Detection and Analysis of Unresolved Multiplet Chromatographi,c Peaks Vaclav Cejka,l Merlin H. Dipert, Sylvanus A. Tyler, and Peter D. Klein2 Dioision of Biological and Medical Research, Argonne National Laboratory, Argonne, Ill. The recognition and description of multiple components in a chromatographic peak requires specialized quantitative techniques when these components represent conformational or positional isomers of the same molecular species. The diagnostic characteristics of the dispersion and displacement differences between experimental and reference peaks are used to demonstrate the detection and determination of up to four components in a single peak. Proceduresfor determining the intra-component separation factors are illustrated, together with an evaluation of the range and accuracy of these techniques. A new method of detecting the presence of a doublet peak in the absence of a suitable reference peak with a single component is presented.
THE PARAMETERS that characterize the intrinsic ability of a chromatographic system to distinguish two compounds and to establish purity without equivocacy have been discussed by Klein and coworkers ( I , 2). Using these parameters and published estimates of their magnitude, Klein evaluated a variety of chromatographic systems and showed that the use of internal reference standards and the collection of fractions permitted determination of separation factors as low as 1.0001 and of intrinsic purities as high as 99.99 (3). Heretofore, attention has been devoted to the magnitude of difference that could be detected between two distinct and unique species-e.g., the 3H and 14C forms of the same molecule (+-or to chromatographic differences arising from positional isomers of the same isotope in the same structuree.g., proline-l-I4C w. proline-5-14C (5). During chromatographic investigations of tritium-labeled leucine on ion exchange columns and of tritium and radio-carbon-labeled cortisone on partition columns, evidence of the presence of radiochemical and conformational isomerism was encountered. This paper is concerned with the diagnostic characteristics that permit recognition of such multiplet forms within a chromatographic peak and with the computational procedures required to obtain the separation factors between the isomeric forms. Model chromatographic systems, generated by computation, are used to illustrate the detection and analysis of up to four components in a single peak. These computations further extend the ability of chromatographic systems to probe the fine structure of energy levels displayed in the characteristics of a single molecular species undergoing chromatographic migration.
x
1 Visiting Scientist, Argonne National Laboratory 1966-67. Present address Department of Medicine, University of Amsterdam, Bimnengasthuis, Amsterdam (C) The Netherlands. 2 Author to whom inquiries should be directed.
(1) P. D. Klein, and S. A. Tyler, ANAL.CHEM., 37, 1280 (1965). (2) P. D. Klein, D. M. Simborg, and B. A. Kunze-Faulkner, Con-
ference on Preparation and Storage of Marked Molecules, EURATOM, 1966, In Press. (3) P. D. Klein, Separation Sci., 1, 511 (1966). (4) P. D. Klein, D. W. Simborg, and P. A. Szczepanik,Pure Appl. Chem., 8, 357 (1964). (5) P. D. Klein and P. A. Szczepanik, ANAL. CHEM.,39, 1276 (1967). 1614
ANALYTICAL CHEMISTRY
60439
THEORETICAL
Properties of the Reference Peak. Measurement of small differences in peak characteristics requires the use of an internal reference with the following properties : (a) the reference compound must be externally distinguishable from the compound being analyzed; (b) the differences in mobility between reference and sample must be small in comparison to the isomeric differences within the analyzed peak; and (c) the reference peak should not have more isomeric forms (and preferably less) than the analyzed peak. It follows from these stipulations that an isotopic substitution in the sample is the minimal change yielding an appropriate reference compound. If this substitution is a stable atom, mass spectrometric measurements will be used to monitor the peak ; if the substituted atom is radioactive, conventional counting techniques are used. Because most applications of this technique have involved comparisons between tritium-labeled and radiocarbon-labeled compounds, the generic term isotope ratio will be used to indicate relative concentrations of the two compounds, even though specific activity measurements or any other forms of comparison are equally valid. Terminology and Designation of Components. The terminology herein continues the use of statistical notation of chromatographic peak characteristics adopted in our previous developments (2, 3, 6). The mobility, M , corresponds to the midpoint of the peak, where 50x of the peak has been eluted, and is equivalent to the corrected retention time or volume; the peak dispersion, U, is the conventional measure of the breadth of a Gaussian peak; and n is the area under the peak. The mathematical techniques, for obtaining M , r, and the standard errors of each have been described by us, as well as equations for the computation of the displacement between two peaks from the isotope ratio, 4, and the algorithm for the generation of a Gaussian peak from given values of M , u, and n ( 4 , 7). Computer programs for executing these algorithms are available upon request. Individual components of the sample peak are numbered in order of emergence by odd-numbered subscripts on the mobilities and dispersions-e.g., MI, 61, M s , u3, etc.-while components of the reference peak are given even-numbered subscripts, The mobility of a compound peak--e.g., Ml,3,5,7 -is written as Mi7 and the dispersion of such a peak is designated u17. The four types of comparisons to be considered are shown in Figure 1. Three displacement terms are involved in these comparisons : (a) AM, the overall displacement of the sample peak from reference peak; (b) AM’, the displacement between two isomeric components present in the sample peak alone; and (c) AM”, the displacement between isomeric forms present in both sample and reference. In these com-
(6) P. D. Klein and B. Kunze-Faulkner, ANAL. CHEM.,37, 1245
(1965).
(7) P. D. Klein, in “Advances in Chromatography” J. C. Giddings and R.A. Keller, Eds. Vol. 3, Dekker, N. Y.,1966, p 3.
TYPE
-.____ REPRESENTATION PARAMETER
SINGLET- SINGLET
H/I)
&
AM:
Mj-Mz
Xn
.
8 CUMUL
x
:- 3
6 -
AM (2x2)
Me
M4
M3
=M~-M,
4 -
-
= M3 -MI
4
A M ' : 2X,
M4
Mq - M z
MI+MJ+M~+MI
MI M3 M 5 MI
+
- -%!% 2
2
AM": 2
hM QUADRUPLETDOUBLET 1412)
:
:
-- M2tM4 2
M,-M,
A M I ' = e k e = M ~ - M ~M :~ - M ~M: 4
30
100
INPUT:
110
i
10
I
L
1
,-2
z!
,..., 90
V-5
j
,
,
100
-3
110
ML
M I 101.00 O U T P U T l M p 100.00
IO0
I
0.1
ML
IUI
Figure 1. Categories of analyses
I
0
90
-~2
- 2
5.00 "2 5.00
AM
{
1.00
I
1
Figure 2. Representation of singlet/singlet analysis parisons, it is stipulated that when the sample and reference peak areas are normalized, all components of a given peak are of equal size, so that: nl
- = I
for a singlet peak,
n2
nl nz
=
3 = 'Iz
for a doublet peak,
n2
While this stipulation appears restrictive, the analysis is specifically directed toward those isomeric forms that may be expected to exist in nearly equal proportions in the peak. Test analyses show that the values for AM' and AM'' are quite stable for limited excursions of component size from those designated; removal of this restriction is being studied. Each type of peak shown in Figure 1 will be analyzed in four stages; first, the basic description of the peak relationships; second, the appearance of the sample peak being analyzed and its diagnostic features ; third, procedures for measurement of the peak parameters; and fourth, the range over which each of the parameters is measurable by this technique. Analysis of a Singlet Peak by a Singlet Reference Peak. When a single sample peak is displaced from a single reference peak, the isotope ratio 4 is given by the equation at the top of Figure 2. The natural logarithm of 4 is linear with elapsed volume or fraction number (x)with a slope equal to the displacement between the two peaks divided by the square of the dispersion. If the dispersions of the two peaks are not identical, the correction terms
an intercept (0 ES 50z elution) equal to the mobility M of any peak so mapped, and the reciprocal of the slope is equivalent to the dispersion u. The bottom of Figure 2 shows the inputs used to generate the sample and reference peaks by computation, the recovered (observed) mobilities and dispersions, and three estimates of the displacement between the two peaks. These three estimates are obtained from the probit difference and from the isotope ratio without and with correction for any differencesand dispersion; all are identical. This set of congruencies (straight line function of 4 and probit, identical sample and reference dispersions, and identical estimates of A M ) constitutes the demonstration of a singlet peak by the chromatographic system used. The limits of detection for AM are dependent upon the standard errors of M I and MZ and of the peak dispersion-typical limiting values for A M range downward from 0.1 to 0.01 and depend upon the efficiency of the column (3). Analysis of a Doublet Peak by a Singlet Reference Peak. The presence of two components in the sample peak analyzed requires the sum of two exponential terms to characterize (shown by the equation in Figure 3). Each term relates the concentration of a single component to the reference peak. The semi-log map of this expression is concave upward. Curvature in the probit plot is less pronounced because the
+
CUMUL X
6t where iqz1 is the arithmetic average of the sample and reference mobilities, must be subtracted from the natural log of before computing the slope. The center left diagram shows the appearance of the sample peak and the isotope ratio. When the cumulative per cent elution of this chromatographic peak is plotted as the normal probability transform (probit) us. elapsed volume, a straight line, shown center right, is obtained. (The individual steps in this transformation have been illustrated in Figure 1 of Reference 7). Such a line has
PROBIT
.* I
f
701 /
90
4 1
30
+
i
1 --2 1
-3 90
ML 97 10s
U=5
INPUT: IO0
OUTPUT'
MI, 101.00 Me 100.00
100 ML
110
5 3 6.47
rz 5.00
1; ::b I
"
1.00
I
Figure 3. Representation of doublet/singlet analysis VOL. 40, NO. 1 1 , SEPTEMBER 1968
e
1615
AM
>
2.0
2.0
,
. :.
1.5
ii0
SU: 1 ;kl CUMUL
X
100
110
0.0
1.0
0.8
30
so
0.5
PROBIT
..............
IO
1.0
:
0.6
so
100 ML
110
-2
0.1
,,::::
,,,,I
, , , , , ,6,l,,;:, l
HL
0.4
90
Figure 4. Representation of a doublet/doublet analysis peak dispersion appears to be increased. If the points are compared to the reference peak (straight line, compensated for the displacement between the peaks), the discrepancy is more evident. The computed characteristics of the peaks also indicate a greater dispersion for the analyzed peak. The displacement is larger than that determined from the probit when the uncorrected isotope ratio is used, and smaller when the correction is made. Quite apart from the fact that the sum of two exponential terms is not the formal equivalent of the single exponential term that incorporates the correction for differences in dispersion, the existence of such differences is sufficient evidence for multiplet forms in the analyzed peak. Dispersion processes in the column must affect all species of the same molecule in the same way, resulting in a dispersion value that is represented by a singlet peak. Peak broadening cannot affect one form of the molecule selectively, hence broadening is itself prima facie evidence of heterogeneity or multiplet forms. When the analysis of a doublet is conducted with a singlet reference peak, the latter provides an estimate of the dispersion value for each of the components in the compound peak. It would then be possible to determine the separation between the two components if the relationship between dispersion and separation were known for a compound peak. Accordingly, doublets of equal size and dispersion but separated by various values for AM' were synthesized by computation (6), combined fraction by fraction and the compound peak
IO0 ML
110
Figure 5. Visibility of sigmoidal isotope ratio resulting from doublet/doublet peak comparisons as a function of the distance separating the two peaks was subjected to probit analysis to obtain the dispersion. The values listed in Table I show that the compound dispersion increases in a manner dependent upon both AM' and u ~ .When the per cent increase in u1 ( A u z ) was plotted against AM'/ul, a linear relationship in which
yields the displacement from the equation : Log(Auz) = 1.077
+ 0.961 log
[?I2
(3)
It is possible to use Equation 3 to estimate the minimal displacement between two components of a doublet peak that can be detected by a chromatographic system with a singlet reference peak. With a previous convention (3) that a quantity 6u, equal to 4 X the standard error of u, is the minimum change that can be detected in the dispersion, it follows that 6 u z or 6u/100 u1 substituted in Equation 3 yields the minimal displacement as (AM'/uJ2. Based upon considerations previously reported by Klein (3) for ion exchange columns whose 6 u z was 1.35zit ,is possible to detect a displacement between components of a doublet as small as 0.34 u in magnitude. A displacement equal to u could be detected in a system in which the standard error of the dispersion was 3 or less. Analysis of a Doublet Peak by a Doublet Reference Peak. The analysis described in Figure 4 involves a fundamental change in the reference compound from a singlet to a doublet peak. This condition, which corresponds to the existence of two isomeric forms for each component involves the loss of considerable resolution and requires that the intraisomeric separation factor AM" be larger than when a singlet peak is available for comparison if it is to be detected. The characteristics of such an analysis that distinguish it from the singlet/singlet case are the sigmoidal shape of the isotope ratio, together with identical dispersions, and the difference in computed displacement by use of probit L;S. isotope ratio techniques. In contrast to previous examples, both methods which use the isotope ratio give the same estimate of the dis-
z
Table I. The Inthence of AM' on Band-Broadening as Measured by Probit Analysis u1
AM'lai 0.2 0.4 0.6 0.8 1.o 1.2 1.4 1.6 1.8 2.0
1616
3.0 3.0159
4.0 4.0214
3.1361 ..*
4.1815
3.5067
4.6757
...
...
... ...
4.3207
5.7610
...
... ...
ANALYTICAL CHEMISTRY
... ...
...
...
5.0 5.0264 5.1042 5.2269 5.3945 5.6070 5.8525 6.1428 6.4481 6.8038 7.2797
2.0
d, 0Q
1.5
0.8
I .2
1,s
'r
... ..
LOG Q
I
s
jIOOO 11.050
s 0
1.0 0.8
PROBIT
CUMUL %
- .
40900
0.6
I10
100
ML
ML M13 I0000
0.4
100
AM =I
CUTPUT' MZ I O 0 CO
02'585
~ 1 53 02
85 AM
5 85
[i3
Figure 7. Representation of doublet peak analysed by reference to its synthetic singlet counterpart Figure 6. Appearance of sigmoidal shape in doublet/doublet comparisons as intra-peak separation increases at constant peak separation placement. This displacement, however, is larger than that obtained from the probit analysis. The curvature of the probit line for the analytical peak, which is the same as in the doublet/singlet case, is now more difficult to discern because the reference probit line is both decreased in slope as well as curved in the same fashion. The same relationship governs the dispersion of the individual doublet peaks and their separation from one another as in the case of the doublet/singlet case. Accordingly a a2 (or a,) and a corresponding AM" can be computed with Equation 3 and these can be substituted into the formula of Figure 4 to obtain the best fit by the method of least squares. It should be noted that the demonstration of a double intrapeak displacement requires the presence of an inter-peak displacement to become visible. When M13 is identical with M24,the isotope ratio is constant across the peak, as shown in Figure 5 and the same intra-peak displacement becomes more pronounced the larger the value of AM. On the other hand, the existence of AM" at constant AM is not readily apparent until the ratio (AM"/c) reaches 1.2-1.6 (Figure 6). The interdependence of AM and AM" in the behavior of the isotope ratio suggests that the absence of a sigmoidal shape-ie., apparent singlet/singlet peak comparison-is no assurance of the absence of isomerism in both peaks. A fitting technique based upon the isotope ratio and the bandbroadening relationship (Equation 3) is therefore useful only when the isomerism is already evident and does not constitute a detection procedure. It is more important to have some means of testing a peak for unresolved and unsuspected isomerism in the absence of a singlet reference peak and to apply this test to both experimental and reference peak alike. Independent confirmation of isomerism from both peaks then warrants the use of the isotope ratio approach. Such a test appears to be provided by the peak generated by the computer from the values for M and u obtained by probit analysis on either the experimental or reference peak. When an experimental doublet peak is compared to a singlet reference peak having the same midpoint and dispersion, all extrinsic differences are eliminated. In the singlet/singlet comparison, this leads to a straight line with zero slope for
Note altered scale on isotope ratio, center left the isotope ratio. However, Figure 7 shows that the presence of two populations in the experimental peak gives rise to a curvature in the isotope ratio characteristic of an equation with four roots, and whose amplitude is determined by the displacements between the two populations. An illustration of the change in this amplitude with various values of AM"/u is provided in Figure 8. The equation in Figure 7 can be used in conjunction with Equation 3 to obtain the best estimate of the separation between the components of the doublet. In this case, the ratio of experimental to synthetic fractions obtained by dividing the observed fraction size by the computed fraction size gives a hybrid isotope ratio. The values of I$ predicted by the equation in Figure 7 are then obtained for progressive estimates of u and Xr or AM" and compared with the hybrid ratios until the criterion of minimum squared
+
0.95
I ' 90
100
110
ML
Figure 8. Change in amplitude of isotope ratio in hybrid comparison as a function of separation between components of doublet peak VOL. 40, NO. 1 1 , SEPTEMBER 1968
1617
L
c 1.0
0
/
1
i
I
6
4
/
I
4
2 -2
I
k
/
i
-3 IO0
90
110
0.1
90
HL
I
\ d
2,o’
0001‘
’ ’ ’
’
5.5
’ ’
I
’
‘
6.0
VI
1
’
‘
Figure 9. Minimum squared deviation of hybrid isotope ratio as a function of u1 value selected deviation is fulfilled. The contour of the error function in the vicinity of the correct dispersion for the singlet shows a minimum which becomes progressively shallower a t larger and larger values for X2/u as shown in Figure 9. The presence of a single minimum with a sharp drop in error and narrow limits about the proper value for LT indicate that a stable, accurate, solution may be obtained with this fitting technique. When a series of synthetic doublet peaks were subjected to this analysis, the ability of the search pattern to find the correct displacement extended down to values of X2 of 2.0 when the dispersion of each component was 5.0, equivalent to a A M ” / . of 0.8. The search pattern approximated values as low as 1.0 for X2 but with decreased accuracy, as might be expected from the amplitudes shown in Figure 8. Analysis of a Quadruplet Peak by a Doublet Reference Peak. It is possible to distinguish one other composite peak; that in which each of the isomeric forms of one species also exists as a pair of positional isomers, as illustrated in the chromatographic input in Figure 10. This combination of peak displacements AM’ and AM” gives rise to a sigmoidal isotope ratio as in the doublet/doublet case, but the two dispersions are different indicative of an unmatched doublet in the analytical peak which is absent in the reference peak. The sigmoidal shape of the isotope ratio is present only when AM’ is one half the size of A M ” ; as AM’ grows with respect to AM”, the sigmoid shape is gradually converted to a parabolic shape ~
110
ML
Figure 10. Representation of quadruplet/doublet analysis
1
t u
100
characteristic of the doublet/singlet case, as shown in Figure 11. To analyze such a quadruplet peak, it is first necessary to know the joint effect of the two displacement terms upon the dispersion of the compound peak. A 6-X-6 table of AM’ and AM” values are synthesized and analyzed for the effect on the dispersion g 1 7 , with the results shown in Table 11. These values satisfy the relationship
1.072
+ 0.962 log [E r2 +
“““1 u2
and because AM’ can be also estimated from the relationship
1.072
+ 0.962 log
[“y] -
Table 11. Band-Broadening by Quadruplet Peaks: Influence of AM‘ and AM” AM’/a AM”/u 0.0 0.2 0.4 0.8 1.2 1.6
1618
0.2
0.4
0.8
1.2
1.6
7.890 8.382 9.738 15.168 23.804 35.136
17.050 17.398 18.694 23.804 31.846 42.512
28.962 29.596 30.800 35.916 42.925 52.968
Au%
0.OOO 0.528 2.084 7.890 17.050 28.962
ANALYTICAL CHEMISTRY
0.528 1.052 2.582 8.358 17.484 29.362
2.084 2.584 4.042 9.716 18.694 30.382
(5)
it is possible to reduce the quadruplet band-broadening equation to two unknowns, AM’ and u2 as in the doublet/doublet case. In this instance we have chosen to use the fitting technique based upon the isotope ratio equation shown in Figure 10, although the method used to fit the doublet/doublet peak by use of a synthetic reference peak would apply equally well. The capabilities of such a fitting technique were investigated for the combinatorial possibilities of small, medium, and large values of XI and A?. The behavior of the minimum squared deviation in a typical search pattern is shown for the
~~
0.0
(4)
'i 0.5
'9
e e
+.I2
0
0.2
0.4
0.8
0.8
1.2
Figure 11. Change in appearance of isotope ratio in quadruplet/ doublet analysis as a function of AM' us. AM"
example XI = 1.0, XZ = 6.0 in Figure 12. It displays the same characteristics of the previous search pattern, namely a sharp, single minimum in the vicinity of the correct dispersion value. The matrix shown in Figure 13 compares the theoretical points for X1 and Xz with those found by analysis. It indicates that solution for Xi, as in the doubletisinglet case, is limited only by the significance of the dispersion differences (u17- 6 2 4 ) . Analysis for Xz is comparable to the technique using a synthetic peak, down to values of 3.0, but becomes progressively worse below this value, and in particular, at large values for X1. Analysis based on the quadruplet/doublet isotope ratio appears most reliable at high values of X2--i.e., where AM"/u is 1.2 or larger. DISCUSSION
The chromatographic peaks presented here are deliberately divorced from the consideration of specific, real chromatographic situations, to be dealt with in a later publication. It is our purpose in this report to make clear the fundamental relationships between the composition of a multiplet peak and that of its reference peak, the features that serve to diagnose the presence of multiple forms and the techniques available to determine the characteristics of the components within a multiplet peak. Such techniques will be used only if their reliability can be demonstrated, and this requires first, that the fitting procedures lead to a stable solution; second, that the error function of the fitting equation be known; and third, that the range and accuracy of the fitting technique be mapped with error-free chromatograms of known composition. Fortunately, the characterizing equations in each instance lead to unique solutions in those situations where the parameters are large enough to be measured. In other words, if the separation factor between two components of a doublet is large enough to be visualized by one of the techniques described, the search pattern can readily find and determine it with accuracy. We have not attached the standard errors (obtained from the derivative of the isotope ratio equations in the region of the solution) associated with these analyses of known, synthetic chromatograms because these are so small; these will of course become significant in the analysis of real chromatograms. However, greater importance is attached to the mapping of range and accuracy of these equations illustrated by the search patterns. These provide a priori demonstrations of the capabilities and limitations of these techniques and permit them to be used in a diagnostic role-ie., the existence of multiplet forms is established by the measurement of a significant difference between their migration rates, not by logical explanations of why they should be present. It may be expected that the application of these techniques will reveal a number of unsuspected instances of
=I
Figure 12. Minimum squared deviation of quadruplet/ doublet isotope ratio cs. value of ~1 selected multiplet peaks, both in chromatographic systems and in other systems approximating a gaussian distribution. It is significant, in our estimation, that the extreme upper limits of separation factors studied were still less than those resulting in visible inflections in the peak shape, and this fact reinforces an earlier proposal (3) that estimates of purity defined by a single chromatographic peak be accompanied by estimates of Sr and AM, Perhaps the most interesting facilitation is the substitution of a computer generated intrinsic reference peak for the extrinsic form. This substitution, derived from the computed characteristics of the experimental peak, achieves two useful
6
'
I I I ~ I
- - - -'pI - - - - - - -? I I
II I
I I
I I
.'I 4 l
I
T----9I I
1 I
'
0
4b
b
_ - - e -
/
/
/
&'
/
I .O0
1.0
@
4
3.0
6.0
Figure 13. Ability of search pattern to find component parameters in analysis of quadruplet peak with reference doublet
Solid circles, test input characteristics, open circles, values found by search pattern VOL 40, NO. 1 1 , SEPTEMBER 1968
1619
objectives; first, it frees the experimenter from the problem of obtaining a suitable reference standard in those cases where alternatively labeled forms may not be available, and second, it provides the mathematical certainty that the reference population contains a single population. To our knowledge, this reflexive procedure of generating a hybrid ratio between the fractions of an experimental distribution and a computed distribution with the same determinant characteristics has not been used as a diagnostic tool before. Its ability to dis-
cern separations between two components as low as 0.8 u indicates the possibility of analyzing doublet peaks in which the components differ in migration rate by as little as 13 of the peak width. This should provide the opportunity to measure extremely small differences in energy levels of molecular species undergoing chromatography. RECEIVED for review March 19, 1968. Accepted June 24, 1968. This work was supported by the U. S. Atomic Energy Commission.
Analysis of Ethylene Oxide and Propylene Oxide Adducts of Alkylphenols or Alcohols by Nuclear Magnetic Resonance, Gas-Liquid Chromatography, and Thin-Layer Chromatography Procedures F. John Ludwig, Sr. Research Laboratory, Petrolite Corp., 369 Marshall
Ave.,St. Louis, Mo. 63119
NMR methods have been developed to distinguish pCH3
I
RC,H,O(CH,CH,O),(CH,CHO),H CH3
(I)
from
p-RCGH,O-
(CH2CHO),(CH2CH20),H (11) and, in conjunction with infrared and H I decomposition-GLC procedures, to determine X , y, and the empirical formula of R. Individual components of the ethylene oxide, propylene oxide, or ethylene oxide-propylene oxide adducts of p-alkylphenols which contain fewer than 10 polyether units were separated as the trimethylsilyl ethers by means of gas-liquid chromatography on SE-52 Chromosorb G columns, and GLC-molecular weight distributions were calculated. Thin-layer chromatographic systems were developed to separate components of I and II with the sum ( x y ) less than 10,to estimate the molecular weight range of polypropylene glycols, and to separate different classes of oxyalkylates from each other. Gel permeation chromatography with Sephadex LH-20 was shown to be a promising method of measuring the molecular weight distributions in oxyalkylates and of separating oxyalkylate mixtures on the basis of molecular weight differences.
+
THEMOST IMPORTANT CLASS of nonionic surfactants consists of the reaction products of ethylene oxide (EtO) and/or propylene oxide (Pro) with compounds such as p-alkylphenols, glycols, or fatty alcohols which contain a reactive hydrogen atom. The commercial adducts, which are frequently termed oxyalkylates, are mixtures with rather broad molecular weight distributions ( I ) . Different products are obtained if E t 0 and P r o are added in two separate steps or as a mixture (2). The structural parameters which must be measured to characterize an oxyakylate containing both E t 0 and P r o groups include the following: (a) the composition of the starting material; (b) the total number of moles of oxide added per reactive hydrogen atom; (c) the weight or mole per cent of E t 0 and P r o ; (d) the order of addition of the oxides-Le., (1)
N. Shachat and H. L. Greenwald in “Nonionic Surfactants,”
,M.. J. Shick, Ed., Marcel Decker, New York, N. Y . ,1967, Chapter L. (2) I. R. Schmolka, ibid., Chapter 10.
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ANALYTICAL CHEMISTRY
as a mixture or consecutively as Pro, E t 0 or EtO, P r o ; and (e) the molecular weight distribution. Nadeau and Siggia (3) have recently reviewed instrumental methods of analysis of nonionic surfactants. Nadeau and Waszeciak ( 4 ) have summarized methods of separation of these products. Several investigators have used proton NMR spectrometry to measure primary hydroxyl/secondary hydroxyl ratios in polyalkylene oxides (5-8). Manatt et al. ( 9 ) employed l9F NMR spectrometry to determine these ratios in polyalkylene oxides which had been converted to trifluoroacetate esters. Gas-liquid chromatography (GLC), column chromatography, gel permeation chromatography, and thinlayer chromatography (TLC) have been used by numerous investigators to measure molecular weight distributions of oxyethylated p-alkylphenols or alcohols, and of polypropylene glycols (10-23). (3) H. G. Nadeau and S. Siggia, ibid., Chapter 26. (4) H. G. Nadeau and P. H. Waszeciak, ibid., Chapter 27. 36, 1981 (1964). (5) T. F. Page and W. E. Bresler, ANAL.CHEM., (6) A. Mathias, Anal. Chim, Acta, 31, 598 (1964). 37, 431 (1965). (7) V. W. Goodlet, ANAL.CHEM., (8) A. Mathias and N. Mellor, ibid., 38, 472 (1966). (9) S. L. Manatt, D. D. Lawson, J. D. Ingham, J. D. Rapp, and J. P. Hardy, ibid., p 1063. (10) L. C. Case and N. H. Rent, Polymer Letters, 2,417 (1964). (1 1) L. Gildenberg and J. R. Trowbridge, J. Amer. Oil Chem. SOC., 42,69 (1965). (12) J. K. Weil, A. J. Stirton, and E. A. Barr, ibid., 43, 157 (1966). (13) J. Tornquist. Acta Cheni. Scand., 20, 572 (1966). (14) M. K. Withers, J. Gus C/iromatogr., 6, 242 (1968). (15) J. Schaefer, R. J. Katnik, and R. J. Kern, J . Anier. Chem. SOC., 90, 2476 (1 968). (16) K. Burger, Z. Anal. Chew., 196 (4), 259 (1963). (17) J. Borecky, CoNeci. Czech. Cliem. Commim., 30, 2549 (1965). (18) N. E. Skelly and W. B. Crummett, J. Chromutogr., 21, 257 (1966). (19) K. Burger, Z. Anal. Cliem., 224,421 (1966). (20) K. Burger, ibid., p 425. (21) R. J. Morris and H. E. Persinger, J . Polymer Sei., Part A , 1, 1041 (1963). (22) M. Hori, K. Kondo, and T. Yoshida, Tukedu Kenykusho Nempo, 24, 194 (1965). (23) E. J. Quinn, H. W. Osterhoudt, J. S. Heckles, and D. C . Ziegler, ANAL.CHEM., 40, 547 (1968).
