Chemists Detect Silk Forgeries Using Enantiomeric Ratios of Amino Acids

Chemists are occasionally called upon to solve some baffling mysteries. It’s part of the attraction of the job. I’ve been an organic chemist for over fifteen years, and I’ve been involved with lots of forensic analysis. Just last month I helped to develop a method of cleaning an abandoned meth lab, so that the house became habitable again; it required lots of surface sampling and trace analysis. A chemists training involves hundreds of hours using sophisticated instrumentation. One of the most powerful tools we have is something called Mass Spectrometry, or MS. It allows us to assign an accurate molecular weight to an unknown chemical. Since we know how much each atom weighs (information that comes from the Periodic Table), we can use MS to assign a molecular formula to a particular compound. If the analysis comes back with a molecular weight of 131.93, we can predict that the compound is probably chloroform. There’s not much else it could be, with that particular molecular weight. Since MS only takes about 20 minutes o run, it’s a quick and accurately way of determining the identity of an unknown sample. I’ve used MS constantly and it’s an invaluable tool.

Of course, the situation becomes more complex if the unknown sample is a mixture of molecules. In the “real world”, most unknown samples are a mixture – it’s rare that a pristine single compound is sent in for analysis. As a result, most samples are first separated by some type of chromatography or electrophoresis. Chromatography separates samples based on polarity, which is loosely defined as how unequally the electrons in the molecule are shared. A very polar molecule such as an acid has multiple oxygen atoms that are “greedy” (electronegative) and the carbon-oxygen bond electrons spend much of their time on the oxygens. Acids are very polar and so they usually can be easily separated using chromatography. The problem with chromatography is that very large molecules – especially large molecules that have multiple charges on them – stick irreversibly to the separation medium. One example of a complex unknown sample that is full of large, multiply charged molecules is a protein. Proteins are comprised of long chains of amino acids, joined end to end. Since each amino acid has multiple polar groups, and since a protein can be hundreds (even thousands) of amino acids long, a protein is impossible to send through a regular chromatography column. It has to be acid digested first, so that the bonds between each individual amino acid can be broken and the collection of separate acids can then be analyzed using something like capillary electrophoresis (CE), which separates molecules based on their size to charge ratio. The separated components can then be analyzed using MS and identified based on their mass. I’ve used CE-MS in the past to analyze digested proteins, and it’s an excellent tool for the job.

One recent example of CE-MS that caught my eye was a paper published in Analytical Chemistry, a journal of the American Chemical Society. The authors (a group from Maryland) published a new method of dating silk samples. Normally, an organic material such as silk is dated using radioactive carbon dating. The carbon dating requires a relatively large sample and it’s not very precise, especially for newer materials. The new method published in this paper discussed monitoring the D:L ratio of amino acids in order to date silk. This requires a little bit of explanation. Silk is a protein excreted by silk worms. Like all proteins, it’s made up of a long chain of amino acids joined to each other. Each amino acid can exist in one of two orientations, either “D” or “L”. They’re mirror images of each other. An example would be your hands; your left hand has similarities to your right hand, but you can’t superimpose the two. They’re mirror images and the same principle applies to molecules. When the silk worms make the silk, the proteins inside the fibers are all L-amino acids; it’s a consequence of their genetics. A freshly prepared silk fiber will be 100% L-amino acids. As time goes on (over hundreds of years), the silk ages and the L-amino acids become D-amino acids. After 1000 years, this conversion is about 25% complete; after 2000 years, it’s about 50% complete.

The authors from the Analytical Chemistry article discuss how they were able to take microscopic samples from ancient silks (much smaller samples than is required for carbon dating) and perfomed an acid digestion step to break down the protein into the component amino acids. This mixture was then analyzed using CE-MS. The CE step separated each of the amino acids from each other so that they entered the mass spectrometer one at a time; the retention time for the D amino acids was different from the L amino acids. The mass spectrometer then reported the accurate weight of each molecular fragment, which allowed the researchers to determine which amino acid was present. At the end, the researchers were able to compile the results into a D:L ratio. A graph could then be plotted against control samples (with known historical ages) in order to date the unknown samples. This was an exciting article to read because it was a nice example of the power of instrumental analysis. As someone who spends hours every day performing these types of techniques, I was happy to read about another successful implementation of CE-MS. The silk analysis is much faster and also more accurate than carbon dating, and I fully expect it to become the standard method of dating silk samples, which should help museums and artifact hunters alike.

This source of this article can be found at:

Moini, M., et al. “Dating silk by capillary electrophoresis mass spectrometry”. Analytical Chemistry 2011, 83, 7577

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