Lecture 14

How can I find my needle needle in the haystack?? haystack??

Advanced Chromatographic Methods

HPLC System and Column

Common HPLC Detectors

Sample Load and Injection

Chromatography Column Chemistry Silica-Based Silica Based Supports

Commonly Observed Masses from Siloxane Based Resin: 222, 296, 370, 455

Scanning Electron Micrographs of Silica Based Stationary Phases Silica-Based

Aggregate of spherical particles SA: 150 m2/g Porosity: 50%

Spongelike structure on surface SA: 300 m2/g Porosity: 70%

Monolithic Columns
Cross-Section of Monolithic Column

Back Pressure in Columns

Tortuous Flow

Through Pores

Chromatography How It Works...

Techniques for Separating Peptides and Proteins

SizeSize -Exclusion Chromatography (SEC) ReversedReversed -Phase HPLC (RP(RP-HPLC) Hydrophobic Interaction Chromatography (HIC) Hydrophilic Interaction Chromatographt (HILIC) IonIon -Exchange Chromatography (IEC) Capillary Electrophoresis (CE) Affinity Chromatography (AC)

Column Selections for Small Mass Molecules

Column Stationary Phase Selection

Particle Size

Pore Size
Small Molecules < 2000 Da 60-150 Large Molecules > 2000 Da 300

Reversed-Phase Separations for Small Molecules (C18 Stationary St ti Ph Phase) )

III I
III.

I II, I, II III
I.

Three Separation Mechanisms C18

II.

II

Stationary Phase

Si Si Si O Si O Si O R R O O R Si O R O O R O OH Si Si O O R R O OH O Si O O Si O R R O O OH Si O O O Si O R Si

Silica Support

Mobile Phase Selection for HPLC

Mobile Phase Selection in Chromatography

Commonly Used Buffer in LC-MC

***List of Mass Spec Compatible Buffers***
Typical Concentration Range: 10-100mM Concentration

Acetic A ti acid id Formic acid Ammonium acetate Ammonium formate Ammonium bicarbonate Ammonium hydroxide Triethylamine (TEA) acetate salts Trifluoracetic acid (TFA) (can suppress ionization)

!!!Do Not Use Non Non-Volatile Volatile Additives and Buffers!!!

Phosphates Surface active agents Detergents Inorganic acids
(e.g. sulphuric acid, nitric acid, phosphoric acid and hydrochloric acid).

Typical Gradient Conditions for RP-HPLC

RP-HPLC Gradient Profile and Separation

Influence of Gradient Optimization

Hydrophilic Interaction Chromatography (HILIC)

Complimentary selectivity to RPRP-HPLC Able to retain molecules not easily retained using other phases Nucleotides Amino Acids (Basic Groups) Amines Peptides Phenols Carbohydrates Gycosylated compounds Phosphorylated compounds High organic mobile phase promotes enhanced MS sensitivity Reduced analysis time by eliminating sample preparation steps (i.e., fractionation/drying down/reconstituting samples)

Retention in RPC vs HILIC

http://www.nestgrp.com/pdf/Pp1/HILIC_example.pdf

How does HILIC work?

http://www.waters.com/WatersDivision/pdfs/HILIC

HILIC Stationary y Phase Selection

There are several types yp of p polar sorbents being g used in HILIC methods.

Cyano (most polar) Polymeric HILIC Silica A id Amide Poly (hydroxyethyl aspartame) Amino (least polar) Polysulfoethyl Aspartamide SCX

proteomics, nn-terminal variant analysis, neuroprptides, growth factors, CNBr Peptides, and synthetic peptides

Separation Methods for HILIC LC-MS

Proteins and Peptides

Phospholipids

10mM TEA, TEA pH 2 2.8, 8 808085% acetonitrile

Sugars and Oli i Oligisaccharides h id

15mM ammonium formate, formate pH 6.5, 9595-50% acetonitrile gradient.

No salt necessary, 8080-85% acetonitrile Salt gradient in 75% acetonitrile. (Cysteines and glycines are retained more than alanines and threonines in these methods.)

Drugs Small Molecules Drugs, Molecules, and Metabolites

80% acetonitrile

Oligonucleotides

RP-MS vs HILIC MS

HILIC-M H MS

RP P-MS

http://www.flux.ch/download/4_5/Rheos_HILIC_Montreux2004.pdf

Ultra High Pressure HPLC

The optimum p linear velocity, y, which offers the maximum column efficiency, y, is shown in the 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 is maintained 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 analysis time shortened without sacrificing the column efficiency efficiency. The use of smaller 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

Ultra High Pressure HPLC and Mass Spec

F ll Compatible Fully C tibl with ith MS
High pressure fittings, column, and autosampler are necessary

1250 psi

8000 psi

8000 psi

Ultra High Pressure HPLC

3200psi

10,000psi

www.jasco.nl/xtreme-lc

Columns Selection for Macromolecules

Challenges g in Protein Separations p

Stationary Phases

Protein Behavior

Multiple peaks from single component Asymmetric peaks Batch-to Batchto-batch inconsistencies Reproducibility L Low recovery

Conformational changes Denaturation Digestion of peptide residues Unpredictable behavior Broad peaks

Separation Mechanism for Macromolecules

Ad Adsorption-Desorption ti D ti
Adsortion Desorption

Protein

Support St ti Stationary phase h

Hydrophobic Interaction Chromatography

Common additives that enhance hydrophobicity: Sodium Sulfate, Potassium Sulfate Ammonium Sulfate Sulfate, Sulfate, Sodium Chloride Chloride, Ammonium Chloride Chloride, Sodium Bromide

Size Exclusion Chromatography

Maintain Native Conformation

PDB: 2CPG

Ion-Exchange Chromatography (Cationic)

www.astom-corp.jp/en/en-main2-neosep

Ion-Exchange Chromatography (Anionic)

www.gly.uga.edu/schroeder/geol6550/CM19.h

Ion-Exchange Stationary Phases (Cationic and Anionic)

Alternative Stationary Phases for Protein Separation

NonNon -Porous/Macroporous/Mololithic

Favorable mass transfer kinetics Increased recovery Decrease carrycarry-over effect/ ghosting and cross crosscontamination

Polymeric y

Stability under acidic and alkaline conditions No silanol groups that lead to undesired i interactions/peak i / k asymmetry

Useful Websites for Information LC-MS

http://www.forumsci.co.il/HPLC/lcms_page.html www.waters.com www.lcms.com l www.thermo.com

Contact Information D ll Moton Dwella M Nelson N l dnelso28@jhmi.edu Office: 410-955-3022