Why Do You Think There Is

8/4/2019 Why Do You Think There Is

1/25

Why do you think thereis some variation?[ANS] Both Rf

values are the same foraspartic acid but thereissome variation for

leusine & lysine.This may due to:1.>The grease on our

fingers contaminatedonchromatographic


2/25

paper will affect greatly

on the amino acidsthatare lesspolar.2.>The measurement error

of the distancetravelled bysolventand/or amino acid.3.>

The shape ofchromatographic paperis not exactlysquare-

shaped, so that oneside of the paper may


3/25

be lifted and thespeed

of the solvent travel inone side is faster.4.>The different proportion

of chemicals (measured by

measuring cylinder) may be used betweenthe researchers and us,

andthe temperaturesmay also different, thusR


4/25

f

valuesobtained aredifferent.2.> Why do Rf

values change when adifferent solventisused?

[ANS] Each amino acidhas different polarityand each solvent

hasdifferent polarity,too. So, when


5/25

a different solvent is

used, aminoacids willtravel at a differentspeed.For example, if a

more polar solvent isused, the morepolaramino acid will

travel at a fasterspeedand has a largerR

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


6/25

used, the non-

polaramino acid willtravel faster and theslowest one should be

thepolar amino acid.3.> Why is it soimportant to avoid

touchingthechromatographicpaper with your

fingers?


7/25

[ANS] There are some

secretion on our fingers- amino acidsandgrease.Amino acids

on our fingers will resultinthe unexpectedcolour

zone appears onchromatographic paperafter sprayingninhydrin

solution. This will makeus confused and


8/25

moredifficult to obtain

the appropriate Rf

values.Grease on our

fingers will affect non-polar amino acids most,

since they are more

likely to dissolve ingrease than morepolardeveloping

solvent, they will move


9/25

at a higher speed.

Hencethe Rf

values are

affected.Moreover,there may have somechemicals on our

fingers,e.g. ammonia,water, etc. These willaffect the polarity of

thedeveloping solventand thus the Rf


10/25

values. KCl

http://hk.geocities.com/fatherofchemistryPrecaution1.> Small area

with concentrated spotof amino acid shouldbeapplied to obtain a

deeper colourof spots.2.> Theedges of

chromatographic papershould not touch


11/25

thesides of the beaker

since he solution atedges will move fasterdueto capillary

action.The spots willbend and Rf

value is affected.3.>The beaker should besaturated with the

solvent beforeputtingthe chromatographicpaper in. If


12/25

the atmosphere is

notsaturated, there isadiffusion gradientbetween

chromatographic paperandair, some solventmay vapourize from

chromatogram andresults inlong tail ofspots.4.> The origin

line should be markedby pencil, not ball


13/25

pen,since the ball pen

ink can dissolve inthe developing solventand theline fades

graduallyor even anadditional spot can beobserved.5.> Ninhydrin

solution should notspray too muchonchromatogram since

it will blur and spreadsthe coloured spots.


14/25

It'sbecause the solvent

that areused todissolve ninhydrincan also dissolve

aminoacids.DiscussionPaperchromatography is

useful for separationandidentification ofmany substances, e.g.

amino acids, dyes, etc.Itsprinciple is based on


15/25

the difference of the

relative adherence ofasolute between astationary phase and a

mobile phase. Sincepaperconsists ofcellulose that contains

a large number of OHgroups, alayer of waterwill be permanently

attached to the paper.This layer


16/25

is the stationary phase.

Under capillary action,the developingsolventwill move upwards,

which is the mobilephase. Thesoluteparticles are

moved upwards withthe solvent anddistributebetween the

stationary phaseand mobile


17/25

phase.Paper

chromatography can beused for analysis ofsolutesbecause at

a constanttemperature,each solute has a

particularretentionfactor (Rf

) in a particular set ofmobile phase &


18/25

stationaryphase. Base

on the Rf

values, the solutes can

be identified.The higherthe Rf

, the faster is the

movement ofthe solute.If the soluteparticles is less polar

than the stationaryphase(i.e. less soluble


19/25

in stationary phase),

it will move up withthe mobilephase (lesspolar). Otherwise, if

the solute particles ishighly polar, itwill onlystay in the stationary

phase. In other words,the differenceinpartition coefficients of

the solute particlesenable the


20/25

soluteparticles to move

with different speeds.In this experiment,leusinehas the highest

solubility in mobilephase (least polar) thaninstationary phase thus

its motion isfastest. Aspartic acidhas thelowest solubility

in mobile phase (mostpolar) than in


21/25


22/25

the chromatogram by

ninhydrin solution toform acoloured spot.The colour of the

complex formedbetween ninhydrin&aspartic acid is deep

blue whereas that ofleusine and lysinearepurple. Other

organic solutes mayalso be located by


23/25

ultraviolet light(if it

gives fluorescence inUV light) or by iodinevapour (I

2willdissolve in organiccompound which results

in a brown spot).2-waychromatography (i.e.turn the chromatogram

for 90and obtaina secondchromatography) may


24/25

be applied for

furtherchromatographyto obtain a more distinctspot.If the

paper medium isreplaced with silica gelor aluminacoated on a

glass plate, it's calledthin layerchromatography. In

thiscase the separationis based on the


25/25

effect of both

adsorption andpartition.

Why Do You Think There Is

Documents

Transcript of Why Do You Think There Is