A Comparative Analysis of Ink Composition under Acidic, Basic, And Neutral Hydrolysis Conditions Using Thin-layer Chromatography (TLC)

Forensic science plays a vital role in investigating crimes and criminals and gathering evidence. A document analysis is considered a crucial aspect in the field where the document is in question. One of the significant aspects of the document examination was ink analysis, which helps forensic experts to determine whether a document has been altered by any physical or chemical methods, fabricated, or tampered with, especially in cases such as forgery, documents that require authentication, and other criminal cases involving written or printed evidence. Ink is a liquid or paste that contains dyes and pigments, it was widely used for writing or drawing by using a pen or brush for making a text, design, image or colored surfaces. Ink is usually composed of solvent, pigment, dye, resins, solubilizer, surfactant, fluorescence and other materials [1]. Various analytical techniques are used for ink analysis in the case of document examination. This study used thin-layer chromatography (TLC) as an instrument for ink analysis because of its capability to separate and identify the mixture of dyes and pigments found on the ink and for its efficiency and cost-effectiveness. TLC, which allows for the separation of ink components based on their solubility and their interaction with the stationary phases [2]. In this study, Reynolds 045 fine carbure blue ballpoint pen ink was used for analysis. Usually, ballpoint pen ink is composed of oil and dyes. This will contribute to the ink thickness, and the oil components in the ink are responsible for the quick drying of ink and the reason for the resistance of the ink in water [3]. This study focuses on the chemical breakdown of ink composition. Ink degradation was the main reason for the chemical breakdown of ink. Ink degradation can occur due to various environmental and chemical factors, which will alter or affect the ink composition and its stability over time. The most common cause of ink degradation is chemical hydrolysis, which means that when we add water, it reacts with organic molecules to break the molecules and form two or more smaller organic molecules. By using this chemical hydrolysis method, the ink in the document can be chemically altered, erased, forged, or tampered with [4]. This study analyzed the chemical breakdown of ink composition under various chemical hydrolysis conditions such as (acidic, basic, and neutral) using thin-layer chromatography as an instrument.

Three chemical hydrolysis conditions were analyzed in this study:

1. Ink hydrolyzed by acid:

Nitric acid (HNO?) was utilized for this procedure, which has (pH <7). Since it breaks bonds and supplies protons (H+) ions, particularly in the ink's dyes and pigments.

2. Ink hydrolyzed by base:

sodium hydroxide (NaOH) was utilized in this procedure, which has (pH >7). The bonds in the ink composition may be broken by the release of (H-) ions from NaOH. 

3. Neutral hydrolysis of ink:

This was done to determine the ink's water solubility and stability using distilled water, which has (pH of 7). For TLC analysis, the ink was chemically extracted from a Reynolds 045 fine carbure blue ballpoint pen, which is commercially available. The ink sample is subjected to three chemical hydrolysis conditions (acidic, basic, and neutral). The extracted ink was applied on the per-coated silica gel TLC plate (stationary phase) by using capillary action [5], and then the plate was developed using a mixture of the solvent system “(i.e., ethyl acetate: ethanol: distilled water - 70:35:30 ml”) [6][7], which was termed the mobile phase. TLC allows for the separation of components based on their affinity towards the stationary phase when it comes in contact with the mobile phase. Components with higher affinity for the stationary phase move slower, and components with lower affinity for the stationary phase move faster [5]. The retention factor (RF) value is an important parameter in a TLC analysis, and it is a direct measurement method to measure the amount of materials and mixtures in each spot. The RF value was calculated by measuring the ratio of the distance traveled by the solute to the distance traveled by the solvent front. [8].

“RF = Distance of center of spot from the baseline

           Distance of solvent front from the baseline” [8]

In this study, the RF value for each component that is separated from the ink samples is measured, and the RF value of the standard (non-hydrolyzed) ink sample was compared with those hydrolyzed ink samples, which were hydrolyzed under acidic, basic, and neutral conditions. Analyzing these changes in RF value will help the experts to authenticate the document containing ink by identifying chemical alteration or tampering in the ink. The result will help forensic document examiners to distinguish between genuine and altered documents by authenticating them. This study provides valuable insights into how different chemical factors affect or alter the composition of the ink; from this, the examiner could easily identify chemically altered ink. This study will help to authenticate the document under question while in legal proceedings [9].

Significance:

In this analysis, the document examiner can determine whether any specific chemical agents are subjected to the ink compositions. In forensic analysis, this study helps to determine whether the ink in the document has been chemically altered, erased, forged or tampered with. “Application of TLC in ink analysis is a simple, rapid, cost-effective and efficient technique” [10]. Apart from the forensic investigation, this study helps to understand the chemical breakdown of ink composition under various chemical hydrolysis conditions such as (acidic, basic and neutral).

METHODOLOGY

Solvent preparation (mobile phase)

The mobile phase is the component that helps the analyte to interact with the stationary phase [11]. For this research, a solvent system composed of “ethyl acetate, ethanol, and distilled water in a ratio of 70:35:30 was used” [6][7] due to its efficiency in separating and identifying the compounds present in the ink.

Ink sample preparation

Ink sample 1

The blue ink was obtained from a commercially available Reynolds 045 fine carbure ballpoint pen, and the ink was extracted in “ethanol as an extraction solvent” [11] [12] This study focused on the chemical breakdown of the ink composition. For this, three chemical hydrolyzed conditions were used;

Ink sample 2 - The ink was hydrolyzed under an acidic solution:

For this, 1 ml of concentrated nitric acid (HNO?) was diluted in 9 ml of distilled water to create a total of 10 ml of reagent. A mixture of 5 ml of extracted ink and 5 ml of the prepared acidic reagent was taken in the plate.

Ink sample 3 - The ink was hydrolyzed under a basic solution:

For this, 1 ml of 10n NaOH (sodium hydroxide) was diluted in 9 ml of distilled water to create a total of 10 ml of reagent. A mixture of 5 ml of extracted ink and 5 ml of the prepared basic reagent was taken in the plate.

Ink sample 4 - The ink was hydrolyzed under neutral conditions:

For this, the mixture of 5 ml of distilled water and 5 ml of extracted ink sample was taken in the plate.

TLC Procedure

Place the solvent system (“i.e., ethyl acetate: ethanol: distilled water) in the proportion of 70:35:30” [6][7], in the TLC chamber. Prepared ink samples are spotted on the pre-coated silica gel TLC plate about 2 cm above the bottom of the plate with the help of capillary tubes. Four distinct ink samples are spotted separately to each TLC plate, sized (5 × 10 cm). The plate was placed in the developing chamber that contained the solvent system (mobile phase) at room temperature. It took 30-45 minutes for the solvent system to reach the top of the plate. Once the solvent traveled within a cm of the top of the plate or 3/4 in the TLC plate, the plate was removed from the chamber. The solvent front and the chromatographic spots were observed under daylight (direct detection method) and marked using a pencil and recorded as a photograph under daylight. Finally, the qualitative analysis of thin-layer chromatography was measured by calculating the respective retention factor of each spot obtained in the plate [6].

RESULT AND DISCUSSION

Figures 2 to 5 show the results of the TLC separation of the ink composition of the prepared standard (non-hydrolyzed) and hydrolyzed ink samples using the developing solvent system of “(i.e., ethyl acetate: ethanol: distilled water) in the ratio of (70:35:30)” [6][7].