Mixed culture: for example, real effluenct where you don't know exactly what kind of humic you have.
Methods that include chromatography (e.g. LC-OCD) are time-consuming and expensive. A fast and cheap method is desired, even if it is less accurate, still would be preferred.
Unfortunately due to the complex nature of the beast, your best bet is to use a gravimetric analysis. Adjust the pH of your effluent to pH 11. Mix for about 6 h. Then centrifuge to remove non solubles (an effluent will not contain humin because it is insoluble anyway). Next adjust the pH to pH = 1 with conc. HCl and let stand for about 6 hours under N2. Any humic present will precipitate. You can separate the humic from the fulvic fraction by centrifugation. You can deash the humic if desired using an HCl/HF wash (5 ml conc. HCl/ 5 ml conc. HF in 1 L total DI H2O). The fulvic fraction will contain fulvic acid and other "stuff". If you want to know the content of pure FA you will have to use a DAX-8 resin to separate. If you want the full procedure see my RG page and view the "New Standardized Method" paper.
Well, since humics are complex supramolecular aggregates, rather than a discrete molecules, it can be very deceptive to speak about "concentration", moreover, when you seek a quick method.
I would try a UV-VIS spectrophotometry, even though you can have many disruptive compounds in your effluent. Then, there is a possibility to try E4/E6 ratio (absorbances at 465 and 665 nm), but this is also not considered reliable any more.
If you have enough of your effluent, I would occupy myself with the standard IHSS extraction procedures and in the end, I would get a yield of humic acid and a yield of fulvic acid. However, this is everything but quick.
you can try to do like this
1. by UV-VIS spectrophotometry
2. analysis by The three-dimensional extraction-emission matrix fluorescence(3DEEM)or EEM. it is a vaild mathod to analysis the functional group of humic substances
see for example
Senesi, N.; D’Orazio, V. Fluorescence spectroscopy.
In Encyclopedia of Soils in the Environment, Hillel, D., Ed. Elsevier: Oxford, 2005
; pp 35-52.
It is not easy to measure humic substances. Even with the proposed protocol by IHSS it is difficult to obtain quantitative results. UV-VIS and Fluorescence are simple and fast methods with the capability to quantify humic compounds but they suffer from unavailable standards. Suwaneee humic or fulvic acids standards from IHSS are available but these compounds are extracted from a small water reservoir not really representative of freshwater, marine, natural waters or even wastewaters.
You can obtain really quantitative results only if you extract humic substances with the IHSS protocol and use the extracted compounds as standards for your calibration curve in UV-VIS or Fluorescence analysis. An alternative to evaluate the humic content will be to use IHSS standards in your UV-VIS analysis and express your results in term of mg (or other mass unit) equivalent of IHSS compounds. Moreover, you can take a UV-VIS spectra of your sample and compare it to the UV-VIS spectra of IHSS standards to check the similarity between your sample and the standard. If you normalise the two spectra and do an overlay of the two spectra your will see clearly the similarity or difference in the spectra and in the nature of organic matter that you analyse.
Our colleague Zheng suggested three-dimensional excitation-emission matrix fluorescence. This technique is mostly qualitative and is used to discuss the nature or the presence of fluorophores present in the samples. It is better to use excitation or emission fluorescence spectra to try quantitative analysis of humic compounds in liquid sample. Personally I would choose the UV-VIS analysis instead the Fluorescence.
We found it is very difficult to dry humic substances. I suggest to use freeze drying. This is the method we use in our lab because humic subtances are very hygroscopic and the method is easy to use even if it is time consuming.. After drying we use a dessicator to store humic or fulvic acids.
Unfortunately due to the complex nature of the beast, your best bet is to use a gravimetric analysis. Adjust the pH of your effluent to pH 11. Mix for about 6 h. Then centrifuge to remove non solubles (an effluent will not contain humin because it is insoluble anyway). Next adjust the pH to pH = 1 with conc. HCl and let stand for about 6 hours under N2. Any humic present will precipitate. You can separate the humic from the fulvic fraction by centrifugation. You can deash the humic if desired using an HCl/HF wash (5 ml conc. HCl/ 5 ml conc. HF in 1 L total DI H2O). The fulvic fraction will contain fulvic acid and other "stuff". If you want to know the content of pure FA you will have to use a DAX-8 resin to separate. If you want the full procedure see my RG page and view the "New Standardized Method" paper.
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