1.

1 Colour Reactions
Car bohydr ates ar e wi del y pr eval ent i n the pl ant ki ngdom, compr i si ng the mono-, di -,
ol i go-, and pol ysacchari des. The common monosacchari des are gl ucose, fructose, gal actose,
ri bose etc. The di sacchari des, i.e., the combi nati on of two monosacchari des i ncl ude sucrose,
l actose and mal tose. Starch and cel l ul ose are pol ysacchari des consi sti ng of many monosacchari de
resi dues. Cel l ul ose i s the most abundant organi c compound on thi s pl anet si nce i t forms part of
the cel l wal l i n pl ants.
Al dehydes (CHO) and ketones ( = CO) are acti ve groups i n carbohydrates. Carbohydrates
contai n many hydroxyl groups as wel l . The number of hydroxyl groups vari es wi th the number
of carbon atoms. Monosacchari des contai n the free al dehyde or ketone group. Some di sacchari des
have the free al dehyde group (mal tose) and some do not have the free ones (sucrose). The
pol ysacchari des, starch and cel l ul ose, are pol ymers of monosacchari des l i nked through the
acti ve groups.
The chemi cal properti es of sacchari des vary dependi ng upon the number of hydroxyl groups
and the presence or absence of CHO/= CO groups. These vari ati ons are the basi s i n the
devel opment of col our reacti ons to i denti fy the sacchari des.
Some si mpl e tests used to i denti fy the presence/absence of certai n sacchari des are l i sted
bel ow:
REAGENTS
H I odine solution: Add a few crystal s of i odi ne to 2% potassi um i odi de sol uti on ti l l the
col our becomes deep yel l ow.
H Fehlings reagent A: Di ssol ve 34.65 g copper sul phate i n di sti l l ed water and make up to
500 mL.
H Fehlings reagent B: Di ssol ve 125 g potassi um hydr oxi de and 173 g Rochel l e sal t
(potassi um sodi um tartrate) i n di sti l l ed water and make up to 500 mL.
1
+D=FJAH 1
+)4*0;,4)6-5
2 Biochemical Methods
H Benedicts qualitative reagent: Di ssol ve 173 g sodi um ci trate and 100 g sodi um carbonate
i n about 500 mL water. Heat to di ssol ve the sal ts and fi l ter, i f necessary. Di ssol ve 17.3 g
copper sul phate i n about 100 mL water and add i t to the above sol uti on wi th sti rri ng
and make up the vol ume to 1 L wi th water.
H Barfoeds reagent: Di ssol ve 24 g copper acetate i n 450 mL boi l i ng water. I mmedi atel y
add 25 mL of 8.5% l acti c aci d to the hot sol uti on. Mi x wel l , Cool and di l ute to 500 mL.
H Seliwanoffs reagent: Di ssol ve 0.05 g resorci nol i n 100 mL di l ute (1:2) hydrochl ori c aci d.
H Bials reagent: Di ssol ve 1.5 g orci nol i n 500 mL of concentrated HCl and add 20 to 30
drops of 10% ferri c chl ori de.
The reacti ons of carbohydrates are gi ven i n Tabl e 1.1.
TABLE 1.1: Reactions of carbohydrates
Experi ment Observati on Remarks
The col our for med i s due to
the reacti on of al pha-naphthol
wi th furfural and/or i ts deri va-
ti ves formed by the dehydrati on
of sugars by concentrated sul -
phur i c aci d. Al l car bohydr ates
r eact posi ti vel y wi th thi s r ea-
gent.
1. Molischs Test
Add two dr ops of Mol i s ch s
r eagen t (5% 1-n aph th ol i n
al cohol ) to about 2 mL of test
sol uti on and mi x wel l .
I ncl i ne the tube and add about
1 mL of concentrated sul phuri c
aci d al ong the si des of the tube.
Obs er v e th e col ou r at th e
juncti on of the two l i qui ds.
2. I odine Test
Add a few dr ops of i odi n e
sol uti on to about 1 mL of the
test sol uti on.
3. Fehlings Test
To 1 mL of Fehl i ngs sol uti on A,
add 1 mL of Fehl i ngs sol uti on
B and a few drops of the test
sol uti on. Boi l for a few mi nutes.
A red-cum-vi ol et ri ng appears
at th e j u n cti on of th e two
l i qui ds.
Appearance of deep bl ue col our.
Thi s i ndi cates the pr esence of
starch i n the sol uti on.
The bl ue col our i s due to the for-
mati on of star ch-i odi ne com-
pl ex.
The bl ue al kal i ne cupri c hydrox-
i de pr esent i n Fehl i ngs sol u-
ti on, when heated i n the pres-
ence of reduci ng sugars, gets re-
duced to yel l ow or red cuprous
oxi de and i t gets pr eci pi tated.
Hence, for mati on of the col -
oured preci pi tate i ndi cates the
presence of reduci ng sugars i n
the test sol uti on.
For mati on of y el l ow or
browni sh-red preci pi tate.
Carbohydrates 3
4. Benedicts Test
To 2 mL of Benedi cts reagent
add fi v e dr ops of th e tes t
sol uti on. Boi l for fi ve mi nutes
i n a water bath . Cool th e
sol uti on.
Formati on of red, yel l ow or green
col our /pr eci pi tate.
As i n Fehl i ngs test, the reduc-
i ng sugar s because of havi ng
potenti al l y fr ee al dehyde or
keto group reduce cupri c hydrox-
i de i n al kal i ne sol uti on to r ed
col oured cuprous oxi de. Depend-
i ng on the sugar concentrati on
yel l ow to green col our i s devel -
oped.
Onl y monosacchar i des answer
thi s test. Si nce Bar foeds r ea-
gent i s weakl y aci di c, i t i s r e-
duced onl y by monosacchari des.
5. Barfoeds Test
To 1 mL of the test sol uti on add
about 2 mL of Barfoeds reagent.
Boi l i t for one mi nute and al l ow
to stand for a few mi nutes.
6. Seliwanoffs Test
To 2 mL of Sel i wanoff s reagent
add two dr ops of test sol uti on
and heat the mi xtur e to just
boi l i ng.
For mati on of br i ck -r ed
pr eci pi tate.
Appearance of deep red col our. I n concentrated HCl , ketoses
under go dehydr ati on to yi el d
furfural deri vati ves more rap-
i dl y than do al doses. These de-
ri vati ves form compl exes wi th
resorci nol to yi el d deep red col -
our.
I t i s a ti med col our r eacti on
speci fi c for ketoses.
7. Bials Test
To 5 mL of Bi al s r eagent add
23 mL of sol uti on and war m
gentl y. When bubbl es ri se to the
surface cool under the tap.
8. Test for non-reducing sugars
such as sucrose:
(a) Do Benedi cts test wi th the
test sol uti on.
(b) Add 5 drops of concentrated
HCl to 5 mL of test sol uti on
i n another test tube. Heat for
fi ve mi nutes on a boi l i ng
water bath.
Appearance of green col our or
pr eci pi tate.
I t i s speci fi c for pentoses. They
get converted to furfural . I n the
presence of ferri c i on orci nol and
furfural condense to yi el d a col -
oured product.
No ch ar acter i s ti c col ou r
for mati on.
Appear ance of r ed or yel l ow
col our.
I ndi cates the absence of reduc-
i ng sugars i n the gi ven sol uti on.
I ndi cates the for mati on of r e-
duci ng sugars from non-reduc-
i ng sugars after hydrol ysi s wi th
aci d.
(Contd.)
4 Biochemical Methods
Add 10% s odi u m h y dr ox i de
s ol u ti on to gi v e a s l i gh tl y
al kal i ne sol uti on (test wi th red
l i tmus paper ). Now per for m
Ben edi cts tes t wi th th i s
hydrol ysed sol uti on.
9. Mucic Acid Test
Add a few drops of conc. HNO
3
to the concentrated test sol uti on
or s u bs tan ce di r ectl y an d
evaporate i t over a boi l i ng water
bath ti l l the aci d fumes ar e
expel l ed. Add a few dr ops of
water and l eave i t overni ght.
10. Osazone Test
To 0.5 g of phenyl hydr azi ne
h y dr och l or i de add 0.1 g of
sodi um acetate and 10 drops of
gl aci al aceti c aci d. To th i s
mi x tu r e add 5 mL of tes t
sol uti on and heat on a boi l i ng
water bath for about hal f an
hour. Al l ow the tube to cool
sl owl y and exami ne the crystal s
under a mi croscope.
Formati on of crystal s. The both end carbon groups are
oxi di zed to car boxyl i c gr oups.
The resul tant sacchari c aci d of
gal actose i s cal l ed muci c aci d
whi ch i s i nsol ubl e i n water.
The ketoses and al doses r eact
wi th phenyl hydr azi ne to pr o-
duce a phenyl hydrazone whi ch
i n turn reacts wi th another two
mol ecul es of phenyl hydrazi ne to
form the osazone.
Gl ucose, fructose and mannose
pr oduce needl e-shaped yel l ow
os azon e cr y s tal s , wh er eas
l actos azon e i s mu s h r oom-
s h aped. Di ffer en t os azon es
s h ow cr y s tal s of di ffer en t
s h apes . Mal tos e pr odu ces
fl ower-shaped crystal s.
NOTES:
1. For osazone test, the reacti on mi xture shoul d be between pH 5 and 6. Fructose takes 2 mi n to
form the osazone whereas for gl ucose i t i s 5 mi n. The di sacchari des take a l onger ti me to form
osazones. Di ssachari des form crystal s onl y on cool i ng.
2. When a mi xtur e of car bohydr ates i s pr esent i n the test sampl e, chr omatogr aphi c methods
shoul d be empl oyed to i denti fy the i ndi vi dual sugars.
READING
1. Sadasi vam, S. and Theymol i Bal asubramani an (1985). Practical Manual (Undergraduate), Tami l
Nadu Agri cul tural Uni versi ty, Coi mbatore, p. 2.
1.2 Determination of Reducing Sugars
by Nelson-Somogyi Method
Sugars wi th reduci ng property (ari si ng out of the presence of a potenti al al dehyde or keto
group) are cal l ed reduci ng sugars. Some of the reduci ng sugars are gl ucose, gal actose, l actose
Carbohydrates 5
and mal tose. The Nel son-Somogyi method i s one of the cl assi cal and wi del y used methods for
the quanti tati ve determi nati on of reduci ng sugars.
PRINCIPLE
The reducing sugars when heated with alkaline copper tartrate reduce the copper from the
cupric to cuprous state and thus cuprous oxide is formed. When cuprous oxide is treated with
arsenomolybdic acid, the reduction of molybdic acid to molybdenum blue takes place. The blue
colour developed is compared with a set of standards in a colorimeter at 620 nm.
MATERIALS
Al kal i ne Copper Tar tr ate
(i) Di ssol ve 2.5 g anhydrous sodi um carbonate, 2 g sodi um bi carbonate, 2.5 g potassi um
sodi um tartrate and 20 g anhydrous sodi um sul phate i n 80 mL water and make up to
100 mL.
(ii) Di ssol ve 15 g copper sul phate i n a smal l vol ume of di sti l l ed water. Add one drop of
sul phuri c aci d and make up to 100 mL.
Mi x 4 mL of B and 96 mL of sol uti on A before use.
Arsenomolybdate reagent: Di ssol ve 2.5 g ammoni um mol ybdate i n 45 mL water. Add 2.5 mL
sul phuri c aci d and mi x wel l . Then add 0.3 g di sodi um hydrogen arsenate di ssol ved i n 25 mL
water. Mi x wel l and i ncubate at 37C for 2448 hours.
Standard glucose solution: Stock: 100 mg i n 100 mL di sti l l ed water.
Working standard: 10 mL of stock di l uted to 100 mL wi th di sti l l ed water [100 g/mL].
PROCEDURE
1. Wei gh 100 mg of the sampl e and extract the sugars wi th hot 80% ethanol twi ce (5 mL
each ti me).
2. Col l ect the supernatant and evaporate i t by keepi ng i t on a water bath at 80C.
3. Add 10 mL water and di ssol ve the sugars.
4. Pi pette out al i quots of 0.1 or 0.2 mL to separate test tubes.
5. Pi pette out 0.2, 0.4, 0.6, 0.8 and 1 mL of the worki ng standard sol uti on i nto a seri es of
test tubes.
6. Make up the vol ume i n both sampl e and standard tubes to 2 mL wi th di sti l l ed water.
7. Pi pette out 2 mL di sti l l ed water i n a separate tube to set a bl ank.
8. Add 1 mL of al kal i ne copper tartrate reagent to each tube.
9. Pl ace the tubes i n a boi l i ng water for 10 mi nutes.
10. Cool the tubes and add 1 mL of arsenomol ybol i c aci d reagent to al l the tubes.
11. Make up the vol ume i n each tube to 10 mL wi th water.
12. Read the absorbance of bl ue col our at 620 nm after 10 mi n.
13. From the graph drawn, cal cul ate the amount of reduci ng sugars present i n the sampl e.
CALCULATION
Absorbance corresponds to 0.1 mL of test = x mg of gl ucose
10 mL contai ns =
0.1
x
10 mg of gl ucose
= % of reduci ng sugars
6 Biochemical Methods
READINGS
1. Somogyi , M. (1952). J . Biol. Chem., 200, 245.
2. Kri shnaveni , S.; Theymol i Bal asubramani an and Sadasi vam, S. (1984). Food Chem., 15, 229.
1.3 Estimation of Reducing Sugar by
Dinitrosalicylic Acid Method
For sugar esti mati on an al ternati ve to Nel son-Somogyi method i s the di ni trosal i cyl i c aci d
methodsi mpl e, sensi ti ve and adoptabl e duri ng handl i ng of a l arge number of sampl es at a
ti me.
MATERIALS
H Dinitrosalicylic Acid Reagent (DNS Reagent)
Di ssol ve by sti rri ng 1 g di ni trosal i cyl i c aci d, 200 mg crystal l i ne phenol and 50 mg sodi um
sul phi te i n 100 mL 1% NaOH. Store at 4C. Si nce the reagent deteri orates due to sodi um
sul phi te, i f l ong storage i s requi red, sodi um sul phi te may be added at the ti me of use.
H 40% Rochel l e sal t sol uti on (Potassi um sodi um tartrate).
PROCEDURE
1. Fol l ow, steps 1 to 3 as i n Nel son-Somogyi s method to extract the reduci ng sugars from
the test materi al .
2. Pi pette out 0.5 to 3 mL of the extract i n test tubes and equal i ze the vol ume to 3 mL
wi th water i n al l the tubes.
3. Add 3 mL of DNS reagent.
4. Heat the contents i n a boi l i ng water bath for 5 mi n.
5. When the contents of the tubes are sti l l warm, add 1 mL of 40% Rochel l e sal t sol uti on.
6. Cool and read the i ntensi ty of dark red col our at 510 nm.
7. Run a seri es of standards usi ng gl ucose (0500 g) and pl ot a graph.
CALCULATION
Cal cul ate the amount of reduci ng sugars present i n the sampl e usi ng the standard graph.
READING
1. Mi l l er, G.L. (1972). Anal. Chem., 31, p. 426.
1.4 Determination of Glucose by
Glucose Oxidase Method
Gl ucose i s a wi del y di stri buted si mpl e sugar wi th an acti ve al dehyde group. Esti mati on of
gl ucose by gl ucose oxi dase gi ves the true gl ucose concentrati on el i mi nati ng the i nterference
by other reduci ng sugars.
PRINCIPLE
Glucose oxidase catalyses the oxidation of alpha-D-glucose to D-glucono-1, 5 lactone (gluconic
Carbohydrates 7
acid) with the formation of hydrogen peroxide. The oxygen liberated from hydrogen peroxide by
peroxidase reacts with the O-dianisidine and oxidises it to a red chromophore product.
Gl ucose + O
2
Gl ucose
Oxi dase
H
2
O
2
+ Gl uconi c Aci d
H
2
O
2
+ O-di ani si di ne
Peroxi dase

Red-col oured product

MATERIALS
H Glucose Oxidase Peroxidase Reagent
Di ssol ve 25 mg O-di ani si di ne compl etel y i n 1 mL of methanol . Add 49 mL of 0.1 M
phosphate buffer (pH 6.5). Then add 5 mg of peroxi dase and 5 mg of gl ucose oxi dase to
the above prepared O-di ani si di ne sol uti on.
H Standard: Di ssol ve 100 mg gl ucose i n 100 mL water. Di l ute 10 mL of thi s stock to
100 mL to obtai n the worki ng standard.
PROCEDURE
1. To 0.5 mL of deproti ni sed pl ant extract (deprotei ni zati on i s not necessary i n sampl es
wi th very l ow protei n content) add 0.5 mL di sti l l ed water and 1 mL gl ucose oxi dase-
peroxi dase reagent.
2. I nto a seri es of test tubes pi pette out 0 (bl ank), 0.2, 0.4, 0.6, 0.8 and 1 mL of worki ng
standard gl ucose sol uti on and make up the vol ume to 1.0 mL wi th di sti l l ed water. Then
add 1 mL of gl ucose oxi dase-peroxi dase reagent.
3. I ncubate al l the tubes at 35C for 40 mi nutes.
4. Termi nate the reacti on by the addi ti on of 2 mL of 6 N-HCl .
5. Read the col our i ntensi ty at 540 nm.
CALCULATION
From the standard graph, cal cul ate the amount of gl ucose present i n the sampl e preparati on.
READINGS
1. Mal i k, C.P. and Si ngh, M.B. (1980). Plant Enzymology and Histoenzymology, Kal yani Publ i shers,
New Del hi , p. 278.
2. Kri shnaveni , S.; Theymol i Bal asubramani an and Sadasi vam, S. (1984). Food Chem., 15, 229.
1.5 Determination of Total Carbohydrate
by Anthrone Method
Carbohydrates are the i mportant components of storage and structural materi al s i n the pl ants.
They exi st as fr ee sugar s and pol ysacchar i des. The basi c uni ts of car bohydr ates ar e the
monosacchari des whi ch cannot be spl i t by hydrol ysi s i nto more si mpl er sugars. The carbohy-
drate content can be measured by hydrol ysi ng the pol ysacchari des i nto si mpl e sugars by aci d
hydrol ysi s and esti mati ng the resul tant monosacchari des.
PRINCIPLE
Carbohydrates are first hydrolysed into simple sugars using dilute hydrochloric acid. I n hot
acidic medium glucose is dehydrated to hydroxymethyl furfural. This compound forms with
anthrone a green coloured product with an absorption maximum at 630 nm.
8 Biochemical Methods
MATERIALS
H 2.5 N HCl
H Anthrone reagent: Di ssol ve 200 mg anthrone i n 100 mL of i ce-col d 95% H
2
SO
4
. Prepare
fresh before use.
H Standard glucose: StockDi ssol ve 100 mg i n 100 mL water. Worki ng standard10 mL
of stock di l uted to 100 mL wi th di sti l l ed water. Store refri gerated after addi ng a few
drops of tol uene.
PROCEDURE
1. Wei gh 100 mg of the sampl e i nto a boi l i ng tube.
2. Hydr ol yse by keepi ng i t i n a boi l i ng water bath for thr ee hour s wi th 5 mL of
2.5 N HCl and cool to room temperature.
3. Neutral i se i t wi th sol i d sodi um carbonate unti l the effervescence ceases.
4. Make up the vol ume to 100 mL and centri fuge.
5. Col l ect the supernatant and take 0.5 and 1 mL al i quots for anal ysi s.
6. Prepare the standards by taki ng 0, 0.2, 0.4, 0.6, 0.8 and 1 mL of the worki ng standard.
0 serves as bl ank.
7. Make up the vol ume to 1 mL i n al l the tubes i ncl udi ng the sampl e tubes by addi ng
di sti l l ed water.
8. Then add 4 mL of anthrone reagent.
9. Heat for ei ght mi nutes i n a boi l i ng water bath.
10. Cool rapi dl y and read the green to dark green col our at 630 nm.
11. Draw a standard graph by pl otti ng concentrati on of the standard on the X-axi s versus
absorbance on the Y-axi s.
12. From the graph cal cul ate the amount of carbohydrate present i n the sampl e tube.
CALCULATION
Amount of carbohydrate present i n 100 mg of the sampl e
=
mg of gl ucose
100
Vol ume of test sampl e

NOTE:
Cool the contents of al l the tubes on i ce before addi ng i ce-col d anthrone reagent.
READING
1. Hedge, J.E. and Hofrei ter, B.T. (1962). I n: Carbohydrate Chemistry, 17 (Eds. Whi stl er R.L. and
Be Mi l l er, J.N.), Academi c Press, New York.
1.6 Phenol Sulphuric Acid Method
for Total Carbohydrate
The phenol sul phuri c aci d method to esti mate total carbohydrates i s descri bed bel ow.
PRINCIPLE
I n hot acidic medium glucose is dehydrated to hydroxymethyl furfural. This forms a green
coloured product with phenol and has absorption maximum at 490 nm.
Carbohydrates 9
MATERIALS
H Phenol 5%: Redi sti l l ed (reagent grade) phenol (50 g) di ssol ved i n water and di l uted to
one l i tre.
H Sul phuri c aci d 96% reagent grade.
H Standard glucose: Stock100 mg i n 100 mL of water. Worki ng standard10 mL of
stock di l uted to 100 mL wi th di sti l l ed water.
PROCEDURE
1. Fol l ow the steps 1 to 4 as gi ven i n anthrone method for sampl e preparati on.
2. Pi pette out 0.2, 0.4, 0.6, 0.8 and 1 mL of the worki ng standard i nto a seri es of test
tubes.
3. Pi pette out 0.1 and 0.2 mL of the sampl e sol uti on i n two separate test tubes. Make up
the vol ume i n each tube to 1 mL wi th water.
4. Set a bl ank wi th 1 mL of water.
5. Add 1 mL of phenol sol uti on to each tube.
6. Add 5 mL of 96% sul phuri c aci d to each tube and shake wel l .
7. After 10 mi n shak e the contents i n the tubes and pl ace i n a water bath at
2530C for 20 mi n.
8. Read the col our at 490 nm.
9. Cal cul ate the amount of total carbohydrate present i n the sampl e sol uti on usi ng the
standard graph.
CALCULATION
Absorbance corresponds to 0.1 mL of the test = x mg of gl ucose
100 mL of the sampl e sol uti on contai ns =
0.1
x
100 mg of gl ucose
= % of total carbohydrate present.
READINGS
1. Duboi s, M.; Gi l l es, K.A.; Hami l ton, J.K.; Rebers. P.A. and Smi th, F. (1956). Anal. Chem., 26, p. 350.
2. Kri shnaveni , S.; Theymol i Bal asubramani an and Sadasi vam, S. (1984). Food Chem., 15, p. 229.
1.7 Estimation of Starch by Anthrone Reagent
Starch i s an i mportant pol ysacchari de. I t i s the storage form of carbohydrate i n pl ants abundantl y
found i n roots, tubers, stems, frui ts and cereal s. Starch, whi ch i s composed of several gl ucose
mol ecul es, i s a mi xture of two types of components namel y amyl ose and amyl opecti n. Starch i s
hydrol ysed i nto si mpl e sugars by di l ute aci ds and the quanti ty of si mpl e sugars i s measured
col or i metr i cal l y.
PRINCIPLE
The sample is treated with 80% alcohol to remove sugars and then starch is extracted with
perchloric acid. I n hot acidic medium starch is hydrolysed to glucose and dehydrated to
hydroxymethyl furfural. This compound forms a green coloured product with anthrone.
10 Biochemical Methods
MATERIALS
H Anthrone: Di ssol ve 200 mg anthrone i n 100 mL of i ce-col d 95% sul phuri c aci d.
H 80% ethanol .
H 52% perchl ori c aci d.
H Standard glucose: Stock100 mg i n 100 mL water. Worki ng standard10 mL of stock
di l uted to 100 mL wi th water.
PROCEDURE
1. Homogeni ze 0.10.5 g of the sampl e i n hot 80% ethanol to remove sugars. Centri fuge
and retai n the resi due. Wash the resi due repeatedl y wi th hot 80% ethanol ti l l the
washi ngs do not gi ve col our wi th anthrone reagent. Dry the resi due wel l over a water
bath.
2. To the resi due add 5.0 mL of water and 6.5 mL of 52% perchl ori c aci d.
3. Extract at 0C for 20 mi n. Centri fuge and save the supernatant.
4. Repeat the extracti on usi ng fresh perchl ori c aci d. Centri fuge and pool the supernatants
and make up to 100 mL.
5. Pi pette out 0.1 or 0.2 mL of the supernatant and make up the vol ume to 1 mL wi th
water.
6. Prepare the standards by taki ng 0.2, 0.4, 0.6, 0.8 and 1 mL of the worki ng standard and
make up the vol ume to 1 mL i n each tube wi th water.
7. Add 4 mL of anthrone reagent to each tube.
8. Heat for ei ght mi nutes i n a boi l i ng water bath.
9. Cool rapi dl y and read the i ntensi ty of green to dark green col our at 630 nm.
CALCULATION
Fi nd out the gl ucose content i n the sampl e usi ng the standard graph. Mul ti pl y the val ue
by a factor 0.9 to arri ve at the starch content.
READINGS
1. Hodge, J.E. and Hofrei ter, B.T. (1962). I n: Methods in Carbohydrate Chemistry, (Eds. Whi stl er,
R.L. and Be Mi l l er, J.N.), Academi c Press, New York.
2. Thayumanavan, B. and Sadasi vam, S. (1984). Qual. Plant Foods Hum. Nutr., 34, p. 253.
1.8 Determination of Amylose
Starch i s composed of two components, namel y amyl ose and amyl opecti n. Amyl ose i s a l i near
or non-branched pol ymer of gl ucose. The gl ucose uni ts are joi ned by -1-4 gl ucosi di c l i nkages.
Amyl ose exi sts i n coi l ed form and each coi l contai ns si x gl ucose resi dues.
PRINCIPLE
The iodine is adsorbed within the helical coils of amylose to produce a blue-coloured complex
which is measured colorimetrically.
MATERIALS
H Di sti l l ed ethanol .
Carbohydrates 11
H 1 N NaOH.
H 0.1% phenol phthal ei n.
H I odine reagent: Di ssol ve 1 g i odi ne and 10 g KI i n water and make up to 500 mL.
H Standard: Di ssol ve 100 mg amyl ose i n 10 mL 1 N NaOH; make up to 100 mL wi th
water.
PROCEDURE
1. Wei gh 100 mg of the powdered sampl e, and add 1 mL of di sti l l ed ethanol . Then add
10 mL of 1 N NaOH and l eave i t overni ght.
2. Make up the vol ume to 100 mL.
3. Take 2.5 mL of the extract, add about 20 mL di sti l l ed water and then three drops of
phenol phthal ei n.
4. Add 0.1 N HCl drop by drop unti l the pi nk col our just di sappears.
5. Add 1 mL of i odi ne reagent and make up the vol ume to 50 mL and read the col our at
590 nm.
6. Take 0.2, 0.4, 0.6, 0.8 and 1 mL of the standard amyl ose sol uti on and devel op the col our
as i n the case of sampl e.
7. Cal cul ate the amount of amyl ose present i n the sampl e usi ng the standard graph.
8. Di l ute 1 mL of i odi ne reagent to 50 mL wi th di sti l l ed water for a bl ank.
CALCULATION
Absorbance corresponds to 2.5 mL of the test sol uti on
= x mg amyl ose 100 mL contai ns
=
2.5
x
100 mg amyl ose = % amyl ose.
NOTES:
1. The sampl e suspensi on may be heated for 10 mi n i n a boi l i ng water-bath i nstead of overni ght
di ssol uti on.
2. The amount of amyl opecti n i s obtai ned by subtracti ng the amyl ose content from that of starch.
READINGS
1. McCready, R.M.; Guggol z, J.; Si l i vi era, V. and Owens, H.S. (1950). Anal. Chem., 22, p. 1156.
2. Jul i ano, B.O. (1971). Cereal Sci. Today, 16, 334.
3. Thayumanavan, B. and Sadasi vam, S. (1984). Plant Foods Hum. Nutr, 34, p. 253.
1.9 Estimation of Cellulose
Cel l ul ose, a major structural pol ysacchari de i n pl ants, i s the most abundant organi c compound
i n nature, and i s composed of gl ucose uni ts joi ned together i n the form of the repeati ng uni ts of
the di sacchari de cel l obi ose wi th numerous cross l i nkages. I t i s al so a major component i n
many of the farm wastes.
PRINCIPLE
Cellulose undergoes acetolysis with acetic/nitric reagent forming acetylated cellodextrins which
get dissolved and hydrolyzed to form glucose molecules on treatment with 67% H
2
SO
4
. This
12 Biochemical Methods
glucose molecule is dehydrated to form hydroxymethyl furfural which forms green coloured
product with anthrone and the colour intensity is measured at 630 nm.
MATERIALS
H Acetic/Nitric reagent: Mi x 150 mL of 80% aceti c aci d and 15 mL of concentrated ni tri c
aci d.
H Anthrone reagent: Di ssol ve 200 mg anthrone i n 100 mL concentrated sul phuri c aci d.
Prepare fresh and chi l l for 2 h before use.
H 67% sul phuri c aci d.
PROCEDURE
1. Add 3 mL aceti c/ni tri c reagent to a known amount (0.5 g or 1 g) of the sampl e i n a test
tube and mi x i n a vortex mi xer.
2. Pl ace the tube i n a water-bath at 100C for 30 mi n.
3. Cool and then centri fuge the contents for 1520 mi n.
4. Di scard the supernatant.
5. Wash the resi due wi th di sti l l ed water.
6. Add 10 mL of 67% sul phuri c aci d and al l ow i t to stand for 1 h.
7. Di l ute 1 mL of the above sol uti on to 100 mL.
8. To 1 mL of thi s di l uted sol uti on, add 10 mL of anthrone reagent and mi x wel l .
9. Heat the tubes i n a boi l i ng water-bath for 10 mi n.
10. Cool and measure the col our at 630 nm.
11. Set a bl ank wi th anthrone reagent and di sti l l ed water.
12. Take 100 mg cel l ul ose i n a test tube and pr oceed fr om Step No. 6 for standar d.
I nstead of just taki ng 1 mL of the di l uted sol uti on (Step 7) take a ser i es of vol umes
(say 0.42 mL cor r espondi ng to 40200 g of cel l ul ose) and devel op the col our.
CALCULATION
Draw the standard graph and cal cul ate the amount of cel l ul ose i n the sampl e.
READING
1. Updegroff, D.M. (1969). Anal. Biochem., 32, p. 420.
1.10 Estimation of Hemicellulose
Hemi cel l ul oses are non-cel l ul osi c, non-pecti c cel l wal l pol ysacchari des. They are regarded as
bei ng composed of xyl ans, mannans, gl ucomannans, gal actans and ar abi nogal actans.
Hemi cel l ul oses are categori zed under unavai l abl e carbohydrates si nce they are not spl i t by
the di gesti ve enzymes of the human system.
PRINCIPLE
Refluxing the sample material with neutral detergent solution removes the water-solubles and
materials other than the fibrous component. The left out material is weighed after filtration and
expressed as Neutral Detergent Fibre (NDF).
Carbohydrates 13
MATERIALS
H Neutral Detergent Solution
Wei gh 18.61 g di sodi um ethyl enedi ami ne tetr aacetate and 6.81 g sodi um bor ate
decahydrate. Transfer to a beaker. Di ssol ve i n about 200 mL of di sti l l ed water by heati ng
and to thi s, add a sol uti on (about 100200 mL) contai ni ng 30 g of sodi um l auryl sul phate
and 10 mL of 2-ethoxy ethanol . To thi s add a sol uti on (about 100 mL) contai ni ng 4.5 g
of di sodi um hydrogen phosphate. Make up the vol ume to one l i tre and adjust the pH
to 7.0.
H Decahydronaphthal ene.
H Sodi um sul phi te.
H Acetone.
PROCEDURE
1. To 1 g of the powdered sampl e i n a refl uxi ng fl ask add 10 mL of col d neutral detergent
sol uti on.
2. Add 2 mL of decahydronaphthal ene and 0.5 g sodi um sul phi te.
3. Heat to boi l i ng and refl ux for 60 mi n.
4. Fi l ter the contents through si ntered gl ass cruci bl e (G-2) by sucti on and wash wi th hot
water.
5. Fi nal l y gi ve two washi ngs wi th acetone.
6. Transfer the resi due to a cruci bl e, dry at 100C for 8 h.
7. Cool the cruci bl e i n a desi ccator and wei gh.
CALCULATION
Hemi cel l ul ose = Neutral detergent fi bre (NDF) Aci d detergent fi bre (ADF)
NOTE:
See Li gni n for determi ni ng aci d detergent fi bre.
READING
Goeri ng, H.D. and Vansoest, P.J. (1975). Forage Fibre Analysis, U.S. Deptt. of Agri cul ture, Agri cul tural
Research Servi ce, Washi ngton.
1.11 Determination of Fructose and I nulin
Fructose, a keto-hexose (cal l ed as frui t sugar), i s usual l y accompani ed by sucrose i n frui ts l i ke
appl e. Honey i s a ri ch source of fructose.
PRINCIPLE
The hydroxymethyl furfural formed from fructose in acid medium reacts with resorcinol to give
a red colour product.
MATERIALS
H Resorcinol reagent: Di ssol ve 1 g resorci nol and 0.25 g thi ourea i n 100 mL gl aci al aceti c
aci d. Thi s sol uti on i s i ndefi ni tel y stabl e i n the dark.
14 Biochemical Methods
H Dilute HCl: Mi x fi ve parts of conc. HCl wi th one part of di sti l l ed water.
H Standard fructose solution: Di ssol ve 50 mg of fructose i n 50 mL water. Di l ute 5 mL of
thi s stock to 50 mL for a worki ng standard.
PROCEDURE
1. To 2 mL of the sol uti on contai ni ng 2080 g of fructose add 1 mL of resorci nol reagent.
2. Then add 7 mL of di l ute hydrochl ori c aci d.
3. Pi pette out 0.2, 0.4, 0.6, 0.8 and 1 mL of the worki ng standard and make up the vol ume
to 2 mL wi th water. Add 1 mL of resorci nol reagent and 7 mL of di l ute HCl as above.
4. Set a bl ank al ong wi th the worki ng standard.
5. Heat al l the tubes i n a water-bath at 80C for exactl y 10 mi n.
6. Remove and cool the tubes by i mmersi ng i n tap water for 5 mi n.
7. Read the col our at 520 nm wi thi n 30 mi n.
8. Draw the standard graph and cal cul ate the amount of fructose present i n the sampl e
usi ng the standard graph.
Inulin
I nul i n i s a pol ymer made of fructose uni ts wi th -2-1 l i nkage. I t i s found i n oni on, garl i c
and i n many other pl ant parts.
Sampl e Extracti on
Gri nd the sampl e and extract i n 80% ethanol for si x hours to remove free sugars. Dry the
sampl e and take 500 mg i n a 100 mL coni cal fl ask. Add 20 mL of water and heat i t i n a water
bath at 90C for 10 mi n. Col l ect the extract and then add 70 mL of water. Repl ace the fl ask for
another 30 mi n wi th occasi onal shaki ng to di ssol ve the fructosan, then remove and cool i t at
room temperature. Combi ne the extracts and fi l ter the sol uti on i f i t i s not cl ear and make up to
100 mL i n a standard fl ask.
To esti mate the i nul i n content i n the extr act fol l ow the pr ocedur e gi ven for fr uctose
esti mati on. The amount of i nul i n i s expressed i n terms of fructose concentrati on.
READING
Ashwel l , G. (1957). I n: Methods in Enzymol. 3 (Eds. Col owi ck, S.J. and Kapl an, N.O.), Academi c
Press, New York, p. 75.
1.12 Estimation of Pectic Substances
Pecti c substances abundantl y exi st i n the mi ddl e l amel l a of the pl ant cel l s. There are three
types of pecti c substancespecti c aci ds, pecti n and protopecti n. Pecti c aci d i s an unbranched
mol ecul e made up of about 100 uni ts of D-gal acturoni c aci d resi dues. The monomers are l i nked
through 14 l i nkages. Pecti n i s an extensi vel y esteri fi ed pecti c aci d. Several carboxyl groups
exi st as methyl esters. Pecti c aci d i s water sol ubl e whereas pecti n forms a col l oi dal sol uti on.
Protopecti n i s a l arger mol ecul e than pecti c aci d and pecti n. Duri ng ri peni ng of frui ts, conversi on
of protopecti n i nto pecti c aci d and pecti n takes pl ace. The pecti ns i n frui ts vary i n thei r methoxyl
content and i n jel l yi ng power.
Carbohydrates 15
Two methods are descri bed bel ow for the esti mati on of pecti n: one gravi metri c and the
other, col ori metri c.
I. Gravimetric Method
PRINCIPLE
Pectin is extracted from plant material and saponified. I t is precipitated as calcium pectate by
the addition of calcium chloride to an acid solution. After thoroughly washing to eliminate
chloride ions, the precipitate is dried and weighed.
MATERIALS
H 1 N Acetic acid (Di l ute 30 mL of gl aci al aceti c aci d to 500 mL wi th water).
H 1 N Calcium chloride solution: Di ssol ve 27.5 g anhydrous CaCl
2
i n water and di l ute to
500 mL.
H 1% Silver nitrate: Di ssol ve 1 g AgNO
3
i n 100 mL water.
H 0.01 N HCl
H 0.05 N HCl
H 0.3 N HCl
PROCEDURE
1. Wei gh 50 g of bl ended sampl e i nto a 1 L beaker and add 300 mL 0.01 N HCl . Boi l for
30 mi n and fi l ter under sucti on. Wash the resi due wi th hot water and col l ect the fi l trate.
2. To the resi due add 100 mL 0.05 N HCl , boi l for 20 mi n fi l ter, wash and col l ect the
fi l trate.
3. To the resi due now add 100 mL 0.3 N HCl , boi l for 10 mi n, fi l ter, wash and col l ect the
fi l trate.
4. Pool the fi l trates. Cool and make to vol ume (500 mL).
5. Pi pette out 100200 mL al i quots i nto 1 L beakers.
6. Add 250 mL water and neutral i ze the aci d wi th 1 N NaOH usi ng phenol phthal ei n
i ndi cator. Add an excess of 10 mL of 1 N NaOH wi th constant sti rri ng and al l ow i t to
stand overni ght.
7. Add 50 mL 1 N aceti c aci d and after 5 mi n, add 25 mL 1 N cal ci um chl ori de sol uti on
wi th sti rri ng. Al l ow i t to stand for 1 h.
8. Boi l for 1 to 2 mi n.
9. Fi l ter through a pre-wei ghed Whatman No. 1 fi l ter paper (see note 1).
10. Wash the preci pi tate wi th al most boi l i ng water unti l the fi l trate i s free from chl ori de.
11. Test the fi l trate wi th si l ver ni trate for chl ori de.
12. Transfer the fi l ter paper wi th the cal ci um pectate, dry overni ght at 100C i n a wei ghi ng
di sh, cool i n a desi ccator and wei gh.
CALCULATION
The pecti n content i s expressed as % cal ci um pectate
% cal ci um pectate =
Wt. of cal ci um pectate 500 100
mL of fi l trate taken Wt. of smapl e for esti mati on
16 Biochemical Methods
NOTES:
The fi l ter paper for Step No. 9 shoul d be prepared as descri bed bel ow:
1. Wet the fi l ter paper i n hot water, dry i n oven at 102C for 2 h. Cool i n a desi ccator and wei gh i n
a covered di sh.
2. The theoreti cal yi el d of cal ci um pectate from pure gal acturoni c anhydri de i s 110.6%.
II. Colorimetric Method
PRINCIPLE
Galacturonic acid is reacted with carbazole in the presence of H
2
SO
4
and the colour developed
is measured at 520 nm.
MATERIALS
H 60% Ethyl al cohol (Mi x 500 mL 95% al cohol and 300 mL water).
H 95% Ethyl al cohol .
H Puri fi ed ethyl al cohol (Refl ux 1 L of 95% ethyl al cohol wi th 4 g zi nc dust and 2 mL conc.
H
2
SO
4
for 15 h and di sti l l i n al l gl ass di sti l l ati on apparatus. Redi sti l l wi th 4 g zi nc dust
and 4 g KOH).
H 1 N and 0.05 N Sodi um hydroxi de.
H H
2
SO
4
(Anal yti cal grade).
H 0.1% Carbazole reagent: Wei gh 100 mg recrystal l i zed carbazol e, di ssol ve and di l ute to
100 mL wi th puri fi ed al cohol .
PROCEDURE
1. Wei gh 100 mg pecti n (see notes secti on for the preparati on of pecti n) and di ssol ve i n
100 mL of 0.05 N NaOH.
2. Al l ow i t to stand for 30 mi n to deesteri fy the pecti n.
3. Take 2 mL of thi s sol uti on and make up to 100 mL wi th water.
4. Pi pette out 2 mL of deesteri fi ed pecti n sol uti on and add 1 mL carbazol e reagent. A
whi te preci pi tate wi l l be formed.
5. Add 12 mL conc. H
2
SO
4
wi th constant sti rri ng.
6. Cl ose the tubes wi th rubber stopper and al l ow to stand for 10 mi n to devel op the col our.
7. To set a bl ank add 1 mL of puri fi ed ethyl al cohol i n the pl ace of carbazol e reagent.
8. Read the col our at 525 nm agai nst bl ank, exactl y 15 mi n after the addi ti on of aci d.
STANDARD
Wei gh 120.5 mg gal acturoni c aci d monohydrate (from a sampl e vacuum dri ed for 5 h at
30C) and transfer to a 1 L vol umetri c fl ask. Add 10 mL 0.05 N NaOH and di l ute to vol ume wi th
water. After mi xi ng, al l ow i t to stand overni ght. Di l ute 10, 20, 40, 50, 60 and 80 mL of thi s
standard sol uti on to 100 mL wi th water. Take 2 mL of these sol uti ons for col our devel opi ng and
pr oceed as i n the case of the sampl e. Dr aw a standar d cur vethe absor bance versus
concentr ati on.
Carbohydrates 17
CALCULATION
Read the concentrati on of the anhydrogal acturoni c aci d correspondi ng to the readi ng of
the sampl e, and cal cul ate as fol l ows:
% anhydrogal acturoni c aci d =
g of anhydr ogal acturoni c aci d i n the al i quot Di l uti on 100
mL taken for esti mati on Wt. of pecti n sampl e 1,000,000
NOTES:
1. Carbazol e i s recrystal l i zed from tol uene.
2. An al ternate procedure adopted for col our devel opment i s as fol l ows:
Take 12 mL of conc. H
2
SO
4
i n a test tube, cool i n an i ce-bath, and add 2 mL of the deesteri fi ed
pecti n sol uti on and agai n cool . Heat the contents i n a boi l i ng water-bath for 10 mi n, cool to 20C
and add 1 mL of 0.15% carbazol e reagent i n puri fi ed ethyl al cohol . Al l ow i t to stand for 25 5
mi n at room temperature to devel op the col our. Read the absorbance at 520 nm. Standards
shoul d al so be treated si mi l arl y.
III. Extraction and Purification of Pectin
1. Bl end the fresh sampl e. I f the materi al i s dry gri nd.
2. Transfer 100 g macerated sampl e (10 g dry ti ssue) to a pre-wei ghed 1 L beaker contai ni ng
400 mL water.
3. Add 1.2 g freshl y ground sodi um hexametaphosphate and adjust to pH 4.5.
4. Heat wi th sti rri ng at 9095C for 1 h. Check the pH i n every 15 mi n and mai ntai n at pH
4.5 wi th ci tri c aci d or NaOH. Repl ace water l ost by evaporati on at i nterval s. However,
do not add water at the l ast 20 mi n.
5. Add 4 g fi l ter ai d and 4 g ground paper pul p. Fi l ter rapi dl y through a fast fi l ter paper
coated wi th 3 g moi stened fast fi l ter ai d.
6. Col l ect at l east 200 mL of the fi l trate i n a prewei ghed contai ner. Cool as rapi dl y as
possi bl e. Now, note the wei ght of the fi l trate.
7. I f the fi l trate contai ns l ess than 0.2% pecti n, concentrate the fi l trate under vacuum to
attai n thi s concentrati on.
8. To three vol umes of ethanol , i sopropanol or acetone contai ni ng 0.5 N HCl , pour the
cool ed, wei ghed fi l trate. The sl urry shoul d be at pH 0.71. Sti r for 30 mi n.
9. Centri fuge or fi l ter. Wash the preci pi tate wi th the same sol vent contai ni ng HCl . Then,
wash repeatedl y wi th 70% al cohol or acetone unti l the preci pi tate i s essenti al l y chl ori de-
free or the pH i s above 4.
10. Dehydrate the preci pi tate further i n 400 mL acetone. Dry overni ght in vacuo wi th a
sl ow stream of dry ai r passi ng through the oven.
11. Wei gh the preci pi tate and use thi s pecti n for anal ysi s.
12. The dri ed pecti n shoul d be free from ammoni a for whi ch a smal l sampl e of the pecti n i s
heated wi th 1 mL of 0.1 N NaOH and ammoni acal odour can be noti ced or tested wi th
a moi stened l i tmus paper. I f ammoni um i ons are present wash wi th aci di fi ed 6% al cohol ,
fol l owed by neutral al cohol to remove the aci d and dry.
18 Biochemical Methods
READING
Ranganna, S. (1979). Manual of Analysis of Fruit and Vegetable Products, Tata McGraw-Hi l l Publ .
Co. Ltd., New Del hi , p. 634.
1.13 Estimation of Crude Fibre
Crude fi bre consi sts l argel y of cel l ul ose and l i gni n (97%) pl us some mi neral matter. I t represents
onl y 6080% of the cel l ul ose and 46% of the l i gni n. The crude fi bre content i s commonl y used
as a measure of the nutri ti ve val ue of poul try and l i vestock feeds and al so i n the anal ysi s of
vari ous foods and food products to detect adul terati on, qual i ty and quanti ty.
PRINCIPLE
During the acid and subsequent alkali treatment, oxidative hydrolytic degradation of the native
cellulose and considerable degradation of lignin occur. The residue obtained after final filtration
is weighed, incinerated, cooled and weighed again. The loss in weight gives the crude fibre
content.
MATERIALS
H Sulphuric acid solution (0.255 0.005 N): 1.25 g concentrated sul phuri c aci d di l uted to
100 mL (concentrati on must be checked by ti trati on).
H Sodium hydroxide solution (0.313 0.005 N): 1.25 g sodi um hydroxi de i n 100 mL di sti l l ed
water (concentrati on must be checked by ti trati on wi th standard aci d).
PROCEDURE
1. Extract 2 g of ground materi al wi th ether or petrol eum ether to remove fat (I ni ti al
boi l i ng temperature 3538C and fi nal temperature 52C). I f fat content i s bel ow 1%,
extracti on may be omi tted.
2. After extracti on wi th ether boi l 2 g of dri ed materi al wi th 200 mL of sul phuri c aci d for
30 mi n wi th bumpi ng chi ps.
3. Fi l ter through musl i n and wash wi th boi l i ng water unti l washi ngs are no l onger aci di c.
4. Boi l wi th 200 mL of sodi um hydroxi de sol uti on for 30 mi n.
5. Fi l ter through musl i n cl oth agai n and wash wi th 25 mL of boi l i ng 1.25% H
2
SO
4
, three
50 mL porti ons of water and 25 mL al cohol .
6. Remove the resi due and transfer to ashi ng di sh (prewei ghed di sh W
1
).
7. Dry the resi due for 2 h at 130 2C. Cool the di sh i n a desi ccator and wei gh (W
2
).
8. I gni te for 30 mi n at 600 15C.
9. Cool i n a desi ccator and rewei gh (W
3
).
CALCULATION
% crude fi bre i n ground sampl e =
2 1 3 1
Loss i n wei ght on i gni ti on (W W) (W W)
100
Wei ght of the sampl e

READING
Maynard, A.J. (Ed.) (1970). Methods in Food Analysis, Academi c Press, New York, p. 176.
Carbohydrates 19
1.14 Estimation of Pyruvic Acid
Pyruvi c aci d or pyruvate i s an i mportant metabol i c i ntermedi ate. I t i s greatl y produced i n the
termi nal step of gl ycol ysi s and funnel s to TCA cycl e for further oxi dati on for rel easi ng the
chemi cal energy. I t can be determi ned fol l owi ng the procedure gi ven bel ow:
PRINCIPLE
The DNPH (2,4-dinitrophenyl hydrazine) reacts with pyruvate after the addition NaOH giving
a brown colored hydrazone product which can be estimated colorimetrically at 510 nm.
MATERIALS
H Phosphate buffer pH 9.4
A: 0.2 M sol uti on of monobasi c sodi um phosphate NaH
2
PO
4
H
2
O (27.8 g i n 1000 mL).
B: 0.2 M sol uti on of di basi c sodi um phosphate (53.65 g of Na
2
HPO
4
.7H
2
O i n 1 L or 17.7
g of Na
2
HPO
4
.12H
2
O i n 1 L).
19 mL of A and 81 mL of B, di l uted to a total of 200 mL
Store i n refri gerator.
H Pyruvate, Standard
Di ssol ve 22 mg sodi um pyruvate i n 100 mL water i n a standard fl ask.
H 2, 4-Dinitrophenyl hydrazine (DNPH)
Di ssol ve 19.8 mg of DNPH i n 10 mL of conc. HCl and make to 100 mL wi th water.
Store i t i n an amber bottl e at room temperature.
H Sodium hydroxide 0.8 N
Di ssol ve 16 g sodi um hydroxi de i n one l i tre water.
H Plant extract
Gri nd 6 g of pl ant materi al i n 15 mL of phosphate buffer. Centri fuge at 25,000 g for 15
mi n. Use the supernatant as pl ant extract.
PROCEDURE
1 Pi pette out 50 L, 75 L, 100 L, 150 L, 200 L of pyruvate standard sol uti on and
0.5 mL, 1.0 mL, 1.5 mL, and 2.0 mL of sampl e extract i nto test tubes and make up the
vol ume to 2.0 mL wi th phosphate buffer (pH 7.4).
2. Set a bl ank wi th no pyruvate sol uti on.
3. Add 0.5 mL of DNPH sol uti on to each tube.
4. I ncubate at 37C for 2030 mi n.
5. Add 5 mL of NaOH sol uti on to each tube, mi x wel l and i ncubate for 10 mi n at room
temper atur e.
6. Record the absorbance at 610 nm.
7. Draw the standard graph and cal cul ate the amount of pyruvi c aci d present i n the sampl e
usi ng the graph.