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Figure 1
Figure 2
Figure 4
Figure 4d shows the contribution of stagnant mobilephase mass transfer to molecular spreading. With
porous column-packing particles, the mobile phase
contained within the pores of the particle is stagnant or
unmoving. In d we show one such pore, for particle 5.
sample molecules move into and out of these pores
by diffusion.
Figure 5
TABLE 1
Peak Tailing: Causes and Cures
Causes
Sample solvent stronger
than the mobile phase
Cure
Dissolve sample in mobile
phase or at least reduce the
strength of the sample solvent
as much as possible
Reduce the amount (mass)
of sample injected. Refer to
Table 2 for recommended
sample injection size for
different column configurations
Adsorption of acids on
silica
Increase salt
concentration in the mobile
phase
25 mM to 50 mM is usually
sufficient
Reduce the pH of the
mobile phase to < 3.0
Add a competing organic
acid.
1% acetic acid or 0.1% TFA is
usually sufficient
Column void
As sample overload
occurs, the front of
peaks will become
sharper, the back of the
peak will tail,and the
retention time will
decrease slightly. If the
peak shape looks
somewhat like a right
triangle, sample
overload is likely
occurring.
TABLE 2
Recommended Maximum Sample
Mass to Inject onto an HPLC Column
Column i.d.(mm)
a)
b)
c)
d)
e)
10.0
4.6
3.0
2.1
1.0
35 - 65
4.5 - 9.0
2.0 - 4.0
1.0 - 2.0
0.2 - 0.4
TABLE 3
Commonly Used Buffers for
Reversed Phase HPLC
Buffer
pKa
Buffer Range
UV Cutoff (nm)
Phosphate
2.1
7.2
12.3
1.1 - 3.1
6.2 - 8.2
11.3-13.3
200
Acetate
4.8
3.8 -5.8
210
Citrate
3.1
4.7
5.4
2.1 - 4.1
3.7- 5.7
4.4 - 6.4
230
Tris
8.3
7.3 - 9.3
205
Triethylamine
11.0
10.0 - 12.0
200
Optimization of Separation
Resolution Values
Figure 11
66
68
Parameter
Organic solvent
Mobile phase pH
Usage
Solvent strength
and additives
Stationary phase
Temperature
Mobile phase pH
Column temperature
Efficiency
The efficiency of a chromatographic peak is a
measure of the dispersion of the analyte band
as it travelled through the HPLC system and
column.
In an ideal world , chromatographic peaks
would be pencil thin lines however , due to
dispersion effects the peaks take on their
familiar Guassian shape.