What are the difference between analysis using High Performance Size Exclusion (HPSEC) with Organic Carbon (OC) detector and HPSEC with ultra violet (UV) detector?
will those instruments detect different organic matter?
thank you
Hi,
First something about the terms: the term "HPSEC" generally refers to size exclusion chromatography using a UV detector (in most of the cases a DAD; diode array detector). Especially wavelengths in the range of 260 nm are used frequently. Using an organic carbon detector (OCD), the term "LC-OCD" is more appropriate. Often, if an LC-OCD analysis is performed, one ore multiple UV signals are recorded in parallel. However, the practical execution of both methods "HPSEC" and "LC-OCD" can vary significantly.
Second, I'll try to answer your question. Obviously, an LC-OCD analysis will provide most information (certainly if parallel UV measurement is performed) and allows for detection of all organic carbon (carbon is measured directly). Additionally, the carbon content is fractionated in different molecular weight (MW) fractions. Fractions can include hydrophobic, hydrophilic, Bio-polymers, humic substances, low MW neutrals, low MW acids. How much of the total dissolved organic carbon (DOC) each fraciton represents can be accurately estimated using the surface area under the curves (different peaks will be generally present). A HPSEC analysis will only detect that part of the dissolved organic matter (DOM) that will absorb at a certain wavelength. As such, a significant fraction of the DOM will not be detected in some cases. Especially the chromophoric part of DOM will determine the result. The chromophoric part in natural water (and wastewater) is mainly composed of unsaturated or aromatic moities of the dissolved organic matter (in natural water, DOM is natural organic matter (NOM)). These double bonds or aromatic moities will absorb strongly in the UV region (especially in the 250-260 nm region). As such, HPSEC provides rough information on the fingerprint of a certain water. At least, if the chromophoric part is concerned. Additionally, the analysis will provide information on the MW distribution, which is comparable to LC-OCD (except that the y-axis is an absorbance signal instead of carbon units). It is much more difficult to derive quantitative information from HPSEC chromatograms, as opposed to LC-OCD chromatograms (the latter is extensively calibrated). An important disadvantage of LC-OCD, however, is that the technique is cost and labor intensive.
Both methods are calibrated with MW standards, LC-OCD is also calibrated using a carbon standard (generally standard solutions of dryed potassium phtalate).
To conclude: 1) LC-OCD allows for fractionation and determination of the carbon content of each fraction. If UV measurements are performed in parallel, also similar information as HPSEC is provided; 2)HPSEC provides information on the molecular weight distribution for the chromophoric part. The absorbance peaks are generally related to concentration, although this relation depends on water characteristics and cannot be quantitatively determined. HPSEC is can be used to compare different waters or water samples taken at different treatment times.
I hope this was helpful.
Wim
To Mr.Wim,
thanks for your answer. this was helpful.
so, OC detector will detect all organic compound, UV detector will detect organic compound that will absorb at certain wavelength.
how about inorganic compound (such as,nitrate)?because i read that LC-UVD can detect nitrate.therefore,my understanding is inorganic compound cold be detected at low UV wavelength (210nm)..is it correct?
Indeed, nitrate and nitrite will absorb at lower wavelengths such as 210nm. However, As a size exclusion column is used, this peak will not interfere with the peaks of organic compounds. Retention times will differ significantly.
But, i think use HPSEC, LC-OCD are just a rough characterisation methods.
because the fractionation doesn't represent the specific component.
it is very difficult to classify what component shown in peak of UV and in OC detector.
then finally, i just classified based on molecular weight (ex.high MW is aromatic/biopolymers, low MW is aliphatic, intermediate MW is humic and fulvic acids). is it correct?
thank you that you dont mind to share with me. i try to learn this topics
Indeed, only groups (MW classes) of compounds with similar properties/molecular weight are detected (e.g. humic acids, building blocks, ...). Individual compounds cannot be detetected due to the complexity of "real water". However, LC-OCD allows for a relatively accurate and repeatable fractionation. The fractionation based on MW you applied is thus correct. Generally, compounds with MW >20.000Da are biopolymers, compounds with MW
Actually, our instrument is HPSEC use 3 detectors, OCD, UV and FEEM.
Now, i try to compare what fractions were generated in each detector.
At least, i hope those detectors will support each others to give information about what fractions in my sample.My research is about identification of organic compounds in nitrification process. it seems too complicated because we dont use LC-OCD (the machine was produced by Dr.Huber in German,right? ). So, it is difficult to get quantitative value of each compound.
Anyway, thanks for your information
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