Importance of Ultra-pure Water for Sample Collection in Vitriolage Cases- Forensic Concern for Corrosives Examination

Vitriolage, a heinous crime committed with intention to disfigure a person has come into view as an extremely brutal gender-based crime especially against young women in India and also another part of world mainly in South Asia over the past few years. India is facing an alarming situation of crime against women such as rape, domestic violence, dowry deaths etc. In recent year‟s cases of vitriolage are on the rise in India. By understanding the gravity of crime, section 326A and 326B was incorporated in the prevailing Indian Penal Code and came into effect from 3 February 2013. Forensic Science Laboratory, Delhi receives a number of cases pertaining to acid attacks every year. We present here five such cases which were received for forensic examination. The exhibits were analyzed using Ion Chromatography apart from classical tests in order to strengthen the forensic reports and play vital role in conviction of the accused. This paper encompasses the necessity and importance of controlled samples treated with the quality of water used in analysis, importance of packing of exhibits and role of investigating agencies/police in collection of exhibits, which play crucial role in proving the sanctity of the forensic examination.

treated as guilty, which causes psychological problems in victims such as depression, insomnia, loss of self-confidence, fear of outside world. They lose their jobs as no one wants them as employees 1 .
By understanding the austerity of the issue, section 326A and 326B were incorporated in the prevailing Indian Penal Code and came into effect from 3 February 2013 1 . The FIR is registered under Section 326A and pertains to voluntarily causing grievous hurt by the use of acid; on the other hand, 326B pertains to voluntarily throwing or attempting to throw acid in someone else. They say that the sustained an injury in her right eye and severe burns on her face as of communal violence and riots that happened recently [14][15][16] . Rehabilitation programs by various organizations are operative these days and give a ray of hope to acid attack survivors to earn livelihood and lead a normal life. Some acid attack survivors are helping to rehabilitate other victims by their NGOs while some organizations are raising funds to launch initiatives that could make acid attack survivors earn their livelihood, thus saving them from emotional trauma and social stigma that follows an acid attack. Orange Café and Restaurant is one such example that is owned and run by acid attack survivors 17,18 . The reasons for such huge figures of acid attack are many: the patriarchal mindset, psychological immaturity, inability to accept denial and rejection, jealousy, hatred etc. The figures can be lowered by quick medical and forensic examination of victim, speedy trials and stringent laws with strict implementation. "A 21 year old acid attack survivor said that she was attacked by her husband and his parents when she failed to fulfill the dowry demands raised by them back in 2008. She was held down and acid was poured on her face. She was struggling to get her attacker punished since 2008 and when years after a court sentenced him, it was only for 7 years and he is still out on bail and roams freely" 1,16,19 . Forensic Science Laboratory, Govt. of NCT of Delhi receives a number of cases pertaining to acid attacks every year. We present here five such cases (Table   no.1) which were received for forensic examination. The exhibits (clothes, skin, hair, footwear, soil etc.) were analyzed using Ion Chromatography apart from classical tests in order to strengthen the medico-legal reports and played vital role in conviction of the accused. The important analytical and instrumental techniques and their significance are mentioned below in nutshell.   HPLC/LCMS Analysis: In Forensics or any kind of sensitive analysis, Water is both the most widely used analytical laboratory reagent and the least-well characterized. While chromatographers take great care to assure the purity of salts, organic solvents, and other HPLC mobile phase components, they often take water quality for granted. High-purity water comprises by far the largest Case no.

1.
A 21 year old girl met an unfortunate acid attack while she was walking back home after completing her shift of work. On her way two bike riding boys crossed her and the boy sitting behind threw a corrosive agent on her.
Acid burn wound were found over her right arm, forearm, neck, cheeks and mandible.

2.
A 23 year old girl (student) was going to attend her English speaking classes in morning; two boys on motor cycle came and threw acid on her.
Mild burning on both legs anteriorly and laterally.

3.
On receiving information of acid throwing, when IO reached the scene of crime it was found that one girl and a women were taken to the hospital as they were attacked by two persons using acid.
Multiple superficial burns over both arms, right and left side of face, both foot and both legs of one victim and multiple superficial burns over breast were found on other victim.

4.
An information was registered by police that a 35 year old lady who worked as a sweeper at a hospital was poured with acid by her husband Superficial burns all over body, face & chest wall, breasts, anterior abdominal wall, upper back, upper limbs and small patchy burn over ears were found.

5.
Information was registered by police that a lady was attacked by a person using urine mixed with acid by the intention of harming her.
Superficial burns on legs.
mobile phase component for most reversed phase HPLC and LC-hyphenated methods. Because of its wide utilization and because of the volumes used in sample preparation and liquid chromatography, extreme care must be taken with the water quality. HPLC performance has been constrained by a system pressure limit of 6000 psi in most systems, which limited the particle size to 3 μm or larger, thereby limiting the speed and efficiency of separation that could be achieved.
With the advent of UHPLC systems, the following benefits offered by smaller particle size can be achieved: higher resolution, faster analyses by using higher flow rates or shorter columns, higher sensitivity because the peaks are taller and narrower, easy method transfer from standard HPLC within the same column family, less eluent consumption per sample because equilibration and run times are shorter. So it"s recommended to use high-purity water with the resistivity of 18.2 and TOC of less than 5 ppb so that no interferences would be the part of analysis which has been introduced by water 20 . So we consolidate the water quality has to be of very high quality like Ultra-pure Water to carry out such analysis and sample collection and preparation should only be in Ultra-pure water 21 .
 Molecular Biology: In forensic experiments, sensitive biological experiments, such as cell culture, require Ultra-pure water, free of contaminants like pyrogens.
To carry out such analysis, water has to be pyrogen-free water, Technology present inside the water purification system, an initial germicidal 254 nm UV lamp is installed in-line in the pre-treatment system, and a second germicidal 254 nm UV lamp is set up in the storage reservoir. The role of both these lamps is to control and prevent bacterial contamination in the water purification chain. A dual wavelength [185 /254 nm] UV lamp is incorporated in polishing systems to reduce the TOC level via photo-oxidation processes. This UV lamp also emits UV at 254 nm and, therefore, additionally has a germicidal effect. Finally, monitoring of TOC levels is also performed by a dual wavelength UV lamp, installed at the outlet of the water purification system to ensure water quality. So carry out any molecular biology experiment with pyrogen free application, it"s recommended to use highly pure water 22 .
This paper encompasses the necessity and importance of controlled samples treated with the quality of water used in analysis, importance of packing of exhibits and role of investigating agencies/police in collection of exhibits, which play crucial role in proving the sanctity of the forensic examination. In the current study we are trying to bring focus towards the sample collection and importance of water in the entire process of sample analysis which starts from sample collection. We would be discussing the various cases having sample collections displaying importance of sample collection and becomes area of prominence. was found in all cases, accused were known to the victim and there were earlier threats to kill them or disfigure their face by using acid.
In this study, we have tried to bring out the impact of sample collection with the right quality of water from the site where the alleged vitriolage took place, on the results of exhibits and finally to the implication it has on the victim/accused.

Reagents and apparatus
Chemicals: Sodium hydroxide, Nitric acid, Hydrocholric acid, Sulphuric acid, Ammonium hydroxide, Silver nitrate, Barium chloride, Diphenylamine and Ultra-pure (Milli Q) water were obtained from MERCK. Glassware: Beaker, Conical and Funnel were obtained from BOROSIL.

Sample handling and preparation
The primary concerns when collecting samples for analysis using any measurement technique is that the sample collected is representative of the total sample matrix, and that no contamination occurs during the sampling process. Also, appropriate storage and preservation of the sample is required, in order that the final sample analysis is representative of the analyte concentrations present when the sample was originally taken. Solid samples, such as soils and sludge, are not directly amenable to IC analysis and require additional sample pretreatment. The choice of extracting solution is dependent on both the sample matrix and the nature of the solute ions.

Extraction from clothes
First possible area of having acid was identified and then cut 125mm piece from several places and chopped/shredded into fine pieces. After that chopped pieces were dipped into 20ml of Ultra-pure Water (Milli Q) at room temperature for overnight.
Sample was filtered by using whatman no. 42 filter paper.
Other exhibits (bag, slippers, stone, mud etc.) were also extracted by above said procedure. After filtration all the exhibits were kept at 4 0 C and for stabilizing by NaOH at pH 12 for further analysis (holding time of 20-30 days).

Test for sulphate ions: barium chloride test
Few drops of dilute HCl and 2 ml. of BaCl 2 solution were added to 2 ml of filtrate then white precipitate was obtained which was insoluble in warm dilute HCl and in dilute HNO 3 but moderately soluble in boiling concentrated HCl showed the presence of sulphate Ions.

Test for nitrate ions: diphenylamine reagent test
Small amount of diphenylamine reagent was taken in a test tube then small portion of the filtrate was added carefully to the side of the test tube, blue ring at the junction indicates the presence of nitrate.

pH paper test
The pH paper was soaked in ultrapure water and then again dipped in the ultrapure water extract of the exhibits. The pH less than 7 indicates the presence of acid.

Litmus paper test
Again for confirmation of presence of acidic moiety the blue litmus paper was dipped in ultrapure water extract of exhibits. The change in color from blue to red indicates the presence of acid.

Test for sodium ions: uranyl zinc acetate test
Small amount of filtrate was taken in a test tube then neutralized it with acetic acid.
After that, few drops of uranyl zinc acetate reagent was added, then stir with the glass rod. Absence of yellow colored precipitate indicated that sample did not contain sodium ions. (Presence of yellow precipitate indicates sodium ions).

Test for potassium ions: dipicrylamine reagent test
The filter paper was soaked in sodium dipicrylaminate reagent and dried. After that a drop of neutralized filtrate was placed. Then paper was put in 0.1N Nitric acid. No formation of red flakes or ring at site of the spot indicates absence of potassium.
(Presence of red colored ring of formation of red colored flakes at the site of spot indicated presence of potassium).

Test for lead ions
One ml of dilute nitric acid and one ml of potassium iodide solution were added into one ml of filtrate in a micro test tube. Absence of bright yellow precipitate indicated absence of lead ions (a bright yellow colored precipitate appears, on boiling, precipitate dissolves out and on cooling golden yellow colored spangles appearsindicates presence of lead). Test for lead ion is carried out especially in case battery acids are used.

Ion chromatography 24
Conductivity detection: Conductivity detection is a non-selective detection principle; in this case both direct and indirect detection determinations are possible. As aqueous electrolytes are frequently used as the mobile phase in ion chromatography, the detector must be able to respond to the relatively small changes in the total conductivity of the eluent caused by the analyte ions. By the use of so-called suppression techniques the inherent conductivity of certain eluents can be dramatically reduced in the case of strong acid anions it is thus possible to considerably improve the sensitivity. Table 2 shows the requirements for sample preparation and the holding times for cations and anions usually determined by IC. Holding times can be increased by adjusting to pH 12 with sodium hydroxide (solution to be prepared in Ultra-pure water).
Filtration: As is the case with all liquid chromatographic methods, samples analyzed by IC should be free of particulates to avoid blockages or damage to connecting tubing, column end frits, and other hardware components. Samples are typically filtered through a 0.45 mm (or less) membrane based filter. Rinsing filters with 20mL of deionized water prior to filtration of the sample has been shown to remove most inorganic contaminants.

Measurement with chemical suppression parameters
The following parameters have been set for measurements:

Eluent preparation
Dissolve 191 mg sodium carbonate (anhydrous) and 143 mg sodium hydrogen carbonate in 980 mL Ultra-pure water. Then add 20 mL acetone.

Results and discussion
Positive results for Hydrogen ion (H + ) was observed by using pH indicator paper and litmus paper in all exhibits except exhibit D which is control sample in case no.2 &3.  (Figures 1a-b, Figures   2a-b, Figures 3a-b, Figures 4a-b, Figures 5a-b, Figure 6 and  (Table 3).

4.
A. One ladies purse (with some ladies item, one tobacco pouch, one spoon, one pocket diary) B. One empty lunch box C. One dupatta in burn condition        We have observed that generally sample collected from such sites are pieces of clothes, skin swabs, hair etc. As per the guidelines, skin pieces or biological tissues, swabs have to be prepared in the Ultra-pure water to avoid any contamination and similarly other samples have to be taken care with specificity without introducing any contaminations which can easily come from the first time used water for cleaning the samples or swabbing. We are emphasizing on the water quality as this will be the first reagent which will come in contact with the sample of analysis and will decide the fate of further analysis. We can easily imagine that if the water quality is compromised at this stage, things can be really disguising in terms of results. As it is describes in Table 3, there are certain methodologies are required to have the analysis done for such sample and for which it becomes important keep a check on the water quality.

in Exhibits
The information and knowledge about such sample collections at this level is either very low or not there at all and we see the impacts of such mishandlings of the samples on the results by the judiciary. In the entire process there will be a lot of analysis will be required to reach an decision so it becomes very important to have the samples analyze accurately and precisely at one go.

Impact of Ultra-pure water on forensic analysis of exhibits 25,26
Classically it has been observed, single/double distillation water has been used for titration but considering the critical nature of exhibits along with the forensic responsibility and stake of such analysis, to carry out the analysis where sample is less in quantity and results has to be accurate. Holistic approach here was to use the ultrapure water instead of distilled water was due to inconsistency of ions/organics/other contaminants levels in distilled water which will vary from location, weather, geography wise and even within the same location the sample taken for test with different lots of distilled water will introduced contaminant, which will results in inconsistent results. Establishing how much ions are there in Distilled water is impractical and will not hold true universally due to inconsistent feed water used to produce distilled water due to various factor. Due to no monitoring mechanism (conductivity/TOC Monitor) on distillation technique, it will be difficult every time see the quality ensuring the good or bad water for sampling. Another factor is distilled water produced hot and has to cool down before use as heat may alter the results. Now, its important to note that during the cooling process, distilled water absorbs atmospheric gaseous like CO2, Nitrogenous, which will be detrimental for the pH of water and in turn, shall not be used for the sampling work. This amount of ions in distilled water even with one sample is less and other sample is high and that will create variation whereas the ultra-pure water eliminates the same inconsistency. Small practical example here is water in Deccan area like Hyderabad will be full of silica etc. but whereas water in northern region, water will have high organic load. Also, On the other hand, ultrapure water through proper laboratory water purification system is bound to give consistent grade of quality ensuring consistent results. This happens as system has their alerts and alarms to tell when water quality degrades.
Water should be free of unwanted ions and charged organics which can shield the ions of interest in titration analysis. So its been recommended to have ion free water like Ultra-pure Water.

Results
In cases pertaining to Violence against women, it"s necessary to collect and preserve the biological, non-biological/chemicals and other samples very carefully because any negligence in sample collection make alteration in nature of crime or may destroy the exhibits which would otherwise had been very useful in investigation.
The importance of water is been also mentioned for the different applications during and after sample collection from the crime sites. As discussed earlier common physical evidences encountered in such cases are: container used to throw the chemical, clothes of the victim, clothes of the accused, swabs from the scene of crime etc. The acid or alkali recovered (if any) from the scene of crime must be taken in an all glass container, labelled with and must bear a caution in bold letters "CONTAINS ACID/ALKALI" and sealed by the Investigating Officer, put in duly labelled cloth bags.If the samples are packed in plastic containers then there is chance of leaching of plastic martial as it may react with samples which contains acid in itself and may alter the results. Each exhibit must be packed in separate container to avoid cross contamination.

Conclusion
The parity. To study the difference in Ultrapure water and distilled water, we would be need to introduce the Liquid Chromatography in tandem mass spectrometry as we have to touch the PPT levels. With the given resources, we could able to bring the attention to the topic. This will be wishfully our next step to introduce more sensitive techniques on the subject.