Advisory Panel Jonathan W. Amy Richard A. Durst G. Phillip Hicks
INSTRUMENTATION Donald R. Johnson Charles E. Klopfenstein Marvin Margoshes
Harry L. Pardue Howard J. Sloane Ralph E. Thiers
Double-Wavelength Spectroscopy T. J. PORRO Perkin-Elmer Corp., Norwalk, CT 06852
Photometric measurement of a material by passing radiation of two different wavelengths through the same sample before reaching the detector can extend the proven effectiveness of uv-vis absorption spectroscopic techniques by overcoming limitations in selectivity, thereby reducing interferences by one dimension
T^LECTRONIC
(uv-vis)
absorption
-L' spectrophotometry has been an effective structurally diagnostic analytical tool for over 30 years, primarily because of its high signal-to-noise ratio and inherent sensitivity (high absorptivity of most absorbing species). This high sensitivity manifests itself in providing to users of the technique a high measurement precision and ultimate analytical accuracy. It is not our intention to review uv-vis absorption spectroscopy since anyone acquainted with it can attest to its utility over the years, particularly as a quantitative tool. However, it might serve a useful purpose to indicate that electronic absorption spectroscopy holds a unique place in the history of analytical instrumentation as applied to the general solution of chemical problems. Prior to the emergence of infrared spectroscopy as a structure elucidation aid, electronic absorption spectroscopy
was employed most effectively in the late thirty's and forty's to unravel the identity of organic molecules, particularly the many steroids and alkaloids which the organic chemists were endeavoring to prepare synthetically. Biochemistry is deeply indebted to uvvis absorption spectroscopy for its initial progress owing to all the reasons already stated, but particularly because the universal physiological solvent, water, had no appreciable absorption in the electronic region of the spectrum, thus allowing direct measurements. However, in spite of these useful advantages, two major limitations have served to restrict its use primarily to the quantitative analysis of clear solutions of low-molecular-weight solutes. One limitation, which we shall label as one of poor selectivity, results from the inherently severe overlap of the vibrational bands within an electronic transition. Thus, for many absorbing species the vibrational bands show up as indistinct shoulders on the side of more prominent absorption peaks. This characteristic causes electronic spectra in their normal presentation of absorbance (or transmittance) vs. wavelength (or frequency) to be relatively poor compared to, say, infrared spectra for the identification of pure unknown materials or for the discrimination among components of a mixture. This is in spite of the fact that the uv-vis spectrum contains vibrational and rotational, as well as electronic, information. The second major limitation, which we shall call one of sensitivity, results from the relatively poor transmission of radiation between 200 and 800 nm through solute and solvent combinations other than those of clear lowmolecular-weight solutions. By this we mean that except for such solutions, radiation in the stated range tends to be scattered quite effectively by many important materials including high-
molecular-weight polymers and proteins, colloidal suspensions, powders, paper, cloth, smoke, and the like, which comprise a sizable percentage of materials requiring absorption measurements. This effective scattering reduces the amount of light reaching the detector (photomultiplier), thereby causing the spectrophotometer system to be a relatively insensitive light collector, thus negating the fundamental sensitivity advantage previously mentioned. The analytical chemist has handled these problems over the years either by getting his sample into an acceptable solution for uv-vis spectrophotometry or by searching for another analytical technique before deciding that there was no immediate answer to his problem. Chance, in his brilliant work on the study of the mechanism of cell respiration by mitochondria, developed two separate instruments, a nonscanning double-wavelength instrument to overcome the selectivity limitation in the analysis of cytochrome mixtures (1) and a scanning double (split) beam spectrophotometer designed to measure small absorption differences between highly turbid mitochondria suspensions (2). Several valuable methods based on Chance's dual-wavelength spectroscopy techniques have been reported (-3-7), and Shibata and coworkers have applied the double-wavelength technique to the analysis of mixtures both organic and inorganic (8). A detailed description of the instruments and their use, mainly for turbid preparations, is available in the literature (8-13). In addition, the use of derivative spectroscopy instrumentation to overcome the selectivity problem has been described (14. 1">). In spite of the demonstrated power of the double-wavelength spectroscopy technique in the field of biochemical research, its exploitation as a general analytical tool remains below the
ANALYTICAL CHEMISTRY, VOL. 44, NO. 4 , APRIL 1972 • 93 A
Instrumentation
LIGHT SOURCE
MONOCHROMATOR
(A) DOUBLE-BEAM PHOTOMETRY
MONOCHROMATOR
MONOCHROMATOR
(B) TWO-WAVELENGTH PHOTOMETRY Figure 1.
Sample arrangements
threshold level, possibly because of the preoccupation many potential users have with their immediate analytical priorities. The purpose of this article is to re view the basic principles, instrumenta tion, and fundamental applications of double-wavelength spectroscopy, in the hope that these techniques may in the future be more rapidly applied to the many analytical problems extant. Principle, Definition, and Types
The amount of fundamental infor mation obtained in an instrumental measurement does not vary with the type of readout. However, the im mediate usefulness of the informa tion can and does depend upon the method of data presentation. For ex ample, a uv transmittance spectrum provides the same information as a corresponding absorbancc curve, but the latter is more directly useful in analytical situations since concentra tion is proportional to absorbance. Double-wavelength spectroscopy provides information from two wave lengths per unit time, and all other factors being equal, the resultant data should be more useful than data from a double beam absorbance (single94 A
•
wavelength) measurement. This is the fundamental principle underlying the application of double-wavelength spec troscopy. In fact, depending on the particular double-wavelength type em ployed, the measurement compensates for the presence of one parameter, be it an interfering impurity, scattering sample, or an indistinct shoulder on the side of a band. These benefits can be summarized as an improvement in the selectivity characteristic of the measurement. Thus, literally, two wavelengths are better than one. The second major limitation of uvvis absorption spectroscopy which we identified was one of sensitivity owing to the relatively poor light gathering power of conventional spectrophotom eters when measuring any non-clear liquid on solids. Though this problem has no fundamental relationship to double-wavelength spectroscopy, the development of instrumentation to solve the absorption overlap or selec tivity problem associated with these types of samples also includes the solu tion to the sensitivity problem. Sev eral of the double-wavelength instru ments available, including the one de scribed in this paper, do provide this important capability, and one of the
ANALYTICAL CHEMISTRY, VOL. 44, NO. 4, APRIL 1972
applications discussed demonstrates this capability. Let us define double-wavelength spectroscopy as we use it and identify several types used. In subsequent sec tions, double-wavelength instrumenta tion will be described, and selected ex amples illustrating a number of ana lytical benefits will be discussed. Double-wavelength spectroscopy re fers to the photometric measurement of a material by passing radiation of two different wavelengths through the same sample before reaching the detector. This technique, which has been re ferred to also as two-wavelength or dual-wavelength spectroscopy, can be compared with the more common double {or split) beam absorption spectroscopy which measures the ratio of light of the same wavelength trans mitted (or absorbed) by a sample and reference material in separate cells. These two techniques are illustrated schematically in Figure 1. Note that for all types of two-wavelength spec trophotometry, the sample is posi tioned close to the detector to better compensate for any turbidity or scat tering of the sample. Derivative absorption spectroscopy refers to a measurement in which the first derivative of either transmittance (T) or absorbance (A) per interval of wavelength (Δλ) is displayed on a recorder. Several conventional spec trophotometers have been modified to perform this function by displaying the readout of the signal of a tachom eter (speed measuring device) at tached mechanically to the pen assem bly of a chart recorder. A doublewavelength spectrophotometer pro vides the derivative function by scan ning the two monochromators with a fixed small wavelength difference be tween them and is, therefore, a spe cial example of double-wavelength spectroscopy. Several examples of this important type are given in the Ap plications Section. Additional types of double- or twowavelength measurements in use in clude the following : Sample and reference beams are set at different wavelengths (nonscanning). This type is used in analytical situa tions where the interfering substance highly overlaps the analyte or when the effect of turbidity must be mini mized. An example of the latter is given later. Sample and reference beams arc set at different wavelengths (nonscanning), and the output at each wavelength is measured independently. This mode is particularly suitable in reaction kinetics where absorbance changes of two spe cies can be monitored simultaneously. Sample beam scanning and refer-
Instrumentation
Figure 2.
Double-wavelength spectrophotometer optical schematic
ence beam fixed mode are used in de termining the spectral characteristics of a highly turbid sample when no suitable reference can b e prepared. Instrumentation There are a number of instruments available as of this writing t h a t pro vide one or all of the double-wave length functions indicated above in ad dition to the double beam scanning capability. I t is not our intention t o provide a comprehensive review of these here b u t rather to describe one such type to illustrate the utility and power of double-wavelength spec troscopy. The optical layout of such an in strument, the Perkin-Elmer Model 356, is shown in Figure 2. Light from a tungsten-iodine (I) or deuterium (D) source passes into a Czerny-Turner monochromator through slit (S x ) to form an image of the mask on gratings Gi and G 2 . T h e dispersed radiation of both is focused by the collimator mirror ( M 3 ) , split, chopped, and sent separately through the secondary sam ple compartment used to minimize fluorescence effects of clear solutions. Bilateral optical attenuators Οχ and 0 2 , situated at the pupil image of each beam, are used to v a r y the intensity of radiation of each beam continuously to compensate for intensity differences, depending on the sampling situation.
1.0 r 0.9 -
OPERATION MODE WAVELENGTH SCANNING SPEED 60nm/min. SCALE A 0-1 BAND PASS 1.0 nm
0.8 0.7
MIXED SAMPLE
ΰ °· 6
ISOPHTHALIC ACID (lOmg/IOOml)
ζ
ΙΟ 0.5
TEREPHTHALIC ACID (0.5 m g / 1 0 0 ml)
CO CD
"*
0.4 0.3 0.2 0.1 Ο
J-
320
Figure 3.
300
280 260 WAVELENGTH ηm
240
Absorption spectra of isophthalic and terephthalic acid
96 A • ANALYTICAL CHEMISTRY, VOL. 44, NO. 4, APRIL 1972
Instrumentation
OPERATION MODE
λ|/λ 2
SCALE BAND PASS
ΑΟ-Ι ΑΟ-Ο.Ι 3nm
ISOPHTHALIC ACID CONCENTRATION α. 2S mg/100 ml b. 2 0
^ b
λ 1278.5 nm λ g 264.0 nm
Ο-·3-r
,o.b c
λ , 278.5 λ2263.5
λ, 278.0 ^263.0
c. 15
ΔΟ.α 0.1
d. 10
"Τ
-a.b λ .278.0
k^ttO Figure 4 . acid
-2-jL—d
ο_±±Λ
λ ,277.5 Χ22625
λ . 277.
Δ 0.0. 0.1
ο Τ
Optimization of parameters for two-wavelength analysis of terephthalic
Mirrors ( Μ 7 - 1 and M 7 _ 2 ) either con verge the two beams through a single cell in the primary sample compart ment for all double-wavelength modes (dual beam) or diverge the beams so that they pass through two cells for double (split) beam spectrometry and then each image on the 2-in. diameter end-windowed detector. The time separated sample, reference, and zero signals are amplified, demodu lated, and compared such that the sample signal is measured against a fixed voltage reference signal, con trolled by a dynode feedback circuit, which makes the preamplifier output zero when no photo signal is received. The compared signals are displayed on a recorder linearly in transmittance or linearly in absorbance through a log converter. The electronic pro cessing of the photometric system is the same whether the two beams are similar in wavelength or not. Since each of the gratings can be inde pendently driven linearly in wave length separately or synchronously, double beam (same wavelength) and the various double-wavelength modes previously mentioned, including the derivative, can be achieved conve niently. A light gathering capability for in creased sensitivity is achieved through the combination of a high intensity tungsten source, the ability to place a sample as close to detector as several millimeters in the primary sample com partment, and the use of a large endwindowed photomultiplier having low noise characteristics.
Analytical Applications
Analysis of Highly Overlapping TwoComponent Mixtures by Two-Wave length Technique. A common situa tion exists in which the spectrum of the analytc and an interfering com ponent, possibly an isomer, have quite
similar spectra resulting in severe over lap or interference. This is illustrated in Figure 3, as described in an article by Shibata et al. (8), which shows the solution spectra of the analyte, ter ephthalic acid, and an interfering iso mer, isophthalic acid, and a mixture of the two. Conventional double or single beam (single-wavelength) tech niques require a two-component anal ysis for this type of problem, whereas appropriate use of the double-wave length technique can reduce the prob lem to a one-component analysis. The two conditions to be met are as fol lows. Choose two wavelengths such that the absorbance of the interfer ing component at both wavelengths is identical or their absorbance difference is exactly zero. Also, the sample wave length (λ2) should be a sensitive mea sure of the analyte. Figure 4 sum marizes for the present sample, op timization of the instrumental analytic conditions; λι is 277 nm and λ2 is 262.5 nm, as determined by trial and error, such that Α (λ2 — λι) = 0 for all concentrations of isophthalic acid be tween 0 and 25 mg/100 ml. Figure 5 is a linear working curve through zero absorbance resulting from the mea surement of the absorbance difference at the two specific wavelengths for varying amounts of terephthalic acid. These data demonstrate that tereph thalic acid can be analvzed directlv
0.0075 ISOPHTHALIC ACID 2 0 mg/100 ml (CONSTANT)
0.005
Figure 5. Working curve of terephthalic acid
Ν I
f-
r-
< 0.0025 -
001
0.02
0.03 0.04
0.05
CONCENTRATION (mg/100 mi) TEREPHTHALIC ACID
ANALYTICAL CHEMISTRY, VOL. 44, NO. 4, APRIL 1972 • 99 A
Instrumentation
1.0-
UJ
υ
Figure 6. Absorp t i o n spectra o f a n erbium salt in presence o f a ce rium salt
ζ
< m CE ο en m
