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Dual Wavelength Spectrophotometric Detector for High Performance Liquid Chromatography Kuang-Pang Li" and John Arrington Depariment of Chemistry, University of Florida, Gainesville, Florida 326 1 1
Peak overlapping is very common in multicomponent elution even with optimized high performance liquid chromatography. Quantitization of the seriously interfered components is not feasible with conventional detection methods. We report here the utilization of dual wavelength spectrophotometry (DWS) as a high resolution, high precision, and high selectivity detector for the remedy of this problem. The basic principles of the novel detection method are illustrated and demonstrated with examples of polycyclic aromatic hydrocarbon analysis.
Multiple component determination with high pressure liquid chromatography (HPLC) depends mainly upon the separation efficiency of the chromatographic system. By properly selecting t h e mobile and stationary phases and operational conditions, such as recycling, solvent programming, temperature or flow programming, multicolumn technique, etc., very high efficiency may be obtained. However, even under the most optimal conditions, peak-overlapping is still very common in the chromatography of structural related compounds. I t takes a lot of patience and great care to reveal t h e analytical information about these hard-to-resolve components. In these cases, if the detector itself also provides means of differentiation for the components in the eluent, it will add a new dimension of resolution to the chromatographic system. When properly designed, the means of differentiation may arise from the slight difference in any physical or chemical properties of the components. Here we report the utilization of t h e small spectral difference for t h e differentiation of structurally closely related compounds. This is accomplished by coupling the HPLC to a spectrometer operating in the dual wavelength mode. Careful selection of the two wavelengths enables us t o flip the elution peak of either component in a highly overlapping profile. As a result, not only is the resolution increased but also the identity of the components may be revealed. Methods of quantitation for the individual components are also discussed.
PRINCIPLES In normal operation of an analytical chromatographic system using an absorption detector, the Beer-Lambert law may be strictly applied for solute detection. Since absorbance is an additive parameter, the instantaneous signal of an eluting profile containing n components is the sum of the absorbance of t h e individual components, t h a t is,
is compensated with proper zero adjustment. Variation due to light scattering by both solvent and solute molecules and t h a t due to flow and lamp current fluctuation are not compensated for. Moreover, components which do not absorb a t the wavelength, usually a spectral line from a mercury lamp, e.g., 254 nm or 280 nm, will not be detected and components which absorb a t that wavelength will be indifferently registered as predicted in Equation 1. Therefore, if the resolution is much less than unity, precise quantitation of the individual components is almost impossible from the resulting chromatogram. Differing from the conventional double-beam arrangement, the dual wavelength spectrometry developed by Chance (1-3) employs two different wavelengths, A, and A*, which are sent through the sample cell along the same light path in a time-sharing manner. The difference in absorbances a t these wavelengths is amplified and measured. As a result, fluctuations due to light scattering, source, and flow variations can be minimized. Components which do not absorb a t one wavelength but absorb a t the other can also be registered. The elution signal observed can thus be expressed as,
AA = A , ,
-
A,, = b C p , C , i=l
where p, = is the compound absorptivity of the zth component and can be either positive or negative depending on the values of (U,)Al and ( u J X 2 .When @,is positive, the elution peak of the ith component will appear on one side of the base line. I t will show up on the other side if 8, is negative. Therefore, by proper selection of the two wavelengths, one can make the elution peaks of some components above and the others below the base line. Such alternating coding of the signal provides not only higher resolution than the chromatographic system alone can possibly attain but also additional information about the identity of the components. This is often possible because two different molecules having similar retention times could not possibly have an identical spectrum over the entire UV-visible region. With the present chromatographic technology, the number of components in an overlapping peak is seldom greater than 3, Le., n 5 3. We will concentrate on the cases where n = 2 and n = 3. Assuming in both cases the elution of each component is Gaussian in shape, t h a t is,
n
A , = bC (a,)xC1
(1)
1=1
where (a,), is the molar absorptivity of the ith component a t a wavelength A, b is t h e cell length of the detector, and A , is the transient absorbance observed during elution. If the dimension used for b is cm and that for C,is molarity, M , ( u J A will have a unit cm-' M-I. Conventionally, absorption is measured against a reference which is usually an air cell. Absorption of the mobile phase 0003-2700/79/035 1-0287$0 1. O O / O
where i = 1, 2, or 3. Cp is the initial concentration of the ith component in the sample, and u and f are t h e volume of sample injected and the flow rate, respectively. The retention time of the ith component is represented by the symbol tR, and t h e corresponding standard deviation by u c . Substitution of Equation 3 into Equation 2 gives, for n = 2, C 1979 American Chemical Society
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(4)
A
where
where i = 1,2. The parameters y1 and y2 can be either positive or negative depending on the values of PI and &, so can AA. Assuming that the selection of wavelengths gives positive p1and negative pz values, and that tR1is slightly smaller than t ~the~second , term on the right hand side of Equation 4 is therefore negative. In a certain range of t , e.g., t < tR1,the absolute value of this term is smaller than that of the first term, because of the rapid decrease of the exponential with the increase of the argument, AA is positive. It stays positive until bylClo is just compensated for by byzCzo. After that LA goes negative, passes through a minimum, and approaches zero from the negative side as t increases. This results in a wave-like elution curve with the peak and valley related t o the relative amounts of the two components, respectively. T o reveal the concentrations of the components in the original sample, the area under the elution profile is needed. Since the Gaussian function is a normalized function, integration of Equation 4 yields,
5
5
ELL'II3::
:IT,
2s
2:
n:l
i
=c57,;--T,
= 25:.:
-2.
) m
= 295.3
7,n
i
= 28e.c nm
i
= 297.: m
Combining Equations 4 and 5 , one obtains,
C1@=
CZ@=
f(PnAA - YZAAT) bu(Y1Pz - Y l P 2 ) f(YIAAT -
bu(y1Pz -
PllA) Y2P1)
(6) (7)
Since the 0's and y's can be obtained from calibration, both CIo and Cz@can be obtained from the measurable values of AA and AAT. In the case where three components are overlapping, one can choose two wavelengths which makes P2 negative while p1and p3 are positive. T h e concentration of all three components can be measured by measuring AAT and two AA values, e.g., a peak and a valley or two peaks.
EXPERIMENTAL Reagent grade methyl alcohol was obtained from Mallinckrodt Inc., Paris, Kentucky. Polycyclic aromatic hydrocarbons (PAH) were purchased from the following sources and were used without further purification: triphenylene, phenanthrene, and anthracene from Aldrich Chemical Company, Inc., Milwaukee, Wis.; benzo[elpyrene from Columbia Organic Chemicals Co. Inc., Columbia, S.C.; chrysene from City Chemical Corporation, New York, N.Y.; pyrene from Eastman Kodak Co., Rochester, N.Y.; 1,2-benzanthracene from Research Organic/Inorganic Chemical Corp., Belleville, N.J.; and benzo[a]pyrene from Switzerland. Stock solutions of the PAH were prepared in methanol and were kept refrigerated no longer than two weeks. Synthetic samples were prepared daily by mixing the stock solutions in proper proportion and diluted to volume with methanol. An ALC/GPC 203 (from Waters Associates, Inc., Milford, Mass.) high pressure liquid chromatograph equipped with a pBondpak C18column (P/N27324) and a loop injector was used. The HPLC is coupled to an Aminco DW-2 UV-visible spectrophotometer by direct connection of the outlet of the HPLC detector to the inlet of a flow cell (Precision Cells, Inc. 8495, 0.25 mL) in the spectrophotometer. The HPLC was operated isocratically in the reversed phase mode with 74% methanol in distilled water. The column temperature was maintained at 37 "C by circulating constant temperature water through a jacket around the column. The signal output from the HPLC detector
3
5
15
LO ELUTIOY X M E ,
23
nln.
Chromatograms of polycyclic aromatic hydrocarbons: A. Conventional chromatogram recorded at 254 nm. Peak 1, impurity; 2, phenanthrene; 3, anthracene; 4,pyrene; 5, triphenylene; 6, chrysene; 7 , benz[a]anthracene; 8, benzo[e]pyrene; and 9, benzo[a]pyrene. B, C, and D. Chromatograms obtained dual wavelength spectrophotometrically at different wavelengths Figure 1.
was recorded with a strip chart recorder (OmniScribe series B-5000, Houston Instrument, Austin, Texas) at a chart speed of 1 cm/min. The chart speed of the DW-2 was set at 1 in./100 s.
RESULTS AND DISCUSSION A typical chromatogram of a mixture containing 8 PAH was reproduced in Figure 1A. I t is seen t h a t the separation of pyrene, benzo[e]pyrene and benzo[a]pyrene is satisfactory. The microparticulate column, however, fails to resolve either phenanthrene from anthracene, or triphenelene, chrysene, and benzlalanthracene from each other. T h e overlapping of chrysene with benz[a]anthracene is particularly serious. Similar observations have been reported with different stationary and mobile phases ( 4 4 ) . None of these investigations using the CIS reverse phase column has succeeded in a complete resolution of the PAH. Quantitation of these seriously overlapping species is not feasible with the conventional absorbance measurement method.
ANALYTICAL CHEMISTRY, VOL. 51, NO. 2, FEBRUARY 1979
289
A
=I
c:-I
C il
257.0
A2
266.5 m
MI
Figure 2. Computer simulations of PAH chromatograms
A fluorescence detector may provide quantitative information about either or both components in an overlapping peak only if they fluoresce strongly and differently. But there
are far less fluorescent than nonfluorescent compounds and fluorescence is a spectral property which depends heavily on the environment the molecules are in and on the presence of
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Table I. Parameters Used for Chromatogram Simulation retenmolar absorptivities,' (M cm)-' tion peak concentration, times,b width,b 250 254 257 266.5 288 297.5 components M X 104 min min nm nm nm nm nm nm 6423 4 817 1.03 x 10-4 8.15 0.25 64 600 64 231 24086 11 562 phenanthrene 356 531 44 560 534 8.70 0.25 2 0 0 0 0 0 2 0 0 5 2 0 0.53, anthracene 2 831 4 247 11 800 11 800 13146 15169 11.20 0.29 3.26 pyrene 15980 9 816 2 169 73053 79902 132995 0.83, 14.05 0.50 triphenylene 9 1 316 1 6 4 0 0 0 9 360 9 132 15.5 43 375 68487 1.33 0.50 chrysene 90 170 7419 0.50 33102 36525 38809 45656 15.8 benz [alanthracene 1.57 21.2 20 816 23970 24601 2 7 124 54 249 1 1 9 8 5 0.67 1.24 benz [ elpyrene 38689 43 736 42894 50464 36166 66865 23.7 0.75 1.37 benz [ alpyrene ' Calculated from Sadtler UV spectra. Obtained from the following chromatographic conditions: Temp = 37 'C, flow rate = 1.21 mL/min, mobile phase = 3 5 mL H , 0 / 1 0 0 mL CH,OH, solvent = CH,OH (Mallinckrodt), column packing = p Bondapak C,,, pressure = 2300 psi. quenching or sensitizing species. These facts make the fluorometer a very selective but less versatile detector in liquid chromatography. T h e HPLC-DWS system reported here has the selectivity t h e fluorescence detector has and the versatility it does not have. In addition, its ability to determine simultaneously both components in a highly overlapping peak is very unique. Because of the additional dead volume of the flow cell, post column mixing in the DWS is believed to be much worse than t h a t in the HPLC detector. Even under such an adverse condition, the resolution of this novel differentiating detection technique is seen t o be much better than t h e conventional method. Resolution can be even better if the analytical column is directly coupled to the DWS. Since t h e chart speed of the DWS is different from t h a t used for t h e HPLC, it is not easy t o line them up for comparison. So we extract the chromatographic information from t h e chromatogram and spectral information from Sadtler spectra (see Table I) and substitute them into Equations 1 a n d 2. By means of a computer we can simulate chromatograms under different conditions (Figure 2). These simulations help a lot in confirming the identity of the PAH. I t is seen from Table I that phenanthrene. anthracene, and triphenylene have greater molar absorptivities a t 254 nm than a t 266.5 nm but the rest of the PAH absorb more strongly a t 266.5 nm. If one chooses XI = 254 nm and X2 = 266.5 nm, the elution profile of phenanthrene, anthracene, and triphenylene will be on the positive side while the others will be on the negative side. Providing t h a t all PAH concentrations are comparable, the largest negative peak in the resulting chromatogram will be chrysene because it has the greatest difference in absorptivity (Figure 2B). When the wavelength A, is changed from 234 nm to 257 nm, t h e absorptivities of phenanthrene and anthracene decrease drastically, as do their elution peaks (Figure 2C), whereas the triphenylene peak is enlarged significantly. Now, if t h e two chosen wavelengths are 250 and 288 nm, benz[a]anthracene will be flipped negative and pyrene positive as shown in Figure 2D. However, the best wavelengths for determination of benzanthracene, benzo[e]pyrene, a n d benzo[a]pyrene in t h e multiple component mixture are 288 and 297.5 nm. At these wavelengths (Figure 2E) the interference from triphenylene and chrysene is negligibly small and t h e elution signals are of considerable size. All these simulations match with the real chromatograms (Figure 1, B-D) with fantastic resemblance in both peak shape and relative peak sizes. This implies t h a t such simulation may be very useful in real sample determination. With a computer-interfaced system, such matching may be reached in real time. Even though a complete analysis may not be feasible in this manner, the identity and quantity of the concerned components may be revealed.
A
_ i
Figure 3. Chromatograms of triphenylene and chrysene. Wavelength used: A, = 257 nm and A, = 266.5 nm. Amount of triphenylene injected: 38.1 ng; amount of chrysene introduced: A, 69.7 ng; B, 139.4 ng; C, 209.1 ng; D, 278.8 ng; and E, 348.5 ng
Figure 3 shows chromatograms of a series of binary mixtures of triphenylene and chrysene. T h e amount of triphenylene is kept constant and that of chrysene is varied. T h e elution profiles recorded conventionally a t 254 nm were reproduced in Figure 3A, whereas those displayed in Figure 3B are chromatograms obtained dual wavelenth spectrophotometrically at 257 and 266.5 nm. At these two wavelengths, the compound absorptivities of triphenylene and chrysene are of opposite signs. .4s a result, the elution of chrysene appears in a valley. The depth of the valley is seen to be proportional to the concentration of chrysene. If this amplitude is plotted against the amount of chrysene injected onto the column, a straight line is obtained. The line does not pass through the origin because of the interference of triphenylene. Referring back to Equation 4, it is seen that the intercept of this line with t h e ordinates is corresponding t o t h e quantity of burLClo/f . Dividing this quantity by the concentration of triphenylene one obtains the value of b u y l / f . In t h e actual calculation, we used the absolute amount, in nanograms, instead of concentration. A conversion factor must be included. But this factor is later eliminated automatically. As long as we keep the units consistent, there is no need to worry about the conversion. The area of the elution profile is measured with a compensating polar planimeter (Keuffel & Esser Co., New York). I t is also proportional t o the concentration as indicated in Equation 4. The intercept yields bu@,/f in the same manner as yi.Once these parameters are found, a reduced parameter corresponding to the quantity ( b u / f ) [ - y l - l A-~ $ l l A ] can be
ANALYTICAL CHEMISTRY, VOL. 51, NO. 2, FEBRUARY 1979
Table 11. Calibration of Chrysene in the Presence of TriphenylenB amount introduced ng,
( bvlf 1['YiAAT -
A A X 100
69.7 139.4 209.1 278.1 348.5
-0.40 -1.15 -1.95 -2.60 -3.45
slope
-1.084
X
A A ~ P , A A ] X 10' +0.031 -0.021 -0.074 -0.115 -0.172 -7.182 X
1.13 2.22 3.42 4.40 5.67 1.617 X
10-3
intercept
3.56 X
7.99 X 10-2 0.9983
-1.44 X
10-3 10-7 0.9991 correlation 0.9990 Amount of triphenylene introduced is 38.1 ng.
A
h 1.0 mv
100
!y
291
Equation 7. This line may serve as the calibration curve for t h e quantitization of chrysene in an unknown sample. T h e same calibration procedure can be applied to triphenylene in the presence of chrysene or t o other binary mixtures such as chrysene and benzanthracene even though the components are nonresolvable in the conventional manner (Figure 4,A and B). Computer simulations demonstrate that this flip-over is often possible unless the two components have identical elution profiles. I t is most convenient t o measure a peak height or valley depth when the peak and valley are of comparable size. However, in most times, particularly in real sample analysis, the interfering component may be much more concentrated than the component of interest. To overcome this problem, one can either use a standard addition method to enlarge the peak size of that component or choose wavelengths such that t h e AA of the interfering component is partially, if not completely, diminished without giving up too much sensitivity for the component of interest. T h e Aminco DW2 spectrophotometer has the highest sensitivity range of 0.005 absorbance unit. We have been using the full-scale range of 0.1 and 0.05 throughout this work. With these ranges, we can easily quantitize triphenylene, chrysene, and benzanthracen a t the 50-ng level or less. We expect that 1-5 ng of these PAH can be determined in the presence of their mutual interferences with the present setup. T h e limit of detection can be lowered if the absorption light path is increased. This is advantageous because post-column mixing is not as critical in this method as in the other detection techniques. T h e volume of t h e flow cell can be enlarged without seriously affecting the quality of the chromatogram. Although the basic principles of this method were developed based on Gaussian profiles, components with non-Gaussian elution can be determined as well.
LITERATURE CITED
9
Figure 4. Chromatograms of chrysene and benz[ alanthracene. Wavelengths used: 266.5 and 288 nm. Curve A: chrysene, 306 ng, benz[a]anthracene, 190 ng; Curve B, chrysene, 306 ng; benz[a]anthracene, 379 ng; and Curve C, chrysene, 306 ng; benz[a]anthracene, 474 ng
evaluated and tabulated in Table 11. If this parameter is plotted against t F c o n c e n t r a t i o n of chrysene, a straight line is again constructed. This line will pass through the origin with a slope equal t o ( b u / f ) * [yl& - y2pl] as predicted from
(1) B. Chance, Rev. Sci, Instr., 73, 158 (1942). (2) B. Chance, Rev. Sci. Instr., 22,634 (1951). (3) B. Chance, Science, 720,767 (1954). (4) B. S.Das and G. H. Thomas, Anal. Chem., 50, 967 (1978). (5) M. Dong, D. C. Locke and E. Ferrand, Anal. Chem., 48,368 (1976). (6) M. A . Fox and S. W. Staley, Anal. Chem., 48,992 (1976).
RECEIVED for review August 16, 1978. Accepted November 10, 1978. T h e authors gratefully acknowledge the National Science Foundation (Grant No. M P 575-02520) for partial support of this work.
