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1Pipet 250 μL of urine into an empty 1.5 mL tube.
2Add 500 μL of ethyl acetate.
3Vortex the sample for 15 seconds.
4Centrifuge for 1 minutes at 3000 rpm.
5Collect 450 µl of the upper layer and transfer this into an empty 1.5 mL Eppendorf tube.
6Collect your sample into an eppendorf tube, and dry it in a speed vacuum (Eppendorf concentrator 5301). Please check with a lab assistant before turning on the speed vac.

What should the properties of the solvent be?

  1. Lower boiling point than water

Correct = no
If the solvent you use is miscible with water, you will end up with a mixture of urine, parabens and solvent. Whereas the goal is to extract the parabens from the mixture. Hence a solvent that is miscible with water is not suitable.

  1. Higher boiling point than water

Correct = yes

  1. Lower density than water

Correct = no
Why does the boiling point matter? Remember what the goal is: to extract the analytes from the urine into a organic solvent. So as long as the solvent is liquid at room temperature, you’re allright.

  1. Higher density than water

Correct = no
Why does the boiling point matter? Remember what the goal is: to extract the analytes from the urine into a organic solvent. So as long as the solvent is liquid at room temperature, you’re allright.

  1. Miscible with water

Correct = no
Well yes it is important to realize what the density of the solvent is, as this determines if it will form the upper or the bottom layer in a two phase system (two layers of liquid). However, it does not determine whether it is suitable.

  1. Immiscible with water

Correct = no
Well yes it is important to realize what the density of the solvent is, as this determines if it will form the upper or the bottom layer in a two phase system (two layers of liquid). However, it does not determine whether it is suitable.

  1. Analytes should dissolve in the solvent rather than in water

Correct = yes

  1. Analytes should dissolve in water rather than in the solvent

Correct = no
The goal is to extract the parabens from the urine, as urine is an aqueous solution, a solvent in which they rather dissolve than water is suitable.

Feedback if correct:
As urine is an aqueous solution, you need a solvent that does not mix with water and in which the analytes (the molecules of interest, thus the parabens) rather dissolve than in water, in order to extract them.


What happens during this vortexing step?

  1. 100% of the parabens dissolve in the organic solvent

Correct = no
You have chosen a wrong option. You have looked up the partition coefficients of the chemicals of interest, what does this mean again?

LogP is defined as the log( [solute]octanol / [solute]water )

So if LogP of a paraben = 3, this means that the concentration of this paraben in octanol will be 103 that of its concentration in water.

  1. Parabens partion themselves over the organic solvent and the water

Correct = yes

  1. A solution is obtained

Correct = no
You have chosen a wrong option. The principle on which liquid liquid extraction relies is that the solvents do not mix. Therefore you will not obtain one solution.

  1. An emulsion is obtained

Correct = yes

Feedback if correct

During this step, the urine sample and the organic solvent are vigorously mixed to ensure optimal partitioning of the parabens into the organic solvent.

You can predict what fraction is actually partitioned in the organic solvent using the LogP values you have looked up earlier.

LogP is defined as the log( [solute]octanol / [solute]water )

So if LogP of a paraben = 3, this means that the concentration of this paraben in octanol will be 103 that of its concentration in water.


  • A proper solvent for this extraction is ethyl acetate. The urine and ethyl acetate will not mix because of a difference in [pH / polarity / density / boiling point / viscosity].
    Feedback if incorrect:
    This is not the reason that these liquids do not mix. Take ethanol and water for example. These substances differ in density however they do mix!
  • When these two liquids are mixed and centrifuged (or left to stand), they will separate.
    The density of ethyl acetate is: ___ g/cm³ (0.902)
    Feedback if incorrect:
    This is not the density of ethyl acetate. Look it up on the internet. Also pay attention to the units! Density is often expressed in kg/m3, but here we ask for g/cm3.
  • The density of the aqueous urine is: ___ g/cm³ (~ 1)
    Feedback if incorrect:
    This is not the density of urine. As it is aqueous, its density will be close to that of water (however all solutes do affect its density).
  • Hence the layer of interest is the [top / bottom] layer.
    Feedback if incorrect:
    Compare the densities. They are expressed per 1 cm3, which makes it easy to compare. Which substance is lighter? Which substance is heavier? The heaviest substance will sink to the bottom.

Feedback if all Correct
Ethyl acetate and water will not mix because of their difference in polarity. Water is polar, ethyl acetate is non-polar.
ρethyl acetate = 0.902 g/cm³
ρurine ~ 1 g/cm³
Hence the bottom layer will be urine, the top layer the ethyl acetate in which the analytes are extracted.

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  • Skill levelIntroduction video
  • CategoryAnalytical chemistry

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This video is created by Leiden Academic Centre for Drug Research (LACDR), Faculty of Science at Leiden University under a open Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. When using this video in its original version please refer to www.labprep.video. When adapting the video, mention the source ‘adapted based on the original version that is created by the labprep.video team’. It is not allowed to use the video for commercial purposes without consultation with the creators. You can contact us via info@labprep.video.

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