3. CENTRIFUGAL WASHING AND RECOVERY AS AN IMPROVED METHOD
3.2. Materials and methods
3.2.1. Black Liquor Characterization
Black liquor was obtained from a kraft mill whose furnish was a mixture of softwood and hardwood. Its pH was measured to be 12.90 – 13.00, and the solid content was determined based on the TAPPI 650 om-05 method, with the following modifications; ca 10 g of black liquor was added to pre-weighed sawdust in Petri dishes, followed by evaporation in an oven set at 105 °C overnight. The final mass of the dried samples was then obtained following the TAPPI method mentioned. The inorganic matter was determined based on TAPPI T 211 om- 93 method to determine the ash content. Total lignin in the black liquor was determined by UV-Vis spectrophotometry, whereby black liquor was diluted with NaOH and its absorbance measured at 280 nm. The absorptivity coefficient used to calculate the total lignin content in black liquor was 24.60 L.g-1.cm-1. Elemental analysis of the black liquor was also determined by CHNS/O analysis.
3.2.2. Lignin Precipitation from Black Liquor
Lignin was isolated by precipitation with H2SO4, according to the procedure described below.
The bulk black liquor sample obtained from the mill was stirred for 3 hours to allow homogeneity. 100 mL of black liquor was sampled from the bulk sample, and 6M H2SO4 was
added to the black liquor drop-wise while stirring until pH = 9 was reached (where most lignin is supposed to be precipitated according to Ohman and Theliander (2007)). The solution was left stirring for 1 hour to allow completion of the reaction. After this period, the sample did not show distinct signs of precipitation, and the solution appeared to be still black, although slightly viscous. The pH of the solution was thus further decreased by the addition of more H2SO4
until a visible sign of precipitation was observed (pH ~ 6), and some gas evolution. The final pH of the solution was established at 4. The solution was left stirring for a further 1 hour at the final pH.
Lignin was recovered from the solution by centrifugation at 3600 rpm for 20 minutes. The mother liquor was recovered, and the dissolved lignin content was determined by UV-Vis spectrophotometry. The recovered lignin was subsequently washed with acid-water a number of times (4-5 times) (or until the supernatant was clearer), each time utilising a centrifuge to recover the washed precipitate. The concentration of the acid-water utilised during the washing process decreased with the number of washes (up to neutrality). For 15 mL centrifuge tubes used in this study, 8 – 10 mL was added to the tube for each wash. Finally, lignin was recovered by filtration with a Buchner funnel under vacuum suction.
Two methods for collection of the precipitated lignin were evaluated, viz.
i) Filtration of the precipitated lignin.
ii) Centrifugation of the precipitated lignin prior to filtration
The obtained lignin product was air-dried overnight and further dried in a vacuum oven at 40 °C.
3.2.3. Analytical Procedures
UV-Vis and Elemental Analysis
The mother liquor supernatant obtained from the initial recovery step following the precipitation was analysed for dissolved lignin by UV-Vis spectrophotometry at 280 nm with a quartz cuvette with a path length of 1 cm, on a Varian 50 CONC spectrophotometer. Elemental analysis (CHNS/O) of the lignin precipitate (and black liquor) was performed on a Thermo Scientific Flash 2000 instrument.
FTIR
The lignin precipitate obtained was characterised by Fourier transform infrared (FTIR) spectroscopy equipped with attenuated total reflectance (ATR) sampling accessory on a Perking Elmer Spectrum 100 Series instrument. The data capturing settings for each spectrum were 10 scans, range: 4000 – 300 cm-1 and resolution: 4 cm-1.
NMR
The structural features of the acetylated lignin (acetylation method found in the section for SEC analysis) samples were studied by NMR spectrometry using a Bruker Avance III spectrometer at 30 °C, where for 1H spectra the following parameters were used: frequency of 600 MHz with 16384 data points, 13.36 μs pulse and pulse delay of 1.328 s. DMSO-d6 was used as a solvent. 13C NMR spectra were obtained using a frequency of 150 MHz, 32768 data points, 13.35 μs and 1.328 s pulse delay. Chloform-d4 was also used as a solvent for another 13C spectrum.
Py-GC/MS
Pyrolysis of approximately 200 μg of finely ground lignin sample was carried out for 10 s at 610 °C on a Frontier Lab EGA/PY-3030D multi-shot pyrolyzer, followed by gas chromatography-mass spectroscopy (GC/MS) performed on a Shimadzu Gas Chromatography GC-2010 Plus linked to a GCMS-Q2010 mass spectrometer. The injector temperature of the GC/MS was kept at 280 °C, and the MS transfer interface temperature was at 300 °C. The oven temperature was programmed from 40 °C to 300 °C at a heating rate of 6 °C/min, with the final temperature held for 15 min. The eluted compounds were identified by mass spectra comparison with NIST libraries.
SEC
Size Exclusion Chromatography was used to determine the molecular weight distribution of the lignin samples. The lignin samples were subjected to acetylation in order to enhance their solubility in the mobile phase solvent.(Tejado et al., 2007; Glasser et al., 1993). The
acetylation reaction, which causes the substitution of the lignin hydroxyl functional groups by acetyl groups, was carried out by placing the lignin precipitate in a mixture of acetic anhydride and pyridine (1:1 v/v), with a final reaction mixture concentration of 0.033 g/mL. The reaction mixture was stirred at room temperature for 72 h (or longer to allow complete acetylation).
Ethanol, equivalent to the solvent mixture, was added to stop the reaction, followed by evaporation under pressure at 60 °C. Continuous addition and evaporation of ethanol allowed for complete removal of the solvent mixture. The concentrate was dissolved in 20 mL chloroform and washed with water. The chloroform phase was dried over MgSO4 and finally concentrated under reduced pressure at 50 °C. The resulting acetylated lignin was dried in a vacuum oven at 40 °C to constant weight and then dissolved in tetrahydrofuran (THF), to a concentration of 7 mg/L. The solution was filtered with a PTFE syringe filter prior to injection of 200 μL of the samples. SEC was performed on an HPLC pump (Rheos 2000) with THF at a flow rate of 1 mL/min. The eluted material was detected using a multi-angle laser light scattering (MALLS) detector (Wyatt Dawn EOS) in tandem with a UV detector (Perkin Elmer) and refractive index (Wyatt Optilab-T-rex) detector. The chromatography media consisted of SEC-columns (PSS – SDV, 104 and 106 Å connected in series, both 300 x 8 mm) in an oven (KNAUER Jetstream) at 35 °C. The system was also equipped with a guard column (PPS – SDV, 20 microns, 50 x 8 mm). The universal calibration curve for the instrument was based on polystyrene standards in the range 580 to 350 Daltons (Polymer Laboratories).
3.3. Results and discussions