Anal. Chem. 1985, 57, 1810-1814
1810
Photoelectrochemical Detector for High-Performance Liquid Chromatography and Flow Injection Analysis William R. Lacourse* and Ira S. Krull* Department of Chemistry a n d Barnett Institute, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts 02115
Karl Bratin Analytical Research Department, Pfizer Central Research, Pfizer & Co., Inc., Groton, Connecticut 06340
Photoelectrochemical detection (PED) combines photochemistry with amperometry. The union allows one to take advantage of the new and/or altered electrochemical properties of photogenerated excited states, lntermedlates or products. A flow-through, thin-layer amperometric cell modifled to Irradiate the warklng electrode surface with optical energy is mated to a high-performance liquid chromatography (HPLCPED) or flow injection analysis (FIA-PED) system. The PED is responsive to alkyl and aryl ketones and aldehydes. The response is linear over 3 to 4 orders of magnitude, replicate injection reproduciblllty is better than 2 % standard deviation, and minimum detection limits for conjugated carbonyl derivatlves are in the 10-2 ng range with a signal to noise ratio of 3 times the background.
In photoelectrochemical detection (PED), light, mainly ultraviolet (UV), is used to produce a species having different electrochemical properties than the original analyte undergoing irradiation. Initial work in PED goes back to the 1960s, when Perone and co-workers used flash photolysis to generate unstable, short-lived, radicals or radical anions from benzophenone or tris(oxa1ato)ferrate that were detected electrochemically (1-5). This work was followed in the mid 1970s by Johnson using photolysis and a rotating ring-disk electrode on the same compounds (6,7). Although similar, these studies were carried out in static or stirred systems, as opposed to a continuously irradiated, flow-through, thin-layer cell design. Only recently has Weber published on work termed photoelectroanalytical chemistry, which involves an amperometric, flow-through cell (8, 9). This work differs in that all the detection chemistry is centered about a ruthenium complex intermediate, Ru(bpy),2+, to accept light energy, undergo excitation, be oxidized or reduced in solution by the analyte, and be detected electrochemically. The experimental conditions in PED are designed to detect photochemically excited species, intermediates, and/or products directly without any solution intermediate. When light is absorbed, the ground-state analyte is transformed to the excited state via electronic promotion. The excited state is both a better oxidizing agent (an electron may fill the lower energy, empty orbital) and a better reducing agent (the electron is more easily removed from its higher energy orbital). Thus, the electrochemical properties should be altered vs. the ground state. Under pseudo-first-order conditions using a diffusion-controlled rate constant in water of 4 x IOg L mol-' s-l for a 100 amu molecule with a diffusivity constant of cm2 s-l, a lifetime of IO4 to IO-' s is needed to have 1 mmol L-l of analyte react with the electrode. Molecules or species derived from photogenerated excited states (singlets or triplets), intermediates, and/or products having the required lifetimes may be detected by continuously 0003-2700/85/0357-1810$01.50/0
irradiating the electrode surface of a flow-through, thin-layer amperometric cell.
EXPERIMENTAL SECTION Apparatus. Figure 1illustrates the high-performance liquid chromatographwith photoelectrochemical detection (HPLC-PED) instrumentation used in all of these studies. The system consists of a Model 590 solvent delivery pump with microprocessor control (Waters Associates, Milford, MA), a Rheodyne 7010 injector with a 7011 needle port insert (Rheodyne Corp., Cotati, CA) modified for sample deoxygenation, a BAS Model LC-4B dual electrode amperometric system (EC) for liquid chromatography (Bioanalytical Systems, Inc., West Lafayette, IN), a modified BAS glassy carbon single/dual working electrode with a silver/silver chloride reference electrode, a 500-W Hg arc lamp with a Model p-510-D power supply (George W. Gates and Co., Long Island, NY) mounted on a 3-ft optical rail (Edmund Scientific Co., Barrington, NJ), and a Model D5217-1A1 dual pen Omniscribe recorder (Bausch & Lomb Co., Houston Instrument Division, Austin, TX). Waters Associates stainless steel fittings and ferrules were used for all of the HPLC-PED connections, except where the EC detector cells required BAS fittings. HPLC mobile phases were degassed and filtered before use with 0.45-pm filters, catalog no. HATF 04700, and a solvent filtration kit, catalog no. xxl0 047 30 (Millipore Corp., Bedford, MA). The mobile phase reservoir was continuously refluxed at 60-70 O C with a constant head of helium over the mobile phase. The mobile phase was further deoxygenated with a 15 cm X 4.6 mm i.d. stainless steel column packed with 400 mesh granular zinc (Fisher ScientificCo., Pittsburgh, PA) (IO). All injectionswere made with the injection system modified for reductive electrochemical analysis (11). The sample was placed in the sample vial and degassed for 3 min. The sample was drawn through the sample loop (25, 50, or 200 pL) using a Model 9011 HPLC syringe (Hamilton Corp., Long Beach, CA). The samples were filtered prior to injection with a LS025 Millipore disposable filter (Millipore Corp., Bedford, MA). HPLC columns utilized in these studies were a 10-pm pBondapak CN 25 cm X 4.6 mm i.d. stainless steel column or a 10-pm Radial-PAK pBondapak CN column with Model RCM-100 column compression system (Waters Associates, Inc., Bedford, MA). The modified cell is depicted in Figure 2. The stainless steel auxiliary was machined to accept a 7 mm diameter hole opposite the working electrode, a rectangular 8 mm X 25 mm fused quartz window was mounted on the flow-through face of the auxiliary electrode and sealed in place with Duro-Brand epoxy cement (Loctite Corp., Cleveland,OH). The complete thin-layer cell was mounted opposite the arc lamp on the optical rail. The light was focused with a double plano-convex, 35 mm, fused quartz lens assembly (Oriel Corp., Stamford, CT). The entire system was surrounded by a grounded, flat-black colored, aluminum box, in order to reduce UV-light hazards to laboratory personnel, and act as a Faraday cage to reduce electrical noise. For flow injection analysis (FIA),the column was removed from the system. Reagents. All organic and inorganic reagents were obtained from commercial sources, such as, Aldrich Chemical Co. (Milwaukee, WI), Pfaltz & Bauer Co. (Stamford,CT), Fisher Scientific 0 1985 American Cpmical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 9, AUGUST 1985
n
Table I. Solvent Compatibility in FIA-PEDa l%organic/aqueous PURGE GAS
+Ne
I
solution
WASTE C
I
dll
OPTICAL BENCH
Figure 1.
1811
Schematic diagram of the HPLC-PED analytical system. I
+OUTLET
INLETAUXILIARY
EL E CTR ODE
water methanol ethanol propanol 2-propanol acetonitrile acetone ethyl acetate dioxane tetrahydrofuran dimethylformamide mobile phase
electrochemical activity lamp on lamp off no
no
no no no no
no
no
no no no
yesb Yes yesb Yes
Yes
Yes
Yes no
Yes no
no no no
JaC1, 8 mL/ a FIA-PED conditions usel 20% MeOH/O. n min, -0.8 V, 200 pL injections, glassy carbon electrode (ox.,ative), 100 nA/V. b2-3 nA response at a concentration of 1%by volume in the mobile ahase.
Table 11. Solvent Effects on Benzophenone Response in FIA-PED
THIN LAYER
MEMBRANE
tJ
GLASSY CARBON ELECTRODE
Specially modified flow-through,thin-layer, BAS type single electrode amperometric cell for HPLC or FIA photoelectrochemlcal detection. Figure 2.
Co. (Pittsburgh, PA) and J. T. Baker Chemical Co. (Phillipsburg, NJ). The HPLC solvents were obtained from Waters Associates, Inc. (Milford, MA), or MCB Chemical Co. (Cincinnati, OH), and the latter as the Omnisolv-brand of HPLC grade solvents. Procedure. Before the experiment was started, the electrode was polished with polishing alumina on a soft pad, Model PK-2 polishing kit from BAS. After cleaning, the thin-layer cell was assembled and mounted opposite the light source on the optical rail. The cell and light source were aligned by pumping a solution of 10 pmol L-l benzophenone in 50150 methanol/water, v/v, through the cell. The signal was maximized by adjusting the positioner holding the cell. The benzophenone solution was washed out of the thin-layer cells. Caution should be observed when working with high-intensity UV radiation.
RESULTS AND DISCUSSION A thorough discussion of the mechanism of detection will be covered in a later publication. Initial PED experiments were designed to characterize, understand, and optimize the detector response. The PED was to be mated with HPLC, and the liquid phase of the PED must be compatible with both the electrochemical detection system and reversed phase chromatography. Table I illustrates that the commonly used reversed phase solvents are compatible with the PED; acetonitrile and ethyl acetate may be used as organic modifiers at low levels. The percent of the organic modifier present also affects the response of the analyte. With methanol as the organic component, the response increases as the percent of organic increases. The increase appears to reach a plateau at 50% organic. This may be due to a decrease in polarity of the solvent system which influences the amount of de-
mobile phase MeOH/HOH, 50/50, 0.1 M NaCl (v/v) EtOH/HOH, 50/50, 0.1 M NaCl (v/v) n-PrOHIHOH, 50/50, 0.1 M NaCl (v/v) i-PrOHIHOH, 50/50, 0.1 M NaCl (v/v)
ppm analytea peak height peak area 1.66 1.59 (1.0)
2.03 (1.3) 2.56 (1.6)
6.84 6.19 (0.9) 6.35 (0.9) 9.65 (1.4)
a Ppm, parts per million, FIA mobile phase flow rates identical, equimolar amounts benzophenone injected, equal volumes injected, all other conditions identical. Numbers in parentheses indicate ratio of response compared to response of MeOH/HOH, 50/ 50, 0.1 M NaCl (v/v) system.
tectable species present via changes in the photochemistry. With benzophenone as a probe analyte, Table I1 shows that the analyte response is independent of the organic solvent used. The plateau region of the response, 50150, v/v, organic/aqueous, is chosen to eliminate the effect of polarity upon the detection mechanism. A large number of inorganic and organic anions have been studied under FIA-PED conditions. The anions are needed both as electrolytes for the EC detection system and as buffers for the chromatography system. The results in Table I11 demonstrate that of the 30 anions studied, a t two widely different potentials (+0.8 V and -0.8 V), very few show any response with irradiation of the electrode surface, with the exception of species such as chromate, carbonate, periodate, and dichromate. Therefore, ammonium acetate and phosphate buffers for reversed phase chromatography can be used with the PED system. For most of the studies presented in this paper a mobile phase of methanol and water with a 0.1 M sodium chloride concentration was used. Of the responsive analytes used, all showed a 100% dependency on having the electrode irradiated. Using FIA-PED, the response of benzophenone showed a square root dependence upon the distance from the light source. Assuming the power of the lamp and the area irradiated is constant, the intensity of the light output decreases as the reciprocal of the distance squared. By selection of the normal working distance of the electrode as the base point, the theoretical line is determined by applying the known intensity relationship and calculating the response if it were a function of the intensity falling upon the electrode. The experimental plot follows the theoretical plot of light intensity vs. distance, Figure 3. Such results indicate that a photochemically generated species is involved in the detection process. The formation of the species being detected is a direct function of the intensity of the light
ANALYTICAL CHEMISTRY, VOL. 57, NO. 9, AUGUST 1985
1812
Table 111. Anion Response in FIA-PED" +0.8 V -0.8 V . . lamp off lamp on lamp off lamp on
anion species mobile phase fluoride chloride bromide iodide nitrite nitrate cyanide sulfate chromate carbonate thiocyanate arsenate oxalate hydrogen phosphate perchlorate hypophosphite periodate iodate bicarbonate acetate hydrogen phthalate dichromate formate bisulfite sulfite dihydrogen phosphate chlorate ammonium acetate
no no no no Yes Yes no Yes no no no Yes no no no no no no no no no no no no Yes Yes no no no
no no no no Yes yes no yes no yes yes Yes no no no no no Yes yes no no no yes no
yes yes no no no
-I3
130-
no no no no no no no yes no no no no no no no no no Yes yes
no no yes Yes no no no no no no
no no no no yes(?) yes(?) no yes no no Yes
no no no no no no Yes
Yes
yes no Yes yes no no no no no no
120-
P
-12
110-
\
-11
-10
100-
-
-9
90, \
-2 8070-
-8
\
\
\
@
I
2
-7:
\
.-cn
\
60-
's\
-6
50-
' q 40Pk.Ht
-
-3
20-
-2
IO-
-I I
0 0 0'2 0'4 0'6 0'8 I O 1'2 1'4 1'6 1'8 $0 F l o w R a t e (rnl/rnin)
RESPONSE [ nanoornpr)
I
A,
:"1
40
30
40 501
a
.20
L 20-
IO
100
io
1'1
15
ih
(4
Ib
(6
i7
~b ~ ' e Z!O 2'1
2'2
1 3 2'4
is IS
DISTANCE FROM LAMP ( cm)
Flgure 3. Plot of response vs. lntenslty for benzophenone In 20% MeOH/80% water: 0.1 M NaCl at 20 ppm level, 0.8 mL/mln, SOO-ML Injection, glassy carbon electrode, -0.8 V (oxidative), and 500 nA/V. Solid ilne Is experimental, and dashed line is theoretical.
irradiating the solution passing over that surface per unit time. The actual light flux, which is affected by scatter, defocusing, channel parameters, and the mobile phase, reaching the electrode surface cannot be measured. The response of the analyte is also dependent upon the variables of the electrochemical cell, which may be affected by the irradiation. Figure 4 shows the response of benzophenone vs. flow rate. Under FIA conditions, the current, or peak height, is nearly independent of the flow rate. The thin-layer gasket used to establish the flow through channel also influences the response. The peak height decreases continuously with increasing gasket thickness. This may be due to a combination of increasing cell dead-volume and/or changes in the photon flux effected by changes in the cell's dimensions. Area response, which
0
Flgure 4. Plot of response vs. flow rate under FIA-PED conditions using benzophenone at 20 ppm level. Conditions are simliar to those of Figure 3. Solid line Is peak height response, and dashed line is area response.
1'04
W
-4
I '
"Mobile phase 20% MeOH/80% HOH; 0.1 M NaC1, 0.8 mL/ min, 200 p L injections, FIA-PEDmode, glassy carbon electrode, 500 nA/V, andvte concentration ca. 1 DDt for d l sDecies.
1304
'.-.
-5
30-
01
8
0
\\
0)
a
0
ANALYTICAL CHEMISTRY, VOL. 57, NO. 9, AUGUST 1985
Table V. Effect of Extended Olefinic Conjugation on FIA-PED Response of Ketones"
Table IV. Organic Analyte Responses in FIA-PED"
PED responses at 0.0 V (response vs. lamp acetophenone) off
organic compound mobile phase acetone 2-cyclohexen-1-one acetophenone 2,3-butanedione cinnamaldehyde mesityl oxide benzaldehyde indanone o-aminobenzaldehyde 1,4-~yclohexanedione fructose 1-alanyl-1-phenylalanine cyclopentanone cyclohexanone 3-pentanone 2-pentanone 4-methyl-2-pentanone 5-methyl-2-hexanone p-methoxybenzaldehyde benzaldehyde p-nitrobenzaldehyde formaldehyde acetyl acetone (AcAc) FeIn(AcAc) 4-nitrobenzophenone 4-(trifluoromethyl) benzophenone 2-(trifluoromethy1)benzophenone 3-(trifluoromethy1)benzophenone 4,4'-dimethylbenzophenone
no no Yes no no no no no no no no no no no no no no no no no no no no no no no no no no no
1813
(1.00)
organic compound
FIA-PED responseb (relative to acetophenone)
cyclohexanone 3-pentanone 2-cyclohexen-1-one acetophenone
1.33 X lo4 6.68 X 1.79 x 10-3 1.00
"Mobile phase was 50% MeOH/50% 0.2 M NaCl, 0.5 mL/min, 20-pL injection, glassy carbon electrode, oxidative mode, 0.0 V. Relative to acetophenone. Table VI. Quantitative Parameters of Some Carbonyl Compounds by HPLC-PED
(1.28 X (1.33 X (4.11 X (5.66 X (3.27 X (3.74 x (1.24 X (1.39 X (5.88 X (1.40 X (1.46 X (2.09 X (1.22) (1.35) (1.74) (4.34)
lo4) lo4) lo4)
compound
potential, V
lo4)
acetone
0.00
10-1) 10-I)
benzaldehyde
0.00
acetophenone
0.00
lo4) loba)
10-l)
-0.60 benzophenone
"Mobile phase 50% MeOH/50% 0.2 M NaCl, 0.8 mL/min, 20pL injections, glassy carbon electrode, equimolar injections of all compounds used.
0.00
line equations"
R = 1.15[C] + 67.3(1580 ppm - MDL) R = 51.7[C] + 35.7(20 ppm __ MDL) R = 189.9[C] + 102(20 -DD _m MDL) R = 145.5[C] + 72.6(20 ppm MDL) R = 131.1[C] + 75.7(15 ppm MDL)
MDL,b PPm corr coeff (200 pL) 0.998
1580
0.998
52.3
0.999
40.8
0.985
41.0
0.996
28.0
""R" is the response as peak height in millimeters. This may be converted to microamps by multiplying the peak height by 0.04. [C] is the concentration in pg/rnL-'. bMDL is the minimum detection limit in Darts Der billion, 200-ULiniections. Table VII. Comparison of Minimum Detection Limits for HPLC-PED VS. HPLC-UV
compound name benzaldehyde acetophenone benzophenone
minimum detection limits, PPb HPLC-PED" HPLC-UVb 50 40 30
20 (247 nm) 40 (243 nm) 30 (253 nm)
"Mobile phase is 50% MeOH/50% 0.2 M NaCl, 0.8 mL/min, 10 pm CN column, 200-pL injection, glassy carbon electrode, oxidative, 0.00 V, 20 nA/V. b0.2 AUFS, Schoeffel variable wavelength detector.
- . . - / - - r
0
i
1 - r -
2 4 rnin
Lamp off
6
-L-
L
TT --
0 2 4 6 8 1 0 rn in Lamil
on
A I
I
I
1
I
0
2
4
6
8
min
L a m p off
Figure 6. FIA-PED response of benzophenone (20 ppm) with and without irradiation. Conditions are similar to those in Figure 3.
is occurring. The current reaches a maximum near 0.0 V for most compounds. These HDV's are of the detected species, which is not the ground-state molecule. If dual electrode detection, either series or parallel, is combined with lamp on/off irradiation of each or both electrodes, the selectivity of the system may be greatly increased. The working electrode surface also affects the response. As the surface is changed from glassy carbon to gold-mercury, the signal increases 4-fold, but the standard deviation of the signal increases 10-fold. The background noise and increased standard deviation of the signal makes the metal electrodes (gold/mercury and platinum) analytically impractical. The glassy carbon electrode was used for all PED experimentation. With optimized conditions of flow rate, gasket thickness, solvent composition (organic and electrolyte), light intensity, and applied potential, the response of a large number of compounds is shown in Table IV. Most of the compounds
1814
ANALYTICAL CHEMISTRY, VOL. 57, NO. 9, AUQUST 1985
B E NZ A LDE HY DE ( 03.7 w m
1
\
L A M P ON
L A M P OFF
,
0
4
0
1
S 12
1
1
so does the response. The response of the analyte gives a direct logarithmic correlation to the quantum efficiency of phosphorescence. This again indicates the presence of a photogenerated species involved in the detection process. Further evidence that a photogenerated state is involved is that at both -0.6 and 0.0 V the response completely disappears when the oxygen scrubber column is removed from the system. Oxygen is both a singlet and a triplet quencher. Figure 6 illustrates the lamp on/lamp off response of a 20 ppm solution of benzophenone injected using FIA-PED. The compound gives no response with the lamp off, but it responds with the lamp on. Figure 7 demonstrates the lamp off/lamp on response for four carbonyl derivatives in the HPLC-PED mode of operation. Again, the selectivity of the system must be emphasized. Table VI gives the line equations, correlation coefficients, linear dynamic range, and minimum detection limits for these same compounds. The relative standard deviation of five replicate injections of a standard analyte is less than 2%. The analyte responses have excellent linearity, the linear dynamic range is 3 to 4 orders of magnitude, and the minimum detection limits are comparable to UV detection; see Table VII. The minimum detection limit of acetophenone is depicted in Figure 8.
1
ACKNOWLEDGMENT
I6 20 24 28
min
Flgure 7. HPLC-PED response of a mixture of carbonyl compounds with and without Irradiation. Mobile pase is 50% MeOH/50% 0.2 M NaCl at 0.8 mLlmin, 200-hL injection, glassy carbon electrode, 0.00 V (oxldatlve), and 500 nA/V.
The authors gratefully acknowledge the assistance and discussions of D. Locke, and we also wish to acknowledge the continued interest and technical support of Bioanalytical Systems, Inc., especially R. E. Shoup and P. T. Kissinger.
LITERATURE CITED
I
I
L 0 4 8 1 2
0 4 8 1 2
min.
min. L A M P OFF
L A M P ON
Figure 8. HPLGPED response of acetophenone’s minimum detection limit with and without irradlatlon. Conditions are similar to those of Figure 7, except sensitivity Is 20 nA/V.
responsive thus far are carbonyl compounds or derivatives. The ability to undergo n-r* excitation as opposed to r-a* excitation is evident in that compounds such as anthracene, fluorene, carbazole, and naphthalene, which easily undergo r-a* transitions, do not respond under FIA-PED analysis. Table V illustrates that as the degree of conjugation increases,
(6) (7) (8) (9)
Perone, S. P.; Birk, J. R. Anal. Chem. 1988, 38, 1589-1593. Birk, J. R.; Perone, S. P. Anal. Chem. 1988, 40, 498-500. Kirschener, G. L.; Perone, S. P. Anal. Chem. 1972, 44, 443-451. Jamisson, E. A.; Perone, S. P. J . Phys. Chem. 1972, 78, 830-839. Patterson, J. I. M.; Perone, S. P. J . Phys. Chem. 1973, 7 7 , 2437-2440. Johnson, D. C.; Resnlck, E. W. Anal. Chem. 1972, 44, 637-640. Lubbers, J. R.; Resnick, E. W.; Gaines, P. R.; Johnson, D. C. Anal. Chem. 1974, 46, 865-873. Weber, S. 0.; Morgan, D. M.; Elbicki, J. M. Clin. Chem. ( Wlnsfon-Sa/em, N . C . ) 1983, 29, 1665-1672. Morgan, D. M.; Elbicki, J. M.; Weber, S. G. Anal. Chem. 1986, 5 7 ,
-
-
1746-1 .. . .751. . ..
(10) MacCrehan, W. A.; May, W. E. Anal. Chem. 1984, 56, 625-628. (1 1) “Installation/OperationsManual for LC-4A Amperometric Controller”; Bioanalyticai Systems, Inc.: West Lafayette, IN; p 6-5.
RECEIVED for review February 11, 1985. Accepted April 16, 1985. This work was supported, in part, by a Barnett Innovative Research Grant, Northeastern University, and by a grant from the Analytical Research Department of Pfizer & Co., Inc., Groton, CT, to Northeastern University. We are indeed grateful for these sources of financial assistance. This is contribution no. 239 from the Barnett Institute of Chemical Analysis at Northeastern University.
