Rapid Separation and Determination of Nonionic Surfactants of the Polyethylene Glycol-Monoalkyl Phenyl Ether-Type by Column Liquid Chromatography J. F. K. Huber, F. F. M. Kolder, and J. M . Miller’ Laboratory of Analytical Chemistry, Uniuersity of Amsterdam, The Netherlands
The purpose of this work was to develop a fast method of analysis for nonionic surfactants which could be used in routine analysis and water pollution studies. This has been achieved for a series of nonylphenolethylene oxide adducts by column liquid-liquid chromatography using a 23-cm column and ultraviolet detection. Adducts varying in chain length from 1 to 20 ethylene oxide units have been separated and a series of commercial products has been characterized. Typical separations take 5 to 30 minutes depending on the resolution desired and the number of components in the sample. The relative precision is 0.4% and the limit of detection i s estimated to be about 0.2 jLg.
NONIONIC SURFACTANTS are the second largest group of surfactants produced in the United States (1). They are used as laundry detergents as well as emulsifying agents in products like cosmetics and paints. The main types are polyoxyethylene and polyoxypropylene adducts such as the alkyl phenol ethyoxylates:
-
R*O-(CH?-CH,-O),
H
I n the manufacture of these products the alkyl phenol is reacted with an excess of ethylene oxide (EO) and a mixture of oligomers of varying chain length ( x ) is produced. The distribution of chain lengths should be Poisson (2) and there is some evidence t o support this prediction (2-6). Commercial products are mixtures of oligomers which are conveniently denoted by the number of moles of ethylene oxide reacted with one mole of phenol, e.g., 5 EO. A fast and simple method of analysis does not exist for nonionic surfactants at the present time (1). Fast, sensitive methods would be useful in biodegradation studies and water pollution analysis as well as routine quality control. Analysis methods have been reviewed in the book edited by Shick (7,and several papers are included in the proceedings of a 1964 International Congress (8) also published in 1967. On leave from Drew University, Madison, N.J. (1) M. Mausner, J. H. Benedict, K. A. Booman, T. E. Brenner, R. A. Conway, J. R. Duthie, and L. J. Garrison, J. Amer. Oil Chem. Soc., 46, 432 (1969). (2) S . A. Miller, B. Bann, and R. D. Thrower, J. Chem. Soc., 1950, 3623. (3) J. Kelly and H. L. Greenwald, J. Phys. Chem., 62, 1096 (1958). 38, 1755 (1966). (4) K. Konishi and S. Yamaguchi, ANAL.CHEM., ( 5 ) R. L. Mayhew and R. C: Hyatt, J. Amer Oil Chem. Soc., 29, 357 (1952). (6) H. G. Nadeau, D. M. Oaks Jr., W. A. Nichols, and L. P. Carr, ANAL.CHEM.,36, 1914 (1964). (7) “Nonionic Surfactants,” M. J. Shick, Ed., Marcel Dekker, New York, N.Y., 1967. (8) “Chemistry, Physics, and Applications of Surface Active Substances,” Proceedings of 4th International Congress, 1964, F. Asinger, Ed., Gordon and Breach Science Publishers, London, 1967, pp 399, 421, 497.
Virtually all of the more recent work has been in chromatography, including combinations of several chromatographic techniques. Planar chromatographic methods (thin layer and paper) have become widely used (9-14) although they suffer from the inherent difficulties in quanptation. Gas chromatographic methods can be used with the short chain lengths (13, 15) but the formation of volatile derivatives is usually required. Most of the separations by liquid-solid (14, 16) and gel permeation (13, 17, 18) chromatography have been slow. Only the recent work of Bombaugh et al. (19) realizes the fast speed of modern high pressure liquid chromatography (LC). LC methods have also been used to isolate nonionics from water (8, 11, 14, 20-22). Usually ion exchange resins are used. These methods are not directly related t o this work which deals only with the separation of the components of commercial mixtures, but they are of interest in water pollution studies. Since modern column L C has not been fully exploited in the separation of nonionics, this was the method chosen for this work. Both liquid-solid and liquid-liquid phase systems were studied and a fast, sensitive method of analysis was sought. The optimum experimental conditions can be estimated, and the separation evaluated, from the concepts of resolution and peak capacity. Resolution. The definition of resolution for two compounds i and j , Rji and its dependence on the process parameters in linear chromatography is given in Equation l (23).
(9) S. Hayano, T. Nihongi, and T. Asahara, Tenside 5 (3/4), 80 (1968). (10) E. S . Lashen and K. A. Booman, Purdue Uniu., B i g . Bull, Ext. Ser. No. 129, Pt. 1, p 211 (1967). (1 1) M. Mutter, 5th International Symp. Column Chromatography, Lausanne 1969, publ. as supplement to Chimia, 24, 263 (1970). (12) S . J. Patterson, C. C. Scott, and K. B. E. Tucker, J . Amer. Oil Chem. Soc., 45, 528 (1968). 40, 1620 (1968). (13) F. J. Ludwig Sr., ANAL.CHEM., (14) G. L. Selden and J. H. Benedict. J. Amer. Oil Chem. SOC., 45,652 (1968). (151 Dr. J. von Pollerberrr. Fette Seifen Anstrichrn.. 69, 179 (1967). (16) R. Wickbold, ibid., 70, 688 (1968). (17) K. J. Bombaugh, W. A. Dark, and R. F. Levangie, J . Chromatogr. Sci., 7 , 4 2 (1969). (18) K. J. Bombaugh, J . Chromatogr., 53, 27 (1970). (19) K. J. Bombaugh, R. F. Levangie, R. N. King, and L. Abrahams, J. Chromntogr. Sci., 8, 657 (1970). (20) Q.W. Osburn and J. H. Benedict, J. Amer. Oil Chem. Soc., 43, 141 (1966). (21) M. J. Rosen, ANAL.CHEM.,35, 2074 (1963). (22) V. S. Varlamov, Z . Y. Leshchenko, and N. I. Ishutina, Maslob.-Zhir. Prom., 34 (12), 21 (1968). (23) J. F. K. Huber, 5th International Symp., Column Chromatography, Lausanne 1969, publ. as supplement to Chimia, 24, 24 (1970).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
105
where
fRj
and
tR(
=
retention times of the components j and 1 (lR3
= =
-
> tRi).
retention time of a nonretarded component standard deviation of the elution function of i fRz
-
fRo
-
capacity ratio;
i.e., the
fRo
=
= =
ratio of the amount of component i in the stationary and the mobile phases at equilibrium. theoretical plate height of component i which is the relative increase of the spatial variance in the column length of the column selectivity factor; i.e., the ratio of the distribution coefficients K, and K i of compounds i and i.
The first two terms in Equation 1 are specific, i.e., they depend on the type of compound. The selectivity factor can vary over a wide range and depends on the nature of the phase system and the temperature. The choice of the phase system is the most critical decision in chromatography (24). The second term, l/(l ~ / K J H,’ 2, is less specific. It varies between zero and about 2mm-1’2 in modern LC (25). To approach the desirable upper limit of two, the capacity ratio must be sufficiently high (e.g., K > 3) and the plate height should be as small as possible. H can be decreased by using small porous particles (26) o r by using particles with a porous layer around a nonpermeable core (27). Further, H depends on the mobile phase velocity and has a minimum a t a very low velocity (e.g. < 0.1 mmjsec) in column LC because of the small diffusion coefficients in liquids (28). However, increasing the mobile phase velocity results in only small increases in H when small porous particles (or particles with a thin porous layer) are used. Thus, high efficiency can be obtained at high speed (23, 24, 27). Peak Capacity. The speed of a chromatographic separation can be characterized by the peak capacity (29) which is defined as the number of peaks which can be separated with a given resolution in a given time. It must not be overlooked that the peak capacity increases with the time by a power less than one, as long as the process parameters (temperature, composition of eluent, etc.) are held constant. I n other words, more peaks can be resolved at an earlier stage of the elution than in the same time interval later. The peak capacity can be estimated empirically in order to characterize the speed of a separation (23).
+
EXPERIMENTAL
Apparatus. The liquid chromatograph was made up of commercially available components except for the column and its connections, the injection port, and the detector cell which were custom made. Previous publications have described in detail the pumping and damping system (26)
and the injection port and detector cell (30,31). The stainless steel precolumn was 1 X 50 cm and the analytical column was made of two equal sections of precision bore glass tubing each 0.27 X 13.5 cm. The length of the packing in each column was about 11.5 cm, the rest of the space being used for low volume connections of the type previously described (26). The detector was an ultraviolet spectrophotometer (Zeiss 1 (30). PMQ 11) with a cell of 1.0-cm length and 7 . 5 ~ volume The wavelength used was 277 nm which is the approximate wavelength of maximum absorption of polyethylene glycol nonylphenyl ethers (3) and the slit width was 2.0 mm. F o r some experiments, a differential refractometer (Waters R-4) was aso used in series after the ultraviolet detector. Chromatograms were recorded on a logarithmic recorder (Servogor type RE 514.9) which is linear in absorbance from 0 t o 1. Full scale deflection corresponding t o 0 % T was 10 mV. For the identification of a peak a double focusing mass spectrometer (AEI MS9-02) was used. The inlet temperature was varied between 100 and 185 “C. Materials. Three different materials were used as solid supporp and/or adsorbents. One is a silica (Spherosil, 1750 A, Pechiney-Saint-Gobain) and the other two are diatomaceous earth products (Hi-Flow Super Cell, JohnsManville, and Kieselguhr, Merck). The sizes of the particles were 2-5 p , 5-10 p and 20-30 p , respectively. Spherosil and Kieselguhr had to be ground to get small enough particles. All were size-graded using a particle size separator (Alpine Model 100 MZR). Microscopic examination of the particles revealed that the sizes quoted should be considered as only approximate. Similar, but coarser, materials were used in the precolumns. The stationary phases polyethylene glycol 400 (PEG-400) and 1,2,3-tris-(2 ’-cyanoeth0xy)propane (Fractonitril, Merck) were gas chromatographic grade. All solvents were reagent grade. The surfactant samples were commercial products of Chemische Werke Hiils A G and Shell Chemical Company. All were liquids except 14 EO which was a semisolid. Procedures. Both the batch and filtration techniques used in gas chromatography (32) were used to coat the solid supports with 10 to 20% of the liquid stationary phase. The columns were packed dry by admitting small amounts of packing and then compressing after each addition with a fluorocarbon-tipped rod using moderate pressure. The most efficient ( H = 0.1 m m at 1.5 mmjsec) columns were made from Spherosil but they also had the highest pressure drop. Precolumns were packed similarly. For liquid-liquid systems the precolumns contained 2 0 z stationary phase o n Kieselguhr, and for liquid-solid systems they contained Spherosil. The reservoir, precolumn, and analytical column were thermostated at 22 “C for most of the work. Samples were injected through a self-sealing silicone rubber septum using a 10-pl syringe (Hamilton 701N) without interrupting the flow. They were dissolved in a solvent, preferably the mobile phase. For liquid-liquid systems, the mobile phase was presaturated with the stationary phase. RESULTS AND DISCUSSION
Screening of Phase Systems. The aim was to find a phase system in which the distribution isotherms for polyethylene (24) J. F. K. Huber, J. Cliromatogr. Sei., 9, 72 (1971). (25) J. F. K. Huber, J. A. R. J. Hulsman, and C. A. M. Meijers, J . Cliromatogr., in press. (26) J. F. K. Huber, J. Cliromatogr. Sci., 7, 85 (1969). (27) J. J. Kirkland, ibid., p 7. (28) J. F. K. Huber and J. A. R. J. Hulsman, Anal. Cliim. Acta, 38, 305 (1967). (29) J. C. Giddings, ANAL.CHEM.,39, 1027 (1967). 106
(30) J. F. K. Huber, J . Cliromatogr. Sei., 7, 172 (1969). (31) J. F. K. Huber, C. A. M. Meijers, and J. A. R. J. Hulsman, ANAL.CHEM., 44, 111 (1972); “Advances in Chromatography 1971,” A. Zlatkis, Ed., Chromatography Symposium, University of Houston, Houston, Texas, 1971, p 230. (32) L. S. Ettre and A. Zlatkis, “The Practice of Gas Chromatography,” Interscience, New York, N.Y., 1967, pp 200-202.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
Table I. Phase Systems Studied Stationary bed Mobile phase
Porous solid
A. Liquid-solid systems
I. ccl4
Liquid
Spherosil Kieselguhr
11. CClJi-octane, l/l (w/w)
B. Liquid-liquid systems
Kieselguhr Spherosil Kieselguhr Spherosil and SuperCel Spherosil and SuperCel Spherosil and SuperCel
111. CCla/i-octane, l / l (w/w)
IV. Ternary (nonpolar phase)“ V. &Octane VI. &Octane VII. i-Octane/CClr, 2/1 (viv) VIII. i-Octane/CCla, 1/2 (v/v)
Water Ternary (polar phase)h Fractonitril PEG-400 PEG-400 PEG-400
97.8% i-octane, 2.21 % ethanol, 0.084% water (by weight).
* 64.2% ethanol, 34.3% water, 1.59% i-octane (by weight) ( 2 4 ) .
0 a
m
P yi
n
a m
m
a
[r
2
4
6
a
Ti m e - minutes
10
12
Figure 1. Chromatogram of 5 E O surfactant. Phase system I1 (Table I) saturated with water Conditions: column 23 cm; inlet pressure 22 atm; sample size 6 fi1
glycol-nonylphenyl ethers were linear over a wide range and the selectivity factors were maximal. Table I lists the phase systems which were studied. G o o d results were not obtained with liquid-solid systems. Spherosil had a very active surface which strongly adsorbed samples resulting in a very slow elution. The retention times of subsequent samples were markedly decreased as the surface of the adsorbent became deactivated by the previously retained samples. The same was found with Kieseluhr but to a lesser extent. The adsorbed samples could be displaced by injecting polar solvents like methanol. Attempts t o deactivate the surface with small amounts of water were unsuccessful. Figure 1 shows a typical chromatogram in which severe tailing is present. It was concluded that the presence of water in the phase system results in poor chromatograms (tailing).
This observation was borne out by the two liquid-liquid systems which contained water (systems 111 and IV in Table I), I n both cases, peaks were badly tailed. Perhaps the nonlinear behavior is due t o the detergent action of these samples. In any case, water was avoided as a part of the phase system, but this does not necessarily preclude the possibility of working with samples isolated from water as in pollution work since the samples can be extracted into organic solvents. The best results were obtained with those systems using PEG-400 as the stationary phase. The three different mobile phases (systems VI, VII, VIII) represent the extremes in “polarity” which were necessary to permit the elution in a reasonable time of sample components ranging from 1 t o over 20 E O units. Figures 2, 3, and 4 show typical chromatograms. Using 2,2,4-trimethylpentane (i-octane) as the mobile phase, samples ranging from 3 EO to 7 EO can be well separated in a reasonable time (Figure 2). When the mobile phase is made more “polar” with CCll (Figure 3), some of the shorter oligomers are no longer separated, but samples up to about 14 EO can be run in 30 minutes or less. With a still larger proportion of CCll in the mobile phase (Figure 4), there is no resolution for these samples, but the total distribution of chain lengths can be seen. Other mixtures of i-octane and CCI?could also be used as mobile phases to achieve the desired separation for a given sample. Thus by optimizing column LC, more compounds (19) can be separated in less time (13,14,16-18). Test of Efficiency and Peak Capacity. From previous work it was known that columns with low theoretical plate heights at high fluid velocities could be made in LC (23. 24, 27). The expected performance was obtained for the PEG400/2,2,4-trimethylpentanesystem. Figure 5 shows a n example of the low value of H a n d its small increase with fluid velocity. Since small H-values at high fluid velocities are achieved, a large peak capacity can be expected. For a resolution of R,, = 6, the peak capacity was estimated to be 8 a t 5 minutes and 18 at 25 minutes. It must be emphasized that the peaks in the chromatograms shown in Figures 2 and 3 correspond t o mixtures which arise from isomers of nonylphenol. Therefore the widths of these superimposed peaks are larger than for pure components. Characterization of Samples. The numbers assigned to the individual peaks in Figures 2 and 3 represent the number of EO units (x) in each oligomer. Their identification was based o n peak number 6 in sample 5 EO (Figure 2) whose
ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
107
A 1
Figure 2. Chromatograms of nonionic surfactants with phase system VI (Table I) A
3 EO; B = 5 EO; C = 7 EO. Numbers refer to number of EO units (x). Conditions: column 23 cm; inlet pressure 42 atm; av linear velocity, 4.3 mmjsec. Flow rate = 1.3 ml/min
4
=
1 1
11
4
8
molecular weight was determined by mass spectrometry. The other chromatographic peaks were assigned by assuming that they differed from each other by only 1 EO unit (see e.g., 3, 6,9,15). The parent peak in the mass spectrum was at 484 corresponding to x = 6 and a molecular formula C2,Has0,. The rest of the spectrum was very similar to that observed by Harless and Crabb (33). The major fragment ion at 399 indicates that the nonyl chain is highly branched at the carbon adjacent to the benzene ring. This information would indicate that this detergent is not likely to be very easily biodegraded. A differential refractometer was used as a second detector to investigate the possibility that nonultraviolet absorbing compounds might be present in the commercial samples. In particular, polyethylene glycols could be formed in the polymerization. No substances were detected at full sensitivity. Several explanations are possible. The glycols are either present at concentrations too low to be detected by refractive index in a solvent already saturated with PEG-400, or they may be retained so long that they were not detected. Finally, it was noted that the chromatograms in Figure 4 appeared to be Poisson in shape. The products of the EOphenol polymerization reaction are predicted to have such a distribution (2). These chromatograms in which the individual oligomers are unresolved provide the necessary data to verify the Poisson distribution if it is assumed that all oligomers have the same molar absorptivities at 277 nm. Those oligomers that have been investigated do have nearly equal molar absorptivities (3, 34), so an attempt was made to com-
12
I
t6 Time- minutes
20
28
24
32
I8
4 14
A
I..,". 17
18
18
5
10 10 T i m o ~ m l n u l o15 15 s Timo~mlnulos
20 20
20
26 26
,
Figure 3. Chromatograms of nonionic surfactants with phase system VI1 (Table I) (33) H. R. Harless and N. T. Crabb, J. Amer. Oil Chem. SOC.,46, 238 (1969). (34) N. T. Crabb and H. E. Persinger, ibid., 45, 611 (1968). 108
A = 7 EO; B = 9 EO; C = 14 EO. Numbers refer to number of EO units ( x ) . Conditions: column 23 cm; inlet pressure 36 atm; av linear velocity, 2.6 mmjsec. Flow rate = 0.76 mlimin
ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
A
0
2
Figure 4.
6
4 Time-minutes
a
Chromatograms of nonionic surfactants with phase system VI11 (Table I)
A = 5 EO; E = 9 EO; C = 14 EO. Conditions: column 23 cm; inlet pressure 15 atm; av linear velocity, 0.90 mmisec. Flow rate = 0.26 mljmin
where t
= time
= average residence time C, = output concentration of n mixers C, = impulse height of the input n = No, of mixers = 3 'T
f!
,
.
0
D
0.6
,
o,a
1.2
LINLAR MLOCITV
,
20
1.6
(rnrn/sc)
Figure 5. Typical plot of H linear velocity
US.
average
Phase system VI (Table I). Solid support: 2-5 p n Spherosil; sample, nitrobenzene ( K = 1.0)
Figure 6 . Computer drawn Poisson curve. (See text for symbols) pare chromatogram C in Figure 4 with a computer generated Poisson distribution. An analog computer (Telefunken R A 742) simulating three ideal mixers in series was used to produce a curve of the following function :
This curve is shown in Figure 6. It matches almost completely peak C in Figure 4. Thus this observation supports the assumption that the chain length of the oligomers have a Poisson distribution. Routine Analysis. The phase systems using PEG-400 as the stationary phase provide rapid routine analyses. The mobile phase composition can be chosen to suit the sample, the desired resolution and/or the time of analysis. These systems have been found stable and reproducible. They have been used at ambient temperature without control over several days. Columns which have been taken out of service and air dried have been reused with the same or different eluents without adverse effects. Some difficulty was experienced initially in finding a suitable solvent for the higher molecular weight samples. For example, 7 EO is not very soluble in i-octane, and chloroform was used to dissolve it since chloroform is commonly used t o extract detergents from water. However, when chloroform was used as the solvent for the sample, the chromatograms were less well resolved and repeated use caused a permanent deterioration of column performance and a n increase in retention times. It was soon discovered that the chloroform was stripping off the stationary phase (PEG) from the top of the column. Saturating the chloroform with PEG should solve the problem, but PEG-400 and chloroform are miscible so this was not possible. The mixtures of CCI, and i-octane used as eluents can be saturated with PEG and they have been found to be suitable solvents for these samples even when ioctane alone is the mobile phase. Repeated injections of the 2/1 (v/v) mixture of i-octane/CCI, had no noticeable effect on column performance when i-octane was the mobile phase. Quantitation. Repeatability was measured by repeated sampling of 6 pl of a solution of 3 EO in eluent using phase system VI (Table I). The heights of peaks 2 and 3 were measured, and the relative standard deviation was 1.8 %. In order to exclude the statistical error due to sampling, the
ANALYTICAL CHEMISTRY, VOL. 44,
NO. 1, JANUARY 1972
109
ratio of the peak heights was calculated. The relative precision improved to 0.43 %. An average sensitivity, S, was calculated from the total peak area of all peaks ( A ) and the injected amount (Q) according to Equation 3 : A
=
SQ
(3)
The sensitivity was found to be 11 mmZ/pg. An estimate of the limit of detection can be made according to Equation 4 :
uQ0 defines the detection limit and uAOis the limiting value of the standard deviation of the peak area (ua) at zero sample size (base-line noise). For an integration time of 2 minutes and a chart speed of 10 mm/min, uAOamounts to about 20 mm* using the logarithmic recorder. With a linear recorder and zero suppression, a 10-times smaller value can be obtained (35) giving a value of 0.2 pg for UQO.
RECEIVED for review July 22, 1971. Accepted August 31, 1971. (35) J. F. K. Huber, C . A. M. Meijers, and J. A. R. J. Hulsman, J. Chromatogr., in press.
110
ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
