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Typical Peptide Mapping Conditions

Peptide Mapping Conditions and Method Development/Optimization

Typical operating conditions for acquiring peptide mapping data are given in Table 1.

Instrument:   UHPLC
Column:   250 mm L x 2.1 mm ID x < 2 μm FPP or < 3 μm SPP dp, C18
Mobile Phase A:   0.05% TFA
Mobile Phase B:   0.05% TFA in acetonitrile
Flow rate:   300 μL/min
Gradient:   0-2 min: 1% B, 2-35 min: 1-45% B
Column temp.:   60 °C
UV:   214 and 280 nm

Table 1: Typical peptide mapping conditions (FPP = fully porous particle and SPP = superficially porous particle).

In summary the separation is performed on a UHPLC using:

Long (250 mm) but narrow (2.1 mm) column packed with either sub-2 μm fully porous particles (FPP) or modern sub-3 μm superficially porous particles (SPP) bound with C18 alkyl chain ligands as the stationary phase.

Starts with very high aqueous, sometimes as high as 100%, care should be exercised here as not all columns are able to operate under such polar mobile phase conditions. The organic is then ramped up to mid concentrations, typically around 50%, over approximately 30 minutes.

Mobile Phase
Standard mobile phases are 0.05 – 0.1% TFA (trifluoroacetic acid) in aqueous as the polar (A) solvent and 0.05 – 0.1% TFA in acetonitrile (MeCN) as the organic (B) solvent.

Flow rate
This very much depends on the ID of the column, but volumetric flow rates of 200 – 300 μL/min for 2.1 mm ID columns are the norm. Wider ID columns will require higher volumetric flow rates to maintain a suitable linear velocity.

Elevated temperatures are common with 60 °C often favored.

In routine production and QC environments dual wavelengths are typically monitored – 214 and 280 nm
Each of these parameters will be reviewed in the following section so their impact and optimization is fully understood.

Whilst UHPLC instruments are preferable, they are not essential. A standard 400 bar rated HPLC system can be used with the long, narrow columns packed with small particles, as long as elevated temperatures and low volumetric flow rates are used to keep the operating back pressures below the 400 bar upper limit, especially at the start of the run when the mobile phase is predominately aqueous.

However, UHPLC instruments are generally preferred; due to the increased efficiency afforded by the narrower system capillary tubing and optimized extra column volumes, alongside the increased detector scanning speeds facilitating more accurate detection of the highly efficient and narrow peaks.

Acetonitrile (MeCN) is the organic modifier of choice and is employed nearly universally. Its reduced viscosity, lower polarity, and lower UV cut-off (imperative when detecting at < 220 nm) provide numerous and sufficient benefits.

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