GB2171926A - A method and apparatus for reagent addition to a flowing liquid carrier stream - Google Patents

Abstract

Apparatus 24 and method for introducing reagent 32 to liquid chromatographic effluent carrier 11 or to the carrier resulting from flow injection analysis through permeation transfer through a porous membrane 42 of a predetermined porosity (volume average pore diameter 125 to 5000 ANGSTROM ) and area (0.2 to 150 mm<2>) for increased detection sensitivity and reduced sample bandspreading. Advantageously, the effluent carrier is conducted through a self-pressurized or externally pressurized reagent addition device 17. <IMAGE>

Description

SPECIFICATION A method and apparatus for reagent addition to a flowing liquid carrier stream The invention relates to an improved method and apparatus by which a reagent can be added to a flowing stream of a liquid carrier from a chromatographic column to enhance the sensitivity of detection and similarly to a flowing stream of a liquid carrier in the field of flow injection analysis.

The term "liquid carrier" signifies a liquid eluent when employed with a chromatographic column and includes the effluent from said chromatographic column.

Membrane reagent addition devices have been used to improve detection in modern high performance liquid chromatography (HPLC). One example of a membrane reagent addition device is disclosed in U.S.

Patent-No. 4,448,691. The membrane reagent addition device taught by that patent utilized hollow fiber membranes with a small pore size and relatively long lengths for the permeation transfer of mobile reagent into chromato-graphic column liquid carrier effluent.

The long lengths of hollow fibers required small pore sizes in the membrane to prevent excessive leakage of chromatographic column liquid carrier effluent into the mobile reagent.

Long lengths of membrane, with large pore sizes, suffered from effluent pressure drops along the length of the hollow fiber resulting in excessive effluent leakage across the membrane at the beginning section of the hollow fiber.

These-problems were partially solved by externally pressurizing the mobile reagent chamber in order to eliminate the initial excessive effluent leakage. However, when mobile reagent pressure was increased to- prevent excessive effluent leakage at the beginning of the fiber, excessive reagent permeation occurred at the end section of the hollow fiber causing significant bandspreading.

A need has long existed for a device which can limit bandspreading and still be associated with conventional liquid chromatography apparatus. An object of the invention is to limit bandspreading to less than 1000 yl.

Another object of the present invention is to provide improved liquid chromatographic and flow injection apparatus characterized by the development of an improved membrane post-column reagent addition device which utilizes a short length of porous hollow fiber membrane or a membrane in sheet form to minimize band-spreading along the length of the membrane. An object of the present invention is to provide an apparatus and method where only a minor amount, less than about 10 percent, of the liquid carrier is lost into the mobile reagent and substantially constant amounts of the mobile reagent permeate into the liquid carrier.

Yet another object of the invention is to provide an apparatus and a method for reducing band-spreading during mobile reagent addition into the liquid carrier from a chromatographic column or flow injection analyzer, and which essentially limits band-spreading to no greater than about 50 FI.

Still another object of the present invention is to provide an improved method and apparatus which achieves essentially constant and "pulseless" metering of the mobile reagent into the liquid carrier using a short hollow fiber porous membrane.

In an alternative embodiment of the method invention, the method can include the additional step of maintaining a difference between the average pressure inside the membrane and the average pressure of the reagent outside the membrane in a range of less than about 10 psig (70 kPa ).

In yet another embodiment of the method invention, the method can include the step of maintaining a pressure drop of the liquid carrier along the length of the membrane which is less than 10 psig ( 70 kPa ).

In still another embodiment of the method invention, the method can include the further step of externally pressurizing the reagent chamber.

Another problem experienced by known reagent addition devices is the tendency for pressure to be higher on the liquid carrier side of the membrane than on the reagent side. This pressure differential is caused by the structural features of the known devices and often exacerbated by the often used reaction coil or packed bed in fluid communication with the post-column reagent addition device and the detector.

The reaction coil or, in the alternative, the packed bed serves to homogenize the constituents of the carrier and in some cases, increase the time necessary to complete reaction between the carrier components. The reaction coil or the packed bed causes an increase in the flow resistance of the carrier stream and thereby reduces reagent permeation into the carrier stream in the known reagent addition device. In addition, the membranes of the known reagent addition devices tend to burst because of the excessive pressure differentials within the membranes.

In an attempt to overcome these problems, the reagent side of the membrane has been externally pressurized by applying air pressure to the reagent reservoir or alternatively, hydrostatically pressurizing the reagent reservoir with a pump. This apparent solution to the problems associated with known reagent addition devices has been troubled with safety problems, i.e., the air pressure used to pressurize the reagent is a safety hazard. More particularly, the stored energy of the compressed air can explode the device, spewing reagent throughout the neighboring area.

To overcome the above problems with known reagent addition methods and apparatus, the present invention also provides an improved device and method for safety adding a reagent to a liquid carrier from a chromatographic column with reduced potential for serious accidents. The invention more partic ularly provides an improved membrane reagent addition device which does not require external pressurization.

It is a significant advantage of the present invention to utilize a device which does not require external pressure applied to the mobile reagent The fact that the increase in pressure on the reagent comes from the interior of the container, that is, the increase in pressure results from the transfer of carrier into the mobile reagent, thereby increasing the pressure on the mobile reagent, reduces the possibility of explosion or leakage of hazardous chemicals in the lab area. Harmful back pressure on the membrane is controlled by using preferably a self-contained, hermetically sealed vessel and controlling carrier flow rate.

Accordingly, another object of the present invention is to provide an automatic, self-pressurizable quantity of mobile reagent for controlled permeation transfer of the reagent into the carrier.

A further object is to provide a method and apparatus, which in contrast with prior art methods and apparatus, reduces the complexity of the necessary hardware, and significantly reduces the cost to use and maintain the apparatus.

A final object of the invention is to provide an apparatus for use in liquid chromatographic and flow injection analysis which is compact and of significantly less cyst to use and maintain than presently known apparatus.

The term "mobile reagent" means a chemical species or combination of species essentially the sole purpose of which, when introduced through the membrane into the carrier, is to react chemically, directly or indirectly, with a sample species of interest or an interferring sample species less than perfectly resolved with respect to the sample species of interest, to-produce measurable enhancement in the detection of the species of interest, or a monitored proportional derivative thereof, compared to the absence of the membranel reagent combination. For example, a monitored proportional derivative can be the well known derivative agent orthophthaldehyde (o-PA) which reacts with primary gamines in the presence of thiol compounds.

In addition, the term "mobile reagent" means a chemical species or combination of species essentially the sole purpose of which, when introduced through the membrane, is to condition a carrier for enhanced detection of a sample species of interest. The phrase "condition a carrier" refers to a method for changing the carrier, such as changing carrier pH, or adding a solvent to carrier which increases the kinetics of the derivatization reaction; e.g., the addition of acetone for the determination of peroxides iodometrically when the carrier is hexane.

"Mobile" refers to reagent in a state by which it may be permeated or transported through the wall or a wall portion of the membrane.

The term "membrane" refers to a porous reagent permeable membrane having the capability of partitioning a carrier stream from the reagent, and the property to transport mobile reagent in permeation contact with one wall surface of the membrane while rejection from transport, at least a detectable amount of the sample species of interest, or a derivative proportional thereto. The term membrane also refers to a membrane which permits permeation transfer of a reagent species, preferably with a molecular weight of less than 100,000. It is preferred that the membrane be a tubular or hollow fiber membrane, with an internal diameter within the range of from 20 to 2000 microns, and most preferably from 200 to 1000 microns and a length of less than 15 cm, preferably 0.1 to 5 cm. However, the membrane can also be a flat sheet.

The term "porous reagent permeable membrane" also refers to a membrane with a volume average pore diameter within the range of from 125 A to 5000 , and preferably from 175 to 2000 , most preferably from 200 A-to 1000 and with structural characteristics that do not promote severe leakage and cause the method of the invention to become inoperative. The term "volume average pore diameter" (Dp) is a term well known in the art and relates to the statistical distribution of total pore volume of the macroreticular resin with respect to varying pore diameter.The method employed herein for determining "volume average pore diameter" is the known technique of mercury porousimetry as described in "Advanced Experimental Techniques in Powder Metallurgy", Vol. 5, Plenum Press (1970). The term also refers to a membrane which has an area, in contact with the eluent, in the range of from 0.2 mmz to 150 mm2, preferably from 1.0 mm2 to 75 mmz, and most preferably from 1.5 mm2 to 50 mm2.

The present invention resides in an apparatus for reagent addition to a flowing stream comprising, a supply of a liquid carrier, means for flowing a stream of the liquid carrier to a membrane reagent addition device, means for adding a sample to the carrier stream, means for adding a mobile reagent to the carrier stream, and a detector for detecting directly versus indirectly components of said carrier, said membrane reagent addition device comprising: a container defining a chamber having an inlet and an outlet port for said carrier stream, said chamber containing said mobile reagent; and said means for adding the mobile reagent to said liquid carrier comprising a porous membrane disposed within the chamber for partitioning the carrier from the mobile reagent for permeation transfer of the mobile reagent into the carrier, characterized by the feature that said membrane has an average pore diameter of from 125 A to 5000 A and an area in contact with said eluent or effluent of from 0.2 mm2 to 150 mm2.

It is preferred to use a container or vessel with means for pressurizing the mobile reagent which optimally completely fills a hermetically sealed chamber of the container. Typically, pressurisation is achieved by permeation transfer of the carrier into the mobile reagent within the chamber to therby increase the pressure of the mobile reagent so that the reagent pressure is approximately the same on both sides of the membrane. For example, when a carrier at a pressure of 100 psig ( 700 kPa ) is used, the permeation transfer of the carrier will increase the pressure of the mobile reagent correspondingly to about 100 psig ( 700 kPa ). This equalization of pressure on both sides of the membrane effectively allows the mobile reagent to be transported through the membrane into the carrier.

A further aspect of the invention includes a plurality of self-pressurized membrane reagent addition devices in fluid communication with one another, with utility for adding a plurality of reagents to the carrier.

One membrane or a plurality of membranes can be used in conjunction with the reagent addition device in conjunction with prior developed liquid chromatographic methods and apparatus. Membranes useful in the present invention may be either isotropic or anisotropic in structure.

The porous membranes used in the method and apparatus of the invention must not only maintain the ability to contain and transport a carrier liquid, but also tolerate various liquids to which the membrane is exposed. Materials which are particularly useful as membrane material are porous polypropylene membranes, such as Celgard (Trade Mark; Celanese Corporation). The membrane material useful in the present invention preferably has a pore size such that the membrane can transfer component species having a molecular weight of less than 100,000.

Further objects and advantages of the invention will, in part, be pointed out and, in part, be apparent from the following detailed description when taken together with the accompanying drawings wherein: Figure 1 is a schematic view of an apparatus for performing liquid chromatography using a hollow fiber membrane reagent addition device in accordance with the principles and teachings of the present invention.

Figure 2 is an enlarged cross-sectional view of a flat membrane reagent addition device constructed in accordance with the principles and teachings of the present invention.

Figure 3 is a chromatogram obtained using the reagent addition device of the present invention.

Figure 4 is a chromatogram obtained without the use of the apparatus of the present invention.

Figure 5 is a chromatogram obtained by using the reagent addition device of Figure 1 for flow injection analysis of a series of sample injections containing calcium ions.

Figure 6 is chromatogram obtained by using the apparatus of the present invention employing a mobile reagent of 30 weight percent ammonium hydroxide.

Figure 7 is a chromatogram obtained by using the apparatus of the invention but replacing the mobile reagent in the membrane reagent addition device with a carrier fluid.

Figure 8 is a chromatogram obtained by using the apparatus of the present invention, and wherein a mixer-delay means is additionally disposed between the membrane reagent addition device and the detector.

Figure 9 is a chromatogram obtained by using the apparatus of the present invention wherein the mobile reagent within the membrane reagent addition device is externally pressurized.

Referring to Figure 1, there is shown a schematic view of an apparatus of the invention, employing a chromatographic column 10. The chromatographic column comprises a separating means typically in the form of a particulate packing or gel through which a sample is eluted or carried to separate the sample into component species. A variety of separating means may be used as described extensively, e.g., by Snyder petal., "Introduction to Modern Liquid Chromato-graphy", 2nd Edition, (1979), pp.740-746.

Preferred means to introduce a carrier to the chromatographic column 10 comprises a reservoir 12 containing a carrier 11. The carrier 11 is withdrawn from the reservoir 12 by a pump 16 operatively connected to conduit 8. The pump is equipped with an optional pulse damping/pressure measuring means 18 for conveying the carrier to the chromatographic column 10.

Preferred means for introducing a sample into the carrier stream comprises e.g., a syringe 17 for injecting the sample in injection valve 20 disposed between the reservoir 12 and column 10. the sample and carrier flow through column 10, and component species thereof ultimately are chromatographically displaced into the chromatographic column carrier exiting the column. An optional guard column 6 can be disposed between column 10 and sample injection valve 20.

Chromatographic column effluent carrier is then introduced to a membrane post-column reagent addition device 24. Mobile reagent 32 is added, by membrane permeation, to the carrier in device 24 and then passed through conduit 56 to an optional reaction delay coil 40 or a functionally equivalent element such as a packed bed column to provide added reaction time for the species to be derivatized. Delay coil 40 is preferably maintained at a controlled temperature by suitable conventional temperature control means. The conditioned carrier is then passed from delay coil 40 to a detector 26 ofa type suited for the detection of component species resulting from liquid chromatographic displacement.

Detector 26 detects the desired property of the carrier such as light absorbance, fluorescence, or a similar property. The detected data is then transferred via conductor 50 from the detector 26 to a suitable recorder 28, such as a strip chart recorder, integrater, computer or the like. In one embodiment, recorder 28 can further direct the detected data via conductor 50, to a display unit 29 for visualization of the data obtained by the detector.

The membrane post-column reagent addition device 24 consists of a leak-proof, two-part container having a body22 and a lid 25 Lid 25 optionally contains a port 69 capable of receiving mobile reagent from an external source, and alternatively, capable of receiving means for applying external pressure to the interior of body 22, thereby increasing the pressure on reagent 32. The device 24 forms a chamber 23 for containing mobile reagent 32. In a preferred design, the vessel is constructed of stainless steel or a similarly inert material capable of sustaining a pressure of from 100 to-20,000 psig (700 to 140 x 103 kPa).

Device 24 preferably has a threaded leak-proof lid 25 engagable with threads provided on the container body 22. An O-ring 36 can be disposed between the lid 25 and body 22 in order to ensure a leak-proof seal. Lid 25 has an inlet port 7 and an outlet port 5 between which are disposed a pair of support conduits 38 and 52. A connector 44 connects the conduit 8 to the device 24. Conduit 56 is connected in a similar fashion by connector 46 to outlet port 5 in the lid 25 for conveying conditioned fluid to detector 26 through the optional delay coil 40.

A tubular membrane 42 is connected to the support conduit 38, preferably with the aid of a sealant 41.

Tubular membrane 42 is connected at the other end to support conduit 52 and preferably sealed with sealant 41. Sealant 41 is preferably a Weldwood brand waterproof wood glue, i.e. a recorcinol formaldehyde type resin, which is capable of withstanding exposure to organic solvents, or Dow Corning RTV Silicone Rubber bathtub caulk which is capable of withstanding exposure to water, The support conduits 38 and 52 are suitably connected to the inlet port 7 and outlet port 5, re-spectively, by means of connectors 27. The conduits and connectors are preferably made of stainless steel. The tubular membrane 42 is disposed within the mobile reagent 32.

In a preferred embodiment of the device 24, the tubular membrane 42 is disposed within the center of the support conduits 38 and 52. The sealant is injected into the support conduits using a blunt hypodermic needle to thereby seal the hollow fiber membrane within the conduits 38 and 52. The sealant is then allowed to cure.

To further facilitate the permeation transfer of column effluent carrier into the reagent and reagent into the column effluent carrier, a magnetic stirrer 48 is disposed in a depression 49 in the lower portion of the body 22.

Following reagent addition, the conditioned carrier fluid is passed out of device 24 to detector 26 and is then passed to a waste reservoir 68 for disposal. In an alternative embodiment, the conditioned carrier fluid need not pass through the delay coil 40. Liquid chromatographic detectors useful in the practice of the invention include photometers and spectrophotometers, used together with suitable reagents which alter the relative light absorbance of sample species in the carrier. An alternative detector includes a flu -orometer used together with suitable reagents which result in fluorescing derivative products. Other liq uid chromatographic detectors which can be used in the invention include, for example, differential refractometers, electrochemical detectors, radioactive detectors, or conductivity detectors.

Figure 2 shows a second embodiment of a membrane reagent addition device 24 having a channel 66 formed in the lid 25 between inlet and outlet ports 5 and 7 for passing carrier from the chromatographic column 10 to the detector 26. At a central portion of the channel 66, the channel is open to the chamber 23 in the body 22. A flat membrane 54 is disposed over this opening completely covering the opening between the channel 66 and the chamber 23. The flat membrane54 is positioned to cover the opening in the lid 25 and is secured by, for example, pins 60 and 62.

The invention is further illustrated by reference to the specific teaching examples below.

Example 1 The separation of various nitrophenols described in U.S. Patent No. 4,451,374 using a silica based reverse phase column requires a carrier of a pH of less than 7 to 8. At a pH greater than this, the column degrades rapidly due to silica dissolution. However, in waste water analysis, detection is improved when the pH of the carrier is greater than 8. This example illustrates a separation at a pH of 6.1 and then, prior to detection, the conversion of the carrier pH to 9.2 for optimum detection using a self-pressurized reagent addition device.

In general, the analytical system used is described in Figure 1. The pump used was an Altex Model 110. The analytical column used was a Merck 10 micron RP-18, 4 mm I.D., 25 cm in length. A Rheodyne Model 7010 sample injection valve fitted with a 100 Fl loop was also used. The detector used was a Kratos Spectraflow Model 773 set for detection at 410 mm. The integrator - recorder used was a Hewlett Packard Model 3380-A. The carrier used was 60 percent acetonitrile- -0 percent water, containing 0.02 M ammonium acetate, 0.005 Mtetrabutylammonium hydroxide adjusted to a pH of 6.1 with a small amount of glacial acetic acid. The carrier flow rate was 1 ml per minute. The carrier pressure at the injection valve was 1,200 psig (8300 kPa) and at the membrane device the carrier pressure was about 50 psig (350 kPa). The delay means between the reagent addition device and the detector was a 4.6 x 100 mm column filled with 140/200 mesh (105/74 micron) stainless steel granules. The reagent used was con-centrated ammonium hydroxide (29 percent strength). The membrane used was sealed in a 1 mm l.D., 1.6 mm O.D., stainless steel tube as shown in Figure 1 The exposed length of the membrane was 40. mm.

The membrane was Celanese Celgard porous polypropylene 0.4 mm l.D., 0.45 mm O.D., designated by the product code X-20 having eliptical pores of a width of about 200 A and a length of about 2000 .

When the carrier pump is turned on, the reagent pressure slowly rises to about 50 psig (350 kPa) over a time span of about one-half hour and then stabilizes at 50 psig (350 kPa). The carrier emerging from the detector slowly changes pH from 6.1 to 9.2 pH over the first one-half hour of operation and then remains at a pH of 9.2. At this time, sample injection can be made, and the chromatogram reproduced in Figure 4 results.

For comparison, the chromatogram reproduced in Figure 3 results when column effluent carrier is directly sent to the delay column without flowing through the reagent addition device 24 containing the membrane. In this configuration, the carrier emerging from the detector has a pH of about 6.1.

When the membrane device is not used in the system the sensitivity of determination for 2-OH and especially 4-OH is low, (see Figure 3). In fact, no 4-OH peak is evident in the chromatogram. When the membrane device is used, sufficient ammonium hydroxide is added to the carrier stream to raise the pH to 9.2, resulting in an improved sensitivity 2-OH detection and 4-OH detection as shown in Figure 4.

Since 34 microliters of reagent are needed to raise the pH of one milliliter carrier from 6.1 pH to 9.2 pH it is estimated that the reagent permeation rate into the carrier using the membrane device is 34 microliters per minute.

The device of the present invention contributed no detectable additional bandspreading to the chromatographic peaks. The DN and 2-OH peaks in Figure 3 or 4 are only about 1450 microliters wide which is a decrease in bandspreading caused by previous devices of about 450 microliters.

Example 2 The ortho-phthaldehyde/2-mercaptoethanol reaction is a particularly important reagent addition reaction used, e.g., in the detection of primary amines. This experiment illustrates a specific example of this reaction as used in the mode of the invention.

The system of Example 1 was used with the following changes: the carrier was 75 percent water, 25 percent acetonitrile, containing 1 gram of sodium acetate per liter of carrier. The column was a Du Pont Zorbaxe ODS, 4.6 mm I.D. by 25 cm length. The reagent was composed of 1000 ppm each of orthophthaldehyde and 2-mercaptoethanol in 250 ml of water containing 6.6 g of boric acid. The reagent solution was adjusted to a pH of 10.3 with 10 percent sodium hydroxide solution. The detector was an LDC Fluorometer Model II. The size of the sample injection valve loop was reduced to a 20 pUl volume.

Ortho-phthaldehyde reacts with primary amines in the presence of 2-mercaptoethanol to produce derivatives that are fluorescent at about 455 nanometers when excited at about 370 nanometers. Analysis of the effluent carrier from the device indicated that the rate of reagent permeation into carrier was about 7.5 microliters per minute. The injected samples contained 1000 ppm each of N-butylamine, or glycine, or L-leucine or L-tryptophan.

Chromatographic peaks were observed for each sample injection. However, when the carrier went directly to the delay column, without flowing through the membrane device, no chromatographic peaks were observed with injections of the samples above.

Example 3 Peroxides and other relatively strong oxidants will oxidize I- to 12 to form highly colored i-3 in the presence of excess i-. The reaction is useful, e.g., to determine the presence of peroxides or other strong oxidants in industrial process streams and products.

The system of'Example 2 was used for the determination of peroxides with the following changes: the carrier was 50 percent isopropanol, 50 percent water. The detector was a Kratos Spectroflow 773 set for detection at 357 nanometers. The reagent was 10 percent tetrabutyl ammonium iodide in carrier. The samples injected were 200 ppb of peracetic acid or 35 ppm acetyl peroxide both dissolved in carrier. No analytical column was used. Instead, a 0.5 meter length of 0.76 mm I.D. by 1.6 mm O.D. conduit replaced the column to generate the approximately 1000 al of bandspreading that could be expected if a column had been used. As such then, this example also demonstrates the utility of the present invention for the important analytical technique known as Flow Injection Analysis (FIA).

Response peaks were observed for each injection of sample. However, when the carrier went directly to the delay column, without passing through the membrane device, no response peaks were observed for the injection of the same samples.

Thus, this example demonstrates the utility of the present invention for the flow injection analysis of peroxide.

Example 4 This example is the determination of a complex polyamine compound (Purifloc C-31, Trade Mark; The Dow Chemical Company). Copper can complex with polyamine compounds and the complex can be determined at 275 nm.

The system of Example 3 was used with the following changes: the carrier was 80 percent water, 20 percent acetonitrile, containing 0.2M NaCI, and adjusted to a pH of 4 using 1M HCI. The injection loop was increased to a volume of 100 microliters. The detector wavelength was changed to 275 nm. Reagent was 0.1 percent cupric chloride in carrier. The sample was 100 ppm Purifloc C-31 in carrier.

A response peak was observed for the injection of the sample. However, when the carrier went directly to the delay column, without passing through the membrane device, no response peak was observed for the injection of the sample.

Copper can complex with polyamine compounds and the complex can be determined at 275 nm. The response peak observed with the use of the present invention indicates that this complexation occurred in this example and allowed the determination of the polyamine compound.

Example 5 The system of Example 4 was used for the determination of hydroxyl containing compounds with the following changes: the carrier was 50 percent t-butyl -alcohol, 50 percent water. The detector was changed to an electrochemical type known as the Chromatix CMX-20 using a nickel electrode. The reagent was 10 percent tetraethylammonium hydroxide in carrier. The samples were 38 ppm formaldehyde in carrier, 100 ppm ethylene glycol in carrier, 10 ppm glucose in carrier and 25 ppmglycerol in carrier.

Many hydroxy containing compounds, but not t-butyl alcohol, react with Ni (FIJI) at the electrode under basic pH conditions (pH from 10 to 12). The resulting Ni (II) is then converted back to Ni (III) by the detector and the current needed to do this is a function of the concentration of hydroxy compound injected.

A response peak was observed for the injection of each sample. However, when the carrier went directly to the delay column, without passing through the membrane device, no response peak was observed for the injection of these same samples. The response peaks observed with the use of the present invention indicates that sufficient base was introduced into the carrier for the determination cited in this example.

Analysis of the carrier emerging from the detector indicated that about 12 microliters per minute of reagent permeated into the carrier using the membrane device, resulting in a carrier pH of about 11.9.

Example 6 The system of Example 5 was used for the determination of calcium and magnesium ions with the following changes: the carrier was 0.3M sodium chloride (prepared using Baker Ultrex grade NaCI which has a low concentration of impurity calcium and magnesium salts) in deionized water The carrier flow rate was 1.1 ml per minute. The detector was the Kratos Spectraflow 773 set for 565 nanometers. The reagent was 0.023M ortho-cresolphthalene complexone sodium salt (OCPC) in saturated sodium borate in deionized water The delay column was removed and the membrane device was connected directly to the detector by means of 60 cm of 0.76 mm l.D. x 1.6 mm O.D. conduit. The samples contained calcium or magnesium ions at the concentration of 10 ppm, 5 ppm, 2.5 ppm or 1.25 ppm in carrier.

Many divalent metal ions (including calcium and magnesium ions) chelate with OCPC to form strongly absorbing complexes. However, OCPC itself has a lower absorbance at 565 nanometers.

Response peaks were observed for the injection of each sample. However, when the carrier went directly to the detector, without passing through the membrane device, no response peaks were observed for the injection of these same samples. The response peaks observed with the use of the present invention indicate that sufficient OCPC was introduced into the carrier for the determinations cited in this example.

Figure 5 shows an example of system performance from a cold start-up at zero minutes for a series of injections of calcium ions. The data in Figure 5 indicates that the system is approximately at equilibrium and ready for use about 10 minutes after start-up.

The system was used intermittently over a 12 day period for 64 hours, i.e., 8 hours a day for 8 days over about 2 weeks with 2 intervening weekends, without replacing the reagent solution. Table I shows data from this study.

Table I Data Over 12 Days Time With 64 Hours of Actual Use First Day Last Day AU AU Background Absorbance of Carrier at 565 Nanometers 0.20 0.14 in Absorbence Units (AU) Peak Height For a 100 Microliter Injection of 0.24 0.16 a 10 ppm Calcium lon Sample in Absorbence Units The data in Table I indicate an overall decrease in sensitivity and background absorbance of about 30 percent for the time period studied. However, during each days use, results were stable +2 percent to +5 percent relative, with no apparent pattern of drift. Since it is common practice to recalibrate an analytical system at least every day, the long term drift of the system is acceptable for most applications.If it can be assumed that the decrease in background absorbance and responsiveness is due to reagent dilution in use, the system permeated OCPC reagent at about 10 microliters per minute.

This example demonstrates the long term reliability of the present invention.

Example 7 The separation and detection of various nitrophenols using a silica based ion-exchange column requires reagent addition for acceptable component detection in certain waste waters.

The experiment was conducted in a non--pressurized (atmospheric pressure) reagent addition device and used the following solutions and apparatus. The carrier is 35 volume percent methanol and 65 volume percent water. This carrier also contains 0.08 M sodium perchlorate and 0.04 M ammonium acetate.

The pH of the carrier is finally adjusted to 6.0 by adding acetic acid. The carrier pump was an Alted Model 110A set for a flow rate of 1.0 ml per minute. The injection valve is a Rheodyne Model 7120 with a 20 > t injection loop. The analytical column used is a Whatman Partisil SAX 10/25 (4.6 mm x 250 mm).

The effluent carrier from the analytical column is conducted to the inlet port of the membrane reagent addition device. The outlet port of said device is connected to a Laboratory Data Control Model 1203 detector set for a detection wave-length of 410 nm. The detector signal was sent to a Hewlett Packard Model 3380A recorder/integrator.

The support conduits of the reagent addition device were two 5 cm sections of 1 mm I.D. x 1.6 mm O.D. stainless steel conduit. These sections of conduit were "L" shaped so that their ends faced each other. The gap between them was closed and the carrier circuit completed by sealing a 3.8 cm length of 0.4 mm l.D. x 0.45 mm O.D. polypropylene reagent permeable hollow membrane (Celgard MHFX20) into the ends of the "L" shaped stainless steel conduits. The gap, com-pleted by the hollow membrane was about 2.5 cm, i.e., 2.5 cm of hollow membrane was exposed to the reagent. The reagent was about 30 weight percent ammonium hydroxide. The sample contained a mixture of 2.2 ppm dinitro-ortho-secondarybutyl phenol (DN), 2.3 ppm 2-hydroxy-3-secondarybutyl-5 nitrobenzene-1--sulfonate (2-OH) and 2.7 ppm 4-hydroxy-3-nitro--secondarybutylbenzene-1-sulfonate (4-OH) in water.

When an sample injection was made, the chromatogram shown in Figure 6 resulted. When the reagent in the membrane reagent addition device was replaced with carrier, the chromatogram shown in Figure 7 resulted. The use of a reagent of 30 weight percent ammonium hydroxide resulted in a pH shift of the carrier from 6 to 9. At a pH of about 9, both the 4-OH and 2-OH components showed beneficially high detectability as is shown when comparing Figure 6 and Figure 7.

Example 8 Added to the system of Example 7 was a coil of 0.25 mm l.D. x 1.6 mm O.D. Teflon conduit. The coil was disposed between the reagent addition device and the detector with a length sufficient to give a back pressure inside the hollow fiber membrane of about 15 psig (100 kPa). The reagent used in this device was about 30 weight percent ammonium hydroxide. When a sample was injected, the chromatogram shown in Figure 8 resulted. The carrier back pressure in the porous hollow fiber membrane prevented sufficient reagent from permeating into the carrier stream to change the carrier pH significantly.

A compressed air line was attached to the reagent filling port 69 of the reagent addition device and the reagent chamber was pressurized to about 16 psig (110 kPa). When a sample was injected, the chromatogram shown in Figure 9 resulted. Pressurizing the reagent chamber to a pressure nearly the same as the carrier pressure within the hollow fiber membrane resulted in sufficient reagent permeation to alter the carrier pH to about 9 and thereby significantly increase the absorbance of the 4-OH and especially the 2 OH compounds as is shown when comparing Figure 8 with Figure 9.

When the above experiments are performed without the use of the analytical column flow injection analysis is performed.

Claims (22)

1. Apparatus for reagent addition to a flowing stream comprising, a supply of a liquid carrier, a membrane reagent addition device, means for flowing a stream of the liquid carrier from said supply to said device, means for adding a sample to said carrier stream, and a detector for detecting directly versus indirectly compounds of said carrier, said membrane reagent addition device comprising: a container defining a chamber having an inlet and an outlet port for said carrier stream, said chamber containing a mobile reagent; and a porous membrane disposed within the chamber for partitioning the carrier from the mobile reagent for permeating transfer of the mobile reagent into the carrier, said membrane having a volume average pore diameter of from 125 Â to 5000 and an area in contact with said carrier or effluent of from 0.2 mm2to 150 mm2.

2. An apparatus as claimed in Claim 1, wherein said membrane has a volume average pore diameter of from 175 A to 2000 and an area of from 1.0 mm2 to 75 mm2.

3. An apparatus as claimed in Claim 2, wherein said membrane has a volume average pore diameter of from 200 A to 1000 A and an area of from 1.5 mm2 to 50 mm2.

4. An apparatus as claimed in any one of the preceding claims, wherein said porous membrane is selected from at least one tubular membrane, hollow fiber membrane, and flat sheet membrane.

5. An apparatus as claimed in Claim 4, wherein said membrane comprises hollow fiber having an internal diameter of from 20 to 2000 micrometres and a length of less than 15 cm.

6. An apparatus as claimed in Claim 5, wherein said membrane has an internal diameter of from 200 to 1000 micrometres and a length of from 0.1 to 5 cm.

7. An apparatus as claimed in any one of the preceding claims, wherein said porous membrane comprises polypropylene.

8. An apparatus as claimed in any one of the preceding claims, wherein said membrane permits permeation transfer of component species having a molecular weight of less than 100,000.

9. An apparatus as claimed in any one of the preceding claims, wherein said chamber is hermetically sealed for self-regulation of the reagent pressure in the chamber.

10. An apparatus as claimed in Claim 9, comprising a plurality of self-pressurized reagent addition devices in fluid communication with one another.

11. An apparatus as claimed in any one of Claims 1 to 8, including a port in said reagent addition device for externally pressurizing the mobile reagent disposed in said chamber.

12. A method for reagent addition to a flowing stream of a carrier liquid comprising the steps of: introducing a sample into said carrier stream and passing said stream through a reagent solution de vice.; introducing a mobile reagent into the carrier stream in said reagent addition device and detecting the sample constituents in said stream, wherein said reagent is introduced into the carrier stream by permeation transfer in which one surface of a porous membrane is contacted by said carrier and the opposite surface is contacted by the mobile reagent and selecting said membrane with a volume average pore diameter of from 125 to 5000 and an area in contact with said carrier of from 0.2 mm2 to 150 mm2.

13. A method as claimed in Claim 12, including the step of selecting said pdrous membrane from at least one tubular membrane, hollow fiber membrane, and flat sheet membrane, and positioning said membrane in a chamber in said reagent addition device in permeation contact with said mobile reagent.

14. A method as claimed in Claim 12 or Claim 13, including the step of hermetically sealing said chamber, contacting said carrier with one surface of said porous membrane, and contacting the opposite surface of the membrane with said mobile reagentwhereby the addition and subtraction of said carrier to the mobile reagent effected by permeation transfer through the membrane self-regulates the pressure in said chamber and mobile reagent is transferred from said space through the membrane into the carrier.

15. A method as claimed in any one of Claims 12, 13 or 14, including the step of maintaining a difference between the average pressure Of the carrier in contact with said membrane and the average pressure of the reagent in contact with the opposite surface of the membrane of less than 10 psig (70 kPa).

16. A method as claimed in any one of Claims 12 to 15, comprising the step of maintaining a pressure drop of the carrier along the length of the membrane reagent addition device of less than 10 psig (70 kPa).

17. A method as claimed in Claim 12 or Claim 13, including the step of externally pressurizing the mobile reagent in said reagent addition device.

18. A method as claimed in any one of Claims 12 to 17, including the step of passing the carrier stream and sample through a chromatographic column having a stationary phase for displacing the sample, and conveying the effluent carrier from the column to the reagent addition device.

19. An apparatus as claimed in Claim 1 and substantially as hereinbefore described with reference to and as illustrated in Figure 1.

20. An apparatus as claimed in Claim 19 and substantially as hereinbefore described with reference to and as illustrated in Figure 2.

21. A method as claimed in any one of Claims 12 to 18, wherein the membrane is as defined in any one of Claims 2, 3 and 5 to 8.

22. A method as claimed in Claim 12 and substantially as described in any one of the Examples.