Bridging the Analytical Gap – Comprehensive Analysis of Cellooligosaccharides by HPTLC Thank you for the introduction…..Titel nochmal, und weiter Josua Timotheus Oberlerchner Department of Chemistry Division of Chemistry of Renewable Resources

…BRIEF INTRODUCTION TO CELLULOSE... Main component of plant cell walls- the most abundant organic molecule Used on large scale (paper & fibre industry, cosmetic& food additives,.....) Key component for lignocellulose biorefinery Oligomeric cellulose still a neglected sidestream in pulp and paper Compound Solubility [g*L-1] Intrinsic viscosity [dl*g-1] Glucose—DP1 909 at 25 °C - Cellobiose—DP2 147 0.027 Cellotriose—DP3 soluble 0.030 Cellotetraose—DP4 78 0.036 Cellopentaose—DP5 40 (slowly soluble in warm water, 4.8 at 25°C) 0.038 Cellohexaose—DP6 10 (slowly soluble in warm water) 0.047 Celloheptaose—DP7 1 (slowly soluble in warm water) Was ist Cellulose, warum damit beschäftigen Welche Rolle Cellulose in der Bioraffiniere, warum alle cellulose wollen Hat schon eine Anwendung (Papier, Fasern, regenarted…) Hemis nein. NAWAROS-> Cellulose (weil ja schon anwednungen oligos ---- Dann cellooligos save the world Dann gleich die Analytik Josua Timotheus OBERLERCHNER

SEC-MALLS: Size Exclusion Chromatography coupled to Multi angle laser light scattering detection MALLS signal decreases within lower molar masses No standards available Mw [g*mol-1] 102 103 2*103 - 104 5*104 - 106 106 – 5*106 Oligomeric fraction GC(MS) CE(MS) SEC-MALLS HPAEC Oberlerchner, J., Rosenau, T., Potthast, A. (2015) Overview of Methods for the Direct Molar Mass Determination of Cellulose, Molecules 20:6, 10313-10341.

Acetolylsis of cellulose HPLC to prep. HPLC for the production of cellooligos Acetolylsis of cellulose Separation MALDI TOF MS Monodisperse Cellooligmers perOAc DP2-DP20 HSQC-NMR Analytical Preparative L [mm] 250 D [mm] 4.6 40 Cross section area [cm2] 0.1661 12.566 F [ml*min-1] 0.6 45.36 v [cm*min-1] 3.61 Zweckmair, T., Oberlerchner, J., Böhmdorfer, S., Bacher, M., Sauerland, V., Rosenau, T., Potthast, A. (2016) Preparation and analytical characterisation of pure fractions of cellooligosaccharides, Journal of Chromatography A 1431, 47-54.

Monitoring preparation of standards via HPTLC Josua Timotheus OBERLERCHNER

HPTLC-MALDI-ToF-MS Imaging Josua Timotheus OBERLERCHNER

Direct HPTLC-MALDI-TOF MS coupling Josua Timotheus OBERLERCHNER

New standards - new possibilities Extending Size Exclusion Chromatography towards Oligomeric Region Better understanding of celllulose structure Model compounds for enzymtic studies Absolute quantification of cellooligomer in biorefinery streams Oberlerchner, J., Zweckmair, T., Kindler, A., Koch, S., Rosenau, T., Potthast, A. (2016) Filling the gap: SEC-calibration of the low molar mass range of cellulose with cellooligomers, Journal of Chromatography A, 1471, 87-93.

HPTLC-Densitometry method for cellooligomers Sample lyophilisation Sample acetylation HPTLC Merck AMD Plates, prewashed, MP: Toluene:Ethylacetat 7:3 (v/v)+ 1% Acetone, development to 60 mm Derivatization with Anisaldehyd-Sulfuric Acid Densitometrie at 366nm Data processing and quantification Separation up to a DP of 12 (3560 g*mol-1) Acetone as modifier – sharper bands Quantification based on peak height Josua Timotheus OBERLERCHNER

The TLC-Oligo-Ladder Oligo ladder perOAc DNA ladder g*mol-1 390 678 966 1255 1543 1831 2120 2408 2696 2984 3273 3561 Similar to a DNA ladder in PCR experiments Mixture of Cellooligomers with accurat knowldge of each individual compount Qualitative and quantitative standard Less space ocuppied for references  More space for samples Oligo ladder perOAc DNA ladder Josua Timotheus OBERLERCHNER

Mean value and standard deviation Oligo ladder applied on plate with samples Mean value and standard deviation   CA1 CA3 CA4 CA6 MV [µg] RSD [µg] DP10 0,97 0,02 0,52 0,25 0,35 0,57 0,03 DP9 1,58 0,14 0,80 0,33 0,06 0,83 DP8 2,79 0,42 0,63 0,88 0,18 DP7 1,52 0,26 0,54 0,19 0,58 0,00 DP6 0,74 0,53 0,59 DP5 2,33 0,27 0,11 0,38 0,07 DP4 2,44 0,45 0,10 0,22 0,32 0,01 DP3 2,05 0,39 0,12 DP2 0,20 0,05 Poster Nr. 7 Quantitative analysis of monosaccharides from lignocellulosic material Josua Timotheus OBERLERCHNER

Summary Preparation of cellooligomers yielded valuable standard compounds Direct mass spectrometry from plates (MALDI Coupling) New HPTLC method for cellooligomers Introduction of the Oligo-Ladder Mw [g*mol-1] 102 103 2*103 - 104 5*104 - 106 106 – 5*106 Oligomeric fraction GC(MS) CE(MS) SEC-MALLS HPAEC

The NAWARO Group Division of Chemistry of Renewable Resources = Chemie nachwachsender Rohstoffe And in the end I would thank my great working group at BOKU Unibvesity in Vienna!

Financial support by: Josua Timotheus OBERLERCHNER Chemistry of Renewable Resources Josua Timotheus OBERLERCHNER

Calibration curves Josua Timotheus OBERLERCHNER

HPTLC for optimizing total hydrolysis Oberlerchner, J., Böhmdorfer, S., Zweckmair, T., Koch, S., Kindler, A., Rosenau, T., Potthast, A. (2015) Optimizing Acid Hydrolysis Conditions for Pulp and Paper using Quantitative HPTLC, 18th ISWFPC, Vienna, Austria, September 9-11th. In Proocedings Vol. II of the 18th ISWFPC.

Limits of SEC-MALLS no light scattering information for low-molar masses Mw [g*mol-1] 102 103 2*103 - 104 5*104 - 106 106 – 5*106 Oligomeric fraction GC(MS) CE(MS) SEC-MALLS HPAEC Oberlerchner, J., Zweckmair, T., Kindler, A., Koch, S., Rosenau, T., Potthast, A. (2016) Filling the gap: SEC-calibration of the low molar mass range of cellulose with cellooligomers, Journal of Chromatography A, 1471, 87-93..