Sample preparation is a crucial procedure in the entire analysis process. It helps prevent contamination, improves accuracy, and minimizes the risk of distorted results. Different matrices need different sample preparation techniques. As with the matrix, sample preparation follows the analytical method used.
For analysts using chromatographic methods, several sample preparation methods are available such as liquid-liquid extraction (LLE), solid phase extraction (SPE), solid-liquid extraction (SLE), and others. We have summarized these techniques for you to choose.
Sample Preparation Methods
- Liquid-liquid Extraction (LLE)
LLE sample preparation technique (liquid-liquid extraction) is the most well-known and widely used method compared to others. Most chromatography users encounter with LLE method when they are in college. LLE is used to obtain analytes from a liquid matrix with the help of a liquid solvent.
LLE works to separate compounds that have different solubility in two different solvents. The separation uses a separating funnel (the separated funnel). The separated funnel will separate compounds with different polarities. It can be applied if distillation is impossible due to the formation of an azeotrope which can inhibit the distillation process.
Samples like antibiotics, vitamins, flavoring agents, salts, and petroleum products, can be prepared using the LLE method. Accordingly, the LLE method is suitable for GC or HPLC instruments. Some advantages of the LLE method include: the extraction method is quite simple, the glassware is inexpensive, organic solvents can be recycled, and the carboxylic acids extracted have high purity. Meanwhile, the weakness of this method is that it takes a lot of time to dissolve the sample. Due to the shaking process, if it is overpowering, it will form an emulsion that is increasingly difficult to break. In addition, it consumes a lot of organic solvents, uses a lot of glassware, and produces quite a lot of waste.
- Solid-Liquid Extraction (SLE)
SLE or solid-liquid extraction is a separation process that dissolves a solid matrix with a liquid solvent. The working principle of SLE is the same as when we are brewing coffee, where the coffee grounds are dissolved in hot water to get the caffeine compound. Meanwhile, we use a Soxhlet extractor to obtain analytes in the sample in the lab. Like LLE sample preparation, SLE is suitable for HPLC or GC instruments.
SLE is generally applicable in the food, pharmaceutical, and chemical industries. In addition, to obtain oil from oil ores and wash metal salts from ores (ores). Pros of the SLE is that there is no agitation during the dissolution process, so an emulsion can’t form. Additionally, inexpensive reagent cost, has high recovery, accurate results, high sample throughput, less solvent and glassware usage compared to LLE, and no need to wash the separatory funnel (the separate funnel). However, SLE has several downsides, improper on samples with a hard texture and a complicated process because it has to be evaporated at a rotary evaporator to obtain a thick extract.
- Solid-Phase Extraction (SPE)
The emergence of the SPE sample preparation technique (solid phase extraction) updated the LLE method. The principle of separating analytes in SPE is that the stationary phase in the SPE column retains analytes contained in the solution. Then, the analyte is taken back using a solvent to obtain a more concentrated concentration. Separation of compounds using a cartridge.
Some advantages of SPE include: the time needed for SPE extraction is relatively fast, only about 30 minutes, the operation is also easy, and the system is automated. In addition, SLE is better recovery than LLE, abolishing emulsion formation, less use of organic solvents, and increased selectivity and reproducibility.
SPE differs from LLE in terms of analyte separation. In SPE, analytes separate by interaction with a solid stationary phase. SPE is available in three types: normal phase, reversed phase, and ion exchange. These three phases use for polar, non-polar, and charged compounds. The SPE methods are for environmental, industrial, pharmaceutical, food, and marine fields. Commonly, it is applied for separation in matrices of urine, blood, eggs, honey, breast milk, and red wine. This extraction can be used before further analysis using GC or HPLC instruments. Some researchers stated that the weaknesses of the SPE procedure include a time-consuming, complicated method, and cartridge replacement is also a troublesome thing.
- QuEChERS
Concerns over the excessive use of glassware, high labor requirements, and low throughput have motivated researchers to find more efficient extraction methods. The QuEChERS extraction appeared around 2003. QuEChERS stands for Quick, Easy, Cheap, Effective, Rugged, and Safe. This method is a dispersive SPE that overcomes the problems of SPE, especially the cost of cartridges.
QuEChERS provides several advantages: high recovery, accurate results, high sample throughput, low solvent and glassware usage, reduced labor, and lower reagent costs. However, for samples with low moisture or high-fat content, the purification effect is not ideal, the extraction efficiency is low, and the purification process has drawbacks. This sample preparation applies to detect pesticide residues in food, olive oil, cereals, milk, fish, and drugs in the blood. QuEChERS is intended for analysis on GC.
- Accelerated Solvent Extraction (ASE)
ASE is an extraction method that extracts organic compounds from a solid or semi-solid matrix with a liquid solvent. Samples suitable for this extraction include honey, pesticides, TPH, hesperidin in orange peels, and samples containing dioxins.
The advantages of ASE sample preparation, namely the automated system, faster extraction time compared to Soxhlet and Sonication extraction, easy to use, requiring 50 – 90% less solvent than other extraction methods, and no human resources.
Preparation for Volatile Substances
- Static Headspace (SHS)
Headspace is the gas phase or vapor portion of the sample in the closed chromatography vial. SHS is used for the extraction of volatile and semi-volatile analytes in liquids. SHS method can apply to extract solid or liquid samples in GC instruments. In SHS, the specimen is placed in a sealed vial and heated. The volatile components migrate out of the sample matrix and into the headspace of the vial. Part of the headspace is then sampled and transferred to the GC for analysis.
You can apply this method for blood alcohol levels, residual solvents in pharmaceuticals, flavors in food and beverages, and fragrances in perfumes and detergents. On the other hand, SPS is often used as a complementarity screening method for high-concentration samples. This sample preparation is effortless and fast. Accordingly, the solvents and reagents used are relatively low.
- Purge and Trap (P+T)
The Purge and Trap technique analyzes volatile organic compounds using GC instruments. P+T developed in the 1970s in Cincinnati. There are three main processes in P+T, namely extraction (purging), simultaneous adsorption (trapping), and subsequent desorption (heating).
P+T involves purging an inert gas through a sample containing volatile organic compounds (VOCs) to an adsorbent trap. Then the VOC is absorbed from the Trap by heating and transferring the analyte to the GC column. This technique is ideal for extracting and concentrating VOCs from water, soil, and silt. P+T is more sensitive than SHS. Also, more samples can be analyzed. Hence, suitable for trace analysis with detection limits in ppb to ppt. However, P+T hardware is more complex than SHS.
- Solid-Phase Micro Extraction (SPME)
SPME was discovered in 1987 by Pawliszyn and Liu. SPME sample preparation technique is a modification of SLE preparation. SPME is solvent-free sample preparation. SPME uses layers of silica fibers to target volatile and semi-volatile compounds from samples, for instance, blood, serum, wastewater, groundwater, and meat.
Applications of this extraction method include environmental, biological, pharmaceutical, food and beverage, flavors and perfumes, forensics, and toxicology. Some of the advantages of this extraction method include being solvent free, easy to automate, not damaging the sample, applying to almost all matrices, the fibers used are reusable and inexpensive, small size fibers make them suitable for fieldwork, and compatible with GC or HPLC instruments.
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