Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/02—Devices for withdrawing samples
  • G01N1/22—Devices for withdrawing samples in the gaseous state
  • G01N1/2226—Sampling from a closed space, e.g. food package, head space
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
  • G01N1/40—Concentrating samples
  • G01N1/4022—Concentrating samples by thermal techniques; Phase changes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/02—Devices for withdrawing samples
  • G01N1/22—Devices for withdrawing samples in the gaseous state
  • G01N1/2226—Sampling from a closed space, e.g. food package, head space
  • G01N2001/2238—Sampling from a closed space, e.g. food package, head space the gas being compressed or pressurized
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
  • G01N2015/019—Biological contaminants; Fouling

Definitions

• the present invention relates to a method and a device for microbiological and / or particulate sampling and analysis of a cryogenic fluid, said fluid being stored or conveyed in the liquid state in a pressurized enclosure.
• cryogenic fluids such as nitrogen, carbon dioxide, oxygen, nitrous oxide and argon , used in the food, pharmaceutical, electronic and medical fields.
• cryogenic fluids such as nitrogen, carbon dioxide, oxygen, nitrous oxide and argon
• the particulate and microbiological control of cryogenic fluids is proving more and more often essential in certain industries to ensure the absence of chemical and / or biological contaminants, or of solid particles in food, pharmaceutical, electronic and medical products. made from or using these fluids.
• the detection and quantification of possible contaminants must also be able to be carried out with the greatest rigor and precision possible, both in terms of storage vessels (transport tanks, tanks, bottles, ...) and distribution (conveying lines, supply lines, ...) of the cryogenic fluid considered.
• document WO-A-92 05420 describes a process for withdrawing a fluid (gas or liquid) from a network under high pressure (up to 350 bars), in particular a network of lubricant, intended to recover a gaseous sample at ambient pressure, a priori 100% representative of the purity of the fluid circulating in the high pressure network.
• a fluid gas or liquid
• a network of lubricant intended to recover a gaseous sample at ambient pressure, a priori 100% representative of the purity of the fluid circulating in the high pressure network.
• an improved device for implementing this process further comprises a chamber with two lamellae spaced apart with little play, making it possible to create, by prolonged circulation (several months) of the fluid between these lamellae, biofilm type surface contamination. The subsequent analysis of this biofilm makes it possible to deduce the presence of contaminants in the fluid analyzed.
• This device as well as that for implementing the method described in WO-A-92 05420, requires a long time of use and does not make it possible to
• the aim of the present invention is to propose a microbiological and chemical sampling and analysis method of the aforementioned type, allowing the qualitative and quantitative analysis of the microbial as well as particulate load of a cryogenic liquid, without vaporization thereof. before sampling, nor prolonged circulation of the liquid in an associated sampling device.
• the subject of the invention is a method of the aforementioned type, in which successively the enclosure is connected to a sampling and analysis device comprising a selected filter compatible with the nature of the fluid to be analyzed, as well as with the temperature and pressure for storing or conveying the liquid to be sampled, a quantity which is determined from the cryogenic fluid in the liquid state is sent to the filter; and the filter is recovered for microbiological and / or particulate analysis.
• a sampling and analysis device comprising a selected filter compatible with the nature of the fluid to be analyzed, as well as with the temperature and pressure for storing or conveying the liquid to be sampled, a quantity which is determined from the cryogenic fluid in the liquid state is sent to the filter; and the filter is recovered for microbiological and / or particulate analysis.
• the filter and the part of said device upstream of the filter are sterile. before connection of the sampling and analysis device to the enclosure, said device is sterilized, at least in part, by thermal input or else by using a gaseous mixture containing ethylene oxide and carbon dioxide.
• said device is free of water.
• the sampling and analysis device comprises, in its part downstream of the filter, a nozzle for venting the cryogenic fluid, said nozzle is at calibrated discharge flow rate, and the quantity of cryogenic fluid sampled and analyzed is determined as a function of the sampling duration and of the storage or delivery pressure of the cryogenic fluid in the liquid state.
• the sampling and analysis device comprises, in its part downstream of the filter, a vaporizer and a gas flow meter and the quantity of cryogenic fluid sampled and analyzed is determined as a function of the sampling duration and of the value measured by the flowmeter.
• the sampling and analysis device comprises, located between the vaporizer and the gas flow meter, a second filter capable of filtering the gas from the vaporizer.
• the device comprises two vaporizers in series making it possible to carry out two successive stages of expansion of the gas.
• the liquid sampled and analyzed is under a pressure between 1 and 200 bars and at a temperature between -273 ° C and 30 ° C.
• the cryogenic fluid is nitrogen, carbon dioxide, oxygen, nitrous oxide, helium or argon.
• the invention further relates to a device for sampling and microbiological analysis of a cryogenic fluid, said fluid being stored or conveyed in the liquid state in a pressure vessel, which device comprises a selected filter compatible with nature of the fluid to be analyzed, as well as with the temperature and the pressure for storing or conveying the liquid to be sampled, means for connection to the enclosure arranged upstream of the filter, and means for discharging the fluid at the gaseous state disposed downstream of the filter, said connection and evacuation means being adapted to maintain the cryogenic fluid in the liquid state from the enclosure at least to the filter inclusive.
• the device comprises means for determining the quantity of cryogenic fluid sent to the filter for analysis.
• Said evacuation means comprise a nozzle for venting the cryogenic fluid, and said nozzle has a calibrated evacuation rate.
• Said evacuation means comprise a vaporizer and a gas flow meter.
• the device comprises, located between the vaporizer and the gas flow meter, a second filter capable of filtering the gas from the vaporizer.
• the device comprises two vaporizers in series capable of carrying out two successive stages of expansion of the gas.
• FIG. 1 is a perspective view of the device according to the invention, connected to a bottle for storing a cryogenic fluid in the liquid state;
• FIG. 2 is an exploded perspective view of a member of the device of Figure 1;
• FIG. 3 is a schematic view of a second embodiment of the device according to the invention, connected to a storage tank for a cryogenic fluid in the liquid state;
• FIG. 4 illustrates another embodiment of the invention implementing a filtration in two stages: filtration of the liquid under pressure and filtration of the gas obtained by vaporization at the outlet of the vaporizer;
• FIG. 5 illustrates an embodiment of the invention particularly advantageous in the case of liquid C0 2 implementing a double expansion.
• FIG. 1 are represented both a bottle 1 for storing liquid carbon dioxide under a pressure of 50 bars, and a device 2 for sampling and microbiological analysis of carbon dioxide, suitable for use on the bottle 1
• the device 2 essentially comprises:
• connection means 6 comprise, from upstream to downstream, that is to say in the direction indicated by the arrow S in FIG. 1, a tubular connection 10 adapted to be fixed in leaktight manner at the outlet of the bottle 1, and a closing valve 12, for example a flywheel valve or a quarter-turn valve.
• the venting means 8 comprise, from upstream to downstream, a closing valve 14, similar to the valve 12, and a nozzle 16 for evacuation to the air.
• the maximum evacuation flow authorized by the nozzle 16 is much lower than the output flow from the bottle 1.
• a safety rupture disc can be arranged between the valve 14 and the nozzle 16, in the form of '' a part of a thin-walled tube adapted to rupture in the event of overpressure.
• connection means 6 and the venting means 8 are suitable for, when the valves 12 and 14 are open, send, through the filtration member
• the device 2 in fact ensures that the cryogenic fluid leaving the bottle 1 in the liquid state vaporizes only at the level of the nozzle 16, the entire upstream part of the device 2 being under very close temperature and pressure conditions those of bottle 1.
• the nozzle 16 is a calibrated nozzle which makes it possible to know, as a function of the analysis time and the pressure of the liquid analyzed, the quantity of liquid withdrawn. For example, for a calibrated discharge flow nozzle sized at 3 liters / min and under 50 bar pressure, it was established by the
• the filtering member 4 comprises a filter support 20, shown alone in detail in FIG. 2, provided with a filter or a membrane for fixing contaminants contained in the liquid passing through the filter, the latter being described below.
• the filter support 20 comprises, from upstream to downstream, an inlet adapter 22, an inlet tray 24, a support grid 26 for the filtration membrane, an outlet tray 28 and an outlet adapter 30.
• the plates 24 and 28 have a series of screw receiving holes 32 adapted to keep the plates tight against each other by trapping the grid 26. Seals 34 are further provided for sealing the elements with respect to each other.
• This filter support 20 is adapted to withstand high pressures up to 200 bars, and is for example distributed by the company MILLIPORE.
• the filter or membrane used in the filtering member 4 is adapted to physically retain contaminants without degrading them, in the sense that these contaminants are not fixed in an irreversible manner, for example by chemical degradation as in a filter for the elimination of particles (the contaminants are therefore reversibly trapped).
• this filter is selected so as to be compatible with the nature of the cryogenic fluid to be analyzed, as well as with the temperature and the pressure of the liquid analyzed.
• the filter is chosen to be hydrophilic in nature, the carbon dioxide molecule being bipolar.
• the selected filter must be able to withstand these temperature and pressure conditions, without deterioration.
• the selected filter is further adapted to support, where appropriate, a sterilization step, described in detail below.
• a bubble point test carried out after sterilization and application of the liquid to be analyzed, makes it possible to check the resistance of the membrane to such treatment.
• This test known per se, consists of measuring the minimum pressure required to expel a liquid retained in a capillary from the filter by its surface tension, this pressure being inversely proportional to the diameter of the capillary.
• the bubble point test is carried out by pre-wetting the filter and gradually increasing the pressure applied upstream of the filter, then observing the appearance of gas bubbles downstream of the filter.
• the pressure at which a continuous bubble flow occurs is called bubble point pressure or bubble point. If this bubble point is significantly lower than that specified by the filter manufacturer, the filtration membrane is damaged. However, if the bubble point is greater than or equal to the manufacturer's specification, the integrity of the filter is confirmed.
• sampling and analysis device 2 The operation of the sampling and analysis device 2 will be described below, detailing in parallel an example of implementation of the sampling and analysis method according to the invention applied to carbon dioxide in bottles in the figure. 1.
• liquid carbon dioxide filtration member 4 is assembled, by placing one of the aforementioned filters on the grid 26 and by fixing the plates 24 and 28 one against the other by the screws 32. One fixes then sealingly on the member 4 the means 6 for connection to the bottle 1 and the means 8 for venting.
• the device 2 thus assembled is then sterilized by passage through autoclaving (typically about thirty minutes at 121 ° C.). After autoclaving, the device 2 is dried by prolonged passage of sterile dry air or alternatively by passage through an oven (typically 3 hours at a temperature of 80 to 100 ° C).
• Another possibility for sterilization is to use a method known as “dry”, using a sterilizing gas type “Stéroxal 10 ®", that is to say a gas mixture composed of 10% oxide ethylene and 90% carbon dioxide. More precisely, the interior of the device 2 is scanned by a stream of high pressure Stéroxal® of the order of 10 bars, then the valves 12 and 14 are successively closed so as to trap the Stéroxal® under a pressure of at least 6 bars inside the device 2. This trapped gas is allowed to act for several hours at room temperature before using the device 2.
• a sterilizing gas type “Stéroxal 10 ®” that is to say a gas mixture composed of 10% oxide ethylene and 90% carbon dioxide.
• the sterilized device must be free of water because it can be brought to operate at temperatures below 0 ° C, which would cause immediate freezing of the water on the surface of the filter and would prevent filtration of the cryogenic fluid. in the liquid state.
• the device 2 is placed on the bottle 1.
• the latter is then opened, then successively the valve 12 then slowly the valve 14 so as to circulate carbon dioxide in the liquid state in the device 2, at the outlet of which the carbon dioxide vaporizes in the air.
• Carbon dioxide is thus allowed to escape for a given time of a few minutes, 2 to 5 minutes for example, before successively closing the valves 14 and 12.
• the device 2 can then be removed from the bottle and disassembled in order to recover the filter of the filter holder 20.
• This filter is then subjected to microbiological analyzes, for example by depositing it on a nutrient agar which will allow the culture of microbiological contaminants, the abiotic contaminants being stable over time and fixed on the filtered.
• microbiological analyzes for example by depositing it on a nutrient agar which will allow the culture of microbiological contaminants, the abiotic contaminants being stable over time and fixed on the filtered.
• the hydrophilic nature of the filter ensures rapid and simple cultivation.
• the method according to the invention thus makes it possible to carry out, after sterilization of the device 2, a rapid pressure sample, directly from the cryogenic fluid in the liquid state.
• Microbiological culture makes it possible to identify microbiological contaminants such as bacteria, yeasts or molds, as well as to count these contaminants. As moreover we know the quantity of liquid withdrawn, we can quantitatively analyze the level of biological contamination of the fluid.
• the device according to the invention is simple to use and compact.
• the calibrated nozzle can be replaced by a standard nozzle, the determination of the quantity of liquid analyzed s '' by weighing before and after sampling.
• Figure 3 is shown a second embodiment of a device 40 for sampling and microbiological analysis of nitrogen in the liquid state.
• the device 40 comprises means 6 for connection to a reservoir 42 for storing liquid nitrogen at 12 bars, and a filtration member 4.
• the device 40 differs from the device 2 by means 44 for evacuation different from the venting means 8.
• These means 44 comprise, from upstream to downstream, a valve 46 similar to the valve 14, a vaporizer 48 and a gas flow meter 50, l downstream end 52 of the means 44 being for example constituted by a vent.
• the means 44 are therefore suitable for evacuating the liquid nitrogen leaving the filtering member 4 by vaporizing it, thus making it possible to measure its quantity using the gas flow meter 50.
• the sampling and analysis process using the device 40 is substantially similar to that described above. Since the nitrogen molecule is apolar, a hydrophobic filter is selected, thus avoiding the risks of plug formation in the filtering member.
• the use of such a hydrophobic filter moreover involves a step of recovering the microorganisms before they are cultured on an aqueous-based agar.
• the hydrophobic membrane which carries filtered contaminants is placed in a sterile physiological liquid; it is subjected to ultrasound to unhook all the contaminants fixed on this membrane, then the contaminants to be cultured are recovered on an agar, for example by filtering the physiological liquid on a hydrophilic membrane.
• the filter supports are removed and the filters treated according to the desired analysis: - immersion in an acid and oxidizing solution (for example HNO3 + H 2 O 2 ), in order to dissolve the metals, which are then assayed by a technique suitable for the determination of metals in aqueous solution;
• an acid and oxidizing solution for example HNO3 + H 2 O 2
• FIG. 4 illustrates another mode of implementation of the invention implementing a filtration in two stages:
• the second filter makes it possible to stop the contaminants which would not have been stopped upstream at the stage of the liquid filtration.
• the device therefore comprises the two filters 4 and 63 separated by an evaporator (coil 61 in a hot water bath 60) sized according to the desired flow rate.
• the evaporator associated with the flow meter 64 makes it possible to measure the volume of gas withdrawn.
• a first trigger typically around 6 bars, which allows the maximum calories to be absorbed for the vaporization of the liquid, without the formation of carbon dioxide snow, a second expansion at atmospheric pressure and supply of heat for the simple rise in temperature of the gas formed.
• a variant could consist in the use of a capillary well dimensioned in diameter and length, depending on the desired flow rate. This arrangement makes it possible to use the pressure gradient all along the capillary to vaporize at relatively high pressure without again risking the formation of carbon dioxide snow.
• FIG. 5 Such a double expansion structure is illustrated in FIG. 5, with the presence of two graduated needle valves 73 and 74, each upstream of one of the vaporization coils 71/72, the whole being again located between the torque of filters 4 and 63 (not shown in Figure 5).
• valve 74 upstream of valve 74 outside the bath: 0 ° C, 6 bars;
• valve 74 downstream of valve 74 out of the bath before reaching the second coil: -10 ° C, 1 bar; - upstream of filter 63 outside the bath: 30 ° C, 1 bar.

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• Engineering & Computer Science (AREA)
• Biomedical Technology (AREA)
• Molecular Biology (AREA)
• Physics & Mathematics (AREA)
• Chemical & Material Sciences (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Immunology (AREA)
• Pathology (AREA)
• Sampling And Sample Adjustment (AREA)
• Apparatus Associated With Microorganisms And Enzymes (AREA)

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• 2002
  • 2002-04-02 FR FR0204069A patent/FR2837924B1/fr not_active Expired - Fee Related
• 2003
  • 2003-03-31 EP EP03738214A patent/EP1493010A2/de not_active Withdrawn
  • 2003-03-31 AU AU2003244730A patent/AU2003244730A1/en not_active Abandoned
  • 2003-03-31 WO PCT/FR2003/000995 patent/WO2003083446A2/fr not_active Ceased

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