毛细管电泳法基于Fe(Ⅱ)与Fe(Ⅲ)的比值识别签字笔字迹的相对书写时间

可疑文件中墨水笔迹的相对时间鉴定对法庭科学、刑事案件的侦破和历史文献的整理都具有重要意义。该文建立了一种识别墨迹相对年代的毛细管电泳（CE）新方法。采用络合剂邻菲罗啉（1，10-phen）和反式-1，2-环己二胺四乙酸（CDTA）分别与Fe（Ⅱ）和Fe（Ⅲ）络合，然后用CE测定Fe（Ⅱ）和Fe（Ⅲ）的峰面积，通过比较从可疑笔迹中提取的Fe（Ⅱ）和Fe（Ⅲ）的峰面积比与从整个文档中提取出的Fe（Ⅱ）和Fe（Ⅲ）的峰面积比，判断整篇文字是否同时书写。实验首先对两种络合剂与两种价态铁离子的特异性络合进行了研究，结果表明1，10-phen与Fe（Ⅱ）、CDTA与Fe（Ⅲ）具有特异性络合。初步研究还表明：由于商用墨水pH值较低，墨水中的Fe（Ⅱ）在墨水瓶中比较稳定，因此Fe（Ⅱ）在墨水瓶中的氧化可以忽略不计；但当墨水书写在纸张上时，墨水中的硫酸会逐渐被纸张的纤维素所消耗，从而导致Fe（Ⅱ）在纸张中被逐渐氧化；在老化过程中Fe（Ⅱ）和Fe（Ⅲ）的峰面积比发生了变化，书写的时间越长，Fe（Ⅱ）和Fe（Ⅲ）的峰面积比就越小。该技术的成功应用依赖于找到一种合适的提取笔迹墨水的方法和CE分离测定Fe（Ⅱ）和Fe（Ⅲ）的方法。样品前处理程序如下：剪取1 cm长的墨水迹线，剪碎后放入2 mL的EP管中，加入0.5 mL 5 mmol/L的1，10-phen萃取1 min，再加入0.5 mL 20 mmol/L的CDTA振荡10 min，10000 r/min离心15 min，取上清液进行CE分离检测。CE条件如下：熔融石英毛细管（40.2 cm（有效长度30 cm）×75 μm i.d.），100 mmol/L硼酸-硼砂缓冲溶液（pH 9.2），压力上样（1.379 kPa，上样时间5 s），分离电压20 kV，检测波长254 nm，温度控制在25℃。最后，对两种不同的墨水进行了测定，以评价所建方法的适用性，结果表明所建方法对于鉴别可疑文件中真伪笔迹的相对年代具有重要的指导意义。

Identification of the relative age of iron gall ink in handwriting by capillary electrophoresis using the Fe(Ⅱ)/Fe(Ⅲ) ratio

Identifying the relative age of iron gall ink in the handwriting on a questioned file is highly significant for court science, because it serves as important evidence for solving criminal cases and in confirming the authenticity of historical documents. This is because many criminal cases involve analysis of forged documents to conclude whether an entire document is as old as purported, or whether the entire text in the document was written at the same time. In this paper, a novel approach based on capillary electrophoresis (CE) to estimate the relative age of iron gall ink-written texts is discussed. Two kinds of chelating agents, 1, 10-phen and CDTA, were used for the simultaneous determination of Fe(Ⅱ) and Fe(Ⅲ) by CE. The stability constants of Fe(Ⅱ)-(phen)₃ ]²⁺ and Fe(Ⅱ)-CDTA]^2- complexes are log β₃ =21.3 and log K =18.2, respectively, while the corresponding values of Fe(Ⅲ)-(phen)₃ ]³⁺ and Fe(Ⅲ)-CDTA]^- complexes are log β₃ =14.1 and log K =29.3. First, specific binding between the two kinds of chelating agents and the ferrous/ferric ions was investigated. The results confirmed specific binding between Fe(Ⅱ) and 1, 10-phen as well as that between Fe(Ⅲ) and CDTA. Preliminary studies also showed that Fe(Ⅱ) in the iron gall ink was relatively stable in the ink tank due to the low pH of commercial inks; hence, the oxidation of Fe(Ⅱ) in the tank was considered to be negligible. However, when the gall ink was exposed on a paper, sulfuric acid in the ink was gradually consumed by the cellulose of paper, thus causing gradual oxidation of Fe(Ⅱ) in the written text. Changes in the peak area ratio of Fe(Ⅱ) and Fe(Ⅲ) with aging were monitored: the older the ink in the writing, the smaller is the Fe(Ⅱ)/Fe(Ⅲ) ratio. Hence, the Fe(Ⅱ)/Fe(Ⅲ) ratio could be used for estimating the relative age of iron gall ink in writing. The Fe(Ⅱ)/Fe(Ⅲ) ratio was determined by CE, and the ratio extracted from the questioned handwriting ink was compared with that extracted from the entire document to confirm whether the entire text written at the same time. The keys to the success of this technique are establishing a suitable procedure for extracting the Fe(Ⅱ) and Fe(Ⅲ) species in the handwriting ink and a CE separation procedure. The optimized sample pretreatment procedure is as follows: (1) an ink-drawn line of 1 cm length was cut and placed in a 2 mL Eppendorf tube; (2) then, 0.5 mL of 5.0 mmol/L 1, 10-phen was added to the EP tube for chelation with Fe(Ⅱ), and the mixture was subjected to vibration on a vortex mixer; (3) within 60 s, 0.5 mL of 20 mmol/L CDTA was added to the sample tube for chelation with Fe(Ⅲ); (4) the tube was strongly vibrated for 10 min on a vortex mixer; (5) after centrifugation at 10000 r/min for 15 min, the supernatant was decanted into another tube for CE analysis. The optimized conditions for the CE analysis are as follows: 100 mmol/L of pH 9.2 H₃ BO₃ -Na₂ B₄ O₇ buffer, 20 kV applied voltage, sample injection (1.379 kPa, 5s), fused-silica capillary dimensions 40.2 cm×75 μm i.d. (30 cm to the detector), and 254 nm detection wavelength. Meanwhile, small amounts of 1, 10-phen and CDTA were added to the buffer solution to ensure stability of the formed complexes during the CE run in the capillary and to maintain the metal ions in their original oxidation state. Finally, two kinds of iron gall ink samples were tested to evaluate the applicability of the developed method. The Fe(Ⅱ)/Fe(Ⅲ) ratios of ink sample 1 and ink sample 2 changed from 1.79 to 0.45 and from 2.67 to 0.3, respectively, from the 1st day to the 75th day after writing. The results demonstrate that the developed method can be used to highlight fraudulent insertion of information and provide important guidance for the forensic analysis of the relative age of gall ink in handwriting.