Crown Ether Catalyzed Derivatization of Carboxylic Acids and Phenols with Pentafluorobenzyl Bromide for Electron Capture Gas Chromatography Bruce Davis Psychiatric Research Division, University Hospital, Saskatoon, Saskatchewan, Canada S7N 0 W8
Quantitative derlvatiratkn of a wlde variety of carboxylic a c k and phenols with pentafluorobenrylkomMe has been achieved using 18-crown-6 as a catalyst, with a potasslum base In organic solvents. The hlgh reactivity of the acid anion toward dlsplacement of the bromkle ion obviated the necesdty of uslng a large excess of reagent and the subsequent removal of the excess before electron-capture gas chromatography. By empioylng a relatively strong potasslum base such as potassium carbonate, both carboxyilc aclds and phenols were derlvatired, whereas a weak base such as potasdum cyanide resulted In derivatiratkn of carboxylic acMs but not of phenols. The PFB derivatives of a number of ailphatlc and arylalkyl adds have been successfully separated by gas chromatography on a 7-ft glass column packed with 4.0% OV-17 on Gas Chrom Q. The minimum detectable amount for standard soiutlons is about 0.3 pmoi.
Electron-capture gas chromatography (EC-GC) has proved to be of great utility in the quantitative determination of carboxylic acids, phenols, and phenolic and catecholic acids in biological materials (1-10) and in the environment (11,12). High sensitivity can be achieved by derivatization with fluorinated reagents. The acid metabolites of the catecholamines have been quantitated by EC-GC after derivatization with fluorinated acid anhydrides and an alcohol (1-10, 13). The pentafluorobenzyl (PFB) derivatives of a number of aliphatic acids and phenols have been prepared and their gas chromatographic properties investigated (11, 12); derivatization occurs when the acid or phenol in strongly basic acetone or alcohol is heated with pentafluorobenzyl bromide for some hours. More recently, Ehrsson (14) and Gyllenhaal e t al. (15)have described derivatizations with PFB bromide in which aliphatic acids in a basic aqueous medium are extracted by means of a positively-charged counterion into an organic phase containing the PFB bromide. In a poorlysolvating organic phase, the anion of the acid or phenol becomes highly reactive in the nucleophilic displacement of bromide ion from the reagent. However, for the short chain acids including the phenylacetic acids (16), the partition ratio between the organic and aqueous phases is extremely low, resulting in inefficient extraction of the acids into the organic phase and thus in very slow or no reaction with the derivatizing agent. This paper describes a fast, simple and virtually quantitative one-phase derivatization of a wide variety of acids and phenols with FFB bromide in poorly-solvating organic' solvents, catalyzed by the addition of a crown ether and a basic potassium salt. The synthesis of acetate esters from potassium acetate and alkyl bromides catalyzed by 18-crown-6 (17,18) and the crown ether-catalyzed derivatization of fatty acids by a,p-dibromoacetophenone (19) have been previously described. EXPERIMENTAL Apparatus. Analyses were carried out on a Hewlett-Packard Model 5710A gas chromatograph equipped with a 63Nielectron 832
ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977
Table I. Reaction Conditions for Derivatization of m-Hydroxyphenylacetic Acid with Pentafluorobenzyl Bromide
Solvent Acetonitrile
Temperature, Time, " C min 20
Ethyl acetate
20
Dimethylformamide
40 20 80
5 10 25 50 60 60 60 30 60 90 15 30 90 120 5 35 120 10 30
40 80
90 120
Benzene
40 80 20 40 80
Heptane
Peak area 3.8 4.1
4.5 4.0 4.8 2.7 3.9 3.0 3.9 12.0 3.7 5.5 4.4 10.0 0.1
0.5 1.2 1.1
obscured by artifacts 0
0
capture detector and using 5.14% methane in argon as carrier gas. The mass spectra were recorded at 70 eV on an AEI-MSSO2S double focusing mass spectrometer. Reagents. Pentafluorobenzyl bromide (Pierce, Rockford, Ill.) and 18-crown-6 (1,4,7,10,13,16-hexaoxacyclooctadecane) and 15-crown-5(1,4,7,10,13-pentaoxacyclopentadecane)(PCR Inc., Gainesville, Fla.) were used without further purification. All solvents were pesticide grade, distilled from glass. The potassium salts (acetate, cyanide, bicarbonate, carbonate, and hydroxide) were reagent grade. The liquid phases OV-17,OV-25,and OV-101 (Pierce) and the solid supports Gas Chrom Q (AlltechAssociates, Arlington Heights, Ill.), Anakrom Q and SD (Analabs Inc., North Haven, Conn.), and Chromosorb 750 (Johns-Manville, Denver, Colo.) were packed in silylated glass columns. Procedure. Acetonitrile, benzene, ethyl acetate, dimethylformamide, and heptane solutions containing PFB bromide (3.0 pL/mL) and 18-crown-6(3.0mg/mL) were prepared and could be stored for weeks without deterioration. To derivatize up to 10 pg of carboxylic acids and/or phenols, 50 pL of the derivatizing solution was introduced into a 0.3-mL Reacti-vial containing the dry acids or phenols, a few grains of powdered potassium carbonate were added, and the mixture shaken occasionally. Powdered potassium acetate, bicarbonate or cyanide are added if one wishes only the carboxylic group of a phenolic acid to be derivatized. Reaction times and temperatures for various solvents are presented in Table I for m-hydroxyphenylacetic acid, but they apply to other acids as well. An aliquot (0.5 to 1.0 wL) of the reaction solution was injected directly onto a in. X 7 f t glass column packed with 4.0% OV-17 on Gas Chrom Q (see Figures 1 and 2 for chromatograph conditions). For the phenolic and catecholic acids, a mixed derivative may sometimes be desirable and, in such cases, the carboxylic acid
group was first esterified (5 min at 25 "C) with 20% HC1-methanol (or other alcohol) (prepared by adding acetyl chloride, 1.0 mL, dropwise to methanol, 4.0 mL), the methanol was evaporated, and then the phenolic groups were derivatized as described above, using potassium carbonate as the base. Standard solutions of the acids were prepared in ethyl acetate; acetic acid should be dissolved in benzene or ether because of the presence of acetic acid in ethyl acetate. Aqueous solutions of the acids were saturated with sodium chloride and extracted with ethyl acetate. The solvent was evaporated, the residue dissolved in benzene, dried with sodium sulfate, and chromatographed on a short column of silica ( 5 mm in a disposable pipet). The acids were washed from the column with ethyl acetate (some aliphatic acids were also eluted with benzene), the ethyl acetate was evaporated in a stream of nitrogen and the residue was further dried at 100 "C for 60 min (unless the acid is volatile at 100 "C). Rigorous drying is not necessary if large (i.e. microgram) quantities are to be derivatized. Calibration curves were prepared in two ways. In the first, 2 pg of each of the arylalkyl acids and caproic acid was derivatized in benzene at 40 "C for 90 min and then successively diluted to give five solutions which would have concentrations 8 ng/pL, 2.7 ng/pL, 800 pg/pL, 270 pg/pL, and 80 pg/pL if derivatizationwere complete. In the second way, five different quantities of the acid, 20 ng, 60 ng, 200 ng, 600 ng, 2 pg were derivatized and diluted to give the same range of concentrationsas above, assuming 100% derivatization. For both series, log peak area was plotted vs. log picograms injected.
RESULTS AND DISCUSSION Derivatization of acids and phenols with pentafluorobenzyl bromide catalyzed by 18-crown-6in the presence of a suitable potassium base is fast, simple to carry out, requires little or no workup, and is virtually quantitative. The crown ether functions by complexing the potassium cation (20,21),leaving the acid or phenol anion "naked' in poorly solvating solvents and therefore extremely reactive in the nucleophilic displacement of bromide ion from the reagent. The compounds included in this investigation are: acetic, propionic, butyric, valeric, isovaleric, caproic, octanoic, oleic, lactic, hippuric, benzoic, mandelic, phenylacetic, phenylpropionic, indoleacetic, m- and p-hydroxyphenylacetic, m- and p-hydroxymandelic, homovanillic and 3,4-dihydroxyphenylacetic acids. The reaction conditions of time, temperature, solvent, and reactant concentrations have been optimized. Concentrations of the crown ether and PFB bromide as low as 0.1% are sufficient for derivatization, although when the concentration of nucleophile is also low (
