advanced chromatography hplc system

7/27/2019 Advanced Chromatography HPLC System

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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

advanced chromatography hplc system

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