서울여자대학교 유기화학실험 Column chromatography 결과 레포트

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1. Column chromatography

Column chromatography는 혼합물로부터 원하는 물질을 mobile phase (eluent)에서 Stationary phase (column material)에 대한 흡착력의 차이를 이용하여 분리하는 것이다. Eluent를 column에 통과시키면 혼합물에 존재하는 ingredient들이 stationary phase를 따라 움직이는데 각 ingredient의 stationary phase에 대한 adsorption 정도가 다르기 때문에 column을 빠져나오는데 걸리는 retention time이 각각 다르다. 이러한 차이를 이용하여 원하는 성분 물질을 분리할 수 있다.
2. Retention

Retention은 chromatographic system으로 물질이 이동하는 속도를 측정한 것을 말한다. 화합물이 eluent으로 용리 되는 HPLC 또는 GC와 같은 연속 현상 시스템에서, Retention은 일반적으로 Retention time Rt 또는 tR, 주입과 검출 사이의 시간으로 측정된다. TLC와 같은 interrupted development system에서 retention은 지연 계수 Rf로 측정되며, 화합물의 run length를 eluent 전면의 run length로 나눈 값이다.
3. Plate theory

크로마토그래피의 Plate theory 는 Archer John Porter Martin 과 Richard Laurence Millington Synge 에 의해 개발되었다. Plate theory 는 크로마토그래피 시스템, 이동상 및 정지상을 평형 상태로 나타낸다. 분배 계수 K는 평형을 기반으로 하며 다음 방정식으로 정의된다.
4. Column Chromatography

Column chromatography에서는 stationary phase와 solid adsorbent가 vertical glass column에 위치하고 mobile phase인 solvent는 column의 위에서 가하여 아래로 흘러내리게 한다. 이 때, gravity를 이용하는 것과 external pressure를 가해주는 두 가지 경우가 있는데 압력을 가해주는 방식이 좀 더 빠르게 column을 흘러나오게 할 수 있는 방법이다.
5. Stationary phase

Column chromatography의 가장 일반적인 고정상은 silica gel이며, 다음으로 가장 일반적인 것은 알루미나이다. 고정상은 보통 미세하게 분쇄된 분말 또는 겔이고 또는 증가된 표면을 위해 미세 다공성이지만, EBA 에서는 유동층이 사용된다.
6. Mobile phase

이동상 또는 용리제는 column을 통해 화합물을 이동시키는데 사용되는 용매를 말한다. Chromatography를 수행하기 위한 시간 및 용리약의 양을 최소화하기 위헤 관심있는 화합물의 보유 인자 값이 대략 0.2 내지 0.3 이 되도록 선택해야한다.
7. Categories of Column Chromatography

일반적인 chromatography의 분류로는 Mobile Phase Classification, Solvent에 따른 분류, Column packing material에 따른 분류가 있다.
8. Adsorbent

유기화학에서 absorbent로는 주로 alumina보다는 silica gel을 사용하는데, 이 absorbent의 mesh size에 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.
9. Compound-adsorbent interaction

유기물은 쌍극자-쌍극자/수소결합/쌍극자-유도쌍극자/반데르발스힘에 따라 absorbent와 결합한다. polar한 silica gel은 극성 작용기를 가진 compound일 수록 흡착이 잘되고, 반면 eluent 속 concentration 은 작아진다.
10. Experimental procedure and observation

실험 과정에서 관찰한 내용으로는 hexane: ethyl acetate = 3 : 1의 eluent가 내려오면서 옅은 노란색 band가 점점 내려오는 것, test tube22번부터 사라졌던 spot이 나오고 test tube 24번부터 vanilla 향이 나는 것 등이 있다.

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1. Column chromatography

Column chromatography is a powerful analytical technique used to separate and purify a wide range of chemical compounds. It involves the use of a stationary phase, typically a solid adsorbent material packed into a column, and a mobile phase, which is a liquid or gas that flows through the column. The separation of compounds is based on their differential interactions with the stationary phase, which can be influenced by factors such as polarity, size, and charge. Column chromatography is widely used in various fields, including organic chemistry, biochemistry, and environmental analysis, to isolate and identify specific compounds from complex mixtures. The technique is known for its high resolution, versatility, and ability to handle a wide range of sample sizes. Understanding the principles and applications of column chromatography is crucial for many scientific and industrial processes.
2. Retention

Retention in column chromatography refers to the degree to which a compound is retained or held back by the stationary phase during the separation process. The retention of a compound is influenced by various factors, such as the nature of the stationary phase, the mobile phase composition, the interactions between the analyte and the stationary phase, and the physical and chemical properties of the compound itself. Understanding and controlling the retention behavior of compounds is essential for optimizing the separation efficiency and achieving the desired separation outcomes. Factors that affect retention include the polarity, size, and charge of the analyte, as well as the pH, ionic strength, and organic modifier content of the mobile phase. Careful selection and manipulation of these parameters can lead to improved resolution, selectivity, and sensitivity in column chromatographic analyses.
3. Plate theory

The plate theory is a fundamental concept in column chromatography that describes the separation efficiency of the technique. It is based on the idea that the column can be divided into a series of theoretical plates, each of which represents a stage of equilibrium between the stationary and mobile phases. The more theoretical plates a column has, the higher its separation efficiency. The number of theoretical plates is influenced by factors such as the column length, particle size and packing of the stationary phase, and the flow rate of the mobile phase. Optimizing these parameters can lead to improved resolution, peak shape, and sensitivity in chromatographic separations. The plate theory provides a quantitative framework for understanding and predicting the performance of column chromatography, and it is widely used in the design and optimization of chromatographic methods.
4. Column Chromatography

Column chromatography is a versatile and widely used separation technique in analytical chemistry, organic chemistry, and biochemistry. It involves the use of a stationary phase, typically a solid adsorbent material packed into a column, and a mobile phase, which is a liquid or gas that flows through the column. The separation of compounds is based on their differential interactions with the stationary phase, which can be influenced by factors such as polarity, size, and charge. Column chromatography is known for its high resolution, versatility, and ability to handle a wide range of sample sizes. It is used for a variety of applications, including the purification of organic compounds, the separation of complex mixtures, the analysis of environmental samples, and the isolation of biomolecules. Understanding the principles and optimization of column chromatography is crucial for many scientific and industrial processes, as it allows for the efficient separation, identification, and quantification of a wide range of chemical and biological compounds.
5. Stationary phase

The stationary phase in column chromatography is a crucial component that plays a key role in the separation of compounds. It is typically a solid adsorbent material, such as silica gel, alumina, or ion-exchange resins, packed into a column. The nature and properties of the stationary phase, such as its polarity, particle size, and surface area, can significantly influence the separation efficiency and selectivity of the chromatographic process. The interactions between the analyte molecules and the stationary phase, which can be based on adsorption, partition, or ion-exchange mechanisms, determine the retention and elution behavior of the compounds. Careful selection and optimization of the stationary phase is essential for achieving the desired separation, resolution, and sensitivity in column chromatography. The development of new and improved stationary phases, as well as the understanding of their underlying principles, continues to be an active area of research in the field of analytical chemistry.
6. Mobile phase

The mobile phase in column chromatography is the liquid or gas that flows through the column, carrying the sample components with it. The choice and composition of the mobile phase are crucial for the separation and elution of the analytes. The mobile phase can influence the interactions between the analytes and the stationary phase, affecting the retention and separation of the compounds. Factors such as the polarity, pH, ionic strength, and organic modifier content of the mobile phase can be adjusted to optimize the separation. The mobile phase should be carefully selected and optimized to achieve the desired selectivity, resolution, and sensitivity in the chromatographic analysis. The use of gradient elution, where the mobile phase composition is varied during the separation, can further enhance the separation efficiency and allow for the analysis of complex mixtures. Understanding the role and optimization of the mobile phase is essential for the successful application of column chromatography in various fields of analytical chemistry.
7. Categories of Column Chromatography

Column chromatography can be categorized into several types based on the underlying separation mechanism and the nature of the stationary and mobile phases. The main categories include: 1. Adsorption chromatography: Separation based on the differential adsorption of analytes onto the stationary phase. 2. Partition chromatography: Separation based on the differential partitioning of analytes between the stationary and mobile phases. 3. Ion-exchange chromatography: Separation based on the exchange of ions between the analytes and the charged stationary phase. 4. Size-exclusion chromatography: Separation based on the differences in the size and molecular weight of the analytes. 5. Affinity chromatography: Separation based on the specific interactions between the analyte and a ligand immobilized on the stationary phase. Each category of column chromatography has its own unique characteristics, advantages, and applications. Understanding the principles and selecting the appropriate type of column chromatography is crucial for achieving efficient and selective separations in various fields, such as organic chemistry, biochemistry, environmental analysis, and pharmaceutical research.
8. Adsorbent

The adsorbent is a crucial component in column chromatography, as it serves as the stationary phase and plays a key role in the separation of compounds. Adsorbents are typically solid materials with a high surface area, such as silica gel, alumina, or ion-exchange resins. The choice of adsorbent depends on the nature of the analytes and the desired separation mechanism. Factors such as the polarity, pore size, and surface chemistry of the adsorbent can significantly influence the retention and separation of the compounds. The interactions between the analytes and the adsorbent, which can be based on adsorption, partition, or ion-exchange, determine the elution behavior and the resolution of the separation. Careful selection and optimization of the adsorbent, as well as the mobile phase composition, are essential for achieving efficient and selective separations in column chromatography. The development of new and improved adsorbents, as well as the understanding of their underlying principles, continues to be an active area of research in the field of analytical chemistry.
9. Compound-adsorbent interaction

The interaction between the analyte compounds and the adsorbent material in column chromatography is a crucial factor that determines the separation efficiency and selectivity. These interactions can be based on various mechanisms, such as adsorption, partition, ion-exchange, or size exclusion, depending on the nature of the analytes and the stationary phase. The strength and selectivity of the compound-adsorbent interactions are influenced by factors like polarity, charge, size, and functional groups of both the analyte and the adsorbent. Understanding and controlling these interactions is essential for optimizing the separation process and achieving the desired resolution and selectivity. Strategies such as modifying the mobile phase composition, adjusting the adsorbent properties, or using specialized adsorbents can be employed to fine-tune the compound-adsorbent interactions and enhance the chromatographic performance. Continuous research and development in this area have led to the advancement of column chromatography techniques, enabling their widespread application in various fields of analytical chemistry, organic synthesis, and biochemistry.
10. Experimental procedure and observation

The experimental procedure and observation in column chromatography are crucial for the successful separation and purification of compounds. The typical steps involved in a column chromatography experiment include: 1. Preparation of the stationary phase: Packing the adsorbent material into the column and ensuring proper packing and equilibration. 2. Sample preparation: Dissolving the sample in a suitable solvent and applying it to the top of the column. 3. Elution: Passing the mobile phase through the column, allowing the compounds to separate based on their interactions with the stationary phase. 4. Fraction collection: Collecting the eluted fractions at specific time intervals or based on observed changes in the mobile phase composition. 5. Analysis and identification: Analyzing the collected fractions using techniques like UV-Vis spectroscopy, mass spectrometry, or nuclear magnetic resonance to identify and characterize the separated compounds. Careful observation and documentation of the experimental parameters, such as flow rate, mobile phase composition, and elution profiles, are essential for understanding the separation process and optimizing the chromatographic method. The ability to interpret the experimental data and correlate it with the underlying principles of column chromatography is crucial for the successful application of this technique in various fields of research and industry. Continuous improvement and innovation in experimental procedures and instrumentation have contributed to the advancement of column chromatography as a powerful analytical tool.

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