Liquid chromatography and its applications in carbohydrate
analysis of lignocellulose samples Puu-0.3130 Instrumental Analysis
in Surface, Polymer, and Nanoscience Lokanathan Arcot
Slide 2
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
Slide 3
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
Slide 4
Principles of chromatography
Slide 5
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
Slide 6
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
Slide 7
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
Slide 8
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)
Slide 9
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
Slide 10
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
Slide 11
Types of chromatography Adsorption chromatography Solid
stationary phase Liquid or gas mobile phase Solute is adsorbed on
the surface of solid particles
Slide 12
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
Slide 13
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
Slide 14
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
Slide 15
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
Slide 16
General aspects of high performance liquid chromatography
Slide 17
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
Slide 18
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
Slide 19
HPLC systems An HPLC in our labColumns in the column
compartment
Slide 20
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
Slide 21
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
Slide 22
Determination of carbohydrate composition of lignocellulosic
samples by HPAEC-PAD HPAEC High Pressure Anion Exchange
Chromatography PAD Pulsed Amperometric Detection
Slide 23
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
Slide 24
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
Slide 25
HPAEC-PAD system in our laboratory Sampler Columns Eluent
bottles Detector Pump Back- pressure regulator
Slide 26
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) + + + + + + + + +
Slide 27
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
Slide 28
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
Slide 29
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
Slide 30
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:
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
Slide 33
Size exclusion chromatography of lignocellulosic samples For
determining molecular size (Weight)
Slide 34
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
Slide 35
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
Slide 36
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
Slide 37
A typical GPC-system Meira, G. & Vega, J. & Yossen, M.
Gel Permeation and Size Exclusion Chromatography. Analytical
Instrumentation Handbook. 2005. p.828.
Slide 38
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
Slide 39
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
Slide 40
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
Slide 41
From time-concentration signal to molar mass distribution:
Calibration Plot
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
Slide 44
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
Slide 45
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
Slide 46
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.
Slide 47
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
Slide 48
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
Slide 49
GPC-chromatograms Chromatogram detected by the RI-detector
Calculated molar mass distribution Note: small molecules elute
later than the large ones!
Slide 50
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
Slide 51
Characterization by average numbers and polydispersity index
MnMn MwMw MzMz M z+1 Average molar mass numbers are used to
describe molar mass distributions
Slide 52
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
Slide 53
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
Slide 54
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
Slide 55
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.
Slide 56
Summary of Size Exclusion Chromatography Separation/Conc. Mw
Quantification technique Separation based on difference in size
Sample preparation non-destructive dispersion
Slide 57
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