Anal. Chem. 1987, 59, 1458-1461
1458
Low-Dispersion Chemiluminescence Detection for Packed Capillary Liquid Chromatography G.J. de Jong* Department of Analytical Chemistry, Free University, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands N. Lammers a n d F. J. S p r u i t
Research Laboratories, Duphar, P.O. Box 2, 1380 A A Weesp, The Netherlands C. Dewaele a n d
M.Verzele
Laboratory of Organic Chemistry, State University of Gent, Krijgslaan 281-S4, B-9000 Gent, Belgium
The peroxyoxalate chemlhdnescence detection system has been used for packed capillary liquld chromatography. Packed capillary fused-sillca columns of 0.32 mm 1.d. were shown to have a few important characteristics in comparison wlth conventional columns, Le., a minlmum in the H-u curve at relatively low flow rates and an about two times higher penneaMHty. Well-known advantages of this type ol columns as higher mass sensltlvlty and lower solvent consumption are also demonstrated. A very sunable system for mixing of the mobile phase and the reagents, Ws( 2,4dlnltrophenyl)oxalate and hydrogen peroxide, has been developed. A zero dead volume is combhed wlth an efHclent cdlectlon of the emitted llght by mlxlng In a flow cell Installed in an integrating sphere. The intluence of various reactlon parameters on the SenSnMty and the band broadening of the detection system was Investigated. Under certaln condltlons, the band broadening a, Is lower than 50 nL which is often negligible even In packed capillary ilquld chromatography. The detection llmlt of a dansylated drug wlth a secondary-amine functional group Is about 100 fg.
The potential of the reaction of bis(2,4,6-trichlorophenyl) oxalate (TCPO) or a similar oxalate and hydrogen peroxide has been demonstrated for the chemiluminescence detection of dansyl amino acids (1-4), fluorescamine-labeled catecholamines (5), dansyl derivatives of a drug with a secondary-amine functional group ( 6 ) ,a steroid (7) and a number of primary alkylamines (8), many polycyclic aromatic hydrocarbons (9-11), and 3-aminoperylene derivatives of carboxylic acids (12). The sensitivity of this detection mode for column liquid chromatography (LC) was shown to be often 10-100 times higher than that of conventional fluorescence detection. The influence of the flow-cell volume on sensitivity and on band broadening is remarkable (6,13).For an increase in the volume of the flow cell, a gain in signal is often observed. Moreover, the chemiluminescence detection causes less band broadening than a fluorescence detector and other detectors when going to larger cells. These effects can be attributed to the decay of the chemiluminescence signal, which is dependent on the oxalate and the composition of the solvent obtained after mixing of the reagents and the LC eluate (13). The chemiluminescence detector system is, therefore, very suitable for miniaturized LC as was demonstrated in some papers (4, 1I,13)for systems with microbore columns (1mm i.d.). In this paper, the use of the chemiluminescence detector for packed capillary columns is described. Special attention
wm paid to the mixing of the LC eluate and the reagents because in a system with relatively low flow rates a (nearly) zero dead volume is required for the detection of a large part of the emitted light. The goal was to combine an efficient collection of the emitted light with a low external band broadening. Moreover, some attractive characteristics of packed capillary columns will be reported.
EXPERIMENTAL SECTION Reagents. TCPO and bis(2,Cdinitrophenyl)oxalate (DNPO) were prepared by the method of Mohan ad Turro (14). Dansyl chloride was purchased from Merck (Darmstadt,Federal Republic of Germany). All other chemicals were of analytical reagent grade. Dansyl derivatives of a drug with a secondary-amine functional group, of the amino acid tryptophane, and of a carboxylic acid obtained by hydrolysis of the drug mebeverine (veratric acid, 4- [ethyl[@-methoxyphenyl)methylethyl]amino] butyl ester hydrochloride) were used as test compounds. The dansylation of the amines was carried out with dansyl chloride according to ref 6 and the dansylation of the acid with dansylethanolamine,which can easily be obtained from dansyl chloride, according to ref. 15. Column Liquid Chromatography. The LC pump was a Haskel type DSTV 25 (Haskel, Burbank, CA). The injection port was a Rheodyne four-port valve type 7520 (Rheodyne,Berkeley, CA) with an internal 0.5-pL loop. The columns were fused-silica capillaries of 20-50 cm X 0.32 mm i.d. packed by a slurry technique with 10-pm RSil-C18-HL-D (Alltech-RSL, Eke, Belgium) at a pressure of about 7000 psi. The packed material was retained by using a 2-pm PTFE frit. In front of the column a 2-pm stainla-steel frit was inserted in the injection port and the column was connected to the injector with a Vespel ferrule. Acetonitrile-0.01 M imidazole buffer pH 7.5 (65:35 (v/v)) was used as the mobile phase at various flow rates. Detection System. The scheme of the chemiluminescence detection system is shown in Figure 1. A capillary (column end tubing) of about 100 pm i.d. was inserted against the end-frit of the column and fiied with epoxy glue. This capillary was brought through a mixing tee (Valco Instruments, Houston, TX) by which the solutions of TCPO or DNPO and hydrogen peroxide were added. A Perfusor ED 2 syringe pump (Braun, Melsungen, F.R.G.) with two glass syringes was used to deliver the solutions of the reagents. Between the DNPO syringe and the mixing tee a forcing ball valve was placed in order to prevent back flush. A high-pressure fiiter of 2.0 pm was inserted in the line of hydrogen peroxide. A 70-pL flow cell (PTFE, 0.3 mm i.d.) was also fixed to the mixing tee and installed in the integrating sphere of a Zeiss spectrophotometer, Model PMQ 11-RA 3 (Zeiss, Oberkochen, F.R.G.). The column end tubing ended in the flow cell and the reagents and the LC eluate were mixed in the first part of the flow cell. RESULTS AND DISCUSSION Packed Capillary Columns. The use of packed capillary columns has been described in some papers (16-20) and
0003-2700/87/0359-1458$01.50/00 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 10, MAY 15, 1987
Table I. Back Pressure (in Bar) for Conventional a n d Packed Capillary Columns" packing material
250 X 4.6 mm
250 X 0.32 mm
ratio of pressures
10 pm RSil-C18-HL-D 5 pm Spherisorb-ODS 2 pm ROSil-C18-D 1 pm ROSiI-C18-D
23 49 437 1190
13 26 228 602
1.77 1.88 1.91 1.97
1450
column (f.s.1 320 ,urn i.d. frit
column end tubing (f.s.1 DNPO
Below 350 bar the values were really measured. Higher figures were obtained by recalculation (linear extrapolation) for 25 cm column length. Chromatographic conditions: mobile phase, acetonitrile-water (9O:lO (v/v)); flow rate, 0.826 mL/min for the 4.6 mm column and 4 pL/min for the 0.32 mm fused-silica column or a linear flow rate of 1.728 mm/s in both cases.
demonstrated advantages are high plate numbers, high mass sensitivity, and reduced solvent consumption. Another interesting aspect is the possibility for in-column fluorescence detection (20-23). In this way, a higher sensitivity can be obtained than with detection in a (capillary) flow cell after the elution. However, the number of applications of packed capillary columns is limited so far due to a lack of commercially available apparatus. Figure 2 shows the Van Deemter curve for a column of 27 cm X 0.32 mm i.d. packed with 10-pm particles. The plate heights have been calculated from chromatograms obtained with the chemiluminescence detection system for a series of dansylated compounds. The contribution of the column to the variance of the peaks was found by linear extrapolation (F = 0.999) of the total variance with respect to the square of the retention volumes (24). At the same time, the band broadening of the detection system was obtained because the contribution of the injection to the band broadening was made negligible by the use of an injection solvent containing more water than the mobile phase. This H-u curve is clearly different from that of stainless steel columns with a larger diameter. The plate height decreases up to about 5 mm/min, and for stainless steel columns packed with the same material, the minimum in the curve is a t about 40 mm/min with polycyclic aromatic hydrocarbons at test compounds. A part of the difference seems to be caused by the influence of the test compounds, because for columns packed with 5-pm ROSil-Cl8-D and polycyclic aromatic hydrocarbons as test compounds the minimum linear flow rates were about 20 and 60 mm/min for columns of 15 cm X 0.32 pm and 15 cm X 4.6 mm, respectively (25). The contribution of the C term in the Van Deemter equation seems more important for the fusedsilica columns as is demonstrated by the increase of the plate height at higher flow rates. The lower optimal flow rate is caused at least partly by the higher C term. The optimal reduced plate height of 1.3 is very low. This is equivalent to a chromatographic efficiency of 77% (26). Calculation of the
integrating sphere -flow cell \
Figure 1. System for mixlng of the LC eluate of the capillary column and the reagents for the chemllumlnescence detection. 1503
-
50
100
150
u(rnrn/min)
Flgure 2. H-u curve for a capillary column of 27 cm X 0.32 mm i.d. packed with 10-pm RSII-C18-HL-D: mobile phase, acetonitrile-0.01 M imidazole buffer pH 7.5 (65:35 (v/v)); reaction system, 0.01 M DNPO in acetonitrile (1 15 pL/mln), 1 M hydrogen peroxMe In tetrahydrofuran (115 pL/min); detector, Zeiss PMQ I I R A 3 with integratlng sphere and 70-pL cell (see Figure 1).
minimum plate height with the second moment (27) of the peak gives an about a factor of 2 higher plate height because the asymmetry of the peak is then also included. The particle
Table 11. Influence of Some Parameters on t h e Sensitivity and t h e Band Broadening of t h e Chemiluminescence Detection System" parameter oxalate concn of DNPO concn of imidazole in H20zsolution concn of H,O, _ DNPO:H2OZ(flowrates)
compared systems
re1 signal
DNPO in acetonitrile TCPO in ethyl acetate 2 mM 16 mM 0 mM 5 mM 25 mM 1M 1:3 3:1
90 30 30 130 90 10 30 90 80 70
re1 background
7 1 1 11 7 6 1 7 6 8
udet (nL)
detection limit, pg
420 1500
3 7 6 2 3 23 6 3 3 4
210
890 420
