84276694-Quantitative-Analysis-Nelson-s-Assay.docx

Quantitative Analysis of Carbohydrates Using Nelson’s Assay Marcus Natividad, Barbara Ngo, Lexley Ong and Jane Jenelle Quilaneta Group 8 2C Pharacy Biocheistry Laboratory ABSTRACT !

INTROUCTION "his experient ais to a#e the students able to deterine the aount o$ reducing sugars speci$ically glucose using Nelson%s "est! Carbohydrates are$ound the ost abundant class o$ organic copounds in living organiss! "hey srcinate as products o$ photosynthesis, an endotheric reductive condensation o$ carbon dioxide re&uiring light energy and the pigent chlorophyll! n CO2 ' n (2O ' energy )



Cn(2nOn ' n O2

*s noted here, the $orulas o$ any carbohydrates can be +ritten as carbon hydrates, Cn(2O-n, hence their nae! "he carbohydrates also #no+n as saccharides, are a a.or source o$ etabolic energy, both $or plants and $or anials that depend on plants $or $ood! *side $ro the sugars and starches that eet this vital nutritional role, carbohydrates also serve as a structural aterial cellulose-, a coponent o$ the energy transport copound *"P, recognition sites on cell sur$aces, and one o$ three essential coponents o$ /N* and 0N*13! Carbohydrates are divided into three general classes depending on the nuber o$ carbohydrate olecules they contain! Monosaccarides are siple sugars that cannot be hydroly4ed! Oligosaccharides are those that contain 2)5 onosaccharide units! Polysaccharides contains ore than 5 onosaccharide unit123

atos +ith an aldehyde group at the end and a reducing sugar! :t occurs +idely in plants and in the blood o$ an and other anials! Glucose is an iportant source o$ energy $or the body since it can be utili4ed directly +ithout any intervening digestive process1;3! One o$ the processes used to deterine glucose is the Nelson%s ethod! "he Nelson%s ethod, ore o$tenly called the Nelson)9oogyi ethod, is a +idely)used classical ethod $or &uantitative deterination o$ reducing sugars! :t deonstrates ho+ uch glucose is liberated $ro glycogen during en4yatic and acid hydrolysis 1<3! !"#!RI$!NTA% A& Sa'(le Used 9aple =sed> Nelson%s reagent *, Nelson%s reagent B, *rsenoolybdate reagent, Glucose standard, distilled +ater B& #ro)edures *&

Qualitative Analysis Using Nelson’s Assay& :n Nelson%s *ssay, Nelson%s reagent +as ade by ixing 2!7 L Nelson%s * +ith 5!7 L Nelson%s B! ?ight test tubes +ere labeled and the easured aounts o$ standard glucose +ere trans$erred into the test tubes! "he $ollo+ing aount o$ standard glucose +as sho+n in the table belo+>

Carbohydrates can be de$ined as copounds that have reactive aldehyde or #etone $unctional group and ultiple hydroxyl groups! "he ost coon carbohydrate is glucose C6(2O6-173!

ig! 9tructure o$ /)Glucose

"able ! Protocol $or glucose standard curve Tube No&

Glucose, or /)glucose, is classi$ied as an aldohexose, a onosaccharide +ith six carbon

+lu)ose Standard ,'%-

istilled .ater ,'%-

Un/no0n sa'(le ,'%-

"able 2! Coputed volue o$ standard glucose

*

5

!5

5

1

5!

5!@5

5

2

5!2

5!85

5

3

5!<

5!A5

5

4

5!6

5!65

5

1

5

2

5

5!8

6

!5

7

5

5!75 5!<5

Test tube no *

5

5!65

5!<

3 4

*$ter preparing the test tubes, !5 L o$ Nelson%s reagent +as added to each test tube! "he test tubes +ere heated siultaneously in a boiling +ater bath $or 25 inutes! :t +as then reoved and placed in a bea#er o$ +ater to cool do+n! "hen, !5 L o$ arsenoolybdate reagent +as added to each test tube and +as sha#en occasionally $or 7 inutes until the Cu 2O precipitate dissolved! "hen the absorbance o$ the standards and un#no+n +as easured against the blan# reagent at <85 n! inally, the concentration o$ the un#no+n +as deterined by constructing a glucose standard curve by plotting absorbance readings against solutions!

concentration

o$

standard

5 6

Nelsons *ssay is used to establish a standard curve $or glucose! 9aples containing accurately #no+n concentrations o$ glucose are sub.ected to this assay, absorbance readings recorded, and the data plotted as a standard curve! :t should be borne in ind that this ethod is a general test $or reducing sugars and does not distinguish bet+een reducing onosaccharides such as glucose- and reducing disaccharides such as altose"o $ind out the concentration o$ the standard glucose +hich is used in the Nelson%s *ssay! "he e&uation belo+ +as used>

Concentration

=

volumeof standard volumeof the solution

herein the volue o$ the standard glucose +as coputed using ratio and proportion +hich is sho+n in "able 2!

5g D EExEEE L 5!5L F xD 5 5!g D EExEEE L 5!5L F xD5!5 5!2g D EExEEE L 5!5L F xD5!52 5!<g D EExEEE L 5!5L F xD5!5< 5!6g D EExEEE L 5!5L F xD5!56 5!8g D EExEEE L 5!5L F xD5!58 !5g D EExEEE L 5!5L F xD5!

"he obtained volue o$ standard glucose is used $or the coputation o$ concentration o$ the standard glucose solution in the di$$erent test tubes! "able ;! Concentration o$ Glucose 9tandard Test Tube No& * 1

R!SU%TS AN ISCUSSION

8olu'e of standard glu)ose

2 3 4 5 6

+lu)ose Standard ,'g9'%5 ;L 5!5 ;L 5!52 D ;L 5!5< D ;L 5!56 D ;L 5!58 D ;L 5! D ;L

D 5 D ;!;;x5); 6!6Ax5

);

5!5; 5!52 5!526A 5!5;;

"able ; sho+s the coputed value o$ concentration o$ the standard glucose "he total volue o$ the solution is deterined by adding the volue o$ the standard glucose, volue o$ the distilled +ater, volue o$ the Nelson%s reagent and the volue o$ the arsenoolybdate reagent! "he total volue $or each test tube in this experient is ; L! "he aount o$ carbohydrates present in a given saple is easured by Nelson%s Method +hich is based on the capacity o$ the $ree

reducing groups o$ sugars in a carbohydrate saple to reduce Cu'2 in an al#aline solution! :n this deterination, the aount o$ $ree reducing sugars in the saple is directly related to the olybdenu blue $ored via series o$ oxidation reductions, and is easured colorietrically!

directly proportional to the concentration o$ the standard glucose solution! "his eans that as the concentration increases, the absorbance also increases!

Glucose Standard Cu rve 2 1.5

Absorbance

1 0.5 0 0

0.01

0.02

0.03

0.04

Glucose standard (mg/mL)

igure ;! Glucose 9tandard Curve

igure 2! 9aples used in Nelson%s *ssay igure 2 sho+s that Cu '2 is reduced to Cu ' by

"his sho+s the glucose standard curve that +as constructed by plotting the absorbance reading against the concentrations o$ the standard solutions! "he line or the Hbest $it lineI

the reducing activity o$ the sugar! :tresults in blue reduced- arsenoolybdous acid!

represents the ideal absorbance readings relative to the concentration!

"able
:t +as done using the linear regression e&uation>

Test Tube no *

Absorban)e

1

!;5A

2

!;8

3

!A@<

4

!85

5

!8A

5!@@A

y =mx + b herein y is e&ual to the absorbance, x represents the concentration o$ the glucose standard and  is the slope o$ the line "he linear e&uation $or glucose standard curve is yD8!;56x

x=

y 18.306

concentration= 6

!8@;

7

!8A

:n table <, +e +ere able to easure the aount o$ light transitted at <85 n o$ the standard solutions and o$ the un#no+n saple! "he absorbance is

absorbance

concentration=

18.306

1.817 18.306

concentration=0.099

mg mL

Referen)es 13http>+++2!cheistry!su!edu$acultyreusc hvirttxt.lcarbhyd!ht 123Crisostoo *! et!al!255-! Laboratory Manual in General Biochemistry !Jue4on City> CK? Publishing

:nc!do!eduhndb#supportochelabtech!htl 255; 1;3http>sta$$!science!nus!edu!sgdbsyhhlab;! ht 1<3http>+++!esu!edu.$reean;Ache;Al Lab25$olders;Alcarbpro;Alcarbpro!ht 173Boyer, 0odney! Concepts in Biocheistry! "hird! (obo#en, Ne+ ersey> ohn iley and 9ons Pte Ltd, 2556! Print!