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2007 vol. 4
Easy Transfer of HPLCMethods to UHPLC
Using Fully Scalable Pinnacle™ DB Columns
By Rick Lake, Pharmaceutical Innovations Chemist
• Methods on Pinnacle™ DB columns are easily transferred from 3 and 5μm to 1.9μm, allowing faster analysis without
losing separation quality.
• Pinnacle™ DB columns are 100% Restek manufactured–from base silica to final packed column.
• Restek offers the widest selection of stationary phases for UHPLC—more choices mean better selectivity for your
analytes.
Ultra High Pressure Liquid Chromatography
(UHPLC) is a rapidly growing technique that pro-
duces significantly faster analysis times compared
to conventional HPLC. While transferring HPLC
methods to UHPLC can increase sample through-
put, comparable method parameters must be used
to maintain equivalent separations. Here we
review which column properties and operating
conditions should remain consistent and which
need to be optimized in order to maintain selectivity.
In this example, we will perform a scale-down
method transfer for sulfonamides (Figure 1). For
optimal selectivity and faster analysis times, we
used a Pinnacle™ DB Biphenyl stationary phase
for this application (Figure 2). When performing a
scale-down procedure, column pore size, carbon
load, and support material must remain the same.
Changes to other parameters can be made using a
few simple calculations. Let’s go through them
sequentially.
Adjusting Column Size
The first calculation determines the appropriate
column length. Keeping the same column length
while decreasing the particle size increases the
number of theoretical plates. Therefore, column
length can be shortened without losing resolution.
By adjusting the column length properly, using
Equation 1, we can maintain the same separation.
Adjusting Injection Volume
Once we have determined the proper column
length, we can calculate injection volume.
Decreasing the column internal diameter and
length decreases the overall column volume and
sample capacity. Therefore, we must alter the
injection volume as described in Equation 2. Note
that since overall column volume has decreased, it
is important to match the sample solvent to the
starting mobile phase composition. Mismatched
sample solvents can cause irreproducible retention
times, efficiencies, and even changes in selectivity.
Adjusting Flow rate
Next, flow rate must be adjusted to maintain com-
parable linear velocity through a column with
smaller internal diameter. To maintain the same
linear velocity (which is important in maintaining
Figure 1
Chemical structures for example sulfonamides.
Figure 2
A 1.9μm Pinnacle™ DB Biphenyl column is more selective
for sulfonamides than a conventional C18 column.
LC_PH0461
Column:
A. Pinnacle™ DB Biphenyl
Cat.#:
9409565
Dimensions:
150mm x 4.6mm
Particle size: 5µm
Pore size:
140Å
Column:
B. Conventional C18
Dimensions:
150mm x 4.6mm
Particle size: 5µm
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
Peak List:
1. sulfadiazine
2. sulfathiazole
3. sulfamerazine
4. sulfamethazine
5. sulrfachlorpyridizine
6. sulfamethoxazole
7. sulfamethoxine
LC_PH0460
Sample:
Inj.:
10µL
Conc.:
100µg/mL
Sample diluent: starting mobile phase (80:20 A:B)
B. C18 Selectivity
A. Biphenyl Selectivity
Sulfadiazine
Sulfamethoxazole