Gelatinization, or Science Makes Me Stay Up All Night

For the second time since I began my lab work, I have arrived at the dreaded gelatinization stage of sample processing.  I say dreaded because this is easily the most time consuming step and requires the samples be regularly checked at odd hours (e.g. 11:30 pm and 2 am last night).

Explaining gelatinization requires a little background about what exactly I do with all of the little
pieces of walrus bone I have gathered over the last semester.  The end goal of the entire process is to analyze the carbon and nitrogen stable isotopes of the collagen in the bones.  Collagen is a structural protein and is found in abundance in connective tissues and in bones.  Unfortunately, all that pesky calcium carbonate (the hard part of the bone) makes it difficult to get to the collagen.  So, after cleaning the bones in a sonic bath and removing any fats/oils using a chloroform-methanol solution, I get rid of the calcium carbonate.

This is a relatively easy process, if a little slow, and involves our old friend hydrochloric acid (or HCl).  Everyone probably knows about HCl as it is fairly ubiquitous, found everywhere from the high school chemistry class to the inside of your stomach.  The acid dissolves the calcium carbonate in the bone, creating calcium chloride, carbon dioxide, and water.  The reaction looks something like this:

This process can be a little slow, taking anywhere from a couple of weeks to a month or more.  As the calcium carbonate dissolves, little bubbles of C02 appear, indicating that the process is working.  I continue adding acid little by little, until all of the hard structure is gone and the bone is soft and rubbery.  Once the demineralized bones have been rinsed back to neutral pH, they are ready for gelatinization.

This step makes use of a machine called a vortex evaporator, which is designed for an entirely different purpose but is very useful for this process.  This machine continuously vibrates and heats the bone samples (colloquially known as the "shake and bake" step), causing the collagen to unravel from its complex and very stable structure, forming gelatin (hence the name "gelatinization").

During this process, the solid pieces of bone dissolve into the solution until they are entirely gone and only a few little tiny white pieces can be seen.  If they stay on the machine too long, however, the collagen will continue to degrade until it is useless and the consequences can be disastrous.  This is why the samples must be checked.... and checked... and checked... and... well you get the idea.  As a result, for the 2 or 3 days (or sadly, sometimes more) that this process is ongoing, I make the 20 minute drive to the lab many times at ungodly hours to ensure the process goes smoothly.

All complaints aside, the process really isn't all that bad.  Once a samples has gelatinized, I pour it into a syringe and squirt it through a very fine filter into a vial then place it in the freezer.  Once all samples have been filtered and frozen, they are put on the freeze drier for 24 hours.  Magically, the clearish liquid turns into a lacy, white, cotton candy-like substance, which is the final product of the process.  I then weigh out a tiny (~0.2 mg) piece, submit it for analysis on the mass spectrometer and VOILA! Science!

For those of you that made it this far and looked at all the boring black and white images, here is a disgruntled looking underwater walrus:

Photo credit: Pete Barrett, National Geographic