Experiment 6

High Pressure Liquid Chromatography Analysis

of Chloroquine malaria treatment tablet

1- Define the separations science.

- A collection of techniques for separating complex mixtures of analytes.

2- Do all separations processes are analytical techniques?

- Most separations are not an analytical technique in their own right, until combined with an analytical
detector (often a type of spectrometer).

3- What are the analytical branches that discussed in this class?

- Chromatography and electrophoresis.

4- Write down the different types of separation with an example for each one.
- Complete separation;
(a + b + c + d + ……) (a) + (b) + ( c ) + (d) +……
- Partial separation
( a + b + c + d + ……) (a) + ( b + c + d+ …..)
- Enrichment
( a + b + c + d + ……) ( a + b ) + ( b + a) + ……..

5- Explain the 100-years history of separation.

- Russian chemist and botanist Michael Tswett coined the term “chromatography”.
- Chromatography was the first major “separation science”.
- Tswett worked on the separation of plant pigments, published the first paper about it in 1903, and
tested>100 stationary phases.
- Separated chlorophyll pigments by their color using CaCO3 (chalk), a polar “stationary phase”, and
petroleum ethers/ethanol/CS2.

6- The figure below shows the original Tswett’s adsorption chromatography apparatus.

7- Explain how Tswet treated the plant pigments? Give an example.

- Tswet washed a plant pigments down through a glass tube filled with calcium carbonate (limestone )
using petroleum ether.
Example:
- Approximately analogue plant pigment chromatography:
- The sample is dandelion leaf extract (in acetone).
- Elution is from left to right, on paper.
8- What are the Basic Types of Separations?
- Liquid Column Chromatography
- Liquid-Liquid (partition) chromatography (LLC)
- Stationary and mobile phases (immiscible)
- Liquid-Solid (adsorption) chromatography (LSC)
- Ion exchange chromatography (IEC)
- Exclusion chromatography (EC)
- Gas-Liquid chromatography (GLC)
- Gas-Solid chromatography (GSC)

9- Write down the processes a sample goes through in the Liquid Column Chromatography.

Pumps, Mixers
Column Detector (s) Detector
and Injectors

10- Name the indicated parts in the Liquid Column Chromatography instrument.

- The figure above shows The Agilent 1100, a typical modern LC system.

11- How are the Packed LC columns usually made?

- They are usually made of stainless steel and carefully filled with material, are the heart of the LC
experiment.

- The stationary phase fills the column. Its properties are critical to the separation.

12- How we can characterize the chromatographic retention? What is the retention time t ?
R
- A way to characterize chromatographic retention is to measure the time between injection and the
maximum of the detector response for the analyte.
 - This parameter, which is usually called the retention time tR, is inversely proportional to the eluent flow
rate.

The time required for an unretained compound to be eluted, or the time required for one column volume (VM)
of mobile phase to pass through the column.

13- Define the retention volume V

R.
- The product of the retention time and the eluent flow rate (F) is called the retention volume VR and
represents the volume of the eluent passed through the column while eluting a particular analyte
VR = tRF

Chromatograms and Electropherograms

A chromatogram or electropherogram shows detector response to analyte presence/concentration

14- Define the Dead time (volume).

- It is the “mobile phase holdup time”, or the time it takes for an unretained analyte to reach the detector.

15- Why interested in Chloroquine and primaquine in this experiment?

- Chloroquine and primaquine are the first-line treatment recommended by World Health Organization for
malaria caused by Plasmodium vivax.
- Since the problem of counterfeit or substandard anti-malarias is well established all over the world, the
development of rapid and reliable methods for quality control analysis of these drugs is essential.
 16- What are the components used in this experiment?
- Eluent (mobile phase): acetonitrile–0.1% trimethylamine (pH=3.0) phosphoric acid used to fix the pH
(20:80, v/v) as a mobile phase.
Detector: UV at 260 nm.
The injection volume was set between 10 µL
Chloroquine Tablet
200 mg concentration of chloroquine
Preparation of chloroquine standard calibration curve in 1:1 (V/V) methanol: 0.1 % of TEA.
1- The working standard solution (c = 20.16 mg/25 mL) 1:1 (V/V) methanol: 0.1 % of TEA .
2- 6 calibration standards were prepared for the calibration curve.
3-Appropriate volumes of the working standard solution were pipetted into separate vials, diluted to a volume
of 1,000 µL with methanol and mixed, resulting in the following concentrations: 0.5, 1, 2, 4, 8, 10 mL of the
stock were completed to 10 mL.

17- Why we used the phosphoric acid?

- Used to fix the pH.