Determination of Magnesium Isotopic Composition of Plant Chlorophylls Based on HPLC and MC-ICP-MS
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摘要:
叶绿素a和叶绿素b是植物光合作用时吸收光能的主要色素，其中叶绿素b能帮助叶绿素a扩展吸收光谱促使其吸收更多光能，叶绿素a和叶绿素b比例的改变有助于植物适应光照变化。准确测定叶绿素a和叶绿素b的镁同位素值对研究叶绿素形成过程中镁的生物合成路径等问题具有重要意义，其中将叶绿素a和叶绿素b分离并收集是使用多接收电感耦合等离子体质谱仪(MC-ICP-MS)准确测定两者镁同位素值的关键。高效液相色谱(HPLC)是分离叶绿素的常用仪器，但目前HPLC分离叶绿素的方法主要聚焦于细菌和藻类叶绿素分离。因此，需开发一套能将植物叶绿素a和叶绿素b分离的方法，且该方法分离的样品适用于MC-ICP-MS的镁同位素测定。本文基于665nm检测波长和C18色谱柱(7.6mm×250mm，5μm)，以三因素三水平正交设计优化HPLC条件，分析影响样品运行因素间的关系，得出符合要求的参数条件。经过优化的HPLC方法柱温为25℃，流速为1mL/min，流动相为甲醇-丙酮（80∶20，V/V）。结果表明：通过外标法定量分析叶绿素a和叶绿素b所得标准曲线在5～50mg/L浓度范围内的相关系数均大于0.9996，检测限为0.40～1.09mg/L，定量限为1.22~3.31mg/L，相对标准偏差(RSD)小于8.10%，样品加标回收率介于91.92%～111.11%。采用该方法对样品进行分离后，再利用MC-ICP-MS对前处理后的样品进行镁同位素测定，标准-样品间插法测定的数据表明镁同位素测试结果可靠，证明本文建立的方法为植物叶绿素a和叶绿素b的分离提供了技术支撑，且分离样品可用于镁同位素测定。
Abstract:The accurate determination of magnesium isotope values of chlorophyll a and chlorophyll b is particularly important for the study of magnesium biosynthesis pathways during chlorophyll formation. HPLC is required before MC-ICP-MS can be utilized, but HPLC lacks a method for separating chlorophylls. Therefore, it is necessary to develop a set of methods for separating plant chlorophyll a and chlorophyll b. The samples separated by this method are suitable for magnesium isotope determination by MC-ICP-MS. Based on the detection wavelength of 665nm and C18 column (7.6mm×250mm, 5μm), the HPLC conditions were optimized by three-factor and three-level orthogonal design to obtain the required parameters. The correlation coefficients of chlorophyll a and chlorophyll b standard curves in the concentration range of 5−50mg/L were greater than 0.9996, the detection limits were 0.40−1.09mg/L, the quantification limits were 1.22−3.31mg/L, the relative standard deviation was less than 8.10%, and the recoveries were 91.92%−111.11%. The magnesium isotope data of the isolated samples also showed that the column temperature of 25℃, flow rate of 1mL/min, and methanol-acetone (80∶20, V/V) mobile phase were reliable as HPLC separation conditions. The method established in this paper provides technical support for the separation of chlorophyll a and chlorophyll b in plants, and the separated samples can be used for magnesium isotope determination. The BRIEF REPORT is available for this paper at
http://www.ykcs.ac.cn/en/article/doi/10.15898/j.ykcs.202208300161 . -
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表 1 叶绿素分离条件正交试验设计与结果
Table 1. Designs and results of orthogonal test on chlorophyll separation conditions
正交试验编号 影响因素 正交试验结果 A柱温
(℃)B流速
(mL/min)C流动相
(V/V)叶绿素a
保留时间（min）叶绿素a和叶绿素b
分离度1 A1:25 B1:0.8 C1:甲醇-丙酮
(90∶10)22.08 6.22 2 A1:25 B2:1.0 C2:甲醇-丙酮
(80∶20)13.34 4.60 3 A1:25 B3:1.2 C3:甲醇-丙酮
(70∶30)8.53 3.19 4 A2:30 B1:0.8 C2:甲醇-丙酮
(80∶20)15.16 4.85 5 A2:30 B2:1.0 C3:甲醇-丙酮
(70∶30)9.44 3.51 6 A2:30 B3:1.2 C1:甲醇-丙酮
(90∶10)13.42 4.99 7 A3:35 B1:0.8 C3:甲醇-丙酮
(70∶30)10.93 3.49 8 A3:35 B2:1.0 C1:甲醇-丙酮
(90∶10)14.77 5.40 9 A3:35 B3:1.2 C2:甲醇-丙酮
(80∶20)9.37 4.47 叶绿素a和叶绿素b
分离度T1 14.01 14.56 16.61 T2 13.35 13.51 13.92 T3 13.36 12.65 10.19 $ \overline{{T}}_1 $ 4.67 4.85 5.54 $ \overline{{T}}_2 $ 4.45 4.50 4.64 $ \overline{{T}}_3 $ 4.45 4.22 3.40 R 0.22 0.63 2.14 叶绿素a
保留时间T1 43.95 48.17 50.27 T2 38.02 37.55 37.87 T3 35.07 31.32 28.90 $ \overline{{T}}_1 $ 14.65 16.06 16.76 $ \overline{{T}}_2 $ 12.67 12.52 12.62 $ \overline{{T}}_3 $ 11.69 10.44 9.63 R 2.96 5.62 7.13 注：Ti代表各因素相同水平实验结果之和，
$ \mathit{\overline{T}_i} $ 代表实验结果的平均值，相同因素下Ti越大，表明该水平对实验结果影响越大；R代表极差，是各水平最大平均值与最小平均值之差，R值越大，表明该因素对实验结果影响越大。
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表 2 方法检测限和定量限
Table 2. Detection limit and quantification limit of the method
叶绿素类别 方法检测限
(mg/L)方法定量限
(mg/L)叶绿素a 0.40 1.22 叶绿素b 1.09 3.31
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表 3 方法精密度
Table 3. Precision of the method
叶绿素类别 浓度(mg/L) RSD
(%)叶绿素a 9.00 8.79 9.09 9.04 9.16 8.44 2.99 16.86 16.31 16.07 14.44 17.58 14.94 7.32 35.38 35.49 32.31 35.47 34.06 35.62 3.79 叶绿素b 8.86 8.12 8.50 8.02 8.82 7.79 5.30 15.33 14.71 14.11 12.71 15.45 13.05 8.10 36.24 36.16 33.35 36.00 34.92 36.78 3.51
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表 4 样品加标回收率
Table 4. Spiked recoveries of the samples
植物种类 叶绿素种类 取样量
（µg）加标量
（µg）7次实测值（µg） 样品加标回收率（%） 水稻 叶绿素a 2.11 2.50 4.80 4.82 4.87 4.82 4.78 4.87 4.81 107.60 108.40 110.40 108.40 106.80 110.40 108.00 叶绿素b 0.82 0.99 1.80 1.73 1.83 1.74 1.87 1.88 1.92 98.99 91.92 102.02 92.93 106.06 107.07 111.11 玉米 叶绿素a 1.88 2.19 4.09 4.21 4.26 4.22 4.24 4.23 4.26 100.91 106.39 108.68 106.85 107.76 107.31 108.68 叶绿素b 0.48 0.66 1.14 1.12 1.16 1.21 1.19 1.17 1.18 100.00 96.97 103.03 110.61 107.58 104.55 106.06
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表 5 本实验样品镁同位素值与文献报道值的对比
Table 5. Comparison of magnesium isotope values in this experiment and reported values in literatures
样品类型 样品或叶绿素类别 序号 δ26Mg(‰) 2SD(‰) δ25Mg(‰) 2SD(‰) n 数据来源 海水 海水 1 −0.78 0.05 −0.41 0.02 3 本文实验 2 −0.84 0.11 −0.42 0.09 4 ［35］ 3 −0.83 0.05 / / 3 ［36］ 水稻 叶绿素a 4 −0.40 0.07 −0.22 0.06 3 本文实验 叶绿素b 5 −0.88 0.07 −0.48 0.05 3 本文实验 玉米 叶绿素a 6 1.69 0.13 0.87 0.04 3 本文实验 叶绿素b 7 −0.64 0.06 −0.34 0.03 3 本文实验 水芹 叶绿素a 8 −0.414 0.12 −0.21 0.08 5 ［11］ 9 −0.440 0.19 −0.22 0.14 5 ［11］ 玉米 叶绿素a 10 2.16 0.16 1.12 0.12 5 ［11］ 11 2.75 0.20 1.56 0.16 5 ［11］ 常春藤 叶绿素a 12 −0.022 0.119 −0.027 0.095 4 ［14］ 13 0.014 0.029 −0.012 0.022 1 ［14］ 叶绿素b 14 −0.455 0.090 −0.262 0.074 4 ［14］ 15 −0.380 0.029 −0.232 0.026 1 ［14］
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