chromatography final

8/18/2019 Chromatography Final

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Experiment 6A, 6B: Chromatography

Rachelle Anne Victorio

CHM 342.003

03 February 2016

Ms. Emily abusa


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Purpose:

!o "i#e $ractice in se$aratin" com$onents o% a mi&ture usin" chromato"ra$hy. !o

'etermine (hich sol#ent (oul' be best %or se$aratin" a $air o% relate' com$oun's that

'i%%er sli"htly in $olarity. !o i'enti%y com$oun')s* $resent in an un+no(n sam$le.

Table of Chemicals:

Molecular

Formula

tructural Formula Molecular

!eight

Boiling "

Melting

#ensity

Methylene

Chlori$e

CH2Cl2 ,4.-3 "mol /-6.C

3-.6C

1.33 "m

Fluorene C13H10 166.223 "mol 2-C116

C

1.2 "m

Fluorenol C13H10 1,2.22 "mol 36.511.0

C12 to

1 C

1.11

"m

Fluorenone C13H, 1,0.1-2 "mol 342C

,3.oC

1.13 "cm

%o$ine

Crystals

72 23.,0,-4

"mol

1,4C114

C

11.2 "

Ben&oin C14H122 212.24 "mol 344oC132o

C

1.31 "m

Ben&il (C₆H₅CO)₂ 210.23 "mol 346/34,oC

-4/-6o

C

1.23

"mol

'exane C6H14 ,6.1, "mol /-.3 C

6,. C

0.66 "m


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Acetone C3H6O ,.0, "mol 6oC/-oC 0.- "m

(utlines of Proce$ure:

Experiment 6A: Thin )ayer Chromatography

7n this e&$eriment8 you (ill use thin/layer chromato"ra$hy )!C* to se$arate a mi&ture o% three

com$oun's9 fluorene, fluorenol, an$ fluorenone*

ase' on the results (ith +no(n sam$les o% these com$oun's8 you (ill 'etermine (hich

com$oun's are %oun' in an un+no(n sam$le. :sin" !C to i'enti%y the com$onents in a sam$le

is a common a$$lication o% this techni;ue.

Preparing the TLC:

!echni;ue 20 'escribes the $roce'ure use' %or thin/layer chromato"ra$hy

1* +se a -cm x .*/ cm T)C plate 0!hatman ilica 1el Plates 2o* 33- 4445 . !hese $lates

ha#e a %le&ible bac+in" but shoul' not be bent e&cessi#ely. !hey shoul' be han'le' care%ully or

the absorbent may %la+e o%%. Also8 they shoul' be han'le' only by the e'"es< the sur%ace shoul'not be touche'.

4. :sin" a lea' $encil )not a $en*8 li"htly 'ra( a

line across the $late )short 'imension* about 1 cm

%rom the bottom )see %i"ure.*

3. :sin" a centimeter ruler8 mo#e its in'e& about

0.6 cm in %rom the e'"e o% the $late an' li"htly

mar+ o%% %i#e 1/cm inter#als on the line. !hese are

the $oints at (hich the sam$les (ill be s$otte'.

4. =re$are %i#e micro/$i$ettes to s$ot the $late.

>!he $re$aration o% these $i$ettes is 'escribe'

an' illustrate' in !echni;ue 208 section 20.4.

. =re$are a !C 'e#elo$ment chamber (ith

methylene chlori'e )see techni;ue 208 %i"ure 20.*. A bea+er co#ere' (ith aluminum %oil or

(i'e/mouth scre( ca$ bottle is suitable container to use. )see tec. 208 %i". 20.4*. >!he bac+in" on

the !C $lates is to thin8 so i% it touches the %ilter $a$er liner o% the 'e#elo$ment chamber at any


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$oint8 sol#ent (ill be"in to 'i%%use onto the absorbent sur%ace o% that $oint. !o a#oi' this8 be sure

that the %ilter $a$er liner 'oes not "o com$letely aroun' the insi'e o% the container. A s$ace o%

2. inches must be $ro#i'e'.

6. n the $late8 startin" %rom le%t to ri"ht, spot fluorene, fluorenol, an$ fluorenone, the

unno7n mixture, an$ the stan$ar$ reference mixture 7hich contains all three compoun$s*8* For each o% the %i#e sam$les8 use a 'i%%erent micro$i$ette to s$ot the sam$le on the $late. >!he

correct metho' o% s$ottin" is 'escribe' in !ec. 208 sec 20.4.

,. !a+e u$ $art o% the sam$le in the $i$ette )'on?t use a bulb8 ca$illary action (ill 'ra( u$

li;ui'*.

-. A$$ly the sam$le by touchin" the $i$ette li"htly to the thin/layer $late. >!he s$ot shoul' not

be lar"er than 2mm in 'iameter. 7t (ill usually be su%%icient to s$ot each sam$le once or t(ice. 7%

you nee' to s$ot the sam$le more than once8 allo( the sol#ent to e#a$orate com$letely bet(een

successi#e a$$lications an' s$ot the $late in e&actly the same $osition each time.

10. @a#e the sam$le in case you nee' the !C.

Developing the TLC Plate:

1. =lace the !C $late in the 'e#elo$ment chamber8 ma+in" sure that the $late 'oes not come in

contact (ith the %ilter $a$er liner.

2. Remo#e the $late (hen the sol#ent %ront is 1/2 cm %rom the to$ o% the $late.

3. :sin" a lea' $encil8 mar+ the $osition o% the sol#ent %ront.

4. @et the $late on a $iece o% $a$er to(el to 'ry.

. hen the $late is 'ry8 $lace the $late in a Bar containin" a %e( io'ine crystals )see tec. 208 sec8

20.*8 ca$ the Bar8 an' lea#e it in the car until the s$ots be"in to a$$ear.

6. Remo#e the $late %rom the Bar an' li"htly outline all the s$ots that became #isible (ith the

io'ine treatment.

. :sin" a ruler8 mar+e' in millimeters8 measure the 'istance that each s$ot has tra#ele'8 relati#e

to the sol#ent %ront.

,. Calculate the RF #alues %or each s$ot )tec 208 sec 20.-*.

-. E&$lain the relati#e $ositions o% the three com$oun's in terms o% their $olarities. 7'enti%y the

com$oun' or com$oun's %oun' in the un+no(n mi&ture.

10. ith your instructor?s o$tion8 submit the !C $late (ith your re$ort.

Experiment 6B: electing the correct ol9ent for Thin)ayer Chromatography

7n e&$eriment 6A8 it (as state' (hat the sol#ent to use %or 'e#elo$in" !C $late. 7n some

Compoun$s: Ben&oin an$ Ben&il


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ol9ents: Acetone, Methylene Chlori$e, 'exane

Preparation:

1. A $air o% com$oun's (ill be assi"n to run on !C8 you selectin" an o(n $air.

2. btain about 0. m o% the three solutions9 one solution o% each o% the t(o in'i#i'ualcom$oun's an' a solution containin" both com$oun's.

3. =re$are three thin/layer $lates in the same (ay in E&$eriment A8 e&ce$t each $late shoul' be

10 & 3.3 cm.

4. hen mar+in" them (ith a $encil %or s$ottin"8 ma+e three mar+s 1 cm a$art.

. =re$are three micro/$i$ettes to s$ot the $lates.

6. =re$are three !C 'e#elo$ment chambers )li+e e&$eriment A*8 (ith each chamber

containin" one o% the three sol#ents su""este' %or your $air o% com$oun's.

Developing the TLC plate:

1. n each $late8 s$ot the t(o in'i#i'ual com$oun's an' the mi&ture o% both com$oun's.

2. For each o% the three sam$les8 use a 'i%%erent micro$i$ette to s$ot the sam$le on the $lates.

3. =lace each !C $late in one o% the three 'e#elo$ment chambers8 ma+in" sure that the $late

'oes not come in contact (ith the %ilter $a$er liner.

4. Remo#e each $late (hen the sol#ent %rom is 1/2cm %rom the to$ o% the $late.

. :sin" a lea' $encil8 mar+ the $osition o% the sol#ent %ront.

6. @et the $late on a $iece o% $a$er to(el to 'ry.

. hen the $late is 'ry8 obser#e it un'er a short/(a#elen"th :V lam$8 $re%erably in a 'ar+ene'

hoo' or a 'ar+ene' room.

,. ith a $encil8 li"htly outlines any s$ots that a$$ear.

-. e&t8 $lace the $late in a Bar containin" a %e( io'ine crystals8 an' lea#e it in the Bar until to

s$ots became #isible (ith the io'ine treatment.

10. :sin" a ruler8 mar+e' in millimeters8 measure the 'istance that each s$ot has tra#ele' relati#e

to the sol#ent %rom.

11. Calculate the R % #alues %or each s$ot. >ith the instructor?s instruction8 submit the !C

$lates (ith the re$ort.

12. Dee$ in min'9 hich o% the three sol#ents resol#e' the t(o com$oun's success%ully For the

t(o sol#ents that 'i' not (or+8 e&$lain8 in terms o% their $olarities8 (hy they %aile'.


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(bser9ations:

!he %irst $art o% the e&$eriment in an intro'uction to the thin layer chromato"ra$hy. !C

(as use' to se$arate the three com$oun's9 %luorene8 %luorenol8 an' %luorenone. !he e&$eriment

starte' by $re$arin" the 'e#elo$ment chamber (hich consists o% a$$ro&imately about 1/3m o%methylene chlori'e insi'e the me'ium bea+er8 an' a %ilter $a$er (ra$$e' aroun' the insi'e o% the

bea+er. !he bea+er chamber (as then co#ere' by a %oil to a#oi' contamination. !C $a$er (as

$re$are' (ith the instructe' measurement 'istances. :sin" a $i$ette8 s$ottin" (as starte' %rom

le%t to ri"ht )%luorene8 %luorenol8 %luorenone8 the un+no(n mi&ture8 the mi&ture o% three

com$oun's*. !he un+no(n that use' in this e&$eriment is8 un+no(n 2. A%ter s$ottin"8 the !C

$a$er (as $lace' insi'e the chamber8 ma+in" sure that the $late (on?t come in contact (ith the

%ilter $a$er. e#elo$in" the !C $late until the sol#ent )methylene chlori'e* reach the sol#ent

%ront8 (hich is about 1/2cm %rom the to$ o% the $late8 ta+es about 10/1 minutes. At %irst8 the

!C $late (as $lace' insi'e a Bar (ith a %e( io'ine crystals. @$ots startin" to a$$ear a%ter about

6/10 minutes. !he $late (as remo#e' %rom the io'ine Bar an' (ere trie' to #ie( the s$ots un'erthe :V li"ht. More s$ots are #isible un'er the :V li"ht. !he $ossible reason behin' the s$ots not

#ery #isible on the io'ine crystal8 is that it ta+es time %or it to 'e#elo$. !he %arthest s$ot is

%luorene ) mm*8 then %luorenone )3 mm* an' lastly %luorenol )1- mm*. !he un+no(n mi&ture

sho(s that it?s the combination o% the three com$oun's )%luone8 %luorenol8 %luorenone*.

!he same $rocess (as use' on the e&$eriment 6. Ho(e#er this time8 benGoin an'

benGyl (as use'8 an' the sol#ents are acetone8 methylene chlori'e8 an' he&ane. 7n this

e&$eriment8 the three sol#ents are teste' to %in' out (hich one is the best !C sol#ents (ill (or+

best on se$aratin" the com$oun's. urin" the e&$eriment8 obser#ation sho(s that acetone

e#a$orate' ;uic+ly an' (hen the !C $lates are obser#e' un'er the :V li"ht8 the com$oun's

mo#es on the same rate. !his conclu'es that acetone isn?t the best sol#ent. !he !C $late %romthe sol#ent he&ane (as then obser#e' un'er the :V li"ht8 it sho(s that the s$ots 'i'n?t mo#e at

all8 there%ore it?s not the best sol#ent. !he !C $late %rom the methylene chlori'e sho(s the best

se$aration o% the s$ots )com$oun's*. @ome s$ots (ere sho(n 'ue to contamination. Ho(e#er8

the com$oun's s$ots are still #isible. y obser#ation8 benGyl tra#ele' the %arthest on the $late

(ith a 'istance o% 43 mm8 then benGoin (ith a 'istance o% 2 mm.


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;esults an$ #iscussion:

Part 6A: Fluorene, Fluorenol, an$ Fluorenone in Methylene Chlori$e

1*!he %irst ima"e belo( sho(s the !C $a$er un'er the :V li"ht. Measurements o% the 'istances

are obtaine' an' recor'e' belo(9

:sin" the 'istances that (ere measure' %rom the !C $a$er8 the R % #alues are calculate' an'

sho(n on the table belo(9

Compoun$: ; f


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Flurenone5 -*68 3 mm mm 0.6

0.5 Mixture

0Fluorene, Fluorenol,

Fluorenone5

*--

-*/.

-*68

)* mm mm 1.00

1- mm mm 0.3

3 mm mm 0.6

)2*

7n this thin layer chromato"ra$hy e&$eriment8 the three com$oun's use' (ere %luorene8

%luorenol8 an' %luorenone. Methylene chlori'e (as use' as a sol#ent %or all o% these. !he R %

#alues %or %luorene is 1.00. ase' on the structure o% %luorene8 (hich is a com$letely %lat an'

non/$olar com$oun'8 an' methylene chlori'e8 a mo'erately $olar molecule8 the R % #alue

calculate' (as accurate. !his is because the methylene chlorise (as too $olar in com$arison to

the %luorene to be an i'eal sol#ent8 so the %luorene trans%erre' all the (ay to the to$ o% the

chromato"ra$hy $a$er.

!he R % #alues %or %luorenol (as 0.3. @ince the %luorenol is a #ery non/$olar molecule because o%

the $henyl "rou$s8 an' the methylene chlori'e has mo'erate $olarity8 it is $re'icte' that the R %

#alues %or the %luorenol (oul' be #ery lo(.

!he R % #alues %or %luorenone (as 0.6. !his is e&$ecte' because methylene chlori'e an'

%luorenone ha#e similar $olarities8 (hich allo(s the methylene chlori'e to mo#e %luorenone

about hal%(ay u$ the chromato"ra$hy $a$er.

)3*

!he un+no(n 2 are com$ose' o% the mi&ture o% the three com$oun's )%luorene8 %luorenol8

%luorenone*. !he R % #alues that are calculate' are9 %luorene9 1.008 %luorenol9 0.38 %luorenone9

0.6. !he R % calculations %or all com$oun's are Bust the same as the one calculate' on in'i#i'ualtest. !he same results are calculate' on the mi&ture com$oun's.


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Part 6B:

)1*

Ben&oin an' Ben&il are the com$oun's use' in this e&$eriment8 an' the sol#ents use' are9

acetone, methylene chlori$e8 an' hexane.

!he %ollo(in" $lates are results %rom the e&$eriment9

Benzoin, Benzil and

mixture in Acetone Benzoin, Benzil mixture in Hexa

Benzoin, Benzil andmixture in Methylene


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:sin" the !C $lates8 the 'istances are measure' an' sho(n belo(9

045

!he t(o com$oun's teste' in this e&$eriment $art 6 (ere benGoin an' benGil. !he sol#ents

(ere teste' in acetone8 methylene chlori'e8 an' he&ane. !he %irst test (as in acetone. As sho(n

on the $ictures on the $re#ious $a"e8 the com$oun's )benGoin8 benGyl8 mi&ture*8 all mo#e' at thesame rate. !he secon' test (as in methylene chlori'e8 as sho(n in the $icture8 the com$oun's

mo#e' hal%(ay u$ the $a$er $late8 an' se$aratin" on the last one )mi&ture* neatly. !he last test is

in he&ane8 as sho(n in the $icture8 the s$ots )com$oun's* 'i'n?t mo#e at all.

!his e&$eriment sho(s that the best sol#ent %or benGoin an' benGil %or !C is methylene

chlori'e.

!he table belo( sho(s the R % #alues %or he&ane9

;f


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enGil 0.6 43 mm66 mm 0.6

Mi&ture )enGoin

an' enGil*

0.3,8 0.6 2 mm66 mm 0.3,

43 mm66 mm 0.6

y usin" the R % #alues8 obser#ation sho(s that benGil tra#ele' %urther than benGoin. enGil

ha#in" a 0.6 R % #alue an' benGoin ha#in" a R % #alue o% 0.3,.

)3*

!he table belo( sho(s the R % #alues %or acetone.

;f


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an' enGil*

!he R % #alues sho(s that the com$oun's )benGoin an' benGil* 'i'n?t mo#e at all (hen usin"

He&ane as the sol#ent. He&ane is too non/$olar8 an' (hen the sol#ents is too lo( o% a $olarity8

the com$onent (ill not mo#e enou"h8 an' a"ain se$aration o% the com$oun's (on?t occur.Conclusion:

!hin layer chromato"ra$hy )!C* may be use' to se$arate similar com$oun's. !C can be use'

to hel$ 'etermine the number o% com$onents in a mi&ture8 i'enti%y the com$oun's8 an' the

$urity o% the com$oun's. !C consists o% three ste$s/ s$ottin"8 'e#elo$ment an' #isualiGation.

@$ottin" consists o% usin" a micro/$i$ette to trans%er a small amount o% solution at the one en' o%

the !C $late. e#elo$ment consists o% $lacin" the bottom o% the !C $late into a bea+er

consistin" o% a 'e#elo$ment sol#ent8 (hich then tra#els u$ the $late by ca$illary action. 7t is

im$ortant to use the ri"ht sol#ent (hen 'oin" this e&$eriment. !he last ste$ is #isualiGation8 there

are t(o (ays to obser#e the $late s$ots )se$aration o% com$oun's*8 one is insi'e the Bar (ith a

%e( io'ine crystals8 an' another one is un'er the :V li"ht. 7n this e&$eriment session8 it is more

con#enient to Bust use the :V li"ht8 this is because the io'ine #a$or #isualiGation8 because this

ta+es lon"er time an' some s$ots are not #isible8 ho(e#er by usin" the :V li"ht8 all s$ots are

#isible. !he s$ots are then circle' (ith a $encil an' 'istances are measure'. R % #alues are

calculate'. R % #alue is e;ual to the 'istance tra#ele' by the substance 'i#i'e' by the 'istance

tra#ele' by the sol#ent %ront. R % #alues 'e$en's on the $olarity o% the sol#ent8 the more $olar the

com$oun'8 the %urther an' ha#e a lar"er R % . !he less $olar8 the com$oun's tra#el a shorter

'istance an' ha#e a smaller R % .

Postlab =uestions:

1. Each of the solvents given should effectively separate one of the following mixture y TLC.

!atch the appropriate solvent with the mixture that you would expect to separate well with that

solvent. "elect your solvent from the following: hexane# methylene chloride# or acetone.

a$ %&phenylethanol and acetophenone

$ 'romoen(ene and p&xylene

c$ 'en(oic acid# %#)&dinitroen(oic acid# and %#)#*&Trinitroen(oic acid .


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Victorio 13

:se methylene chlori'e %or 2/$henylethanol an' aceto$henone.

:se he&ane %or romobenGene an' $/&ylene.

:se acetone %or enGoic aci'8 284/'initrobenGoic aci'8 an' 28486/!rinitrobenGoic aci'.

+. Consider the following errors that could be made when running TLC.

Indicate what should be done to correct the error.

a. A two-component mixture containing 1-octene and 1,4-dimethlben!ene

ga"e onl one spot with an #f "alue of $.%&. The sol"ent used was acetone.

b. A two-component mixture containing a dicarboxlic acid and tricarboxlic

acid ga"e onl one spot with an #f "alue of $.$&. The sol"ent used was

hexane.

c. 'hen a TLC plate was de"eloped, the sol"ent front ran o( the top of the

plate.

a. Acetone $olarity )elution sol#ent* is too hi"h8 there%ore ma+in" the com$oun's mo#es on the

same rate. !o correct the error8 try a ne( elution sol#ent mi&ture (hich is less $olar an' the

sin"le s$ot shoul' se$arate more e%%iciently.

b. !he R % #alue o% 0.0 means the com$onents har'ly mo#e' at all. !here%ore8 he&ane is too non/

$olar an' coul'n?t trans%er into the !C $late. !o correct the error8 try to use a more $olarsol#ent.

c. hen the !C $late (as 'e#elo$e' an' the sol#ent %ront ran o%% the to$ o% the to$8 to correct

this error8 use a ne( !C $late an' run the e&$eriment once a"ain.

chromatography final

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