I don’t know if what I have written down is right. I can’t find anything explain
ID: 713006 • Letter: I
Question
I don’t know if what I have written down is right. I can’t find anything explaining TLC and RF values in a biochemistry sense with positive and negative charges CHEM 453L- LAB 4 Excel Template INSTRUCTIONS FOR TABLE-Report distances to the nearest Q.i emg Have Table 1. Known Amino Acid Data from TLC Plates c Excel perform all of the necessary computations. Be careful to indicate the appropriate number of digits Buffer A (Acidic Solvent CALCULATED VAL UES buffer B (Basic Solvent System) H-2 (ula pH paper) 2 (via pH paper) Addic Solvent Hydropathy Migration DistanceMigration Distance Migration DistanceMigration Distance- Std # | Identity Solvent Front (am Amino Acid (om) 8 30 Solvent Front (cm 14.48 448 14.48 14.48 14.48 14.48 1448 Amino Acid (om) 1.6 1.8 3.5 14.99 14.99 14.99 14.99 14.99 7.90 6.50 4.90 6.20 6.30 9.20 055 0.41 0.15 0.55 6.10 4.40 2.30 4,40 G.AS 0.34 0.43 0.44 0.64 daspartic acid 2.0 1.0 1.0 0.29 leucine 14.99 Relative R,-Each value in the colurnn by the smallest value in the column
Explanation / Answer
Let me first explain you the concept of TLC and Rf values and the correlation with pH.
Chromatography is used to separate mixtures of substances into their components. All forms of chromatography work on the same principle.
They all have a stationary phase (a solid, or a liquid supported on a solid) and a mobile phase (a liquid or a gas). The mobile phase flows through the stationary phase and carries the components of the mixture with it. Different components travel at different rates.
Thin layer chromatography is done exactly as it says - using a thin, uniform layer of silica gel or alumina coated onto a piece of glass, metal or rigid plastic. The silica gel (or the alumina) is the stationary phase. The stationary phase for thin layer chromatography also often contains a substance which fluoresces in UV light - for reasons you will see later. The mobile phase is a suitable liquid solvent or mixture of solvents.
Different compounds in the sample mixture travel at different rates due to the differences in their attraction to the stationary phase and because of differences in solubility in the solvent. pH also plays a role in case of a mixture of amino acids. Since, amino acid are charged species, and also the buffer or solvent medium used is charged ( acidic or basic), interactions between the two play a major role.
In an acidic buffer (it is positively charged due to H+ ions), positively charged amino acids like lysine will feel repulsive forces and would be adsorbed on the stationary phase (silica) quickly being the first ones explaining the lowest Rf. On the other hand, negatively charged amino acids like glutamic acid will have favourable attractive interactions with H+ of acidic buffer and hence would adsorb on silica only after changing the polarity of mobile phase to destabilize these interactions explaining th higher rf. For, uncharged or non polar amino acids like valine, alanine, leucine, etc. since they have no favourable interactions with the mobile phase and also not with the stationary polar phase (silica), they are always adsorbed the last explaining the highest rf.
In case of basic buffer (negatively charged due to OH-), the same concept applies to explain the lowest rf of aspartic acid (negatively charged), higher rf of lysine (positively charged) and highest rf of valine, alanine type non polar amino acids.
Hope that gives you the answers to both the questions :)
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