Column+Chromatography

=Column Chromatography =


 * Dylan Trost**

Technique:
Column chromatography is purification technique where certain compounds of sample mixture can be separated based on a certain characteristics, such as charge, hydrophobicity, and size. The matrix of the column effects the criteria for which the mixture is separated. The matrix usually consist of a absorbent (s ilica gel (SiO2) or alumina (Al2O3)  ) and a solvent. The affinity of the solvent used to the compounds present in the sample determines the rate a which compounds will pass though the column. Figure 1 illustrates the basic idea behind column chromatography using a polarity as an example is, the more polar substances in a sample will be attracted to the polar matrix and pass though the column slowly. The more non-polar substances will pass though the column at a faster rate and can be collected and separated from the sample mixture earlier. The purpose of the technique is to separate certain components based on many different matrices available (Alberts, Johnson, Lewis, Raff, Roberts, and Walter, 2002). In 1978, W. Clark Still invented a new form of column chromatography in which pressure is used to drive the solvent through, leading to more separations. This is known as flash column chromatography. The setup is similar to figure 1, except some sort of pressure, usually gas, is applied to the top. This can be seen in figure 2. 

History:
Chromatography was first utilized by Mikhail Tsvett, a Russian botanist. He wanted to separate plant pigments, and used calcium carbonate powder as his matrix. The resulting colored bands, he called a chromatogram. The technique was ignored for many years, but rediscovered by Richard Willstatter, a German organic chemist. He was also studying chlorophyll. The technique is now considered very versatile for organic chemists (“Chromatography,” 2012).

Experiments that Utilize Column Chromatography:
1. Deng, Y. //et al.// Acaricidal activity of petroleum ether extract of neem (Azadirachta indica) oil and its four fractions separated by column chromatography against Sarcoptes scabiei var. cuniculi larvae in vitro. //Experimental Parasitology// 130, 475–477 (2012). -In this paper column chromatography was used to isolate and purify dryofragin and aspidin BB from Dryopteris fragrans. These compounds are herbs that can be used to wash hair for getting rid of scurf and relieving itching. The results of this research paper saw dryofragin and aspidin BB to increase by 8.39- and 5.99-fold with recovery yields of 91.22% and 75.64%. 3.

2. Li, X.-J. //et al.// Preparative separation of dryofragin and aspidin BB from Dryopteris fragrans extracts by macroporous resin column chromatography. //Journal of Pharmaceutical and Biomedical Analysis// 61, 199–206 (2012). -This study used column chromatography to extract oil of neem in to four fractions. Neem oil has medicinal properties, including antifertility, antifungal, antibacterial, immunostimulant, antipyretic and acaricidal activities, which extraction and prufication of the oil is useful. The results showed that the oils were successfully extracted into 4 different fractions with a Acaricidal activity of 68.3% and 100.0% in the F2 and F4 was confirmed

3. Yang, W. //et al.// A strategy for efficient discovery of new natural compounds by integrating orthogonal column chromatography and liquid chromatography/mass spectrometry analysis: Its application in Panax ginseng, Panax quinquefolium and Panax notoginseng to characterize 437 potential new ginsenosides. //Analytica Chimica Acta// 739, 56–66 (2012). -In this paper column chromatography is used to discover natural compounds found in herbal medicines. Specifically ginsenosides from panax ginseng (PG), Panax quinquefolium (PQ), and Panax notoginseng (PN). The results of this study found 437 potential new ginsenosides.

4. Zhang, K., Wang, X., Huang, J., Liu, Y. (2012). Purification of L-alpha glycerylphosphorylcholine by column chromatography. Journal of Chromatography A, 1220, 108-114. -This study used column chromatography to separate colorless L-alpha glycerylphosphorylcholine. They obtained 99.8% purity. It was a novel preparation and the conclusion was that it was in agreement with the standard. The study provided a cost-effective, environmentally friendly and simple preparation for L-alpha glycerylphosphorylcholine.

5. Zhang, Y., Jiao, J., Liu, C., Wu, X., Zhang, Y. (2008). Isolation and purification of four flavone C-glycosides from antioxidant of bamboo leaves by macroporous resin column chromatography and preparative high-performance liquid chromatography. Food Chemistry, 107, 1326-1336. -The study used column chromatography to prepare four flavone C-glycosides from bamboo leaves. The study concluded that this was a good method for preparing and quantifying these glycosides from bamboo leaves.

6. Zeng, F., Liu, W., Jiang, H., Yu, H., Zeng, R.J., Guo, Q. (2011). Separation of phthalate esters from bio-oil derived from rice husk by a basification-acidification process and column chromatography. Bioresource Technology, 102, 1982-1987. -The study used column chromatography to separate phthalate esters from rice husks. They found that more than 80% were in only two of the fractions and also that the formation of the esters was a result of a radical reaction with substituted benzene and other molecules.

7. One recent 2013 paper studied a 30 kDa protein, High mobility group B1, commonly expressed in animal cells. This protein functions as a chromatin structure regulator, while also regulating gene expression. OUtside of the nucleus, it regulates immune response. Not much was known about the binding nature of HMGB1. In this study, column chromotography was used to isolate and identify HMGB1 biding molecules ( Rouhia et al. 2013).

Rouhiainen, A., Tukiainen, H., Siljander, P., & Rauvala, H. (2013). Identification of HMGB1-Binding Components Using Affinity Column Chromatography.

References:
Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P. (2002). Manipulating Proteins, DNA, and RNA. In S. Gibbs (Ed.), Molecular Biology of the Cell (481-82). New York: Garland Science.

Deng, Y. //et al.// Acaricidal activity of petroleum ether extract of neem (Azadirachta indica) oil and its four fractions separated by column chromatography against Sarcoptes scabiei var. cuniculi larvae in vitro. //Experimental Parasitology// 130, 475–477 (2012).

3.

Li, X.-J. //et al.// Preparative separation of dryofragin and aspidin BB from Dryopteris fragrans extracts by macroporous resin column chromatography. //Journal of Pharmaceutical and Biomedical Analysis// 61, 199–206 (2012).

Yang, W. //et al.// A strategy for efficient discovery of new natural compounds by integrating orthogonal column chromatography and liquid chromatography/mass spectrometry analysis: Its application in Panax ginseng, Panax quinquefolium and Panax notoginseng to characterize 437 potential new ginsenosides. //Analytica Chimica Acta// 739, 56–66 (2012).

<span style="font-family: Arial,Helvetica,sans-serif;">Zeng, F., Liu, W., Jiang, H., Yu, H., Zeng, R.J., Guo, Q. (2011). Separation of phthalate esters from bio-oil derived from rice husk by a basification-acidification process and column chromatography. Bioresource Technology, 102, 1982-1987. <span style="font-family: Arial,Helvetica,sans-serif;">Zhang, K., Wang, X., Huang, J., Liu, Y. (2012). Purification of L-alpha glycerylphosphorylcholine by column chromatography. Journal of Chromatography A, 1220, 108-114. <span style="font-family: Arial,Helvetica,sans-serif;">Zhang, Y., Jiao, J., Liu, C., Wu, X., Zhang, Y. (2008). Isolation and purification of four flavone C-glycosides from antioxidant of bamboo leaves by macroporous resin column chromatography and preparative high-performance liquid chromatography. Food Chemistry, 107, 1326-1336.