Pulsed amperometric detection of aliphatic alcohols in liquid

Activated pulsed amperometric detection of cysteine at platinum electrodes in acidic media. Matthew E. Johll , Douglas G. Williams , Dennis C. Johnson...
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Anal. Chem. 1991, 63, 134-139

134

Pulsed Amperometric Detection of Aliphatic Alcohols in Liquid Chromatography William R. Lacourse* and Dennis C. Johnson

Department of Chemistry, Iowa State University, Ames, Iowa 50011 Maria A. Rey and Rosanne W. Slingsby

Dionex Corporation, P.O. Box 3603, Sunnyvale, California 94088-3603

The quantitative determination of aliphatic alcohols in liquid chromatography has been hindered by the lack of a sensitive detector. Aliphatic alcohols have no inherent chromophore or fiuorophore and are considered to be eiectroinactive under constant applied potential. The determination of underlvatired aliphatic alcohols by reversed-phase polymer-based chromatography with pulsed amperometric detection (PAD) is direct, sensitive, and simple. PAD was applied to the determination of simple alcohols at Pt and Au working electrodes under acidic and basic conditions, respectively. The combination of mixedmode chromatography with a chromophoric (UV-vis) detector and a nonchromophoric detector (PAD) is a powerful analyticai technique.

INTRODUCTION Numerous aromatic compounds are detected easily by oxidation at inert electrodes (i.e., Au, Pt, and glassy carbon) ( I ) . The effect of aromaticity upon the reaction mechanism is to stabilize the free-radical products of one-electron oxidation steps by 9-resonance. In contrast, this stabilization is not available for aliphatic organic compounds (e.g., amines and alcohols). This results in rates of oxidation that are often very slow, even though the reactions are favored thermodynamically ( 2 ) . Furthermore, the absence of *-bonding results in a poor chromophore and/or fluorophore. Alternatively, stabilization of free-radical intermediates can be achieved via adsorption to the surface of noble-metal electrodes that have unsaturated d-orbitals. The electrocatalytic benefit of the stabilization of free-radical intermediates is often accompanied by rapid fouling of the electrode surface by accumulated oxidation products (3). Hence, the general opinion of nonreactivity for aliphatic organic compounds on Au and Pt frequently is a result of surface fouling. Pulsed amperometric detection (PAD) overcomes fouling of electrodes by combining amperometric detection with alternated anodic and cathodic polarizations to clean and reactivate the electrode surface. PAD efficiently exploits the electrocatalytic activity of the clean, noble-metal electrode surface to oxidize aliphatic molecules. Although pulsed waveforms have been applied to carbon electrodes for maintenance of electrode surface activity ( 4 , s),detection of aliphatic organic compounds has not been successful probably because of carbon’s inability to stabilize free-radical intermediates. PAD was introduced in 1981 for the detection of simple alcohols at Pt electrodes (6). Although the viability of aliphatic alcohol determination was shown, this work used PAD with flow injection due to the lack of highly efficient pH-stable columns. Since that time, liquid chromatography (LC)-PAD has been applied to the direct detection of sugar alcohols, monosaccharides, oligosaccharides, amino glycosides, amino

alcohols, amino acids, and several sulfur compounds by using Pt and Au electrodes. Recently, a review of LC-PAD and its applications has been published ( 7 ) . Although the LC separation of aliphatic alcohols is easily achieved by using reversed-phase chromatography, their quantitative determination is hindered due to the lack of an inherent chromophore, fluorophore, or dc-active electrophore. Derivatization to benzoate esters is often used to improve the detection properties of aliphatic alcohols (8,9). On the other hand, reversed-phase chromatography with PAD is highly amenable to the determination of aliphatic alcohols. Determinations of alcohols in a variety of sample matrices by LCPAD are simple and direct. As described in one application in this paper, the combination of PAD, a nonchromophoric detector, with UV-vis detection allows for multiple determinations within a single chromatographic experiment. Described here are the development, optimization, and application of LC with PAD for the determination of alcohols and polyalcohols. The versatility of PAD a t Pt and Au working electrodes under acidic and alkaline conditions, respectively, is emphasized and illustrated by chromatographic examples. EXPERIMENTAL SECTION Reagents. All solutions were prepared from reagent-grade chemicals. Triply distilled water was further purified in a Millipore Milli-Q system (Millipore Corp., Bedford, MA). Acetonitrile was HPLC grade (Fisher Scientific, Springfield, NJ). All mobile phases were filtered before use with 0.45-ym Nylon-66 filters (Rainin Corp., Woburn, MA) and a solvent filtration kit (Rainin). Apparatus and Procedures. Voltammetric data were obtained at a Pt (ca. 0.18 cm2)or a Au (ca. 0.18 cm2) rotating disk electrode (RDE) (Pine Instrument Co., Grove City, PA) with a Model RDE4 potentiostat (Pine). Separations using acidic eluants were performed in an IonPac ICE-AS1 separator column (Dionex Corp., Sunnyvale, CA) in a Model 4500 isocratic/gradient chromatography system (Dionex). Isocratic chromatography was accomplished with a mobile phase of 50 mM HCIO, at a flow rate of 0.8 mL min-’, unless otherwise noted. PAD was performed with a Model PAD-I1 (Dionex) system at a Pt working electrode with a Ag/AgCl reference electrode. Gradient separations utilized an OmniPac PCX-500 column (Dionex) at a flow rate of 1 mL min-’. Eluants were 90% ACN (eluant A) and water (eluant B). The gradient program consisted of starting at 20% eluant A for 5.0 min and ramping linearly to 95% eluant A at 14.0, and at 14.1 min, the column was equilibrated with the weaker eluant for ca. 7 min. Postcolumn addition of 0.3 M NaOH was accomplished with a Model DQP reagent pump (Dionex) at 1 mL min-’, which was connected after the analytical column by a “mixing tee”. A 4-ft beaded reaction coil was placed after the mixing tee to facilitate the mixing of the reagent and eluent streams with minimal band broadening. PAD was performed at a Au working electrode with a Ag/AgCl reference electrode. Spectrophotometric detection at 254 nm was performed with a Model VDM-I1 detector (Dionex) placed between the analytical column and the mixing tee.

0003-2700/91/0363-0134$02.50/0 C 1991 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 63, NO. 2, JANUARY 15, 1991

II I

t ia Figure 1. Voltammetric response ( / - E ) for ethanol at a Pt RDE. Conditions: 50 mM HCIO,, 400 rev min-' rotation speed, 200 mV s-l scan rate, AglAgCi reference. Solutions: (-) supporting electrolyte with dissolved 02, (-) supporting electrolyte degassed, (---) 17.1 mM ethanol. Data collection was performed with a chart recorder (Bausch & Lomb Co., Austin, TX) or an IBM-compatible computer with Advanced Computer Interface (Dionex) and AI-450 software (Dionex). All samples were prepared by diluting a weighed portion of sample, diluting to the proper level with mobile phase, and filtering before injection. All injection volumes were 50 pL. RESULTS AND DISCUSSION Voltammetry. Although applications of pulsed electrochemical detection have typically been performed at Au electrodes, simple alcohols show virtually no electrochemical response at Au in acidic media. In contrast, Pt electrodes show good response for aliphatic alcohols even in 1 M HC104. The current-potential (i-E) response is shown in Figure 1 for a Pt RDE in 50 mM HCIOl with (- - -) and without (-) ethanol in the absence of dissolved 02. The residual response for the supporting electrolyte (-) exhibits anodic waves on the positive potential scan in the regions ca. -0.3 to +0.05 V (A) for oxidation of adsorbed hydrogen, ca. +0.5 to +1.4 V (B)for oxide formation, and E > +1.3 V (C) for O2evolution. The cathodic peak at ca. +0.6 to +0.1 V (D) results from dissolution of the oxide formed on the positive scan. The cathodic wave at ca. +0.05 to -0.3 V (E) is the formation of adsorbed hydrogen, and onset of hydrogen evolution at ca. -0.35 V (F) is observed on the negative scan. Cathodic reduction of dissolved O2is observed in the region of ca. E < +0.6 V (G)on the positive and negative scans. For the presence of ethanol (- - -), an anodic wave is observed on the positive scan in the region of ca. +0.1 to +0.75 V (H) in Figure 1, where alcohols are observed to be oxidized to the corresponding acid, and in the region ca. +0.75 to 1.4 V (I) for the anodic desorption of adsorbed ethanol and intermediates simultaneously with the formation of surface oxide on the Pt electrode (B). The absence of signal on the negative scan in the region ca. +1.4 to +0.4 V indicates the absence of activity of the oxide-covered surface for ethanol oxidation. When the oxide is cathodically dissolved on the negative scan to produce peak D, the surface reactivity for ethanol is im-

135

I

I

Figure 2. Voltammetric response (&E) for ethanol at a Au RDE. Conditions: 9% ACN/150 mM NaOH, 400 rev min-' rotation speed, 200 mV s-l scan rate, Ag/AgCI reference. Solutions: (-) supporting (-) supporting electrolyte degassed, (- - -) electrolyte with dissolved 02, 171 mM ethanol.

mediately returned and an anodic peak (J)is observed for the oxidation of ethanol. Anodic waves H, I, and J-areall observed to increase in signal with increases in the ethanol concentration. The net anodic current in wave H (Figure 1) for the detection of the alcohol functionality increases with increases in rotation speed yet exhibits little change with variations in scan rate. These observations support the conclusion that the mechanism producing wave H for ethanol is primarily under mass-transport control. In contrast, wave I increases with increases in potential scan rate yet shows a negligible change as a result of variation in rotation speed. These observations indicate that the anodic reaction for wave H is under the control of an electrode surface process. This is consistent with the proposed mechanism of an oxide-catalyzed desorption/ oxidation of adsorbed ethanol and stabilized intermediates on the electrode surface (IO, 11). The addition of acetonitrile to the supporting electrolyte virtually eliminates the electrochemical response for ethanol on Pt in acidic media. This observation is concluded to be the result of an interference by acetonitrile (ACN) with the adsorption mechanism of the alcohol. Acetonitrile is strongly adsorbed at Pt electrodes, and the adsorbed ACN is concluded to block surface sites required for a preadsorption step in the anodic detection of the alcohol. Under basic conditions in the presence of ACN, both Pt and Au electrodes exhibit a weak, surface-controlledresponse for the oxidation of ethanol. The response on the Pt electrode occurs simultaneously with the formation of surface oxide and in the region of dissolved O2 reduction. In contrast, the signal on the Au electrode is in a region with minimal oxide formation and with minimal contribution from reduction of dissolved 02.Thus, in the presence of ACN, a Au electrode under alkaline conditions is preferred for the most sensitive detection of aliphatic alcohols. The i-E response is shown in Figure 2 for a Au RDE in 9% ACN/150 mM NaOH with (---) and without (-) ethanol. The residual response for the supporting electrolyte (--) ex-

136

ANALYTICAL CHEMISTRY, VOL. 63, NO. 2, JANUARY 15, 1991 PERCENT C O M P 0 S I T I O h !%I I03 0

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hibits anodic waves on the positive scan in the regions of ca. +0.2 to +0.8 V (A) for oxide formation and E > +0.8 V (B) for O2 evolution and a cathodic peak on the negative scan in the region of +0.3 to -0.6 V (C) for the dissolution of the oxide formed on the positive scan. Cathodic reduction of dissolved 0, is observed in the region of -0.1 to -0.8 V (D) for both the positive and negative scans. The sharp, well-defined peak typical of a Au electrode at high pH tends to be rounded out and deformed by the presence of the acetonitrile. For the presence of ethanol (- - -), an anodic wave is observed on the positive scan in the region of -0.1to +0.4 V (E),where alcohols are observed to be oxidized. The absence of any anodic signal on the negative scan agrees with the conclusion of the absence of activity of the oxide-covered surface for ethanol oxidation. Scan rate and rotation speed data suggest that wave E is under the control of an electrode surface process. Chromatography. In liquid chromatography, aliphatic alcohols are generally separated under reversed-phase conditions. Polymeric-based stationary phases with mixed-mode separation capabilities are effective for the separation of simple alcohols in the reversed-phase-only mode. Tolerance for a wide range of pH conditions allows flexibility in choosing chromatographic conditions compatible with electrochemical detection requirements. A second mode of separation (Le., ion exclusion or ion exchange) offers additional chromatographic selectivity when ionic analytes are also present in the sample. Ion-exclusion columns contain styrene-based, fully sulfonated resin, which is normally used for ion-exclusion separations of weak acids. Separations that typically use these types of columns are accomplished via Donnan exclusion (acid strength and degree of ionization), steric exclusion (size), and adsorption/partitioning (hydrophobicity). In this application, the alcohols and polyalcohols are not ionizable. Retention of smaller alcohols (e.g., ethanol) is based mainly on inclusion, whereas larger alcohols are retained by adsorptive interactions with the resin. Separations of very hydrophobic alcohols (e.g., decanol) on this type of resin are impractical due to excessively long retention times. In addition, this type of resin swells in organic solvents, which precludes the use of solvent gradients. The capacity factor (k') for ethanol on an ion-exclusion column (Le., ICE-AS1 column) is nearly independent of pH (HCIO, concentration) and ionic strength (NaN03 concentration), as shown in Figure 3. The absolute difference in k ' between using HClO, and NaNO, is attributable to dissimilarity in the hydrated ionic volumes of the counterions.

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Figure 4. LC-PAD for 13 aliphatic alcohols at a R working electrode. Conditions were the same as in Figure 3, except the mobile phase was 50 mM HCIO,. Samples: (a) adonitol, 45 ppm; (b) erythitol, 36 ppm; (c) glycerol, 9 ppm; (d) ethylene glycol, 10 ppm; (e) methanol, 30 ppm; (f) ethanol, 45 ppm; (9) 2-propanol, 177 ppm; (h) 1-propanol, 202 ppm; (i) 2-butanol, 202 ppm; (j) 2-methyl-1-propanol, 120 ppm; (k) 1-butanol, 122 ppm; (I) 3-methyl-l-butanol, 364 ppm; (m) 1-pentanol, 365 ppm.

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Figure 5 . Comparison of blank injections using LC-PAD (A) without and (B) with woven reactor in-line. Conditions were the same as in Figure 4.

The larger hydrated ionic volume of H+ as compared to Na+ is reflected in a larger stationary-phase volume/pore size and, hence, a greater retention time for ethanol. The dashed line (Figure 3) illustrates the change in k'as one converts from the Na+ form to the H+ form of the stationary phase at a constant ion concentration. This change in retention time cannot be utilized for gradient chromatography due to the requirement of long equilibration times. In addition to offering the greatest selectivity for these columns, acidic mobile phases are ideally suited for PAD at a Pt working electrode. The LC-PAD separation of 13 polyalcohols, primary alcohols, and secondary alcohols is shown in Figure 4 t o illustrate the range and applicability of 100% aqueous separation of aliphatic alcohols. These types of separations are especially useful for more hydrophilic alcohols such as glycols. Under these conditions, any imbalance in the O2 level between the mobile phase and injection matrix is readily detected (see Figure 5A). Therefore, a woven Teflon mixing reactor (ca. 1-min delay time), which connects the column to the thin-layer electrochemical cell (Dionex), serves to minimize injected dissolved O2 peaks by equilibrating the column eluant with atmospheric oxygen (Figure 5B).

ANALYTICAL CHEMISTRY, VOL. 63, NO. 2, JANUARY 15, 1991

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Table I. Waveform for Pulsed Amperometric Detection (PAD) at Pt (A) and Au (B) Working Electrodes

ti

t

T

2 PA

1 TIME

section A potential, mV vs Ag/AgCl time, ms

section B potential, mV vs Ag/AgCl time, ms

El, +300

El, +lo0

E2, +1400 E3, -400

td, 280 ti, 20" tl, 300 t2, 120 t3, 420

E2, +lo00 Ea, -800

tdr 520 ti, 200 ti, 720 t*, 120 ts, 300

" Integration time is 16.7 ms of a 20-ms Deriod.

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As the ICE-AS1 column is incompatible with organic modifiers, gradient chromatography can be performed on an OmniPac 500 series column (12),such as the PCX-500. These columns are compatible with organic solvents, stable at pHs 0-14, and have both reversed-phase and cation-exchange functionalities. As neutral compounds, the alcohols are separated solely by the reversed-phase character of the column. As expected, the capacity factor is determined to decrease as the concentration of acetonitrile was increased. The gradient separation of 13 alcohols is shown in Figure 6. Alternatively, an IonPAC NS1 polymeric, reversed-phase column or a mixed-mode OmniPac PAX-500 with anion-exchange functionality can also be used to give identical separations to the PCX-500. Gradient chromatography with organic modifiers is essential for the separation of larger, less polar alcohols (e.g., 3-phenyl-1-propanol and 1-tetradecanol) from numerous matrix components. LC-PAD.For separations on the ion-exclusion column in acidic media, a Pt electrode is preferred. Table IA describes the optimized triple-step waveform for the pulsed amperometric detection of ethanol on Pt in 50 mM HCIO1. Upon stepping to the detection potential (El),the anodic signal is integrated for 16.7 ms (ti) after a delay of 280 ms ( t d ) . The signal for ethanol is detected simultaneous with the reduction of dissolved 0,;hence, the integration time is kept to a minimum to reduce noise caused by the randomly fluctuating

Figure 6. LC-PAD of 13 aliphatic alcohols at a Au working electrode. Chromatography: OmniPAC PCX-500 column, 18-85.5 % ACN gradient at 1.0 mL min-', postcolumn base addition of 0.3 M NaOH at 1.0

mL min-I, 50-pL injection. Detection: PAD, Au electrode; waveform, see Table I. Samples: (a) ethanol, 1840 ppm; (b) 1-propanol, 460 ppm; (c)2-methyC2-propen-l+l, 46 ppm; (d) cyclopentanoi, 460 ppm; (e) phenylmethanoi, 69 ppm; (f) 1-phenylethanol, 115 ppm; (9) 3phenyl-1-propanol, 115 ppm; (h) P-ethyl-1-hexanol, 460 ppm; (i) 1decanol, 460 ppm; 0) 1-undecanol, 920 ppm; (k) ldodecanol, 920 ppm; (I) 1-tridecanol, 920 ppm; (m) 1-tetradecanol, 920 ppm.

oxygen signal. Since the electrode activity diminishes due to inhibition by the fully developing surface oxide and by fouling of the surface by adsorbed oxidation products, electrode activity is renewed by subsequent positive and negative potential steps to achieve anodic (E,) and cathodic (E3)polarizations, respectively. In order to fully remove "cleaning" oxide from the electrode surface, the duration of the cathodic step ( t 3 ) should be at least 3 times the duration of the anodic step (t2). For gradient separations where acetonitrile is used as the organic modifier, a Au electrode under basic conditions gives the best results. Table IB lists the optimized triple-step waveform for the pulsed amperometric detection of ethanol on Au in 9% ACN/150 mM NaOH. Upon stepping to the detection potential (El),the anodic signal is integrated for 200 ms (ti) after a delay of 520 ms (ta). The signal for ethanol is detected without the simultaneous reduction of dissolved 02, and the long delay helps to reduce capacitance currents

Table 11. Quantitative Parameters of Aliphatic Alcohols at a Pt Electrode in 0.05 M HClO, compound

ppm, LOD"

sorbitol adonitol erythitol glycerol ethylene glycol methanol ethanol 2-propanol 1-propanol 2-butanol 2-methyl-1-propanol 1-butanol 3-methyl-1-butanol pentanol

10, 0.3 10, 0.3 10, 0.2 10, 0.2 10, 0.1 20, 0.1 20, 0.2 200, 1.1 50, 0.3 200, 2.1 50, 0.7 50, 1.2 50, 2.2 100, 2.3

linear range pA = a(ppm) + b a b 143 f 0.7 163 f 0.8 194 i 0.7 264 f 0.7 329 f 1.3 383 f 3.2 235 f 2.4 18 f 0.1 117 f 2.3 18 f 0.1 49 f 0.7 59 f 0.5 20 f 0.1 19 f 0.2

21 f 5.2 20 f 6.3 15 f 5.1 29 f 5.7 0 f 10 115 f 54 43 f 41 8 f 11 109 f 101 7 f 11 25 f 30 15 f 23 -4 f 4.3 -9 f 13

R2

repeatability %rsd (ppm; n)

0.999 94 0.999 93 0.999 97 0.999 98 0.999 95 0.999 72 0.999 58 0.999 93 0.998 04 0.999 94 0.99901 0.999 60 0.999 91 0.999 77

1.5 (10; 10) 1.1 (10;6) 1.2 (10; 6) 0.9 (10; 6) 0.9 (10; 6) 0.7 (10; 6) 0.8 (10; 6) 6.7 (10; 6 ) 3.3 (10; 6) 5.4 (10; 6) 3.6 (10; 6) 3.9 (10; 6) 2.8 (10; 6) 4.6 (10; 6)

"LOD = limit of detection determined for a signal-to-noise ratio of 3 from the lowest injected concentration. Average noise 13 nA.

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ANALYTICAL CHEMISTRY, VOL. 63, NO. 2, JANUARY 15, 1991

Table 111. Quantitative Parameters of Aliphatic Alcohols at a Au Electrode in X % ACN/0.15 M NaOH

linear range nA = a(ppm) + b

"OD

compound

ppm, LOD"

a

b

R2

repeatability %rsd (ppm; n)

ethanol 1-propanol 2-methyl-2-propen-1-01 cyclopentanol phenylmethanol 1-phenylethanol 3-phenyl-I-propanol 2-ethyl-1-hexanol

3680, 10 230, 5 20, 0.06 230, 0.8 70, 0.1 60, 0.2 120, 0.2 1840, 7

0.16 f 0.002 0.25 f 0.001 17.0 f 0.25 1.5 f 0.01 13.0 f 0.18 5.7 f 0.03 4.6 f 0.05 0.23 f 0.003

-9 f 6.8 0 f 0.1 0 f 4.4 0 2.1 2 f 10 0 f 1.1 3 f 5.1 1 f 4.5

0.999 24 0.999 99 0.999 58 0.999 88 0.999 40 0.999 96 0.999 60 0.999 39

7.6 (115; 4 ) 9.1 (30; 4) 11 (3; 4) 3.1 (30; 4) 2.6 (4; 4) 4.3 (7; 4 ) 2.4 (7; 4) 11 (60; 4)

*

= limit of detection determined for a signal-to-noise ratio of 3 from lowest injected concentration. Average noise 1.3 nA.

j6

f 1

d

'OOt 0' methano

IO glyco'

glycerol erythitol ALCOHOL

adonitol

sorbi:ol

Figure 7. Effect of the number of alcohol functionalities on (-) and (---) mass response factors.

i ,

molar 7 0

IC

0

to virtually nil. The weak signal is enhanced by increasing the integration time to 200 ms. As with the Pt electrode, surface activity is maintained by the application of alternated anodic ( E 2 )and cathodic (E3)polarizations. Table I1 lists the quantitative aspects of alcohol detection on a Pt electrode in acidic media. Regression analysis of calibration data indicates that all the alcohols tested are linear from 10 ppm to their limit of detection (LOD) with some maintaining linearity upward to 200 ppm. Limits of detection range from 0.1 ppm (160 nmol) for methanol to 2 ppm (1100 nmol) for pentanol. The repeatability of six or more injections for each alcohol ranges from 0.7% to 6.7% RSD (10 ppm each) with deviations being larger for those compounds nearer their detection limits. Figure 7 indicates that, as the number of alcohol groups on the molecule increases, the molar response reaches a plateau after the first three. This may be attributable to temporal limitations of the molecules at the electrode surface and steric hindrance. As the number of methylene groups increase (methanol to pentanol), the molar (255 f 1.9 to 38 f 1.8 nA/nmol) and mass (7.95 i 0.06 to 0.26 i 0 . 0 2 nA/nG) response factors decrease. Table I11 lists the quantitative parameters of simple alcohols on Au under alkaline conditions in the presence of ACN. In comparison to the results of alcohol detection at a Pt electrode in acidic media, it is apparent that the sensitivity is drastically reduced at Au in basic media. In addition, the Au electrode system (1.3-nA peak-to-peak noise) is an order of magnitude less noisy than that of the Pt system (13-nA peak-to-peak noise). The loss in sensitivity is attributable to the presence of ACN, which is strongly adsorbed to the Au electrode surface. The adsorbed acetonitrile blocks surface sites usually available for the anodic detection of the alcohol. These results are in agreement with previous conclusions that preadsorption of the analyte is required for the detection of the alcohol functionality (13). Signal attenuation attributed to the presence of ACN results in poorer detection limits; e.g., the

20

10

0r-r-&-3-0 TIME i m n l

Figure 8. Detection of alcohols in various samples by LC-PAD. Conditions were the same as in Figure 4. Samples were (A) toothpaste, (B) liquid cold formula, (C) brandy, and (D) wine cooler. Compounds detected were (a) glycerol, (b) sorbitol, (c) propylene glycol, (d) ethanol, (e) sugars, and (f) methanol. i b

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Figure 9. Cephalosporin antibacterial assay by cation-exchangeheversed-phase chromatography with (A) UV-vis detection and (B) PAD in series. Conditions were the same as in Figure 6, except the gradient system was 18-67.5% ACN and a constant 30 mM HCIO,. Compounds detected were (a) p-toluenesulfonic acid, (b) cefazolin, (c) 1,6-hexanediol, and (d) 1,4-~yclohexanediol.

LOD for ethanol is 10 ppm for the Au/base system and 0.1 ppm for the Pt/acid system. Although the more hydrophobic alcohols (i.e., decanol, undecanol, dodecanol, tridecanol, and

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Anal. Chem. 1991. 63. 139-146

tetradecanol) are separated and detected, quantitation is hindered by poor solubility and the formation of micelles. Applications. Since a prerequisite for PAD reactivity is adsorption of the analyte, the presence of other surface-adsorbable substances, as well as electroactive compounds, can act as interferences. Therefore, general selectivity is achieved via chromatographicseparation prior to PAD. This conclusion does not preclude additional selectivity from control of detection parameters. The assay for alcohols was applied to several matrices to illustrate the analytical utility of the procedure. Separation on an ion-exclusion column with direct detection is illustrated in Figure 8 for various aliphatic alcohols and polyalcohols in toothpaste (A), liquid cold formula (B), brandy (C), and wine cooler (D). The selectivity for alcohols in acidic media at a Pt electrode contributes to decreased time for sample preparation and simplified chromatograms. The versatility of separations on mixed-mode ion-exchange columns with selective detection is illustrated in Figure 9 by the simultaneous detection of ionic and neutral species in a pharmaceutical preparation. This experiment utilizes a UV detector and PAD in series after a PCX-500 column. Under acidic conditions, the cephalosporin antibacterial consists of a cation (i.e., cefazolin) and neutral and anionic compounds (i.e., lB-hexanediol, 1,4-cyclohexanediol,and p-toluenesulfonic acid). The neutral and anionic compounds are separated by the reversed-phase character of the column, while the cationic compound is separated by a combination of cation exchange

and reversed-phase mechanism. Figure 9 shows that the p-toluenesulfonic acid and the cefazolin are both detected by UV at 254 nm (A), and the two diols, which do not have a chromophore, are easily detected by PAD (B). In addition, the cefazolin has a PAD signal, which has a higher limit of detection than UV, but may be utilized for added selectivity. LITERATURE CITED Kisslnger, P. T. In Laboratory Techniques in Electroanalytical Chemistry; Klssinger, P. T., Heineman, W. R., Eds.; Marcel Dekker: New York, 1984; pp 631-32. Adams, R. N. Electrochemistry at SolM Electrodes; Marcel Dekker: New York, 1969. Gllman, S . I n E/ectroana/ytica/ Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1967; Vol. 2, pp 111-92. Fleet, B.; Little, C. J. J. Chromtogr. Sci. 1874, 72, 747. Van Rooljen, H. W.; Poppe. H. Anal. Chim. Acta 1881, 730,9. Hughes, S.; Meschi, P. L.; Johnson, D. C. Anal. Chim. Acta 1881. 732,1. Johnson, D. C.; Lacourse, W. R. Anal. Chem. 1890. 62,589A. Nachtmann, F.; Budna. K. W. J. Chromtogr. 1977, 736,279. Jupille, T. J. Chromatogr. Sci. 1978, 77, 160. Beden, B.; Cetin, I.;Kahyaoglu. D.;Takky, D.; Lamy, C. J. Catal. 1987, 704, 37.

Ocon, P.; Alonso, C.; Celdran, R.; Gonzalez-Velasco,J. J. Electroanal. Chem. 1888, 206, 179.

Slingsby, R. W.; Rey, M. J. Li9. Chromatogr. 1880, 73(1), 107. Lacourse, W. R.; Jackson, W.A.; Johnson, D. C. Anal. Chem. 1888, 67, 2486.

RECEIVED for review July 20,1990. Accepted October 8,1990. The financial support of Dionex Corporation is acknowledged with gratitude.

Statistical Treatment for Rejection of Deviant Values: Critical Values of Dixon’s “ Q ” Parameter and Related Subrange Ratios at the 95% Confidence Level David B. Rorabacher

Department of Chemistry, Wayne State University, Detroit, Michigan 48202

CrHlcal values at the 95% Confidence level for the two-tailed 0 test, and related tests based upon subrange ratlos, for the statlstkal rejectlon of outlying data have been Interpolated by applying cublc regresslon analysls to the values orlglnally published by Dlxon. Corrections to errors In Dixon’s orlglnal tables are also Included. The resultant values are judged to be accurate to wlthln f0.002 and corroborate the fact that correspondlng crltlcal values published In recent slatlstlcal treatlses for analytical chemlsts are erroneous. I t Is recommended that the newly generated 95% crltlcal values be adopted by analytical chemlsts as the general standard for the rejection of outller values.

Analytical chemists depend upon the generation and interpretation of precise experimental data. As a result, they are especially cognizant of the value of statistics in data treatment, and a number of statistical treatises have recently been published that are specifically written for the professional analytical chemist ( I ) . Included in each of these publications is a brief section dealing with tests for the rejection of grossly deviant values (outliers). Although many statistical tests have been proposed to deal with this topic [Barnett and Lewis (2) discuss 47 different equations designed for this purpose], it 0003-2700/91/0363-0139$02.50/0

is interesting to note that these treatises, as well as essentially all analytical chemistry textbooks published in the U.S.during the past decade (3),have settled on the use of Dixon’s Q test (and variants thereof) ( 4 ) as the primary method for testing for the rejection of outlying values. Each of the recent statistical treatises written for analytical chemists has attempted to include critical values of Q for the 95% confidence level, values that were not included in Dixon’s publications. However, not only do the 95% confidence values differ in each treatise but all compilations contain significant errors. The most legitimate set of 95% values is that presented by Miller and Miller (4 In I10) ( l a ) ,which they attribute to King (5),but no such values are listed in King’s article, and the values of Miller and Miller differ by amounts varying from 0.002 to 0.007 from the 95% values presented in the current manuscript. Anderson ( I b ) describes the equations corresponding to the two-tailed tests for Dixon’s parameters designated as rl0 (for 3 5 n 5 lo), rll (for 8 5 n Ilo), and rZ1(for 11 In I13) and purportedly lists critical values for the 90%,95%, and 99% confidence levels for these sample sizes, but the values actually listed in his table are Dixon’s values for one-tailed tests. Thus,as applied to two-tailed tests, Anderson’s confidence levels should be labeled 80%, 9070, and 98% (vide infra). Caulcutt and Boddy (IC), while describing only the equation for the Q (i.e., rl0)ratio, accurately list both 0 1991 American Chemical Society