- Email: [email protected]
Photochemical reaction detection in HPLC
trends
in
ana&ical
chemistry,
vol.
I,
no.
361
15, 1982
However, in the future IDMS will play an important role in calibrating other analytical methods as well as for the certification of standard reference materials.
References 1 de BiZvre, P. (1978) Adv. Mass Spectrom. 7, 395 2 Heumann, K. G. (1980) Toxicol. Environ. Chem. Rev. 3, 111 3 Turnbull, A. H. (1963) S urf ace Zonisation Techniques in Mass Spectrometsy: AERE Report 4295, Harwell 4 Heumann, K. G., Baier, K., Beer, F. and Schindlmeier, W. (1980) Adv. Mass Spectrom. 8, 3 18 5 Jochum, K. P., Seufert, M. and Best, S. (1981) Z. Anal. CIem.
309, 308 6 Dalrymple, G. B. and Lanphere, M. A. (1969) Potassium-Argon Dating, Freeman and Co., San Francisco
Photochemical
reaction
7 Reamer, D. C. and Veillon, C. (1981) Anal. Chem. 53, 2166 8 Schulten, H. R. (1979) Int. J. Mass Spectrom. Ion Phys. 32, 240 9 Smith, D. H., Christie, W. H. and Eby, R. E. (1980)Znt. J. Mass
Spectrom. Ion Phys. 36, 301 10 Shields, W. R., Murphy, T. J., Garner, E. L. and Dibeler, V. H. (1962) J. Am. Chem. SOL 84, 1519 11 Yergey, A. L., Vieira, N. E. and Hansen, J. W. (1980) Anal.
Chen. 52, 1811 12 Houk, R. S., Fassel, A. L. and Taylor,
V. A., Flesch, G. D., Svec, H. J., C. E. (1980) Anal. Chem. 52, 2283
Gray,
Professor Heumann received his Ph.D. from the Technical University of Dawnstadt in 1969. Since 1974 he has been Professor at the Institute of Inorganic &&try, University of Regensburg, Universitilstrasse 31,840O Regensburg, F.R. G.
detection’
in HPLC
The versatility of post-column reaction detection in chromatogra hy is greatly enhanced by exploiting the principles otp organic photochemistry. Improvements in photochemical reactor design will result in further advantages. John W. Birks University of Colorado, Boulder, CO, U.S.A.
Roland W. Frei Free University, Amsterdam, The Netherlands Post-column derivatization in high performance liquid chromatography (HPLC) has become a popular means of increasing the sensitivity and selectivity of detection for trace components of complex matrices. The technique has many advantages over pre-column derivatization, e.g. the reaction need not be complete or well defined so long as it is reproducible, and the analysis time is considerably reduced as a result of the elimination of additional sample handling steps. Many successful applications of post-column derivatization have been described in recent reviews’s*. Photochemical reactions may be employed as a special type of post-column derivatization, and dramatic improvements in both sensitivity and selectivity have been demonstrated here as well. In the simplest case, photons are the only reagent added, and many examples of improved detection - particularly in the pharmaceutical area - have been demonstrated. For example, tamoxifen and clomiphene are stilbene derivatives (I), which upon irradiation with UV light are transformed into the corresponding phenanthrenes (II): -
The increased rigidity and aromaticity of the phenanthrene molecule results in a much enhanced absorption coefficient and a higher fluorescence quantum yield. Selectivity for such analyses is very high since any interfering species must (1) co-elute with the analyte, (2) absorb light from the lamp, and (3) undergo a reaction to produce a fluorescent compound. Biological tissues and fluids have been found to be particularly free of interferences. Post-column photochemical derivatization should be applicable to the determination ofnearly all analytes for which batch photochemical methods have already been developed. In addition, the use of photochemical reagents greatly increases the applicability of the technique. For example, the use of a sensitizer to absorb light and initiate a photochemical reaction allows the detection of compounds such as aliphatic alcohols, ethers, aldehydes and saccharides - compounds that have only weak chromophores or none at al13*4. Post-column photochemical reaction detection may be seen as part of an expanding interest in the application of well-known principles of organic photochemistry to HPLC detection. Other recent developments include applications of chemiluminessourcesg’lo, and energy cence5-8, chemical excitation transfer phosphorescence” to achieve improved sensitivities and a wide variety of selectivities.
Photochemical
0 165~9936/82/oooO~/SOl.~
reactor design
The optimal photochemical reactor has probably not yet been constructed. Of the few designs that have appeared in the literature, most incorporate a medium or high pressure arc lamp (Hg, Xe or Xe-Hg)12-14, although low pressure mercury lamps15 and fluorescent black lights3*4 have also been used. The spectral output @ 1982 Elsevier Scientific Publishing Company
trends in analytical chemistry, vol. I, no. 15< 1982
362 of the lamp should, of course, overlap with the absorption spectrum of the analyte or sensitizer. The high intensity of a Xe-Hg arc lamp over the entire UV-visible region makes it a generally useful excitation source. Optical filters can, in principle, be used to increase the selectivity of the photochemical reaction, although this has yet to be demonstrated. A disadvantage of arc lamps is that they produce a great deal of heat and infrared radiation, and thus cooling of both the lamp and reaction coil are required. High temperatures within the reaction coil can result in undesirable side reactions, including polymerization. Furthermore, temperature fluctuations adversely affect the reproducibility. The housing of an arc lamp must be resistant to the possible explosion of the lamp, and the toxic ozone gas produced by the photolysis of oxygen must be vented. The design of a fourth generation photochemical reactor that incorporates all of these features is shown in Fig. 1 16. The reactor makes use of a polytetrafluoroethylene (PTFE) reaction coil. PTFE has been found to transmit light by an internal reflection mechanism in which light passes through the pores of the polymer. Some reactor designs have utilized quartz capillaries. However, these are both expensive and fragile. They are not readily available in different geometries (coil and helix diameter, length), and tight connections are difficult to make. The surprisingly high transmission of light in the 200-300 nm spectral region by PTFE makes this material nearly ideal for photochemical reactors, especially since the tubing is inexpensive and readily available in a range of internal diameters. Also, it has been shown that the use of PTFE coils results in chromatographic peaks having better symmetry and less tailing than for quartz capillaries13. The only disadvantage of PTFE reaction coils is that the material is permeable to oxygen, so that the sensitivity of detection may be reduced for certain analytes if the reaction coil is exposed to air. In the reactor design of Fig. 1, the PTFE coil is immersed in a circulating water bath which serves both to regulate the temperature and to exclude oxygen. Also, metal salts may be added to the water bath to serve as an optical filter.
Fig. 1. Photochemical reactor. 1, fan; 2, outlet for circulating liquid; 3, capillary; 4,filter compartment; 5, additional cooling via pressurized air; 6, quartz cylinders; 7, liquid inlet. The light source is a 200 W Hg-Xe lamp operated at a current of IOA. The PTFE reaction coil is cooled by a continuous flow of water through thejacketsformed by two quartz and onepyrex cylinders.
for (1) small capillary lengths, (2) small internal diameters and (3) low flow rates. For a fixed reactor volume (m*L) and flow rate (and thus reaction time), the variance is reduced by using longer capillaries of smaller diameter. For this reason, it has been pointed out that for reaction times of up to 20 min, small tr
I”5 ec
t
90
80
x=01)
70
60
50
Band broadening considerations Any post-column reactor will contribute at least a small amount to the dispersion of the chromatographic peaks and thus reduce the resolution of the chromatography. The contribution to the variance for a coiled tubular reactor is given by: (TV 2=K-
7Tr4L 4. 24 Dm
Here r is the internal radius of the capillary, L is the capillary length, 4 is the liquid flow-rate, Dm is the molecular diffusion coefficient for the analyte in the solvent, and K is a proportionality factor which depends on the flow profile in the capillary. It is a function ofthe dimensionless Dean and Schmidt numbers. From equation (1) we see that band broadening is minimized
40
3c
2c
10
Fig. 2. Plots of reaction time, t, v. radius of the reaction capillary, r, that satisfy equation (2) for different K values. Flow segmentation is advantageous when the point (r,t) lies above the appropriate curve.
363
trends in analytical chemistry, vol. 1, no. 1.5, 1982
0
5
7
0
5
h
7
tlmo(mbn
0
5
7
1
Fig. 3. Chromatograms of clobazam (c) and desmethylclobazam (d) after photochemical reaction for non-segmented, liquid segmented and air segmented systems. Capillary, PTFE of 0.5 mm I.D.; reaction time, 19 s.
diameter (r = 20 pm) coiled tubing could be used as the post-column reactor with minimal dispersion17. However, for reasons of convenience (back pressure, clogging, material cost, etc.) most workers prefer the use of larger diameter, = 0.2-2.0 mm I.D. tubing. Coiling the capillary also reduces band broadening by breaking up the parabolic velocity profile and thus reducing the value of K in equation (1). The use of a capillary knitted from PTFE tubing has been used in one photochemical reactor design to minimize peak broadening15. Reaction coils up to 20 m in length allowed reaction times ofup to 3.75 min in the detection of clobazam and fenbendazole and their major metabolites in man. We have found that 12 m of 0.3 mm I.D. PTFE tubing may be knitted such that the dispersion is the same as for one meter of loosely coiled tubing of the same diameter. A very effective means ofcurtailing band broadening
in post-column reactors is flow segmentation, a method that has long been used in clinical analyzers. In this technique the HPLC eflluent is divided into small segments by introducing air (or inert gas) bubbles or segments of an immiscible liquid. After sufficient reaction time the two phases are separated with only a small loss (typically IO-20%) of the analytical phase. Since nearly all of the band broadening in a segmented reactor results from the phase separator, the variance is nearly independent of reaction time, making very long reaction times (many minutes) possible. A theoretical expression comparing band broadening for segmented and non-segmented reactors has recently been derived”. For small injection volumes and negligible contribution to the band broadening by connective tubing, mixing tees and the detector, it has been found that flow segmentation is advantageous when t,r* > y
0 I*poxy-quinon~l
ho,02
ph.sep.
Values for the variance of the phase separator, a,‘, ph.sep., typically fall in the range 70-250 pL* with an average value of approximately 150 pL*. Plots of reaction time v. capillary radius that satisfy equation (2) are shown in Fig. 2. For typical numerical values, namely Dm = 0.5-2.5. lop3 mm* s-l, K = 0.25 and 4 = lo-20 PL s-l, we have ta’ > 0.03-0.6 mm’s. Thus, for capillary radii of 0.15 mm, 0.25 mm and 0.40 mm, segmentation is advantageous when the reaction times exceed 1.3-26 s, 0.5-10 s and 0.24 s, respectively. As an example, Fig. 3 shows chromatograms of the drug clobazam and its major metabolite, desmethylclobazam, for non-segmented detection and
FLUORESCENT
?’
a,*,
POLVMERISI
Iepoxy-hydroquinonel
I
ho
.
&9x I
0
trioriran* K, -CHROHENOL !compound
I&ar~2R5) II ?)
o-quinom
4
methidelAmOx=475nml R
OH-
H’ II 0-
Kl$
llVDROQUlNONE Icompound
Fig. 4. Proposed photochemical
17)
reactions of Vitamin KL
rmi-qulnme
IX,,,=
60Onml
hydropcroxide
eon R
trends in analytical chemistry, uoi. I, no. 15, 1982
364 for both solvent (hexane) and air segmentation using a 0.5 mm I.D. PTFE capillary (tr = 19 s) as the photochemical reaction coil’* It is evident that the peak heights for the non-segmented system are distinctly smaller than are those in either of the segmented systems, and the resolution for the non-segmented system is much poorer.
Applications Post-column photochemical reaction detection is still in its infancy as an analytical technique. The first application of a post-column photochemical reactor was published in 1976 by Iwaoka and Tannenbaum”. They selectively detected N-nitroso compounds by photohydrolysis to produce nitrite, which was then determined colorrmetrically using the Griess reaction. The technique allowed the determination of nonvolatile nitrosamines in food products in the lo-100 rig/g range. A similar idea was pursued by Hall and Rodgers, who developed the photoconductivity detector for HPLC, now marketed by Tracer In this detector the photolytic decomInstruments*‘. position of halogen and many nitrogen and sulfur compounds results in ionic products which are subsequently detected in a conductivity cell using the bipolar pulse measurement technique. Detection limits are in the low picogram range for many compounds.
+/+
/
The use of an on-line photochemical reaction to form fluorescent or UV-absorbing products was first demonstrated by Twitchett et al. in 1978*l. They determined cannabinol in urine by converting it into a highly fluorescent derivative upon irradiation. The detection limit was about 0.5 ng. By injecting a second sample, but with the lamp off, any background due to naturally fluorescing compounds could be determined. For the forensic drug lysergic acid diethylamide (LSD), they demonstrated the opposite use of UV irradiation This naturally fluorescent to achieve selectivity. compound is first chromatographed and detected with the UV lamp off. Upon irradiation of 320 nm, the compound is converted into the non-fluorescent derivative, and thus a repeat injection with the lamp on allows the drug to be distinguished from other fluorescent compounds with similar chromatographic properties. The post-column photochemical derivatization of a number of pharmaceuticals has now been reported, and these studies are summarized in Table I. A recent study in which Vitamin K homologues were determined illustrates a number of important considerations in photochemical reaction detection16. To begin with, the photochemistry ofcomplex molecules is seldom simple. This is illustrated by Fig. 4, which summarizes the literature on the photochemistry of Vitamin Kl solutions. The reaction products are highly dependent on solvent composition, the presence or absence of oxygen, pH and irradiation time. The Vitamin K homologues do not exhibit native fluorescence; however, in batch photolysis experiments it was found that at least three fluorescent products were formed. It was also found that in the presence of small concentrations of ascorbic acid the only isolable fluorescent product is the hydroquinone KHZ. The ascorbic acid acts as a chemical reductant and protects the KHZ from further oxidative decomposition. Because the hydroquinone is highly fluorescent and can be reproducibly formed in good yield, its optimum excitation and emission wavelengths were chosen for the analytical procedure. Purging the solvent system with nitrogen improved the sensitivity by up to 50%, as would be expected, considering the various reactions with 02 shown in Fig. 4. A general observation in photochemical reaction detection is that there exists an optimal residence time in the photochemical reactor. This is a result of photochemical degradation of the desired reaction product (e.g. the photochemical reaction ofKHn shown in Fig. 4). This optimum may be found by making plug injections of the analyte (no column) and measuring the signal as a function of flow rate. As is shown in Fig. 5, the optimal reaction time is highly dependent on the chosen reaction conditions. For Vitamin Kl the long reaction time required to obtain the optimal signal obviated the use of a segmented reactor. Fig. 6 compares the chromatograms obtained by air-segmented photochemical reaction and fluorescence detection with non-segmented UV absorption
+\ r;------r*\
Fig. 5. Reaction kinetics for the formation of KHzfrom vitamin K I showing different optimal reaction times for different reaction conditions. 0-0 in CHSOH + ascorbic acid (air cooled reactor); x-x in CHIOH + ascorbic acid + pH 3.8 buffer (air cooled reactor); +-•+ in CH30H + ascorbic acid + pH 3.8 buffer (water cooled reactor); @-O n CHDOH + ascorbic acid + pH 3.8 buffer (water cooled reactor and air segmented Jlow system).
trends in analytical chemistry, vol. 1, no. 15, 1982
TABLE
Analyte
365
I. Photochemical
Photochemical reaction parameters
Detection limit
Clobazam
200 W high pressure Xe-Hg, 3.8 m PTFE (0.3 mm I.D.) 15 s
20 Pg
Clobazam
15 W low pressure Hg, 20 m knitted PTFE (0.5 mm I.D.), 54 s
130 Pg
reaction
detection
methods
for pharmaceuticals
Linearity demonstrated
Chromatographic conditions
2.5-90
ng/ml
30 pg/ml
0.7-l
Ref.
Sample
matrix
and other notes
Column: 15 cm X 4.6 mm, 5-pm LC-18 (Supelco) Eluent: methanol0.01 M sodium acetate (6:4) -
13,22
A detection limit of 50 ng was obtained for the metabolite desmethylclobazam, and the detection of these species in serum is also demonstrated.
Column: 10 cm X 4.6 mm LiChrosorb-RP8, 10 /*m (Brownlee Labs)
15
Desmethylclobazam and two other metabolites are also separated and detected in pure solutions.
Column: 25 cm X 4.6 mm Zorbax silical, 6 pm (Du Pont)
14
An ether extraction allows the determination at levels below 1 ng/ml of plasma. Plasma levels were measured for a group of patients undergoing clomiphene therapy.
Column: 15 cm X 4.6 mm, 5 pm LC-18 Supelcosil (Supelco)
23
Chromatogram of human serum spiked with 8 ppm of demoxepam.
24
A simple ether extraction of urine plasma and serum allows the detection of DES at low ppb levels.
15
Chromatograms of serum extracts spiked with fenbendazole and its three principal metabolites are also demonstrated at levels down to 10 ng/ml.
Eluent: 0.1 M tetraethylammonium phosphate buffer pH 7.0methanol (16:13) Clomiphene
Medium pressure Hg, 3 m PTFE (0.3 mm I.D.) 10 s
Demoxepam
200 W high pressure Xe-Hg, 3.8 m PTFE (1.1 mm I.D.), air segmentation, 110 s
60 pg
l-45
ng/ml
lo-10,000
100Pg
ng/ml
Eluent: MeOH-O.1 phosphate buffer
M
(PH 8) (3:2) Diethylstilbestrol (DES)
100 W medium pressure Hg, 0.7 m fused silica (0.25 mm I.D.) 2 s
-
0.2 ng
2.5-10
ng
Column: 25 cm x 4.5 mm RP8 (Perkin-Elmer) Eluent: CH&Nwater (l:l), pH 3.5
Fenbendazole
15 W low pressure Hg, 20 m knitted PTFE (0.5 mm I.D.) 78 s
1.2 ng
0.7-70
pg/ml
Column: 12 cm x 4.6 mm LiChrosorb RP8, 7 pm (Knauer) Eluent:
0.033
M
HsPOd-methanol Phenothiazines: mesoridazine sulphoridazine thioridazine
200 W high pressure Xe-Hg, 7.6 m PTFE (0.3 mm I.D.) 25 s
0.5 ng
0.15-l
1.5
pg/ml
(1 :l )
Column: 10 cm X 4.6 mm, 5 pm LiChrosorb RP8 (Merck)
13
Chromatogram of extract of serum spiked to 2.3 ppm with mesoridazine and 6 ppm of thioridazine. The fluorescence signal enhancement factors after irradiation were 8-10 for thioridazine and 2-3 for mesoridazine and sulphoridazine
23
Detection limit of 10 ng/ml for phenothiazines extracted for serum. Irradiation enhanced the fluorescence sensitivity for four of the phenothiazines by about a factor of five, but had no effect on thiodiphenylamine.
Eluent: CHsOH7.6 M sodium acetate (4:1), 0.01% ammonium peroxodisulphate Phenothiazines: fenergan largactil levopromazine nedaltran thiodiphenylamine
200 W high pressure Xe-Hg, 7.6 m PTFE (0.3 mm I.D.) 35 s
40-100
pg 2-100
ng/ml
Column: 10 cm x 4.6 mm, Polygosil 60-10 CN (Marcherey, Nagel and Co.), Eluent: CH30H sodium
-2.6 M acetate (4:l)
trends in analytical chemistry, vol. I, no. M, 1982
366 detection for a 400 pg sample of Kl. The improvement in sensitivity by about a factor of four may seem small in light of the effort required. However, the selectivity for the photoreduction method allows the detection of normal levels of Kl in serum by a simple extraction followed by reversed phase chromatography and without an intermediate clean-up step. All of the applications described in Table I require absorb light tq initiate the that the analyte A photochemical reduction photochemical reaction. similar to that described above for Vitamin Kl has recently been used to detect a variety of compounds that have only very weak chromophores in the 0 I
1 I
2 I
i
3 I
L r
min.
5 r
UV-visible region, or which have none at a114. In this detection scheme the compound anthraquinone-2,6is photoreduced to the hydrodisulfonate (AQDS) quinone in the presence of analytes having C-H bond strengths less than about 94 kcal/mol. The mechanism of photoreduction of isopropanol is shown in Fig. 7. For this reaction scheme the analyte need not absorb light. the AQDS reagent absorbs light and Rather, undergoes rapid intersystem crossing to the triplet state. This triplet excited species can abstract the o-hydrogen to produce a semiquinone radical (QH*) and the (CH3)&OH* radical. Subsequent reactions lead to the fully reduced and highly fluorescent hydroquinone, QH2. Using a fluorescent black light and a reaction time of 26 s, detection limits in the range 5-30 ng were achieved for a variety of aliphatic alcohols, aldehydes, ethers and saccharides. As it this post-column photochemical reaction stands, technique is more than an order of magnitude more sensitive than a conventional refractive index detector, while providing the benefit of considerable selectivity.
Future trends
7”” 2C*nm
I
0
2
L
P
FLUOR. 320/420
nm
,..,,
6
8
10
12
1L min.
Fig. 6. Chromatograms of 4OOpg of Vitamin Klfor direct UV detection (248 nm) and post-column photochemical reduction followed by Jluorescence (320 ex. I420 em.) detection of the KHz product.
Many improvements in photochemical reactor design are possible. As mentioned earlier, the selectivity for a particular photochemical reaction could be enhanced by using a monochromatic light source. Provisions for varying the intensity of the source would allow an additional degree of freedom in the signal optimization. Because the instantaneous concentrations of short-lived species such as radicals and electronically excited molecules depend on the light intensity, the branching ratios to various products may also be affected by source intensity. Thus, depending on the kinetics of the particular reaction system, the optimum product yield may occur either for a high intensity source and short reaction time or for a low intensity source and long reaction time. In the latter case, further exploitation of the segmentation principle in order to minimize band broadening would be called for. It is even possible that modulation of the source, allowing time for dark reactions to occur in the absence of the highly reactive short-lived species, would increase the yield of desired products in some cases. The ability to regulate the temperature within the reaction cell over a wide range would provide further control over the photochemical reactions. It is also desirable to miniaturize photochemical reactors, making use of smaller point sources with more efficient coupling of the radiant energy to the reaction cell. Simple photochemical reactors tailored for specific reactions could also be developed. For example, a fluorescent black light may be used as effectively for some reactions as a high pressure Xe-Hg lamp, with the advantage of great savings in cost and reduction in complexity. It seems likely that many more analytes will be found whose detection sensitivities are enhanced by a direct photochemical reaction. However, we expect that the major progress in photochemical reaction detection
trenk ia analyticalchemistry.vol. 1. no. 15, 1982
AQ + hu+AQ*
(singlet, S)
AQ*( S 1 -
AQ* (triplet,T)
AQ*(T 1 +
KH,),CHOH
KH,),
FOH + AQ -
2AQH*-
367
-
AQH* + KH,),
AQH* + (CH&
GOH
C=O
AQ + AQH,
Fig. 7. Mechanism for the photoreduction of anthraquinone by isopropanol. AQ is anthraquinone; AQH* is the semiquinone radical; AQHz is 9,10-dihydroxyanthracene.
will result from the application of new photochemical reagents and the coupling of photochemical reactors to other detection methods, such as electrochemical and chemiluminescence detection. We have described above the photoreduction of AQDS as a means of detecting ‘hydrogen-donating’ compounds. In an alternate detection scheme for such compounds, AQDS acts as a sensitizer in a photo-oxygenatiot?. The reaction mechanism for ethanol is shown in Fig. 8. Rather than exclude oxygen, as is necessary for the photoreduction, the HPLC mobile phase is saturated with oxygen by continuously bubbling 02 through the solvent reservoir. Hydrogen peroxide is a major product of the reaction and may be detected by its chemiluminescent reaction with luminol. An important difference between the photo-oxygenation (Fig. 8) and photoreduction (Fig. 7) mechanisms is that in the photo-oxygenation the AQDS is not consumed, but rather, acts as a catalyst. This suggests what is potentially a very sensitive method of detecting analytes that, like AQDS, can sensitize the photo-oxygenation of an alcohol. Using an alcohol/water mobile phase saturated with 02, it should be possible to detect even very weak sensitizers at low concentrations, since the reaction of the triplet-excited analyte with the alcohol would be quite favourable in comparison to the Tl + So deactivation and because long reaction times in the photochemical reactor would allow very high many carbonyl quantum yields of Hz0 2. Certainly compounds could be detected in this way and probably many nitrogen compounds as well. Q+
hu%%*
%* + CH,CH,OH
-
CH CHOH y’-,i.zi
02
3l
00.
02
t Q+HOO. 2 CH$OOH
+ H202
CH$HO
+ 02 +
v2
CH,CHO
+
Y
HO0 .
HOO. J
HO0 . 1 HOO.
Q+HOO.
“,2
02 +
H202
H202
02
+
H202
Fig. 8. Mechanism for the sensitizeddhoto-oxygenation of ethanol in neutral aqueous solution. Q is a sensitizer such as AQDS.
In some cases additional selectivity might be obtained by coupling a photochemical reactor to an appropriate electrochemical detector. For example, the photo-oxidation products of some compounds could be detected by subsequent reduction in a polarographic detector. Phenothiazines comprise one class of pharmaceuticals that might be more selectively detected in biological matrices using this approach.
Acknowledgement J. W. B. thanks the Nederlandse Organisatie voor Zuiver-Wetenschappelijk Onderzoek (Z.W.O.) for a visitor’s grant. This work was partially supported by a grant from the National Science Foundation (CHE-7915801).
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Frei, R. W. (1982) Chromatographia 15, 161 Stewart, J. T. (1982) Tremis Anal. Chem. 1, 170 Gandelman, M. S. and Birks, J. W. (1982)J. Chromatogr. 242,21 Gandelman, M. S. and Birks, J. W. (submitted for publication) Neat-y, M. P., Seitz, W. R. and Hercules, D. M. (1974) Anal. Lett. 7, 583 Birks, J. W. and Kuge, M. C. (1980) Anal. Chem. 52, 897 Shoemaker, B. and Birks, J. W. (1981)J. Chromatogr. 209, 251 Veazey, R. L. and Nieman, T. A. (198O)J. Chromatogr. 200, 153 Kobayashi, S. and Imai, K. (1980) Anal. Chem. 52, 424 Kobayashi, S., Sekino, J., Honda, K. and Imai, K. (1981) Anal. Biochem. 112, 99 Donkerbroek, J. J., van Eikema Hommes, N. J. R., Gooijer, C., Velthorst, N. H. and Frei, R. W. (1982) Chromatographz’a 15,218 Twitchett, P. J., Williams, P. L. and Moffat, A. C. (1978)J. Chromatogr. 149, 683 Scholten, A. H. M. T., Welling, P. L. M., Brinkman, U. A. Th. and Frei, R. W. (198O)J. Chromatogr. 199, 239 Harman, P. J., Blackman, G. L. and Phillipou, G. (1981) J. Chromatogr. 225, 13 1 Uihlein, M. and Schwab, E. (1982) Chromatographia 15, 140 Lefevere, M. F., Frei, R. W., Scholten, A. H. M. T. and Brinkman, U. A. Th. (in press)
17 Tijssen, R. (1980) Anal. Chim. Actu 114, 71 18 Scholten, A. H. M. T., Brinkman, U. A. Th. and Frei, R. W. (1981) J. Chromatogr. 205, 229 19 Iwaoka, W. and Tannenbaum, S. R. (1976) IARCSci. Publ. 14, D. J. Dixon J. B. and Ehrlich, B. J. (1979) J. 20 ibpvich, Chromatogr. Sci. ‘17, 643 ’ P. J., Williams, P. L. and Moffat, A. C. (1978) J. 21 Twitchett, Chromatogr. 149, 683 22 Scholten, A. H. M. T., Brinkman, U. A. Th. and Frei, R. W. (1980) Anal. Chim. Acta 114, 137 23 Brinkman, U. A. Th., Welling, P. L. M., de Vries, G., Scholten, A. H. M. T. and Frei, R. W. (1981) J. Chromatogr. 217, 463 24 Rhys Williams, A. T., Winfield, S. A. and Belloli, R. C. (1982) J. Chromatogr. 235, 461 John Birks earned his Ph.D. at the University of California at Berkeley in 1974. Following three_years as Assistant Professor in the Department of Chemistry of the University of Illinois at Urbana-Champaign, he joined the faculty at the University of Colorado, Boulder, CO 80309, U.S.A. where he is Associate Professor of Chemistry and Fellow of the Cooperative Institute for Research in Environmental Sciences (CIRES). Thispastyear he has been on sabbatical leave at the Max Planck Institute for Chemistry in Main<, West Germany and the Free University, Amsterdam, The Netherlands. Roland Frei received his diploma in chemistry in Switzerland and his Ph.D. in analytical chemistry in the U.S.A. Since 1977 he has been Professor at the Department of Analytical Chemistry at the Free University, de Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
in
ana&ical
chemistry,
vol.
I,
no.
361
15, 1982
However, in the future IDMS will play an important role in calibrating other analytical methods as well as for the certification of standard reference materials.
References 1 de BiZvre, P. (1978) Adv. Mass Spectrom. 7, 395 2 Heumann, K. G. (1980) Toxicol. Environ. Chem. Rev. 3, 111 3 Turnbull, A. H. (1963) S urf ace Zonisation Techniques in Mass Spectrometsy: AERE Report 4295, Harwell 4 Heumann, K. G., Baier, K., Beer, F. and Schindlmeier, W. (1980) Adv. Mass Spectrom. 8, 3 18 5 Jochum, K. P., Seufert, M. and Best, S. (1981) Z. Anal. CIem.
309, 308 6 Dalrymple, G. B. and Lanphere, M. A. (1969) Potassium-Argon Dating, Freeman and Co., San Francisco
Photochemical
reaction
7 Reamer, D. C. and Veillon, C. (1981) Anal. Chem. 53, 2166 8 Schulten, H. R. (1979) Int. J. Mass Spectrom. Ion Phys. 32, 240 9 Smith, D. H., Christie, W. H. and Eby, R. E. (1980)Znt. J. Mass
Spectrom. Ion Phys. 36, 301 10 Shields, W. R., Murphy, T. J., Garner, E. L. and Dibeler, V. H. (1962) J. Am. Chem. SOL 84, 1519 11 Yergey, A. L., Vieira, N. E. and Hansen, J. W. (1980) Anal.
Chen. 52, 1811 12 Houk, R. S., Fassel, A. L. and Taylor,
V. A., Flesch, G. D., Svec, H. J., C. E. (1980) Anal. Chem. 52, 2283
Gray,
Professor Heumann received his Ph.D. from the Technical University of Dawnstadt in 1969. Since 1974 he has been Professor at the Institute of Inorganic &&try, University of Regensburg, Universitilstrasse 31,840O Regensburg, F.R. G.
detection’
in HPLC
The versatility of post-column reaction detection in chromatogra hy is greatly enhanced by exploiting the principles otp organic photochemistry. Improvements in photochemical reactor design will result in further advantages. John W. Birks University of Colorado, Boulder, CO, U.S.A.
Roland W. Frei Free University, Amsterdam, The Netherlands Post-column derivatization in high performance liquid chromatography (HPLC) has become a popular means of increasing the sensitivity and selectivity of detection for trace components of complex matrices. The technique has many advantages over pre-column derivatization, e.g. the reaction need not be complete or well defined so long as it is reproducible, and the analysis time is considerably reduced as a result of the elimination of additional sample handling steps. Many successful applications of post-column derivatization have been described in recent reviews’s*. Photochemical reactions may be employed as a special type of post-column derivatization, and dramatic improvements in both sensitivity and selectivity have been demonstrated here as well. In the simplest case, photons are the only reagent added, and many examples of improved detection - particularly in the pharmaceutical area - have been demonstrated. For example, tamoxifen and clomiphene are stilbene derivatives (I), which upon irradiation with UV light are transformed into the corresponding phenanthrenes (II): -
The increased rigidity and aromaticity of the phenanthrene molecule results in a much enhanced absorption coefficient and a higher fluorescence quantum yield. Selectivity for such analyses is very high since any interfering species must (1) co-elute with the analyte, (2) absorb light from the lamp, and (3) undergo a reaction to produce a fluorescent compound. Biological tissues and fluids have been found to be particularly free of interferences. Post-column photochemical derivatization should be applicable to the determination ofnearly all analytes for which batch photochemical methods have already been developed. In addition, the use of photochemical reagents greatly increases the applicability of the technique. For example, the use of a sensitizer to absorb light and initiate a photochemical reaction allows the detection of compounds such as aliphatic alcohols, ethers, aldehydes and saccharides - compounds that have only weak chromophores or none at al13*4. Post-column photochemical reaction detection may be seen as part of an expanding interest in the application of well-known principles of organic photochemistry to HPLC detection. Other recent developments include applications of chemiluminessourcesg’lo, and energy cence5-8, chemical excitation transfer phosphorescence” to achieve improved sensitivities and a wide variety of selectivities.
Photochemical
0 165~9936/82/oooO~/SOl.~
reactor design
The optimal photochemical reactor has probably not yet been constructed. Of the few designs that have appeared in the literature, most incorporate a medium or high pressure arc lamp (Hg, Xe or Xe-Hg)12-14, although low pressure mercury lamps15 and fluorescent black lights3*4 have also been used. The spectral output @ 1982 Elsevier Scientific Publishing Company
trends in analytical chemistry, vol. I, no. 15< 1982
362 of the lamp should, of course, overlap with the absorption spectrum of the analyte or sensitizer. The high intensity of a Xe-Hg arc lamp over the entire UV-visible region makes it a generally useful excitation source. Optical filters can, in principle, be used to increase the selectivity of the photochemical reaction, although this has yet to be demonstrated. A disadvantage of arc lamps is that they produce a great deal of heat and infrared radiation, and thus cooling of both the lamp and reaction coil are required. High temperatures within the reaction coil can result in undesirable side reactions, including polymerization. Furthermore, temperature fluctuations adversely affect the reproducibility. The housing of an arc lamp must be resistant to the possible explosion of the lamp, and the toxic ozone gas produced by the photolysis of oxygen must be vented. The design of a fourth generation photochemical reactor that incorporates all of these features is shown in Fig. 1 16. The reactor makes use of a polytetrafluoroethylene (PTFE) reaction coil. PTFE has been found to transmit light by an internal reflection mechanism in which light passes through the pores of the polymer. Some reactor designs have utilized quartz capillaries. However, these are both expensive and fragile. They are not readily available in different geometries (coil and helix diameter, length), and tight connections are difficult to make. The surprisingly high transmission of light in the 200-300 nm spectral region by PTFE makes this material nearly ideal for photochemical reactors, especially since the tubing is inexpensive and readily available in a range of internal diameters. Also, it has been shown that the use of PTFE coils results in chromatographic peaks having better symmetry and less tailing than for quartz capillaries13. The only disadvantage of PTFE reaction coils is that the material is permeable to oxygen, so that the sensitivity of detection may be reduced for certain analytes if the reaction coil is exposed to air. In the reactor design of Fig. 1, the PTFE coil is immersed in a circulating water bath which serves both to regulate the temperature and to exclude oxygen. Also, metal salts may be added to the water bath to serve as an optical filter.
Fig. 1. Photochemical reactor. 1, fan; 2, outlet for circulating liquid; 3, capillary; 4,filter compartment; 5, additional cooling via pressurized air; 6, quartz cylinders; 7, liquid inlet. The light source is a 200 W Hg-Xe lamp operated at a current of IOA. The PTFE reaction coil is cooled by a continuous flow of water through thejacketsformed by two quartz and onepyrex cylinders.
for (1) small capillary lengths, (2) small internal diameters and (3) low flow rates. For a fixed reactor volume (m*L) and flow rate (and thus reaction time), the variance is reduced by using longer capillaries of smaller diameter. For this reason, it has been pointed out that for reaction times of up to 20 min, small tr
I”5 ec
t
90
80
x=01)
70
60
50
Band broadening considerations Any post-column reactor will contribute at least a small amount to the dispersion of the chromatographic peaks and thus reduce the resolution of the chromatography. The contribution to the variance for a coiled tubular reactor is given by: (TV 2=K-
7Tr4L 4. 24 Dm
Here r is the internal radius of the capillary, L is the capillary length, 4 is the liquid flow-rate, Dm is the molecular diffusion coefficient for the analyte in the solvent, and K is a proportionality factor which depends on the flow profile in the capillary. It is a function ofthe dimensionless Dean and Schmidt numbers. From equation (1) we see that band broadening is minimized
40
3c
2c
10
Fig. 2. Plots of reaction time, t, v. radius of the reaction capillary, r, that satisfy equation (2) for different K values. Flow segmentation is advantageous when the point (r,t) lies above the appropriate curve.
363
trends in analytical chemistry, vol. 1, no. 1.5, 1982
0
5
7
0
5
h
7
tlmo(mbn
0
5
7
1
Fig. 3. Chromatograms of clobazam (c) and desmethylclobazam (d) after photochemical reaction for non-segmented, liquid segmented and air segmented systems. Capillary, PTFE of 0.5 mm I.D.; reaction time, 19 s.
diameter (r = 20 pm) coiled tubing could be used as the post-column reactor with minimal dispersion17. However, for reasons of convenience (back pressure, clogging, material cost, etc.) most workers prefer the use of larger diameter, = 0.2-2.0 mm I.D. tubing. Coiling the capillary also reduces band broadening by breaking up the parabolic velocity profile and thus reducing the value of K in equation (1). The use of a capillary knitted from PTFE tubing has been used in one photochemical reactor design to minimize peak broadening15. Reaction coils up to 20 m in length allowed reaction times ofup to 3.75 min in the detection of clobazam and fenbendazole and their major metabolites in man. We have found that 12 m of 0.3 mm I.D. PTFE tubing may be knitted such that the dispersion is the same as for one meter of loosely coiled tubing of the same diameter. A very effective means ofcurtailing band broadening
in post-column reactors is flow segmentation, a method that has long been used in clinical analyzers. In this technique the HPLC eflluent is divided into small segments by introducing air (or inert gas) bubbles or segments of an immiscible liquid. After sufficient reaction time the two phases are separated with only a small loss (typically IO-20%) of the analytical phase. Since nearly all of the band broadening in a segmented reactor results from the phase separator, the variance is nearly independent of reaction time, making very long reaction times (many minutes) possible. A theoretical expression comparing band broadening for segmented and non-segmented reactors has recently been derived”. For small injection volumes and negligible contribution to the band broadening by connective tubing, mixing tees and the detector, it has been found that flow segmentation is advantageous when t,r* > y
0 I*poxy-quinon~l
ho,02
ph.sep.
Values for the variance of the phase separator, a,‘, ph.sep., typically fall in the range 70-250 pL* with an average value of approximately 150 pL*. Plots of reaction time v. capillary radius that satisfy equation (2) are shown in Fig. 2. For typical numerical values, namely Dm = 0.5-2.5. lop3 mm* s-l, K = 0.25 and 4 = lo-20 PL s-l, we have ta’ > 0.03-0.6 mm’s. Thus, for capillary radii of 0.15 mm, 0.25 mm and 0.40 mm, segmentation is advantageous when the reaction times exceed 1.3-26 s, 0.5-10 s and 0.24 s, respectively. As an example, Fig. 3 shows chromatograms of the drug clobazam and its major metabolite, desmethylclobazam, for non-segmented detection and
FLUORESCENT
?’
a,*,
POLVMERISI
Iepoxy-hydroquinonel
I
ho
.
&9x I
0
trioriran* K, -CHROHENOL !compound
I&ar~2R5) II ?)
o-quinom
4
methidelAmOx=475nml R
OH-
H’ II 0-
Kl$
llVDROQUlNONE Icompound
Fig. 4. Proposed photochemical
17)
reactions of Vitamin KL
rmi-qulnme
IX,,,=
60Onml
hydropcroxide
eon R
trends in analytical chemistry, uoi. I, no. 15, 1982
364 for both solvent (hexane) and air segmentation using a 0.5 mm I.D. PTFE capillary (tr = 19 s) as the photochemical reaction coil’* It is evident that the peak heights for the non-segmented system are distinctly smaller than are those in either of the segmented systems, and the resolution for the non-segmented system is much poorer.
Applications Post-column photochemical reaction detection is still in its infancy as an analytical technique. The first application of a post-column photochemical reactor was published in 1976 by Iwaoka and Tannenbaum”. They selectively detected N-nitroso compounds by photohydrolysis to produce nitrite, which was then determined colorrmetrically using the Griess reaction. The technique allowed the determination of nonvolatile nitrosamines in food products in the lo-100 rig/g range. A similar idea was pursued by Hall and Rodgers, who developed the photoconductivity detector for HPLC, now marketed by Tracer In this detector the photolytic decomInstruments*‘. position of halogen and many nitrogen and sulfur compounds results in ionic products which are subsequently detected in a conductivity cell using the bipolar pulse measurement technique. Detection limits are in the low picogram range for many compounds.
+/+
/
The use of an on-line photochemical reaction to form fluorescent or UV-absorbing products was first demonstrated by Twitchett et al. in 1978*l. They determined cannabinol in urine by converting it into a highly fluorescent derivative upon irradiation. The detection limit was about 0.5 ng. By injecting a second sample, but with the lamp off, any background due to naturally fluorescing compounds could be determined. For the forensic drug lysergic acid diethylamide (LSD), they demonstrated the opposite use of UV irradiation This naturally fluorescent to achieve selectivity. compound is first chromatographed and detected with the UV lamp off. Upon irradiation of 320 nm, the compound is converted into the non-fluorescent derivative, and thus a repeat injection with the lamp on allows the drug to be distinguished from other fluorescent compounds with similar chromatographic properties. The post-column photochemical derivatization of a number of pharmaceuticals has now been reported, and these studies are summarized in Table I. A recent study in which Vitamin K homologues were determined illustrates a number of important considerations in photochemical reaction detection16. To begin with, the photochemistry ofcomplex molecules is seldom simple. This is illustrated by Fig. 4, which summarizes the literature on the photochemistry of Vitamin Kl solutions. The reaction products are highly dependent on solvent composition, the presence or absence of oxygen, pH and irradiation time. The Vitamin K homologues do not exhibit native fluorescence; however, in batch photolysis experiments it was found that at least three fluorescent products were formed. It was also found that in the presence of small concentrations of ascorbic acid the only isolable fluorescent product is the hydroquinone KHZ. The ascorbic acid acts as a chemical reductant and protects the KHZ from further oxidative decomposition. Because the hydroquinone is highly fluorescent and can be reproducibly formed in good yield, its optimum excitation and emission wavelengths were chosen for the analytical procedure. Purging the solvent system with nitrogen improved the sensitivity by up to 50%, as would be expected, considering the various reactions with 02 shown in Fig. 4. A general observation in photochemical reaction detection is that there exists an optimal residence time in the photochemical reactor. This is a result of photochemical degradation of the desired reaction product (e.g. the photochemical reaction ofKHn shown in Fig. 4). This optimum may be found by making plug injections of the analyte (no column) and measuring the signal as a function of flow rate. As is shown in Fig. 5, the optimal reaction time is highly dependent on the chosen reaction conditions. For Vitamin Kl the long reaction time required to obtain the optimal signal obviated the use of a segmented reactor. Fig. 6 compares the chromatograms obtained by air-segmented photochemical reaction and fluorescence detection with non-segmented UV absorption
+\ r;------r*\
Fig. 5. Reaction kinetics for the formation of KHzfrom vitamin K I showing different optimal reaction times for different reaction conditions. 0-0 in CHSOH + ascorbic acid (air cooled reactor); x-x in CHIOH + ascorbic acid + pH 3.8 buffer (air cooled reactor); +-•+ in CH30H + ascorbic acid + pH 3.8 buffer (water cooled reactor); @-O n CHDOH + ascorbic acid + pH 3.8 buffer (water cooled reactor and air segmented Jlow system).
trends in analytical chemistry, vol. 1, no. 15, 1982
TABLE
Analyte
365
I. Photochemical
Photochemical reaction parameters
Detection limit
Clobazam
200 W high pressure Xe-Hg, 3.8 m PTFE (0.3 mm I.D.) 15 s
20 Pg
Clobazam
15 W low pressure Hg, 20 m knitted PTFE (0.5 mm I.D.), 54 s
130 Pg
reaction
detection
methods
for pharmaceuticals
Linearity demonstrated
Chromatographic conditions
2.5-90
ng/ml
30 pg/ml
0.7-l
Ref.
Sample
matrix
and other notes
Column: 15 cm X 4.6 mm, 5-pm LC-18 (Supelco) Eluent: methanol0.01 M sodium acetate (6:4) -
13,22
A detection limit of 50 ng was obtained for the metabolite desmethylclobazam, and the detection of these species in serum is also demonstrated.
Column: 10 cm X 4.6 mm LiChrosorb-RP8, 10 /*m (Brownlee Labs)
15
Desmethylclobazam and two other metabolites are also separated and detected in pure solutions.
Column: 25 cm X 4.6 mm Zorbax silical, 6 pm (Du Pont)
14
An ether extraction allows the determination at levels below 1 ng/ml of plasma. Plasma levels were measured for a group of patients undergoing clomiphene therapy.
Column: 15 cm X 4.6 mm, 5 pm LC-18 Supelcosil (Supelco)
23
Chromatogram of human serum spiked with 8 ppm of demoxepam.
24
A simple ether extraction of urine plasma and serum allows the detection of DES at low ppb levels.
15
Chromatograms of serum extracts spiked with fenbendazole and its three principal metabolites are also demonstrated at levels down to 10 ng/ml.
Eluent: 0.1 M tetraethylammonium phosphate buffer pH 7.0methanol (16:13) Clomiphene
Medium pressure Hg, 3 m PTFE (0.3 mm I.D.) 10 s
Demoxepam
200 W high pressure Xe-Hg, 3.8 m PTFE (1.1 mm I.D.), air segmentation, 110 s
60 pg
l-45
ng/ml
lo-10,000
100Pg
ng/ml
Eluent: MeOH-O.1 phosphate buffer
M
(PH 8) (3:2) Diethylstilbestrol (DES)
100 W medium pressure Hg, 0.7 m fused silica (0.25 mm I.D.) 2 s
-
0.2 ng
2.5-10
ng
Column: 25 cm x 4.5 mm RP8 (Perkin-Elmer) Eluent: CH&Nwater (l:l), pH 3.5
Fenbendazole
15 W low pressure Hg, 20 m knitted PTFE (0.5 mm I.D.) 78 s
1.2 ng
0.7-70
pg/ml
Column: 12 cm x 4.6 mm LiChrosorb RP8, 7 pm (Knauer) Eluent:
0.033
M
HsPOd-methanol Phenothiazines: mesoridazine sulphoridazine thioridazine
200 W high pressure Xe-Hg, 7.6 m PTFE (0.3 mm I.D.) 25 s
0.5 ng
0.15-l
1.5
pg/ml
(1 :l )
Column: 10 cm X 4.6 mm, 5 pm LiChrosorb RP8 (Merck)
13
Chromatogram of extract of serum spiked to 2.3 ppm with mesoridazine and 6 ppm of thioridazine. The fluorescence signal enhancement factors after irradiation were 8-10 for thioridazine and 2-3 for mesoridazine and sulphoridazine
23
Detection limit of 10 ng/ml for phenothiazines extracted for serum. Irradiation enhanced the fluorescence sensitivity for four of the phenothiazines by about a factor of five, but had no effect on thiodiphenylamine.
Eluent: CHsOH7.6 M sodium acetate (4:1), 0.01% ammonium peroxodisulphate Phenothiazines: fenergan largactil levopromazine nedaltran thiodiphenylamine
200 W high pressure Xe-Hg, 7.6 m PTFE (0.3 mm I.D.) 35 s
40-100
pg 2-100
ng/ml
Column: 10 cm x 4.6 mm, Polygosil 60-10 CN (Marcherey, Nagel and Co.), Eluent: CH30H sodium
-2.6 M acetate (4:l)
trends in analytical chemistry, vol. I, no. M, 1982
366 detection for a 400 pg sample of Kl. The improvement in sensitivity by about a factor of four may seem small in light of the effort required. However, the selectivity for the photoreduction method allows the detection of normal levels of Kl in serum by a simple extraction followed by reversed phase chromatography and without an intermediate clean-up step. All of the applications described in Table I require absorb light tq initiate the that the analyte A photochemical reduction photochemical reaction. similar to that described above for Vitamin Kl has recently been used to detect a variety of compounds that have only very weak chromophores in the 0 I
1 I
2 I
i
3 I
L r
min.
5 r
UV-visible region, or which have none at a114. In this detection scheme the compound anthraquinone-2,6is photoreduced to the hydrodisulfonate (AQDS) quinone in the presence of analytes having C-H bond strengths less than about 94 kcal/mol. The mechanism of photoreduction of isopropanol is shown in Fig. 7. For this reaction scheme the analyte need not absorb light. the AQDS reagent absorbs light and Rather, undergoes rapid intersystem crossing to the triplet state. This triplet excited species can abstract the o-hydrogen to produce a semiquinone radical (QH*) and the (CH3)&OH* radical. Subsequent reactions lead to the fully reduced and highly fluorescent hydroquinone, QH2. Using a fluorescent black light and a reaction time of 26 s, detection limits in the range 5-30 ng were achieved for a variety of aliphatic alcohols, aldehydes, ethers and saccharides. As it this post-column photochemical reaction stands, technique is more than an order of magnitude more sensitive than a conventional refractive index detector, while providing the benefit of considerable selectivity.
Future trends
7”” 2C*nm
I
0
2
L
P
FLUOR. 320/420
nm
,..,,
6
8
10
12
1L min.
Fig. 6. Chromatograms of 4OOpg of Vitamin Klfor direct UV detection (248 nm) and post-column photochemical reduction followed by Jluorescence (320 ex. I420 em.) detection of the KHz product.
Many improvements in photochemical reactor design are possible. As mentioned earlier, the selectivity for a particular photochemical reaction could be enhanced by using a monochromatic light source. Provisions for varying the intensity of the source would allow an additional degree of freedom in the signal optimization. Because the instantaneous concentrations of short-lived species such as radicals and electronically excited molecules depend on the light intensity, the branching ratios to various products may also be affected by source intensity. Thus, depending on the kinetics of the particular reaction system, the optimum product yield may occur either for a high intensity source and short reaction time or for a low intensity source and long reaction time. In the latter case, further exploitation of the segmentation principle in order to minimize band broadening would be called for. It is even possible that modulation of the source, allowing time for dark reactions to occur in the absence of the highly reactive short-lived species, would increase the yield of desired products in some cases. The ability to regulate the temperature within the reaction cell over a wide range would provide further control over the photochemical reactions. It is also desirable to miniaturize photochemical reactors, making use of smaller point sources with more efficient coupling of the radiant energy to the reaction cell. Simple photochemical reactors tailored for specific reactions could also be developed. For example, a fluorescent black light may be used as effectively for some reactions as a high pressure Xe-Hg lamp, with the advantage of great savings in cost and reduction in complexity. It seems likely that many more analytes will be found whose detection sensitivities are enhanced by a direct photochemical reaction. However, we expect that the major progress in photochemical reaction detection
trenk ia analyticalchemistry.vol. 1. no. 15, 1982
AQ + hu+AQ*
(singlet, S)
AQ*( S 1 -
AQ* (triplet,T)
AQ*(T 1 +
KH,),CHOH
KH,),
FOH + AQ -
2AQH*-
367
-
AQH* + KH,),
AQH* + (CH&
GOH
C=O
AQ + AQH,
Fig. 7. Mechanism for the photoreduction of anthraquinone by isopropanol. AQ is anthraquinone; AQH* is the semiquinone radical; AQHz is 9,10-dihydroxyanthracene.
will result from the application of new photochemical reagents and the coupling of photochemical reactors to other detection methods, such as electrochemical and chemiluminescence detection. We have described above the photoreduction of AQDS as a means of detecting ‘hydrogen-donating’ compounds. In an alternate detection scheme for such compounds, AQDS acts as a sensitizer in a photo-oxygenatiot?. The reaction mechanism for ethanol is shown in Fig. 8. Rather than exclude oxygen, as is necessary for the photoreduction, the HPLC mobile phase is saturated with oxygen by continuously bubbling 02 through the solvent reservoir. Hydrogen peroxide is a major product of the reaction and may be detected by its chemiluminescent reaction with luminol. An important difference between the photo-oxygenation (Fig. 8) and photoreduction (Fig. 7) mechanisms is that in the photo-oxygenation the AQDS is not consumed, but rather, acts as a catalyst. This suggests what is potentially a very sensitive method of detecting analytes that, like AQDS, can sensitize the photo-oxygenation of an alcohol. Using an alcohol/water mobile phase saturated with 02, it should be possible to detect even very weak sensitizers at low concentrations, since the reaction of the triplet-excited analyte with the alcohol would be quite favourable in comparison to the Tl + So deactivation and because long reaction times in the photochemical reactor would allow very high many carbonyl quantum yields of Hz0 2. Certainly compounds could be detected in this way and probably many nitrogen compounds as well. Q+
hu%%*
%* + CH,CH,OH
-
CH CHOH y’-,i.zi
02
3l
00.
02
t Q+HOO. 2 CH$OOH
+ H202
CH$HO
+ 02 +
v2
CH,CHO
+
Y
HO0 .
HOO. J
HO0 . 1 HOO.
Q+HOO.
“,2
02 +
H202
H202
02
+
H202
Fig. 8. Mechanism for the sensitizeddhoto-oxygenation of ethanol in neutral aqueous solution. Q is a sensitizer such as AQDS.
In some cases additional selectivity might be obtained by coupling a photochemical reactor to an appropriate electrochemical detector. For example, the photo-oxidation products of some compounds could be detected by subsequent reduction in a polarographic detector. Phenothiazines comprise one class of pharmaceuticals that might be more selectively detected in biological matrices using this approach.
Acknowledgement J. W. B. thanks the Nederlandse Organisatie voor Zuiver-Wetenschappelijk Onderzoek (Z.W.O.) for a visitor’s grant. This work was partially supported by a grant from the National Science Foundation (CHE-7915801).
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Frei, R. W. (1982) Chromatographia 15, 161 Stewart, J. T. (1982) Tremis Anal. Chem. 1, 170 Gandelman, M. S. and Birks, J. W. (1982)J. Chromatogr. 242,21 Gandelman, M. S. and Birks, J. W. (submitted for publication) Neat-y, M. P., Seitz, W. R. and Hercules, D. M. (1974) Anal. Lett. 7, 583 Birks, J. W. and Kuge, M. C. (1980) Anal. Chem. 52, 897 Shoemaker, B. and Birks, J. W. (1981)J. Chromatogr. 209, 251 Veazey, R. L. and Nieman, T. A. (198O)J. Chromatogr. 200, 153 Kobayashi, S. and Imai, K. (1980) Anal. Chem. 52, 424 Kobayashi, S., Sekino, J., Honda, K. and Imai, K. (1981) Anal. Biochem. 112, 99 Donkerbroek, J. J., van Eikema Hommes, N. J. R., Gooijer, C., Velthorst, N. H. and Frei, R. W. (1982) Chromatographz’a 15,218 Twitchett, P. J., Williams, P. L. and Moffat, A. C. (1978)J. Chromatogr. 149, 683 Scholten, A. H. M. T., Welling, P. L. M., Brinkman, U. A. Th. and Frei, R. W. (198O)J. Chromatogr. 199, 239 Harman, P. J., Blackman, G. L. and Phillipou, G. (1981) J. Chromatogr. 225, 13 1 Uihlein, M. and Schwab, E. (1982) Chromatographia 15, 140 Lefevere, M. F., Frei, R. W., Scholten, A. H. M. T. and Brinkman, U. A. Th. (in press)
17 Tijssen, R. (1980) Anal. Chim. Actu 114, 71 18 Scholten, A. H. M. T., Brinkman, U. A. Th. and Frei, R. W. (1981) J. Chromatogr. 205, 229 19 Iwaoka, W. and Tannenbaum, S. R. (1976) IARCSci. Publ. 14, D. J. Dixon J. B. and Ehrlich, B. J. (1979) J. 20 ibpvich, Chromatogr. Sci. ‘17, 643 ’ P. J., Williams, P. L. and Moffat, A. C. (1978) J. 21 Twitchett, Chromatogr. 149, 683 22 Scholten, A. H. M. T., Brinkman, U. A. Th. and Frei, R. W. (1980) Anal. Chim. Acta 114, 137 23 Brinkman, U. A. Th., Welling, P. L. M., de Vries, G., Scholten, A. H. M. T. and Frei, R. W. (1981) J. Chromatogr. 217, 463 24 Rhys Williams, A. T., Winfield, S. A. and Belloli, R. C. (1982) J. Chromatogr. 235, 461 John Birks earned his Ph.D. at the University of California at Berkeley in 1974. Following three_years as Assistant Professor in the Department of Chemistry of the University of Illinois at Urbana-Champaign, he joined the faculty at the University of Colorado, Boulder, CO 80309, U.S.A. where he is Associate Professor of Chemistry and Fellow of the Cooperative Institute for Research in Environmental Sciences (CIRES). Thispastyear he has been on sabbatical leave at the Max Planck Institute for Chemistry in Main<, West Germany and the Free University, Amsterdam, The Netherlands. Roland Frei received his diploma in chemistry in Switzerland and his Ph.D. in analytical chemistry in the U.S.A. Since 1977 he has been Professor at the Department of Analytical Chemistry at the Free University, de Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.