Introduction
Although present as in a trace amount, speleothem organic matter (OM) is a sensitive recorder of the overlying ecosystem (
McGarry and Baker, 2000;
Xie et al., 2003;
Ban et al., 2008;
Blyth et al., 2008;
Gázquez et al., 2012). Luminescence spectrophotometry is widely applied to characterize speleothem OM due to its non-destructive and convenient manipulation features (e.g.,
Baker et al., 1996;
Baker and Genty, 1999;
McGarry and Baker, 2000;
van Beynen et al., 2001;
Baker, 2005;
Hartland et al., 2010). During the fluorescence analysis of cave drip water using conventional methods, the complex nature of dissolved organic matter (DOM) in drip water allows for the influence of minor components on major components. In addition, fluorescence intensity and wavelength are affected by pH (Miano et al., 1988;
Senesi et al., 1991a,
b;
McGarry and Baker, 2000;
Patel-Sorrentino et al., 2002). Here we describe an improved method for fluorimetric determination of DOM in drip water that is accomplished by adding ascorbic acid (AA; vitamin C), with the aim of eliminating the influence of minor components on major components, and of maintaining a relatively high fluorescence intensity over a wide range of pH.
Materials and methods
Drip water samples were collected from the Heshang Cave (30°27′N, 110°25′E, 294 m above sea level). For a detailed description of the cave, see Hu et al. (
2008a,
b). The drip water was collected at the former growth site of the HS-4 stalagmite, which was collected in 2001. During each field trip, the drip water was collected using a glass bottle that had been cleaned sequentially using diluted HCl, non-fluorescent detergent, and ultrapure water. Once transported to the lab, the water samples were kept frozen. The samples were collected during September 2012 to June 2013.
After warming to room temperature, an aliquot (10 mL) of drip water was transferred to a 25 mL test tube and a measured amount of AA was added. The solution was thoroughly mixed and allowed to stand for 1 hr. Fluorescence analysis was conducted with a Perkin-Elmer LS-55 Luminescence Spectrophotometer using a 150 W Xe lamp. The synchronous fluorescence intensity (SFI) was measured using a 1 cm quartz cell with wavelength scanning from 200 nm to 500 nm in steps of 45 nm.
Results and discussion
For synchronous fluorescence analysis, it is important to choose the optimal wavelength difference (Δλ) between excitation and emission. Using the samples (adding 0.01 g AA) collected in March 2013, we compared Δλ values ranging from 1 to 150 nm at intervals of 10 nm. The results show that when Δλ was below 40 nm or above 50 nm, the SFI was lower or the peak shape was poor (Fig. 1). After comparing Δλ from 40 to 50 in steps of 1 nm, we finally set the Δλ value at 45 nm.
Using the wavelength difference of 45 nm, we assessed the influence of adding AA to the drip water. Before adding AA, the synchronous fluorescence spectrum of the water collected in March 2013 showed some minor peaks between 200 nm and 300 nm (Fig. 2(a)). After adding 0.01 g AA to 10 mL drip water, these minor peaks between 200 nm and 300 nm disappeared (Fig. 2(b)). As a comparison, adding AA to pure water did not show any obvious spectral peak in the scanning range (Fig. 2(c)). The results reveal that AA can quench the fluorescence effect from some compounds and thereby eliminate their influence on the major peaks during the fluorescence analysis of DOM in cave drip water.
Fluorophores in DOM can be separated into two groups, one with electron withdrawing substituents and the other with electron donating substituents (
Senesi et al., 1991b). Peaks at 200–300 nm in aquatic solutions probably originate from protein-like fluorophores, whereas fulvic-like fluorophores normally have maximal fluorescence intensity at 300–400 nm excitation wavelengths (
Hudson et al., 2007). Consequently, the disappearance of minor peaks at 200–300 nm after adding AA probably results from the reaction between AA and protein-like fluorophores. AA is a strong reducing agent (
Yang et al., 1997;
Arya et al., 2000). In contrast, our results suggest that the reactivity between AA and fulvic/humic acids is quite weak, so adding AA does not affect the SFI in drip water.
AA is a weak acid (
pKa=4.10 at 25 °C). Adding AA to drip water can change its pH. A number of studies have shown that fluorescence intensity increases with pH, probably due to changes in the fluorescence characteristics of the acidic functional groups and changes in the conformation of the organic compounds (
Coble, 1996;
Mobed et al., 1996;
McGarry and Baker, 2000). Our results show that SFI values reach high and near constant values when the concentration of AA is between 0.1 and 1.0 mg/mL (Fig. 3). When the AA concentration was higher than 1.0 mg/mL, SFI decreased sharply, corresponding to a decrease in pH to below 4. The study clearly shows that adding AA in the proper amount can maintain relatively high fluorescence intensity over a wide pH range (pH 9–4).
With the optimized experimental conditions, we selected two water samples, collected on March 2013 and September 2012, respectively, to check the reproducibility of our method. Using the same procedure for both water samples resulted in quite a low standard error (<0.36%; Fig. 4).
Conclusions
In this study, we considered the influence of AA on the fluorescence intensity and peak shape during analysis of DOM in cave drip water. Using water samples collected from the Heshang Cave for a case study, the results show that adding AA can quench the minor peaks, at 200–300 nm, but does not affect the intensity of the main peaks at 300–500 nm. This phenomenon probably results from the reductive reaction between AA and the protein-like substances in the drip water. Thus our improved method for fluorescence analysis on drip water has the advantage of eliminating the influence of the protein-like compounds on the predominant fluorophors at 300–500 nm. In addition, adding AA can maintain relatively high fluorescence intensity over a wide pH range (pH 9–4).
Higher Education Press and Springer-Verlag Berlin Heidelberg
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