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.

Review of Derry et al. (2005)

Derry, L.A.; Kurtz, A.C.; Ziegler, K.; Chadwick, O.A. 2005. Biologic control of terrestrial silica cycling and export to watersheds. Nature 433.

Background and problem statement. Germanium/silicon (Ge/Si) ratios in streams are always lower than the primary bedrock from which they drain. Kurtz et al. (2002) found that the excess Ge is stored in secondary minerals. Therefore, higher Ge/Si values should be found in older soils. However, dissolved Ge and Si in rivers can come from both primary and secondary minerals, which can skew the interpretation. Primary minerals will export more Si to streams than secondary minerals. Thus, these primary minerals should have a high dissolved total Si (termed [Si]) and a low Ge/Si. Conversely, secondary minerals (clays) should have a low [Si] and a high Ge/Si. The authors term this the Murnane, Stallard, Froelich (MSF) model, after the authors who proposed the idea. This model can be seen in Fig. 1, or in my recreation above. Since the authors are concerned with two sources of Si in the study streams (primary vs. secondary mineral weathering), they have termed the high Ge/Si and low [Si] (derived from clays; old soils) sources as component 2. Low Ge/Si and high [Si] (derived from primary minerals) sources are termed component 1. Thus, armed with the two measurements and the hyperbolic curve in the figure below, it is possible to ascertain what the percentage of each component is present in the stream.

Despite this novel approach, the MSF model does not consider the role of biologic processes in the Si cycle. It assumes direct control by mineral weathering. [Si] should be controlled by the amount of weathering and Si export to streams. Likewise, Ge/Si should be controlled by the state of the weathering: older soils with more secondary minerals such as clays will be Ge enriched (which means a higher Ge/Si). But the ultimate pathway which Si takes before it reaches the stream may be highly influenced by vegetation. Plant uptake of DSi, precipitation of Si as phytoliths, and subsequent deposition of the phytoliths back into the soil may ultimately control the rate of Si dissolution and export. Further, a sizable amount of Si may be stored in the soil as phytoliths.

Goals. “To test the predictions of the MSF model and to investigate the impact of biogenic silica cycling on stream export.”

Study area. A chronosequence of Hawaiian streams.

Methods. The authors use two measurements: Ge/Si and total DSi [Si]. The authors recognized that DSi in the streams must be of two components (see above). The reason for this is simple: the Ge/Si value from fresh basalts is ~0.2 x 10-6 mol/mol. But the Ge/Si values recorded for the Hawaiian streams did not agree with the basalt Ge/Si. In fact, a mixing was recorded: Ge/Si of 0.2 x 10-6 mol/mol and [Si] >600 µM for component 1; and a Ge/Si of 2.6 x 10-6 mol/mol and [Si] ≤25 µM for component 2. As can be seen on the graph above, these components don’t quite fit together. The MSF model predicts that component 1 must be originating from bedrock weathering at the soil-regolith interface or from young soils which aren’t yet depleted of Si. On the other hand, component 2 should be originating from weathered soils, where the dissolution of clays and secondary minerals is dominant.

To test the MSF model, the authors recorded Ge/Si and [Si] in a chronosequence of Hawaiian streams. In other words, stream 1 drained a young watershed, stream 2 drained an older one, and so on. The chronosequence ranged from 0.3 to 4100 kyr (see Kurtz et al. 2002). The soils in the young watersheds have low Ge/Si and high [Si], as expected. Soil water solutions were extracted, and tested for Ge and Si.

Results. At sites older than 20 kyr, something strange was noted. Below 15 cm, DSi concentrations range from 6 to 45 µM, which is expected. The streams had concentrations of 100 µM or more, which is in contradiction of the MSF model. However, the top 15 cm of the soil did have roughly equal DSi concentrations as the streams. Thus, most of the DSi in the old soils is found in the topsoil, even though DSi is easily leached. Other studies had found this oddity as well, but had attributed the high Si values in the topsoil to dust import.

At the young sites (0.3 kyr), DSi is high: 200-600 µM. This is expected from young soils. However, the DSi in the topsoil is extremely high; in some soils it approaches the point of DSi saturation. Ge/Si values are lowest in these young topsoils. As with the old soils, something seems to be pumping Si into the topsoil.

At the old sites, the soils below 15 cm are in agreement with the MSF model as component 2. At the young sites, below 15 cm, Ge/Si is too high and does not agree with that found in streams. To put this another way, the lower soils of the old sites are in agreement with the MSF, but the upper parts have too much [Si] (Si enriched). At the young sites, the upper soils are in agreement, but the lower soils have abnormally high Ge/Si (Si depleted). The young soils are only in agreement with the MSF model in the upper layers.

These high [Si] and low Ge/Si findings in the upper soils at all sites are the result of phytolith entrainment. Next, the authors apply their data to form the mixing model (% component 1, % component 2), which I’ll skip.

As one may guess, the amount of Si exported to streams cannot exceed the supply in a long term fashion. The authors found an export of DSi at 150-5400 mol ha-1 yr-1 in the streams. In the upper zones of the soils, they found an export 400-9400 mol ha-1 yr-1. There seems to be a large surplus, so where is all the excess going? The authors propose that there is a rapid cycling of Si in the upper soils. Namely, any excess which is not exported to streams will probably be dissolved and taken up by plants: internal cycling.

Discussion. These data suggest that most Si in streams has passed through the internal vegetation Si cycle. This means that Si directly from mineral weathering passing into streams is only of a minor constituent. The authors go on to suggest that the phytoliths and other BSi is much more prone to dissolution than primary and secondary minerals.

Tropical humid soils are usually Si depleted, meaning that most of the Si is trapped in the vegetation. Any which is deposited in the soil is rapidly recycled. The high amount of [Si] in the upper soils acts as a buffer to toxic Al levels.

Conclusions. This is one of a series of papers which came out in 2005 detailing the biologic control of plants on Si.