Trace determination of sugar acids (gluconic acid) in sea water by liquid chromatography

AnalytrcaCka Acta, 271(1993)25-29 ElsevlerScience Pubhshers B V , Amsterdam

Trace determination of sugar acids (gluconic acid) in sea water by liquid chromatography Shigeto Nakabayashr, Isao Kudo, Kenshr Kuma and Katsuhrko Matsunaga Department of Chemutry, Faculty of Fdzenes, Hokhxdo

Unrverst& Hakodate 041 (Japan)

Kryoshi Hasebe Department of Chmustry, Faculty of Sctence, Hokhdo

Unwemty, Sapporo MO (Japan)

(Received 28th Apn11992, revised manuscnpt received 10th August 1992)

A hqmd chromatographlc (LC) method wth fluorescence detection was developed mth sufficient sensltlvlty to deternune organic acids m sea water and sedunents Sugar acids, except for uromc and a-keto acids, have not previously been measured IIIsea water because of then low concentrations After desalting wth a column, glucomc acid was esterLfied with 9-anthryldmxomethane The reaction muture contammg the glucomc acid denvatlve was directly chromatographed by LC mung an octadecylsllane reversed-phase column and fluorescence detection The peak areas were hnearly related to glucomc acid concentration The detetion limit was less than 10 nM The reproduabhty and recovery were 8% at 200 nM glucomc acid and 99 8%, respectively kYeywora3 Fluonmetry, bqmd chromatography, Glucomc acid, Sea water, Sugar acids, Waters

In previous studies [1,21, Fe(H) was found m surface sea water durmg sprmg blooms and the presence of Fe(H) may meet an non demand for phytoplankton growth It was proposed that Fe(H) is produced by photochemical reactions with Fe(II1) and organic substances under sunlight Moreover, laboratory experiments showed that the rmportant orgamc substances that reduce Fe(II1) to Fe(H) are sugar acids such as glucomc, glucanc and glucuromc acid [2] Among these sugar acids, glucomc actd 1s especrally btochemitally important as rt 1s an intermediate of glucose metabohsm [31 and may be present m sea water For this reason it 1s important to know the amount Correspondenceto K Matsunaga, Department of Chemistry, Faculty of Fisheries, Hokksldo Umversity, Hakodate 041

(Japan)

of sugar acids present m sea water which can contrrbute to the reductton of non However, except for uromc acids [4,5] and cw-keto acids [6], these sugar acids have never been measured m sea water Lrqurd chromatographrc (LC) methods usually employ ultravrolet or refractwe mdex detectors wrth low sensitrvrties (usually > 1 mm01 1-l) and also necessrtate preconcentratron by liquid-hquid extraction or an ron-exchange separation prror to LC analysis If possible, additronal separation procedures should be avoided, pnmanly to nnmmlze possrble contammation where nanomolar concentratrons of sugars are involved The method proposed here 1s sample and by usmg a fluorescence detector the detection lumt of sugar acids has been lowered In order to prepare fluorescent derrvatives, the samples are treated with 9-anthrylduuomethane

0003-2670/93/$06 00 0 1993 - Elsevler Science Publishers B V All nghts reserved

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S Nakabayashr et al /Anal. Chm. Acta 271 (1993) 25-29

(ADAM), which 1s a prelabelhng reagent for fatty acids, reactmg with the carboxyl group (COOH) The esters produced with ADAM and carboxyl radical emit fluorescence [7] Glucomc acid contams a carboxyl group, and a method for the determination of glucomc acid by LC was mvestlgated m this study

EXPERIMENTAL

Apparatus

The LC system conslsted of a Shlmadzu (Kyoto) LCdA chromatograph with an SCG6B system controller, FCV3AL low-pressure rationing valves, SIG9A automatic sampler, RF-535 fluorescence detector and a C-R3A data processor Chromatographlc separation was carried out on a 250 X 4 6 mm Ed column (Tosoh, TSKgel ODS-80Tm, octadecylsllane reversed-phase type) Separation of sugar acids from sea water was accomplished on a 300 x 8 mm 1 d column (Ultron PS-80H, strong acidic cation-exchange resin type, Chromato Packmg Centre, Kyoto) with an SPD-6AV W-vlslble spectrophotometrtc detector (Shlmadzu) Reagents and mater&s

9Anthryldlazomethane (ADAM) was purchased from Funakoshl Chemical Industry (Tokyo) The reagent was prepared by dlssolvmg 6 3 mg of ADAM m 1 ml of acetone Although It has been reported that this solution was stable for about 1 week at -20°C [8], it was prepared Just pflor to use Chromatographlc-grade organic solvents were obtamed from Kanto Chemical (Tokyo) and sugar acids from Sigma (St Lams, MO) Stock standard solutions (10 mM) were prepared m distilled water and stored at 4°C All standards were stable and lower concentration standard solutions were prepared by diluting the stock standard solutions with distilled water (pH 4 6, adjusted with HC104) Just prior to use

sample, the sugar acids were separated from the sea water to avoid salt interference For this separation, the pH of the filtrate was adJusted to 4-5 wth HClO, and a 200~~1 ahquot of the filtrate was injected into an Ultron PS-8OH column and eluted vvlth dlsttied water (pH 4 6, adjusted with HClO,) at a constant flow-rate of 05 ml mm-l at 50°C A volume of 2 ml of eluate was collected between 11 and 15 mm after injection The detlvatlzatlon of the sugar acid fraction with ADAM was carried out as follows A 25-~1 volume of methanol and 25 ~1 of ADAM solutron were added to 25 ~1 of the eluate After allowing the solution to stand for 60 mm at room temperature m the dark, it was filtered (0 45 pm) with nitrogen pressure A lo- or 20-~1 ahquot of the filtrate was injected into the LC column (TSKgel ODS-8OTM) and eluted with methanol-water (1 + 1, v/v) at a constant flow-rate of 10 ml mm-’ at 50°C The fluorescence of the ADAM ester was measured at 412 nm, with excitation at 365 nm The concentration was calculated using a data processor The compounds of mterest were eluted from the TSKgel ODS-80Tm column v&hm 25 mm with the aqueous methanol However, some strongly retained substances were not eluted Therefore, It was necessary to perform the followmg exchange to elute these compounds before the next sample mJectlon methanol-water (1 + 1, v/v) for 25 mm, acetone-water (7 + 3, v/v) for 35 mm, then methanol-water (1 + 1, v/v) for 30 mm to equilibrate the analytical column with the starting mobile phase One analytical cycle was completed wth 90 mm These exchanges of mobile phases were controlled by an SCG6B system controller and FCV3AL low-pressure rationing valves

RESULTS AND DISCUSSION

Separatwn of sugar a&s from sea water Procedure

A sea-water sample was filtered through a precleaned 0 45pm M&pore membrane filter Before the addltlon of ADAM to the sea-water

Figure 1 shows a chromatogram of 1 mM gluconic acid m sea water A 200-~1 volume of 1 mM glucomc acid was inJected mto the column, eluted with distilled water (pH 4 6) and detected spec-

S Nakabayashr et al /Anal

27

Chtm Acta 271 (1993) 25-29

I. 0

I.

I.

I.

E

IO

20

30

40

ADAM,

I

50

~1

Fig 3 Effect of the addltlon of ADAM solution on glucomc acid peak area Reaction time, 1 h at room temperature in the dark (pH 4 6)

I‘\ .

0

5

10 15 20 RetentionTime, PI”

25

Fq 1 Chromatogram of 1 mM glucomc acrd in sea water Volume mjected, 200 ~1 Peaks 1 and 2 are mhlbltors and glucomc acid, respectively

trophotometrlcally at 210 nm The pH of the mobile phase was adjusted to 4 6 Hnth HClO,, as ADAM reacts Hrlth sugar acids (carboxyl group) wlthm the pH range 4-5 Glucomc acid was eluted at a retention tnne of 13 mm, resulting m its complete separation from salts Figure 2 shows the recovery of glucomc acid in each eluate fraction Each eluate fractxon was collected durmg every 0 25-mm penod Approxlmately 100% glucomc acid was recovered m the fractions of retention tune from 1175 to 14 25 mm Hence, for collection of sugar acids from sea-water samples eluates were collected m the interval from 11 to 15 mm

Effect of aa!dttwnof ADAM The study was carried out with 1 PM glucomc acid The conditions for the reaction vvlth ADAM were basically referred those m a previous method for oxalic acid [8] The conditions for sugar acid derlvatlzatlon were optlmlsed Figure 3 shows the effect of the addition of ADAM solution on the glucomc acid peak area (area/Fmol glucomc acid) The peak area of glucomc acid was constant when > 20 ~1 of ADAM were added Thus, 25 ~1 of ADAM were sufficient to react with glucomc acid m methanol Effect of pH on reaction of sugar aczd with ALMM The pH of the reaction mixture was varied from 2 5 to 7 8 by addition of HClO, or NaOH With a l-h reaction time at room temperature m the dark, the glucomc acid ester response dlmmlshed below pH 4 and above pH 5 (Fig 4) These effects may be due to decomposition of ADAM at low pH and to the lack of reaction between

I. 2

Retentnn

Time,

mh

Fig 2 Recovery of duuxuc acid m each elute fraction Each eluate fraction was collected durmg every 0 25mm penod

I

I.

3

4

a.

a.

5

6

I

1

.I 8

PA

Fig 4 Effect of pH on reaction of glucomc acid HrlthADAM Reactlon tune, 1 h at room temperature m the dark

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S Nakabayasht et al /AnaL ChurnActa 271 (1993) 25-29

ADAM and COO- at higher pH Based on these results, the pH adopted was between 4 and 5 Reactton tune of sugar aed with ALMM The reaction time of glucomc aad with ADAM was varied from 5 mm to 16 h prior to chromatography The reaction m methanol was completed within 60 mm and the reactlon mixture was stable for 12 h at room temperature (Fig 5) This result IS smular to that observed for the reaction of ADAM wrth oxahc acid, which was faster m methanol than m a low-polarity solvent such as ethyl acetate [8] L C profile A chromatogram of sugar acid esters [glucomc (11, glucarlc (2) and glucuromc acid (311 is shown m Fig 6 Glucomc acid ester was eluted at 19 0 mm under these conditions and glucanc acid and

L

a

I 300

200

loo Time. mm

, 0

5

10

15

Retention Time,

20

25

mln

Fig 6 Chromatogram of sugar acid standards Peaks 1,2 and 3 are glucomc, glucarlc and ducuromc aad, respectwely

glucuromc acid appeared at 20 5 and 212 mm, respectively Hence glucomc acid was separated from glucarlc and glucuromc acids The other lsomerrc carboxyhc acids were well separated from glucomc acid Calibration A cahbratlon graph was plotted of peak area, computed by the data processor, against the concentration of glucomc acid m sea water usmg standard additions The relatlonshlp between peak area and concentration was lmear m the range O-l 0 PM with the regresslon equation A (peak area) = 7 5 x lo3 + 2 57 x 105C (PM) The correlation coefflaent was 0 994 (n = 6) The detection hnut of glucomc acid ester with fluorescence detection was < 10 nM based on the mmlmum area of the integrator The blank was estl-

TABLE 1 Recovery

of gh~mc

acid added to sea water WHI nM)

Amount found (nM)

I 0

I 5

10

I5

20

Time, h

Fig 5 Effect of reaction tame of glucomc acid urlth ADAM at pH 4 6 and stabdlty of the ester

Orlgmal sample (A) a

After addltlon (B) a

181 198 194 164 171 172 Mean 18OOk136

270 299 261 284 263 2% Mean 279 8 f 15 4

a B-A=998*205

nM, recovery=998%

S Nakabayadu et al /And

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Chm. Acta 271 (1993) 25-29

water sample that contained 850 nM of glucomc acid In conclusion, a method for the determmatlon of sugar acids m sea water has been established and the presence of dissolved glucomc acid m sea water during a sprmg bloom was confirmed The occurrence of glucomc acid m sea water may be slgmflcant, as it may catalyse the photoreductlon of Fe(II1) to Fe(I1) This method may be also applicable to the measurement of other carboxyhc compounds m natural waters The dlstnbutlon of sugar acids and the relatlonshlp wth Fe(H) m sea water during a spring bloom ~111be reported m detail elsewhere ,

0

5

10

15

Retentlon Time,

20

25

mln

The authors thank M Kolde of Scripps Instltutlon of Oceanography for helpful comments on the manuscript

Fig 7 Typlcal chromatogram of a denvatlzed sea-water sample Volume injected 20 ~1 REFERENCES

mated to be 30 nM using sea water subjected to UV irradiation for 6 h to destroy organic substances The reproduclbdlty and recovery were 8% at 200 nM glucomc acid and 99 8%, respectively as shown m Table 1 Measurement of dwsolved glucomc and m sea water Dissolved glucomc acid was measured m surface sea-water samples during a phytoplankton bloom at Funka Bay, Japan Figure 7 shows an example of a chromatogram obtained for a sea-

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