Lab Report 2 HPLC

SEPARATION OF SAMPLES BY LIQUID CHROMATOGRAPHY
IN HPLC SIMULATOR SOFTWARE

Sofia Peñafiel Vicuña
Department of Chemistry, School of Chemical and Engineering Sciences, Research
University of Experimental Technology YACHAY TECH, Hacienda San José.
Urcuqui, Ecuador
INTRODUCTION
HPLC (High-Performance (former Pressure) Liquid Chromatography) is a technique

used to separate, identify, and quantify each component in a mixture. Like in other
chromatographic technique the separation is carried out on stationary phase using
mobile phase. HPLC, is the most widely used analytical separation technique. The
reasons for its success are its sensitivity, its easy adaptation to exact quantitative
determinations, its suitability to automate it, its ability to separate non-volatile or
thermolabile species, but above all its wide applicability to important substances in
industry, many fields of science and for society in general. Some examples of these
materials are amino acids, proteins, nucleic acids, hydrocarbons, carbohydrates, drugs,
terpenoids, pesticides, antibiotics, steroids, organometallic species and a variety of
inorganic substances.
Focused on stationary phase polarity, we can have two types of HPLC: the normal–
phase chromatography, which separates analytes based on their affinity for a polar
stationary surface, and the Reversed-phase high pressure liquid chromatography, which
uses a hydrophobic stationary phase (non-polar). In this practice, we will focus on RP-
HPLC technique which HPLC simulators provided in this material. Reversed-phase
chromatography employs a polar (aqueous) mobile phase. As a result, hydrophobic
molecules in the polar mobile phase tend to adsorb to the hydrophobic stationary phase,
and hydrophilic molecules in the mobile phase will pass through the column and are
eluted first. Hydrophobic molecules can be eluted from the column by decreasing the
polarity of the mobile phase using an organic (non-polar) solvent such as acetonitrile,
which reduces hydrophobic interactions.
OBJECTIVE:
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.
MATERIALS AND REAGENTS:

Computer with Java JRE and HPLC simulators. There would not be any reagents for
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this practice
PROCEDURE:
In this practice, we will focus on RP-HPLC technique which HPLC simulators provided
in this material. Using the simulator, we would vary some parameters that influence the
separation and we would record the results.
ANALYSIS AND RESULTS

Before we started using the simulator, a theoretical exercise was made to compare with
the results obtained with the program.
Theoretical Exercise
1) Draw structures of the following compounds
3-
Benzophenone Propiophenone Acetophenone Ketoprofeno
Nitrophenol
2) Based on their structures, predict qualitatively their retention order from

least to most retained
Write your prediction here:

Elution Reverse Phase- Normal phase
order HPLC. HPLC
First 3-Nitrophenol Benzophenone
Second Ketoprofeno Propiophenone
Third Acetophenone Acetophenone
Fourth Propiophenone Ketoprofeno
Fifth (last) Benzophenone 3-Nitrophenol
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:
100%/0% water/acetonitrile to 50%/50% water/acetonitrile and to 0%/100% (%v/v)

water/acetonitrile
100% water 0% acetonitrile: water, compared to acetonitrile, has a higher polarity,

therefore, the stationary phase would retain more the non-polar compounds, increasing
the retention times.
50% water 50% acetonitrile: as acetonitrile is less polar than water, it would carry up
more the non-polar solutes, decreasing the retention times.
0% water 100% acetonitrile: without water, retention times would be decreased

considerably. However, this could cause the peaks to overlap, which is not good for the
separation.
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 response.
On table 1, we can notice that for each component, retention times increase as the
concentration of acetonitrile decreases. This is consistent with the predictions made in
the theoretical exercise. We can also notice that for high concentrations of water (40%
acetonitrille) as solvent, we have superposition of the peaks for propiophenone and
ketoprofen. This is caused due to the simililarity of polarity of both compounds,
2.- Use following parameter and take note of your observations:
a) The flow rate was increased significantly (2.0 mL/min to 6.0 ml/min)
Compound Rt (min)
2.0 mL/min 6.0 ml/min
Acetophenone 2,3759 0,4504
Benzophenone 15,7106 2,9038
Propiophenone 2,4405 0,9725
Ketoprofen 3,1306 1,2457
3 nitrophenol 0,8564 0,3518
Here, we can see that, changing the flow rate from 2.0 to 6.0 mL/min, we obtained
similar results (same number of peaks) on the separation of the components, but in a
shorter time for 6mL/min and higher retention time.
b) The diameter of the packing particles was increased (3.0 µm to 6.0 µm)
Retention time/ min

Compound
3.0 µm 6.0 µm
We can see that the retention times decreased significantly after the particle size was
increased, causing the peaks to be more close together, which can cause problems in the
separations.
c) The length of the column is doubled (100 mm to 200 mm)
Retention time
Compound
100mm 200mm
2.- Keep changing in a logical way the gradient to separate until at least those two
peak are separate with 2.1 min. save at least 3 chromatograms with your results
when the third is with the biggest separation in the maximum of 8 min.
Acetonitrile
Figure 1: Acetonitrile: 75% Figure 2: Acetonitrile: 50%
Figure 3: Acetonitrile: 25%
Retention time
Compound 75% Acetonitrile 50% Acetonitrile 25% Acetonitrile
Acetophenone 3,56 4,46 6,25
Benzophenone 5,94 8,04 17,31
Propiophenone 4,66 6,07 9,24
Ketoprofen 4,87 6,52 12,31
3 nitrophenol 3,36 4,17 6,2
Peaks were greatly separated with lower concentration of acetonitrile.

a Do the same using methanol instead of acetonitrile.
Methanol
Figure 13: Methanol: 25% Figure 14: Methanol: 50%
Compound Retention time
25% Methanol 50% Methanol
Better separation of the peaks with lower concentration of methanol.

b Now double the column size to readapt gradient, what are your conclusions.
Using 50% of methanol and 50ºC we obtained:
100mm
Figure 15: HPLC with a 100mm column
200mm
Figure 15: HPLC with a 100mm column
c Change particle size in column properties on one hand from 3 µM to 10 µM and

then to 0.5 µM. What is your observation?
The data obtained are shown in the next table. To compare the effects of particle size
variation, we maintain the temperature at 26ºC, the flow rate at 6 mL/min and we use
three size values.
Size of Particle 0,5μm 3μm 10μm
Number of Plates N 373815 5538 569
Height of Plate H 2,675E-4 cm 1,806E-4 cm 1,758E-3 cm
The number of plates N and the height of the plate H are a measure of the efficiency of
the separation. Efficiency is greater for big N and lower H. Therefore, as it is shown in
the table, the efficiency increased as the size of the particle decreased.
CONCLUSIONS:
Reversed Phase High Pressure Liquid Chromatography is a sustainable method of
separation for organic compounds, as the ones used in this practice. However, in order
to obtain better results, it is important to take into account the different factors that can
increase or decrease the separation efficiency. Some of the factors that influence the
results of the HPLC separation are, the type of eluent, flow rate, particle size, column
size, etc. A HPLC simulator can be used in order to evaluate the effect of these factor in
the separation and, therefore, choose the best conditions to perform a high efficient
separation.
The results of this practice showed better results for a mixture of water 65% -
acetonitrile 35%, and water 75% - methanol 25%, which had better retention time
intervals, this is, not too short because the separation is very poor; and not too large, that
makes the separation process inefficient. Efficiency also improved with lower particle
size, larger column size and a suitable flow rate.
REFERENCES
Chemistry Department Laboratory guide of Inorganic Chemistry (II) (2017). Escuela de
Ciencias Químicas e Ingeniería.
Skoog, D., Holler, F., & Crouch, S. Principles of instrumental analysis (6th ed., pp.761-
855). Santa Fe: Cengage Learning.
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SEPARATION OF SAMPLES BY LIQUID CHROMATOGRAPHY

IN HPLC SIMULATOR SOFTWARE

HPLC (High-Performance (former Pressure) Liquid Chromatography) is a technique

MATERIALS AND REAGENTS:

ANALYSIS AND RESULTS

2) Based on their structures, predict qualitatively their retention order from

Write your prediction here:

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

100%/0% water/acetonitrile to 50%/50% water/acetonitrile and to 0%/100% (%v/v)

100% water 0% acetonitrile: water, compared to acetonitrile, has a higher polarity,

0% water 100% acetonitrile: without water, retention times would be decreased

Practice part with HPLC simulator

2.- Use following parameter and take note of your observations:

Acetophenone 2,3759 0,4504

Benzophenone 15,7106 2,9038

Propiophenone 2,4405 0,9725

Ketoprofen 3,1306 1,2457

3 nitrophenol 0,8564 0,3518

Retention time/ min

Acetophenone 2,3759 3,67

Benzophenone 15,7106 5,44

Propiophenone 5,2306 4,52

Ketoprofen 6,7213 4,57

3 nitrophenol 1,8243 3,52

Acetophenone 3,67 3,67

Benzophenone 5,44 4,79

Propiophenone 4,52 3,85

Ketoprofen 4,57 3,97

3 nitrophenol 3,52 2,87

Figure 1: Acetonitrile: 75% Figure 2: Acetonitrile: 50%

Compound 75% Acetonitrile 50% Acetonitrile 25% Acetonitrile

Acetophenone 3,56 4,46 6,25

Benzophenone 5,94 8,04 17,31

Propiophenone 4,66 6,07 9,24

Ketoprofen 4,87 6,52 12,31

3 nitrophenol 3,36 4,17 6,2

Peaks were greatly separated with lower concentration of acetonitrile.

Figure 13: Methanol: 25% Figure 14: Methanol: 50%

Compound Retention time

Acetophenone 4,0418 1,0794

Benzophenone 36,1556 4,2591

Propiophenone 9,0584 1,7976

Ketoprofen 16,0889 2,2880

3 nitrophenol 2,5728 0,9911

Better separation of the peaks with lower concentration of methanol.

Using 50% of methanol and 50ºC we obtained:

Figure 15: HPLC with a 100mm column

c Change particle size in column properties on one hand from 3 µM to 10 µM and

Size of Particle 0,5μm 3μm 10μm

Number of Plates N 373815 5538 569

Height of Plate H 2,675E-4 cm 1,806E-4 cm 1,758E-3 cm

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