Figure 11.2 Blow down evaporation using N2.
PRE-CONCENTRATION AND CLEAN-UP PROCEDURES 151
acid-alkaline partition; acetonitrile-hexane partition; sulfur clean-up;
and alkaline decomposition.
11.3.1 Column Chromatography
Adsorption chromatography is often used to separate relatively non- polar organic compounds in environmental sample extracts; adsorption chromatography separates the extract components according to an equilibrium between the adsorbent (i.e. the stationary phase), the organic compounds (i.e. those under analysis and unwanted matrix components), and the eluent, that is the organic solvent used to elute the organic compounds [Practical point: sample extracts are initially concentrated using a procedure outlined in Section 11.2]. Then, the column is eluted with a range of organic solvents of differing polarity [Practical point:
a non-polar solvent is often used first, e.g. hexane]. A range of adsorbents is used for column chromatography including silica gel, florisil and alumina.
Silica gel:this is weakly acidic, amorphous silica used for clean-up of compounds containing ionic and non-ionic functional groups [Practical point:to activate the silica gel, heat to 150–160C for a few hours prior to use; silica gel is best if it contains between 3–5% w/w water. Beware when using methanol or ethanol as the eluent, as issues can arise in the ability of silica gel to function].
Florisil:this is based on magnesium silicate (with an acidic character) and is used for the clean-up of extracts for GC containing pesticides, organochlorine compounds, esters, ketones, phtahlic esters, nitros- amines, organophosphate pesticides as well as aliphatic and aromatic hydrocarbons [Practical point: commercially available Florisil should already be activated; if not it needs to be heated to 667C. Also check out batch-to-batch elution solvent consistency. Beware that some pesticides may decompose in ethylether on Florisil].
Alumina:Three types of alumina are available: basic (pH 9–10); neutral;
and acidic (pH 4–5).Basic Aluminais used for basic and neutral compounds (e.g. alcohols, hydrocarbons and steroids) which are stable in alkali [Prac- tical point:ethyl acetate cannot be used as an eluent; this is because esters are unstable in alkali and decompose. In addition, acetone cannot be used as an eluent; amidol condensation reactions can occur leading to diacetone alcohol formation]. Neutral Aluminais used for aldehydes, ketones, qui- nines and esters whileacidic Aluminais used for acidic pigments (e.g. dyes) or acidic compounds which are adsorbed by basic or neutral alumina.
11.3.1.1 Procedure for Column Clean-up of Polycyclic Aromatic Hydrocarbons from a Soil Extract
Initially, a column (e.g. 200 mm18 mm) is prepared by adding either basic alumina (10 g of 150 mesh; Sigma Aldrich) or Florisil (10 g of 60–100 mesh;
Fluka) with additional anhydrous Na2SO4(11 g) placed on top. Then, the column is eluted with 50 mL of hexane (and discarded). Just prior to air exposure of the Na2SO4, the soil extract (e.g. in hexane) is added [Practical point: a soil sample was extracted using PFE (see Section 8.2) using a dichloromethane–acetone solvent mixture. Then, the extract is evaporated to dryness under a stream of N2(see Section 11.2) and reconstituted in 2 mL of hexane]. Then again, and just prior to exposure to the air of the Na2SO4, a further 15 mL of hexane is added and the eluate discarded (this is repeated once more). Finally, the column is eluted with approximately 20 mL of dichloromethane into a volumetric flask and retained. Finally, an internal standard is added (e.g. 60mL of a 2mg/mL 4,4-difluorobiphenyl solution) and dichloromethane to give a final volume of 25 mL.
Partition Chromatography In reversed phase column chromatography [Practical point:the column could be a solid-phase extraction cartridge, see Section 9.3] clean-up is done using a non-polar stationary phase (e.g. C18) and a polar solvent (e.g. methanol–water). The sample extract is added to the column (in water) and eluted with solvent mixtures (e.g. methanol–
water or acetonitrile–water). Reversed phase chromatography is used for clean-up of polar organic compounds.
Gel Permeation Chromatography In gel permeation chromatography (GPC) sample extracts are separated by molecular size on a column of fixed pore size. In reality larger molecules elute the quickest. GPC is normally used to remove lipids, proteins and natural resins from samples.
Ion-exchange Chromatography Ion exchange chromatography is used to separate compounds that have fully ionisable functional groups. Sample extracts are added to the column, containing an ion exchange resin, and eluted using an electrolyte solution.
11.3.2 Acid-Alkaline Partition
Acid-alkaline is used to separate basic, neutral and acid compounds by adjustment of the pH (of the aqueous sample extract) [Practical point:
PRE-CONCENTRATION AND CLEAN-UP PROCEDURES 153
phenols can be extracted into organic solvent from an aqueous extract (pH 2); then, the phenols are reverse extracted by water (pH 12–13). Finally, the aqueous extract is acidified (<pH 2) and re-extracted by organic solvent. In this situation, only phenols will be extracted. In a similar manner basic compounds for example amines, can be separated by pH reversal.
11.3.3 Acetonitrile-Hexane Partition
Acetonitrile-hexane partitioning is used to remove lipids from sample extracts. [Practical point: the compounds of interest partition into acetonitrile while lipids partition into the hexane phase].
11.3.4 Sulfur Clean-up
This procedure is used to remove sulfur from the sample extract; this is done by addition of copper powder [Practical point:in Soxhlet extrac- tion, copper powder can be added to the sample in the thimble to effect in situremoval of sulfur].
11.3.5 Alkaline Decomposition
Alkaline decomposition is used to extract organic compounds, which are stable in alkaline solution (e.g. PCBs), from biological samples (which contain lipids). [Practical point: the samples are refluxed in an alkaline ethanolic solution; this saponifies the lipids]. Then, the extract is extracted using liquid–liquid extraction (see Section 9.2) [Practical point:the addition of salt aids recovery of the target compounds in liquid–liquid extraction].