BioHPLC Column Selection Guide Cover - Agilent Technologies

More documents

Recommendations

Info

Method Development Typically, a water/acetonitrile with 0.1% TFA gradient is used to elute all components of interest. A typical high resolution gradient on a 300Å pore size column requires 30-50 min. A Poroshell column requires a shorter analysis time and a higher flow rate and still provides exceptional resolution. To improve resolution, increase the gradient time, decrease column length, or increase flow rate. For best peak shape and recovery at any pH, it is important to completely solubilize a sample. Highly acidic or neutral solvents can be used with ZORBAX 300StableBond and Poroshell 300SB, while neutral solvents and dilute bases can be used with ZORBAX 300Extend-C18. 00 www.agilent.com/chem/biohplc Start at Low pH with Simple Aqueous/Organic Gradient Water/phosphate Buffer Dilute acid (TFA, Acetic Acid or HCI) Neutral pH, 6-8 M guanidine-HCI or isthiocyanate 5% HOAc/6 M urea Dilute acid + aqueous/organic solvents (ACE, MeOH, THF) Dilute base (ammonium hydroxide) DMSO or 0.1%-1% in DMSO Formamide StableBond 300SB - up to 80 ºC Weakest Strongest Separations of proteins and peptides are influenced by temperature and higher column temperature can dramatically improve both resolution and recovery of proteins and hydrophobic and aggregating peptides. Poroshell 300SB - up to 80 ºC If an optimized, low pH method does not provide an ideal separation, then mid or high pH mobile phase can be used. At high pH, selectivity is often very different because acidic amino acids become negatively charged and some basic amino acids may lose their charge. ZORBAX 300Extend-C18 is an excellent choice for mid to high pH separation. <strong>Column</strong>: ZORBAX 300Extend-C18 4.6 x 150 mm, 5 µm 773995-902 Optimize Sample Solubility Solvent Choices to Solubilize Proteins and Peptides Increase the Temperature Optimize Mobile Phase pH Try Mid and High pH if Low pH does not work Mobile Phase: A: 20 mM NH 4OH in H 2O B: 20 mM NH 4OH in 80% ACN Gradient: 5-60% B in 30 min Temperature: 25-30 ºC (
Reversed-Phase LC/MS Methods Method Development Starting <strong>Column</strong> Choices for Analytical Separations of Peptides, Polypeptides, and Proteins MW
Page 1 and 2:
Agilent BioHPLC Column Selection Gu
Page 3 and 4:
BIOCOLUMN SELECTION GUIDELINES From
Page 5 and 6:
Column Selection Flow Chart Biocolu
Page 7 and 8:
Protein Column Selection Guide Appl
Page 9 and 10:
Separation of charge variants of hu
Page 11 and 12:
Increased resolution for peptide ma
Page 13 and 14:
Preparative scale purification of L
Page 15 and 16:
DNA and RNA Oligonucleotide Column
Page 17 and 18:
Broad Distribution Biomolecules Car
Page 19 and 20:
ReveRseD-PHase HPLC Confidently per
Page 21 and 22:
Sterically Protected 300StableBond
Page 23 and 24:
Improved reproducibility of monoclo
Page 25 and 26:
Short-chain ZORBAX 300SB-C3 is stab
Page 27 and 28:
ZORBAX 300Å StableBond Hardware De
Page 29 and 30:
LC/MS analysis of angiotensin on Ex
Page 31 and 32:
Tips & Tools ZORBAX 300Å Extend-C1
Page 33 and 34:
Poroshell 300 columns separate prot
Page 35 and 36:
Monoclonal IgG1 chains: Separation
Page 37 and 38:
Poroshell 120 • 120Å pore size f
Page 39 and 40:
0 µm 0 µm 0 µm A B C Scanning el
Page 41 and 42:
Exploiting chemical stability - TFA
Page 43 and 44:
Temperature as a tool to enhance ma
Page 45 and 46:
ZORBaX amino acid analysis (aaa) Co
Page 47 and 48:
High resolution of 24 amino acids u
Page 49 and 50:
Ion-Exchange Column Selection Appli
Page 51 and 52: Consistent ion-exchange MAb separat
Page 53 and 54: Column Specifications Tips & Tools
Page 55 and 56: Weak cation-exchange chromatography
Page 57 and 58: PL-SAX Strong Anion-Exchange Column
Page 59 and 60: Analysis of representative whey pro
Page 61 and 62: Standard protein separation Column:
Page 63 and 64: Agilent Bio-Monolith HPLC Column Se
Page 65 and 66: Baseline expansion of a separation
Page 67 and 68: size exclusion Chromatography (seC)
Page 69 and 70: Column Specifications Agilent Bio S
Page 71 and 72: Comparison of Agilent Bio SEC-3 and
Page 73 and 74: Column Length size exclusion Chroma
Page 75 and 76: Column Specifications Agilent Bio S
Page 77 and 78: Calibration curves - Bio SEC-5 Colu
Page 79 and 80: ProSEC 300S • Mechanically robust
Page 81 and 82: Overlay of UV and light scattering
Page 83 and 84: Retention volume versus log (MW) fo
Page 85 and 86: Agilent PL aquagel-OH SEC Columns f
Page 87 and 88: Heparin Column: 2 x PL aquagel-OH 3
Page 89 and 90: Polyethylene Glycol/Oxide standards
Page 91 and 92: aFFiniTY CHROMaTOGRaPHY Agilent Bio
Page 93 and 94: Rapid human polyclonal IgG quantifi
Page 95 and 96: Multiple Affinity Removal System af
Page 97 and 98: Illustration of high abundance prot
Page 99 and 100: mRP-C18 High-Recovery Protein Colum
Page 101: MeTHOD DeveLOPMenT ZORBAX Column Me
Page 105 and 106: Method Development Ionic Strength:
Page 107 and 108: Method Development After the initia
Page 109 and 110: Tips & Tools Agilent offers a varie
Page 111 and 112: Human serum: Low abundance protein
Page 113 and 114: 2-D LC/MS Analyses Using ZORBAX Cap
Page 115 and 116: ZORBAX Bio-SCX Series II Capillary
Page 117 and 118: ZORBAX HPLC Capillary Columns (glas
Page 119 and 120: Microbore HPLC for sensitive peptid
Page 121 and 122: BioPharmaceutical Lifecycle Reverse
Page 123 and 124: ZORBAX 300Å StableBond ZORBAX Prep
Page 125 and 126: PLRP-S Prep to Process Application
Page 127 and 128: Prep to Process PLRP-S Size (mm) Pa
Page 129 and 130: PL-SAX AND PL-SCX FOR PREP TO PROCE
Page 131 and 132: Prep to Process PL-SAX and PL-SCX D
Page 133 and 134: Crude peptide screen Column: VariTi
Page 135 and 136: CARTRIDGE COLUMN SYSTEMS Cartridge
Page 137 and 138: USP DESIGNATIONS - BIOHPLC COLUMNS
Page 139 and 140: BioHPLC Columns Literature Title Ag
Page 141 and 142: BioHPLC Columns Literature Title An
Page 143 and 144: BioHPLC Columns Literature Title Co
Page 145 and 146: BIOPHARM DEFINITIONS A active start
Page 147 and 148: dimer A polymer made up of two iden
Page 149 and 150: K-L ligase An enzyme that causes mo
Page 151 and 152: S secondary structure In proteins,
Page 153 and 154:
Poroshell 300 Citations Matilainen,
Page 155 and 156:
Agilent 1260 Infinity Bio-inert Qua
show all

BioHPLC Column Selection Guide Cover - Agilent Technologies

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?