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Lecture 14
Advanced ChromatographicMethods
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HPLC System and Column
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Common HPLC Detectors
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Sample Load and Injection
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Chromatography Column Chemistry
-
Commonly Observed
Masses from Siloxane
Based Resin:
222, 296,370, 455
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Monolithic Columns
Cross-Section of
Monolithic
Column
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Back Pressure in Columns
Through
Pores
Tortuous
Flow
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ChromatographyChromatography
How It Works...How It Works...
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Techniques for Separating Peptides andTechniques for Separating Peptides and
SizeSize--Exclusion Chromatography (SEC)Exclusion Chromatography (SEC)
ReversedReversed--Phase HPLC (RPPhase HPLC (RP--HPLC)HPLC)
Hydrophilic Interaction Chromatographt (HILIC)Hydrophilic Interaction Chromatographt (HILIC)
IonIon--Exchange Chromatography (IEC)Exchange Chromatography (IEC)
Capillary Electrophoresis (CE)Capillary Electrophoresis (CE)
Affinity Chromatography (AC)Affinity Chromatography (AC)
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Column Selections for Small MassColumn Selections for Small Mass
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Column Stationary Phase Selection
Particle Size
Small Molecules < 2000 Da
Large Molecules > 2000 Da
300 60-150
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Reversed-Phase Separations for Small
18 a onary ase
III.III
I.
, ,
Three SeparationMechanisms
I
C18
II.
Si O
O
Si
OSi
OSi
O SiO
O
OHO
O
O
O
OH
O
Si
OH
O
O
Si
Si
SiSi
Si
R
O
Si
O
R
O
O Si
O
R
O
R
R R R O
RO
R
Silica Support
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Mobile Phase Selection for HPLC
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Mobile Phase Selection in
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Commonly Used Buffer in LC-MC***List of Mass Spec Compatible Buffers***
Typical Concentration Range: 10-100mM Concentration
ce c ac
Formic acidAmmonium acetate
Ammonium bicarbonate
Ammonium hydroxide
Trifluoracetic acid (TFA) (can suppress ionization)
- PhosphatesSurface active agents
Inorganic acids
(e.g. sulphuric acid, nitric acid, phosphoric acid and hydrochloric acid).
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Typical Gradient Conditions for RP-HPLC
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RP-HPLC Gradient Profile and Separation
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Influence of Gradient Optimization
C
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Hydrophilic Interaction ChromatographyHydrophilic Interaction Chromatography
Complimentary selectivity to RPComplimentary selectivity to RP--HPLCHPLC
phasesphases NucleotidesNucleotides
AminesAmines
PeptidesPeptides
CarbohydratesCarbohydrates
Gycosylated compoundsGycosylated compounds
High organic mobile phase promotes enhanced MS sensitivityHigh organic mobile phase promotes enhanced MS sensitivity
Reduced analysis time by eliminating sample preparationReduced analysis time by eliminating sample preparationsteps (i.e., fractionation/drying down/reconstituting samples)steps (i.e., fractionation/drying down/reconstituting samples)
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Retention in RPC vs HILIC
http://www.nestgrp.com/pdf/Pp1/HILIC_example.pdf
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How does HILIC work?How does HILIC work?
http://www.waters.com/WatersDivision/pdfs/HILIC
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HILIC Stationar Phase SelectionHILIC Stationar Phase Selection
There are several t es of olar sorbents beinThere are several t es of olar sorbents bein
used in HILIC methods.used in HILIC methods. Cyano (most polar)Cyano (most polar)
Polymeric HILICPolymeric HILIC
SilicaSilica m em e
Poly (hydroxyethyl aspartame)Poly (hydroxyethyl aspartame)
Polysulfoethyl Aspartamide SCXPolysulfoethyl Aspartamide SCX
proteomics, nproteomics, n--terminal variant analysis, neuroprptides,terminal variant analysis, neuroprptides,
growth factors, CNBr Peptides, and synthetic peptidesgrowth factors, CNBr Peptides, and synthetic peptides
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Separation Methods for HILIC LC-MS
Proteins and PeptidesProteins and Peptides
--
PhospholipidsPhospholipids
, . ,, . ,
85% acetonitrile85% acetonitrile Sugars andSugars and
,,
pH 6.5, 95pH 6.5, 95--50% acetonitrile50% acetonitrilegradient.gradient.
g sacc ar esg sacc ar es
No salt necessary, 80No salt necessary, 80--85%85%
acetonitrileacetonitrile
, ,, ,
and Metabolitesand Metabolites
80% acetonitrile80% acetonitrile
OligonucleotidesOligonucleotides
Salt gradient in 75%Salt gradient in 75%
acetonitrile. C steinesacetonitrile. C steines
and glycines are retainedand glycines are retainedmore than alanines andmore than alanines and
threonines in thesethreonines in these
methods.)methods.)
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RP-MS vs HILIC MS
-MS
ILIC-
R S
http://www.flux.ch/download/4_5/Rheos_HILIC_Montreux2004.pdf
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Ultra High Pressure HPLCThe o timum linear velocit which offers the maximum column efficienc is shown inthe graph.
Reducing the particle size offers lower HETP values resulting in higher column
efficiency. For columns packed with 1.8 m particles, when the linear velocity of the
mobile phase is increased to two or three times its original value, the HETP value ismaintained at a much lower level compared to a similar increase when using columns
with larger particles. This demonstrates that the flow rate can be increased and the
.
particles, however, requires increased pumping pressure. The X-LCtmis capable of
delivering solvents at pressures up to 15,000 psi.www.jasco.nl/xtreme-lc
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Ultra High Pressure HPLC and Mass Specu y ompa e w
High pressure fittings, column, and autosampler are
necessary
1250 psi 8000 psi
8000 psi
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Ultra High Pressure HPLC
3200psi
10,000psi
www.jasco.nl/xtreme-lc
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Columns Selection for MacromoleculesColumns Selection for Macromolecules
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Challen es in Protein SeparationsChallen es in Protein Separations
Multiple peaks fromMultiple peaks from ConformationalConformational
Asymmetric peaksAsymmetric peaks DenaturationDenaturation
BatchBatch--toto--batchbatch
inconsistenciesinconsistencies
Digestion of peptideDigestion of peptide
residuesresidues
ReproducibilityReproducibility UnpredictableUnpredictablebehaviorbehavior
ow recoveryow recovery Broad peaksBroad peaks
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Separation Mechanism for Macromolecules
sorp on- esorp on
Adsortion Desorption
Supporta onary p ase
Hydrophobic Interaction ChromatographyCommon additives that enhance hydrophobicity: Sodium Sulfate, Potassium
, , , ,
Bromide
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Size Exclusion Chromatography
Maintain NativeConformation
P D B : 2 C P GP D B : 2 C P GP D B : 2 C P GP D B : 2 C P G
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Ion-Exchange Chromatography
(Cationic)
www.astom-corp.jp/en/en-main2-neosep
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Ion-Exchange Chromatography
www.gly.uga.edu/schroeder/geol6550/CM19.
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Ion-Exchange Stationary Phases
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Alternative Stationary Phases for ProteinAlternative Stationary Phases for Protein
SeparationSeparation
NonNon--Porous/Macroporous/MololithicPorous/Macroporous/Mololithic
Favorable mass transfer kineticsFavorable mass transfer kinetics
Increased recoveryIncreased recovery
Decrease carryDecrease carry--over effect/ ghosting and crossover effect/ ghosting and cross--
PolymericPolymeric
Stability under acidic and alkaline condit ionsStability under acidic and alkaline condit ions
No silanol groups that lead to undesiredNo silanol groups that lead to undesired
interactions/peak asymmetryinteractions/peak asymmetry
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Useful Websites for Information
LC-MS
http://www.forumsci.co.il/HPLC/lcms_page.html
www.waters.com
www. cms.com
www.thermo.com
Contact Information
Dwe a Moton Ne [email protected]
Office: 410-955-3022