Name: John Christopher L. Luces
Date performed: January 11, 2011
Co-worker: hresda!e "ndrada
Date su#mitted: January 21, 2011
$emm Domini%ue Jarani!!a
&'(&)*+&N N0. C)+"/)"( *.
J&C*&3 - o !earn the techni%ues techni%ues of paper paper chromato4ra chromato4raphy phy and and thin thin !ayer chromato4rap chromato4raphy hy - o app!y chromato4ra chromato4raphic phic methods methods in the the separati separation on of the compound compoundss of a mi5ture mi5ture - o ide ident ntif ify y an an unkn unknow own n #y compa compari rin4 n4 ) f 6a!ue 6a!ue and other characteristics with those of a standard.
**.
&)&*C"L " "C7/)8ND
Chromato4raphy is a wide!y used method that a!!ows the separation, identification and determination of the chemica! components in comp!e5 mi5tures. *t is a techni%ue in which the components co mponents of a mi5ture are separated #ased on differences in the rates at which they are carried throu4h a fi5ed or stationary phase #y a 4aseous or !i%uid mo#i!e phase. he stationary phase is fi5ed in p!ace either in a co!umn or on a p!anar surface. he mo#i!e phase mo6es in a definite direction o6er or throu4h the stationary phase carryin4 with it the ana!yte mi5ture, where the samp!e interacts with the stationary phase and is separated. he mo#i!e phase may #e a 4as, !i%uid, or a supercritica! f!uid. i4h-performance !i%uid chromato4raphy 9(LC is the most 6ersati!e and wide!y used type of e!ution chromato4raphy. *n !i%uid chromato4raphy, chromato4raphy, the mo#i!e phase is a !i%uid so!6ent containin4 the samp!e as a mi5ture of so!utes. he types of (LC are often c!assified #y separation mechan ism or #y the type of stationary phase. hese inc!ude 91 partition chromato4raphy, 92 adsorption chromato4raphy, 9; ione5chan4e, 9 si chira! chromato4raphy. (artition chromato4raphy is a!so ca!!ed !i%uid-!i%uid chromato4raphy in which the stationary phase is a second !i%uid that is immisci#!e with the !i%uid mo#i!e phase. *ts separations may # e attri#uted to the differences in the so!u#i!ity of the samp!e in the stationary and mo#i!e ph ases. (aper chromato4raphy is an e5amp!e of !i%uid-!i%uid or partition chromato4raphy. *t is a usefu! techni%ue in the separation and identification of different p!ant pi4ments. 3ince the fi!ter paper is made of hi4h!y purified ce!!u!ose, it a#sor#s and retains water mo!ecu!es stron4!y. he fi!ter paper with ce!!u!ose and the #ound water compose the stationary phase. he so!6ent is then the mo#i!e phase. he separations #ecome f!a6ora#!e due to different affinities affinities of the components. "fter "fter de6e!opment, the spots correspondin4 to different compounds may #e !ocated #y their co!or, u!tra6io!et !i4ht, ninhydrin or #y
treatment with iodine 6apors. he paper remainin4 after the e5periment is known as the chromato4ram. he components which ha6e #een separated differ in the retention factor. )etention factor may #e defined as the ratio of the distance tra6e!!ed #y the su#stance to the distance tra6e!!ed #y the so!6ent. *n adsorption chromato4raphy, the mo#i!e phase is usua!!y an or4anic so!6ent and the stationary phase is fine!y di6ided partic!es of si!ica or a!umina. *n adsorption chromato4raphy, the on!y 6aria#!e that affects the distri#ution coefficient of ana!ytes is the composition of the mo#i!e phase, in co ntrast with partition chromato4raphy where the po!arity of the stationary phase can a!so #e 6aried. hin !ayer chromato4raphy is a form of so!idp!i%uid adsorption chromato4raphy. *t in6o!6es a stationary phase consistin4 of a thin !ayer of adsor#ent ma teria!, usua!!y si!ica 4e!, a!uminium o5ide, or a ce!!u!ose immo#i!i
?:1 96@6 pet-ether-acetone ' 1. 9 cm 2.8 cm
)f
Co!or
6.20 cm 6.20 cm
0.5 0.45
/reen
9:1 (v/v) pet-ether-acetone
?:1:1 96@6@6 pet-ether-diethy! etheracetone ' )f Co!or 1.;
>.= cm
0.2
e!!ow
9:1:1 (v/v/v) pet-ether-diethyl ether-acetone
/reen
. "na!ysis of the Component Dyes of !ack *nk #y LC 3o!6ent 3ystem: >:2:2 96@6@6 n-#utano!-ethano!-N; 3amp!e: /-&C 3pot No. 1 2 ;
' 2. 2.? ;.A
.? .? .?
)f 0.? 0.=? 0.BA
' ;.2 ;.> .?
.? .? .?
) 0.>= 0.B; 1
Co!or !ack (ink e!!ow
$"&)C"3&L 3pot No. 1 2 ;
G-!"#
$a%erca&tel
Co!or (urp!e e!!ow !ue
C. *dentification of "mino "cids #y (aper Chromato4raphy 'olvent 'y&tem: 1:2 (v/v) 2 ammonim hydro*ide-i&opropyl alcohol otal di&tance travelled %y &olvent &y&tem: +.2 cm ,i&aliation method: 'prayin 2 ninhydrin &oltion in acetone to the paper (chromatoram)
henylalanine ()
yro&ine ()
&partic acid ()
nno3n
rial1
rial2
rial1
rial2
rial1
rial2
rial1
rial2
(cm)
6.8
6.8
6.
6.5
5.1
5.2
6.9
6.+
(cm)
+.2
+.2
+.2
+.2
+.2
+.2
+.2
+.2
7
0.95
0.95
0.88
0.90
0.+1
0.+2
0.96
0.9
"olor
ar violet
ar ,iolet
iht ,iolet
iht ,iolet
iht ,iolet
iht ,iolet
ar violet
ar ,iolet
verae 7
0.95
0.89
8nknown is: (heny!a!anine
*.
D*3C833*N
0.49
0.95
(aper chromato4raphy can #e used to identify and separate p!ant pi4ments in !ea6es. he p!ant samp!e that was used is Codiaeum variegatum. he so!6ents that were used are ?:1 96@6 pet-ether-acetone (polar) and ?:1:1 96@6@6 pet-ether-diethy! ether- acetone 9nonpo!ar. *n the first set-up where in (&" was used as a so!6ent, 2 pi4ments were identified whi!e in the other so!6ent 9(&D", on!y one co!or was determined 94reen. ased on the )f 6a!ues o#tained in setup ", the ye!!ow pi4ment is more po!ar than the 4reen pi4ment. his is #ecause the ye!!ow pi4ment rose hi4her in the po!ar so!6ent 9(&" whi!e it didnt appear in the nonpo!ar so!6ent 9(&D". ased on the princip!e !ike disso!6es !ikeE since ye!!ow pi4ment is more po!ar than the 4reen pi4ment it is more so!u#!e to the po!ar so!6ent that is why it rose hi4her on it. "!so, the ye!!ow pi4ment has !ower affinity to stationary phase and hi4h affinity to mo#i!e phase. hus the so!6ent system ?:1 96@6 pet-ether-acetone is more ad6anta4eous and more appropriate to #e used as so!6ent in the Codiaeum variegatum !ea6es than any other so!6ent. *n 3etup , on!y the 4reen pi4ment is 6isi#!e. his indicates that the 4reen pi4ment is !ess po!ar #ecause it appeared in the nonpo!ar so!6ent. e!!ow pi4ment was not 6isi#!e #ecause it didnt disso!6e in the nonpo!ar so!6ent. *n the ana!ysis of dyes in /-tech ink, the ye!!ow dye rose the hi4hest then the pink and the #!ack dye in the !owest position. 3ince the so!6ent used is po!ar, and the stationary phase 9LC p!ate with si!ica 4e! is po!ar, the dyes can #e arran4ed accordin4 to increasin4 po!arity. e!!ow F (ink F !ack. "nd a!so, the dyes can #e arran4ed in increasin4 so!u#i!ity, that is accordin4 to their rise and )f 6a!ue as in !ackG (inkG e!!ow. 3o the most po!ar is the most so!u#!e in the so!6ent whi!e the !east po!ar is the !east so!u#!e. n the other hand, in the $a#er caste!!e ink, a!so three co!ors were determined #!ue, ye!!ow and purp!e. his shows that the ink component in $a#er caste!!e is different in that of /-tech. *n the ink of $ a#er caste!!e, the #!ue dye rose the hi4hest then the ye!!ow and !ast!y the purp!e !ie in the !owest position. 3ince the so!6ent used is po!ar, and a!so the stationary phase is po!ar, Hust !ike in /-tech the d yes can #e arran4ed accordin4 to increasin4 po!arity. !ueFe!!owF (urp!e here were three known amino acids and one unknown amino acid tested under paper chromato4raphy. he de6e!opment and p!acement of spots are accordin4 to the so!u#i!ity, po!arity and affinity of the amino acids to the so!6ent 1:2 96@6 2 I ammonium hydro5ide-isopropy! a!coho!. yrosine has a !i4ht 6io!et spots measurin4 >.;->.= cm a#o6e the ori4ina! samp!e. he )f is therefore 0.AA and 0.? respecti6e!y. "spartic acid, on the other hand, a!so has !i4ht 6io!et co!or #ut is darker than that of tyrosine and has the !owest rise with the )f of 0.?. f the four amino acids, pheny!a!anine and the unknown are the most so!u#!e to the 4i6en so!6ent #ecause they ha6e tra6e!!ed@ risen the hi4hest. "s we!!, they are amino acids 4i6in4 the hi4hest )f 6a!ues. "!so, they ha6e hi4her affinity to the mo#i!e phase 9so!6ent as they tra6e!!ed easi!y and farther with it. n the other han d, they ha6e !ower affinity towards the stationary phase 9chromato4raphic paper #ecause they dont interact stron4!y with the paper #ut rather proceeds faster with the so!6ent. "spartic acid has the !owest rise. 3o it is the !east so!u#!e to the so!6ent. "!so it has the hi4hest affinity to the stationary phase. ut has the !owest affinity to the mo#i!e phase. ased on the co!or and so!u#i!ity of the amino acids, it can #e identified that the unknown is pheny!a!anine. oth the pheny!a!anine
and the unknown ha6e dark 6io!et spots and ha6e )f 6a!ue appro5imate!y 0.?=. he same )f 6a!ues indicates that the amino acids ha6e the same so!u#i!ity to the so!6ent system used. .
C"LC8L"*N3
R f =
distancetravelled by compound distance travelled by solvent
¿ the origin( x ) ¿¿ origin ( y )¿
. 'eparation o lant iment& 1.) 9:1 (v/v) pet-ether-acetone 2.) 9:1:1 (v/v/v) pet-ether-diethyl-acetone 'pot ;o.1 'pot ;o 2. * < 1 90 cm < 2.8 *< 1. cm y< 6.20 cm < 6.20 y< 6.5 cm 7 < 1.9cm / 6.20cm 7 < 2.8/6.20 cm 7 < 1.cm / 6.5cm 7 < 0.5 cm 7 < 0.45 7 < 0.2 =.
/-&C !ack ' 2. .? )f 2.@.? )f 0.? $"&) C"3&LL (urp!e ' ;.2 .? )f ;.2@.? )f 0.>=
(ink ' 2.? .? )f 2.?@.? )f 0.=?
e!!ow ' ;.> .? )f ;.>@.? )f 0.B;
e!!ow ' ;.A .? )f ;.A@.? )f 0.BA
!ue ' .? .? )f .?@.? )f 1
C. he same method was used in ca!cu!atin4 )f in C 9*dentification of "mino "cids #y (aper Chromato4raphy
1. a. #.
c.
*. K8&3*N3 hat wou!d #e the effect of the fo!!owin4 errors in the chromato4raphic workM he so!6ent !e6e! in the de6e!opin4 cham#er is hi4her than the spotted samp!e ": here wi!! #e no resu!t #ecause the spot wi!! smear out and wi!! #e disso!6ed #y the so!6ent oo much samp!e is app!ied to the paper ": *f too much samp!e is app!ied to the paper, a !ar4e spot on the de6e!oped chromato4ram wi!! appear and com#ine with others and may run to4ether makin4 co!or identification difficu!t. he paper is a!!owed to remain in the cham#er after the so!6ent front has reached the top of the p!ate.
": *f this wou!d happen, the so!6ent front wi!! reach the ma5imum point , and the 6a!ue of ) f wi!! 6ary, so the rea! 6a!ueE wi!! #e s!i4ht!y unre!ia#!e, since the continues to 4oes up and ' remains. 2. hy is it necessary to co6er the de6e!opin4 cham#er ti4ht!y durin4 the de6e!opment of the chromato4ramM ": o a6oid rapid e6aporation of the 6o!ati!e so!6ents in the de6e!opin4 cham#er, the cham#er must #e co6ered ti4ht!y. his is #ecause most of the so!6ents used are 6o!ati!e and 6o!ati!e so!6ents tend to e6aporate faster and easier than non-6o!ati!e ones "!so, if the cham#er is not co6ered ti4ht!y, there is a tendency that the chromato4raphic paper wi!! #ecome dry and it wou!d #e difficu!t to !ocate the so!6ent front since the so!6ent has 4one to the 4as phase. ;. *dentify your unknown. &5p!ain c!ear!y how you made this identification. ": he unknown is the "mino "cid- pheny!a!anine. *t was identified throu4h comparin4 the hei4ht of the de6e!opment of the unknown samp!e with the de6e!opment of that of other amino acid. *ts hei4ht of the de6e!opment of the samp!e is the same with that of (heny!a!anine. asica!!y, the unknown was identified with respect to the )f of known "mino "cids. 4. Can LC or paper chromato4raphy #e used to separate and identify 6ery 6o!ati!e su#stancesM &5p!ain
your answer. ": hin-Layer Chromato4raphy is not for the separation and identification of 6ery 6o!ati!e su#stances. *t is #ecause 6o!ati!e su#stances e6aporate %uick!y, thus the so!6ent used ea si!y #ecomes dry. herefore, LC cant #e used to separate and identify 6ery 6o!ati!e su#stances. =. hy were you re%uired to hand!e the chromato4raphic paper on!y at its corners in part CM ": he chromato4raphic paper shou!d on!y #e hand!ed to its sides. 3o as not to a#sor# unnecessary moistures and thus to reduce contamination. "!so, not touchin4 the chromato4raphic paper wou!d pre6ent the contact to our skin which has a!so amino acids present.
