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Consider this: You want to determine the hormone-like effect of xenobiotic compounds found in blood serum. 2,3,7,8-TCDD (”Dioxin”). Cl. O. Cl. Cl. O. Cl. Problem 1: Your serum contains a mixture of both hormones and xenobiotic compounds. Xenobiotic substance.

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Presentation Transcript
slide1

Consider this:You want to determine the hormone-like effect of xenobiotic compounds found in blood serum

2,3,7,8-TCDD (”Dioxin”)

Cl

O

Cl

Cl

O

Cl

Problem 1:Your serum contains a mixture of both hormones and xenobiotic compounds

Xenobiotic substance

Estradiol (Female sex hormone)

Naturally occuring hormone

Xenobiotic compound:A substance foreign to a living system.

The job and the problem

You want to do this by using a test (an assay) that responds to hormones and hormone-like substances (the CALUX assay)

Problem 2:The test gives a false positive if hormones are present.

Environmental Studies - Spring 2005 HPLC Lab (Environ.- & Occu. Med.)

slide2

Sample preparation strategy

Two steps are necessary:

Removal of interfering substances from serum sample

Removal of hormones from the clean serum sample

Environmental Studies - Spring 2005 HPLC Lab (Environ.- & Occu. Med.)

slide3

Serum

Wash

Elute

Water

Methanol

Serum

Waste

Sample

Waste

TCDD

Estradiol

Removal of interfering substances from serum sample

Solid phase extraction (SPE)

This is called sample preparation

To remove water, salts and proteins from serum

What is left after this step is a sample of hormones and ”other things”

Environmental Studies - Spring 2005 HPLC Lab (Environ.- & Occu. Med.)

slide4

Imagine a tube filled with tiny particles like grains of sand – completely packed

Imagine that we have a continous flow of liquid through the tube

Sample with a mixture of TCDD and Estradiol

Now we put a mixture of TCDD and Estradiol on top of the tube

Flow

The particles in the tube interacts with the mixture

Sample

Removal of hormones from the clean serum sample

High pressure liquid chromatography (HPLC)

Because of the flow the mixture moves down the tube

Interaction is not equally strong for all components

Environmental Studies - Spring 2005 HPLC Lab (Environ.- & Occu. Med.)

slide5

Estradiol

TCDD

Sample with a mixture of TCDD and Estradiol

Flow

Removal of hormones from the clean serum sample

High pressure liquid chromatography (HPLC)

Imagine a tube filled with tiny particles like grains of sand – completely packed

Imagine that we have a continous flow of liquid through the tube

Now we put a mixture of TCDD and Estradiol on top of the tube

Because of the flow the mixture moves down the tube

The particles in the tube interacts with the mixture

Interaction is not equally strong for all components

Some components moves slower

Separation complete

Environmental Studies - Spring 2005 HPLC Lab (Environ.- & Occu. Med.)

slide6

Thank you

Environmental Studies - Spring 2005 HPLC Lab (Environ.- & Occu. Med.)

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