Instrumentation T. D. Harris Bell Laboratories Murray Hill, N.J. 07974
High-Sensitivity Spectrophotometry Quantitative analysis by solution spectrophotometry has been in contin uous practice for more than a century (1). Instrumental design has remained conceptually stable since the early part of this century. However, the ad vent of stable electronics and reliable lasers has spawned a group of tech niques for measuring absorbance that provides a quantum leap in spectrophotometric sensitivity. The excep tional sensitivity available from a wide variety of methods is illustrated in Table I. This high sensitivity has not yet been given enough attention by the analytical community. The great majority of newly published spectrophotometric procedures for trace anal ysis continue to assume a minimum measurable absorbance of 1 X 10~2 (2). In this paper, a representative set of these new high-sensitivity methods is examined, and comparisons are made of their relative strengths and weak nesses and the circumstances for which each can best be applied. The discussion is restricted to those meth-
Table I. Absorption Detection Limits in Solution
Conventional transmission
1 Χ "ΙΟ-5
Laser intracavity
5 X 10~6
Interferometry
1 X 10~7
Photothermal deflection
1 X 10" 7
Photoacoustic
1 X 1(T 7
Thermal lens
8 X 1(T 8
ods useful for absorbances of 1 X 1 0 - 4 or less in fluid solution and to the UV/ VIS portion of the spectrum. Absorbance methods can be sepa rated into two groups: methods that measure transmission, including con ventional spectrophotometry, and those that measure the power ab sorbed by the sample, the so-called calorimetric techniques. The six meth ods (three from each group) to be used as examples in this paper are listed in Table II.
Figure 1. Experimental arrangement for filling and taking spectra with long hollow fibers. Source spectral light cou pling correction is made by first recording a spectrum with a 0003-2700/82/0351-741 A$01.00/0 © 1982 American Chemical Society
Detection limit ( M )
Method
A second property important in characterizing absorbance-measuring techniques is the distinction between peak-sensing methods, such as wave length modulation, and continuum ab sorption methods, such as calorimetry. A peak-sensing method is any tech nique that measures the change in ab sorbance with wavelength rather than the absorbance itself. Spectrally broad or flat changes in transmission of 0.02% or less can arise from several sources and are therefore difficult to unambiguously attribute to absorp tion. As a result, high-sensitivity transmission measurements are in practice peak-sensing methods. This factor together with the problem of a reliable reference makes measurement of neat solvent absorption by trans mission especially subject to error. Criteria for Comparison
Table III lists eight criteria by which any proposed absorbance mea surement can be evaluated. These properties are equally divided be-
long fiber and then removing all but a small length and re cording again. The difference is the transmission spectrum of the filled hollow fiber ANALYTICAL CHEMISTRY, VOL. 54, NO. 6, MAY 1982 · 741 A
Transmission Methods
Table II.
Spectrophotometric Methods Transmission
Calorimelric
Conventional spectrophotometry
Thermocouple
Wavelength modulation
Thermal lens
Laser intracavity
Photoacoustic
Table III. Criteria for Comparison of Spectrophotometric Methods Instrumental factors
Sample factors
Tuning range
Solvent physical properties
Pathlength effects
Solvent absorbance
Reflection losses
Molecular scatter
Flow effects
Particulate scatter
tween sample- and instrument-depen dent factors and can be used to deter mine which methods are most applica ble in different situations. A brief explanation will clarify the eight items in Table III. Tunability al lows measurement at the optimum wavelength for a large variety of analytes. The ability to tune also makes it possible to distinguish the analyte ab sorbance from interferences, especial ly dissolved impurities that vary wide ly from sample to sample. However, the usefulness of single-wavelength measurements cannot be ignored, as evidenced by the popularity of singlewavelength chromatographic detec tors. The pathlength dependence of the signal varies widely with measure ment method. Some techniques show considerably increased signal with in creased pathlength while others show no change at all. This is an important consideration, especially when small sample volume or experimental com plexity favors a small pathlength. Sur face reflections become important when experimental conditions make it difficult to keep surface reflection pre cisely constant throughout a measure ment sequence. Response to flow is important for detection in chromatog raphy and flow injection applications. Those measurements tolerant of flow are finding wider applicability due to the advantages of flow injection as a sample-handling method (3). The last four properties deal direct ly with sample characteristics. The de pendence of instrumental response on the matrix, the solvent in our case, is
most important for the calorimetric methods. The heat capacity, thermal expansion coefficient, thermal con ductivity, and the temperature depen dence of the refractive index are im portant in one or more of the thermal methods. The signal intensity for a fixed absorbance can differ by a factor of 50 among commonly available sol vents. The solvent absorbance can be comparatively large, so the ability to distinguish the analyte absorbance from the solvent absorbance may de termine the appropriate method. However, the solvent concentration will normally be at least 100 times greater than the most concentrated solutes, so the absorbance of the sol vent is generally a large but constant background. Finally, the response of the methods to scatter should be considered. Transmission methods respond to all types of transmission loss and there fore to scatter. As with solvent absorb ance, molecular scattering will likely be constant from reference to sample, unless there is a large change in solute concentration. Most calorimetric methods do not respond to elastic scattering loss. However, inelastic scattering results in energy deposition identical to absorption. Raman scat ter, for example, will yield an equiva lent absorption response of 2 X 10~8· Many methods are approaching this detection capability (4). In addition, molecular scattering processes are strongly wavelength dependent and may give steeply sloping baselines. Particulate scatter is analogous to im purity absorbance in its variability be tween samples. For transmission methods the result is a variable base line offset. Many calorimetric meth ods claim immunity from solid parti cle scattering. This claim is valid if the scattering loss is small, but even the most transparent solids absorb light, giving a large calorimetric response in some cases. In addition, if a focused laser beam is used, even small particu lates can block a significant part of the beam and disrupt the measurement. With these considerations in mind, each of the listed techniques will be discussed, beginning with the trans mission methods.
As stated, transmission methods are inherently difference techniques. The measurement of small absorbances re quires detection of a very small differ ence between two large signals. The fundamental limit is then the uncer tainty in the signal with the greatest noise. From a theoretical standpoint, the minimum uncertainty is the statis tical noise, or shot noise, of the input beam. With a few specialized excep tions, this shot noise is not the factor that limits experimental uncertainty. The fundamental limitations to trans mission measurements have been thoroughly discussed elsewhere (5). There has been recent work on over coming the sample-dependent anoma lies, such as beam deflection and defocusing (6*). Two approaches to the small-difference problem can be taken. One must either increase the difference or reduce the noise. In creasing the difference can be accom plished by increased pathlength or by placing the sample inside a laser cavi ty. Reducing the noise has a statistical limit but can be enhanced by limiting the bandwidth of detected noise, as in wavelength modulation spectrometry. First and foremost, at low absorb ance, the magnitude of the transmis sion loss scales linearly with pathlength. Therefore, if increased pathlength is a viable option to yield a measurable signal, then it is probably the most straightforward instrumental option. The analyst, however, should be prepared for an increase in optical artifacts. Any interference from mo lecular scatter or particulate scatter will also increase proportionally. Differences in surface reflections can be a serious problem for all trans mission methods. A change in reflec tion of 0.001% is equivalent to an ab sorbance of 4 Χ 10 - 4 . One first must consider the mismatch between sam ple and reference cells if the instru ment calls for both. The author is not aware of any cells sufficiently matched for the detection limits of concern here. Thus, high-sensitivity methods require both the reference solution and the sample solution to be mea sured in the same cell. A slight difference in refractive index between the solutions will result in a substantial error in absorbance. This error will be manifested as a constant offset above the baseline. If the analyte has a well-known spec trum, as is usually the case in analysis, this offset will probably be identified as such, provided the measurement method is sufficiently tunable. How ever, if the absolute transmission spectrum of a relatively transparent analyte is sought, this change could result in serious error. The calorimet-
ANALYTICAL CHEMISTRY, VOL. 54, NO. 6, MAY 1982 · 743 A
ric techniques, if properly calibrated, offer a simpler and more reliable method to measure net solvent absorbance. Long Path Cells. Multipass cells offer very long pathlength and have gained considerable popularity for gasphase infrared analysis. The enormous volume of these cells will likely pre vent a similar device from being used for liquids. However, the spatial co herence of lasers makes it possible to use simple long pathlength cells of reasonable volume. A device for this purpose has been constructed and tested for gases (7) but not for liquids. A 70-pass equivalent length was achieved in gases with only moderate effort. A similar liquid cell of modest 20-cm length would yield a 14-m equivalent path. For this pathlength an intensity difference of 0.5% would result from an absorbance of 1.5 X 10~ 6 cm - 1 —less than most solvents. Other examples of extremely long pathlength cells are hollow glass and quartz fibers. Stone has published sev eral reports of spectra taken with liq uid-filled hollow core fibers (8-10). An ingenious device for this purpose is shown in Figure 1. Pathlengths from 4.5 m to 130 m were generated without difficulty. Since these fibers are usual ly less than 100 μιη in diameter, a 10-m cell would have a volume of less than 0.1 mL. Problems could be en countered in filling, rinsing, and refill ing the same fiber and in extracting impurities from the fiber itself. How ever, disposable cells several meters long are quite attractive and deserve serious consideration. Since the effec tive pathlength is dependent on the refractive index of both the fiber and the liquid, accurate determination of absolute absorbance in hollow fiber cells is difficult, but peak detection is straightforward. Laser Intracavity Absorption. The last method for making the signal larger is the laser intracavity ap proach. This method is not yet widely used and is conceptually different from the others. Laser intracavity absorption en hancement has been known since at least 1967, with the invention of the dye laser. It is based on three proper ties of lasers, only two of which apply in most analytical situations. Lasers consist of a light-amplifying medium surrounded by mirrors (Figure 2). The usual case is to make one of the mir rors partially transmissive and the other a total reflector. This oscillator, so called because of the multiple round-trip path of the radiation, natu rally provides for multiple passes of the light through a sample placed in the optical train. In addition, the laser exhibits a threshold. This means that if more than a given loss, say 10%, is
Figure 2. Schematic configuration for quantitative intracavity absorption enhance ment measurements. Mi, M2 are mirrors incurred in one round trip, the device ceases to operate. The threshold arises from the finite gain of the amplifica tion medium, in our case the same 10%. The net result is that a precipi tous drop in output power results when the accumulated loss is in creased from 9.9% to 10.1%. Therefore, an incremental loss of 0.2% can gener ate a nearly 100% change in intensity, from a great deal to almost none. The third property of lasers is the competi tion effect that takes place when the absorber line width is narrower than the laser line width. Even the broadest spectrum lasers exhibit a bandwidth
