Page 12 - BR20828_E Accucore 0713S

delivering the gradient to the column. For
instance for a pump with 800µL dwell
volume, running at 400µL/min flow rate, it will
take two minutes for the gradient to reach
the head of the column. Conversely, a pump
with 80µL dwell volume, running at the same
flow rate, will deliver the gradient to the
head of the column in 0.2 minutes.
Solid-core particle packed
columns robustness
The robustness and reproducibility of a
chromatographic separation is dependent on
the column stability and lifetime but also on
operational parameters such as mobile
phase pH, temperature and sample
cleanliness. Common causes of column
instability can be either chemical or physical.
For instance, use of extremes of pH in the
mobile phase can lead to degradation of the
column through chemical attack of the
bonded stationary phase or dissolution of
the base silica. Another aspect of column
stability is the ability of the packed bed to
resist pressure changes such those
experienced inline sample preparation
techniques such as TurboFlow
chromatography.
The tight control of the particle size
distribution on solid-core materials allows for
highly uniform and mechanically stable
packed beds which can withstand a very high
number of injections. The robustness of the
bonded phase will determine the column’s
stability under different mobile phase pHs
and temperature. At low mobile phase pH,
the bonded phase can be lost through
hydrolysis of the organosilane bond and at
high pH the mobile phase can dissolve the
silica support resulting in collapse of the
stationary phase. The advanced bonding
technology used for Accucore columns
generates robust bonded phases that are
resistant to extremes of pH and also
temperature. Figures 12 and 13 demonstrate
Accucore C18 column stability at pH 1.8 and
10.5 respectively. Over 30,000 column
volumes of mobile phase were run through
the column in each instance using a gradient
method which is equivalent to 5.5 days of
continuous operation. Monitoring of capacity
factor of the test mixture components over
this period reveals no loss of retention for
any of the analytes, which would be
expected if bonded phase cleavage had
occurred. The pH range for the RP-MS and
aQ phases is 2 – 9 and 2 – 8 for the Phenyl-
Hexyl, PFP and HILIC phases.
Most LC separations are performed at 25 to
40°C, however, temperature is a
useful method development
parameter. The use of higher
temperatures has advantages: mass
transfer is improved because analyte
diffusivity is increased, thus the
peaks obtained are sharper, which
provides better peak height and
therefore better signal-to-noise ratio,
improving the sensitivity of the
analysis. Also at high temperatures,
solvent viscosity is lower, which
allows the use of higher flow rates to
increase speed, without loosing
efficiency. One limiting factor is
column stability, where thermal
degradation of the bonded surface
Figure 13: Accucore column stability at pH 10.5. Experimental conditions: Column - Accucore C18 2.6µ m, 100
x 2.1m; Mobile phase: A – Water + 0.1% Ammonia, B – Methanol + 0.1% Ammonia; Gradient: 15%B for 1
min, then to 100%B by 8 min, hold at 100%B for 3 min, return to 15%B and hold for 5 min for re-equilibration;
Flow rate: 400µ L/min; Injection volume: 1µ L; Temperature: 30°C; Detection: UV at 254nm (0.1s rise time,
20Hz); Order of elution: 1. Uracil (t0), 2. 4-Chlorocinnamic acid, 3. Procainamide, 4. 4-Pentylbenzoic Acid, 5.
N-Acetylprocainamide, 6. Di-isopropyl phthalate, 7. Di-n-propyl phthalate.
Figure 12: Accucore column stability at pH 1.8. Experimental conditions: Column - Accucore C18 2.6µ m, 100 x
2.1mm; Mobile phase: A – Water + 0.1% Trifluoroacetic Acid, B – Methanol + 0.1% Trifluoroacetic Acid; Gradient:
25%B for 0.75 min, then to 100%B by 10 min, hold at 100%B for 2 min, return to 25%B and hold for 5 min for re-
equilibration; Flow rate: 400µ L/min; Injection volume: 1µ L; Temperature: 30°C; Detection: UV at 254nm (0.1s rise
time, 20Hz); Order of elution: 1. Uracil (t0), 2. Acetaminophen, 3. p-Hydroxybenzoic acid, 4. O-Hydroxybenzoic acid,
5. Amitriptyline, 6. Nortriptyline, 7. Di-isopropyl phthalate, 8. Di-n-propyl phthalate.
Figure 11: Effect of detector sampling rate on the peak height
and peak area.