[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court science, because it serves as important evidence for solving criminal cases and in confirming the authenticity of historical documents.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 β 3=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 β 3=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 H3BO3-Na2B4O7 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.