Importance of sample pretreatment
Sample pretreatment is a time-consuming and error-prone step in instrumental analysis (especially chromatographic analysis). The quality of sample treatment directly affects the final result of chromatographic analysis. Therefore, in order to improve the efficiency of analysis and determination, improving and optimizing the sample preparation methods and techniques for chromatographic analysis is an important issue. Since some samples belong to complex matrix systems, containing components such as proteins, oils, carbohydrates, pigments, etc., the complex matrix background will cause great trouble to the extraction, separation, purification and determination of the target compounds to be analyzed. Therefore, sample pretreatment is not only complex and difficult, but also has a decisive role in the accuracy, reliability and sensitivity of the analysis results.
Percentage of sample pretreatment time consumption
For LC/MS/MS highly sensitive instruments, proper sample pretreatment is crucial to reduce matrix interference and enrich components.
Principles of sample pretreatment
Avoid chemical changes in components during preparation; prevent and avoid contamination of pre-determined components; minimize the introduction of irrelevant compounds into the preparation process; and make it as simple and easy as possible.
Purpose of sample pretreatment
Remove particles; reduce interfering impurities; concentrate trace components; improve detection sensitivity and selectivity; improve separation effect; protect chromatographic columns and instruments; solvent replacement.
Development trend of sample pretreatment
▶Common sample pretreatment includes: Digestion method: a method of placing the sample with acid, oxidant, catalyst, etc. in a reflux device or a closed device, heating and decomposing and destroying organic matter. Wet digestion method
1. Nitric acid digestion method (for clearer aqueous solution samples) 2. Nitric acid-perchloric acid digestion method (digestion of samples containing difficult-to-oxidize organic matter) 3. Nitric acid-sulfuric acid digestion method (nitric acid: sulfuric acid = 5:2, often adding a small amount of hydrogen peroxide) 4. Sulfuric acid-phosphoric acid digestion method (helps to eliminate the interference of Fe3+ ions during determination) 5. Sulfuric acid-potassium permanganate digestion method (commonly used to determine aqueous solution samples of mercury) 6. Nitric acid-hydrogen peroxide digestion method: Some people use this method to digest biological products to determine nitrogen, phosphorus, potassium, boron, arsenic, fluorine and other elements 7. Multi-component digestion method: A three- or more acid or oxidant digestion system is required. Dry ash method (high temperature decomposition method)
1. The ash method does not use or uses a small amount of chemical reagents to decompose samples, and can handle larger weighing samples, so it is beneficial to improve the accuracy of trace element determination. 2. The ashing temperature is generally 450-550℃, which is not suitable for processing samples with volatile components, and the ashing time is also relatively long. 3. According to the type of sample and the properties of the components to be measured, different crucibles and ashing temperatures are selected. Commonly used crucibles are quartz, platinum, silver, nickel, iron, porcelain, polytetrafluoroethylene and other properties. The principle is that the crucible does not react with the sample and is stable at the processing temperature. 4. Usually, no other reagents are added to the ashing biological samples, but in order to promote decomposition and inhibit the volatilization loss of certain elements, an appropriate amount of auxiliary ashing agent is often added. After the sample is completely ashed, it is dissolved in dilute nitric acid or hydrochloric acid for analysis and determination.
Conventional pretreatment methods Extraction and enrichment 1. Extraction method 1. Oscillation extraction method (vegetables, fruits, grains) 2. Tissue crushing extraction (extracting organic pollutants from animal and plant tissues) 3. Soxhlet extraction (commonly used to extract organic pollutants such as pesticides, petroleum, phenylhydrazine and pyrene from biological and soil samples) 2. Volatilization and evaporation concentration The volatile separation method uses the high volatility of certain components or converts the components to be measured into volatile substances, and then uses inert gas to take them out to achieve the purpose of separation. Evaporation concentration refers to heating the water sample on a hot plate or in a water bath to slowly evaporate the water, so as to reduce the volume of the water sample and concentrate the components to be measured. 3. Distillation method uses the different boiling points of the components of the water sample to separate them from each other; when determining volatile phenols, cyanides, and fluorides in water samples, they must first be pre-distilled and separated in an acidic medium; distillation has three functions: digestion, enrichment, and separation. 4. Ion exchange method uses ion exchangers to exchange reactions with ions in the solution for separation. Ion exchangers can be divided into inorganic ion exchangers and organic ion exchangers (ion exchange resins). ㈤ Coprecipitation method: The phenomenon that a poorly soluble compound in a solution carries out certain coexisting trace components in the process of forming a precipitate. The principle of coprecipitation is based on surface adsorption, the formation of mixed crystals, the interaction and inclusion of heteroelectron nuclei colloidal substances, etc. 1. Coprecipitation separation using adsorption: Common carriers include Fe (OH) 3, Al (OH) 3, Mn (OH) 2 and sulfides, etc. 2. Coprecipitation separation using the formation of mixed crystals 3. Coprecipitation separation using organic coprecipitants ㈥ Adsorption method: Use porous solid adsorbents to adsorb one or several components in the water sample on the surface to achieve the purpose of separation. Commonly used adsorbents include activated carbon, alumina, molecular sieves, large mesh resins, etc. The polluted components adsorbed and enriched on the surface of the adsorbent can be desorbed by organic solvents or heated for determination. ㈦ Chromatography Chromatography is divided into column chromatography, thin layer chromatography, paper chromatography, etc., and adsorbents are divided into inorganic adsorbents and organic adsorbents. ㈧ Sulfonation and saponification Sulfonation: The interfering substances such as fats and waxes in the extract can undergo sulfonation reaction with concentrated sulfuric acid to generate highly polar sulfonic acid compounds, which are separated from the pesticides in the extract as the sulfuric acid layer separates. The sulfonation method uses the saponification reaction of oils and fats with strong alkali to generate fatty acid salts and separate them. ㈨ Low-temperature freezing method is based on the principle that the solubility of different substances in the same solvent varies with temperature to separate them from each other. ㈩ Principle of extraction: The distribution coefficient of substances in different solvent phases is different, so as to achieve the separation and enrichment of components. Types of conventional liquid-liquid extraction Extraction of organic substances: Organic substances separated in the aqueous phase are easily extracted by organic solvents Extraction of inorganic substances: First, a reagent is added to combine with the ionic components in the aqueous phase to generate a substance that is uncharged and easily soluble in organic solvents. The reagent, organic phase, and aqueous phase together form an extraction system. According to the different types of extractables generated, it can be divided into chelate extraction system, ion-association complex extraction system, ternary complex extraction system, and synergistic extraction system. Overview of solid phase extraction (SPE) It is developed by combining liquid-solid extraction and column liquid chromatography technology. SPE is a column chromatography separation process, which has many similarities with high performance liquid chromatography (HLPC) in terms of separation mechanism, stationary phase and solvent selection. The particle size of SPE filler (>40μm) is larger than that of HLPC (3-10μm). Therefore, SPE can only be used to separate compounds with very different retention properties. SPE technology with low separation efficiency is mainly used to process samples. The purpose of SPE is to remove substances that interfere with subsequent analysis from the sample; enrich trace components and improve analytical sensitivity; change the sample solvent to match the analytical method; in-situ derivatization; sample desalting; and facilitate the storage and transportation of samples. Installation SPE column: The filler particle size is different from the HLPC column filler, and the rest is the same. The most used is the C18 phase. This type of filler is highly hydrophobic and shows retention for most organic matter in the aqueous phase; other materials with different selectivity and retention properties are also used. SPE phases with active groups or coated with active compounds can be used to analyze derivatization reactions. SPE disk: very similar to membrane filters. The disk extractor is a PTFE disc containing filler or a glass fiber sheet loaded with filler; the filler accounts for about 60% to 90% of the total SPE disk, and the thickness of the disk is about 1mm. The difference from the former is the bed thickness/diameter (L/d) ratio. Suitable for enriching trace pollutants from water. Solid Phase Microextraction (SPME) Offline and Online SPE Offline SPE 1. SPE and analysis are performed independently, and SPE only provides suitable samples for subsequent analysis. 2. In order to ensure sufficient contact between the sample solution and the filler, the solvent flow cannot be too high. 3. It can be completed by automated instruments. The automatic SPE instrument consists of a column rack, a plunger pump, a liquid reservoir, a pipeline and a sample processor. Online SPE is also known as online purification and enrichment technology, which is mainly used for the establishment of HLPC analysis SPE method Column pretreatment purpose: 1. Remove impurities that may exist in the filler; 2. Solventize the filler and improve the reproducibility of solid phase extraction Sample addition 1. To prevent the loss of analytes, the sample solvent concentration should not be too high; 2. When extracting with reverse phase mechanics, water or buffer is used as the solvent, and the amount of organic solvent does not exceed 10% (V/V); 3. To overcome the loss of analytes during sample addition, weak solvents can be used to dilute the sample, reduce the sample volume, increase the amount of filler in the SPE column, and select adsorbents that have strong retention of the analyte. Elution and collection of analytes (another case is that impurities are retained while analytes pass through the column) (Solid phase extraction with solid dispersion media) 1. For reverse phase extraction columns, the cleaning solvent is water or buffer containing an appropriate concentration of organic solvent; 2. To determine the optimal concentration and volume of the cleaning solvent, add the sample to the SPE column, clean it with 5 to 10 times the volume of the SPE column bed, collect and analyze the effluent in turn, and obtain the elution profile of the cleaning solvent for the analyte. Increase the strength of the cleaning solvent in turn, and determine the appropriate strength and volume of the cleaning solvent according to the elution profile of the analyte at different strengths; 3. Purpose of elution and collection: to completely elute the analyte and collect it in the smallest volume fraction, while retaining as many impurities as possible that are more strongly retained than the analyte on the SPE column; 4. To increase the concentration of the analyte or adjust the solvent properties for subsequent analysis, the collected analyte fraction can be blown dry with nitrogen and then dissolved in a small volume of solvent. Application of SPE Environmental Analysis 1. The concentration of analytes in environmental samples such as surface water is very low, and the analyte must be enriched before analysis. 2. The composition of biological fluids is complex and contains a large amount of protein. Before analysis, the sample needs to be pretreated to remove the protein. Drug analysis Clinical analysis Food and beverage analysis Solid phase microextraction (SPME) Solid phase microextraction integrates "sampling, extraction, concentration, and injection" and can be used in conjunction with gas chromatography or high performance liquid chromatography for sample pretreatment technology. Solid phase microextraction theory Equilibrium theory: During the adsorption process, an adsorption equilibrium is established between the solid and liquid or gas phase. Within a certain period of time, due to the slow mass transfer process, the equilibrium is not fully reached. The selectivity of the coating material extraction mainly depends on the performance of the coating material. According to the principle that the analyte is easily extracted by a solid phase with similar polarity, a suitable SPE coating is selected. The most commonly used substances for solid phase coatings are polymethylsiloxane (PDMS) and polyacrylate (PA), both of which can be used for gas chromatography and liquid chromatography. The former is mostly used for non-polar compounds such as volatile compounds, polycyclic aromatic hydrocarbons and aromatic hydrocarbons, and the latter is mostly used for polar compounds such as triazines and phenolic compounds. The solid phase layer can be coated on the quartz fiber in a non-bonded, bonded or partially cross-linked form. Adding some polymers to the coating can increase the surface area of the coating and improve the efficiency of SPME. 1. Polydimethylsiloxane-divinylbenzene (PDMS-DVB), used for aromatic hydrocarbons and volatile compounds. 2. Polyethylene glycol-divinylbenzene (CW-DVB), used for polar compounds such as alcohols. 3. Polyethylene glycol-template resin (CW-TPR), used for ionized surfactants 4. Quartz fiber coated with graphite carbon black, used to analyze trace pollutants in water and air. 5. Establishment of carbon nanotube and titanium dioxide nanotube methods 1. Maintain the consistency of sampling conditions. 2. Factors affecting sampling include sampling time, temperature, fiber depth, etc. 3. Maintain a linear relationship between the response value and the initial concentration of the analyte. The sample concentration cannot be too high and the sample volume cannot be too small, so that the extraction is within the linear range of the adsorption isotherm. 4. Adding electrolytes to the sample can increase the ionic strength of the solution, thereby reducing the solubility of the analyte and improving the extraction efficiency; changing the pH of the sample has a greater effect on the extraction rate of acidic and alkaline substances. Note: The effect of adding salt in microextraction is sometimes different from that of conventional liquid-liquid extraction, and the experimental conditions need to be optimized. 5. Stirring can shorten the extraction time. Microwave extraction (MAE) Microwave extraction has short extraction time, good selectivity, high recovery rate, low reagent usage, low pollution, can use water as an extractant, and can automatically control sample preparation conditions; it has fewer applications and is currently used in the extraction of polycyclic aromatic hydrocarbons, pesticide residues, organometallic compounds, active ingredients in plants, harmful substances, metals in minerals, drugs in blood, and pesticide residues in biological samples. Principles and characteristics of microwave extraction methods Absorb microwaves (water, ethanol, acid, alkali and salts) High efficiency of microwave extraction: 1. Direct action of microwaves on the separated substances; 2. It is more advantageous to use polar solvents than non-polar solvents for microwave extraction; 3. The use of closed containers allows microwave extraction to be carried out at a temperature much higher than the boiling point of the solvent, significantly improving the efficiency of microwave extraction Reflect microwaves (metallic substances) Transmit microwaves (non-polar substances) Microwave extraction equipment and methods (the main components are specially manufactured microwave heating devices, extraction containers and pressure and temperature control devices equipped according to different requirements) Multi-cavity 2450MHz: Multiple samples can be prepared at one time, it is easy to control the extraction conditions, and the extraction is fast. Conventional microwave extraction method: Mix polar solvents or a mixture of polar solvents and non-polar solvents with the extracted samples, put them into microwave sample preparation containers, and heat them in a microwave sample preparation system under a closed state. Control the extraction pressure or temperature and time according to the requirements of the extracted components; at the end of heating, filter the sample, and the filtrate is directly measured, or measured after corresponding treatment. Under normal circumstances, the microwave extraction heating time is about 5 to 10 minutes. The total volume of the extraction solvent and sample shall not exceed 1/3 of the volume of the sample preparation cup. Single-mode focusing 2450MHz: No pressure and temperature control is required, the sample preparation volume is large, only one sample can be prepared at a time, and the extraction time is long. Supercritical fluid extraction (SCF)
Supercritical fluid (SCF) is a fluid whose temperature and pressure are both higher than the critical point. Its own characteristics are: 1. Its diffusion coefficient is smaller than that of gas, but one order of magnitude higher than that of liquid; 2. Its viscosity is close to that of gas; 3. Its density is similar to that of liquid, and a slight change in pressure can lead to a significant change in its density; 4. Changes in pressure or temperature can lead to phase changes. Basic principle In the supercritical state, the supercritical fluid is contacted with the substance to be separated, so that it can selectively extract the components of polarity, boiling point and relative molecular mass in turn, and the density and dielectric constant of the supercritical fluid increase with the increase of the pressure of the closed system, and the polarity increases. The components of different polarities can be extracted step by step by using program boost. The solubility of supercritical CO2: 1. Lipophilic and low-boiling components can be extracted at low pressure (104kPa); 2. The more polar groups a compound has, the more difficult it is to extract; 3. The higher the relative molecular mass of the compound, the more difficult it is to extract. Modifier CO2 is a non-polar solvent, and generally a polar solvent is added to improve its solubility in CO2, so it is called a modifier. The more commonly used ones are methanol, acetone, ethanol, ethyl acetate, etc. The effect of the modifier is limited. While changing the solubility of the supercritical fluid, it will also weaken the capture effect of the extraction system, resulting in an increase in co-extracts, which may interfere with analytical determination. The amount of modifier used should be small, generally not exceeding 5%. The application of supercritical fluid extraction technology has great advantages in the extraction of natural substances; it can be used in conjunction with GC, IR, MS, LC, etc. to become an efficient analytical method.
