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Determination of Riboflavin by Spectrophotometry

M.D. Berry, Partner – A.N. Other, Group A

Sept !"#

$nyro%uction

Ab&orption &pectro&copy i& a u&eful techni'ue (hich allo(& the &tu%y of chemical& in anin tact &y&tem. $n nparticular thi& techni'ue can be u&e% to %etermine the concentration&

of chemical&, or to %etermine the i%entity of an un)no(n chemical *Berry, +#.

-n)no(n chemical& can be i%entifie% &ince the (avelenth& at (hich %ifferent functional

roup& (ithin a chemical ab&orb a particular (avelnth of liht vary. /hi& re&ult& in%ifferent chemical entitie& havin %i&tinct pattern& of ab&orption acro&& a (i%e rane of

(avelentyh&. /hi& pattern of %i&tinct ab&orption& can act a& a uni'ue finerprint allo(in

the i%entification of an un)no(n &ample. /o %etermine the concentration of an )no(n

chemical, a calibration curve of )no(n concentration& mu&t fir&t be prepare%. Such acurve (ill &ho( a linear relation&hip bet(een ab&orption an% concentration over a (i%e

rane of value&. /hi& relation&hip i& iven by the Beer01ambert la( (hich &tate& that theamount of ab&orption i& proportional to the number of ab&orbin molecule& pre&ent.

Mathematically thi& i& iven a&

/he molar e2tinction coefficient i& a con&tant for a %efine% &et of parameter&. /hu&, if the

 path lenth i& )ept con&tant, the ab&orption (ill vary a& a function of chane& in

concentration. $n the current e2periment the path lenth i& hel% con&tant by u&in acuvette of uniform (i%th *3.+4cm.

/he aim of the current e2periment i& to obtain an ab&orption &pectra for a )no(nconcentration of riboflavin, an% from thi& %etermine the ab&orption ma2imum * ma2.

Once the ab&orption ma ha& been %etermine%, the concentration of an un)no(n &olution

of riboflavin (ill be eluci%ate%, by con&truction of a calibration curve at the lamb%a ma2value.

Metho%&

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All proce%ure& (ere %one accor%in to the laboratory manual *Berry, +#. Briefly, an

ab&orption &pectrum for a #mM &olution of riboflavin (a& %etermine% by mea&urin the

ab&orbance at 3nm interval& bet(een 56nm an% 7nm. At each (avelenth the&pectrophotometer (a& calibrate% to 8ero ab&orbance u&in a blan) &olution of .3M

 pho&phate buffer *p9 4..

A calibration curve (a& con&tructe% for riboflavin at concentration& ranin from to

#uM u&in a lamb%a ma2 value of 66#nm. /hree %ilution& of an un)no(n riboflavin

concentration (ere prepare% an% the ab&orbance of each %etermine% a& %e&cribe% above.

Re&ult&

/he ab&orption& &pectrum of riboflavin i& &ho(n in :iure 3. :rom thi& the ab&orption

ma2imum (a& %etermine% to be 66;nm.

A calibration curve for varyin concentration& of riboflavin i& &ho(n in fiure +. :romthi& the un)no(n concentration (a& %etermine% to be 6# =. 9o(ever, thi& probably relate&

to the %ifference& in con%ition& u&e% in the t(o &tu%ie&. =?PAND an%f e2plain.

:rom the calibration curve *fiure + the un)no(n concentration (a& %etermine% to be 8.

/he calculate% lamb%a ma2 ('a&