BIOCHEM EXP 1 FORMAL REPORT.docx

8/19/2019 BIOCHEM EXP 1 FORMAL REPORT.docx

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EXPERIMENT 1PREPARATION OF BUFFER SOLUTIONS AND THE DETERMINATION OF THE pH USING DIFFERENT

LIQUID INDICATORS (COLORIMETRICALLY) AND USING THE pH METER (ELECTROMETRICALLY)

Caña, Emmeline D., Dalmacio, Juan Severino B., Guzman, Gerome B., Doria,Reina Mari Q., Elazegui, Monina J., Empaynado, Aira oui!e E.

GR"#$ %& o' (C)Medical *ec+nology General Bioc+emi!ry a-oraoryaculy o' $+armacy

Abstrat

*+i! e/perimen re0uire! +e re!earc+er! o prepare di''eren -u''er !oluion and o deermine +e p1 o' +e-u''er! and !ample! u!ing di''eren li0uid indicaor! and u!ing +e p1 meer. $rimary p+o!p+ae -u''er!oluion o' p+ 2 3a! a!!igned o +e group o -e prepared, 3+ic+ i! al!o uilized in +e e/perimen. 4 3a!prepared u!ing $+o!p+oric acid 51&$"67 a! +e 3ea8 acid and $rimary !odium p+o!p+ae di+ydrae59a1($"6

.(1("7 a! con:ugae -a!e. ;.2< g 9a"1 pelle! 3ere di!!olved in ;.=><& ml o' p+o!p+oric acid.*+e p1 o' +e -u''er !oluion 3a! deermined u!ing +e p1 meer and manipulaed drop3i!e u!ing =M9a"1. *+e p1 meer !+o3ed accurae p1 reading! o' +e prepared -u''er !oluion. "n +e o+er +and, +ep1 o' +e prepared -u''er !oluion 3a! deermined -a!ed on +e color! produced upon applying +e acid)-a!e indicaor! (Thymol blue, Bromophenol blue, Bromocresol green, Bromocresol purple, Phenol red,Methyl orange, Phenolphthalein7. Colorimeric deerminaion o' p1 !+o3ed +e di''eren color c+ange! an

acid)-a!e indicaor undergoe! 3+en added o a !oluion o' cerain p1. *+i! propery o' an acid)-a!eindicaor ena-le! one o ideni'y di''eren !u-!ance! -y narro3ing +eir p1 range. *+i! ena-le! one oideni'y a !peci'ic !u-!ance a! di''eren !u-!ance! e/+i-i di''eren p1 level!.

I!tr"#$t%"!

Acid! and -a!e! compo!e +e e!!enial componen! inour daily li'e. rom cleaning agen! 5-a!ic7 o 'rui!5acidic7, +e!e conain acid! and -a!e!. 1ou!e+oldammonia, a cleaning agen, and i0uid $lum-er aree/ample! o' -a!e!. According o *+oma! M. o3ry

and Jo+anne! 9. Bron!ed, acid! relea!e proon!5+ydrogen ion!7 in +eir reacion! 3+erea! -a!e! are!u-!ance! 3+ic+ accep! proon!. Srong acid!relea!e proon! readily and almo! compleely in diluea0ueou! !oluion! -u 3ea8 acid! do no !o +a, ae0uili-rium, in mo! ca!e!, le!! +an ;? o' a 3ea8acid i! ionized o yield proon!. Srong -a!e! +ave agrea capaciy 'or acceping proon! 3+erea! 3ea8-a!e! are poor accepor! o' proon!.

1 re'er! o a mea!ure o' +e +ydrogen ionconcenraion o' a !oluion. Soluion! 3i+ a +ig+concenraion o' +ydrogen ion! +ave a lo3 p1 and!oluion! 3i+ lo3 concenraion! o' 1@ ion! +ave a+ig+ p1. *+ere'ore, p1 i! al!o u!ed a! a mea!ure o' +e acidiy or -a!iciy o' a !oluion. Ma+emaically, p1i! e/pre!!ed a! +e negaive log in -a!e ; o' +ea0uaed +ydrogen ion concenraion.p1 )log 1@*+e p1 o' !oluion! are imporan in +e -iomedical!cience! 'or +e!e rea!on!. ir!, +e proper'uncioning o' -iomolecule! depend o an imporandegree on +e conrol o' p1. Second, c+ange! a! !malla! .; or .( p1 uni can cau!e !igni'ican mea-olic

di!ur-ance! in cerain cell!, i!!ue!, and organ!Becau!e o' +e p1 !en!iiviy o' many -iomolecule!conrol o' p1 al!o i! imporan 'or +e !ucce!! o!everal procedure! u!ed in +e -iomedical la-oraory*+e!e include +e !eparaion, puri'icaion, and a!!ay

'or -iological aciviy o' !everal -iomolecule!. *+e p1can -e mea!ured elecromerically andcolorimerically. "ne 3ay o' mea!uring p1 i! -y u!inga device called p1 meer, 3+ic+ con!i!! o' a pair oelecrode! conneced o a meer capa-le o' mea!uring!mall volage!, on +e order o' millivol!. A volage3+ic+ varie! 3i+ +e p1, i! generaed 3+en +eelecrode! are placed in a !oluion. *+i! volage iread -y +e meer, 3+ic+ i! cali-raed o give +e p1"n +e o+er +and, Acid)-a!e indicaor! can al!o -eu!ed o narro3 do3n +e p1 range o' a !u-!ance*+e colour)c+ange inerval o' an indicaor i! +e p1range, 3+ere emp+a!ized colour c+ange a8e! place

and i 3a! deermined in +e e/perimen.

*+ere are cerain !oluion! +a re!i! c+ange in p1even 3+en 3e add o +em acid! or -a!e!. *+e!e arecalled -u''er!. A -u''er i! a !oluion 3+ic+ conain! a3ea8 con:ugae acid)-a!e pair +a can re!i! dra!icc+ange! in p1 upon +e addiion o' !mall amoun! o' a!rong acid or -a!e. A -u''er re!i!! c+ange! in p1-ecau!e i conain! -o+ an acidic !pecie! o neuralize

) ion! and a -a!ic one o neuralize 1@ ion!. 4 i! are0uiremen +a +e componen! o' a -u''er mu! no



con!ume eac+ o+er. or +i! rea!on, -u''er! areprepared -y mi/ing a 3ea8 acid or a 3ea8 -a!e 3i+ a!al o' +a acid or -a!e. *+e o-:ecive! o' +e 'ollo3ing e/perimen are a! oprepare di''eren -u''er !oluion! and deermine +ep1 o' +e -u''er! and !ample! colorimerically u!ingdi''eren li0uid indicaor! and elecromerically u!ing+e p1 meer.

EXPERIMENTAL

& C"'p"$!#s Tst#

Di!illed 3aer, $+o!p+oric acid 51&$"67, $rimary!odium p+o!p+ae di+ydrae 59a1($"6.(1("7, =M 1Cl,=M 9a"1, Acid)-a!e indicaor! 5*+ymol -lue,Bromop+enol -lue, Bromocre!ol green, Bromocre!olpurple, $+enol red, Me+yl orange, $+enolp++alein7

B& Pr"#$rMt*"#"+",-

1& Prparat%"! ". ra,!ts A volume o' <m conc. 1Cl 5= M7 3a! mea!uredu!ing a graduaed cylinder o prepare a volume o' <m o' =M 1Cl. *+e reagen 3a! ran!'erred o areagen -ole +a 3a! la-eled accordingly.

Compuaion!5;(.(M75/7 5=.M75.(<7/ .;( or ;(m conc. 1Cl

/& Prparat%"! ". B$..r s"+$t%"!

*+e -u''er !oluion 5$+o!p+aeF pa H.(;F

p1 27 3a! prepared u!ing +e 'ollo3ingguideline!

*a-le ;. Guideline!IDaa 'or -u''er !oluionpreparaion

A ma!! o' ;.2< g 9a"1 pelle! 3a! 3eig+ed u!ing ananalyical 3eig+ing !cale. 4 3a! +en ran!'erred ino

a -ea8er 3i+ a cerain volume o' di!illed 3aer odi!!olve +e pelle!. 4n ano+er -ea8er, a volume o' ;.=><& ml 5;> drop!7 $+o!p+oric acid 51&$"67 3a!mea!ured u!ing a !erological pipee and an a!piraor.A'er preparing +e !oluion!, +e acid 3a! mi/ed 3i++e 9a"1 !oluion. *+e mi/ure 3a! +en ran!'erredo +e (<m volumeric 'la!8. Di!illed 3aer 3a!added ino +e volumeric 'la!8 unil i reac+ed +e(<m mar8. *+e prepared !oluion 3a! !ored ino a<m am-er -ole +a 3a! la-eled properly.

Compuaion! o' +e Bu''er Soluion

1&$"6 )))K 1($"6) @ 1

@

1ender!on 1a!!el-ac+ e0uaion p1 pa @ logcon:. -a!eacid 2. H.(; @log51($"6

)1&$"6 7 51($"6

)1&$"6 7 anilog 2. L H.(; 51($"6

)1&$"6 7 =.;=<><

e / 1&$"6

=.;=<></ 1($"6

)

*oal Mole! 5n*7 or Mole!-u''er 5.(<5.;M7 .(< mole!

Mole!-u''er 5Mole!acid7@5Mole!con:. -a!e7.(< mole! / @ =.;=<></

&.62=2 / ;)& Mole!1&$"6 =.;=<>< .(;&H& Mole!1($"6

Molariy o' 1&$"6

2< g 1&$"6 ; g 1&$"6 ; mole >2 g ;.H g ; cm& ; cm& ; mole ;6. H6<mole

Nolume 1&$"6 .(< mole5;6.H6< moleI7 5;I;ml7;.=><& ml 1&$"6

Ma!! o' 9a"1Mole 9a"1 Mole oal @ Mole con:. -a!e.(< @ .(&;H& .6=&H& mole

.6=&H& mole 9a"1 / 6 g ; mole ;.2< g9a"1

0& E+tr"'tr% Dtr'%!at%"! ". pH

*+i! in!rumen indicae! +e +ydrogen ionconcenraion in a e! !oluion -y re!ponding o +epoenial developed -y an elecrical cell. *+e p1 mee3a! cali-raed a p1 H -y !oa8ing +e elecrode inneural reagen! li8e di!illed 3aer. *+e elecrode 3ali'ed ou o' +e neural !oluion and dried u!ing ai!!ue paper. *+e elecrode 3a! +en immer!ed in +eprepared -u''er !oluion. *+e !andard -u''er !+ould

+ave a p1 3i+in 3o 5(7 p1 uni! o' +e e/peced p1o' +e e! !oluion. *+e -ul- o' +e elecrode 3a!compleely covered 3i+ !oluion. #pon immer!ing +eelecrode ino +e -ea8er conaining +e prepared-u''er !oluion, +e reading !opped 'lucuaing a +ereading H.6. Amoun! o' 9a"+ 3ere added drop3i!eo -ring +e p1 level up o +e de!ired level, 2. A'e2); drop! o' +e !rong -a!e, +e reading 3aalready 2 and +e de!ired p1 level 3a! aained. *+eelecrode 3a! care'ully removed 'rom +e -u''e!oluion, rin!e i 3i+ di!illed 3aer u!ing a 3ae-ole and +en dried u!ing a i!!ue paper.

Oea8acid

Con:ugae-a!e

Nolume Conc. p1 pa1&$"6

1&$"6 1($"6); (<m .;M 2.

H.(;



& C"+"r%'tr% Dtr'%!at%"! ". pH

Eig+ e! u-e! 'or eac+ -u''er !oluion! 3ereprepared and properly la-eled 3i+ +e acid)-a!eindicaor!. *+e 'ollo3ing acid)-a!e indicaor! 3ereuilized *+ymol -lue, Bromop+enol -lue,Bromocre!ol green, Bromocre!ol purple, $+enolred, Me+yl red, Me+yl orange, and$+enolp++alein. Eac+ e! u-e 3a! +en 'illed 3i+;m 5(2 drop!7 o' +e prepared -u''er !oluionu!ing a !erological pipee and an a!piraor. Acerain amoun 5( drop!7 o' acid)-a!e indicaor 3a!dropped in +e corre!ponding la-eled e! u-e!.*+e e! u-e! 3ere !+a8en and +e color 3a!recorded.

RESULTS AND DISCUSSION

1& E+tr"'tr% Dtr'%!at%"! ". pH 4n +e proce!! o' adding 2); drop! o' +e !rong-a!e, +e reading 3a! already 2 and +e de!ired p1level 3a! aained. 4 !+o3ed 'lucuaion! inreading! 3i+ +e addiion o' 9a"1. *+i! +appen!

-ecau!e +e elecrode i! !en!iive o c+ange in +econcenraion! o' 1@ and "1) ion!. 4n +amanner, +e p1 meer i! more accurae in readingp1 level! compared o a p1 paper.

/& C"+"r%'tr% Dtr'%!at%"! ". pH

Colorimeric Analy!i! u!e! +e variaion a! amean! o' deermining +e p1 !ince +einen!iy o' +e color o' a !oluion c+ange!3i+ i! concenraion or p1. Re!earc+ !ay+a +e color may -e due o +e in+erenpropery o' a !u-!ance in +e !oluion, or +e'ormaion o' a produc a! a re!ul o' +eaddiion o' a !uia-le reagen or acid)-a!eindicaor. *+e p1 o' a !oluion can -deermined -y comparing +e color inen!iieo' +e !oluion 3i+ un8no3n p1 3i+ +e

inen!iie! o' +e !oluion! 3i+ 8no3n p1Cerain organic !u-!ance! c+ange color indilue !oluion 3+en +e +ydrogen ioconcenraion reac+e! a paricular value. oe/ample, p+enolp++alein i! a colourle!!u-!ance in any a0ueou! !oluion 3i+ a+ydrogen ion concenraion greaer +a;./;)2 M 5p1 le!! +an 2.7. 4n !oluion3i+ a +ydrogen ion concenraion le!! +an;./;)2 M 5p1 greaer +an 2.7p+enolp++alein i! red or pin8. Su-!anceli8e p+enolp++alein, 3+ic+ can -e u!ed o

deermine +e p1 o' a !oluion, are calledacid)-a!e indicaor!. Acid)-a!e indicaor! areei+er 3ea8 organic acid!, 1A, or 3eaorganic -a!e!, B"1, 3+ere +e leer! A or B!and 'or comple/ organic group.

*+e e0uili-rium in a !oluion o' +e acid)-a!eindicaor me+yl orange, a 3ea8 acid, can -erepre!ened -y +e e0uaion

1A ↔ 1@ @ A)

red yello3

or in!ance, +e anion o' me+yl orange i!yello3, and +e non)ionized 'orm i! red. 4'acid i! added o +e !oluion, +e increa!e in+e +ydrogen ion concenraion !+i'! +ee0uili-rium o3ard +e red 'orm in accordance3i+ +e la3 o' ma!! acion.

*+e indicaorP! colour i! +e vi!i-le re!ul o'+e raio o' +e concenraion! o' +e 3o!pecie! A) and 1A. 4n +e ca!e o' me+ylorange, 3+en 1@ +a! +e !ame numerical



value a! a, +e raioo' A) o 1A i!e0ual o ;, meaning+a <? o' +eindicaor i! pre!enin +e red acid 'ormand <? in +eyello3 ionic 'orm,and +e !oluion

appear! orange incolour. O+en +e+ydrogen ionconcenraionincrea!e! o a p1 o' &.;, a-ou >? o' +e indicaor i!pre!en in +e red'orm and ;? in +eyello3 'orm, and +e!oluion urn! red. 9o c+ange in color i!vi!i-le 'or any 'ur+er increa!e in +e+ydrogen ion concenraion.

*+e addiion o' a -a!e o +e !y!em 3ill increa!e +e+ydro/ide ion concenraion and decrea!e +e+ydrogen ion concenraion and !+i'! +e e0uili-riumo3ard +e yello3 'orm. A a p1 o' 6.6 a-ou >? o'+e indicaor i! in +e yello3 ionic 'orm, and a 'ur+erdecrea!e in +e +ydrogen ion concenraion doe! noproduce a vi!i-le colour c+ange. *+e p1 range-e3een &.; 5red7 and 6.6 5yello37 i! +e colour)c+ange inerval o' me+yl orangeF +e pronouncedcolour c+ange a8e! place -e3een +e!e p1 value!.

Simply pu, +e colour)c+ange inerval o' anindicaor i! +e p1range, 3+erepronounced colourc+ange a8e! placeF +e-order! o' +i! inervalcan -e e!imaed -ypa); and pa@;. 4n

addiion, acid)-a!eindicaor! al!o !+o3molecular c+araceri!ic!o' a !u-!ance. Colorc+ange! in molecule!can -e cau!ed -yc+ange! in elecroncon'inemen. Morecon'inemen ma8e! +elig+ a-!or-ed -luer

5dar8er7, and le!! ma8e! i redder 5lig+er7. *+e'ollo3ing a-le pre!en! +e e/peced color c+ange!+a 3ould occur depending on +e given pA value!.

*+e a-le a-ove -ecame +e -a!i! in order opredic more or le!! +e oucome o' +ee/perimenal procedure. *+e re!earc+er!3ere a!8ed o prepare a p+o!p+ae -u''er3i+ a p1 level o' 2. #pon adding +e acid)-a!e indicaor! o +i! paricular -u''er, +e'ollo3ing 3ere +e re!uling color!.

TABLE INFO HERE

The following table presents theresulting colors upon the addition ofthe acid-base indicators to thedierent buer solutions and todistilled water. The legend below mustbe referred to in order to interpret thetable of results.

Legend:R-Red

Y-YellowPY-Pale Yellow

GY- Golden Yellow YG- Yellow Green

O-OrangeRO- Red OrangeL- Light lue

-lue

Acid-BaseIndicator

pHRange

pkaValue

s

ExpectedColor

change

Th!mol ".# - #.$% $.& -

'.(

".) %$.'

Red* Yellow*lue

romophenollue

).& - +.( ,.& Yellow*luendigo

romocresolGreen

).$ - /.+ +., Yellow*lue

romocresolPurple

/.# - (.$ (.) Yellow*Pin0Purple

Phenol Red (.+ - $.& ,.' Yellow*Pin0Purple

1eth!l Red +.+ - (.# /." Red* Yellow

1eth!lOrange

)." - +.+ )., Red* Yellow

Phenolphthalein

$.& -"&.&

'.+ 2olorless*Pin0

Acid – BaseIndicator

p3 $.& uer

Th!mol Light !ellow

romophenol blue La4ender

romocresol green 5aded blue

romocresol purple Purple

Phenol red Pin0ish red

1eth!l orange Yellow orange

Phenolphthalein 2olorless



Acid-Base

Indicator

pH 2.5 pH .! pH5.!

pH ".! pH".5

pH #.! pH$2.!

%istilled&ater

'h()ol

R Y PY Y Y

GY

GY

Bro)ophenolBlue

Y PY

Bro)ocresol

*reen

Y GY G 7 7 7

LY

Bro)ocresol

+urple

P PY GY 6 P7P P

P

+henolRed

O GY P PR 7P P

Y

,eth(lRed

LP 7P LP LY P Y Y

LP

,eth(lrange

RRO O

OO O O

O



+henolphtalein

2 2 2 22-P LP

7P

2

table of e8pected color outcomes:

BIOCHEM EXP 1 FORMAL REPORT.docx

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