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Pharmaceutical
efficiencies), flow rates must be decreased. Also, since smaller particle sizes
give rise to higher optimal linear velocities, isocratic flow rates should be cal-
culated with particle size taken into account. In this example, a gradient elu-
tion was used and, therefore, particle size was not included in the equation.
Equation 3 can be used to estimate the adjusted flow rate needed for equiva-
lent chromatography. Also, note that since <2µm particle sizes are less affect-
ed by flow rate, faster flow rates can be used in isocratic systems without detri-
mental effects on peak efficiency.
Adjusting Time Program
After determining the proper column length, injection volume, and flow rate,
we can calculate the time needed for gradient or step elutions. As an analyti-
cal method is scaled down, the time program also needs to be scaled down to
keep the phase interactions the same. Time can be adjusted using Equation 4.
Conclusion
After determining the equivalent conditions for scaling-down the analysis of
sulfonamides, we can see that the separations are equivalent, while the analy-
sis time was greatly reduced (Figure 3). By following the procedure described
here to ensure that the columns are equivalent, scaling analytical procedures
from HPLC to UHPLC can easily be accomplished using Pinnacle™ DB
columns.
Equation 1
Adjusted column length can
easily be calculated when scaling from
HPLC to UHPLC.
Equation 2
Changing column dimensions
requires an adjusted injection volume.
Equation 3
Changing column internal
diameter requires using an adjusted flow
rate.
Equation 4
When scaling down a gradient
method, the time program needs to be
adjusted.
Figure 3
Restek’s Pinnacle™ DB 1.9μm columns can easily be scaled
from HPLC to UHPLC and vice versa.
A. Pinnacle DB Biphenyl
1.9μm, 50 x 2.1mm
B. Pinnacle DB Biphenyl
5μm, 50 x 4.6mm
Scaling down
methods saves
analysis time!
Column:
A. 1.9µm Pinnacle™ DB Biphenyl
Cat.#:
9409252
Dimensions:
50mm x 2.1mm
Particle size: 1.9µm
Column:
B. Pinnacle™ DB Biphenyl
Cat.#:
9409565
Dimensions:
150mm x 4.6mm
Particle size: 5µm
Pore size:
140Å
Peak List:
1. sulfadiazine
2. sulfathiazole
3. sulfamerazine
4. sulfamethazine
5. sulrfachlorpyridizine
6. sulfamethoxazole
7. sulfamethoxine
LC_PH0462
LC_PH0461
Pinnacle™ DB Biphenyl Columns (USP L11)
Physical Characteristics:
1.9µm Column, 2.1mm
cat. #
price
50mm
9409252
$460
5µm Column, 4.6mm
cat. #
price
150mm
9409565
$356
particle size:
1.9µm, & 5µm, spherical
pore size: 140Å
carbon load: 8%
endcap: yes
pH range: 2.5 to 7.5
temperature limit: 80°C
Sample:
Inj.:
10µL
Conc.:
100µg/mL
Sample diluent: starting mobile phase (80:20 A:B)
For a full product listing, including guard cartridges for these
columns, visit our website at
www.restek.com.
Conditions:
Mobile phase: A: 0.1% formic acid in water
B: 0.1% formic acid in acetonitrile
Time(min.)
%B
0.0
20
1.0
20
6.0
80
8.0
80
Flow:
1.0mL/min.
Temp.:
30ºC
Det.:
UV @ 254nm