Post Laboratory Assignment

PostLaboratory Assignment

Separating&amp Identifying a Mixture of Amino Acids by TLC &amp IEXChromatography

Separating&amp Identifying a Mixture of Amino Acids by TLC &amp IEXChromatography

Ionexchange chromatography is among the most efficient methods used inseparating charge particles. Mostly, ion exchange chromatography(IEX) is performed inform of column chromatography. Also, there arethin layer chromatographic (TLC) methods that are mainly based on ionexchange principle. This report presents results and analysisobtained when separating and identifying mixture of amino acids basedon IEX and TLC chromatography.

Resultsand Discussion

Question1

Beloware the results obtained from analytical separation of the amino acidcomponents of the unknown mixture. Table 1 below presents a summaryof the results obtained.

Table1: AminoAcid Standards by TLCa

Lane

dspot (mm)

Rfb

Spot Descriptionc

Amino Acid Assignment

E

450

0.600

In the middle, purple ring, magenta and medium intensity

Glutamate

F

540

0.720

Hazy purple, solid, furthest

Phenylalanine

G

360

0.480

Maroon with Medium intensity, medium distance

Glycine

K

250

0.333

Deep royal purple, lowest on TLC

Lysine

L

530

0.706

Hollow circle, light purple (darker than E), 2nd furthest

Leucine

S

380

0.507

Hollow purple same as G but perfect circle and in middle distance

Serine

  1. Thin-layer chromatography was performed on {silica gel} plates using {PAW Solvent} as the developing solvent.

  2. Footnote ‘b’ is the ratio to the solvent front or retardation factor. Expressed as distance travelled by the spot to the distance moved by the solvent front. The value should be less than 1

Figure1: Movementof Different Amino Acids through the Solvent

  1. Set of Amino acids that appear to vary quantitatively

Allamino acids have carboxyl group and amino group within the samecarbon chain. The difference in elution is normally because of thedifferences in the side chains, R groups, which vary in electriccharge structure. Therefore, the interaction of amino acid with thesilica gel, stationary phase varies based on ‘R’ group. Typicaladsorbents are considered to be more polar. The strength of elusionis directly proportional to the polarity of the solvent.

Serineand Phenylalanine

Serinehas an ‘R’ group that is polar. Other amino acids likePhenylalanine have the ‘R’ as benzene ring that can be presentedas –CH2(C6H5). The ‘R’ group is non-polar, hence hydrophobic. Thenon-polar amino acids tend to lower solubility levels in the solvent,while the highly polar amino acids tend to bind strongly to theadsorbent. The more the amino acid is bound to the solvent, theslower it moves through the TLC plate. Therefore, it can be concludedthat non-polar compounds such as phenylalanine has higher elusionrate that polar compounds such as glycine.

Fromthe expression of Rfabove, it can be concluded that non polar compounds have higher Rfvalue than non-polar amino acids.

  1. Pair of Amino acids where qualitative data should be considered in making any definitive distinction

Theother 2 set of amino acids distinguishable through qualitativeanalysis of TLC are Leucine and Glutamate

Leucinehas a non-polar side chain ‘R’ while Glutamate has a polar sidechain ‘R’. This implies that Rfis higher for Glutamate than for Leucine, hence more elusion power.

  1. Glutamate displayed a higher Rf value than glycine in this experiment.

      1. Considering that glutamate is classified as a “polar charged” amino acid and glycine as an “uncharged” amino acid, from the explanation above, the Rf results should be vice versa, with Glutamate having lower values than glycine, which is nonpolar. This is not the case as Glutamate has 0.600 and glycine has 0.507.

      2. This may cannot be so much of a surprise since though Glutamate is polar, the side chain carry carboxylic group (–CH2CH2COOH) and basic -NH2 group that renders ‘R’ neutral, hence hydrophilic when compared to Glycine, which is the simplest amino acid, whose ‘R’ is a hydrogen atom.

      3. Hypothesizing the rationale for this “anomalous” result and assume pH,

ThepH affects the Rfvalue of an amino acid. The neural ‘R’ have high Rfvalue than either acid or basic pH group.

Question2

Question2: Creating a Table using Spot Number. Unknown amino acid D is acomponent of F, E and G.

Spot No.

dspot (mm)

Rfb

Spot Descriptionc

Amino Acid Assignment

360

0.480

Maroon with Medium intensity, medium distance

Glycine

450

0.600

In the middle, purple ring, magenta and medium intensity

Glutamate

540

0.720

Hazy purple, solid, furthest

Phenylalanine

Table2: Separationof Unknown 4

    1. Principle of separation of components in the amino acid mixture

      1. For the unknown substance 4, dynamic equilibrium orinciple will be used to necessitate the separation of the mixture that is made up of amino acids F, E and G. These amino acids are phenylalamine, Glutamate and glycine. Due to benzine ring, phenylalamine is non-polar. Also, glycine is nonpolar will Glutamate is polar but has the neutral ‘R’ group due to –CCOH and NH2 groups that makes it nonpolar. Therefore, during TLC using PAW solvent and silica gel as stationary phase. The chemical dynamic equilibrium, the three amino acids, FG and E, will have different attraction rates to the stationary and mobile phases. F, being the most unattractive to the solvent will be first to drain off, followed by Glutamate and finally glycine.

      2. Chemical Dynamic Equilibrium in Chromatography

Inchromatography, two phases are involved. These are mobile andstationary phases. For a certain chemical sample, dynamic equilibriumexists between the two phases. As a result, the overall separation isdependent on how strongly a certain chemical is attracted to thestationary and mobile phases.

    1. The components of the unknown mixture were separated from the mixture

Theunknown substance contained a mixture of 3 components. These arephenylalanine, Glycine and Glutamate

      1. Qualitative justification

Thequalitative determination entails localization of substances throughuse of Rfvalue. Due to the presence of the benzene ring in phenylalanine, theRfvalue should be comparatively higher than for the others due topresence of non-polar bonds. The Rfvalue should therefore decrease or increase based on the level ofpolarity of the substances involved. Also, the pH level seems toaffect the Rfvalue. Ideally, Rfvalue should be between 0.1 and 0.8.

      1. Quantitative justification

Quantitativeevaluation entails use of calibration equipment. In this case, thearea of the spot is measured, or photometric evaluation carried out.First, the size of spot four was observed and compared with the rest,hence the conclusion that unknown 4 is a combination of F, G and E.

Question3

TLCanalytical method provides base essential information that can beused and derived for use in other applications

  1. Importance of TLC in IEX expectations

Incation exchange, the separation process is dependent on reversibleion exchange in solution with the ions that are boundelectrostatically to the insoluble support media.

Incation exchange, two functional groups are attached covalently tosupport beads.

Addingsample ions to the column binds the charges that are opposite to thefunctional group. Bound molecules are eluted sequentially increasingthe number of counter ions. The TLC analysis entails the dynamicchemical equilibrium of the stationary and mobile phases. This actsas a background to Cation Exchange column since they both deal withexchange of ions between phases. While in TLC may not describe thestationary and mobile phases in terms of charges, the exchange thattakes place is more or less the same as that in cation exchange.

  1. Effectiveness of Cation Exchange in determining presence of glutamate

Ionexchange entails the reversible adsorption of molecules that arecharged to the immobilized ion groups within the matrix of oppositecharges. Currently, IEX is the most effective method in determinationof the amino acids. The cation exchange method depends onelectrostatic attraction of the oppositely charged ions. Therefore,where TLC fails cation exchange is effective since the amino acid ismade up of positive and negative ions. The separation of thecolumn’s dimension in cation-exchange can be easily increased inprecision to enhance high resolution separation between the aminoacids.