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(24) Pace, C. F.; Maple, J. R. Anal. Chem. 1985, 57. 940-945. (25) Pace, C. F.; Maple, J. R. J . Opf. SOC.Am. 8 1985, 2 , 1582-1588. (26) Pace, C. F. Ph.D. Dissertation, The University of New Mexico, Albuquerque, NM, 1986. (27) O’Donnell, C . M.; Harbaugh, K. F.; Fisher, R. P.; Winefordner, J. D. Anal. Chem. 1973, 45, 609-611. (28) Pohland, A. E.; Yang, G. C. J . Agric. Food Chem. 1972, 2 0 , 1093- 1099. (29) Brinkman. U. A. T.; Reymer, H. G. M. J . Chromatogr. 1978. 127, 203-243. (30) Pace, C. F.; Thornberg, S. M.; Maple, J. R. Appl. Spechosc. 1988, 42, 891-896. (31) Wiilis, B. G.; Woodruff, W. H.; Frysinger, J. R.; Margerum, D. w.; Pardue, H. L. Anal. Chem. 1970, 42, 1350-1355.
(32) Meiling, G. E.; Pardue, H. L. Anal. Chem. 1978. 50, 1611-1616. (33) Colmsjo, A. L.; Zebuhr, Y. U.; Ostman, C. E. Anal. Chem. 1982, 54, 1673-1677. (34) Qrrigues, P.; Ewald, M. Anal. Chem. 1983. 5 5 , 2155-2159. (35) Wolf, H. C.; Port, H. J . Lumin. 1978. 12/13, 33-46. (36) El-Sayed, M. A. In Molecular Luminescence; Lim, E. C., Ed.; W. A. Benjamin: New York, 1969; pp 715-736. (37) Harbaugh, K. F.; O’Donnell, C. M.; Winefordner, J. D. Anal. Chem. 1974, 4 6 , 1206-1209.
RECEIVED for review November 10,1988. Accepted January 23, 1989.
On-Column Capillary Flow Cell Utilizing Optical Waveguides for Chromatographic Applications Alfred0 E. Bruno,* E r n s t Gassmann, Nico PericlBs, a n d Klaus Anton
Central Analytical Department, CIBA GEIGY Ltd., CH-4002 Basel, Switzerland
The high separatlon efficiencies provided by various chromatographic technlques employlng microbore and capillary columns, countered by current detection llmltatlons, present new challenges In detection cell design. I n thls contrlbution we descrlbe a device sultable for on-column simultaneous absorption and fluorescence detection that utlllzes optical waveguides. The deslgn of this cell is based upon the aMlty of a capmary to functlon as a strongly focuskrg lens along one of Its axes. I n order to use thls attribute to the fullest advantage, a three-dimensional ray tracing algorithm, which sbnuiates the optlcal phenomena at the Interfaces and In the travellng media, was developed. Thus, from a trial set composed of commercially available components, we were able to choose optkal flber/capillary tube geometric configurations that yielded theorellcai transmittances of ca. 90%. Subsequent lmphmtatbn of the cell In capillary supercrltical flukl chromatography led to verlflcatlon of these flndlngs. Statlc and dynamlc refractive index effects, which are known to dlstort recording signals, were carefully Investigated and mlnimlzed. Two guldeHnes based upon experimental and theoretical observations are formulated-the first statlng the maxhnum source mer dlameter for a given choke of cap#lary tube and the second relating the minlmum dlameter of the co#ectlng fiber to an already chosen source Hber and caplllary tube-enabilng rapld selection of the cell optical hardware.
INTRODUCTION The development of small optical detectors for microbore and capillary chromatographic techniques is gaining considerable momentum (1-3). The demand for improvements in presently available models or, these improvements being inadequate, for the design of new devices is a direct consequence of today’s advanced state of the art of separation methods such as microbore liquid chromatography(4),capillary supercritical fluid chromatography (CSFC) (4, 5 ) , and capillary zone electrophoresis (CZE) (6). The high resolution delivered by these techniques is often lost at the detection stage as a result of coupling with insufficiently sensitive detection devices. 0003-2700/89/036 1-0876$01.50/0
Furthermore, the performance of these techniques increases with decreasing capillary tube inner diameters, thus compromising concentration sensitivity. The fiist problem one encounters when designing an optical on-column flow cell is the focusing of a considerable amount of light into the small light/solute interaction volume defined by the capillary tube inner diameter. Lasers undoubtedly provide the best light source for this purpose. Virtually the entire laser beam (exclusing Fresnel losses, which are unavoidable) can be readily focused into a tiny spot inside the capillary orifice. Excellent sample selectivity and sensitivity fluorescence (8,9),light have been reported for absorption scattering ( l o ) ,refractive index (2,1 1 ) , and thermal lens (12) methods employing coherent laser light. Unfortunately, due to their relatively high degree of sophistication, laser-based detection methods are primarily suitable for research-oriented laboratories. For industrial analyses, where chromatography is widely used, we designed a flow cell which, while retaining many of the virtues of laser detection methods, utilizes conventional lamps as the light source. The efficient focusing of spatially incoherent light from conventional lamps into capillary tubes with internal diameter i.d. ca. 5100 pm is not trivial. On-column absorption measurements are commonly made simply by intercepting the optical path of an absorption spectrophotometer with the capillary tube (3,13,14). A pair of masking slits ensure that only light that has passed through the capillary tube reaches the photosensitive detector. A major drawback of this experimental arrangement is the associated alignment procedure, which must be carried out exceedingly carefully to ensure good reproducibility and a high signal-to-noise (S/N) ratio. Optical fibers, originaUy developed primarily for the purpose of communication technology, considerably simplify the construction of small on-column detection cells. In fact, a few cells based upon these fibers are now emerging (15-17). The most recent and promising detector of this type, developed by Foret et al. (17),consists of a pair of optical fibers that bypass the optical path of a UV-visible spectrophotometer. One fiber, the source fiber, transfers light from the source to the capillary tube while a pair of fibers, the collecting and emission fibers, carry the absorption ( 17) and fluorescence
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(9) signals to the correspondingphotodetectors. A metal frame holds the two fibers directly against the outer wall of the capillary tubecoaxially for operation in the absorption mode and orthogonally for fluorescence measurements. In both configurations the fiber pair remains perpendicular to the capillary tube cylindrical axis. Good reproducibility and high SIN ratio are among the advantages reported for this method (17). The flow cell described here, an improved version of the previously d i s d design, is suitable for on-column detection in microbore and capillary separation techniques. It is compact, is easily mounted, has a wide application range, and can withstand pressures as high as 500 bar. The success of this device-it demonstrates very high illumination and transmittance-is a result of choosing the best poasihle combination of optical fibers, capillary tubes, and auxiliary materials from commercially available components. The normally tedious process of trial and error was largely eliminated by use of a computer program that simulates the optical pbenomena occurring a t all optical fiber/capillary tube interfaces and in all media through which the light travels. The most significant feature of this mathematical model is the use of a three-dimensional, rather than planar (3,18,19),approach to the actual mathematical problem. The computed transmittance and relative absorbance parameters are thus of real quantitative value, and the normalized theoretical results are readily comparable to experimental observahles.
THEORY The quality of a simulation algorithm depends upon the extent to which mathematical model employed is able to mimic the actual phenoma studied. The two mathematical models (A and B) incorporated into our ray tracing algorithm assume that the volume of the illumination is adequately represented by an array of single rays emerging from equally spaced origins located at the surface of the source fiber. The algorithm has been written in Turbo Pascal 4.0 for an IBM AT computer. The ray coordinates a t all optical boundary regions are calculated according to classical optics (Snell's laws) (21-24). Synovec (18)treated the case in which the incident light striking into the capillary tube is provided by an unfocused laser beam and is thus parallel and coherent. As shown in Figure 1 there are four possible cases that may arise depending upon the initial ray coordinates, angles of
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incidence a t the various interfaces, and refractive index of the traveling media (n). Case 1 describes incoming rays entering a t an angle 0, (formed by an imaginary line perpendicular to the tangent of the capillary tube outer surface) larger than the critical one 0, (i.e., ON > Bc = arcsin (n'/n)). Such rays are therefore totally reflected a t the capillary tube surface. Case 2 depicts rays that enter the capillary at angles less than the critical angle Bc and travel through the capillary wall without intercepting the innermost interface. Case 3 represents rays reflected at the fused silica innermost intezface; i.e., they strike a t an angle larger than the critical angle for this interface. Case 4, the most desirable situation, arises when the rays travel through both the orifice and wall of the capillary tube. Rays that do not strike the capillary tube a t all comprise a trivial case not considered further for obvious remns. In Figure 1,rays numbered 1-8, It12,13, and 14-30 correspond to cases labeled 1 , 2 , 3 and 4, respectively. The parallel rays shown inside the source fiber have been drawn only as a guide. The parameters used in the computation of the diagram are nl(air) = 1.00, n2(fused silica) = 1.45845, ns(H20) = 1.3333, and BNA = 12.5O. The dimensions of the optical components are indicated in the figure axes. Model A. In the selected coordinate system, the optical fibers and capillary tube are coaxial with the and axes, respectively. The coordinate origin is set a t the center of the tube, and rays travel from left to right. The ray tracing diagram in Figure 1 shows an (X, Y, Z = 0)cross section computed by using this model. For simplicity, only rays emerging from positions with positive (y) m r d i n a t e s are indicated in the figure. In this model, a single ray model, light emerging from the source fiber diverges from the (XIoptical axis until it intercepts the capillary tube. The maximum allowed ray divergence angle occurs a t the edge of the source fiber, where it has the value BNA (typically 12.5") according to its numerical aperture (NA = sin [SNA]). These angles gradually decrease until they reach Oo at the optical fiber axis IX, Y = 0,z = 01. Model B. In order to better mimic the incoherent light power distribution in the so-called "near field" (25), in this model, a multiple-ray model, each ray emerging from the source fiber is decomposed into 10 equally spaced secondary diverging rays in the (X, y) plane. The angles of divergence among these i rays are chosen to range from positive to negative BNA, thus defining a cone of twice this value (i.e., Ol0 - 0, = 2BNA).The distances between the individual secondary angles are therefore one tenth of the total cone aperture (i.e. Bi - B,l = 2BNA/1O). A ray tracing diagram corresponding to this more elaborate model is shown in Figure 2. For simplicity, only five starting rays are shown in the figure; actual runs employ ca.1ray/lO e. The computationalparameters and component dimensions are identical with those of Figure 1. Light Integration Method. In order to compute cell transmittance and relative absorbance, one must consider the light power distribution in the near field of the source fiber output surface. Provided that the fiber in question is coupled to a Lambertian source, as it is in this case, the output power distribution very closely approximates the index profile of the fiber (23,25). Because we only used step-index fibers having constant refractive index profiles, light integration is rather simple. The reduction of the actual 3-D problem into a pseudoplanar one is easily accomplished by exploiting the symmetry of the system along the capillary tube and optical fibers axes. Thus, a t the source fiber end (i.e., (X= -0D/2I, where OD is the capillary tube outer diameter o.d.), all rays with the 881316 ( Yj coordinate, for all starting (ZIvalues, will travel the same optical path inside the capillary tube (see Figure 3). However,
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Flgure 2. Optical ray tracing diagram for five primary rays traveling through a cylindrical flow cell, computed by using model B.
Flgure 4. Cross sectional view of the flow cell: (a) a cut along the (Z) axis (capillary tube axis) and (b) a cross sectbn at ( X . Y . 2 = 0). Some wmponents have been drawn in expanded format. Key: 1, capillary column; 2. anodized aluminum ring chnp; 3. high-pressure union assembly mnsisting of (Sa)5-mm stainless steel kit. (3b) plastic ferrule, and (3c) 5-mm stainless steel nut: 4, heat-shrink tubing; 5, plastic O-ring; 6. anodized aluminum cell body; 7. detection capillary; Ea, plastic light shield; 8b. light shield groove; 9, fiber-optic sockets: loa, stainless steel guae pin; lob. groove; 11. fiber-optic connector: 12. optical fiber. As the j rays cross each interface the I j intensities are subject to Fresnel losses (21-23), which in turn are a function of the striking angle. The find light integration along the axis provides cell transmittance directly. The integration is carried out a t X = OD/2 (the collecting fiber surface input end) by using
{v
T = x I j ( X = OD/2, y j ) J
0
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Flgure 3. View of the positive quadrant of the Source fiber surface with SIX rays emerging from ( X = OD/2, Y , Z = 0) (dots). i t is imperative that all starting rays emerging from the capillary tube strike the collecting fiber; otherwise, distortions in the recording signal are t o be expected (see below). The fortuitous symmetric properties of the system also reduce the problem of integration of the total emerging light to consideration of only one quarter of the total source fiber surface. For this purpose we have arbitrarily chosen the quarter with positive (Y,ZI ray coordinates shown in Figure 3. With these considerationstaken into account, the individual intensities I, assigned to the model A starting rays drawn in Figure 3 are given by the right angle triangle relationship as 1. I = (R2 - Y.2)1/2 J (1) where R is the source fiber core diameter and Yj the (yl coordinates of the j ray. The intensity of each of the secondary rays (model B) is simply one tenth of that given by eq 1. T o compute actual useful observable parameters, it is further necessary to normalize the intensities I j according to
Z I j X = -OD/2, Yj)= 1.0 J
(2)
(3)
In order to minimize the transmittance of rays travelling according to cases 1, 2, and 3 while optimizing the transmittance of case 4 rays, eq 3 has been computed for each ray path case separately. The model can be used to estimate the "effective" path length of absolute absorbance measurements. For example, most light rays will pass through the capillary tube near the axis of the tube. Those rays will have a path length in the sample given by the capillary diameter. On the other band, those rays that pass far from the axis of the capillary will have much shorter optical path lengths. A n "effective" path length would he useful in estimating the absorbance to be expected from a given concentration of analyte passing through the cell.
EXPERIMENTAL SECTION Flow Cell Design. A schematic of the on-column UV-visible detection cell is given in Figure 4. After an appropriate fused silica detection capillary W E ) ,I, is chosen, the protective polyimide coating is removed, e.g., by using heat or KOH solution, to produce a 2-5m-long optical window. The analytical capiuary column 1 (or a transfer capillary) is joined to one end of the detection capillary by means of plastic double-ferrule3b, which fits over the capillaries like a sleeve. The ferrule is enclosed by stainless steel nut 3c (a modified Vici ZU1 union) and bolt 3a (a modified Dynased Knaur 420 7520250 connector), resulting in a union capable of withstanding up to 5M) bar of internal pressure. This same configurationcan also be used when it is necessary or desirable for the two capillaries to have different diameters, in
ANALYTICAL CHEMISTRY,
which case the ferrules must be modified. The opposite end of the detection capillary is likewise connected to a transfer capillary (not shown). The unions are then covered by heat-shrink tubing 4, and the entire assembly is placed into the cell body 6-a twcl-partanodized aluminum “shell”50 mm long and 16 mm wide held together by means of guide pin 10a on one side, groove 10b in the other, and a pair of external aluminum ring clamps 2 (the purpose of the heat-shrink tubing is to ensure that the unions, and thus the capillaries, are held snugly, but flexibly, in the cell body, thus eliminating the possibility of the unions sliding away from the body as a result of high pressure). Plastic O-ring 5, which fits around the detection capillary and into a groove in the cell body, is important for centering the capillary in the cell assembly. Plastic light shield 8a, which fits into corresponding groove 8b when the cell body is closed, ensures the absence of interfering light. The source and collecting optical fibers had core diameters of 200,320,400,500, and 600 pm with an NA of 0.216 (Superguide G, Fiberguide Ind. and Polymicro Technologies). Their lengths ranged from 50 cm to 1.5 m. They were reinforced with black heat-shrink tubing 12, mounted into SMA type connectors 11 (Suhner 9840 type OlA, 03B and 04B, depending upon fiber diameter), abraded, polished, and finally connected to the cell by means of two pairs of externally threaded ports 9, located along the (X, y) axis of the cell body (surroundingthe optical window). The arrangement of the optical fibers around the cell window determines the mode of operation-in order to measure absorption at a single wavelength, the source fiber is mounted such that it is coaxial with the collecting fiber; to measure absorbance at two different wavelengths, two source/collecting fiber pairs are used (the source and collecting fiber from each pair still coaxial, but the two pairs orthogonal to each other); and to measure fluorescence, the source and emission fibers are orthogonal. Fluorescence and absorbance measurements are carried out simultaneously by using a single source fiber coupled to both a coaxial and an orthogonal Collecting fiber. Double pass absorption is possible by connectingthe collecting fiber (mounted on an (X) port) to one of the two remaining ( y) ports. The entire mounting procedure takes about 2 h and is accomplished with the aid of a microscope. Apparatus. The CSFC measurements were carried out by using a commerical instrument (CCS 5000 SFC/GC from Computer Chemical Systems) equipped with a 50-pm frit restrictor (Lee Scientific) and a 50-pm, 10-m-longDB-5 column (df = 0.25). For the sample injection a 0.1-pL rotary valve and a 1 0 1 split ratio were used. A modified model of the Spectroflow 783 absorption spectrophotometer (AB1 Analytical, Kratos Division) served as the detector. The original sample and reference cells were removed to enable the mounting of the light and bypassing source and of the source and reference fibers, which had been previously polished and fitted into SMA type connectors. A black aluminum block specially machined to accept these connectors was mounted at the detector head. The reference light fiber was directly coupled to the spectrophotometer photodiode, and in order to be within the absorbance offset of the instrument, the source and reference light waveguides had identical core diameters. Fluorescence measurements were made with a modified F-1000 Merck-Hitachifluorometer. Data acquisition was carried out with the Xtra Chrom I1 chromatographic software package (Nelson Analytical) through HP 310-760 ad/da interfaces.
RESULTS AND DISCUSSION Figure 5 shows the application of the flow cell to a typical CSFC separation of a standard mixture of polycyclic aromatic hydrocarbons. Traces a and b correspond to absorbance and fluorescence measurements, respectively, which were taken simultaneously. Trace c was recorded subsequently by using a universal flame ionization detector (FID), enabling the detection of substances that neither absorb nor fluoresce at the working wavelength. This arrangement facilitates peak identification and also lowers the concentration detection limits. The cell optical hardware used to record the chromatograms shown in Figure 5 consists of a 530 pm i.d., 700 pm 0.d. capillary tube coupled to a 400-pm source. Fibers of 600-pm
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Flgure 5. CSFC separation of a mixture of (1) naphthalene (10.31 mg/mL), (2) fluorene (1.72 mg/mL),(3) anthracene (1.6 mg/mL), (4) methylanthracene (10.6 mg/mL), (5) pyrene (10.7 mg/mL), (6) 2nitrafluorene (25.0 mg/mL), and (7) crysene (1.5 mg/mL) dissolved in CHCI,: (a) UV, (b) fluorescence, and (c) F I D detection.
diameter were used to collect the absorption and the emission signals at 254 and 380 nm, respectively. T o enhance fluorescence efficiency, the emission from the two coaxial { y) ports was collected. In chromatogram c the solvent peak is denoted by 0. The a/b/c signal intensity ratios are 1.73/ 1.00/1.22. The initial column pressure of 141 bar was held constant for 2.5 min and then steadily increased at a rate of 21 bar/min until it reached 423 bar, at which point it was again held constant. Linearity and Sensitivity. The linearity behavior of the flow cell was investigated by varying the concentration of anthracene [Ant] dissolved in methylene chloride. These solutions were injected into a self-made packed-column SFC apparatus, and the absorbance was measured. The data was fit to the following linear function suggested by Scott (28): log A = log C
+ J log [Ant]
(4)
where A is the detector response (i.e., photodiode output voltage or absorbance), C is a proportionality constant, and J is the response index, which takes a theoretical value of unity for an ideal, linear detection response. Absorbance data was collected for 11 different values of [Ant] ranging from 500 ng/mL to 0.1 mg/mL, each repeated five times. A linear regression performed on this data yielded a response index of J = 0.93 (correlation coefficient 0.99)-clw to the expected theoretical value. The detection limit for anthracene under these experimental conditions was found to be [Ant] = 1.6 ng/mL, with an S/N ratio of 3 and a noise level of 5.0 X au. The so-called “static” linearity test, which is expected to be independent of injection and column effects, were also performed according to the method described in the literature (13, 29). For this test, triplicate runs of 11 solutions of anthracene in ethanol ([Ant] = 8.2 ng/mL to 16.4 Mg/mL) were carried out. As above, slight deviations from ideal behavior were observed. These deviations, which are consistent with previous observations (3, 18, 30, 31) and are attributed to changes in the refractive index (RI) as the concentration of anthracene is varied, can be reduced by proper cell design. Diffraction effects, which can distort the recording signal, were neither anticipated nor observed. Light emerging from the soume fiber is spatially incoherent, has a broad bandwidth, and in comparison to the dimensions of the capillary tubes used, has a small wavelength. For thin capillary tubes (i.d.
