0% Brij-35 curve (Figure 4B) shows that there is a gradual increase in absorbance after the system holdup volume. This indicates that some additional surfactant continues to adsorb onto the column. In fact, steady-state conditions were not achieved until an appreciable volume of surfactant had been passed through the column. Taken together, these results indicate that Brij-35, unlike ionic surfactants, coats the stationary phase not only below the CMC but also above it. If this proves to be generally correct for polyoxyethylene nonionic surfactants, then the MLC multipartition theory may have to be modified in order to take
ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985
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A 10%
.321
8%
Used Column
+
&- . 3 0
6%
*
'241
b
+
*
0
'
0
0
New Column
,22
I
.20 0
I
.I
I
I
I
I
.2
.3
.4
.5
.6
ii (cm/sec)
Figure 5. Van Deemter flow-efficiency plots for (0)a new Radical-PAK C-18 column and (+) Brij-35 exposed "used" C-18column: 30:70 (v/v)
acetonitrl1e:water mobile phase; test solute, benzene.
1
1
, , , ,
I,,,. 1 0
, , , , 8 0
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
D O
120
150
111
S l D
2'0
270
En0
Mi"",-
Figure 4. (A) Breakthrough curves obtained for aqueous mobile phases M (2 CMC) and 2.0, 4.0, 6.0, 8.0, and 10.0% containing 2.0 X (w/v) Brij-35 on a prevlously exposed (see Experimental Section and text) column. The arrow indicates the system holdup volume. (B) Expanded absorbance scale presentation of the 4.0% Brij-35 breakthrough curve from A. Conditions were as follows: 10 cm C-18 Radial-PAK column; flow rate, 2.0 mL/min; absorbance of Brij-35 monitored at 200 nm.
into account the changing nature of the nonionic surfactant modified stationary phase. Further work to determine the adsorption behavior of a series of nonionic polyoxyethylene ether surfactants is in progress in our laboratory. The effects of Brij-35 modification of the stationary phase are illustrated in Figure 2. These figures present a separation of the reverse-phase test mix with a conventional RP-HPLC mobile phase [30:70 (v/v) acetonitri1e:water mixture] before (Figure 2A) and after (Figure 2B) the C-18 column has been exposed to aqueous Brij-35. Coating the column with Brij-35 has the general effect of reducing all k ' values because the modified stationary phase is now primarily polyoxyethylene, which is more polar than the bonded C-18 phase. In addition to reduced retention, the selectivity of the stationary phase changes when coated with Brij-35. This is demonstrated by a different benzene, methyl benzoate, and dimethyl terephthalate elution order (compare Figure 2A and 2B). Although we were unable to completely wash the Brij-35 from the column packing even after eluting with a 30:70 (v/v) acetonitri1e:water mixture for 24 h (see Figure 2C), Brij-35 can be essentially desorbed from the C-18 stationary phase material if extensively eluted with 100% methanol. Chromatographic Efficiency. The most significant deficiency of micellar mobile phases reported to date has been a lack of chromatographic efficiency (10, 11, 21). Several reasons for reduced MLC efficiency compared to traditional RP-HPLC mobile phases have been proposed, including poor wetting of the stationary phase (10) and restricted mass transfer between the various microscopic phases (i.e., water,
micelle, and stationary phases) (11). While Brij-35 offers nothing new in terms of increased column efficiency, it does provide additional insight into the causes of reduced MLC efficiency. The data which will be described in this section verify the premise of a surfactant coated stationary phase discussed in the previous section of the paper and show that this stationary phase modification is a significant cause of reduced chromatographic efficiency. To study efficiency, the column and extracolumn contributions to band broadening must be defined. The column contributions for our system were defined by experimentally determing the chromatographic efficiency (HETP) as a function of mobile phase average linear velocity (p). A number of rate equations have been proposed which define the relationship between HETP, p, and the physical characteristics of the column and mobile phase (31-34). Although the equations differ in many respects, one characteristic common to all is the stationary phase contribution to the chromatographic efficiency. The stationary phase mass transfer contribution is approximated by the slope of the Van Deemtertype flow-efficiency curve at the higher linear velocities (31-34). As the stationary phase coating increases, the slope of the flow-efficiency curve will also increase, indicating higher resistance to mass transfer (32,341. Such is the case with the flow-efficiency relationship determined for a (2-18 column before and after exposure to Brij-35 (Figure 5). It is important to note that the column, mobile phase [30:70 (v/v) acetonitri1e:water mixture], and probe compound (benzene) are identical, therefore any difference in the two flow-efficiency curves is caused by the previous exposure of the column to Brij-35. Comparison of the two curves reveals that after exposure to Brij-35, the overall column efficiency is significantly reduced, the stationary phase-mass transfer portion of the curve is much steeper, and the capacity factor of each test solute is reduced (see Figure 2B). These facts support the premise that Brij-35 is coated onto the C-18 stationary phase increasing the effective stationary phase film thickness, producing a more polar stationary phase, and reducing chromatographic efficiency. The reduced efficiency which results from adsorbtion of Brij-35 on the packing material is not surprising if one considers the contributions to the theoretical plate height as shown in eq 1, where C,, Cd, C,, C,, and C,, are plate height coefficients due to Eddy diffusion, longitudinal diffusion, mobile-phase mass transfer, stationary-phase mass transfer, and stagnant mobile-phase mass transfer, respectively, and the variables include the particle diameter d,, mobile-phase
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/
Before Solvent Extraction
velocity u,solute diffusion coefficient in the mobile phase D,, solute diffusion coefficient in the stationary-phase layer D,, and the thickness d f of the stationary-phase layer that coats the column particle surface (34). It should be noted that the fourth term of eq 1 is of particular importance to this discussion since it directly relates H E T P to the square of the stationary-phase film thickness and inversely relates H E T P to the solute stationary-phase diffusion coefficient. For well-designed column packings, this term is considered to be negligible (32,34). However, this term is considered significant for column packings which have a thick stationary phase or poor stationary-phase diffusion, such as some of the original bonded-phase materials that had thick polymeric layers of stationary phase (34). The Brij-35 coated column is somewhat analogous to the original poorly designed RPLC packing materials in that the Brij-35 layer adsorped onto the packing material significantly increases the effective stationary-phase film thickness and produces poor stationary-phase diffusion. This premise is supported by the following facts: (i) The amount of Brij-35 adsorbed onto the packing material exceeds the original C-18 carbon content. The original carbon content is 12% by weight and a typical column was found to contain 1.6 g of the packing material. Therefore, approximately 0.19 g of C-18 stationary phase is originally present in the column. A conservative estimate of the amount of Brij-35 adsorbed on the column based on data presented in Figure 4A is 0.26 g. (ii) The extended chain length of a Brij-35 monomer is approximately four times that of a C-18 chain brush. (iii) The viscosity of a 30% Brij-35 solution is 95 f 5 cP. The first two points leave little doubt that Brij-35 adsorption significantly increases the effective stationary-phase film thickness. The exact increase, however, can only be calculated if the true Brij-35 and C-18 chain conformations are known. The third point above addresses the poor stationary-phase diffusion, which is inversely related to the stationary-phase viscosity. It is difficult to predict the amount of hydration in the Brij-35 modified C-18 stationary phase. However, the equivalent of a 30% Brij-35 solution as an estimate for the degree of hydration and stationary-phase viscosity does not seem unreasonable since the driving forces responsible for micellization are also responsible for formation of the combined C-18/Brij-35 stationary phase (50). Having established that Brij-35 is coating the stationary phase, a logical question which follows is: “How stable is the modified phase?“ This question was addressed by dynamically “extracting” the column with acetonitrile/water for approximately 24 h. The result (Figure 6) indicated a partial stripping of the Brij-35 from the stationary phase, hence a flatter flow-efficiencycurve. The associated selectivity changes (Figure 2C) observed were shifts to longer retention times for all test mixture components except benzene which shifted toward the shorter, original benzene retention time. More concisely, the net selectivity change is toward the original, less polar C-18 selectivity (Figure 2A). The effect of an alcohol modifier on MLC chromatographic efficiency was also studied. Dorsey et al. (10) have reported that the addition of various alcohols, particularly propanol, to the micellar mobile phases of ionic surfactants increases efficiency because stationary phase wetting is improved. Modification of a 6.0% Brij-35 mobile phase with 0-12% added ethanol resulted in essentially the same efficiency for all mobile phases studied, which is in contrast to the results reported for ionic surfactants (10, 11).
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p (crnlsec) Figure 6. Plot showing the variation of HETP with mobile phase linear velocity for benzene on a Brij-35 exposed RadicaCPAK C-18 column (+) before and (0)after extraction (“stripping”) by washing with a 30:70 (v/v) acetonitri1e:water mixture for 24 h. Mobile phase, 30:70 (v/v) acetonitr1le:water mixture. .65
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Flgure 7. Relationship of theoretical piate height, HETP, to mobile phase linear velocity, for benzene as test solute on the Radical-PAK C-18 column using either (X) 1585 (v/v) ethanokwater, ( 6 )aqueous 6.0% Brij-35, or ( 0 )6.0% Brij-35 in a 15:85 (vlv) ethanoi:water mixture as the mobile phase. The average capacity factors for benzene in these three mobile phase systems were 52, 27.4, and 20,
a,
respectively. Surprisingly, the addition of 15% ethanol to a 6.0% Brij-35 mobile phase slightly decreased the efficiency (Figure 7 ) . The change in micelle aggregation numbers associated with the alcohol addition is a possible explanation for the slight decrease. The aggregation number is reduced from 40 to 23 almost doubling the number of micelles present in the mobile phase and potentially increasing the mobile phase viscosity. For ionic surfactants, it has been reported that increases in the micelle concentration in the mobile phase reduce the observed efficiency (11). Loss of chromatographic efficiency with an increase in mobile phase viscosity is also a well-known phenomenon (33,34). Measurement of aqueous mobile phases containing 15% ethanol, 6.0% Brij-35, and 6.0% Brij-35/15% ethanol yielded relative viscosities of 1.5, 1.8, and 2.7 cP, respectively, confirming this hypothesis. Effect of Organic Modifier Concentration on Retention. The dependence of the capacity factor for benzene on the concentration of ethanol in an aqueous 6.0% Brij-35 micellar mobile phase was determined. It was found that there is a linear decrease of k’ with an increase in the ethanol concentration in the 0-1270 added ethanol range. The capacity factors for benzene on the C-18 column ranged from
ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985
Minutes
Flgure 8. Chromatograms showing the separation of the components from the Brij-35 extraction of (dashed line) smoking tobacco sample 1 and (dotted line) smoking tobacco sample 2. The separation of an
aldehyde reference mix (solid line) confirms the presence of vanillin (peak A) and ethylvanillin (peak B). Conditions were as follows: Radicai-PAK C-18 column; 6.0% Brij-35 micellar mobile phase; flow rate, 1.0 mL/min; UV detection at 280 nm. 24 with 2.0% added ethanol to 20 with 12.0% added ethanol in the Brij-35 mobile phase. Similar effects have been reported when organic modifiers are added to ionic micellar mobile phase systems (11, 12, 16, 19). Consequently, considerable control of selectivity in any separation attempted in this new MLC mode can be achieved by judicious and careful addition of relatively small amounts of organic modifiers to the Brij-35 micellar mobile phases. The addition of ethanol increases the hydrophobic character of the bulk liquid in the mobile phase and concomitantly shifts the solute equilibrium from the micelle to bulk liquid (see Table 11) as well as from the stationary phase to bulk liquid phase. While the mode of separation is still predominately micellar in nature, it should be recognized that the micellar structural assembly is perturbed by the addition of any organic modifier, such as ethanol. As was previously shown, the addition of organic modifiers can cause changes in micellar parameters [such as CMC, aggregation number (Table I), and partial molar volume]. In fact, if the ethanol concentration becomes greater than approximately 22%, no Brij-35 micelles form (39). Detection Sensitivity. One of the unique advantages of micelle solutions and micellar mobile phases is the possibility of observing enhanced fluorescence and room temperature liquid phosphorescence detection sensitivity (18,19,21,51). Although we have not studied the effect of a Brij-35 mobile phase on luminescence detection for the ASTM test mix solutes, we have completed a preliminary study of the effect of Brij-35 on UV detection. The molar absorptivity for three test solutes (benzene, benzophenone, and benzyl alcohol) in four different mobile phases (15:85 ethanokwater mixture, 3070 acetonitri1e:water mixture, 6% aqueous Brij-35, and 6% Brij-35 in 18:85 ethano1:water mixture) indicates that there is no significant difference in micellar and nonmicellar performance with respect to UV detection at 254 nm. Selected Application: Analysis of Smoking Tobacco with Brij-35. The study of micellar liquid chromatography to date has been largely academic in nature. Although micellar mobile phases are quite interesting from a mechanistic standpoint, some practical analytical utility must be demonstrated before they can be considered as an alternative to the more traditional hydroorganic mobile phases. A practical requirement of any micellar mobile phase is compatibility with the sample matrix or sample preparation matrix. One approach to sample preparation, which assures matrix compatibility, is the use of Brij-35 as the extraction solvent. Potential advantages of a Brij-35 extraction solvent include
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a much lower cost than traditional LC grade solvents, adjustable solvent strength, nonflammability, and easy waste disposal. To demonstrate the analytical utility of Brij-35, two samples of smoking tobacco were extracted with a 30% aqueous Brij-35 solution. An aliquot of each extract was then chromatographically separated with a 6.0% aqueous Brij-35 mobile phase, chosen arbitrarily. Although no attempt was made to optimize the separation, qualitative differences in the chromatograms are obvious (Figure 8). Comparison to an aromatic aldehyde standard mixture enabled verification of vanillin and ethylvanillin as two of the extract components. The chromatographic precision for these two components determined from ten replicate injections was excellent (i.e., the average peak height, standard deviation, and relative standard deviation were 113.87,0.83, and 0.73%, respectively, for vanillin and 429.05, 1.88, and 0.44%, respectively, for ethylvanillin).
CONCLUSIONS Aqueous micellar solutions of the nonionic surfactant Brij-35 have been demonstrated to successfully function as mobile phases in reversed-phase high-performance liquid chromatography. The chromatographic behavior of test solutes eluted with this micellar mobile phase can be adequately described by the Armstrong-Nome partition theory. The Brij-35 appears to adsorb onto the C-18 stationary phase at concentrations well above its critical concentration. This behavior is in contrast to that exhibited by charged ionic micellar systems. Compared to conventional hydroorganic mobile phases, selectivity, cost, safety, and disposal advantages are possible with the nonionic micellar Brij-35 mobile phase. The main disadvantages of the Brij-35 mobile phases in LC are the higher column back pressure and reduced chromatographic efficiency. Our work indicates that mass transfer from the stationary phase plays a major role in the decreased efficiency of nonionic micellar LC. The fact that nonionic micelles can be utilized as the mobile phase in LC in a manner analogous to that previously reported for charged ionic surfactant micelles is important in several respects. First, it extends the pool of charge-type micelles available for consideration as the mobile phase for a particular separation problem. Since there are no formal electrostatic interactions possible between nonionic micelles and solutes, this could favorably affect both the selectivity and resolution achievable in many instances compared to that possible using ionic micelles. Additionally, the binding constants determined by the chromatographic technique for such nonionic micelle-solute systems factors out any electrostatic contribution to the binding interaction. This could be useful in solute binding studies. ACKNOWLEDGMENT The authors wish to thank W. C. Hamlin, Jr. (R. J. Reynolds Tobacco Co.), for some surface area analyses, S. K. Parks (WFU) for making some of the cloud point determinations, H. N. Singh (WFU) for making some of the CMC measurements, and D. W. Armstrong (Texas Tech University), J. G. Dorsey (University of Florida), R. M. Jones (WFU), and C. L. Malehorn (WFU) for their helpful comments with regard to this work. Registry No. Brij-35, 9002-92-0; vanillin, 121-33-5; ethylvanillin, 121-32-4;benzyl alcohol, 100-51-6;benzaldehyde, 100-52-7; acetophenone,98-86-2;methyl benzoate, 93-58-3;benzene, 71-43-2; dimethyl terephthalate, 120-61-6. LITERATURE CITED (1) Armstrong, D. W.; Henry, S. J . J . L i q . Chromatogr. 1980, 3 , 657-662. (2) Armstrong, D. W. Am. Lab (Fairfield, Conn.) 1981, 13, 14-20. (3) Armstrong, D. W. In "Solution Behavior of Surfactants"; Mlttal, K. L., Fendler, E. J., Eds.; Plenum Press: New York, 1982; Vol. 2, pp 1273-1 282.
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(4) Armstrong, D. W.; Nome, F. Anal. Chem. 1981, 53, 1662-1666. (5) Armstrong, D. W.; Stine, G. Y. J . Am. Chem. SOC. 1983, 105, 6220-6223. (6) Armstrong, D. W.; Stine, G. Y. J . Am. Chem. SOC. 1983, 105, 2962-2964. (7) Armstrong, D. W., personal communication. (8) TerweljGroen, C. P.; Heemstra, S.;Kraak, J. C. J . Chromatogr. 1978, 161, 69-82. (9) Arunyanart, M.; Cline Love, L. J. Anal. Chem. 1984, 56, 1557-1561. (10) Dorsey, J. G.; DeEchegaray, M. T.; Landy, J. S. Anal. Chem. 1983, 55, 924-928. (11) Yarmchuk, P.; Welnberger, R.; Hirsch, R. F.; Cline Love, L. J. J . Chromatogr. 1984, 283, 47-60. (12) Mullins, F. G. P.; Kirkbright, G. F. Analyst (London) 1984, 109, 1217-1221. (13) Armstrong, D. W.; Stine, G. Y. Anal. Chem. 1983, 55, 2317-2320. (14) Pramauro, E.; Pellzzettl, E. Anal. Chlm. Acta 1983, 154, 153-158. (15) Yarmchuk, P.; Welnberger, R.; Hlrsch, R. F.; Cline Love, L. J. Anal. Chem. 1982, 54, 2233-2238. (16) Kirkbright, G. F.; Mullins, F. G. P. Analyst (London) 1984, 109, 493-496. (17) Landy, J. S.;Dorsey, J. G. J . Chromatogr. Sci. 1984, 22, 68-70. (18) Armstrong, D. W.; Hlnze, W. L.; Bul, K. H.; Singh, H. N. Anal. Lett. 1981, 14, 1659-1667. (19) Weinberner, R.; Yarmchuk, P.; Cline Love, L. J. Anal. Chem. 1982. 54, 155%-1558. (20) Dorsey, J. G.; Khaledi, M. G.; Landy, J. S.;Lin, J. L. J . Chromatogr. 1984, 316, 163-167. (21) Armstrong, D. W. Sep. Purif. Methods, in press. (22) Wall, R. A. J . Chromatogr. 1980, 194, 353-363. (23) Ghaemi, Y.; Wall, R. A. J . Chromatogr. 1980, 198, 397-405. (24) Ghaeml, Y.; Wall, R. A. J . Chromatogr. 1981, 212, 271-281. (25) Amano Pharmaceutical Co., Ltd. Jpn. Kokai Tokkyo Koho JP 58,215,554, Dec 1983, 3 pp; Chem. Abstr. 1984, 101, 3 5 5 6 5 ~ . (26) Carson, S. D.; Konigsberg, W. H. Anal. Biochem. 1981, 116, 398-401. (27) Regnier, F. E. “Receptor Protein Purification”; Alan R. Liss, Inc.: New York, 1984. Chang, J. P. “Abstracts of Papers”; 6th International Symposium on Column Liquid Chromatography, New York, NY, 1984; Abstr. No. 2a-
83. Barford, R. A.; Sllwinski, E. J. Anal. Chem. 1984, 56, 1554-1556. Subcommittee E-19.06 Task Group on Llquid Chromatography of the American Society for Testing and Materials. J . Chromatogr. Sci. 1981, 19, 338-348. Krstulovic, A. M.; Brown, P. R. “Reversed-Phase Hlgh PerformanceLiquid Chromatography”; Wiley: New York, 1982; pp 16-24. Hamilton, R. J.; Sewell, P. A. “Introduction to High Performance Liquid
Chromatography”; Chapman & Hall: London, 1977; pp 16-27. (33) Karger, B. L.; Snyder, L. R.; Horvath, C. “An Introduction to Separation Sclence”; Wiley: New York, 1973; pp 135-146. (34) Synder, L. R.; Kirkland, J. J. “Introduction to Modern Liquid Chromatography”; Wiley: New York, 1979; Chapter 5. (35) Tyler, W. E. “Skew and Column Efflciency Program”; Exxon Research and Engineering Co.: Linden, NJ. (36) Mukerjee, P.; Mysels, K. J. “Critical Micelle Concentrations of Aqueous Surfactant Systems”; Natl. Stand. Ref. Data Ser. ( U S . Natl. Bur. Stand.) 1971, NSRDS-NBS 36, 6-16, (37) Mukerjee, P. J . Phys. Chem. 1962, 66, 1733-1735. (38) Kalyanasundaran, K.; Thomas, J. K. I n “Micellization, Solubilization, and Microemulsions”; Mittal, K. L., Ed.; Plenum Press: New York, 1977; Vol. 2, pp 569-588. (39) Becker, P. J . Colloid Sci. 1985, 2 0 , 728-729. (40) Mathai, K. G.;Ottewlll, R. H. Trans. Faraday Soc. 1966, 62, 750-758. (41) Kuno, H.; Abe, R. Kolloid-2. 1961, 177, 40-44. (42) Corkill, J. M.; Goodman, J. F.; Tate, J. R. Trans. Faraday SOC. 1966, 62, 979-966. (43) Abe, R.; Kuno, H. Kolloid-Z. 1962, 181, 70. (44) Rosen, M. J. “Surfactants and Interfacial Phenomena”; Wiley: New York, 1976; Chapter 2. (45) Attwood, D.; Florence, A. T. “Surfactant Systems”; Chapman & Hall: New York, 1983; Chapters 1 and 9. (46) Sorel, R. H. A.; Hulshoff, A.; Wiersema, S.J . Li9. Chromatogr. 1981, 4 , 1961-1985. (47) Hung, C. T.; Taylor, R. B. J . Chromatogr. 1981, 209, 175-190. (48) Knox, J. H.; Hartwick, R. A. J . Chromatogr. 1081, 204, 3-21. (49) Hung, C. T.; Taylor, R. B. J . Chromatogr. 1980, 202, 333-345. (50) Clunie, J. S.; Ingram, B. T. I n “Adsorption from Solution at the Solid/ Liquid Interface”; Parfitt, G. D., Rochester, C. H., Eds.; Academic Press: New York, 1983; Chapter 3. (51) Hinze, W. L.; Singh, H. N.; Baba, Y.; Harvey, N. G. Trends Anal. Chem. 1984, 3 , 193-199.
RECEIVED for review January 29,1985. Accepted May 24,1985. The authors thank the R. J. Reynolds Tobacco Co., Winston-Salem, NC, for their generous support of this research. This work was presented at the 17th Middle Atlantic Regional of the American Chemical Society, Middle Atlantic Regional Meeting of the Amercian Chemical Society, White Haven, PA, April 8, 1983, Abstract No. 89, and at the 4th Annual Liquid Chromatography Symposium, Research Triangle Park, NC, October 5, 1983, Presentation No. 12.
Hydro-Organic and Micellar Gradient Elution Liquid Chromatography with Electrochemical Detection Morteza G. Khaledi’ and John G. Dorsey* Department of Chemistry, University of Florida, Gainesuille, Florida 32611 The parameters affecting base-iine shifts in gradient eiutlon wlth electrochemlcal detection are dlscussed. These parameters are applied potentlal, moblle phase conductance, pH, flow rate and vlscoslty, resistance of the electrochemlcal cell, moblle phase electroactlve lmpurltles, electrode sensltlvlty, temperature, and solvent type. The magnltude of residual current change Is reported for water-methanol gradients at different potentlals. As expected, the magnitude of the base-line shin is far greater at high potentlals where masslve oxldatlon of water occurs. The extent of base-line shlfts caused by mlcellar concentratlon gradients at dlfferent potentials, pH, and lonlc strengths ls also studied. The size of mlcellar gradlent induced base-llne shifts can be greatly reduced, especially at high potentials, by balancing the pH and the conductance of the two micellar solutlons. The Improved compatlblllty of micellar gradlents with electrochemlcal detectors Is a great advantage over that of hydro-organic moblle phases. Present address: D e p a r t m e n t of Chemistry, U n i v e r s i t y of N e w Orleans, N e w Orleans, LA 70148.
One of the major drawbacks of electrochemical (EC) detectors is their limited compatibility with gradient elution ( I , 2). It is not uncommon to read in LCEC detection literature: “EC detectors are often incompatible with gradient elution” (1). Some even believe that gradient elution cannot be used with EC detectors (2,3). Their logic is based on the fact that background current is dependent on solvent and electrolyte composition, so any change in mobile phase composition would shift the base line. Recently, however, some authors have reported: “Contrary to previous reports, the compatibility of LCEC with gradient elution is excellent” ( 4 ) . This is based on the successful experience of some workers in using gradient elution with EC detectors (4-6). Different experimental conditions have caused this contradiction. Undoubtedly in order to answer the problem, it is necessary to study the parameters involved, the importance of each in contributing to the shift, and the possibility of decreasing the gradientinduced base-line shift to an “acceptable” value by adjusting the contributing factors. Despite the large number of publications on LCEC, to our knowledge, no one has addressed the issue in detail. Some workers have briefly mentioned the parameters involved; however, their efforts are far from a
0003-2700/85/0357-2190$01.50/0@ 1985 American Chemical Society
