# The Evaluation of the Detection of Cr(VI) in Leather
## 3 Results  
The colorimetric method recognises that the reaction between Cr(VI) and DPC is not instantaneous, and a reaction time of 900 \(\pm\) 300 s (15 \(\pm\) 5 min) is stipulated to ensure the reaction is sufficiently complete during quantitative analysis [11]. Figure 1 shows how absorbance (ABS) (540 nm) of a carbazone complex solution varies as a function of time and confirms that a minimum of 300 s is needed for the reaction to stabilise. However, there is no suggested guideline in annex C for the chromatographic method [12]. The recommended procedure in annex C results in a retention time of approximately 275 s, of which this work has found that the eluted Cr(VI) was in contact with DPC for only approximately 60-120 s [12]. To ensure accuracy at all concentrations, it is recommended that a retention time of at least 15 min is designed into a chromatographic method involving a post-column reaction with DPC. Concerns of statistical variance in responses from the colorimetric (part 1) methodology have been previously reported [16]. These are thought to be caused by:  
* Oxidation of Cr(III) during extraction at pH 8. * Reduction of Cr(VI) during extraction in the presence of a protein material. * Dyes contributing to the absorbance at 540 nm. * Interference from overlapping Cr(III) absorbances [17]. Model extraction trials involving known amounts of Cr(VI), Cr(III), and bovine gelatine were used to examine the extraction process, removing possible variance caused by chromium extraction from a leather matrix. Using the peak for carbazone at 540 nm, the concentration of Cr(VI) was determined by measuring the absorbance difference between two samples of a solution where one contains acidified DPC, as outlined in BS EN ISO 17075-1:2017. The model solutions containing only Cr(III) (with or without gelatine) were found to give false positive results for Cr(VI) during the extraction period (Figure 2A). This can be cross-referenced with Figure 2B, which provides evidence that this apparent positive result for Cr(VI) is because of the coalescence of the two Cr(III) peaks at 410 and 630 nm. If Cr(VI) were to be formed during extraction, a peak at 540 nm as seen in Figure 3B would be resolvable. The reason for the apparent value of Cr(VI) to continually increase as a function of extract time is likely because of a general increase in the base line absorbance due to olation of Cr(III) [18]. Olation causes turbidity, resulting in irregularity between the DPC-sample and the control, thus producing a false positive. Filtering the extraction solution can help this issue, but olation continues over time [18]. The presence of protein can cause the same effect due to turbidity; however, this can be resolved with cellulose nitrate (CN, 0.45 \(\upmu\)m pore size) membrane filtration. Figure 1: Absorbance vs. time of a solution of DPC and Cr(VI) at 540 nm. Figure 2: (**a**) Apparent Cr(VI) concentration (mg kg\({}^{-1}\)) of a Cr(III) and gelatine solution vs. extraction time (minutes), (**b**) UV spectra of the same solution after 115 min: with DPC (Blue), without DPC (Red). The concentration of Cr(VI) in a solution containing only Cr(VI) (with or without gelatine) remains consistent during the extraction process as highlighted in Figure 3A, which suggests that Cr(VI) does not decrease during the extraction process. This observation is not surprising considering the extraction process is at pH 8.0 \(\pm\) 0.1, and the oxidative properties of Cr(VI) require acidic conditions [19]. Figure 3B shows the Cr(III)-carbazone peak is clearly resolvable versus the control. This statistical reliability could also be due to Cr(VI) not undergoing olation and thus avoids the issues caused by turbid solutions. Model extraction solutions with known concentrations of Cr(III), Cr(VI), and gelatine (see Appendix A Figure A1) also showed increases in concentration of Cr(VI) as a function of extraction time. The apparent increase in Cr(VI) concentration correlated with the increases seen in Figure 2A.