Anal. Chem. 1084, 56, 483-486
483
On-Column Fluorescence Detector for Open-Tubular Capillary Liquid Chromatography Edward J. Guthrie and James W. Jorgenson* Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27514
The deslgn and constructlon of an on-column fluorescence detector speclflcally suited for open-tubular capillary llquld chromatography are presented. Some advantages of uslng a portlon of the chromatographiccolumn for the detector flow cell are discussed. An Inherent sensltlvlty advantage of detectors operatlng on a partltionlng reglon of the chromatographic column Is also dlscussed. The detectlon llmlt for perylene, a representatlve fluorophor, Is 10 pg Injected on column. The detector response Is llnear over a tested range approaching 4 orders of magnltude.
During the last few years a great deal of research interest has been centered on improving liquid chromatography (LC) column efficiency. Theoretical and experimental evidence has supported the pursuit of smaller column diameters, either small bore packed columns, packed capillary columns, or open tubular capillary columns (I). The columns of this type reported in the literature have diameters ranging from tenths of a millimeter for the small bore packed columns down to micrometers for open tubular columns. Knox and Gilbert (2) have predicted that open-tubular LC columns will have to have internal diameters in the range of 10-30 pm to produce efficiencies comparable to packed columns. Jorgenson and Guthrie (3)have predicted that the optimum open-tubular capillary LC columns will be approximately 2 pm internal diameter, 2 m long, and generate in excess of lo6 theoretical plates. The internal volume of such a column will be only 6 nL. Extending the calculations of Jorgenson and Guthrie, a chromatographic peak of lo6 theoretical plates and a capacity factor of 10 under optimal operating conditions would elute in a peak volume of only 0.3 nL ( 4 ~ ) . Commercially available LC detectors have cell volumes as small as a few tenths of a microliter. When this detector volume is compared to the predicted peak volume just calculated for an “optimum” capillary column, it becomes clear that detector technology is a limiting factor confronting development of open-tubular columns. There are different ways to confront this impasse. The simplest approach is to use make-up flows to decrease the residence time of the effluent in the detector cell. This method is somewhat unsatisfactory since make-up flows inherently decrease sensitivity by diluting the solute concentration at the detector. As an alternative, existing LC detectors could be miniaturized to reduce their cell volumes. The literature contains miniaturized UV detectors by Hirata, Novotny, Tsuda, and Ishii (4),by Krejci, Tesarik, and Pajurek (5),by Ishii and Takeuchi (6),and by Tsuda, Hibi, Nakanishi, Takeuchi, and Ishii (7), a miniaturized fluorescence detector by Hirata and Novotny (8)) a laser-induced fluorescence detector by Folested, Johnson, Josefason, and Galle (9),and miniaturized electrochemical detectors by Hirata, Lin, Novotny, and Wightman (lo),by Slais and Krejci (11),and by Novotny (12).Finally, new, sensitive detection methods for LC could be developed. New LC detectors of this type include the flame photometric detector (FPD) by McGuffin and 0003-2700/84/0356-04S3$01.50/0
Novotny (13),a sheathed flow fluorescence detector by Hershberger, Callis, a d Christian (14),and a flame ionization detector by Krejci, Tesarik, Rusik, and Pajurek (15). A different approach has been initiated by Yang (16),who has developed a UV detector system which utilizes a segment of the fused silica capillary column for its detector cell. This sytem, which Yang termed “on-column detection”, offers a few appealing advantages. First, there is no band broadening caused by column-detector connections, detector cell void volume, or make-up flows. Another advantage is that the detector cell volume becomes a function only of the column internal diameter and the illuminated length. Since the detector cell is a portion of the column tubing material itself, the cell “volume” decreases or increases with the column diameter. In fact, the term “detector cell volume” becomes superfluous since the resolution limiting criterion of the detector is no longer cell volume at all, but instead the spatial resolution or “window” of the detector. The length of column illuminated in the detector is the critical factor of optical, on-column detector designs. A chromatographic peak of lo6 theoretical plates on a 2 pm internal diameter, 2 m long column would be 8 mm wide (4u) at the detector. A final advantage arises from purely mechanical aspects. The problem of accurately attaching the 2 pm bore of a capillary LC column with a comparable size detector without unacceptable misalignment or dead volume would be infinitely tedious if not impossible. On-column detectors eliminate such problems very simply and neatly. These advantagesmake any on-column detector design very appealing. The UV detector designed by Yang was operated on segments of fused silica capillary column which had been stripped of stationary phase. While this might be necessary for some detection systems, we will show that a sizable sensitivity advantage can be gained by illuminating a section of column containing stationary phase. We will show that the increased sensitivity of a detector utilizing this design, which we prefer to term “true” on-column detection, arises in part from minimizing dilution effects. Furthermore, a detector of this design is simplier to use, when compared to Yang’s method, since the coated column can be used as is, without any steps to remove stationary phase. Our laboratory has developed a true on-column fluorescence detector. The illuminatedcolumn length and hence the spatial resolution can be varied from zero to several millimeters. The minimum detectable injected amounts for representative fluorophors are in the 10 pg range, representing 40 fg in the detector window at the peak maximum. The detector has good linearity over dynamic ranges exceeding a t least 4 orders of magnitude and has been used to obtain chromatograms for separations performed on columns of internal diameters as small as 9 pm. The on-column fluorescencedetector has also been used extensively as a detector for capillary electrophoresis.
EXPERIMENTAL SECTION Fluorescence Detector. A schematic diagram of the optical layout of the on-column fluorescence detector is shown in Figure 1. The detector utilizes a 100-W, high-pressure, short arc, Hg 0 1984 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 3, MARCH 1984
PMT
PMT
ter
arc support r
L,
Ll
L
moin body
photovoltaic cell
A & , capillary y i r j T [\
’L!-
Flgure 1. Optical layout of on-column fluorescence detector.
T-k,
Figure 3. Detector block. DETECTOR
ELECTROMETER
w t
bolonce
FILTER
oai4meni
Figure 2. Detector electronics, including noise compensating system and filters.
lamp (HBO 100 W/2, OSRAM Corp./Macbeth Sales Corp., Newburgh, NY) as a UV source. The lamp output is collimated by lens L,, which is a 50 mm focal length, 3.8 cm diameter fused silica lens (no. 6304, Oriel Corp., Stamford, CT), is then passed through a 3.8 cm diameter water-filled liquid filter (no. 65194, ORIEL Corp., Stamford, CT) used to trap IR wavelengths which might overheat and destroy subsequent filters and a UV transmission filter (360 nm, 03FCG001, Melles Griot, Irvine, CA), and then is focused down to a 1 mm diameter spot at the capillary by lens Lz which is a 3.8 cm diameter, 50 mm focal length lens (no. 61961, Oriel Corp., Stamford, CT). The lamp housing, filter holders, and lens holders were all obtained from ORIEL Corp., Stamford, CT. The emitted fluorescent light is collected at 90° to both the excitation beam and capillary by a “Suprasil”syntheticfused silica, plano-convex, spherical collection lens, 15.0 mm diameter, 25.0 mm focal length (no. 01LQP177, Melles Griot, Irvine, CA) and passed through a pair of low fluorescence UV-blocking filters (420 nm cutoff, no. 5217, and 470 nm cutoff, no. 5219, ORIEL Corp., Stamford, CT). The transmitted light is then detected by a photomultiplier tube (no. 1P21,Hamamatsu TV Co., Ltd., Japan), which is housed in a Pacific 50B PMT housing. (Pacific Precision Instruments, Concord, CA). The PMT electrometer is a Pacific Model 110 Photometer (Pacific Precision Instruments, Concord, CA). The PMT is operated with a 400-V bias potential. Lamp noise and drift are compensated by an opposed mounted, UVsensitive, photovoltaic cell (PVC) (SD 290-13-13-242, Silicon Detector Corp., Newbury Park, CA) whose output is amplified and subtracted from the PMT electrometer output prior to the chart recorder. Amplification of the reference cell output is adjusted via a balance adjustment until base line noise is minimized. High-frequency noise is further suppressed by low pass filtering. A block schematic of the electronics is shown in Figure 2. The PVC electrometer was designed and constructed in-house. The detector body was machined from a 7.6 X 5.1 cm aluminum block. The beam entrance is a 1.9 cm diameter hole in the block face. The column is supported by two 6.3 mm 0.d. stainless steel tubes whose tips have been tapered to allow optimal illumination of the capillary and collection of the emitted fluorescent light. The support tubes enter from either side of the block, and the positions of the two support tubes are adjustable, enabling the user to vary the illuminated column length from zero to several millimeters (see Figure 3). The collection lens is supported in a brass tube mounted in the top of the detector body. The lens height above the capillary is adjustable to optimize the efficiency of the collecting optics. The photovoltaic cell is mounted in a hole bored through the bottom of the detector block directly beneath the capillary. The detector block itself is mounted on
solvent
vent sample VALVE I
4
k+@-
i
waste
VALVE
TEE (reservoir)
Flgure 4. Chromatographic injection system.
an x-y-z positioner (Daedal, Inc., Harrison City, PA), which is used to position the capillaryat the focus of the excitation light beam. Chromatograph. A block diagram of the chromatographic system is shown in Figure 4. Nitrogen gas pressure provides column head pressure. The system utilizes a static splitting injection technique. A 0.5-mL portion of sample solution is introduced to the tee at the column head by switching valve 1, a four-port, two-way valve (Valco Valve Co., Houston, TX), and opening valve 2, a shut-off valve (Scientific Systems, Inc., State College, PA). The sample is then injected aa a plug onto the head of the column under solvent pressure by closing valve 2 and re-turning valve 1. After the desired injection duration, the remaining sample solution is flushed from the tee by opening valve 2. Once the tee is flushed, valve 2 is closed and the chromatographic separation begins. The details of this injector design were published previously (17). Columns. The capillary column used for evaluation of the detector was of Pyrex type 7740 borosilicate glass, 3.3 m long, with 63 pm internal diameter drawn from capillary-bore tubing on a Shimadzu GDM-1B glass drawing machine (Shimadzu, Kyoto, Japan). The stationary phase was chemically bonded octadecylsilane. The details of the column fabricationtechnique have been previously described (18). Reagents. The mobile phase was 50% by volume acetonitrile (J. T. Baker Chemical Co., Phillipsburg, NJ) in distilled water. The ODS bonded phase was made with trichlorooctadecylsilane obtained from Petrarch Systems, Inc., Levittown, PA. Riboflavin and 9-methylanthracene were obtained from the Sigma Chemical Co., St. Louis, MO. Perylene was obtained from Aldrich Chemical Co., Inc., Milwaukee, WI.
RESULTS AND DISCUSSION It was decided to evaluate the on-column fluorescence detector by using a group of solutes which would demonstrate the detector’s performance over a range of capacity factors. A compromise between retention (capacity factor) and solubility was found by wing a 50% HzO, 50% CH&N mobile phase and three solutes: riboflavin (k’ = 0), 9-methylanthracene (k’= 0.2), and perylene (k’= 0.5). The chromatographic peaks obtained were sharp and symmetrical (see Figure 5). Benzo[ghi]perylene, included in Figure 5, was not
ANALYTICAL CHEMISTRY, VOL. 56, NO. 3, MARCH 1984
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485
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Table I. Standards: Retention Time (tr),Capacity Factor (k’)in 50/50 (v/v) Acetonitrile/H,O, Linear Range, Slope ( r ) by Linear Least Squares Regression, Standard Deviation ( ur) of Calculated Slope, Minimum Detectable Amount (MDA) compound t,, s k’ linear range, g/mL r or MDA, Pg 0.98 2x 7 315 0.0 2 x 10-*-2 x riboflavin 0.94 5 x 10-5 16 370 0.2 2 X 10‘7-4X 9-methylanthracene 0.97 8 X 10.’ 14 472 0.5 3 X 10-*-7 X loe6 perylene
B
0
5
IO
TIME (min.) Figure 5. Chromatogram of standards on 64 pm i.d. X 3 m long column, mobile phase = 50/50 (v/v) aceton%rlle/water:(A) riboflavin; (6)9-methylanthracene;(C) perylene; (D) benzo[ghl]peryiene.
included as a test solute due to its very low solubility in the 50% HzO, 50% CH&N mobile phase. We chose to quantitate the chromatograms by manual measurement of the peak heights since (with peaks of this type) this method is very simple and reasonably accurate. The injector system reproducibility was tested by making replicate injections of a dilute solution of riboflavin. The relative standard deviation of the seven injections was 2%. Nearly saturated solutions of each solute in mobile phase solvent were prepared and a series of standard solutions were then prepared by making sequential 1:2 dilutions of the prepared stock solutions. The initial dissolution of the solute crystals was facilitated by adding a few drops of tetrahydrofuran to the weighed crystals before addition of the solvent. Replicate injections of the serial dilutions were then made as quickly as possible to minimize any nonlinearity effects which might have been created by the solutes decomposing in solution. R. P. W. Scott (18) has stated that if a response, y, arises as a function of concentration, c, then a function
y = Ac‘ (1) where A and r are constants, will describe the response curve. Therefore, the equation log y = log A r log c (2) should describe a straight line with a slope, r. Scott arbitrarily proposed that for a “linear” dependence of response on concentration, r should fall in the range 0.98-1.02. Values outside this range imply degrees of nonlinearity. Standard curves for “nonlinear” responses can be utilized provided r is uniform over the analytical concentration range and provided r can be accurately determined. The standard lines for three test solutes in log-log format were calculated by using a linear least-squares fit algorithm. The r values (slopes) calculated for the three standard lines, as well as the calculated standard
+
deviations of the r(ur)values are shown in Table I. Riboflavin and perylene show good linearity over the tested ranges. 9-Methylanthracene was found to rapidly photodecompose in solution and this fact may be responsible for the lower r value found for this substance. The calculated r had a low relative standard deviation indicating that a calibration curve for this substance could still be used with good accuracy. Linear Dynamic Range. The linear dynamic ranges were close to 4 orders of magnitude for riboflavin and 9-methylanthracene and nearly 3 orders of magnitude for perylene (see Table I). In all cases, the upper limits of the linear dynamic ranges were a result of the solubility limits of the solutes in the CH3CN:H20 (5050)mobile phase, not any detector nonlinearity at high concentrations. Even though these limits could have been extended by increasing the acetonitrile content of the mobile phase, we deemed it more important to demonstrate the on-column fluorescence detector’s capabilities with retained solutes. The log-log plots of all three solutes were linear at the high concentrations. This was to be expected because the major source of nonlinearity in fluorescence detectors is attributed to inner-filter effects. Since the path length of this detector is very short (the column internal diameter), one would expect this detector to show very little inner-filter effect and hence excellent linearity over a wide concentration range. Sensitivity. The detector sensitivity was evaluated for each of the three compounds. The minimum detectable amount (MDA) for the three materials was determined as the injected amount which produced a signal twice the noise level. These all fell in the 10 pg range (injected on column), as shown in Table I. In the case of perylene, its 10 pg MDA represents 30 fg (or 140 amol) of material inside the detector “cell” at the peak maximum of the MDA. This was calculated by using the method outlined by Locke, Uhingra, and Baker (19) and a 1-mm cell window. Yang has clearly demonstrated the advantages of detectors which utilize a portion of the capillary column as the detector cell. Foremost of these advantages is the elimination of extracolumn effects arising from the connectors and detector cell dead volumes and the elimination of diluting make-up flows. An on-column detector becomes a virtual necessity when one considers capillary columns in the 10 pm range. One simply cannot consider efficiently attaching and aligning an extracolumn detector to such a small internal diameter column. Our experience with the on-column fluorescence detector has shown that a choice exists as to whether such detectors should be operated in a partitioning region of the column (i.e., with stationary phase present), or in a nonpartitioning region (Le., stripped of stationary phase) as described by Yang. A true on-column detector (one operating in a partitioning region of the column) has a very unique sensitivity advantage. All postcolumn detectors suffer loss of sensitivity due to a dilution of solute in the column effluent as k’increases. This phenomenon is part of what has been termed the “General Elution Problem”. The effect arises as a result of detecting the solute after the stationary phase, i.e., in a nonpartitioning region. The phenomenon can be demonstrated by a simple diagram. Referring to Figure 6, two plane surfaces are taken normal to the chromatographicflow, one plane (p) in a region containing stationary phase, and one plane (p?in a region
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 3, MARCH 1984
P
P’ STAT ION A RY PHASE
I
//////////A
Figure 6. Pictorial representation of column at stationary phase iuminus.
beyond the stationary phase. The mobile phase velocity is designated u, the cross sectional area of the column is designated A , and c and c’represent the solute concentrations at p and p‘, respectively. If the solute has a partition coefficient, k’, then-the solute mass flow rate at p is mass U -(P) =C 1 + , (3) time while a t p’, the solute mass flow rate is mass -(p? time
U
= Cf-A (4) l + k Since these solute mass flow rates must be equal (i.e., for a constant flux of solute, no mass accumulates or is depleted after “steady state“ is attained), then n
C’UA =
1 kfUA 1
+ k’
1
+
The concentration, c’, at p’will always be a fraction, 1/(1 k’), of the concentration, c, at p . Therefore, an on-column detector will have a factor of 1 k’better sensitivity than a postcolumn detector. Increased analyte concentration is not obtained without other effects. The result of increased concentration is that the peak is contained in a narrower spatial region. This is obvious since concentration is defined mass - -mass concn = -(7) volume wA
+
where w is the width of the solute zone (peak) and A is the column cross sectional area. If the solute mass and the cross sectional area are constant, it is obvious that w ,the peak width, will be inversely dependent on the concentration. If c’ =
1 -
1 + k’
+ k’)
Knox, J. H. J . Chromatogr. Sci. 1980, 78, 453-461. Knox, J. H.; Giiber, M. T. J . Chromatogr. 1979, 786, 405-418. Jorgenson, J. W.; Guthrie, E. J. J . Chromatogr. 1983, 255,335-348. Hirata, Y. Novotny, M.; Tsuda, T.; Ishii, Daklo Anal. Chem. 1979, 57, 1807-1809. Krejcl, M.; Tesarik, K.; Pajurek, J. J . Chromatogr. 1980, 797, 17-24. Ishii, D.; Takeuchi, T. J . Chromatogr. Sci. 1980, 78, 462-472. Tsuda, T.; Hibl, K.; Nakanlshl, T.; Takeuchl, T.; Ishii, D. J . Chromatogr. 1978, 758,227-232. Hirata, Y.; Novotny, M. J . Chromatogr. 1979, 786, 521-528. Folestad, S.; Johnson, R.; Josefason, B.; Gaiie, B. Anal. Chem. 1982, 5 4 , 925-929. Hirata, Y.; Lin, P. T.; Novotny, M.; Wightman, R. M. J . Chromatogr. 1980, 187, 287-294. Siais, K.; Krejcl, M. J . Chromatogr. 1982, 235,21-29. Novotny, M. J . Chromatogr. Sci. 1980, 7E3 473-478. McGuffin, V. L.; Novotny, M. Anal. Chem. 1981, 53,948-951. Hershberger, L. W.; Caiiis, J. B.; Christian, G. D. Anal. Chem. 1979, 57. 1444-1446. KreJci, M.;Tesarik, K.; Rusik, M.; Pajurek, J. J . Chromatogr. 1981, 218. 167-178. Yang, F. J. HRC C C , J . High Resolut. Chromatogr. Chormatogr. Commun. 1981, 4 , 83-85. Jorgenson, J. W.; Guthrie, E. J. J . Chromatogr. 1983, 255,335-348. Scott, R. P. W. J . Chromatogr. Libr. 1977, 2,Chapter 2. Locke, D. C.; Dhingra, B. S.; Baker, A. D. Anal. Chem. 1982, 5 4 , 447-450. Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53,1298-1302.
(8)
then the peak width, w ‘, in the nonpartitioning zone will be w’= w(l
+
LITERATURE CITED
and therefore c ’ = c-
partitioning region. This means that the spatial resolution capabilities of an on-column detector must be a factor 1 k’ better than the corresponding postcolumn detector. Fortunately, adequate spatial resolution is easily obtainable with simple focusing of the excitation beam. The chemical environment of a fluorophor can be significant in determining the fluorescence excitation and emission wavelengths and the quantum efficiency. This fact is important to the on-column detector since the detected solute is in a partitioning region. For a given solute of capacity factor k’, a fraction 1/(1 + k? of the solute molecules will be in the mobile phase while a fraction k’/(l + k? will reside in the stationary phase. Since these environments are usually radically different, molecules in the two regions may exhibit significantly different fluorescence characteristics. It is important to consider, then, that any change in k ‘arises from a change in the relative amounts of analyte in the two phases and that such a change may alter the spectral and quantitative characteristics of the analyte. We do not feel that the current detector design has the optimum obtainable signal to noise levels. It is our belief that the major portion of the noise arises from arc wander in the arc lamp and mechanical vibration of the capillary relative to the excitation beam. We believe that a beam splitter placed between the capillary and the UV blocking filters, with the reference photovoltaic cell placed in the split beam instead of below the capillary, should offer improved compensation for noise arising from the above sources and should greatly improve the signal to noise ratio. The detector with the SIN improvementsoutlined should be applicableto 2 pm diameter columns. Our laboratory has also used the on-column fluorescence detector successfully in open-tubular high-voltage electrophoresis. The details of this application have been published elsewhere (20).
(9)
A solute zone (peak) will be a factor 1 + k’narrower in the
RECEIVED for review August 4,1983. Accepted November 28, 1983. Support for this work was provided by the donors of Petroleum Research Fund, administered by the American Chemical Society, and the University Research Council of the University of North Carolina.
