Aqueous Workup

Using a separatory funnel (“sep funnel”)

Once a reaction is complete, it is usual to add something to "end" the reaction. This might be something to physically "quench" the reaction (e.g. by providing a proton source to the reaction) or it might simply dilute the reaction. This quench or dilution often forms the first stage of a workup, prior to an aqueous extraction taking place.

Photo of a round-bottomed flask clamped to the frame of a fume-hood, using a boss and clamp, with colourless liquid in the flask. A gloved hand is pouring colourless liquid from a measuring cylinder into the flask.

To carry out the work-up, a separating funnel will be used. First, an appropriately sized separating funnel should be selected. This should have a capacity of at least double the volume of the solvent that you are extracting from. Additionally, it is important to ensure that the lid fits onto the separating funnel securely. The funnel will be turned upside down and shaken as part of the work up process and therefore you do not want the lid to fall off.

Photo of the top part of a separating funnel, showing a Teflon lid securely sat in the funnel.

The separating funnel needs to be supported whilst in use. This can be either by placing it in a metal ring stand or clamping carefully on its ground glass joint.

Photo of a ring-stand, attached to the frame at the back of the fume hood with a boss. A separating funnel is sat in the ring stand, with a Teflon lid securely in the top of the separating funnel.
Photo of the top part of a separating funnel, clamped at the ground glass joint, to the frame in a fume hood by a boss and clamp. A Teflon stopper is securely sat in the top of the separating funnel.

Once the funnel has been secured, the tap should be closed, and a conical flask placed underneath the tap to catch any drips, in case the tap is not secure.

A photo of a close-up of the bottom part of the separating funnel, showing that the tap is closed by being in a horizontal position. A clean conical flask is placed underneath the spout of the separating funnel.
A photo showing the full set-up of a separating funnel, with the separating funnel sat in a ring-stand, that is clamped to the frame in a fume hood with a boss and clamp. The tap of the separating funnel is closed and a conical flask is underneath the spout.

The reaction mixture and extraction solvent can be transferred into the separating funnel using a glass funnel. The reaction flask can then be rinsed out with a little of the extraction solvent and this added into the separating funnel. The glass funnel can then be removed. 

A photo of a ring-stand, attached to the frame of a fume hood with a boss and clamp, with a separating funnel sat in it. A glass funnel is in the top of the separating funnel, and a gloved hand is pouring liquid out of a round-bottom flask and into the separating funnel using the glass funnel.

If left to settle, two layers should form in the flask. The separating funnel should never be more than two-thirds full as there needs to be room to mix the layers and room for solvent vapour. If the volume of mixture is too large, it should either be done in batches or a larger separating funnel used.

A photo of a close up of a separating funnel with liquid in, where there are two layers of liquid, the top layer is clear blue, and the bottom layer is clear red. The tap on the separating funnel is closed.
A photo of a close up of a separating funnel with liquid in, where there are two layers of colourless liquid. The tap on the separating funnel is closed.

Each photo from this point will have 2 photos, one of the workup carried out using dyes for visualisation, with the blue dye showing the organic layer and the red dye showing the aqueous layer, and one of the workup done as it would be typically in a lab, with colourless solvents.

To ensure an efficient extraction, the two layers need to be thoroughly mixed. However, mixing the layers can cause the formation of pressure in the funnel, so care needs to be taken. The lid should be placed securely on the funnel, and then it should be removed from it support. The funnel should be held in both hands, with one hand around the top of the funnel, holding onto the lid and the other around the base by the tap, and it can then be inverted.

A photo of a pair of gloved hands holding a separating funnel, with one hand holding the lid securely on, and the other holding the funnel near the tap. There is blue and red liquid in the separating funnel.
A photo of a pair of gloved hands holding a separating funnel, with one hand holding the lid securely on, and the other holding the funnel near the tap. There is colourless liquid in the separating funnel.

The funnel should be vented, by holding the funnel inverted, pointing the spout towards the back of the fume hood, and opening the tap to release the pressure. If there is a pressure build-up in the flask, a small ‘hiss’ may be heard when the tap is opened. Once the tap has been closed, the funnel can be gently shaken for a few seconds, and then the funnel should be vented again. The shaking and venting should continue until no ‘hiss’ can be heard when the flask is vented. Once the last vent has occurred, the separating funnel should be returned to its support. 

The lid of the separating funnel should be removed, and layers should be allowed to settle. A clear line should be seen between the two layers.

A clean, labelled conical flask should be placed under the funnel, and it should be ensured that the stopper has been removed from the top. The tap should be opened carefully, and the interface should be watched as the first layer is allowed to drain slowly into the conical flask.

The tap should be closed when the interface between the layers passes through.

The top layer can then be drained out of the funnel into another clean, labelled conical flask. Alternatively, the top layer can be poured out of the neck of the funnel, into the conical flask, to avoid any contamination by residual material in the tap and funnel.

If another extraction or wash is required, the appropriate layer can be returned to the separating funnel using a glass funnel, and the new extraction or wash solvent can be added and the separation process repeated. 


If the layer containing the product is the less dense layer, and therefore remains in the separating funnel after the bottom layer has been drained out, there is no need to put the top layer into a conical flask, instead the next extraction or wash solvent can be added straight into the separating funnel.


Once the final separation of the work-up has been completed, the correct layer can be taken to the next step, in the case of an organic layer, this is usually drying over magnesium sulfate to remove any water.