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Liquid chromatography and its applications in carbohydrate analysis of lignocellulose samples Puu-0.3130 Instrumental Analysis in Surface, Polymer, and Nanoscience Lokanathan Arcot

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Chemical analysis of a lignocellulose sample Biomass consists of a complex matrix of polymers having different chemical composition and molecular size and shape Chemical composition of the carbohydrates in biomass can be analyzed e.g. by high performance anion exchange chromatography Molecular size of carbohydrates can be analyzed by size exclusion chromatography Cellulose Hemicellulose Lignin Extractives

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Content Principles of chromatographyGeneral aspects of high performance liquid chromatography Determination of carbohydrate composition of lignocellulosic samples by HPAEC-PAD Size exclusion chromatography of lignocellulosic samplesSummary Following Harris, Daniel C., Quantitative Chemical Analysis, 8th Edition, 2010

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Principles of chromatography

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What is chromatography Column Eluent Solutes A & B Eluate Solutes are separated in a chromatograph column or columns The concentration of solutes is determined by a detector when they elute out from the column(s) Solute A Solute B

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Components of chromatography Chromatography: A separation technique Column Eluent Solutes A & B Eluate Stationary Phase: Solid or liquid support which responsible for separation of solutes based on relative difference in mobility/affinity Mobile Phase: Liquid or gas phase that transports solute molecules across a stationary phase Objective: Separating mixtures of solute molecules/ polymer

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Retention time Basis of separation in chromatography Column Eluent Solutes A & B Eluate The interval between the instant of injection and the detection of the component is known as the retention time. Which one has higher t a A or B ? Solute A Solute B A has higher t a

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Simple examples of chromatography Thin Layer Chromatography: -Thin layer of stationary phase -Transport of mobile phase due to capillary force Column Chromatography: -Tube filled with stationary phase -Transport of mobile phase due to gravity -Special type HPLC (thin column, high pressure)

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Column packing: open and packed columns Packed columns are filled with (usually spherical) beads and the stationary phase is on the surfaces of these beads. In open (or capillary) columns the stationary phase is on the wall of the column The chromatography columns have either open or packed structure

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Types of chromatography Adsorption chromatography Partition chromatography Ion-exchange chromatography Size exclusion chromatography Affinity chromatography = How the different solutes are separated in the columns What kind of detection, eluent, or samples are used

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Types of chromatography Adsorption chromatography Solid stationary phase Liquid or gas mobile phase Solute is adsorbed on the surface of solid particles

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Types of chromatography Partition chromatography Liquid stationary layer bound to a solid surface Liquid or gas mobile phase Solute is separated by different retention times between stationary and mobile phases

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Types of chromatography Affinity chromatography Solid stationary phase containing covalently attached specific molecules Solute interacts selectively with solutes in mobile phase High selectivity e.g. towards selected proteins

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Types of chromatography Ion-exchange chromatography Solid stationary phase containing charged groups attached to a solid phase, which is often a resin Charged stationary phase attracts the solutes with opposite charge Liquid mobile phase Example shown here Anion exchange or cation exchange ? Cations are being exchanged So, cation exchange chromatography

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Types of chromatography Size exclusion chromatography Solid stationary phase Liquid (or gas) mobile phase Separation is based on the size of the solute Ideally no interaction between the solute, mobile phase, and stationary phase

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General aspects of high performance liquid chromatography

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HPLC: High-Performance Liquid Chromatography (or sometimes High-Pressure Liquid Chromatography) Versatile analysis technique Wide variety of organic, inorganic, and biological samples It can be used for compounds that have insufficient volatility for gas chromatography Sample preparation is typically easy, and often only the dilution to desired concentration is required Analysis of one injection takes 1-60 minutes

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Schematic of an HPLC-system (1)Solvent reservoirs (2)Solvent degasser (3)Gradient valve (4)Mixing vessel for delivery of the mobile phase (5)High-pressure pump (6)Switching valve in "inject position" (7)Sample injection loop (8)Pre-column (9)Analytical column (10) Detector (i.e. IR, UV) (11) Data acquisition (12) Waste or fraction collector. http://en.wikipedia.org/wiki/File:HPLC_apparatus.svg

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HPLC systems An HPLC in our labColumns in the column compartment

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HPLC columns Packed columns are used in HPLC-systems Typical packing material is microporous silica particles High operation pressure: 7-40 MPa (70-400 bar), sometimes up to 100 MPa (1000 bar) Increases with increasing flow rate and with decreasing particle size Precolumn (or guard column) is recommended Protects longer and more expensive analytical column(s) Pure (and expensive) analytical grade eluents are used

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Concentration sensitive detectors Requirements for good detector High sensitivity Linear response Concentration sensitive detectors Refractive index detectors Practically all compounds can be detected Limited sensitivity Absorbance Better sensitivity than RI-detector UV/VIS, fluorescence Advanced detectors detect several wavelengths simulateneously Electrochemical detectors

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Determination of carbohydrate composition of lignocellulosic samples by HPAEC-PAD HPAEC High Pressure Anion Exchange Chromatography PAD Pulsed Amperometric Detection

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Determination of the chemical composition of a lignocellulose sample The information about the molecular size and shape is lost in hydrolysis Pretreatment, e.g. hydrolysis Chromatography analysis

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Sample preparation for sugar analysis Polysaccharides must be hydrolyzed to monosaccharides prior to analysis NREL/TP-510-62618 300 mg of dry sample is hydrolyzed in 72 wt% H 2 SO 4 at 30 C for 60 minutes Diltution to 4 wt% and hydrolysis in an autoclave at 121 C for 60 minutes Dilution to desired concentration and filtration into vials using syringe filters

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HPAEC-PAD system in our laboratory Sampler Columns Eluent bottles Detector Pump Back- pressure regulator

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HPAEC column HPAEC: High performance anion exchange chromatography Positively charged groups in the stationary phase resin interact with the dissociated groups of the solutes Used primarily for analysis of inorganic ions (ion chromatography, IC) but also for carbohydrate analysis Alkaline eluent for sugar analysis In our lab 100 mmol/L NaOH(aq) + + + + + + + + +

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Question: how does retention time change if the pH is increased and why? + + + + + + + + + H2OH2O H2OH2O H2OH2O H2OH2O H2OH2O H2OH2O H2OH2O OH - HPAEC-column pKa (reducing sugar): ~12.5 High pH : Sugars ve charge

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Retention of various sugars in CarboPac PA 20 HPAEC-column at different pHs Weitzhandler et al. J. Biochem. Biophys. Methods 60, 309-317 Increasing pH Decreasing t a Increasing NaOH pHpKa (applicable here) - pH increase -Ionic Strength increase -Screening causes decrease in Anion-cation interaction (Dominant) Hence decrease in t a

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Pulsed amperometric detection (PAD) Electrochemical Pulsed Amperometric Detection (PAD) has a superior sensitivity Pico and femtomol concentrations Carbohydrates are oxidized on the detector by electric potential A proton is removed causing electric current detected by the detector Current is integrated for a short interval (e.g. 0.6 s) and these integrals are plotted against time Detectors electric potential cycle Obtained chromatogram

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Suppressor Na + OH - Cl - Na + H+H+ Cl - H+H+ Na + H2OH2O Conductive ions such as Na + would interfere with the electrochemical detection of ions and therefore they are removed in a suppressor prior to the detector. Column Suppressor Eluent: NaOH + H 2 OEluent: H 2 O To detectorFrom sampler NaOH(aq) NaCl(aq) Analysis of NaCl by a HPAEC-system:

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Sugar analysis HPAEC-PAD chromatography Arabinose Rhamnose Galactose Glucose Xylan Mannose Standards: Unknown sample:

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Summary of HPAEC-PAD chromatography Separation/ Concn Quantification technique Separation based on difference in affinity for stationary phase: Anion-Cation interactions Sample preparation Acid hydrolysis into sugars Quantification using known standards

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Size exclusion chromatography of lignocellulosic samples For determining molecular size (Weight)

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Types of chromatography (recap) Size exclusion chromatography Solid stationary phase Liquid (or gas) mobile phase Separation is based on the size of the solute Ideally no interaction between the solute, mobile phase, and stationary phase

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Size exclusion chromatography Stationary phase porous gel, hence name GPC GPC: gel-permeation chromatography Size-exclusion chromatography (SEC) Is used to analyze the molar mass distribution (MMD) of samples Macromolecules Proteins Separation based on the molecular size of solutes in solution Ideally no interaction between the solutes and packing material Requires non-degradative dissolution of samples Solvent for Cellulosic materials Lithium chloride / N,N-dimethylacetamide solvent (LiCl/DMAc) Ionic liquids

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Molar mass determination Non-degradative dissolution Dissolved polymers in solution Size-based separation in size exclusion column; usually it does not give (detailed) information about the chemical composition

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A typical GPC-system Meira, G. & Vega, J. & Yossen, M. Gel Permeation and Size Exclusion Chromatography. Analytical Instrumentation Handbook. 2005. p.828.

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Packing of GPC-columns Polystyrene/divinylbenzene particles most widely for organic eluents Particle size and pore size are the main parameters affecting the separation: Narrow pore size distribution gives high resolution for a specific molar mass range Columns with mixed pore size distributions are applicable for wide molar mass ranges Pore size: 5010 -10 - 110 -6 m Particle size: 310 -6 - 2010 -6 m

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Selection of size exclusion columns http://www.chem.agilent.com/Library/brochures/5990-7994-GPCorganics-Apr11-9lo.pdf Particle size (m) Plate number (1/m) Pressure loss at 1 mL/min (bar) 20> 17,0003 10> 35,00010 5> 50,00030 5> 50,00050 3> 80,00050 Smaller particle size increases the resolution of columns but raises operation pressure Applicable molar mass range depends on the used column

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Separation in GPC-columns Detector signal in time Sepration is based on molecular size in solution Large molecules elute first because there are less accessible pores for them

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From time-concentration signal to molar mass distribution: Calibration Plot

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Comparing Calibration Plot Sample chromatogram : Mw of Sample molecules Calibration Plot Sample Chromatogram

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From time-concentration signal to molar mass distribution Concentration sensitive detector is always needed In addition, a method to convert the elution time to molar mass is required Narrow standard calibration (narrow Mw distribution) Using molecules not chemically identical to sample molecule Extrapolation of range of Mw Broad standard calibration Using chemically identical (same) as in sample molecule Direct measurement with out extrapolation Light-scattering detector

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Calibration by narrow standards Calibration is done by known narrow standards Polystyrene Pullulan Calibration curve is obtained Often 3 rd degree polynomial function Signal in time Known MMDs Calibration curve

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Problems with narrow standard calibration Different polymers have different conformation Requires that standards are available, usually they are not Pullulan is widely used for cellulose analysis Calibration is valid for one polymer only at a time Problem with polymer mixtures Pullulan Cellulose

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Correction factor for cellulose samples when pullulan calibration is used Berggren, R.; Berthold, F.; Sjoholm, E.; Lindstrom, M. Improved methods for evaluating the molar mass distributions of cellulose in kraft pulp. J. Appl. Polym. Sci. 2003, 88, 1170-1179.

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Multi-angle Laser Light Scattering Detector (MALLS) Absolute method: it gives weight-weighted average molar mass without calibration Based on static light scattering Zimm equation J. Chem. Phys. 16, 1093-1099 Together with separation by GPC it gives the absolute MMD Also mean radius in solution is obtained with a multi-angle detector

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Sample preparation for the GPC-analysis of cellulose samples 1.Samples (50 mg) are activated by a solvent exchange sequence) 1.Overnight h in water 2.Acetone wash + min. 6 h in acetone 3.Overnight in pure DMAc 2.Dissolution in 5 mL of 90 g/L LiCl/DMAc (N,N-dimethylacetamide) for one night (concentration: 10 mg/mL) 3.Dilution (DF 10) to 1 mg/L and mixing 4.Filtration with 0.2 m syringe filters into vials Manifold used for solvent exchanges Properly dissolved samples form clear solution Only samples with a low lignin content will dissolve. Often delignification is required prior to dissolution

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GPC-chromatograms Chromatogram detected by the RI-detector Calculated molar mass distribution Note: small molecules elute later than the large ones!

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Molar mass distribution (MMD) Molar mass distribution (molecular weight distribution, MWD) is a histogram: the mass of a selected molar mass range can be obtained from the corresponding area below the molar mass distribution curve Its area is commonly normalized to unity The unit of ordinate (y-axis) in the figure below is: Sometimes called as Differentia mass fraction Caution is recommended if the MMD is transferred to a linear base because also the values of y-axis have to recalculated

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Characterization by average numbers and polydispersity index MnMn MwMw MzMz M z+1 Average molar mass numbers are used to describe molar mass distributions

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Comparison of MMDs with sugar analysis by HPAEC-PAD Deconvolution by 2 Gaussians (1 for hemicelluloses + 1 for cellulose) GPC-RIHPAEC-PAD Cellulose: 72.9%Glucan:73.1% Hemicellulose: 26.3% Xylan:25.3% Hemicelluloses Cellulose

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Band broadening Band broadening affects the shape (width) of the peaks in chromatography In GPC the position and shape (not the area or height) is crucial and the band broadening has direct influence on the analysis results Real MMD Observed MMD An example of the effect of band broadening for a sample with a low PDI of 1.05

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Causes of band broadening Flow rate Band broadening Factors causing band broadening: A)Static diffusion B)Eddy dispersion: multiple pathways C)Mass transfer kinetics between the mobile and static phases Effect of flow rate on band broadening A C B Total A BC

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Error from band broadening in GPC Detected signal for perfectly monodisperse cellobiose sample in GPC Calculated molar mass distribution greatly over- estimated polydispersity Depending on the type of columns and sample, the band broadening may cause an overestimation of polydispersity and the minimum and maximum molar masses in the sample.

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Summary of Size Exclusion Chromatography Separation/Conc. Mw Quantification technique Separation based on difference in size Sample preparation non-destructive dispersion

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Summary Liquid chromatography offers versatile analyses for wide variety of samples Hydrolysis to mononers HPAEC-PAD Chemical compositions Size exclusion chromatography Non-degradative dissolution Molecular size