ZORBAX Strategy for Reversed-Phase Method Development of Proteins and Peptides

Low pH

Mid and High pH

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300SB columns are wide-pore columns with unbeatable lifetime in TFA containing mobile phases. This makes them an ideal first choice for separations of peptides and proteins. C8 is an excellent starting bonded phase because of its moderate hydrophobicity

• C18 and C8 are generally selected for peptides and protein digests but can also be used for proteins
• C3, C4 and CN are generally selected for larger, hydrophobic polypeptides and proteins but can also be used for peptides.

Poroshell 300SB columns use an innovative particle technology to deliver rapid protein separations. Short analysis times with efficient peaks are easily obtained with Poroshell columns. Using the 300SB bonded phases also gives these columns exceptional lifetime with TFA containing mobile phases.

• C18 is a good starting bonded phase choice for most peptides, polypeptides and proteins to maximize retention
• C8 is generally selected for moderate size proteins but can be used with any sample
• C3 is generally selected for antibodies or large proteins but can be used for any sample

Start at low pH with simple aqueous / organic gradient

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 30-60 minutes. A Poroshell column requires a shorter analysis time and a higher flow rate and still provides exceptional resolution. Then to improve resolution, increase the gradient time, decrease column length, or increase flow rate.

Optimize sample solubility

For best peak shape and recovery at any pH, it is important to solubilize a sample completely. 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.

Solvent Choices to Solubilize Proteins and Peptides

Water/phosphate buffer Weakest Dilute acid (TFA, Acetic Acid or HCl) Neutral pH, 6-8M Guanidine-HCl or isothiocyanate to 5% HOAc/6M Urea Dilute acid + aqueous/organic solvents (ACN, MeOH, THF, IPA) Dilute Base (Ammonium hydroxide) DMSO or 0.1%-1% TFA in DMSO Strongest Formamide

Raise the Temperature

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. StableBond 300SB – up to 80ºC Poroshell 300SB – up to 80ºC

Optimize Mobile Phase pH

Try mid and high pH if low pH does not work 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 loose their charge. ZORBAX 300Extend-C18 is an excellent choice for mid to high pH separation.
Column: 300Extend-C18, 4.6 x 150 mm, 5 mm
Part Number: 773995-902
Mobile Phase: A: 20 mM NH4OH in H2O
B: 20 mM NH4OH in 80% ACN
Flow Rate: 1 mL/min
Temperature: 25 - 30ºC (not >60ºC)
Gradient: 5 – 60% B in 30 minutes