SRISHAKTHII

NSTI
TUTEOF
ENGINEERI
NGANDTECHNOLOGY
COI
MBATORE641062

DEPARTMENTOFFOODTECHNOLOGY

BT8261
BI
OCHEMI
STRYLABORATORY

LABORATORYRECORDBOOK

APRI
L2019

Name :………………………………………………………………
Reg.No. :………………………………………………………………
Degr
ee :B.Tech(
FoodTechnol
ogy
)
Year
/Sem. :I/I
I
 SRISHAKTHII
NSTI
TUTEOFENGI
NEERI
NGANDTECHNOLOGY
COI
MBATORE641062

DEPARTMENTOFFOODTECHNOLOGY

Thi
sist
ocer
ti
fyt
hatt
hisBT8261BI
OCHEMI
STRYLABORATORYr
ecor
dwor
kisdone

byMr
./Ms._
___
___
___
___
___
___
___
___
___
___
___
__f
ort
hecour
seB.Tech FOOD

TECHNOLOGYdur
ingFi
rstYear/SecondSemest
erofAcademi
cyear2018–2019.

St
aff–i
n–Char
ge HoD/FT

Regi
sterNo.………………………………….
.

Thi
srecor
dissubmi
tt
edf
orSECONDSemest
erB.TechPr
act
icalExami
nat
ions

ofAnnaUni
ver
sit
yconduct
edon_
___
___
___
___

I
NTERNALEXAMI
NER EXTERNALEXAMI
NER

BT8261Bi
ochemi
str
yLabor
ator
y 2/2
 ANNAUNI
VERSI
TY,
CHENNAI
SYLLABUS
(
R2017)

BT8261 BI
OCHEMI
STRYLABORATORY LTPC
0042

AI
M

Tolearnandunderst
andt heprinci
plesbehindt hequal
it
ati
veandquanti
tat
iveesti
mati
on
ofbiomol
ecul
es(protei
ns,carbohydrat
es,l
ipids,metabol
it
esetc.
,
)andlabor
atoryanal
ysi
s
oft
hesamei nthebodyfluids.

EXPERI
MENTS

1.Gener
algui
del
i
nesf
orwor
kingi
nbi
ochemi
str
ylab(
theor
y)

2.Uni
tsofv ol
ume,weight
,densi
tyandconcent
rat
ionmeasur
ement
sandtheirr
angein
bi
ologi
calmeasurements.Demonstr
ati
on ofpr operuse ofv
olume and wei
ght
measurementdev
ices.

3.Accur
acy
,pr
eci
sion,
sensi
ti
vi
tyandspeci
fi
cit
y(t
heor
y)

4.Pr
epar
ati
onofbuf
fer–t
it
rat
ionofaweakaci
dandaweakbase.

5.Quali
tat
ivet
est
sforcar
bohy
drat
es – di
sti
ngui
shi
ng r
educi
ng f
rom non-
reduci
ng
sugar
sand

ket
ofr
om al
dosugar
s.

6.Quant
it
ati
vemethodf
orami
noaci
dest
imat
ionusi
ngni
nhy
dri
n–di
sti
ngui
shi
ngami
no
fr
om i
minoaci
d.

7.Pr
otei
nest
imat
ionbyBi
uretandLowr
y’smet
hods.

8.Pr
otei
nest
imat
ionbyBr
adf
ordandspect
roscopi
cmet
hods.

9.Ext
ract
ionofl
i
pidsandanal
ysi
sbyTLC.

10.
Est
imat
ionofnucl
eicaci
dsbyabsor
banceat260nm andhy
per
chr
omi
cef
fect(
demo)
.

11.
Enzy
mat
icassay
:phosphat
asef
rom pot
ato.

12.
Enzy
mat
icassay
:est
imat
ionofgl
ucosebyGOD-
POD met
hod af
terhy
drol
ysi
sof

3/
3
 st
archwi
thaci
dandspeci
fi
cit
yoft
heenzy
mat
icmet
hod.

TOTAL:60PERI
ODS

BT8261Bi
ochemi
str
yLabor
ator
y 4/4
 I
NDEX

Expt Page Facul

ty
Dat
e Ti
tl
e Mar
ks
No. No. si
gnat
ure

1 Gener
al guidel
i
nes f or wor
king i
n
bi
ochemi
str
ylab(
theor
y)

2 St
udyofuni
tandmeasur
ement
s

3 Accur
acy
,pr
eci
sion,sensi
ti
vi
tyandspeci
fi
cit
y
(t
heor
y)
Pr
epar
ati
onofbuf
fer– Ti
tr
ati
onofaweak
4
aci
dandaweakbase
Quali
tati
ve tests for carbohydr
ates –
5 Dist
ingui
shi
ng reduci
ng f
rom non-reduci
ng
sugarsandket
of r
om al
dosugars

Quanti
tati
ve met hod f or ami no acid
6 est
imation usi
ng ni
nhydr
in – Di
sti
ngui
shi
ng
aminofrom imi
noacid

Pr
otei
n est
imat
ion by bi
uret and l
owr
y’s
7
met
hods
Pr
otei
n est
imat
ion by br
adf
ord and
8
spect
roscopi
cmet
hods

9 Ext
ract
ionofl
i
pidsandanal
ysi
sbyTLC

Est
imat
ionofnucl
eicaci
dsbyabsor
banceat
10
260nm andhy
per
chr
omi
cef
fect(
demo)

11 Enzy
mat
icassay
:phosphat
asef
rom Pot
ato

Enzymat
icassay:Estimati
on ofgl ucoseby
GOD-POD methodafterhydrol
ysi
sofst ar
ch
12 wit
h aci
d and speci
fi
cit
yoft he enzymati
c
method

Expt
.No:
1
GENERALGUI
DELI
NESFORWORKI
NGI
NBI
OCHEMI
STRYLAB
Dat
e:

AI
M

5/
5
Tost
udyt
hegener
algui
del
i
nesf
orwor
kingi
nbi
ochemi
str
ylabor
ator
y.

GUI
DELI
NES
Ther
ear
etwomaj
orconcer
nst
oconsi
derwhenwor
kingi
nabi
ochemi
str
ylabor
ator
y.
Fi
rsti
s saf
ety
:thi
s can nev
erbe ov
eremphasi
zed.The second i
s ef
fi
ciencyi
nthe
l
abor
ator
ywor
k.Al
thought
hel
att
erv
erymuchdependsont
hei
ndi
vi
dual
sdoi
ngt
he
exper
iment
s,t
her
ear
egener
alr
ulesst
udent
sar
eadv
isedt
ofol
l
ow:
 Keept
hebenchesandshel
vescl
eanandwel
l
-or
gani
zed.
 Av
oid cont
ami
nat
ing t
hechemi
cal
s;useonl
ycl
eangl
asswar
eand spat
ulas;l
abel
gl
asswar
einuse.
 Pl
any
ourexper
iment
sbef
orest
art
ingt
ocar
ryt
hem out
.
 Payat
tent
iont
oot
her
sint
hel
abor
ator
y.

SAFETYI
NTHELABORATORY
St
udent
swor
kingi
nabi
ochemi
str
ylabor
ator
ymustal
way
sbeawar
ethatt
hechemi
cal
s
usedar
epot
ent
ial
l
ytoxi
c,i
rr
it
ati
ngandf
lammabl
e.Suchchemi
cal
sar
ehazar
ds,howev
er,
onl
ywhent
heyar
emi
shandl
ed.St
udent
swhocomet
othel
abor
ator
ysessi
onmusthav
ea
compl
eteunder
standi
ngoft
hel
abor
ator
ypr
ocedur
est
ocar
ryoutandbef
ami
l
iarwi
th
bot
hthephy
sicalandchemi
cal
proper
ti
esofchemi
cal
sandr
eagent
stobeused.Si
ncet
he
car
elessnessont
hepar
tofonest
udentcanof
tencausei
njur
ytoot
herst
udent
s,onemust
hav
easpeci
alconcer
nfort
hesaf
etyofcl
assmat
es.St
udent
smustbef
ami
l
iarwi
th
gener
alsaf
etypr
act
ices,
faci
l
iti
esandemer
gencyact
ion.

SAFETYRULESI
NGENERAL
 Donotwor
kal
onei
nthel
abor
ator
y.
 Unaut
hor
izedexper
iment
sar
enotal
l
owed.
 Eat
ing,
dri
nki
ngandsmoki
ngi
nthel
abor
ator
yar
est
ri
ctl
ypr
ohi
bit
ed.
 Becomef
ami
l
iarwi
tht
hel
ocat
ionandt
heuseofst
andar
dsaf
etyf
eat
uresi
nthe
l
abor
ator
y.Thel
abor
ator
yisequi
pped wi
thf
ir
eext
ingui
sher
s,ey
ewashes,saf
ety
shower
s,f
umehoodsand f
ir
st-
aid ki
ts.Anyquest
ionr
egar
ding t
heuseoft
hese
f
aci
l
iti
esshoul
dbeaddr
essedt
oyouri
nst
ruct
or.
 Speci
alcar
eforey
epr
otect
ioni
srequi
red.Saf
etygl
assesmustbeusedwhencer
tai
n
pr
ocedur
esar
ebei
ngcar
ri
edout
.Thei
nst
ruct
orwi
l
lcal
lthest
udent
s'at
tent
iont
o
t
hosepr
ocedur
es.Theuseofcont
actl
ensesi
snotr
ecommended,si
ncet
heyr
educe
t
her
ateofsel
f-
cleansi
ngoft
heey
e.

BT8261Bi
ochemi
str
yLabor
ator
y 6/6
SPECI
ALSAFETYRULES
 Whi
l
eheat
ingasol
uti
ononeshoul
dmakesur
enott
oov
erheati
t;t
her
efor
e,v
igor
ous
mi
xi
ngoft
hesol
uti
onbyshaki
ngorst
ir
ri
ngi
srequi
red.Themout
hoft
hegl
asswar
e
cont
aini
ngt
hesol
uti
ont
obeheat
edshoul
dnev
erbepoi
ntedt
owar
dany
one.
 Handl
i
ng of st
rong aci
ds and bases r
equi
res speci
alat
tent
ion.When di
l
uti
ng
concent
rat
edaci
ds,
theaci
dshoul
dbepour
edi
ntot
hewat
erandnev
ert
heopposi
te.
 Thepi
pet
tesshoul
dnev
erbef
il
ledwi
thsol
uti
onsoft
oxi
csubst
ances,bi
ologi
calf
lui
ds,
st
rongaci
dsandbasesbymout
hsuct
ion.Useei
theraut
omat
icpi
pet
teorpi
pet
tepumps.
 Vol
ati
l
eli
qui
dsandsol
i
dst
hatar
etoxi
cori
rr
it
ati
ngshoul
dbehandl
edunderf
ume
hoods.
 Whi
l
ehandl
i
ngf
lammabl
eli
qui
dssuchaset
her
,al
cohol
s,benzene,
nakedf
lame(
bur
ner
s,
mat
ches)mustnotbei
nuse.Theabov
eli
qui
dsmustnotbest
orednearr
adi
ati
ngheat
sour
ces,
suchast
hel
abor
ator
yov
en.
 Bef
oreusi
ngel
ect
ri
cal
appl
i
ances,
makesur
etheyar
egr
ounded.
 Fl
askswi
thf
lat
-bot
tomsort
hinwal
l
sshoul
dnotbedesi
ccat
ed.
 Bef
orel
eav
ingt
hel
abor
ator
y,el
ect
ri
calequi
pmentshoul
dbet
urnedof
f,andgasbur
ner
s
ext
ingui
shed.Not
apwat
ershoul
dbel
eftr
unni
ng.

RULESTOFOLLOW I
NTHECASEOFACCI
DENTSANDI
NJURI
ES

CHEMI
CALSPLATTERSI
NTO THEEYE:Fi
rstt
heey
eli
dshoul
dbeopenedbyusi
ngt
he
t
humbandt
hepoi
nti
ngf
inger
.Then,byusi
ngt
heey
ewashki
t,t
heey
eshoul
dber
insed
wi
thl
argeamount
sofwat
er.Whenanaci
doral
kal
i
nesol
uti
onget
sint
oey
e,t
heey
e
shoul
dber
insedwi
th1% NaHCO3 or1% bor
icaci
d,r
espect
ivel
y.Thev
ict
im shoul
dbe
t
akent
othedoct
orassoonaspossi
ble.
BURNI
NG:Thebur
nedspotont
heski
nshoul
dnotbet
reat
edwi
thwat
er;r
ather
,aspeci
al
bandageshoul
dbeused.Seedoct
ori
fnecessar
y.
POI
SONI
NG:Pr
omptmedi
cal
treat
mentshoul
dbeobt
ained.

RESULT
Gener
algui
del
i
nesf
orwor
kingi
nthebi
ochemi
str
ylabor
ator
ywer
est
udi
ed.

Expt
.No:
2 STUDYOFUNI
TSANDMEASUREMENTS

7/
7
Dat
e:

AI
M
Tost
udyt
heuni
tsandi
tsmeasur
ement
s.

DEFI
NITI
ONS
VOLUME:Vol
umei
sthespaceoccupi
edbyl
i
qui
d,gasorasol
i
d.TheSIuni
tofv
olumei
s
Cubi
cmet
er.
WEI
GHT:Wei
ghti
sthemeasur
ementt
hatgi
vest
hei
nfor
mat
ionaboutt
hef
orcewi
thwhi
ch
t
heobj
ecti
spul
l
edbyt
heear
th.TheSIuni
tisKi
l
ogr
am(
Kg)
.
MASS:Massofanobj
ecti
stheamountofmat
tercont
ainedi
nthatobj
ect
.Massi
sthe
i
nvar
iantpr
oper
tyofal
lobj
ect
s.
DENSI
TY:I
tist
hequant
it
ythatr
elat
esmassandv
olume.Densi
ty(
D)i
sdef
inedasmass(
m)
peruni
tvol
ume(
V)
D=m/
V
UNI
TSOFMASS:
 Gr
am -gm

 Ki
l
ogr
am -Kg

 Mi
l
li
gram -mg

 Nanogr
am -ng

 Pi
cogr
am -pg

 Mi
crogr
am -µg

 1kg =1000gm =1000000mg

 1gm =1000mg=100000µg

 1mg =1000ng=1000000pg

 1ng =1000pg

UNI
TSOFVOLUME:
 1mi
l
li e =10-3mol
mol e=1mi
cr e=10-6mol
omol e

 e=10-9mol
1nanomol e=1pi e=10-12mol
comol e

BT8261Bi
ochemi
str
yLabor
ator
y 8/8
  1ml
=1000l
i
tre(
micr
oli
tr
es)=1000µl

UNI
TSOFCONCENTRATI
ON:
Sol
uti
on:
ASol
uti
oni
sahomogenousmi
xtur
eoft
woormor
esubst
ances.
Quant
it
yofsol
ute
Concent
rat
ionofaSol
uti
on= x100
Volumeofsol
uti
on

PHASEOFEXPRESSI
NGCONCENTRATI
ON:
PERCENTAGEBYWEI
GHT:I
tist
hewei
ghtofsol
uteasaper
cent
ageoft
otalwei
ghtof
sol
uti
on.
Weig
htofsolut
e
Per
cent
agebyWei
ghtofaSol
uti
on= x100
Vol
umeofsoluti
on
MOLEFRACTI
ON:Mol
efr
act
ion(
X)ofasol
utei
sdef
inedast
her
ati
oofnumberofmol
es
t
otot
alnumberofmol
esoft
hesol
uti
on.
Mol
esofthesolut
e
Mol
eFr
act
ion(
X)=
Mole
softhesoluti
on

MOLARI
TY:Mol
ari
tyoft
hesol
uti
oni
sdef
inedast
henumberofmol
esofsol
uteperl
i
treof
t
hesol
vent
.
No:
ofmolesofsol
ute
Mol
ari
ty=
Li
treofsol
vent
MOLALI
TY:Mol
ali
tyoft
hesol
uti
oni
sdef
inedast
henumberofmol
esofsol
uteper
ki
l
ogr
am oft
hesol
vent
.
No:ofmol
esofs
olut
e
Mol
ali
ty=
Ki
logr
amofsol
vent
NORMALI
TY:Nor
mal
i
tyoft
hesol
uti
oni
sdef
inedast
henumberofequi
val
entofsol
uteper
l
i
treoft
hesol
uti
on.
Equiva
lentweightofsol
ute
Nor
mal
i
ty=
Vol
umeofs ol
utioninli
tre

RESULT

9/
9
Uni
tsandmeasur
ement
swer
est
udi
ed.

Expt
.No:
3 ACCURACY,
PRECI
SION,
SENSI
TIVI
TYANDSPECI
FICI
TY
Dat
e:

AI
M
Tost
udyt
hedef
ini
ti
onsofaccur
acy
,pr
eci
sion,
sensi
ti
vi
tyandspeci
fi
cit
y.

DEFI
NITI
ONS

ACCURACY:Accur
acyi
sthecl
osenessoft
hemeanofasetofr
epl
i
cat
eanal
ysi
stot
he
t
ruev
alueofsi
mpl
e.I
tisexpr
essedi
nter
msofer
rorwhi
chi
sthedi
ff
erencebet
weent
rue
v
alueandobser
vedv
alue.
PRECI
SION:Pr
eci
sioni
sther
epr
oduci
bil
i
tyofamet
hod.I
text
entt
owhi
chanumberof
r
epl
i
cat
emeasur
ement
sofasampl
eagr
eedwi
thoneanot
herandi
tisaf
fect
edbyt
he
r
andom er
rormet
hod.Pr
eci
sioni
sexpr
essednumer
ical
l
yint
ermsofst
andar
ddev
iat
ion
st
ati
sti
cal
l
yisr
epr
esent
edasco-
eff
ici
entofv
ari
ati
oni
nwhi
ch,
S
D
V= x100
Mean
wher
e,
V=Var
iat
ion
SD=St
andar
ddev
iat
ion
SENSI
TIVI
TY:Sensi
ti
vi
tyi
sdef
inedast
heabi
l
ityt
odet
ectsmal
lamountoft
estsubst
ance
f
orexampl
ethesmal
l
err
eadi
ngaf
terzer
ocanbeconst
ant
lydet
ect
edandmeasur
edt
he
sl
opeoft
hecal
i
brat
ioner
rori
sconv
ent
ional
wayofexpr
essi
ngsensi
ti
vi
ty
SPECI
FICI
TY:Speci
fi
cit
yisdef
ined asabi
l
ityt
o det
ectonl
ythe t
estsubst
ance,t
he
speci
fi
cit
ywi
l
lresul
tint
heposi
ti
ver
esul
t.I
fthemet
hodi
squant
it
ati
ve,i
tisi
mpor
tantt
o
appr
eci
atet
hatspeci
fi
cit
yisof
tenl
i
nkedwi
thsensi
ti
vi
ty.

BT8261Bi
ochemi
str
yLabor
ator
y 10/10
RESULT

Expt
.No:
4 PREPARATI
ONOFBUFFER–TI
TRATI
ONOFAWEAKACI
DANDA
WEAKBASE
Dat
e:

AI
M
Tot
it
rat
etheweakaci
dwi
thst
rongbaset
odet
ectt
hepKav
aluesofweakaci
d.
Theweakaci
dis0.
1NCH3COOHandbase0.
1NNaOH.

PRI
NCI
PLE
ThepHi
stheuni
ttomeasur
etheact
ivi
tyoft
hesol
uti
ons.I
tisdef
inedast
he
negat
ivel
ogar
it
hm ofhy
drogeni
onconcent
rat
ion.
pH=-
l H+]
og[
Accor
dingt
oHender
son–Hassel
bal
chequat
ion,
[A-
]
pH=pKa+l
og
HA
pKacanbeobt
ainedf
rom t
hegr
aphbyt
aki
nghal
foft
hev
aluef
ormt
hepoi
ntatwhi
ch
t
her
eisagr
adual
incr
easeofpH.Hal
fthev
alueaf
terext
endi
ngonX-
axi
sisgi
venpKa.
Whenanaci
dist
it
rat
edwi
thabase,
att
hemi
dpoi
ntoft
it
rat
ion,
theconcent
rat
ionof
pr
otondonorandpr
otonaccept
orandar
eequal

APPARATUSREQUI
RED
 Pi
pet
te

 pHmet
er

 Beakerandagl
assr
od

REAGENTSREQUI
RED
 0.
1NCH3COOH

 0.
1NNaOH

11/
11
PROCEDURE
1.St
andar
dizet
hepHmet
er.20ml
of0.
1NCH3COOHi
spi
pet
teouti
ntoabeaker
.

2.2ml
of0.
1NaOHi
saddedt
othesol
uti
onandst
ir
redwel
l
.ThepHi
smeasur
ed.

3.Thepr
ocedur
eisr
epeat
edwi
thaddi
ti
onof2ml
of0.
1NNaOHt
il
li
treachespH11or
t
her
eisashar
pincr
easei
npH.

4.Agr
aphi
sdr
awn,
plot
ti
ngt
hemeasur
edv
aluesofpHal
ongX-
axi
s(t
it
rat
ioncur
ve)

5.Fr
om t
het
it
rat
ioncur
ve,
pKav
alueoft
heweakaci
dcanbedet
ermi
ned.Att
hispoi
nt
pH=pKa(
whent
it
rat
ioni
shal
fcompl
ete)

Thebuf
ferdescr
ibedi
nther
eact
ionsar
esui
tabl
eforuseei
theri
nenzy
mat
icor
bi
ochemi
cal
studi
es.Theaccur
acyoft
het
abl
eiswi
th±0.
05pHat23˚
c

CI
TRATEBUFFER
A=0.
1M sol
uti
onofci
tr
icaci
d(21.
01gi
n100ml
)
B=0.
1M sol
uti
onofsodi
um ci
tr
ate(
29.
4gofC6H5O7)
NO3.
H2Oi
n1000ml
.
Theuseofsal
twi
thi
n5.
5H2Oi
snotr
ecommended.Xml
ofA+yml
ofB,
di
l
utedt
otot
al100ml
.
X Y pH X y pH

ACETATEBUFFER
STOCKSOLUTI
ON
A=0.
2M sol
uti
onofacet
icaci
d(11.
5ml
of1000ml
)

B=0.
2M sol
uti
onofsodi
um acet
ate
(
16.
4gofC2H3O2NaCO3,
27.
2gofC2H3O2 Na.
3H2Oi
n1000ml
.)
xml
ofA+yml
ofBdi
l
utedt
het
otal
of100ml

BT8261Bi
ochemi
str
yLabor
ator
y 12/12
 X Y pH X y pH

PHOSPHATEBUFFER
STOCKSOLUTI
ON
A=0.
2M sol
uti
onofmonobasi
csodi
um phosphat
e(27.
8gof1000ml
)
B=0.
2M sol
uti
onofdi
basi
csodi
um phosphat
e
(
56.
5gofNaHPO4.
7H2Oor71.
7gofNa3HPO4.
2H2O)
xml
ofA+yml
ofBdi
l
utedt
oat
otal
of200ml
.

X Y pH X y pH

SUCCI
NATEBUFFER
STOCKSOLUTI
ON
A=0.
2M sol
uti
onofmonobasi
csodi
um phosphat
e(23.
6gi
n1000ml
)
B=0.
2M NaOH(
sodi
um hy
droxi
desol
uti
on)
25ml
ofA+xml
ofBdi
l
utedt
oat
otal
of100ml
.

X pH X pH x pH

13/
13
TRI
S(HYDROXYMETHYL)AMI
NEMETHANETRI
SBUFFER
STOCKSOLUTI
ON
A=0.
2ml
sol
uti
onoft
ri
s(hy
droxy
lmet
hyl
ami
nemet
hane211.
2gi
n1000ml
)
B=0.
2M Hcl
(hy
drochl
ori
caci
dsol
uti
on)
50ml
ofA+xml
ofBdi
l
utedt
oat
otal
of100ml
.

X pH X pH

OBSERVATI
ON
S.
No Vol
umeofAcet
icaci
d Vol
umeofNaOH pH
(
ml) (
ml)

BT8261Bi
ochemi
str
yLabor
ator
y 14/14
RESULT

15/
15
ThepKav
alueoft
heweakaci
dis_
___
___
___
__

Expt
.No:
5 QUALI
TATIVETESTSFORCARBOHYDRATES-
DI
STI
NGUISHINGREDUCINGANDNONREDUINGSUGARS
Dat
e: ANDKETOFROM ALDOSUGARS

AI
M
Toi
dent
if
ythesugarpr
esenti
nthegi
vensampl
ebyper
for
mingqual
i
tyanal
ysi
sfor
car
bohy
drat
es.Al
sodi
sti
ngui
shbet
weenr
educi
ng&non-
reduci
ngsugar
sandket
o&al
do
sugar
s.

QUALI
TATI
VETEST

BENEDI
CT’
STEST
PRI
NCI
PLE:Reduci
ngsugar
shav
eei
theraf
reeal
dehy
deorket
onegr
oupasapat
hof
mol
ecul
arst
ruct
ure.Thi
sfr
eeorpot
ent
ialf
reegr
oupofal
dehy
deorket
onei
samonoor
di
sacchar
idei
sreadi
l
yoxi
dizedbymi
l
kyal
kal
i
nesol
uti
onofcupr
ici
onsofBenedi
ct’
s
r
eagent educedCu2+ t
.Theyr oCu2Obyal
l
owi
ngt
hebr
ickr
edpr
eci
pit
ate.Car
bohy
drat
e
t
hatcanr
educeoxi
dat
ionar
ecal
l
edr
educi
ngsugar
s.Al
lmonosacchar
idesal
mostar
e
r
educi
ngsugar
sli
keLact
oseandMal
tosear
ereduci
ngbutsucr
osei
snot
.Benedi
ctt
esti
s
asi
mpl
etestf
orr
educi
ngsugar
.
REAGENT:Di
ssol
ve100gmsofNa2cl
3and173gm ofsodi
um ci
tr
atedehy
drat
einf
inal
v
olumeof850mlofH2Osl
owl
ywi
thst
ri
ng.Addasol
uti
onof17.
3gmsofCuSO4 pent
a
hy
drat
ein100ml
ofl
i
trewat
er.Br
ingt
hev
olumet
o1l
i
tre.
PROCEDURE:Per
for
mthet
eston1%Car
bohy
drat
esol
uti
oni
nthet
estt
ube. Add2mlof
car
bohy
drat
eand5mlofBenedi
ct’
sreagentandshakeeacht
estt
ube.Pl
acei
tinwat
er
bat
hfor5-
6mi
nut
es.Add2-
3dr
opsof3M Hclt
o2mlof1%sucr
osesol
uti
onandst
arch
sol
uti
on(
1%)separ
atel
yandheat
edi
nboi
l
ingwat
erbat
hfor5mi
nut
esf
orsucr
oseand30
mi
nut
esperst
arch.Per
for
m Benedi
ct’
stestandnot
ethecol
ourchange.Compar
eyour
r
esul
tswi
thandwi
thoutt
reat
mentandr
ecor
dtheobser
vat
ion.

BARFOED'
STEST

PRI
NCI
PLE:Thi
sreagenti
sweakl
yaci
dicand i
sonl
yreduced bymonosacchar
ides.
Pr
olongedboi
l
ingmayhy
drol
yzedi
sacchar
idest
ogi
vef
alseposi
ti
ver
eact
ions.Col
ourof

BT8261Bi
ochemi
str
yLabor
ator
y 16/16
Cu2Oi
smor
ebr
ickr
edt
hant
heor
angebr
ownobt
ainedwi
thBenedi
ctst
est
.
METHOD:1mloft
estSol
uti
oni
saddedwi
th2mlofBar
foed'
sreagentboi
l
edf
or1mi
n.
Br
ickr
edcol
ouri
sobser
vedwi
thmonosacchar
ides.Pol
ysacchar
idesanswert
hist
estonl
y
af
terhy
drol
ysi
s.

BI
AL’
STEST

PRI
NCI
PLE:Whenpent
osesar
eheat
edwi
thcon.HCl
,fur
fur
ali
sfor
medwhi
chcondenses
wi
thr
esor
cinol
int
hepr
esenceoff
err
ici
onst
ogi
veabl
uegr
eencol
our
.
METHOD:To1mloft
estSol
uti
on,add2-
5mloft
her
eagentandheati
ttoboi
l
ing.Abl
ue
gr
eencol
ouri
ndi
cat
est
hepr
esenceofpent
ose.

SELI
WANOFF'
STEST

PRI
NCI
PLE:Ket
osear
edehy
drat
edmor
erapi
dlyt
hanal
dosest
ogi
veaf
urf
uralder
ivat
ive
whi
cht
hencondensewi
thr
esor
cinolt
ofor
m ar
edcompl
ex,pr
olongedheat
ingmustbe
av
oided.
METHOD:1mloft
estSol
uti
oni
saddedwi
th2mloft
hisr
eagentt
hecont
ent
sar
eboi
l
edi
n
awat
erbat
hfor1mi
n.Ther
eact
ioni
sgi
venbyf
ruct
ose,Sucr
oseandot
herf
ruct
ose
cont
aini
ngcar
bohy
drat
es.
TESTFORSUCROSE:Sucr
osei
stheonl
ycommonnon-
reduci
ngdi
ssacchar
ide.Sot
hati
t
doesnotr
educeonal
kal
i
nesol
uti
onorf
orm anosazone.Ther
efor
eiti
shy
drol
ysedi
naci
d
Sol
uti
ont
ogl
ucoseandf
ruct
ose,
whi
char
ethent
est
ed.
HYDROLYSI
SOFSUCROSE:5ml
ofSucr
osei
stakenand2ml
ofcon.HCl
isaddedheat
ed
f
or5mi
ninboi
l
ingwat
er.Thensol
i
dNa2CO3i
saddedt
il
ltheef
fer
vescencecases.Then
t
her
educt
iont
estandt
heSel
i
wanof
f'
stestar
eper
for
medt
othehy
drol
ysedSol
uti
on.

I
ODI
NETEST
PRI
NCI
PLE:TheuseofLugol
’
siodi
ner
eagenti
susef
ult
odi
sti
ngui
shst
archandgl
ycogen.
pol
ysacchar
idesr
eact
swi
thI
odi
nesol
uti
onandmonosachhar
idesy
iel
dsnocol
ourwi
th
I
odi
ne.Thet
estsol
uti
onr
emai
nchar
act
eri
sti
cbr
owny
ell
owcol
our
.St
archandgl
ycogen
consi
stsofbr
anchesar
emadeupofl
i
nearchai
nsofgl
ucoseuni
tswhi
char
eint
hef
orm
ofhel
i
cal
coi
l
s.I
odi
neat
om f
it
sint
ohel
i
cesandst
arch-
iodi
neorgl
ycogeni
odi
necompl
ex.
Compl
exst
archi
nthef
orm ofamy
lase3-amy
lopect
inhasf
ewerbutl
owerbr
ancehest
han
gl
ycogenaccommodat
ingmor
eiodi
neat
om i
nabr
anch.Thi
saccount
sfort
hedi
ff
erence
i
ncol
ouri
niodi
neagent
.
LUGOL’
SIODI
NEREAGENT: I
tist
ranspar
entbr
ownl
i
qui
dconsi
sti
ngof10par
tsofKI

17/
17
t
o5par
tsofi
odi
net
o8.
5mlofdi
sti
l
ledwat
er.Whencompl
etel
ydi
ssol
ved,adddei
oni
sed
0.
5gofi
odi
necr
yst
als.Wheni
odi
nei
scompl
etel
ydi
ssol
vedanddei
oni
sedwat
ert
ofi
nal
v
olume(
25ml
)
PROCEDURE:Add2-
3dr
opsofLugol
’
siodi
nesol
uti
ont
o2.
5mlofsapl
etobet
est
ed.
St
archgi
vesapur
plecol
our
.Br
owni
shbl
uecol
ouri
sposi
ti
vet
estf
orgl
ycogen.Br
owni
sh
y
ell
owcol
ouri
nnegat
ive

TABULATI
ON
Mar
k(+)si
gnf
orposi
ti
ver
eact
ionsand(
-)si
gnf
ornegat
iver
eact
ion

est
est

est
est

st

est
st

st

f'
net

st
oed'
ct

wanof
Benedi

al'
odi

Car
bohy
drat
e

Bi
Bar

i
I

Sel
Gl
ucose
Fr
uct
ose
St
arch
Sucr
ose
Mal
tose

DI
STI
NGUI
SHBETWEENREDUCI
NGANDNON-
REDUCI
NGSUGAR

PRI
NCI
PLE
Thecupr
icorcopperpr
esenti
nal
kal
i
necoppersul
phat
esol
uti
oni
sreducedt
ocopper
hy
drox
idewhi
chi
sunst
abl
eandf
ormsCu2O whi
chappear
sasacol
our
edpr
eci
pit
ate.
Sucr
osei
snon-r
educi
ngsugaronaci
dhy
drol
ysi
sfor
msami
xtur
eofr
educi
ngsugar
.

PROCEDURE
1.Take2-
3ml
ofunknownsugarsol
uti
on.

2.Thenadd2ml
ofBenedi
ct’
sreagent
.

3.Boi
litf
or5mi
nut
esi
naboi
l
ingwat
erbat
handt
hencooli
tatr
oom
t
emper
atur
e.

BT8261Bi
ochemi
str
yLabor
ator
y 18/18
 4.Redpr
eci
pit
atei
sfor
medwhi
chi
ndi
cat
est
hepr
esenceofr
educi
ngsugar
.

DI
STI
NGUI
SHBETWEENALDOSEANDKETOSESUGAR

PRI
NCI
PLE
HotHclr
eact
swi
thket
oset
ofor
m dehy
drat
edcompoundst
hatr
eact
swi
thr
esor
cinolt
hat
condenseswi
thSel
i
vanof
f’
sreagentt
ocausedar
kandr
eddi
shbr
owncol
ourket
oses,
from
5hy
droxymet
hylf
urf
urolwhi
chr
eact
swi
thr
esor
cinolt
ogi
vecol
ourwi
thi
ntwomi
nut
es.
Al
so,
hexosesr
eact
swi
tht
hesamet
ofor
mthepr
oductbutsl
owl
y.

PROCEDURE
Pl
ace2-
3dr
opsof1%car
bohy
drat
esol
uti
ont
obet
est
edi
nasepar
atet
estt
ube.
Add1mlofsel
i
wanof
f’
sreagentandl
eav
eiti
nawat
erbat
hfor1mi
nut
e. Recor
dthe
col
oury
ouobser
vei
neacht
estt
ubeatt
heendof1mi
nut
eandf
ourmi
nut
es.
Ket
ohexoses sel
i
wanof
f’
sreagentCher
ryr
edcol
our
Ket
ohexoses sel
i
wanof
f’
sreagentBl
uishgr
eencol
our
Ket
ohexoses sel
i
wanof
f’
sreagent Pi
nkcol
oursl
owl
yappear
s
Di
sacchar
ides sel
i
wanof
f’
sreagent Nocol
ourchange

19/
19
RESULT
i
) Sugarpr
esenti
nthesampl
ewas_
___
___
_.
i
i
) Redpr
eci
pit
atei
sfor
medt
hati
ndi
cat
est
hepr
esenceofr
educi
ngsugar
.
i
i
i) Col
ourobser
vedi
ndi
cat
ethepr
esenceofal
dhoseorket
ose

Expt
.No:
6
QUANTI
TATI
VEMETHODFORAMINOACI
DESTI
MATIONUSING
NI
NHYDRI
N–DISTI
NGUI
SHINGAMI
NOFROM I
MINOACID
Dat
e:

AI
M
Todi
sti
ngui
shbet
weenami
noandi
minoaci
dusi
ngNi
nhy
dri
n

PRI
NCI
PLE
Ni
nhy
dri
nact
sasapower
fuloxi
diz
ingagent
.Itcausesoxi
dat
ivedecar
boxy
lat
ionand
deami
nat
ionofami
noaci
dpr
oducesCO2,ammoni
aandal
dehy
de.I
tget
sreduced.Then
t
her
educedf
orm ofni
nhy
dri
nreactwi
thonemol
ecul
eofni
nhy
dri
ntoy
iel
dRuhemas’
s
pur
plecol
our
edcompl
ex.Howev
erPr
oli
neandhy
droxy
lwi
l
lgi
vey
ell
owcol
ourwhi
chhav
e
t
hesecondar
yami
nogr
oup.

PROCEDURE
1.Take2-
5ml
ofami
noaci
dsol
uti
oni
nacl
eangl
asst
estt
ube.

2.Addf
ewdr
opsofconcent
rat
edni
nhy
dri
n(1mgof1000ml
)

3.Heatt
het
estt
ubeat100˚
cforaf
ewseconds.

BT8261Bi
ochemi
str
yLabor
ator
y 20/20
RESULT
Ami
noaci
dappear
sPur
plewhi
l
eimi
noaci
dappear
syel
l
owi
ncol
our
.

Expt
.No:
7
PROTEI
NESTI
MATI
ONBYBI
URETANDLOWRY’
SMETHODS
Dat
e:

AI
M
Toest
imat
etheamountofpr
otei
ningi
vensampl
ebyBi
uretmet
hod.

PRI
NCI
PLE
Compoundcont
aini
ngt
woormor
epept
idebondt
akechar
act
eri
sti
cpur
plecol
ourwhen
t
reat
edwi
thcoppersul
phat
einal
kal
i
nemedi
um.Thepur
plecol
ourcompl
exi
sduet
othe
f
ormat
ionofco-
ordi
nat
ioncompl
exbycopperat
omswi
thni
tr
ogenat
om f
oreachoft
he
t
owpept
idechai
nsandf
ort
hef
ormat
ionofcol
our
edcompl
exabout1-
25mgofpept
idei
s
r
equi
red.Thepupl
ecol
ouri
scol
ori
met
ri
cal
l
yreadat540nm.

REAGENTSREQUI
RED
I
. STOCKSOLUTI
ON(
BSA)SOLUTI
ON:

Di
ssol
ve5gofBSAandmakeupt
hev
olumet
o100mlwi
thdi
sti
l
ledwat
er.
Theconcent
rat
ioni
s5g/
100ml
or50mg/
ml.
I
I. WORKI
NGSTANDARDSOLUTI
ON:

10mlofst
ockst
andar
dsol
uti
oni
sdi
l
utedt
o100mlwi
thdi
sti
l
ledwat
er.
Theconcent
rat
ioni
s5mg/
ml.
I
II
. BI
URETREAGENT:

Toast
andar
dfl
ask,Di
ssol
ve1.
5gofCuS04 and6gofNak-t
art
arat
ein
about500mlofdi
sti
l
ledwat
er. Tot
hisadd300mlof10% NaOH.
Makeupt
hev
olumeupt
othemar
kinast
andar
dfl
ask.

21/
21
PROCEDURE
i
. 0.
2mloft
hewor
kingst
andar
dsol
uti
oni
spi
pet
teoutwi
thconcent
rat
ionof
20-
100µg/
mli
ntoaser
iesoft
estt
ubesl
abel
edasS1t
oS5.

i
i
. Makeupt
hegi
venunknownsampl
ewi
thdi
sti
l
ledwat
erf
rom t
hat0.
2mland
0.
4ml
unknownsol
uti
onar
etakeni
ntwot
estt
ubel
abel
edT1andT2.

i
i
i. Makeupt
hev
olumeofal
ltestt
ubest
o1ml
usi
ngdi
sti
l
ledwat
er.

i
v. 1ml
ofdi
sti
l
ledwat
eri
sser
vedasbl
ankandl
abel
l
edasB.

v
. Pi
pet
teout5mlofBi
uretr
eagenti
ntocel
l
soft
het
estt
ubesi
ncl
udi
ngbl
ank
andi
ncubat
ingat67˚
cfor10mi
n.

v
i. Thepur
plecol
ourdev
elopedwasmeasur
edcol
ori
met
ri
cal
l
yat540nm

v
ii
. Thei
ntensi
tyoft
hecol
ourf
ormedi
sdi
rect
lypr
opor
ti
onalt
otheamountof
pr
otei
npr
esenti
nit
.

OBSERVATI
ON
TEST VOLUME OF CONCN. OF DI
STI
LLED BI
URET OD
TUBE PROTEI
N PROTEI
N H2O REAGENT 540nm
WORKI
NG X(
µg) (
ml)
STANDARD
(
ml)

BT8261Bi
ochemi
str
yLabor
ator
y 22/22
RESULT
Theamountofpr
otei
npr
esenti
n200ml
oft
hegi
vensampl
eis_
___
___
___
__
AI
M
Toest
imat
etheamountofpr
otei
nbyLowr
y’smet
hod.

PRI
NCI
PLE
Pr
otei
nswhent
reat
edwi
thal
kal
i
necoppersul
phat
ereagentl
eadst
othef
ormat
ionof
Cu2+i
onsf
rom compl
exwi
thpr
otei
ntoal
kal
i
nesol
uti
on.Thecompl
exi weenCu2+i
sbet ons
f
rom compl
exwi
thpr
otei
ntoal
kal
i
nesol
uti
on.Thecompl
exi weenCu2+ andf
sbet our
ni
tr
ogenat
oms,t
wof
rom eachoft
headj
acentpept
idechai
ns.Thet
etr
aami
necopper
compl
exr
educesFol
i
n’sr
eagentbyt
ransf
erofel
ect
ront
ophosphomol
ybol
i
caci
d.
Theaddi
ti
onoff
oli
nphenolr
eagentr
esul
tsi
nfor
mat
ionofi
ntensebl
uecol
ourt
hrought
he
r
educt
ionofmol
ybdat
e.
Mo6+ Mo4+
Ther
educt
ioni
sbr
oughtbyt
rypt
ophanandt
yroxi
ner
esi
duesi
npr
otei
n.

REAGENT’
SREQUI
RED
STANDARDPROTEI
NSOLUTI
ON:Wei
gh100mgBSA(
Bov
ineSer
um Al
bumi
n),di
ssol
vei
t
andmakeupt
o100ml
usi
ngdi
sti
l
ledwat
er.
WORKI
NG STANDARD SOLUTI
ON:10mlofst
ocksol
uti
on,st
andar
dpr
otei
nsol
uti
oni
s
di
ssol
vedi
n100ml
ofdi
sti
l
ledwat
er.

OBSERVATI
ON
S.
NO REAGENTS B S1 S2 S3 S4 S5 T1 T2

23/
23
 I
NCUBATI
ONATROOM TEMPERATUREFOR10MI
NUTES

I
NCUBATI
ONATROOM TEMPERATUREFOR30MI
NUTES

ALKALI
NECOPPERSULPHATEREAGENT:
ReagentA:
1%CuSo4
ReagentB:2%Sodi
um pot
assi
um t
art
arat
e
ReagentC:2%Na2CO3i
n0.
1NNaOH
98mlofr
eagentC,1mlofr
eagentAand1mlofr
eagentBwer
emi
xedf
orusei
susedas
Al
kal
i
neCuSo4r
eagent
.
FOLI
N’SPHENOLREAGENT:Ther
eagenti
sdi
utedi
nther
ati
oof1:
2wi
thdi
sti
l
ledwat
er
f
reshl
yjustbef
oreuse.

PROCEDURE
Thest
andar
dpr
otei
nsol
uti
onofconcent
rat
ion0.
2mlt
o1mlwaspi
pet
teouti
ntot
est
t
ubesl
abel
l
edasS1t
oS5.Vol
umesi
nal
lthet
estt
ubeswasmadeupt
o1ml
usi
ngdi
sti
l
led
wat
er.Oneoft
heset
estt
ubeshad1ml
ofwat
eral
oneandser
vesasbl
ank.
4.
5mlofal
kal
i
necoppersul
phat
ereagenti
saddedt
oal
ltubesandmi
xwel
l
.Thet
est
t
ubesar
eincubat
edatr
oom t
emper
atur
efor10mi
n.0.
5mlofdi
l
utedf
oli
n’
sphenolr
eagent
wasaddedt
oal
ltestt
ubes,
mixedwel
landi
ncubat
edatr
oom t
emper
atur
efor30mi
nut
es.
Thei
ntensi
tyofbl
uecol
ourdev
elopedi
smeasur
edat640nm. Thest
andar
dgr
aphi
s
cal
cul
atedt
aki
ngconcent
rat
iononx-
axi
sandopt
ical
densi
tyony
-axi
s.

BT8261Bi
ochemi
str
yLabor
ator
y 24/24
RESULT
Theamountofpr
otei
npr
esenti
n100ml
ofunknownsol
uti
onwasf
oundt
obe_
___
___
__

Expt
.No:
8 PROTEI
NESTI
MATI
ONBYBRADFORDAND
SPECTROSCOPI
CMETHODS
Dat
e:

AI
M
Toest
imat
etheamountofpr
otei
npr
esenti
ntheunknownsampl
ebyBr
adf
ord’
smet
hod.

PRI
NCI
PLE
Pr
otei
ninv
olv
esi
nnon-
cov
alentbi
ndi
ngoft
hedy
ecoomassi
ebr
il
li
antbl
uewhi
chi
san
aci
dicdy
e,bi
ndst
obasi
car
omat
icami
noaci
dspr
esenti
nthepr
otei
ns.Thedy
eispal
e
r
edi
ncol
ourandt
ransf
ormst
obl
uewheni
tbi
ndswi
thpr
otei
n.Thebl
uecol
ourdev
eloped
i
scol
ori
met
ri
cal
l
ymeasur
edat550nm.

REAGENTSREQUI
RED
1.STOCKSTANDARD BSA:Di
ssol
ve100mgofBSAandmakei
tupt
o100mlwi
th
phosphat
ebuf
fersal
i
nesol
uti
on.Theconcent
rat
ioni
s1mg/
ml.

2.WORKI
NG STANDARD:10mlofst
ocksol
uti
oni
sdi
l
utedt
o100mli
nphosphat
e
buf
fersal
i
nesol
uti
on,
concent
rat
ioni
s100µg/
ml

3.COOMASSI
EBRI
LLI
ANTBLUE:Di
ssol
ve100mgofcoomassi
ebr
il
li
antbl
uei
n50ml
of95% et
hanol
,100mlofconcent
rat
edor
thophosphor
icaci
dandt
hev
olumei
s
madeupt
o200ml
indi
sti
l
ledwat
erandst
oredi
namberbot
tl
es.

4.COOMASSI
EBRI
LLI
ANTBLUEDI
LUTI
ON:Mi
x1v
olumeofconcent
rat
eddy
eint
o4

25/
25
 v
olumesofdi
sti
l
ledwat
er.

OBSERVATI
ON
Test Vol
ume Concent
rat
io Vol
ume Vol
ume Vol
ume Opt
ical
Tubes of nofWor
king of e Mi
of H2O of Dy xthe densi
ty
Wor
king St
andar
d unknown (
ml) (
ml) cont
ent
St
andar
d (
ml) sol
uti
on wel
l
(
ml) (
ml) and
keep
t
het
est
t
ube
atr
oom
t
emp.
(
10mi
n)

PROCEDURE
Pi
pet
teout0.
2–1mlofwor
kingst
andar
dinconcent
rat
ionof20-
100µgi
naser
iesoft
est
t
ubes.Makeupt
hev
olumeupt
o4mli
ndi
sti
l
ledwat
er.0.
4mlof
giv
enunknownsol
uti
oni
s
al
so madeupt
o 4ml
. 1mlofdy
eisaddedt
o al
lthet
ubesandar
ekeptatr
oom
t
emper
atur
efor10mi
nut
esaf
termi
xi
ng.Measur
ethebl
uecol
ourdev
elopedi
sdi
rect
ly
pr
opor
ti
onalt
o amountofpr
otei
npr
esenti
nit
. Dr
aw ast
andar
d gr
aphbypl
ott
ing
concent
rat
ionofpr
otei
n(X-
axi
s)Vsopt
ical
densi
ty(
Y-axi
s)

BT8261Bi
ochemi
str
yLabor
ator
y 26/26
RESULT
Theamountofpr
otei
npr
esenti
ntheunknownsampl
eis_
___
___
___
__

Expt
.No:
9
EXTRACTI
ONOFLI
PIDSANDANALYSI
SBYTLC.
Dat
e:

AI
M
Tosepar
ateandi
dent
if
ythel
i
pidsi
neggy
olkbyt
hinl
ayerchr
omat
ogr
aphy
.

MATERI
ALSREQUI
RED
 TLCPl
ate

 TLCchamber

 Fi
l
terpaper

 Capi
l
lar
ytubes

 Eggy
olk

 Chl
orof
orm

 Met
hanol
mixt
ure(
2:1)

 Chl
orof
orm met
hanol
acet
icaci
dmi
xtur
e(25:
15:
4)

 Sol
ventt
ank

PRI
NCI
PLE

27/
27
Thei
mpel
l
ingf
orceofmobi
l
epHcar
ri
erwi
thi
tthel
i
pidmol
ecul
eforwhi
chi
thasaf
fi
nit
yis
f
avour
edbysol
ubi
l
ityofl
i
pidmol
ecul
esi
nmobi
l
ephase.Ther
etar
dingf
orceatst
ati
onar
y
phasehol
dbackt
hel
i
pidmol
ecul
eswi
thwhi
chi
tint
eract
s.Thebal
ancebet
weent
het
wo
f
orcesdi
ff
ersf
ort
hedi
ff
erentmol
ecul
eswhi
chi
mpl
i
esdi
ff
erentmobi
l
iti
esandhencet
he
separ
ati
onofmol
ecul
es,
Di
sta
ncemove
dbya
na l
yte
Rf=
Di
sta
ncemove
dbys
olvent

Rfv
aluesar
eusedt
ocompar
etheunknownsampl
ewi
thknownst
andar
dandi
dent
if
y
mol
ecul
epr
esenti
ngi
vensampl
eRfv
aluesdi
ff
erf
orv
ari
oussol
ventmi
xtur
esused.Li
pids
i
nbi
ologi
calmat
eri
alsi
spr
esentascompl
exmi
xtur
e,underf
ract
ionat
ion,
itsepar
atesi
nto
numberbyagr
oupofsol
venti
nter
act
ion.
Yol
kisar
ichsour
ceofv
ari
etyofi
mpor
tantcompoundl
i
kepr
otei
n,l
i
pids,gl
ycer
ide,
chol
est
erol
,phospol
i
piset
c. Eggl
i
pidsbel
ongt
otwomaj
orcl
asses,phosophol
i
pids
bel
ongt
otwomaj
orcl
asses,
phosphol
i
pidsandnonphosophol
i
pids.
Resol
uti
onofcompoundscanbecar
ri
edoutusi
ngt
heTLCsol
venti
ssel
ect
edaccor
ding
t
othel
i
pidmembr
anei
nter
act
ionunderi
nvest
igat
ionbutgener
all
yli
pidsar
esepar
ated
wi
thnonpol
arsol
vent
s.Anumberofsepar
ati
ngmedi
acanbeusedandact
ualchoi
ceof
adsor
bentdependsongr
oupofl
i
pidsandsol
ventempl
oyedsi
l
icai
swel
lknownadsor
bent
whi
chhassi
l
icahy
droxi
degr
ouponi
tssur
faceandwi
del
yusedi
nthesepar
ati
onofl
i
pids.
The basi
s ofsepar
ati
on i
s adsor
pti
on and par
ti
ti
on. The si
l
ica has abi
l
ityt
o hol
d
mol
ecul
esoni
tssur
face.
Theadsor
pti
onpr
ocessi
nvol
veswor
kofweak– noni
oni
cfor
ces,at
tract
ionf
orceof
hy
drogenbondi
ngf
orces,
att
ract
ionofweakbondi
ngf
orces.Noni
oni
cfor
cesonsi
l
icagel
pl
ates,asmobi
l
ephase mov
esacr
ossoneedget
oopposi
tesi
de.I
ttr
ansf
eranyanal
yte
pl
acedont
hel
ayeratt
her
atedet
ermi
nedbydi
stur
bingco-
eff
ectbet
weenst
ati
onar
yand
mobi
l
ephases.Anal
ytemov
ement
sceaseswhenpl
atei
sremov
edf
rom mobi
l
ephase
r
eser
voi
r.Themeasur
eofanal
ytei
sexpr
essedbyRJ.

PROCEDURE
SAMPLEPREPARATI
ON:Take2mlofy
olkandadd6mlof1% Nacli
naependor
ftube.
Shakewel
landcent
ri
fugeat500r
pm f
or5mi
n.Di
scar
dthel
owerl
ayer
,col
l
ect2mlof
cl
earsuper
nat
anti
n anot
hert
ube and add 2mlof1% NaCland 2mlofmet
hanol
chol
orof
orm mi
xtur
eandmi
xwel
lcent
ri
fugeat500r
pm f
or5mi
n.Di
scar
dtheupperphase
car
eful
l
yanduset
hel
owerphasef
orexper
iment
.Spott
hel
owerphaseator
igi
n(i
.e)1cm

BT8261Bi
ochemi
str
yLabor
ator
y 28/28
f
rom t
hebot
tom ofTLCusi
ngcapi
l
lar
ytube

Di
sta
ncemove
dbya
na l
yte
Rf=
Di
sta
ncemove
dbys
olvent

Keept
hepl
atesi
nthesol
ventt
ankcont
aini
ngchl
orof
orm met
hanolacet
icaci
dmi
xtur
eas
mobi
l
ephasesat
urat
e,t
het
ankusi
ngaf
il
terpaperdi
ppedi
nsol
ventmi
xtur
etoav
oid
i
rr
egul
arr
un.Af
tert
her
uni
sov
erl
ocat
edt
hespotusi
ngi
odi
nev
apour
.Not
ethatRJv
alue
shoul
dnotbegr
eat
ert
han1Measur
ethesol
ventf
rontandmeasur
ethesubst
ancesof
v
ari
ousspotf
rom t
heor
igi
nandcal
cul
ateRfv
alue.
Rfv
alueofv
ari
ouscomponent
sar
eappr
oxi
mat
edas0.
9forphosphot
idy
let
hanolami
ne,
0.
5forphosphot
idy
lchol
i
ne,0.
2forsphi
ngomy
li
n0.
1forl
ysophosphat
idy
lchol
i
neasper
t
hest
andar
ds.Somet
imesphosphot
idy
lchol
i
necannotbedet
ect
ed,
butseemst
omov
e.

RESULT
Thust
hev
ari
ousl
i
pidswer
esepar
atedandRfv
aluewasnot
edf
oreachl
i
pidusi
ngThi
n
l
ayerchr
omat
ogr
aphy
.

Expt
.No:
10 ESTI
MATI
ONOFNUCLEI
CACI
DSBYABSORBANCEAT260NM
ANDHYPERCHROMI
CEFFECT(
DEMO)
Dat
e:

AI
M
Toest
imat
etheamountofDNApr
esenti
nthegi
venunknownsol
uti
onbydi
pheny
lami
ne
met
hod.

PRI
NCI
PLE
WhenDNAi
str
eat
edwi
thdi
pheny
lami
neundert
heaci
diccondi
ti
onabl
uishgr
eencol
ored
compl
exi
sfor
medwhi
chhasanabsor
pti
onpeakat595nm.
Thi
sreact
ioni
sgi
venby2
deoxy
pent
osei
ngener
al.I
naci
dicsol
uti
ondeoxy
pent
osear
econv
ert
edi
ntoahi
ghl
y
r
eact
iveβhy
droxy
lleav
uli
nical
dehy
dewhi
chr
eact
swi
thdi
pheny
lami
negi
vesbl
uishgr
een
col
oredcompl
ex.Thecol
ouri
ntensi
tywasmeasur
edusi
ngar
edf
il
terat595nm.

REAGENTREQUI
RED
1.St
ockSt
andar
dSol
uti
on:
50mgofDNAwasdi
ssol
vedi
n50ml
ofSal
i
neSodi
um ci
tr
ate
buf
fer
.Concent
rat
ion1mg/
ml

29/
29
2.
Wor
kingSt
andar
dSol
uti
on:
5ml
ofst
ocksol
uti
onsol
uti
onwasdi
l
utedt
o50ml
wit
h
di
sti
l
ledwat
er.Concent
rat
ion100µg\
ml

3.Di
pheny
lami
neReagent
:10gofpur
edi
pheny
lami
newasdi
ssol
vedwi
th25ml
of
concent
rat
ionsul
phur
icaci
dwhi
chwasmadeupt
o1ml
wit
hgl
aci
alaci
dicaci
dthe
sol
uti
onmustbepr
epar
edf
reshl
y.

4.Buf
fer
edSal
i
neph7.
4:0.
14NSodi
um chl
ori
deand0.
02M sodi
um ci
tr
ate.

5.UnknownSol
uti
on:
Thegi
venunknownsol
uti
oni
smadeupt
o100ml
wit
hdi
sti
l
ledwat
er.

PROCEDURE
0.
5-2.
5mlofwor
kingst
andar
dsol
uti
oni
spi
pet
tedouti
nto5t
estt
ubesl
abel
edas s1-
s5
wher
econcent
rat
ionr
angi
ngf
rom 50-
250µg.
1ml
and2ml
ofunknownsol
uti
oni
spi
pet
tedouti
ntot
wot
estt
ubeu1andu2.
Thev
olumei
nal
ltestt
ubesi
smadeupt
o3mlwi
thdi
sti
l
ledwat
erand3mlofdi
sti
l
led
wat
eral
oneser
veasabl
ank.
4mlofdi
pheny
lami
ner
eagentwasaddedt
oal
lthet
ubes.Thet
ubeswer
ekepti
na
boi
l
ingwat
erbat
hat36°
Cfor20mi
n.Thet
ubeswer
ethancool
edandt
hebl
uish
col
ourdev
elopedi
sreadat595nm.
Ast
andar
dgr
aphi
sdr
awnt
aki
ngconcent
rat
ionofDNAonx-
axi
sandabsor
pti
onofy
-
axi
s.Fr
om t
hest
andar
dgr
apht
heamountofDNApr
esenti
ntheunknownsol
uti
on
i
scal
cul
ated.

BT8261Bi
ochemi
str
yLabor
ator
y 30/30
RESULT
TheamountofDNApr
esenti
nthegi
venunknownsol
uti
oni
sfoundt
obe

Expt.
No:11 ENZYMATI
CASSAY:
PHOSPHATASEFROM POTATO
Dat
e:

AIM
Todeter
minet
heaci
dphosphat
aseact
ivi
tyi
npot
atoesusi
ngSodi
um-
β-Gl
ycer
ophosphat
e
asSubst
rat
e

PRINCIPLE
Phosphatases liberat
e inor
gani
c phosphate fr
om or ganic phosphate ester. Acid
phosphatasehy drol
asesanumberofphosphomonoest ersandphosphorpr oteins.Acid
phosphatasescanbeassay edbymeasur
ingtheamountofi norgani
cphosphor
usr eleased
fr
om Sodi um-β-Glycer
ophosphat
e.The phosphorous r
eleased is est
imat
ed by Fi ske
Subbaraomet hod.

REAGENTS

1.Ci
tr
atebuf
fer(
pH5.
6):

1M Ci
tr
icaci
d:Di
ssol
ve5.
25gmsofci
tr
icaci
dindi
sti
l
ledwat
erandmakeupt
o250ml

31/
31
wit
hdisti
ll
edwater
1M tr
isodi
um cit
rat
e:Dissol
ve14.7gmsoftrisodi
um cit
rat
eindist
il
ledwat
erandmakeup
to500ml
Mix137ml ofci
tr
icacidwit
h363ml oft
ri
sodium cit
rat
esoluti
onandsetuppH5.6buff
er
sol
uti
ons.
(0.
02M Sodium-
β-Glycer
ophosphat
einci
tr
atebuf f
er.
)

2.10%TCA(
Tri
Chl
oroacet
icaci
d)

ENZYMEEXTRACT
Cleanandwasht hepotat
oeswi thdist
il
ledwaterandpeelouttheski
n.Wei
ghabout
200gmsbyusi ngroughbalance.Cutitint
osmal lpi
ecesthenadd200mlofi
cecold
di
still
edwaterandhomogeni zedinagr i
nder.Fi
lt
erthehomogenat
eusingacl
othand
storedthef
il
tr
ateinaref
ri
gerator
.

PROCEDURE
Take5mlofBuf f
ersubst ratecleananddr yt
hetesttubethenpr ei
ncubatefor30mi nut
es
0
at37C.Att heendoft hei ncubati
onperiodadd2.5ml of10%TCAandmi xwell
.Keepthe
testtubesf
oranotherminut eatroom temperat
ureandf i
l
terthecontents.Take1mlofthe
fi
ltr
atefort
hedenaturati
onofi norgani
cphosphorousbyFiske-Subbar
aomet hod

Setupacontr
olsi
mul
taneousl
ybyaddi
ngmlofTCAt
o5mlofbuf
fersubst
rat
efol
l
owed
bymlofenzy
meandproceedsaspert
est
.

To deter
minet hepr
otein contentoft heenzy
meext r
actbybi
uretmethod.Fort hi
s
experi
ment,t
ake1mlofext ractadds1mlofwat erandadd3mlofbi
uretreagentand
fol
lowsasperthebi
uretest
imat i
onprocedur
e.

BT8261Bi
ochemi
str
yLabor
ator
y 32/32
RESULT
Theamountofinorgani
cphosphorouspresentinthegi
venunknownsampleis_
___
___
___
mgofi nor
gani
cphosphatefor
medper1ml ofenzyme/30mi nut
es.
Expt.
No:12 ENZYMATI CASSAY: ESTIMATI ONOFGLUCOSEBYGOD- POD
METHODAFTERHYDROLYSI SOFSTARCHWI THACI
DAND
Date: SPECIFICI
TYOFTHEENZYMATI CMETHOD

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