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Question: In this experiment you are given a mixture of seven coloured compounds that vary in their size and charge that you must separate using chromatography. You are provided with a number of different chromatographic media and an assortment of solutions for eluting samples from your columns. Your task is to devise an efficient and economical method for the separation of all seven components. You are to design your experiment such that you have minimum loss of each component. Present your method in a flow chart form. Clearly indicate in your flow chart when you have purified each of the seven coloured compounds from the mix. Hint: Remember you are a researcher, you want to take as few steps as possible Below are the components of the mixture and tools you may use to devise a separation that will result in the separation of all seven compounds into separate samples. The 7 components of the mixture are: compound colour MW (Da) Ionic Character at pH 6 or pI Blue dextran blue >500,000 Very strong anion Yellow dextran yellow 10,000 Strongly anionic Purple actin purple 44,000 pI of 5.3 Cytochrome c orange 12,400 Strong cation, pI of 10 Vitamin B12 red 1357 Weak cation DNP-glycine yellow 241 Weak anion , pI of 3 myoglobin red 15,600 Very weak cation Chromatographic Media available Sephadex G, 75 equilibrated in 0.05 M NaAc, pH 6 This medium has a fractional range of G- 3000–80,000 CM-Sephadex 75 equilibrated in 0.05 M NaAc, pH 6 This medium has a fractional range of 3000 to 80,000. Resin functional group positively charge, weak CM-cellulose (functional group negatively charged, weak) DEAE-cellulose (functional group positively charged, weak) Solutions available 0.05M sodium acetate, pH 6 0.1 M sodium acetate, pH 6 0.5 M sodium acetate, pH 6 1.0 M sodium acetate pH 6 2.0 M sodium acetate, pH 6 1M HCl 1M NaOH
To devise an efficient and economical method for the separation of all seven components, you can use a combination of size-exclusion chromatography (SEC) and ion-exchange chromatography (IEX). Flowchart: 1. Start with the mixture of seven compounds. 2. Perform size-exclusion chromatography (SEC) using a Sephadex G-75 column equilibrated in 0.05 M sodium acetate (NaAc), pH 6. - Blue dextran (MW > 500,000) will be excluded and eluted first. - Yellow dextran (MW 10,000), purple actin (MW 44,000), cytochrome c (MW 12,400), vitamin B12 (MW 1,357), DNP-glycine (MW 241), and myoglobin (MW 15,600) will be separated based on their molecular weights. 3. Collect fractions from the SEC step and identify which fractions contain the remaining six compounds. 4. Perform ion-exchange chromatography (IEX) on the fractions containing multiple compounds, using the appropriate chromatographic media and solutions. a. For fractions containing weak cations and anions, perform IEX using CM-cellulose (-). - Elute with a gradient of sodium acetate solutions, starting from 0.05M to 2.0M NaAc, pH 6. - DNP-glycine (weak anion) and vitamin B12 (weak cation) will be separated based on their charge interactions. b. For fractions containing strong anions and cations, perform IEX using DEAE-cellulose (+). - Elute with a gradient of sodium acetate solutions, starting from 0.05M to 2.0M NaAc, pH 6. - Yellow dextran (strong anion) and cytochrome c (strong cation) will be separated based on their charge interactions. c. For the fractions containing proteins close to their isoelectric point (pI), perform IEX using CM-Sephadex 75 (+). - Elute with a gradient of sodium acetate solutions, starting from 0.05M to 2.0M NaAc, pH 6. - Purple actin (pI 5.3) and myoglobin (very weak cation) will be separated based on their charge interactions. 5. Collect purified fractions of each of the seven colored compounds from the IEX steps (3a, 3b, and 3c). By following this method, you can effectively separate and purify each of the seven colored compounds with minimal loss and in the least number of steps.
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