Maintaining Integrity in Sample Preparation

Published: 01st Jun 2012

by Dr Claire Stone


Environmental samples comprise a wide range of matrices, each presenting the laboratory with analytical challenges. Analytical techniques and guidance documents exist to direct the laboratory to use the most appropriate sample preparation technique, but it is also crucial that the end user of the analytical data generated is aware of the preparation techniques which may be employed, and those that may be inappropriate for the sample matrix or test.

There is a continual need from the industry for laboratories to achieve lower and lower limits of detection while offering cost effective analysis. This drive has led to laboratories investing in high quality scientific instruments which enable high throughput and routine low limits of detection.

Sample sizes

New types of scientific instrumentation have driven lower costs by decreasing the sample volumes and reagent volumes required, an effect which has led to laboratories requiring less sample to carry out the same analytical testing. This effect is particularly prevalent in the case of soil testing. While analysing small sample volumes has associated risks to do with ensuring a representative sample is taken, it does ensure the emphasis on representative sampling is made by the person on site, rather than the laboratory carrying out the ‘cone and quarter’ or ‘riffling’ techniques.

Sample preservation

Sample preservation from sampling to analysis is the key to maintaining sample integrity. Often thought of as simply adding preservative chemicals to samples, guidance on the use of samples is supplied by each laboratory and although preservatives are not necessarily the same for all laboratories, this is not the only method. Both soil and water must be sampled in an appropriate manner into containers which are fit for the analysis being undertaken.

The types of containers required are typically advised by the laboratory based on the sample type and analysis requirements. Each laboratory will provide the appropriate sample containers for their analytical techniques and will have undertaken trials to ensure the containers used are suitable. A good preservative for most types of analysis, both for soil and water samples, is cooling. Laboratories typically provide cool boxes or purpose designed boxes which hold frozen ice packs and are used for sample storage and shipping.

These help to provide a stable and cool environment for shipping. Some purpose built boxes are also designed to reduce the risk of container breakage and sample cross contamination; however, it is important that the containers are correctly sealed and are relatively free of surface contamination beforehand. Taking a lead from the USEPA (US Environmental Protection Agency) sampling and sample integrity guidelines, the use of trip blanks, spikes and duplicates is a good way to ensure the samples are adequately preserved and shipped.

Trip blanks are typically a ‘clean’ sample provided by the laboratory. This sample is not opened in the field and is simply retested on receipt at the laboratory and is not to be confused with a field blank, which is opened in the field and exposed to ambient cross contamination. The use of a spiked sample is a good way to check samples have not been degraded. A sample is spiked with a known concentration of an analyte or analytes of interest, and then treated the same as a trip blank. The analytical data generated is checked against the known concentration at the beginning and assessed with regards to the acceptable method bias.

While sample duplicates are used routinely, this is less to ensure sample integrity but to instead ensure sample homogeneity and that the sample data is representative. There are also increasing requirements for those taking samples to understand and take into account recommended guidance on deviating samples with respect to sample containers, holding times and conditions and the use of preservatives for both soils and waters.


Soils from contaminated land sites typically have inclusions within the soil matrix. These inclusions may be volatile or oily, inert or even reactive. Depending on the size and nature of these, different preparative methods may be required. The purpose of preparing soil samples for analysis is to ensure they are as homogenous as possible. Analytical data generated is representative of the sample submitted for analysis and although laboratories may choose to accomplish this in different ways the process is essentially the same.

The sample is dried, then sieved to remove large inclusions such as stones, and the resulting finer fraction is ground to a fine consistent particle size, typically ≤ 250 µm3. Sample drying may be accomplished by air drying; with temperatures not exceeding 30° C, air assisted drying; with forced air flow and temperatures not exceeding 30° C or even freeze drying. The sieving to remove large inclusions may be done at anywhere between two and ten millimeters fraction size, depending on the preference of the testing facility.

The sample crushing procedure may be achieved by hand or mechanical grinding, through either a jaw crusher or a mill. Although each laboratory will have their own preference and procedures, these will all be validated and shown to be appropriate for their analytical methods. This has previously been discussed at length in the Soil Sample Preparation article in AWE International, June 2011.

Soil sample preparation

The use of different sample preparation procedures can have a pronounced effect on analytical data. A good example to consider is the testing of total metals and any subsequent bioaccessibility analysis. The concentration of target metals may be initially indicated by on site analysis using hand held X-Ray Fluorescence (XRF) instruments. This gives an indication of the concentrations in a non-homogenised on site sample.

Laboratories may also test samples using XRF but in this case laboratory instruments rather than hand held instruments are used. In this instance, a dried homogenised sample (particle size < 250 µm) is then analysed as a whole dry sample. The analytical technique is non destructive and does not suffer from extraction efficiency matrix effects; however, the method does not have a high throughput, and suffers high running costs compared to other analytical techniques used. It may suffer from solid phase matrix interferences but this may be reduced using matrix matched calibration standards.

Laboratories often employ extraction of a dried homogenised sample using acid and then analysis of the aqueous extract by ICP-OES (inductively coupled plasma – optical emission spectrometry). Most laboratories use aqua-regia, a mix of hydrochloric and nitric acids; however, depending on the choice of acid, the extraction efficiency may be affected by matrix effects. These three methods may yield different total metal concentrations.

These concentrations may exceed guideline values and hence additional information of bioaccessibility may be required. All bioaccessibility models and methods have a specific sample preparation requirement: the sample must be dried and then sieved to obtain the < 250 µm test portion. This portion is chosen as it is the fraction most likely to adhere to children’s hands and therefore pose most risk. The sample preparation is designed to obtain the finer portion for analysis without employing any force that could not be applied by a small child, such as crushing or grinding.

The test portion generated is then analysed for both bioaccessible and total metals. Depending on the fraction where the metal is found, this may lead to either a higher or lower total concentration. Considering these examples it is evident the difference that sample preparation can have on relatively simple analyses. It is therefore important that these are understood, as the effect on more complicated analyses would be compounded. It is a requirement that all laboratories report their basic approach on sample preparation, including any fractions removed from the test portion.

Although there are guidance documents available it is recognised that they are not applied consistently throughout the industry.


Water is often assumed to be a homogenous matrix and little consideration is given to components within the sample which may affect testing and results. With the instatement of the Environment Agency MCERTS for waters performance standard, there is now good guidance available on sampling and appropriate performance criteria for samples.

Water samples are often pre-treated and preserved in the field by using the appropriate equipment or containers supplied by a laboratory. There is little consideration in the industry of pre-treatment or sampling for samples with non-aqueous phases present. In some cases it may be appropriate to sample the aqueous phase, in some cases the organic phase and in some cases it may not be possible to split them in the field, hence this responsibility is passed on to the laboratory. The presence of a phase is often compounded by colloidal or suspended solids in the sample.

For these reasons, it is important to understand how samples are prepared for analysis so that any impact of these can be understood, and where possible taken into account.

Water sample preparation

A good example of where to use water sample preparation is when considering petroleum hydrocarbons and poly-aromatic hydrocarbons, yet it should be borne in mind that not all laboratories use the same preparation protocol. For testing of volatile analysis often there is no preparation and a small volume of sample is taken whole and analysed. This is less problematic than when analysing large volumes of sample for non volatile fractions.

An added advantage of volatile analysis is that it tends to yield instrumental fingerprints, or chromatograms, that are typically easier to interpret. This is mainly due to reducing the matrix effect as one is dealing essentially with the gaseous phase during analysis.

Extraction techniques

Non volatile fractions may be extracted from the sample in a variety of ways including solid phase extraction (SPE), liquid: liquid extraction (LLE) and direct analysis. A clean sample with no sheen or sediment may be considered differently to those where either or both are present. SPE techniques are often used within the water industry for analysing ‘clean’ samples.

Put simply, the process involves passing the sample through a cartridge or column which has been specially designed to attract the compounds of interest. As the sample passes through, the column becomes loaded with the compounds of interest. When a sufficient volume has been filtered, a solvent is added to flush or ‘elute’ the compounds from the column into the solvent and then this is analysed. Depending on the choice of SPE column the technique can be selective for a range of chemically similar analytes that are contained within a multi analyte matrix. This is typically not used for dirtier samples as the column may be overloaded, resulting in poor recovery or even blocking the column with organic phases or particulates.

LLE uses a large volume of sample relative to a smaller volume of solvent and can be applied to: • Samples which have organic phases • Samples which have sediment • Samples where these have been removed

The solvent is added to the water sample and agitated in some way, often by shaking. The solvent is chosen so that compounds of interest preferentially transfer to this phase. At the end of the extraction period the solvent is recovered, pre-concentrated and then analysed. Most laboratories will employ similar solvents during LLE but a combination of solvent mixtures can be prepared to deal with difficult to extract analytes.

Direct analysis may be appropriate where the organic phase is that of interest. It is difficult to provide fully quantitative results using this method and for this reason direct analysis is most often used to provide information on the composition rather than the concentration. Essentially, a small volume of the organic phase is taken and diluted with a portion of the solvent used in the standard preparation method, and this is then analysed. Considering these three preparation methods, it is clear that physical composition and any pre-treatment of the sample will have a significant effect on the analytical data obtained for the sample.

Each laboratory will have criteria for any preparation methods they use before carrying out sample extraction and analysis. These will be fit for purpose based on their methodologies; however, it is worth considering that not all laboratories have the same criteria and in some cases it may be appropriate for the user of the sample data to direct the laboratory as to which phase of the sample they require analysing.


It is clear that there are a variety of approaches that can be taken in the preparation and pre-treatment of environmental samples. These different approaches may lead to inconsistency if data is compared between different laboratories. A recent survey carried out by the Environmental Industries Commission (EIC) shows that despite the availability of guidance documents there are still differences across the industry between laboratories.

It is important that consultants or contractors understand how their laboratory processes samples for analysis, so that they are able to give instruction when targeting a particular phase for analysis.


1. BS 10175:2011 The Investigation of Potentially Contaminated Sites 2. USEPA SW-846 Test Methods for Evaluating Solid Waste, Physical/Chemical Methods 3. Environment Agency Methods for the Examination of Waters and Associated Materials, ‘The preparation and pre-treatment of potentially contaminated soils prior to chemical analysis’ (2006) 4. Environmental Laboratory Testing in AWE International, March 2011 5. BS ISO 12404:2011 Soil quality. Guidance on the selection and application of screening methods 6. US EPA. Method 6200 – Field Portable X-Ray Fluorescence Spectrometry for the determination of elemental concentrations in soil and sediment, 2007, rev.0 7. US EPA. Method 6010C – Inductively coupled plasma – Atomic emission spectrometry, Feb. 2007, rev.03 8. Environment Agency Methods for the Examination of Waters and Associated Materials, ‘The determination of metals in solid environmental samples (2006)’ 9. Ruby MV, Davis A, Schoof R, Eberle S, Sellstone CM (1996). Estimation of lead and arsenic bioavailability using a physiologically based extraction test. Environmental Science and Technology 30, 422-43 10. Environment Agency Performance Standard for Laboratories Undertaking Chemical Testing of Soil, 2012, Version 4. 11. Environment Agency Performance Standard for Organisations Undertaking Sampling and Chemical Testing of Water, 2009, Version 1.1 12. US EPA. Method 8270D – Semivolatile Organic Compounds by Gas Chromatography/Mass Spectrometry (GC/MS), Feb. 2007, rev.04

Published: 01st Jun 2012 in AWE International

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