CA1163830A - Liquid chromatographic method and apparatus with hollow fiber device for post-column derivatization - Google Patents
Liquid chromatographic method and apparatus with hollow fiber device for post-column derivatizationInfo
- Publication number
- CA1163830A CA1163830A CA000381805A CA381805A CA1163830A CA 1163830 A CA1163830 A CA 1163830A CA 000381805 A CA000381805 A CA 000381805A CA 381805 A CA381805 A CA 381805A CA 1163830 A CA1163830 A CA 1163830A
- Authority
- CA
- Canada
- Prior art keywords
- reagent
- chromatographic column
- effluent
- hollow fiber
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/84—Preparation of the fraction to be distributed
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/24—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the treatment of the fractions to be distributed
- B01D15/245—Adding materials to the effluents
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J19/24—Stationary reactors without moving elements inside
- B01J19/2475—Membrane reactors
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J4/00—Feed or outlet devices; Feed or outlet control devices
- B01J4/001—Feed or outlet devices as such, e.g. feeding tubes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J4/00—Feed or outlet devices; Feed or outlet control devices
- B01J4/04—Feed or outlet devices; Feed or outlet control devices using osmotic pressure using membranes, porous plates
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00049—Controlling or regulating processes
- B01J2219/00051—Controlling the temperature
- B01J2219/00159—Controlling the temperature controlling multiple zones along the direction of flow, e.g. pre-heating and after-cooling
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- 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
- G01N1/38—Diluting, dispersing or mixing samples
- G01N2001/381—Diluting, dispersing or mixing samples by membrane diffusion; Permeation tubes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/84—Preparation of the fraction to be distributed
- G01N2030/8423—Preparation of the fraction to be distributed using permeable separator tubes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/84—Preparation of the fraction to be distributed
- G01N2030/8429—Preparation of the fraction to be distributed adding modificating material
- G01N2030/8435—Preparation of the fraction to be distributed adding modificating material for chemical reaction
Abstract
Abstract of the Disclosure Reagent is added to liquid chromatographic effluent to increase detection sensitivity of sample bands, or to enhance sensitivity with respect to interfering bands which overlap sample bands of interest, using one or more hollow fibers immersed within mobile reagent which is permeated through the walls of the fibers and, thus, ultimately diffused into the column effluent.
29,226-F
29,226-F
Description
LIQUID CHROMATOGRAPHIC METHOD AND
APPARATUS WITH HOLLOW FIBER
DEVICE FOR POST-COLUMN DERIVATIZATION
The invention relates to the field of li~uid chromatography and, more particularly, to an improved method and apparatus by which reagent is added to chromatographic column effluent to enhance sensitivity of detection.
The very significant potential of using post-column reactors to improve detection in modern liquid chromatography (HPLC) has long been recognized, but little applied. For example, in the publicat:ion by Gfeller et al., "Post-Column Derivatization in High--Performance Liquid Chromatography Using the Air Segmentation Principle: Application to Digitalis Glycosides," Journal of Chromatography 142 (1977), pp.
271-281, the authors state:
"Although the use of such reaction tech-niques after column chromatographic separation has been known for more than a decade with classical column techniques (e.g., amino acid analyzers), little has appeared in relation to modern HPLC. One reason is the many tech-nical problems [ ] that still have to be solved."
29,226-F -1-~k
APPARATUS WITH HOLLOW FIBER
DEVICE FOR POST-COLUMN DERIVATIZATION
The invention relates to the field of li~uid chromatography and, more particularly, to an improved method and apparatus by which reagent is added to chromatographic column effluent to enhance sensitivity of detection.
The very significant potential of using post-column reactors to improve detection in modern liquid chromatography (HPLC) has long been recognized, but little applied. For example, in the publicat:ion by Gfeller et al., "Post-Column Derivatization in High--Performance Liquid Chromatography Using the Air Segmentation Principle: Application to Digitalis Glycosides," Journal of Chromatography 142 (1977), pp.
271-281, the authors state:
"Although the use of such reaction tech-niques after column chromatographic separation has been known for more than a decade with classical column techniques (e.g., amino acid analyzers), little has appeared in relation to modern HPLC. One reason is the many tech-nical problems [ ] that still have to be solved."
29,226-F -1-~k
-2- 1 16 3 8 30 A~ong specific difficulties that are described in the literature are problems directly involved with state of the art post-column reactor designs. Thus, Snyder et al., in "Introduction to Modern Liquid Chromatography", 2nd Ed. (1979), p. 740, state:
"The adaptation of reaction detection to modern LC columns requires careful attenti on to [] the design of equipment, because extra column effects can be serious. For these 10reasons reaction detectors have so far found rather limited use in modern LC."
Equivalent conclusions are also expressed by Frei et al., "Reaction Detectors in HPLC", Journal of Chromatographic Science, Vol. 17, March 1979, pp.
15152-159, wherein the authors state: "The construction of proper reaction detectors comprises a constant struggle against band broadening." In still another recent publication by Jupille, " W - Visible Absorption Derivatization in Liquid Chromatography", Journal of Chromatographic Science, Vol. 17, March, 1979, pp.
160-167, the author listed among disadvantages of the design or state of the reactors: "a need for hardware modification (with attendant loss in flexibility); and [ ] a risk of band broadening due to post-column mixing volume resulting in loss of resolution."
By way of further explanation, the often mentioned problem of avoiding band spreading is inter-related to various factors, among which is the mode for metering reagent. ~ny lack in consistency of metering produces fluctuations in reagent concentration in the effluent, which shows up as "noise" in the chromatograph developed ~y the detector.
29,226-F -2-The problem is especially severe where highly concentrated reagent is used, since minute fluctuations in metering can produce high background "noise" levels that severely hamper the sensitivity of detection.
While one of the choices of the prior art is to use concentrated reagent to avoid band spreading by sample dilution, the gain may, nevertheless, be offset at least partially by increased backgrc7lnA r.^__e 'cv-cls.
Band spreading is also caused by diffusion of sample bands into one another as a function of time.
Prior devices inherently appear to obtain poor reagent/-effluent mixing, hence, extending the time factor. The deficiency is particularly shown by the description in the literature of reagent/effluent mixing devices as means for promoting faster reactions.
Prior solutions to these and other related problems can thus be said to often involve serious drawbacks, e.g., increased "noise". What may be equally as objectionable is the addition or too much complex eguipment such as air segmentation methods to the apparatus, which has already been characterized as involving too many inherently disadvantageous "hardware modifications" with an "attendant loss of flexibility".
Accordingly, an objective of the invention is to provide an improved liquid chromatographic method and apparatus characterized by the development of an improved post-column reactor which requires little in the way of hardware modifications.
A particular object of the invention is to provide an improved method and apparatus which 29,226-F -3-,~
achieve a substantially constant "pulseless" metering of reagent, and, in addition, achieve significantly improved diffusion of the reagent into the chromato-graphic column effluent.
It is a further object to provide such apparatus and method wherein sample dilution is minimized without the need foL resoriing tO the addition of highly concentrated forms of reagent as a means or requirement, or resorting to other objectionable process or apparatus restrictions.
Still a further objective hereof is to provide such method and apparatus, which in contrast with prior art methods and apparatus, are of significantly less cost to use and to maintain.
The term "hollow fiber membrane" means an extremely small tube or fiber having an internal diameter of from 20 to 1,000 microns, preferably, from 50 to 500 microns, and which has the property to trans-port mobile reagent in permeation contact with the exterior wall portion of the fiber (or saturated within the fiber wall matrix), while rejecting from transport, at least a detectable amount of a sample species of interest, or a derivative proportional thereto, flowing as a component of liquid chromatographic effluent through the internal bore of the hollow fiber membrane.
"Reagent" means a chemical species or com-bination of species which, when introduced through the hollow fiber membrane into chromatographic column effluent, reacts chemically, directly or indirectly, with a sample species of interest (or an interfering 29,226-F -4-~63830 sample species which is less than perfectly resolved with respect to a sample species of interest) to produce a measurable enhancement in the detection of said species of interest, or a monitored proportional derivative thereof, compared to the absence of the hollow fiber membrane/reagent combination.
"Mobile" refers to a .eayc.l~ in a S~d~ by which it may be permeated or transported through the wall or a wall portion of the hollow fiber membrane.
The above-stated objects of the invention are achieved by a liquid chromatographic apparatus comprising a chromatographic column, injector means for adding a sample solution to the chromatographic column, means for adding eluent to the chromatographic column whereby the sample is eluted through the chromatographic column and component species thereof appear in chromatographically displaced form in the effluent of the chromatographic column characterized by a post-column reactor comprising a hollow fiber membrane through which the effluent of the chromatographic column is fed to a liquid chroma-tographic detector, said hollow fiber being in permeation contact with a mobile reagent for permeation transfer of the reagent into the effluent of the chromatographic column.
A further aspect of the invention is a method of analyzing samples by liquid chromatographic comprising the steps of adding a sample solution to a chromatographic column, adding an eluent to the chromatographic column effective to chromatographically displace species of 29,226-F -5-the sample from the chromatographic column, whereby chromatographically displaced sample species appear ultimately in the effluent of the chromatographic column characterized by feeding the effluent from the chromatographic column through the internal bore of a hollow fiber membrane, ultimately to a liquid chromatographic detector, and prior to detection, using the hollow fibe~ .eïl~arle Lor permeaiion trans-fer of a mobile reagent into the effluent of the chromatographic column to enhance the sensitivity of detection.
An essential feature of the invention is the use of a single or multiple hollow fiber membranes in conjunction with prior developed liquid chromatographic methods and apparatus. In the preferred device, mul-tiple fibers are potted at each end into a tube sheet for purposes of isolating the inner portion or inner bore of the fibers and also to provide means for con-necting the inner portion of the fibers to standard liquid chromatographic fittings. The center section of the hollow fiber "bundle", which may be coiled to conserve space, is immersed within a mobile reagent. As such, the device is readily combined with standard and commercial forms of liquid chromatographic apparatus with minimal need for basic modification in the hard-ware.
Hollow fiber membranes useful in the practice of the invention are characterized by a molecularly porous wall structure obtained as an inherent property of the material used to make the fiber, or obtained as 29,226-F -6-1163E~30 _7--a product of the manufacturing process, or a combination of both. The invention makes no claim, as such, to inventing the hollow fiber membrane structure per se.
Typically used in practicing the method and apparatus of the invention are thus commercial hollow fiber membranes developed for other applications, and which are adaptable to the invention.
Among desired properties of the hollow fiber membrane (in addition to the ability to contain and transport reagent and resist transport of at least part of the sample) is its tolerance to contact with various liquids to which it is exposed. Particularly useful in this respect are porous cellulose membranes prepared such as by the method of U.S. Patent 3,546,209.
~15 Such membranes may be either isotropic or anisotropic in structure. The transport properties of this type of membrane are produced typically by fabricating discretely sized pores piercing the fiber wall, e.g., by "pushing aside" the membrane material to form the pore structure. As such, the permeation character-istics are basically that of size selection and may be broadly applied to permeate widely diverse reagent species.
Synthetic polymeric membranes, such as produced ~5 typically from polyolefins and also silicone rubber, as well as a considerable group of other polymeric materials, may be adapted for use in the invention. Particularly useful are charged ion-exchange hollow fiber membranes produced by sulfonation or amination processes in order to obtain "Donnan exclusion" rejection properties of ionic speciés. Permeation through such membranes is often typically through a "tortuous" path defined in the molecular spaces between polymer molecules. The 29,226-F -7-.~
~163830 transport and selective properties of the solid matrix polymeric hollow fiber is thus often very specific and thus requires careful selection with reagent and sample in mind. Specific examples of this type of the membrane are detailed below with respect to some of the reagents.
The "leakage" characteristics of hollow fiber memhranes use~ ir. the lnvellt~ al-e often~imes not highly critical. For example, leakage through the fiber wall of components other than sample is (although often not inconsequential) not a phenomenon that will severely hamper the analysis. Thus, eluents used in the invention are generally characterized as non-inter-fering or substantially non-interfering. Reagent diluents, where used, may be selected for the same properties. Accordingly, leakage of these character-ized components, assuming there is a net transfer one way or the other, may, at most, produce a dilution on the sample in the chromatographic effluent. Assuming a net loss of chromatographic effluent through such transfer, a desirable concentration of the sample may in fact result from beneficial leakage phenomena pro-ducing higher detection sensitivity.
In addition, while it would be desired to have a hollow fiber membrane that rejected sample in the perfect sense, such is neither an absolute or essential requirement. While too much loss of sample can be consequential to the sensitivity of the analysis, substantial loss of sample does not necessarily mean that severe detrimental results will occur. Thus, for illustrative example, 90 percent leakage of the sample would not nécessarily preclude a highly sensitive analy-sis from being performed where the gain of sensitivity 29,226-F -8-1~63830 g by the method is on the order of a thousandfold, which is not an unusual result possible with reagent addition methods. Since the amount of reagent necessary to achieve highly dramatic improvement in detection sensi-tivity can be very small, the selective transportproperties of the membrane may in fact favor transport of the sample. That is, a greater leakage of sample than p~rm~t~ on nf reagent may be experienced by use of a particular membrane, while at the same time obtaining enhancement in sensitivity of detection. Thus, selective transport properties of the hollow fiber membrane, favoring the reagent, is not an essential requirement of the invention in its broadest sense.
In respect to band spreading produced by dilution factors, or adding liquid volume to the chro-matographic effluent, the invention has a very important advantage over prior art reagent addition methods.
Thus, while it is contemplated that there is often a significant leakage of the non-active component of the reagent into the chromatographic effluent, since other than perfect permselective qualities are present in hollow fibers, there is at the same time the mentioned ability of the fiber to extract fluid from the effluent, so that a net exchange of fluid volume is involved.
For membranes which possess good sample rejection properties, it is thus possible to minimize dilution volume without resorting to difficult procedures that severely restrict the method.
In order to minimize band spreading due to geometry and flow pattern factors, it is desirable that if multiple hollow fibers are used, that each fiber produce the same resistance to flow and, 29,226-F -9-consequently, as the effluent stream is divided and flows in the multiple passages of the individual fibers, flow proceeds at a uniform rate. Thus, with flow dwell time in the individual fibers being substantially constant, the tendency for band spreading is greatly reduced. This is typically achieved by using multiple fibers of the same internal diameter and length.
Qbviously, to ^vercomc the effect entirely, fibers could be matched to produce exactly equivalent flow characteristics.
Also, as a general rule, the larger the fiber, the greater the tendency to produce band spreading by inherent fluid drag at the fiber wall/flow interface, hence producing an effect that if accentuated enough or prolonged for long enough, can cause loss of resolution.
The effect is minimized by using multiple fibers of extremely small diameters which, by means of larger surface area, can pass the reagent more quickly, fre-~uently permitting the use of a shorter path of travel and hence minimal band spreading based on diffusion of sample bands into one another as a factor of time. The rate of diffusion of the reagent in the chromatographic effluent is also effectively improved by using small--diameter multiple hollow fiber membranes. Hence, significant advantages in critical parameters are achieved by the use of a post-column reactor design based on the use of multiple hollow fibers having sample rejection properties, and which are connected in parallel between the chromatographic column and detector.
Highly suited for use in the multiple fiber device are fibers of between about 100-300 ~m 29,226-F -10-"
(micrometers) in internal diameter for use in conventional bore liquid chromatography. In conjunc-tion with micro-bore liquid chromatography such as described by Scott et al., Journal of ChromatographY, Vol. 169, pg. 51 (1979) extremely small fibers in the range as low as the current manufacturing limit of about 20 ~m I.D. would be usable. The wall thickness of the fibers is not always highly critical to rate of permeability as explained in U.S. Patent 3,808,267.
Generally, the wall thickness of fibers as used in the invention is from 5 to 250 ~m. The length of the fibers can also vary, depending on permeation and/or diffusion rates through the fiber wall. Generally, it is contemplated to use fibers of from 10 to 200 centi-meters in length. The restriction on length (andminimum I.D.) is ultimately a function of pressure drop through the fibers and back pressure on the fibers.
Too much back pressure can rupture the fibers, thus limiting the pumping pressure. This can be compensated for, however, by equilibrating the pressure about the fibers with the internal pressure, such as by maintain-ing the fibers in a pressurized vessel. By taking the latter precautions, the length of the fibers may be increased to the point that resolution of the chroma-tographically displaced species is not too adverselyaffected.
The fibers or a portion thereof are in permea-tion contact with the mobile reagent. The temperature, differential pressure with the effluent, and concentra-tion of the reagent can si~nificantly affect permeationrates in accordance with known membrane transfer phenomena.
29,226-F -11-The reagent comprises, e.g., a pure liquid, pure gas or solution of reagent in a diluent or carrier whereby the reagent is mobile and may thus permeate the fiber wall.
In some cases, an inert diluent such as methylene chloride is used which modifies the membrane such as by diffusion into the membrane to increase the rate of per-meation of the reagent, also in accordance with known mem~rane phenomer.a. Other types of liquids, such as water or acetonitrile, are typically used as the reagent diluent.
In addition, it is also possible to use reagent, particularly ionic species, attached to active ion exchange sites of solutions or gels of ion-exchange resin, or agitated ion exchanged beads, in order to pro-duce mobile reagent. This embodiment may be used, forexample, with a charged ion-exchange hollow fiber mem-brane, whereby the reagent ion is exchanged with the exchangeable ion of the membrane and, thus, ultimately dif~uses into the chromatographic column effluent. The embodiment is particularly useful wherever it would be desired that the ions similarly extracted from the chromatographic effluent, by the charged hollow fiber membrane, would be detrimental to detection. A means would thus be provided for minimizing return leakage.
In addition, the embodiment can be used to prevent transfer of the relatively large polymeric counter ion of a given ionic reagent particularly where the counter ion would interfere or otherwise be detrimental to the analysis.
Two forms of reagent reservoirs or containers are particularly contemplated. The preferred form is generally described as a static reservoir, although the 29,226-F -12-1163~330 reagent may be agitated or stirred in order to prevent a concentration differential from occurring. Alterna-tively, a dynamic flow of the reagent, wherein con-tinuously fresh reagent is pumped into contact with the fibers may be used. The latter embodiment has advantages where contamination of the reservoir by leaklng chromato-graphic effluent would be detrimental to the analysis.
In such an embodiment, the fibers are placed, e.g., co-axially within a preferably flexible tube container to define an annular space between the fiber and inner wall of the tube. The fibers are potted at each end to isolate the inner bores of the fibers for connection to chromatographic fittings similarly as with respect to a static reservoir. Spaced tees are connected at the end of the tube container and the reagent is continuously pumped into the annular space, most effectively in counter flow to the flow of the effluent from the chromatographic column.
Because of less complexity, and typically very comparable performance to the dynamic reservoir embodiment, the static reservoir is to be preerred.
Because the dilution of leaking sample into the rela-tively large reservoir of reagent produces an extremely dilute solution of a possible interfering species, the effect at most may be reflected as a slightly varying base line over a period of use. Ordinarily such would thus not justify the additional complexity associated with a dynamic reservoir embodiment.
The reaction kinetics of reagent addition methods, as well as prior developed reagents, are considered background technology to the invention and useful in its practice. Thus, the specific reagents 29,226-F -13-~i63~30 and reactions developed for detection purposes and used in the invention are drawn from prior art sources. A
fairly detailed listing of reactions considered suitable for use in post-column effluent reaction procedures is given by Snyder et al., supra, pages 740-746, and the similar teachings of the prior cited references by Gfeller et al.; Frei et al.; and Jupille, together with a publ'ication by Vance Nau et al., "Application of Microporous Mernbranes to Chemiluminescence", Analytical ChemistrY, Vol. 51, No. 3, March 1979, pp. 424-428.
Favorable reaction conditions necessary to promote the reaction must of course be present with this invention as with other reagent addition modes.
Thus, reactions which require a major proportion of water in the final reaction mixture will generally require that aqueous eluents be used. The latter requirement is generally met with respect to ion--exchange and reverse-phase liquid chromatographic separation methods, or similar liquid chromatographic methods which employ an aqueous eluent or mobile phase in which the reagent is at least partially miscible.
Similarly, for liquid chromatographic techniques, such as classical normal phase chromatography, the invention is typically limited to the selection of reagents that can proceed in or require an organic solution in which the reagent is at least partially miscible. For slow reactions, elevated temperatures are imparted to the reaction mixture such as by means of a heated reaction delay loop which provides added residence time.
Liquid chromatographic detectors useful in the practicé of the invention are particularly advan-tageously photometers, spectro-photometers and fluoro-meters used together with reagents which alter or produce 29,226-F -14-11~i3830 light absorbance of sample species in the chromato-graphic effluent or which produce fluorescing deriva-tive products. Among other liquid chromatographic detectors beneficially used in the mode of the inven-tion are, for example, differential refractometers,electrochemical detectors, radioactive detectors, and conductivity detectors.
Further.objects and advantages of the inven-tion will 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 chromatograhy using post-column reagent addition in accordance with the principles and teachings of the present invention.
Figure 2 is an enlarged cross-sectional view showing additional details of the post-column reactor used in the Figure l apparatus; and Figure 3 is a view of a reactor similar to Figure 2 showing a modified form of the post-column reactor.
Referring to Figure 1, there is shown a chromatographic column 10 comprising a housing contain-ing a separating means in the form of a particulate packing or gel through which a sample solution is eluted to separate the sample into component species. Diverse types of separating means may be used to construct a suitable chromatographic column, as described exten-sively by Snyder et al.
'~ 29,226-F -15-1~L63~3t~
A preferred means for adding an eluent solu-tion or mobile phase to the chromatographic column 10 comprises an eluent reservoir 12 containing an eluent solution 14, the latter which is withdrawn from the reservoir by a pump 16 equipped with an optional pulse damping coil 18.
A preferred means for adding a sample comprises a syringe loadable sample injection valve 20. Sample added to the system at valve 20 is swept through the apparatus by the pumped eluent solution through an optional guard column 22 to chromatographic column 10.
The sample is eluted through column 10, and component species thereof thus ultimately appear chromatograph-ically displaced in the chromatographic column effluent which is delivered to the reagent addition device or post-column reactor 24, described in further detail below.
The reagent addition device or reactor is optionally followed by a reaction delay coil (not shown) or functionally equivalent element used whenever neces-sary to provide added reaction time vis-a-vis the species to be derivatized. Optionally, both or either the reagent solution or delay coil is maintained at a controlled temperature by suitable temperature control means (not shown) such as a temperature controlled plate which heats the solution or fluid in which the delay coil is immersed;
and/or which heats the reagent solution. The delay coil is ultimately followed by a detector 26 of a type suited for liquid chromatography.
In the detector, the effluent produces an electrical signal proportional to the property monitored 29,226-F -16-11~i3830 such as, for example, light absorbance, fluorescence, and which signal is conducted from the detector to a recorder 28 such as, for example, a strip chart recorder, an integrator, and the like, all of which are well-known to the art.
Referring to Figure 2, a preferred form of reagent addition device, (based on a silllpii~ity facior and acceptable performance for a great number of reagent addition reactions) is the "static" reservoir design which comprises a reservoir or reagent container 30, preferably made of glass or a similar inert material.
Contained in the reservoir is a mobile reagent 32. The reagent is periodically changed through a screw-cap or other suitable closure 34. A hollow fiber or fibers 36 are suspended in the reagent between an effluent-in feed connection 38 and an effluent-out feed connection 40.
Each connection is made by drilling openings 42 in the closure 34. An adapter 44 is mounted in each open-ing by use of general purpose epoxy glue, shown at 42.
Tubing 48 or supplying the chromatographic column effluent iB attached to a threaded nipple 50 on the adapter 44 by using a standard connecting nut 52 and ferrule 54. The out feed is similarly connected by tubing 56 to the detector 26 through a similar connecting nut 52 and ferrule 54.
A preferred form of fixing the ends of the hollow fiber or fibers into the female part of adapter 44 is to mold a itting 58 about the end portions 60 of the fibers. This fitting is made by using a rubber compound, such as Silastic brand mold making rubber J-RTV, to make a mold, using as the form for the mold, the commercial 29,226-F -17-fitting which is to be duplicated. For an epoxy system, the fibers are threaded through the mold by drilling a small diameter hole for threading purposes. An epoxy, such as, Dow 331 Epoxy Resin/Ancamine LT Hardener is poured into the mold and cured, with the fibers "wetted", or dry depending whether fiber swelling is expected in the end use application. After removal from the mold, the fiber ends are tri~med as re~iLed. A fiDer coating layer 62, such as, Silastic 730 RTV Fluorosilicone Sealant (from Dow Corning Corp.) is preferably applied in the area immediately adjacent the fitting to avoid fiber point stress, and thus minimize damage as may occur such as in the physical handling of the fibers.
The epoxy resin system is useful for aqueous reagent and eluent systems, certain aromatic solvents, and some hydrocarbons. For reagent solutions or eluents which chemically attack epoxies, there is used in preference and for longer life, a Silastic Brand J-RTV
material for constructing the fittings 58 which otherwise is manufactured similar to the epoxy fittings. Fittings 58 are also suitably manufactured using a hollow commer-cial fitting through which the fibers are passed and which is filled with a sealant material, such as, 730 RTV
sealant which is subsequently cured. The latter thus basically consist of commercial fittings with the ends of the fibers potted inside the hollow portions of the fittings by an in situ curing process using the described RTV material, or a substitute in situ curable substance.
A preferred embodiment of a reagent addition device having the fibers immersed in a counter flowing mobile reagent is illustrated in Figure 3. This device 29,226-F -18-~1638;~0 is''c'onstructed of a center section of stainless steel tubing or tubular containing means or jacket 68 through which a fiber or a bundle of hollow fibers is inserted by suction or by gluing the end of a length of thread to a fiber bundle and pulling the fibers through the jacket using water as the lubricant. A tee or tee fit-ting 70 is affixed to opposite ends, respectively, of the jacket 68 using tube nutC 72 wi~h f~,ru es 74 tG
make the attachment.
Exposed portions of the hollow fibers (out-wardly of each tee) are dried and inserted into sealing tubes 76, respectively, which are preferably made of stainless steel. A section of about six inches of the fibers is left exposed between the sealing tubes and the tees 70 and the exposed fiber sections are coated with a suitable sealant, such as, Silastic 732 RTV sili-cone rubber, as indicated at areas 66, after which the sealing tubes are coupled to the tees using tube nuts 78 and ferrules 80. Additional RTV sealant is injected into the sealing tubes using a blunt 20 gauge needle to completely fill each sealing tube but taking care not to force excess rubber into the tees. The RTV sealant is allowed to cure for about 10 minutes to promote initial bonding and curing is completed with the fiber "wetted" or dry as the end-use application demands. A
razor blade is used to cut the fiber ends off so that they are flush with the end of the sealing tube. The device may then be coupled into the apparatus of Figure 1 by using reducing union assemblies 82. Similar connec-tions are provided to the tees 70 for supplying thereagent through tubing 84 and 86 to a reservoir and pump (not shown), respectively. These latter connec-tions may consist a tube element 88 joined at one end -- 29,226-F -19-1~63830 to each tee 70 through a tube nut 90 and ferrule 92;
and at the opposite end joined, respectively, to the reagent inlet and outlet 84, 86 through a reducing union assembly 94. The device as assembled defines contiguous flow channels comprising, respectively, the collective bores of the hollow fibers, and the spaces exterior of the fibers within the jacket 68 and which communicate, respectively, wlth the chromatographic column 10 and the reagent supply source (not shown).
The described reagent addition devices operate by receiving the effluent from the chromatographic column 10 which is routed internally through the hollow fibers. Simultaneously, the mobile reagent solution (static or in the dynamic form) flow on the exterior surfaces of the hollow fibers, thus causing permeation of the reagent into the chromatographic effluent.
The invention is still further illustrated by reference to the specific teaching examples below.
ExamPle 1 - Hollow Fiber Membranes Various hollow fiber membranes are used to construct reagent addition devices for use in the invention using the preferred static reservoir design.
These include:
Device (A) A device constructed of hollow fiber strands initially (prior to sulfonation) of 380 ~m O.D. and 300 ~m I.D., prepared by extrusion through a spinnerette in a known manner per se. The fiber is formed of Product Code No. 4005 low density polyethylene, commercialy 29,226-F -20-~63830 available from The Dow Chemical Company. The fibers for purposes of sulfonation are wound on a glass mandrel (cage) and secured with Teflon tape. Since sulfonation weakens the fibers, it is desired that portions of the fibers where the fittings are to be attached are not sub-ject to the sulfonating process. This is accomplished by looping discrete sections at a 90 angle to the circum-ferenti.ally wolln~ ib~r secti^nc '^ for... a loop offset from the mandrel end. Using a Teflon cord attached to the loop, the fibers are suspended in a 3-neck/one liter flask equipped with a condenser and heating mandrel.
A sufficient volume of a ten percent (10%) solution (v/v) of chlorosulfonic acid in methylene chloride is added to the flask to immerse the fibers (but not the looped end) and the solution is heated to reflux conditions (42C) and sulfonated for approximately 30 minutes. The fibers are retrieved and placed in methylene chloride and soaked for l/2 hour followed by washing with deionized water. The loops are cut to provide unsulfonated ends for potting in fittings 58. Capacity is approximately 1 meg/gm. The device as constructed uses preferably 7 sulfonated fiber strands each 8 inches long.
Device (B) A device constructed using 4 hollow fiber strands 8 inches long, 300 ~m O.D., 230 ~m I.D., obtained under the trade designation SR-~/751-7010 from Bio-Rad Laboratories. The fibers are believed to com-prise a copolymer of 40% ~-methylstyrene/60% polymethyl siloxane.
Device (C) A device prepared using the basic sulfonation procedure and material of Device (A), supra, prepared 29,226-F -21-~1~3~30 .
using 16 fiber strands each 8 lnches long, with a capacity of about 0.5 meq/gm, and final dimensions of approximately 375 ~m O.D., and 300 ~m I.D.
Device (D) Similar fibers to Device (A) are used in this device using 9 fiber strands, each about 10 inches long, sulfonatQd to achieve a capacl~-y- of about 0.7 meq/gm.
Device_(E) A device prepared from microporous cellulose hollow fibers, obtained commercially as a product of Spectrum Medical Industries, Inc., designated by Manu-facturers Order No. 132272, and sold under the trademark SPECTRA/POR hollow fibre (HF) membrane. The device consists of 10 fiber strands, each 6 inches long, described as having a molecular weight cutoff in the range of from 500 to 2000.
Example 2 ~ NitroPhenol Detection Separation of various nitrophenols using a silica based column, re~uires acid eluent pH conditions (about pH 6) for acceptable component resolution and/or for reason of column pH limitations. ~hile the nitro-phenols absorb at 280 ~m, organic interferences in the sample matrix produce poor detection sensitivity.
This experiment illustrates an excellent solution to this analysis problem is available by producing a pH change through treating the chromato-graphic column effluent w-th NH3 reagent, in order to produce derivative sample species which will absorb at 29,226-F - -22-~16~
a distinct wavelength that avoids matrix interferences.
The experiment uses Device (A), and as the reagent, a solution of 30% ammonium hydroxide in water.
The experiment is conducted under the further conditions as follows: Aqueous eluent of 12 volume per-cent methanol, .08 M sodium perchlorate, .04 M ammonium aceta~e; adjllsted to pH 6.1 wi'h glacial acetic acid is pumped at 1.8 ml/min using a Milton Roy mini-pump through a Rheodyne 7120 sample injection valve with a 100 ~1 sample loop. The sample consisting of an identified organic matrix spiked with nitrophenols of interest is added to a Whatman Partisil Sax 10/25 chromatographic column, and detected using a LDC 1203 (Laboratory Data Control) photometer at 410 nm detection wavelength. A
Spectro-Physics SP-4100 computing integrator is used for signal processing and computation.
Typical nitrophenols detectable by the system include 2,4,6-trinitrophenol, 2-sec butyl-4-6-dinitro--phenol, 3-sec buty-2-hydroxy 5-nitrobenzene sulfonic acid and 3-sec butyl-4 hydroxy 5-nitrobenzene sulfonic acid. Detection limits for these compounds are at sub-ppm levels with no sample preparation. At this detection wavelength and pH change, the system is found to be very selective.
In a similar experiment to the above, 2,4,6--trinitrophenol species of interest is detected in admixture with co-eluting 2-sec-butyl-4,6-dinitro--phenol, using as the reagent 2 M HCl in water. The chromatographic eluent is modified to a pH of about 2 29,226-F -23-~1~3~30 (eluent pH 6.1). The pH adjustment selectively attenu-ates the dinitrophenol peak, to produce resolution and detection of the trinitrophenol species of interest in the sub-ppm range.
Example 3 - Fluorescamine Addition The fluorescamine reaction is a particularly important reagent ~ddit~on reaction used, e.g., in the detection of primary amines. This experiment illus-trates a specific example of the fluorescamine reaction as used in the mode of the invention.
The experiment is conducted using as the reagent additive device, Device (B), and as the reagent in which the fibers are immersed, a 2 mg/ml solution of fluorescamine in acetonitrile. A Varian 8500 chromato-graphic pump is used to pump 30 volume percent aceto-nitrile in water eluent solution containing .01 M
ammonium acetate at 1 ml/min. A Rheodyne 7120 Injector (Z0 ~1 loop) is used to add a sample standard (1 ppm a-amino toluene in water) to a Waters ~-C-18 analytical HPLC column. The sample is detected using a duPont 836 fluorescence detector operated at 390 nm, 475 nm, excitation and emission wavelengths, respectively. A
strong peak (offscale) is obtained at the ppm sample concentration level.
ExamPle 4 - Ninhvdrin Reaction The ninhydrin reaction is an excellent tool used in the detection, e.g., of amino acids, and is commonly utilized in commercial amino acid analyzers to develop blue color species derivatives which may be detected by a visible light photometer. The reaction can be beneficially utilized in the mode of the inven-tion as illustrated in this Example.
29,226-F -24-~i63B30 Under the conditions of this experiment, eluent of .02 M citric acid in water adjusted to pH 4.0 with NaOH, is pumped by a Milton-Roy minipump at 0.52 ml/min through a Rheodyne 7120 injection valve equipped with a 200 ~1 loop where periodic injections of 50 ppm glycine are made to simulate peaks coming off of an analytical separation column. Device (C) is employed for th~ re~en-t addition purpose, using as the reagent, 100 ml of 10% ninhydrin solution in water. A hot plate is used to maintain the reagent at a temperature of 90C ~ 5C. The ninhydrin treated solution is subse-quently passed through a heated delay coil (2 meter, coiled Teflon tubing maintained at 90C) to provide a
"The adaptation of reaction detection to modern LC columns requires careful attenti on to [] the design of equipment, because extra column effects can be serious. For these 10reasons reaction detectors have so far found rather limited use in modern LC."
Equivalent conclusions are also expressed by Frei et al., "Reaction Detectors in HPLC", Journal of Chromatographic Science, Vol. 17, March 1979, pp.
15152-159, wherein the authors state: "The construction of proper reaction detectors comprises a constant struggle against band broadening." In still another recent publication by Jupille, " W - Visible Absorption Derivatization in Liquid Chromatography", Journal of Chromatographic Science, Vol. 17, March, 1979, pp.
160-167, the author listed among disadvantages of the design or state of the reactors: "a need for hardware modification (with attendant loss in flexibility); and [ ] a risk of band broadening due to post-column mixing volume resulting in loss of resolution."
By way of further explanation, the often mentioned problem of avoiding band spreading is inter-related to various factors, among which is the mode for metering reagent. ~ny lack in consistency of metering produces fluctuations in reagent concentration in the effluent, which shows up as "noise" in the chromatograph developed ~y the detector.
29,226-F -2-The problem is especially severe where highly concentrated reagent is used, since minute fluctuations in metering can produce high background "noise" levels that severely hamper the sensitivity of detection.
While one of the choices of the prior art is to use concentrated reagent to avoid band spreading by sample dilution, the gain may, nevertheless, be offset at least partially by increased backgrc7lnA r.^__e 'cv-cls.
Band spreading is also caused by diffusion of sample bands into one another as a function of time.
Prior devices inherently appear to obtain poor reagent/-effluent mixing, hence, extending the time factor. The deficiency is particularly shown by the description in the literature of reagent/effluent mixing devices as means for promoting faster reactions.
Prior solutions to these and other related problems can thus be said to often involve serious drawbacks, e.g., increased "noise". What may be equally as objectionable is the addition or too much complex eguipment such as air segmentation methods to the apparatus, which has already been characterized as involving too many inherently disadvantageous "hardware modifications" with an "attendant loss of flexibility".
Accordingly, an objective of the invention is to provide an improved liquid chromatographic method and apparatus characterized by the development of an improved post-column reactor which requires little in the way of hardware modifications.
A particular object of the invention is to provide an improved method and apparatus which 29,226-F -3-,~
achieve a substantially constant "pulseless" metering of reagent, and, in addition, achieve significantly improved diffusion of the reagent into the chromato-graphic column effluent.
It is a further object to provide such apparatus and method wherein sample dilution is minimized without the need foL resoriing tO the addition of highly concentrated forms of reagent as a means or requirement, or resorting to other objectionable process or apparatus restrictions.
Still a further objective hereof is to provide such method and apparatus, which in contrast with prior art methods and apparatus, are of significantly less cost to use and to maintain.
The term "hollow fiber membrane" means an extremely small tube or fiber having an internal diameter of from 20 to 1,000 microns, preferably, from 50 to 500 microns, and which has the property to trans-port mobile reagent in permeation contact with the exterior wall portion of the fiber (or saturated within the fiber wall matrix), while rejecting from transport, at least a detectable amount of a sample species of interest, or a derivative proportional thereto, flowing as a component of liquid chromatographic effluent through the internal bore of the hollow fiber membrane.
"Reagent" means a chemical species or com-bination of species which, when introduced through the hollow fiber membrane into chromatographic column effluent, reacts chemically, directly or indirectly, with a sample species of interest (or an interfering 29,226-F -4-~63830 sample species which is less than perfectly resolved with respect to a sample species of interest) to produce a measurable enhancement in the detection of said species of interest, or a monitored proportional derivative thereof, compared to the absence of the hollow fiber membrane/reagent combination.
"Mobile" refers to a .eayc.l~ in a S~d~ by which it may be permeated or transported through the wall or a wall portion of the hollow fiber membrane.
The above-stated objects of the invention are achieved by a liquid chromatographic apparatus comprising a chromatographic column, injector means for adding a sample solution to the chromatographic column, means for adding eluent to the chromatographic column whereby the sample is eluted through the chromatographic column and component species thereof appear in chromatographically displaced form in the effluent of the chromatographic column characterized by a post-column reactor comprising a hollow fiber membrane through which the effluent of the chromatographic column is fed to a liquid chroma-tographic detector, said hollow fiber being in permeation contact with a mobile reagent for permeation transfer of the reagent into the effluent of the chromatographic column.
A further aspect of the invention is a method of analyzing samples by liquid chromatographic comprising the steps of adding a sample solution to a chromatographic column, adding an eluent to the chromatographic column effective to chromatographically displace species of 29,226-F -5-the sample from the chromatographic column, whereby chromatographically displaced sample species appear ultimately in the effluent of the chromatographic column characterized by feeding the effluent from the chromatographic column through the internal bore of a hollow fiber membrane, ultimately to a liquid chromatographic detector, and prior to detection, using the hollow fibe~ .eïl~arle Lor permeaiion trans-fer of a mobile reagent into the effluent of the chromatographic column to enhance the sensitivity of detection.
An essential feature of the invention is the use of a single or multiple hollow fiber membranes in conjunction with prior developed liquid chromatographic methods and apparatus. In the preferred device, mul-tiple fibers are potted at each end into a tube sheet for purposes of isolating the inner portion or inner bore of the fibers and also to provide means for con-necting the inner portion of the fibers to standard liquid chromatographic fittings. The center section of the hollow fiber "bundle", which may be coiled to conserve space, is immersed within a mobile reagent. As such, the device is readily combined with standard and commercial forms of liquid chromatographic apparatus with minimal need for basic modification in the hard-ware.
Hollow fiber membranes useful in the practice of the invention are characterized by a molecularly porous wall structure obtained as an inherent property of the material used to make the fiber, or obtained as 29,226-F -6-1163E~30 _7--a product of the manufacturing process, or a combination of both. The invention makes no claim, as such, to inventing the hollow fiber membrane structure per se.
Typically used in practicing the method and apparatus of the invention are thus commercial hollow fiber membranes developed for other applications, and which are adaptable to the invention.
Among desired properties of the hollow fiber membrane (in addition to the ability to contain and transport reagent and resist transport of at least part of the sample) is its tolerance to contact with various liquids to which it is exposed. Particularly useful in this respect are porous cellulose membranes prepared such as by the method of U.S. Patent 3,546,209.
~15 Such membranes may be either isotropic or anisotropic in structure. The transport properties of this type of membrane are produced typically by fabricating discretely sized pores piercing the fiber wall, e.g., by "pushing aside" the membrane material to form the pore structure. As such, the permeation character-istics are basically that of size selection and may be broadly applied to permeate widely diverse reagent species.
Synthetic polymeric membranes, such as produced ~5 typically from polyolefins and also silicone rubber, as well as a considerable group of other polymeric materials, may be adapted for use in the invention. Particularly useful are charged ion-exchange hollow fiber membranes produced by sulfonation or amination processes in order to obtain "Donnan exclusion" rejection properties of ionic speciés. Permeation through such membranes is often typically through a "tortuous" path defined in the molecular spaces between polymer molecules. The 29,226-F -7-.~
~163830 transport and selective properties of the solid matrix polymeric hollow fiber is thus often very specific and thus requires careful selection with reagent and sample in mind. Specific examples of this type of the membrane are detailed below with respect to some of the reagents.
The "leakage" characteristics of hollow fiber memhranes use~ ir. the lnvellt~ al-e often~imes not highly critical. For example, leakage through the fiber wall of components other than sample is (although often not inconsequential) not a phenomenon that will severely hamper the analysis. Thus, eluents used in the invention are generally characterized as non-inter-fering or substantially non-interfering. Reagent diluents, where used, may be selected for the same properties. Accordingly, leakage of these character-ized components, assuming there is a net transfer one way or the other, may, at most, produce a dilution on the sample in the chromatographic effluent. Assuming a net loss of chromatographic effluent through such transfer, a desirable concentration of the sample may in fact result from beneficial leakage phenomena pro-ducing higher detection sensitivity.
In addition, while it would be desired to have a hollow fiber membrane that rejected sample in the perfect sense, such is neither an absolute or essential requirement. While too much loss of sample can be consequential to the sensitivity of the analysis, substantial loss of sample does not necessarily mean that severe detrimental results will occur. Thus, for illustrative example, 90 percent leakage of the sample would not nécessarily preclude a highly sensitive analy-sis from being performed where the gain of sensitivity 29,226-F -8-1~63830 g by the method is on the order of a thousandfold, which is not an unusual result possible with reagent addition methods. Since the amount of reagent necessary to achieve highly dramatic improvement in detection sensi-tivity can be very small, the selective transportproperties of the membrane may in fact favor transport of the sample. That is, a greater leakage of sample than p~rm~t~ on nf reagent may be experienced by use of a particular membrane, while at the same time obtaining enhancement in sensitivity of detection. Thus, selective transport properties of the hollow fiber membrane, favoring the reagent, is not an essential requirement of the invention in its broadest sense.
In respect to band spreading produced by dilution factors, or adding liquid volume to the chro-matographic effluent, the invention has a very important advantage over prior art reagent addition methods.
Thus, while it is contemplated that there is often a significant leakage of the non-active component of the reagent into the chromatographic effluent, since other than perfect permselective qualities are present in hollow fibers, there is at the same time the mentioned ability of the fiber to extract fluid from the effluent, so that a net exchange of fluid volume is involved.
For membranes which possess good sample rejection properties, it is thus possible to minimize dilution volume without resorting to difficult procedures that severely restrict the method.
In order to minimize band spreading due to geometry and flow pattern factors, it is desirable that if multiple hollow fibers are used, that each fiber produce the same resistance to flow and, 29,226-F -9-consequently, as the effluent stream is divided and flows in the multiple passages of the individual fibers, flow proceeds at a uniform rate. Thus, with flow dwell time in the individual fibers being substantially constant, the tendency for band spreading is greatly reduced. This is typically achieved by using multiple fibers of the same internal diameter and length.
Qbviously, to ^vercomc the effect entirely, fibers could be matched to produce exactly equivalent flow characteristics.
Also, as a general rule, the larger the fiber, the greater the tendency to produce band spreading by inherent fluid drag at the fiber wall/flow interface, hence producing an effect that if accentuated enough or prolonged for long enough, can cause loss of resolution.
The effect is minimized by using multiple fibers of extremely small diameters which, by means of larger surface area, can pass the reagent more quickly, fre-~uently permitting the use of a shorter path of travel and hence minimal band spreading based on diffusion of sample bands into one another as a factor of time. The rate of diffusion of the reagent in the chromatographic effluent is also effectively improved by using small--diameter multiple hollow fiber membranes. Hence, significant advantages in critical parameters are achieved by the use of a post-column reactor design based on the use of multiple hollow fibers having sample rejection properties, and which are connected in parallel between the chromatographic column and detector.
Highly suited for use in the multiple fiber device are fibers of between about 100-300 ~m 29,226-F -10-"
(micrometers) in internal diameter for use in conventional bore liquid chromatography. In conjunc-tion with micro-bore liquid chromatography such as described by Scott et al., Journal of ChromatographY, Vol. 169, pg. 51 (1979) extremely small fibers in the range as low as the current manufacturing limit of about 20 ~m I.D. would be usable. The wall thickness of the fibers is not always highly critical to rate of permeability as explained in U.S. Patent 3,808,267.
Generally, the wall thickness of fibers as used in the invention is from 5 to 250 ~m. The length of the fibers can also vary, depending on permeation and/or diffusion rates through the fiber wall. Generally, it is contemplated to use fibers of from 10 to 200 centi-meters in length. The restriction on length (andminimum I.D.) is ultimately a function of pressure drop through the fibers and back pressure on the fibers.
Too much back pressure can rupture the fibers, thus limiting the pumping pressure. This can be compensated for, however, by equilibrating the pressure about the fibers with the internal pressure, such as by maintain-ing the fibers in a pressurized vessel. By taking the latter precautions, the length of the fibers may be increased to the point that resolution of the chroma-tographically displaced species is not too adverselyaffected.
The fibers or a portion thereof are in permea-tion contact with the mobile reagent. The temperature, differential pressure with the effluent, and concentra-tion of the reagent can si~nificantly affect permeationrates in accordance with known membrane transfer phenomena.
29,226-F -11-The reagent comprises, e.g., a pure liquid, pure gas or solution of reagent in a diluent or carrier whereby the reagent is mobile and may thus permeate the fiber wall.
In some cases, an inert diluent such as methylene chloride is used which modifies the membrane such as by diffusion into the membrane to increase the rate of per-meation of the reagent, also in accordance with known mem~rane phenomer.a. Other types of liquids, such as water or acetonitrile, are typically used as the reagent diluent.
In addition, it is also possible to use reagent, particularly ionic species, attached to active ion exchange sites of solutions or gels of ion-exchange resin, or agitated ion exchanged beads, in order to pro-duce mobile reagent. This embodiment may be used, forexample, with a charged ion-exchange hollow fiber mem-brane, whereby the reagent ion is exchanged with the exchangeable ion of the membrane and, thus, ultimately dif~uses into the chromatographic column effluent. The embodiment is particularly useful wherever it would be desired that the ions similarly extracted from the chromatographic effluent, by the charged hollow fiber membrane, would be detrimental to detection. A means would thus be provided for minimizing return leakage.
In addition, the embodiment can be used to prevent transfer of the relatively large polymeric counter ion of a given ionic reagent particularly where the counter ion would interfere or otherwise be detrimental to the analysis.
Two forms of reagent reservoirs or containers are particularly contemplated. The preferred form is generally described as a static reservoir, although the 29,226-F -12-1163~330 reagent may be agitated or stirred in order to prevent a concentration differential from occurring. Alterna-tively, a dynamic flow of the reagent, wherein con-tinuously fresh reagent is pumped into contact with the fibers may be used. The latter embodiment has advantages where contamination of the reservoir by leaklng chromato-graphic effluent would be detrimental to the analysis.
In such an embodiment, the fibers are placed, e.g., co-axially within a preferably flexible tube container to define an annular space between the fiber and inner wall of the tube. The fibers are potted at each end to isolate the inner bores of the fibers for connection to chromatographic fittings similarly as with respect to a static reservoir. Spaced tees are connected at the end of the tube container and the reagent is continuously pumped into the annular space, most effectively in counter flow to the flow of the effluent from the chromatographic column.
Because of less complexity, and typically very comparable performance to the dynamic reservoir embodiment, the static reservoir is to be preerred.
Because the dilution of leaking sample into the rela-tively large reservoir of reagent produces an extremely dilute solution of a possible interfering species, the effect at most may be reflected as a slightly varying base line over a period of use. Ordinarily such would thus not justify the additional complexity associated with a dynamic reservoir embodiment.
The reaction kinetics of reagent addition methods, as well as prior developed reagents, are considered background technology to the invention and useful in its practice. Thus, the specific reagents 29,226-F -13-~i63~30 and reactions developed for detection purposes and used in the invention are drawn from prior art sources. A
fairly detailed listing of reactions considered suitable for use in post-column effluent reaction procedures is given by Snyder et al., supra, pages 740-746, and the similar teachings of the prior cited references by Gfeller et al.; Frei et al.; and Jupille, together with a publ'ication by Vance Nau et al., "Application of Microporous Mernbranes to Chemiluminescence", Analytical ChemistrY, Vol. 51, No. 3, March 1979, pp. 424-428.
Favorable reaction conditions necessary to promote the reaction must of course be present with this invention as with other reagent addition modes.
Thus, reactions which require a major proportion of water in the final reaction mixture will generally require that aqueous eluents be used. The latter requirement is generally met with respect to ion--exchange and reverse-phase liquid chromatographic separation methods, or similar liquid chromatographic methods which employ an aqueous eluent or mobile phase in which the reagent is at least partially miscible.
Similarly, for liquid chromatographic techniques, such as classical normal phase chromatography, the invention is typically limited to the selection of reagents that can proceed in or require an organic solution in which the reagent is at least partially miscible. For slow reactions, elevated temperatures are imparted to the reaction mixture such as by means of a heated reaction delay loop which provides added residence time.
Liquid chromatographic detectors useful in the practicé of the invention are particularly advan-tageously photometers, spectro-photometers and fluoro-meters used together with reagents which alter or produce 29,226-F -14-11~i3830 light absorbance of sample species in the chromato-graphic effluent or which produce fluorescing deriva-tive products. Among other liquid chromatographic detectors beneficially used in the mode of the inven-tion are, for example, differential refractometers,electrochemical detectors, radioactive detectors, and conductivity detectors.
Further.objects and advantages of the inven-tion will 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 chromatograhy using post-column reagent addition in accordance with the principles and teachings of the present invention.
Figure 2 is an enlarged cross-sectional view showing additional details of the post-column reactor used in the Figure l apparatus; and Figure 3 is a view of a reactor similar to Figure 2 showing a modified form of the post-column reactor.
Referring to Figure 1, there is shown a chromatographic column 10 comprising a housing contain-ing a separating means in the form of a particulate packing or gel through which a sample solution is eluted to separate the sample into component species. Diverse types of separating means may be used to construct a suitable chromatographic column, as described exten-sively by Snyder et al.
'~ 29,226-F -15-1~L63~3t~
A preferred means for adding an eluent solu-tion or mobile phase to the chromatographic column 10 comprises an eluent reservoir 12 containing an eluent solution 14, the latter which is withdrawn from the reservoir by a pump 16 equipped with an optional pulse damping coil 18.
A preferred means for adding a sample comprises a syringe loadable sample injection valve 20. Sample added to the system at valve 20 is swept through the apparatus by the pumped eluent solution through an optional guard column 22 to chromatographic column 10.
The sample is eluted through column 10, and component species thereof thus ultimately appear chromatograph-ically displaced in the chromatographic column effluent which is delivered to the reagent addition device or post-column reactor 24, described in further detail below.
The reagent addition device or reactor is optionally followed by a reaction delay coil (not shown) or functionally equivalent element used whenever neces-sary to provide added reaction time vis-a-vis the species to be derivatized. Optionally, both or either the reagent solution or delay coil is maintained at a controlled temperature by suitable temperature control means (not shown) such as a temperature controlled plate which heats the solution or fluid in which the delay coil is immersed;
and/or which heats the reagent solution. The delay coil is ultimately followed by a detector 26 of a type suited for liquid chromatography.
In the detector, the effluent produces an electrical signal proportional to the property monitored 29,226-F -16-11~i3830 such as, for example, light absorbance, fluorescence, and which signal is conducted from the detector to a recorder 28 such as, for example, a strip chart recorder, an integrator, and the like, all of which are well-known to the art.
Referring to Figure 2, a preferred form of reagent addition device, (based on a silllpii~ity facior and acceptable performance for a great number of reagent addition reactions) is the "static" reservoir design which comprises a reservoir or reagent container 30, preferably made of glass or a similar inert material.
Contained in the reservoir is a mobile reagent 32. The reagent is periodically changed through a screw-cap or other suitable closure 34. A hollow fiber or fibers 36 are suspended in the reagent between an effluent-in feed connection 38 and an effluent-out feed connection 40.
Each connection is made by drilling openings 42 in the closure 34. An adapter 44 is mounted in each open-ing by use of general purpose epoxy glue, shown at 42.
Tubing 48 or supplying the chromatographic column effluent iB attached to a threaded nipple 50 on the adapter 44 by using a standard connecting nut 52 and ferrule 54. The out feed is similarly connected by tubing 56 to the detector 26 through a similar connecting nut 52 and ferrule 54.
A preferred form of fixing the ends of the hollow fiber or fibers into the female part of adapter 44 is to mold a itting 58 about the end portions 60 of the fibers. This fitting is made by using a rubber compound, such as Silastic brand mold making rubber J-RTV, to make a mold, using as the form for the mold, the commercial 29,226-F -17-fitting which is to be duplicated. For an epoxy system, the fibers are threaded through the mold by drilling a small diameter hole for threading purposes. An epoxy, such as, Dow 331 Epoxy Resin/Ancamine LT Hardener is poured into the mold and cured, with the fibers "wetted", or dry depending whether fiber swelling is expected in the end use application. After removal from the mold, the fiber ends are tri~med as re~iLed. A fiDer coating layer 62, such as, Silastic 730 RTV Fluorosilicone Sealant (from Dow Corning Corp.) is preferably applied in the area immediately adjacent the fitting to avoid fiber point stress, and thus minimize damage as may occur such as in the physical handling of the fibers.
The epoxy resin system is useful for aqueous reagent and eluent systems, certain aromatic solvents, and some hydrocarbons. For reagent solutions or eluents which chemically attack epoxies, there is used in preference and for longer life, a Silastic Brand J-RTV
material for constructing the fittings 58 which otherwise is manufactured similar to the epoxy fittings. Fittings 58 are also suitably manufactured using a hollow commer-cial fitting through which the fibers are passed and which is filled with a sealant material, such as, 730 RTV
sealant which is subsequently cured. The latter thus basically consist of commercial fittings with the ends of the fibers potted inside the hollow portions of the fittings by an in situ curing process using the described RTV material, or a substitute in situ curable substance.
A preferred embodiment of a reagent addition device having the fibers immersed in a counter flowing mobile reagent is illustrated in Figure 3. This device 29,226-F -18-~1638;~0 is''c'onstructed of a center section of stainless steel tubing or tubular containing means or jacket 68 through which a fiber or a bundle of hollow fibers is inserted by suction or by gluing the end of a length of thread to a fiber bundle and pulling the fibers through the jacket using water as the lubricant. A tee or tee fit-ting 70 is affixed to opposite ends, respectively, of the jacket 68 using tube nutC 72 wi~h f~,ru es 74 tG
make the attachment.
Exposed portions of the hollow fibers (out-wardly of each tee) are dried and inserted into sealing tubes 76, respectively, which are preferably made of stainless steel. A section of about six inches of the fibers is left exposed between the sealing tubes and the tees 70 and the exposed fiber sections are coated with a suitable sealant, such as, Silastic 732 RTV sili-cone rubber, as indicated at areas 66, after which the sealing tubes are coupled to the tees using tube nuts 78 and ferrules 80. Additional RTV sealant is injected into the sealing tubes using a blunt 20 gauge needle to completely fill each sealing tube but taking care not to force excess rubber into the tees. The RTV sealant is allowed to cure for about 10 minutes to promote initial bonding and curing is completed with the fiber "wetted" or dry as the end-use application demands. A
razor blade is used to cut the fiber ends off so that they are flush with the end of the sealing tube. The device may then be coupled into the apparatus of Figure 1 by using reducing union assemblies 82. Similar connec-tions are provided to the tees 70 for supplying thereagent through tubing 84 and 86 to a reservoir and pump (not shown), respectively. These latter connec-tions may consist a tube element 88 joined at one end -- 29,226-F -19-1~63830 to each tee 70 through a tube nut 90 and ferrule 92;
and at the opposite end joined, respectively, to the reagent inlet and outlet 84, 86 through a reducing union assembly 94. The device as assembled defines contiguous flow channels comprising, respectively, the collective bores of the hollow fibers, and the spaces exterior of the fibers within the jacket 68 and which communicate, respectively, wlth the chromatographic column 10 and the reagent supply source (not shown).
The described reagent addition devices operate by receiving the effluent from the chromatographic column 10 which is routed internally through the hollow fibers. Simultaneously, the mobile reagent solution (static or in the dynamic form) flow on the exterior surfaces of the hollow fibers, thus causing permeation of the reagent into the chromatographic effluent.
The invention is still further illustrated by reference to the specific teaching examples below.
ExamPle 1 - Hollow Fiber Membranes Various hollow fiber membranes are used to construct reagent addition devices for use in the invention using the preferred static reservoir design.
These include:
Device (A) A device constructed of hollow fiber strands initially (prior to sulfonation) of 380 ~m O.D. and 300 ~m I.D., prepared by extrusion through a spinnerette in a known manner per se. The fiber is formed of Product Code No. 4005 low density polyethylene, commercialy 29,226-F -20-~63830 available from The Dow Chemical Company. The fibers for purposes of sulfonation are wound on a glass mandrel (cage) and secured with Teflon tape. Since sulfonation weakens the fibers, it is desired that portions of the fibers where the fittings are to be attached are not sub-ject to the sulfonating process. This is accomplished by looping discrete sections at a 90 angle to the circum-ferenti.ally wolln~ ib~r secti^nc '^ for... a loop offset from the mandrel end. Using a Teflon cord attached to the loop, the fibers are suspended in a 3-neck/one liter flask equipped with a condenser and heating mandrel.
A sufficient volume of a ten percent (10%) solution (v/v) of chlorosulfonic acid in methylene chloride is added to the flask to immerse the fibers (but not the looped end) and the solution is heated to reflux conditions (42C) and sulfonated for approximately 30 minutes. The fibers are retrieved and placed in methylene chloride and soaked for l/2 hour followed by washing with deionized water. The loops are cut to provide unsulfonated ends for potting in fittings 58. Capacity is approximately 1 meg/gm. The device as constructed uses preferably 7 sulfonated fiber strands each 8 inches long.
Device (B) A device constructed using 4 hollow fiber strands 8 inches long, 300 ~m O.D., 230 ~m I.D., obtained under the trade designation SR-~/751-7010 from Bio-Rad Laboratories. The fibers are believed to com-prise a copolymer of 40% ~-methylstyrene/60% polymethyl siloxane.
Device (C) A device prepared using the basic sulfonation procedure and material of Device (A), supra, prepared 29,226-F -21-~1~3~30 .
using 16 fiber strands each 8 lnches long, with a capacity of about 0.5 meq/gm, and final dimensions of approximately 375 ~m O.D., and 300 ~m I.D.
Device (D) Similar fibers to Device (A) are used in this device using 9 fiber strands, each about 10 inches long, sulfonatQd to achieve a capacl~-y- of about 0.7 meq/gm.
Device_(E) A device prepared from microporous cellulose hollow fibers, obtained commercially as a product of Spectrum Medical Industries, Inc., designated by Manu-facturers Order No. 132272, and sold under the trademark SPECTRA/POR hollow fibre (HF) membrane. The device consists of 10 fiber strands, each 6 inches long, described as having a molecular weight cutoff in the range of from 500 to 2000.
Example 2 ~ NitroPhenol Detection Separation of various nitrophenols using a silica based column, re~uires acid eluent pH conditions (about pH 6) for acceptable component resolution and/or for reason of column pH limitations. ~hile the nitro-phenols absorb at 280 ~m, organic interferences in the sample matrix produce poor detection sensitivity.
This experiment illustrates an excellent solution to this analysis problem is available by producing a pH change through treating the chromato-graphic column effluent w-th NH3 reagent, in order to produce derivative sample species which will absorb at 29,226-F - -22-~16~
a distinct wavelength that avoids matrix interferences.
The experiment uses Device (A), and as the reagent, a solution of 30% ammonium hydroxide in water.
The experiment is conducted under the further conditions as follows: Aqueous eluent of 12 volume per-cent methanol, .08 M sodium perchlorate, .04 M ammonium aceta~e; adjllsted to pH 6.1 wi'h glacial acetic acid is pumped at 1.8 ml/min using a Milton Roy mini-pump through a Rheodyne 7120 sample injection valve with a 100 ~1 sample loop. The sample consisting of an identified organic matrix spiked with nitrophenols of interest is added to a Whatman Partisil Sax 10/25 chromatographic column, and detected using a LDC 1203 (Laboratory Data Control) photometer at 410 nm detection wavelength. A
Spectro-Physics SP-4100 computing integrator is used for signal processing and computation.
Typical nitrophenols detectable by the system include 2,4,6-trinitrophenol, 2-sec butyl-4-6-dinitro--phenol, 3-sec buty-2-hydroxy 5-nitrobenzene sulfonic acid and 3-sec butyl-4 hydroxy 5-nitrobenzene sulfonic acid. Detection limits for these compounds are at sub-ppm levels with no sample preparation. At this detection wavelength and pH change, the system is found to be very selective.
In a similar experiment to the above, 2,4,6--trinitrophenol species of interest is detected in admixture with co-eluting 2-sec-butyl-4,6-dinitro--phenol, using as the reagent 2 M HCl in water. The chromatographic eluent is modified to a pH of about 2 29,226-F -23-~1~3~30 (eluent pH 6.1). The pH adjustment selectively attenu-ates the dinitrophenol peak, to produce resolution and detection of the trinitrophenol species of interest in the sub-ppm range.
Example 3 - Fluorescamine Addition The fluorescamine reaction is a particularly important reagent ~ddit~on reaction used, e.g., in the detection of primary amines. This experiment illus-trates a specific example of the fluorescamine reaction as used in the mode of the invention.
The experiment is conducted using as the reagent additive device, Device (B), and as the reagent in which the fibers are immersed, a 2 mg/ml solution of fluorescamine in acetonitrile. A Varian 8500 chromato-graphic pump is used to pump 30 volume percent aceto-nitrile in water eluent solution containing .01 M
ammonium acetate at 1 ml/min. A Rheodyne 7120 Injector (Z0 ~1 loop) is used to add a sample standard (1 ppm a-amino toluene in water) to a Waters ~-C-18 analytical HPLC column. The sample is detected using a duPont 836 fluorescence detector operated at 390 nm, 475 nm, excitation and emission wavelengths, respectively. A
strong peak (offscale) is obtained at the ppm sample concentration level.
ExamPle 4 - Ninhvdrin Reaction The ninhydrin reaction is an excellent tool used in the detection, e.g., of amino acids, and is commonly utilized in commercial amino acid analyzers to develop blue color species derivatives which may be detected by a visible light photometer. The reaction can be beneficially utilized in the mode of the inven-tion as illustrated in this Example.
29,226-F -24-~i63B30 Under the conditions of this experiment, eluent of .02 M citric acid in water adjusted to pH 4.0 with NaOH, is pumped by a Milton-Roy minipump at 0.52 ml/min through a Rheodyne 7120 injection valve equipped with a 200 ~1 loop where periodic injections of 50 ppm glycine are made to simulate peaks coming off of an analytical separation column. Device (C) is employed for th~ re~en-t addition purpose, using as the reagent, 100 ml of 10% ninhydrin solution in water. A hot plate is used to maintain the reagent at a temperature of 90C ~ 5C. The ninhydrin treated solution is subse-quently passed through a heated delay coil (2 meter, coiled Teflon tubing maintained at 90C) to provide a
3-minute delay for the reaction to occur. The reactor effluent is monitored using a Perkin-Elmer LC-55 W /VIS
absorption photometer operated at 600 nm. The peak of blue absorbance due to the ninhydrin-amino acid reaction product is recorded on a Sargent Welch Model SG Recorder.
Excellent peaks are produced showing detection limits at the ppm level. Approximately 75% of the amino acid i8 estimated to have reacted under the conditions employed. No discoloration of the ninhydrin reagent solution is noted during the course of consecutive injections of sample .
Example 5 - Iodide Reaction Peroxides and other relatively strong oxidants will oxidize I to I2 to form highly colored I3 in the presence of excess I . The reaction is useful, e.g., to deterrnine the presence of peroxides or other strong oxidants in industrial process streams and products.
As an illustration of the use of this reaction in the inventive mode, eluent of 100 mg/liter (NH4)2MoO4 29,226-F -25-1~ti38~0 in water, pH 5, is pumped at l ml/min into reagent additive Device (D), using as the reagent, stirred 1.0 M potassium iodide in water maintained at 54C ~ 0.2C.
Repeated injections of aqueous hydrogen peroxide samples are made using a Rheodyne sample injection valve, 50 ~l sample loop size (a separating column is not used in this experiment).
The sample is detected using a duPont 837 Visible W photometer set at 375 nm. The recorded data is shown in the following Table.
TABLE
Sample Size Attenuation Peak Heiqht Att'n x Peak 31.6 ppm 32x 208 6650 100 ppm 128x 126 16130 316 ppm 256x 108 27650 10 ppm 32x 70 2240 10 ppm 16x 139 2224 3.1 ppm 16x 49 784 3.1 ppm 8x 98.5 788 3.1 ppm 4x 197.5 790 1.0 ppm 4x 62.5 250 0.3 ppm 4x 19.5 78 29,226-F -26-~6;~830 The detection limit, calculated from the data, is estimated to be about 0.1 ppm. The method is considered to show linearity up to about 50 ppm hydrogen peroxide.
Example 6 - Cellulose Fibers The iodide reaction may similarly be applied to ~tect sp~cies e.g., N02,C103, bleach and C12. This example illustrates the detection of sodium nitrite, using the cellulose hollow fiber Device (E), and using as the reagent, 0.1 N potassium iodide in water.
In order to demonstrate the feasibility of the reactor--detector combination, sample standards of 100 to 1000 ppm, 20 ~1 sample injection size, are injected into pumped water eluent flowing at 1 ml/min, fed through Device (E), and the effluent of the Device monitored by a Perkin-Elmer LC-55 photometer set at 360 nm. Although not considered optimized, excellent detector sensitivity response is achieved, producing an estimated detection sensitivity comparable to that of hydrogen peroxide in the preceding example.
29,226-F -27-
absorption photometer operated at 600 nm. The peak of blue absorbance due to the ninhydrin-amino acid reaction product is recorded on a Sargent Welch Model SG Recorder.
Excellent peaks are produced showing detection limits at the ppm level. Approximately 75% of the amino acid i8 estimated to have reacted under the conditions employed. No discoloration of the ninhydrin reagent solution is noted during the course of consecutive injections of sample .
Example 5 - Iodide Reaction Peroxides and other relatively strong oxidants will oxidize I to I2 to form highly colored I3 in the presence of excess I . The reaction is useful, e.g., to deterrnine the presence of peroxides or other strong oxidants in industrial process streams and products.
As an illustration of the use of this reaction in the inventive mode, eluent of 100 mg/liter (NH4)2MoO4 29,226-F -25-1~ti38~0 in water, pH 5, is pumped at l ml/min into reagent additive Device (D), using as the reagent, stirred 1.0 M potassium iodide in water maintained at 54C ~ 0.2C.
Repeated injections of aqueous hydrogen peroxide samples are made using a Rheodyne sample injection valve, 50 ~l sample loop size (a separating column is not used in this experiment).
The sample is detected using a duPont 837 Visible W photometer set at 375 nm. The recorded data is shown in the following Table.
TABLE
Sample Size Attenuation Peak Heiqht Att'n x Peak 31.6 ppm 32x 208 6650 100 ppm 128x 126 16130 316 ppm 256x 108 27650 10 ppm 32x 70 2240 10 ppm 16x 139 2224 3.1 ppm 16x 49 784 3.1 ppm 8x 98.5 788 3.1 ppm 4x 197.5 790 1.0 ppm 4x 62.5 250 0.3 ppm 4x 19.5 78 29,226-F -26-~6;~830 The detection limit, calculated from the data, is estimated to be about 0.1 ppm. The method is considered to show linearity up to about 50 ppm hydrogen peroxide.
Example 6 - Cellulose Fibers The iodide reaction may similarly be applied to ~tect sp~cies e.g., N02,C103, bleach and C12. This example illustrates the detection of sodium nitrite, using the cellulose hollow fiber Device (E), and using as the reagent, 0.1 N potassium iodide in water.
In order to demonstrate the feasibility of the reactor--detector combination, sample standards of 100 to 1000 ppm, 20 ~1 sample injection size, are injected into pumped water eluent flowing at 1 ml/min, fed through Device (E), and the effluent of the Device monitored by a Perkin-Elmer LC-55 photometer set at 360 nm. Although not considered optimized, excellent detector sensitivity response is achieved, producing an estimated detection sensitivity comparable to that of hydrogen peroxide in the preceding example.
29,226-F -27-
Claims (6)
1. Liquid chromatographic apparatus com-prising a chromatographic column, injector means for adding a sample solution to the chromatographic column, means for adding eluent to the chromatographic column whereby the sample is eluted through the chromatographic column and component species thereof appear in chromato-graphically displaced form in the effluent of the chromatographic column characterized by a post-column reactor comprising a hollow fiber membrane through which the effluent of the chromatographic column is fed to a liquid chromatographic detector, said hollow fiber being in permeation contact with a mobile reagent for permeation transfer of the reagent into the effluent of the chromatographic column.
2. The apparatus of Claim 1 characterized in that said post-column reactor comprises multiple hollow fiber membranes.
3. The apparatus of Claim 1 or 2 character-ized by a reservoir containing the mobile reagent, wherein the reservoir is of a type non-continuously replenished by reagent.
29,226-F -28-
29,226-F -28-
4. A method of analyzing samples by liquid chromatography comprising the steps of adding a sample solution to a chromatographic column, adding an eluent to the chromatographic column effective to chromato-graphically displace species of the sample from the chromatographic column whereby chromatographically displaced sample species appear ultimately in the effluent of the chromatographic column characterized by feeding the effluent from the chromatographic column through the internal bore of a hollow fiber membrane, ultimately to a liquid chromatographic detector, and prior to detection, using the hollow fiber membrane for permeation transfer of a mobile reagent into the effluent of the chromatographic column to enhance the sensitivity of detection.
5. The method of Claim 4 characterized by using multiple hollow fiber membranes connected between the chromatographic column and detector and immersed within mobile reagent.
6. The method of Claim 4 wherein said mobile reagent is contained within a non-continuously replen-ished reservoir.
29,226-F -29-
29,226-F -29-
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US18352780A | 1980-09-02 | 1980-09-02 | |
| US183,527 | 1980-09-02 | ||
| US27412781A | 1981-06-16 | 1981-06-16 | |
| US06/465,976 US4451374A (en) | 1980-09-02 | 1983-02-14 | Liquid chromatographic method and post-column effluent treatment for detection and separation at optimized pH |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1163830A true CA1163830A (en) | 1984-03-20 |
Family
ID=27391693
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000381805A Expired CA1163830A (en) | 1980-09-02 | 1981-07-15 | Liquid chromatographic method and apparatus with hollow fiber device for post-column derivatization |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US4451374A (en) |
| EP (1) | EP0058168B1 (en) |
| JP (1) | JPH0239742B2 (en) |
| AU (1) | AU532872B2 (en) |
| CA (1) | CA1163830A (en) |
| WO (1) | WO1982000773A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4672042A (en) * | 1981-12-21 | 1987-06-09 | Orion Research Puerto Rico, Inc. | Method of and apparatus for chromatographic analysis |
| CA1220053A (en) * | 1983-10-01 | 1987-04-07 | Toyo Soda Manufacturing Co., Ltd. | Method for chromatographic analysis and apparatus therefor |
| USRE38435E1 (en) * | 1983-12-28 | 2004-02-24 | Daicel Chemical Industries, Ltd. | Separating agent |
| USRE34457E (en) * | 1983-12-28 | 1993-11-30 | Daicel Chemical Industries, Inc. | Separating agent |
| US4628726A (en) * | 1984-03-29 | 1986-12-16 | Etd Technology, Inc. | Analysis of organic compounds in baths used in the manufacture of printed circuit board using novel chromatographic methods |
| US4694682A (en) * | 1984-03-29 | 1987-09-22 | Etd Technology, Inc. | Analysis of organic additives in plating baths using novel chromatographic methods in a mass balance approach |
| US4999098A (en) * | 1984-10-04 | 1991-03-12 | Dionex Corporation | Modified membrane suppressor and method for use |
| AU4750885A (en) * | 1984-10-04 | 1986-04-10 | Dionex Corporation | Ion exchange membrane for chromatographic reagent addition |
| DE3678194D1 (en) * | 1985-01-25 | 1991-04-25 | Toray Industries | METHOD AND DEVICE FOR PRODUCING A THIN OBJECT. |
| US4837161A (en) * | 1985-03-04 | 1989-06-06 | The Dow Chemical Company | Method for reagent addition to a flowing liquid carrier stream |
| JPS61212758A (en) * | 1985-03-04 | 1986-09-20 | ザ ダウ ケミカル カンパニー | Method and device for adding reagent to flowing liquid carrier flow |
| GB2171926B (en) * | 1985-03-04 | 1988-12-21 | Dow Chemical Co | A method and apparatus for reagent addition to a flowing liquid carrier stream |
| US4715217A (en) * | 1985-06-06 | 1987-12-29 | The Dow Chemical Company | Determining organic compounds using a membrane |
| JPS62282260A (en) * | 1986-05-31 | 1987-12-08 | Tosoh Corp | Analysis of anion |
| US4966695A (en) * | 1988-02-04 | 1990-10-30 | Henry Joshua | High pressure liquid chromatography column jacket |
| US4819478A (en) * | 1988-03-04 | 1989-04-11 | The Dow Chemical Company | Membrane assisted flow injection analysis |
| EP0361234B1 (en) * | 1988-09-26 | 1995-05-10 | Waters Investments Limited | Process and apparatus for preparing samples for ion analysis |
| US4957620A (en) * | 1988-11-15 | 1990-09-18 | Hoechst Celanese Corporation | Liquid chromatography using microporous hollow fibers |
| DE4234728A1 (en) * | 1992-10-15 | 1994-04-21 | Peter Prof Dr Bartholmes | Process for the recovery and the buffering and / or concentration of dissolved macromolecules of a macromolecule mixture |
| US5980834A (en) * | 1996-07-25 | 1999-11-09 | The United States Of America As Represented By The Secretary Of Commerce | Sample storage devices |
| EP0820804B1 (en) * | 1996-07-26 | 2005-12-14 | Metrohm Ag | Method and apparatus for preparing samples |
| US6100522A (en) * | 1998-06-15 | 2000-08-08 | Amway Corporation | Interface for liquid chromatograph and mass spectrometer |
| US20020155033A1 (en) * | 2000-10-06 | 2002-10-24 | Protasis Corporation | Fluid Separate conduit cartridge |
| AUPR094600A0 (en) * | 2000-10-23 | 2000-11-16 | Usf Filtration And Separations Group Inc. | Fibre membrane arrangement |
| US20070071649A1 (en) * | 2001-09-10 | 2007-03-29 | Marcus R Kenneth | Capillary-channeled polymer fibers as stationary phase media for spectroscopic analysis |
| CN100564332C (en) * | 2004-09-28 | 2009-12-02 | 科聚亚公司 | Sulfonation nitrophenyl phenolic as polymerization retarder |
| US7691952B2 (en) * | 2004-09-28 | 2010-04-06 | Chemtura Corporation | Sulfonated nitrophenols as polymerization inhibitors |
| US11287403B2 (en) | 2016-01-07 | 2022-03-29 | Board Of Regents, The University Of Texas System | Ion chromatography system and methods utilizing a weak acid or weak base extraction device |
| CN105758982A (en) * | 2016-02-25 | 2016-07-13 | 杭州飞山浩科技有限公司 | Device and method for measuring nitrite ions in milk powder |
Family Cites Families (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3367850A (en) * | 1964-12-07 | 1968-02-06 | Exxon Research Engineering Co | Method and apparatus for determining moisture content of hydrocarbon fluids |
| CH462501A (en) * | 1965-03-26 | 1968-09-15 | Ceskoslovenska Akademie Ved | Method and device for avoiding the remixing of individual components of a substance mixture capable of an indication reaction with an indication reagent after its separation |
| US3472627A (en) * | 1965-04-07 | 1969-10-14 | Ceskoslovenska Akademie Ved | Method and apparatus for separating number of samples displaced in a tubing of small internal diameter |
| NL136034C (en) * | 1965-12-22 | |||
| GB1199440A (en) * | 1966-10-13 | 1970-07-22 | Ceskoslovenska Akademie Ved | Method of and Device for Removing Gas Bubbles in Capillary Reactors. |
| DE1798208C3 (en) * | 1968-09-07 | 1973-10-04 | Jenaer Glaswerk Schott & Gen, 6500 Mainz | Separation column for liquid chromatography and process for its preparation |
| US3751879A (en) * | 1971-04-26 | 1973-08-14 | Instrumentation Specialties Co | Apparatus for reducing the dissolved gas concentration in a liquid |
| FR2240754B1 (en) * | 1973-08-17 | 1976-06-18 | Erap | |
| US3954678A (en) * | 1974-07-11 | 1976-05-04 | E. I. Du Pont De Nemours And Company | Semipermeable microcapsules containing a silica gel |
| US4181853A (en) * | 1976-12-10 | 1980-01-01 | Varian Associates, Inc. | Liquid chromatography system with packed flow cell for improved fluorescence detection |
| US4233030A (en) * | 1977-03-08 | 1980-11-11 | National Research Development Corporation | Methods and apparatus for liquid chromatography |
| US4251218A (en) * | 1978-08-07 | 1981-02-17 | Orion Research Incorporated | Method of adjusting pH |
| CA1162416A (en) * | 1980-01-16 | 1984-02-21 | Timothy S. Stevens | Ion analysis method and apparatus |
-
1981
- 1981-07-10 JP JP56502539A patent/JPH0239742B2/ja not_active Expired - Lifetime
- 1981-07-10 AU AU73763/81A patent/AU532872B2/en not_active Ceased
- 1981-07-10 EP EP81902122A patent/EP0058168B1/en not_active Expired
- 1981-07-10 WO PCT/US1981/000926 patent/WO1982000773A1/en active IP Right Grant
- 1981-07-15 CA CA000381805A patent/CA1163830A/en not_active Expired
-
1983
- 1983-02-14 US US06/465,976 patent/US4451374A/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| AU7376381A (en) | 1982-03-31 |
| WO1982000773A1 (en) | 1982-03-18 |
| EP0058168A1 (en) | 1982-08-25 |
| JPS57501544A (en) | 1982-08-26 |
| US4451374A (en) | 1984-05-29 |
| EP0058168A4 (en) | 1983-01-14 |
| EP0058168B1 (en) | 1987-05-06 |
| AU532872B2 (en) | 1983-10-13 |
| JPH0239742B2 (en) | 1990-09-06 |
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