1. 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

2. 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

3. DO30 DR202 DY42 DB27Naturalised dyes LACTOSE

4. Commercial acid dyes Commercial acid dyes O37Y49 R249 B113

5. 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

6. 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

7. 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

8. 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

9. B.1, B.2, B.4 Dyes characterization UV-Vis 200250300350400450500550600650700750800 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 Wavelength [nm] Absorbance DO30 dyeing bath DO30 washing 1 DO30 washing 2 DO30 washing 3 0.0 0.5 Washing water 3 Washing water 2 Washing water 1 A Tanning solution -61% -79% -83% 442

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

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

12. Colorimetry B.1, B.2, B.4 CM2600d KONICA MINOLTA 360-740 nm α = 10° D65 d. 5 mm SAMPLESCISCELabLab DR202 acid 48.1139.6513.6648.0839.5913.64 DY42 acid 81.47-7.625.7981.35-7.5625.75 DB27 acid 53.56-13.29-22.3553.4-13.29-22.39 DO30 acid 62.9225.7339.2762.925.6539.22 Red acid 52.4648.5611.4852.3348.6211.56 Yellow acid 78.02-10.6957.2477.85-10.6257.18 Blue acid 32.123.76-22.1131.963.7922.21 Orange acid 55.6832.8140.0755.5632.8740.19 DR202 neutr 45.1140.0814.8145.0440.0614.81 DY42 neutr 80.83-7.4530.1880.72-7.4130.14 DB27 neutr 51.54-13.61-21.9751.39-13.61-22.02 DO30 neutr 60.9327.4440.4160.9227.3740.35 Red neutr 54.348.46-11.3854.1948.52-11.45 Yellow neutr 80.71-11.0647.9880.6-10.9447.58 Blue neutr 35.823.48-23.835.733.54-23.84 Orange neutr 64.62.1438.0464.524.1238.03

13. Dyed leather characterization Colorimetry B.1, B.2, B.4 CM2600d KONICA MINOLTA 360-740 nm α = 10° D65 d. 5 mm SAMPLESCISCELabLab DR202 2% acid 48.1139.6513.6648.0839.5913.64 DR202 5% acid 41.1838.9814.7341.063914.74 DR202 10% acid 39.5937.2714.3939.4537.3514.43 DR202 15% acid 37.2536.114.5337.1136.1614.57 DY42 2% acid 81.47-7.625.7981.35-7.5625.75 DY42 5% acid 80.16-7.2540.2380.04-7.2340.19 DY42 10% acid 78.81-649.7178.7-5.9849.68 DY42 15% acid 77.84-4.625477.7-4.6154.02 DB27 2% acid 53.56-13.29-22.3553.4-13.29-22.39 DB27 5% acid 49.7-10.88-25.8349.59-10.86-25.87 DB27 10% acid 44.54-9.46-25.3444.49-9.45-25.37 DB27 15% acid 40.93-7.58-24.9340.8-7.58-25.01 DO30 2% acid 62.9225.7339.2762.925.6539.22 DO30 5% acid 57.9731.7344.0657.9131.744.02 DO30 10% acid 53.636.0945.1453.5936.0345.08 DO30 15% acid 53.2836.3944.8353.2136.3744.9

14. FT-IR CHARACTERIZATION 0500100015002000250030003500 0 0.02 0.04 0.06 0.08 0.1 0.12 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

15. FT-IR CHARACTERIZATION 0500100015002000250030003500 0 0.02 0.04 0.06 0.08 0.1 0.12 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

16. 162016251630163516401645165016551660 Wavenumbers [1/cm] 0.954 0.96 0.966 0.972 0.978 0.984 0.99 0.996 1.002 1.008 1.014 1.02 Absorbance BLANK DO30-HCl DY42-HCl DR202-HCl DB27-HCl 1654 cm -1  1650 cm -1 1634 cm -1  1637 cm -1 Naturalised dyes FT-IR CHARACTERIZATION B.1, B.2, B.4

17. 16201625163016351640164516501655166016651670 0.965 0.97 0.975 0.98 0.985 0.99 0.995 1 1.005 1.01 Wavenumbers [1/cm] AbsorbanceBLANK ORANGE 37 YELLOW 49 RED 249 BLUE 113 FT-IR CHARACTERIZATION Commercial dyes B.1, B.2, B.4

18. FT-IR CHARACTERIZATION Principal Component Analysis 0500100015002000250030003500 0 0.02 0.04 0.06 0.08 0.1 0.12 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

19. FT-IR CHARACTERIZATION Principal Component Analysis -2024 -1.5 0.0 1.5 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 01 0 1 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

20. FT-IR CHARACTERIZATION Principal Component Analysis -2024 -1.5 0.0 1.5 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 01 0 1 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

21. FT-IR CHARACTERIZATION Principal Component Analysis -2024 -1.5 0.0 1.5 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 01 0 1 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 1  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

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

23. 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

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

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

26. COLLAGEN CROSS-LINKING TGA Gas: N 2 200 mL/min Heating: 30-700°C, 10 °C/min B.1, B.2, B.4

27. COLLAGEN CROSS-LINKING TGA T01T01 T02T02 T onset % finSAMPLE T01T01T01T01 T onset T02T02T02T02 % fin Leather77.4302.2334.322.2892 BLANK72.7299.933820.2782 DB27 2% 70.6300.6339.520.9291 DO30 2% 76.6298.233721.5649 DY42 2% 74297.7337.620.2527 DR202 2% 74.1299.133921.639 Blue76.1299.2335.422.3998 Orange75.4302335.222.1174 Yellow71.9300.6333.520.4022 Red73299.133621.37 DB27 15% 72.7299.3330.922.6931 DO30 15% 77.7297.4334.622.4728 DR202 15% 79299.9330.622.1285 DY42 15% 76.5299.5331.522.5689 B.1, B.2, B.4

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

29. 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)

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