Hydrolysis of Proteins and Chromatography HC CHEMISTRY HC CHEMISTRY Proteins Hydrolysis of Proteins and Chromatography After completing this lesson you should be able to : During digestion, enzyme hydrolysis of dietary proteins can produce amino acids. The structural formulae of amino acids obtained from the hydrolysis of proteins can be identified from the structure of a section of the protein. Chemical analysis In chromatography, differences in the polarity and/or size of molecules are exploited to separate the components present within a mixture. Interpretation of the results of chromatography. Learners are not required to know the details of any specific chromatographic method or experiment. Depending on the type of chromatography in use, the identity of a component can be indicated either by the distance it has travelled or by the time it has taken to travel through the apparatus (retention time).

Protein hydrolysis COPY Proteins are broken down during digestion. Digestion involves the hydrolysis of proteins to form amino acids + 2H2O Protein Amino acids

Identifying amino acids by chromatography COPY Identifying amino acids by chromatography In the lab a protein can be hydrolysed back to its constituent amino acids by refluxing with concentrated hydrochloric acid for several hours. Amino acids can be identified by the use of paper (or thin layer) chromatography. A piece of chromatography paper is spotted with some amino acids suspected as being present and also with the hydrolysed protein.

Identifying amino acids by chromatography COPY Identifying amino acids by chromatography By comparing the position of the spots of the known amino acids with that of the hydrolysed protein, the amino acids in the protein can be identified. Add your results to the diagram The hydrolysed fruit juice contained? Scholar animation available

COPY Chromatography Is a technique used to separate and identify the components of a mixture. Works by allowing the molecules present in the mixture to distribute themselves between a stationary and a mobile medium. Molecules that spend most of their time in the mobile phase are carried along faster.

Gas Liquid Chromatography COPY Gas Liquid Chromatography Here the mobile phase is an unreactive gas ( eg Nitrogen) flowing through a tube. And the stationary phase is an involatile liquid held on particles of a solid support.

COPY In the animation below the red molecules are more soluble in the liquid (or less volatile) than are the green molecules.

COPY In practice the Column is contained in a thermostatic oven. About 1μL of liquid is injected into one end of the column. As each component reaches the other end it is detected and registered on a chart recorder. The Retention Time is characteristic of a particular substance. (for the same column, temperature, gas flow etc.) The area under each peak indicates the relative quantities.

Injection port Recorder Oven Detector Column Nitrogen cylinder

Chromatogram of petrol Suggest identities of some of the unlabelled peaks.

Thin Layer Chromatography COPY Thin Layer Chromatography Here the mobile phase is a liquid Flowing past a thin layer of powder on a solid support. Substances that are less attracted to the solid or are more soluble in the liquid move faster. And so move further up the plate by the time that the process has been stopped by taking the plate out of the liqiud. - larger Rf

Rf = distance moved by substance distance moved by solvent front COPY Rf = distance moved by substance distance moved by solvent front For substances that are very soluble in the liquid Rf will be close to .... 1 For substances that are rather insoluble in the liquid Rf will be close to ....