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

doi:10.3724/SP.J.1123.2020.05033

Abstract

Abstract:

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court science, because it serves as important evidence for solving criminal cases and in confirming the authenticity of historical documents.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.

Key words: capillary electrophoresis (CE), Fe(Ⅱ), Fe(Ⅲ), handwriting, iron gall ink, relative age, chelation

CAI Yu, CAO Chengxi, ZHUO Xianyi, LI Honggen, FAN Liuyin. Identification of the relative age of iron gall ink in handwriting by capillary electrophoresis using the Fe(Ⅱ)/Fe(Ⅲ) ratio[J]. Chinese Journal of Chromatography, 2020, 38(10): 1249-1255.

Figures/Tables 6

Fig. 1 Oxidation of ferrous ions in the gall ink bottle

Fig. 2 Specific binding between two complexing agents and Fe(Ⅱ), Fe(Ⅲ) ions a. 1, 10-phen and Fe(Ⅱ): 40 μL of 10 mmol/L standard Fe(Ⅱ) solution with a small amount of ascorbic acid (antioxidant) with the simultaneous addition of 1.0 mL of 5.0 mmol/L 1, 10-phen and 1.0 mL of 10 mmol/L CDTA; b. CDTA and Fe(Ⅲ): 50 μL of 10 mmol/L standard Fe(Ⅲ) solution with the simultaneous addition of 1.0 mL of 5.0 mmol/L 1, 10-phen and 1.0 mL of 10 mmol/L CDTA.Conditions: 40.2 cm (effective length 30 cm)×75 μm i.d. (375 μm o.d.) capillary; 100 mmol/L pH 9.2 sodium borate acid buffer; voltage, 20 kV; injection, 1.379 kPa 5 s; detection wavelength, 254 nm; temperature, 25 ℃. Peak identifications: 1. [Fe(phen)3 ]2+ ; 2. free 1, 10-phen; 3. [Fe(Ⅲ)-CDTA]- ; 4. benzoic acid; 5. ascorbic acid.

Fig. 3 Influence of addition order of 1, 10-phen and CDTA on the complexation of Fe(Ⅱ) and Fe(Ⅲ) a. Addition of 1.0 mL of 10 mmol/L CDTA to the mixture of Fe(Ⅱ) and Fe(Ⅲ) (solution A); b. subsequent addition of 1.0 mL of 5.0 mmol/L 1, 10-phen to solution A; c. addition of 1, 10-phen, followed by CDTA. Conditions: mixed standard solution: 40 μL of 10 mmol/L Fe(Ⅱ) and 50 μL of 10 mmol/L Fe(Ⅲ); other conditions are the same as those in Fig. 2 . Peak identifications: 1. [Fe(phen)3 ]2+ ; 2. free 1, 10-phen; 3. [Fe(Ⅲ)-CDTA]- ; 4. benzoic acid.

Fig. 4 Influence of addition order of 1, 10-phen and CDTA on the complexation of Fe(Ⅱ) and Fe(Ⅲ) a. Addition of 0.5 mL of 5 mmol/L 1, 10-phen to the mixture standard solution of Fe(Ⅱ) and Fe(Ⅲ) (20 μL of 10 mmol/L Fe(Ⅱ) and 40 μL of 10 mmol/L Fe(Ⅲ), solution A); b. subsequent addition of 1.0 mL of 10 mmol/L CDTA to solution A. The separation conditions are the same as those in Fig. 2 . Peak identifications: 1. [Fe(Ⅱ)-(phen)3 ]2+ ; 2. free 1, 10-phen; 3. [Fe(Ⅲ)-CDTA]- ; 4. benzoic acid.

Table 1 Effect of time interval between the addition of complexing agents on equilibration (n =3)

Time interval/s	A₁ /A₂	RSD/%
A₁ : peak area of [Fe (Ⅱ)-(phen)₃ ]²⁺ ; A₂ : peak area of [Fe (Ⅲ)-(CDTA)]^- .
10	1.0469	0.15
30	1.0484	0.31
60	1.0502	0.43
180	1.0577	0.25
300	1.4535	6.94

Fig. 5 Electropherograms of two gall ink handwriting samples a. sample of gall ink handwriting written on 26th Sept., 2018; b: sample of gall ink handwriting written on 10th Dec., 2018. The conditions are the same as those in Fig. 2 . Peak identifications: 1. [Fe(Ⅱ)-(phen)3 ]2+ ; 2. [Fe(Ⅲ)-(CDTA)]- .

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