Lab 4 HPLC

High Performance Liquid Chromatography
(HPLC)
LABORATORY MANUAL
LABORATORY 4 : IDENTIFICATION OF GLYCEROL USING HIGH
PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
Objectives:
i. To indentify the grycerol production from transesterification process.

ii. To quantify the glycerol production from transesterification process by comparing to
standard.
iii. To compare the amount of glycerol theoretically and experimentally.
Introduction:
Glycerol is a colorless, odorless liquid with a sweet taste. It is viscous at room temperature
and non-toxic in low concentrations. Glycerol can be obtained from transesterification
process. Transesterification is a process of transforming triglycerides in vegetable oils into a
mixture of fatty acid esters and glycerol by the reaction with alcohol in the presence of base
catalyst. In this reaction, glycerol is the by product. However, glycerol is used in a number of
industrial applications, in the pharmaceutical industry, in cosmetics and personal care
products, in the production of resins, detergents, plastics and tobacco and as a humectant in
food.
This is a chemical reaction in which 1 mole of triglycerides (vegetable oil or animal fats)
reacts with 3 moles of alcohol to produce 3 moles of alkyl esters (biodiesel) and 1 mole of
glycerol. This is a reversible, equilibrium reaction and requires excess alcohol to push it
toward the products side.
Figure 1: Transesterification reaction
In order to separate mixture components, HPLC takes advantages of partitioning between a

mobile and stationary phase under a uniform pressure that is typically between 500 to 5000
psi. High pressure is required to obtain a reasonable flow rate through the column. The
process begins when a small amount of liquid sample is injected into the column that has a
stream of liquid flowing through (which is known as the mobile phase). In partition
chromatography, the column is packed with particles that are coated with the stationary
phase. The polarity of the component and the type of HPLC being performed determines
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which phase the component is more attracted to. If the component is more attracted to the
mobile phase, it will flow out of the column and have a shorter retention time. If the
component is more attracted to the stationary
phase, the component will be retained and will, therefore, have a longer retention time.
Selecting the mobile phase (or solvent) is one of the most important steps when performing
HPLC and is selected based on polarity. Solvent polarity relates to the ability of the
components to partition into that phase.
The apparatus consists of a container of the mobile phase, a pump capable of pressures up to
4000 psi or greater, a valve for injecting the sample (usually 10 to 500 μL volumes), the
column (sometimes thermostatted), a detector, electronics associated with the detector, and a
recorder. A schematic of the HPLC instrument can be seen in Figure 2. This instrument in
this lab used a C column.
Figure 2: Schematic Diagram of a High-Performance Liquid Chromatograph. (1) Solvent

reservoirs, (2) Solvent degasser, (3) Gradient valve, (4) Mixing vessel for delivery of the
mobile phase, (5) High-pressure pump, (6) Switching valve in "inject position", (6')
Switching valve in "load position", (7) Sample injection loop, (8) Pre-column or guard
column, (9) Analytical column, (10) Detector (i.e. IR, UV), (11) Data acquisition, (12) Waste
or fraction collector.
UV-visible absorbance is the most commonly used mode of detection. Such detectors enable
the component (or effluent) from the column to flow through an 8 to 10 μL
spectrophotometric cell for detection of compounds at a particular wavelength (often in the
ultraviolet, < 400nm, where many organic molecules absorb). Electrochemical and
fluorescence detectors often are used to achieve lower detection limits. The other commonly
used detector is based on a measurement of the differential refractive index.
Materials
1000ppm glycerol standard, glycerol sample collected from Experiment Lab 3, acetonitrile
(ACN), purified water.
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Apparatus
25 ml volumetric flask, autosample vial, HPLC with UV detector.
Procedure:
(A) Standard and sample preparation

1. To prepare the diluent, 78 % acetonitrile is mixed with 22 % of methanol.
2. From 1000 ppm glycerol standard, prepare a blank (diluent) and 5 calibration
standard solutions at concentration of 50, 100, 150, 200 and 250 ppm using the
diluent.
3. From Experiment 3, collect the glycerol by product. You may rinse the product with
water to ensure the full extraction of glycerol from FAME product.
(B) Glycerol analysis using HPLC
1 Switch on the HPLC instruments and PC.

2 Open the HPLC online software.
3 Set up the mobile phase of ACN:H2O (78:22)
4 Set the method as follow:
Column: Inersile amino (250 mm x 4.6 mm)
Flowrate: 1.0 ml/min
Column temperature: 30 °C
Auto sampler temperature: 5 °C
Injection volume: 100 µL
UV Detector wavelength: 191 nm
Evaporation temperature: 40 °C
Nebulization temperature: 70 °C
N2 gas flow rate: 0.9 L/min
Run time: 10 min
5 Set the sequence starting with blank, 5 standards from low to high concentration sequence
and followed by sample.
6 Run the analysis.
7 Plot a calibration curve of peak area against concentration of standard. Determine the
concentration of glycerol sample. Report your results.
6.0 DISCUSSION:
1. Identify which peak is glycerol. Lable in your chromatogram.

2. Based on your result, what is the retention time of glycerol?
3. Using the calibration curve, determine the concentration of glycerol in g/L.
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4. Describe whether you used peak height, peak area, or both to estimate the concentration.
Discuss the advantages to the method that you choose.
5. What is the range of standard concentration has you prepare? Give reason why do you
choose the standard concentration range?
6. Discuss the errors potentially made during this experiment.
7. Discuss how retention times depends on ACN and the pH of the mobile phase. What
factors contribute to the choice of mobile phase composition and pH in the present
analysis.
8. If you have already performed Experiment 3, compare the percentage of glycerol obtained
in Experiment 3 based on volume of product, theoretical result and the result obtained
from HPLC analysis.
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High Performance Liquid Chromatography

i. To indentify the grycerol production from transesterification process.

Figure 1: Transesterification reaction

In order to separate mixture components, HPLC takes advantages of partitioning between a

Figure 2: Schematic Diagram of a High-Performance Liquid Chromatograph. (1) Solvent

(A) Standard and sample preparation

(B) Glycerol analysis using HPLC

1 Switch on the HPLC instruments and PC.

1. Identify which peak is glycerol. Lable in your chromatogram.

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