CA1162417A - Method and apparatus for on-column detection in liquid chromatography - Google Patents
Method and apparatus for on-column detection in liquid chromatographyInfo
- Publication number
- CA1162417A CA1162417A CA000393664A CA393664A CA1162417A CA 1162417 A CA1162417 A CA 1162417A CA 000393664 A CA000393664 A CA 000393664A CA 393664 A CA393664 A CA 393664A CA 1162417 A CA1162417 A CA 1162417A
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- Prior art keywords
- column
- detection
- detector
- accordance
- segment
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Classifications
-
- 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
- G01N30/60—Construction of the column
- G01N30/6052—Construction of the column body
- G01N30/6073—Construction of the column body in open tubular form
- G01N30/6078—Capillaries
-
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- 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
- G01N30/60—Construction of the column
- G01N30/6052—Construction of the column body
- G01N30/6082—Construction of the column body transparent to radiation
Abstract
Abstract Method and Apparatus for On-Column Detection in Liquid Chromatography On-column detection by optical means is accomplished with a flexible silica column. The flexible fused silica column has an inner diameter less than 500µm, an external protective coating with a stripped portion near the end of the column.
The stripped portion is placed in the working path of an optical detector in order to detect and resolve the sample.
The stripped portion is placed in the working path of an optical detector in order to detect and resolve the sample.
Description
1~62~7 Method and Apparatus for On-Column Detection in Liquid Chromatoyraphy This invention relates to sample detection in ` 5 the field of chromatography and, more particularly, relates to a method and apparatus for on-column I detection in high resolution liquid chromatography.
In the field of chromatography, separation of species is accomplished by flowing a sample contain-ing multiple species in a carrier gas or liquid I through a column containing a stationary phase.
¦ The species within the sample are separated on the basis of their relative transit time through I the column. The species are detected at different ! 15 time intervals by means such as ionization detectors, spectrophotometric detectors, spectrofluorometric detectors, electrochemical detectors and the ~ike.
Since these detectors are attached to the end oE
chromatographic columns an interconnection between the column and the detector is required. ~heseinterconnections invariably introduce mixing .
.. ~
1~6ZA~L7
In the field of chromatography, separation of species is accomplished by flowing a sample contain-ing multiple species in a carrier gas or liquid I through a column containing a stationary phase.
¦ The species within the sample are separated on the basis of their relative transit time through I the column. The species are detected at different ! 15 time intervals by means such as ionization detectors, spectrophotometric detectors, spectrofluorometric detectors, electrochemical detectors and the ~ike.
Since these detectors are attached to the end oE
chromatographic columns an interconnection between the column and the detector is required. ~heseinterconnections invariably introduce mixing .
.. ~
1~6ZA~L7
-2 effects and dead volume which result in the loss of resolution between peaks especially if the peaks are close together and especially when the cross-section of the interconnection tube becomes much larger than the cross-section of the column. This problem has become exacerbated as the field o chromatography has progressed to the use of micro-columns having inner-diameters of 500 microns or less. In such cases, it has become increasingly difficult, if not impossible, to provide inter-connections which do not introduce substantial mixing effects, since, even though the absolute volumes are small, the cross-sectional areas oE the column, interconnection and flow cell may be much different.
In addition, it has been di~ficult to fabricate and attach detectors with volumes as small as those found within the columns themselves.
Microcapillary liquid chromatography has rapidly progressed to the use of columns having inner diameters in the range of 30--60 microns. See e.g., D. Ishii, et al. "Study of Open-Tubular-Micro-Capillary Liquid Chromatography", J. Hi~h Resolution Chromatography & Chromatography Communications, June 1979, p. 371. The use of such columns leads to severe discontinuities between the column and the detector. This discontinuity in inner diameters may be of the following type: a column having inner diameter of 30-60 microns may be succeeded by connectors having inner diameters of 130 microns and 70 microns, respectively, and a detector having an inner diameter of 170 microns (D. Ishii, supra, p.
373); and a capillary column having an inner diameter of 60 microns followed by an interconnection haviny an inner diameter of 150 microns and a detector having an inner diameter o~ 300 microns (P. ~'suda, Z4~7
In addition, it has been di~ficult to fabricate and attach detectors with volumes as small as those found within the columns themselves.
Microcapillary liquid chromatography has rapidly progressed to the use of columns having inner diameters in the range of 30--60 microns. See e.g., D. Ishii, et al. "Study of Open-Tubular-Micro-Capillary Liquid Chromatography", J. Hi~h Resolution Chromatography & Chromatography Communications, June 1979, p. 371. The use of such columns leads to severe discontinuities between the column and the detector. This discontinuity in inner diameters may be of the following type: a column having inner diameter of 30-60 microns may be succeeded by connectors having inner diameters of 130 microns and 70 microns, respectively, and a detector having an inner diameter of 170 microns (D. Ishii, supra, p.
373); and a capillary column having an inner diameter of 60 microns followed by an interconnection haviny an inner diameter of 150 microns and a detector having an inner diameter o~ 300 microns (P. ~'suda, Z4~7
-3-"Studies of Open Tubular Micro-Capillary Liquid Chromatography", J. Chromatogr., V. 158, P. 227, 229 (1978~). It is evident that even though the inner diameters involved are small, there are great differences among the successive links so that at the interface severe mixing efEects and dead volume effects will be experienced. Sueh ef~ects necessarily reduce the resolution between peaks.
Fused silica columns are now being used in place of capillary columns in gas chromatography and in liquid chromatography. See F. J. Yang, "Fused Silica Open Tubular Column for Liquid Chromatography", J. High Resolution Chromatography and Chromatography Communications, p. 589, November, 1980, and refer-ences cited therein. The advantage of fused silicacolumns is that they can be drawn on heating to exceedingly small diameters and active absorbents can be internally bonded for use in producing chromatographic separation. In addition, fused silica columns have excellent mechanical strength and optical properties and when drawn down to sufficiently small diameters, as described subse-quently, are flexible. For a discussion of the utility of narrow diameter glass columns, see T. Tsuda, et al. U.S. Patent No. 4,207,188, "Open Tubular Capillary Colurnn for High-Speed Micro-Liquid Chromatography".
On-column flourescence detection has previously been accomplishedO In B. F. Lloyd, "Nitrogen Heter-acycle and Polynuclear Hydrocarbon Flourescence andAdsorption Effects in the Presence of Silica Gel -- Applications in High-Pressure Liquid and Microcolumn Chromatography'l, The Analyst, v. 100, pp 529, 531 ~1975), a 3.5 mm outer diameter, l mm inner diameter , 35 microcolumn is constricted at one end by heating to ~L6;~7 ,g_ have an inner diameter of approximately .1 mm. The cell, i.e., the constricted region, was packed with a dry absorbent which was retained between cotton or glass fibre plugs. As sample was flowed through the constricted region at flow rates of up to 2 ~1 per minute the flourescence emanating from the absorbent was monitored by a microscope. In this instance of on-column detection, khe column was rigid since it was 1000~ m in inner diameter and was not transparent due to the inclusion o packing material.
Summary of the Invention On-column detection is accomplished by optical means with a flexible fused silica column. The flexible column is internally activated, has an inner 15 diameter less than 500~ m, and an external protective coating with a portion thereof stripped near the end of the column. The stripped portion is placed in the working path of an optical detector while a sample is flowed through the column in order to detect and 20 resolve the sample.
Brief Description of the Drawings For a more complete understanding of the method and apparatus of the present invention, reference may be made to the accompanying drawings which are 25 incorporated herein by reference and in which:
FIGS. la-le illustrate the steps for fabricating a microcapillary column suitable for on-column detection in accordance with the present invention;
FIG. 2 depicts an on-column ultraviolet detection 30 apparatus used in conjunction with the apparatus of the present invention;
~.
1~6%~7 FIG. 3 is a chromatogram of components of a sample which were separated by the apparatus of the present invention;
FIG. 4 is a graph illustrating the contribution to the loss of resolution produced by the on-column detector of the present invention for two retained components with partition constants o 1 and 10, respectively; and FIG. S is a chromatogram of benzene as detected b~ the apparatus of the present invention.
Description of the Preferred Embodiments The conventional wisdom has been that it is not feasible to detect the separation of constituents within a sample while it is yet flowing at the end of a chromatographic column. This was not believed to be feasible especially for visible optical detection because conventional column materials have not had acceptable optical properties. An exception is the flourescence detection of the Lloyd article discussed above. For visible optical detection, however, soda lime and borosilicate glass have had unacceptably high optical absorption and metal columns have been opaque. In addition, ordinary glass columns have not been able to operat~ at pressures up to 750 atmospheres which are required for high resolution liquid chromatography. The method of the present invention, however/ utilizes fused silica both for a continuous chromatographic column and for an in-tegral detector flow cell. Fused silica is much stronger than other glasses due to the internal bonding. As a consequence~ it requires temperatures of 1800-2000C to be drawn as compared with ~00C for ~oda lime glass and 700C for pyrex. Fused silica columns ~; which are drawn down to have inner diamete~s to less
Fused silica columns are now being used in place of capillary columns in gas chromatography and in liquid chromatography. See F. J. Yang, "Fused Silica Open Tubular Column for Liquid Chromatography", J. High Resolution Chromatography and Chromatography Communications, p. 589, November, 1980, and refer-ences cited therein. The advantage of fused silicacolumns is that they can be drawn on heating to exceedingly small diameters and active absorbents can be internally bonded for use in producing chromatographic separation. In addition, fused silica columns have excellent mechanical strength and optical properties and when drawn down to sufficiently small diameters, as described subse-quently, are flexible. For a discussion of the utility of narrow diameter glass columns, see T. Tsuda, et al. U.S. Patent No. 4,207,188, "Open Tubular Capillary Colurnn for High-Speed Micro-Liquid Chromatography".
On-column flourescence detection has previously been accomplishedO In B. F. Lloyd, "Nitrogen Heter-acycle and Polynuclear Hydrocarbon Flourescence andAdsorption Effects in the Presence of Silica Gel -- Applications in High-Pressure Liquid and Microcolumn Chromatography'l, The Analyst, v. 100, pp 529, 531 ~1975), a 3.5 mm outer diameter, l mm inner diameter , 35 microcolumn is constricted at one end by heating to ~L6;~7 ,g_ have an inner diameter of approximately .1 mm. The cell, i.e., the constricted region, was packed with a dry absorbent which was retained between cotton or glass fibre plugs. As sample was flowed through the constricted region at flow rates of up to 2 ~1 per minute the flourescence emanating from the absorbent was monitored by a microscope. In this instance of on-column detection, khe column was rigid since it was 1000~ m in inner diameter and was not transparent due to the inclusion o packing material.
Summary of the Invention On-column detection is accomplished by optical means with a flexible fused silica column. The flexible column is internally activated, has an inner 15 diameter less than 500~ m, and an external protective coating with a portion thereof stripped near the end of the column. The stripped portion is placed in the working path of an optical detector while a sample is flowed through the column in order to detect and 20 resolve the sample.
Brief Description of the Drawings For a more complete understanding of the method and apparatus of the present invention, reference may be made to the accompanying drawings which are 25 incorporated herein by reference and in which:
FIGS. la-le illustrate the steps for fabricating a microcapillary column suitable for on-column detection in accordance with the present invention;
FIG. 2 depicts an on-column ultraviolet detection 30 apparatus used in conjunction with the apparatus of the present invention;
~.
1~6%~7 FIG. 3 is a chromatogram of components of a sample which were separated by the apparatus of the present invention;
FIG. 4 is a graph illustrating the contribution to the loss of resolution produced by the on-column detector of the present invention for two retained components with partition constants o 1 and 10, respectively; and FIG. S is a chromatogram of benzene as detected b~ the apparatus of the present invention.
Description of the Preferred Embodiments The conventional wisdom has been that it is not feasible to detect the separation of constituents within a sample while it is yet flowing at the end of a chromatographic column. This was not believed to be feasible especially for visible optical detection because conventional column materials have not had acceptable optical properties. An exception is the flourescence detection of the Lloyd article discussed above. For visible optical detection, however, soda lime and borosilicate glass have had unacceptably high optical absorption and metal columns have been opaque. In addition, ordinary glass columns have not been able to operat~ at pressures up to 750 atmospheres which are required for high resolution liquid chromatography. The method of the present invention, however/ utilizes fused silica both for a continuous chromatographic column and for an in-tegral detector flow cell. Fused silica is much stronger than other glasses due to the internal bonding. As a consequence~ it requires temperatures of 1800-2000C to be drawn as compared with ~00C for ~oda lime glass and 700C for pyrex. Fused silica columns ~; which are drawn down to have inner diamete~s to less
4~
r --6--than 500~ m are flexible and may be coiled with large lengths fitting in small volumes. This fused silica is now commercially available for use in gas chroma-tography columns with inner diameters of less than 300 microns. See, e,gO, R. Dandeneau, et al~, "Flexible Fused Silica Columns", American Laboratory, June 1979; Alltech Associates, Product Bulletin No.
37, p. 1 (1980). The use of the end of a fused silica column itself as a flow cell for detection in the present invention enhances sensitivity and preserves chromatographie resolution because coneen-tration is high and sharp peaks are seen.
Some ingenious approaehes to low dead volume detectors which have been developed and reported are (1) laser fluorimetry, G. J. Diebold and R.N. ~are, "Laser Fluorimetry: Subpicogram Detection of Alfa-toxins Using High-Pressure Liquid Chromatography", Scienee 196, 1439-1441 (1977~; (2) miniaturized W
deteetors, D. Ishii, et al. "A Study of Micro-High-Performance Liquid Chromatography to Development ofTechnique for Miniaturization of High-Performance Liquid Chromatography", J. Chromatogr, 144, 157-168 (1977); (3) Y. Hirata, et al., "Packed Mierocapillary Columns With Different Selectivities for Liquid Chromatography", Anal. Chem. 51, 1807-1809 (1979);
(4) miniaturized electro-ehemical detectors, Y.
Hirata, et al., "Small-Volume Electrochemical Detector for Mierocolumn Liquid Chromatography", J. Chromatogr.
181, 287-294 (1980); (5) a sheath flow fluorometric detector, L.W. Hershberger, et al., "Sub-Microliter Flow-Through Cuvette for Fluorescence Monitoring of High Performance Liquid Chromatographic Effluents", Anal. Chem. 51, 1444-1446 (1979); and (6) the free falling drop fluorometrie detector, F. Martin, et 1~
al., "The Free-Falling Drop Detector - A Novel Fluor-escence Detector for High Performance Liquid Chroma-tography", Clin. Chem. 22, 1434-1437 (1979). Among these, the UV and spectro~fluorometric detectors are the most trouble-free and most frequently used.
The availability of low dead volume UV and spectr-ofluorometric detectors are an important factor in permitting small-bore column LC to become a powerful separation technique for routine applications.
Yet, a limitation to these low dead volume detectors is that the flow cell volumes between 0.1 and 0.3 L are far from the optimum desirable for small-bore column LC. For example, in small-bore column liquid chromatography, it has been theoretically derived that detector flow cell volumes of 1 - 10 nl are required for achieving less than 1~ loss of peak resolution on a l0 to 50~ m diameter open tubular column, for a retained peak with partition ratio k = 10 and retention time = l hour. See J. Knox, et al. "Kinetic Optima~ation of Straight Open-TubuJar Liquid Chromatography", J. Chromatogr., vO 186, pp 405-418 (1979). In addition, the extra band broadening caused by column to detector connec-tions and interface tubing significantly affect performance. The method and apparatus of the present invention serves to eliminate effects due to column-to-detector interconnection and detector dead volume effects due to mixing.
Method The method of the present invention is illus-trated in the Figures and particularly in FIGS.
la-lf. A fused silica column 10, shown in cross-section in FIG. la, is selected with a wall thickness J 11 on the order of 24 mm and an inner diameter I~
4~
less than 20 mm. The ~used silica column 10 is then drawn under thermal treatment in a iber optics drawing machine at temperatures on the order o ` ~ 2000C. The drawing rate (ratio of extrac~ion rate/
insertion rate) can be varied rom grea~er than one up to 1000. As this rate is increased bo~h the inner I diame~er and the wall thickness decreases. The reduced inner diameter will typically be less than 500lJm and the reduced wall thickness 11' will be between 20 and 150~ m. Such columns are of the type which are commercially available for gas chromato-graphy as described above. Larger diameter columns are typically not flexible and are therefore not encom-passed within the method of the present invention.
The drawn column 10' is flexible but fragile. For protection a polymer coating 12 such as polyimide or a metallic coating is applied externally; typically the external coating has a thickness between 10 and 150 ~ m.
As shown in FIGS. ld and le, the fused silica column is activated either by coating the interior with a stationary phase 13 or by chemically bonding a stationary phase 13 to the interior surface of the column or by packing the columns with activated microparticles 14. At the lowest inner column diameters (~hose approaching the diameters of the microparticles), coatings may be more practible than packings, although packing techniques such as disclosed in K. Mochizuki, et al., "Column for use in High Speed Liquid Chromatography", U.S. Patent No. 4,059,523 may be used. The art of coating or packing is well established and most conventional approaches may be used. See L. R. Snyder, et al., Introduction to Modern Liquid Chromatography, 9.2, "Column Packings" p. 287 et seq. Next, as shown in 2~7 FIG. lf, the external protective coating is removed along a short segment of the coated or packed fused silica microcapillary column. Removal i5 accom-plished by scraping, dissolving or thermally decomposing the coating. This segment is of the order of 1 cm or less and is sufficient to permit access by a visible light optical detector through the column. In efect, a flow cell is produced without having to interconnect separate pieces. The volume of this cell may be as small as .24 nL when the inner diameter of the column is 10~ m. The exposed segment of the fused silica column 15' is then placed in the optical path of an ultraviolet photodetector, such as a Jasco UVIDEC III or a lS Varichrome UV-50l as shown in FIG. 2. In an alternative embodiment the activating layer 13 is also removed along the end of the fused silica column up to and including the short segment to eliminate any absorption of visible light by the thin film. Removal is accomplished by external application of heat or by dipping the end of the column in a suitable solvent. For packed columns the activated microparticles 14 are kept from the flow cell by a frit positioned upstream.
Structure The apparatus for accomplishing on-column detection in accordance with the present invention is shown in FIGS. 2 and 3. In FIG. 2 a UV detector such as a Jasco UVIDEC III is utilized. The UV
detector comprises a light source 20, focusing optics 21, monochromator 22 having an exit slit 25 and a photodetector 24. In lieu o a flow cell, a passage-way is provided or the used sillca column 10'.
The exposed segment 15 ls placed across the optlcal ., 4~7 path so that photodetector 24 receives light that has passed through a full diameter of the column which has a sample flowing through it. The flow rate is not a limiting factor and may be as great as 10 ml/min. In an alternative embodiment a multiple wavelength UV detector such as Varian Varichrome UV-50 may be employed. The system arrangement is the same with the wavelengths being selected to permit several substances to be detected simultaneously.
Since there is negligible mixing in the on-column detector of the present invention, the extra-column band broadening contribution to the percent loss of resolution, ~R, can be expressed by RC-R ~ 1 % ~ R = O 100~ - [~ ~ ~] 2 ) ~ 100~ (1) ~c HL
where Rc,R = resolution measured for the column and the combination of column and detector, respectively h,H = the height equivalent to a theore-tical plate for the flow cell and the column, respectively ~,L = the entrance/exit slit length for the flow cell and the column length, respectively.
For a circular cross-sectional column, as an example, h, and H can be expressed by equations (2) and (3), respectively.
2Dm ~ r2U
h = _ _ (2) U 2~Dm H~ m ~ 6k' ~ llk' ). r U ~ 2k' df U (3) U 24(1 ~ k~)2 Dm 3(1 + k~)2 D
where r = column radius U = the average linear flow veloc;ty for the mobile phase Dm & Dl = are the diffusivities for the solute molecules in the mobile and the stationary phases, respectively df - the stationary phase film thickness k' = the partition ratio for the solute molecules = true retention time/
column void time.
Using these equations, the contribution of the on--15 column detector of the present invenkion to %
for two retained components with partition ratios, k', of l and lO on a column with length up to 15 meters is given in Figure 4. Flow rate and Dm are assumed to be 1 ~ L/min and l X 10-5 cm2/sec, respectively. The length of the detector is 0.3 cm.
Column diameter is assumed to be 150~ m. For conven-tional optical detectors the associated flow cells may make a significant contribution, on the order of tens of percent, to loss of resolution. As shown in FIG. 4, even for a partition ratio approaching l, (a lower limit for a chromatographic column perform-ing good separation) there is a contribution by the on-column detection "cell" to loss of resolution of the order of hundredths of a percent. As 3G expected, the higher the partition ratio, the less the contribution to loss o~ percent resolution.
The percent loss in peak resolution due to the on-column detector is in fact calculated to be less '., 1 162~7 than 0.02% Eor retained components with partition constants between 1 and 10 on columns wi~h lengths longer than 1 meter. This is negligible but will be even less if the diameter of the column is less than lS0~ m. An example of nicely resolved components is shown in FIG. 3A ~he mobile phase was 70% aceton-itrite and 30~ water. The column was 60 cm in length and 200 ~m in inner diameter and composed of fused silica. It was packed with 5 ~m particles having octadecylsiloxane bonded thereto. The 10w ra~e was
r --6--than 500~ m are flexible and may be coiled with large lengths fitting in small volumes. This fused silica is now commercially available for use in gas chroma-tography columns with inner diameters of less than 300 microns. See, e,gO, R. Dandeneau, et al~, "Flexible Fused Silica Columns", American Laboratory, June 1979; Alltech Associates, Product Bulletin No.
37, p. 1 (1980). The use of the end of a fused silica column itself as a flow cell for detection in the present invention enhances sensitivity and preserves chromatographie resolution because coneen-tration is high and sharp peaks are seen.
Some ingenious approaehes to low dead volume detectors which have been developed and reported are (1) laser fluorimetry, G. J. Diebold and R.N. ~are, "Laser Fluorimetry: Subpicogram Detection of Alfa-toxins Using High-Pressure Liquid Chromatography", Scienee 196, 1439-1441 (1977~; (2) miniaturized W
deteetors, D. Ishii, et al. "A Study of Micro-High-Performance Liquid Chromatography to Development ofTechnique for Miniaturization of High-Performance Liquid Chromatography", J. Chromatogr, 144, 157-168 (1977); (3) Y. Hirata, et al., "Packed Mierocapillary Columns With Different Selectivities for Liquid Chromatography", Anal. Chem. 51, 1807-1809 (1979);
(4) miniaturized electro-ehemical detectors, Y.
Hirata, et al., "Small-Volume Electrochemical Detector for Mierocolumn Liquid Chromatography", J. Chromatogr.
181, 287-294 (1980); (5) a sheath flow fluorometric detector, L.W. Hershberger, et al., "Sub-Microliter Flow-Through Cuvette for Fluorescence Monitoring of High Performance Liquid Chromatographic Effluents", Anal. Chem. 51, 1444-1446 (1979); and (6) the free falling drop fluorometrie detector, F. Martin, et 1~
al., "The Free-Falling Drop Detector - A Novel Fluor-escence Detector for High Performance Liquid Chroma-tography", Clin. Chem. 22, 1434-1437 (1979). Among these, the UV and spectro~fluorometric detectors are the most trouble-free and most frequently used.
The availability of low dead volume UV and spectr-ofluorometric detectors are an important factor in permitting small-bore column LC to become a powerful separation technique for routine applications.
Yet, a limitation to these low dead volume detectors is that the flow cell volumes between 0.1 and 0.3 L are far from the optimum desirable for small-bore column LC. For example, in small-bore column liquid chromatography, it has been theoretically derived that detector flow cell volumes of 1 - 10 nl are required for achieving less than 1~ loss of peak resolution on a l0 to 50~ m diameter open tubular column, for a retained peak with partition ratio k = 10 and retention time = l hour. See J. Knox, et al. "Kinetic Optima~ation of Straight Open-TubuJar Liquid Chromatography", J. Chromatogr., vO 186, pp 405-418 (1979). In addition, the extra band broadening caused by column to detector connec-tions and interface tubing significantly affect performance. The method and apparatus of the present invention serves to eliminate effects due to column-to-detector interconnection and detector dead volume effects due to mixing.
Method The method of the present invention is illus-trated in the Figures and particularly in FIGS.
la-lf. A fused silica column 10, shown in cross-section in FIG. la, is selected with a wall thickness J 11 on the order of 24 mm and an inner diameter I~
4~
less than 20 mm. The ~used silica column 10 is then drawn under thermal treatment in a iber optics drawing machine at temperatures on the order o ` ~ 2000C. The drawing rate (ratio of extrac~ion rate/
insertion rate) can be varied rom grea~er than one up to 1000. As this rate is increased bo~h the inner I diame~er and the wall thickness decreases. The reduced inner diameter will typically be less than 500lJm and the reduced wall thickness 11' will be between 20 and 150~ m. Such columns are of the type which are commercially available for gas chromato-graphy as described above. Larger diameter columns are typically not flexible and are therefore not encom-passed within the method of the present invention.
The drawn column 10' is flexible but fragile. For protection a polymer coating 12 such as polyimide or a metallic coating is applied externally; typically the external coating has a thickness between 10 and 150 ~ m.
As shown in FIGS. ld and le, the fused silica column is activated either by coating the interior with a stationary phase 13 or by chemically bonding a stationary phase 13 to the interior surface of the column or by packing the columns with activated microparticles 14. At the lowest inner column diameters (~hose approaching the diameters of the microparticles), coatings may be more practible than packings, although packing techniques such as disclosed in K. Mochizuki, et al., "Column for use in High Speed Liquid Chromatography", U.S. Patent No. 4,059,523 may be used. The art of coating or packing is well established and most conventional approaches may be used. See L. R. Snyder, et al., Introduction to Modern Liquid Chromatography, 9.2, "Column Packings" p. 287 et seq. Next, as shown in 2~7 FIG. lf, the external protective coating is removed along a short segment of the coated or packed fused silica microcapillary column. Removal i5 accom-plished by scraping, dissolving or thermally decomposing the coating. This segment is of the order of 1 cm or less and is sufficient to permit access by a visible light optical detector through the column. In efect, a flow cell is produced without having to interconnect separate pieces. The volume of this cell may be as small as .24 nL when the inner diameter of the column is 10~ m. The exposed segment of the fused silica column 15' is then placed in the optical path of an ultraviolet photodetector, such as a Jasco UVIDEC III or a lS Varichrome UV-50l as shown in FIG. 2. In an alternative embodiment the activating layer 13 is also removed along the end of the fused silica column up to and including the short segment to eliminate any absorption of visible light by the thin film. Removal is accomplished by external application of heat or by dipping the end of the column in a suitable solvent. For packed columns the activated microparticles 14 are kept from the flow cell by a frit positioned upstream.
Structure The apparatus for accomplishing on-column detection in accordance with the present invention is shown in FIGS. 2 and 3. In FIG. 2 a UV detector such as a Jasco UVIDEC III is utilized. The UV
detector comprises a light source 20, focusing optics 21, monochromator 22 having an exit slit 25 and a photodetector 24. In lieu o a flow cell, a passage-way is provided or the used sillca column 10'.
The exposed segment 15 ls placed across the optlcal ., 4~7 path so that photodetector 24 receives light that has passed through a full diameter of the column which has a sample flowing through it. The flow rate is not a limiting factor and may be as great as 10 ml/min. In an alternative embodiment a multiple wavelength UV detector such as Varian Varichrome UV-50 may be employed. The system arrangement is the same with the wavelengths being selected to permit several substances to be detected simultaneously.
Since there is negligible mixing in the on-column detector of the present invention, the extra-column band broadening contribution to the percent loss of resolution, ~R, can be expressed by RC-R ~ 1 % ~ R = O 100~ - [~ ~ ~] 2 ) ~ 100~ (1) ~c HL
where Rc,R = resolution measured for the column and the combination of column and detector, respectively h,H = the height equivalent to a theore-tical plate for the flow cell and the column, respectively ~,L = the entrance/exit slit length for the flow cell and the column length, respectively.
For a circular cross-sectional column, as an example, h, and H can be expressed by equations (2) and (3), respectively.
2Dm ~ r2U
h = _ _ (2) U 2~Dm H~ m ~ 6k' ~ llk' ). r U ~ 2k' df U (3) U 24(1 ~ k~)2 Dm 3(1 + k~)2 D
where r = column radius U = the average linear flow veloc;ty for the mobile phase Dm & Dl = are the diffusivities for the solute molecules in the mobile and the stationary phases, respectively df - the stationary phase film thickness k' = the partition ratio for the solute molecules = true retention time/
column void time.
Using these equations, the contribution of the on--15 column detector of the present invenkion to %
for two retained components with partition ratios, k', of l and lO on a column with length up to 15 meters is given in Figure 4. Flow rate and Dm are assumed to be 1 ~ L/min and l X 10-5 cm2/sec, respectively. The length of the detector is 0.3 cm.
Column diameter is assumed to be 150~ m. For conven-tional optical detectors the associated flow cells may make a significant contribution, on the order of tens of percent, to loss of resolution. As shown in FIG. 4, even for a partition ratio approaching l, (a lower limit for a chromatographic column perform-ing good separation) there is a contribution by the on-column detection "cell" to loss of resolution of the order of hundredths of a percent. As 3G expected, the higher the partition ratio, the less the contribution to loss o~ percent resolution.
The percent loss in peak resolution due to the on-column detector is in fact calculated to be less '., 1 162~7 than 0.02% Eor retained components with partition constants between 1 and 10 on columns wi~h lengths longer than 1 meter. This is negligible but will be even less if the diameter of the column is less than lS0~ m. An example of nicely resolved components is shown in FIG. 3A ~he mobile phase was 70% aceton-itrite and 30~ water. The column was 60 cm in length and 200 ~m in inner diameter and composed of fused silica. It was packed with 5 ~m particles having octadecylsiloxane bonded thereto. The 10w ra~e was
5.4 ~ L/minute the inlet pressure was 350 atmospheres.
The volume of the flow cell was .09 ~'~. Tha clean separation of components a-f is indicated in Table I
below.
Label Identity Elution Time ~min) . ...
a benzene 10 b toluene 10.7 c naphthalene 12 d flourene 17.8 e phenathacene 20.5 f pyrene 29 _ .
FIG. 5 shows the detection limit for a 6 NL
on-column flow cell configured on the end of a 213 cm column. The detection limit was 0.24 nanograms of benzene which converts to a minimum detectable concentration of 1.5 nanogramsJmicroliter~
The flow rate was 1,06~ I,/min. The peak height was o the order of one thousand times the noise.
,..
.
The volume of the flow cell was .09 ~'~. Tha clean separation of components a-f is indicated in Table I
below.
Label Identity Elution Time ~min) . ...
a benzene 10 b toluene 10.7 c naphthalene 12 d flourene 17.8 e phenathacene 20.5 f pyrene 29 _ .
FIG. 5 shows the detection limit for a 6 NL
on-column flow cell configured on the end of a 213 cm column. The detection limit was 0.24 nanograms of benzene which converts to a minimum detectable concentration of 1.5 nanogramsJmicroliter~
The flow rate was 1,06~ I,/min. The peak height was o the order of one thousand times the noise.
,..
.
Claims (10)
1. A method for high resolution on-columnn detec-tion in high resolution liquid chromatography, comprising:
providing a fused silica microcapillary column having an inner diameter less than 500µ m;
activating the interior of said column with a chromatographically active material;
applying a protective coating to the exterior of said column;
removing a segment of said protective coating to expose the underlying segment of said fused silica column;
providing an optical chromatographic detector;
positioning said exposed segment of said column in the optical path of said detector;
flowing a sample containing species to be ident-ified through said column; and detecting separated species as they flow through said exposed segment of said column.
providing a fused silica microcapillary column having an inner diameter less than 500µ m;
activating the interior of said column with a chromatographically active material;
applying a protective coating to the exterior of said column;
removing a segment of said protective coating to expose the underlying segment of said fused silica column;
providing an optical chromatographic detector;
positioning said exposed segment of said column in the optical path of said detector;
flowing a sample containing species to be ident-ified through said column; and detecting separated species as they flow through said exposed segment of said column.
2. A method for on column detection in accordance with claim 1 wherein said step of activating the interior of said column is accomplished by the step of packing said column with a chromatographically active material and wherein said packing is removed within the volume of said exposed segment.
3. A method for on-column detection in accordance with claim 1 wherein said step of activating the interior of said column is accomplished by the step of coating the inner wall of said column with a chromatographically active material.
4. A method for on-column detection in accordance with claim 3 in combination with the step of removing said chromatographically active material along said exposed segment.
5. A method for on-column detection in accordance with claim 1 wherein said step of providing a chromatographic detector is accomplished by the step of providing an ultraviolet absorbance detector.
6. Apparatus for on-column detection in high resolution liquid chromatography comprising:
a flexible fused silica column having an inner diameter less than 500µ m, said column being chroma-tographically activated and also having an external protective coating, said external coating having a segment exposed along the end of its length; and an optical chromatographic detector configured to receive said column so that said exposed segment lies in the optical detection path of said detector.
a flexible fused silica column having an inner diameter less than 500µ m, said column being chroma-tographically activated and also having an external protective coating, said external coating having a segment exposed along the end of its length; and an optical chromatographic detector configured to receive said column so that said exposed segment lies in the optical detection path of said detector.
7. Apparatus for on-column detection in accordance with claim 5 wherein said exposed segment is less than one centimeter in length.
8. Apparatus for on-column detection in accordance with claim 6 wherein said optical chromatographic detector is an ultraviolet detector.
9. Apparatus for on-column detection in accordance with claim 7 wherein said column is packed with a chromatographically active material.
10. Apparatus for on-column detection in accordance with claim 7 wherein the inner surface of said column is coated with a chromatographically active material.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US06/223,445 US4375163A (en) | 1981-01-08 | 1981-01-08 | Method and apparatus for on-column detection in liquid chromatography |
| US223,445 | 1981-01-08 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1162417A true CA1162417A (en) | 1984-02-21 |
Family
ID=22836526
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000393664A Expired CA1162417A (en) | 1981-01-08 | 1982-01-06 | Method and apparatus for on-column detection in liquid chromatography |
Country Status (5)
| Country | Link |
|---|---|
| US (1) | US4375163A (en) |
| JP (1) | JPS57136161A (en) |
| CA (1) | CA1162417A (en) |
| DE (1) | DE3151962A1 (en) |
| GB (1) | GB2090768B (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5940253A (en) * | 1982-08-27 | 1984-03-05 | ザ・パ−キン−エルマ−・コ−ポレイシヨン | Column of chromatography |
| GB2128099B (en) * | 1982-10-04 | 1986-07-02 | Varian Associates | Narrow bore microparticle column packing process and product |
| JPS6235244A (en) * | 1985-08-09 | 1987-02-16 | Sato Yakugaku Kenkyusho:Kk | New high-performance liquid chromatography |
| AT396998B (en) * | 1985-12-09 | 1994-01-25 | Ottosensors Corp | MEASURING DEVICES AND PIPE CONNECTION AND METHOD FOR PRODUCING A MEASURING DEVICE AND METHOD FOR CONNECTING TUBES TO A MEASURING DEVICE OR FOR THE PRODUCTION OF PIPE CONNECTIONS |
| JPH0526529Y2 (en) * | 1986-03-06 | 1993-07-05 | ||
| JPS63183542U (en) * | 1987-05-18 | 1988-11-25 | ||
| US4908676A (en) * | 1987-12-18 | 1990-03-13 | Bio-Recovery Systems, Inc. | Sensors for dissolved substances in fluids |
| GB8803993D0 (en) * | 1988-02-20 | 1988-03-23 | Phase Separations Ltd | Tubing assembly |
| US4854700A (en) * | 1988-06-15 | 1989-08-08 | The Dow Chemical Company | Column holder for on-column photometric detection |
| JP2745568B2 (en) * | 1988-09-30 | 1998-04-28 | 株式会社島津製作所 | Adjustment method and adjustment mechanism for capillary cell detector |
| EP0396163B1 (en) * | 1989-04-14 | 1994-01-19 | Kontron Instruments Holding N.V. | Capillary flow cell |
| US4940883A (en) * | 1989-04-24 | 1990-07-10 | Northeastern University | Window burner for polymer coated capillary columns |
| US5269930A (en) * | 1990-07-13 | 1993-12-14 | Isco, Inc. | Apparatus and method for supercritical fluid extraction |
| US5614089A (en) * | 1990-07-13 | 1997-03-25 | Isco, Inc. | Apparatus and method for supercritical fluid extraction or supercritical fluid chromatography |
| US5690828A (en) * | 1990-07-13 | 1997-11-25 | Isco, Inc. | Apparatus and method for supercritical fluid extraction |
| US5932095A (en) | 1990-07-13 | 1999-08-03 | Isco, Inc. | Multi-chambered supercritical fluid extraction cartridge |
| US5653885A (en) * | 1990-07-13 | 1997-08-05 | Isco, Inc. | Apparatus and method for supercritical fluid extraction |
| US5601707A (en) * | 1990-07-13 | 1997-02-11 | Isco, Inc. | Apparatus and method for supercritical fluid extraction or supercritical fluid chromatography |
| US5250195A (en) | 1990-07-13 | 1993-10-05 | Isco, Inc. | Apparatus and method for supercritical fluid extraction |
| US5635070A (en) * | 1990-07-13 | 1997-06-03 | Isco, Inc. | Apparatus and method for supercritical fluid extraction |
| JP3181609B2 (en) * | 1990-12-28 | 2001-07-03 | 株式会社島津製作所 | Photodetector for capillary chromatography |
| US5235409A (en) | 1991-08-13 | 1993-08-10 | Varian Associates, Inc. | Optical detection system for capillary separation columns |
| US5114551A (en) * | 1991-09-30 | 1992-05-19 | Bio-Rad Laboratories, Inc. | Multi-point detection method for electrophoresis and chromatography in capillaries |
| US6204919B1 (en) | 1993-07-22 | 2001-03-20 | Novachem Bv | Double beam spectrometer |
| US5938919A (en) * | 1995-12-22 | 1999-08-17 | Phenomenex | Fused silica capillary columns protected by flexible shielding |
| US5641893A (en) * | 1996-02-22 | 1997-06-24 | University Of Kentucky Research Foundation | Chromatographic separation apparatus |
| DE19806640C2 (en) * | 1998-02-18 | 2000-11-16 | Degussa | Process for capillary chromatographic separation of substance mixtures using a new sensor |
| US6544396B1 (en) | 2000-07-20 | 2003-04-08 | Symyx Technologies, Inc. | Multiplexed capillary electrophoresis system |
| US6531041B1 (en) | 2000-07-20 | 2003-03-11 | Symyx Technologies, Inc. | Multiplexed capillary electrophoresis system with rotatable photodetector |
| US6572750B1 (en) * | 2000-07-21 | 2003-06-03 | Symyx Technologies, Inc. | Hydrodynamic injector |
| US6462816B1 (en) | 2000-07-21 | 2002-10-08 | Symyx Technologies, Inc. | Parallel capillary electrophoresis system having signal averaging and noise cancellation |
| JP4520621B2 (en) * | 2000-11-01 | 2010-08-11 | 信和化工株式会社 | Chromatographic separation column, solid phase extraction medium, and chromatographic sample injection system |
| US20070071649A1 (en) * | 2001-09-10 | 2007-03-29 | Marcus R Kenneth | Capillary-channeled polymer fibers as stationary phase media for spectroscopic analysis |
| JP2006120598A (en) * | 2004-09-21 | 2006-05-11 | Toshiba Corp | Fuel cell system |
| WO2006127590A2 (en) * | 2005-05-20 | 2006-11-30 | The Government Of The United States As Represented By The Secretary Of The Department Of Health And Human Services | Microfluidic detection cell for stimulated radiation measurements |
| US7651762B2 (en) * | 2007-03-13 | 2010-01-26 | Varian, Inc. | Methods and devices using a shrinkable support for porous monolithic materials |
| US20090173146A1 (en) * | 2008-01-07 | 2009-07-09 | Matthias Pursch | Temperature programmed low thermal mass fast liquid chromatography analysis system |
| US9946058B2 (en) * | 2010-06-11 | 2018-04-17 | Nikon Corporation | Microscope apparatus and observation method |
| US9086421B1 (en) * | 2010-07-29 | 2015-07-21 | Entanglement Technologies, Inc. | Device and method for cavity detected high-speed diffusion chromatography |
| JP5939781B2 (en) * | 2011-12-09 | 2016-06-22 | 日本分光株式会社 | High pressure flow cell, flow cell assembly, fluorescence detector and supercritical fluid chromatograph |
| JP2015213866A (en) * | 2014-05-09 | 2015-12-03 | キヤノン株式会社 | Fluid control system |
| US10451593B2 (en) * | 2016-08-04 | 2019-10-22 | Aa Holdings, Ltd. | Detection system and method with nanostructure flow cell |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CH448566A (en) * | 1965-05-05 | 1967-12-15 | Ceskoslovenska Akademie Ved | Optical device for evaluating concentration gradients in liquids flowing through the capillary lines of analyzers |
| US3518009A (en) * | 1966-08-18 | 1970-06-30 | Technicon Corp | Colorimeter flow cell |
| JPS5489691A (en) * | 1977-12-24 | 1979-07-16 | Nippon Bunko Kogyo Kk | Open tube type capillary column for micro rapid liquid chromatography and production thereof |
| US4199260A (en) * | 1978-08-21 | 1980-04-22 | Technicon Instruments Corporation | Apparatus and method for determining the concentration in a sample |
| US4293415A (en) * | 1979-04-27 | 1981-10-06 | Hewlett-Packard Company | Silica chromatographic column |
-
1981
- 1981-01-08 US US06/223,445 patent/US4375163A/en not_active Expired - Fee Related
- 1981-12-17 GB GB8138122A patent/GB2090768B/en not_active Expired
- 1981-12-30 DE DE19813151962 patent/DE3151962A1/en not_active Ceased
-
1982
- 1982-01-06 CA CA000393664A patent/CA1162417A/en not_active Expired
- 1982-01-07 JP JP57000646A patent/JPS57136161A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| DE3151962A1 (en) | 1982-08-12 |
| GB2090768B (en) | 1984-06-27 |
| US4375163A (en) | 1983-03-01 |
| JPS57136161A (en) | 1982-08-23 |
| GB2090768A (en) | 1982-07-21 |
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