Derivatisation techniques for the analysis of resin acids in pulp and paper industry effluent using high performance liquid chromatography

The environmental significance of resin acids originating from pulp and paper industry effluent has led to a detailed investigation of techniques used for their analysis. The chromatographic separation of a number of resin acids (based on those found in Pinus radiata) and their coumarin derivatives was investigated using High Perform- ance Liquid Chromatography (HPLC) with a view to developing a sen- sitive quantitative technique for analysis of total resin acids in pulp and paper industry effluent and in waterways receiving those effluents. Seven resin acids (sandaracopimaric, isopimaric. levopimaric, palust- ric, dehydroabietic, abietic. and neoabietic acid) were reacted with 4-bromomethyl-7-methoxycoumarin (4BrMMC) or 4-bromomethyl-7-acetoxy- coumarin (4BrMAC) to form (7-methoxy-coumarin-4-yl)methyl (MMC) esters and (7-acetoxy-coumarin-4-yl)methyl (MAC) esters respectively. The molar absorptivity of the resin acid MMC esters at 318 nm was shown to be the same for each resin acid ester, thus making it poss- ible to measure them quantitatively by HPLC even though they were not all chromatographically separated. Fluorescence quantum yield meas- urements of the MMC esters however showed a variation in fluorescence response that depended on the resin acid. There was also a variation in fluorescence quantum yield with solvent and water content of the solvent. These two factors precluded the use of the fluorescent properties of the MMC esters for quantitative analysis. Like the MMC esters, the molar absorptivity of the resin acid MAC esters was the same for each resin acid ester, thus enabling use of the chromophore at 310 nm for quantitative analysis. The resin acid MAC esters possessed no fluorescence. but reaction with alkaline methanolic solution was shown to hydrolyse the acetoxy group of the coumarin compound while maintaining the ester linkage to the resin acid. Fu1the1more the fluorescence response of each of the seven resin acids was the same in alkaline media. In the course of these studies, both 4BrMMC and 4BrMAC were found to decompose rapidly when exposed to light with up to half the reagent decomposing in only 30 minutes. The consequences of this photolabil- ity were explored to ascertain the probability of reactions with resin acids during the derivatisation reaction. Similar decomposit- ion products were observed for both coumarin compounds. In the presence of oxygen. 4-methyl-. 4-formyl-. 3-bromo-4-methyl-, and 3-bromo-4-formyl- derivatives of both methoxy and acetoxy coumarin were formed. In the absence of oxygen. and using a high intensity mercury discharge lamp light source. 4BrMMC was shown to produce 4-methyl-, and 3-bromo-4-methyl-7-methoxycoumarin as well as a photo- dimer. The latter two compounds eluted after the 4BrMMC peak thus posing a potential inte1ference problem for HPLC analysis. It was concluded that for the purposes of the analytical technique, it was important to protect solutions containing the coumarin derivatisation reagent from light during all stages of the derivatisation process apart from volumetric transfer of solutions. Derivatisation procedures rep01ted in the literature for similar types of reactions rely on the use of heat and crown ether catalysts to achieve rapid quantitative derivatisation. The effect of tempera- ture, crown ether catalyst, solvent and ratio of derivatisation reagent to resin acid were studied. Quantitative conversion to both the methoxy- and acetoxy-coumarin esters was obtained at room temp- erature, without catalyst. in either acetonitrile or acetone in I 0-15 mins., provided a 10-20 molar excess of derivatisation reagent was present. Application of the technique to extracts from water samples contain mg a variety of other organic compounds and resin acid concentrate- ions was pursued. Apart from the coumarin derivative of dehydroa- bietic acid. all of the resin acid coumarin esters studied co-eluted on a C-8 reverse phase HPLC column. A gradient elution programme based on acetonitrile and water was optimised to obtain adequate resolution of the resin acid coumarin esters from other components present m the effluent extracts. The method was applied to the analysis of resin acids in a river estuary into which pulp and paper effluent was discharged. Based on extraction of 100 ml samples. a detection limit of 0.005 mg C1 was calculated for the technique using 4BrMMC as the derivatisation reagent and UV absorption for detection. A detection limit of 0. OOO 1 mg C 1 was calculated for the techniques using 4BrMAC with fluorescence detection of the hydrolysed ester derivative. The lower detection limit associated with 4BrMAC esters offers a significant improvement over current gas chromato- graphy methods using flame ionisation detection.