1.

1 Colour Reactions
Ca r bohydr a t es a r e widely pr eva lent in t he pla nt kingdom, compr ising t he mono-, di-,
oligo-, and polysacchar ides. The common monosacchar ides ar e glucose, fr uct ose, galact ose,
r ibose et c. The disacchar ides, i.e., t he combinat ion of t wo monosacchar ides include sucr ose,
lact ose and malt ose. St ar ch and cellulose ar e polysacchar ides consist ing of many monosacchar ide
r esidues. Cellulose is t he most abundant or ganic compound on t his planet since it for ms par t of
t he cell wall in plant s.
Aldehydes (CHO) and ket ones ( = CO) ar e act ive gr oups in car bohydr at es. Car bohydr at es
cont ain many hydr oxyl gr oups as well. The number of hydr oxyl gr oups var ies wit h t he number
of car bon at oms. Monosacchar ides cont ain t he fr ee aldehyde or ket one gr oup. Some disacchar ides
have t he fr ee aldehyde gr oup (malt ose) and some do not have t he fr ee ones (sucr ose). The
polysacchar ides, st ar ch and cellulose, ar e polymer s of monosacchar ides linked t hr ough t he
act ive gr oups.
The chemical pr oper t ies of sacchar ides var y depending upon t he number of hydr oxyl gr oups
and t he pr esence or absence of CHO/= CO gr oups. These var iat ions ar e t he basis in t he
development of colour r eact ions t o ident ify t he sacchar ides.
Some simple t est s used t o ident ify t he pr esence/absence of cer t ain sacchar ides ar e list ed
below:
REAGENTS
H Iodine solution: Add a few cr yst als of iodine t o 2% pot assium iodide solut ion t ill t he
colour becomes deep yellow.
H Fehlings reagent A: Dissolve 34.65 g copper sulphat e in dist illed wat er and make up t o
500 mL.
H Fehlings reagent B: Dissolve 125 g pot assium hydr oxide and 173 g Rochelle salt
(pot assium sodium t ar t r at e) in dist illed wat er and make up t o 500 mL.
1
Chapter 1
CARBOHYDRATES
2 Biochemical Methods
H Benedicts qualitative reagent: Dissolve 173 g sodium cit r at e and 100 g sodium car bonat e
in about 500 mL wat er. Heat t o dissolve t he salt s and filt er, if necessar y. Dissolve 17.3 g
copper sulphat e in about 100 mL wat er and add it t o t he above solut ion wit h st ir r ing
and make up t he volume t o 1 L wit h wat er.
H Barfoeds reagent: Dissolve 24 g copper acet at e in 450 mL boiling wat er. Immediat ely
add 25 mL of 8.5% lact ic acid t o t he hot solut ion. Mix well, Cool and dilut e t o 500 mL.
H S eliwanoff s reagent: Dissolve 0.05 g r esor cinol in 100 mL dilut e (1:2) hydr ochlor ic acid.
H Bials reagent: Dissolve 1.5 g or cinol in 500 mL of concent r at ed HCl and add 20 t o 30
dr ops of 10% fer r ic chlor ide.
The r eact ions of car bohydr at es ar e given in Table 1.1.
TABLE 1.1: Reacti ons of carbohydrates
Exp er i ment Obser va t i on Rema r k s
Th e col ou r for med i s du e t o
t he r eact ion of alpha-napht hol
wit h fur fur al and/or it s der iva-
t ives for med by t he dehydr at ion
of sugar s by concent r at ed sul-
phur ic acid. All car bohydr at es
r eact posit ively wit h t his r ea-
gent .
1. Molischs Test
Add t wo dr ops of Mol i s ch s
r ea gen t (5% 1-n a ph t h ol i n
alcohol) t o about 2 mL of t est
solut ion and mix well.
Incline t he t ube and add about
1 mL of concent r at ed sulphur ic
acid along t he sides of t he t ube.
Obs er ve t h e col ou r a t t h e
junct ion of t he t wo liquids.
2. Iodine Test
Add a few dr ops of i odi n e
solut ion t o about 1 mL of t he
t est solut ion.
3. Fehlings Test
To 1 mL of Fehlings solut ion A,
add 1 mL of Fehlings solut ion
B and a few dr ops of t he t est
solut ion. Boil for a few minut es.
A r ed-cum-violet r ing appear s
a t t h e j u n ct i on of t h e t wo
liquids.
Appear ance of deep blue colour.
This indicat es t he pr esence of
st ar ch in t he solut ion.
The blue colour is due t o t he for -
ma t ion of st a r ch-iodine com-
plex.
The blue alkaline cupr ic hydr ox-
ide pr esent in Fehlings solu-
t ion, when heat ed in t he pr es-
ence of r educing sugar s, get s r e-
duced t o yellow or r ed cupr ous
oxide and it get s pr ecipit at ed.
Hen ce, for ma t ion of t h e col-
our ed pr ecipit at e indicat es t he
pr esence of r educing sugar s in
t he t est solut ion.
F or ma t i on of yel l ow or
br ownish-r ed pr ecipit at e.
Carbohydrates 3
4. Benedicts Test
To 2 mL of Benedict s r eagent
a dd fi ve dr ops of t h e t es t
solut ion. Boil for five minut es
i n a wa t er ba t h . Cool t h e
solut ion.
Format ion of red, yellow or green
colour /pr ecipit at e.
As in Fehlings t est , t he r educ-
ing suga r s beca use of ha ving
pot en t i a l l y fr ee a l deh yde or
ket o group reduce cupric hydrox-
ide in alkaline solut ion t o r ed
colour ed cupr ous oxide. Depend-
ing on t he sugar concent r at ion
yellow t o gr een colour is devel-
oped.
Only monosacchar ides answer
t his t est . Since Bar foeds r ea-
gent is weakly acidic, it is r e-
duced only by monosacchar ides.
5. Barfoeds Test
To 1 mL of t he t est solut ion add
about 2 mL of Bar foeds r eagent .
Boil it for one minut e and allow
t o st and for a few minut es.
6. S eliwanoff s Test
To 2 mL of Seliwanoff s r eagent
add t wo dr ops of t est solut ion
a nd hea t t he mixt ur e t o just
boiling.
F or ma t i on of br i ck -r ed
pr ecipit at e.
Appear ance of deep r ed colour. In concent r at ed HCl, ket oses
under go dehydr at ion t o yield
fur fur al der ivat ives mor e r ap-
idly t han do aldoses. These de-
r ivat ives for m complexes wit h
r esor cinol t o yield deep r ed col-
our.
It is a t imed colour r eact ion
specific for ket oses.
7. Bials Test
To 5 mL of Bials r eagent add
23 mL of solut ion and war m
gent ly. When bubbles r ise t o t he
sur face cool under t he t ap.
8. Test for non-reducing sugars
such as sucrose:
(a) Do Benedict s t est wit h t he
t est solut ion.
(b) Add 5 dr ops of concent r at ed
HCl t o 5 mL of t est solut ion
in anot her t est t ube. Heat for
five minut es on a boiling
wat er bat h.
Appear ance of gr een colour or
pr ecipit at e.
It is specific for pent oses. They
get conver t ed t o fur fur al. In t he
pr esence of fer r ic ion or cinol and
fur fur al condense t o yield a col-
our ed pr oduct .
No ch a r a ct er i s t i c col ou r
for mat ion.
Appea r a n ce of r ed or yellow
colour.
Indicat es t he absence of r educ-
ing sugar s in t he given solut ion.
Indicat es t he for mat ion of r e-
ducing sugar s fr om non-r educ-
ing sugar s aft er hydr olysis wit h
acid.
(Cont d.)
4 Biochemical Methods
Add 10% s odi u m h ydr oxi de
s ol u t i on t o gi ve a s l i gh t l y
alkaline solut ion (t est wit h r ed
l i t mu s pa per ). Now per for m
Ben edi ct s t es t wi t h t h i s
hydr olysed solut ion.
9. Mucic Acid Test
Add a few dr ops of conc. HNO
3
t o t he concent r at ed t est solut ion
or s u bs t a n ce di r ect l y a n d
evapor at e it over a boiling wat er
ba t h t i l l t h e a ci d fu mes a r e
expelled. Add a few dr ops of
wat er and leave it over night .
10. Osazone Test
To 0.5 g of ph en yl h ydr a zi n e
h ydr och l or i de a dd 0. 1 g of
sodium acet at e and 10 dr ops of
gl a ci a l a cet i c a ci d. To t h i s
mi xt u r e a dd 5 mL of t es t
solut ion and heat on a boiling
wa t er ba t h for a bout ha lf a n
h ou r . Allow t h e t u be t o cool
slowly and examine t he cr yst als
under a micr oscope.
For mat ion of cr yst als. The bot h end car bon gr oups ar e
oxidized t o car boxylic gr oups.
The r esult ant sacchar ic acid of
galact ose is called mucic acid
which is insoluble in wat er.
The ket oses and aldoses r eact
wit h phenylhydr a zine t o pr o-
duce a phenylhydr azone which
in t ur n r eact s wit h anot her t wo
molecules of phenylhydr azine t o
for m t he osazone.
Glucose, fr uct ose and mannose
pr oduce needle-shaped yellow
os a zon e cr ys t a l s , wh er ea s
l a ct os a zon e i s mu s h r oom-
s h a ped. Di ffer en t os a zon es
s h ow cr ys t a l s of di ffer en t
s h a pes . Ma l t os e pr odu ces
flower -shaped cr yst als.
NOTES:
1. For osazone t est , t he r eact ion mixt ur e should be bet ween pH 5 and 6. Fr uct ose t akes 2 min t o
for m t he osazone wher eas for glucose it is 5 min. The disacchar ides t ake a longer t ime t o for m
osazones. Dissachar ides for m cr yst als only on cooling.
2. When a mixt ur e of car bohydr at es is pr esent in t he t est sample, chr omat ogr aphic met hods
should be employed t o ident ify t he individual sugar s.
READING
1. Sadasivam, S. and Theymoli Balasubr amanian (1985). Practical Manual (Undergraduate), Tamil
Nadu Agr icult ur al Univer sit y, Coimbat or e, p. 2.
1.2 Determination of Reducing S ugars
by Nelson-S omogyi Method
Sugar s wit h r educing pr oper t y (ar ising out of t he pr esence of a pot ent ial aldehyde or ket o
gr oup) ar e called r educing sugar s. Some of t he r educing sugar s ar e glucose, galact ose, lact ose
Carbohydrates 5
and malt ose. The Nelson-Somogyi met hod is one of t he classical and widely used met hods for
t he quant it at ive det er minat ion of r educing sugar s.
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 k a l i ne Cop p er Ta r t r a t e
(i) Dissolve 2.5 g anhydr ous sodium car bonat e, 2 g sodium bicar bonat e, 2.5 g pot assium
sodium t ar t r at e and 20 g anhydr ous sodium sulphat e in 80 mL wat er and make up t o
100 mL.
(ii) Dissolve 15 g copper sulphat e in a small volume of dist illed wat er. Add one dr op of
sulphur ic acid and make up t o 100 mL.
Mix 4 mL of B and 96 mL of solut ion A befor e use.
Arsenomolybdate reagent: Dissolve 2.5 g ammonium molybdat e in 45 mL wat er. Add 2.5 mL
sulphur ic acid and mix well. Then add 0.3 g disodium hydr ogen ar senat e dissolved in 25 mL
wat er. Mix well and incubat e at 37C for 2448 hour s.
S tandard glucose solution: S tock: 100 mg in 100 mL dist illed wat er.
Working standard: 10 mL of st ock dilut ed t o 100 mL wit h dist illed wat er [100 g/mL].
PROCEDURE
1. Weigh 100 mg of t he sample and ext r act t he sugar s wit h hot 80% et hanol t wice (5 mL
each t ime).
2. Collect t he super nat ant and evapor at e it by keeping it on a wat er bat h at 80C.
3. Add 10 mL wat er and dissolve t he sugar s.
4. Pipet t e out aliquot s of 0.1 or 0.2 mL t o separ at e t est t ubes.
5. Pipet t e out 0.2, 0.4, 0.6, 0.8 and 1 mL of t he wor king st andar d solut ion int o a ser ies of
t est t ubes.
6. Make up t he volume in bot h sample and st andar d t ubes t o 2 mL wit h dist illed wat er.
7. Pipet t e out 2 mL dist illed wat er in a separ at e t ube t o set a blank.
8. Add 1 mL of alkaline copper t ar t r at e r eagent t o each t ube.
9. Place t he t ubes in a boiling wat er for 10 minut es.
10. Cool t he t ubes and add 1 mL of ar senomolybolic acid r eagent t o all t he t ubes.
11. Make up t he volume in each t ube t o 10 mL wit h wat er.
12. Read t he absor bance of blue colour at 620 nm aft er 10 min.
13. Fr om t he gr aph dr awn, calculat e t he amount of r educing sugar s pr esent in t he sample.
CALCULATION
Absor bance cor r esponds t o 0.1 mL of t est = x mg of glucose
10 mL cont ains =
0.1
x
10 mg of glucose
= % of r educing sugar s
6 Biochemical Methods
READINGS
1. Somogyi, M. (1952). J . Biol. Chem., 200, 245.
2. Kr ishnaveni, S.; Theymoli Balasubr amanian and Sadasivam, S. (1984). Food Chem., 15, 229.
1.3 Estimation of Reducing S ugar by
Dinitrosalicylic Acid Method
For sugar est imat ion an alt er nat ive t o Nelson-Somogyi met hod is t he dinit r osalicylic acid
met hodsimple, sensit ive and adopt able dur ing handling of a lar ge number of samples at a
t ime.
MATERIALS
H Dinitrosalicylic Acid Reagent (DNS Reagent)
Dissolve by st irring 1 g dinit rosalicylic acid, 200 mg cryst alline phenol and 50 mg sodium
sulphit e in 100 mL 1% NaOH. St ore at 4C. Since t he reagent det eriorat es due t o sodium
sulphit e, if long st orage is required, sodium sulphit e may be added at t he t ime of use.
H 40% Rochelle salt solut ion (Pot assium sodium t ar t r at e).
PROCEDURE
1. Follow, st eps 1 t o 3 as in Nelson-Somogyis met hod t o ext r act t he r educing sugar s fr om
t he t est mat er ial.
2. Pipet t e out 0.5 t o 3 mL of t he ext r act in t est t ubes and equalize t he volume t o 3 mL
wit h wat er in all t he t ubes.
3. Add 3 mL of DNS r eagent .
4. Heat t he cont ent s in a boiling wat er bat h for 5 min.
5. When t he cont ent s of t he t ubes ar e st ill war m, add 1 mL of 40% Rochelle salt solut ion.
6. Cool and r ead t he int ensit y of dar k r ed colour at 510 nm.
7. Run a ser ies of st andar ds using glucose (0500 g) and plot a gr aph.
CALCULATION
Calculat e t he amount of r educing sugar s pr esent in t he sample using t he st andar d gr aph.
READING
1. Miller, G.L. (1972). Anal. Chem., 31, p. 426.
1.4 Determination of Glucose by
Glucose Oxidase Method
Glucose is a widely dist r ibut ed simple sugar wit h an act ive aldehyde gr oup. Est imat ion of
glucose by glucose oxidase gives t he t r ue glucose concent r at ion eliminat ing t he int er fer ence
by ot her r educing sugar s.
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.
Glucose + O
2
Glucose
Oxidase
H
2
O
2
+ Gluconic Acid
H
2
O
2
+ O-dianisidine
Peroxida se

Red-colour ed pr oduct
MATERIALS
H Glucose Oxidase Peroxidase Reagent
Dissolve 25 mg O-dianisidine complet ely in 1 mL of met hanol. Add 49 mL of 0.1 M
phosphat e buffer (pH 6.5). Then add 5 mg of per oxidase and 5 mg of glucose oxidase t o
t he above pr epar ed O-dianisidine solut ion.
H S tandard: Dissolve 100 mg glucose in 100 mL wat er. Dilut e 10 mL of t his st ock t o
100 mL t o obt ain t he wor king st andar d.
PROCEDURE
1. To 0.5 mL of depr ot inised plant ext r act (depr ot einizat ion is not necessar y in samples
wit h ver y low pr ot ein cont ent ) add 0.5 mL dist illed wat er and 1 mL glucose oxidase-
per oxidase r eagent .
2. Int o a ser ies of t est t ubes pipet t e out 0 (blank), 0.2, 0.4, 0.6, 0.8 and 1 mL of wor king
st andar d glucose solut ion and make up t he volume t o 1.0 mL wit h dist illed wat er. Then
add 1 mL of glucose oxidase-per oxidase r eagent .
3. Incubat e all t he t ubes at 35C for 40 minut es.
4. Ter minat e t he r eact ion by t he addit ion of 2 mL of 6 N-HCl.
5. Read t he colour int ensit y at 540 nm.
CALCULATION
From t he st andard graph, calculat e t he amount of glucose present in t he sample preparat ion.
READINGS
1. Malik, C.P. and Singh, M.B. (1980). Plant Enzymology and Histoenzymology, Kalyani Publisher s,
New Delhi, p. 278.
2. Kr ishnaveni, S.; Theymoli Balasubr amanian and Sadasivam, S. (1984). Food Chem., 15, 229.
1.5 Determination of Total Carbohydrate
by Anthrone Method
Car bohydr at es ar e t he impor t ant component s of st or age and st r uct ur al mat er ials in t he plant s.
They exist as fr ee sugar s and polysacchar ides. The basic unit s of car bohydr at es ar e t he
monosacchar ides which cannot be split by hydr olysis int o mor e simpler sugar s. The car bohy-
dr at e cont ent can be measur ed by hydr olysing t he polysacchar ides int o simple sugar s by acid
hydr olysis and est imat ing t he r esult ant monosacchar ides.
PRINCIPLE
Carbohydrates are first hydrolysed into simple sugars using dilute hydrochloric acid. In 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: Dissolve 200 mg ant hr one in 100 mL of ice-cold 95% H
2
SO
4
. Pr epar e
fr esh befor e use.
H S tandard glucose: St ockDissolve 100 mg in 100 mL wat er. Wor king st andar d10 mL
of st ock dilut ed t o 100 mL wit h dist illed wat er. St or e r efr iger at ed aft er adding a few
dr ops of t oluene.
PROCEDURE
1. Weigh 100 mg of t he sample int o a boiling t ube.
2. Hydr olyse by keeping it in a boiling wa t er ba t h for t hr ee hour s wit h 5 mL of
2.5 N HCl and cool t o r oom t emper at ur e.
3. Neut r alise it wit h solid sodium car bonat e unt il t he effer vescence ceases.
4. Make up t he volume t o 100 mL and cent r ifuge.
5. Collect t he super nat ant and t ake 0.5 and 1 mL aliquot s for analysis.
6. Pr epar e t he st andar ds by t aking 0, 0.2, 0.4, 0.6, 0.8 and 1 mL of t he wor king st andar d.
0 ser ves as blank.
7. Make up t he volume t o 1 mL in all t he t ubes including t he sample t ubes by adding
dist illed wat er.
8. Then add 4 mL of ant hr one r eagent .
9. Heat for eight minut es in a boiling wat er bat h.
10. Cool r apidly and r ead t he gr een t o dar k gr een colour at 630 nm.
11. Dr aw a st andar d gr aph by plot t ing concent r at ion of t he st andar d on t he X-axis versus
absor bance on t he Y-axis.
12. Fr om t he gr aph calculat e t he amount of car bohydr at e pr esent in t he sample t ube.
CALCULATION
Amount of car bohydr at e pr esent in 100 mg of t he sample
=
mg of glucose
100
Volume of test sample

NOTE:
Cool t he cont ent s of all t he t ubes on ice befor e adding ice-cold ant hr one r eagent .
READING
1. Hedge, J .E. and Hofr eit er, B.T. (1962). In: Carbohydrate Chemistry, 17 (Eds. Whist ler R.L. and
Be Miller, J .N.), Academic Pr ess, New Yor k.
1.6 Phenol S ulphuric Acid Method
for Total Carbohydrate
The phenol sulphur ic acid met hod t o est imat e t ot al car bohydr at es is descr ibed below.
PRINCIPLE
In 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%: Redist illed (r eagent gr ade) phenol (50 g) dissolved in wat er and dilut ed t o
one lit r e.
H Sulphur ic acid 96% r eagent gr ade.
H S tandard glucose: St ock100 mg in 100 mL of wat er. Wor king st andar d10 mL of
st ock dilut ed t o 100 mL wit h dist illed wat er.
PROCEDURE
1. Follow t he st eps 1 t o 4 as given in ant hr one met hod for sample pr epar at ion.
2. Pipet t e out 0.2, 0.4, 0.6, 0.8 and 1 mL of t he wor king st andar d int o a ser ies of t est
t ubes.
3. Pipet t e out 0.1 and 0.2 mL of t he sample solut ion in t wo separ at e t est t ubes. Make up
t he volume in each t ube t o 1 mL wit h wat er.
4. Set a blank wit h 1 mL of wat er.
5. Add 1 mL of phenol solut ion t o each t ube.
6. Add 5 mL of 96% sulphur ic acid t o each t ube and shake well.
7. Aft er 10 mi n s h a ke t h e con t en t s i n t h e t u bes a n d pl a ce i n a wa t er ba t h a t
2530C for 20 min.
8. Read t he colour at 490 nm.
9. Calculat e t he amount of t ot al car bohydr at e pr esent in t he sample solut ion using t he
st andar d gr aph.
CALCULATION
Absor bance cor r esponds t o 0.1 mL of t he t est = x mg of glucose
100 mL of t he sample solut ion cont ains =
0.1
x
100 mg of glucose
= % of t ot al car bohydr at e pr esent .
READINGS
1. Dubois, M.; Gilles, K.A.; Hamilt on, J .K.; Reber s. P.A. and Smit h, F. (1956). Anal. Chem., 26, p. 350.
2. Kr ishnaveni, S.; Theymoli Balasubr amanian and Sadasivam, S. (1984). Food Chem., 15, p. 229.
1.7 Estimation of S tarch by Anthrone Reagent
St arch is an import ant polysaccharide. It is t he st orage form of carbohydrat e in plant s abundant ly
found in r oot s, t uber s, st ems, fr uit s and cer eals. St ar ch, which is composed of sever al glucose
molecules, is a mixt ur e of t wo t ypes of component s namely amylose and amylopect in. St ar ch is
hydr olysed int o simple sugar s by dilut e acids and t he quant it y of simple sugar s is measur ed
color imet r ically.
PRINCIPLE
The sample is treated with 80% alcohol to remove sugars and then starch is extracted with
perchloric acid. In hot acidic medium st arch is hydrolysed t o glucose and dehydrat ed t o
hydroxymethyl furfural. This compound forms a green coloured product with anthrone.
10 Biochemical Methods
MATERIALS
H Anthrone: Dissolve 200 mg ant hr one in 100 mL of ice-cold 95% sulphur ic acid.
H 80% et hanol.
H 52% per chlor ic acid.
H S tandard glucose: St ock100 mg in 100 mL wat er. Wor king st andar d10 mL of st ock
dilut ed t o 100 mL wit h wat er.
PROCEDURE
1. Homogenize 0.10.5 g of t he sample in hot 80% et hanol t o r emove sugar s. Cent r ifuge
and r et ain t he r esidue. Wash t he r esidue r epeat edly wit h hot 80% et hanol t ill t he
washings do not give colour wit h ant hr one r eagent . Dr y t he r esidue well over a wat er
bat h.
2. To t he r esidue add 5.0 mL of wat er and 6.5 mL of 52% per chlor ic acid.
3. Ext r act at 0C for 20 min. Cent r ifuge and save t he super nat ant .
4. Repeat t he ext r act ion using fr esh per chlor ic acid. Cent r ifuge and pool t he super nat ant s
and make up t o 100 mL.
5. Pipet t e out 0.1 or 0.2 mL of t he super nat ant and make up t he volume t o 1 mL wit h
wat er.
6. Pr epar e t he st andar ds by t aking 0.2, 0.4, 0.6, 0.8 and 1 mL of t he wor king st andar d and
make up t he volume t o 1 mL in each t ube wit h wat er.
7. Add 4 mL of ant hr one r eagent t o each t ube.
8. Heat for eight minut es in a boiling wat er bat h.
9. Cool r apidly and r ead t he int ensit y of gr een t o dar k gr een colour at 630 nm.
CALCULATION
Find out t he glucose cont ent in t he sample using t he st andar d gr aph. Mult iply t he value
by a fact or 0.9 t o ar r ive at t he st ar ch cont ent .
READINGS
1. Hodge, J .E. and Hofr eit er, B.T. (1962). In: Methods in Carbohydrate Chemistry, (Eds. Whist ler,
R.L. and Be Miller, J .N.), Academic Pr ess, New Yor k.
2. Thayumanavan, B. and Sadasivam, S. (1984). Qual. Plant Foods Hum. Nutr., 34, p. 253.
1.8 Determination of Amylose
St ar ch is composed of t wo component s, namely amylose and amylopect in. Amylose is a linear
or non-br anched polymer of glucose. The glucose unit s ar e joined by -1-4 glucosidic linkages.
Amylose exist s in coiled for m and each coil cont ains six glucose r esidues.
PRINCIPLE
The iodine is adsorbed within the helical coils of amylose to produce a blue-coloured complex
which is measured colorimetrically.
MATERIALS
H Dist illed et hanol.
Carbohydrates 11
H 1 N NaOH.
H 0.1% phenolpht halein.
H Iodine reagent: Dissolve 1 g iodine and 10 g KI in wat er and make up t o 500 mL.
H S tandard: Dissolve 100 mg amylose in 10 mL 1 N NaOH; make up t o 100 mL wit h
wat er.
PROCEDURE
1. Weigh 100 mg of t he powder ed sample, and add 1 mL of dist illed et hanol. Then add
10 mL of 1 N NaOH and leave it over night .
2. Make up t he volume t o 100 mL.
3. Take 2.5 mL of t he ext r act , add about 20 mL dist illed wat er and t hen t hr ee dr ops of
phenolpht halein.
4. Add 0.1 N HCl dr op by dr op unt il t he pink colour just disappear s.
5. Add 1 mL of iodine r eagent and make up t he volume t o 50 mL and r ead t he colour at
590 nm.
6. Take 0.2, 0.4, 0.6, 0.8 and 1 mL of t he st andar d amylose solut ion and develop t he colour
as in t he case of sample.
7. Calculat e t he amount of amylose pr esent in t he sample using t he st andar d gr aph.
8. Dilut e 1 mL of iodine r eagent t o 50 mL wit h dist illed wat er for a blank.
CALCULATION
Absor bance cor r esponds t o 2.5 mL of t he t est solut ion
= x mg amylose 100 mL cont ains
=
2.5
x
100 mg amylose = % amylose.
NOTES:
1. The sample suspension may be heat ed for 10 min in a boiling wat er -bat h inst ead of over night
dissolut ion.
2. The amount of amylopect in is obt ained by subt r act ing t he amylose cont ent fr om t hat of st ar ch.
READINGS
1. McCr eady, R.M.; Guggolz, J .; Silivier a, V. and Owens, H.S. (1950). Anal. Chem., 22, p. 1156.
2. J uliano, B.O. (1971). Cereal S ci. Today, 16, 334.
3. Thayumanavan, B. and Sadasivam, S. (1984). Plant Foods Hum. Nutr, 34, p. 253.
1.9 Estimation of Cellulose
Cellulose, a major st r uct ur al polysacchar ide in plant s, is t he most abundant or ganic compound
in nat ur e, and is composed of glucose unit s joined t oget her in t he for m of t he r epeat ing unit s of
t he disacchar ide cellobiose wit h numer ous cr oss linkages. It is also a major component in
many of t he far m wast es.
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: Mix 150 mL of 80% acet ic acid and 15 mL of concent r at ed nit r ic
acid.
H Anthrone reagent: Dissolve 200 mg ant hr one in 100 mL concent r at ed sulphur ic acid.
Pr epar e fr esh and chill for 2 h befor e use.
H 67% sulphur ic acid.
PROCEDURE
1. Add 3 mL acet ic/nit r ic r eagent t o a known amount (0.5 g or 1 g) of t he sample in a t est
t ube and mix in a vor t ex mixer.
2. Place t he t ube in a wat er -bat h at 100C for 30 min.
3. Cool and t hen cent r ifuge t he cont ent s for 1520 min.
4. Discar d t he super nat ant .
5. Wash t he r esidue wit h dist illed wat er.
6. Add 10 mL of 67% sulphur ic acid and allow it t o st and for 1 h.
7. Dilut e 1 mL of t he above solut ion t o 100 mL.
8. To 1 mL of t his dilut ed solut ion, add 10 mL of ant hr one r eagent and mix well.
9. Heat t he t ubes in a boiling wat er -bat h for 10 min.
10. Cool and measur e t he colour at 630 nm.
11. Set a blank wit h ant hr one r eagent and dist illed wat er.
12. Take 100 mg cellulose in a t est t ube and pr oceed fr om St ep No. 6 for st andar d.
Inst ead of just t aking 1 mL of t he dilut ed solut ion (St ep 7) t ake a ser ies of volumes
(say 0.42 mL cor r esponding t o 40200 g of cellulose) and develop t he colour.
CALCULATION
Dr aw t he st andar d gr aph and calculat e t he amount of cellulose in t he sample.
READING
1. Updegr off, D.M. (1969). Anal. Biochem., 32, p. 420.
1.10 Estimation of Hemicellulose
Hemicelluloses ar e non-cellulosic, non-pect ic cell wall polysacchar ides. They ar e r egar ded as
being composed of xyla ns, ma nna ns, glucoma nna ns, ga la ct a ns a nd a r a binoga la ct a ns.
Hemicelluloses ar e cat egor ized under unavailable car bohydr at es since t hey ar e not split by
t he digest ive enzymes of t he human syst em.
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 S olution
Weigh 18.61 g disodium et hylenediamine t et r aacet at e and 6.81 g sodium bor at e
decahydr at e. Tr ansfer t o a beaker. Dissolve in about 200 mL of dist illed wat er by heat ing
and t o t his, add a solut ion (about 100200 mL) cont aining 30 g of sodium laur yl sulphat e
and 10 mL of 2-et hoxy et hanol. To t his add a solut ion (about 100 mL) cont aining 4.5 g
of disodium hydr ogen phosphat e. Make up t he volume t o one lit r e and adjust t he pH
t o 7.0.
H Decahydr onapht halene.
H Sodium sulphit e.
H Acet one.
PROCEDURE
1. To 1 g of t he powder ed sample in a r efluxing flask add 10 mL of cold neut r al det er gent
solut ion.
2. Add 2 mL of decahydr onapht halene and 0.5 g sodium sulphit e.
3. Heat t o boiling and r eflux for 60 min.
4. Filt er t he cont ent s t hr ough sint er ed glass cr ucible (G-2) by suct ion and wash wit h hot
wat er.
5. Finally give t wo washings wit h acet one.
6. Tr ansfer t he r esidue t o a cr ucible, dr y at 100C for 8 h.
7. Cool t he cr ucible in a desiccat or and weigh.
CALCULATION
Hemicellulose = Neut r al det er gent fibr e (NDF) Acid det er gent fibr e (ADF)
NOTE:
See Lignin for det er mining acid det er gent fibr e.
READING
Goering, H.D. and Vansoest , P.J . (1975). Forage Fibre Analysis, U.S. Dept t . of Agricult ure, Agricult ural
Resear ch Ser vice, Washingt on.
1.11 Determination of Fructose and Inulin
Fr uct ose, a ket o-hexose (called as fr uit sugar ), is usually accompanied by sucr ose in fr uit s like
apple. Honey is a r ich sour ce of fr uct ose.
PRINCIPLE
The hydroxymethyl furfural formed from fructose in acid medium reacts with resorcinol to give
a red colour product.
MATERIALS
H Resorcinol reagent: Dissolve 1 g r esor cinol and 0.25 g t hiour ea in 100 mL glacial acet ic
acid. This solut ion is indefinit ely st able in t he dar k.
14 Biochemical Methods
H Dilute HCl: Mix five par t s of conc. HCl wit h one par t of dist illed wat er.
H S tandard fructose solution: Dissolve 50 mg of fr uct ose in 50 mL wat er. Dilut e 5 mL of
t his st ock t o 50 mL for a wor king st andar d.
PROCEDURE
1. To 2 mL of t he solut ion cont aining 2080 g of fr uct ose add 1 mL of r esor cinol r eagent .
2. Then add 7 mL of dilut e hydr ochlor ic acid.
3. Pipet t e out 0.2, 0.4, 0.6, 0.8 and 1 mL of t he wor king st andar d and make up t he volume
t o 2 mL wit h wat er. Add 1 mL of r esor cinol r eagent and 7 mL of dilut e HCl as above.
4. Set a blank along wit h t he wor king st andar d.
5. Heat all t he t ubes in a wat er -bat h at 80C for exact ly 10 min.
6. Remove and cool t he t ubes by immer sing in t ap wat er for 5 min.
7. Read t he colour at 520 nm wit hin 30 min.
8. Dr aw t he st andar d gr aph and calculat e t he amount of fr uct ose pr esent in t he sample
using t he st andar d gr aph.
Inulin
Inulin is a polymer made of fr uct ose unit s wit h -2-1 linkage. It is found in onion, gar lic
and in many ot her plant par t s.
Sa mp l e Ext r a ct i on
Gr ind t he sample and ext r act in 80% et hanol for six hour s t o r emove fr ee sugar s. Dr y t he
sample and t ake 500 mg in a 100 mL conical flask. Add 20 mL of wat er and heat it in a wat er
bat h at 90C for 10 min. Collect t he ext r act and t hen add 70 mL of wat er. Replace t he flask for
anot her 30 min wit h occasional shaking t o dissolve t he fr uct osan, t hen r emove and cool it at
r oom t emper at ur e. Combine t he ext r act s and filt er t he solut ion if it is not clear and make up t o
100 mL in a st andar d flask.
To est imat e t he inulin cont ent in t he ext r act follow t he pr ocedur e given for fr uct ose
est imat ion. The amount of inulin is expr essed in t er ms of fr uct ose concent r at ion.
READING
Ashwell, G. (1957). In: Methods in Enzymol. 3 (Eds. Colowick, S.J . and Kaplan, N.O.), Academic
Pr ess, New Yor k, p. 75.
1.12 Estimation of Pectic S ubstances
Pect ic subst ances abundant ly exist in t he middle lamella of t he plant cells. Ther e ar e t hr ee
t ypes of pect ic subst ancespect ic acids, pect in and pr ot opect in. Pect ic acid is an unbr anched
molecule made up of about 100 unit s of D-galact ur onic acid r esidues. The monomer s ar e linked
t hr ough 14 linkages. Pect in is an ext ensively est er ified pect ic acid. Sever al car boxyl gr oups
exist as met hyl est er s. Pect ic acid is wat er soluble wher eas pect in for ms a colloidal solut ion.
Pr ot opect in is a lar ger molecule t han pect ic acid and pect in. Dur ing r ipening of fr uit s, conver sion
of pr ot opect in int o pect ic acid and pect in t akes place. The pect ins in fr uit s var y in t heir met hoxyl
cont ent and in jellying power.
Carbohydrates 15
Two met hods ar e descr ibed below for t he est imat ion of pect in: one gr avimet r ic and t he
ot her, color imet r ic.
I. Gravimetric Method
PRINCIPLE
Pectin is extracted from plant material and saponified. It 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 (Dilut e 30 mL of glacial acet ic acid t o 500 mL wit h wat er ).
H 1 N Calcium chloride solution: Dissolve 27.5 g anhydr ous CaCl
2
in wat er and dilut e t o
500 mL.
H 1% S ilver nitrate: Dissolve 1 g AgNO
3
in 100 mL wat er.
H 0.01 N HCl
H 0.05 N HCl
H 0.3 N HCl
PROCEDURE
1. Weigh 50 g of blended sample int o a 1 L beaker and add 300 mL 0.01 N HCl. Boil for
30 min and filt er under suct ion. Wash t he r esidue wit h hot wat er and collect t he filt r at e.
2. To t he r esidue add 100 mL 0.05 N HCl, boil for 20 min filt er, wash and collect t he
filt r at e.
3. To t he r esidue now add 100 mL 0.3 N HCl, boil for 10 min, filt er, wash and collect t he
filt r at e.
4. Pool t he filt r at es. Cool and make t o volume (500 mL).
5. Pipet t e out 100200 mL aliquot s int o 1 L beaker s.
6. Add 250 mL wat er and neut r alize t he acid wit h 1 N NaOH using phenolpht halein
indicat or. Add an excess of 10 mL of 1 N NaOH wit h const ant st ir r ing and allow it t o
st and over night .
7. Add 50 mL 1 N acet ic acid and aft er 5 min, add 25 mL 1 N calcium chlor ide solut ion
wit h st ir r ing. Allow it t o st and for 1 h.
8. Boil for 1 t o 2 min.
9. Filt er t hr ough a pr e-weighed What man No. 1 filt er paper (see not e 1).
10. Wash t he pr ecipit at e wit h almost boiling wat er unt il t he filt r at e is fr ee fr om chlor ide.
11. Test t he filt r at e wit h silver nit r at e for chlor ide.
12. Tr ansfer t he filt er paper wit h t he calcium pect at e, dr y over night at 100C in a weighing
dish, cool in a desiccat or and weigh.
CALCULATION
The pect in cont ent is expr essed as % calcium pect at e
% calcium pect at e =
Wt. of calcium pect at e 500 100
mL of filt rat e t aken Wt . of smaple for est imat ion
16 Biochemical Methods
NOTES:
The filt er paper for St ep No. 9 should be pr epar ed as descr ibed below:
1. Wet t he filt er paper in hot wat er, dr y in oven at 102C for 2 h. Cool in a desiccat or and weigh in
a cover ed dish.
2. The t heor et ical yield of calcium pect at e fr om pur e galact ur onic anhydr ide is 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% Et hyl alcohol (Mix 500 mL 95% alcohol and 300 mL wat er ).
H 95% Et hyl alcohol.
H Pur ified et hyl alcohol (Reflux 1 L of 95% et hyl alcohol wit h 4 g zinc dust and 2 mL conc.
H
2
SO
4
for 15 h and dist ill in all glass dist illat ion appar at us. Redist ill wit h 4 g zinc dust
and 4 g KOH).
H 1 N and 0.05 N Sodium hydr oxide.
H H
2
SO
4
(Analyt ical gr ade).
H 0.1% Carbazole reagent: Weigh 100 mg r ecr yst allized car bazole, dissolve and dilut e t o
100 mL wit h pur ified alcohol.
PROCEDURE
1. Weigh 100 mg pect in (see not es sect ion for t he pr epar at ion of pect in) and dissolve in
100 mL of 0.05 N NaOH.
2. Allow it t o st and for 30 min t o deest er ify t he pect in.
3. Take 2 mL of t his solut ion and make up t o 100 mL wit h wat er.
4. Pipet t e out 2 mL of deest er ified pect in solut ion and add 1 mL car bazole r eagent . A
whit e pr ecipit at e will be for med.
5. Add 12 mL conc. H
2
SO
4
wit h const ant st ir r ing.
6. Close t he t ubes wit h r ubber st opper and allow t o st and for 10 min t o develop t he colour.
7. To set a blank add 1 mL of pur ified et hyl alcohol in t he place of car bazole r eagent .
8. Read t he colour at 525 nm against blank, exact ly 15 min aft er t he addit ion of acid.
STANDARD
Weigh 120.5 mg galact ur onic acid monohydr at e (fr om a sample vacuum dr ied for 5 h at
30C) and t r ansfer t o a 1 L volumet r ic flask. Add 10 mL 0.05 N NaOH and dilut e t o volume wit h
wat er. Aft er mixing, allow it t o st and over night . Dilut e 10, 20, 40, 50, 60 and 80 mL of t his
st andar d solut ion t o 100 mL wit h wat er. Take 2 mL of t hese solut ions for colour developing and
pr oceed a s in t he ca se of t he sa mple. Dr a w a st a nda r d cur vet he a bsor ba nce versus
concent r at ion.
Carbohydrates 17
CALCULATION
Read t he concent r at ion of t he anhydr ogalact ur onic acid cor r esponding t o t he r eading of
t he sample, and calculat e as follows:
% anhydr ogalact ur onic acid =
g of anhydr ogalacturonic acid in t he aliquot Dilution 100
mL taken for estimat ion Wt . of pect in sample 1,000,000
NOTES:
1. Car bazole is r ecr yst allized fr om t oluene.
2. An alt er nat e pr ocedur e adopt ed for colour development is as follows:
Take 12 mL of conc. H
2
SO
4
in a t est t ube, cool in an ice-bat h, and add 2 mL of t he deest er ified
pect in solut ion and again cool. Heat t he cont ent s in a boiling wat er -bat h for 10 min, cool t o 20C
and add 1 mL of 0.15% car bazole r eagent in pur ified et hyl alcohol. Allow it t o st and for 25 5
min at r oom t emper at ur e t o develop t he colour. Read t he absor bance at 520 nm. St andar ds
should also be t r eat ed similar ly.
III. Extraction and Purification of Pectin
1. Blend t he fr esh sample. If t he mat er ial is dr y gr ind.
2. Tr ansfer 100 g macer at ed sample (10 g dr y t issue) t o a pr e-weighed 1 L beaker cont aining
400 mL wat er.
3. Add 1.2 g fr eshly gr ound sodium hexamet aphosphat e and adjust t o pH 4.5.
4. Heat wit h st ir r ing at 9095C for 1 h. Check t he pH in ever y 15 min and maint ain at pH
4.5 wit h cit r ic acid or NaOH. Replace wat er lost by evapor at ion at int er vals. However,
do not add wat er at t he last 20 min.
5. Add 4 g filt er aid and 4 g gr ound paper pulp. Filt er r apidly t hr ough a fast filt er paper
coat ed wit h 3 g moist ened fast filt er aid.
6. Collect at least 200 mL of t he filt r at e in a pr eweighed cont ainer. Cool as r apidly as
possible. Now, not e t he weight of t he filt r at e.
7. If t he filt r at e cont ains less t han 0.2% pect in, concent r at e t he filt r at e under vacuum t o
at t ain t his concent r at ion.
8. To t hr ee volumes of et hanol, isopr opanol or acet one cont aining 0.5 N HCl, pour t he
cooled, weighed filt r at e. The slur r y should be at pH 0.71. St ir for 30 min.
9. Cent r ifuge or filt er. Wash t he pr ecipit at e wit h t he same solvent cont aining HCl. Then,
wash r epeat edly wit h 70% alcohol or acet one unt il t he pr ecipit at e is essent ially chlor ide-
fr ee or t he pH is above 4.
10. Dehydr at e t he pr ecipit at e fur t her in 400 mL acet one. Dr y over night in vacuo wit h a
slow st r eam of dr y air passing t hr ough t he oven.
11. Weigh t he pr ecipit at e and use t his pect in for analysis.
12. The dr ied pect in should be fr ee fr om ammonia for which a small sample of t he pect in is
heat ed wit h 1 mL of 0.1 N NaOH and ammoniacal odour can be not iced or t est ed wit h
a moist ened lit mus paper. If ammonium ions ar e pr esent wash wit h acidified 6% alcohol,
followed by neut r al alcohol t o r emove t he acid and dr y.
18 Biochemical Methods
READING
Ranganna, S. (1979). Manual of Analysis of Fruit and Vegetable Products, Tat a McGr aw-Hill Publ.
Co. Lt d., New Delhi, p. 634.
1.13 Estimation of Crude Fibre
Cr ude fibr e consist s lar gely of cellulose and lignin (97%) plus some miner al mat t er. It r epr esent s
only 6080% of t he cellulose and 46% of t he lignin. The cr ude fibr e cont ent is commonly used
as a measur e of t he nut r it ive value of poult r y and livest ock feeds and also in t he analysis of
var ious foods and food pr oduct s t o det ect adult er at ion, qualit y and quant it y.
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
cont ent .
MATERIALS
H S ulphuric acid solution (0.255 0.005 N): 1.25 g concent r at ed sulphur ic acid dilut ed t o
100 mL (concent r at ion must be checked by t it r at ion).
H S odium hydroxide solution (0.313 0.005 N): 1.25 g sodium hydr oxide in 100 mL dist illed
wat er (concent r at ion must be checked by t it r at ion wit h st andar d acid).
PROCEDURE
1. Ext r act 2 g of gr ound mat er ial wit h et her or pet r oleum et her t o r emove fat (Init ial
boiling t emper at ur e 3538C and final t emper at ur e 52C). If fat cont ent is below 1%,
ext r act ion may be omit t ed.
2. Aft er ext r act ion wit h et her boil 2 g of dr ied mat er ial wit h 200 mL of sulphur ic acid for
30 min wit h bumping chips.
3. Filt er t hr ough muslin and wash wit h boiling wat er unt il washings ar e no longer acidic.
4. Boil wit h 200 mL of sodium hydr oxide solut ion for 30 min.
5. Filt er t hr ough muslin clot h again and wash wit h 25 mL of boiling 1.25% H
2
SO
4
, t hr ee
50 mL por t ions of wat er and 25 mL alcohol.
6. Remove t he r esidue and t r ansfer t o ashing dish (pr eweighed dish W
1
).
7. Dr y t he r esidue for 2 h at 130 2C. Cool t he dish in a desiccat or and weigh (W
2
).
8. Ignit e for 30 min at 600 15C.
9. Cool in a desiccat or and r eweigh (W
3
).
CALCULATION
% cr ude fibr e in gr ound sample =
2 1 3 1
Loss in weight on ignition (W W ) (W W )
100
Weight of t he sample

READING
Maynar d, A.J . (Ed.) (1970). Methods in Food Analysis, Academic Pr ess, New Yor k, p. 176.
Carbohydrates 19
1.14 Estimation of Pyruvic Acid
Pyr uvic acid or pyr uvat e is an impor t ant met abolic int er mediat e. It is gr eat ly pr oduced in t he
t er minal st ep of glycolysis and funnels t o TCA cycle for fur t her oxidat ion for r eleasing t he
chemical ener gy. It can be det er mined following t he pr ocedur e given below:
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 solut ion of monobasic sodium phosphat e NaH
2
PO
4
H
2
O (27.8 g in 1000 mL).
B: 0.2 M solut ion of dibasic sodium phosphat e (53.65 g of Na
2
HPO
4
.7H
2
O in 1 L or 17.7
g of Na
2
HPO
4
.12H
2
O in 1 L).
19 mL of A and 81 mL of B, dilut ed t o a t ot al of 200 mL
St or e in r efr iger at or.
H Pyruvate, S tandard
Dissolve 22 mg sodium pyr uvat e in 100 mL wat er in a st andar d flask.
H 2, 4-Dinitrophenyl hydrazine (DNPH)
Dissolve 19.8 mg of DNPH in 10 mL of conc. HCl and make t o 100 mL wit h wat er.
St or e it in an amber bot t le at r oom t emper at ur e.
H S odium hydroxide 0.8 N
Dissolve 16 g sodium hydr oxide in one lit r e wat er.
H Plant extract
Gr ind 6 g of plant mat er ial in 15 mL of phosphat e buffer. Cent r ifuge at 25,000 g for 15
min. Use t he super nat ant as plant ext r act .
PROCEDURE
1 Pipet t e out 50 L, 75 L, 100 L, 150 L, 200 L of pyr uvat e st andar d solut ion and
0.5 mL, 1.0 mL, 1.5 mL, and 2.0 mL of sample ext r act int o t est t ubes and make up t he
volume t o 2.0 mL wit h phosphat e buffer (pH 7.4).
2. Set a blank wit h no pyr uvat e solut ion.
3. Add 0.5 mL of DNPH solut ion t o each t ube.
4. Incubat e at 37C for 2030 min.
5. Add 5 mL of NaOH solut ion t o each t ube, mix well and incubat e for 10 min at r oom
t emper at ur e.
6. Recor d t he absor bance at 610 nm.
7. Dr aw t he st andar d gr aph and calculat e t he amount of pyr uvic acid pr esent in t he sample
using t he gr aph.