Collagen extraction

Organic bone collagen was extracted and the isotopic composition was found utilizing isotopic ratio mass spectrometry. Collagen is an organic component of the bone, and like other archaeologically recovered organic material, is subjected to the elements of nature that can potentially contaminate and alter its structure. The quality of its preservation must therefore be carefully monitored before analysis. An attempt was made to actively reduce the influence of diagenetic effects by selecting only certain portions of the bone. The intention was to avoid bones that visually appeared to be decomposed, overly porous or unsuited for analysis due to the lack of intact organic substrate. This visual selection proved to be the least

effective method for determining destruction through diagenesis. Inevitability, skeletal material that gave the optic impression that no protein could possibly have survived turned out to be exceptionally well preserved and virtually free of diagenetic assault. By contrast, bone samples derived from anatomically well preserved skeletons, looking almost like fresh bones, were sometimes unusable for isotopic analysis due to the advanced state of protein decomposition.

Cortical sections of a long bone diaphysis, preferentially from the anterior femoral surface, were chosen as a sampling region. The femur diaphysis was used because it is less porous and has a thicker shell of compact bone than other elements, which make it less susceptible to diagenetic effects and produces a larger amount of bone material relative to sample size. A crescent shaped sliver of cortical bone, weighing approximately 3-4 g, produced by two separate band saw cuts angled inwards towards one another, was taken from the anterior diaphysis and mechanically scraped of gross impurities with a scalpel. The sample was pre- washed under running tap water and subsequently cleansed ultrasonically in distilled water. This cleaning instrument was especially effective in removing dirt and other impurities locked in pores, holes, seams and the fine labyrinth-like network of the bone’s endosteal matrix. In the event, that the femur was not viable for sampling, the tibia, humerus, radius or ribs, in this order of preference, were used as substitutes.

The sample was then dried at room temperature for several days. The dry bone sample was then converted to powder form in a pulverizing machine. 500 mg of this bone powder were weighed out on an A&D Instruments HR-120-EC electronic scale and placed in a labeled Teflon test tube fitted with a screw on cap. 10 ml of 1 M HCl was carefully (to avoid foaming) added to the Teflon test tube containing the 500 mg sample. The Teflon test tube containing the mixture was then placed on a shaker for 20 min to remove the mineral component of the bone sample. The test tube was capped off to avoid sample loss; however, it was important that the cap was not screwed down tight, which would otherwise produce an airtight container. Pressure resulting from the chemical processes involved built up rapidly; therefore, the cap had to be loosely applied to facilitate a pressure release.

By means of a vacuum driven suction apparatus equipped with a hose and tipped with a pipette, the HCl and dissolved mineral fraction of the sample were removed from the remaining solid precipitate. Caution was taken not to suction off any portion of the sample, as this tended to happen abruptly. To neutralize the sample and raise the pH to 6 or approximately that of distilled water, the sample was left in the Teflon test tube, filled with distilled water and capped off. In contrast to the previous step, this time, tightly. The distilled

water and solid were then briefly placed on a vortex to ensure thorough dispersion of the acidic solid in the watery solution.

The mixture was then centrifuged at 3000 rpm for 5 min with a Sigma Laboratory 4K15 centrifuge. The centrifugal action resulted in the formation of a distinct pellet-like sample. Utilizing the same suctioning equipment, the distilled water solution containing the washed out HCl was removed. This step was repeated at least five times before the first litmus test was conducted. After the litmus test indicated that a pH of 6 had been reached, the solution was suctioned off.

In a step necessary to remove traces of the contaminant humic acid, the Teflon test tubes containing the pellet were filled with approximately 10 ml of a 0.125 M NaOH solution. The test tubes were capped, and once again, in such a manner as to allow built up pressure to escape, and placed on the shaker for a period of 20 hours. The test tubes containing the pellets mixed with the NaOH solution were centrifuged for 5 min at 3000rpm. The solution was suctioned off and, as before, the pellet was rinsed with distilled water, mixed briefly on the vortex and subsequently centrifuged to achieve a pH of approximately 6. This step was also repeated at least 5 times before conducting the first litmus test.

The pellet, still in the Teflon tube, was treated with 10 ml of 0.001 M HCl (pH 3). The test tubes were then placed in a warm water bath (90°C) for at least 10 hours, but no longer than 17 hours. The warm acid functioned to gelatinize the pellet by breaking the helical structure of the protein.

The collagen solute was filtered off using a glass funnel equipped with a screen filter (pore size 5 µm) and a vacuum. The solubilized gelatin was then poured into small, labeled glass vessels, leaving the remaining pellet exposed on filter. The glass vessels containing the gelatin solution were covered with a piece of perforated aluminum foil and lyophilized in a Christ Alpha 1-4 freeze drying apparatus under a vacuum bell for 72 hours at -52°C.

Taking care to avoid contamination, a 1 mg sample was weighed out on the electronic scale and wrapped in square of tin foil and placed in an Eppendorf-cup. The samples were subsequently tested at the GeoBio-Center at the LMU, Munich.