Characterize NOM in typical china s water using HPLC-SEC with continuous wavelength UV absorbance detector

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1 Characterize NOM in typical china s water using HPLC-SEC with continuous wavelength UV absorbance detector Mingquan Yan Associate Professor (CESE, Peking Univ.) Gregory Korshin Professor (CEE, Univ. of Washington) Dongsheng Wang Professor (REES, CAS) Costa Mesa, July, 2011

2 Introduction Content Materials, and methods Theory Results and discussion Conclusion

3 1. Introduction HPLC SEC is one of the most widely used techniques to characterize the chemistry of NOM molecules. Without complicated laborious pretreatment, Small inject volume, Availability of equipment, Ease and speed of analysis. In principle, the separation of molecules in a SEC column is based on their size, and compounds with lower MW are eluted later than those with higher MW.

4 Significantifi progress has been achieved in the detectors employed to quantify contributions and properties of varying-mw NOM fractions. multi-angle light scattering (MALS), a absorbance UV detector, fluorescence detector and DOC analyzer, etc

5 On-line absorbance detectors are most commonly used : Availability Relatively low cost, Sensitivity, High level of standardization However, since the UV spectrum of NOM are typically described in the literature as broad and featureless typically only the data at a single wavelength (e.g. 254 nm) are reported to indicate the overall NOM concentration. ti

6 Recent Progress Her et al. (2008) indicated that the ratio of absorbances b of varying-mw fractions measured at 210 and 254 nm is helpful for estimating relative contributions of fulvic acids and microbial biopolymers. Korshin et al. (2009) have evaluated the varying- AMW fractions NOM removal and DBP formation potential by the ratio of slope of NOM absorbance in the wavelength ranges nm to that in nm.

7 Our objects Examine in more detail the performance of HPLC-SEC with a continuous wavelength absorbance detector The absorbance-based indexes (slope and ratio absorption) were introduced to interpret the properties of varying-mw NOM fractions. Assess the error in evaluation of MW distribution based on HPLC-SEC chromatograms

8 2.1 Samples. 2. Materials and Methods Suwannee River humic acid (SRHA) (1R101H) was obtained from the International Humic Substances Society (IHSS). A well characterized commercial humic acid extracted from Yellow River sediment at Tianjin, China (TJHA), were isolated by a procedure recommended by the IHSS for the extraction of these materials from soils (Zhou, 2003) SRHA and TJHA solutions were prepared at final concentration of 5 mg L -1 TOC with Milli-Q water, respectively.

9 2.2. Analyzer HPLC-SEC analysis was carried out using using Dionex UltiMate 3000 HPLC system with Agilent GPC/SEC 79911GF-083 column via gradient elution with a flow rate of 0.5 ml/min, Elution profiles were monitored with a Waters 996 photodiode array detector, which acquired absorbance spectra in the 200 to 445 nm range at a 1.0 nm resolution. where the mobile phase was pre-equilibrated at 25% CH 3 CN/75% 10-mM aqueous H 3 PO 4 for5minutespriorto sample injection, ramped to 45% CH 3 CN/55% 10-mM aqueous H 3 PO 4 over 15 minutes following injection, and finally ramped to 95% CH 3 CN/5% 10-mM aqueous H 3 PO 4 over 5 minutes. Absorbance spectra of HA samples were also recorded in a 5 cm quartz cell on a Perkin-Elmer Lambda 18 UV/vis spectrophotometer with wavelength from 200 to 600 nm.

10 3. Theory Figure 1a, The absorbance spectra of TJHA and SRHA at TOC concentration of 5.0 mg/l. The spectra do not show any prominent peaks and decrease gradually with increase with wavelength. Although lh hthe TOCconcentration of the both samples are 5.0 mg/l, the intensity of them are different significantly. It will lead mistake if the UV is used as an indicator of TOC content in HPLC-SEC due to inconsistent DOM property with elution time.

11 The spectra property of DOM samples is more obvious after the spectra normalized to reference absorbance (e.g. 250 nm). Based on the multi-wavelength detectors, it is helpful for estimating relative contributions of HA fraction with ih certain property. Figure 1b. The spectra normalized to the differential absorbance at 220 nm

12 It is well recognized that UV-visible absorption spectrum for NOM increase approximately exponentially with decreasing wavelength (Twardowski et al. 2004). where a = Napierian absorption coefficient (m -1 ), λ = wavelength (nm), λ ref = reference wavelength (nm), s e (nm - 1 ) is the spectral slope parameter describing the relative steepness of the spectrum.

13 It can be divided into five regions, in each region, the log-transformed spectra decrease linear with wavelength increase nm (S ), nm (S ), nm (S ), > nm (S ), nm (S ), Figure 1c. Natural log-transformed absorption spectra for the HA samples. >350 (S >350 )

14 A review of spectral parameters Spectral parameter TJHA SRHA note S S a Helm et al., 2008; S Twardowski et al., 2004; Carder et al., 1989 S S > S / S b Korshin et al., 2009 S / S Helm et al., 2008; S >365 / S Twardowski et al., 2004 A 203 /A c Korshin et al., 1997; Li et al., 2006 A 210 /A d Her et al., 2008 A 280 /A e Korshin et al., 1997; 2009 A 250 /A f Helm et al.,2008; De Haan and De Boer, 1987; Peuravouri and Pihlaja, 1997 a S, used to semiquantitatively describe the ratio of fulvic acids to humic acids in a sample. Slope of nm and ratio of slopes of nm and nm are inversely related to the MW fractionated by ultrafiltration or gel filtration fraction. b ASI, ratio of slopes of nm and nm, used to evaluate NOM removal and DBPFP. c A 254 /A 203, used to evaluate the degree of activation of the PHA moiety in NOM. A 254 /A 203, used to evaluate the degree of activation of the PHA moiety in NOM. d A 210 /A 254, used to estimate relative contributions of fulvic acids and microbial biopolymers. e A 280 /A 350,like A 203 /A 253, used to evaluate the degree of activation of the PHA moiety in NOM. f A 250 /A 365 (called E2:E3) or A 465 /A 665, used to track changes in the relative size of DOM molecules.

15 4. Results and discussion

16 TJHA SRHA Figure 2. 3-dimension MW profiles of TJHA and SRHA characterized by HPLC-SEC with continuous wavelength detector

17 TJHA SRHA Figure 3. HPLC-SEC chromatograms for TJHA and SRHA at reprensitative wavelength. Four fractions can be found by peak picking methods in both HAs : Peak 1: min; Peak 2: min; Peak 3: min; Peak 4: min.

18 TJHA SRHA Figure 4.Absorption ratios of (a) TJHA and (b) SRHA.

19 Figure 5. Slopes of absorbance spectra of (a) TJHA and (b) SRHA. Five fractions can be found in both HAs : F1: min; humic substances F2: min; fulvic acids or aliphatic diprotic organic acids F3: min; lower MW humic substances, operationally defined building blocks (sub-units of humic substances) F4: min; low MW acids, non-aromatic matters F5: min; low molecular weight neutrals and amphiphilics, proteins and their amino acid building blocks

20 4.2 MW evaluation using spectra index Figure 6. MW estimation indexs (a) A 356 /A 254, (b) S >365. Slope of absorbance at wavelength >365 nm is a better indexes to evaluation molecular weight than A365/A254, which was used in literatures.

21 4.3 Assess the error in evaluation of MW distribution To evaluate the effect of wavelength utilized to estimate MW distribution characterized by HPLC-SEC, the normalized HPLC- SEC spectra at various elution times to absorbance at reference wavelength (e.g., 220 nm) was processed further by subtracting the normalized absorbance spectra of the HAs to absorbance at the same reference. The calculation can be expressed as equation: where RI w,t are the relative intensity at wavelength w and elution time t, (i= nm; w= min); I w,t are the intensity measured by HPLC-SEC at wavelength w and elution time t,(w= nm; t= min); I 220,t are the intensity measured by HPLC-SEC at wavelength 220 nm and elution time t,(t= min); i w is intensity measured by UV/vis spectrophotometer at wavelength w (w= nm). i 220 is intensity measured by UV/vis spectrophotometer at wavelength 220.

$fractions to MW distribution$ using absorption at wavelength

$fraction, such as humic$

22 TJHA SRHA Figure 7. Error in estimating the contribution of various MW fractions to MW distribution using absorption at wavelength 220nm as reference. (a) TJHA, (b) SRHA. In generally, the error is within plus or minus 10%, while for some high purified fraction, such as humic substance or low WM non-aromatic matters, it may be as high as more than 30% for certain wavelength

23 Conclusion In addition to the four fraction were found in HPLC-SEC chromatogram, one fraction with medium MW was found through investigating spectral parameters Slope of absorbance at wavelength >365 nm is a better indexes to evaluation MW than A365/A254, the latter was used in literatures. It may lead to errors up to 30% in evaluation of MW based on 2- dimension chromatograms for purified fractions of NOM, such as humic substances and low MW non-aromatic matters The 3-dimension HPLC-SEC chromatogram is a useful technique to characterize NOM, it can not only characterize the MW distribution of NOM more accurately, but also provide some interesting information of NOM characteristics with different MW. However, further work is needed to understand the UV spectra.

24 Thanks for your attention! scenery in Peking University

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