Following

Try an electric blender with stainless steel blades, that could work.

for conifers materials, we used to cut them to small framents with a razor blade washed with chlorofom/methanol (to avoid lipid contamination) before freeze drying and using agate mortar and pestle and liquid nitrogen. This enhance the specific surface of your samples and thus make freeze drying more efficient. We used solvent extraction afterward though, so i don't know if it would be dry enough for effective grinding and ICP-MS

I suggest you first cut the leaves with stainless steel scissors and, then, you can grind them in an agate pestle and mortar mill. If you can have access to an agate rotary mill, it would be more efficient. I have found that if you don't pre-cut the leaves, the fibers just get elongated and not ground by the pestle. I also found that blending was not quite fine enough to produce an homogeneous sample.

Hi Miranda

If you need to use fresh samples then it should be hard to change it to power.

In my work I use dry samples so first I freeze fdry them and then its very easy to transform them into a powder using some of the material you mentioned.

I read also something about freezing the samples in liquid nitrogem inside a container with agata balls and after hake the container in a mechanical shaker. I guess you can you this methodology for fresh samples.

Well, the first question is what you call a 'sample' . If it is soft plant tissue, the most practical tool are ball mills, were you can use steel balls in small plastic tubes 5 or 10 at one go (with a special holder). The benefit is, you don't have to clean the mill, your sample is all contained and remains contained until weighing in processing for MS. Retsch is one company that sells such mills.

We used an electric coffee mill with stainless blades. Dry the plant material, and these mini grinders will turn it into dust, fairly inexpensive too, like this one:

http://www.zappos.com/krups-203-fast-touch-coffee-grinder-white?ef_id=UVxDOgAAAZS6lwge:20130403145658:s

First dry the sample in a hot air oven at 100 or 110 degrees centigrade to eliminate moisture and try to grind it into a fine powder. Follow the procedures given in Biogeochemistry in Mineral Exploration written by COLIN E. DUNN and edited by M. Hale

What do you want to do with the dried, ground leaves? Digest them in strong acid? Surely you don't feed ground leaves into an ICP-MS. Depending on how aggressive the digestion is, it may not be important to grind the leaves into a fine powder.

Thank you for all the feedback.

One of the problems is that I cannot use any metal-rich tools such as stainless steel blades/mills etc, or even ceramics, as they will contaminate the samples and they won't be usable in the study. At least this is what I have been told by my supervisor and the lab staff.

The samples have been freeze-dried under vacuum for over 200hours in total, so they are dry, but they are still not brittle. Even when crushing them in liquid nitrogen in the mortar and pestle (as this should make them brittle) the stems and leaf veins would not break up.

The samples need to be totally homogenous, as the different portions of the leaf have different concentrations of metals, and using a non-homogenous sample would skew the results.

For irms preparation, we use a mill grinder in our lab. It has interchangeable sieves down to 0.25mm. Have not had any problem with any type of plant after it was first dried. Our mill was purchased from VWR.com. Although i'd probably prefer a ball mill, the mill grinder we have is about 1/2 the cost of a ball mill. In the US, the VWR catalog number is: (43300-188). You'll also have to purchase a "cutting grinding head" as well as a "sieve".

In grad school, I used a home-made grinder. A thin (2cm) pvc tube attached to a rock tumbler motor. This was then placed close enough to another pvc tube of the same length that was able to rotate. Glass jars with samples rotated between the pvc tubes. Samples were placed in round glass jars with ball bearings and very thin metal rods that fit in the jars. Samples went in the glass jars for grinding for a few days. Very inexpensive. Could do multiple samples depending on how long the pvc tube was.

At the Forest Soils Analytical Lab, we begin by drying foliage at 60C, cooling, to room temperature in a dessicator, and then processing the material through a small Wiley mill to pass a 20 mesh sieve, Next, that material is thoroughly mixed and a subsample is processed through a ball mill. This procedure has worked well for us.

Dear Miranda,

I had the same problems with poplar leaves. Ball mills don't perform well at all.

In the end we did it with the

Vibrating Cup Mill PULVERISETTE 9 (Fritsch): In the Vibrating Cup Mill, the grinding is performed by horizontal circular oscillations of the grinding set on a vibrating plate. The grinding set consisting of ring and puck comminute the grinding sample with extremely high pressure, impact forces and friction.

It is a very expensive device, but it grinds the samples within a few seconds!!!!

Look for a lab that has a mill like that.

best wishes.

You can check for metal contamination by comparing different mills.

We had absolutely no problems with that.

we had no contamination problems with the inexpensive coffee bean grinder. Easy enough to check.

I had similar problems when tried to grind my plant samples. And decided do not grind them. The plant samples can be successfully digested with strong nitric acid. Of course, it is necessary to wash the samples carefully just after sampling to remove small soil particles from root and leaf surface (then we will not have problems with digestion of these impurities). As to me, after washing I use air-drying of samples till constant weight.

Have you got access to a cryogenic mill? It works wonders and grinds leafs, leathers and even bones! we use it in our IRMS and TA lab to grind and homogenise difficult samples. You will need liquid nitrogen. good luck.

@ Elisa- what is the use of the liquid nitrogen?

You can think about microwave assisted digestion

To make an fine powder of sample If you autoclave the dried sample at 121 lbs for 20 mins then grind it & sieve then you will get the fine powder.

We grind thousands of plant samples after drying them down using a paint shaker retrofitted to take 15 mL or 50 mL corning tubes with stainless steel beads. We run it for 5 minutes and get a very fine powder. At first we borrowed the shaker from another lab but since we use it so intensely we purchased our own from ebay for $300. The brand that we are using is called Red Devil (http://www.reddevilequipment.com/) I think that it uses a lot of force and with the stainless steel beads may be strong enough to grind your samples. There's also a piece of equipment called GenoGrinder (http://www.opsdiagnostics.com/products/homogenizers/gg2010.htm) that does the same thing, but I think it has less force. In the beginning we used it but the paint shaker was much better. Good luck.

For the grinding of leaves and stems, first of all they must be dry enough and the you may use a grinder which utilizes the action of rotating knives and the leaf or stem material is fed from above/ The ground material passes through a built in filter *(various meshes are available) to get the required particle size. wish you good luck

Hi Miranda

If you do continue to try grinding your leaves, or for any other kind of samples for that matter, the container in which you grind can be problematic. If you are using ICP-MS for trace and rare earth analysis, beware of contamination. Glass jars and beads can contaminate your samples with boron, plastic can transfer Zn, anything made of steel can transfer various metals. Of course there is always the potential for contamination. I usually use ceramic mortar and pestle with liquid nitrogen, (students typically don't like this slow manual method...), but I also agree with the other suggestions of using the power of acid to obtain complete digestion.

For moderate particle size, or as a first step, have you tried a kitchen blender?

If you want to measure trace metals, you can potentially try microwave digesting them using nitric acid (highly purified, trace metal grade). We have used this for homogenizing a variety of samples types, which we then analyze with a ICP-OES.

See this paper: Rodushkin, I., Ruth, T. & Huhtasaari, A ̊. 1999. Comparison of two digestion methods for elemental determinations in plant material by ICP techniques. Analytica Chimica Acta 378: 191–200.

Also, here is a link to a page from Stanford's Environmental Measurements lab that gives general information about microwave digesters:

http://em-1.stanford.edu/Schedule/Mars/Index.htm

You can try drying in hot air oven at 80 deg C for about 8 hours. then try grinding it with agate mortar and pestle. Or the same with freeze drying and grinding. If both of them doesn't work, then you may do dry ashing at 500 deg C and dissolving the residue (ash) it with aqua-regia, dilute, nutralise, filter and make up the volume for directly taking for ICPMS analysis. But you should take precaution not to put any contaminant in the sample. Reagents should be ultra pure, dilution should be carried out with millipore water.

You can add dry ice and powder the tissue. Or freeze using liquid nitrogen nd powder the tissue in a mortar with a pestle.

I agree with Donna and Dinesh comments about the potential contamination of samples if you grindrinding your samples by ceramic, steel or plastic grinders or shakers. You can use agate mortar and pestle and liquid nitrogen.

Hi, I would try freeze dry (lyophilization) and then any of the methods you say.

Hope it helps.

Hi Miranda, I would try liquid-nitrogen and high stirring blade-mixer or ice-breaker; if this still does not work, then you might try to get rid of your resins (all or most of them) by extraction with hexane, and then repeat the pulverisation as you did already. Metals should not pass in hexane, althought I would check this accordingly.

It is not necessary at all to grind the sample. Leaf is easily to be completely digested in hot HNO3. If your sample is too large and have to be grinded for analysis, any method for grinded is OK, but should be careful to avoid introducing any element you concern.

Just add liquid nitrogen (or dry ice) to tissue in a mortar and after a couple of minutes . It So long as you keep it cold by adding liquid nitrogen the tissue will remain hard and you can grind with a pestle.

when you grind your broken plant samples, you can try to move your pestle immediately once some liquid nitrogen was added into your Agate mortar. You try do it several times, and then you will find the best chance. Be careful, samples must be dried.

HI Miranda

Have you tried a coffee grinder whilst the leaves are still frozen.

There have been many good recommendations provided thus far. If the freeze-dry step has not been effective, then the oil/wax/resin content is too high and you will not achieve a good result in the standard procedures. We see this with tobacco leaves and of course peanuts.

Unless you have microwave capability available, then simply using HNO3 even with peroxide is not going to destroy the oils/waxes, etc. even with prolonged heating. The hot digests will appear clear but the oils/waxes will soon solidify. Without availability of microwave or similar digestion bomb, your best approach is to try dry ashing with possibly an ashing aid, pretreatment with some sulfuric acid before wet digestion, or simply work around the undigested components. Pretreatment with sulfuric acid is effective, but may compound the analysis because of purity problems with the acid and transport effects in ICP torches of the acid content on nebulizer efficiency.

If you try dry ashing, we do not exceed 500C and there is loss of some metals, which seems to be a function of the original plant matrix. It might be possible to use a charring technique at lower temperatures and then switch to wet digestion. This will have to be tested with spike recoveries. Of course you would need to use Pyrex or Quartz with accompanying chances of contamination for the elements of interest.

Have you considered something like neutron activation analysis which technically would avoid the issue of tissue grinding? Sample geometry is important to achieve the same neutron flux exposure, but it might be worth considering a subset of samples done by INAA as part of your overall evaluation of a digestion procedure. This depends on your elements of interest and their concentration in the original matrix. It is more expensive as well, depending on availability of facilities.

I think madam if you are using digestion of your sample for ICP_MS then it is not necessary to make fine powder just turn it into pieces and digest using standard method. your results will be more accurate

Not necessary to grind finely...for digestion partial grinding is enough...

I powdered the plants leaf after drying in oven. It is better to dry in the oven at 60C-70C until then grind and follow the standard acid digestion. After filtration you could measure with ICP-MS.

Dear Miranda, Its better to dry all the samples in oven at 60 degree, ground in mill and follow standard method for their digestion (HNO3 & HClO4 1:4). I have tried this method for Cd determination from plant samples.

You don't need a finely ground powder. If you're analyzing plant material by ICP-MS, you need to use a bomb - microwave bombs are more convenient than conventional. In the bomb it doesn't matter if the plant material is finely ground or not. Depending upon which elements you're looking for (you didn't say), you can adjust the acid mixture to completely dissolve the plant matrix.

We routinely grind leaf samples to a fine powder for a variety of analyses including ICP-MS. We use cryogenic milling using instrument from SPEX (such as the Geno/Grinder or Freezer/Mill). By freezing the samples in liquid nitrogen and then using the impact grinder, we achieve a powder consistency every time (as long as the samples remain frozen).

I will try introducing CO2 pellets inside the mill. The CO2 pellets (at -20ºC) are able to maintain the foliar material frozen during the grinding process and you will not have cross contamination in your samples.

The leaf samples can be super cooled in Liquid Nitrogen and ground into power (for fresh leaves) without vacuum. But the Liquid nitrogen has to be poured 2 to 3 times before grinding and also 2 to 3 times while grinding.

I got very good fine powder with a small coffee grinder and LN for sugarcane leaves. The leaves cut into small strips need to become brittle and make a metallic sound prior to grinding. The flash freezing is done in a plastic container then when the LN has just evaporated the leaves are transferred to the coffee grinder. This must be quick.The coffee grinder needs to be filled with leaves at least to half its capacity and cover the blade. Maybe you should not freeze dry as it removes the water needed for LN to work best.

Freeze drying and adding liquid nitrogen into the zirconium oxide vessel prior to the milling.

I have done some work with woody plants and seaweed. The best method I found was to dry the samples overnight-48 hours (60-75oC) and then place the sample in a cryo-mill cooled with LN. We use plastic coated grinding tubes and rods to avoid sample contamination. I found that some of the woody material needed to be pre-milled in an agate pestle and mortar but after this the samples were well powdered. Make sure you leave about 1/3-1/2 of the volume of the tube unfilled for best efficiency.

Put the freeze dried material into a ceramic mortar and add small portions of liquid nitrogen during the grinding. For a total digestion include a small volume of HF in the digestion procedure.

Dear Miranda

Depending on what elements you want to analyze for, the best way of dealing with plant samples is actually to ash them at 400 degrees C. The technique preconcentrates the samples and one achieves best results. However, if you are interested in the more elements that are volatile at low temps e.g. selenium etc, then milling the samples followed by hash digestion would help. Note that even after ashing samples you must digest, dilute and then analyze. Hope this helps.

Dear Miranda,

I agree with Mark. When you are interested in analyzing your samples in ICP-MS, finely grounded powder is not a requisite (in case you are unable to attain it). Moreover, following this you need to digest your samples with concentrated acid which will bring them to a liquid state. So, the coarsely grounded sample will also work out. In addition, you will be taking the same sample in triplicate or more which eventually will reduce your error.

So, Happy Researching !!!

This is less a problem of "micro-homogeneity" of your sample (fine powder), but a question of sample size (sample weight) which must be used for digestion/dissolution to be considered as a representative sample.

P.S. You can use a microwave digestion oven, but a simple hot block or even hot plate acidic digestion should work well.

Jerzy, I thought that would be the case, but I was told that it was categorically not possible to digest a larger amount of sample to make up for the lack of homogeneity in the sample.

Anyhow, what I have found that works best, and didn't require buying a new mill, was to sieve my leaf samples by punching tiny holes in weighing boats (one for each sample to avoid contam) and then using Liquid N to grind the samples in an agate Mortar and pestle, then sieve out the fines, regrind the remains, sieve, regrind, sieve, regrind, until the sample was sufficiently fine. It's tedious but effective.

Jerzy, I thought that would be the case, but I was told that it was categorically not possible to digest a larger amount of sample to make up for the lack of homogeneity in the sample.

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In some cases "not", but in some cases "yes" (I know it, because I was working for several years on slurry sampling AAS {including plant materials} and in

this analytical technique, a micro-homogeneity of the sample is critical

{as we used samples at the 1 to 10 mg level}). However, if you take,

e.g. 0.4 - 0.5 g of the sample, homogeneity may not be

a problem...

You can always check the homogeneity, by preparing a statistically valid number of samples (e.g. a dozen) and after then looking at the repeatability/precision of your results (if your analytical method is very

well established).

I am sorry to say that unless the objective of the experiment is clearly stated, such questions are meaningless.

Miranda said "I can't proceed with the analysis", so I understand that her goal is a typical chemical analysis of leaves (elemental analysis, maybe analysis of some organic stuff)... but, maybe I am wrong. In fact, she should explain her analytical goal more in detail.

I have stated that I am doing analysis by ICP-MS, and that I need to have my samples suitably homogenised so that I can subsample a representative sample set.

As I am not the only person who will be using this sample set, I am preparing the samples by the most conservative methods possible. My objectives are somewhat convoluted, and the ICP-MS is only a small fraction of the analysis that I am doing.

MANUAL on methods and criteria for harmonized sampling, assessment,

monitoring and analysis of the effects of air pollution on forests

http://www.icp-forests.org/pdf/FINAL_Foliage.pdf

I used to add a couple of small pieces of dry ice along with my leaf sample (previously frozen solid at -80C) to a handheld electric coffee grinder. Be sure to use heavy duty gloves while doing this! End result is a beautiful, evenly distributed powder.

Good Luck!

If the leaves contain only ligno-cllulosic materials (biopolymers), then they should become brittle on cryogenic cooling.

In my experience with wood and leaf biomass samples (analyzed with both ICP-AES and ICP-MS), I agree with Sudeepta: you don't necessarily need to mill your samples to a very fine texture to analyze them since you will be digesting the samples in acid. The times I have replicated sample digestion to assess intra-sample variability due to milling, I found very little variability.

Thanks. I found that by alternatively milliing and sieving the samples using a boat with small holes punched into it, and then remilling the coarser material I managed to get a fine enough texture.

Air drying the leaves in a contaminated free environment and grinding with mortar and pestle and dissolution with microwave digester could solve this problem

If your leaf samples contain epicuticular wax, it is a challenge to make fine powder. You may want to pool the leaves, cut into smallest pieces possible, mix them nicely and then take subsamples for various analyses. Each subsample can be processed according to your group's target analyte(s).

I run into this issue all the time. I recently had to grind up marsh grasses for isotopic and biomarker analysis, and it was not fun. Very fibrous, lots of structural material. I found by using liquid nitrogen directly in the mortar and pestle I was able to get a much better grind. Even so some samples I had trouble with but it was night and day vs. just trying to crush them up. you can actually pour just a bit of liquid N2 directly in the bottom of the mortar, and dump your sample right on top of it. As others have suggested cutting the leaves with scissors is a good way to prime them

Prior to the analysis of metals in complex plant materials by ICP-MS, I would suggest digesting the samples with three strong acid sequentially (nitric acid, chlorhydric acid and fluoric acid). This type of total digestion usually allow recovering virtually all metals of of samples. There are protocols available for this type of total digestion. If you are interested to get one , I can send you one by email.

Good luck in your analyses,

Hello Miranda,

Since you will be digesting your samples, you do not need to have them in a powder form. You can either digest them as they are after drying at 105oC to remove moisture ; or you can ash them at a higher temperature and then digest them. For digestion use a mixture of perchloric HF and nitric acids in a teflon beaker until the contents are nearly dry (gel like). Then make the solution to volume so that the final solution is 3% with respect to nitric acid. The digestion must be carried out in a fume hood with an attached scrubber as perchloric and HF acids are lethal. Personal protective gear such as safety glasses, safety shoes, laboratory coat and gloved(double) are mandatory to prevent accidents. For analysis use ICP-OES and/or ICP-MS depending on the concentration of the analytes in question.

Freeze tissue with liquid nitrogen and ggrind in a mortar with pestle.

For biomass samples, there is no need to go as far as HF which is an extremely dangerous acid and necessitates heavy procedures to manipulate. For biomass samples, concentrated nitric acid (50 to 69%) is enough. Digestion is simply done by heating the sample+nitric acid.

For analysis of sample by ICP-MS then need to digest the sample using strong nitric acid.

Hello there,

I do not agree with Gregory. If there is any silica or silicon compounds in the leaf you must use HF for digestion - no other acid except HF can digest silica. These may be in low quantities, but for total digestion they must be accounted for. Another thing is that the silica may be in colloidal form and not visible to the naked eye and when you filter the solution for analysis (when not using HF), the silica remains in the filter paper. So one erroneously assumes that there is no silica in the solution. Therefore, HF, perchloric acid and nitric acid must all be used for total digestion of any sample which may contain silica or silicon compounds.

That is a good point! I haven't given Si compounds in leaves much thought because I mainly work with macronutrients and not with Silica. I am wondering if digesting the SI compounds is important is you are interested in other elements and to be more specific trace metals. I'm guessing that if macronutrients get trapped in these Si compounds, the amounts are probably low compared to the other pools in the leaf but with trace metals, if they get caught up in these compounds, it's another story and not digesting Si may induce a bias.

Do you have any experience in this?

I would also like to add that using HF besides the fact that it's really dangerous and therefore tedious to use, you need to neutralize the HF once the digestion finished by precipitating it before you can analyse the sample. This may also bias analysis if elements get caught up in those precipitates. In Mg stable isotope analysis, HF precipitation is known to fractionate isotopes. So you then have to dissolve the precipitates again in acid.

Why do you want all of it well powdered? 70 % success is fine. filter and prorate concentration.

Thanks and good luck Muller

Hi Gregory,

You don't need to neutralise the HF for analysis at all these days. With ICP-OES, ICP-Ms and AAS, there are nebulisers which are compatible with HF provided it is 1% in solution - ie. they are not glass nebulisers. So there is no problems in using HF and getting total digestion of a sample. I hope this helps.

I think that neutralizing HF is necessary simply for security reasons.

Everytime you manipulated the samples, you would need to have the full protection gear on and be under an adapted fume cabinet.

I'm also pretty sure that most ICP instruments can endure a little HF if their components are made of teflon. However, you can't have sample tubes with HF in them seating open in a room: the HF would be dangerous for anyone in that room who would be breathing it in .Repeatedly, the HF would end up by corroding other components of the ICP instrument and other lab components in the room.

So unless, teh ICP instrument is itself in a fume cabinet, I would say that you need to neutralize the HF.

Yes Our ICP-MS is in the cytotoxic cabinet and we have an efficient fume hood. I do not see a problem. Talk to the instument specialist if you are unsure. They are the people who set up the instruments after taking everything into account.

Cut in small pices and digest with nitric acid with Microwave MARS 6 of CEM Corporation. Write me at [email protected]. I will send you a method to digest leaf samples to read elemtes at ICP MS

you can directly digest with conc. HNO3 after small cutting of leaf sample.

Break up the dried leaves into pieces with agate mortar and attack directly with Aqua Regia (3ml HNO3 + 2ml HCl). The digestion will be complete after 6hrs on a hot plate at 250 degree C

Hi Miranda, back to your original question. You clearly have covered a lot of ground and I have to admit I am surprised that liquid N2 in combination with pestle and mortar did not work. I would have suggested trying a SPEX freezer mill, which uses stainless steel impactors, liquid N2 cooled, but now I am not sure. However, there might be an alternative to pulverizing your samples. Have you tried "washing" your samples in an organic solvent or solvent mix to dissolve the organic sap / resin and thus removing it? Sap / resin is a mix of terpenes and sesquiterpenes that should dissolve readily in hexane or heptane, perhaps with a bit of DCM added. These solvents are apolar and not miscible with water so there should be no risk of loosing any of the trace elements.

Hello Mirinda, hexane will be fine just like Meier suggested. Goodluck!

Hi.

One approach might be to consider your problem from the other end.... what precision do you actually need from the analysis, and what is the maximum amount of material that your digestion method could cope with.

If you're preparing material to use the leaves as, for instance, a certified reference material, then a fine powder would be the end goal. However, if you're looking for indicative levels, say within a batch of leaves, you might be as well to spend less time grinding specific pieces of leaf (which may reduce the amount of sticky material released), and try taking a LARGER sub-sample, but of a LESS FINELY GROUND material. This would give you a less precise, but more accurate measurement of the overall elemental composition within the leaves generally. As I say, a lot depends on the end use of the data.

Regarding the digestion methodology: again, a lot will depend on what elements you're specifically interested in, and what the data will then be used for. A microwave-assisted, high-pressure, high-temperature aqua-regia digest has proven to be a very robust approach in our lab (see Fernando Villanueva's earlier offer of help). However, if you're looking for a total digestion (to include the silicaceous material) you'll probably have to go down the HF route.... but beware of the H&S implications!!

Keep 'fit-for-purpose' in mind.

Best of luck.

John Lewis

If you are trying to understand the localization of metal at different plant parts (I guess)...I dont think stainless still instruments has any problem.........steel is not that easy to mix into your sample i guess

I agree with Sen

Hi Miranda

Porcelain pestle and mortar with liquid nitrogen.. works like a charm. Give me a shout and come through to my lab. I'll show you :)

If that still doesn't work see if you can get hold of a spex mill.. not that many places have them because they are very expensive but they are very effective and used in  a variety of studies where contamination is an issue. 

Liquid nitrogen is a good idea. Ball mill does a better job than the hand with the pestle- and is even safer.

Dear Miranda, I will like to know if this is for elemental analysis?

Following

"Why not use laser ablation icp-ms this way you could also map out the amount of each element across the leaf."

A good proposal, but even today (2019) ICP-MS spectrometers with a laser ablation sample introduction system are expensive and not commonly found.