Separation and Identification of Organic Unknowns

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An organic crude product obtained from a ‘worked up’ reaction mixture will in almost all cases need to be purified further. The work-up, by which this procedure is usually known, simply refers to the isolation of the product from the reaction mixture, free from solvent and spent reagents, and does not imply any purification.

In order to purify an organic compound by separating the impurities, one has to rely on the desired compound having different properties to the impurities. Differences that may be taken advantage of are: differences in solubility, volatility, polarity, shape and functional groups present. For example, crystallisation relies on the differences in solubility between the desired compound and the impurities whereas distillation exploits differences in volatility. Adsorption chromatography separates and purifies compounds according to their adsorption to the chromatographic material, which to a good approximation is related to the polarity of the compounds. Major purification techniques relevant to the laboratory include extraction, crystallisation, distillation and chromatography in all their various forms.

Extraction in the chemical sense means ‘pulling out’ a compound from one phase to another, usually from a liquid or a solid to another liquid. In the organic laboratory, the most common process involves the extraction of an organic compound from one liquid phase to another. The two liquid phases are usually an aqueous solution and an organic solvent, and the technique is known as liquid-liquid extraction or more commonly, as extraction.

A simple extraction is often used in the work-up of an organic reaction mixture, but extraction can also be used to separate and purify organic compounds. Extraction is particularly useful in the separation of water-soluble products (inorganic salts) from water insoluble products (organic compounds). This is usually achieved by taking up the crude product in an organic solvent and extracting the organic solution with water – a procedure usually described as ‘washing’ the organic solution with water.

Washing a solution is the same as ‘extracting’ a solution. The difference is that the ‘washings’ remove undesired material, whereas the ‘extractions’ remove desired material.

Extraction of the acidic and basic components of a given organic mixture can then be achieved by their reaction with a dilute aqueous base or acid as appropriate. Since this relies on an acid-base chemical reaction, the technique is often called chemically active extraction. An extraction protocol for the separation of acidic (AH), basic (B:) and neutral (N) components of a mixture is shown on the following page.

Phase 1: Separation of the organic mixture

The first procedure carried out was to identify a suitable solvent in order to dissolve the crude organic product. The solvent used to dissolve the crude product was dichloromethane as it fulfilled all the main requirements for an extraction solvent: immiscibility with water, different density of water, solubility characteristics, a good stability and volatility so that it can easily be removed from the organic compound by evaporation. Ideally an extraction solvent should also be non-toxic and non-flammable, but these two criteria are less easy to meet. The volume of solvent used to dissolve the organic sample was kept to a minimum (~110ml was used for 11.08g of crude product). The resultant organic solution was then placed in a separating funnel of suitable size where it was washed with water to remove any inorganic salts present. This procedure was repeated twice to ensure all the inorganic salts were removed.

The next separation was to extract organic amines in the mixture by adding dilute hydrochloric acid to the separating funnel. Any organic amines present would now be in the aqueous top layer because they are now present as water-soluble salts. This process was repeated twice in order to make sure all the organic amines present were extracted from the mixture. The resulting aqueous solutions containing the extracted amines were combined and donated as solution (A). The organic solution/phase left was then washed twice with water to remove any traces of hydrochloric acid.

The organic solution was then extracted with dilute sodium hydroxide in order to remove any carboxylic acids. As with extracted amines, any carboxylic acids present in the solution should be present as water-soluble carboxylic acid salts in the aqueous top layer. This procedure was repeated twice in order to remove all carboxylic acids from the solution. The resulting aqueous solutions containing the carboxylic acids were combined and donated as solution (B). The organic solution/phase was then dried by putting it into a conical flask and adding sodium sulphate (anhydrous salt) until the solid became ‘powdery’. Any solvent present was then removed using a rotary evaporator. After the solvent was removed, sticky oil remained and was labelled as product (C). After a week the oil solidified into a brown solid.

Solutions (A) and (B) were then worked up in order to isolate the amines and carboxylic acids present in their respective solutions. Firstly, the amines were isolated from solution (A) by adding 50% sodium hydroxide to the hydrochloric acid solution containing organic amines as a salt. The sodium hydroxide was added drop wise with stirring until the solution was basic to pH paper (it should be noted that if the molecular weight of the amine base was unknown then it could have been calculated from acid-base titration). The amines present were no longer soluble in the solution and precipitated out as a white solid.

The solid was then filtered off and washed with water to remove any traces of sodium hydroxide. The solid was then allowed to air dry for a week and labelled as product (A).

The carboxylic acids were removed by a similar procedure except that hydrochloric acid was used instead of sodium hydroxide (again it can be noted that if the molecular weight of the carboxylic acid was unknown then it could have been calculate from the acid-base titration). The carboxylic acid also precipitated out as a white solid, which was then filtered and washed with water to remove any traces of hydrochloric acid still present. The carboxylic acid was then allowed to air dry for a week and labelled as product (B).

Phase 2: Thin layer chromatography

The crude organic sample provided at the start of the experiment had now been separated into its constituent components. However it was unclear whether these components were present as a single product or mixture requiring purification. Thin layer chromatography (TLC) provided an analytical technique for determining the purity of the constituents and also for preliminary identification purposes.

A very small quantity of each sample was ‘spotted’ onto the baseline of a TLC plate (solid adsorbent) and the solvent was allowed to evaporate rapidly through the absorbent layer by capillary action, leaving the small spot of solute behind. The three individual spots (acid, base and neutral sample) moved up with the solvent at different relative rates. These rates depend on a number of factors, including chemical natures of components of the sample being analysed, the nature of the solvent and the activity of the solvent. Ideally the spots should be less than half way up the plate after running the sample.

After testing various solvent systems, methanol-dichloromethane was found to be the most efficient system and from this it was concluded that all three individual components required purification. Solvent polarity is extremely important, with spots moving faster in more polar solvents. Thus polar solvents should be used for strongly absorbed (polar) compounds, and non-polar compounds should be used for weakly absorbed (non-polar compounds).

The chromatogram generated was then exposed to UV light to determine the distance travelled by the spots. The distance travelled by the centre of a particular spot from the starting point where it was applied, divided by the distance travelled by the solvent front upward from the point of spot application, is called the Rf value and is fairly constant for a given compound chromatographed in a given way. Thus, Rf values may be used as aids in identification; responses to visualizing techniques also assist in such identifications.

The visualized spots are almost always larger than they were before development, owing to diffusion. If the spots were too large (as observed during this analysis), the centres are difficult to locate precisely; the spots may even overlap or merge. In this case the TLC run was repeated using the same solvent system and under the same conditions but a smaller sample/spot was applied.

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