Notas de Aplicaciones
13ene/15

Determinacion de las constantes cineticas de la fosfatasa acida de germen de trigo y efecto del pH sobre su actividad.

Determinación de las constantes cinéticas de la fosfatasa ácida de germen de trigo y efecto del pH sobre su actividad.

Estudio realizado por el Institut Químic de Sarrià.

1. OBJETIVOS

  • Determinación de la actividad enzimática de la fosfatasa ácida de germen de trigo mediante un método colorimétrico discontinuo.
  • Estudio del efecto del pH sobre su actividad.
  • Determinación de la constante de Michaelis (KM) para el p-nitrofenil-fosfato (pNPP), la velocidad máxima (Vmax), y la constante de inhibición (Ki) por fosfato de la fosfatasa.

2. INTRODUCCIÓN

La cinética enzimática estudia la velocidad de las reacciones químicas que son catalizadas por las enzimas (E). Para muchos enzimas que catalizan reacciones con un único sustrato (S), la velocidad de catálisis (V) (moles de producto (P) formado por segundo) varía de forma no lineal con la concentración de sustrato [S], siguiendo una cinética de saturación por sustrato de tipo Michaelis-Menten. La ecuación de Michaelis-Menten (V = Vmax S / (KM + S)) define el comportamiento cinético de estas enzimas. La constante de Michaelis KM se define como la concentración de sustrato [S] a la que la velocidad de la reacción enzimática es la mitad de la Vmax (cuando [S] = KM, entonces V = Vmax / 2). Cuando la [S] es mucho menor que la KM ([S]<<KM), la velocidad de la reacción incrementa de forma lineal con la [S]. Cuando la [S] es mucho mayor que la KM ([S]>>KM), entonces la velocidad es independiente de la [S] y V = Vmax. El valor de KM no sólo es variable en función de la naturaleza de la enzima y del sustrato, sino que también depende de las condiciones de temperatura y pH de la reacción.

Una forma tradicional de calcular los valores de KM y Vmax a partir de datos experimentales consiste en representar gráficamente los inversos de las concentraciones mM de sustrato (eje de abscisas) vs los inversos de la velocidad (eje de ordenadas) (representación de dobles inversos o Lineweaver-Burk). Esta representación de los puntos sigue una recta definida por la ecuación de Lineweaver-Burk (que se deriva de la ecuación de Michaelis-Menten) del tipo “y = mx + c”. A partir de los puntos de intersección de la recta de Lineweaver-Burk con el eje de la X y con el eje de la Y, se puede calcular los valores de KM y Vmax, respectivamente.

La inhibición enzimática se define como la disminución de la velocidad de la reacción catalizada por una enzima debido a la presencia de un inhibidor (I). Los inhibidores pueden actuar alterando los parámetros cinéticos (KM y Vmax), de forma que los valores observados en presencia del inhibidor se denominan KM aparente (KM-ap) y Vmax aparente (Vmax-ap). Las enzimas pueden inhibirse reversiblemente de forma competitiva y no competitiva. Los inhibidores competitivos se unen a la enzima impidiendo la unión del sustrato. Esto conlleva un incremento en la KM en presencia del inhibidor (KM-ap > KM), mientras que la Vmax se mantiene constante. Los inhibidores no competitivos se unen a un sitio regulador distinto al centro activo, y actúan disminuyendo la velocidad máxima (Vmax) de la reacción catalizada por la enzima (Vmax-ap < Vmax) sin alterar la KM (puesto que no alteran la unión de la enzima al sustrato). Consecuentemente, en la inhibición competitiva se observa una disminución del porcentaje de inhibición al incrementar la concentración de sustrato en la reacción, mientras que en la no competitiva el porcentaje de inhibición permanece constante. La constante de inhibición (Ki) mide la afinidad de la enzima por el inhibidor. En el caso de la inhibición competitiva, la Ki se puede deducir a partir de la KM y la KM-ap: KM-ap = KM {1 + ([I] / Ki )}

Las fosfatasas son una familia de enzimas que catalizan la hidrólisis de un grupo fosfato (desfosforilación). Su actividad es sensible al pH del medio, de forma que las fosfatasas alcalinas funcionan de forma óptima en condiciones de pH alcalino, mientras que las fosfatasas ácidas lo hacen en ambientes ácidos. La actividad fosfatasa se puede medir mediante un ensayo enzimático colorimétrico basado en su capacidad de hidrólisis del p-nitrofenilfosfato (pNPP) a p-nitrofenol: p-nitrofenilfosfato (pNPP) + H2O → p-nitrofenol + fosfato.

A diferencia del pNPP, el  producto de la reacción p-nitrofenol es un compuesto amarillo que presenta un máximo de absorbancia a 405 nm en un medio alcalino que se puede monitorizar mediante espectrofotometría.

En este ensayo se determinará la actividad fosfatasa ácida de un preparado de germen de trigo, y se ensayará cómo esta actividad se ve afectada por cambios en el pH y en la [S], y por la presencia de un inhibidor. Por un lado, se ensayará la actividad fosfatasa en distintas condiciones de pH y se determinará el pH óptimo. Por otro, se determinará la actividad fosfatasa a pH óptimo en presencia de distintas concentraciones de pNPP y de fosfato inorgánico, un inhibidor reversible de la fosfatasa, y se determinará las constantes cinéticas KM, Vmax, KM-ap, Vmax-ap y Ki.

3. CONCEPTOS BÁSICOS

  • Ensayo enzimático colorimétrico discontinuo
    • ε = Coeficiente de extinción molar
    • Abs = Absorbancia
    • c = Concentración
    • l = Longitud del paso de luz (path length) (cm)
  • Unidades de actividad enzimática (U, µmol/min), actividad específica (U/mg) y concentración enzimática (U/ml)
  • Representación y ecuación de Michaelis-Menten y de Lineweaver-Burk
  • Velocidad de catálisis (V), velocidad máxima (Vmax) y constante de Michaelis-Menten (KM)
  • Inhibición reversible competitiva y no competitiva
  • KM aparente (KM-ap), Vmax aparente (Vmax-ap) y constante de inhibición (Ki)
  • Ecuación de Lambert-Beer:   Abs = ε · c · l

4. MATERIAL Y REACTIVOS

  • Selecta Spectrophotometer UV-3100. Código 4120021
  • Selecta PH Meter PH-2006. Código 4120600
  • Selecta Microcentrífuga Sigma 1-14. Código 5710014
  • Selecta Vórtex Heidolph Reax Top. Código 5411000
  • Selecta Baño termostático Precisterm. Código 6000387
  • Selecta Agitador magnético  Agimatic-E. Código 7002431
  • Balanza analítica. Código 5830039  
  • Micropipetas.
  • Germen de trigo
  • Ácido cítrico monohidrato
  • p-nitrofenil-fosfato (pNPP)
  • Citrato de trisodio dihidratado
  • Tris-base
  • Ácido clorhídrico
  • Carbonato de sodio
  • Fosfato monopotásico

5. PROCEDIMIENTO

1. PREPARACIÓN DE TAMPONES Y DISOLUCIONES.

Con la ayuda del Selecta PH Meter PH-2006 y del Selecta Agitador magnético con placa calefactora Agimatic-E, preparar las siguientes disoluciones.

1.1.  Preparación de tampones citrato 0,2 M a pH 3, 4, 5 y 6.

1.1.1.     Preparar tampón citrato 0,2 M a pH 3, 4, 5 y 6 mezclando las siguientes soluciones stock en las proporciones adecuadas:

  • Solución A: Ácido cítrico monohidrato 0,2 M en agua desionizada.
  • Solución B: Citrato de trisodio dihidratado 0,2 M en agua desionizada.

1.2.  Preparación de tampones tris 0,2 M a pH 7, 8, y 9. 1.2.1. Preparar tampón tris 0,2 M a pH 7, 8 y 9 utilizando tris-base y ajustando el pH con ácido clorhídrico 6 N.

1.3.  Preparación de carbonato de sodio 0,1 M en agua desionizada.

1.4.  Preparación de fosfato monopotásico 50 mM en agua desionizada.

2. DETERMINACIÓN DE LA ACTIVIDAD FOSFATASA DE GERMEN DE TRIGO. Se ensayará la actividad fosfatasa ácida de un producto parcialmente purificado de germen de trigo. Para ajustar las condiciones del ensayo, se testarán distintas diluciones de la muestra y distintos tiempos de reacción. NOTA: Para preservar la actividad fosfatasa, mantener la muestra stock y las respectivas diluciones en hielo.

2.1.  A partir del preparado de germen de trigo, preparar el siguiente banco de diluciones en agua:

  • 0,5 ml a 1 mg/ml
  • 0,5 ml a 0,5 mg/ml
  • 0,5 ml a 0,25 mg/ml
  • 0,5 ml a 0,125 mg/ml
  • 0,5 ml a 0,05 mg/ml

2.2.  Con la ayuda del Selecta Vórtex Heidolph Reax Top, en un tubo Eppendorf, preparar 1 ml de 50 mM p-nitrofenil-fosfato (pNPP) en agua desionizada. NOTA: El pNPP es muy lábil, y se debe preparar fresco y mantener protegido de la luz.

2.3.  Numerar 16 tubos Eppendorf y añadir los siguientes reactivos:

Tubo Agua Tampón citrato 0,2M pH 5 Germen de trigo
Blanco 200 µl - -
1.1 25 µl 125 µl 50 µl 1 mg/ml
2.1 25 µl 125 µl 50 µl 0,5 mg/ml
3.1 25 µl 125 µl 50 µl 0,25 mg/ml
4.1 25 µl 125 µl 50 µl 0,125 mg/ml
5.1 25 µl 125 µl 50 µl 0,05 mg/ml
1.2 25 µl 125 µl 50 µl 1 mg/ml
2.2 25 µl 125 µl 50 µl 0,5 mg/ml
3.2 25 µl 125 µl 50 µl 0,25 mg/ml
4.2 25 µl 125 µl 50 µl 0,125 mg/ml
5.2 25 µl 125 µl 50 µl 0,05 mg/ml
1.3 25 µl 125 µl 50 µl 1 mg/ml
2.3 25 µl 125 µl 50 µl 0,5 mg/ml
3.3 25 µl 125 µl 50 µl 0,25 mg/ml
4.3 25 µl 125 µl 50 µl 0,125 mg/ml
5.3 25 µl 125 µl 50 µl 0,05 mg/ml

NOTA: Se testará la actividad fosfatasa en cada una de las 5 diluciones de la muestra y en tres tiempos de reacción (10, 15 y 20 min).

2.4.  Mezclar los tubos suavemente, y con la ayuda de la Selecta Microcentrífuga Sigma 1-14, centrifugar brevemente.

2.5.  Preincubar los tubos a 37ºC en el Selecta Baño termostático Precisterm.

2.6.  Iniciar la reacción añadiendo 50 µl de pNPP 50 mM a cada tubo, siguiendo el mismo orden que se indica en la tabla, y con un intervalo de 30 s entre tubo y tubo. Mezclar brevemente con el Selecta Vórtex Heidolph Reax Top tras iniciar la reacción en cada tubo, e incubar a 37 ºC en el Selecta Baño termostático Precisterm.

2.7.  Dejar transcurrir la reacción durante 10 min exactos para los tubos 1.1-5.1, 15 min exactos para los tubos 1.2-5.2, y 20 min exactos para los tubos 1.3-5.3.

2.8.  Añadir 750 µl carbonato de sodio (Na2CO3) 0,1 M a cada tubo para detener la reacción a los tiempos indicados, también a intervalos de 30 s entre tubo y tubo y siguiendo el mismo orden. Mezclar por inversión.

2.9.  Transferir el volumen a una cubeta de espectrofotómetro y medir la absorbancia a 405 nm (A405) de cada una de las reacciones respecto la del blanco en el Selecta Spectrophotometer UV-3100.

2.10.  Para cada dilución de la muestra, representar gráficamente la A405 (eje Y) en función del tiempo de reacción (eje X).

2.11.  Calcular los µmoles totales de p-nitrofenol liberados a tiempo 15 min para cada una de las diluciones de muestra ensayada, utilizando la ecuación de Lambert-Beer y considerando que el coeficiente de extinción molar del p-nitrofenol es de ε =18.000 M−1 cm−1.

2.12.  Calcular las unidades de actividad fosfatasa (U, µmol/min) en cada una de las diluciones de muestra ensayada (a tiempo 15 min). Representar gráficamente la actividad fosfatasa (eje Y) en función de la concentración de las muestras ensayadas (eje X).

2.13.  Para aquellas diluciones de muestra que respondan dentro del rango lineal (a tiempo 15 min), calcular la concentración de actividad fosfatasa (U/ml), y la actividad fosfatasa por mg de preparado de germen de trigo (U/mg).

3. EFECTO DEL PH SOBRE LA ACTIVIDAD ENZIMÁTICA DE LA FOSFATASA. Se ensayará la actividad fosfatasa a distintos pH entre 3 y 9. Para ello, se utilizará los tampones citrato 0,2 M a pH 3, 4, 5 y 6, y los tampones tris 0,2 M a pH 7, 8 y 9.

3.1.  En función de los resultados obtenidos en el punto 2, preparar una dilución de germen de trigo adecuada para el ensayo, y mantener en hielo.

3.2.  Con la ayuda del Selecta Vórtex Heidolph Reax Top, en un tubo Eppendorf preparar 1 ml de 50 mM p-nitrofenil-fosfato (pNPP) en agua desionizada.

3.3.  Numerar 8 tubos Eppendorf y añadir los siguientes reactivos:

Tubo Agua Tampón 0,2M Dilución de germen de trigo
Blanco 200 µl - -
1 25 µl 125 µl  pH = 3 50 µl
2 25 µl 125 µl  pH = 4 50 µl
3 25 µl 125 µl  pH = 5 50 µl
4 25 µl 125 µl  pH = 6 50 µl
5 25 µl 125 µl  pH = 7 50 µl
6 25 µl 125 µl  pH = 8 50 µl
7 25 µl 125 µl  pH = 9 50 µl

3.4  Mezclar los tubos suavemente, y con la ayuda de la Selecta Microcentrífuga Sigma 1-14, centrifugar brevemente.

3.5.  Preincubar los tubos a 37ºC en el Selecta Baño termostático Precisterm.

3.6.  Iniciar la reacción añadiendo 50 µl de pNPP 50 mM a cada tubo, siguiendo el mismo orden que se indica en la tabla, y con un intervalo de 30 s entre tubo y tubo. Mezclar brevemente con el Selecta Vórtex Heidolph Reax Top tras iniciar la reacción en cada tubo, e incubar a 37 ºC en el Selecta Baño termostático Precisterm.

3.7.  Dejar transcurrir la reacción a 37 ºC durante 15 min exactos.

3.8.  Añadir 750 µl carbonato de sodio (Na2CO3) 0,1 M a cada tubo para detener la reacción, también a intervalos de 30 s y siguiendo el mismo orden. Mezclar por inversión.

3.9.  Transferir el volumen a una cubeta de espectrofotómetro y medir la absorbancia a 405 nm (A405) de cada una de las reacciones respecto la del blanco en el Selecta Spectrophotometer UV-3100.

3.10.  Determinar los μmoles de p-nitrofenol producidos en cada reacción a partir de la ecuación de Lambert-Beer y el coeficiente de extinción molar del p-nitrofenol.

3.11.  Calcular las unidades de actividad enzimática (U, μmol/min) fosfatasa en cada una de las condiciones de pH ensayado.

3.12.  Representar gráficamente los resultados situando la actividad enzimática en el eje Y y el pH en el eje X.

4. DETERMINACIÓN DE LAS CONSTANTES CINÉTICAS DE LA FOSFATASA: KM PARA EL P-NITROFENIL-FOSFATO Y Ki PARA LA INHIBICIÓN POR FOSFATO. Para calcular la constante de Michaelis (KM) de la fosfatasa para el p-nitrofenil-fosfato, se determina la actividad de la enzima en distintas concentraciones de sustrato. La constante de inhibición (Ki) por fosfato se determina de la misma manera pero en presencia de una concentración constante del inhibidor fosfato.

4.1.  En función de los resultados obtenidos en el punto 2, preparar una dilución de germen de trigo adecuada para el ensayo, y mantener en hielo.

4.2.  Con la ayuda del Selecta Vórtex Heidolph Reax Top, preparar 1 ml de p-nitrofenil-fosfato (pNPP) 80 mM y 500 µl de pNPP 8 mM en tampón citrato 0,2 M pH 5.

4.3.  Numerar 17 tubos Eppendorf y añadir los siguientes reactivos:

Tubo Tampón citrato 0,2M pH=5 pNPP Agua KH2PO4 50 mM
Blanco 112,5 µl 12,5 μl de 80 mM 125 µl -
1 112,5 µl 12,5 μl   de 8 mM 75 µl -
2 100 µl 25 μl   de 8 mM 75 µl -
3 75 µl 50 μl   de 8 mM 75 µl -
4 50 µl 75 μl   de 8 mM 75 µl -
5 112,5 µl 12,5 μl de 80 mM 75 µl -
6 100 µl 25 μl de 80 mM 75 µl -
7 75 µl 50 μl de 80 mM 75 µl -
8 50 µl 75 μl de 80 mM 75 µl -
9 112,5 µl 12,5 μl   de 8 mM 25 µl 50 µl
10 100 µl 25 μl   de 8 mM 25 µl 50 µl
11 75 µl 50 μl   de 8 mM 25 µl 50 µl
12 50 µl 75 μl   de 8 mM 25 µl 50 µl
13 112,5 µl 12,5 μl de 80 mM 25 µl 50 µl
14 100 µl 25 μl de 80 mM 25 µl 50 µl
15 75 µl 50 μl de 80 mM 25 µl 50 µl
16 50 µl 75 μl de 80 mM 25 µl 50 µl

4.4.  Mezclar los tubos suavemente, y con la ayuda de la Selecta Microcentrífuga Sigma 1-14, centrifugar brevemente.

4.5.  Preincubar los tubos a 37ºC en el Selecta Baño termostático Precisterm.

4.6.  Iniciar la reacción añadiendo 50 µl de la dilución de germen de trigo a cada tubo excepto el blanco, siguiendo el mismo orden que se indica en la tabla, y con un intervalo de 30 s entre tubo y tubo. Mezclar brevemente con el Selecta Vórtex Heidolph Reax Top tras iniciar la reacción en cada tubo, e incubar a 37 ºC en el Selecta Baño termostático Precisterm.

4.7.  Dejar transcurrir la reacción a 37ºC durante 15 min exactos.

4.8.  Añadir 750 µl carbonato de sodio (Na2CO3) 0,1 M a cada tubo para detener la reacción, también a intervalos de 30 s y siguiendo el mismo orden. Mezclar por inversión.

4.9.  Anotar los valores de A405 y para cada reacción, calcular los siguientes parámetros:

  • Concentración inicial de sustrato [S] pNPP en mM.
  • Concentración de inhibidor [I] fosfato en mM.
  • Los μmoles de p-nitrofenol producidos en cada reacción a partir de la ecuación de Lambert-Beer y el coeficiente de extinción molar del p-nitrofenol.
  • La velocidad (V) de reacción: μmoles de p-nitrofenol liberados por minuto (μmol/min).
  • El % inhibición producida por el fosfato para cada concentración de sustrato.
    • % Actividad = (V con inhibidor / V sin inhibidor) x 100
    • % Inhibición = 100 - % Actividad
  • La inversa de la concentración inicial de sustrato (1/[S]) y de la velocidad de reacción (1/V).

4.10.  Representar gráficamente la curva de Michaelis-Menten de concentración de sustrato (eje de la X) vs velocidad (eje de la Y), con y sin inhibidor.

4.11.  Representar gráficamente los inversos de las concentraciones mM de sustrato (eje de la X) vs los inversos de la velocidad (eje la Y), con y sin inhibidor (representación de dobles inversos o Lineweaver-Burk).

4.12.  A partir de los puntos de intersección de la recta de Lineweaver-Burk con el eje de la X y con el eje de la Y, calcular los valores de KM (mM) y la Vmax (µmol/min), respectivamente, en ausencia del inhibidor. Estos valores se pueden deducir por extrapolación a partir de la regresión lineal:

4.12.1.     Cuando y = 0, KM = -1/x. 4.12.2.     Cuando x = 0, Vmax = 1/y.

4.13.     De forma equivalente, a partir de la representación de Lineweaver-Burk calcular los valores de KM aparente (KM-ap) y de Vmax aparente (Vmax-ap) de la enzima en presencia del inhibidor fosfato.

4.14.     En base a los datos de % de inhibición por fosfato y de las variaciones entre KM y KM-ap, y Vmax y Vmax-ap, determinar si la inhibición de la fosfatasa por fosfato es competitiva o no competitiva.

4.15.     En el caso de que la inhibición sea de tipo competitiva, calcular el valor de la constante de inhibición (Ki) (mM) de la fosfatasa por fosfato a partir de la KM, KM-ap, y [I].

6. BIBLIOGRAFÍA

  • BIOQUÍMICA 7ED. L. Stryer / J. Berg / J. Tymoczko.
  • Brouillard, J., and Quellet, L. (1965). Acid phosphatases of wheat germ. Chromatographic analysis. Can. J. Biochem., 43, 1899-1905.
  • Waymack, P.P., and van Etten, R.L. (1991). Isolation and characterization of a homogeneous isoenzyme of wheat germ acid phosphatase. Arch. Biochem.Biophys., 288, 621-33.
10oct/12

METODO KJELDAHL / KJELDAHL METHOD

Kjeldahl method is used in analytical chemistry for the determination of nitrogen content in organic samples which is of great interest in areas such important today as food and environmental.

APPLICATIONS
Since 1883 when John Kjeldahl presented his work, his method has gained wide acceptance and is applied in a wide variety of jobs for food analysis of food, drinks, feed, grain, meat, wastewater, soils for crops and others. Today is the most widely used method for protein analysis and is performed by determining organic nitrogen. This is because different types of proteins coincide all in a similar proportion of such organic nitrogen. In most cases the following calculation factor is used:

Protein content = organic nitrogen content x 6.25

In this technique, proteins and other organic compounds in a food mixture are digested with sulfuric acid in the presence of catalysts. The total organic nitrogen is converted into ammonium sulfate through the digestion. The resulting mixture is neutralized with a base and distilled. The distillate is collected in a solution of boric acid. Borate anions thus formed are titrated with standardized HCL to determine the nitrogen content in the sample.

Generally, the Kjeldahl method has the advantage of being running by unsophisticated equipments and can be performed by less experienced technicians.

ACKNOWLEDGEMENTS

The Kjeldahl method has been officially recognized by a large number of government agencies and associations such as: the International AOAC, EPA, AACC, AOCS, ISO, USDA and others.

PROCEDURE

The method consists of three stages: DIGESTION - DISTILLATION - TITRATION.

DIGESTION occurs in the nitrogen decomposition contained in organic samples by using a concentrated acid solution. This is obtained by boiling the sample in a sulfuric acid concentration. The result is an ammonium sulfate solution.

Ammonia is liberated in the DISTILLATION stage, which is retained in a solution with a known amount of boric acid. Initially a steam distillation is performed by the water steam distillation method, by which distillation obtainment is accelerated.

TITRATION is used in the end to finally assess the amount of ammonium present in the distilled sample.

REACTIONS CARRIED OUT IN THE KJELDAHL METHOD

DIGESTION

catalysts→
(1) n - C -NH2 + mH2SO4 → CO2 + (NH4)2 SO4 + SO2
protein heat→

NEUTRALIZATION AND DISTILLATION

(2) (NH4)2SO4 + 2 NaOH → 2NH3 + Na2SO4+ 2H2O
(3) NH3 + H3BO3 (boric acid) → NH4 + H2BO3- (borate ions)

TITRATION

The borate anion (proportional to the amount of nitrogen) is titrated with standardized HCl (or H2SO4):

(4) H2BO3- + H+ → H3BO3

LABORATORY EQUIPMENTS

In recent years, new equipments are being developed and improving technologies to implement these analytical techniques.

J.P. Selecta, aware of these laboratories’ needs, has devoted a considerable effort to put on the market a new range of equipments, as complete as possible, in order to help the work of developing the Kjeldahl method with the speed, accuracy and reproducibility of results.

The equipments for organic nitrogen determination are composed of three basic elements:

- Bloc-Digest digestion unit.
- Tools for handling (Macro or Micro).
- Pro-Nitro “M”, Pro-Nitro “S” (semiautomatic) and Pro-Nitro “A” (automatic) distillers.

Recently, the new automatic system Auto Digest 20 has been added, which optimizes speed and reliability of laboratory professionals.

DIGESTION PROCESS

A number of interrelated conditions in the digestion process determine the speed of reaction and the decomposition of nitrogen into ammonium sulfate, such as the amount of heat transferred, the quantity of salts to raise the acid boiling temperature, the catalyst employed and the time of digestion. Adjustment of any of these parameters influences the rest. There are studies to determine the parameters needed to obtain optimum conditions depending on the samples matrix. For example, the amount of acid required varies depending on the fat present in the sample. The more quantity of fat, the more acid is required. It also varies with the time of digestion. The longer time, the more acid lost by evaporation.

Digestion time should be determined depending on the amount of recovery by using known matrix samples.

Salts addition is helpful to raise the boiling temperature of H2SO4. Depending on the kind of salts used, the temperature may go from 330°C being just the sulfuric acid, to a one of 400°C, thereby accelerating the rate of decomposition and considerably shortening the digestion time.

To perform digestion, a heating block made ​​of aluminium is normally used, surrounded by a thick layer of thermal insulation and assembled on a stainless steel frame. There are different block sizes for 6, 12 and 20 samples. The heating element is a high electrical resistance which is controlled by an electronic device incorporating a microprocessor which allows the user to choose and memorize several programs working with fully programmable ramps and times. Such programming capability optimizes digestions according to the material used.

DIGESTION IS PERFORMED IN THREE STEPS

  1. Depending on the sample water content, begin the digestion by evaporating the water at 150°C during 15 and 30 minutes.
  2. Perform a second step between 270 and 300°C within a period of 15 to 30 minutes in order to reduce the production of white fumes.
  3. Continue the digestion at 400ºC during 60 and 90 minutes.

Visual Control: The result is a clear transparent liquid with light blue colour, green or yellow depending on the catalyst used. No black residues should remain attached to the tube wall.

SOME EXAMPLES OF PROGRAMMING:

Cheese or meat:
Step 1: 150ºC / 30’ Step 2 : 270ºC / 30’ Step 3: 400ºC / 90’

Cereals:
Step 1: 150ºC / 15’ Step 2 : 300ºC / 15’ Step 3: 400ºC / 60’

J.P. SELECTA’S EQUIPMENTS MOST SUITABLE FOR THE DIGESTION PROCESS ARE THE FOLLOWING: “BLOC-DIGEST” DIGESTION UNIT

FEATURES:

  • Temperature independent control.
  • Bidirectional serial RS-232 connection for temperature recording and digestion program edition with RAT connected to a computer.
  • A software CD is included in the digestion unit.
  • Less sample handling.
  • Uniform heating of the aluminium block.
  • Temperature range from 45 to 450°C.
  • 4 steps memory for 20 programs.
  • Maximum time per step: 600 minutes.
  • Acoustic indication for end of digestion program.
  • Two selectable temperature gradients: Kjeldahl / D.Q.O.
  • Alarm for temperature sensor breakage.
  • Independent temperature control.
  • Bidirectional RS-232 serial connection for temperature recording and digestion program edition with RAT connected to a computer.

A software CD is included in the digestion unit. The software facilitates the digestion editing programs and allows you to track and record the digestor temperature.

EXTRACTION SYSTEM AND GAS NEUTRALIZATION

Specially designed to absorb and neutralize acid gases generated in Kjeldahl digestion processes.

It consists of a "Scrubber" unit that blocks and neutralizes the acidic condensations, and a recirculating water pump that provides a great volume of vacuum for gases suction.

It is essential to place the "Scrubber" unit with the neutralizing solution between the digester and the recirculation pump.

AUTO DIGEST 20 AUTOMATIC EQUIPMENT FOR DIGESTION

The equipment carries out the digestion process fully automated, with rise and fall of the rack holder.

Equipment with metallic structure and automatic sample holder epoxy covered. Tube support rack made ​​of a special dur-al plate chemically treated.

Features:

  • Automatic sample manipulation.
  • Uniform heating.
  • Control automatic control for up to 20 programs of temperature, time, and sample’s elevation after digestion and start/stop of the "Scrubber".
  • RS-232 port for temperature recording and digestion programming from computer.
  • Gas collection system which could be used without extracting cabinets.

It is completely supplied with:

  • 1 metal heating block of 20 positions.
  • 1 automatic lifting system for samples.
  • 1 “Rat-2” programmer for time/temperature processes.
  • 1 tube support rack.
  • 1 gas collector.
  • 20 digestion tubes of 250 ml capacity.

DISTILLATION PROCESS

The digestion product is usually diluted with ammonia-free water to minimize the effects of mixtures containing high proportions of acid / salts.

Most of the NH3 is distilled and caught in the acidic solution during the first 5 to 10 minutes of boiling, but depending on the volume of digestion mixture and on the method followed, from 20 to 140ml of condensate can be collected to obtain a complete nitrogen collection. Sometimes distillation is required to be extended, which produces more water but this does not alter the results when making the titration.

The distillation speed varies with the condenser cooling capacity and with the capacity of generating heat from the heater. The system of heating by water steam accelerates the obtaining of distillation. Using a recipient solution made of boric acid, dosing is not required with accuracy, as titration measures exactly the amount of ammonia by neutralizing 1:1 the complex formed by ammonia and boric acid. In fact, quite boric can be added to ensure the complete absorption of ammonia.

The reception solution must remain at 45°C to avoid loss of ammonia.

J.P. SELECTA’S EQUIPMENTS MOST SUITABLE FOR THE DISTILLATION PROCESS ARE THE FOLLOWING:PRONITRO “M” AND PRONITRO “S” KJELDAHL DISTILLER

Pronitro M is a Kjeldahl with an automation level that provides a simple and safety operation. It is suitable for a laboratory with a small or medium samples volume.

Features:

  • Steam distillation unit.
  • Compact steam generator with safety overtemperature thermostat and overpressure protection pressure switch.
  • Safety door that avoid distillation with door open.
  • Presence detection of digestion/distillation tube. This device avoids NaOH dosing if there’s no tube.
  • Universal adapter for digestion/distillation tubes MACRO (Ø 42 mm) and MICRO (Ø 26 mm).
  • H2O and NaOH reservoirs are placed inside the equipment, which saves space in the laboratory.
  • Stainless steel case and ABS plastic front.
  • Automatic titration adapter kit.

Specifications:

  • Measuring range: from 0,2 to 200 mg of Kjeldahl nitrogen.
  • Programmable distillation time.
  • Nitrogen recovery: > 99,5%
  • Distillation speed: from 35 to 40 ml/minute.
  • Typical distillation time: from 7 to 10 minutes.
  • Water consumption rate: from 80 to 100 litres/h.
  • Steam generator water consumption: 2,5 litres/h.
  • Water reservoir capacity for steam generator: 6 litres.
  • NaOH reservoir capacity: 2 litres.

TITRATION PROCESS

Boric acid captures ammonia gas and forms a boric ammonia complex. When ammonia is captured, colour of the recipient solution changes. Proceed as follows:

• Titrate the distillate with HCl or H2SO4 till colour changes. (End point: pH 4.65)
HCl moles = NH3 moles= N moles in the sample
H2SO4 moles = 2 NH3 moles = 2 N moles in the sample

Actually, different indicators can be used to get a turn as clean and sharp as possible. If it becomes difficult to detect the turning point, it may be useful to use a white reference solution.

CALCULATIONS

When making calculations, we must take into account the recipient solution and the dilution factors used in the distillation process. References may be taken in the published reference methods.

• Make the calculation:

mg N = N x V x 14

Where:

N = Titration acid normality.
V = Consumed acid volume.
14 = Nitrogen atomic weight.

• To switch to protein content correct it by the appropriate factor depending on the nature of the sample (6.25 by default).
• Periodically perform a blank test and subtract it from the result.

% Proteins = P2/P0 x 100 x F

Where:

P2: Nitrogen (mg).
P0: Sample weight (mg).
F: Protein factor.
(6.25 by default)

J.P. SELECTA HAS PRONITRO “A” ANALYZERS WHICH PERFORMS DISTILLATION AND TITRATION.
AUTOMATIC KJELDAHL DISTILLER PRONITRO “A”

Kjeldahl distiller is fully automatic and with a titration system "on-line" (real-time titration). Equipment for a systematic analysis, highly accurate, and with minimal staff, easy and safe. Suitable for laboratory with medium or large sample volume.

Kjeldahl distiller PRO-NITRO “A” titrates distillation while it is obtained (“On-Line” titration), and therefore distillation and titration become a single operation, drastically shortening analysis time.

This type of titration provides an additional advantage: it detects the point at which the sample no longer produces nitrogen, this property is used for stopping distillation at the right moment and thus ensuring that the distillation time is always optimal for a maximum nitrogen recovery and extend distillation no longer than necessary.

The colorimetric evaluation is accepted by the AOAC and needs no periodic calibration.

Features:

  • Steam distillation unit.
  • Automatic «On-line» colorimetric evaluation.
  • Steam generator with overtemperature safety thermostat and overpressure protection pressure switch.
  • Safety door that prevents distillation with door open.
  • Presence detection of digestion/distillation tube. This device avoids NaOH dosing if there’s no tube.
  • Universal adapter for digestion/distillation tubes MACRO (Ø 42 mm) and MICRO (Ø 26 mm).
  • H2O and NaOH, boric acid and HCl reservoirs are placed inside the equipment, which saves space in the laboratory.
  • Drain system of digestion /distillation tube and collector.
  • Automatic distillation stop.
  • Large LCD display of 20 x 4 characters.
  • RS-232 output to print the results.
  • Stainless steel case and ABS plastic front.

Specifications:

  • Measuring range: from 0,2 to 200 mg nitrogen.
  • Nitrogen recovery: > 99,5%.
  • Distillation speed: from 35 to 40 ml/minute.
  • Water consumption rate: from 80 to 100 litres/h.
  • Steam generator water consumption: 2,5 litres/h.
  • Water reservoir capacity for steam generator: 6 litres.
  • NaOH reservoir capacity: 2 litres.
  • Boric acid reservoir capacity: 2 litres.
  • Tritant reservoir capacity: 2 litres.
  • Titrator accuracy: 1,5%.
  • Titrator minimum dose: 0,01 ml.

PRACTICAL EXAMPLE

GROSS PROTEIN DETERMINATION BY KJELDAHL METHOD

1. Principle:

The method consists of mineralizing the sample with a concentrated sulfuric acid and alkalinizing with sodium hydroxide. The ammonia released is carried by distillation and collected on boric acid. Subsequent titration with hydrochloric acid allows calculation of the amount of protein initially present in the sample.

2. Reagents needed:

  • 96% sulfuric acid (d = 1.84).
  • NaOH, 35% solution (w / v).
  • Mixed indicator, especially for ammonia titrations.
  • Catalyst Kjeldahl.
  • 4% boric acid (w / v).
  • 0.25N HCl.
  • Distilled water.
  • Pumice stone in grains.

Note: It is important that all reagents are completely free from nitrogen.

3. Material needed:

  • Resolution balance 0.1 mg.
  • Digestor unit (Bloc-Digest).
  • RAT process programmer.
  • Collector / Extractor fan.
  • Pro-Nitro “M” or Pro-Nitro “A” Distiller.
  • Burette for titration.

4. Digestion:

  • Weigh about 1 gram of sample perfectly ground and homogenized in a nitrogen-free paper and insert it into a digestion tube.
  • Add 10 g of Kjeldahl catalyst, 25 ml of 96% sulfuric acid (d = 1.84), and some pumice granules treated to the sample tube.
  • Place the digestion tubes with the samples in the Bloc-Digest with the flume collector running.
  • Digestion can be performed at a temperature between 350 ... 420°C and at a time which can vary between 1 and 2h.
  • At the end, the resulting liquid is transparent green or blue depending on the catalyst used.
  • Let the sample cool down to room temperature.
  • Avoid precipitation by stirring occasionally.
  • Slowly dose 50 ml of distilled water in each sample tube (be careful of the reaction violence).
  • Let the sample cool down to room temperature.
  • If precipitation occurs, gently stirring or heat.

5. Distillation

Dose 50 ml of boric acid in an Erlenmeyer flask and some drops of mixed indicator. Place the Erlenmeyer flask in the refrigerant extension taking care it has to be submerged in the boric acid.

Once the sample tube and the Erlenmeyer with boric acid are placed, dose 50ml of NaOH and start distillation.

Distillation should be extended long enough to distillate at least 150 ml, approximately from 5 to 10 minutes.

6. Blank test

After distillation of a sample, perform a blank test by using the above described method, but using 5 ml of distilled water.

7. Titration

Titrate the obtained distillate with 0.25N hydrochloric acid until the solution turns from green to violet.

Calculate the amount of nitrogen detected.

% Nitrogen = 14 x (V1-V0) x N / P

% Protein = % Nitrogen x F

Being:

P = Sample weight in g.
V1 = Volume of HCl consumed by titration (ml).
N = HCl normality.
V0 = Volume of HCl consumed by blank titration (ml).
F = Conversion factor to pass from nitrogen content to protein content. For crude protein a value of 6.25 is normally used. For greater accuracy, other conversion factors can be used by distinguishing the protein quality according to the sample nature.

10oct/12

Aspectos relevantes de la tecnología Peltier aplicada a las estufas incubadoras refrigeradas Selecta / Relevant aspects of Peltier technology applied to Selecta refrigerated incubators

From the first day of its activity, J.P. Selecta has tried to be aware of the latest technologies and methodologies in the field of laboratory equipments. Continuing with this innovative eagerness, as soon as the new electronic elements to produce both heat and cold through the principle called Peltier were known, a series of research projects aimed at creating a new refrigerated incubator based on this system started. Early work with Peltier elements for knowing technology started in the early 80's but it was not until 1990 when the first incubator finally hit the market as a world premiere, running only by Peltier elements.

Over these 32 years that we have had our incubators cooled with Peltier system on the market, effectiveness and utility have been proven, being this line of ovens the most prestigious of our brand.

Particularly it is worth mentioning a long list of advantages if compared with traditional ovens made with conventional heating elements and cold groups:

With Peltier technology, heating and cooling processes are conducted into one compact system. Both the excellent adjustment level and the oscillation reduced to the minimum of the temperature obtained occur thanks to the perfect development of refrigeration and heating technique, with a considerable energy saving and environmentally friendly.

Applications:

Biotechnology, Bacteriology, Plasma fractions, Biology, Enzymatic test, Research, Serum studies, Metrology, Botany, Phytopharmacy, Cosmetics, Water analysis, Industry, Agriculture.

Benefits:

BENEFITS
SPECIFICATIONS
at 5ºC at 37ºC at 60ºC
Stability ±0,05 °C ±0,05 °C ±0,05 °C
Homogeneity ±0,35 ±0,30 ±0,75 °C
Set point error ±0,25 °C ±0,20 °C ±0,40 °C

Models
Most important aspects:

  • The energy consumption reduction is up to 90%, when working with an ambient temperature of 22 °C.
  • No air exchange with the environment. Closed refrigeration system.
  • Hermetic refrigeration system.
  • Optimum temperature distribution.
  • Silent.
  • Stable.
  • Vibration-free.
  • Condensations free.
  • High accuracy.
  • Tempered glass inner door.

Peltier System Background
The discovery of thermoelectric phenomenon two centuries ago, and the search for new alternative energy generation, has allowed continued progress in thermoelectric technology in recent years. Since 1834 this is known as the Peltier effect; however, its practical application needed the development of semiconductor materials. The Peltier effect is characterized by the occurrence of a temperature difference between two sides of a semiconductor when a current flows through it. Generally such cells are made with Bismuth for the P type semiconductor face and with Tellurium for the type N face.

Operation Theory

The establishment of a heat flow, opposite to the thermal diffusion, when a material subjected to a temperature gradient is crossed by an electric current, suggests thermoelectric cooling applications. This alternative solution to the traditional refrigeration that uses compression-expansion cycles does not need moving parts, which increases its reliability. Besides being totally quiet, they have a very small size and weight, and they can easily handle bumps and vibrations, they can be used in any position, vertical, horizontal, inclined and also, thanks to them, cooling capacity can be adjusted by simply varying the feed stream.

What makes them even more interesting is the fact that, when reversing the supply polarity, their operation is also reversed, that is to say: the surface that formerly generated cold begins generating heat and the one generating heat, starts generating cold. These properties are essential in applications where the temperature must be controlled very precisely and reliably, such as in the containers used in transporting organs for transplantation or in those in which vibrations are serious disadvantages, for example: guide systems that employ laser, or integrated circuits. Furthermore, the possibility of creating a thermal flow directly from an electrical current makes the use of gases such as freon useless, as they are harmful to the ozone layer.

Moreover, the high reliability and durability of these systems (due to the absence of moving parts) have motivated their use in spacecraft electrical power, as in the Voyager spacecraft, launched into space in 1977. Here the heat flow established between the fissile material PuO2 (the PuO2 is radioactive and disintegrates, and so constitutes a heat source) and the outside passes through a thermoelectric conversion system based on SiGe (silicon and germanium thermocouple), thereby allowing electrical power of the probe (space probes cannot be powered by solar panels beyond Mars, as the solar flux is too weak).

As discussed below, conversion systems that use the thermoelectric effect have a very small output. Currently, their applications are limited to commercial sectors where reliability and durability are more important than price.

Some Typical Uses of Thermoelectric Cooling and Heating

LABORATORY AND SCIENTIFIC EQUIPMENT

Electronics, laser diodes, temperature control plates, and climate process chambers, freezing point reference baths, constant temperature baths, dew point hygrometers, osmometers, microscope stages.

TRANSPORT TECHNOLOGY

Moving Boxes, cabinets or containers for food distribution, medical equipments, and pharmaceuticals, by land, sea or air.

MEDICAL

Mobile or stationary storage of blood or pharmaceutical instruments, hypothermia blankets, coolers, ophthalmic cornea freezers, blood analyzers, preparation and storage of tissues.

MILITARY AND AEROSPACE

Constant temperature portable cases for blood distribution and pharmaceutical supplies, electronic devices, inertial guidance systems for cooling and heating, parametric amplifiers and other equipment on ships, submarines, trucks, aircrafts and spacecrafts.

Ricard Cardus

R & D Department

29sep/11

ENSAYO DE VINOS / WINE TESTS

CRYOTHERMOSTAT WITH STIRRER FOR WINE TESTS

Tartaric stability measurement of wines by Boulton test


J.P. SELECTA HAS DESIGNED A CRYOTHERMOSTAT WITH DRYING REFRIGERATED SYSTEM THAT DOESN’T NEED WATER DUE TO THE PELTIER EFFECT, AND INCORPORATING A STIRRER IN ORDER TO KEEP THE SAMPLE TO BE TESTED AT A STABLE TEMPERATURE.

BOULTON TEST
Tartaric stability measurement of wines: It is an analytical essay consisting of a quick precipitation of tartrate crystals, potassium acid, which are supersaturated in the wine.

The wine sample being analyzed is cooled to 0ºC and causes a quick precipitation of crystals by addition of approximately 10g/l of a potassium acid tartrate reagent powder.

The reduction of potassium is followed by a conductometric method.

When supersaturation is reduced to zero, there is no more precipitation and the conductivity value remains constant. Now the sample has the characteristics of a stable wine and this conductivity is the one which has to be taken into account. The conductivity measurement is very easy to do.

OPERATIONAL TECHNIQUE

  1. Pour 100ml of the wine sample in the beaker, measured with the probe.
  2. Set the stirring rod and place it in the bath over the stirrer.
  3. Insert the conductivity cell and the thermometer, if necessary, in the vessel and start stirring.
  4. If the conductimeter does not include temperature compensator, check the temperature in order it is stabilized at 0ºC.
  5. Then add a gram of potassium acid tartrate and read the conductivity every 2 minutes.
  6. Continue with the readings until you check that conductivity is the same for two or three consecutive times.

NOTES

A) The final conductivity value will be the one corresponding to this stabilized wine.This value will be compared with the cold treatment sample to determine the moment when stability is reached.
B) The difference between conductivity before adding bitartrate and final conductivity provides a potential stability measure with respects to bitartrate. In general, if this difference is lower than 5% of initial value, wine is stable; but if it is higher than 5%, wine is unstable.

Information provided by: Oenology department. Mrs. Valvanera Martínez de Toda Vallilengua – Technical Direction of BODEGAS COSTERS DEL SIO - LLEIDA.
Distributor: SOLTEVI, S.L - Vilafranca del Penedés.

21jun/11

DETERMINACION DE PROTEINAS POR EL METODO DE KJELDAHL / KJELDAHL METHOD FOR PROTEIN DETERMINATION

Francisco Santiago. Engineering Department.

INTRODUCTION

The total protein content in food is made up of a complex mixture of proteins. These exist in a combination with carbohydrates or lipids, which may be physical or chemical. Currently, all methods for determining the total protein content of foods are of empirical nature. An absolute method is isolation and direct weighing of the protein but this method is only used occasionally in biochemical research, as it is difficult and not very practical.

In 1883, the Danish researcher Johann Kjeldahl developed the method currently most used for protein analysis (Kjeldahl method) by organic nitrogen determination. In this technique, proteins and other organic components of food are digested in a mixture with sulfuric acid in the presence of catalysts. The total organic nitrogen is converted by the digestion in ammonium sulfate. The digested mixture is neutralized with a base and distilled later. The distillate is collected in a boric acid solution. Borate anions thus formed are titrated with standarized HCl (or H2SO4) to determine the nitrogen content in the sample.

The analysis result is a good approximation of crude protein content of the food as nitrogen also comes from non-protein components.

Kjeldahl method has undergone several modifications. Originally, potassium permanganate was used to carry out the oxidation process (digestion); however, the results were not satisfactory, so that reagent was discarded. In 1885, Wilforth found that digestion could be speed up by using sulfuric acid and adding a catalyst.  Gunning in 1889 proposed the addition of potassium sulfate, which raises the boiling point of sulfuric acid used in the digestion, in order to reduce the reaction time.

Nowadays, copper sulfate pent hydrated CuSO4.5H2O is mainly used as a catalyst.

REACTIONS CARRIED OUT IN THE KJELDAHL METHOD

DIGESTION

catalysts→
(1) n - C -NH2 + mH2SO4 CO2 + (NH4)2 SO4 + SO2
Protein                                  heat→

NEUTRALIZATION AND DISTILLATION

(2)  (NH2)SO4 + 2 NaOH                        →                     2NH3 + Na2SO4+ 2H2O
(3)  NH3 + H3BO3 (boric acid)                  →                 NH4 + H2BO3- (borate ion)

TITRATION

Borate anion (proportional to the amount of nitrogen) is titrated with standarized HCl (or H2SO4):

(4)    H2BO3- + H+               → H3BO3

MATERIAL
J.P. SELECTA provides the necessary equipment for carrying out the process by the Kjeldahl method.

- 1 electronic burette “Digitrate-Pro 50” of resolution 0.01ml.
Part No 0182026.

-1 precision analytical balance “FA-2204B” of resolution 0,1mg. 
Part No.
5830039.

- 1 Digestion unit (Bloc Digest) for 6, 12 and 20 positions. 
Part No.
4000629, 4000630 and 4000631.

- 1 Scrubber unit. 
Part No. 4001611

 - 1 Water circulation pump.
Part No.
4001612

- 1 Kjeldahl distiller (Pro Nitro M, S or A). 
Part No.
4002627, 4002851 and 4002430.

- Laboratory miscellaneous material.

REAGENTS

Reagents prepared:

It is recommended to use reagents already prepared, specially the titration HCl (or H2SO4). Any error in its preparation may directly affect the determination result.

Use the following reagents, or other equivalent trademarks:

•      Boric acid (powder) 99.5%   PANREAC 141015
•      Mixed indicator 4.8 (or 5) RV   PANREAC 283303

(Methyl Red + Bromocresol Green)

•      Mixed indicator 4.4 RV   PANREAC 282430

(Methyl Red + Methylene Blue)

•      HCl 0.1N SV   PANREAC 171023
•      HCl 0.25N SV   PANREAC 182318
•      H2SO4 0.1N SV    PANREAC 181061
•      H2SO4 0.2N SV    PANREAC 182011
•      Sodium hydroxide 40% RE   PANREAC 171220

(For N determination)

•      Acetanilide 99% (Standard for validation)   PANREAC 151005
•      Ammonium sulfate (Standard for validation)   PANREAC 131140
•      Kjeldahl catalyst 6.25% Cu tabl. 8g   PANREAC 174428

Preparation of ammonia fixative solution.

1.- With mixed indicator 4.8 (or 5):

Prepare the solution of the following formula for 1 litre of fixative solution:

•      Weight 10g of boric acid (powder).   PANREAC 141015
•      Dissolve in 1 litre of distilled water.
•      Add 15ml of mixed indicator 5.    PANREAC 283303

2.- With mixed indicator 4.4:

Prepare the solution of the following formula for 1 litre of fixative solution:

•      Weight 10g of boric acid (powder).   PANREAC 141015
•      Dissolve in 1 litre of distilled water.
•      Add 10ml of mixed indicator 4.4.   PANREAC 282430

PROCEDURE

SAMPLE PREPARATION

•   Triturate, homogenize and mix the sample.
•   Weight between 1 and 2g of sample.
•   In samples with very small nitrogen content, take sample enough to contain at least 5mg of     nitrogen.

DIGESTION

•   Add between 10 and 15ml (macro tube) of H2SO4 96-98% and 1 tablet (8 gm) of catalyst
(For micro tube, the H2SO4 maximum is 5ml).
•   Build a system for fumes extraction or a scrubber with Na2CO3.
•   Perform the digestion in three steps:

1. Depending on the sample water content, begin digestion by evaporating water at 150ºC     during 15-30 minutes.
2. Make a second step between 270 and 300ºC during 15 or 30 minutes to reduce production     of white fumes.
3. Continue the digestion at 400ºC between 60 and 90 minutes.

Some examples:

Cheese or meat:
Step 1: 150ºC / 30’              Step 2: 270ºC / 30’              Step 3: 400ºC / 90’

Cereals:
Step 1: 150ºC / 15’              Step 2: 300ºC / 15’              Step 3: 400ºC / 60’

Visual control: The result is a clear transparent liquid with light blue, green or yellow colour, depending on the catalyst used. There should be no black residues attached to the tube wall.

Note: During digestion, the samples foam production must be controlled. If this is excessive, step 1 should be extended.

DILUTION

•   Take the sample tubes out of the digestor block and let them cool at ambient temperature     (this could be forced by carefully immersing the tubes in a little water).
•   Add about 25ml of distilled water on each tube.
•   Add the water slowly and shaking the tube in order not to solidify the sample. If necessary,     heat slightly the tube (for example, by inserting it in the digestor block still hot).
•   Cool again until it arrives at ambient temperature.
•   To prevent nitrogen losses and violent reactions, do not insert the tube still hot in the distiller.

DISTILLATION

•   Place a 250ml Erlenmeyer flask at the coolant outlet with 50ml of boric acid and some drops     of indicator.
•   Program a dosage of NaOH from 50 to 75ml.
•   Insert the sample tube in the distiller.
•   Distillate it to collect 250ml in the Erlenmeyer flask (50ml boric + 200ml distilled).

Visual control: Once added the NaOH, the sample must have a bluish colour. If not, please add more NaOH.

TITRATION AND CALCULATION

•   Titrate the distillate with HCl or H2SO4, until the colour changes (endpoint: pH 4.65)
HCl moles = NH3 moles = N moles in the sample
H2SO4 moles = 2 NH3 moles = 2 N moles in the sample
•   Do the calculation:

mg N = N x  V   x  14

Where:

N = Titration acid normality.
V = Consumed acid volume.
14 = Nitrogen atomic weight.

•   To change to protein content, correct it by the appropriate factor according to the sample     nature (6.25 by default).
•   Periodically perform a blank test and subtract it from the result.

% proteins =  P2/P0 x 100 x F

Where:

P2: Nitrogen (mg).
P0: Sample weight (mg).
F: Protein factor.
(6.25 by default)

Protein factor in some foods:

Almonds 5,18
Nuts 5,30
Nuts - peanuts 5,41
Jelly 5,55
Soya 5,71
Barley, trenches, rye 5.83
Wheat, whole flour 5,83
Flours (not whole ones) 5,70
Rice 5,95
Corn 6,25
The rest of foods 6,25
Saved 6,31
Milk and milky products 6,38

VERIFICATION OF RECOVERY WITH AMMONIUM SULFATE

This verification is widely used to certify the distiller operation.

You have to prepare a sample whose nitrogen content is known. Then distil, titrate with HCl 0.25N and calculate the detected nitrogen.

The nitrogen percentage detected on the sample is called nitrogen recovery.

Prepare the sample:

Nitrogen content of an ammonium sulfate sample:

Formula: (NH4)2 SO4
Molecular weight: 132.14
Factor: 14 * 2 / 132.14 = 0.212
mg of nitrogen = 0.212 * mg of ammonium sulfate.

•   Weight about 100mg of ammonium sulfate. The exact weight will be P0.
•   Nitrogen exact weight is:

P1 (mg) = P0 x 0,212

•   Distil by adding 25ml of NaOH.
•   Once distilled and titrated, we get the detected nitrogen (mg): P2

P2 = N x V x 14

Where:

N = Titration acid normality (HCl 0.25).
V = Consumed acid volume.
14 = Nitrogen atomic weight.

•   Calculate the recovery:

R (%) = P2 / P1 *100

•   An acceptable recovery is between 99.5 and 100.5 %.

If we use HCl of different normality, prepare samples:

Normality Ammonium sulfate samples

0,05                               20... 40mg

0,1                                  40... 90mg

0,25                               100… 200mg

0,5                                 200… 400mg

VERIFICATION OF RECOVERY WITH ACETANILIDE

This verification is widely used to certify the complete process operation of the Kjeldahl nitrogen analysis, which includes digestion, distillation and titration stages.

You have to prepare a sample whose nitrogen content is known. Then digest, distil, titrate and calculate the detected nitrogen.
The nitrogen percentage detected on the sample is called nitrogen recovery.
Acetanilide is used to do this verification.

Prepare the sample:

Nitrogen content of an acetanilide sample:

Formula: C8H9NO
Molecular weight: 135.17
Factor: 14 / 135.17 = 0.1035
mg of nitrogen = 0.1035 * mg of acetanilide.

•   Weight about 250mg of acetanilide. The exact weight will be P0.
•   Nitrogen exact weight is:

P1 (mg) = P0 x 0,1035

Sample digestion:

•   Place the acetanilide in the tube, add 10ml of 98% sulfuric acid and a Kjeldahl catalyst tablet.
•   Digest at 400ºC for 1h (The result must be a transparent liquid with blue colour).
•   Cool and add 25ml of distilled water on each tube. Take care to avoid spillage. Water reaction     over sulfuric acid is violent.

Distillate:

•   Distil by adding 75ml of NaOH. Use HCI 0.25N for titration.
•   Once distilled and titrated, we get the detected nitrogen (mg): P2

Where:

N = Titration acid normality (HCl 0.25).
V = Consumed acid volume.
14 = Nitrogen atomic weight.
P2 = N x V x 14

•   Calculate the recovery:

R (%) = P2 / P1 *100

•   An acceptable recovery is between 99.5 and 100.5 %.

ERROR FONTS

The main error fonts are:

During the digestion process:

1.- The inclusion of non-protein nitrogen (although the amount of this nitrogen is usually negligible compared with the protein nitrogen one).
2.- Nitrogen loss during digestion:
An excess of sodium or potassium sulfate added to the acid to raise the boiling point may produce decomposition by the heat and therefore a loss of nitrogen.
On the other hand, the catalyst excess (generally copper) may also produce nitrogen loss.
3.- Sample incomplete digestion:
It is usually due to a lack of reaction time or sulfuric acid.

During distillation:

1.- Incomplete neutralization of digested mixture. It is necessary to add enough NaOH to neutralize the sulfuric acid excess resulting from the digestion as well as to transform the entire ammonium formed in the ammonia digestion.
2.- Ammonia loss due to leaks in the distillation circuit.
3.- Ammonia loss due to insufficient cooling in the condenser.

BIBLIOGRAPHY

A.O.A.C. 1980. Association of Official Agricultural Chemists.  Official Methods of Analysis. Washington, D.C.
Nielsen, S.S. 1994. Introduction to the Chemical Analysis of Foods. Ed. Jones and Bartlett Publishers. U.S.A. pp 209-212.
Ranganna, S. 1977. Manual of Analysis of Fruit and Vegetable Products.  Ed. McGraw-Hill Publishing Co. Ltd. New Delhi.
Yeshajahu P. & Meloan C.E. 1987. Food Analysis. Theory and Practice. 2on Ed. AVI U.S.A. pp 753-758.

29abr/11

INCUBATOR CO2

INCUBATORS FOR ANAEROBIC CELL AND TISSUE CULTURES “INCUBATOR CO2”

CO2 Incubators are one of the basic elements of any research laboratory working in the area of cell biology.

Recently, its use is increasing due to the work increase in specific areas.

CO2 Incubators are a great working tool that can facilitate and accelerate the delivery of results, either in traditional biologic science jobs for growing anaerobic cell and tissue cultures, in molecular biology and also in emerging and rapid growth fields such as stem cells, vitro fertilization, new pharmaceuticals, oncology, etc.

To meet the most demanding needs of any advanced laboratory, J.P. Selecta manufactures INCUBATOR CO2 incubators, which comply with the requirements of the upper-medium segment of the market, both for its reliability and durability as for its accuracy and ease of programming.

INCUBATOR CO2 incubator complies with DIN 12880 Class 3.1 Safety standard including a second independent over temperature control, CO2 set point deviation indicator, door open indication, lack of CO2 pressure and lack of power energy.

It also incorporates the latest technologies such as CO2 monitoring by infrared sensor, microprocessor total control, programming and management by a single knob in conjunction with a large alphanumeric display user interactive.

It can be externally controlled via a computer. It also allows processes to be recorded to a “pen-drive” by attaching the optional USB connection accessory and also can be printed with the printer that can be optionally installed.

It includes chamber sterilization, and has a double door, interior reinforced glass with silicone gasket, and outdoor with heating system to prevent condensations on the glass door.

The moisture content remains at a constant 98% RH level, produced by the evaporation of water from the interior cuvette. It also has a very complete alarm system. These alarms are categorized as follows:

  • Door open for more than 20 seconds.
  • Incubator chamber temperature exceeds the alarm temperature set by the user for more than 7 minutes.
  • Low CO2 supply pressure for more than 20 seconds.
  • Measured CO2 concentration value exceeds the maximum fixed by the user. Close the CO2 valve.
  • Lower chamber temperature exceeds the minimum programmed value.
  • Measured CO2 concentration value is lower than the minimum fixed by the user.
  • CO2 valve open for more than 7 minutes in interrupted.
  • Difference between the two probes higher than 2ºC for more than 5 minutes.

Technical Features

- Steel case coated with epoxy.
- Thermal insulation around the used chamber.
- Inner chamber made of stainless steel.
- Interior door of tempered glass with silicone gasket.
- Heated external steel door with electromagnetic lock.
- CO2 input for Ø 6 and 4 mm tube with microfilter.
- Front sampling to analyze the CO2 concentration.
- RS232 connexion
- Controlled humidity level at 98% H.R.
- Control and programming by a single knob.
- LCD alphanumeric screen of 2x40.
- Temperature and CO2 digital control by microprocessor.
- Temperature range from ambient +5ºC to 50ºC.
- Stability ±0.2ºC to 37ºC.
- Homogeneity ±0.5ºC to 37ºC.
- Resolution: 0.1ºC
- Alarm range: from ambient +5ºC to 50ºC.
- CO2 range from 0 to 20%.
- CO2 stability: ±0.3%
- CO2 resolution: 0.1%
- Capacity 150L. with 9 shelf guides.
- External measures: 95cm height x 65cm width x 73cm depth
- Inner measures:65cm height x 50cm width x 46cm depth
- Weight: 110Kg
- Power consumption: 800W

10dic/10

AUTOCLAVES DE LABORATORIO / LABORATORY AUTOCLAVES

STERILIZATION

Sterilization is the process by which destruction or elimination of all forms of microbial life, including bacteria and their structures, is achieved.

Laboratory autoclaves can perform sterilization by using steam at a higher pressure than atmospheric, and thus accumulating the steam temperature and reaching from 105ºC to 134ºC, according to the microorganisms to be destroyed. The steam penetrates the sterilization chamber at the set pressure; when condensating, it releases damp heat and simultaneously heating the material inside.

J.P. SELECTA offers a wide range of desktop, for dentistry and medicine, laboratory autoclaves being able to choose between manual purge, atmospheric or vacuum models, from which we highlight the following laboratory autoclaves:

"PRESOCLAVE II" for capacities of 50 and 80 litres, with electronic temperature, time and atmospheric purge regulation.

"AUTESTER ST DRY PV II" for capacities of 50, 80 and 150 litres, with microprocessor control of processes, drying system and vacuum fractional automatic purge.

This equipment has a RS232 output available for computer parameters printing, USB module for parameters storage and thermal printer with temperature, pressure, time and mode indications.

Only for information, please find below a relation of the different programs used for the different materials to be sterilized:

J.P. Selecta autoclaves meet with the Quality Management System Certificate UNE-EN ISO 9001, AENOR body accredited by ENAC with nº 01/C-SC003 and Safety Standards: EN 61010-1, EN 61010-2-040 and EN 61326.

9dic/10

VALIDACION DEL PROCEDIMIENTO DE ANALISIS DE N-KJELDAHL EN AGUAS RESIDUALES / N-KJELDAHL ANALYSIS PROCEDURE VALIDATION IN WASTEWATER

M. Monras, Degree in Chemistry (Institut Quimic de Sarrià);
Lourdes Margarit, Chemical Engineer (IQS), Analytical Chemistry Department;
Mª José Blanco, Quality Management (IQS);
David Pecanins, Quality Department Engineer (J.P. SELECTA, s.a.)

0. SUMARIO

In this research, the N-Kjeldahl analysis procedure has been validated in wastewater, in a concentration range between 20 and 1000 mg N/L, including the uncertainty calculation associated with the analysis.

All the analyses have been carried out with a JP Selecta “Bloc-digest” digestion unit and with a JP Selecta PRONITRO “A” automatic distillation unit.

1. INTRODUCTION

In the food and environmental field, there’s a great interest in the determination of nitrogen content of a sample. In 1883, the Danish chemist Johan Kjeldahl (1849-1900) first introduced to the “Danish Chemical Society” a method for determination known as “Kjeldahl nitrogen”, which provides the organic nitrogen content plus ammoniacal nitrogen of a sample. (1)

From this date, the Kjeldahl method has been the subject of an ongoing study in the analytical chemistry field and has been optimized in the three stages in which it is based: (2)

1. Sample digestion in acid medium: most of the organic and ammoniacal nitrogen to ammonium ion conversion takes place in this stage. The digestion process efficiency has been improved over the years with the addition of inorganic salts, to increase the sulphuric acid boiling point, the use of digestion blocks (introduced in 1970 by the Swedish chemist Roger Moosberg), the digestion time and temperature optimization and the use of different substances as catalysts.

2. Basification and distillation of the sample: ammonia is released in this stage, which is held in a solution with a known acid amount or with a boric acid solution. Initially, a simple distillation with a stem bath was performed, but now it has been replaced by a steam water distillation, which allows a significant reduction of the analysis time.

3. Acid-base evaluation: this could be a backward evaluation, if the ammonia released has been collected on an acid solution, or a direct evaluation, if the ammonia released has been collected on a boric acid solution. Initially, the evaluation end point was detected by means of an indicator colour change; later on, the automatic values were introduced with the detection of the evaluation end point by potentiometry.

Today, instrumentation in chemical analysis laboratories tends to automation. Thus, there are some equipments in the market that cover several stages of the Kjeldahl method, making it faster and easier of use, such as JP Selecta PRONITRO “A” automatic distiller.

Kjeldahl method is a reference method for ammoniacal and organic nitrogen determination and it is accepted by major international organizations such as International AOAC, EPA, AACC, AOCS, ISO or USDA. Nowadays, any laboratory working in a quality environment must demonstrate reliability of the data generated. Thus, it is necessary that each laboratory validates the analytical methods used.

2. MATERIALS

Reagents and Standards:

  • Ammonium sulphate standard.
  • Acetanilide standard.
  • Catalyst mineralization (it is prepared by intimately mixing 0.1g copper sulphate per gram of potassium sulphate).
  • Concentrated sulphuric acid.
  • Sodium hydroxide solution 40 % (p/v).
  • Hydrochloric acid (standard solutions 0.100 N and 0.500 N).
  • “Ammonium fixing solution” commercially prepared (composition: 1g of boric acid, 0.75mg of methyl red, 1mg of bromocresol green, 1.5mL of methanol, up to 100 mL with water).
  • Deionised water milli-Q quality.

Samples:

- Industrial wastewaters.
- Sewage from a comparison between laboratories.

3. EQUIPMENT

JP Selecta “Bloc Digest” digestion unit.

Unidad de digestión "Bloc-Digest"

- JP Selecta PRONITRO “A” automatic distiller.

JP Selecta PRONITRO “A” automatic distiller is an equipment which performs nitrogen analysis by Kjeldahl method. This equipment measures out the amount of reagent required for the analysis, makes a steam distillation of the sample from the digestion stage and an on-line evaluation of distillate, automatically detecting the end point by colour change.

The equipment allows a tritant reagent dosage up to 30mL and a concentration selection between 0.01 and 0.50N in increments of 0.01N. Thus, by appropriately selecting the tritant acid concentration, one can determine nitrogen in sample range between 0.5 and 200mg. (3)

4. EXPERIMENTAL METHOD

All the analyses in this work have been carried out by using the following methods: (4)

  • Insert in a sample tube, the volume of wastewater for the analysis, 1g of mineralization catalyst and 6mL of concentrated sulphuric acid.
  • Place the sample tube in the digestor block at a temperature slightly above 100ºC during enough time for the water to evaporate.
  • Increase the digestor block temperature to approximately 420ºC and maintain it enough time to produce the sample total digestion. When the sample becomes a transparent clear green-blue colour, we consider the digestion has finished.
  • Samples are cooled down at room temperature and about 25mL of milli-Q water is added.
  • Analyze the sample tube content from the digestion with the JP Selecta PRONITRO “A” automatic distiller, selecting the tritant reagent concentration according to the estimated nitrogen amount in the sample.

5. VALIDATION PROTOCOL (5) (6)

The above described method validation covers the range from 20 to 1000mg N/L. In order to do this, we work in three concentration levels: low (around 20mg N/L), medium (around 500mg N/L) and high level (around 1000mg N/L).

The low concentration level (ad1 sample) is daily prepared from acetanilide standard and milli-Q water. Whereas in the medium (ad2 sample) and high levels (ad3 sample) the concentrations are prepared by adding acetanilide standard to the industrial wastewater sample.

5.1. LINEARITY

The Linearity study is performed with standard solutions of ammonium sulphate.

The obtained results are presented in tables 1 and 2.

Nominal value
Experimental values
N/ mg (1)
HCl/ mL(2)
N / mg(3)
Recov./%
CV/%
white 0.26 --------- --------- ---------
0.50 2.05 0.50 99.9 1.80
1.00 3.74 0.97 97.5 1.58
2.00 7.27 1.96 98.1 1.05
4.00 14.78 4.07 101.7 1.63
5.00 18.32 5.06 101.2 0.56
6.00 21.83 6.04 100.7 0.57
8.00 29.32 8.14 101.7 0.65

Table 1. Results obtained by using HCl 0.02 N as tritant reagent.

Nominal value
Experimental values
N/ mg (1)
HCl/ mL(2)
N / mg(3)
Recov./%
CV/%
White 0.04 --------- --------- ---------
20 2.81 19.4 97.0 1.65
40 5.67 39.4 98.5 1.61
50 7.21 50.2 100.5 0.08
70 9.78 68.2 97.5 1.75
100 14.50 101.3 101.3 0.21
125 18.19 127.1 101.7 0.19
150 21.88 152.9 102.0 0.19
175 25.43 177.8 101.6 0.19
200 29.10 203.5 101.8 0.14

Table 2. Results obtained by using HCl 0.50 N as tritant reagent.

The average values of three consecutive determinations made under the same conditions are shown in each table row.

(1) Amount of nitrogen inserted by means of ammonium sulphate standard solutions.
(2) Amount of tritant reagent used.
(3) Amount of nitrogen derived from experiments in the analysis. This is calculated using the following formula: mg N = (mL HCl used – mL HCl white)*14.007*NHCl

5.2. INTERMEDIATE PRECISION AND RECOVERY

The intermediate precision and recovery study is performed by analysing, on three different days, the following samples sequence: two whites (one analyzed with HCl 0.02N, while the other with HCl 0.10N), a wastewater sample (since knowing its concentration will allow a later on calculation of recovery in every concentration level), a low concentration level sample (ad1 sample), a medium concentration level sample (ad2 sample) and a high concentration level sample (ad3 sample), getting the following results:

(Press the image to ampliate)

Table 3. Results obtained in three different days.

Below you can find the recovery data analysis obtained according to the concentration level, the analysis day and globally.

Low level
(ad1 sample)
Medium level (ad2 sample)
High level
(ad3 sample)
Average
Value
Average/%
97.5 99.0 98.3 98.2
S/%
3.6 1.2 0.8 2.0
CV/%
3.7 1.3 0.8 2.1

Table 4. Statistical parameters according to the concentration level studied.

5.3. QUANTIFICATION LIMIT

To study the quantification limit, we work with ammonium sulphate standards, tritant reagent HCl 0.02 N and three replicates are made. Results obtained are shown in the following table:

Nominal value
Experimental values
N/ mg
HCl/ mL
N / mg
Recov./%
CV/%
white 0.26 --------- --------- ---------
0.1 0.59 0.09 90.6 11.71
0.2 0.91 0.18 90.6 4.09
0.5 2.05 0.50 99.9 1.80

Table 5. Results obtained for the equipment quantification limit determination.

From these data we conclude that the equipment quantifies with a satisfactory accuracy and precision from 0.5mg of Nitrogen. Bearing in mind that the usual sample volume is 50mL, the equipment quantification limit is set to 10mg N/L.

When working with samples, the lowest concentration level studied (ad1 sample) has been of 20mg/L, so when using a sample volume of 50 mL, this means that the equipment quantifies around 1 mg N.

5.4. UNCERTAINTY

From the validation results obtained, uncertainty (u) is calculated associated to analysis, to each concentration level, by using the following formula: (4)

(Press the image to ampliate)

Expanded uncertainty (u) is calculated associated to analysis, as U=k*u, with a coverage factor of k=1.96 for a significance level of 95%.

So, the result of an analysis can be expressed as x ± U*x / 100.

The following table shows the uncertainty calculation values for each concentration level studied:

Concentration level
u standard /%
u accuracy/%
u precision /%
u/%
U (k=1.96)/%
Low level
0.29 2.48 3.67 4.44 8.7
Medium level
0.29 1.02 1.26 1.65 3.2
High level
0.29 1.74 0.80 1.93 3.8

Table 6.

6. ANALYSIS OF A WASTEWATER

A wastewater sample has been tested from a comparison between laboratories. A couple of blanks and four repetitions of the sample have been tested on the same day and under the same operating conditions, according to the procedure described in paragraph 3, using a sample volume of 50mL and as a tritant reagent HCl 0.05 N, with the results shown below:

Sample
Tritant reagent volume/ mL
Concentration/ mgN.L-1
Blank
0.10 --------
Blank
0.08 --------
Replication nº1
1.99 26.61
Replication nº2
1.83 24.37
Replication nº3
1.98 26.47
Replication nº4
1.85 24.65

Table 7. Water analysis results from a comparison between laboratories.

Then, the results obtained are compared to the results provided in the comparative between laboratories.

Results obtained
Result between laboratories (*)
Recov/%
Average/ mgL-1
25.5 27.0 94.4
S/ mgL-1
1.18 3.27
CV/ %
4.62 12.1

Table 8.

(*) Circuit Calitax. Agencia Catalana de l’aigua. October 2002

According to the uncertainty calculation made in the previous section (low concentration level), the result obtained in this analysis can be expressed as 25.5 ± 2.2 mg N.L-1.

7. CONCLUSIONS

1. The equipment provides satisfactory results working with standards in the range between 0.5 and 200 mg of Nitrogen, with recovery values between 97 and 103 %.
2. The accuracy of the analysis method applied to wastewaters is considered satisfactory, as recoveries between 95 and 102% are obtained.
3. The method intermediate precision is also considered satisfactory, since we obtain variation coefficient values of recoveries lower than 5% in all cases. It should also be noted that in medium and high concentration levels, these variation coefficient values are lower than 2%.
4. A result of 25.5 ± 2.2 mg N.L-1 has been obtained in the water analysis from the comparison between laboratories. This range includes the value 27.0 mg N.L-1 considered true.
5. The JP Selecta Automatic Distiller PRONITRO “A” is an easy to use and maintenance equipment, and saves time in Nitrogen determinations by Kjeldahl method.

8. BIBLIOGRAPHY

(1) Thiex, N.J., Manson, H., Anderson, S., Persson, Determinacion of crude protein in Animal Feed, Forage, Grain, and Oilseeds by Using Block Digestion with a Copper Catalyst and Steam Distillation into Boric Acid: Collaborative Study, Journal of AOAC International, 85, 2, 309-317 (2002)
(2)
Ogg, Clyde L., Treatise on analytical chemistry: edited by I.M.Kolthoff and Philip J.Elving with the assistance of B.Sandell, Vol x. Part 2. Kjeldahl Method, ………John Wiley and Sons, New York
(3)
JP Selecta, JP Selecta Automatic Distiller PRONITRO “A” Instructions Manual (2006)
(4)
IQS internal procedure.
(5)
Several authors, 2001, Validación de métodos analíticos, AEFI, Barcelona.
(6)
Miller, J.N., Miller, J.C., 2002, Estadística y quimiometría para química analítica, 4th edition, Ed. Prentice Hall, Madrid.