Anal. Chem. 1086, 58, 1245-1248
The order of magnitude shift of the calibration curves may be due primarily to the well-recognized phenomenon of required antibody-antigen equivalency, which depends upon the initial concentration of both reactants (22). The precision of the method was determined with and without antibody. The blank assay was repeated 8 times over an 8-h period giving 5.1% relative standard 'deviation (RSD). Eight measurements of the antibody assay, containing 0.083 pg/mL anti-lidocaine, were taken over a 4-h period yielding a 4.9% RSD. Platinum is susceptible to poisoning by oxidation and contamination from the oxidation products of NADH. Therefore, the absolute values of the rates were checked for indications of a change in the Pt response with time. No trend of increasing or decreasing response was evident. As a further point of reference a standard HzOz solution was tested during the latter precision study. The HzOz determinations, 11 points, had a 4.1% RSD. The electrochemical determination of anti-lidocaine as discussed here provides an example for rapid, separation-free measurement of antibodies. The coefficient of determination of both regression lines (significant at 99% confidence level) gives merit to the described methods for high and low antibody concentrations. In its simplest form, without preincubation, the assay can be used as a qualitative procedure. With stricter adherence to possible interferences and appropriate care of the platinum probe, the technique offers quantitative measurement of antibodies over restricted concentration ranges at nanogram levels. Registry No. GGPD, 9001-40-5; lidocaine, 137-58-6.
1245
LITERATURE CITED (1) Langone, J. L.; Van Vunakis, H. Methods Enzymol. 1983, 92. (2) Schall, R. F.; Tenoso, H. J. Clin. Chem. (Winston-Salem, N . C . )1981, 27, 1157-1164. (3) Van Vunakis, H.; Langone, J. L. Methods Enzymol. 1980, 70. (4) Crowle, A. J. Adv. Clin. Chem. 1978, 20, 181-224. (5) Blake, C.; Gould, B. J. Analyst (London) 1984, 109, 533-547. (6) Monroe, D. Anal. Chem. 1984, 56, 921A-931A. (7) Rubenstein, K. E. Scand. J . Immunol. Suppl. 7 1978, 8 , 57-62. (8) Shiba, K.; Umezawa, Y.; Watanabe, T.; Ogawa, S.; Fujiwara, S. Anal. Chem. 1982, 52, 1610-1613. (9) D'Orazio, P.; Rechnltz, G. A. Anal. Chlm. Acta 1979, fO9, 25-31. (10) Soisky, R. L.; Rechnitz, G. A. Anal. Chim. Acta 1978, 99, 241-246. (11) Aizawa, M.; Suzuki, S.; Nagamura, Y.; Shinohara, R.; Ishlguro, I. Chem. Lett. 1977, 7 , 779-782. (12) Janata, J. J. Am. Chem. SOC. 1975, 97, 2914-2916. (13) Brontman, S. B.; Meyerhoff, M. E. Anal. Chim. Acta 1984, 162, 363-367. (14) Ngo, T. T.; Lenhoff, H. M. Appl. Blochem. Blotechnol. 1981, 6 , 53-64. (15) Finiey, P. R.; Williams, R. J.; Lichti, D. A. Clin. Chem. (Winston-&/em, N . C . ) 1980, 26, 1723-1726. (18) Wehmeyer, K. R.; Doyle, M. J.; Wright, D. S.; Eggers, H. M.; Halsali, H. B.; Heineman, W. R. J . Lip. Chromatogr. 1983, 6 , 2141-2156. (17) Eggers, H. M.; Haisail, H. B.; Heineman, W. R. Clin. Chem. (WinstonSalem, N . C . ) 1982, 28, 1848-1851. (18) Blaedel, W. J.; Jenkins, R. A. Anal. Chem. 1975, 47, 1337-1343. (19) Cunningham, J. A.; Underwood, A. L. Arch. Biochem. Biophys. 1966, 117, 88-92. (20) Singh, P. U S . Patent 4069 105, Jan 17, 1978 (Syva Co.). (21) Hohman, R. J.; Rhee, S. G.; Stadtman, E. R. R o c . Nafl. Acad. Scl. USA 1980, 77, 7410-7414. (22) Myrvik, Q. L.; Weiser, R. S. Fundamentals of Immunology, 2nd ed.; Lea and Febiger: Philadelphia, PA, 1984.
RECEIVED for review August 27,1985. Resubmitted December 23, 1985. Accepted January 23, 1986. We gratefully acknowledge the financial support of NIH Grant GM-25308.
CORRESPONDENCE ~~
~~
Quenched Peroxyoxalate Chemiluminescence as a New Detection Principle in Flow Injection Analysis and Liquid Chromatography Sir: Peroxyoxalate chemiluminescence is known as a method for the analysis of HzOz (1-4)and certain fluorophores (5-8). Thus far we have confined our attention to the development of a hydrogen peroxide monitor based on the use of solid bis(2,4,6-trichlorophenyl)oxalate (TCPO) and an immobilized fluorophore. Recently, we found that certain easily oxidizable compounds such as sulfite, nitrite, anilines, and organosulfur compounds quench peroxyoxalate chemiluminescence. This phenomenon is of importance for at least two reasons. First, it gives an insight in the role of possible interferences on analytical procedures using the peroxyoxalate reaction. Second, a prelimifiary survey shows that under favorable conditions (relatively high H202 concentration and immobilized fluorophore) the analytical potential of peroxyoxalate chemiluminescence can be extended to the analyses of quenchers. Equations 1-4 presumably apply for the reaction of TCPO, HzOz, and fluorophore (F) in the presence of a quencher (Q); TCP is 2,4,6-trichlorophenol. This mechanism, apart from the quenching step Za, was proposed by Rauhut et al. (9) and modified by McCapra (10) introducing the electron transfer steps 2 and 3. The type of mechanism, known as the chemically induced electron exchange (CIEEL) mechanism, has extensively been studied by Schuster et al. (11,I.Z). Nevertheless uncertainty still prevails not in the least due to the
I
-0
L
-0
-0
I
1
products
3
*
F
--+
* F
F+hu
4
F+heat
4a
difficulties in positively identifying the intermediate C204(13, 14). All these studies, however, indicate that electron transfer
0003-2700/86/0358-1245$01.50/00 1986 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
is the excitation step in the peroxyoxalate chemiluminescence mechanism. The position of the quencher Q in the reaction scheme is not known yet. If we for convenience assume, as has been done in the proposed scheme, that it only reacts with Cz04 in competition with step 2 then the influence of quenching can readily be formulated. From a steady state kinetics calculation the chemiluminescence in absence of quencher can be written as
Io = ~,*[Cz041[Fl (5) wherein h,* is equal to h2k4/(k4 + k4J and thus to k,$f if $f is the fluorescence quantum yield of the fluorophore. In the presence of a quencher the chemiluminescence intensity is reduced from Io to IQ,which is given by
Hence the signal reduction is
In the ratio mode a Stern-Volmer type expression is obtained
If the above mechanism applies, compounds that easily donate an electron are expected to be good quenchers. So in principle they might interfere with the H202determination. On the other hand they are likely to be detectable by quenched chemiluminescence. For a detection system based on quenching, IzZa/k2[F]should be as large as possible. This can be achieved by lowering the concentration of F or by the choice of a fluorophore that has a low kz. Of course k,[F] cannot be chosen too small because then Io will get very low and base line noise will be the major factor determining the detection limit. For the latter purpose the simple solid-state peroxyoxlate reactor developed previously ( 4 ) is very convenient. This apparatus makes use of a dual bed reactor packed with solid TCPO and immobilized aminofluoroanthene and requires only one pump. The presence of an immobilized fluorophore complicates the calculations on the above reaction system because the reaction will be heterogeneous so that a precise definition for [F] cannot be given. The most simple adaption will be the substitution of parameter k , for k,[F], which is expected to depend on the batch of immobilized fluorophore. EXPERIMENTAL SECTION TCPO was prepared according to Mohan and Turro (15),recrystallized twice from uvasol benzene, and dried under reduced pressure for 3 h. Acetonitrile was purchased from Baker and purified as described before (4). Tris buffer (Trizmabase,reagent grade, Sigma) was recrystallized twice from uvasol methanol (Merck). The pH of the buffer was adjusted with nitric acid. 3-Aminofluoranthene was immobilized with controlled pore glass beads (CPG-lo),77 A, pretreated with 5% nitric acid and dried. One gram of the CPG was placed under vacuum to remove air from the pores and treated with 3 g of glycidoxytrimethoxysilane either in aqueous medium (16), in dry toluene (17), or in water-saturated toluene. The resulting intermediate was then coupled with 3-aminofluoranthene as previously described ( 17). Hydrogen peroxide (30%)was obtained from Merck (Darmstadt, FRG). All analytes were analytical grade. Figure 1 shows a block diagram of the chemiluminescence detector. The light was measured with an ORIEL 7070 (Oriel Corp., Stamford, CT) photomultiplier detection system equipped with the solid-state chemiluminescence cell described in a previous paper (4). The cell was dry-packed with immobilized 3-aminofluoranthrene and TCPO in a ratio of 2:l. The obtained signal
Figure 1.
Block diagram of the chemiluminescence detector.
was inverted by the electronic inverter described by Donkerbroek et al. (18). For a good functioning of this inverter the maximum output of the Oriel 7070 was altered from 100 mV to 10 V full scale output. In the FIA and HPLC measurements the flow was delivered by a Kontron 410 HPLC pump (Kontron, Zurich, Switzerland) and a Rheodyne 7120 injection valve equipped with a 20-pL stainless steel injection loop. The chromatographic separation of the anilines was performed with a 150 X 3 mm stainless steel column packed with 5-pm spherical carbon particles type PCG, which was a gift from J. Knox (Edinburgh). The mobile phase for both the FIA and the HPLC experiments was 80:20 acetonitrile-Tris buffer (0.05 M, pH 8.0) in which M hydrogen peroxide was dissolved. In the FIA experiments the analyte was dissolved in mobile phase; in the HPLC experiments the analytes were dissolved in water.
RESULTS AND DISCUSSION The present study was performed with the H2O2monitor described previously (4), in which solid TCPO is packed in a two-layer bed together with 3-aminofluoranthene immobilized on glass beads. As 3-aminofluoranthene is one of the most efficient fluorophores in peroxyoxalate chemiluminescence, on the basis of the proposed mechanism it is not expected to be optimal for the quenched mode because the h,[F] term in eq 8 will be rather high, and thus kQ low. In this preliminary study other fluorophores were not tested. For the choice of the model compounds for the quenched mode we utilized results obtained in our laboratory for phosphorescence quenching of biacetyl in the liquid state (18, 19). Presumably many of them induce phosphorescence quenching via an electron transfer mechanism in which the andyte acts as a donor. Therefore we have chosen compounds such as anilines and sulfur compounds in addition to sulfite and nitrite ions, which may be present in rain- and cloudwater samples and so many interfere with the determination of hydrogen peroxide in such matrices. The mobile phase composition was simply chosen at the optimum of the HzOzdetermination. All measurements were performed in 80:20 acetonitrile-Tris buffer (pH 8). In this preliminary work the effect of changes in the mobile phase composition was not studied. On the basis of the kinetic model we discussed above, one would not expect an influence of the hydrogen peroxide concentration on k,. This was found to be true; for concentrations between and M of HzOz no variation of k~ was observed. The LOD for quenchers, however, does depend on the H,O, concentration, because the magnitude of Io influences the base line noise. At lo4 M peroxide the base line M apshows considerably more noise than a t lo-’ M; peared to be a convenient HzOzconcentration. Under these mobile phase conditions no corrosion was observed from parts of the system such as pump heads, valves, and unions. Furthermore the samples injected into the system appeared to be stable which was investigated by diluting the analyte in the mobile phase and following the response as a function of time. The consequences for the hydrogen peroxide determination with the solid-state peroxyoxalate system can readily be evaluated. Since the influence of quenchers does not vary with the hydrogen peroxide concentration, straight calibration lines will be observed for hydrogen peroxide, even in the presence
ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986 ~
Table I. FIA Detection Limits of Several Analytes compound
3
LOD for 2 0 - ~ Linjection, ng
NO;
2.8"
SO?-
6.4" 5.8" 1.1"
thiohydantoin thiourea ethenyl thiourea thioridazin sulforidazine methimazole aniline 3-ethylaniline 4-isopropylaniline 3.5-dimethylaniline N,N-dimethylaniline N,N-diethylaniline N,N-dipropylaniline N,N-dibenzylaniline benzylamine a-naphthylamine 2-toluidine 4-toluidine m-methyltoluidine
50t
1247
1.6O 7.70 9.B0
1.5; 0.4OC 18.0' 1.46
3.56 3.8' 3.76 3.S6
40.0' >400' >4OOb
45.0b 40.0b 47.0b 8.0b
OMeasured on batch I. bMeasured on batch 11. cMeasured on batch 111. of quenchers. Their slopes, however, will depend on the concentration of quencher. Consequently, it is necessary to apply standard addition procedures in order to circumvent systematic errors in the determination of hydrogen peroxide. Our explorative experiments show that the immobilization procedure of 3-aminofluoranthene on CPG requires an extensive and systematic investigation, Various experimental parameters probably affect the results, i.e., pretreatment of the support, reaction time of silanization, and solvent used for silanization (toluene, water). It is known that under dry conditions only monomer layers are obtained, whereas with water as a solvent polymeric layers are formed due to silanization of the silane. By means of defined addition of traces of water a controlled polymerization can be achieved resulting in a thin layer (20-22). Unfortunately, characterization of our batches appeared to be difficult. Elemental analysis provided nitrogen contents not higher than 0.2%; for these low amounts the accuracy is too bad to provide conclusive information. Another complicating factor is that in both chemiluminescence and fluorescence measurements the transparancy of the material may be dependent on the immobilization procedure. Several batches were tested by measuring the chemiluminescence in absence of quencher in relative units ( l o ) and determining kQ for the model compound 2-mercapto-lmethylimidazol (MMZ), see eq 8. Both Io and kQvaried about 1order of magnitude and furthermore their interrelationship appeared to be more complicated than suggested by the quenching model. In a forthcoming paper the immobilization process will be studied systematically and also the long-term stability of the generated batches will be investigated. Presently we only explore the response of the system for quenching analytes making use of three batches, with respective Io values of 32.0, 60.0, and 9.5 and respective k , values for MMZ of 5.5, 20.3, and 46.6 (in M-'). Of course for the system as a whole the stability is determined by the depletion of the TCPO reactor because during the chemiluminescence reaction the oxalate is consumed. A screening of analytes, i.e., the inorganic anions sulfite and nitrite, some anilines, and some organic sulfur compounds, was performed by means of flow injection analysis (FIA). The limits of detection (LOD) a t a signal to noise ratio of 3 are given in Table I. The relative standard deviation (RSD) of
2 4 6 8 -time
(rnin)
Flgure 2. Chromatogram of a mixture of anilines detected by quenched chemllurninescence: eluent, 80:20 acetonitrile/0.05 M Tris buffer pH 8, M of H,O,; column, 150 X 3 mm packed with 5-pm spherical carbon (type PCG); detector cell, packed one-third with solid TCPO and two-thirds with diluted batch IV (1:2);injected in 20 pL of water; flow rate, 0.6 mL min-I; vertical axis in relative units. Injected amounts: 75 ng of p-isopropylaniline,peak 1; 25 ng of N,Ndimethylaniline,peak 2; 50 ng of N-ethyl-rn-toluidine, peak 3; 75 ng of N,Ndipropylaniline, peak 4.
Table 11. HPLC Detection Limits for Some Anilines" compound
LOD, ng
4-isopropylaniline N,N-dimethylaniline N-ethyl-m-toluidine N,N-dipropylaniline
5.6 2.4 3.6 30.0
"Conditions as stated in Figure 2. these FIA measurements is very similar to that found in the hydrogen peroxide determination; the average RSD (n = 7) at 10 times the LOD was 2.5%. The linearity of the method, using an electronic signal inverter, is 2 to 3 orders of magnitude. This is in line with the range observed with phosphorescence quenching, in which a relationship similar to eq 8 applies (18, 19). The results are promising especially for those compounds difficult to detect sensitively with other means. Within this context we want to emphasize the possibility of improving the results if an optimal batch preparation procedure is available. For MMZ the LOD is decreased from 1.5 to 0.4 ng on going from batches I to I11 (see Table I). T o illustrate the possible use of the quenched chemiluminescence detector in liquid chromatography, in Figure 2 a chromatogram of a mixture of anilines is shown. A column was chosen so that the mobile phase composition could be set equal to those used in the FIA measurements. The lifetime of the reactor under the present conditions is about 3 h; after that period the TCPO layer in the cell becomes depleted. This implies that more than 20 of these chromatograms can be recorded before the TCPO reactor needs to be refilled. For 3-aminofluoranthene, batch I11 was chosen and the corresponding LC detection limits are given in Table 11. Preliminary experiments on the band broadening show a gZvof approximately 4000 pL2 due to the solid-state detector cell. A study on the effect of mobile phase composition on the quenching characteristics and a study on band broadening aspects in order to examine the compatibility of the solid-state reactor with HPLC are currently under way.
CONCLUSION From this explorative study two conclusions can be drawn: (1)Readily oxidizable compounds such as nitrite, sulfite, and certain organics such as anilines and organosulfur compounds influence the sensitivity of the TCPO chemiluminescence method for the determination of H202but do not disturb the linear relationship between the signal and the peroxide concentration. Hence for the analysis of real samples a standard
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Anal. Chem. 1986, 58, 1248-1251
addition procedure shodd be applied. (2) The reported results for a first screening of analytes strongly suggest that quenched peroxyoxalate chemiluminescence has potential as a detection method in HPLC. Presently we are systematically investigating the immobilization procedure for 3-aminofluoranthene on glass, and furthermore the potential of other fluorophores is explored.
ACKNOWLEDGMENT J. Knox (Edinburgh) is greatly acknowledged for the gift of the spherical carbon column material. Registry No. TCPO, 1165-91-9; NO;, 14797-65-0; SO-:, 14265-45-3;thiohydantoin, 5789-17-3; thiourea, 62-56-6; ethenyl thiourea, 1483-58-5;thioridazin, 50-52-2;sulforidazine,14759-06-9; methimazole, 60-56-0;aniline, 62-53-3;3-ethylaniline, 587-02-0; 4-isopropylaniline, 99-88-7; 3,5-dimethylaniline, 108-69-0;N,Ndimethylaniline, 121-69-7;N,N-diethylaniline, 91-66-7;N,N-dipropylaniline, 2217-07-4;N,N-dibenzylaniline, 91-73-6;benzylamine, 100-46-9; a-naphthylamine, 134-32-7;%toluidine,95-53-4; 4-toluidine, 106-49-0; m-methyltoluidine, 108-44-1;p-isopropylaniline, 99-88-7;N-ethyl-m-toluidine, 102-27-2; 3-aminofluoranthene, 2693-46-1.
LITERATURE CITED Williams, D. C.; Huff, G. F.; Seitz, W. R. Anal. Chem. 1976, 48, 1003-1 006. Scott, G.; Seitz, W. R.; Ambrose, W. R. Anal. chim. Acta 1 ~ 8 0 775, , 221-228. Van Zoonen, P.; Kamminga, D. A.; Gooijer, C.; Velthorst, N. H.; Frei, R. W. Anal. Chim. Acta 1985, 767, 249-257. Van Zoonen, P.; Kamminga, D. A.; Gooijer, C.; Velthorst, N. H.; Frei, R. W.; Giibltz, G. Anal. Chim. Acta 1985, 774, 151-161. Kobayashl, S . ; Imakl, K. Anal. Chem. 1980, 52,424-435. Sigvardson, K. W.; Blrks, J. W. Anal. Chem. 1983, 55, 432. Weinberger, R. J . Chromatogr. 1984, 314, 155-165. Grayeski, M. L.; Weber, A. J. Anal. Lett. 1984, 77, 1539-1552. Rauhut, M. M.; Bollyky, L. J.; Roberts, B. G.; Loy, M.; Whiteman, R. H.; Ianotta, A. V.; Semsel, A. M.; Clarke, R. A. J . Am. Chem. SOC. 1967, 89, 6514-6516.
(10) McCapra, F. Prog. Org. Chem. 1973, 8 , 231. (11) Schuster, G. B. Acc. Chem. Res. 1979, 72, 366-373. (12) Schuster, G. B.; Horn, K. A. I n "Chemical and Biological Generation of Excited States"; Adam, W., Cilento, G , Eds.; Academic Press, New York, 1982, Chapter 7. (13) Catherall, C. L. R.; Frank Palmer, T.; Cendall, R. B. J . Chem. Soc., Faraday Trans. 2 1984, 80 823-837. (14) Catherall, C. L. R.; Frank Palmer, T.; Cendall, R. B. J . Chem. SOC. Faraday Trans. 2 1984, 80, 637-849. (15) Mohan, A. G.; Turro, N. J. J . Chem. Educ. 1974, 57,528-529. (16) Chang, S. H.; Gooding, U. M.; Regnier, F. E. Chromatogr. 1976, 720, 321-333. (17) Giibitt, G.; Van Zoonen, P.; Gooijer, C.; Velthorst, N. H.; Frei, R. W. Anal. Chem. 1985, 5 7 , 2071-2074. (18) Donkerbroek, J. J.; Veltkamp, A. C.; Gooijer, C.; Velthorst, N. H.; Frei, R. W. Anal. Chem. 1983, 55, 1886-1893. (19) Gooijer, C.; Velthorst, N. H.; Frei, R. W. TrAC, Trends Anal. Chem. (Pers. Ed.) 1984, 3 , (IO),259-265. (20) Hustings, C. R.; Aue, W. A.; Augl, J. M. J . Chromatogr. 1970, 53, 487-506. (21) Herman, P. P.; Field, C. R.; Abbot, S. J . Chromatogr. Sci. 1961, 79, 470-478. (22) Majors, R . E.; Hopper, M. J. J . Chromatogr. Sci. 1974, 72, 767-778
Piet van Zoonen Dik A. Kamminga Cees Gooijer* Ne1 H. Velthorst Roland W. Frei Department of General and Analytical Chemistry F~~~university De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands Gerald Giibitz Department of Pharmaceutical Chemistry University of Graz Universitatzplatz 10, A-8010, Graz, Austria
RECEIVED for review October 4,1985. Accepted January 21, This work was by Dutch Foundation Of Technical Sciences under Grant ll-20-46/79-O,VCh 11.0137.
Thermal Desorption Modulator for Capillary Liquid Chromatography Sir: Extremely narrow bore columns have significant advantages in liquid chromatography. The use of substantially smaller quantities of stationary phase packing and mobile phase solvent reduces costs and allows the use of exotic materials for better performance (1, 2 ) . Higher efficiency is obtained, which improves resolution, sensitivity, and limits of detection. Despite these advantages, microcolumns are not widely used. Injector and detector volumes must be reduced in proportion to the reduction in mobile phase volumetric flow rate. It is quite possible to reduce the injection volume, but unless the sample pretreatment volumes are also reduced, much of the analyte will be lost during sample preparation. It is also possible to reduce the detector volume, but without very precise and expensive instrument design, detector sensitivity will be Iost. The loss of sample at the injector and loss of sensitivity at the detector result in higher than necessary limits of detection at the bottom end of the calibration curve. The upper end of the calibration curve is limited by column sample capacity, which is proportional to the square of the column diameter. For a sufficiently small column diameter, the calibration curve may not exist as the column sample capacity limit becomes less than the limit of detection. The best way to keep injector and detector volumes in proper proportion to the column is to build them into the 0003-2700/86/0356-1248$0 1.50/0
column itself. On-column detectors are an example of this approach (3, 4). As the column diameter is reduced, the detector volume is automatically reduced proportionately. Injection valves, however, are mechanical devices and cannot be conveniently built into the head of a column. The purpose of an injection valve is not just sample introduction. The primary purpose is imposition of a modulation signal on the sample so that chemical identity information may be encoded in the chemical signal passing through the column (5-7). Divorcing modulation from sample introduction allows the application of many more devices as chromatographic column modulators. Some of these modulators are nonmechanical or chemical devices, which can easily be built into the head of a column. Since the sample stream continuously flows through a chemical modulator, the modulation signal form is not limited to a single pulse but may consist of a long sequence of pulses. Such long modulation signals have much greater sample throughput but result in a multiplexed detector output signal and require complex computation to recover the chromatogram ( 5 ) . A thermal desorption modulator has been described for use with open tubular fused silica capillary columns (5). The head of the column is rapidly heated and cooled to generate a derivative form chemical signal as the sample flowing into the column is alternately driven out of and then readsorbed into 0 1986 American Chemical Society
