Hplc Case

1. Synopsis

The objective of the experiment is to familiarise with the use of a high performance liquid chromatography in forensic drug analysis, perform suitable sample preparation techniques, identify different kinds of steroid hormones present in a sample and determine the concentration of hydrocortisones, testosterone and progesterone in a sample using HPLC.

This is done by dissolving 0.002g of unknown cream sample with 10 ml of methanol and filters cream into a 2ml sample vial. Inject the 3 steroid hormone standards and the cream into HPLC to record retention time for each sample and also identify the steroid hormone present in the cream by comparing the retention time with the standard.

Then prepare 100, 200, 300, 400 mL of the hormone present in the cream, top up with methanol, mix and filter the sample into the sample vial.

Then inject into the HPLC for calibration graph.

The unknown cream sample contains hydrocortisone.

2. Introduction

2.1 High performance liquid chromatography (HPLC)

It is a form of liquid chromatography to separate compounds that are dissolved in solution and provides both qualitative and quantitative information. It detects the different types of compound in a mixture by their elution time under a given set of conditions and the area of each signal is proportional to the amount of the corresponding substance.

HPLC yields excellent separation in a short time, because in the column, the packing material has smaller particle size which gives a much greater surface area for interactions between the stationary phase and the molecules flowing past it. This allows a much better separation of the components of the mixture.

Since the packing material has very small particle holes, a high pressure is essential for forcing the mobile phase through the column,

As a result, HPLC is sometimes referred to as high pressure liquid chromatography.

HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column and a detector.

HPLC Instruments

2.2 Hydrocortisone

A steroid hormone, C21H30O5, produced by the adrenal cortex, that regulates carbohydrate metabolism and maintains blood pressure. It is used as a drug to fight allergies and inflammation.

Hydrocortisone

2.3 Testosterone

Testosterone is a steroid hormone, C19H28O2 from the androgen group, and is found in mammals, reptiles, birds, and other vertebrates. In mammals, testosterone is primarily secreted in the testes of males and the ovaries of females, although small amounts are also secreted by the adrenal glands.

Testosterone

2.4 Progesterone

Progesterone also known as pregn-4-ene-3,20-dione is a C-21 steroid hormone involved in the female menstrual cycle, pregnancy and embryogenesis of humans and other species. Progesterone belongs to a class of hormones called progestogens, and is the major naturally occurring human progestogen.

Progesterone

3. Theory

3.1 HPLC

Compounds are separated by injecting a plug of the sample mixture onto the column. The different components in the mixture pass through the column at different rates due to differences in their partitioning behaviour between the mobile liquid phase and the stationary phase.