EVALUACIÓN

Sample extracts injected into the GC can be contaminated with many interfering compounds. This can have two serious detrimental effects. The first problem is the co-elution of compounds interfering with the detection of the analyte of interest and the second problem is damage to the analytical instruments. The problems associated with extract contamination are loss of detector sensitivity, a shortened column

lifetime, extraneous peaks, and a deterioration in peak resolution and column efficiency (U.S. EPA, 2000). The sewage sludge matrix is one of the most difficult sample matrixes for the analysis of organic analytes due to the level of extract contamination. The cleanup procedures therefore become a crucial step in the analytical process.

The sample extracts may be purified by one or more of the following techniques:

• Partitioning between immiscible solvents

• Adsorption chromatography

• Gel permeation chromatography

• Destruction of interfering substances with acids, alkalies, and oxidising agents

• Distillation

The equipment available to the analyst and the experience in the working laboratory will influence how the work is performed or why one cleanup technique is chosen over another. A summary of the cleanup methods is presented in Table 2-2. Each analyte group of interest often has unique recommended cleanup procedures making the co-analysis of various groups of organic compounds challenging. Almost all of the sample cleanup processes that the U.S. EPA recommends have been performed as part of this research work, therefore a brief discussion of each technique follows.

Table 2-2 summarises the technique and Table 2-3 indicates the techniques applicable to each analyte.

Table 2-2 U.S. EPA sample cleanup methods (3600 series)

Method Name Cleanup Type

3610 Alumina Cleanup Adsorption

3620 Florisil Cleanup Adsorption

3630 Silica Gel Cleanup Adsorption

3640 Gel Permeation Cleanup (GPC) Size-Separation

3650 Acid-Base Partition Cleanup Acid-Base Partitioning

3660 Sulphur Cleanup Oxidation/Reduction

3665 Sulphuric Acid/Permanganate Oxidation/Reduction

Table 2-3 U.S. EPA recommended cleanup techniques for indicated groups of compounds

Analyte Group Determinative Method Cleanup Method Options

Brominated Flame Retardants - -

Organochlorine pesticides 8081 3620, 3640, 3660

PCBs 8082 3620, 3630, 3665

Chlorinated Hydrocarbons 8121 3620, 3640

PCDDs and PCDFs by LR/MS 8280 8280

PCDDs and PCDFs by HR/MS 8318 8318

2.5.1 Alumina Column Cleanup

This method is used with either traditional column chromatography or solid-phase extraction cartridges. Highly porous and granular aluminium oxide – available in three pH ranges (acidic, neutral and basic) – is used. Analytes are separated from the interfering compounds by virtue of their different chemical polarity. The column is packed with alumina and then covered under anhydrous Na2SO4. The extract is then loaded on it. A suitable solvent is selected to elute the analytes. The interfering compound is left adsorbed onto the column. Each of the three pH ranges of alumina has different applications. Basic alumina (pH 9 to 10) is most active at separating basic and neutral compounds: alkali, alkaloids, steroids, alcohols, and pigments (U.S.

EPA, 1996a). However, certain solvents such as acetone or ethyl acetate cannot be used and the basic form of alumina can cause polymerisation, dehydration, and condensation reactions (Patnaik, 1997). The neutral form is less active than the basic grade and is used to separate aldehydes, ketones and the acidic form (pH 4 to 5) is used to separate strong acids and acidic pigments (U.S. EPA, 1996a).

2.5.2 Florisil Column Cleanup

Florisil™ is a form of magnesium silicate with acidic properties. Florisil cleanup may be accomplished using a glass chromatographic column packed with Florisil or using solid-phase extraction cartridges containing Florisil. The column or extraction cartridge is packed with florisil, topped with anhydrous sodium sulfate, and then loaded with the sample to be analysed (Patnaik, 1997). Suitable solvent(s) are passed through the column. The eluate is concentrated for analysis, while the interfering compounds are retained on the column.

It is suitable for cleanup of sample extracts containing the following types of analytes:

organochlorine pesticides, organophosphorus pesticides, phthalate esters,

nitrosamines, haloethers, nitroaromatics, and chlorinated hydrocarbons (U.S. EPA, 1996b). Florisil is also used to separate aromatic compounds from aliphatic-aromatic mixtures, as well as to separate esters, ketones, glycerides, steroids, alkaloids, and some carbohydrates. It can be used to separate out nitrogen compounds from hydrocarbons (U.S. EPA, 1996b).

2.5.3 Gel-Permeation Chromatography

Gel-permeation chromatography (GPC) is a size exclusion cleanup procedure using organic solvents and hydrophobic gels in the separation of synthetic macromolecules.

The packing gel is porous and is characterized by the range or uniformity (exclusion range) of that pore size. GPC is recommended for the elimination from the sample of lipids, polymers, copolymers, proteins, natural resins and polymers, cellular

components, viruses, steroids, and dispersed high-molecular weight compounds. GPC is appropriate for both polar and non-polar analytes, therefore, it can be effectively used to cleanup extracts containing a broad range of analytes (US EPA, 1994).

2.5.4 Silica Gel Cleanup

Silica gel is a regenerative adsorbent of amorphous silica with weakly acidic

properties. It is made by treating H2SO4 with sodium silicate when used for cleanup purposes (Patnaik, 1997). Interfering compounds of different polarity are absorbed onto and retained on the column. There are two types of silica gel: activated and deactivated. The former is prepared by heating silica gel for several hours at 150°C.

It is used to separate hydrocarbons. The deactivated form contains <10% water and is

used to separate plasticizers, steroids, terpenoids, alkaloids, glycosides, dyes, lipids, sugar, esters, and alkali metal cations. (U.S. EPA, 1996c).

2.5.5 Sulphur Cleanup

Sulphur is a typical contaminant in sewage sludge and may mask the region of chromatogram, overlapping with peaks of interest. Sulphur may occur as organic of inorganic sulfur and two separate techniques are used to remove both of these.

Inorganic sulphur is removed by treating the extract with copper. Copper is activated by washing with pre-cleaned hydrochloric acid (50/50 v/v) and the extract is passed through a miniature packed column and eluted with hexane (U.S. EPA, 1996d). A packed column of silver oxide mixed with florisil, eluted with hexane was performed to removed organic sulphur residues.

2.5.6 Permanganate-Sulphuric Acid Cleanup

Interfering substances in the sample extract can be destroyed by treating the extract with a strong oxidizing agent, such as KMnO4 or a strong acid like conc. H2SO4, or a combination of both. The analyte must be chemically stable to these reagents. This method can be applied to dioxins, PBDEs and PCBs but cannot be applied to OCPs as some pesticides are destroyed (i.e. dieldrin) (U.S. EPA, 1998). Concentrated

sulphuric acid was mixed with 10 mL of sample extract (in hexane) and washed in a liquid-liquid style setup up to 7 times, or until all residual color was removed. If the color of humic material was still present after 7 acid washes a base wash was

conducted (conc. potassium hydroxide, KOH) (U.S. EPA, 1994).