SEPARATION OF SAMPLES BY LIQUID CHROMATOGRAPHY IN HPLC

SIMULATOR SOFTWARE
Vvian Yomaira Achig Merino,1 Jordy Mateo Cusangua Vargas,1 Carla
Lizbeth Parra Altamirano,1 and Jonnathan Ariel Castro Angamarca1
1
Instrumental Chemical Analysis , School of Chemical Sciences and Engineering, Yachay Tech University
(Dated: 01/12/2021)

I. INTRODUCTION

High-performance liquid chromatography (HPLC) is an analytical separation technique that involves the high-
pressure flow of a liquid through a column that contains the stationary phase. Where there is a mobile phase: Liquid,
its function is to carry the sample through the stationary phase. Then, the stationary phase can be a solid (LSC) or
a liquid (LLC).

The components with a higher affinity for the stationary phase will move with less speed than those with the lowest
affinity. Molecules of interest in the mobile phase are separated based on their differing physicochemical interactions
with the stationary and mobile phases.The speed differences give the data of separation of the components, where
the interactions can be determined by London dispersion forces, van Der Waals interactions, hydrogen bonds,
electrostatic interactions, which will have a direct implication on the degree of separation; These interactions can be
based on molecular size,is know as size exclusion chromatography, charge which use an ion exchange chromatography,
hydrophobicity is useful to hydrophobic interaction chromatography. The main application of HPLC is the separation
of a wider range of compounds, high MW, polar, and ionic compounds because it helps to separations and purification
in areas as pharmaceuticals, biotechnology, environmental, polymer and food industries.

The main objectives of this practice are optimizing parameters for reversed-phase High-Pressure Liquid Chromatog-
raphy (RP-HPLC) simulation, analyzing and separating compounds from a theoretical mixture sample, understanding
concepts of HPLC, and getting familiar with RP-HPLC chromatograms. Therefore, the practice carries out with Java
JRE and HPLC simulators planted various conditions as, flow rate (ml/min), column length (mm), particle size (um),
to Acetophenone, Benzophenone, Propiophenone, Ketoprofen, 3-nitrophenol to know for each compound according
to the conditions the retention times, number of theoretical plates, the height of theoretical plate (HETP) and back-
pressure and which corresponding chromatogram figures. Using isocratic and gradient elution, where isocratic and
gradient elution describe the properties of the mobile phase and the main difference is that isocratic elution maintains
a constant concentration in the mobile phase while gradient elution maintains a variable concentration in the mobile
phase

II. BACKGROUND

A. Distribution Constant

The distribution constant, K, is defined as the ratio of the concentration of the substance in the two phases at
equilibrium, since Liquid-liquid extraction is an equilibrium process between two immiscible phases, described by a
equilibrium constant. Then, it usually called partition coefficient. Then, it is calculated with:

Cu
k= (1)
Cl
Where:
CU: concentration of analyte in the less dense (upper) phase.
CL: concentration in the more dense (lower) phase).

It can be described as the quantity of A that will be associated with the stationary phase and the quantity of A
that will be associated with the mobile phase.
Separation of samples by liquid chromatography in HPLC simulator software .

B. Retention Factor

A term called the retention factor, k’, is often used to describe the migration rate of an analyte on a column[].
In isocratic separations, analyte retention time can be normalised to retention factor, k, which allows a direct
comparison between columns with different dimensions. This is possible due to the constant mobile phase composition
used in isocratic methods.For gradient chromatography prohibits the use of k. The front of the peak is moving slower
than the tail of the peak, with the increasing organic composition, which creates a focused, narrow peak. Gradients
are typically used to separate a mixture with a wide range of polarity which would be impractical to separate using
isocratic conditions.
Since Kc is a factor that is wholly dependent on a particular column and solvent flow rate, a quantitative measure
of the affinity of a compound for a particular set of mobile and stationary phases. The retention factor, k, can be
derived from Kc and is independent of the column size and the solvent flow rate.

kc ∗ V s
kc = (2)
VM
The retention factor is calculated by multiplying the distribution constant by the volume of stationary phase in the
column and dividing by the volume of mobile phase in the column.

C. Selectivity

The selectivity factor could be described as the separation of two species (A and B) on the column. To separate two
compounds, their respective retention factors must be different, otherwise both compounds would elute simultaneously,
the selectivity factor is the retention rate factor.

KB
α= (3)
KA
Where B is the compound that is retained more strongly by the column and A is the compound with the faster
elution time.

D. The Theoretical Plate Model of Chromatograph

The plate model supposes that the chromatographic column is contains a large number of separate layers, called
theoretical plates. Separate equilibrations of the sample between the stationary and mobile phase occur in these
”plates”. The analyte moves down the column by transfer of equilibrated mobile phase from one plate to the next.

E. The Rate Theory of Chromatography

A more realistic description of the processes at work inside a column takes account of the time taken for the solute
to equilibrate between the stationary and mobile phase (unlike the plate model, which assumes that equilibration is
infinitely fast). The resulting band shape of a chromatographic peak is therefore affected by the rate of elution. It is
also affected by the different paths available to solute molecules as they travel between particles of stationary phase.
If we consider the various mechanisms which contribute to band broadening, we arrive at the Van Deemter equation
for plate height:

HET P = A + B/u + Cu (4)

where u is the average velocity of the mobile phase. A, B, and C are factors which contribute to band broadening.

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Separation of samples by liquid chromatography in HPLC simulator software .

F. Separation Efficiency

To obtain optimal separations, sharp, symmetrical chromatographic peaks must be obtained. This means that
band broadening must be limited. It is also beneficial to measure the efficiency of the column.In order to optimize
separation efficiency, it is necessary to maximize the number of theoretical plates, which requires reducing the plate
height. The plate height is related to the flow rate of the mobile phase.

G. Resolution

Although the selectivity factor, a, describes the separation of band centres, it does not take into account peak
widths. Another measure of how well species have been separated is provided by measurement of the resolution[–
]. The resolution of an elution is a quantitative measure of how well two elution peaks can be differentiated in a
chromatographic separation. The resolution of two species, A and B, is defined as

2[(tr)B − (tr)A]
Rs = (5)
WB − WA
Where B is the species with the longer retention time, and tR and W are the retention time and elution peak width
respectively. If the resolution is greater than one, the peaks can usually be differentiated successfully.

III. PROCEDURE

FIG. 1: Procedure for working with HPLC software

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Separation of samples by liquid chromatography in HPLC simulator software .

IV. RESULTS

Conditions A B C D
Flow rate (ml/min) 2.0 5.0 2.5 2.5
Column length
100 100 200 100
(mm)
Particle size (um) 3.0 3.0 3.0 6.0

TABLE I: Simulator Conditions

A. Isocratic Eluent

1. Isocratic eluent mixture, water/acetonitrile 90-10 % v/v

FIG. 2: Chromatogram for separation of benzophenone, propiophenone, acetophenone, ketoprofen and 3-nitrophenol using
conditions ”a” from Table 1

FIG. 3: Chromatogram for separation of benzophenone, propiophenone, acetophenone, ketoprofen and 3-nitrophenol using
conditions ”b” from Table 1

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Separation of samples by liquid chromatography in HPLC simulator software .

FIG. 4: Chromatogram for separation of benzophenone, propiophenone, acetophenone, ketoprofen and 3-nitrophenol using
conditions ”c” from Table 1

FIG. 5: Chromatogram for separation of benzophenone, propiophenone, acetophenone, ketoprofen and 3-nitrophenol using
conditions ”d” from Table 1

Measurements Conditions
a b c d
HETP (cm) 6.193E-4 8.779E-4 6.519E-4 1.756E-3
Theoretical plates 16147 11391 30679 5696
Back pressure (bar) 154.43 386.07 386.07 48.26
Acetophenone 8.2205 3.2882 13.1528 6.5764
Benzophenone 76.6868 30.6747 122.6989 61.3495
tr (min) Propiophenone 20.6648 8.2659 33.0637 16.5318
Ketoprofen 37.9771 15.1908 60.7633 30.3817
3-nitrophenol 6.1597 2.4639 9.8555 4.29277

TABLE II: Simulator Conditions

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Separation of samples by liquid chromatography in HPLC simulator software .

In the first case, the mobile phase is constituted by a mixture of water/acetonitrile 90-10 % v / v using an
isocratic elution mode, which means that: the mixture of its mobile phase is consistent throughout the test
time.
In the Chromatographic Process, the solute is interacting with either the Mobile Phase or the Stationary Phase.
These interactions can be approximated as a finite and statistically significant number of regions along the
length of the column, and are called ”plates.” The greater the number of these hypothetical regions, the more
equilibrium processes can be said to have occurred, and therefore the more ”efficient” the column will be in
performing chromatography. According to what is observed in the results, comparing the theoretical plates
observed between A and B we could observe a diminution of 4756 but in the case of C, the value increases
significantly and is the same with D.
Also, it is important to analyze the height of the theoretical plate (HETP) as a way of comparing column
performance characteristics; the highest efficiency columns will have the lowest HETP. Analyzing the HEPT
results in conditions A and B it is observed that the value of HETP and the number of theoretical plates
decreases, also an increase in back-pressure is observed, concluding that the efficiency of the column decreases.
Related to the flow rate and the length of the column in I, there is a slight increase in the HETP value, an
increase in the number of theoretical plates, and an increase in back-pressure comparing conditions A and B.
This is due to the increasing the length of the column counteracted the increase in the flow rate, the efficiency
of the column was improved rather than decreased by increasing the linear velocities of the solute.
Analyzing the particle size comparing conditions A and d where the particle size increase from 3.0 to 6.0, there
was an increase in HETP values, a decrease in the number of theoretical plates, and a decrease in back-pressure.
Since the particle size increases as does the flow rate, a fast linear velocity of the solute is achieved. In this way,
the retention time of the components in the stationary phase is reduced, which leads to low column efficiency
and low chromatogram resolution.
In addition, the higher the proportion of water, the polarity of the phase increases, which makes the time
necessary for detachment to be longer. Analyzing the polarity of the compounds and knowing that the polar
compounds separate first due to their low affinity for the stationary phase, and possess lower retention time
values while apolar compounds have higher retention time values due to their affinity for the stationary phase
(hydrophobic solvent) and their peaks appear at the end of the chromatogram, we deduce that, the polarity of
the compounds decreases as follows:
3 − nitrophenol > Acetophenone > P ropiophenone > Ketoprof en > Benzophenone
2. Isocratic eluent mixture, water/acetonitrile 75-25 % v/v

FIG. 6: Chromatogram for separation of benzophenone, propiophenone, acetophenone, ketoprofen and 3-nitrophenol using
conditions ”a” from Table 1

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Separation of samples by liquid chromatography in HPLC simulator software .

FIG. 7: Chromatogram for separation of benzophenone, propiophenone, acetophenone, ketoprofen and 3-nitrophenol using
conditions ”b” from Table 1

FIG. 8: Chromatogram for separation of benzophenone, propiophenone, acetophenone, ketoprofen and 3-nitrophenol using
conditions ”C” from Table 1

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Separation of samples by liquid chromatography in HPLC simulator software .

FIG. 9: Chromatogram for separation of benzophenone, propiophenone, acetophenone, ketoprofen and 3-nitrophenol using
conditions ”d” from Table 1

Measurements Conditions
a b c d
HETP (cm) 6.141E-4 8.532E-4 6.429E-4 1.706E-3
Theorical plates 16284 11721 31109 5860
Back pressure (bar) 155.33 388.31 388.31 48.54
Acetophenone 2.6207 1.0483 4.1931 2.0966
Benzophenone 18.2880 7.3152 29.2608 14.6304
tr (min) Propiophenone 5.9044 2.3618 9.4471 4.7235
Ketoprofen 7.8053 3.1221 12.4885 6.2443
3-nitrophenol 2.1065 0.8426 3.3704 1.6852

TABLE III: Simulator Conditions

In this section, the analyze of Isocratic Elution continue, where there is in the mobile phase a mixture of water
/ acetonitrile at 75-25 % v / v.
Also, in this case, the theoretical plates show the hypothetical stage; in which two phases of a substance (liquid
phase and vapor phase) are in equilibrium, from A to B it decreases by a difference of 4563. However for
condition C increases, and for condition D decreases considerably again with a difference 25249 between C and
D. Here is necessary to emphasize that if the particles are smaller, the pressure necessary to drive the mobile
phase through the column will be greater and it will be more difficult to pack the column uniformly, then,
according to the Back-pressure, there is an increase between A and B, it remains constant in C and decreases
considerably for D.
According to the increase in the length of the column from 100 to 200, the increase in the flow rate is counteracted,
where the efficiency of the column is improved instead of decreasing due to the increase in the linear velocities
of the solute, then for A and C, there was an increase of the HETP value, the number of theoretical plates
increases, and an increase of the back pressure and in the C chromatogram, there is good efficiency but low
selectivity due to the low peaks in resolution.
According to the particle size, between A and D conditions there is an increase in particle size, from 3 to 6, and
an increase in the Flow rate of 0.5. Then, the retention time of the stationary phase decreases, as evidenced
by the retention time for the 5 compounds between conditions A and D of table I. A fast linear velocity of the
solute is then achieved, generating a slight overlap of the peaks, which leads to poor column efficiency and low
chromatogram resolution. Therefore, the size of the particle is more important than the change of the Flow

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Separation of samples by liquid chromatography in HPLC simulator software .

rate according to the chromatograms with the conditions B and D since the retention time increases for each
compound and the chromatogram of B does not have a good resolution despite the efficiency. column compared
to chromatogram D.
A liquid-liquid chromatography is considered when the separation involves a simple partition between two
immiscible liquid phases, one stationary and the other mobile, which reveals their difference according to their
degree of polarity. According to (Brondz, 2002)The more polar or less polar liquid may be immobilized. One
liquid is immobilized in the pores of solid support and acts as the stationary phase. The other liquid, saturated
with the stationary phase, is used as the mobile phase. Each phase in liquid-liquid chromatography may be
regarded as a bulk phase and the analyte is separated by partition between the two liquid . Therefore, the
stationary phase is Hydrophobic and the mobile phase is a polar liquid. To know which compounds are more
polar than others, There is a consideration that the polar compounds separate first and according to the
chromatogram they appear in the first peaks with a shorter retention time. On the other hand, non-polar
compounds have a long retention time and appear at the end of the chromatogram because they have a direct
affinity for the solvent that is hydrophobic. Then, we get that:

3 − nitrophenol > Acetophenone > P ropiophenone > Ketoprof en > Benzophenone

The chromatogram resolution varies on the equilibrium between the stationary mobile phase, which depending
of elution conditions, sample volume, resin properties. In this case, it is considered a good equilibrium between
the stationary phase and the mobile one due to the fact that between the 4 chromatograms. There is a low-
resolution change since it is possible to observe the 5 corresponding peaks of the 5 samples. These present an
evident separation between each peak, then each of the analytes elutes with good time retention and resolution
is adequate because solutes are able to interact for a longer time with the stationary phase and to be retained
in it for a longer time, thus achieving a better separation. It is important consider this aspect because there
are chromatograms that show a single peak, despite having other compounds and in these cases all the analytes
elute at the same time and there is not enough time for the occurrence of the corresponding equilibrium of each
of them between the stationary and mobile phases.
3. Isocratic eluent mixture, water/acetonitrile 60-40 % v/v

FIG. 10: Chromatogram for separation of benzophenone, propiophenone, acetophenone, ketoprofen and 3-nitrophenol using
conditions ”a” from Table 1

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Separation of samples by liquid chromatography in HPLC simulator software .

FIG. 11: Chromatogram for separation of benzophenone, propiophenone, acetophenone, ketoprofen and 3-nitrophenol using
conditions ”b” from Table 1

FIG. 12: Chromatogram for separation of benzophenone, propiophenone, acetophenone, ketoprofen and 3-nitrophenol using
conditions ”c” from Table 1

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Separation of samples by liquid chromatography in HPLC simulator software .

FIG. 13: Chromatogram for separation of benzophenone, propiophenone, acetophenone, ketoprofen and 3-nitrophenol using
conditions ”d” from Table 1

Measurements Conditions
a b c d
HETP (cm) 6.067E-4 8.118E-4 6.288E-4 1.624E-3
Theorical plates 16482 12318 31808 6159
Back pressure (bar) 147.98 369.96 369.96 46.24
Acetophenone 1.0993 0.4397 1.7589 0.8795
Benzophenone 4.6718 1.8687 7.4749 3.7375
tr (min) Propiophenone 1.9655 0.7862 3.1448 1.5724
Ketoprofen 1.9446 0.7779 3.1114 1.5557
3-nitrophenol 0.9724 0.3890 1.5558 0.7779

TABLE IV: Simulator Conditions

In this case the acetonitrile is low, according this in the chromatograms there are three peaks corresponding to 3
compounds only. In conditions A and B, the flow rate increase three times more than A. The theoretical plates
decrease with a difference of 4164 between these conditions, for condition C increases, and for condition D decreases.
Also, the value of HETP increase there is a low efficiency in the column. In the case of A with D and C when
the flow rate increase only 0.5 more than A according D with column length increase 100 more and according the
chromatogram the peaks with C the retention time is longer and peaks are narrower and have a better separation.

Finally, the best results were between cases A and C. In case A we have a shorter retention time than case C,
however, in case C, although the retention time is slightly longer, the chromatogram peaks are narrower and have a
better separation. In addition, the polarity of the compounds decreases from left to right. This refers to the fact
that the more polar compounds have a shorter retention time. Thus, they can be organized as follows:

3 − nitrophenol > Acetophenone > Ketoprof en > P ropiophenone > Benzophenone

B. Gradient Eluent

The Gradient Eluent mode is used for the remaining simulations, meaning that the following eluent mixtures
compositions changes as the measurement is done. Therefore, it directly influences the retention of the used analytes.
The simulations were done with three different gradient mixtures that were:

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Separation of samples by liquid chromatography in HPLC simulator software .

1. Gradient mixture eluent: water/acetonitrile 10 min-75% v/v.

If we compare the HETP, theoretical plates, and retention time values of all the conditions of table V, we can
see that the measurements that were done under b conditions have the lowest values, but this does not mean
that they were the optimal values for the procedure. As we know, the higher is the number of theoretical plates,
the more efficient will be the column; and the lower the HETP values means that the column will be more
efficient. But we also have to ensure that the retention times of the different compounds are different enough
to guarantee a good separation and that the chromatogram peaks are apart from each other.

Conditions
Measurements
a b c d
HETP (cm) 6.0E-4 7,39E-4 6,08E-4 1,48E-3
Theoretical plates 16666 12515 32863 6757
Back pressure (bar) - - - -
Acetophenone 4,1754 2,5085 5,1627 3,7120
Benzophenone 7,2741 5,5121 8,2396 6,8036
tr (min) Propiophenone 5,6085 3,7992 6,6247 5,1201
Ketoprofen 5,8602 4,2611 6,7461 5,4284
3-nitrophenol 3,7489 2,1433 4,7283 3,2958
TABLE V: Simulator Conditions water/acetonitrile 10 min-75% v/v

Taking into consideration just the HETP and Theoretical Plate values, we can say that c conditions provided
the best values. This means that a change in flow rate from 2.0 to 2.5 milliliters per minute and in column length
from 100 to 200 millimeters is the optimal, or at least the better, for this gradient mixture. These parameters
also provide enough difference between retention times.
Saying that c conditions were better is also supported by the chromatograms. As we can see, figure 16 has
its peaks sufficiently spaced apart and the signal values are the highest, giving a chromatogram with good
resolution.

FIG. 14: Chromatogram for separation of benzophenone, propiophenone, acetophenone, ketoprofen and 3-nitrophenol using
conditions ”a” from Table 1

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Separation of samples by liquid chromatography in HPLC simulator software .

FIG. 15: Chromatogram for separation of benzophenone, propiophenone, acetophenone, ketoprofen and 3-nitrophenol using
conditions ”b” from Table 1

FIG. 16: Chromatogram for separation of benzophenone, propiophenone, acetophenone, ketoprofen and 3-nitrophenol using
conditions ”c” from Table 1

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Separation of samples by liquid chromatography in HPLC simulator software .

FIG. 17: Chromatogram for separation of benzophenone, propiophenone, acetophenone, ketoprofen and 3-nitrophenol using
conditions ”d” from Table 1

2. Gradient mixture eluent: water/acetonitrile 10 min-95% v/v

As we change the gradient mixture, we also can see how this affects the simulation values. In the same way, as
the previous simulation, c conditions present the highest values for theoretical plates and a pretty acceptable
value for HEPT, as can be seen in table VI. On the other hand, the retention times for c conditions are not
different enough, as the values of propiophenone and ketoprofen are almost the same, and this can be seen more
properly in figure 20

Conditions
Measurements
a b c d
HETP (cm) 6,00E-4 7,39E-4 6,08E-4 1,48E-3
Theoretical plates 16666 13515 32863 6757
Back pressure (bar) - - - -
Acetophenone 6,6970 2,2623 4,5396 3,2964
Benzophenone 6,1061 4,6337 6,9201 5,7069
tr (min) Propiophenone 4,8290 3,3054 5,6865 4,4133
Ketoprofen 4,9728 3,6250 5,7255 4,6034
3-nitrophenol 3,3589 1,9611 4,1997 2,9638
TABLE VI: Simulator Conditions water/acetonitrile 10 min-95% v/v

Then, a conditions provide a better retention time relation and also present the second-highest theoretical plates
value. This results in a conditions providing the optimal values among the rest of the conditions.

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Separation of samples by liquid chromatography in HPLC simulator software .

FIG. 18: Chromatogram for separation of benzophenone, propiophenone, acetophenone, ketoprofen and 3-nitrophenol using
conditions ”a” from Table 1

FIG. 19: Chromatogram for separation of benzophenone, propiophenone, acetophenone, ketoprofen and 3-nitrophenol using
conditions ”b” from Table 1

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Separation of samples by liquid chromatography in HPLC simulator software .

FIG. 20: Chromatogram for separation of benzophenone, propiophenone, acetophenone, ketoprofen and 3-nitrophenol using
conditions ”c” from Table 1

FIG. 21: Chromatogram for separation of benzophenone, propiophenone, acetophenone, ketoprofen and 3-nitrophenol using
conditions ”d” from Table 1

3. Gradient mixture eluent: water/acetonitrile 20 min-95% v/v

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Separation of samples by liquid chromatography in HPLC simulator software .

FIG. 22: Chromatogram for separation of benzophenone, propiophenone, acetophenone, ketoprofen and 3-nitrophenol using
conditions ”a” from Table 1

FIG. 23: Chromatogram for separation of benzophenone, propiophenone, acetophenone, ketoprofen and 3-nitrophenol using
conditions ”b” from Table 1

FIG. 24: Chromatogram for separation of benzophenone, propiophenone, acetophenone, ketoprofen and 3-nitrophenol using
conditions ”c” from Table 1

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Separation of samples by liquid chromatography in HPLC simulator software .

FIG. 25: Chromatogram for separation of benzophenone, propiophenone, acetophenone, ketoprofen and 3-nitrophenol using
conditions ”d” from Table 1

Conditions
Measurements
a b c d
HETP (cm) 6,00E-4 7,39E-4 6,08E-4 1,48E-3
Theoretical plates 16666 13515 32863 6757
Back pressure (bar) - - - -
Acetophenone 5,1296 2,9557 6,4523 4,5246
Benzophenone 9,9142 7,4387 11,255 9,2673
tr (min) Propiophenone 7,2734 4,7907 8,6743 6,6107
Ketoprofen 7,8348 5,6226 9,0497 7,2499
3-nitrophenol 4,5020 2,4605 5,7940 3,9223
TABLE VII: Simulator Conditions water/acetonitrile 20 min-95% v/v

In this case, the mobile phase consists of a 20-95% v / v water / acetonitrile mixture.
When using the gradient elution mode, it is very important to note that the composition of the eluent mixture
changes during measurement, which directly influences the time and retention of analytes. That is, at the beginning
of the chromatography the solvent begins with a polar tendency; however, as time passes (10 min), the acetonitrile
concentration will increase changing the tendency of the solvent to apolar. Although increasing the flow rate will
decrease the retention time of the analyte, increasing the length of the column does not ensure a clear separation
between the peaks, a clear example of this property can be seen in figure d in which the size of the column has been
increased but the distance between the peaks is still very close.
As can be seen in table VII, the HETP values are very close to each other, the parameter b has the lowest value
with 7.39E-4, which indicates greater efficiency in the chromatography; however, the number of theoretical plates is
much smaller compared to the other parameters, which decreases the efficiency of the chromatography, since a higher
plate value is needed to better define the results.
In this case, Table VII indicates that parameter c has better retention values, this increases the distance between
peaks and allows us to better characterize the analytes. Similarly, the theoretical plates for parameter c is 32863,
which increases the efficiency of the chromatography, this is reflected in its HETP value, which is at 6.08E-4, a value
that oscillates the average of the other parameters.
Regarding the parameters to the retention values obtained, they are very similar to the parameter c, this indicates
that the chromatography has been carried out efficiently but they are not highly efficient, this could generate errors
in the characterization of analytes, increasing costs and time due to their low purity.

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Separation of samples by liquid chromatography in HPLC simulator software .

Parameters b and d can be considered of low efficiency and not suitable for the realization of the chromatography.
In the case of parameter b, the retention value obtained for the compounds Acetophenone and 3-nitrophenol are very
low. Unlike, in the parameter d the retention values are very good but the theoretical avocados are too few to achieve
an efficient chromatography.

V. CONCLUSIONS

HPLC is a rapid technique for the analysis and separation of mixtures. Technological advances related to this
technique allow a wide range of analytes to be analyzed efficiently and precisely which can be achieved by the
equipment of distinct columns with specific characteristics and design (solid, liquid, ionic exchange, size exchange,
etc). This is why various methods have been proposed to establish the best conditions, and characteristic equipment
has been developed for the separation of samples of different natures.

Through the analysis that was made in the different chromatograms it was determined that Isocratic elution
occurs when the mobile phase has a constant concentration and the increase and width of the peak is observed
according to the retention time, which means that the composition of the solvent is constant throughout the
separation procedure. Then, it was possible to establish an adequate prediction of the performance of the col-
umn under the appropriate conditions. In this case, the number of plates is only an indication of whether a
column has been well packed, the height of the HEPT plate, is the distance that the solute moves while the
chromatography is carried out, in this case from a Theoretical point of view the height of the plate can be directly
related to the experimental conditions and the operating parameters and it is considered that for an efficient
column HEPT should be a small number, and the latter can decrease through the kinetic characteristics of the
operation of the column, perhaps by decreasing the flow of the mobile phase (but not below a minimum). The
solute bands gradually widen as they advance through the chromatographic column. Therefore, the resolution of in-
dividual solutes into discrete bands occurs only if the bands widen less than the maximums of the peaks are separated.

The solvent mixture in the gradient allows the variation of the polarity in the solvent increasing the characterization
of the analytes, this benefit allows the samples to be separated more obtaining better peaks in the software. An
important factor is the amount of theoretical plates that have been developed for each factor, this increases the
effectiveness of the sample by lowering the theoretical value of the HETP plate and improving the results. Taking
into account these factors, it can be defined that in Gradient of eluent mixture: water / acetonitrile 10 min-75% v /
v the parameter c defines the chromatography, in the water / acetonitrile mixture 10 min-95% v / v conditions that
provide the optimal values, and with water / acetonitrile 20 min-95% v / v the parameter c gives the best results in
the chromatography. The use of the gradient method improves the separation of the peaks and their appreciation.
This is because, since HPLC is a polarity difference method, it is better applied.

VI. FINAL QUESTIONS

A. Discuss the fundamental differences between adsorption chromatography and partition chromatography.

Adsortion Chromatography

The separation is based on the adsorption process, which is due to polar dipole-dipole intermolecular interac-
tions or hydrogen bonds between the solute and the adsorbent, where the equilibrium at the interface intervenes and
the separation mechanism is based on the difference of adsorption of the components of the mixture on the active
surface of the stationary phase and this phase is more polar than the mobile phase. The mobile phase can be liquid
or gaseous and the stationary phase is solid, due to the effect of surface physical forces such as those of Van der
Waals that retain solutes, which is why it is used in the separation of both liquid and liquid compounds. solids and
various organic compounds. In this case; The most commonly used adsorbents corresponding to the stationary phase
are silica gel (SiO2 ) and alumina (Al2 O3 ), which have polarity as their main characteristic. Compounds that have
a high affinity for it are absorbed on the surface of the stationary phase, and at the same time retain the maximum
time in this phase, in this case, the retention times lengthen as the polarity of the analyte increases. On the contrary,
as there is no affinity between the compound with the stationary phase, there is too rapid an elusion with the mobile
phase.

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Separation of samples by liquid chromatography in HPLC simulator software .

Partition Chromatography

This type of chromatography is based on the difference in solubility that exists between two immiscible phases which
differ in the degree of polarity; where the solution to be separated passes into the mobile phase and the stationary
phase adheres to a solvent phase and it is possible to immobilize the more polar or less polar liquid.
In this case, the stationary phase is a liquid that is trapped, impregnated, or bound on an inert solid. In fact, the
principle of equilibrium is applied where the solute is distributed in two layers in equal portions. Its stationary phase
is liquid and its mobile phase can be liquid or gaseous. Finally, if there is an affinity with the mobile phase, the
maximum time in this phase elapses and there is an elusion of the substance first, as occurs when there is an affinity
with the stationary phase.

B. Discuss the fundamental differences between ion exchange chromatograph and size exclusion
chromatography.

Ion exchange chromatography

It is a purification method that separates a target molecule from other impurities based on the net surface
charge or ionic partition, based on ionic interactions between analytes that are ionic and polar. Then, the stationary
phase has ions where there is a cationic or anionic exchange where they are exchanged with the ions of the analyte,
managing to separate the compounds from the mixture that is being treated. The binding of charged compounds is
reversible and adsorbed compounds are commonly eluted with salt or a pH gradient. There is an ion exchanger with
the opposite charge to the charged compounds, where it retains and absorbs them. On the contrary, there is the
circumvention of the column and that also passes through an empty volume of those compounds that are neutral or
maintain the same charge.

Size exclusion chromatography

The analyte components elute from the chromatographic column according to the size, then this analytical
technique in which molecules of a mixture can be separated according to their size and molecular weight, where the
stationary phase provides a classification of molecules based for the most part on the geometry and molecular size.
This method is also called gel permeation chromatography, in polymer chemistry, and gel filtration, in biochemistry,
then it applied principally with large molecules (macromolecules). The stationary phase consists of resins which
contain some smaller molecules to pass through these pores, it means that the tiny molecules can pass through the
pores of the particles which conformed to the stationary phase making their travel slower and the molecules with a
bigger size cannot penetrate it, so their move faster through this phase, it is said that this kind of molecules elutes
earlier.

C. Comment the main chemical structural criteria to choose HPLC techniques vs Ion Exchange and/or Size
Exclusion Chromatographyâs to carry out given analysis

Several chromatographic mechanisms that we can use to separate the compounds in our sample have been studied
and it has been concluded that depending on the nature of our sample, we will use a specific chromatography method.

1. The HPLC method is based on the solute retention mechanism between the mobile phase and the stationary
phase where the less polar components will have affinity with the stationary phase which is also non-polar; on
the other hand; the more polar compounds will be present in the mobile phase. Thus, HPLC is focus on the
polarities of the compounds that we want to separate.
2. The ion exchange chromatography separation is carried out using an insoluble, porous material with ionic
behavior. Then, the surface of the stationary phase has a charge opposite to that of the ions present in the
sample. Also, another common strategy applied to ion detection is because of their conductivity. Therefore,
we will use the ion exchange method when the sample has ionic character structures or there is a significant
difference between the conductivity of the ions.
3. Size exclusion chromatography is based on the geometry and molecular size of the compounds to be separated,
since this method consists of a porous matrix of spherical particles (beads) that lack reactivity and adsorptive

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Separation of samples by liquid chromatography in HPLC simulator software .

properties. Once the sample has entered the column, molecules larger than the pores cannot diffuse into the
beads, so they elute first. Molecules ranging in size from very large to very small can penetrate pores to varying
degrees depending on their size. It is used to obtain information on how much a sample contains of the respective
molecular weights. Therefore, it is widely used for quality control, such as to identify differences in polymer
properties. Thus, we will use size exclusion chromatography when there is a notable difference in the size of the
compounds to be separated in the sample.

D. Draw the following structures and based in your result comment and justify the retention order
observed from least retained to the most retained

During the development of the experiment, it was observed that the longer the retention time the compound is
more apolar. This is because during HPLC the stationary phase is apolar, and the apolar compounds will have an
affinity of being in this phase, on the other hand, the polar compounds will have an affinity for the mobile phase
which is polar, supporting the formation of hydrogen bonds.
By analyzing its functional groups is possible to know how polar a compound is. Compounds that contain -OH
groups have a greater polar character followed by the ketones and finally the nitrile group. Thus, we have two
compounds that contain an -OH group ( 3-Nitrophenol and Ketoprofen). However, to determine which of both are
more polar it is necessary to take into account their structure, the bonds they have and their intermolecular forces
(the higher the intermolecular forces, the more polar it will be).

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Separation of samples by liquid chromatography in HPLC simulator software .

The 3-nitrophenol molecule has a benzene ring with two substituents (-NO and -OH) in âmetaâ position. This
creates a hydrogen bond between both substituents, increasing its intermolecular strength, and turning it into a
more polar compound than Ketoprofen. On the other hand, there are three compounds that contain a ketone group
(Benzophenone, Propiophenone and Acetophenone). In this case we will base ourselves on the length of the chain,
since the longer the chain is, its polarity tends to decrease. Acetophenone is the second polar compound, followed by
Propiophenone. Finally, between Ketoprofen and Benzophenone, Benzophenone by its symmetry is less polar than
Ketoprofen. Thus, we can order them from the most polar to the least polar as follow:

3 − nitrophenol > Acetophenone > P ropiophenone > Ketoprof en > Benzophenone

E. What do you understand for isocratic and gradient eluent solvent protocols for HPLC? Which one of
those two produce better separation and why?

Isocratic means that the mixture of the mobile phase will remain constant throughout the measurement process.
On the other hand, a gradient indicates a change of composition of the mixture throughout the measurement process,
this influences the analyte retention. The use of any of these can result in acceleration or deceleration of the process.
Now talking about which of these types of eluents will produce a better separation, the answer is gradient elution.
According to Schellinger, besides there were many reasons to avoid the use of gradient elution, it provides overall
faster analysis, narrower peaks, and very similar resolution compared with isocratic elution due to the non-polar
interactions being the main process base.

F. According to Van Deemter equation and the principles acting in chromatography, which strategies
regarding the equipment variables adjustments do you think will be the best to improve chromatogram
resolution

Ven Deemter equation is:

 
B
HET P = A + + Cµ
µ
Where:

• A= Eddy diffusion term

• B= Longitudinal or ordinary diffusion term
• C= Resistance to mass transfer
• µ= Average mobile phase velocity
The main objective is to minimize HETP. The Eddy diffusion term is used to describe how molecules move through
the column, leading to the diffusion of the mixture. This term can be reduced with the reduction of particle size, so
the molecules will move freely through the column reducing the diffusion.
It is well known that the concentration of analyte is lower near the band’s periphery than in the middle. Analyte
diffuses from the center to the periphery. This causes the band to widen. If the mobile phase velocity is high, the
analyte will spend less time on the column, reducing the effects of longitudinal diffusion.
Finally, for resistance to mass transfer we need to know that if the mobile phase has a high velocity and the analyte
has a significant affinity for the stationary phase, the analyte in the mobile phase will migrate ahead of the analyte
in the stationary phase. The analyte band will be widened. Thus, a greater velocity of the mobile phase will result
in a worse broadening.
Summarizing, we have to reduce the particle size to reduce the diffusion, and find a flow velocity that reduces the
effects of longitudinal diffusion and does not increases the resistance to mass transfer.

G. Please write down the order of retention of compounds in D according the polarity of HPLC column
used (normal and reverse)

In the reverse phase, the solvent used is hydrophobic, that is, this solvent has an affinity for apolar analytes. For
this reason, polar compounds separate first and have lower retention time values, while non-polar compounds have

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Separation of samples by liquid chromatography in HPLC simulator software .

higher retention time values due to their affinity for the stationary phase. Then the peaks that appear at the end of
the chromatography correspond to non-polar compounds. Therefore, it is possible to mention that the polarity of the
compounds decreases as follows:

3 − nitrophenol > Acetophenone > P ropiophenone > Ketoprof en > Benzophenone

However, in the normal column using a Hydrophilic solvent in the mobile phase the Benzophenone has the largest
compartment in apolar

Benzophenone > Ketoprof en > P ropiophenone > Acetophenone > 3 − nitrophenol

H. Remembering your experience in TLC practice, would you expect qualitatively good elution times for
the 5 above listed molecules if the mobile phase is changed adjusting to following isocratic solvent mixtures

100%/0% water/acetonitrile No
50%/50% water/acetonitrile Yes
0%/100% (%v/v) water/acetonitrile No

The balance between the solubility of the sample in the Mobile phase and stationary phase are necessary to obtain
high separation efficiency. If we use a mobile phase of 100% acetonitrile or 100% water, the solutes can dilute very
quickly, making it impossible to observe the separation. Unlike 50-50% mobile phase composition, better elution times
will be expected for Benzophenone, Ketoprofen, Propiophenone, Acetophenone, 3-nitrophenol. In the case of 50% /
50% water / acetonitrile, we can see that it will have a short retention time

VII. REFERENCES

Glockner, G. (1989). Chromatographic Cross-fractionation. Comprehensive Polymer Science and Supplements,

313â337. https://doi.org/10.1016/b978-0-08-096701-1.00016-1

Lemmon, J. (2001). Ion Chromatography. Encyclopedia of Materials: Science and Technology, 4280â4283.
https://doi.org/10.1016/b0-08-043152-6/00750-6

Olesik, S. V., Zewe, J. W., Newsome, T. E. (2012). Electrospun Nanofiber-Based Solid-Phase Microextraction Me-
dia. Comprehensive Sampling and Sample Preparation, 533â540. https://doi.org/10.1016/b978-0-12-381373-2.00099-
5
Pohl, C. (2005). 8 Ion chromatography. Separation Science and Technology, 219â254.
https://doi.org/10.1016/s0149-6395(05)80052-2

Schellinger, A. P., Carr, P. W. (2006). Isocratic and gradient elution chromatography: A comparison in
terms of speed, retention reproducibility and quantitation. Journal of Chromatography A, 1109(2), 253â266.
doi:10.1016/j.chroma.2006.01.047

Vedantu. (2020, March 23). Partition Chromatography. Retrieved November 29, 2021, from VEDANTU website:
https://www.vedantu.com/chemistry/partition-chromatography.

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