Variations of Molecular Weight
Estimation by HP-Size Exclusion
Chromatography with UVA
versus Online DOC Detection
N A M G U K H E R , * ,† G A R Y A M Y , †
DAVID FOSS,§ AND JAEWEON CHO‡
Civil & Environmental Engineering, University of Colorado,
ECOT 441, Boulder, Colorado 80309, Wright Water Engineers,
Inc., Denver, Colorado 80211, and Kwangju Institute of
Science and Technology, Kwangju, Korea, 500-712
High performance size exclusion chromatography (HPSEC)
with ultraviolet absorbance (UVA) detection has been
widely utilized to estimate the molecular weight (MW) and
MW distribution of natural organic matter (NOM). However,
the estimation of MW with UVA detection is inherently
inaccurate because UVA at 254 nm only detects limited
components (mostly π bonded molecules) of NOM, and the
molar absorptivity of these different NOM constituents is
not equal. In comparison, a SEC chromatogram obtained with
a DOC detector showed significant differences compared
to a corresponding UVA chromatogram, resulting in
different MW values as well as different estimates of
polydispersivity. The MWs of Suwannee River humic acid
(SRHA), Suwannee River fulvic acid (SRFA), and various
mixtures thereof were estimated with HPSEC coupled with
UVA and DOC detectors. The results show that UVA is
not an adequate detector for quantitative analysis of MW
estimation but rather can be used only for limited
qualitative analysis. The NOM in several natural waters
(Irvine Ranch, California groundwater, and Barr Lake, Colorado
surface water) were also characterized to demonstrate
the different MWs obtained with the two detectors. The
results of the SEC-DOC chromatograms revealed NOM
constituent peaks that went undetected by UVA. Utilizing
online DOC detection, a better representation of NOM MWs
was suggested, with NOM displaying higher weightaveraged MW (Mw) and lower number-averaged MW
(Mn) as well as higher polydispersivity. A method for estimation
of the MWs of NOM fractional components and polydispersivities is presented.
Introduction
Natural organic matter (NOM), ubiquitous in all ground and
surface waters, is comprised of a heterogeneous mixture of
humic and fulvic acids, lignins, carbohydrates, and proteins.
These compounds are chemical and biological products of
plant and animal residues (1, 2). NOM is known to play an
important role in pollutant chemistry and geochemistry
because it can form metal complexes, bind solubilized
* Corresponding author phone: (303)735-2433; fax: (303)492-7317;
e-mail: her@ucsub.colorado.edu.
† University of Colorado.
§ Wright Water Engineers, Inc..
‡ Kwangju Institute of Science and Technology.
10.1021/es015649y CCC: $22.00
Published on Web 00/00/0000
 xxxx American Chemical Society
nonpolar organic compounds, influence colloid stability, and
affect redox behavior in soil (3, 4).
Molecular weight (MW) and MW distributions are important factors in NOM characterization because they relate
to disinfection byproduct formation potential (5), proton and
metal binding, organic pollutant partitioning, adsorption onto
minerals and activated carbon, and NOM persistence and
removal during water treatment (6). MWs have been determined by ultrafiltration (7), field flow fractionation (8), vapor
pressure osmometry (9), analytical centrifugation (10), low
angle X-ray scattering (11), and high performance size
exclusion chromatography (HPSEC) methods (12). NOM MW
estimation by HPSEC can be influenced by numerous factors
such as calibration standards, undesirable column packing/
resin interactions, suitable data handling of chromatograms,
and detection methods (13, 17). In recent years, HPSEC, with
an ultraviolet absorbance (UVA) detector, has been widely
employed due to various advantages (small sample volume,
minimal pretreatment, availability of equipment, and ease
and speed of analysis) (13). To improve accuracy of MW
estimation by HPSEC, numerous investigators have reported
their efforts (13-17). O’Loughlin and Chin (13) examined
the effect of UVA detector wavelength on the determination
of MW distribution of humic and fulvic acids. They found
that both the number-averaged (Mn) and weight-averaged
(Mw) MWs increased with increasing wavelength for humic
substances. Zhou and co-workers (17) showed the effects on
the Mn, Mw, and polydispersivity (F, a measure of the sample
heterogeneity) by the definition of low MW (LMW) cutoff.
They recommended either 2% of the maximum chromatogram height or MW ) 50 as the LMW cutoff, whichever is the
higher value, and 1% of the maximum chromatogram height
as the high MW (HMW) cutoff (17).
Despite these recent and intensive efforts to improve MW
estimation by HPSEC with UVA detection, significant inaccuracy remains due to the unequal molar absorptivities ()
of the organic components contained in NOM. The energies
of the various types of molecular orbitals differ significantly.
The electronic transitions among certain of the energy levels
can be brought about by the absorption of radiation through
σfσ*, nfσ*, nfπ*, and πfπ* (18). However, most UVA by
organic compounds is based on transitions of n or π electrons
to the π* excited state, because the energies required for
these processes bring the absorption peaks into an experimentally convenient spectral region (200-700 nm) (18). Both
transitions require the presence of unsaturated groups
(double or triple bonds contained in NOM) to provide π
orbitals. Therefore most of the compounds that have distinct
chemical bonds show different molar absorptivities.
The HPSEC-UVA-DOC technique has shown effective
separation of different NOM fractions based on MW by both
UVA and DOC online detectors. This system, in addition to
detecting the aromatic NOM moieties with UVA, can also
detect aromatic as well as aliphatic carbon fractions with the
use of the DOC detector (19). The DOC detector has shown
less interference than a UVA detector (254 nm) by inorganics
(e.g., NO3-). The research objectives of this study were to
compare and contrast the MW variation between UVA
detection with the results obtained by the DOC detector and
to suggest an alternative MW representation for NOM
comprised of multiple fractional components.
Materials and Methods
Suwannee River humic acid (SRHA) reference material
(1R101H) and fulvic acid (SRFA) standard material (1S101F)
obtained from the International Humic Substances Society
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9
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were analyzed to compare MWs obtained with the UVA and
DOC detectors. The isolates (15.1 mg of SRHA and 15.3 mg
of SRFA) were first dissolved in 1 L of Millipore Milli-Q (MQ)
water to produce 8 mg C/L. After adjusting the pH to 6.8 with
NaOH solution and filtering with a 0.45 µm cartridge (nylon)
filter, molar absorptivity was measured with a UV spectrophotometer from 500 to 200 nm (UV-160A UV/Visible
Spectrophotometer, Shimadzu). The ionic strength of these
samples was then adjusted to 0.1 M with a concentrated
eluent solution to match the ionic strength of the HPSEC
mobile phase for the MW determination.
Additional samples that were evaluated included an Irvine
Ranch Water District (IRWD), California groundwater, and
a Barr Lake, Colorado surface water (BL-SW). These samples
were also analyzed by HPSEC system to illustrate the MW
differences by detectors and to demonstrate an improved
MW representation. Sample pretreatments also consisted of
0.45 µm cartridge (nylon filter) filtration and ionic strength
adjustment.
The HPLC (LC600 Shimadzu) was coupled with a UVA
detector (SPD-6A Shimadzu) operated at 254 nm and an
online DOC analyzer (Modified Sievers Turbo Total Organic
Carbon Analyzer). The DOC detector was connected to the
UVA detector outlet. The system, adapted based on the
pioneering work of Huber and Frimmel (20, 21), employed
a TSK-50S column (35 µm Toyopearl HW resin) that had a
length of 25 cm and an inner diameter of 2 cm. A detailed
description of this HPSEC system was provided by Her et al.
(19). The HPSEC mobile phase was prepared with a phosphate
buffer (0.0024 M NaH2PO4 + 0.0016 M Na2HPO4, pH 6.8) and
0.025 M Na2SO4, producing an ionic strength of 0.1 M. Helium
gas was sparged into the mobile phase reservoir to eliminate
inorganic carbon and oxygen that can cause interferences or
react with the mobile or stationary phases. The flow rates
were 1 mL/min, and sample injection volume was 2 mL.
UVA and DOC data over time were collected every 6 s by
digital signal processing using a modified Labview software.
When the same operating conditions were applied, the
reproducibility of both UVA and DOC chromatograms
showed indistinguishable traces with multiple injections. The
difference of peak area for three injections is less than 3%
for this system, based on an injected DOC of 5 mg/L.
Polystyrene sulfonate (PSS) standards have been shown
to be more representative of NOM than PEG in SEC
characteristics (hydrodynamic radii, viscosity etc.) (22).
However, this system was calibrated with poly(ethylene
glycol) (PEG) standards ranging from 200 to 10 000 g/mol
because PSS displayed more undesirable interactions than
did the PEG with the Toyopearl HW resin within the column.
A semilog calibration curve (r2 > 0.99) was used to calculate
the MWs. The baseline of the chromatograms was changed
due to tailing and was set as 0 at 2% of the maximum
chromatogram height, based on the approach of Zhou et al.
(17). Mn, Mw, and F (number-averaged MW, weight-averaged
MW, and polydispersivity, respectively) were determined
using the following equations
n
Mw )
n
∑(h ‚M )/∑h
i
i
i)1
n
Mn )
i
n
∑h /∑(h /M )
i
i)1
(1)
i)1
i
i
(2)
i)1
F ) Mw/Mn
(3)
where hi and Mi are the height of the HPSEC chromatogram
and the molecular weight at eluted volume i (16).
Mp,ME (measured peak maximum MW) is defined as the
MW corresponding to the peak maximum location measured
B
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FIGURE 1. Absorptivities based on 1 mol C/L for SRHA, SRFA, albumin,
and sucrose in pure water (pH 7) as a function of wavelength.
by HPSEC when the peak shape shows a Gaussian distribution. However, because the peaks of the samples did not
follow a Gaussian distribution, Mp,ME values were determined
as the MW at the median value of the cumulative weight
chromatograms (corresponding to the centroid of the chromatogram area), instead of the using equations composed
of Mn and Mw (Mp,ME ) xMnMw) (6). To compare the
deviation between measured and calculated MWs for the
mixture samples, the concept of calculated Mp (Mp,CA,
calculated peak maximum MW) was introduced based on
Mp,ME values of individual components. Using an arithmetic
scale, Mp,CA can be determined for the known concentrations
of SRHA and SRFA using the following equation
Mp,CA )
a(Mp,SRHA) + b(Mp,SRFA)
a+b
(4)
where a is the DOC concentration of SRHA, b is the DOC
concentration of SRFA, and Mp,SRHA and Mp,SRFA are MWs at
the median value of the cumulative weight chromatograms.
However, MW distributions were depicted on a logarithmic
scale. Therefore, Mp,CA was calculated using eq 8, which was
derived from equations from 5-7.
log Mp,CA )
log Mp,CA )
a log(Mp,SRHA) + b log(Mp,SRFA)
a+b
(a +1 b)(log(M
p,SRHA)
a
+ log(Mp,SRFA)b)
log Mp,CA ) log((Mp,SRHA)a(Mp,SRFA)b)1/(a+b)
Mp,CA )
a+b
x(Mp,SRHA)a(Mp,SRFA)b
(5)
(6)
(7)
(8)
Results and Discussion
Chin et al. (12) pointed out that MWs measured with HPSEC
generated higher values than those measured with other
methods due to the UVA detector normally used in the HPSEC
system. These researchers found that the higher MW fractions
have a greater molar absorptivity () and that high weight
fractions appeared to be more abundant than they actually
were, while the lower MW fractions (with lower ) appeared
to be lower in concentration. The net result is an overestimation of the humic substance components of NOM (that
have high ) and an under-representation of the nonhumic
constituents. Carbohydrates and proteins, for example, are
additional NOM components. Figure 1 shows the absorptivities based on 1 mol/L of organic carbon of four different
NOM components: SRHA, SRFA, albumin (a protein), and
sucrose (a small simple carbohydrate). As expected, the is
FIGURE 2. Mw and Mn values estimated by UVA and DOC
chromatograms for (a) 8 mg C/L of SRHA and (b) 8 mg C/L of SRFA.
not the same for all samples across all wavelengths. Albumin
shows highest at lower wavelengths, decreasing rapidly
with increasing wavelength. At 254 nm, SRHA and SRFA have
high ; however, albumin and sucrose show low or negligible
at this wavelength. A UVA detector connected to the HPSEC
quantifies the response intensity based on the . As a result,
the MW determined with a UVA detector (at 254 nm) is
primarily the MW of only the high components such as
humic and fulvic acids, leading to inherent inaccuracy, and
over- or underestimation of MWs. Therefore, MW estimation
with a UVA detector is problematical for a hetero-mixture of
NOM, and a UVA detector cannot accurately be used for
quantitative MW measurements of NOM.
The estimated MWs of SRHA and SRFA are displayed in
Figures 2. SRHA and SRFA were selected because they share
similar chemical properties. The MWs (Mn ) 1385 daltons
(Da) and Mw ) 2114 Da) of SRFA by the UVA detector were
similar or slightly higher than reported values (Mn ) 1112 Da
and Mw ) 1950 Da (9); Mn ) 1360 Da and Mw ) 2310 Da (16)),
even with the use of PEG standards, which have been
suggested to underestimate the MW of humic substances
(22). The integrated areas under the DOC chromatograms
for both samples were equal. In contrast, the areas of the
UVA chromatograms for equivalent sample concentrations
displayed significant differences due to the different molar
absorptivities of humic and fulvic acid. This discrepancy
between SRHA and SRFA is clearly seen in the UVA cumulative
responses in Figure 3, portraying cumulative weight derived
from integration of the chromatograms in Figure 2. Cumulative response is assumed to be equivalent to cumulative
weight based on the UVA and DOC signal being proportional
to the concentration (12). The cumulative weight of SRHA
obtained with the UVA detector is much higher than that of
SRFA. Because the chromatograms in Figure 2 were not
Gaussian distributions, Mp,ME values were determined as the
FIGURE 3. UVA and DOC cumulative responses and Mp,ME values
estimated for (a) 8 mg C/L of SRHA and (b) 8 mg C/L of SRFA.
TABLE 1. Comparison of MWs of Mixed Samples (SRHA and
SRFA) by HPSEC-UVA-DOC
Mp (Da)
detector
sample (mg C/L)
Mn
(Da)
Mw
(Da)
G
DOC
SRHA 12 + SRFA 4
SRHA 8 + SRFA 8
SRHA 4 + SRFA 12
SRHA 12 + SRFA 4
SRHA 8 + SRFA 8
SRHA 4+SRFA 12
1544
1393
1272
1809
1680
1534
3084
2759
2436
3102
2851
2531
2.00
1.98
1.91
1.71
1.70
1.65
UVA
Mp,ME Mp,CA
diff
2501
2220
1982
2697
2419
2126
-9
-14
-6
+79
+81
+38
2510
2234
1988
2618
2338
2088
MW of the median value of the cumulative response
chromatograms (Figure 3). Mp,ME values for SRHA and SRFA
with the UVA detector were 2932 and 1865 Da, respectively,
and these values were higher than those measured by the
DOC detector (2821 Da for SRHA and 1770 Da for SRFA).
The MWs of three different mixtures of the isolates (12
mg/L SRHA and 4 mg/L SRFA, 8 mg/L SRHA and 8 mg/L
SRFA, and 4 mg/L SRHA and 12 mg/L SRFA (as DOC)) were
also determined with the UVA and DOC detectors (Table 1).
Mn and Mw values were higher and F values were lower with
UVA detection compared to the values obtained by DOC
detection. Figure 4 shows the cumulative response distributions of the three different mixtures. The DOC cumulative
chromatograms (Figure 4b) have equal total heights for these
three samples, each with the same total DOC concentration
of 16 mg C/L. However, the UVA cumulative chromatograms
(Figure 4a) show different heights at these same total DOC
concentrations. With an increasing portion of SRHA, the total
height of the UVA cumulative chromatogram increased due
to the higher of SRHA compared to SRFA.
The Mp,CA for mixture samples (in Figure 5) was calculated
with eq 8 using the individual Mp,ME values of the SRHA and
SRFA obtained with the UVA and DOC detectors from Figure
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FIGURE 4. Cumulative responses and Mp,ME values for three different mixtures of SRHA and SRFA: (a) with UVA detector and (b) with
DOC detector.
3. As shown in Table 1 and Figure 5, the Mp,ME values obtained
by DOC detection were very similar to the Mp,CA values, within
the error of the DOC detector. However, in contrast, the Mp,ME
values obtained by UVA detection showed slightly more
deviation from the Mp,CA values. Higher Mp,ME than Mp,CA
values were obtained due to the greater contribution of the
higher MW SRHA. As discussed earlier, this discrepancy
results from the differences in .
The MW deviation between SRHA and SRFA with the UVA
detector displayed in Figure 5 showed minor differences likely
attributable to the relatively similar and MW for these NOM
components. Figures 6 and 7 further elucidate the different
MW distributions depending on the detector. Figure 6 shows
the HPSEC-UVA-DOC chromatograms for Barr Lake, a 32 000
acre-foot (3.95 × 107 m3), hypereutrophic reservoir. The Mn
and Mw values measured with the DOC detector were 443 Da
and 5609 Da. However, the values measured with the UVA
detector were significantly different. The component at 13 500
Da showed a very low response in the UVA chromatogram
compared to DOC chromatogram, illustrating the low of
this component. Therefore, the UVA-determined Mw value
was much lower, 2010 Da, and the F value was also
significantly different (12.7 with the DOC detector versus 5.3
with the UVA detector).
Figure 7 presents another example of the MW deviation
between the two detectors using a groundwater sample
collected at 1090 ft depth from the Irvine Ranch Water District
(IRWD). An aliphatic carbon fraction displaying a strong DOC
response and no discernible UVA response was observed at
D
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ENVIRON. SCI. & TECHNOL. / VOL. xx, NO. xx, xxxx
a high MW range (19 600 Da, could represent polysaccharide
like substances (23)) and a low MW range (210 Da). With
these two additional large responses, the DOC detector
resulted in a higher Mw (6300 Da) and a lower Mn (750 Da)
compared with those values measured by UVA (Mw: 3400
Da and Mn: 1770 Da) and also a significantly higher F (8.4
with the DOC detector and 1.9 with the UVA detector).
The higher F indicates greater NOM heterogeneity. Thus,
singular MW representations utilizing Mw and Mn are not
appropriate for NOM that consists of a high F (e.g., 8.40).
Therefore, in the case of multiple MW fractions, each fraction
should be represented independently, possibly including
each fractional concentration.
Figure 8 presents the normalized cumulative response
using the DOC data from Figure 7. To represent the MWs of
multiple components of NOM, each fractional component
should be expressed. Chromatogram peaks were divided into
four different fractions based on the “inflection points” in
Figure 8. Then, Mn, Mw, F, and Mp,ME values were obtained
for each fraction. Each fractional concentration was also
determined by multiplying total concentration by fractional
height. Table 2 shows a comparison of currently accepted
and proposed representations of MWs. The F values, which
ranged from 1.02 to 1.27 for each fraction, are reasonable
values for a single MW constituent. This proposed representation can express MW more specifically and accurately
and provides much greater utility and insight, especially when
evaluating NOM persistence and removal across water
treatment processes.
FIGURE 5. Mp,ME and Mp,CA values obtained by UVA and DOC chromatograms of mixture samples: (a, d) 12 mg C/L SRHA and 4 mg C/L
SRFA, (b, e) 8 mg C/L SRHA and 8 mg C/L SRFA, and (c, f) 4 mg C/L SRHA and 12 mg C/L SRFA.
FIGURE 6. HPSEC-UVA-DOC chromatograms for Barr Lake.
The estimation of MW is an important factor for understanding the physical and chemical properties of NOM (24,
25) and determination of appropriate water treatment process
selection, design, and operation. Even though the molar
absorptivities of NOM components are significantly different,
UVA detection has been widely used for determining MWs.
Instead, for estimating MW based on equitable consideration
of all NOM components, a DOC detector that registers equal
intensity for both aliphatic and aromatic compounds should
be employed. The importance of employing a DOC detector
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FIGURE 7. HPSEC-UVA-DOC chromatograms for IRWD groundwater.
FIGURE 8. Cumulative response distributions (normalized) by DOC detector of IRWD groundwater.
TABLE 2. Comparison of Currently Accepted and Proposed
Representations of MWs (by DOC Detector)
proposed representation
currently
accepted
fraction fraction fraction fraction
representation
1
2
3
4
concn
(mg C/L)
Mn (Da)
Mw (Da)
F
Mp (Da)
2.69
0.77
1.06
0.37
0.49
750
6300
8.40
2460
17780
19200
1.08
18870
2234
2838
1.27
2607
509
528
1.04
517
210
215
1.02
213
in MW determination relates to the measurement of nonaromatic compounds (e.g., polysaccharides) and partially
aromatic compounds (e.g., proteins), which are NOM
constituents, and are important in water treatment processes.
Understanding these aliphatic and partially aromatic compounds is especially significant, as proteins can form
chlorination byproducts and polysaccharides have been
implicated as major foulants in membrane treatment (26).
UVA detection should only be used for qualitative analysis,
F
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ENVIRON. SCI. & TECHNOL. / VOL. xx, NO. xx, xxxx
such as comparison of the aromaticity of NOM components
as a function of MW.
Acknowledgments
We gratefully acknowledge Ionics-Sievers Instruments for
supplying the modified TOC 800 analyzer and Mr. Paul
Kosenka for his continuous technical support.
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Received for review August 16, 2001. Revised manuscript
received May 5, 2002. Accepted June 3, 2002.
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