Size Exclusion Chromatography (SEC)

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.

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).

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 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).

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 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.

(Equation 1)

(Equation 2)

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 molecular weight species of a mixture, causing Mw to show higher values than Mn (Figure 2) [Moore, 1972 #59]. Molecular weights generated by SEC using a UV detector tend to be slightly 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].

Figure 2: Typical locations of number and weight average molecular weight

1SEC Background.doc