Page 46 - BR20828_E Accucore 0713S

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Columns Part Number
Accucore XL C18 4 µm, 150 × 2.1 mm 74104-152130
Accucore XL C18 4 µm, 300 × 2.1 mm
Accucore XL C18 4 µm, 450 × 2.1 mm
Separation Conditions
Instrumentation: Thermo ScientificTM DionexTM UltiMateTM 3000 HPLC system
Column temperature: 30 °C
Injection volume: 2 μL (partial loop)
Flow rate: 0.3 mL/min
UV detection: 254 nm (data rate 20 Hz)
Mobile phase A: 0.1% formic acid in water
Mobile phase B: 0.1% formic acid in methanol
Gradient conditions: Dependent on column length (Table 1a-c)
Tables 1a-c: Gradient timetables for Accucore XL HPLC column lengths of 150 mm (a), 300 mm (b), and 450 mm (c)
Gradient Timetable
Time % B
0.0 5
0.5 5
13.0 75
15.0 75
15.1 5
20.0 5
When total peak capacity (nc ) is calculated based on a
gradient elution, tg is total gradient time and w is the
average peak width. In this case peak width is measured
at the baseline of the peak.
As shown in Equation 1, peak capacity is influenced by
peak width, which is directly related to efficiency. Solid
core particles maximize efficiency and therefore, under
the conditions used for this application, peak capacity
by reducing the degree of eddy and longitudinal
diffusion through the column [2]. This means that
Accucore XL HPLC columns exhibit greater peak
capacities compared to columns packed with fully
porous particles of a similar size.
The separation of highly complex samples is a major
challenge in chromatography and the properties of
the Accucore XL HPLC columns provide a means of
providing highly efficient separations in a short analysis
time. The separation properties, including assessment
of efficiency through peak capacity of an Accucore XL
HPLC column were demonstrated through the
analysis of a complex mixture of a green tea extract. In
addition, the peak capacity to analysis time ratio was
calculated to show the benefits of shortening the
column length on an Accucore XL HPLC column. This
provides improvements in analysis times with some
sacrifice in resolution and peak capacity and presents
the user with a choice between maximizing peak
capacity or reducing their analysis times for their
separation.
Experimental Conditions
Sample Preparation
Green tea extract was removed from a 315 mg capsule
and dissolved in 5 mL methanol / water (50:50 v/v).
The sample was vortexed and centrifuged at 14,000 rpm
for 10 minutes to remove insoluble particulates and
also maximize the number of analytes in the sample for
analysis. The supernatant was transferred and diluted
1:5 in mobile phase A for injection onto the HPLC.
Results and Discussion
Using a gradient composed of 0.1% formic acid in
methanol and water in conjunction with the Accucore
XL HPLC column, separation of a complex green tea
extract was performed. Column lengths of 450 mm,
300 mm, and 150 mm were examined to investigate the
effect of column length on peak capacity, as well as peak
capacity to analysis time ratio. The gradient timetables
were adjusted to keep the % B change per unit column
length constant. Therefore, a similar separation of the
compounds on the different column lengths based on the
critical pair was achieved. The gradient timetables for
three column lengths are shown in Tables 1a-c.
(a) Accucore XL 4 µm, 150 × 2.1 mm
Gradient Timetable
Time % B
0.0 5
1.0 5
26.0 75
30.0 75
30.2 5
40.0 5
(b) Accucore XL 4 µm, 300 × 2.1 mm
Gradient Timetable
Time % B
0.0 5
1.5 5
39.0 75
45.0 75
45.3 5
60.0 5
(c) Accucore XL 4 µm, 450 × 2.1 mm