1. 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

2. …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

3. 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* 5*
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,

4. 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,

5. Monitoring preparation of standards via HPTLC
Josua Timotheus OBERLERCHNER

6. HPTLC-MALDI-ToF-MS Imaging
Josua Timotheus OBERLERCHNER

7. Direct HPTLC-MALDI-TOF MS coupling
Josua Timotheus OBERLERCHNER

8. 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,

9. 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

10. 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

11. 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

12. 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* 5*
106 – 5*106
Oligomeric fraction
GC(MS)
CE(MS)
SEC-MALLS
HPAEC

13. 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!

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

15. Calibration curves
Josua Timotheus OBERLERCHNER

16. 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.

17. Limits of SEC-MALLS no light scattering information
for low-molar masses
Mw [g*mol-1]
102
103 2* 5*
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,