hi to all,
I need to quantify a metal in order to understand if it triggers the binding of a protein to DNA. So I performed an EMSA and I transferred the gel on a 6,6 Nylon membrane positively charged.
The problem is that the membrane is a polymer very hard to dissolve, and I think that a simple solvent is not enough in oder to dissolve it and recover the analytes for the subsequent analysis.
So I tried harsh conditions by using high temperature and the mix usually used to mineralize analyses for ICP MS, so that I kill 2 birds with one stone: in the same time I would have dissolved the membrane (because of the hydroxyls of amide bonds) and I would have mineralized the samples. Unfortunately the membrane was not dissolved and I had very bad results.
The next time I'd like to perform an extraction of what is retained on the membrane by using for example a cloud point extraction with TRITON X and than the usual mineralization of the surfactant phase.
Does any one have experience or does anyone have had similar problems to resolve?
Thans in advance for the suggestions.
ok, thanks!!! so do you think that using so harsh conditions the membrane will dissolve?
1. digestion procedure for metal
and 2. http://www.nylon66membrane.com/MS-Nylon-Membrane-Filter.html
Hi,
Microwave digestion will help for sure. If you have some problems, carbonize it carefully and then digest it like carbon sample. There is a paper on this by the way: "Evaluation of sample preparation methods for polymer digestion and trace elements determination by ICPMS and ICPOES"
Depending on the metal concentration, you can use EDS / WDS to quantify the amount of it in a solid sample. (http://en.wikipedia.org/wiki/Energy-dispersive_X-ray_spectroscopy). See the graph at corresponding wikipedia page. In principal it is not a sacrificial testing, it means you will recover your sample as it is.
Do not know whether it fits your further analysis but Nylon will dissolve in chloroform and some other chlorinated solvents.
what do you mean by "bad results"? high blank or just bad reproducibility?
microwave is helpful for complete digestion of you samples. you need to control the operation blank to get a good results
Evaluation of sample preparation methods for polymer digestion and trace elements determination by ICPMS and ICPOES
J. S. F. Pereira,a C. L. Knorr,a L. S. F. Pereira,a D. P. Moraes,b J. N. G. Paniz,a E. M. M. Flores*a and G. Knappc
Show Affiliations
J. Anal. At. Spectrom., 2011,26, 1849-1857
DOI: 10.1039/C1JA10050E
http://pubs.rsc.org/en/Content/ArticleLanding/2011/JA/c1ja10050e#!divAbstract
I would stay clear of dissolving the membrane and concentrate on digesting the membrane in order to free the trace metals. I would try digesting the membrane in 500uL conc nitric acid at 60 degrees C overnight and dilute to 10mL with water.and analysing this by ICP: MS along with a sample blank (centrifuge if necessary)..
yes, also for me dissolving the membrane is the best solution. the problem is how to dissolve it. i have tried to incubate it at 60 °C overnight in HNO3:H2O2, but nothing happened. so i think i will try with microwaves, were the pressures and the temperatures reached would allow the carbonization/dissolution of the membrane
Depending on the element of interest you could turn the membrane in a muffle furnace at a few hundred degrees Celsius and then dissolve the carbon in conc hydrochloride acid. This would work for the less volatile transition elements
When participating (successfully) in ring analyses for interlaboratory tests on metals in PA containing waste material we used an aqua regia digestion. On the one side a total metal extraction can be achieved. On the other side, PA is partially disintegrated under these conditions and organics are transferred to the extract also, at least in traces. Organics will change the characteristics of the plasma totally, and your extract is not comparable to the standard solution; you will not get reliable results.
Procedure: The material was shredded as fine as possible and treated with a.r. in covered (watch glass) quartz crucibles for about 12 hours at about 90 º C. In order to destroy the organics, we added HNO3 (1 N) to the nearly evaporated a.r. digest and, after filtering, HNO3 (65 %; 7 mL) and HClO4 (100 %) 2 mL) were added. The solution was slowly evaporated on a heating plate to dryness, starting with about 90 º C, ending with about 200 º C (quartz crucibles). Evaporation after adding HNO3 and HClO4 was repeated and finally the digest was taken up in HNO3 (1 N) for analysis. Blinds were included.
If you think, your lab furnace is not too sensitive on fumes and smokes and you do not consider volatile elements, the proposal of C. Harrington is a viable alternative.
In any case, to be on the safe side I would recommend to check the digest solution on possible matrix interferences by the standard addition method (at least partially) when analyzing by ICP-MS.
Hi I think this article will help you pl have a look
http://jb.asm.org/content/194/5/932.full
Article Cellular Iron Distribution in Bacillus anthracis
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