NL1022604C2 - Sensor, for measuring oxygen concentration in gas or liquid, includes membrane comprising alpha-alkene polymer with specific oxygen permeability - Google Patents

Abstract

Sensor membrane (4) is made from a homo- or copolymer of a linear or branched 2-10C alpha -alkene which has a specific minimum oxygen permeability. A sensor comprises a measuring head (1), a membrane module (2), a membrane holder (3) and a membrane made from a homo- or copolymer of a linear or branched 2-10C alpha -alkene. The polymer has an oxygen permeability of at least 10 x 10->1>0> ml.mm/s.cm2>.cm Hg.

Description

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Sensor for the amperometric determination of the amount of oxygen in a gaseous or liquid medium

The present invention relates to a sensor for the amperometric determination of the amount of oxygen in a gaseous or liquid medium.

Such a sensor is known from US 5,239,257, which describes a sensor consisting of an electrode, which comprises an anode and a cathode and which is arranged in a membrane module. The membrane module comprises a housing which is provided on one side with a membrane holder in which a membrane is arranged. The membrane shields the electrode from the medium containing the oxygen, the amount of which can be measured with the aid of the sensor. The membrane consists of three layers: the first and the third layer consist of a material that is resistant to chemical substances and the second layer or intermediate layer consists of a material with a relatively high permeability to oxygen. According to US 5,239,257, the first and third layers are preferably both made of polytetrafluoroethylene. The intermediate layer preferably consists of silicone rubber in which a mesh of stainless steel is included for imparting rigidity to the membrane.

It has been found that the sensor according to US 5,239,257 has various disadvantages when the sensor is used in measuring and controlling the concentration of oxygen. In fermentation processes or culture processes of micro-organisms that are carried out in bioreactors, measuring and controlling the concentration of oxygen is of great importance for an optimal course of these processes. In such processes, the bioreactor is first sterilized, for example, by thermal sterilization for about 20-30 minutes at a temperature in the range of about 120 ° - 135 ° C before the fermentation or culture medium is inoculated. Subsequently, the duration of the fermentation process or the breeding process can run from a few hours to several weeks. It will therefore be clear that the sensor must meet high requirements, such as resistance to the conditions under which the sterilization is carried out and the stability of the signal emitted by the sensor during the duration of the process. But sensors as described in US 5,239,257 have been found to be unsatisfactory in the applications described above: (1) the sensors exhibit a variable and therefore unknown. .t * *) ') £ 0 A "~ - H drift that has been shown to be as high as 15% per week; (2) after I sterilization, the sensors as described in US 5,239,257 only give after a long period of time (6 hours to 1 day) a stable signal, (3) the manufacture of the membrane according to US 5,239,257 is complicated and therefore expensive and further proves to be poorly reproducible, and (4) the response time of the sensors as described in US 5,239,257 is relatively slow, ie that in culture and fermentation processes where oxygen is consumed, the response time is of the same order of magnitude as the time required for the oxygen uptake making control of the culture or fermentation process difficult If the sterilization is carried out with γ radiation (gamma radiation), the I sensor according to US 5,239,257 has the further disadvantage that the first and third layer, which preferably consists of polytetrafluoroethylene, is not resistant to such radiation. .

The present invention provides a solution to the problems described above and relates to a sensor comprising a measuring head (1), a membrane module (2), a membrane holder (3) and a membrane (4), the membrane (4) ) is made from a homopolymer or a copolymer of a linear or branched alpha-olefin with 2 to 10 carbon atoms and wherein the homopolymer or copolymer has an oxygen permeability of at least 10 * 10'10 ml.mm/s.cm2.cm 1 Λ Λ

Hg, preferably of at least 100 * 10 'ml.mm/s.cm.cm Hg.

According to the invention, the sensor is suitable for determining the amount of oxygen in a liquid fermentation or culture medium. A schematic representation of the sensor is shown in Figure 1. The sensor comprises a measuring head (1) consisting of both a cathode (5) and an anode (6) as described in US 5,239,257, with I at the cathode oxygen is converted to OH 'at a potential of -675 mV. The measuring head (1) is preferably made from an inert material, in particular stainless steel, and provided with connecting means, for example an external screw thread (7), in order to be able to connect the measuring head (1) to the membrane module (2) .

The measuring head further comprises an O-ring (8) for hermetically shielding the measuring head (1) when it is mounted on the membrane module (2). The membrane module (2) can consist of one or more different materials, but according to a preferred embodiment, the membrane module (2) and the membrane holder (3) are made of the same material as the membrane. The membrane module (2) comprises, as stated before, a membrane holder (3) in which the membrane (4) is mounted. According to a preferred embodiment, however, the membrane module (2), the membrane holder (3) and the membrane (4) form one whole and this whole is also produced as such from the same material, that is to say the homopolymer or copolymer defined above. The membrane module (2) further comprises connecting means, for example an internal screw thread (9), in order to be able to connect the membrane module (2) to the measuring head (1).

The homopolymer or copolymer preferably has a density of 0.78 - 0.90 g / cm 3 according to ASTM D 1505, in particular 0.80 - 0.85 g / cm 3. The melt index of the homopolymer or copolymer is preferably 0.1 to 100 g / 10 min according to ASTM D 1238, in particular 1 to 50 g / 10 min. The melting point of the homopolymer or copolymer is preferably higher than 150 ° C and in particular higher than 200 ° C. The mechanical properties of the homopolymer or copolymer are preferably as follows: a tensile strength of 150-300 kg / cm 2 according to ASTM D 638 (test conditions 23 ° C, 5 mm / min.); • a tensile strength at break of 125 - 250 kg / cm 2 according to ASTM D 638 (test conditions 23 ° C, 5 mm / min.); • an elongation at break of 10-50% (test conditions 23 ° C, 5 mm / min.); A flexural strength of 250-500 kg / cm 2 according to ASTM D 790 (test conditions 23 ° C, 5 mm / min.); A stiffness (Olson) of 1 - 50 * 103 kg / cm 2 according to ASTM D 790 (test conditions 23 ° C, 5 mm / min.); • a shock resistance (Izod) of 1-50 kg.cm/cm2 according to ASTM D 256 25 (test conditions 20 ° C, 2 min.).

According to the invention, the polymer is in particular a copolymer of 4-methyl-1-pentene marketed by Mitsui Plastics under the trade name TPX. TPX is prepared in the following manner: (1) dimerization of propylene to 4-methyl-1-pentene, (2) polymerization of 4-methyl-1-pentene to a 4-30 methyl-1-pentene copolymer.

According to the invention, it is preferred that the membrane be resistant to a thermal sterilization carried out at a temperature of at least 120 ° C

1 022ft (U "H for at least 20 minutes and / or against γ-radiation sterilization with an H radiation intensity of at least 25 kGray.

The thickness of the membrane is preferably 1 μηι - 1 mm and more preferably 25 - 300 μm and in particular 50 - 200 μηι.

For a good response it is necessary that the measuring head is close to the surface of the membrane (distance between the measuring head and the membrane is preferably less than 100 μηι) and preferably the measuring head presses against the membrane, the force with which the pressure head against the membrane is greater than 1 g / mm2. In particular, this force is between 10-100 g / mm 2.

The invention will be further elucidated with reference to the following examples, which, however, are to be regarded as not limiting the scope of the invention.

Example 1

A sensor as described in US 5,239,257 was provided with a TPX membrane with a thickness of 100 µm. For the test, the pressure of the electrode on the membrane was varied and the tso response times were determined (0 - 100% and 100 - 0%). Air and nitrogen were used for calibration and response times. The tests were conducted with carbon dioxide as the test gas and the results are shown in

Table 1.

Table 1

Cathode pressure Sensitivity Zero current t90 (s)> 0% t90 (s)> 100% (g / mm2) (20 ° C) (20 ° C) 34 57 nA 1.7 nA 32 33 (slope 0.43) (offset 0 13) 65 59 nA 2.0 nA 28 28 (slope 0.45) (offset 0.15)

These results show that the TPX membrane is effective and that the cathode mk has little influence on the performance of the sensor.

25 5

If the sensor was exposed to pure carbon dioxide, a stable signal of 2.9% was obtained after 10 minutes (cathode pressure 65 g / mm 2). When air was subsequently supplied to the sensor, a value of 103% was obtained. When air was continuously supplied, it took 10 minutes to obtain a 100% signal. This shows that carbon dioxide gives the sensor a deviation of ± 3%.

Example 2

Four sensors as described in example 1 were provided with a TPX-10 membrane with a thickness of 100 μηι and a membrane holder with a diameter of 12 mm also made from TPX. Air and argon were used for the calibration. The sensors were exposed to thermal sterilization conditions (121 ° C, 20 minutes). The following results were obtained (see Table 2; Sensors 1 and 2 have low sensitivity).

Π Π /? R η λ - H Table 2 H For sterilization

Sensor Sensitivity Zero current t90 (s)> 0% t90 (s)> 100% (20 ° C) (20 ° C)

Sensor 1 252 nA 3.0 nA 8 7.5 (slope 0.83) (offset 0.10)

Sensor! 200 nA 3.0 nA 15.0 15.0 (slope 0.66) (offset 0.10)

Sensor 3 95 nA 0.98 nA 47 ^ 5 47 ^ (slope 0.72) (offset 0.07)

Sensor 4 82 nA 0.80 nA 37.0 35.5 (slope 0.63) (offset 0.06)

After sterilization

Sensor Sensitivity Zero current t90 (s)> 0% t90 (s)> 100% I (20 ° C) (20 ° C)

Sensor 1 299 nA 0.6 nA 6.8 6 I (slope 0.99) (offset 0.02) I Sensor 2 207.5 nA 3.0 nA '12, 0 12.0 I (slope 0.68) (offset 0.10) I Sensor 3 99 nA 0.61 nA 45.0 45.0 I (slope 0.75) (offset 0.04) I Sensor 4 81.6 (slope 0.48 nA 37.5 33 (I 0.62) (offset 0.03) I It appears from these results that the sensors can be exposed to thermal sterilization conditions without reducing performance. All sensors have an increased sensitivity and a reduced offset and a somewhat lower response time after exposure to the thermal sterilization conditions.

I Example 3 I Two sensors of the Applisens Low Drift type (110 mm; sensor 1 had a normal sensitivity, sensor 2 had a high sensitivity) were provided with an I membrane module and a membrane as described in example 2. These sensors I tn ?? an. - 7 were installed in a 3 liter Applikon bioreactor with a flat bottom. In addition to these two sensors, a sensor 3 of the AppliSens 4-pin DO sensor (corresponding to the sensor described in US 5,239,257) was provided as a reference. For calibration, the sensors were immersed in water (100% air, 0% argon; 32 ° C. Stirring speed 650 rpm). After 15 minutes the following values were obtained (see Table 3). The reactor was then emptied and flushed with argon for 15 minutes to determine the zero flow; these values were determined 1 hour after the sensors were connected to the measuring and control device (see Table 3).

10

Table 3

After immersing in water Sensor Sensitivity

Sensor 1 92 nA (slope 0.68)

Sensor 2 285 nA (slope 0.96)

Sensor 3 45 nA (slope 0.32)

After rinsing with Ar Sensor Zero Current

Sensor 1 5.4 nA (offset 0.41)

Sensor 2 2.1 nA (offset 0.07)

Sensor 3 0.7 nA (offset 0.05)

After calibration, the fermentation medium was composed in the bioreactor, checking whether the addition of each chemical had an effect on the operation of the sensors (the fermentation medium contained the following chemicals: PBS buffer, 500 g sugar, 81 g soybean culture medium; none of these substances were found to have an influence on the performance of the sensor). After the fermentation medium was ready, it was inoculated with 2 g of baker's yeast and fermented for two days at a temperature of 32 ° C with constant air input and stirring speed. The bioreactor was then emptied and filled with water, after which the response / drift (performance test) of all sensors with air (100%) and argon (0%; for 15 minutes) was tested (water temperature was 32 ° C, stirring speed 650 rpm, see v ^ ...

Table 4), so that a change in sensitivity before and after fermentation could be observed. The results are shown in Figures 2 and 3.

Table 4

After immersion in water Sensor Sensitivity

Sensor 1 92 nA (100% 02)

Sensor 2 300 nA (> 100% 02)

Sensor 3 48.2 nA (107.1% 02)

After rinsing with Ar Sensor Zero Current

Sensor 1 5.4 nA (0% 02)

Sensor 2 2.1 nA (0% 02)

Sensor 3 0.7 nA (0% 02)

The data from table 4 shows that the sensitivity of sensors 2 and 3 had increased.

In Figure 2 the relative oxygen concentration during the fermentation is plotted against the time: t = 0 - 30 hours. Start of the fermentation. The relative amount of oxygen in the fermentation medium decreases because the yeast cells multiply and thereby consume oxygen. Sensor 3 gives a signal that is approximately 10% lower than the signal from sensors 1 and 2 (sensor 1: 27.7%, sensor 2: 33.5%, sensor 3: 15 has 19.4% at 30 hours;% relative to the amount of relative oxygen).

t = 30 - 31 hours. Because sensors 1 and 2 gave a larger signal, the air supply was turned off to check whether all sensors still showed the corresponding zero point. Sensor 3 reached the zero point fastest, followed by sensor 2 and then sensor 1. Sensor 1 did not reach the calibrated zero point (sensor 1: 1.5%; sensor 20 2: 0.0%; sensor 3: 0.1% at 31 hours).

t = 31-33 hours. The air supply was resumed and the stirring speed was increased to 900 rpm, so that the fermentation medium was completely saturated with oxygen. In this way it was possible to check whether the sensors could still indicate 100% oxygen and whether drift had occurred (sensor 1: 99.2%; sensor 2: 103.9%; sensor 3: 94.3%).

t = 33 - 57 hours. The stirring speed was reduced to 650 rpm. It appears that the oxygen concentration increases due to a lack of nutrients, (the 5 yeast bacteria are multiplying more and more slowly). At 45 hours there were no more nutrients present and the relative oxygen concentration remained constant as sensors 1 and 2 correctly displayed; sensor 3 indicated an increasing relative oxygen concentration (sensor 1: 86.5%; sensor 2: 92.4%; sensor 3: 82.0% at 57 hours.

In Figure 3, the relative oxygen concentration during the performance test is plotted against time, with sensors 1 and 3 being monitored over a weekend (water temperature was 32 ° C, stirring speed 400 rpm). Sensor 1 gave a constant value between 99% and 100% and the variation of the signal was no more than +/- 0.5%. Sensor 3 did not give a constant value since it increased (drift) from 104% to 107%; the variation of the signal was approximately +/- 0.5%. This performance test shows that sensor 1 shows less drift than sensor 3.

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Claims (18)

A sensor comprising a measuring head (1), a membrane module (2), a membrane holder (3) and a membrane (4), wherein the membrane (4) is made from a homopolymer or a copolymer of a linear or branched α -alkene with 2 to 10 carbon atoms and in which the homopolymer or I copolymer has an oxygen permeability of at least 10 * 10 · 10 1 ml.mm/s.cm2.cm Hg. Sensor according to claim 1, wherein the oxygen permeability is at least 100 I 10 * 10 · 10 ml.mm/s.cm2.cm. Sensor according to claim 1 or claim 2, wherein the membrane module (2), the membrane holder (3) and the membrane (4) form a whole. Sensor according to claim 3, wherein the whole of the membrane module (2), the membrane holder (3) and the membrane (4) are formed from the same material Sensor according to any one of the preceding claims, wherein the homopolymer or the copolymer has a density of 0.78 - 0.90 g / cm 3. Sensor according to any one of the preceding claims, wherein the homopolymer or the I copolymer has a melt index of 0.1 to 100 g / 10 minutes. 7. Sensor as claimed in any of the foregoing claims, wherein the melting point of the homopolymer or the copolymer is higher than 150 ° C. A sensor according to any one of the preceding claims, wherein the homopolymer or the copolymer has a tensile strength of 150 - 300 kg / cm 2. A sensor according to any one of the preceding claims, wherein the homopolymer or the copolymer has a tensile strength at break of 125 - 250 kg / cm 2. I 25 Sensor according to any one of the preceding claims, wherein the homopolymer or the copolymer has an elongation at break of 10 - 50%. Sensor as claimed in any of the foregoing claims, wherein the homopolymer or the copolymer has a flexural strength of 250 - 500 kg / cm 2. 12. Sensor as claimed in any of the foregoing claims, wherein the homopolymer or the copolymer has a rigidity of 1 - 50 * 103 kg / cm 2. A sensor according to any one of the preceding claims, wherein the homopolymer or the copolymer has a shock resistance of 1-50 kg.cm/cm 2. I 1 noo αλ λ - .......... · ιιιπ ·· ^ Μτ ···· π ^ ίτ ^ · .τ ^ '' Μΐ jaaAfci ^ .. iiL-iirrgsTiiiri 'τ- ^: ψρ * "':' ··. ·· j ··.» ·. · ... _ _ ° · · - ^ · '"~“ 4 The sensor of any one of the preceding claims, wherein the polymer is a copolymer. The sensor of any one of the preceding claims, wherein the polymer is a 4-methyl-1-pentene copolymer. Sensor according to any one of the preceding claims, wherein the membrane has a thickness of 1 µm -1 mm. A sensor according to any one of the preceding claims, wherein the measuring head presses against the membrane with a force that is greater than 1 g / mm 2. 18. Sensor according to claim 17, wherein the material is the homopolymer or the copolymer as defined in one or more of claims 1-13. 1 022804 -