A method for separating impurity minerals from biological apatite of unearthed bone in an archaeological site

Abstract

The application discloses a method for separating impurity minerals from biological apatite in unearthed bone skeletons of archaeological sites, and belongs to the field of pretreatment technology of unearthed bone skeleton samples of archaeological sites. The method specifically comprises the following steps: 1) placing pretreated blocky bone skeleton samples in deionized water, and then freezing the samples in an environment with a temperature of-45 DEG C to-55 DEG C for 80 to 100 minutes; transferring the frozen bone skeleton samples to an environment with a temperature of 75 DEG C to 85 DEG C to thaw the samples for 8 to 12 minutes, and completing one freeze-thaw cycle; 2) repeating the above steps for several freeze-thaw cycles to obtain freeze-thaw products; and 3) performing particle size sorting on the freeze-thaw products by means of leaching, and obtaining bone skeleton samples with particle sizes in the ranges of 0.5 to 2 mm, 2 to 5 mm and greater than 5 mm respectively for subsequent analysis. The application combines the thermal expansion difference and ice expansion stress to damage the interface between the impurity minerals and the biological apatite, combines the particle size distribution characteristics, replaces gravity screening with liquid flow screening, avoids triboelectrification and re-adsorption, and realizes precise separation of impurity minerals.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pre-treatment technology of bone samples unearthed from archaeological sites, and particularly relates to a method for separating impurity minerals in biological apatite of bone samples unearthed from archaeological sites. BACKGROUND

[0002] In the field of bioarchaeology, the carbon and oxygen isotope ratios of biological apatite of unearthed bones can reflect the diet, migration of ancient humans or animals and paleoenvironmental information, and are important analysis carriers. However, impurity minerals such as calcite, quartz, muscovite and feldspar often adhere to the surface of biological apatite crystals or in the pores of bone cancellous. The existence of these impurities will interfere with the identification results and reduce the accuracy of isotope testing. With the continuous improvement of testing accuracy, it is urgent to develop efficient pre-treatment technology to remove these impurities.

[0003] At present, the methods for removing exogenous impurity minerals mainly include physical method and chemical method, but both methods have obvious limitations. In the physical method, although ultrasonic cavitation can strip the adhered impurity particles, because part of the particles adhere tightly or penetrate into the bone pores, they cannot be fully exposed to the ultrasonic action environment, resulting in poor treatment effect and difficulty in completely removing the impurities. This shows that the ultrasonic cleaning technology is limited by the strong combination of impurity minerals and biological apatite matrix. This combination has multiple characteristics: first, a heterogeneous interface solid connection is formed through interfacial effects such as electrostatic adsorption and mechanical intercalation; second, there is a significant difference in the thermal expansion coefficient between different mineral components (for example, quartz α = 5.5 x 10 -7 / K, biological apatite α = 13.3 x 10 -6 / K); third, the relative particle size and distribution characteristics of various mineral particles. In the chemical method, acetic acid has been proven to be able to effectively dissolve calcite impurities. However, for other chemically stable oxide and silicate impurities such as quartz (which is the most common contaminant mineral other than calcite), although hydrofluoric acid or concentrated alkali (such as NaOH) has good dissolution effect, the use of such strong corrosive reagents requires special protective facilities, and the cost of waste liquid treatment is high, which is difficult for ordinary archaeological laboratories to popularize and promote.

[0004] In summary, the existing physical and chemical methods have insurmountable limitations in removing exogenous impurity minerals in biological apatite, which not only affects the accuracy of isotope testing, but also limits the in-depth development of bioarchaeology. Therefore, it is urgent to design a new separation technology to remove impurities to meet the growing demand for high-precision analysis. SUMMARY

[0005] The present application aims to solve the problems in the prior art and provide a method for separating impurity minerals in bone biological apatite unearthed from an archaeological site.

[0006] The specific technical scheme adopted by the present application is as follows:

[0007] The present application provides a method for separating impurity minerals in bone biological apatite unearthed from an archaeological site, and the specific steps are as follows:

[0008] S1: Place the pretreated block-shaped bone sample in water, then freeze it in an environment of-45℃ to-55℃ for 80 to 100 minutes; transfer the frozen bone sample to an environment of 75℃ to 85℃ for thawing for 8 to 12 minutes to complete one freeze-thaw cycle;

[0009] S2: Repeat step S1 to obtain a freeze-thaw product after several freeze-thaw cycles;

[0010] S3: The freeze-thaw product obtained in step S2 is subjected to particle size sorting by leaching, and bone samples with particle sizes of 0.5 to 2 mm, 2 to 5 mm, and greater than 5 mm are obtained for subsequent analysis.

[0011] As a preferred, the pretreatment method of the bone sample is specifically as follows:

[0012] S11: Place the block-shaped bone sample to be treated in a test tube containing deionized water, so that the bone sample is immersed in deionized water; repeatedly clean the bone sample with ultrasonic waves to remove surface dust and loose impurities;

[0013] S12: Immerse the cleaned bone sample in a sodium hypochlorite solution for dissolving organic matter, and after 24 hours of standing, wash the bone sample with deionized water to neutral;

[0014] S13: Place the bone sample washed in step S12 in an acetic acid-calcium acetate buffer solution for dissolving carbonate, shake it thoroughly, then stand for 24 hours, and then wash the bone sample with deionized water to neutral to complete the pretreatment of the bone sample.

[0015] Further, the frequency of the ultrasonic waves in step S11 is 40 to 50 kHz, and the cleaning time is 15 to 20 minutes.

[0016] Further, the volume concentration of the sodium hypochlorite solution in step S12 is 2%.

[0017] Further, the concentration of the acetic acid-calcium acetate buffer solution in step S13 is 1 mol / L.

[0018] As a preferred, the freeze-thaw cycle in step S2 is performed 5 to 67 times.

[0019] Preferably, the leaching in step S3 is performed using deionized water, and the water flow rate is controlled at 0.5-1.5 L / min.

[0020] Preferably, the skeletal samples with particle sizes in the ranges of 0.5-2 mm, 2-5 mm and greater than 5 mm obtained after the particle size sorting are subjected to phase analysis using an X-ray diffractometer, and the skeletal samples with impurity mineral contents lower than 5 wt% of the detection limit are used for subsequent analysis.

[0021] Compared with the prior art, the present application has the following beneficial effects:

[0022] The present application innovatively proposes a freeze-thaw cycle separation method for mineral thermal expansion mismatch and water cyclic frost heaving effect. By using the synergistic effect of an ultra-low temperature refrigerator (-50℃, 90min) and a constant temperature metal bath (80℃, 10min), the differences in thermal expansion performance of heterogeneous materials and the periodic stress generated by water ice expansion are fully utilized to effectively damage the combination interface of impurity minerals and biological apatite. Since these two devices are relatively common in ordinary archaeological laboratories, and the use of freezing and thawing functions separately can significantly improve the efficiency of freeze-thaw cycles, they have good application prospects.

[0023] Further, a multi-stage particle size screening system is designed in combination with the relative particle size distribution characteristics (from microns to millimeters) of various mineral particles in archaeological bones. The liquid flow medium screening method effectively avoids the problem of re-adsorption of biological apatite particles and impurities caused by dry sample tribocharging in the traditional gravity screening process, thereby achieving more accurate separation of impurity minerals. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 XRD patterns of skeletal samples under different particle sizes of sample DR12;

[0025] Figure 2 XRD patterns of skeletal samples under different particle sizes of sample DR11. DETAILED DESCRIPTION

[0026] The present application will be further described and explained with reference to the accompanying drawings and specific embodiments. The technical features of each embodiment in the present application can be combined accordingly without conflict. The skeletal samples in the following specific examples come from the impurity-containing bones unearthed from different archaeological sites.

[0027] Example 1

[0028] This example separates the impurity minerals in the skeletal biological apatite of three different skeletal samples, and the specific method is as follows:

[0029] I. Pretreatment of skeletal samples

[0030] (1) Using a cutting machine to cut 3 different block bone samples to about 2g, while cutting the sample, use the cutting machine to gently scrape off the surface contaminants. The sample numbers are DR10, DR11 and DR12 respectively.

[0031] (2) Put 3 different block bone samples to be treated in a test tube containing deionized water, so that the bone sample is immersed in deionized water. Put the test tube in an ultrasonic cleaner, repeatedly clean the bone sample with ultrasonic waves to remove surface dust and loose impurities; the frequency of the ultrasonic waves is 40 kHz, the total power is 1200 W, the cleaning time is 15 minutes, until the solution is clear.

[0032] (3) After washing, immerse the bone sample in a 2% sodium hypochlorite solution, loosely cover the test tube opening with aluminum foil, and after 24 hours, wash the bone sample to neutral with deionized water to remove organic matter in the bone sample.

[0033] (4) Put the bone sample washed in step (3) into 1 mol / L acetic acid-calcium acetate buffer (pH adjusted to 4.8) and shake for 1 minute, loosely cover the test tube opening with aluminum foil, and after 24 hours, wash the bone sample to neutral with deionized water to remove carbonates in the bone sample, complete the pretreatment of the bone sample.

[0034] II. Freeze-thaw cycle treatment

[0035] Put the pretreated block bone sample into deionized water, then into an ultra-low temperature freezer at -50℃ for 90 minutes; transfer the frozen bone sample to a constant temperature metal bath at 80℃ for 8-10 minutes to complete a freeze-thaw cycle. The ultra-low temperature freezer and the constant temperature metal bath can automatically control the temperature, with an accuracy of 1℃ and 0.5℃. Repeat the above freeze-thaw cycle 46 times, a total of 47 freeze-thaw cycles to obtain the freeze-thaw product. According to the crushing condition of the bone sample during freeze-thawing, screen every 10-20 times to retain samples greater than 0.5mm to ensure the amount of sample for subsequent testing.

[0036] III. Particle size sorting

[0037] The freeze-thaw product obtained in step II is sorted by leaching. Use 5mm, 2mm and 0.5mm aperture sample separation screens, and deionized water is used to leach through the screen, with a water flow rate of 1L / min. Bone samples with particle sizes ranging from 0.5 to 2mm, 2 to 5mm and greater than 5mm are obtained, respectively, and then placed in a 70℃ oven for drying for 24h for subsequent X-ray diffraction testing.

[0038] IV. X-ray diffraction analysis

[0039] The bone samples after particle size sorting were subjected to X-ray diffraction phase analysis by using an X-ray diffractometer, and the specific method was as follows: D8 Discover X-ray diffractometer, test conditions: power 42 kV x 100 mA, Cu Kα radiation, scanning range: 5-80° (2θ), step width 0.02°, scanning speed 0.1 s / step, one-dimensional continuous scanning. The results are shown in Table 1.

[0040] Table 1 Phase identification results of different bone samples in Example 1

[0041]

[0042] Note: "◎" indicates that only biological apatite single phase is contained, "X" indicates that exogenous impurity minerals are contained, and "N" indicates that the sample is not obtained

[0043] Comparative Example 1

[0044] In comparison with Example 1, the present comparative example separates the impurity minerals in the bone biological apatite of three different bone samples, and the specific method is as follows:

[0045] I. Pretreatment of bone samples, same as Example 1.

[0046] II. Freeze-thaw cycle treatment, same as Example 1.

[0047] III. Particle size sorting

[0048] The freeze-thaw product obtained in step II. is subjected to particle size sorting by leaching. Deionized water is used to leach through the sieve with a particle size of 0.125 mm and 0.25 mm, and the water flow is controlled at 1 L / min. Bone samples with particle sizes of 0.125-0.25 mm and less than 0.125 mm are obtained, respectively, and then dried in a 70°C oven for 24 h for subsequent X-ray diffraction test.

[0049] IV. X-ray diffraction analysis

[0050] The bone samples after particle size sorting were subjected to X-ray diffraction phase analysis by using an X-ray diffractometer, and the specific method was as follows: D8 Discover X-ray diffractometer, test conditions: power 42 kV x 100 mA, Cu Kα radiation, scanning range: 5-80° (2θ), step width 0.02°, scanning speed 0.1 s / step, one-dimensional continuous scanning. The results are shown in Table 1.

[0051] Table 2 Phase identification results of different bone samples in Comparative Example 1

[0052]

[0053] Note: "◎" indicates that only biological apatite single phase is contained, "X" indicates that exogenous impurity minerals are contained, and "N" indicates that the sample is not obtained

[0054] Comparing Table 2 with Table 1, it can be seen that the product particle size classification after freeze-thaw is 0.125-0.25 mm and particle size less than 0.125 mm cannot separate the impurity minerals in the bone sample, which is not conducive to subsequent analysis. According to the table, the impurity particles of sample DR11 are separated to smaller particle sizes (0.125-0.25 mm and <0.125 mm) after freeze-thaw cycle and particle size sorting, and there is no impurity in the large particle size (0.5-2 mm, 2-5 mm and >5 mm).

[0055] Figure 1 The XRD patterns of the bone samples of sample DR12 at different particle sizes are shown in Table 5. Figure 2 The XRD patterns of the bone samples of sample DR11 at different particle sizes are shown in Table 4. Figure 1 It can be seen that the bone samples with particle size less than 0.125 mm and 0.125-0.25 mm of sample DR12 after freeze-thaw cycle contain impurity minerals (quartz, muscovite), and the bone sample with particle size of 0.5-2 mm contains only hydroxyapatite, without impurity minerals. Figure 2 It can be seen that the bone samples with particle size less than 0.125 mm and 0.125-0.25 mm of sample DR11 after freeze-thaw cycle contain impurity minerals (quartz), and the bone samples with particle size of 0.5-2 mm, 2-5 mm and particle size greater than 5 mm contain only hydroxyapatite, without impurity minerals. It shows that the method provided by the present application can obtain bone samples without impurity minerals, significantly improve the utilization rate of contaminated bone samples, and realize green separation without chemical pollution.

[0056] The above-described embodiments are only a preferred scheme of the present application, and are not intended to limit the present application. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present application. Therefore, any technical scheme obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present application.

Claims

1. A method for separating impurity minerals from bioapatite in skeletal remains unearthed from archaeological sites, characterized in that, The specific steps are as follows: S1: Place the pretreated block bone sample in water, and then freeze it in a constant temperature environment of -45℃ to -55℃ for 80 to 100 minutes; transfer the frozen bone sample to a constant temperature environment of 75℃ to 85℃ to thaw for 8 to 12 minutes to complete one freeze-thaw cycle; S2: Repeat step S1 and perform several freeze-thaw cycles to obtain the freeze-thaw product; S3: The freeze-thaw products obtained in step S2 are sorted by particle size through leaching to obtain bone samples with particle sizes ranging from 0.5 to 2 mm, 2 to 5 mm and larger than 5 mm, respectively, for subsequent analysis; The freeze-thaw cycle described in step S2 is performed 5 to 67 times.

2. The method for separating impurity minerals from bioapatite in skeletal remains unearthed from archaeological sites according to claim 1, characterized in that, The specific pretreatment methods for bone samples are as follows: S11: Place the block-shaped bone sample to be processed in a test tube containing deionized water, so that the bone sample is submerged in the deionized water; repeatedly clean the bone sample with ultrasound to remove surface dust and loose impurities; S12: Immerse the cleaned bone sample in a sodium hypochlorite solution used to dissolve organic matter, let it stand for 24 hours, and then wash the bone sample with deionized water until neutral. S13: Place the bone sample washed in step S12 into an acetate-calcium acetate buffer solution for dissolving carbonates and shake to allow it to react fully. After standing for 24 hours, wash the bone sample with deionized water until it is neutral to complete the pretreatment of the bone sample.

3. The method for separating impurity minerals from bioapatite in skeletal remains unearthed from archaeological sites according to claim 2, characterized in that, In step S11, the frequency of the ultrasonic wave is 40~50 kHz, and the cleaning time is 15~20 minutes.

4. The method for separating impurity minerals from bioapatite in skeletal remains unearthed from archaeological sites according to claim 2, characterized in that, The volume concentration of the sodium hypochlorite solution in step S12 is 2%.

5. The method for separating impurity minerals from bioapatite in skeletal remains unearthed from archaeological sites according to claim 2, characterized in that, The concentration of the acetate-calcium acetate buffer solution in step S13 is 1 mol / L.

6. The method for separating impurity minerals from bioapatite in skeletal remains unearthed from archaeological sites according to claim 1, characterized in that, In step S3, deionized water is used for filtration, and the water flow rate is controlled at 0.5~1.5 L / min.

7. The method for separating impurity minerals from bioapatite in skeletal remains unearthed from archaeological sites according to claim 1, characterized in that, X-ray diffraction was used to perform phase analysis on bone samples with particle sizes ranging from 0.5 to 2 mm, 2 to 5 mm, and larger than 5 mm after particle size sorting. Bone samples with impurity mineral content below the detection limit of 5 wt% were used for subsequent analysis.

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