Anal. Chem. 1987, 59, 49-53
ACKNOWLEDGMENT We thank M. Gordon for helpful discussions. Registry No. DL-Arg, 7200-25-1; D-Arg, 157-06-2;L-Arg, 7479-3; DL-Try, 54-12-6; D-Try, 153-94-6;L-Try, 73-22-3; DL-Met, 59-51-8; D-Met, 34867-4; L-Met, 63-68-3; DL-Phe, 150-30-1;DPhe, 673-06-3; L-Phe, 63-91-2;DL-Sa, 302-84-1;D-Ser, 312-84-5;L-Ser, 56-45-1;D L - T ~80-68-2; , D-Thr, 632-20-2; L-Thr, 72-19-5;DL-TYT, 556-03-6; D-Tv, 556-02-5; L-Tyr, 60-18-4; DL-Norval, 760-78-1; D-Norval, 2013-12-9; L-Norval, 6600-40-4; DL-Norleu, 616-06-8; D-Norleu, 327-56-0; L-Norleu, 327-57-1;DL-Leu, 328-39-2; D-Leu, 328-38-1;L-Leu, 61-90-5; DL-a-ab, 2835-81-6; D-a-ab, 2623-91-8; La-ab, 1492-24-6DL-Ah, 302-72-7;DAla, 33869-2; a ala, 56-41-7; DL-Val, 516-06-3; D-Val, 640-68-6 L-Val, 72-18-4; DL-Pro, 609-36-9; D-Pro, 344-25-2; L-Pro, 147-85-3; DL-(CyS),, 923-32-0; D-(CYS)z, 349-46-2; ~ - ( C y s )56-89-3; ~, DL-ASP, 617-45-8;D - A s ~1783-96-6; , LAP, 56-84-8;DL-CYSac, 3024-83-7; D-CYSac, 35554-98-4;L-CYS ac, 498-40-8; DL-GIu,617-65-2; D-G~u, 6893-26-1;L-G~u, 56-86-0; Cu(II), 7440-50-8; aspartame, 22839-47-0. LITERATURE CITED (1) Chemistry and Biochemistry of the Amino AcMs; Barren, G. C., Ed.; Chapman 8 Hall: London, 1985. (2) Blogeochernisffy of the Amlno AcMs; Hare, P. E., Hoering, T. C., King, K., Jr., Eds.; Wiley: New York, 1980. (3) Bada, J. L. Ann. Rev. Earth Planet Sci. 1985, 13, 241.
49
Davankov, V. A.; Kurganov, A. A.; Bochkov, A. S. I n Advances in Chromatography; Giddlngs, J. C., Grushka, E., Cazes, J., Brown, P. R., Eds.; Marcel Dekker: New York, 1983; Vol. 22, p 71. Gassmann, E.; Kuo, J. E.; Zare, H. N. Science (Washington, D . C . ) 1985, 230, 813. Gilon, C.; Leshem, R.; Tapuhi, Y.; Grushka, E. J . A m . Chem. SOC. 1979, 101, 7612. Tapuhi, Y.; Schmidt, D. E.; Lindner, W.; Karger, B. L. Anal. Biochem. 1981. 115, 123. Shaw, D. J. Nectrophoresis; Academic Press London, 1989. Touche, M. L. D.; Wllliams, D. R. J. Chem. Soc., Datton Trans. 1978, 2001. Giion, C.; Leshem, R.; Grushka, E. J . Chromatogr. 1981, 203, 365. Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya. A.; Ando, T. Anal. Chem. 1984, 56, 111. Terabe, S.;Otsuka, K.; Ando, T. Anal. Chem. 1985, 5 7 , 834. Scott, R. P. W. I n Liquid Chromatography Detectors; Elsevler: Amsterdam, 1977; Part 1, Chapter 3, p 9. Gilon, C.; Leshem, R.; Grushka, E. Anal. Chem. 1980, 52, 1206. Meites, L. I n Handbook of Analyficai Chemistry; McGraw-Hill: New York, 1963; pp 1-13. Hlnckley, J. 0. N. J . Chromatogr. 1975, 109, 209. Brown, J. F.; Hinckley, J. 0. N. J . Chromatogr. 1975, 109, 218. Said, A. S. Theory and Mathematics of Chromatography; Dr. Alfred Huthig Verlag: Heidelberg, 1981. Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298.
RECEIVED for review June 13,1986. Accepted August 11,1986. Support for this work by Beckman Instruments, Inc., and by the Swiss National Science Foundation for P.G. is gratefully acknowledged.
Photoelectrochemical Detection of Benzaldehyde in Foodstuffs William R. LaCourse and Ira S.Krull* Barnett Institute and Department of Chemistry, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts 02115
Photoelectrochemlcal detection (PED) coupled with hlghperfmance Rquld chromatography was used to quantltatlvely determine benzaldehyde In extracts, beverages, and foodstuffs. Photoelectrochemical detectlon Is responsive to alkyl and aryl ketones and aldehydes and offers the advantages of 2-3 orders of magnltude Ilnearlty, 5-1-ng Umlts of detection, and a high degree of selectivity wlthout chemlcai derlvatlzation. Thls Is the first appllcatlon of the PED to sample anaiysis.
High-performance liquid chromatography (HPLC) with electrochemical detection (EC) has gained widespread analytical use over the past decade (1). This has been due, in part, to its high sensitivity and selectivity for specific functional groups. The photoelectrochemical detector (PED), recently introduced by LaCourse and Krull, expands the normal range of electrophores to include alkyl and aryl ketones and aldehydes (2, 3). Photoelectrochemical detection takes advantage of the fact that optical energy is converted to electrochemical energy via electronic promotion. The excited state may be a better oxidizing agent (an electron may fill the lower energy, empty orbital) or a better reducing agent (the electron is more easily removed from its higher energy orbital) depending on the molecule. Thus, the electrochemicalproperties of the excited state should be different from the ground state. The PED uses a conventional thin-layer amperometric detection system, which has been modified such that the working electrode can be continuously irradiated with high-intensity ultraviolet light. As the analyte passes over the working electrode surface, it 0003-2700/87/0359-0049$0 1.50/0
is optically transformed to the excited state. The PED is designed to detect molecules or species derived from photogenerated excited states (singlets and/or triplets), intermediates, and/or products. A detailed discussion of the mechanism of detection will be covered in a forthcoming publication (4). HPLC-PED combines the selectivity of a commercial chromatographic system with the high sensitivity and additional selectivity of electrochemiial detection. Under optimized conditions of flow rate, gasket thickness, solvent composition (organic component and electrolyte), light intensity, and applied potential, the responses of over 50 compounds have been characterized. The responsive compounds were all carbonyl compounds or carbonyl derivatives. It is the goal of this paper to demonstrate the analytical utility of the PED by applying it to “real-world”applications, namely, the analysis of benzaldehyde in a variety of sample matrices. Production of benzaldehyde, or oil of bitter almonds, is in the millions of pounds per year (5). About 50% of the produced benzaldehyde has been used in the manufacture of odorants and flavoring agents. These compounds are used by the soap, perfume, and food industries. Thirty percent is used in the synthesis of various dyes. The remainder is accounted for as an intermediate in the manufacture of pharmaceuticals (analgesics, antipyretics, and antispasmodics)and fine organics. It may also be employed as an ingredient in compounding and dispensing, as well as a flavoring agent in pharmaceuticals. Benzaldehyde is a skin irritant and, in high enough quantities, a central nervous system (CNS) depressant (6). Since benzaldehyde is found in so many products, accurate and sensitive monitoring procedures are needed. In spite of 0 1988 American Chemical Society
50
ANALYTICAL CHEMISTRY, VOL. 59, NO. 1, JANUARY 1987
the existing and growing interest concerning the levels and fate of benzaldehyde and other carbonyl compounds in the environment, there is no simple, direct HPLC method available for the determination of nanogram amounts in complex matrices. Therefore, most of the literature assays involve chemical derivatization methods (7-16). HPLC-PED offers a simple method for the quantitative determination of trace amounts of benzaldehyde. The selectivity afforded by both the chromatographic system and the PED allows for sensitive analysis of benzaldehyde even in complex matrices. EXPERIMENTAL SECTION Apparatus. The HPLC-PED system consisted of a Model 501 solvent delivery pump (Waters Chrom. Div., Millipore Corp., Milford, MA), a Rheodyne 7010 injector with a 7011 needle port insert (Rheodyne Corp., Cotati, CA) modified for sample deoxygenation ( I n ,a LDC UVIII 254-nm ultraviolet detector (LDC Corp., Riviera Beach, FL), a BAS Model LC-4B amperometric system for liquid chromatography (Bioanalytical Systems, Inc., West Lafayette, IN), a modified BAS glassy carbon, single working electrode with silver/silver chloride reference electrode, a Model USSH-1O2D 100-W high-pressure Hg lamp with an LPS 200 power supply (Photon Technology International, Inc., Princeton, NJ) mounted on a 3-ft optical rail (Edmund Scientific Co., Barrington, NJ), and a Model D5217-la1 dual-pen Omniscribe recorder (Bausch & Lomb Co., Houston Instrument Division, Austin, TX). Waters stainless-steel (SS)fittings, ferrules, and tubing were used for all of HPLC-PED connections, except where the EC detector cell required BAS fittings. The SS auxiliary electrode was machined to accept a 7-mmdiameter hole opposite the working electrode; a rectangular &mm x 25-mm fused quartz window was cemented into a recess on the flow-through face of the auxiliary electrode with Duro-Brand epoxy cement (Loctite Corp., Cleveland, OH). By use of a 127-pm gasket to form the thin-layer channel, the completed cell was mounted opposite the arc lamp on the optical rail. The light was focused with an elliptical mirror such that the arc of the Hg lamp was at one focal point and the solution at and above the working electrode surface of the modified cell was at the other focal point. Detailed illustrations of the HPLC-PED instrumentation and modified thin-layer cell can be found in ref 2. HPLC mobile phases were degassed and filtered before use with 0.45-pm filters, catalog no. HAIF 04700, and a solvent filtration kit, catalog no. XXlO 047 30 (Millipore Corp., Bedford, MA). The mobile-phase reservoir was continuously refluxed at 60-70 "C with constant helium purge. The mobile phase was further deoxygenated with a 15-cm X 4.6-mm-i.d. SS column packed with 400-mesh granular zinc (Fisher Scientific Co., Pittsburgh, PA) (18). The samples were filtered with an LS025 Millipore disposable filter (Millipore Corp., Bedford, MA), placed in the sample vial of a modified injector, degassed for 3 min, and drawn through a 200-pL sample loop using a Model 9011 HPLC syringe (Hamilton Co., Reno, NV). All assays were performed by using a 10-pm Radial-PAK pBondapak CN column with a Model RCM-100 column compression system (Waters Chrom. Div., Millipore Corp., Bedford, MA) unless otherwise noted. The entire system was surrounded by a grounded, flat-blackcolored, aluminum box, in order to reduce UV light hazards to laboratory personnel and act as a Faraday cage. Reagents. All organic and inorganic reagents were obtained from commerical sources; Aldrich Chemical Co. (Milwaukee, WI), Pfaltz & Bauer Co. (Stamford, CT), Fisher Scientific Co. (Pittsburgh, PA), and J. T. Baker Chemical Co. (Phillipsburg, NJ). HPLC solvents were the Omnisolv brand from EM Science (Gibbstown, NJ). The benzaldehyde standard was identified by boiling point, infrared spectroscopy, ultraviolet spectroscopy, and nuclear magnetic resonance, all of which agreed with literature values (19-22). Purity of the standard was determined by HPLC-UV (23). One major peak comprising 99.6 f 0.8% (n = 3) of the total area was detected. Procedure. Each day the working electrode surface was polished with polishing alumina on a soft pad (Model PK-2, BAS).
After cleaning, the assembled thin-layer cell was mounted opposite the light source. The response was optimized by pumping a solution of 10 pM benzophenone in 50/50, v/v, MeOH/0.2 M NaCl through the cell. The signal was maximized by adjusting the x-yz positioner holding the cell. The benzophenone solution was washed out of the system with mobile phase. Caution should be observed when working with high-intensity UV radiation. The use of protective goggles is recommended, and bare skin should not be exposed to the irradiation beam. Preparation of Samples and Standards. Pure almond extract (Durkee Famous Foods, Cleveland, OH) samples for PED and direct-UV assay were prepared by diluting a 200-pL aliquot of the sample to 100 mL (solution A), and a 5-mL aliquot of solution A was diluted to 100 mL (solution B). All solutions were made with mobile phase. Similarly, a 200-pL aliquot of Amaretto di Saronno (Illva Saronno, Inc., Edison, NJ) sample was diluted to 100 mL. Pure almond paste (Marcipan Fabrik, Odense, Denmark) samples were prepared by weighing ca. 1.0 g of sample into a 100-mL Erlenmeyer flask. Fifty milliliters of mobile phase was added, and the mixture was shaken for 1 h using a Model S-500 wrist-type shaker (Kraft Apparatus, Inc., Mineola, NY). The sample mixture was passed through a fine, sintered glass funnel. The filtrate was collected in a 100-mLvolumetric flask and diluted to 100 mL. Salada orange pekoe and pekoe cut black tea (Salada Foods, Inc., Little Falls, NY) samples were prepared by leaching ca. 10.0 g of sample in 100 mL of boiling water for 5 min. The mixture was cooled and passed through a fine, sintered glass funnel. The volume of the filtrate was reduced to 45 mL by evaporation at 50 "C. The filtrate was collected in a 50-mL volumetric flask and diluted to volume. Approximately 1.5 g of whole almond (Planters, Nabisco Brands, Inc., East Hanover, NJ) was ground in a mortar and pestle. On gram of crushed almond and 50 mL of mobile phase were added to a 100-mLErlenmeyer flask and shaken for 14-16 h. The sample mixture was passed through a fine, sintered glass funnel, and the filtrate was collected in a 100-mL volumetric flask and diluted to 100 mL. Spiked samples for standard addition analysis were prepared by additions of benzaldehyde standard equal to twice and one-half the amount of benzaldehyde in the original sample aliquot. These amounts were optimum statistically (24). The sample aliquots were then treated as described for each analysis. A weighed aliquot (200 pL) of benzaldehyde standard was diluted to 100 mL (stock solution), and a 2-mL aliquot of stock solution was brought to a volume of 100 mL (stock solution A). Stock solution A, which was ca. 50 ppm, was further diluted to match the concentration of the sample (2 ppm to 50 ppb). Samples and standards were injected alternately and in triplicate. Quantitative results for direct and standard addition methods of analysis were derived by using routine analytical calculations.
RESULTS AND DISCUSSION Assay Development a n d Validation. As shown in Table I, benzaldehyde was determined to be responsive to the PED in flow-injection analysis (FIA). This listing emphasizes the carbonyl specificity of the PED. The response of benzaldehyde can be attributed to its carbonyl functionality, long-lived triplet state, and near-unit quantum efficiency of triplet formation. These are all characteristics necessary for PED detectability. It was necessary for the mobile phase to be compatible with the PED. Optimization and characterization of the PED were discussed in ref 2. As the percent MeOH in the mobile phase increased, the k'value decreased when using a cyano column. This same trend was observed when using i-PrOH with a C-18 column packing. A separation of benzaldehyde from interferences required using a mobile phase of 40:60 MeOH/water, 0.1 M NaCl (k' = 0.78) a t 0.8 mL/min. This mobile phase was used for all work unless otherwise noted. Using 0.1 M NaCl mobile phases has not resulted in any corrosion to the HPLC-PED system. After each use, the HPLC-PED system
ANALYTICAL CHEMISTRY, VOL. 59, NO. 1, JANUARY 1987
Table 11. Single-Blind Study of Spiked Water Samples (Benzaldehyde)
Table I. Responsive Compounds in FIA-PED" PED responses at 0.0 v
compd
lamp off
mobile phase acetone acetophenone acetylacetone (AcAc) 1-alanyl-1-phenylalanine 2-aminobenzaldehyde anthracene benzaldehyde benzophenone 2,3-butanedione carbazole cinnamaldehyde 1,4-cyclohexanedione 2-cyclohexen-1-one cyclohexanone Fe"' (AcAc) fluorene formaldehyde fructose indanone 4-methoxybenzaldehyde naphthalene 4-nitrobenzaldehyde 4-nitrobenzophenone
no
lamp on
6.06 0.24
,
,
0.2
4 ,
,
,
0.0
,
0.61
no no no
2.42
2.50 f 0.12 (103)
"Mobile phase 40:60 MeOH/HOH, 0.1 M NaCl, 0.8 mL/min; 200-pL injections; Radial-pak pBondapak CN column; glassy carbon electrode; 127-rm gasket; +0.3 V, oxidative mode; 10-mV full scale. Samples run in the order shown. bHPLC-UV a t 254 nm.
,
-0.4
10 ppm to 50 ppb. The line equation was nanoamps = 22.1 f 0.9 [ppm] 0.2 f 0.7, with the slope and intercept at the 95% confidence limits (t = 2.16, n - 2 = 13), and r = 0.997. This was a chosen range of quantitation and linearity of response was not determined outside of this range. The limit of detection was determined to be 25 ppb (200 pL, 5 ng) with a signal-to-noise ratio of 3. Fourteen 1.5 ppm solutions of benzaldehyde standard were prepared to determine repeatability of the system at the assay concentration. By use of the same solutions, system repeatability was f2.4% ( n = 14) for the PED and f1.4% (n = 14) for UV. A two-tailed F test showed no significant difference between the two variances at the 5% level. Repeatability drops to f16% near the limit of detection. All statistical methods and tables utilized in this work were taken from ref 26 and 27. A single-blind study of spiked (benzaldehyde) water samples were assayed in order to validate the method. Results, Table 11, showed a 98-10370 recovery of the spiked benzaldehyde for 0.619-12.12 ppm solutions. As the limit of quantitation was approached the recovery increased to 108 and 117% for the 0.24 and 0.12 ppm solutions, respectively. The results found by HPLC-PED were plotted against the spiked values, + 0.014 and a regression line of [ppm]pED = 0.999[ppmlspi~~ (r = 0.999) was determined. Further calculations showed that for 4 df an appropriate t value ( t = 2.78) gave the 95% confidence limits for the intercept and slope as 0.014 f 0.113 and 0.999 f 0.020, respectively. From these values and the correlation coefficient (r) it is clear that the calculated slope and intercept do not differ significantly from the ideal values of 1, 0, and 1, respectively, and therefore there is no evidence for systematic differences between the two sets of data. In addition, a comparison was made between the results obtained by the PED vs. UV (direct) in a similar fashion. A regression line of [ppm]pED = (1.011 f 0.018[ppm]w) + (0.030 f 0.102) ( r = 0.999) was determined. The 95% confidence limits are given using the same parameters as the previous calculations. These results show there is no evidence for systematic differences between the two sets of results. Applications. The assay for benzaldehyde was applied to foodstuffs to illustrate the analytical utility of the assay for complex matrices. The high selectivity and sensitivity of the HPLC-PED for the carbonyl function of benzaldehyde contributes to reduced sample preparation and simplified chromatograms. Almond Extract Food Flavoring. Almond extract food flavoring was determined to contain 18.4 h 0.7 ppth (parts per thousand) of benzaldehyde (Table 111). This agreed, within 98%, with the HPLC-UV assay of 18.7 f 0.6 ppth. PED results were compared to conventional HPLC-UV analysis. Since actual values for the analyte of interest could not be obtained, the HPLC-PED analysis results by external standards were also compared to HPLC-UV results obtained by standard addition data reduction. Standard addition would
+
no no no no no no no no no
,
5.84 f 0.39 (96)
0.26 f 0.18 (108) 11.99 i 0.57 (99) 0.13 f 0.11 (108) 0.62 f 0.43 (102) 2.61 f 0.07 (108)
no
no
.
5.94 f 0.39 (98)
0.26
12.12 0.12
no no no no no no
-0.2
found level, % recovery PED (ppm)" UV (ppm)b f 0.13 (108) 12.17 f 0.84 (100) 0.14 f 0.28 (117) 0.62 f 0.40 (102)
4140
,
spiked level, ppm
no no no
Mobile phase is 50:50 MeOH/0.2 M NaCl, 0.8 mL/min; 20-pL injections; glassy carbon electrode; equimolar injections of all compounds used.
0.4
51
1
1
-0.6
1
1
-0.8
APPLIED VOLTAGE
Flgure 1. Hydrodynamic voltammogram ( I M vs. E ) of benzaldehyde (2.03 ppm) in 40:60 MeOH/HOH, 0.1 M NaCi, at 0.8 mLlmin, with a 200-pL injection, on a glassy carbon electrode being irradiated.
was flushed for 30 min with a 1:l solution of methanol and water. The hydrodynamic voltammogram (HDV) for benzaldehyde is shown in Figure 1. An applied voltage of +0.3 V was chosen for all the assay work. The current was nearing the plateau, and above +0.3 V the background noise and sample background increased to an unfavorable level. The current was oxidative, in that a negative charge flow to electrode occurred. The high current at negative potentials is due to the fact that the response of the HDV is due to the response of a number of intermediates. The ketyl radical and ketyl radical anion undergo oxidation anodically from ca. -1.4 V, and the pinacol undergoes oxidation anodically from -0.3 V (25). Benzaldehyde itself reduces cathodically from ca. -1.5 V (25). Therefore, the HDV was used empirically to determine the best applied potential. Under these conditions, the PED response was linear from
52
ANALYTICAL CHEMISTRY, VOL. 59, NO. 1, JANUARY 1987
Table 111. HPLC-PED Assay Results of Benzaldehyde in Almond Extract Food Flavoring assay no.
HPLC detection method UV-direct, ppthb UV-std addn, ppthb
PED, ppth” 18.7 f 0.5 ( n = 18.2 f 0.3 ( n = 18.4 f 1.1 (n = 18.4 f 0.7 (n =
1 2 3
overall
3) 3) 3) 9)
19.1 f 18.1 f 19.1 f 18.7 f
0.2 0.2 0.3 0.6
( n = 3)
18.9 19.3 19.4 19.2
( n = 3) ( n = 3) ( n = 9)
( n = 9, r = 0.999)
( n = 9, r = 0.999) ( n = 9, r = 0.999) f 0.3 ( n = 3)
nSee Table I1 for experimental parameters; ppth, parts-per-thousand. * HPLC-UV at 254 nm; std addn, standard addition; r , correlation coefficient of standard addition line.
T 1
Table IV. Statistical Comparison of Methods”
HPLC-PED
VS.
almond extract amaretto liqueur almond paste tea
HPLC-UV (direct) t testb F testC 1.10 0.80 0.41 1.60
2 5 nA
HPLCUV (std addn) t testd
1.53
1.89 1.15 1.93 1.78
1.71 4.82 1.09
i 4.L i i i I ( I
0
” HPLC-PED
4
and HPLC-UV (direct) calculations were based on 8 df. HPLC-UV (std addn) calculations were based on 2 df. b P = 0.05 It1 = 2.12. c P = 0.05, IF1 = 4.43. d P = 0.05, It1 = 2.23.
correct for matrix interferences. The results of the PED were compared against results obtained by UV (direct) detection using a t test. The observed value of It1 was less than the critical value, and therefore the mean values can be considered as coming from the same population. A two-tailed F test suggests there is no significant difference between the two variances at the 5% level. In addition, a t test performed on the PED results vs. the UV standard addition results showed no significant differences between the mean values of each. See Table IV for statistical evaluation of all methods discussed. Amaretto Liqueur. A more complex sample matrix was encountered with amaretto liqueur. Figure 2 shows a chromatogram of benzaldehyde in amaretto. This illustration also emphasizes the lamp “on” vs. lamp “off“ selectivity of the PED. Any conventionally EC active components may be present in both chromatograms. Thus,in addition to retention time data, lamp “on” vs. lamp “off“ activity could be used for analyte identification, The amaretto liqueur contained 508 f 12 ppm benzaldehyde. As listed in Table V, these results are in agreement with HPLC-UV analysis by both methods.
8 1 2 1 6 0 4 81216 min. min. (b)
(0)
0
8
4
I2
min. (C)
1 2 3
overall a
PED, ppm 515 f 20 ( n = 3) 503 5 ( n = 3) 507 f 5 ( n = 3) 508 f 12 ( n = 9)
*
HPLC detection method UV-direct, ppm UV-std addn, ppm 520 516 503 513
f 5 (n= f 6 (n = f 3 (n = f 9 (n=
3) 3) 3) 9)
535 510 510 518
( n = 9, r = 0.999) ( n = 9, r = 0.999) (n = 9, r = 0.999) f 14 ( n = 3)
Table VI. HPLC-PED Assay Results of Benzaldehyde in Pure Almond Paste’
1 2 3
overall a
PED, PPm 112 f 106 f 105 f 108 f
See Table I1 for experimental parameters.
1 ( n = 3) 1 (n = 3) 1 (n = 3) 4 ( n = 9)
812 min. (d)
The statistical results shown in Table IV suggest no significant differences between the mean values of all three methods, and there is no significant difference between the variances of the PED results and UV (direct) results. Pure Almond Paste. The analysis of benzaldehyde in pure almond paste helps to support the ability of the PED to perform low-level analyses. The concentration of analyte was found to be 108 f 4 ppm (Table VI). These results agreed well with HPLC-UV analysis by external standard and standard addition analyses (Table IV). The calculated value for the two-tailed F test is greater than the critical value, so there is a significant difference between the variances of the results obtained by PED and UV (direct) analysis. The PED results were found to be more precise. Tea. Tea is an excellent sample, in that, in the drying process many aromatic compounds of varying functionalities
See Table I1 for experimental parameters.
assay no.
4
Figure 2. Chromatogram depicting lamp “on” HPLC-PED response of (a) benzaldehyde standard, (b)amaretto sample, (c) mobile-phase blank, and (d) amaretto sample with lamp “off”. I is the time of injection, and B is benzaldehyde. Conditlons were similar to Figure 1, and the applied voltage was +0.3 V.
Table V. HPLC-PED Assay Results of Benzaldehyde in Amaretto Liqueur” assay no.
0
HPLC detection method UV-direct, ppm UV-std addn, ppm
*
114 6 100 f 5 110 f 5 108 f 8
( n = 3) ( n = 3) ( n = 3) ( n = 9)
113 (n = 9, r 110 (n = 9, r 115 ( n = 9, r 113 f 3 ( n =
= 0.998) = 0.996) = 0.994) 3)
ANALYTICAL CHEMISTRY, VOL. 59, NO. 1, JANUARY 1987
53
Table VII. HPLC-PED Assay Results of Benzaldehyde in Teaa assay
PED, ppb
no.
*
243 11 (n = 3) 249 f 15 (n = 3) 246 f 9 (n = 3) 11 (n = 9) 246
1 2
3 overall
*
HPLC detection method UV-direct, ppb UV-std addn, ppb
*
250 12 (n = 3) 253 f 13 (n = 3) 260 f 5 (n = 3) 254 10 (n = 9)
*
246 (n = 9, r = 0.999) 271 (n = 9, r = 1.000) 262 (n = 9, r = 0.996) 259 12 (n = 3)
*
Mobile phase 25:75 i-PrOHIHOH, 0.1 M NaCl, 0.8 mL/min; 200-gL injections; Radial-PAK gBondapak C18 column; glassy carbon electrode; 127-fim gasket; +0.3 V, oxidative mode; 10-mV full scale. Sample run in the order shown. HPLC-UV at 254 nm. PED results agreed well with HPLC-UV.
ACKNOWLEDGMENT We gratefully acknowledge the assistance and discussions of B. Karcher, K. Bratin, and T. Gilbert. We also acknowledge the con$inued interest and technical support of Bioanalytical Systems, Inc., especially R. Shoup and P. T. Kissinger. Registry No. PhCHO, 100-52-7. I
I
PED
- L A M P ON
PED
-
L A M P OFF
~~
U V - 2 5 4 nm
U V - 214 nm
Figure 3. Comparison of PED and UV detection for whole almond hydrolysis solutions. I is the time of injection, and B is benzaldehyde. HPLC-PED conditions were similar to Figure 2.
are formed. The analysis of benzaldehyde in tea supports and illustrates the sensitivity and selectivity of the PED as compared to UV analysis. Benzaldehyde was determined to be 246 f 11 ppb. This result was in agreement with HPLC-UV results (Table VII). Statistical agreement between techniques was achieved as well; see Table IV. Whole Almond Hydrolysis Solution. Figure 3 compares the chromatograms of benzaldehyde in whole almond hydrolysis solution. Figure 3a,b emphasizes lamp "on" vs. lamp "off" peak identification. The response of the HPLC-PED for benzaldehyde was compared to HPLC-UV detection at 254 nm, Figure 3c. The background and solvent front were more pronounced for HPLC-UV (254 nm), with a reduced peak height for the analyte peak of interest. When the response for the analyte was increased by using 214-nm detection (Figure 3d), the background and solvent front increased dramatically.
CONCLUSION The PED with high-performance liquid chromatography is a highly selective and sensitive analytical technique for the determination of benzaldehyde and other carbonyl-containing compounds. These aspects of the PED allow for reduced sample preparation and simplified chromatograms. The analytical capability to do low-level analysis and handle complex sample matrices has been demonstrated, and the
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RECEIVED for review January 24,1986. Resubmitted August 25, 1986. Accepted September 3,1986. This work was supported by an ACS Analytical Division Fellowship, sponsored by the Society of Analytical Chemists of Pittsburgh, a Gustel Giessen Advanced Research Award, an award from the Research and Scholarship Development Fund from Northeastern University, a Barnett Institute Innovative Research Award, 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. 298 from the Barnett Institute at Northeastern University.
