Si Extraction Steps, May 10, 2009

Extraction Steps 10 May 2009

Lab equipment
*Water bath, 85˚C
*Spectrophotometer, 810 nm
*4-place scale
*Digital pipettes, 100-5000 µl (2)
*Acid bath, 10% HCl
*Glass Erlenmeyer flasks, 250, 500, 1000 ml
*Glass volumetric flasks (class A), 25, 100 ml
*Glass volumetric flask (class A or B), 1000 ml
*Plastic volumetric flask, (class A or B), 500 ml

Chemicals
*H2O
*H2SO4, 100%
*HCl, 32%
*Ammonium molybdate tetrahydrate
*Sodium sulfite
*1-amino-2-naphthol-4-sulfonic acid
*Sodium bisulfite
*Sodium hydroxide pellets

Notes
*All references to H2O assume Milli-Q deionized H2O unless otherwise specified.
*All glass labware should be rinsed 3x in H2O before using (even if it has been previously cleaned).
*Reagents and other liquids should not be stored in glass containers. Exposure to glass should be minimized.
*NaOH solution should never come into contact with glass unless diluted.
*When using pipettes, use the 2x aspirate-dispense procedure.
*Label all reagents!

Reagents
*2 M H2SO4, 1000 ml
-Add 800 ml H2O to 1000 ml volumetric flask.
-Add 108.6 ml 100% H2SO4.
-Fill to 1000 ml mark with H2O. Mix thoroughly.
-Dispense using 5 ml plastic repipettor. 5 ml dispensing volume.
-Reagent typically lasts indefinitely, but do not store in an open container.
*Molybdate, 1000 ml
-Add 27 g ammonium molybdate to 1000 ml flask
-Fill to 900 ml with H2O. Mix until dissolved.
-Add 6.178 ml 100% H2SO4. Fill to 1000 ml with H2O. Mix thoroughly.
-Dispense using 5 ml plastic repipettor. 10 ml dispensing volume.
-Store in opaque container. Do not use after 24 hr.
*Tartaric acid, 20%, 500 ml
-Add 100 g tartaric acid powder to 500 ml flask.
-Fill to 500 ml with H2O. Mix until dissolved.
-Dispense using 5 ml plastic repipettor. 2.5 ml dispensing volume.
-Reagent typically lasts indefinitely, but do not store in an open container.
*Reducing solution, 250 ml
-Add 2 g sodium sulfite and 0.4 g 1-amino-2-naphthol-4-sulfonic acid to 25 ml beaker. -Add 25-30 ml H2O, mix thoroughly using stir button under fume hood. Be careful of the fumes.
-Add 20 g sodium bisulfite to 250 ml flask. Fill to 200 ml with H2O. Mix until dissolved.
-Add sodium sulfite/naphthol mixture to sodium sulfite mixture. Fill to 250 ml with H2O. Mix thoroughly.
-Dispense using 1 ml repipettor. 1 ml dispensing volume.
-Store in refrigerator. Reagent typically lasts a few days. Do not use if precipitate is visible in the solution.
*0.5 M NaOH, 500 ml
-Add 450 ml H2O to 500 ml plastic volumetric flask.
-Add 20 g NaOH pellets. Fill to mark with H2O.
-Mix until dissolved. Fill to mark once again, mix.
-500 ml volume added to soil sample.
-Reagent typically lasts indefinitely, but do not store in an open container.
-Sample preparation and weighing
-Crush soil sample gently with a rubber pistil
-Dry in desiccator at least overnight.
-Weigh 1.3500 g of sample onto 50 ml scintillation bottle cap.
-Transfer weighed sample to 500 ml plastic bottle. Be sure no sample is stuck to the cap.
-Record sample weight and bottle number in notebook.
Dissolution procedure
*Add 500 ml 0.5 M NaOH (preheated to 85˚C) to soil sample. Mix thoroughly. Place in water bath. Record time of NaOH addition.
*Approximately 10 minutes before aliquot extraction, add 10 ml molybdate reagent to 25 ml flask. Add 5 ml 2 M H2SO4 to 100 ml flask, fill approximately ¾ full with H2O. Set aside.
*At specified time, remove sample from water bath. Mix thoroughly. Allow to sit for exactly 1 minute.
*Using the digital pipette, aspirate the appropriate aliquot volume. Dispense back into bottle, then aspirate again. Dispense aliquot into 100 ml volumetric flask.
*Fill flask to mark, mix thoroughly.
*Aspirate appropriate aliquot from 100 ml flask. Dispense into 25 ml flask with molybdate reagent. Mix thoroughly; allow to sit at least 2 minutes, but no more than 10.
*Add 2.5 ml tartaric acid reagent followed immediately by 1 ml reducing solution. Swirl. Fill to mark with H2O. Mix thoroughly.
*Allow to sit for at least ½ hour, but no more than 2.
*Measure Si-reagent mixture using spectrophotometer set to 810 nm. Be sure to thoroughly rinse cuvette with Si-reagent solution before measuring. Record values in notebook.
Dissolution procedure details
*The dissolution procedure typically lasts between 5 and 7 hours.
*Extraction begins at 3 hr and ends between hours 5 and 7.
*Extraction intervals are typically 20 or 30 minutes.
*Aliquot volumes are typically 2 ml for both 1st and 2nd aliquots. Aliquot volumes must be held constant throughout the procedure. Aliquot volumes must be the same as those used in the standard curve.
*Redundant aliquots are usually taken at each extraction (2-3 aliquots per extraction).
*Total ASi measurements though the dissolution procedure, n=20-30, but never less than 20.
*Linear regression is used to extrapolate total ASi. Total error associated with the y-intercept of the linear regression curve should be checked and kept to a minimum.
Standard Curve
*Follow reagent addition procedure outlined above. Si standards are located in cabinet below spectrophotometer.
*Use same aliquot volume as in dissolution procedure.

New Techniques for Si Analysis Part 1

It has been a long time since my last substantial post, for which I apologize. My onkly excuse is that I have been extremely busy with classwork, research, and several side project (which I will write about at some later date). As far as my research goes, I do have some updates to report regarding the Si extraction and analysis procedure.

1. Traditionally I have been using ~0.075 g of soil coupled with 100 ml 0.2M NaOH. I have been experimenting with the same amount of soil, but a much larger volume of NaOH (250 or 500 ml). This allows me to take a much larger aliquot, which lessens problems due to pipetting errors. Interestingly, I have been finding higher values of Si when I use a larger volume of NaOH (see graph). There may be several reasons for this: a) there may be residual Si on the inside of the bottles, the total amount of which may be magnified simply because of the larger aliquot taken; b) the larger aliquot (and NaOH volume) may be causing some weird absorption probem. I have observed in the past that NaOH, when mixed with molybdate before it is totally neutralized by acid, can raise absorption values by ~0.01. This is substantial.

The larger aliquot extracts probably require their own unique standard curve. If the NaOH is causing increased absorbance, or if there is residual Si, then it should be possible to correct for this in the standard curve in two ways. First, by premixing the Si standard with 0.2M NaOH, it will mimic conditions present in the bottle. When an aliquot of the NaOH/Si is added to the reaction flask (and the molybdate and acid reagents), it will be added in exactly the same fashion as the extractions. (By the way, I am now adding the molybdate reagent before I add the Si aliquot (see below). This could be critical, as the NaOH may not be neutralized completely by he acid (especially a larger aliquot). Second, water used to make the NaOH/Si standard should be first stored in one of the reaction bottles, to account for residual Si. Using these two steps, I should be able to determine if the problems outlined above are the real culprits.

2. The amount of 0.5M H2SO4 added to the flask matters. In the past when I was taking small aliquots (in the range of 0.25 ml) and adding 5 ml of H2SO4, this may have been problematic. The key is to keep the pH below 1.5 in the flask, which 5 ml of H2SO4 does, but the ionic strength may have been too great. An ionic strength of 0.5 and above may cause problems with the molybdate reaction. To account for this, I have now adjusted the acid volume in the flask relative to the Si aliquot volume. For example, an Si aliquot of 0.25 ml receives 3.9 ml H2SO4, while an Si aliquot of 1 ml receives 5 ml H2SO4, and so on. In this way, the pH and ionic strength remain low.

3. The mixing order of the reagents matter. In the past I have added the acid to the flask, followed by the Si aliquot, and then the molybdate reagent. This is not a problem for the molybdate, but it is problematic for the Si aliquot. Something happens to the Dissolved Si when it is added to only acid. My guess is that it polymerizes with some other compound present (perhaps the NaOH?). When this happens, it will not be able to combine with the molybdate. As I mentioned earlier, I suspect that unneitralized NaOH can raise the absorbance. Thus, the NaOH may be reacting directly with the molybdate. To account for these problems, I have begun adding the molybdate reagent to the flask before the Si aliquot. This has greatly reduced scatter problems. One would think that adding the Si aliquot before the molybdate would be preferrable, as the NaOH would be neutralized. I have found that this isn't the case.

More soon...

Currently Waiting for Stats to Begin

So I figured I would blog about it; something I have not done since February. Shame on me.

So THAT'S How You Dissolve Silica!

My current Si dissolution procedure:

Extraction Steps 12 February 2008
Items outlined in red have not been attempted yet

Items needed
50 ml Nalgene bottles
100 ml Nalgene bottles
Chemicals outlined by Jones & Dreher
pH meter
5N NaOH
0.5 M H2SO4
Digital pipette
Cuvettes

Reagents: follow the procedures outlines by Jones & Dreher.

Dissolution procedure
Weigh and record a dry 50 ml Nalgene bottle (cap included).

Place approximately 0.38 g soil in the bottle. Weigh and record.

Allow the soil sample to dry at least 2 hours at 70˚C, then weigh and record. Set aside.

Determine plant available Si
Add approximately 50 ml Academic water to another 50 ml Nalgene bottle. Record the time.

Place in the pre-heated 85˚C water bath for at least 0.5 h.

Using the digital pipette, add 48 ml of the heated water to the soil sample bottle. Swirl the mixture gently. Tighten the cap and place it in the water bath for 1 min.

Remove a 1 ml aliquot using the digital pipette. Place the aliquot in a 25 ml (class A) volumetric flask which is pre-filled with 5 ml 0.5 M H2SO4. Flush the pipette tip into the flask with Academic water at least 2x to remove any residual Si. Record the time. Set aside. To determine the absorbance, go to the spectrophotometric procedures section.

Add 2 ml of 5N NaOH to the soil sample bottle. Replace and tighten the cap, and swirl the mixture. Loosen the cap approximately ¼ turn from tight. Place the bottle in the water bath, and turn the agitation speed to 4.5. Record the time.

Remove 1 ml aliquots at pre-determined times (10 min, 0.5 h, and so on up to 5 h). Record the extraction times. Depending on the extraction time, the concentration of Si in the aliquot will be up to 1000 µg. Since this is too high for the spectrophotometric procedures, the aliquot must be diluted.Place the aliquot in a 25 ml (class A) volumetric flask which is pre-filled with 2.5 ml 0.5M H2SO4. Flush the pipette tip into the flask with Academic water at least 2x to remove any residual Si. Bring the solution to mark (meniscus bottom should be at the line). Shake the flask to mix the solution. Transfer the diluted Si solution to a dry 50 ml Nalgene bottle.

At this point, the Si aliquot is diluted 25x. For example, if the original aliquot holds 500 µg Si, its concentration is 500 µg Si ml-1 H2O. When diluted, the flask still holds 500 µg Si, but now its concentration is 20 µg Si ml-1 H2O. However, this is still too high.

Extract 2.5 ml of the diluted Si solution with the digital pipette.

Place the diluted aliquot in another 25 ml (class A) volumetric flask which is pre-filled with 5.0 ml 0.5 M H2SO4. Flush the pipette tip into the flask with Academic water at least 2x to remove any residual Si.

At this point the Si solution is diluted another 10x. Using the scenario outlined above, the 2.5 ml second aliquot (which has a concentration of 20 µg Si ml-1 H2O) will hold 30 µg Si total. When this aliquot is diluted in the second volumetric flask to 25 ml, its new concentration will be 0.2 µg Si ml-1 H2O, well within the range of the spectrophotometric procedures.

Proceed to the spectrophotometric procedures section

Spectrophotometric procedures
Set the spectrophotometer λ to 810 nm. Set the background to 0.200. Allow the machine to warm up for at least 0.5 h.

Add 5 ml of the molybdate reagent to the reaction vessel (the 25 ml volumetric which holds the 2.5 ml Si aliquot along with 5 ml 0.5 M H2SO4). Swirl the mixture. Record the time. Allow the reaction to continue for 5 min.

Add 2.5 ml 20% tartaric acid. Swirl the mixture. Record the time. Allow the reaction to continue for 5 min.

Add 1 ml of the reducing solution. Bring to mark. Place the cap on the reaction vessel and shake the mixture gently. Transfer to a dry 50 ml Nalgene bottle. Record the time. Allow the reaction to continue for 15 min.

Fill a clean cuvette with Academic water and record its absorbance in the spectrophotometer. Empty the same cuvette and fill with the blue solution from the reaction vessel. Empty the solution from the cuvette and refill. Record the absorbance.

Yes, I am Still Alive

It has been a busy few months since my last posting. You know how it goes: I kept meaning to post to the blog, but could never find the time. Well, I still don't have the time, but oh well. Some things are more important that school work. Like procrastinating.

My last few blogs have been reviews of various articles related to silica dissolution methods. Not exactly great reading material, but nevertheless important for my research. Since then, I have continued reading plenty of articles in my attempt to perfect (or at least get a little better at) my own particular dissolution procedure. I have decided to pursue an 85 C water bath dissolution method similar to that outlined by Sauer et al. To that end, I purchased a used water bath on ebay. The nice thing about this particular water bath is that it is also an orbital shaker. Thus, my samples will not only be heated, but also will be agitated. This is a big step, and should greatly speed up dissolution. I have found that using a 0.2 M NaOH solution works best, and have thus far had good results.

I have run into several sticking points however. First is the issue of silica contamination from glassware. The use of glass pipettes, volumetric flasks, etc. can greatly influence the amount of silica actually in the sample. This is especially bothersome since I am working with dissolved silica amounts in the neighborhood of 10 micrograms (0.0000010 gram). Any contamination from glass can greatly influence this. To get around this problem, I have been using as little glassware as possilbe. For example, I have purchased a digital pipette which uses plastic pipette tips instead of glass. I am still forced to use glass volumetric flasks when I add reagents to the silica sample (this is a step used to "color" the silica, so its concentration can be determined). To mitigate contamination in this step, I never add the silica sample to a dry flask. Instead, I always make sure that there is water in the flask to dilute the silica before it comes into contact with the glassware. On top of this, the silica sample is not left in the flask any longer than is necessary. Undoubtedly, there is some silica contamination. But as long as I am consistent with my procedures (i.e. each sample spends the same amount of time in the flasks), any variation in the data should be mitigated.

Review of Johnson et al. (1990)

Johnson, D.L.; Keller, E.A.; Rockwell, T.K. 1990. Dynamic pedogenesis: new views on some key soil concepts, and a model for interpreting Quaternary soils. Quaternary Research, 33, 306-319.

Past researchers have attempted to create a model for soil formation (pedogenesis). Starting with Dokuchaev in the 19th c., climate was viewed as the dominant factor in pedogenesis. Thus, a soil was said to be zonal or monogenic if it was in equilibrium with its driving factor, climate. Johnson et al argue against this, stating that climate can change rapidly through time. Since we know this to be true, how can a given soil ever truly be zonal, or mature? A new model is needed.

As stated above, the authors discredit the monogenic concept, saying that no soil can truly be created by only one factor (climate). This seems fairly obvious, but it bears emphasis: soils are mixtures of solids, liquids, and gases. Various fluxes and processes occur within soils all the time. For example, plant roots can greatly speed up mineral weathering. While one could argue that climate is the ultimate driver of vegetation, I would state that many species (and mosaics) can be present in a given climate. It would be naive to think that they would all behave similarly in regards to soil weathering.

Thus the authors embrace the polygenic concept: that soils are formed from many different things. Further, these things can change through time. The authors give an example of a soil which develops distinct horizons with time. At some point, a new species of plant moves in, which encourages high worm populations. These worms mix (bioturbate) the soil, which blurs the horizons. Thus, a soil can be thought of as progressive (increasing complexity, organization) or regressive (decreasing complexity, organization).

While the authors don't explicitly state that the model of Jenny is incorrect, they may as well have. The Jenny model goes like this: a soil is a function of many factors, including climate, organisms, topography, parent material (rock) and time: S=f(Cl,O,R,P,T). This makes sense, and it is still widely used today. However, the Jenny model must assume that the factors remain more or less steady through time. For example, the climate must remain the same, even though we know it does not. Thus, the Johnson et al model (which they term the Dynamic-Rate Model) is an attempt to address varying factors. Here is the equation:

S = f (D, P, dD/dt, dP/dt)

where

S = degree of soil pedogenesis
D = dynamic vectors (aka more influential factors)
P = passive vectors (aka less influential factors)
dD/dt, dP/dt = change of the vectors any any chosen time

My interpretation of their use of the word 'vector' is, more or less, where the factor is going. As an example, water flux is considered a dynamic vector. How much pedogenesis would X amount of water percolating through the system cause? With this in mind, placing D and P individually in the equation makes them a sort of description of pedogenesis, or the rate or pedogenesis. The variables dD/dt, dP/dt are a bit more abstract. the little d is a calculus term: differential. To make a long and complicated story short, it basically tells you the rate of change of the vector at any chosen time. In other words, where is D going at time X? What about time Y? The sum of dD/dt, dP/dt can be positive or negative. Positive values indicate soil progression; that is, the soil in undergoing increasing complexity and organization. A negative value means regression; just the opposite.

The D and P of the dynamic-rate model is simply a copy of the Jenny model: it accounts for all of the factors and processes which can change a soil. The variables dD/dt, dP/dt are new. They account for changes in the factors through time. Of course, this makes the model infinitely complex: how does a soil scientist account for a large set of factors which can change in any way and at any time?

Old dog, new tricks?

It's the end of another perfectly good summer, and what do you have to show for it? Hopefully some good research data and a pounding hangover. For me, the summer research has been quite a learning experience.

You might recall that my original objective in the lab was to extract all of the phytoliths (more generically called biogenic silica or BSi) from my Nebraska soil samples. This hasn't changed, but the methods have. The old BSi extraction method follows that used by Piperno (2006). It is called the density extraction method, and it goes something like this:

1. Dry and weigh your sample.


2. Add hydrogen peroxide (H202) and hydrochloric acid (HCl) to remove organics and carbonates, respectively.


3. Add sodium metaphosphate to deflocculate the sample.


4. Wet seive the sample through a 53 micron seive to remove larger particles. [There are phytoliths which are larger than 53 microns, but most researchers tend to ignore these, as they are rare.]


5. Perform gravity sedimentation to remove particles smaller than 4 microns. [Again, there are phytoliths and phytolith fragments smaller than 4 microns, but researchers ignore these because it is very difficult to extract them in the density extraction method. Only later did I discover that a large percentage of BSi is actually smaller than 4 microns. This is one of the reasons I ultimately decided to abandon this technique.]


6. Float BSi in heavy liquid, such as sodium polytungstate, set at 2.3 g per cubic cm. Extract with pipette or peristaltic pump.


7. Dry BSi extract and weigh.

There are many reasons why this method should not be used for quantifying total BSi. First, seiving and gravity sedimentation exclude BSi which is >53 or <4>

Based on these drawbacks, I decided to turn elsewhere in my BSi quest. There is another method out there, termed alkali dissolution, which is promising (see my reviews of Jones 1969, Herbauts et al 1994, Saccone 2005). The basic method is as follows:

1. Add H2O2 and HCl to remove organics and carbonates. [This step is recommended by Saccone et al 2005, since it allows for easier dissolution of the silica.]


2. Add sodium carbonate or sodium hydroxide (both alkali solutions) to the sample, and heat for about 5 hours. [Heating and digestion times vary depending upon the researcher.]


3. Once an hour, remove a subsample of the supernatant and run it through the spectrophotometer to determine the dissolved silica concentration.

As time progresses in the digestion process, the concentration of dissolved silica will increase. If this were a perfect world, all of the dissolved silica would be from BSi. But unfortunately silica is a very common mineral, and can be found in many different inorganic forms, such as quartz, allophane, feldspars, aluminosilicates, and many others. These inorganic silicates usually have a lower dissolution rate than BSi. Moreover, the inorganics tend to dissolve at a linear rate through time. BSi, on the other hand, tends to dissolve quickly within the first two hours. This is shown graphically above (this graph is taken from DeMaster 1981). The x-axis is time, while the y-axis is the concentration of dissolved silica. Thus, the graph shows the increase of dissolved silica through time. As you can see, there is a large increase in silica in the first two hours, meaning that something is dissolving quickly (duh, right?). After two hours, the dissolution slows down and progresses at a linear rate. With this graph, DeMaster was able to estimate accurately the amount of BSi. Up to that point, nobody had been able to do this, since dissolved silica is all the same, regardless of the source. But DeMaster worked off of a few assumptions. First, that the BSi would dissolve quickly. Second, the inorganic silicates are going to be dissolving throughout the whole process, even in the beginning. But since the silicates dissolve at a linear and predictable rate, it was easy to determine how much dissoved silica was added from them. All one needs to do is follow the flat part of the curve back to time zero. All of the silica added below this extrapolation would presumably be from the BSi, while all that above the line would be from silicates.

This is a novel approach to determining total BSi, and quite a few researchers have used it, in both soil and aquatic sciences. However, it is not without its drawbacks. The DeMaster graph works best when there are very few inorganic silicate mineral types present. The graph above illustrates the dissolution of only one silicate. But what happens when there are multiple silicates, as is the case in soils?

This is where accuracy can take a back seat to averaging. In the case of soils, one must look at the dissolution curve and try to find the average silicate dissolution curve (hopefully there will be one dominant silicate which controls the curve). There are other options worth looking into as well. First, I wonder if it would be possible to document all of the silicates in a sample, and their relative proportions. If the dissolution rates are known for each silicate, then maybe it would be possible to get more accurate results. I guess all you would really need to know is the most reactive silicate, right after the BSi.

Second, aquatic researchers have made use of silicon-aluminum ratios (Si/Al) to estimate the amount of silica added from silicates. Koning et al (2002) dissolved various aquatic samples in sodium carbonate, and simultaneously measured dissolved silica and aluminum concentrations through time. The underlying idea is that BSi has a very high Si/Al (meaning it has very little Al), while silicates tend to have much lower Si/Al (alot more Al). As with the DeMaster graph above, Koning et al. displayed the change in dissolved silica through time. But they also added the change in dissolved Al as well. Armed with this type of graph and some really scary looking equations, they were able to differentiate BSi from up to three different types of silicates in a sample. The researchers were trying to show that they could identify individual silicates in their samples; they really didn't care about the BSi. But Si/Al ratios can still be used to quantify the total BSi: one simply needs to find the shift from high to low Si/Al ratios in the dissolution curve. It doesn't matter how many silicates are in the sample, so long as they all have lower Si/Al ratios than the BSi.

A third possible method is the use of stable isotopes. Derry et al. (2005) and Kurtz et al. (2002) looked at the ratio of germanium to silica in soils (see my earlier reviews of these articles). In a nutshell, higher Ge/Si indicates a more weathered soil. While I'm not concerned about soil weathering, I am interested in the Ge/Si of soils, because it may be vastly different than that found in BSi. There is no study that I am aware of which has documented the Ge/Si in BSi. Combined with Si/Al, Ge/Si could be a valuable tool. It's only drawback would be the expense.

So all in all, that is what I've been working on this summer. Joe and I have ordered the supplies to begin the dissolution extraction method. A few of the supplies are on backorder, so here I sit and wait. And blog.