NAME:
MERVE
MURAT
08.03.2017 ID: 2004141 SECTION / GROUP: 3 / 1 SUBMITTED TO EMRE EVİN
TITRATION OF AMINO ACIDS PURPOSE The purposes of this experiment are to draw titration cures for !"#cine and un$nown amino acid after titration of these amino acids with %&' (and ')"*+ and to determine the p%a and p, a"ues of un$nown amino acid -# usin. this titration cure
THEORY Titration for amino acids is a techniue that proide chemica" ana"#sis After titration process+ titration cure is formed and this cure proides an important information a-out amino acids such as its p, and p%a a"ues Moreoer+ from this titration cure+ -ufferin. ran.e of the amino acid can -e determined ,n addition+ the num-er of dissocia-"e protons of amino acid can -e predicted -# "oo$in. at the num-er of -uffer re.ion on a titration cure (T'E T,TR ,TR,MET ,METR, R,) )
ETE ETERM RM,,AT AT,& ,&
&
T'E
)&) )&)E ETRA TRAT T,& ,&
A
A), ),
,55&),AT,& )&5TAT5 & A U%&6 AM,& A),+7 nd* Amino acids can -e either accept or "ose more than one proton 8ecause of this+ the# are ca""e ca""ed d po"# po"#pr prot otic ic A"so+ "so+ the# the# are amph amphot oteri ericc owin owin. . to thei theirr ioni ioni9a 9a-"e -"e :;am :;amin ino o and and :; car-ox#"ic .roup< that is+ the# can act as acid or -ase dependin. on the p' ,n addition+ amino acids hae ioni9a-"e .roups that can act wea$ acid or -ase ,f amino acids are disso"ed in water+ the# can -e .enera""# o-sered in isoe"ectric form (o-son = 6inter+ 2014< Titration )ures of Aminoacids+7 nd* Each amino acid hae different p, a"ue This a"ue is the p' where the net char.e of amino acid is eua" to 9ero ,n other words+ amino acid is in the 9witter ion form ,t can -e ca"cu"ated -# ta$in. the aera.e of p%a a"ues for .ien amino acid and p%a a"ues can -e found -# ta$in. the midpoint of -uffer re.ion 6hen the concentration of the unprotonated amino acid eua"s that of the unprotonated form at a .ien p'+ their ratio is one 5o+ this p' can -e ca""ed as p%a ,n addition+ it can -e said that the ioni9a-"e .roup is at its -est -ufferin. capacit# at
when reachin. p%a a"ue< that is+ amino acid so"ution can resist the p' chan.es most effectie"# (Titration )ures of Aminoacids+7 nd*
PROCEDURE pH !"!# $%&'(#%"')*: 1. 2. 3. +. ,. -.
Remoe the coer of ."ass e"ectrode Rinse it with disti""ed water and dr# it with a-sor-ent tissue ,mmerse e"ectrode into p' > -uffer and measure the p' Rinse it with disti""ed water and dr# it with a-sor-ent tissue ,mmerse e"ectrode into p' 10 -uffer and measure the p' Measure the p' of tap water+ disti""ed water+ 00? ')" and 00? %&'+ and record them
G&$'*! "'"#%"')*:
1. 2. 3. +.
@ut 2? m of !"#cine into -ea$er and add 2? m of disti""ed water Mix the so"ution -# usin. ma.netic stirrer Measure the p' of so"ution -efore titration and record it @erform the titration process -# addin. 1 m of %&' to !"#cine so"ution and mix the
,. -.
so"ution -# stirrer Measure the p' of so"ution and record it Repeat ?th and B th steps unti" so"ution comes to -uffer re.ion (unti" so"ution stron."#
7.
resists to p' chan.es* Repeat ?th and B th steps -# addin. 2 m of %&' rather than 1 m unti" so"ution c"ose
8.
to the end point of -uffer re.ion Repeat ?th and B th steps -# addin. 1 m of %&' rather than 2 m unti" the end of -uffer re.ion
U**)* %'*) %$' "'"#%"')*: 1. @ut ?0 m of un$nown amino acid into -ea$er 2. Repeat 4;C step of !"#cine titration
CACUATIONS 4!5"')*5: 1. 6%" )4& %! (!!* "! pH 4p)* %'"')* )9 10 )9 0.1 M N%OH ") 3.0 0.1 M 5)&4"')* )9 '5)!&!$"#'$ G&$'*! p;% %&4!5 9)# G&$'*! %#! 2.+ %* <.8= > M =
n L
D
milimole = M ∗mL
milimole NaOH = 0.1∗10= 1 milimoleGly =0.1∗3.0=0.3 1−0.3 =0.7
mi"imo"e a&' remainin.
At the end of the titration+ 0> mi"imo"e a&' remains ,n other words+ there is no contri-ution to p' -# !"#cine 5o+ p&' can -e ca"cu"ated -# the he"p of remainin. a&' pOH =−loq [ OH ] where
pOH =−loq ( 0.05 )=1.27
[ OH ] =
0.7 mmole = 0.05 M 13 mL
pH =14 −1.27 =12.73
D
2. H) %* )9 0.1 M N%OH %#! #!?4'#! ") "'"#%"! 0.3 @ )9 '5)!&!$"#'$ A#@'*'*! M6 )9 A#@'*'*! '5 17+= > 0.3 m = =0.0017 n= MW 174
mo"e Ar.inine
5o+ we need 0001> mo"e of a&' for titration M =
n L
L=
D
n 0.0017 = = 0.017 L a&' is reuired M 0.1
3. D!5$#'(! "! p#!p%#%"')* )9 10 )9 0.0+, M p)"%55'4 p)5p%"! (499!# pH 7., M6 )9 ; 2HPO+ '5 17+ M6 )9 ;H 2PO+ '5 13- p;%2 7.2=.
[ K 2 HPO 4 ] [ KH 2 PO 4 ]
pH = pKa + log
log
[ K 2 HPO 4 ] = 0.3 [ KH 2 PO 4 ]
D
D
[ K 2 HPO 4 ] =2 [ KH 2 PO 4 ]
[ K 2 HPO 4 ] =2 [ KH 2 PO 4 ] [ KH 2 PO 4 ] + [ K 2 HPO 4 ] =0.045 M
[ K 2 HPO 4 ] [ KH 2 PO 4 ]
7.5=7.2 + log
[
2 KH 2 PO 4
] +[ KH 2 PO 4 ] =0.045
[
3 KH 2 PO 4
D
] =0.045
[ KH 2 PO 4 ] =0.015 M [
0.015 M + K 2 HPO 4
M =
n L
] =0.045 M
n =0.015∗10 =0.15 mole
D
n =0.03∗10 =0.3 mole
m n= MW
[ K 2 HPO 4 ] =0.03 M
D
%'2@&4 is reuired
% 2'@&4 is reuired
m=0.3∗174 =52.2 g
→
m=0.15∗136= 20.4 g
of % 2'@&4 is reuired
of %'2@&4 is reuired
5o+ for preparation of 10 of 004? M potassium phosphate -uffer p' >?+ ?22 . of % 2'@&4 and 204. of %' 2@&4 shou"d -e mixed and then fina" o"ume shou"d -e comp"eted to 10 -# usin. d'2&
pH M!%54#!!*"5: pH T%p %"!# H2O 0.0, N HC& 0.0, N ;OH >3 > 13 12> T!)#!"'$%& M!%54#! >42 F03 14? 12BB T%(&! 1. Theoretica" and measured p' a"ues of rea.ents used in experiment (T.C. ANKARA BÜYÜKŞEHİR BELEDİYESİ ASKİ GENEL MÜDÜRLÜĞÜ İÇME SUYU
KALİTE PARAMETRELERİ KIYASLAMASI + 201>* (Goumans+ 1C>2*
H nM
D 00? H 1M
D M H 00? mo"ar ')"
5o+ 00? mo"ar ' I p' H ;"o.J'IK D H nM
p' H ;"o.(00?*
D 00? H 1M
D p' H 13 for 00? ')"
D M H 00? mo"ar %&'
5o+ 00? mo"ar &' ; p&' H ;"o.J&';K D
p&' H ;"o.(00?*
D p&' H 13
p' H 14 L p&'
D p' H 14 L 13
D
p' H 12> for 00? %&'
T'"#%"')* C4#!5:
TI"#%"I)* $4#! 9)# G&$I*! Glycine 12 10 8
p'
6 4 2 0
Ttrant Vo"ume(m",TER*
G#%p 1. Titration cure for !"#cine (titrants are 0? ')" and 0? %&'* 5tartin. point ca"cu"ated -# usin. two .roup data H (?41I?F0*/2 H ?B H p'
Accordin. to titration cure+ p%a 1 a"ues can -e found p%a1 can -e around 20F mar$ed with p%a2 can -e around CC0 mar$ed with 5o+ p, a"ue is (20F I CC* / 2 H ?CC for !"#cine
p;%
N!" HC& )# ;OH 45!
N!" M HC& )# ;OH 45!
20F 21 14F 10 2 CC0 1? 11? 10 2 T%(&! 2. Accordin. to p%a a"ues of !"#cine+ net m and mM %&' used orma"it# of ')" and %&' is 0?
5o+ N = n∗ M
D
M =
0.5 = 0.5 M 1
')" and %&'
1 M =1000 mM
D
mM =500 for ')" and %&'
M1 V1 H M2 V2
D
? 10 2 21 H M 2 (21I?0*
D
M2 H 14F 10 2 mM ')"
M1 V1 H M2 V2
D
? 10 2 1? H M 2 (1?I?0*
D
M2 H 11? 10 2 mM %&'
TI"#%"I)* $4#! 9)# 4**)* Unknown 12 10 8
p'
6 4 2 0
Ttrant Vo"ume(m",TER*
G#%p 2. Titration cure for un$nown amino acid (titrants are 0? ')" and 0? %&'* 5tartin. point for -oth .roup (p' H >?B*
Accordin. to titration cure+ p%a a"ues can -e found p%a1 can -e around 1?3 mar$ed with p%a2 can -e around B30 mar$ed with p%a3 can -e around C2> mar$ed with 5o+ this amino acid can -e 'istidine 5o+ p, a"ue is (B3 I C2>* / 2 H >>C for 'istidine
p;%
N!" HC& )# ;OH 45!
N!" M HC& )# ;OH 45!
1?3 ?4 2B0 10 2 B30 100 333 10 2 C2> 13 103 10 2 T%(&! 3. Accordin. to p%a a"ues of 'istidine+ net m and mM %&' used orma"it# of ')" and %&' is 0? 5o+ N = n∗ M
D
M =
0.5 = 0.5 M 1
')" and %&'
1 M =1000 mM
D
mM =500 for ')" and %&'
M1 V1 H M2 V2
D
? 10 2 ?4 H M 2 (?4I?0*
M1 V1 H M2 V2
D
? 10 2 100 H M 2 (100I?0*
M1 V1 H M2 V2
D
? 10 2 13 H M 2 (13I?0*
D D
D
M2 H 2B0 10 2 mM ')" M2 H 333 10 2 mM ')" M2 H 103 10 2 mM %&'
DISCUSSION ,n this experiment+ !"#cine and un$nown amino acid titrated with %&' and titration cures of these amino acids were drawn or these cures+ the data set resu"ted from titration with ')" are ta$en from !roup 2 8efore titration procedure+ p' meter ca"i-ration was done with p' > and 10 -uffer rather than 4 and then some p' measurements were performed -# usin. tap water+ disti""ed water+ 00? ')" and 00? ' %&' or ca"i-ration+ the reason of usin. p' > and 10 -uffer is that titrant used durin. experiment is %&' 5o+ ca"i-ration was done with -asic -uffer -ecause %&' is a -asic rea.ent (p' )a"i-ration @rocedure for &ptima" Measurement @recision+7 200C+ Two; @oint )a"i-ration+7 nd* Accordin. to p' measurements of tap water+ disti""ed water+ 00? ')" and 00? %&'+ a"most a"" measurement oer"ap with theoretica" a"ues There is a deiation on"# in disti""ed water measurement (measured a"ue is F03 whereas theoretica" a"ue is >* The possi-"e reason of this deiation is that -ea$er mi.ht not -e washed proper"# with disti""ed water and contamination (chan.e in ion concentration* mi.ht -e occur -ecause of tap water+ or disti""ed water stoc$ mi.ht not -e prepared proper"# ,n addition+ these measurements was done for contro" of ca"i-ration ,n the first titration process+ ?0 m !"#cine and d' 2& mixture titrated with 30 m %&' To reach the second p%a a"ue+ 1? m of %&' was used and CC was o-tained as p%a a"ue This a"ue oer"ap with theoretica" a"ue that is CB (Amino Acids Reference )hart+7 nd*
The titration process with ')" performed -# !roup 2 -# usin. ?0 m !"#cine and d' 2& mixture This mixture titrated with 3C m of ')" To reach the first p%a a"ue+ 21 m of ')" was used and 20F was o-tained as p%a a"ue This a"ue oer"ap with theoretica" a"ue that is 234 (Amino Acids Reference )hart+7 nd* A"so+ its p, a"ue was found as ?CC and this a"ue oer"ap with theoretica" p, a"ue which is ?C> (Amino Acids Reference )hart+7 nd* ,n the second titration process+ ?0 m un$nown amino acid titrated with 2C m of %&' To reach the third p%a a"ue+ 13 m of %&' was used and C2> was o-tained as p%a a"ue The titration process with ')" performed -# !roup 2 -# usin. ?0 m un$nown amino acid This un$nown titrated with 11? m of ')" To reach the first and the R .roup p%a a"ues+ ?4 m and 100 m ')" were used and 1?3 and B30 were o-tained as p%a a"ue respectie"# 8# "oo$in. at these a"ues+ it is predicted that this un$nown amino acid can -e 'istidine -ecause its theoretica" p%a1 is 1F2+ p%a R is B0 and p%a 2 is C1> (Amino Acids Reference )hart+7 nd* A"so+ its p, a"ue was found as >>C and this a"ue oer"ap with theoretica" p, a"ue which is >?C (Amino Acids Reference )hart+7 nd* ,soe"ectric point (p,* app"icationsN ; ; ; ;
@rotein purification and separation usin. isoe"ectric focusin. and 2 e"ectrophoresis+ 5tud# of protein hetero.eneit# (protein truncations+ isoforms+ @TMs+ etc*+ 5tud# of protein;protein interactions+ Ana"#sis of protein fo"din. status (,soe"ectric @oint+7 nd*
REFERENCES Amino Acids Reference )hart (nd* Retrieed March 12+ 201>+ from httpN//wwwsi.maa"drichcom/"ife;science/meta-o"omics/"earnin.;center/amino;acid; reference;charthtm" o-son+ ) M+ = 6inter+ 5 (2014* The ,dentification of Amino Acids -# ,nterpretation of Titration )uresN An Under.raduate Experiment for 8iochemistr# World Jor!"l o#
C$%&'("l Ed(")'o!+ *(4*+ ?CLB1 httpsN//doior./1012BC1/6O)E;2;4;3 ,soe"ectric @oint (nd* Retrieed March 12+ 201>+ from httpN//wwwcreatie; proteomicscom/serices/isoe"ectric;pointhtm p' )a"i-ration @rocedure for &ptima" Measurement @recision (200C* Retrieed March 11+ 201>+ from httpN//too"sthermofishercom/content/sfs/-rochures/T;ph;ca"i-ration; procedure;for;optima";measurement;precision;T;@')A;Epdf
T.C. ANKARA BÜYÜKŞEHİR BELEDİYESİ ASKİ GENEL MÜDÜRLÜĞÜ İÇME SUYU KALİTE PARAMETRELERİ KIYASLAMASI (201>* An$ara Retrieed from httpN//wwwas$i.otr/#u$"e/dos#a/pdf/icmePsu#uPrapor"ari/201>;5u-atpdf T'E T,TR,METR,) ETERM,AT,& & T'E )&)ETRAT,& A A), ,55&),AT,& )&5TAT5 & A U%&6 AM,& A), (nd* Retrieed March 13+ 201>+ from httpN//wwwchemuc"aedu/Q-acher/)'EM14)/'andouts/Titrimetric etermination of the )oncentration and Acid issociation )onstants of an Amino AcidP5p1Bpdf Titration )ures of Aminoacids (Theor#* N 8iochemistr# Virtua" a- , N 8iotechno"o.# and 8iomedica" En.ineerin. N Amrita Vishwa Vid#apeetham Virtua" a- (nd* Retrieed March 14+ 201>+ from httpN//"a-amritaedu/su-H3=-rchHB3=simH133B=cntH1 Two;@oint )a"i-ration (nd* Retrieed March 11+ 201>+ from httpN//facu"t#sdmiramaredu/f.arces/a-Matters/)hemTech/modu"es/phmeter/p'2ptca" htm Goumans+ ' (1C>2* Measurement of p' of disti""ed water Jor!"l o# C$%&'("l
Ed(")'o!+ +,(B*+ 42C httpsN//doior./101021/ed04Cp42C
