they do, I respond that our success was in polarimetry and not in circular dichroism. Although these two phenome na are easily confused, the instrumen tation is very different. Polarimetry re quires working at low light levels, whereas CD involves measuring the small difference between two large sig nals. It became clear to us that CD can be improved by high-frequency modu lation. A subtle aspect is that polariza tion is modulated between the left and right circular components, but every thing else is identical in the optical path. It is, in fact, a double-beam mea surement in a single-beam arrange ment. It took some time to find the correct laser and the correct driving electronics to do the experiment. Rob Synovec was able to suppress noise to one-half of a micro-absorbance unit (12). This provided CD measurements in LC with a detectability about 110 times better than that of commercial instruments. We attempted to extend high-frequency modulation to stan dard absorption measurements, but because we needed to use alternating beams in separate optical paths, true compensation was not possible. At the microabsorbance level, even a dust par ticle passing through one beam and not the other causes noise. The factor-oftwo improvement over conventional
in indirect fluorescence. Performance is therefore degraded. However, 104 is still quite an improvement over the 102 level inherent in the laser intensities. At that stage, detectability in indi rect fluorometry for anions was compa rable to, but not superior to, detectabil ity resulting from indirect photometry or conductivity. We knew that more work lay ahead of us. The next step had to involve chromatography. For a given dynamic reserve, detectability is pro portional to the concentration of the fluorophore in the eluent. In ion chro matography, this is the fluorescing eluting ion. Standard columns simply have too large a capacity to allow the use of eluent concentrations much be low 1 mM. We accepted the challenge and began to dynamically modify our own columns to achieve low capacities. Toyohide Takeuchi eventually found the right combinations. Eluent concen trations as low as 1 μΜ could be used. By using open tubular capillary col umns, the noise levels were equivalent to 1 pg of CI" or N0 3 ~ injected (14). Actually, we were able to improve the concentration detectability by only 1 order of magnitude. The smaller vol umes and our weak (8 mW) UV laser beam further degraded the dynamic re serve. Furthermore, the relatively large i.d. of our capillary column did not fa-
spectrometers does not justify all the complications introduced into the in strument. Still, we were able to partially over come the intensity fluctuations in laser beams in general. This immediately made some improvements in the polar imeter (4). It also brought new life to our pursuit of indirect detection meth ods. The challenge was to develop indi rect fluorescence. Perhaps one could transfer the advantages of fluorometry, such as low detectability and small vol umes, to nonfluorescing species as well. Sun-il Mho subsequently developed a double-beam fluorometer suitable for LC {13). Whereas in normal fluorome try we try to reduce the background as much as possible, indirect fluorometry depends on having a large background. The double-beam arrangement is es sential for good detection. The dynam ic reserve in fluorometry has only ap proached 104, whereas we were able to achieve 105 in absorption, 106 in CD, and 108 in polarimetry. This demon strates the subtle features of modula tion. The reference beam provides compensation only if the reference cell and the sample cell are exactly matched. Vibrations, heat-induced turbulence, photobleaching, light scat tering, and other phenomena are not identical in the two capillary flow cells
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240 Alnhd Drivv · P.O Box 11Γ>12 · Pittsburgh. ΡΛ 152:5S Î412) (,:} 7nM) · Tde.x S12 'ilh ΡΧΤΗΛΡΛΗ CIRCLE 42 ON READER SERVICE CARD ANALYTICAL CHEMISTRY, VOL. 60, NO. 7, APRIL 1, 1988 · 447 A
