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Advanced Chromatography HPLC System
Advanced Chromatography HPLC System
Commonly Observed Masses from Siloxane Based Resin: 222, 296, 370, 455
Monolithic Columns
Cross-Section of Monolithic Column
Tortuous Flow
Through Pores
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)
Pore Size
Small Molecules < 2000 Da 60-150 Large Molecules > 2000 Da 300
I II, I, II III
I.
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
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)
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)
http://www.nestgrp.com/pdf/Pp1/HILIC_example.pdf
http://www.waters.com/WatersDivision/pdfs/HILIC
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
Phospholipids
No salt necessary, 8080-85% acetonitrile Salt gradient in 75% acetonitrile. (Cysteines and glycines are retained more than alanines and threonines in these methods.)
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
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
1250 psi
8000 psi
8000 psi
10,000psi
www.jasco.nl/xtreme-lc
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
Protein
PDB: 2CPG
www.astom-corp.jp/en/en-main2-neosep
www.gly.uga.edu/schroeder/geol6550/CM19.h
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