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2007 vol. 2
Pharmaceutical
In HPLC column terminology, particle size refers
to the mean diameter of the silica spheres used as
the support material to which the stationary phase
is bonded. Until recently, the practical particle size
limit was around 3μm; smaller particles created
backpressures above the limit of conventional LC
systems. The advent of LC systems capable of han-
dling higher backpressures (>10000psi) now allows
chromatographers to realize the benefits of sub-
2μm particle size columns. Smaller particles give
rise to greater column efficiencies and a wider
range of usable flow rates, resulting in better resolu-
tion and higher sensitivity with a significantly faster
overall analysis time. Figure 1 and Table 1 illustrate
the excellent peak shape and higher efficiency
characteristic of a 1.9μmPinnacle™DB C18 column,
compared to competitive columns.
To demonstrate the substantial gain in sample
throughput that is possible on a small particle col-
umn, we assayed a series of parabens under condi-
tions that give comparable linear velocities on both
a C18 column with conventional dimensions and
on a 1.9μm Pinnacle™ DB C18 column (Figure 2B
& C). Similar resolution was achieved in a much
shorter analysis time on the 1.9μm Pinnacle™ DB
C18 column. We also doubled the flow rate on the
1.9μm Pinnacle™ DB C18 column: the resolution
and peak efficiencies again were comparable, but
the analysis time was cut in half (Figure 2A). This
illustrates the considerable effect that small particles
can have on chromatographic separations; a much
wider range of usable flow rates translates into sig-
nificantly faster analysis times–in this case 10-fold
faster, with no loss in chromatographic quality.
Explaining the Small Particle Advantage
(continued from page 3)
Column
Efficiency (n/m)
Pressure (psi)
Asymmetry
1.9μm Pinnacle
™
DB
217,619
4,500
1.10
Competitor A
177,999
4,400
1.13
Competitor B
188,508
4,300
1.09
Data from the biphenyl peak of a reversed phase test mix.
Table 1
1.9μm Pinnacle™ DB C18 column offers the highest efficiency
of all columns tested.
Figure 2
1.9μm Pinnacle™ DB columns offer a wider range of usable
flow rates, dramatically increasing sample throughput–with no loss
in resolution.
Figure 1
Excellent peak symmetry and
efficiency on a 1.9μm Pinnacle™ DB C18
column.
Sample: Inj.: 2μL, HPLC Reversed Phase Test Mix #1 (cat.# 35005),
Sample diluent: water:methanol (25:75), Sample temp.: ambient,
Column: Pinnacle
™
DB C18, Cat. #: 9414252, Dimensions: 50 x 2.1mm,
Particle size: 1.9μm, Pore size: 140Å, Mobile phase: water:acetonitrile
(45:55), Flow: 0.4 mL/min., Temp.: 25°C, Det.: UV @ 254nm
Peak List:
1. benzene
0.02 mg/mL
2. naphthalene 0.50 mg/mL
3. biphenyl
0.06 mg/mL
Performance: (Calculations for biphenyl)
Efficiency: 217,619 n/m
Asymmetry: 1.10
Pressure: 4,500 psi
LC_EX0427
A) 1.9μm Particle Size, 1.0mL/min.
C) 5μm Particle Size, 1.0mL/min.
LC_PH0435
All chromatograms; Column: Pinnacle
™
DB C18, Pore size: 140Å; Sample Conc.: ~100 μg/mL each
component in mobile phase (50:50 0.1% acetic acid:acetonitrile), Temp.: Ambient, Det.: UV @ 254nm
A) Inj.: 2μL, Cat. #: 9414252; Dimensions: 50 x 2.1 mm; Particle size: 1.9μm
B) Inj.: 2μL, Cat. #: 9414252; Dimensions: 50 x 2.1 mm; Particle size: 1.9μm
C) Inj.: 10μL, Cat. #: 9414565; Dimensions: 150 x 4.6 mm; Particle size: 5μm
methyl paraben ethyl paraben
propyl paraben
butyl paraben
B) 1.9μm Particle Size, 0.5mL/min.
LC_PH0437
methyl paraben ethyl paraben
propyl paraben
butyl paraben
LC_PH0436
methyl paraben ethyl paraben
propyl paraben
butyl paraben
0