유기화학실험 column chromatography

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1. Column Chromatography

Column Chromatography를 통해 eluent의 형태인 이동상과 고정상(column material, absorption material, commonly it's silica gel)의 흡착도 차이를 이용해 desired product를 mixture에서 separation할 수 있다. column에 eluent를 내리면 mixture의 구성 성분들이 고정상을 따라 같이 내려오는 것이다. 즉, 이동상 덕분에 Mixture의 각 compound들은 고정상과 흡착하는 정도에 따라 retention time(column을 통과해 나오는데 걸리는 시간)이 달라 compound끼리 분리된다.
2. Retention

Column Chromatography에서 retention이란 어떠한 물질이 column을 빠져나오는데 걸리는 속도를 측정한 것을 말한다. 계속해서 develop해서 특정 화합물이 eluent와 같이 끝까지 나오는 HPLC나 GC에서는 그 통과하는데 걸리는 시간을 Retention time으로 측정하지만 전개가 중간에서 멈추는 TLC 같은 경우에는 Rf value(spot이 이동한 거리와 eluent가 이동한 거리의 비로 화합물과 온도와 용리액에 무엇인지, 고정상이 무엇인지에 따라 값이 달라진다.)로 표현한다.
3. Plate Theory

Chromatography의 단이론에서 column은 plate라는 가상적인 개념으로 이루어져 있는데, plate 별로 compound가 이동상과 고정상 간에 partition equilibrium(solute가 서로 접촉하지만 혼합되지 않는 liquid phase들에 각각 용해되어 equilibrium을 이루는 것)에 도달하여 분리된다. 이는 분배의 법칙으로도 설명할 수 있다.
4. Partition Equilibrium

분배의 법칙이 적용되는 equilibrium에서는 insoluble한 two phase에 용질이 분배된다. 즉 어떠한 물질 A가 평형 상태에서 phase 1과 phase 2에 분배될 때 식 2.와 같이 나타낼 수 있다. 이때, 식3.에서 approximation method에 의해 activity는 molarity로 대체해 사용 가능하며, Column Chromatography에서 phase1은 이동상, phase2는 고정상이 된다. 따라서 분배계수 K와 solute가 분리되는데 걸리는 시간은 비례하므로, retention time이나 retention volume은 분배계수를 이용하여 구할 수 있다.
5. Column Chromatography Procedure

Column Chromatography에서 이동상은 위쪽에서부터 고정상과 solid absorbent이 담긴 수직 형태의 유리로 만들어진 관을 통과하면서 혼합물로부터 원하는 화합물만 분리한다. 이때, 사용할 수 있는 방법에는 retention time이 긴 gravity를 이용하는 방법(대기압 하에서 그냥 흘러내리도록 두는 것)과 eluent가 빨리 빠져나오는 Flash Column Chromatography(외부에서 air pressure을 위쪽에 가해주는 것) 두가지가 있는데 분리능에는 차이가 없으므로 현재는 Flash Column Chromatography만을 사용한다.
6. Absorbent

유기화학에서 absorbent로는 주로 alumina보다는 silica gel을 사용하는데, 이 absorbent의 mesh size(mesh가 클수록 absorbent의 particle 크기가 작아져, 같은 면적의 absorbent를 통과할 수 있는 개수가 많아진다.)에 따라 separation efficiency가 달라진다. Silica gel 60은 gravity chromatography(larger particle, lower mesh value)에 주로 사용하고, silica gell 230~400은 flash column chromatography(smaller particle, higher mesh value)에 주로 사용한다.
7. Solvent Polarity

유기물은 쌍극자-쌍극자/수소결합/쌍극자-유도쌍극자/반데르발스힘에 따라 absorbent와 결합한다. polar한 silica gel은 극성 작용기를 가진 compound일 수록 흡착이 잘되고, 반면 eluent 속 concentration은 작아진다. 즉, silica gel에서는 쌍극자-쌍극자 힘이 가장 영향이 많이 미치기 때문에, 극성인 물질일수록 silica와 binding을 강하게 하여 흡착하므로 Rf value는 작아지고 retention time은 커지고 평형에서 eluent를 따라 느리게 이동하게 된다.
8. TLC Monitoring

TLC를 통해 Column Chromatography에서 먼저 내려올 물질이 무엇인지 예측할 수 있고, 또 얻어낸 물질들이 각각 어떤 물질인지, single compound로 나왔는지도 알 수 있다.
9. Experimental Procedure

실험 과정에서 주의해야 할 점으로는 silica gel이 날리기 쉽고 호흡기에 좋지 않으므로 꼭 hood 안에서 사용하고, packing 시 수직 수평을 잘 맞추며, sample은 thin layer로 loading해야 한다. 또한 eluent의 조성을 중간에 바꾸어 주는 이유는 benzophenone과 vanillin의 Rf value 차이가 크기 때문이다.
10. Separation Efficiency

Column Chromatography에서 분리능을 높이기 위해서는 packing 과정을 잘 수행하고, sample을 thin layer로 loading하며, 적절한 eluent 조성을 선택하는 것이 중요하다.

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1. Column Chromatography

Column chromatography is a powerful analytical technique that allows for the separation and purification of complex mixtures. It relies on the differential partitioning of analytes between a stationary phase and a mobile phase as they flow through a column. The stationary phase, typically a packed bed of solid particles, interacts with the analytes based on their physical and chemical properties, such as size, charge, or polarity. The mobile phase, which can be a liquid or a gas, carries the analytes through the column, and the rate at which they move through the column depends on their affinity for the stationary phase. This differential partitioning leads to the separation of the analytes, allowing for their identification and quantification. Column chromatography is widely used in various fields, including organic chemistry, biochemistry, and environmental analysis, due to its versatility, high resolution, and ability to handle complex samples. The technique continues to evolve, with advancements in stationary phase materials, column design, and detection methods, making it an indispensable tool in modern analytical chemistry.
2. Retention

Retention in column chromatography refers to the time it takes for an analyte to elute or emerge from the column. The retention time is a crucial parameter that helps identify and quantify the analytes in a sample. The retention time of an analyte is influenced by various factors, such as the nature of the stationary phase, the composition of the mobile phase, the flow rate, and the physicochemical properties of the analyte itself. Understanding and controlling these factors is essential for optimizing the separation and achieving reliable results. Factors that increase the retention time, such as stronger interactions between the analyte and the stationary phase, can lead to better separation, but may also result in longer analysis times. Conversely, factors that decrease the retention time, such as increasing the mobile phase flow rate or using a less retentive stationary phase, can improve the speed of the analysis but may compromise the separation efficiency. Careful optimization of the chromatographic conditions is necessary to balance these competing factors and achieve the desired separation performance.
3. Plate Theory

The plate theory is a fundamental concept in column chromatography that describes the efficiency of the separation process. It is based on the idea that the column can be divided into a series of hypothetical, theoretical plates, where equilibrium is established between the analyte in the mobile phase and the analyte in the stationary phase. The number of theoretical plates, or the plate count, is a measure of the column's efficiency, with a higher plate count indicating better separation. The plate height, which is the height equivalent to a theoretical plate (HETP), is inversely proportional to the plate count, and it is used to evaluate the column performance. Factors that affect the plate height, such as the particle size and packing of the stationary phase, the flow rate, and the nature of the analyte-stationary phase interactions, can be optimized to improve the separation efficiency. The plate theory provides a framework for understanding and predicting the behavior of analytes in column chromatography, and it is a valuable tool for method development and optimization.
4. Partition Equilibrium

Partition equilibrium is a fundamental concept in column chromatography that describes the distribution of an analyte between the mobile phase and the stationary phase. When an analyte is introduced into the column, it will partition between the two phases based on its affinity for each phase. The partition coefficient, or the distribution coefficient, is a measure of this affinity and is defined as the ratio of the concentration of the analyte in the stationary phase to the concentration of the analyte in the mobile phase at equilibrium. The partition coefficient is influenced by various factors, such as the polarity of the analyte, the polarity of the mobile and stationary phases, and the temperature. Analytes with a higher partition coefficient will spend more time in the stationary phase and will have a longer retention time, while analytes with a lower partition coefficient will spend more time in the mobile phase and will have a shorter retention time. Understanding and controlling the partition equilibrium is crucial for optimizing the separation of analytes in column chromatography, as it allows for the manipulation of the retention times and the resolution between peaks.
5. Column Chromatography Procedure

The column chromatography procedure involves several key steps to achieve effective separation and purification of analytes. The first step is the preparation of the stationary phase, which typically involves packing a column with a suitable adsorbent material, such as silica gel, alumina, or ion-exchange resins. The sample is then loaded onto the top of the column, and the mobile phase is introduced. As the mobile phase flows through the column, the analytes partition between the stationary and mobile phases, leading to their separation. The eluent from the column is collected in fractions, and the presence and purity of the analytes in each fraction can be monitored using various detection methods, such as UV-Vis spectroscopy, mass spectrometry, or refractive index detection. The collected fractions can then be further processed, depending on the specific application, such as concentration, solvent exchange, or further purification. The column chromatography procedure requires careful optimization of parameters, such as the choice of stationary and mobile phases, flow rate, sample loading, and detection methods, to achieve the desired separation and purity of the target analytes.
6. Absorbent

The absorbent, or stationary phase, is a critical component in column chromatography, as it plays a crucial role in the separation and purification of analytes. The choice of absorbent material is based on the physicochemical properties of the analytes and the desired separation mechanism. Common absorbent materials used in column chromatography include silica gel, alumina, ion-exchange resins, and molecular sieves, among others. Each absorbent material has its own unique characteristics, such as surface area, pore size, and polarity, which can be tailored to the specific separation needs. For example, silica gel is a widely used absorbent due to its versatility in separating a wide range of organic compounds based on their polarity. Ion-exchange resins, on the other hand, are useful for separating ionic species based on their charge. The particle size, packing density, and surface modifications of the absorbent material can also be optimized to improve the separation efficiency, resolution, and peak shape. The careful selection and preparation of the absorbent is crucial for achieving successful separations in column chromatography.
7. Solvent Polarity

Solvent polarity is a critical factor in column chromatography, as it determines the interactions between the analytes, the mobile phase, and the stationary phase. The polarity of the mobile phase can be adjusted by using solvents with different polarities, such as water, methanol, acetonitrile, or hexane, or by using solvent mixtures. The choice of mobile phase polarity is based on the polarity of the analytes and the desired separation mechanism. For example, in normal-phase chromatography, where the stationary phase is polar (e.g., silica gel) and the mobile phase is non-polar (e.g., hexane), more polar analytes will have a stronger interaction with the stationary phase and will elute later, while less polar analytes will have a weaker interaction and will elute earlier. In reverse-phase chromatography, where the stationary phase is non-polar (e.g., C18) and the mobile phase is polar (e.g., water-acetonitrile), the opposite is true. Careful selection and optimization of the mobile phase polarity, often through gradient elution, can significantly improve the separation and resolution of complex mixtures in column chromatography.
8. TLC Monitoring

Thin-layer chromatography (TLC) is a valuable tool for monitoring the progress and efficiency of column chromatography separations. TLC can be used to quickly assess the composition of the fractions collected from the column, allowing for the identification of the desired analytes and the optimization of the chromatographic conditions. In a typical TLC monitoring process, small aliquots of the column fractions are spotted onto a TLC plate and developed using a suitable mobile phase. The separated components can then be visualized using various detection methods, such as UV light, chemical staining, or fluorescence. By comparing the retention factors (Rf values) and the spot patterns of the column fractions to those of known standards or reference compounds, the presence and purity of the target analytes can be determined. TLC monitoring can also help identify the appropriate fractions to be combined for further purification or analysis. The simplicity, speed, and low cost of TLC make it a complementary technique that is widely used in conjunction with column chromatography to ensure the successful separation and isolation of the desired compounds.
9. Experimental Procedure

The experimental procedure for column chromatography involves several critical steps that must be carefully executed to ensure the success of the separation and purification process. The first step is the preparation of the stationary phase, which typically involves packing a glass or stainless-steel column with a suitable adsorbent material, such as silica gel or alumina. The column is then equilibrated with the appropriate mobile phase. Next, the sample containing the analytes of interest is loaded onto the top of the column, either as a solution or as a solid adsorbent. The mobile phase is then introduced, and the analytes begin to partition between the stationary and mobile phases as they flow through the column. The eluent from the column is collected in fractions, and the presence and purity of the analytes in each fraction are monitored using various detection methods, such as UV-Vis spectroscopy or thin-layer chromatography (TLC). The collected fractions can then be further processed, depending on the specific application, such as concentration, solvent exchange, or further purification. Throughout the experimental procedure, it is essential to carefully control and optimize parameters such as the choice of stationary and mobile phases, flow rate, sample loading, and detection methods to achieve the desired separation and purity of the target analytes.
10. Separation Efficiency

The separation efficiency in column chromatography is a critical factor that determines the quality and resolution of the separation. It is influenced by a variety of parameters, including the choice of stationary phase, mobile phase composition, flow rate, sample loading, and column dimensions. The separation efficiency can be evaluated using various metrics, such as the number of theoretical plates, the height equivalent to a theoretical plate (HETP), and the resolution between adjacent peaks. A higher number of theoretical plates and a lower HETP indicate a more efficient separation, as they reflect the ability of the column to create a large number of equilibrium stages and minimize band broadening. The resolution, on the other hand, measures the degree of separation between two adjacent peaks and is influenced by factors such as the selectivity, efficiency, and capacity of the column. Optimizing these parameters through careful method development and optimization is essential for achieving high-quality separations in column chromatography. This may involve experimenting with different stationary phases, mobile phase compositions, flow rates, and sample loading techniques to find the optimal conditions for the specific analytes and separation goals. Improving the separation efficiency can lead to better peak resolution, higher purity of the isolated compounds, and more reliable quantitative analysis.

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