8.

Espectrofometra: Espectros de absorcin y

cuantificacin colorimtrica de biomolculas
Nieves Abril Daz1, J. Antonio Brcena Ruiz1,
Emilio Fernndez Reyes1, Aurora Galvn Cejudo1,
Jess Jorrn Novo1, Jos Peinado Peinado1,
Fermn Toribio Melndez-Valds1, Isaac Tnez Fiana2

     
  

        
         
    

   &

       
       
      
         

       ! " # $ ! %     

 &
' (

    

      )   * 
 + 
, -     .   ! " # # " %     *

RESUMEN

La espectrofotometra UV-visible es una tcnica analtica que permite

determinar la concentracin de un compuesto en solucin. Se basa en
que las molculas absorben las radiaciones electromagnticas y a su vez
que la cantidad de luz absorbida depende de forma lineal de la
concentracin. Para hacer este tipo de medidas se emplea un
espectrofotmetro, en el que se puede seleccionar la longitud de onda de
la luz que pasa por una solucin y medir la cantidad de luz absorbida por
la misma.
En esta prctica se darn a conocer las caractersticas generales y
conceptos relacionados con la espectrofotometra. A continuacin y tras
la explicacin del funcionamiento del espectrofotmetro se harn
espectros de absorcin con el fin de determinar la longitud de onda
donde la muestra presenta la mxima absorcin, y tras realizar las
correspondientes rectas de calibrado se cuantificarn las
concentraciones de distintas biomolculas.
/ 0 1 0 2 3 0 4 5 1 0 6 7 8 9 : ; < = : > ? @ A > B C = > ? ; D A C > ? @ A > B @ < E < = A D F C = G > B @ = < D H I < = < B

F ; J F @ C = < B = F @ C > ; K F @ > E A : = > K < L

M 2 3 7 6 N 0 O P 3 0 4 7 Q R 1 7 0 S 0 4 L 9 8 9 : ; < = : > ? @ A > B T U 9 V 8 T U 9 D A ? < > ? C A J A = A ? > B W X Y 8

I E > Z A ? D < ? < ? [ @ E F H C A K < B W X Y \ ] 8 I < = D > = F K [ @ A K > K F E I E > Z A ? D < ? < ? [ @ E F H C A K < B ^ 8

A ? C F ? ; A K > K K F E [ _ B Y U Y ` a 9 8 Y U Y > I C < E U b U F C A E F ? K A > D A ? < B J U Y V 8 R U ? A C = < I F ? < E B V c a 8

V F = < d A K > ; > B e 8 C = > ? ; D A C > ? @ A > B f g 8 E [ _ [ E C = > Z A < E F C > L

1. INTRODUCCIN Y OBJETIVOS
` E F ; C [ K A < > ? A Z F E : A < h [ G D A @ < K F @ [ > E h [ A F = : A < D < E i @ [ E > = F h [ A F = F E > [ C A E A _ > @ A H ?

K F C i @ ? A @ > ; > ? > E G C A @ > ; h [ F J F = D A C > ? ; [ K F C F = D A ? > @ A H ? @ [ > E A C > C A Z > j @ [ > ? C A C > C A Z > k

> ; G @ < D < ; [ @ > = > @ C F = A _ > @ A H ? I G ; A @ < U h [ G D A @ > j : A < E H l A @ > L f ? < K F E < ; D i C < K < ;

D m ; ; F ? @ A E E < ; k > @ @ F ; A : E F ; k n C A E F ; j [ C A E A _ > K < ; F ; E > F ; J F @ C = < ; @ < J G > k F ? l F ? F = > E k j

E > F ; J F @ C = < ; @ < J G > [ E C = > Z A < E F C > U Z A ; A : E F k F ? J > = C A @ [ E > = L o F J [ F K F ? A K F ? C A I A @ > = j

@ [ > ? C A I A @ > = : A < D < E i @ [ E > ; F ? ; < E [ @ A H ? j F ? D [ F ; C = > ; : A < E H l A @ > ; k @ < ? F E F D J E F <

p
K F = F > @ C A Z < ; F ; J F @ G I A @ < ; h [ F = F > @ @ A < ? > ? @ < ? F E @ < D J [ F ; C < > > ? > E A _ > = j I < = D > ?

[ ? J = < K [ @ C < @ < E < = F > K < h [ F J F = D A C F K F C F @ C > = E < F ? D [ F ; C = > ; @ < D J E F q > ; L

` E I [ ? K > D F ? C < K F E > F ; J F @ C = < ; @ < J G > ; F K F : F > E > @ > J > @ A K > K K F E > ; D < E i @ [ E > ;

J > = > > : ; < = : F = = > K A > @ A < ? F ; k F ? C = F F E E > ; E > ; = > K A > @ A < ? F ; K F ? C = < K F E F ; J F @ C = < f g U

Z A ; A : E F L r > ; E < ? l A C [ K F ; K F < ? K > K F E > ; = > K A > @ A < ? F ; h [ F [ ? > D < E i @ [ E > J [ F K F

> : ; < = : F = j E > F I A @ A F ? @ A > @ < ? E > h [ F ; F > : ; < = : F ? K F J F ? K F ? K F E > F ; C = [ @ C [ = >

> C H D A @ > j K F E > ; @ < ? K A @ A < ? F ; K F E D F K A < s J \ k C F D J F = > C [ = > k I [ F = _ > A H ? A @ > k

@ < ? ; C > ? C F K A F E i @ C = A @ > t k J < = E < h [ F K A @ u > C i @ ? A @ > @ < ? ; C A C [ j F [ ? Z > E A < ; <

A ? ; C = [ D F ? C < J > = > E > K F C F = D A ? > @ A H ? j @ > = > @ C F = A _ > @ A H ? K F : A < D < E i @ [ E > ; L

r > ; D < E i @ [ E > ; J [ F K F ? > : ; < = : F = F ? F = l G > E [ D A ? < ; > j > E D > @ F ? > = E > F ? I < = D >

K F F ? F = l G > A ? C F = ? > L ` ; C < J F = D A C F J < ? F = F ? I [ ? @ A < ? > D A F ? C < @ A @ E < ; Z A C > E F ; @ < D < E >

I < C < ; G ? C F ; A ; F ? J E > ? C > ; j : > @ C F = A > ; L v [ > ? K < E > E [ _ s @ < ? ; A K F = > K > @ < D < F ? F = l G > t

F ; > : ; < = : A K > J < = [ ? > D < E i @ [ E > ; F < = A l A ? > [ ? ; > E C < K F ; K F [ ? F ; C > K < F ? F = l i C A @ <

: > ; > E < I [ ? K > D F ? C > E k ` w k > [ ? F ; C > K < K F D > j < = F ? F = l G > s F ; C > K < F d @ A C > K < t k ` ] L x

; H E < ; F > : ; < = : F = m E > F ? F = l G > h [ F J F = D A C > F E ; > E C < > E F ; C > K < F d @ A C > K < L v > K >

D < E i @ [ E > C A F ? F [ ? > ; F = A F K F F ; C > K < ; F d @ A C > K < ; s < : > ? K > ; t h [ F E > K A ; C A ? l [ F K F E

= F ; C < K F D < E i @ [ E > ; L v < D < @ < ? ; F @ [ F ? @ A > k E > > : ; < = @ A H ? h [ F > K A ; C A ? C > ;

E < ? l A C [ K F ; K F < ? K > J = F ; F ? C > [ ? > D < E i @ [ E > U F ; C < F ; k ; [ F ; J F @ C = < K F > : ; < = @ A H ? U

@ < ? ; C A C [ j F [ ? > ; F y > K F A K F ? C A K > K K F E > D A ; D > L V < = n E C A D < k E > D < E i @ [ E > F ? I < = D >

F d @ A C > K > E A : F = > E > F ? F = l G > > : ; < = : A K > u > ; C > F E F ; C > K < F ? F = l i C A @ < I [ ? K > D F ? C > E L

Figura 1 Diagrama de niveles de energa en z

una molcula

z

{ |

| }

E2 - E1 = hQ
 ~ | | |  | | ~ ~

~ |  | | |   |  z

 ~  |  | | | ~ |

| ~  ~ | ~  ~ } |

 | z

` ? F ; J F @ C = < ; @ < J G > F E C i = D A ? < E [ _ ? < ; H E < ; F > J E A @ > > E > I < = D > Z A ; A : E F K F

= > K A > @ A H ? F E F @ C = < D > l ? i C A @ > k ; A ? < C > D : A i ? > E > ; I < = D > ; f g F ^ k h [ F ; < ?

A ? Z A ; A : E F ; L ` ? F ; J F @ C < I < C < D F C = G > K F > : ; < = : > ? @ A > ; F [ C A E A _ > ? E > ; = F l A < ? F ; K F E

[ E C = > Z A < E F C > s f g @ F = @ > ? < k K F b U T ? D t j F E Z A ; A : E F s T U ? D t L

400 nm 700 nm

Luz visible
Longitud de onda (metros)

Rayos gama Rayos X ultravioleta infrarrojo microondas

Frecuencia (hertz)

energa (electrn-voltios)

Figura 2.

 ; F K F I A ? F @ < D < F E = > ? l < K F E < ? l A C [ K F ; K F < ? K > K F b > T

? D
La regin UV L ` ; [ ? > = F l A H ? K F F ? F = l G > D [ j > E C > L V = < Z < @ > K > y < > E < q < u [ D > ? < > ; G @ < D <

h [ F D > K [ = > @ < D n ? L r < ; @ < D J [ F ; C < ; @ < ? K < : E F ; F ? E > @ F ; > A ; E > K < ; k C = A J E F ;

F ? E > @ F ; k F ? E > @ F ; J F J C G K A @ < ; k ; A ; C F D > ; > = < D m C A @ < ; k l = [ J < ; @ > = : < ? A E < ; j < C = < ;

u F C F = < m C < D < ; C A F ? F ? ; [ D m d A D > > : ; < = : > ? @ A > F ? E > = F l A H ? f g k J < = E < h [ F i ; C >

F ; D [ j A D J < = C > ? C F J > = > E > K F C F = D A ? > @ A H ? @ [ > E A C > C A Z > j @ [ > ? C A C > C A Z > K F

@ < D J [ F ; C < ; < = l m ? A @ < ; L a A Z F = ; < ; I > @ C < = F ; U @ < D < J \ k @ < ? @ F ? C = > @ A H ? K F ; > E j F E

K A ; < E Z F ? C F U h [ F > E C F = > ? E > @ > = l > K F E > ; D < E i @ [ E > ; k J = < Z < @ > ? K F ; J E > _ > D A F ? C < ;

K F E < ; F ; J F @ C = < ; f g L

r > I [ F ? C F K F = > K A > @ A H ? [ E C = > Z A < E F C > F ; [ ? > E m D J > = > K F K F [ C F = A < L

` ? E > > J = F @ A > D < ; F E @ < E < = Z A ; A : E F K F [ ? > ; < E [ @ A H ? j h [ F

@ < = = F ; J < ?
regin visible K F > E > ; E < ? l A C [ K F ; K F < ? K > K F E [ _ h [ F C = > ? ; D A C F k ? < h [ F > : ; < = : F L ` E

@ < E < = h [ F > : ; < = : F F ; F E @ < D J E F D F ? C > = A < K F E @ < E < = h [ F C = > ? ; D A C F L

V < = C > ? C < k J > = > = F > E A _ > = D F K A @ A < ? F ; K F > : ; < = @ A H ? F ; ? F @ F ; > = A < [ C A E A _ > = E >

E < ? l A C [ K K F < ? K > F ? E > h [ F > : ; < = : F E [ _ E > ; < E [ @ A H ? @ < E < = F > K > L

r > I [ F ? C F K F = > K A > @ A H ? Z A ; A : E F ; [ F E F ; F = [ ? > E m D J > = > K F C [ ? l ; C F ? < j ? <

J = < J < = @ A < ? > ; [ I A @ A F ? C F F ? F = l G > J < = K F : > q < K F ? D L

1.1. Transmitancia y Absorbancia

v [ > ? K < [ ? = > j < K F E [ _ K F [ ? > K F C F = D A ? > K > E < ? l A C [ K K F < ? K >

^ ^

K F A ? C F ? ; A K > K ^ A ? @ A K F J F = J F ? K A @ [ E > = D F ? C F ; < : = F [ ? > K A ; < E [ @ A H ?

K F [ ? @ < D J [ F ; C < h [ G D A @ < h [ F > : ; < = : F E [ _ < k F E

@ < D J [ F ; C < > : ; < = : F = m [ ? > J > = C F K F E > = > K A > @ A H ?

cromforo
A ? @ A K F ? C F s ^ t j

K F q > = m J > ; > = F E = F ; C < s ^ t k K F I < = D > h [ F ; F @ [ D J E F 8 ^ ^ ^

r > s

T
t K F [ ? > ; [ ; C > ? @ A > F ? ; < E [ @ A H ? F ; E > = F E > @ A H ? F ? C = F E >

@ > ? C A K > K K F E [ _ C = > ? ; D A C A K > h [ F E E F l > > E K F C F @ C < = [ ? > Z F _ h [ F u > > C = > Z F ; > K < E >

D [ F ; C = > k ^ j E > @ > ? C A K > K K F E [ _ h [ F A ? @ A K A H ; < : = F F E E > k ^ k j ; F = F J = F ; F ? C >

% T = It/Io x 100
? < = D > E D F ? C F F ? C > ? C < J < = @ A F ? C < 8

r > C = > ? ; D A C > ? @ A > ? < ; K > [ ? > D F K A K > I G ; A @ > K F E > = F E > @ A H ? K F A ? C F ? ; A K > K

A ? @ A K F ? C F j C = > ? ; D A C A K > > E J > ; > = J < = E > D [ F ; C = > L r > = F E > @ A H ? F ? C = F e j E >

@ < ? @ F ? C = > @ A H ? ? < F ; E A ? F > E k J F = < > ; [ D F [ ? > = F E > @ A H ? E < l > = G C D A @ > A ? Z F = ; > L

r > s t F ; [ ? @ < ? @ F J C < D m ; = F E > @ A < ? > K < @ < ? E > D [ F ; C = > J [ F ; C <

h [ F ? <
absorbancia A ; A ? K A @ > E > @ > ? C A K > K K F E [ _ > : ; < = : A K > J < = E > D A ; D > k j ; F K F I A ? F @ < D < F E

A = log 1/T = -log T = -log It/ Io

E < l > = A C D < K F b e k F ? @ < ? ; F @ [ F ? @ A > 8

v [ > ? K < E > A ? C F ? ; A K > K A ? @ A K F ? C F j C = > ? ; D A C A K > ; < ? A l [ > E F ; s ^ < ^ C t k E >

C = > ? ; D A C > ? @ A > F ; K F E b F A ? K A @ > h [ F E > D [ F ; C = > ? < > : ; < = : F > [ ? >

K F C F = D A ? > K > E < ? l A C [ K K F < ? K > k j F ? C < ? @ F ; 9 Z > E F E < l b L

 r > @ > ? C A K > K K F E [ _ > : ; < = : A K > K F J F ? K F = m K F E > K A ; C > ? @ A > h [ F > C = > Z A F ; > E > E [ _ >

C = > Z i ; K F E > ; < E [ @ A H ? K F E @ = < D H I < = < j K F E > @ < ? @ F ? C = > @ A H ? K F i ; C F

1.2. Ley de Lambert-Beer

` ; C > E F j F d J = F ; > E > = F E > @ A H ? F ? C = F > : ; < = : > ? @ A > K F E [ _ D < ? < @ = < D m C A @ > s K F

E < ? l A C [ K K F < ? K > I A q > t j @ < ? @ F ? C = > @ A H ? K F [ ? @ = < D H I < = < F ? ; < E [ @ A H ? 8

A = log I/Io = Hcl

r > > : ; < = : > ? @ A > K F [ ? > ; < E [ @ A H ? F ; K A = F @ C > D F ? C F J = < J < = @ A < ? > E > ; [

@ < ? @ F ? C = > @ A H ? > D > j < = ? n D F = < K F D < E i @ [ E > ; D > j < = A ? C F = > @ @ A H ? K F E > E [ _ @ < ?

F E E > ; U B C > D : A i ? K F J F ? K F K F E > K A ; C > ? @ A > h [ F = F @ < = = F E > E [ _ J < = E > ; < E [ @ A H ? >

A l [ > E @ < ? @ F ? C = > @ A H ? k @ [ > ? C < D > j < = K A ; C > ? @ A > = F @ < = = F E > E [ _ J < = E > D [ F ; C = > D m ;

H
D < E i @ [ E > ; ; F F ? @ < ? C = > = m U B j J < = n E C A D < k K F J F ? K F K F k [ ? > @ < ? ; C > ? C F K F

J = < J < = @ A < ? > E A K > K U K F ? < D A ? > K > @ < F I A @ A F ? C F K F F d C A ? @ A H ? U h [ F F ; F ; J F @ G I A @ > K F

H
@ > K > @ = < D H I < = < L v < D < 9 F ; > K A D F ? ; A < ? > E k E > ; K A D F ? ; A < ? F ; K F K F J F ? K F ? K F

E > ; K F @ j E L r > ; F l [ ? K > D > l ? A C [ K s E t ; F F d J = F ; > ; A F D J = F F ? @ D D A F ? C = > ; h [ F E >

J = A D F = > s @ t ; F u > @ F k ; A F D J = F h [ F ; F > J < ; A : E F k F ? X k @ < ? E < h [ F E > ; K A D F ? ; A < ? F ;

w w

H
K F = F ; [ E C > ? ; F = X @ D L ` ; C F @ < F I A @ A F ? C F > ; G F d J = F ; > K < k F ? C i = D A ? < ; K F

[ ? A K > K F ; K F @ < ? @ F ? C = > @ A H ? D < E > = s < [ ? ; [ : D n E C A J E < > J = < J A > K < t k ; F K F ? < D A ? >

H
@ < F I A @ A F ? C F K F F d C A ? @ A H ? D < E > = s t L v [ > ? K < k J < = K F ; @ < ? < @ F = ; F F E J F ; <

D < E F @ [ E > = K F E ; < E [ C < k E > @ < ? @ F ? C = > @ A H ? K F E > K A ; < E [ @ A H ? ; F F d J = F ; > F ? < C = > ;

H
[ ? A K > K F ; K A ; C A ? C > ; K F X k J < = F q F D J E < l r k E > ; K A D F ? ; A < ? F ; K F = F ; [ E C > ? ; F =

w w

K A ; C A ? C > ; k J < = F q F D J E < l r @ D k j > E @ < F I A @ A F ? C F > ; G F d J = F ; > K < ; F K F ? < D A ? >

H
@ < F I A @ A F ? C F K F F d C A ? @ A H ? F ; J F @ G I A @ < s t L

r > E F j K F r > D : F = C U F F = ; F @ [ D J E F J > = > ; < E [ @ A < ? F ; K A E [ A K > ; B J > = > Z > E < = F ; K F

H
@ > E C < ; k Z > = G > @ < ? E > @ < ? @ F ? C = > @ A H ? k K F : A K < > I F ? H D F ? < ; K F K A ; J F = ; A H ? K F E >

E [ _ k > l = F l > @ A H ? K F D < E i @ [ E > ; k @ > D : A < ; K F E D F K A < k F C @ L

1.3. Instrumentacin para la medicin de absorbancias de la luz visible y

ultravioleta: espectrofotmetro UV-visible
r > D F K A @ A H ? K F > : ; < = : > ? @ A > K F E > E [ _ J < = E > ; D < E i @ [ E > ; ; F = F > E A _ > F ? [ ? < ;

> J > = > C < ; E E > D > K < ; F ; J F @ C = < I < C H D F C = < ; L 9 [ ? h [ F J [ F K F ? Z > = A > = F ? K A ; F y < k F ?

F ; J F @ A > E @ < ? E > A ? @ < = J < = > @ A H ? K F < = K F ? > K < = F ; J > = > F E > ? m E A ; A ; K F K > C < ; k C < K < ;

E < ; F ; J F @ C = < I < C H D F C = < ; @ < ? ; C > ? k ; F l n ? ; F A ? K A @ > F ? E > I A l [ = > k K F 8

b L f ? > I [ F ? C F K F F ? F = l G > = > K A > ? C F 8 E m D J > = > K F K F [ C F = A < j C [ ? l ; C F ? < L

L f ? D < ? < @ = < D > K < = J > = > E > ; F E F @ @ A H ? K F = > K A > @ A < ? F ; K F [ ? > K F C F = D A ? > K >

E < ? l A C [ K K F < ? K > 8 I A E C = < ; k J = A ; D > ; k = F K F ; K F K A I = > @ @ A H ? L

L f ? @ < D J > = C A D F ? C < K < ? K F ; F > E < q > [ ? = F @ A J A F ? C F C = > ? ; J > = F ? C F s @ [ : F C > ; <

C [ : < ; t h [ F @ < ? C F ? l > E > D [ F ; C = > V [ F K F ? ; F = K F Z A K = A < k @ [ > = _ < < J E m ; C A @ <

C = > ? ; J > = F ? C F L V > = > D F K A = F ? f g ; F K F : F ? [ ; > = E > ; K F @ [ > = _ < < ; G E A @ F I [ ? K A K < k

J < = h [ F F E Z A K = A < ? < C = > ? ; D A C F E > = > K A > @ A H ? f g L

T L f ? K F C F @ C < = K F E [ _ j [ ? > D J E A I A @ > K < = @ < ? Z F = C A K < = K F E > ; ; F y > E F ; E [ D A ? < ; > ;

F ? ; F y > E F ; F E i @ C = A @ > ; L

L f ? = F l A ; C = > K < = < ; A ; C F D > K F E F @ C [ = > K F K > C < ; L

 a F ; K F F E J [ ? C < K F Z A ; C > < J F = > C A Z < k F E J = A D F = J > ; < F ; ; F E F @ @ A < ? > = E > I [ F ? C F K F

E [ _ j E < ? l A C [ K K F < ? K > > E > h [ F ; F Z > > = F > E A _ > = E > D F K A K > L \ > j

F ; J F @ C = < I < C H D F C = < ; K F [ ? ; < E < u > _ s @ < ? [ ? > ; < E > @ F E K A E E > J > = > > E < q > = E > @ [ : F C >

@ < ? E > D [ F ; C = > t j K F K < : E F u > _ s @ < ? K < ; @ F E K A E E > ; J > = > K < ; @ [ : F C > ; t B F ? ? [ F ; C = <

@ > ; < ; F C = > : > q > = m @ < ? E < ; K F [ ? ; < E < u > _ L

o F D A K F J = A D F = < E > > : ; < = : > ? @ A > K F E K A ; < E Z F ? C F s @ < ? < @ A K < @ < D < : E > ? @ < t j > E

h [ F ; F E F > ; A l ? > F E Z > E < = K F @ F = < D F K A > ? C F F E > q [ ; C F K F E D > ? K < k K F I < = D > h [ F E >

A ? C F ? ; A K > K A ? @ A K F ? C F j C = > ? ; D A C A K > ; F > ? A l [ > E F ; s ^ ^ t k j J < = C > ? C < E > > : ; < = : > ? @ A >

F ; @ F = < L 9 @ < ? C A ? [ > @ A H ? ; F J < ? F F ? E > @ F E K A E E > E > @ [ : F C > @ < ? E > D [ F ; C = > j ; F E F F E >

> : ; < = : > ? @ A > K F i ; C > L

ESPECTROFOTMETRO
OH

Detector
NO 2

Muestra
Rendija salida

Prisma dispersin

Rendija entrada

Fuente luminosa

Ajuste de longitud de onda

Compartimento
muestra Ajuste de 100% T

ON/OFF
Ajuste 0% T

1.4 Obtencin de un espectro de absorcin

` E F ; J F @ C = < K F > : ; < = @ A H ? F ; [ ? > = F J = F ; F ? C > @ A H ? l = m I A @ > h [ F A ? K A @ > @ > ? C A K > K K F

H O
E [ _ > : ; < = : A K > s t > K A I F = F ? C F ; Z > E < = F ; K F L

9 J > = C A = K F [ ? > ; < E [ @ A H ? K A E [ A K > K F [ ? @ < D J [ F ; C < k @ [ j > > : ; < = : > ? @ A > D m d A D >

F ? C = > K F ? C = < K F E = > ? l < K F D F K A K > K F E F ; J F @ C = < I < C H D F C = < k ; F Z F = m F E Z > E < = K F

> : ; < = : > ? @ A > > K A I F = F ? C F ; E < ? l A C [ K F ; K F < ? K > I = F ? C F > [ ? : E > ? @ < h [ F @ < ? C F ? l > F E

K A ; < E Z F ? C F K F E > ; < E [ @ A H ? K F E > D [ F ; C = > > @ > = > @ C F = A _ > = L 9 J > = C A = K F E F ; J F @ C = < K F

O
> : ; < = @ A H ? ; F < : C F ? K = m F E Z > E < = K F > E h [ F F E @ < D J [ F ; C < J = F ; F ? C > E > D > j < =

O O
> : ; < = : > ? @ A > s t L a A @ u < ; F [ C A E A _ > = m > E > u < = > K F u > @ F = K F C F = D A ? > @ A < ? F ;

@ [ > E A C > C A Z > ; j @ [ > ? C A C > C A Z > ; K F E @ < D J [ F ; C < L

` E F ; J F @ C = < K F > : ; < = @ A H ? K F [ ? @ = < D H I < = < K F J F ? K F k I [ ? K > D F ? C > E D F ? C F k K F E >

F ; C = [ @ C [ = > h [ G D A @ > K F E > D < E i @ [ E > L


 Clorofila 
Absorcin de los

Clorofila
del cloroplasto

~  ~ | } ~ 
pigmentos

~  ~   ~  ~

Carotenoides

  | ~  ~ z

} ~ |  ~ 

~ 

 

| } ~  z

Longitud de onda (nm)

Y < < : ; C > ? C F k u > j [ ? > l = > ? @ > ? C A K > K K F I > @ C < = F ; h [ F < = A l A ? > ? Z > = A > @ A < ? F ; F ?

O H
E < ; Z > E < = F ; K F j k F ? C = F E < ; h [ F ; F A ? @ E [ j F F E J \ k E > J < E > = A K > K K F E ; < E Z F ? C F

< D < E i @ [ E > ; Z F @ A ? > ; j E > < = A F ? C > @ A H ? K F E < ; @ = < D H I < = < ; Z F @ A ? < ; B j @ > K > [ ? <

> I F @ C > K F I < = D > J > = C A @ [ E > = L V < = F q F D J E < k Z > = A > @ A < ? F ; < = A l A ? > K > ; J < = @ > D : A < ; K F

J \ ; < ? K F : A K > ; > E F I F @ C < K F i ; C F ; < : = F E > A < ? A _ > @ A H ? K F E @ < D J [ F ; C < L 9

@ < ? C A ? [ > @ A H ? ; F D [ F ; C = > ? @ < D < F q F D J E < E < ; F ; J F @ C = < ; K F > : ; < = @ A H ? K F \ Y e o

s [ ? = F > @ C A Z < F D J E F > K < J > = > E > K F C F = D A ? > @ A H ? K F F ; J F @ A F ; < d A K > ? C F ; t j

@ < D J = < : m ? K < ; F h [ F J < = F ; J F @ C = < C < D F C = G > ; F J [ F K F ; F l [ A = F E F I F @ C < h [ F F q F = @ F ?

F E J \ j E < ; < d A K > ? C F ; L

& &

 
  
 

          

  
    ,   
  
      
        

p  
 
  
              

~  ~    |  ~


     p 

| } ~  | ~  ~ } |  | ~  ~  ~ 

  p  p

 } ~   | z   ~ z

1.5 Curvas de calibrado


 V > = > < : C F ? F = [ ? > @ [ = Z > K F @ > E A : = > K < K F [ ? @ < D J [ F ; C < ; F J = F J > = > ? ; < E [ @ A < ? F ;

K F K A I F = F ? C F ; @ < ? @ F ? C = > @ A < ? F ; K F E D A ; D < k K F C F = D A ? m ? K < ; F J > = > @ > K > [ ? > K F

O
F E E > ; F E Z > E < = K F > : ; < = : > ? @ A > > L ` ; C < ; Z > E < = F ; K F > : ; < = : > ? @ A > ; F

= F J = F ; F ? C > ? F ? F E F q F K F > : ; @ A ; > ; s F q F K F d t j E < ; K F @ < ? @ F ? C = > @ A H ? F ? F E F q F K F

< = K F ? > K > ; s F q F K F j t L o F < : ; F = Z > = m h [ F k > : > q > ; @ < ? @ F ? C = > @ A < ? F ; k F E > [ D F ? C < K F

@ < ? @ F ? C = > @ A H ? ; F @ < = = F ; J < ? K F @ < ? [ ? A ? @ = F D F ? C < E A ? F > E F ? E > > : ; < = : > ? @ A > s _ < ? >

K F @ [ D J E A D A F ? C < K F E > E F j K F r > D : F = C U F F = t L 9 @ < ? @ F ? C = > @ A < ? F ; > E C > ; E >

E A ? F > E A K > K ; F J A F = K F j ; F < : ; F = Z > h [ F E > E G ? F > ; F > J E > ? > k J < = E < h [ F E > ; D F K A K > ;

; < ? J < @ < I A > : E F ; L

H
r > = F J = F ; F ? C > @ A H ? K F r > D : F = C U F F = k 9 @ E k ? < ; J F = D A C A = m @ > E @ [ E > = F E Z > E < =

K F E @ < F I A @ A F ? C F K F F d C A ? @ A H ? D < E > = k h [ F @ < = = F ; J < ? K F > E > J F ? K A F ? C F K F E > = F @ C > L

1.6. Objetivos

Espectos de absorcin
o F Z > ? > = F > E A _ > = F ; J F @ C = < ; K F > : ; < = @ A H ? K F K A ; C A ? C > ; ; [ ; C > ? @ A > ; J > = >

O
K F C F = D A ? > = ; [ j @ < D J = < : > = h [ F [ ? @ > D : A < F ? E > F ; C = [ @ C [ = > K F E > D < E i @ [ E > k

J < = F q F D J E < ; [ = F K [ @ @ A H ? k ; [ J < ? F [ ? > D < K A I A @ > @ A H ? F ? ; [ F ; J F @ C = < L

Ley de Lambert-Beer
o F > J E A @ > = m E > E F j K F r > D : F = C U F F = j C = > ; u > @ F = [ ? > = F @ C > K F @ > E A : = > K < J > = >

K < ; ; [ ; C > ? @ A > ; k ; F @ > E @ [ E > = m l = m I A @ > D F ? C F F E Z > E < = K F E < ; @ < = = F ; J < ? K A F ? C F ;

@ < F I A @ A F ? C F ; K F F d C A ? @ A H ? D < E > = L

Determinaciones colorimtricas especficas de compuestos

o F > J E A @ > = m ? = F > @ @ A < ? F ; F ; J F @ G I A @ > ; J > = > E > K F C F = D A ? > @ A H ? @ < E < = A D i C = A @ > K F

; [ ; C > ? @ A > ; L

2. LISTADO DEL MATERIAL NECESARIO

2.1. Material de uso general

` ; J F @ C = < I < C H D F C = < L

9 l A C > K < = K F C [ : < ; K F F ? ; > j < L

> E > ? _ > L

J \ D F C = < L

e A q F = > ;

2.2. Material para cada grupo


= > K A E E > ; @ < ? C [ : < ; K F F ? ; > j < L

 [ F l < K F J A J F C > ; s L k b L k L k L k b L j L D E t L

V = F J A J F C > ; s K < ; q [ F l < ; t L

V = < : F C > ; s b j D E t L

g > ; < ; K F J = F @ A J A C > K < s b j D E t L


 W = > ; @ < E > Z > K < = @ < ? > l [ > K F ; C A E > K > L

F @ A J A F ? C F ; J > = > D [ F ; C = > ; : A < E H l A @ > ; K F D E L

V > J F E K F I A E C = < L

V > J F E K F J > = > I A E D L

< C [ E > K < = J > = > Z A K = A < L

V A J F C > ; V > ; C F [ = K F J E m ; C A @ < s @ [ > C = < t L


[ > ? C F ; K F E > C F d L

v [ : F C > ; K F J E m ; C A @ < J > = > D F K A = F ? F E F ; J F @ C = < I < C H D F C = < L

2.3. Reactivos

Espectos de absorcin
o < E [ @ A H ? K F R U ? A C = < I F ? < E k D X F ? > l [ > K F ; C A E > K > L

o < E [ @ A H ? K F Y > c \ k Y L

o < E [ @ A H ? K F R U ? A C = < I F ? < E K F @ < ? @ F ? C = > @ A H ? K F ; @ < ? < @ A K > L

o < E [ @ A H ? K F I E > Z G ? D < ? < ? [ @ E F H C A K < s W X Y t k X F ? > l [ > K F ; C A E > K > L

a A C A < ? A C < ; H K A @ < L

Determinacin colorimtrica de glucosa y nitrito mediante reacciones

especficas

E [ @ < ; >


E [ @ < ; > < d A K > ; >

V F = < d A K > ; >

W F ? < E

T > D A ? < U > ? C A J A = A ? >

e > D J H ? I < ; I > C <

Y A C = A C < J < C m ; A @ <

o [ E I > ? A E > D A K >

a A @ E < = [ = < K F Y U ? > I C < E U b U F C A E F ? K A > D A ? <

@ A K < @ E < = u G K = A @ <