Inexpensive and convenient methods for qualitative analysis of heavy metals in Eucalyptus tissue and mine tailings are described. Traditional published methods of thin layer chromatography show promise for detecting cations of Mercury and aluminum in plant tissue and mine tailings. Arsenic can also be included if suitable detection methods are developed. It should be possible to visualize the distribution of aluminum in the plant tissue thin sections by means of Morin treatment and UV microscopy.
Methods and materials
Silica gel (Sigma H) was coated on 10x20cm glass plates at a thickness of 0.25mm using a Stahl-Desaga spreader. When acid washed Silica gel is called for, the gel is soaked in conc HCl - Acetone 1:5, followed by decanting, and three washes with double distilled water. Sigmacell 200 was coated in a similar manner, but was never acid washed. Lanes were scraped with a pointed object and straightedge, followed by heating on a woodstove to activate the gel layer.
Micropippette columns are constructed from a pasteur glass pipette, a small paper disk and Silica gel 60. The paper disk is punched to fit the inside of the pipette, and rammed down to the taper with a dowel. Silica gel is filled to the halfway point followed by several washes with solvent.
Eucalyptus roots and stems were broken into small pieces and roasted at 290 deg C until vapors ceased to evolve. Vapor was condensed onto the glass surface of a retort, or test tube and later re-dissolved in chloroform or HNO3 with HCl (aqua regia). In one experiment, hexane was used to pre-extract essential oils. Charcoal from this digest is also treated with aqua regia.
The dissolved solids containing heavy metals, and other plant products were concentrated by boiling and spotted onto a Silica gel plate according to Stahl and eluted with one of several solvent systems, which usually consisted of an organic solvent plus pH buffer.
Control ions were made from a variety of available materials. No attempt was made to obtain high purity standards. (As an example, Lead was obtained by scraping a small amount of the shield material from an old piece of Lead covered electrical wire and dissolving it in aqua regia, Nickel was obtained by dissolving a a coin in Nitric acid.) Most were dissolved in water or HCl. These were spotted alongide the unknowns in separate lanes.
The standard procedure as published breaks down the ions into groups by treatment with thioacetamide and centrifugation to separate the Copper group followed by additional treatments with thioacetamide at alkaline pH to divide out the (NH4)2S group. The resulting solutions containing the ions of interest are analyzed as follows:
Copper group is separated in n-butanol-1.5N HCl-acetonylAcetone (100+20+0.5) migration sequence is Hg>Bi>Cd>Pb>Cu on MN Silica Gel S-HR
(NH4)2S group is separated on MN Silica Gel S-HR with Acetone-Conc HCl- acetonylAcetone (100+1+0.5) migration sequence is Fe>Zn>Co>Mn>Cr>Ni>Al
In these experiments, the thioacetamide treatment was skipped in the interest of finding a less cumbersome method that could be used in the field.
Mixtures of heavy metals can be directly analyzed be pre- treating the samples with Dithizone in chloroform. Sn Cu Hg Pb Cd Zn are applied as the dithizonates on Silica Gel G and eluted with Benzene. The Dithizone derivatives have colors that are charactaristic and no spray reagents are required. Undreacted Dithizone is present on the final plates, as well as oxidation products that are located very close to the Hg spot.
Another method is much simpler, and was found to produce the easiest separation. Acetone-25% HNO3 (70+30) and Cellulose are used to separate Hg>Bi>Be>Cu>Ni>Ce according to the literature. In these tests, additional ions were included and good separations were achieved.
Several reagent sprays were formulated from published methods and used to visualize the ions in question. After spraying, in most cases Ammonia fumes are used to enhance or modify the reactions.
PAN result PAN- 1-(2-Pyridylazo)-2-napthol for Cd2+, Co2+(light blue), Cu2+(violet), Mn2+(pink-violet), Pb2+,Ni2+(violet), Zn2+(magenta), and UO2++ ions used as 0.25% solution in ethanol followed by ammonia vapor. Some reaction with Hg (pink) and Fe (violet).
Dithiooxamide- for Pb++,Co++,Cu++,Mn++,Ni++,Hg++,Bi++ ions used as 0.5% solution in ethanol followed by ammonia vapor. Produces a rapid darkening for Copper.
Alizarin result Alizarin - for cations. Used in a .2% solution in ethanol followed by ammonia vapor. One oxidation state of Hg has a blue color.
Potassium Iodide (produces a red spot with Hg if in high conc.) spray used as 2% solution followed by ammonia vapor followed by Na2S spray (dark spot for Hg).
8-Hydroxyquinoline for Ba++, Sr++ and Ca++ used as a solution of 0.5g in 60ml ethanol and 40ml water followed by ammonia. Observe under long wave UV.
Quercetin for cations of the Copper and ammonium sulphide group used as 0.2% solution in ethanol followed by ammonia vapor and UV light.
Diphenylcarbazide for Ag+, Pb++, Hg++, Cu++, Sn++, Mn++,Zn++, and Ca++ was used in 2% soln in ethanol followed by ammonia vapor. Additional heating to 80 degrees centigrade will produce a blue violet color from Mercuric acetate adducts.
Dithizone direct method Dithizone produces colored derivatives and can be sprayed directly.
Results and discussion
When N-propanol-1.5N HCl-AcetonylAcetone or Acetone-Conc HCL- AcetonylAcetone were used as solvent systems on Silica gel H or Acetone- 25% HNO3 was used for elution on layers of Sigmacell 200 then sprayed with PAN, dithio-oxamide, Diphenylcarbazide, Alizarin, Morin, Postassium iodide, Sodium Sulfide solution, Dithizone, Quercetin, 8-Hydroxyquinoline, colored spots and bands appeared that where characteristic in position and color for each of the test ions. Careful examination of the plates at each stage of the process yielded useful diagnostic information.
Improvements in pre-separating the Eucalyptus extracts included attempts at roasting using crucible, test tubes, and glass retorts. The oily residues(note 1) from the roasting process could be shown to contain some Mercury, but the greatest amounts were found in the charcoal residue. If the roasting temperatures were kept below 300 degrees centigrade, the Mercury didn't vaporize, and appeared on the tlc plates. If the temperatures exceeded 300 degrees, only aluminum remained in the ash, along with other ions that were not identified.
Arsenic could not be visualized with any of the spray reagents used, but in some cases, masking of background color could be used to identify the location of Arsenic on the plate. It was not possible to confirm the presence of Arsenic in any of the roasts using this method.
Morin result Aluminum was best visualized using Morin as an indicator as it produced intense fluorescence under UV. It was possible to identify Aluminum in the charcoal residue without question. Some reagents produced distinctive colors, or noreaction, allowing for a process of elimination. Aluminum suffers from blocking by Copper on several of the solvent systems, but the intensity of the fluorescence using Morin is sufficient to reveal a fringe of aluminum just surrounding the dark zone produced by the Copper, in an eclipse like manner.
Copper produced distinct color reactions and was easily identified. It could be detected with every system used, and from every sample tested. Reaction time with Diphenylcarbazide was rapid and was diagnostic. Copper could be visualized just above and partly overlapping the spot for Iron.
Most of the reagents produced a colored spot with Mercury, Na2S produced a dark spot, that did not seem to be affected by the presence of plant oils. The amount of Mercury in these samples is much less than that of Aluminum. When heavily loaded, the extracted Mercury can be found leading all other ions in the sample lane, but merged together in a cluster in some solvent systems. By using each spray reagent (on separate plates) one can identify each component of the cluster by position and color. In this manner it was possible to rule out Ni, Co, Bo, but Manganese and Iron are candidates for several unidentifiable spots that often overlap Mercury in these assays. When KI and Na2S are used, however, only one dark area appears where the Mercury should be, its shape modified by the overlap of the now unseen substances. In the straight ore extracts, there are a number of substances overlapping the Mercury area, color indicating that the predominant ion being Iron. In these plates, the Iron completely blocks the signal from the Mercury.
Another system involves the pre-reaction of the sample with Dithizone in Chloroform. The colored reaction products are then spotted on Silica gel G and eluted with benzene. The Dithizone reaction products are colored, and no sprays are needed to visualize the spots. In this case, it was possible to detect Mercury in the root samples where there was contamination with Copper or other materials due to the difference in separation characteristice of the Dithizone product. The Mercury complex appeared close to the solvent front as a distinct orange spot.
Pre-concentration of samples is necessary to bring out a dark spot on the plate. One method is so use a simple piece of cellulose paper and solvent to pre-chromatograph a large amount of extract. The solvent front area of the paper is cut off and re-eluted at 90 degrees to the first run, bringing all the Mercury to the end of the paper strip. This can then be spotted with a bit of reagent, and the color observed, or washed out and concentrated for spotting on a regular tlc plate. Also, columns of Silica gel made up from pipettes could be used to pre-elute the extract. These methods are still in development, and show great promise.
Summary of analysis of root and ore: Chromium, Boron, Nickel and Zinc, can be ruled out in ore and root samples.
Possibly Manganese (pink) and quite likely Iron (dark blue) can be detected in ore using PAN, or Alizarin as indicator, and may be present in small amounts in root as well.
Aluminum is detectable in ore and root when plates are sprayed with Morin solution and observed under UV, and by color reactions with PAN and Alizarin, and by lack of color reaction with other reagents.
Mercury is detectable in ore and root using KI, Na2S, Alizarin, Dithizone directly, or pre-derivitivized, Dithio-oxamide, or Diphenylcarbazide. Hg content of root is low and difficult to detect, except for Alizarin reaction which seems quite sensitive for only one of the Hg oxidation states.
One or more of these methods can be readily adapted into a stable system for routine screening of specimens, at low cost, and with readily available materials.
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Methods of preparation of organic samples must take into consideration different degrees of binding between plant essential oils and heavy metals. These adducts need to be explored in greater detail.