Pre-Column for Preconcentrating Substances to Be Chromatographed as Well as Device for a Liquid Chromatography System and Coupling Member for Said Device

The invention will now be described in greater detail with reference to preferred embodiments which are shown in the drawings, in which FIGS. 1, 2 and 3 are longitudinal cross-sectional views of pre-columns according to the invention; FIGS. 4, 5 and 6 are a plan view and cross-sectional views of a coupling member for a liquid chromatography system; FIG. 7 is a sectional view of a device for a liquid chromatography system having a coupling member and pre-columns in accordance with the invention, and FIG. 8 shows the same device as FIG. 7, this time as a plan view.

The pre-column shown in the longitudinal sectional view of FIG. 1 consists of one assembly and is made of stainless steel. The cylindrical central part 1 converges conically towards the two ends, the part 2 converging more strongly in the direction of the upstream end 3 than the part 4 in the direction of the downstream end 5. The upstream end 3 has internal screwthread into which a nipple having external screwthread can be screwed. An inlet tube for the liquid to be chromatographed can be connected to said nipple. The downstream end has external screwthread with which the pre-column can be screwed into a coupling member, as shown for example in FIG. 6, and has a projecting circumferential edge or ridge 6 which forms a mount for a gasket.

FIGS. 2 and 3 are cross-sectional views of pre-columns according to the invention which are composed of two parts, namely vessel-shaped parts 70 and 54, respectively, and covers 48 and 55, respectively. The cover of the pre-column shown in FIG. 2 has a nipple 71 to which an inlet tube can be connected through which liquid can reach the interior of the column via central bore 72. The cover has external screwthread 73 with which it can be screwed into the vessel-shaped part having internal screwthread 74 up to a gasket 75 which fits in a mount 76 of the vessel-shaped part. The gasket 75 in which a filter of sintered metal 50 is mounted, is shown on an enlarged scale in the drawing. The vessel-shaped part 70 at the downstream end has an external circumferential ridge 77 which forms a mount for a gasket, and has screwthread 78 with which the column can be screwed into a coupling member, for example, as shown in FIGS. 7 and 8. Through recesses 79 and 79a in the cover and tap-shaped apertures 80 and 80a in the vessel-shaped part, which apertures have internal screwthread, the parts of the bipartite column shown in FIG. 3 can be screwed one on top of the other by means of bolts. The gaskets 81 and 82 fall into the circumferential grooves 83 and 84 recessed respectively in the cover and vessel-shaped part, while a filter or filters present in a filter holder 85 is or are kept in its or their place between the gaskets. The cover has a circumferential edge 86 which adjoins the outer edge of the vessel-shaped part. An internally screwthreaded connection aperture 60 for a nipple for connection to an inlet tube is present in cover 55. Internally the column tapers conically towards the two ends, towards the upstream end at an angle of 75.degree., towards the downstream end at an angle of 27.degree.. Finally, the vessel-shaped part of the column at the downstream end again has externally a circumferential ridge 87 and screwthread 88.

FIG. 4 is a plan view of a coupling member of a device for a liquid chromatography system to which a pre-column can be connected at 96. As is clearly visible in FIGS. 5 and 6 which are cross-sectional views of the same device taken on the lines V--V and VI--VI, respectively, of FIG. 4, the coupling member comprises two concentric disks 7 and 8, also of stainless steel, disk 7 being rotatable about a bearing boss 9. The disks are clamped one on the other by the same boss 9 which is constructed as a bolt and is screwed through disk 7. Circular gaskets (for example, 10 and 11) of teflon which have an approximately circular cross-section are present between the two disks in grooves recessed in one of the disks. The circular gaskets are present between the disks and are provided around the bores (see hereinafter) in the disks. The connection aperture for the pre-column 96 has external screwthread so that the column can be screwed into it up to a sealing gasket 12 of reinforced teflon. On the other side of the gasket the aperture 96 is narrowed to form a bore having a smaller diameter, while a filter of sintered metal 90 which is disk-shaped is present between the connection aperture and the bore. On the outer edge of the same disk in which the connection aperture for the pre-column is recessed, a handle 13 is provided. Said disk furthermore comprises a nipple 14 for the connection to an inlet tube for conditioning liquid. A conditioning liquid is used to condition the separating column or columns prior to the chromatography process. On the other disk of the coupling member a handle 15 is connected by means of two bolts, which handle can be fixed at its end 16 remote from the disk in a stand. An insert 18 for the connection to a separating column and having a central bore is present in a mount 17 of the same disk. This latter disk moreover comprises a nipple 19 for the connection to an outlet tube for waste liquid. The disk furthermore comprises an abutment plate and pin 20, 21, as a result of which the rotational distance of the disks relative to each other necessary to make the apertures in the disks communicate with each other can more easily be determined.

The device shown in FIGS. 7 and 8 for a liquid chromatography system comprises a coupling member 22 consisting of two concentric stainless steel disks which are rotatable about a shaft 23 which has bearings at each end and in which disks apertures are recessed. The same teflon gaskets are present as described before the FIGS. 4-6 embodiment. The upper disk 24 is connected to a flange 26 of the central shaft 23 by means of bolts 25. The upper disk is rotatable relative to the lower disk, the shaft also rotating. Six apertures are recessed in the upper disk and are provided with screwthread so that either pre-columns (at 29, 30 and 31) or, as at 32, inserts 33 having central bores and comprising nipples or to which nipples can be connected can be screwed into them. All apertures are continued inwardly as bores of reduced diameters. Apertures 34 are also recessed in the lowermost disk and form mounts for inserts 35 which have a central bore and to which nipples or separating columns are or can be connected. The mounts in the disks are of the same shape as those described with reference to FIGS. 4-6. The upper disk can be rotated with respect to the lower disk by means of a handle 36. The lower disk 28 furthermore comprises a locking device for the upper disk 24, comprising a resilient arm 37 having a pawl 61 which falls into apertures recessed in the outer edge of the upper disk. Therewith the upper disk can be fixed in a given position relative to the lower disk. The fixing can be removed by pulling the pawl with the knob 38 against the action of the spring out of an aperture in the outer edge of the upper disk.

At its outer edge the lower disk is screwed into a stand 39, comprising two diametrically oppositely located rod-shaped elements 40 extending parallel to the central shaft 23. At some distance above the coupling member 22, two circular stainless steel plates 41, 42 are connected to the central shaft 23, the uppermost 41 of which being connected to the rod-shaped elements 40 of the stand by means of connection bars or spokes 43. The plates are sealingly clamped one on top of the other, circular teflon gaskets being provided between the plates as was the case with the disks of the coupling member. The lower plate 42 is rigidly secured to the bearing shaft 23 by means of bolts 44, and hence rotates with the upper disk 24 of the coupling member 23. Both plates 41 and 42 comprise nipples 27, 27a which are connected to bores provided in the plates. The nipples in the lower plate 42 can be connected by means of liquid tubes to the nipples or pre-columns in the upper disk 24 of the coupling member 22; the nipples in the upper plate 41 can be connected to reservoirs containing different liquids, for example, conditioning liquid, liquid to be chromatographed and various elution liquids. The device shown in FIGS. 7 and 8 is excellently suitable for an automatic chromatography process, because between conditioning, loading the pre-column or columns with trace components to be separated, and various elutions, no inlet tubes need to be detached. Any desired liquid can be introduced into one of the pre-columns or separating columns by a simple rotation of the upper coupling disk.

In the device shown in FIGS. 7 and 8, three stainless steel pre-columns according to the invention are provided which are screwed into the upper disk 24 of the coupling member 22. The column 45 screwed into aperture 29 of said disk is of the type as shown in FIG. 2. The columns 46 and 47 screwed into the apertures 30 and 31 of disk 24 are of the type shown in FIG. 3. Column 45 comprises a cover 48 having a screw connection and connection nipple. The carrier material is enclosed in the column between two filters 50, 51 of sintered metal. In the conical part of the column on the side of the upstream end, an inert filler 52 is present on the filter. The column is sealingly connected to the elongated bore 62 of the aperture 29 recessed in the upper coupling disk 24 by means of a teflon gasket 53. Column 46 consists of two parts (54 and 55) which are connected together by means of bolts 56. As in column 45, filters of sintered metal 57, 58 are also present in column 46, while the sealing to the upper coupling disk 24 is formed by a teflon gasket 59. The column communicates at the downstream end with aperture 63 in disk 24.

Of course, various modifications of the abovedescribed devices are possible. For example, the number of apertures recessed in the disks of the coupling member and in the circular plates may be varied at will and may be suitable for various connections, for example, inserts or screw connections. Many other modified embodiments obvious to those skilled in the art are also possible without departing from the scope of the present invention.

When using a pre-column according to the invention, first the carrier material, for example a hydrophobated silica gel, for example Corasil, Nucleosil, Zorbax or Lichroprep is enclosed between two filters of sintered metal in the column. An inert filler, in this case glass beads, is provided on the filter in the conical part on the side of the upstream end of the column. The pre-column is connected to the coupling device shown in FIGS. 5-6 or in FIGS. 7-8. In the device shown in FIGS. 7-8 more pre-columns are used according to the invention. The device further comprises one or more separating columns which are preferably filled with the same type of carrier material. For conditioning the pre-columns and separating columns, conditioning liquid is rinsed through the columns and collected in a waste vessel. The precolumn is then loaded with the material to be chromatographed for example, a urine sample, by causing this material to flow through the pre-column which is connected to an outlet tube. After loading, the pre-column is connected to a separating column, after which the column is eluted. During this elution a so-called gradient elution is preferably used, which means that during the elution process the composition of the eluent is varied. The elution rate, i.e. the rate at which the eluent is pumped through the columns, depends on the dimensions of pre-column(s) and separating column(s), on the material to be chromatographed, the carrier material and the elution liquid. The elution is preferably carried out at room temperature; during the elution the pressure in the columns and coupling device may increase to 20 to 30 MPa, even to 40 MPa. The detection of the trace components may take place in various manners, for example, by means of spectroscopic methods such as UV-spectroscopy. In most of the cases, however, too many chromophorous "impurities" are present in the eluate to be able to successfully use a UV-measurement. Therefore preferably a radioactivity-measurement is used. In that case in metabolites examination, the chemical compound to be examined, usually a medicament, is labelled radioactive, for example with radioactive labelled carbon or hydrogen: .sup.14 C or .sup.3 H. By measuring the emitted radioactivity, both the chemical compound itself and also possible metabolites thereof which comprise the labelled fragment, can be detected. This detection may take place "on-line" in the eluate flow, or "off-line", i.e. in the collected eluate fractions. The former method is more rapid, the "off-line" detection method usually gives more accurate results.

In order to be able to detect Isoxsuprine and metabolites hereof in urine, 200 ml of urine were subjected to the above-described chromatography process. Isoxsuprine or p-hydroxy-.alpha.-[1-{(1-methyl-2-phenoxyethyl)amino}ethyl]benzylalcohol is a substance having a vaso-dilating activity. The medicament and the metabolites hereof were present in traces in the sample to be chromatographed, namely in an overall quantity of a few micrograms. For the pre-concentration a stainless steel column according to the invention was used of the type as shown in FIG. 3 having a capacity of 27 ml. As a filler for the pre-column was used Nucleosil 30 C 8, a hydrophobated silica gel. The separating column had an inside diameter of 9 mm and a length of 50 cm, and was filled with Zorbax 7 C 8, also a hydrophobated silica gel. Isoxsuprine was labelled with .sup.3 H. The gradient elution was carried out with a mixture of water and methanol having a methanol content increasing from 0 to 100%. The elution rate was approximately 5 ml per minute. The trace components were detected off-line the chromatogram shown in FIG. 9 being obtained. In this chromatogram the measured radioactivity RA in counts per minute is plotted against time in minutes. The peak denoted in the chromatogram by I is of Isoxsuprine, the other peaks represent metabolites of Isoxsuprine.

For comparison the above experiment was repeated with a straight pre-column usual for this purpose, instead of a pre-column according to the invention. The straight pre-column had an inside diameter of 9 mm which remains the same and a length of 15 cm. In practice a longer column could not be used because in that case during loading the pressure build-up in the column became too large. The experiment was further carried out with the same apparatus, the same type of material and in the same reaction conditions as described above for a pre-column according to the invention. The resulting chromatogram is shown in FIG. 10. It is clear from the chromatogram that so-called "ghost effects" occur: at GE.

The pre-column according to the invention was also used in solving various other separating problems, as appears from the chromatograms shown in FIGS. 11, 12, 13 and 14. A single coupling device, as shown in FIGS. 4-6, or the six-fold device, as shown in FIGS. 7-8, was used.

The chromatograms of FIGS. 11 and 12 have been obtained by column chromatography of samples which comprise [2-{4-(3-trifluoromethylphenyl)-1-piperazinyl}ethyl]-urea, a compound having antiagressive activity, and metabolites hereof. FIG. 11: stainless steel pre-column as shown in FIG. 2; capacity 1.5 ml; filler Nucleosil 30 C 8. Separating column: stainless steel; length 500 mm; inside diameter 9 mm; filler Nucleosil 7 C 8. Provided sample: 22 ml of blood plasma of a dog. Elution: gradient elution with a mixture of water and methanol having a methanol content increasing from 0 to 100%. The compound was labelled with .sup.14 C. Detection: off-line; measured radioactivity RA in counts per minute. The peak at T is of the active compound: the other ones are of metabolites. FIG. 12: stainless steel pre-column as shown in FIG. 3; capacity 27 ml; filler Nucleosil 7 C 8. Separating column: stainless steel; length 500 mm; inside diameter 9 mm; filler Nucleosil 7 C 8. Provided sample: 620 ml of urine of a dog. Elution: gradient elution with a mixture of water and methanol having a methanol content increasing from 0 to 100%. The compound was labelled with .sup.14 C: Detection: on-line. The peak at T is of the active compound; the other ones are of metabolites. The gradient profile of the eluent in % methanol is denoted by GP.

FIG. 13 shows a chromatogram obtained by chromatography of a urine sample to which 0.5 ppb of DES had been added. DES or diethylstilbestrol is a substance having oestrogenic activity. Stainless steel pre-column according to the invention having a capacity of 13 ml; filler Lichroprep RP2, a hydrophobated silica gel. Separating column: stainless steel; length 250 mm; inside diameter 9 mm; filler Nucleosil 7 C 8. Provided urine sample: 50 ml. Elution: gradient elution with water-methanol having a methanol content increasing from 0 to 100%.

DES was labelled with .sup.3 H. Detection: off-line. The peak at D is of DES.

The chromatogram of FIG. 14 has been obtained by column chromatography of a urine sample of a rat containing traces of N-(2,6-difluorobenzoyl)-N'-(4-trifluoromethylphenyl) urea and metabolites hereof; this urea compound can be used as an insecticide in agriculture, horticulture and forestry. Stainless steel pre-column as shown in FIG. 3; capacity 27 ml; filler Nucleosil 30 C 8. Separating column: stainless steel; length 500 mm, inside diameter 9 mm; filler Zorbax 7 C 8. Provided urine sample: 121 ml. Elution: gradient elution with a mixture of water and methanol having a methanol content increasing from 0 to 100%.

The compound was labelled with .sup.14 C. Detection: on-line. The peak at P is of the active compound, the other ones are of metabolites. The gradient profile of the eluent in % methanol is denoted by GP.