PROJECT: LIFE12 ENV/IT/352 «BIONAD» <<Naturalised dyes replacing commercial colorants for environmentally friendly leather dyeing and water recycle>> EMILIA BRAMANTI ICCOM-CNR, Pisa 12 Months Progress Meeting – SERICHIM premises, TORVISCOSA (UDINE), January 29th 2015

Actions of the last 6 months B.1 BIOKIMICA, INESCOP, ICCOM-CNR (0-6 months) Demonstration of the use of traditional chemical acid dyes in leather dyeing B.2 BIOKIMICA, INESCOP, (ICCOM-CNR) Demonstration of the quality features of dyed leather with acid dyes B.4 BIOKIMICA, (ICCOM-CNR) (3-9 months) Demonstration of naturalised dyes in leather dyeing at laboratory level

Naturalised dyes:a new chemical bridge linking the chromophore and lactose  DB27 LACTOSE DR202 DY42

Commercial acid dyes: the chromophore and many unknown impurities B113 R249

Leather dyeing procedure B.1, B.4 1. Formic acid at 55°C Heating at 55°C 400 µL MilliQ + 4 mg formic acid Magnetic stirring for 0.5 h Washing with 4 mL MilliQ for 3 times and drying at room T Magnetic stirring at 20°C for 1 h FTIR and TG analysis 2 mg/mL 2% (dye/leather) 0.2 g 0.2 g of chrome tanned leather specimen were put in a plastic tube with a 2 mL aqueous solution of dye at C1 concentration (4 mg, 2% w/w), C2 (5% w/w), C3 (10% w/w) and C4 (15% w/w). The dyeing bath was heated at 55°C and 0.4 mL of water and 4 mg of formic acid were added The w/w percentage of an ADs chromophore is approximate, due to the presence of additives in commercial products [2]. Instead, NDs arechemically pure products and the percentage refers to the actual weighted amount of dye.

Dyed leather characterization B.1, B.2, B.4 Naturalised dyes Commercial acid dyes 2% 5% 10% 15%

Dyed leather characterization B.1, B.2, B.4 Naturalised dyes Commercial acid dyes Formic acid Neutralization NDs penetrate across the leather sample better than ADs and also in mild, laboratory stirring conditions.

FT-IR CHARACTERIZATION B.1, B.2, B.4 AMIDE II CN ν, NH δ ~1550 cm-1 0.12 AMIDE I C=O ν ~1650 cm-1 0.1 0.08 Absorbance 0.06 COOH ν ~1730 cm-1 0.04 ATR spectrum 128 scans Untreated leather 0.02 3500 3000 2500 2000 1500 1000 500 Wavenumbers [1/cm]

FT-IR CHARACTERIZATION B.1, B.2, B.4 AMIDE I1 ~1654 cm-1 AMIDE II CN ν, NH δ ~1550 cm-1 0.12 AMIDE I2 ~1636 cm-1 0.1 AMIDE I C=O ν ~1650 cm-1 0.08 Absorbance 0.06 COOH ν ~1730 cm-1 0.04 ATR spectrum 128 scans Untreated leather 0.02 3500 3000 2500 2000 1500 1000 500 Wavenumbers [1/cm]

COLLAGEN CROSS-LINKING FT-IR B.1, B.2, B.4 ~1654 cm-1/~1690 cm-1 Collagen cross-linking type Cross-linking in dyed leather samples are due to the interaction/reaction with dyes and/or with formic acid employed in the dyeing procedure. Il livello di cross liniking del collagene è stato valutato studiando il rapporto tra la banda corrispondente alla piridinolina e alla deidro diidrossi norleucina. La piridinolina è un cross linker del collagene costituito da uno ione idrossi piridinio legato a 3 residui di lisina. Questo tipo di cross-linker si forma nel collagene e nell’elastina a partire da residui di lisina e idrossilisina in una reazione catalizzata dalla lisil ossidasi. Un aumento di questo rapporto indica un maggior contenuto di piridinolina e quindi una struttura del collagene più compatta grazie all’elevato grado di cross-linking.

COLLAGEN CROSS-LINKING - FT-IR Absorbance ratio (1654/1690 cm-1) from the FTIR spectra of leather samples dyed with NDs and Ads at C1 concentration. The dotted lines represent the ratio values of the blank-1 and blank-2 samples. In naturalised dyes the cross-linking is higher Weak-chrome tanned leather specimen processed with the dyeing procedure in the absence of colorants Naturalised Commercial Weak chrome tanned leather

The Amide I peak fitting of FTIR spectra: collagen conformational analysis The interaction of NDs and ADs with leather proteins and the spectral differences observed in the 1654/1690 cm-1 ratio of Amide I band of FTIR spectrum, were studied more in detail applying a peak fitting analysis to the vibrational C=O stretching frequencies associated to the amide I

The Amide I peak fitting of FTIR spectra: collagen conformational analysis Higher helix percentages (48%) Higher 1660/1690 cm-1 ratio values Higher cross-linking 2% NDs Two phenomena may contribute to high cross-linking and higher helix percentage: the treatment with formic acid The presence of the lactose unit in the dye structure. which may favor the hydrogen bonding within the amino acid residues of collagen, contributing to the stabilization of helix structure and to the increase of cross-linking ADs >5% NDs Lower helix percentages (38%) Lower 1660/1690 cm-1 ratio values lower cross-linking Stacking interactions between chromophore rings in excess [46] that surround collagen fibers avoid the intermolecular covalent crosslinking and stretch the protein structure because of their steric hindrance

Characterization using Thermo Gravimetric Analysis (TGA) Gas: N2 200 mL/min Heating: 30-700°C, 10 °C/min

TGA: same trend  B.1, B.2, B.4 Naturalised 2% Higher cross-linking=higher thermal stability Commercial 2% Naturalised 15% The correlation between the thermal stability of the samples analysed and the higher values of the 1660/1690 cm-1 ratio found in the FTIR study supports the correlation of this ratio with crosslinked structures, in agreement with the literature data [32-34].

Conclusions NDs may be competitive with the traditional AD because of their eco-friendly properties better penetrating capacity at low concentrations (about 2%) give more cross-linked structures (better quality features to the leather in terms of resistance and flexibility?) Improved biodegradability properties in the presence of common bacteria strains (e.g., Escherichia coli). This would offer the opportunity to treat dyeing effluents in an eco-friendly manner, re-use the water for further dyeing cycles, cutting the costs associated to water management.

Action C1. Environmental impact of acid dyes within leather manufacture Determination of metals in traditional (metalized) dyes which are employed in the dyeing process in leather industry. The aim is the assessment of the concentration of metal ions in the final process solution for the evaluation of the environmental impact of wastes resulting from the dyeing process. The determination of metals has been performed on the solid chemical products and on the waste solutions at the end of the dyeing process.

Chemicals and procedures Four acid dyes have been selected for testing experiments: ‐ ACID YELLOW 194 ‐ ACID ORANGE 142 ‐ ACID RED 357 ‐ ACID BLUE 158 The acid dyes have been tested in both the dyeing of sheep hide and calf hide samples. The dyeing solutions (calf hide: 20 g/L; sheep hide : 32 g/L). The single dyes solutions have been employed and also in combination among them. Metal analysis and quantification have been performed by atomic spectrometry (FAAS), ICP-OES.

Action C1. Environmental impact of acid dyes within leather Manufacture. Determination of metals in solid dyes Co and Cr were the principal metal components in the dyes (>> 1%). All the other metals were present at trace level,

Action C1. Environmental impact of acid dyes within leather Manufacture. Determination of Cr and Co dyeing waste solution of Sheep Hide Residual Co in waste solution: 2.1 ± 0.34 % Residual Cr inwaste solution: 11.2 ± 3.7 %.

Action C1. Environmental impact of acid dyes within leather Manufacture. Determination of Cr and Co dyeing waste solution of Calf Hide Residual Co in waste solution: 23 ± 11 % Residual Cr inwaste solution: 59.6 ± 21 %.

FUTURE ACTIONS for ICCOM C.3 Monitoring of quality improvement for leather dyed with naturalised dyes C.6 Monitoring of quality assessment for leather dyed with naturalised dyes