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Sample retention time will vary depending on the interaction between the stationary
phase, the molecules being analyzed, and the solvent, or solvents used. As the sample
passes through the column it interacts between the two phases at different rate, primarily
due to different polarities in the analytes. Analytes that have the least amount of
interaction with the stationary phase or the most amount of interaction with the mobile
phase will exit the column faster.
Objective
Equipment
Theoretical Exercise
Exploring the effects of solute structure and organic mobile phase modifiers on
retention and resolution.
1) Draw structures of the following compounds, do you remember any of those
structure?
2) Based on their structures, predict qualitatively their retention order from least
to most retained
Observation: In isocratic elution mode is used in which the composition of the mobile
phase remains constant. The best retention time and peaks separation correspond to a
range of 50 and 25% of acetonitrile. In the first case at 50% of acetonitrile the compounds
are separated. But in the 25% the peaks of the chromatogram show us more efficiency to
perform this fractions of the solvent. The overlapping in chromatogram makes the system
more inefficient, and the mass transfer between phases will be more difficult taking
account the brand broadening in the experimental process.
2) The use of the gradient separation increase the efficiency of the system. This
mode allows to get separate peaks in the analysis. The elution gradient is
programmable to define vary of composition on the mobile phase from a
minimum to a pre-set maximums within a time interval.An interval of 5 minutes
was programmed, initiating in 5% of acetonitrile until reaching 95% of
acetonitrile. The results are presented below:
The number of plates and efficiency increase when increasing the flow rate, or what is
the same, the plate size decreases. This can be explained because the size of particle can
flow through the solution on the column. In the next part, the particle size will is going
to be more explained.
Particle Size
To increase the efficiency and have a good brand broadening there are some parameters
that can be influenced by the packaging type, length, and particle size. Decrease of
particle size of the column support leads to a noticeable improvement in the efficiency of
said column. To compare the effects of particle size variation, three size values were used,
maintaining the gradient elution at T = 106 C and a flow range of 6.
A plate is the minimum slice of column length where the system is at equilibrium. A
higher quantity of plates means more efficiency. It is visible the fact that reducing the
size of the particle, the efficiency determined by the number of plates is increased. This
is reflected on the bandwidth:
Decrease in particle size leads to a decrease in the bandwidth, thus decreasing the size of
the plate, increasing the number of plates, and the resolution of the process is greater.
How is knew the brand broadening allows to recognize the parameters that influence the
performance of HPLC, the particle size of the Fig. 6 show the difference between changes
in particle size in the peaks of chromatogram. The peaks with less broadening, represents
high efficiency and resolution for interpreting analysis of data.
Conclusion:
HPLC is a useful technique to separate, identify and quantify components in a
mixture. There are parameters that can affect the efficiency and good resolution
of the process like: eluent, temperature, properties of column.
There are different modes to analyze the mixtures: isocratic and elution gradient.
The more efficient is the eluent gradient because have a low retention times and
the broadening of compounds in chromatogram are more effective to get a good
results in the performance of experiment.
The temperature affects the resolution of the experiment because it decrease the
retention time, and produce the decrease of broadening in chromatogram. Also,
the major number of plates with small size of bandwidth, produce that the system
has a high efficiency due the great separation between components.
Finally the small particle allows to get separation between compound in the
chromatography column to achieve a good separation and resolution on results in
the experimental process.
References:
HiQ for High performance liquid chromatography (HPLC). (2017). HiQ. Retrieved from:
http://hiq.lindeas.com/en/analytical_methods/liquid_chromatography/high_perfo
rmance_liquid_chromatography.html