03-semana-clase sesión 5 1s2015
DESCRIPTION
clase de bioquimicaTRANSCRIPT
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1 Semestre 2015
EspectrofotometraCuantitativa
Bioqumica Clnica I
TME-067
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Procedimiento
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Tabla 1
TUBO
BSA(mL) H2O
(mL)R.C.A.(mL)
R.F. A Conc.
0,4 mg/mL MP (mL) (mL) (mg/mL)
1B ---- --- 1,0 1,0 4,0
2S 0,1 --- 0,9 1,0 4,0
3S 0.2 --- 0,8 1,0 4,0
4S 0,3 --- 0,7 1,0 4,0
5S 0,4 --- 0,6 1,0 4,0
6S 0,5 --- 0,5 1,0 4,0
7MP --- 0,5 MP1 0,5 1,0 4,0
8MP --- 0,5 MP2 0,5 1,0 4,0 -
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Procedimiento
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Patrn = Protena = BSA
[ ] Inicial BSA = 0,4 mg/mL
Volumen Final Tubos = Vf = 6 mL
Calculo de [ ] BSA en cada tubo:
ffii c * v c v *
Ejemplo: Tubo 2S
Cf = (Vi * Ci) = (0.1 mL * 0,4 mg/mL) = 0.0067 mg/mL
Vf 6 mL
Clculos
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TUBO
BSA(mL) H2O
(mL)R.C.A.(mL)
R.F. A Conc.
0,4 mg/mL MP (mL) (mL) (mg/mL)
1B ---- --- 1,0 1,0 4,0 0
2S 0,1 --- 0,9 1,0 4,0 0.0067
3S 0.2 --- 0,8 1,0 4,0 0.0133
4S 0,3 --- 0,7 1,0 4,0 0.0200
5S 0,4 --- 0,6 1,0 4,0 0.0267
6S 0,5 --- 0,5 1,0 4,0 0.0333
7MP --- 0,5 MP1 0,5 1,0 4,0 ?
Tabla 1
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TUBO
BSA(mL) H2O
(mL)R.C.A.(mL)
R.F. A Conc.
0,4 mg/mL MP (mL) (mL) (mg/mL)
1B ---- --- 1,0 1,0 4,0 0 0
2S 0,1 --- 0,9 1,0 4,0 0.136 0.0067
3S 0.2 --- 0,8 1,0 4,0 0.257 0.0133
4S 0,3 --- 0,7 1,0 4,0 0.356 0.0200
5S 0,4 --- 0,6 1,0 4,0 0.484 0.0267
6S 0,5 --- 0,5 1,0 4,0 0.611 0.0333
7MP --- 0,5 MP1 0,5 1,0 4,0 0.567 ?
Tabla 1
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TUBO
BSA(mL) H2O
(mL)R.C.A.(mL)
R.F. A Conc.
0,4 mg/mL MP (mL) (mL) (mg/mL)
1B ---- --- 1,0 1,0 4,0 0 0
2S 0,1 --- 0,9 1,0 4,0 0.136 0.0067
3S 0.2 --- 0,8 1,0 4,0 0.257 0.0133
4S 0,3 --- 0,7 1,0 4,0 0.356 0.0200
5S 0,4 --- 0,6 1,0 4,0 0.484 0.0267
6S 0,5 --- 0,5 1,0 4,0 0.611 0.0333
7MP --- 0,5 MP1 0,5 1,0 4,0 0.567 ?
Tabla 1
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-0,005 0,000 0,005 0,010 0,015 0,020 0,025 0,030 0,035
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
Concentracin BSA (mg/mL)
Absorb
ancia
(U
.A.)
Curva Calibrado BSA: Mtodo de Lowry
Curva Calibrado
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-0,005 0,000 0,005 0,010 0,015 0,020 0,025 0,030 0,035
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
Y = A + B * X
Parameter Value Error
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A 0,00817 0,00571
B 18,03989 0,28286
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R SD N P
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0,99951 0,00788 6
Concentracin BSA (mg/mL)
Absorb
ancia
(U
.A.)
Curva Calibrado BSA: Mtodo de Lowry
Curva Calibrado
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Anlisis de valores de regresin lineal: Y = A + B * X
Parmetro Valor Error
A 0,00817 0,00571
B 18,03989 0,28286
R 0,99951
N 6
Ecuacin de la Recta: Y = A + B * X
Ley de Lambert-Beer: A = Ao + l c
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Absorbancia MP1 = 0.567 => Y = A + B * X
0.567 = 0,00817 + 18,03989 * X
X = 0.0310 mg/mL en el tubo, [ ] diluida.
[ ] Real = [ ] diluida * Vol. Final Tubo
Vol. Inicial Prot.
[ ] Real = 0.0310 mg/mL * 6 mL = 0.372 mg/mL
0.5 mL
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Procedimiento
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Clculos
Patrn = Protena = BSA
[ ] Patrn BSA = 1,0 mg/mL
Absorbancia Patrn BSA = 0,500
Absorbancia MP= 0,250
[ ] Protena = Absorbancia MP * [ ] Patrn BSA
Absorbancia Patrn BSA
[ ] Protena = 0,250 * 1,0 mg/mL
0,500
[ ] Protena = 0,5 mg/mL