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Why do you think there is some variation?

[ANS] Both R

values are the same for aspartic acid but there issome variation for leusine & lysine. This may due to:1.> The grease on our fingers contaminated onchromatographic

paper will affect greatly on the amino acids thatare lesspolar.2.> The measurement error of the distance travelled bysolvent and/or amino acid.3.> The shape of chromatographic paper is not exactlysquareshaped, so that one side of the paper may

be lifted and thespeed of the solvent travel in one side is faster.4.> The different proportion of chemicals (
measured by

measuring cylinder

) may be used between the researchers and us, andthe temperatures may also different, thus R

valuesobtained are different. 2.> Why do R


values change when a different solvent isused? [ANS] Each amino acid has different polarity and each solvent hasdifferent polarity, too. So, when

a different solvent is used, aminoacids will travel at a different speed.For example, if a more polar solvent is used, the more polaramino acid will travel at a faster speedand has a larger R

value.On the contrary, if a less polar solvent is

used, the nonpolaramino acid will travel faster and the slowest one should be thepolar amino acid. 3.> Why is it so important to avoid touching thechromatographic paper with your fingers?

[ANS] There are some secretion on our fingers - amino acids andgrease.Amino acids on our fingers will result in the unexpectedcolour zone appears on chromatographic paper after sprayingninhydrin solution. This will make us confused and

moredifficult to obtain the appropriate R


values.Grease on our fingers will affect nonpolar amino acids most, since they are more likely to dissolve in grease than more polardeveloping solvent, they will move

at a higher speed. Hencethe R


values are affected.Moreover, there may have some chemicals on our fingers,e.g. ammonia, water, etc. These will affect the polarity of thedeveloping solvent and thus the R

values. KCl m/fatherofchemistryPre caution1.> Small area with concentrated spot of amino acid shouldbe applied to obtain a deeper colour of spots.2.> The edges of chromatographic paper should not touch

thesides of the beaker since he solution at edges will move faster dueto capillary action.The spots will bend and R

value is affected.3.> The beaker should be saturated with the solvent beforeputting the chromatographic paper in. If

the atmosphere is notsaturated, there is adiffusion gradient between chromatographic paper andair, some solvent may vapourize from chromatogram and results inlong tail of spots.4.> The origin line should be marked by pencil, not ball

pen,since the ball pen ink can dissolve in the developing solvent and theline fades graduallyor even an additional spot can be observed.5.> Ninhydrin solution should not spray too much onchromatogram since it will blur and spreads the coloured spots.

It'sbecause the solvent that areused to dissolve ninhydrin can also dissolve amino acids.DiscussionPaper chromatography is useful for separation andidentification of many substances, e.g. amino acids, dyes, etc. Itsprinciple is based on

the difference of the relative adherence of asolute between a stationary phase and a mobile phase. Since paperconsists of cellulose that contains a large number of OH groups, alayer of water will be permanently attached to the paper. This layer

is the stationary phase. Under capillary action, the developingsolvent will move upwards, which is the mobile phase. The soluteparticles are moved upwards with the solvent and distributebetween the stationary phase and mobile

phase.Paper chromatography can be used for analysis of solutesbecause at a constant temperature, each solute has a particularretention factor (R

) in a particular set of mobile phase &

stationaryphase. Base on the R


values, the solutes can be identified.The higher the R


, the faster is the movement of the solute.If the solute particles is less polar than the stationary phase(i.e. less soluble

in stationary phase), it will move up with the mobilephase (less polar). Otherwise, if the solute particles is highly polar, itwill only stay in the stationary phase. In other words, the differencein partition coefficients of the solute particles enable the

soluteparticles to move with different speeds. In this experiment, leusinehas the highest solubility in mobile phase (least polar) than instationary phase thus its motion is fastest. Aspartic acid has thelowest solubility in mobile phase (most polar) than in

stationaryphase thus it moves slowest. After the mobile phase moved acertain distance, the spots (after drying) may be cut out andextracted with an appropriate solvent.If the solute is colourless such as amino acid, it can be locatedby spraying

the chromatogram by ninhydrin solution to form acoloured spot. The colour of the complex formed between ninhydrin& aspartic acid is deep blue whereas that of leusine and lysine arepurple. Other organic solutes may also be located by

ultraviolet light(if it gives fluorescence in UV light) or by iodine vapour (I


willdissolve in organic compound which results in a brown spot).2-way chromatography (i.e. turn the chromatogram for 90and obtain a second chromatography) may

be applied for furtherchromatography to obtain a more distinct spot.If the paper medium is replaced with silica gel or aluminacoated on a glass plate, it's called thin layer chromatography. In thiscase the separation is based on the

effect of both adsorption andpartition.