Earthworms produce balls of calcium carbonate which are visually very attractive.
|Successive close ups of a calcium carbonate ball excreted by an earthworm, taken using a scanning electron microscope. The ball is about 1 m in diameter|
We've tried to look at what controls the stability of the ACC using bulk analyses but to no avail. The granules are so heterogeneous that we can't see a relationship between granule composition and the amount of ACC that they contain. What we need then is a technique that is spatially explicit. Beamline B22 at the Diamond light source is the answer. This is an infrared beamline and you can use infrared (FTIR) to spot calcite and ACC.
First you have to polish your granules to get a nice flat surface.
Polished slice through a calcium carbonate granule and a close up
We can then do infrared spectroscopy (actually Fourier transform infrared spectroscopy or FTIR) to map out chemically or structurally different regions distributed across the grain. These are identified by how the areas interact with infrared light. Different wavelengths of the light are absorbed or reflected. We focussed on two regions that are both present in calcite but only one of which is present in ACC. We can map out the relative intensity of the different regions in our sample.
This map shows the relative intensity of a peak characteristic of all calcium carbonate
This map shows the relative intensity of a peak characteristic of calcite BUT NOT ACC
From the above two images we'd argue that there is calcite in the top right of the image and ACC in the bottom right. To check this we can look at the ratio of the two maps.
Here we can see that the ratio of the ACC to calcite peak is high in the bottom left and low top right
That is all very well but it relies on our judgement which at times is questionable (see previous post on the polka!). So next we do some statistics to show that we're not just seeing things. The first thing we did is called cluster analysis. This groups bunches of similar spectra together.
Cluster analysis shows that there is a group of similar spectra bottom left and top right (different coloured zones)
That is all well and good but we'd like to know if these distinct zones correspond to ACC and calcite. So we have done something called component regression. We use some standard spectra - one for ACC, one for calcite - and see whether these spectra match the ones in our maps.
A component regression map using an ACC standard. The spectra at the bottom of our map are similar to the ACC standard
And for good measure a component regression map using a calcite standard showing that our spectra are more calcite-like at the top
So it looks to us that we have located the ACC and calcite in our slices. What we need to do now is to map these elementally to see if there is an elemental control on the ACC stability, i.e. is the ACC stabilised because of an unusually high (or low) content of a particular element, Mg is a potential candidate. We'd also like to see if the different zones of the calcium carbonate have a different organic compound signal. We may be able to do this with our FTIR data but there are potentially problems with contamination as we use an organic-containing resin to make the thin slices of the granules that we map.
Incidentally we want to know why the ACC is stable to learn about crystallisation processes, there is lots of interest out there in what controls the way that calcium carbonate crystallises. This can have relevance to the control of industrial scale and the production of pigments for example.
So all told this has been an extremely successful period of beam time. Many thanks to the beamline scientists Mark Frogley, Katia Wehbe and Gianfelippe Cinque for all their help and support. Thanks also to the very many scientists making Diamond work and also to the non-science staff and in particular the cooks, mass catering isn't easy!