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University of Research and Technological Investigation YACHAY TECH

School of Chemical Sciences and Engineering Department of Chemistry

Chemical Analysis II Separation of samples by liquid chromatography in HPLC simulator
Michelle Beatriz Chicaiza Lema (0502998867)
Group: Jos Andino, Johana Gmez, Michelle Chicaiza
Urcuqu, October 2 2017
Separate, identify & quantify components dissolved in a liquid solvent with a high
analytical resolution. The sample carried by a moving gas stream of Helium or Nitrogen

High Performance Liquid Chromatography (HPLC) is a form of column chromatography

that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high
pressure through a column with chromatographic packing material (stationary phase).
The sample is carried by a moving carrier gas stream of helium or nitrogen. HPLC has
the ability to separate, and identify compounds that are present in any sample that can be
dissolved in a liquid in trace concentrations as low as parts per trillion. Because of this
versatility, HPLC is used in a variety of industrial and scientific applications, such as
pharmaceutical, environmental, forensics, and chemicals.

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.


Optimization of parameters for Reversed-phase chromatography High Pressure

Liquid Chromatography (RP-HPLC) to separate a given compound from a
theoretical mixture sample.
Understand concepts of HPLC and get familiar with RP-HPLC chromatograms.


Java JRE and HPLC simulators


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

2) Based on their structures, predict qualitatively their retention order from least
to most retained

3) Remembering your experience in TLC practice, how would you expect

qualitatively the elution times for these molecules if the mobile phase was
changed adjusting it to following conditions:
a. 100%/0% water/acetonitrile
Taking into account that TLC the stationary phase is polar and the mobile
phase is 100% water, which is polar. The most polar molecules of practice,
the elution time is fast due to the polar interactions between TLC phases.
Like dissolves like. The first eluted compound will be benzophenone
b. 50%/50% water/acetonitrile
In this case the elution time of the polar and apolar molecules will be
depending of molecular interactions between the stationary phases. Also
is important mention that acetonitrile is solvent in water.
c. 0%/100% (%v/v) water/acetonitrile
Taking into account that TLC the stationary phase is polar and the mobile
phase is 100% acetonitrile which dissolves organic compounds and apolar
solvents. The most apolar molecules of practice, the elution time is fast
due to interactions between TLC phases. Like dissolves like. In this case
the first eluted compound is 3-nitrophenol
Practice part with HPLC simulator
1) From the theoretical exercise introduce compounds into the simulator, adjust
parameters that you were asking about and compare them with your previous
a) 100% acetonitrile on water as mobile phase.

Figure 1chromatogram and results 100% of acetonitrile

b) 50% acetonitrile on water as mobile phase.

Figure 2:chromatogram and results 50% of acetonitrile

In the chromatogram is observable that the elution times is very acceptable to

perform the experiment with the 50% acetonitrile on water as mobile phase at
40 C. The first two chromatographic bands corresponding to 3-nitrophenol,
acetophenone compounds.
c) 25 % acetonitrile on water as mobile phase.

Figure 3 chromatogram and results 25% of acetonitrile

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:

Figure 4 Elution gradient cromatogram

Observations: The retention time with the use of elution gradient comparing with
isocratic is notable, because the peaks are closer to recognize to compare and
analyses the true values in performance of experiment. Also the retention time is
very important because, allows to get result in less time with high resolution of
the system.
Temperature for HPLC is not so significant parameter as in chromatography. However,
it has to be considered as an increase in column temp results in a decrease in retention
time. To illustrate the effect of temperature in the separation, the table below shows a
comparison between the retention times at different temperatures. The temperatures have
been set at 30C, 70C and 106C keeping the gradient elution, in the next table. The
retention factor decrease 1 or 2% each increased centigrade grade. It also decreases the
pressure and viscosity. Additionally, change peak spacing, give narrower, taller peaks as
seen in the chromatogram with T = 106 C (Fig 5).

Figure 5 Chromatogram 106C

Flow Rate and Column Efficiency
The next equation relate the parameters like the plate height H (cm) and the number of
plates or theoretical quantity of plates N to increase the efficiency of performance of
experimental procedure.The efficiency of the column increases the greater the number of
plates N, and the lower is the height H of the plate. For other case flow rate is a parameter
that directly affects the efficiency of the column because it can modify the height H of
the theoretical plate as well as the number of plates N. These parameters can increase the
resolution and efficiency. For illustrating this effect, a comparison has been prepared
between two different flow values by maintaining the gradient elution at T = 106 C:

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.

Figure 6 Comparision of chromatograms with diferent size

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.
HiQ for High performance liquid chromatography (HPLC). (2017). HiQ. Retrieved from: