PROJECT: LIFE12 ENV/IT/352 «BIONAD» > > DAVIDE PELLEGRINI ICCOM-CNR, Pisa 6 Months Progress Meeting – BIOKIMICA premises, Santa Croce sull’Arno (PI), June 13th 2014

Actions 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) (0-6 months) 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

DO30 DR202 DY42 DB27Naturalised dyes LACTOSE

Commercial acid dyes Commercial acid dyes O37Y49 R249 B113

Leather dyeing procedure 0.2 g 4 mg/2 mL 2% (dye/leather) Magnetic stirring at 20°C for 1 h 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 1. Formic acid at 55°C B.1, B.4

Leather dyeing procedure Magnetic stirring at 20°C for 1 h Neutralization with 50µL K 2 CO 3 (pH = 7-8) Magnetic stirring for 10 min 2. Neutralization Washing with 4 mL MilliQ for 3 times 0.2 g B.1, B.4 4 mg/2 mL 2% (dye/leather) Heating at 55°C 400 µL MilliQ + 4 mg formic acid Magnetic stirring for 0.5 h

Dyes characterization UV-Vis Naturalised dyes Commercial acid dyes 1  Dyeing bath 2  Washing 1 3  Washing 2 4  Washing 3 B.1, B.2, B.4 FORMIC ACID PROCEDURE

Naturalised dyes Commercial acid dyes 1  Dyeing bath 2  Washing 1 3  Washing 2 4  Washing 3 B.1, B.2, B.4 Dyes characterization UV-Vis NEUTRALIZATIONPROCEDURE

B.1, B.2, B.4 Dyes characterization UV-Vis Wavelength [nm] Absorbance DO30 dyeing bath DO30 washing 1 DO30 washing 2 DO30 washing Washing water 3 Washing water 2 Washing water 1 A Tanning solution -61% -79% -83% 442

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

Naturalised dyes Commercial acid dyes Formic acid Neutralization B.1, B.2, B.4 Dyed leather characterization

Colorimetry B.1, B.2, B.4 CM2600d KONICA MINOLTA nm α = 10° D65 d. 5 mm SAMPLESCISCELabLab DR202 acid DY42 acid DB27 acid DO30 acid Red acid Yellow acid Blue acid Orange acid DR202 neutr DY42 neutr DB27 neutr DO30 neutr Red neutr Yellow neutr Blue neutr Orange neutr

Dyed leather characterization Colorimetry B.1, B.2, B.4 CM2600d KONICA MINOLTA nm α = 10° D65 d. 5 mm SAMPLESCISCELabLab DR202 2% acid DR202 5% acid DR202 10% acid DR202 15% acid DY42 2% acid DY42 5% acid DY42 10% acid DY42 15% acid DB27 2% acid DB27 5% acid DB27 10% acid DB27 15% acid DO30 2% acid DO30 5% acid DO30 10% acid DO30 15% acid

FT-IR CHARACTERIZATION Wavenumbers [1/cm] Absorbance AMIDE I C=O ν ~1650 cm -1 AMIDE II CN ν, NH δ ~1550 cm -1 ATR spectrum 128 scans Untreated leather COOH ν ~1730 cm -1 B.1, B.2, B.4

FT-IR CHARACTERIZATION Wavenumbers [1/cm] Absorbance AMIDE II CN ν, NH δ ~1550 cm -1 ATR spectrum 128 scans Untreated leather COOH ν ~1730 cm -1 AMIDE I1 ~1654 cm -1 AMIDE I2 ~1636 cm -1 AMIDE I C=O ν ~1650 cm -1 B.1, B.2, B.4

Wavenumbers [1/cm] Absorbance BLANK DO30-HCl DY42-HCl DR202-HCl DB27-HCl 1654 cm -1  1650 cm cm -1  1637 cm -1 Naturalised dyes FT-IR CHARACTERIZATION B.1, B.2, B.4

Wavenumbers [1/cm] AbsorbanceBLANK ORANGE 37 YELLOW 49 RED 249 BLUE 113 FT-IR CHARACTERIZATION Commercial dyes B.1, B.2, B.4

FT-IR CHARACTERIZATION Principal Component Analysis Wavenumbers [1/cm] Absorbance AMIDE I1 ~1654 cm -1 AMIDE I2 ~1636 cm -1 VARIABLES Peak heights of AI1/AIIAI2/AIICOOH/AII B.1, B.2, B.4 AMIDE II CN ν, NH δ ~1550 cm -1 COOH ν ~1730 cm -1 AMIDE I C=O ν ~1650 cm -1

FT-IR CHARACTERIZATION Principal Component Analysis DO30-HCl DO30-HCl Yellow Orange Red Blue Yellow Orange Red Blue DY42-HCl DR202-HCl DB27-HCl DY42-HCl DR202-HCl DB27-HCl PC1 (69%) PC2 (29%) Leather Blank Blank AI1/AII AI2/AII PC1 PC2 COOH/AII PCA Variables: AI1/AII, AI2/AII, COOH/AII Var. tot. = 98% Matrix: covariance Score plot Loading plot Formic acid procedure Neutralization procedure B.1, B.2, B.4

FT-IR CHARACTERIZATION Principal Component Analysis DO30-HCl DO30-HCl Yellow Orange Red Blue Yellow Orange Red Blue DY42-HCl DR202-HCl DB27-HCl DY42-HCl DR202-HCl DB27-HCl PC1 (69%) PC2 (29%) Leather Blank Blank AI1/AII AI2/AII PC1 PC2 COOH/AII PCA Variables: AI1/AII, AI2/AII, COOH/AII Var. tot. = 98% Matrix: covariance Score plot Loading plot 1 2 Formic acid procedure Neutralization procedure B.1, B.2, B.4

FT-IR CHARACTERIZATION Principal Component Analysis DO30-HCl DO30-HCl Yellow Orange Red Yellow Orange Blue DY42-HCl DR202-HCl DY42-HCl DR202-HCl DB27-HCl PC1 (69%) PC2 (29%) Leather Blank Blank AI1/AII AI2/AII PC1 PC2 COOH/AII PCA Variables: AI1/AII, AI2/AII, COOH/AII Var. tot. = 98% Matrix: covariance Score plot Loading plot  High AI1/AII, AI2/AII Formic Acid procedure (and RED neutralization) 2  Low AI1/AII, AI2/AII Neutralization procedure (and DB27-HCl and blue formic acid procedure) Formic acid procedure Neutralization procedure B.1, B.2, B.4 Blue Red DB27-HCl

FT-IR CHARACTERIZATION Principal Component Analysis DY42-HCl Formic acid DY42-HClNeutralization AMIDE II INCREASES B.1, B.2, B.4

COLLAGEN CROSS-LINKING FT-IR ~1654 cm -1 /~1690 cm -1 Collagen cross-linking type Pyridinoline (Pyr) Dehydro-dihydroxy- norleucine (deH-DHLNL) B.1, B.2, B.4

Commercial Naturalised Naturalised dyes Rich in Pyr type cross-linking COLLAGEN CROSS-LINKING FT-IR B.1, B.2, B.4

Naturalised dyes 5%, 10%, 15% Same behavior of commercial dyes 2% COLLAGEN CROSS-LINKING FT-IR B.1, B.2, B.4

COLLAGEN CROSS-LINKING TGA Gas: N mL/min Heating: °C, 10 °C/min B.1, B.2, B.4

COLLAGEN CROSS-LINKING TGA T01T01 T02T02 T onset % finSAMPLE T01T01T01T01 T onset T02T02T02T02 % fin Leather BLANK DB27 2% DO30 2% DY42 2% DR202 2% Blue Orange Yellow Red DB27 15% DO30 15% DR202 15% DY42 15% B.1, B.2, B.4

COLLAGEN CROSS-LINKING TGA B.1, B.2, B.4 T01T01 T02T02 T onset % fin Naturalised 2% Commercial 2% Naturalised 15%

FUTURE ACTIONS B.1 BIOKIMICA, INESCOP, ICCOM-CNR (0-6 months) Demonstration of the use of traditional chemical acid dyes in leather dyeing HPLC-DAD  characterization of dyes bath and washing solutions GC-MS  characterization of the volatile fraction of the dyed leather C.1 ICCOM-CNR, UNIFI, INESCOP, BIOKIMICA (0-12 months) Monitoring on the enviromental impact of acid dyes within leather manufacture Atomic spectroscopy  determination of the metal content of dyeing wastewaters (Cr, Co, Cu, Fe)

BIONAD ICCOM-CNR, PISA Emilia Bramanti Alessandro D’Ulivo Massimo Onor Manuela Cempini Davide Pellegrini Emanuela Pitzalis