Post on 07-Apr-2018
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
8/4/2019 Why Do You Think There Is
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
8/4/2019 Why Do You Think There Is
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
8/4/2019 Why Do You Think There Is
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
8/4/2019 Why Do You Think There Is
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
8/4/2019 Why Do You Think There 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?
8/4/2019 Why Do You Think There Is
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
8/4/2019 Why Do You Think There Is
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
8/4/2019 Why Do You Think There Is
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
8/4/2019 Why Do You Think There Is
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
8/4/2019 Why Do You Think There Is
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
8/4/2019 Why Do You Think There Is
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
8/4/2019 Why Do You Think There Is
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.
8/4/2019 Why Do You Think There Is
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
8/4/2019 Why Do You Think There Is
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
8/4/2019 Why Do You Think There Is
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
8/4/2019 Why Do You Think There Is
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 &
8/4/2019 Why Do You Think There Is
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
8/4/2019 Why Do You Think There Is
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
8/4/2019 Why Do You Think There Is
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
8/4/2019 Why Do You Think There Is
21/25
8/4/2019 Why Do You Think There Is
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
8/4/2019 Why Do You Think There Is
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
8/4/2019 Why Do You Think There Is
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
8/4/2019 Why Do You Think There Is
25/25
effect of both
adsorption andpartition.