Chemical Wonders: Experimental Techniques (Part 1) - Separation and Purification: Chromatography and Recrystallization

- Reposting from my blog on minds.com - 

Identifying and analysing compounds is an important part of chemistry, the modern laboratory provides various analytical techniques. These methods and techniques can be roughly grouped depending on their usage:

• in determining molecular structure;
• in investigating the bonds of the analyte, how and which atoms are connected in the molecule; and what the oxidation states of each element in the analyte;
• in determining molecular formulae and analysing the composition of the analyte.

Separating and purifying compounds

Once you have made your compound and before you begin to analyse the structure and molecular formula, you must first separate your product and purify it. It is essential to separate and purify your analyte from other products in your reaction mixture because you would get an inaccurate reading.

Gas Chromatography

Gas chromatography (GC) involves very small amounts of gas, and is typically used to separate particularly volatile components from a reaction mixture. GC depends entirely on the various interactions between the components in the mobile phase (a carrier gas such as Helium, He and the sample gas) and the stationary phase containers (silica,SiO₂; or alumina, Al₂O₃). Typically, GC is combined with mass spectrometry (MS) in a machine called a GC-MS instrument, so that the sample is separated, purified and an MS reading is made at once. 

The small sample of gas is injected directly into the coiled-shaped chromatography column, which is generally a capillary or microbore column. The temperature of the column can vary depending on the gas, from 50 °C to 250 °C. The gas may separate in any number of ways that aren't necessarily permanent:

• the gas could condense in the stationary phase;
• on the other hand, the compound could remain in the gas phase;
• the gas could even dissolve on the liquid surface present on the surface of the stationary phase.

The time it takes for these compounds to be eluted (washing the substance with a solvent present in the stationary phase. In this case, 'washing' the sample gas from the carrier gas) from the chromatography column is called the retention time. The retention time of a substance depends on various factors, e.g.:

• If the sample's components do not interact very well with the chromatography column, the retention time will be short. This is usually the case when the chromatography oven is heated at high temperatures, exciting the sample molecules so that they evaporate readily; OR because they are moving so rapidly they aren't able to interact with the stationary phase.
• Depending on the boiling point of the sample; for example, a sample with a higher boiling point than the liquid in the stationary phase will spend more time in the liquid state at the beginning of the coil. 
•  If the sample is more strongly absorbed by the column. In other words, the the solubility of the gas will determine how long the gas is carried by the carrier gas; a higher solubility means a shorter time in the gaseous phase and a higher retention time.

 Liquid Chromatography (LC)

Column liquid chromatography is a common method of purifying a product after synthesising it. This separation technique involves the mobile phase being a liquid, while the stationary phase is absorbed into the chromatography column or is stuck to a glass plate. There are many ways to do liquid chromatography, some examples are open-column or liquid chromatography combined with mass spectrometry (LC-MS).

The stationary phase of LC uses a crude material such as silica (SiO₂) or alumina (Al₂O₃), with a particle diameter of around 20 μm, which then absorbs the . Depending on the substance, the chromatography column is eluted after the sample has been absorbed. This allows different components of the reaction mixture to separate as they each have different solubilities. As such, the solvent that is used allows the sample to flow out of the column already separated from the reaction mixture. 

The retention factor (Rf) is the value that represents the ratio of the distance travelled down the column by the analyte to the solvent used. The solvent and the analyte species form an equilibrium; either the stationary and mobile phase are given preference, and can be represented by an equilibrium constant (K) and the activity (a):

K = a_stationary / a_mobile  

When the column is eluted with the solvent, different components of the reaction mixture are separated (some more easily than others), eventually separating the analyte in fractions. These fractions are collected as the exit the column. In open column chromatography, these fractions are usaually eluted using gravity or pressure ("flash LC"). Colored compounds are often detected using mass spectrometers or a UV spectrometer.

High-Performance Liquid Chromatography

 This particular type of chromatography is different from the usual LC, as induces high pressure (up to 40MPa) to the mobile phase and the stationary phase utilises very small particles, ranging from 3 to 10μm in diameter. The pump that induces the pressure alters the flow rate and is dependant on the analyte passing through the column. HPLC is often used to separate fullerenes, columns specifically designed for the separation of fullerenes are commercially available, such as Cosmosil^TM Buckyprep columns.

Once the sample enters the chromatography column and is eluted, fractions are monitored using a type of detector (such as UV-VIS, IR, MS, fluorescence etc.). Data in HPLC is recorded in terms of the absorbance, A, over the retention time.

Analytical machines typically have 3 to 25 cm long by 2-4 mm wide chromatography columns. There are two types of HPLC chromatography columns:

• Reversed-phase: Where the surface of the column is hydrophobic and combined with a polar solvent, usually aqueous MeOH, MeCN or tetrahydrofuran (THF). The sample fractions are then eluted based on decreasing polarity. 
• Normal-Phase: Uses solvents that have low polarity or are non-polar, and fractions are eluted based on increasing polarity; non-polar fractions elute first. The stationary phase comprises of a polar substance such as silica.

Recrystallization

 When the product of a reaction is a solid, it needs to be separated from the solution it formed in. This is normally the final stage of purification, separating the analyte from minor impurities, such as water.

• To begin, you need to know what your solid easily dissolves in at relatively high temperatures but not in low temperatures. Generally the rule is "like dissolves like," so for instance: polar and ionic/polar solids are soluble in polar solvents; while non-polar solids do not (they are soluble in non-polar solvents). The ideal solvent in one in which the impurities do not dissolve in.
•   The impure sample mixed with the solvent is heated and then filtered to remove the impurities.
• When the filtered solution cools down, it becomes saturated and the sample begins to crystallize as its solubility decreases. Rapid recrystallization produces very fine crystals, the longer it takes to cool, the larger the crystals become (generally). The purpose of this process is to minimize the impurities present in the crystal lattice: a purer the lattice leads to a more ordered (and well defined shape) crystal lattice. 
• After the crystals form, you filter the solution containing the crystals with a vacuum, a buchner flask is an example of a tool used for this purpose (see below). 

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The next section will focus on the analysis of the elements present in a compound, which allows us to predict the chemical formula of the analyte in question. 

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Links provided bring you to some of the info I used from the web. the second-year university textbook, "Inorganic Chemistry," 4ED, by Housecroft & Sharpe, Pearson Ed. LTD, Chapter 4, was used as a guide to write this post. You can buy it here.