Size Exclusion Chromatography (SEC)
Background
SEC is used to determine the molecular weight distribution of natural organic matter. It
accomplishes this by pushing water through a resin column. The lower molecular weight
components are retained longer than the higher molecular weight components. The NOM is
detected by a spectrophotometric detector at a wavelength of 254 nm. The standard curve for
this process is performed with the use of polystyrene microspheres of varying size. The SEC
column can also be followed by a DOC analyzer to correlate peaks in TOC with peaks in UV
absorbance.
A significant hurdle that needs to be overcome in this process is fouling of the SEC columns
which are very expensive. The “Waters” column needs a 20 l loop to minimize fouling. This
volume is however, too small for the DOC analyzer to produce a good peak. Hence, the
“German” column is utilized, due to the higher sample size capacity of 200 l.
Another concern is the eluent used. Standard methods specifies a NaCl eluent. If the DOC
detection is used with the Sievers DOC machine, the NaCl eluent can not be used because of
chloride interferences. The NaCl eluent provides better separation of components. If the NaCl
eluent can not be used, a sulfate eluent can be substituted.
1
116101658.doc
MOLECULAR WEIGHT (from Dundorf Thesis)
The determination of NOM molecular weight can offer some significant insights into
differences between NOM sources, treated waters and the quantification of its elements.
Establishing a molecular weight can aid in determining general molecular formulas and molar
concentration values for NOM.
Molecular weight, especially with NOM, is a continuum. A single average value for
molecular weight is a solid starting point, but it can often be of limited use as it is one number
among many on this continuum (Figure 1).
0.1
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
10000
1000
100
10
Molecular Weight (daltons)
Figure 1: A typical graph of molecular weight
There are two common techniques that have been employed for molecular weight
determination: membrane filtration and size exclusion chromatography. The key component in
establishing the molecular weight with either technique is the correlation between size and mass.
For example, aromatic and aliphatic compounds may have the same molecular weight, but very
different steric configurations. The ideal calibration standard is one that has similar structural
properties to the sample being analyzed; achieving this with NOM can be difficult. The reality
of the connection between size and molecular weight is that they are not the same, but only an
approximation of each other. Molecular weight, especially for NOM, should be considered an
estimate, apparent, or approximate molecular weight.
Membrane filtration uses a series of membranes of known molecular weight cutoffs to
sieve out each size range of organic matter. A benefit of this process is that the molecular weight
of particulate and colloidal NOM can be determined. This is not the case in size exclusion
chromatography (SEC) where all NOM has to be dissolved. The possibility of membrane
rejection by electrostatic repulsion may make the actual molecular weight cutoff smaller than the
2
116101658.doc
physical pore size. Factors such as the possibility of membrane fouling and transmembrane
pressure must also be considered. In addition the fact that the molecular weight measurements
are only in ranges between two molecular weight cutoffs instead of a continuum make the results
not as ideal as SEC achieves.
SEC for the determination of organic matter molecular weight is a technique that began
in the early 1980’s and is continuing to be refined today. A typical SEC setup consists of a high
performance liquid chromatography (HPLC) pump capable of flows around 1 ml/min. and
pressures of 0 to 200 psi, a gel chromatography column and detector (Figure 2).
HPLC Pump
1.000
0.005
Packed Detector
column
Figure 2: Typical SEC setup
The initial work with NOM began with the use of an inline UV detector. Recent research
advancements by Her, et Al and Huber, et Al. has made use of inline DOC detectors. The basis
for the separation process is the lower molecular weight molecules are retained longer than the
higher molecular weight components. Sample conditions of the eluent and sample such as: pH,
ionic strength and consequential charge interactions with the packing material must be
considered [Amy, 1987 #58]. A standard curve is generated using compounds of known
molecular weight, typically ranging from 1,000 to 10,000 daltons. The use of polystyrene
sulfonate (PSS) microspheres and a UV detector have shown to correlate well to humic and
fulvic acids [Chin, 1994 #19]. The assumption is that the UV absorbance of PSS microspheres is
the same as the absorbance of NOM. However, NOM is not a homogeneous substance, but a
heterogeneous mixture that is different with each sample. Each sample will have a different UV
absorbance. Hence, the relationship between the UV absorbance of PSS microspheres and NOM
is only approximate. The inaccuracy of this approximation, which can be up to 20%, is a
significant debate on how reliable this technique of UV detection is [Her, 2001 #56].
New developments in SEC technology for NOM analysis have come from the
incorporation of inline TOC machines. These techniques eliminate differences in UV
3
116101658.doc
absorbance found with changes in NOM structure. Instead, samples are measured on an organic
carbon basis which incorporates the total DOC component of NOM. There is still a need to use a
standard that approximates the structure/size of the NOM and the associated error must be
considered.
Although molecular weight in natural waters is best represented by a continuum and
more relative changes (i.e. across treatment processes), efforts have been made to assign single
numbers to represent molecular weight. The two most used numbers are the number average
molecular weight (Mn) and weight average molecular weigh (Mw) (Equation 1 & 2). It is
important to note that these two equations for Mn and Mw are not equal. The right equation by
Chin, et Al. is more commonly used in our labs.
 (n M )  n


n
n

i
Mn
i
i
i
i
i
 (n M )  (n M


 (n M )  n
i
2
i
i
i
i
)
i
i
i
(Equation 1)

 M 
 i
i
Mw
i
i
i
(Equation 2)
i
i
Mi = molecular weight
ni = number of molecules of molecular weight Mi
(usually presented as a segment height or signal response at a
certain elution time)
Mn tends to emphasize the lower molecular weight compounds. Mw tends to favor the higher
0.04 2)
molecular weight species of a mixture, causing Mw to show higher values than Mn (Figure
0.03
[Moore, 1972 #59]. Molecular weights generated by SEC using a UV detector tend to0.02
be slightly
0.01
0
higher than molecular weights generated by other methods. This is due to the higher molar
absorptivity of higher molecular weight fractions and the lower molar absorptivity of lower
molecular weight fractions [Chin, 1994 #19].
4
116101658.doc
0.1
0.09
Median Mn
0.08
Mw
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
00
1000
1000
2000
2000
3000
3000
4000
4000
Molecular Weight (daltons)
Figure 2: Typical locations of number and weight average molecular weight
5
116101658.doc