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What is a sample preparation process?

Sample preparation refers to the series of actions taken to transform a sample from its original state to a form that is suitable for analysis in a laboratory setting. The exact process can vary widely depending on the type of sample and the analysis to be performed. However, the main goal of sample preparation is to isolate the desired analytes (the components of interest) from the sample matrix (the bulk of the material), remove any contaminants or interfering substances, and convert the analytes into a form that is compatible with the analytical technique to be used.

Generally, different steps are involved in a sample preparation:

  1. Sampling / sample division: The first step involves collecting a representative portion of the material or substance to be analyzed. This is crucial for ensuring that the results are reflective of the whole sample.
  2. Drying / embrittlement: Size reduction and homogenization of sticky, moist, elastic or tough samples can be challenging. Drying makes the sample more brittle and facilitates the grinding process. Embrittling elastic or tough samples, which cannot be pulverized at room temperature, has the same effect.
  3. Cleanup / sieving: This step is necessary to remove any substances that might interfere with the analysis. Techniques like chromatography or the use of selective adsorbents can be employed to purify the sample.
  4. Size Reduction / homogenization: For solid samples, this might involve grinding or crushing to reduce the particle size, making the analytes more accessible for further processing.
  5. Extraction: This step involves separating the analytes from the sample matrix. Various extraction methods can be used, including solvent extraction, solid-phase extraction (SPE), and solid-phase microextraction (SPME), depending on the nature of the sample and analytes.
  6. Concentration / compaction: Often, the analytes need to be concentrated to be detectable by the analytical instrument. This can involve evaporating solvents, centrifugation, or filtration to reduce the volume of the sample and increase the concentration of the analytes.
  7. Derivatization: In some cases, especially for gas chromatography (GC) or certain types of spectroscopy, the analytes may need to be chemically modified to make them more volatile, detectable, or analytically stable. This process is known as derivatization.
  8. Transfer to Analysis Vessel: Finally, the prepared sample is transferred to a vessel or container suitable for the analytical instrument, such as a chromatography vial, an autosampler container, or a specimen cup for spectroscopic analysis.

The specific techniques and equipment used in sample preparation depend on the sample type (e.g., solid, liquid, gaseous), the matrix complexity, the analytes of interest, and the analysis technique (e.g., chromatography, spectroscopy, mass spectrometry). Proper sample preparation is critical for obtaining accurate, reliable, and reproducible analytical results. Retsch offers instruments for different steps of the sample preparation process:

Sampling and sample division, drying or embrittlement, clean-up and sieving, size reduction and homogenization, extraction as well as concentration and compaction. Each part of the sample preparation process is described in the following sections.

1. Sampling and sample division

To ensure reproducibility in sample preparation, it is essential to extract a sub-sample that truly represents the bulk material. This means the sub-sample must share the same properties as the bulk. Given that most samples are inhomogeneous mixtures, care must be taken to avoid segregation due to varying particle sizes, especially during transport. If the entire sample isn't processed, a representative portion must be taken. The quantity of the sample is crucial; it must be sufficient for analysis and proportionate to the total sample size and grain size. These factors dictate the minimum quantity needed for the sub-sample to accurately reflect the bulk.

Some important questions should be clarified in advance:

a. Which quantity is required to ensure representativeness of the original sample?
b. From which part of the original material should the sample be taken?

A. 1. Which quantity is required?

Some industry-specific standards provide guidelines and directions on the correct sampling process, for example DIN 51701-2:1985 in the coal industry. It contains, for example, the formula

G [kg] = 0.07 [kg/mm] x z [mm]

which indicates how much sample “G” must be extracted from a bulk sample with maximum particle size “z” to obtain a representative quantity. Taking a coal sample with a maximum particle size of 5 cm as an example, the following calculation applies:

G [kg] = 0.07 kg/mm x 50 mm
G = 3.5 kg

Consequently, the extracted sample amount for a sample with max. 50 mm particles should be at least 3.5 kg to make sure it is representative. For other materials than coal a different density should be taken into account.

B. 2. From which part is the sample taken?

The sub-sample taken from the original sample must accurately represent the entire bulk, which requires careful consideration of the sampling location (middle, top, bottom, inner, or outer part of the heap). Factors such as moisture content can vary within the heap, potentially affecting analytical results. For example, the inner parts may be moister than the outer parts. Additionally, particle composition may differ between the top and bottom due to segregation. Sampling large volumes, like ship or train loads, poses challenges. To achieve a representative sub-sample, it's necessary to collect samples from multiple locations and combine them. Alternatively, samples can be drawn directly from the material flow in a production unit.

1.1. 縮分器

Once the sample is in the laboratory, the sample amount may be too large for the subsequent steps in the sample preparation process. Therefore, further sample division may be required. The selection of the division method and the instrument depends on the sample material and quantity. Dry, free flowing samples can be fed via vibratory feeders to rotary tube dividers and sample dividers with a rotating dividing head.

1.2. サンプルスプリッタ

Sample splitters are used for materials with a low flowability. Manual random sampling is only acceptable if the sample is homogeneous with regards to material and grain size. However, without preliminary examination, this is difficult to ascertain.

The importance of sampling is demonstrated in the figure: Even if the analysis is carried out correctly, random sampling (e.g. with a scoop) leads to varying results which are not reproducible although the samples come from the same initial material. As shown, three different samples taken from the same initial material show variations of up to 20 % for the fraction below 2 mm. Therefore, it is essential that sampling is carried out with utmost care.

Random sampling with scoop; three correct sieve analyses lead to three different results
 

Random sampling with scoop; three correct sieve analyses lead to three different results

Professional sample dividers with a marginal standard deviation should be used for the extraction of representative sub-samples. The figure shows the qualitative sampling errors of the different methods. It can clearly be seen that rotary tube sample dividers produce the smallest qualitative variation (A). They achieve the highest degree of reproducibility and are clearly superior to all other methods.

Qualitative sampling errors (standards deviations) of the different sampling methods
 
 

Qualitative sampling errors (standards deviations) of the different sampling methods

The standard deviation e.g. in a plastic sample analyzed for its moisture content can be decreased drastically by correct sample division using a sample divider
 

The standard deviation e.g. in a plastic sample analyzed for its moisture content can be decreased drastically by correct sample division using a sample divider

2. Why does drying or embrittlement help in sample preparation?

Grinding moist or wet sample materials can lead to undesirable side effects. Such materials are prone to clogging the mill's ring and bottom sieves, potentially causing a machine blockage. This results in material loss during the sample preparation process and requires significant time and effort for cleaning. There are exceptions, however, such as colloidal grindings in ball mills that necessitate adding a liquid. Fresh fruits and vegetables can be processed in knife mills without losing material. Typically, moist samples must be dried (like leaves) before size reduction. The chosen drying method and temperature must not alter the sample's properties, particularly volatile components. These samples are usually air-dried at room temperature. RETSCH's TG 200 is designed for rapid and gentle drying, employing fluidized bed drying, akin to industrial dryers, and can dry many products in just 5 to 20 minutes.

Drying ovens from Carbolite Gero

The ideal solution for removing moisture from material.

Embrittlement (with liquid nitrogen or dry ice) helps to improve the sample´s breaking behavior, which is beneficial for sample preparation. Therefore, temperature-sensitive materials, such as some types of plastics, have to be cooled directly before they can be subjected to preliminary or fine size reduction. One way is to embrittle the sample in liquid nitrogen before grinding. At a temperature of -196°C even soft rubber becomes so hard and brittle that it can be ground without problems. Another possibility is to mix the sample with dry ice. However, materials which must not become moist should not directly be treated with cooling agents. The reason for this is that the steam in the air is frozen and is precipitated as water when it unfreezes. Cooling agents should not be used in closed grinding tools as evaporation causes overpressure.

Cooling a jar in liquid nitrogen
 

Cooling a jar in liquid nitrogen

3. Metal Separation and Sieving

Samples such as industrial waste, recyclable waste and secondary fuels often contain metallic components which cannot be pulverized with laboratory mills. On the contrary, metallic objects such as steel nails or iron screws can damage the grinding tools which can lead to a considerable deterioration of the mill's performance. Therefore, it is necessary to separate the metal components before switching to the next step in sample preparation: grinding. If required, the fractions have to be evaluated separately. Undesired particles like metal parts can also be removed by sieving, if their sizes differ from the particles which need to be analyzed. Here, Retsch sieving machines are the devices of choice. RETSCH sieve shakers and sieving machines not only cover a wide measuring range. Thanks to different sieving motions and sieve sizes, the RETSCH sieve shaker range is suitable for almost any bulk material. Our sieve shakers and sieving machines produce exact and reproducible results and comply with the requirements for the test materials monitoring according to DIN EN ISO 9000 ff.

4. Size reduction and homogenization

A laboratory mill is engineered for pulverizing or grinding small material samples for compositional, physical property analysis, or other testing objectives. They are capable of preparing samples to a uniform size or texture, ensuring consistency for precise evaluations. Various sample mill models exist, each tailored for distinct materials and particular uses. Selecting an appropriate sample mill is contingent on the material's characteristics and the intended result of the sample processing. For analytical techniques like AAS, NIR, ICP, or XRF, it is crucial that the sample is thoroughly homogenized to a suitable level of analytical fineness. Only through consistent sample preparation dependable and accurate analyses are assured. RETSCH provides an extensive selection of the sample mills and crushers for the coarse, fine, and ultrafine size reduction of virtually any substance. The wide range of grinding instruments and accessories guarantees that our devices facilitate contamination-free and reliable sample preparation before laboratory testing.

RETSCH Grinding Mills

Retsch offers a wide range of grinding mills designed to accommodate a variety of applications.

5. Retsch benefit for the extraction step

The chemical extraction of analytes from ground samples is a pivotal sample preparation step in analytical chemistry, environmental science, and biochemistry, aimed at isolating specific compounds for detailed analysis. This process is essential for monitoring environmental pollution e.g. with pesticides, assessing nutrient levels in soils, detecting hazardous substances, and conducting research in various scientific fields.

The choice of extraction solvent and technique is critical and is determined based on the nature of the analytes of interest and the matrix of the ground sample. Common techniques include solid-phase extraction (SPE), liquid-liquid extraction (LLE), solid-phase microextraction (SPME), and supercritical fluid extraction (SFE), among others.

The extraction process typically involves the solubilization of the target analytes into a suitable solvent, separating them from the solid matrix and other non-target components. Following extraction, the analytes may undergo further purification and concentration steps before analysis using techniques such as gas chromatography (GC), liquid chromatography (LC), or mass spectrometry (MS) to quantify and identify the compounds of interest.

The QuEChERS method, an acronym for "quick, easy, cheap, effective, rugged and safe," streamlines sample preparation for pesticide residue analysis. Testing has shown that results from the QuEChERS method are comparable to those from other methods. During homogenization, it's important to keep the sample from overheating, as some pesticides are volatile. Cooling the sample not only prevents this but also enhances the material's breaking behavior, leading to better fineness and homogeneity. The extraction process involves taking 10 g of the food or soil sample and extracting it with 10 ml of acetonitrile. To eliminate ghost peaks in chromatograms, the extraction is performed with a salt mixture, typically sodium chloride and magnesium sulphate in a 1:2 ratio. To transfer pesticides into the organic phase, the sample is shaken with the acetonitrile and salt for 1 to 3 minutes. A laboratory mill, like RETSCH’s Mixer Mill MM 400, can be used to agitate the mixture in a 50 mL Falcon tube at up to 30 Hz, ensuring thorough and reproducible mixing, which is beneficial for the extraction process.

6. Concentration and compaction

In this crucial step of sample preparation Retsch can support with its Pellett Press PP40. The press compacts the powder to tablets with smooth surfaces, which are often required for XRF analyses. The accuracy of XRF analysis depends on the interaction of X-ray beams with the sample surface, and several factors related to the surface condition can impact the quality of the data obtained:

  • A smooth surface ensures that the X-rays interact uniformly with the sample. Rough or irregular surfaces can cause scattering and absorption of X-rays in an uneven manner, leading to inaccurate or inconsistent results.
  • Smooth surfaces help to achieve reproducible results across multiple measurements or samples. Variability in surface texture can introduce measurement inconsistencies, making it difficult to compare results.
  • In bulk samples, a smooth and finely polished surface minimizes the effects of particle size variation. Larger particles on a rough surface might not be fully penetrated by the X-ray beam, leading to underrepresentation of their elemental composition in the analysis.
  • For solid samples, good surface contact between the sample and the detector window is crucial. A smooth surface ensures closer contact, which is particularly important for light elements that have low-energy X-ray emissions, as these emissions are easily absorbed by air gaps or inconsistencies in surface contact.
  • On a rough surface, elevated points can create shadow regions that are not adequately exposed to the X-ray beam, resulting in an incomplete analysis of the sample's elemental composition.

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Sample Preparation Process - FAQ

Why is sample preparation important?

Before samples can be analyzed using advanced scientific equipment and instruments, they must be properly treated and prepared. This preliminary step is an important stage of the overall analysis process as it helps to prevent contamination, improve accuracy and minimize the risk of result distortion.

What is the first step in sample preparation?

The first step of sample preparation is collection. In simple terms, this is the extraction of a representative sample from a larger source. That could be anything from a cell suspension sample to a food sample. It is important to minimize sample loss, avoid contamination and ensure consistency.