ACE Catalogue 2006/7

300Å
ACE 300Å Wide Pore HPLC Columns
The Benefits of Ultra-Inert Stationary Phases for the
Reversed-Phase HPLC of Biomolecules
Benefit #1 – Increased Sensitivity
TFA or trifluoroacetic acid is used as a mobile phase
additive for reversed-phase separations of peptides and
proteins. This additive is typically used to improve both
the peak shape and resolution of complex mixtures of
peptides and proteins. As shown in Figure 20 , the use of
0.1% TFA in the mobile phase enables a column packed
with an active stationary phase to give peak widths
comparable to those obtained from a new generation
Figure 20. Sensitivity and Peak Shape as a Function of TFA Concentration
0.1% TFA
0.01% TFA
0.005% TFA
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ACE 5 C18-300
Ultra-inert silica
Column: 250 x 4.6mm, 5µm C18 300Å
Mobile Phase: A: TFA in H 2O
B: TFA in MeCN
(% TFA as specified above)
10% to 55% B in 37.5 mins.
Flow Rate: 1.5ml/min
Detection: UV 200nm
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20
column made from ultra-inert stationary phase. However,
as the TFA concentration is lowered to 0.01% and finally
0.005%, peak widths on the ultra-inert phase stay the
same, but degrade on the active stationary phase. The
ability to analyse peptides and proteins using very low
levels of TFA is beneficial for high sensitivity detection by
mass spectrometry. TFA complexes with polypeptides and
can enhance selectivity. However, this same complexation
lowers sensitivity in the mass spectrometer.
Compounds: 1. Gly-Tyr
2. Oxytocin
3. Angiotensin II
4. Neurotensin
Vydac 218TP
Lower purity ‘active’ silica
Columns based on lower quality silica (chromatograms on right) show a dramatic loss in performance as TFA concentration is
lowered. Columns from ultra-inert silica such as ACE maintain performance when TFA concentration is decreased.
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