Application of chromatographic techniques to assess degree of soil pollution with Polycyclic Aromatic Hydrocarbons (PAHs)

The aim

Isolation of polycyclic aromatic hydrocarbon (PAH) fraction from soils contaminated on various levels and analysis of particular compounds.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are chemicals characterized by relatively high toxicity, often being of carcinogenic character. The most carcinogenic compounds are benzo(a)pirene, benzo(a)anthracene, dibenzo(a,h)anthracene, benzo(b)fluoranthene, benzo(k)fluoranthene and indeno(1,2,3-c,d)pirene. PAHs are metabolized in the living organisms to metabolites binding effectively to DNA (e.g. epoxide derivatives), which in turn lead to highly probable carcinogenic effects.

Figure: Selected polycyclic aromatic hydrocarbons

PAHs are present in the environment always as mulitcomponent mixtures and their composition is source-dependent. They are formed during incomplete combustion of the fossil fuels, during incineration of waste, as well as a consequence of various industrial activities associated with the conversion of oil and coal. The most significant source of these pollutants in the urban areas is provided by car transportation. The equally important, especially during the winter season, are individual heating systems.

Near 90% of total PAHs in the environment is present in soils. Concentrations of these entities in soils are on the trace levels (ppm or ppb)*, therefore their analytical procedures comprise of several steps. Each of them is equally important for successful analysis, thus each step (especially those which can not be later on corrected or repeated) must be proceed with high carefulness and precision.
*ppm – parts per million, ppb – parts per billion

Nowadays liquid extraction is the most popular way to isolate hydrocarbons from soils. Extraction is then conducted with solvents of low polarity that are able to dissolve hydrocarbons: e.g. n-hexane, cyclohexane, toluene, dichloromethane, acetone and mixtures of thereof.

Most of extracted compounds from soil are of unpolar or low polar character whereas polar compounds are usually in minority. In the non-contaminated soils they are mainly plant originating chained hydrocarbons whereas in polluted areas mixtures of saturated and aromatic hydrocarbons in the ratio depending from the contamination rate.

The very frequent way to isolate PAH fraction from organic extracts is application of liquid adsorption chromatography. In this system separation occurs due to a different adsorption potential of various compounds on the adsorbent (in chromatography called 'stationary phase'). The higher the affinity of the substance to adsorbent, the longer it is retained and therefore leave the chromatographic column later on. Therefore, more solvent is necessary (in chromatography called 'mobile phase') to effectively elute such compound from the column (in chromatography called 'higher retention volume'). In some cases interactions of substances and adsorbent are so strong, it is necessary to apply solvents with much higher elution strength (usually more polar ones).

Silica gel is one of the most popular inorganic adsorbents. Its surface is covered with active silanol groups ( ≡ Si-OH) responsible for adsorption. Since silanol groups quite easily adsorb polar molecules (e.g. water), moisture can drastically reduce its activity. Therefore, to achieve good adsorption of aromatic or aliphatic hydrocarbons activated (water free) silica gel is applied.

Identification and quantification of PAHs is conducted through analysis in gas chromatography (GC). In this technique separation of substance mixture is proceed in the gas mobile phase whereas stationary phase comprise of highly boiling liquid chemically bound on the inner column. Substances are being separated in this system through the differences in their affinity to the stationary phase as well as through their differences in the boiling points.

The most common detector used in GC analysis of hydrocarbons is flame ionization detector (FID).

Chemicals

• Silica gel MN-Kieselgel 60 – particle size 0.08 mm
• Petroleum ether p.a.
• Toluene p.a.
• Dichloromethane p.a.
• Anhydrous sodium sulfate p.a.
  

Materials and instruments

• Metal spoon
• Glass flask with closure to load a soil sample
• Ceramic mortar
• Weighting vails 5-10 ml – 2 pcs.
• Erlenmeyer flask 25 ml – 2 pcs.
• Erlenmeyer flask 100 ml – 3 pcs.
• Glass funnel – 2 pcs.
• Filter paper
• Florence flask 100 ml – 2 pcs., 50 ml – 2 pcs.
• Tubular glass column with stopcock, inner diameter 1 cm, minimal lenght 15 cm
• Graduated cylinder 10 ml – pcs., 25 ml – 2 pcs, 50 ml – 1 pcs.
  

Hazards

Procedure

Take two soil samples from 0 to 5 cm depth, 50 g each. Samples shall be taken with a metal spoon and placed into the glass flask with closure. One of the samples shall be taken from uncontaminated territory (e.g. park or forest distanced from potential contamination sources) and the second from the place evidently endangered with pollution (e.g. car parking, side of the road).

After bringing to the laboratory, place thin layer of soil onto filter paper, then remove fragments of plants and stones. Leave in dark to let it air-dry. Grind dry soil in the mortar. Further, estimate soils dry weight. Using analytical balance and tarred weighting vials weight 2-3 g of soil subsamples with accuracy not lower than 1 mg. Place vials with soils into the oven and heat with 105°C until reaching constant mass.

Calculate percentage of water in the soil from the following equation:

% H₂O = 100% x (m_{air-dried soil} [g] - m_{oven dried soil} [g]) / m_{air-dried soil} [g]

Weight ca. 5 g (±0.01g) of soil from contaminated area and ca. 25 g (±0.01g) of soil from unpolluted area. Place weighted samples in Erlenmeyer flasks (100 ml) and add 50 ml of mixture of petroleum ether and dichloromethane (3:2, v:v). Place both flasks in ultrasonic bath for 15 min or mix on the magnetic stirrer for 20 min. Decant the suspension and filter the extract through the paper filter supported with anhydrous sodium sulfate. Collect the filtrated extract in a florence flask (100 ml). Add fresh portion (25 ml) of the solvent mixture to the soil and run the extraction procedure once again. Filter the extract through the same filter and collect the filtrated extract to the same Florence flask. Then add 0.2 ml of toluene and vaporize it with the rotavapor not exceeding 30 °C of water bath to the final volume of added toluene (thin layer of “oil” on the flask interior). After that add 0.5 ml of petroleum ether. In case of difficulties with extract solubility mildly warm the flask in the water bath.

Weight two portions of silica gel (2.5 g each) in Erlenmeyer flasks (25 ml) and place it to the oven for 8 h in 150 ° C. Close both flasks and after cooling them down add to each a portion of petroleum ether to reach solvent layer of ca. 1 cm thickness above the gel. Mix flasks gently to remove formed air bubbles. Until use keep both flasks closed to avoid the evaporation of the solvent.

Place the circle of the filter paper onto column frit, then place the flask directly under the outlet in order to collect the solvent from the column. Mix mildly one of silica gel portions and pour it slowly (to avoid aerating) to the column through the funnel. After loading the gel and letting it to settle, place circle of the filter paper on top of the adsorbent and flash the column with 6 ml of petroleum ether (or more until reaching constant height of the gel – ca. 6 cm) and close the outlet.

Remember - the adsorbent MUST be always covered with a layer of solvent!

If heated before, cool down the extract to reach room temperature. Open the stopcock and let out petroleum ether to leave not more than 1-2 mm of the solvent above the sorbent. Then add the extract to the column by gently injecting it with the syringe or pipette onto column inner wall just above the adsorbent layer. Place the graduated cylinder (25 ml) at the column outlet.

When extract will start to enter adsorbent and the height of the liquid above the gel layer will reach 1-2 mm, pour gently onto column inner wall 1 ml of petroleum ether and wait until it will reach again 1-2 mm above the sorbent. Repeat this action again. Then pour (again very gently) remaining amount of petroleum ether (according to the detailed instruction below) and start collection of the fraction rich in aliphatic hydrocarbons. After this, place the second cylinder at the outlet and switch the solvent to the petroleum ether / toluene mixture (9:1, v:v) in order to collect the second fraction containing PAHs.

Separation conditions

• Activated silica gel
• The height of the adsorbent 6 cm
• The volume of the first fraction rich in aliphatic hydrocarbons eluted with petroleum ether – 12 ml
• The volume of the second fraction rich in PAHs eluted with petroleum ether and toluene (9:1, v:v) – 25 ml
• If height of the adsorbent is different than 6 cm, volumes of collected fractions shall be changed proportionally.
• Place PAHs fraction in florentine flask (50 ml) and concentrate it on the rotavapor to the volume of 1 ml. Obtained concentrate shall be stored in the refrigerator after flask was rounded with alumina foil.
• The same shall be proceed with the second extract but using fresh portion of silica gel.
  

Analysis of PAHs

The final analysis of PAHs (after addition of 2-methylantracene as the internal standard) will be handled by means of gas chromatography.

Results are presented in the form of chromatograms:

• Gas chromatogram of the PAHs isolated from the contaminated soil
• Gas chromatogram of the PAHs isolated from the unpolluted soil
• Gas chromatogram of the standard mixture of PAHs

are given to make final calculations

Read the times, in which particular signals representing various PAHs are present at the chromatogram of the standard mixture (in chromatography they are called 'retention times'). Table 1 presents detection coefficients for particular PAHs, which are needed for further quantitative calculations. On the basis of retention times identify particular compounds in the chromatograms 1 and 2. Then measure heights of identified chromatographic signals (so-called 'peaks'). Write your results in the table 1.

Calculate content of particular PAHs in the fraction using the following equation

m_analite(i) = m_int.stnd. x f_analite(i) x W_analite(i) / W_int.stnd.

where

m_analite(i) - mass of particular PAH in the fraction,
m_int.stnd. - mass of the internal standard,
f_analite(i) - detection coefficient for particular PAH,
W_analite(i) - height of the chromatographic signal of particular PAH [mm],
W_int.stnd. - height of the chromatographic signal of internal standard [mm].

Table 1 Results of measurements for PAHs in soils

Considering that fraction isolation was made with 80% yield and taking into the account the water content calculate how much of particular PAHs were present in the soil. Present the results as mg/kg dry weight.

Calculate the total PAHs calculation again expressed as mg/kg dry weight.

Compare obtained results with standard values for soil quality. Assess the level of soils contamination

Quality standards presented below qualify soils according, between others, to their PAHs content (Directive of the Polish Environment Ministry Dz. U. No. 165, 2002, 1359).

Following groups of soils are defined:

• Group A - soils from the protected areas (water and nature conservation law)
• Group B – agricultural, forest and park soils
• Group C – soils from the industrial and transportation areas, exploitation sites

Table 2 presents quality standards for soils due to particular PAHs content (in mg/kg dry weight). It shall be noticed that for the group A the depth of the soil core is neglected, whereas for groups B and C variability of PAHs limits are due to the depth of the core.

Tab. 2. Reference values for nine PAHs (mg/kg d.w.) in the surface soils
from 3 groups.

The total concentration of PAHs is understood as a sum of concentrations of the following compounds: naphthalene, chrysene, benzo(a)anthracene, benzo(a)pirene, benzo(a)fluorantene and benzo(ghi)perylene. According to the presented quality standards soil is considered as contaminated when at least one compound exceeds the given level.

Prepared by: Faculty of Chemistry, University ob Gdansk, Poland