An Investigation of Freshwater Humic Material Using Liquid Chromatography/ Mass Spectrometry

B. K. Dimock

ABSTRACT

Size exclusion chromatography with inline UV/visible absorbance and electrospray mass spectrometry (SEC/ES-MS) was used to characterise humic materials (HM) in both whole water and extracted samples. A high ionic strength eluent was employed to compare the averaged molecular weights of the HM determined through traditional SEC calibration and through the mass spectra. SEC analysis revealed that the examined HM had weight-averaged molecular weights (M_w) of approximately 4000 to 6000 g/mol, whereas the M_w values determine through the mass spectra were 3 to 6 times smaller indicating that the humic ions entering the mass spectrometer were multiply charged. A low ionic strength eluent was employed for separation and mass spectral characterisation of dissolved organic carbon (DOC) components within whole water samples.

INTRODUCTION

Freshwater humic material plays a vital role as it binds both toxic and essential metals within its structure. Most humic material originates from the degradation of terrestrial and aquatic plants, and, thus, constitutes a size continuum of charged heterogeneous compounds that are well suited for analysis through SEC/ES-MS. Recent studies have illustrated the effectiveness of the electrospray interface to separate the large, multiply charged ions from their aqueous matrix, thus allowing the mass spectral analysis of the intact humic ions (McIntyre et al., 1997; Fievre et al., 1997; Brown and Rice, 2000; Persson et al., 2000). This novel application has the unique ability to separate, characterise and quantify humic material in unperturbed freshwater samples.

METHODS

All analysis was performed using the Hewlett Packard Series 1100 LC/MSD system. A Polymer Laboratories PL aquagel-OH Mixed (300x7.5 mm) was employed inline with the ES-MS. All whole water samples were taken from watersheds in the Muskoka-Haliburton area of Ontario between February to August 1999. Samples were filtered with 0.45 µm syringe cartridges. A reference humic acid solution (20 mg/L) was made by dissolving Aldrich Na humic acid in de-ionised water. Aqueous polystyrene sulfonate (PSS) molecular weight standards and benzoic acid were used for SEC calibration under high ionic strength conditions outlined below.

High Ionic Strength Conditions: All solutions were made up in de-ionised, carbon-filtered water. A 50 mM NH₄CH₃COO in 10/90 methanol/water eluent was pumped at a flow rate of 0.5mL/min. Post-column addition of 1 % (v/v) NH₄OH in 50/50 water/isopropanol was teed into the column outflow at a flow rate of 0.3 mL/min using a Waters Model 590 HPLC pump. A sample injection volume of 300 µL was employed. Each sample run was 40 minutes in duration. The diode array detector (DAD) was set to record a spectrum from 190 to 600 nm every 0.8 seconds. The ES-MS was set to scan from 50 to 2250 M/z every 8.8 seconds with the fragmentor voltage set at 70 V. The following electrospray chamber conditions were employed for negative ion formation: 11 L/min drying gas flow rate, 350 °C drying gas temperature, 40 psig nebulizer pressure and 3500 V capillary voltage. All conditions for positive ion mode were the same except for employing a 4000 V capillary voltage.

Low Ionic Strength Conditions: For the low ionic strength conditions the following elution program was employed: 0-10 minutes with100% 20 µM NH₄OH at 0.5 mL/min; 11-20 minutes with 100% 10 mM CHOOH at 0.5 mL/min.; and 21-45 minutes with100% 20 µM NH₄OH at 0.5 mL/min. The formic acid leached off any bound cations that were retained on the column after sample elution. A rinse of 20 minutes with 1 mL/min 20 µM NH₄OH was applied to ensure the column had re-equilibrated after each sample run. Both the DAD and the ES-MS were set to the same conditions as outlined for the high ionic strength conditions.

Calculations: A log-linear plot of standard molecular weight versus retention time at 254 nm peak maximum yielded a linear calibration curve with an R² of 0.995. Number (M_n) and weight-averaged (M_w) molecular weights for samples were calculated from SEC 254 nm absorbance chromatogram as described in Chin et al. (1994). In a similar manner, the number (M/z_n) and weight-averaged (M/z_w) mass to charge ratios for each sample was determined from the average mass spectra as described in Lattimer et al. (1989). If the molecular ions are singly charged, M_n should be equivalent to M/z_n and M_w to M/z_w. The apparent charge (z_app) is determined by dividing M_w by M/z_w. The polydispersity of the sample was determined by dividing M_w by M_n.

RESULTS AND DISCUSSION

The major challenge interfacing SEC and ES-MS is harmonising the optimal conditions of both techniques. Aqueous SEC typically employs eluents with relatively high concentrations (i.e. >0.1M) of non-volatile salts to minimise “non-size exclusion” effects such as cation exchange and anion exclusion. Unfortunately, high concentrations of salts tend to suppress analyte ion formation within the electrospray chamber, thus resulting in a high background and low sensitivity (Figure 1A). Though low ionic strength eluents are not conducive to molecular weight determination through SEC, their use allows the separation of DOC components as function of both molecular charge and size in an ionic environment more typical to freshwater.

The results present in Table 1 were calculated from 254nm absorbance chromatograms (e.g. Figure 1B) and the mass spectra (e.g. Figure 2B) obtained using the high ionic strength conditions. The M_w and M_n values determined for Aldrich humic acid are comparable to those reported by Chin et al. (1994); that is, 4100 and 1630 g/mol. The whole water DOC contains smaller organic acids and, thus, the determined M_w and M_n are generally lower than the comparable humic and fulvic acid fractions. The humic acid fraction is slightly larger, is more polydisperse (i.e. M_w/M_n) and is less highly charged (i.e. z_app) compared to the fulvic acid fraction. The argument for the presence of multiply charge HM and not just fragment ions in the ES-MS is

Table 1: Molecular weight data calculated from analysis with SEC or ES-MS

Method	Parameter	Dickie Lake - Inlet #10 (Sampled Feb. 1999)			Aldrich Humic Acid	Polystyrene Sulfonate MW Standards
		Whole Water	Humic Acid Fraction	Fulvic Acid Fraction		MW 7950	MW 1800
SEC	Mw	4876 (± 8%)	6185 (±10%)	5889 (± 12%)	4005 (± 5%)	7515 (±3%)	1946 (±8%)
	Mn	2309 (± 3%)	2292 (± 14%)	2691 (± 12%)	1601 (± 12%)	2962 (± 25%)	1721 (± 7%)
	Mw/Mn	2.1	2.7	2.2	2.5	2.5	1.1
MS - negative	M/zw	1031 (± 1%)	1254 (± 2%)	963 (± 3%)	1151 (± 3%)	1263 (± 1%)	996 (± 5%)
	M/zn	654 (± 1%)	860 (± 7%)	622 (± 4%)	793 (± 5%)	1125 (± 0.2%)	745 (± 6%)
	Mw/Mn	1.6	1.5	1.5	1.5	1.1	1.3
	Zapp	4.7	4.9	6.1	3.5	5.9	2.0
MS - positive	M/zw	848 (± 4%)	1360 (± 8%)	873 (± 7%)	1235 (± 4%)	ND	ND
	M/zn	531 (± 3%)	950 (± 17%)	552 (± 6%)	820 (± 6%)	ND	ND
	Mw/Mn	1.6	1.4	1.6	1.5	ND	ND
	Zapp	5.7	4.5	6.7	3.2	ND	ND

supported by the presence of PSS 1800 ions with 2, 3 and 4 negative charges (spectra not shown).

For low ionic strength conditions, separation of the DOC components is function of both molecular size and charge. The latter mechanism is a consequence of the polyhydroxyl functionality of the column packing that tends to repulse negatively charged species and reversibly bind small, positively charged molecules. This technique is less perturbing and more sensitive to the actual size and chemical functionality of the DOC components as the eluent ionic strength is more typical of the freshwater environment. For example, the broad peak between 9 and 14 minutes (Figure 1), is initially the humic acid fraction, followed by the predominant fulvic acid fraction. The humic acid elutes first as it is slightly larger in molecular size and has a net negative charge, whereas the fulvic acid is smaller and has an amphiprotic nature. The large, narrow peak at 16 minutes is likely the largely ignored hydrophilic DOC component, as it is not present in either the fulvic or humic acid fractions.

Future work will focus on developing a “fingerprinting” methodology for elution time versus mass spectra data matrices to compare and characterise freshwater DOC as function of ecosystem and season.

REFERENCES

Brown TL, Rice JA (2000), Anal. Chem. 72: 384-390.

Chin Y-P, Aiken G, O’Loughlin E (1994), Environ. Sci. Technol. 28: 1853-1858.

Fievre A, Solouki T, Marshal AG, Cooper WT (1997), Energy & Fuels 11: 554-560.

Lattimer RP, Harris RE, Schulten H-R (1989), In: Determination of Molecular Weight (AR Cooper, Editor), New York, John Wiley & Sons, pp. 391-411.

McIntyre C, Batts BD, Jardine DR (1997), J. Mass Spectrom. 32: 328-330.

Persson L, Alsberg T, Kiss G, Odham G (2000), Rapid Commun. Mass Spectrom. 14: 286-292.