Anal. Chem. 1995,67, 1293-1295
HPLC-FPD Instrumentation for the Measurement of the Atmospheric Dimethyl Sulfide Precursor pl( Dimethylsulf0nio)propionate A. 0. Howard* and D. W. Russell Chemistty Department, University of Southampton, Southampton, Hampshire, SO17 lBJ, U.K.
dependent on salinity,14-16day length,I7temperature,16J8and light Instrumentation has been specilkally developed for the inten~ity.’~ measurementof /3-(dimethy1sulfonio)propionate (DMSP), While the release of dimethyl sulfide has been extensively used a compound implicated as a major precursor of atmoto measure the supposed levels of DMSP, the technique is flawed spheric dimethyl sulfide. The instrument links the sepain that other naturally occurring sulfonium compounds may also ration and identificationpossible by ion-exchangeHPLC yield DMS with alkali.lg This is rarely acknowledged, and the with selective postcolumn hydrolysis and quantitative results of such measurements are frequently cited as being flame photometric detection. Compound selectivity is derived solely from DMSP. Several such tertiary sulfonium therefore gained from three independent characteristics: compounds have so far been found including 4(dimethylsulfonio)retention time behavior, base hydrolysis resulting in the 2 - m e t h o x y b ~ t y r a t eSmethylmethionine,21*zz ,~~~~~ and (dimethylsulproduction of a volatile sulfur compound, and sulfurfonio)-5pentanoate23 (this may however be a product of the action spec& detection. The instrumentation described in this of the 6 N HCl used in the extraction on a side chain of paper therefore provides the basis for the iirst reliable g l u c o e r u ~ i n . ~ ~ identification and quantitation of DMSP at trace levels. Recently, direct methods of DMSP detection have been The 3a detection of the prototype instrument, using a 200 these include fast atom bombardment mass spectrom pL injection volume, was 2.2 x lo-’ mol of DMSP ~ I I -developed; ~ etry,8,10,25 gas chromatography of Sdemethylated silyl derivatives or an absolute injected mass of 6 ng of DMSP. This of sulfonium compound^?^ and ‘H NMRZ5 These are, however, corresponds to 36 pg of Ss-’.The instrument has been relatively insensitive (lowest detected concentration 20 mmol kg-1 employed in the measurement of DMSP in marine macfresh weight) and are mainly only used for characterization, the roalgae. The environmental significance of dimethyl sulfide (DMS) is wide ranging. In 1972 this compound was proposed as the important “missing l i n k in the sulfur cycle, linking the hydrosphere and the atmosphere.’ In the atmosphere, DMS is rapidly photooxidized to methanesulfonic acid, sulfur dioxide, and sulfate, contributing to the acidity of precipitation? Since Haas3f i s t identified dimethyl sulfide as the “odoriferous principle” evolved from the macroalgae Polysiphoniu fastigutu in 1935, there has been a wealth of papers concerning DMS and its likely biological precursor P-(dimethylsulfonio)propionate (DMSP). DMSP was first extracted and characterized from P. fastigata by Challenger and Simpson in 1948.4 A presumptive test for DMSP was developed based on the evolution of DMS on treatment with cold alkali4 This indirect method has since been used almost exclusively, the DMS evolved being swept into a flame photometric detector (FPD). The procedure was later modified to include a cryogenic trap to concentrate the DMS prior to detecti0n.j Using this method, DMSP has been identified and quantified in phytoplankton,6 ~ e a w a t e rgrasses!-” ,~ and a wide range of marine m a c r ~ a l g a e . ~DMSP ~ J ~ content was found to (1) Lovelock, J. E.; Maggs, R J.; Rasmussen, R. A. Nature 1972,237, 452453. (2) Malin, G.; Turner, S. M.; and Liss, P. S.; J. Phycol. 1992,28, 590-597. (3) Haas. P.; J Biochem. 1935,1297-1302. (4) Challenger, F.; Simpson, M. I.; J. Chem. SOC.1948,43,1591-1597. (5) Andreae, M. 0.; Barnard, W. R; Anal. Chem. 1983,55,608-612. (6) Ackman, R. G.; Tocher, C. S.; Mchchlan, J.J Fish.Res.,Board Can. 1966, 23,357-364.
0003-2700/95/0367-1293$9.00/0 0 1995 American Chemical Society
presumptive test being resorted to for most quantitative measurements and to achieve lower detection limits.8 This paper reports the development of prototype instrumentation which has been specifically developed for the measurement Malin, G.; Turner, S.; Liss, P.; Holligan, P.; Harbour, D. Deep Sea Res.1993, 40, 1487-1508. Paquet, L.; Rathinasabapathi, B.; Saini, H.; Gage, D. A; Hanson, A. D. Plant Physiol. 1993,102 (5), 163. Pakulski, J. D.; Kiene, R. P. Mar. Ecol. Prog, Ser. 1992,81, 277-287. Hanson, A D.; Rivoal, J.; Gage, D. A Abstracts ofPapem, 202nd National Meeting of the American Chemical Society, New York, Aug 25-30, 1991; American Chemical Society: Washington, DC, 1991; 202, 158. Larher, F.; Hamelin, J.; Stewart, G. R Phytochemistry 1977,16, 20192020. Blunden, G.; Gordon, S. M. Prog. Phycol. Res. 1986,4, 39-75. Reed, R. H. Mar. Biol. Lett. 1983,4,173-181. Reed, R H. J. Exp. Mar. Biol. and Ecol. 1983,68, 169-193. Dickson, D. M. J.; Wyn Jones, R G.; Davenport, J. Planta 1982,155,409415. Kirst, G. 0.;Theil, C.; Wolff, H.; Nothnagel, J.; Wanzek, M.; Ulmke, R Mar. Chem. 1991,35, 381-388. Karsten, U.; Wienke, C.; Kirst, G. 0. Plant, Cell Enuiron. 1990,13, 989993. Karsten, U.; Wienke, C.; Kirst, G. 0. Polar Biol. 1992,12, 603-607. Challenger, F. Aspects of the Organic Chemistry of Sulfur; Butterworths Scientific Publications: London, 1959. Sciuto, S.; Piatelli, M.; Chillemi, R Phytochemistry 1982,21, 227-228. McRorie, R. A; Sutherland, G. L.; Lewis, M. S.; Barton, A D.; Glazener, M. R.; Shive, W.J Am. Chem. SOC.1954,76, 115-118. White, R J. Mar. Res. 1982,40,529-535. Larher, F.; Hamelin, J. Phytochemistry 1979,18, 1396-1397. Hanson, A. D.; Huang. Z.-H.; Gage, D. A. Plant Physiol. 1993,101, 13911393. Storey, R.; Gorham, J.; Pitman, M. G.; Hanson, A D.; Gage, D. J. Exp. Bot. 1993,44,1551-1560.
Analytical Chemistry, Vol. 67,No. 7,April 1, 1995 1293
Filter \ PhotomultiDlier
L1-l
~
_
_
_
~
_
~
~
Table 1. Variation of DMSP Retention Time with the pH of the Eluent
PH
retention time (min)
3.0 3.5 4.2
22.2 15.5 11.1
Amplifier
n
mL min-l) and carried to a gashquid separator where the DMS was purged from solution. Liquid was pumped from the separator Computer Waste to waste. The gas stream then passed through two traps to remove the water vapor that would otherwise extinguish the air/ Figure 1. Schematic representation of the flame photometric HPLC hydrogen flame of the flame photometric detector. The first detection system. drying trap was empty and used to physically condense water vapor and spray, the second contained the chemical drying agent of DMS precursors such as DMSP. The instrument links the anhydrous magnesium perchlorate (14-22 mesh). separation and identification possible by HPLC with selective The gas stream then passed into the air/hydrogen flame of a postcolumn hydrolysis and flame photometric detection. Comcustom-built flame photometric detector. The emission arising pound selectivity is therefore gained from the combination of from 5& species in the flame was monitored by a high-sensitivity chromatographic, chemical, and spectroscopic means. photomultiplier tube (EM1 6256B, run at 800 V) viewing through EXPERIMENTAL SECTION a widebandpass glass filter (Oriel BG12). The signal was Standardsand Reagents. DMSP was synthesised by stirring amplified, damped, and displayed on a chart recorder or computdimethyl sulfide Uanssen 99K+ GC) with acrylic acid (Aldrich) ing integrator. Increased selectivity can be achieved using a at room temperature for 3 days. The DMSP was then precipitated narrow-bandpassinterference filter, but at the expense of sensitivfrom toluene solution by passing hydrogen chloride through the ity. mixture." The product was filtered from the remaining solution, (d) Calibration. The instrument was calibrated by using peak washed with toluene, and then vacuum dried. height or area measurements obtained from standard solutions Analytical reagent grade materials were employed elsewhere and taking into account the intrinsic nonlinearity of all flame unless otherwise stated. photometric detector systems based on the 5& emission. Instrumentation. The apparatus employed in this work was Analysis of Algae. Sample Preparation. Samples of algae specially designed and constructed for the measurement of DMSP. were frozen with liquid nitrogen and then ground to a fine powder It incorporates HPLC of the DMSP followed by postcolumn base in a mortar and pestle. The algae (100 g) was then extracted hydrolysis of the eluting DMSP. This results in the production with methanol (20 mL), the alcoholic extract was then filtered of DMS, which, being a volatile species, can be separated from from the solid, and the residue was rinsed with a further 30 mL the liquid stream and detected using a custom-designed flame of methanol. The filtrate was concentrated and then made up to photometric detector (Figure 1). 10 mL in methanol. An aliquot of the extract was spiked with (a) HPLC Conditions. The HPLC separation of DMSP from DMSP, and both the spiked and the unspiked samples were then coextracted material and its identification was carried out by subjected to a cleanup procedure to remove coextracted pigments, cation-exchange chromatography on a Spherisorb 5 SCX column sugars, and other carbohydrates. The resulting clear aqueous (5 pm packing, 25 cm x 4 mm i.d.). The column was eluted with samples were then analyzed for DMSP using the apparatus aqueous potassium dihydrogen orthophosphate (0.05 m ~ l d m - ~ , described previously. Fuller details of the sample preparation pH adjusted to 5.7 with sodium hydroxide) at a flow rate of 0.8 procedures currently under development, and the levels of DMSP mL min-l. A Du Pont liquid chromatographypump was employed found in a variety of marine algae, will be published at a later to deliver the eluent, and the sample was injected onto the column date. using a Rheodyne 7125 injection valve fitted with a 20 or 200 pL loop. RESULTS AND DISCUSSION (b) Postcolumn Hydrolysis. The HPLC eluent stream (0.8 Instrument Performance. (a) Chromatography of DMSP. mL min-l) was subjected to on-line base hydrolysis by mixing DMSP can be satisfactorilychromatographed on both anion- and with a pumped stream (2.0 mL min-l) of sodium hydroxide cation-exchange chromatography systems. In this work, cationsolution (4 mol dm-3). The rate of hydrolysis was enhanced by exchange chromatography has been largely employed using an passing the mixture through a PTFE tube (length 650 cm, 0.7 aqueous phosphate solution as eluting solvent. The DMSP is well mm i.d.) which was maintained at 80 OC in a temperature-regulated separated from the solvent front, and its retention time can be oven. Higher temperatures increased the apparent yield of DMS controlled by varying the pH of the eluent. but only at the expense of additional water vapor, which resulted Varying the pH alters the degree of ionization of the carboxylic in increased instrument noise and deteriorating signal-to-noise acid group on the DMSP, which in turn controls the net charge characteristics. Incorporation of the derivatization module reand the retention time vable 1). sulted in a small deterioration in the apparent plate count (330A typical HPLC trace, obtained from the system, is shown in 260 plates). Figure 2. (c) DMS Detection. Following the hydrolysis step, the (b) Calibration. The linear regression of a log (concentration) resulting solution was mixed with a nitrogen stream (flow rate 50 vs log (response) plot gives a gradient of -2 (Figure 3), which is 1294 Analytical Chemistry, Vol. 67, No. 7, April 1, 1995
'
r
-
.
B. Fuw extract
Time.
I min
Figure 2. Typical chromatograms showing chromatograms resulting from 200 yL injections of (A) a standard solution containing 1.2 x mol dm-3 DMSP and (B) an extract from the marine macroalgae Fucus. 2.50
-E E
CONCLUSIONS
2.25
I
c
c CI) I-
2 2.00 .m
-n. 0)
0
ol 0 -1
of DMSP in the Fucus was found to be 0.074 f 0.007 mmolkg-l on a fresh weight basis. The recovery of added DMSP was 94 i 14%. The concentration of DMSP in Fucus depends on a number of factors including the sampling season and thermal and osmotic stresses encountered by the sample. The single result quoted in this paper is included for illustrative purposes only and should not be taken as indicative of the general levels to be found in this species. In addition, sample storage may be a difficulty as fresh algae stored at -20 "C for only a few weeks has been observed to smell strongly of DMS and when analyzed contained little or no DMSP. Work is currently underway to investigate more rapid analysis procedures that will obviate the need for sample storage and to study the factors influencing the levels of DMSP in marine phytoplankton and macrophytes.
1.75
1.50
0.6
0.7
Log,,lDMSP
0.8
0.9
concentration/
1.0
lo-'
1.1
1.2
moles dm")
Figure 3. Typical calibration for DMSP (gradient 2.09).
characteristic of the expected squared relationship obtained from emission resulting from the diatomic S, species. (c) Detection Limit. The 30 detection of the instrument, using a 200 pL injection volume is 2.2 x mol of DMSP dm-3 or an absolute injected mass of 6 ng of DMSP. Calculated in terms of sulfur this corresponds to 36 pg of Ss-'. Analysis of Marine Algae. Fucus vesiculosus was collected from the mouth of Langstone Harbour at Eastney, Hampshire, England on 31 January 1994. The algae (125.5 g) was extracted into 100 mL of methanol, and 24 mL of this extract was spiked with 1 mL of a 2.7096 x mol dm-3 DMSP solution. Four aliquots of each were subjected to a cleanup procedure to remove coextracted material and analyzed. The average concentration
Previous methods for DMSP quantitation have largely relied upon DMSP being the only naturally occurring sulfur compound to generate a volatile sulfur-containing species on base hydrolysis. This is known not to be the case. The instrumentation described in this paper provides the basis for the first reliable identiiication and measurements of (dimethy1sulfonio)propionate. The instrument is in prototype form, and design enhancements are expected to lead to significant performance improvements. As described, the current instrumentation has more than sufficient performance for the measurement of DMSP in marine flora and must rank as one of the most selective HPLC detection systems in existence, the specificity of the DMSP detection coming from three independent sources: characteristic retention time behavior, base hydrolysis resulting in the production of a volatile sulfur compound, and sulfur-specific detection The instrumentation described in this work is not completely restricted to the measurement of DMSP, and developments in the HPLC separation will permit the technique to be extended to other compounds that on base hydrolysis yield volatile sulfurcontaining products. Studies are currently underway to extend both the range of compounds that can be determined and the nature of the samples that can be analyzed using this system. Received for review July 18, 1994. Accepted January 4,
1995.8 AC9407151 @
Abstract published in Advance ACS Abstracts, February 1, 1995.
Analytical Chemistry, Vol. 67, No. 7, April 1, 1995
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