Anal. Chem. 1993, 65, 1023-1027
1023
Tandem Detectors To Quantitate Overlapping Chromatographic Peaks Frederick L. Herman Air Products and Chemicals, Inc., 7201 Hamilton Boulevard, Allentown, Pennsylvania 18195
Chromatographersgenerally try to achleve good resolutionof components In a mlxture as a prerequlslte to quantltation. Occasionally, multiple detectors are used to resolve and quantltate chromatographlcally overlapplng components. I n mostcases,however, the detecton exhlblt very hlghselectlvtty for the overlapping components; Le., one detector Is much more senrltlve to a partlcular molecular structural feature contalned In one of the specles belng analyzed. I n some cases, one detector exhlbttsIHtle or no responsefor a particular component In a mMure, andso multiple detect- oftenfunctlon In a compkmentary fashlon. Thk paper describes an analytical method for quantltatlng chromatographlcally overlapping componentsusing multlpledetectorsthat exhlblt comparable, although not Identlcal, selectlvlty for the coelutlng species. Equatlons are derlved for quantltatlng two overlapping components using two detectors wlth elther an Internal or an external standard analytical method. Quantltatlonlo poedble even when the components exhlblt complete overlap. The Internalstandard methodIs applied to the analysisof acrolein/ proplonaklehyde mlxtures using a tandem photoionlzatlon/ flame lonlzation detector. Component concentrations are determlned wlth a relatlve accuracy of 3.4% when both overlapplng components are present at equal concentratlons. When the componentsare present at different concentratlons, greater relative accuracy le obtalned for the more highly concentratedcomponent. Regredon techniques may be used to generallze the method to the analysls of N overlapping components uslng at least N different detectors.
INTRODUCT10N Chromatographers generally try to achieve good resolution of components in a mixture as a prerequisite to quantitation. In the analysis of poorly resolved, complex mixtures by gas chromatography, the resolution of overlapping components is commonly improved by changing the chromatographic phase or by optimizing the chromatographic conditions. However, these solutions are not without their shortcomings. For example, a change of the chromatographic phase may indeed improve the resolution of two overlappingcomponents, but it may also cause a loss in resolution between two otherwise well-resolved components. Likewise, optimization of the chromatography, e.g., by changing the temperature program, may result in longer analysis times. One possible solution to achieving resolution of all componenta in a mixture is the use of two different columns in parallel. For example, Andrawesl used two complementary columns in parallel connected to a single detector to resolve a complex gas mixture. Several componenta coeluted from each column. However, each column resolved the coeluting components from the other column, and thus resolution of all components could be achieved. However, since both columns were connected to a single detector, the columns (1)Andrawes, F. F.; Gibson, E. K., Jr. Anal. Chem. 1979,51,462-463.
had to be tailored to allow for retention time windows to prevent peak overlap from the two different columns. While this solution was effective in this case,the difficulty in tailoring multiple columns to a given mixture will increase with the complexity of the mixture to be analyzed. A more general variation of this technique that would not be limited by retention timeoverlap between the two columna would involve the use of multiple columns in parallel, each connected to ita own detector. The use of multiple detectors in chromatography, i.e., two detectors in series or in parallel to analyze the effluent from a single chromatographic column, is well-known.*-15 In most cases, one detector is more sensitive to a particular molecular structural feature contained in one or more of the molecules being analyzed. In some cases, one detector exhibits little or no response for a particular component in a mixture, and so a complementary detector must be used to analyze for it. Multiple detectors have most often found use in the following situations: (1)to increase the sensitivity of the detection method for a given component or class of components in a mixture; (2) for qualitative identification of a particular functional class of compounds in a complex mixture, the peaks for which are often close in retention time to those of other components; (3) for structural confirmation of a given component by utilizing a functional relationship of the detector responses. The types of applications enumerated above use multiple detectors mainly for qualitative and quantitative analysis of well-resolved chromatograms. Multiple detectors have some times been used in the resolution of overlapping peaks, but this was most often accomplished in situations where the detectors exhibit very high selectivity for the overlapping components. For example, Blankloused dual electrochemical detectors in series for analysis of overlapping peaks in HPLC. In a case where two overlapping components had different oxidation potentials, he was able to resolve them instnunentally by setting the potential for the first electrode such that (2)Bjorseth, A.; Eklund, G. J . High Res. Chromatogr. Chromatogr. Commun. 1979,2,22-26. (3)Earp, R. F.; Cox,R. D. In Identification and Analysis of Organic Pollutants in Air; Keith, L. H., Ed., Butterworth Boston, MA, 1984,pp 159-169. (4)Klemedtason, L.;Simkins, S.;Svensson, B. H. J . Chromatogr. 1986, 361,107-116. (5)Sodergren, A. J. Chromatogr. 1978,160,271-276. (6)Lopez-Avila,V.J.High Res. Chromatogr. Chromatogr. Common. 1980,3,545-550. (7)Lopez-Avila, V.;Northcutt, R. J . High Res. Chromatogr. Chromatogr. Commun. 1982,5,67-74. (8)Driscoll, J.N.;Ford, J.; Jaramillo,L.F.;Gruber,E.T. J . Chromatogr. 1978,158,171-180. (9)Cox, R. D.; Earp, R. F. Anal. Chem. 1982,54,2265-2270. (10)Blank, C.L.J . Chromatogr. 1976,117,35-46. (11)Li, K.-P.; Arrington, J. Anal. Chem. 1979,51,287-291. (12)Webb, P. A.; Ball, D.; Thornton, T. J . Chromatogr. Sci. 1983,21, 447-453. (13)Malinowski, E. R. Factor Analysis in Chemistry, 2nd ed.; John Wiley and Sons: New York 1991;pp 172-185,232-233. (14)Huang, J.; Liu, X.; Liu, P. Hua Hsueh Hsueh Pa0 1981,39,335340; Chem. Abstr. 1981,95(26),231447d. (15)Imamichi, S.;Nishimura, M. Jpn. Kokai Tokkyo Koho 1989, 01088248;Chem. Abstr. 1989,111 (14),126230d.
0003-2700/93/0365-1023$04.00/0 0 1993 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 8, APRIL 15, 1993
only one of the compounds was actively detected, while maintaining the second electrode a t a potential at which both compounds were active. Often, however, two detectors exhibit comparable response for overlapping components. Li and Arringtonll reported on the use of dual-wavelength spectrophotometric detectors for the quantitation of polynuclear aromatics by HPLC. While their technique aided in the quantitation of overlapping peaks, the authors stated that their method is ineffective when the overlapping components exhibit identical elution profiles. Webb et al.12 used dual-wavelength absorbance detectors for the quantitation of amino acids by HPLC. They were able to resolve overlapping peaks by applying an algorithm that utilized absorbance ratio and absorbance difference techniques. While their analyses were highly accurate, these authors used specialized detector circuitry to collect and process the detector signals, which would appear to limit the general applicability of their technique. A number of investigators have used multivariate partial least squares- and principal component-based methods for the resolution and quantitation of chromatographically overlapping components.13 Such methods permit the quantitation of partially overlapping components without resorting to the use of pure component standards. However, the application of these methods may be somewhat limited by their requirement for fairly sophisticated chemometric techniques that may not be generally accessibleto many analysts. Simple methods for quantitation of coeluting Components using multiple detectors that exhibit comparable response for both components have only rarely been reported.14J5 Such simple methods could, in principle, be developed under conditions where (1)the identities of all of the overlapping components are known, (2) response factors can be individually measured for each of the overlapping components by each detector, and (3) the detector response factors for the individual components are sufficiently different from each other. In such cases, even for a pair of perfectly coeluting components, one should be able to quantitatively relate the concentration of the analytes to some function of the detector responses and the response factors of the individual components. Such methods should not require infinite selectivity by the detectors or complex chemometric techniques to be quantitatively useful. Equations are developed in this paper that relate the concentrations of individual coeluting components to the response of multiple detectors and to the individual detector response factors for each of the coeluting components. Equations are described that are useful for both internal and external standard techniques. The method is applied to the resolution of two overlapping components, propionaldehyde and acrolein, using tandem photoionization/flame ionization detection (PID/FID).
200 "C. Helium was used as the carrier gas. Column head pressure was 50 kPa when the column was maintained at 60 O C , affording a carrier gas flow rate between 2 and 3 mL/min. Detector gas flow rates followed the manufacturer's recommendations, i.e., combined carrier plus makeup gas flow of 30 mL/ min, hydrogen flow rate of 35 mL/min, and air flow rate of 165 mL/min. The oven temperature program was as follows: initial temperature of 60 "cfor 10min, temperature ramp at 20 "C/min to a final temperature of 200 "C, holding at that temperature for 5 min. Data collection and integration were performed by Hewlett-Packard Model 3365 ChemStation software (Version A.03.01) operating on a Hewlett-Packard QS/20 computer.
EXPERIMENTAL SECTION Standard solutions of the individual analytes, acrolein and propionaldehyde,alongwith toluene as an internal standard were prepared in n-heptane. Chemicalswere used as received without further purification. Initially prepared solutions were subsequently diluted further with n-heptane to achieve a final solution concentration that would be within the linear range of the detectors. Analysis was accomplished on a 60 m X 0.53 mm X 1.5 pm RT.-1701 column (Restek Corp., No. 12073) fitted with a 5 m X 0.53 mm phenyl methyl silicone-deactivated guard column (Restek No. 10045). The column was contained in a HewlettPackard Model 5890 Series I1 gas chromatographusing a packed column injector. Injections(1.0pL) were made using a HewlettPackard Model 7673 autoinjector. Peak detectionwas via an 01 Analytical Corp. Model 4450 tandem photoionization/flame ionization detector. Injector and detectors were maintained at
Similarly, component Y in the absence of component X could be quantitated by either of the following two equations:
THEORY This section describes the derivation of equations for quantitation of two overlapping peaks using two detectors for both internal and external standard methods of analysis. Nomenclature. There are two components in the sample being quantitated, X and Y, whose peaks overlap in the chromatogram. The internal standard is designated as S. There are two detectors, designated as A and B. Internal Standard Method. In general, the response factor R can be defined for any component and detector with respect to an internal standard as R, = (amtj/areq)/(amuareas) (1) where amh refers to the amount of component i, areai is the integrated detector response of that component, and amh and are% refer to the amount and detector response of the internal standard, respectively. Having determined a response factor from solutions containing known concentrations of components, the amount of a component in an unknown can be quantitated using the internal standard by applying the following equation:
amt, = (area,/area&mkR,
(2)
This method for quantitation of coeluting components initially involves separately determining the response factor of each of the overlapping components with respect to the internal standard for each detector. Thus, four response factors are determined for two overlapping components (one response factor for each component on each of two detectors). Thus, for component X and detector A, in the absence of overlap (for example, if component Y was not present), we could quantitate an unknown using the following equation: amt, = (areai/areat)amtsRi (3) Likewise, component X could be quantitated in the absence of component Y on detector B using the analogous equation: amt, = (areai/areaE)amtsRg
amt, = (area$/areat)amtsR{
(4)
(5)
amty = (area!/areaE)amt,R;
(6) When both coeluting components are present in a sample, each component can no longer be treated separately. Rather, the total area of the combined peak of both components in each detector must be defiied as being equal to the sum of the contributions from each component. For detectors A and B, this affords the following two equations: Are& = area; Are$ = area:
+ areat + area!
(7)
(8)
ANALYTICAL CHEMISTRY, VOL. 65, NO. 8, APRIL 15, 1993
Rearranging eqs 3 and 5, we have
1025
tuting the individual peak areas as defined in eq 21 into eqs 7 and 8 affords
area; = amt,area$(amt,R;)
(9)
area; = amt,are~I(amt,R~) (10) Substituting the areas of the individual components on detector A from eqs 9 and 10 into eq 7 affords Area$ =
amkarea;
+amhared
(11)
am@$ amtsR; Similar to eqs 9 and 10, eqs 12 and 13 may be generated by rearrangement of eqs 4 and 6: area: = amt,areai/(amt,R~)
(12)
area! = amt+eail(amt,Ry") (13) Substituting the areas of the individual components on detector B from eqs 12 and 13 into eq 8 affords Areg =
amkareai
+ amtyarea,B
"1'
A r e ,amts g --
(16)
area, R! Equations 15 and 16 both relate the amount of component X in the mixture to a series of knowns and to the amount of component Y. Equating the right side of these equations with each other
Solving for the amount of component Y in the mixture
*RB
B
area:
am%=(
--Are$R$)amts/[ area,
-1
R! - R$ -
(18)
R;
R!
Similarly, eqs 11 and 14 may be solved for the amount of component Y. These two equations can then be equated, which would result in a single expression containing the amount of component X along with a series of knowns. Solution of this expression for the amount of component X affords the following:
(
amk= Are' area: RB - *RA)amts/[ area;
-
21
(19)
External Standard Method. In the absense of an internal standard, the response factor for a component may be defined as R, = amti/areai
Similarly,solving eqs 22 and 23 individuallyfor amty, setting the two resultant equations equal to each other, and then solving the resultant equation for amtx affords
(14)
ambR; amtsR! Equations 11 and 14 relate a series of knowns (amount of standard, peak areas, and response factors) to two unknowns, the amounts of components X and Y. These two equations containingtwo unknowns may be solved as follows: collecting and rearranging terms, eqs 11 and 14 may be transformed into eqs 15 and 16
amt, = R[:
Solving eqs 22 and 23 individually for amtx, setting the two resultant equations equal to each other, and then solving the resultant equation for amty affords the following:
(20)
Rearranging eq 20 area, = amti/Ri (21) As before, when Componentsoverlap,the combined peak area in each detector is given by the sum of the contributions from each of the overlapping components (eqs 7 and 8). Substi-
RESULTS AND DISCUSSION Quantitationof AcroleinIPropionaldehydeMixtures with a Tandem PhotoionizationIFlame Ionization Detector. Acrolein and propionaldehyde were found by GCI MS to be present in a mixture that was being analyzed routinely, and these components were found to overlap under the chromatographic conditions described in the Experimental Section. The analytical conditions were otherwise satisfactoryfor resolving all of the other mixture components, and so this set of overlapping components was deemed to be appropriate for testing this method. A commerciallyavailable tandem photoionizationlflame ionization detector was used as the detector pair. The internal standard method was chosen for this analysis, with toluene being chosen as the internal standard. The method may be summarized as consisting of the following three steps: 1. Prepare separate solutions containing each of the overlapping components along with the internal standard and obtain the response factor for each detector of each of the overlapping components. 2. Add a known quantity of the internal standard to a known quantity of the sample being analyzed, obtain the chromatogram of the sample, and determine the responses of each detector for the overlapping peak and for the internal standard peak. 3. Compute the component concentrations from the observed peak areas and response factors using the equations in the Theory section (eqs 18 and 19 for the internal standard method). The composition of each of the solutions prepared in this study is shown in Table I. Separate solutions of acrolein and propionaldehyde were prepared in n-heptane, with each solution also containing toluene as the internal standard (solutions 1 and 2 in Table I). The individual detector response factorsfor each of the components relative to toluene were calculated using eq 1, and these resulta are summarized in Table 11. As shown in the table, the FID component response factors are similar, which would be expected considering the similarity in the molecular formula of these compounds. In contrast, the PID response factors, which are related to both the ionization potentials of the molecules and the number of their A electrons,lBdiffer for the two compounds by a factor of 2.5. (16)Casida, M. E.; Casida, K. C. J. Chromatogr. 1980,200, 35-45.
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ANALYTICAL CHEMISTRY, VOL. 85, NO. 8, APRIL 15, 1993
Table I. Composition of Standard Solutions and Mixtures. soln no.
component wt (g) propionaldehyde
acrolein
1 2 3 4 5
toluene
0.2359 0.3291 0.1336 0.2428 0.0549
0.1563 0.1715 0.1757 0.1936 0.1675
0.1478 0.0563 0.2376
After dilution with n-heptane (solvent), final concentrations of each of the components were in the range of 0.07-0.4 wt % .
Table 11. Component Response Factorsa component
SD
re1 SD (%)
0.030 0.060 0.004 0.020
0.3 1.6 0.2 0.8
detector av response factor
PID
acrolein propionaldehyde acrolein propionaldehyde
9.42 3.74 2.32 3.13
PID FID FID
Response factors and statistics based on chromatograms of each of the solutions run in triplicate.
4.0-4
e
6
1 0
1'2
1 4
Figure 1. PID chromatogram of acroleinlproplonaldehyde mixture (solution 3) in n-heptane. Peaks at 7.8, 14.5, and 10.5-12.5 mln are acroleinlproplonaldehyde, toluene, and solvent, respectlvely.
n
4'o=l 3.0-
in each of the solutions in Table I11 and as a percentage of the s u m of the components in Table IV. As can be seen from the results in these tables, the method is reasonably accurate in assessing the concentrations of the components in the equimolar mixture, solution 3. Accuracy is also relatively high for the major component in solutions 4 and 5. However, relative accuracy deteriorates for the minor component in these solutions. This loss in accuracy for the minor component may be rationalized by examination of the equations by which these concentrations were determined. The following equation recasts eq 19 in expanded form: amt, =
tsR!
- Are& -amhR$)/[
-1
y R! - R$
(26)
area; Rx R;: Recall from eq 2 that, for a pure component,
amt, = (areai/areas)amtsRi (2) Assume that component X is the minor component and that its concentration is being determined by eq 26. In the limit, as the concentration of component X approaches zero, the numerator of eq 26 essentially equals the difference between the amount of component Y as determined from the B and A detectors, respectively. As the concentration of component X decreases, its ever-decreasing concentration is being determined as an ever-smaller difference of larger numbers (the concentration of component Y determined from the B and A detectors). The inherent inaccuracy in determining the concentration of a minor component from the difference of two larger values dictates that the relative accuracy of the minor component, as determined by this method, will suffer as its concentration decreases. Alternate ComputationalTechniques. Equations have been derived above for the case in which two detectors are used to quantitate two overlapping components. In general, the method may be extended to analyze for any number of overlapping components as long as there are at least as many detectors as there are overlapping components. Derivation of the equations may be more complex in the general case. However, recall from the Theory section above (e.g., eqs 7 and 11)that, for each detector, the total area of the coeluting peak can be regarded as being equal to the sum of the contributions from each of the coeluting components. For N coeluting components, the value for the combined peak area assumes the following general form N
.o,q
= ' O = I 1
Area, = x k i a m t i
(27)
t=l
c
, , b
.
,
e
.
.
.
( 1 0
.
.
.
, 1 2
.
.
.
,
.
c
14
Flgure 2. FID chromatogram of acrolelnlproplonaldehyde mlxture (solutlon 3) in +heptane. Peaks at 7.8, 14.5, and 10.5-12.5 min are acrolein/propionaidehyde, toluene, and solvent, respectively.
Solutions 3-5 were prepared to simulate mixtures containing both components. Solution 3 is a roughly equimolar mixture, while solutions 4 and 5 contain the individual components in concentrations that are skewed in either direction. The PID and FID chromatograms of solution 3 are shown in Figures 1 and 2, respectively. The peak at 7.6 min contains the overlapping components that are totally unresolved in the chromatogram. The peak at 14.5 min is due to toluene, while the broad peaks from 10.5 to 12.5 min are due to solvent. The amounts of each of the components in samples 3-5 were determined using eqs 18 and 19. Component concentrations are listed as the calculated weight of each component
For the internal standard calculation, the value ki equalsa r e a (amwi).Treatment of the two-component/two-detector case by this computational technique results in two equations of the form of eq 27. For detector A Are& = kiamt,
+ k$amt,
(28)
where and k{ = area;/(amtsR{) A similar equation results for detector B. The values of k in the individual equations are all composed of known quantities, i.e., areas and amounts of standard and previously determined response factors. Likewise, the total detector responses for the coeluting peaks are known after the samplesare chromatographed. Componentamounts may then be calculated using straightforward regression tech-
ANALYTICAL CHEMISTRY, VOL. 65, NO. 8, APRIL 15, 1993
Table 111. Analyzed VS Actual Component Weights (from eqs 18 and 19)a actual wt (9) anal. w t (9) re1 SD (%) soln no. acrolein propionaldehyde acrolein propionaldehyde acrolein propionaldehyde 3 4 5 a
0.1336 0.2428 0.0549
0.1478 0.0563 0.2376
0.1381 0.2489 0.0616
0.1494 0.0602 0.2353
1.0 1.5 1.0
0.5 0.7 2.0
reldev(%) acrolein propionaldehyde 3.4 2.5 12.2
1.1 6.9 1.0
Component weights and statistics based on chromatograms of each of the solutions run in triplicate.
Table IV. Measured vs Actual Mixture Composition (from eqs 18 and 19)a anal. composn ( % ) re1 SD (%) actual composn (%) solnno. acrolein propionaldehyde acrolein propionaldehyde acrolein propionaldehyde 3 4 5 a
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47.5 81.2 18.8
52.5 18.8 81.2
48.0 80.5 20.8
52.0 19.5 79.2
0.2 0.2 0.5
0.2 0.2 0.5
re1 dev ( % ) acrolein propionaldehyde 1.1 0.9 10.6
1.0 3.7 2.5
Mixture compositions and statistics based on chromatograms of each of the solutions run in triplicate.
niques. As long as response factors of the individual components are predetermined, regressiontechniques permit the facile determination of any number of coeluting components from the responses of an equal or greater number of different detectors. Scope and Limitations of the Method. One of the limitations of the tandem detector method as described here is ita requirement for pure component standards for the calculation of response factors. These response factors essentially provide the basis for a single-point calibration curve for each of the overlapping components. Since component concentrations are determined using equations that contain four response factors, errors associated with a singlepoint calibration may be magnified in the analytical result. Accordingly, prudent precautions, Le., workingwith standards that are in the same general concentrationrange as unknowns, and ensuring that the detectors exhibit linear response over the standard and unknown concentration ranges of interest, should be taken to minimize the extent of errors associated with single-point calibrations. As shown above,all of the final analyticalequations contain a denominator that is equal to the difference between two ratios, each ratio representing the selectivity of one of the detectors for the overlapping components. As these ratios approach each other, the denominator approaches zero. In the limit, if the two detectors exhibit equal response ratios for the overlapping components,the denominator equals zero and the analytical result becomes undefined. Further examination of the analytical equations reveals that the method will continue to be operative even if one of the detectors exhibits equal response for both overlapping components. However,if both detectors exhibit this behavior, the method will fall under the limitation discussed above, i.e., a zero denominator with an undefined analytical solution. In the illustrative example used in this paper, quantitation was accomplishedthrough the use of a commercially available
tandem PID/FID configured in series. Tandem PID/ELCD detectors for GC are also commercially available. The series arrangement will be effective as long as the analytes are not destroyed by the first detector or otherwise rendered nonanalyzable in the second detector. If both detectors are destructive, they may be configured in parallel and may be used for quantitating overlapping peaks in exactly the same way as described here. A potential advantage of the series arrangement, however, is the fact that both detectors -see" the total column effluent, which would lead to greater sensitivity than a parallel configurationin which the analytes are split before arriving at the detectors. While the example presented here involves GC analysis, the method is, in principle, adaptable to any other chromatographictechnique. Furthermore, the detectors need not operate on different detection principles. For example, subject to the limitations discussed above, multiple-wavelength optical detectors may be used to discriminate overlapping peaks in HPLC. In fact, multiple diode array detectors are now commonly available on modern HPLC instruments, thereby making this tandem detector technique readily adaptable for HPLC analysis. Its general applicability across many different chromatographic techniques using a wide variety of commercially available detector systems renders this method worthy of consideration as an alternative to chromatographic optimization for the quantitation of overlapping chromatographic peaks.
ACKNOWLEDGMENT Dave Parees, Andy Gilicinski, Tom Bzik, and Bill Cowen, all of Air Producta, are acknowledged for their helpful discussions, encouragement, and support.
RECEIVEDfor review September 1, 1992. Accepted December 31, 1992.
