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Laboratory Manual

High Performance Liquid Chromatography

Manish Kumar

Chemical Engineering

High Performance Liquid Chromatography (HPLC)

The objective of this laboratory session is to get familiar with high performance liquid
chromatography techniques. The general purpose of chromatography is to separate components
in a mixture by moving this mixture through a support for which the different components have
different affinity and hence get separated. Ion chromatography is a mode of chromatography that
uses ion interactions as the main affinity parameter. Several kinds of chromatography techniques
can be defined, depending on the state of the support, also called stationary phase, and the mean
through which the sampled is carried, the mobile phase. In gas chromatography, the mobile
phase is a gas, whereas it is a liquid in High Performance Liquid Chromatography, column
chromatography and thin layer chromatography. Gel permeation chromatography is specific in
that it uses a gel, and not a solid nor a liquid, as stationary phase. Ion chromatography (IC) is a
specific use of HPLC principles, and can be described using the same models. The main
specificities of IC are its stationary phase and pre-detector sample processing.

On an historical point of view, liquid chromatography was first discovered in 1903 by
M.S.Tswett, who used a chalk column to separate the pigments (this is where the chromate
root comes from) of green leaves.

The principle of chromatography is the same for every mode: the sample is moved through a
static phase by the mobile phase. The different component of the mixture can swap from one
phase to another, but the rate of this migration will depend on the affinity of each component for
the stationary phase. Therefore, different affinity will make components need different time to go
through the same length of stationary phase. This time is one of the two characteristic datas
given by chromatography, and is known as retention time. The other important data, which will
be discussed later, is the peak area.
Chromatographic techniques are used to separate the components of a mixture before detection
and analysis, and can be used to determine the composition of a mixture and the quantity of
individual chemical compounds within a mixture. An example would be to determine how much
caffeine there is in a cup of coffee. High Performance Liquid Chromatography (HPLC) is an
analytical technique for the separation and determination of organic and inorganic solutes in
samples. The technique is particularly applicable to biological, pharmaceutical, food,
environmental, and industrial analyses. Chromatographic techniques provide both qualitative and
quantitative information. The quality of the analysis is determined by how well separated the
analytes are and how efficient the separation process is. In order to obtain as much analytical

information as possible from a chromatographic separation, it is important to understand the

theory and practice of operation.

Figure 1: Illustration of the basic chromatographic process.

All chromatographic separations utilize the same basic approach. The sample (a mixture of the
analytes and other compounds, including a solvent) is introduced into a flowing stream called the
mobile phase. The mobile phase carries the sample through a column that contains a second
phase, the stationary phase, which is fixed in place. The analytes partition between the mobile
and stationary phases, and those analytes with stronger attraction to the stationary phase take
longer to travel through the column. A detector that responds to the analytes is placed at the
downstream end of the column. The detector monitors the concentration of the analytes as they
elute from the column, providing a chromatogram.

The various forms of chromatography are generally classified by the nature of the mobile phase.
Gas chromatography utilizes a gas as the mobile phase, while liquid chromatography utilizes a
liquid mobile phase. Reversed-phase chromatography, which is the most popular form of HPLC
and is the mode that will be employed in this lab, utilizes a non-polar stationary phase and a
polar mobile phase. Other modes of liquid chromatography include normal phase (polar
stationary phase), ion exchange, and size exclusion chromatography.


Figure 2: Schematic of an HPLC system.

HPLC instruments consist of a pump, injector, column, detector, and data analysis and control
system (Figure 2). The pump is used to force the liquid mobile phase through the column and
past the detector. The injector is used to introduce the sample into the flowing stream. The
analytes in the sample are (hopefully) separated as they travel through the column, and are then
detected by the detector. All of the components are controlled by a computer, which also
collects, stores and analyzes the signal from the detector. In HPLC, the column is packed with
very small particles (3-10 m), and high pressures are required to pump the solvent through the
system. Thus the pump and all plumbing are designed to generate and withstand high pressure
(up to 6000 psi). The injector must also be engineered to allow the sample to be introduced into a
flowing stream at very high pressures. The detector on the system to be used in this exercise is a
UV/Vis diode array absorbance detector or RI detector.

A typical chromatogram is illustrated in Figure 3. Important data that can be obtained from the
chromatogram are the retention times (t
), peak widths (w), and peak areas. Retention times can
be used to identify compounds, as each compound will (hopefully) have a different affinity for
the stationary phase and a different retention time. Peak widths determine the quality, or
efficiency, of a separation. Narrow peaks are advantageous because this allows the separation of
compounds with similar retention times. The peak area can be used to determine the quantity or
concentration of a given analyte in the mixture. Important figures of merit for a chromatographic
separation are the retention factor (k), the selectivity (), the resolution between peaks (Rs), and
the plate number or efficiency (N):
k =

; o =
; R
) 2
; N = 16 _

= S.S4S_

1 2


Figure 3: Typical chromatogram.



(i) learn the basics of liquid chromatography and to learn how to operate the WATERS
600E HPLC.
(ii) Optimize the separation of two compounds (sugars) using isocratic elution methods.
(iii) Obtain following data from EMPOWER (HPLC software): retention time (T
), peak
widths (w), and peak areas for each peak.
(iv) (iv)Calculate following parameters on the basis of data obtained from HPLC:
Resolution between peaks (R
), and Plate number or efficiency (N) for each peak.
(v) Plot the calibration curve for mixture of both compounds and quantify the
concentration of unknown compound

1. Switch on the HPLC in following sequence: Pump unit > degasser > autosampler > RI
Detector > computer.
2. Prepare 2L of 0.005MH
as mobile phase, and samples of analytes and keep in
ultrasonic bath for degassing purpose.
3. Select a mobile phase stream out of four lets say A and put in the mobile phase.
4. Set the flow rate 4 ml/min through controlling software (EMPOWER) and pass the
mobile phase through bypass valve for 5min for removing the air bubbles from the
5. Set the flow rate according to the separation method and maintain the same flow for 20
min to condition the column.
6. Start purging mobile phase in detector for 15 min.
7. Set auto zero in detector.
8. Keep samples in the autosampler in proper sequence.
9. Develop the method in EMPOWER, which includes several parameters like flow rate,
column temperature, autosampler temperature, sample volume, run time etc.
10. Run the method
11. Take data and chromatograph as PDF file.
12. Repeat procedure for finding the retention times, plotting calibration curves, and
quantifying the concentration of unknown sample.


1. Prepare two tables - first for data obtained from the HPLC and second for calculated
parameters (see in Objectives).
2. Attach the chromatographs obtain from EMPOWER.


Results and Discussion:

1. Mention the name and concentration of unknown compound
2. Discuss the significance of resolution between peaks and efficiency of each peak.


Conclude the experiment.

Lab Report:

The lab report should contain:
Results and Discussion