Page 11 - BR20828_E Accucore 0713S

positional isomers of halogenated
compounds (Figure 9). The Phenyl-Hexyl
phase offers a mixed mode separation
mechanism, with the C6 chain responsible for
hydrophobic interactions and the phenyl ring
responsible for - interactions. The HILIC
stationary phase provides an approach for
the retention of very polar compounds via a
retention mechanism that involves
partitioning, hydrogen bonding and weak
electrostatic interactions [10]. For an example
HILIC separation, see Figure 10.
System considerations
One of the great advantages of solid-core
particle packed columns is that the
backpressures produced often allow the use
of standard HPLC instrumentation. However,
the LC system needs to be optimised in
order to produce efficient chromatography.
In particular, system volumes (connecting
tubing ID and length, injection volume, flow
cell volume in UV) must be minimised,
detector time constant and sampling rate
need to be carefully selected, and when
running fast gradients pump delay volume
needs to be minimal. Failure to consider the
parameters may result in loss of the efficiency
gained by using the solid-core particles [11].
Band broadening, which has a detrimental
effect on the chromatographic performance,
can be caused by high sample volume, it can
occur in the tubing connecting the column to
injector and detector and in the detector
flow cell. These band broadening effects
which occur in the fluidic path of the HPLC
instrument are volumetric effects. Each
contributes an additive variance to the width
of the chromatographic band. In general, the
extra column band broadening, covering the
injection volume, flow cell volume and tubing
volume should not exceed 10% of the total
band broadening. The extra column effects
are more significant for scaled down
separations (as column volume decreases)
and for less retained peaks which have a
lower peak volume. It is therefore critical to
minimise extra column dispersion if high
efficiency separations are required. In
addition to the volumetric effects, the time
constant of the detector (response rate) and
the scan rate may also contribute to the
broadening of the peak, and should be
considered. With solid-core particles peaks
may be of the order of 1-2 seconds in width.
It is important to scan the detector quickly
enough to achieve optimum peak definition,
otherwise resolution, efficiency and analytical
accuracy will be compromised. This is
illustrated in Figure 11, which clearly shows a
loss of peak height and area when less than
ten data points are taken across the width of
the peak. For fast gradients it is also
important to minimise the pump dwell
volume to ensure that there in no delay in
Figure 8: Radar plots for Accucore stationary phases: comparison of the phase selectivities. Tables 2, 3 and 4 for
axis labels.
Figure 9: Separation of 14 positional isomers on Accucore PFP. Experimental conditions: Column - Accucore PFP
2.6µ m, 50mm x 2.1mm; Mobile phase: A – Water + 0.1% Formic Acid, B – Acetonitrile + 0.1% Formic Acid;
Gradient: 15 – 30% B in 7 minutes; Flow rate: 600µ L/min; Temperature: 50°C; Detection: UV at 270nm; Injection
volume: 2µ L. Analytes: 1. 3,4 – Dimethoxyphenol; 2. 2,6 – Dimethoxyphenol; 3. 2,6 – Difluorophenol; 4. 3,5 –
Dimethoxyphenol; 5. 2,4 – Difluorophenol; 6. 2,3 – Difluorophenol; 7. 3,4 – Difluorophenol; 8. 3,5 – Dimethylphenol;
9. 2,6 – Dimethylphenol; 10. 2,6 – Dichlorophenol; 11. 4 – Chloro-3-Methylphenol; 12. 4 – Chloro-2-Methylphenol;
13. 3,4 – Dichlorophenol; 14. 3,5 – Dichlorophenol.
Figure 10. Separation of melamine and cyanuric acid
on Accucore HILIC. Experimental conditions: Column
Accucore HILIC 2.6µ m, 150mm x 4.6mm; Mobile
phase: 90:10 (v/v) Acetonitrile:50mM Ammonium
Acetate, pH 5; Flow rate: 1mL/min; Temperature: 40°C;
Detection: MS at m/z 127, 128, 168 (negative mode 0-3
mins, positive mode 3-10 mins); Injection volume: 5µ L;
Backpressure: 117 bar; Analytes: Cyanuric Acid: m/z
128.1 (-1) Melamine: m/z 127.1 (+1), 168.1 (+1 with
Acetonitrile adduct).