1634
Anal. Chem. 1986, 58,1634-1638
On-Line Thermionic Detection for Narrow-Bore Reversed-Phase Liquid Chromatography Frans A. Maris, Rob J. van Delft, Roland W. Frei, Rent5 B. Geerdink, and Udo A. Th. Brinkman* Section of Environmental Chemistry, Department of Analytical Chemistry, Free University, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
Reversed-phase column liquid chromatography with on-line thermionlc detectlon (LC-TID) can convenlenHy be carried out with methanol-water mlxtures at a flow rate of 20-30 pL/mln. Commerclal GC-TID equipment is used, and an evaporation Interface serves to vaporize the LC effluent. Addition of at least 3 mUmln of nitrogen to the LC effluent helps to reduce the base-llne noise. The setup used allows easy swltchlng from the GC-TID to the LC-TID mode, and vlce versa. Optlmlzatkm of the gas flow through the detector revealed that, compared to GC-TID, a much hlgher air flow Is required. Optimal smsiWty In LC-TID Is achieved at a low background current of about 2 PA. The seiectlvity of the detector in the LC-TID mode Is 1 X lo6 g of C/g of P, and detection Hmits are on the order of 5 pg of P/s. Callbratlon plots are hear over at least 3 orders of magnitude. As an example, the trace-level determination of diazlnon in onion samples Is reported.
It is generally recognized that high-performance liquid chromatography (LC) and gas chromatography (GC) are complementary rather than competitive techniques, each having its own advantages, drawbacks, and specific fields of application. One of the advantages of GC is the availability of a rather wide range of highly sensitive and selective detectors. Over the years, many attempts have been made to use such detectors for LC systems, employing both belt or wire transport and direct effluent introduction devices for proper interfacing. The latter approach has become more popular after the advent of miniaturized LC, which considerably alleviates the numerous technological and detector performance problems inherent in the continuous introduction of aqueous/organic mobile phases into the GC detector cell at the milliliter per minute flow rates typical for conventional-size LC. In the field of column liquid chromatography with GC detectors, recent years have witnessed impressive successes in the case of LC-mass spectrometry (1). Interesting results have also been obtained in LC with on-line electron capture detection (2, 3). A third area has involved the use of element-specific flame ionization and thermionic detectors in LC. Here, most attention has been devoted to the selective detection of phosphorus-containing compounds using a thermionic detector (TID; also called a nitrogen-phosphorus detector). The present-day TID is essentially based on the work of Kolb and Bischoff ( 4 ) who described a detector configuration in which a constant-current source, rather than a flame, is utilized to heat an alkali bead. The bead consists of an alkali metal silicate or a ceramic material coated with an alkali metal salt activator. Although the nature of the TID mechanism is not completely known, it has been amply demonstrated that ionization of the analyte must occur in the relatively lowtemperature plasma (“cold diluted flame”) surrounding the electrically heated bead, rather than in a flame, for specific nitrogen-phosphorus detection (5). LC-TID using a transport interface was first described by Slais and Krejci (6) and, several years later, by Compton and 0003-2700/86/0358-1634$01.50/0
Purdy (7). Further progress in this field was initiated by Novotny and co-workers (8-10) who used packed microcapillary columns coupled via a nebulization interface to a dual-flame TID. Solutes and solvents are first decomposed in a primary flame, and the combustion products are then swept into the analytical flame. The best responses were obtained with mobile-phase flow rates of less than 5 lL/min, with the proportion of water not exceeding 15-20%. Also orthogonal, rather than concentric, nebulization of the column effluent was tested in the LC-TID system, allowing the effective aspiration of higher molecular weight substances, but providing little gain in sensitivity. In our laboratory, we have ample experience with combining normal-phase and reversed-phase LC systems with an electron capture detector (2,3)and a mass spectrometer (11). For the first combination a rather simple evaporation interface is employed to obtain an on-line coupled system. In the present communication, we report on a research project in the field of LC-TID using a modified evaporation interface. The main aim was the development of a system for narrow-bore reversed-phase LC (0.7-1.0-mm4.d. columns; flow-rates, 20-60 bL/min) that does not show serious construction, operation, or maintenance problems.
EXPERIMENTAL SECTION Materials. The solvents were of HPLC grade quality (Baker, Deventer, The Netherlands). Analytical grade triethyl phosphate, triethyl thiophosphate, diethyl chlorothiophosphate, N,N-dimethylaniline, and dimethyl sulfoxide were supplied by Aldrich Europe (Beerse, Belgium). Analytical grade tri-n-butyl phosphate, parathion-ethyl, and fenitrothion were obtained from Merck (Darmstadt, FRG), Chrompack (Middelburg, The Netherlands), and Riedel-de Haen (Seelze, FRG), respectively. Diazinon (180 g/L) was obtained as agriculturally used from Denka Chemie (Voorthuizen, The Netherlands). Methods. The mobile phases for LC were delivered by a Gilson (Villiers-le-Bel,France) Model 302 pump. A homemade membrane pulse damper was used in conjunction with the pump. Samples were directly introduced with a homemade micro injection valve provided with a 50- and a 500-nL internal injection loop. Glass-lined stainless-steel columns (GLT; 200 X 0.7 mm i.d.; S.G.E., Melbourne, Australia) were homepacked with 5-pm LiChrosorb RP-18 (Merck). A Packard (United Technologies, Delft, The Netherlands) Model 427 gas chromatograph with a homepacked 3% OV-101on 80-100 mesh Chromosorb WHP glass column (100 cm x 2 mm i.d.; Chrompack) and a Packard thermionic detector Model 905 were used. The rubidium silicate bead of the TID was electrically heated by a Packard Model 612 detector controller. According to the manufacturer detection limits for the TID are W3g of N/s (for nitrobenzene) and 2 X g of P/s (for tri-n-butyl phosphate). The interface is situated in the detector block, kept at a constant temperature of 300 OC, and coupled to the LC column with a 120-wm fused silica capillary (S.G.E.). RESULTS AND DISCUSSION LC-TID Interfacing. A schematic diagram of the LCTID system used in the present study and the detailed construction of the interface are shown in Figure 1. The evaporation interface is a modified version of the interface used 0 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986
A
A
d+
LCcolumn
N2
I
‘face
rid pump
I
GCcolumn B
Gcoven]
from LC
C
1 s cap 012mm I D
300 ‘c
si steel cap 15cmx025mmlO
H
GC column
Flgwe 1. (A) Schematic design of the GC-TID and LC-TID system. (E) Detail of LC interface.
in a recent LC-electron capture detector study (3) and essentially consists of an aluminum block containing a 15-cm X 0.25-mm-i.d. stainless-steel capillary. As a modification, the connection of the LC column to the interface now goes via a 120-bm-i.d. fused silica capillary that runs through a Valco T-piece mounted at the top of the interface. An appropriate length, between 1 and 5 cm, of the fused silica capillary enters the 0.25-mm-i.d. stainless-steel capillary of the interface. The optimum position of the fused silica capillary is easily found by adjusting its position until the base-line noise is minimum. A further noise reduction can be obtained by adding nitrogen gas via the side entrance of the T-piece. The gas flows around the fused silica tubing into the interface capillary to prevent back diffusion of the mobile-phase vapor and sweeps the vapor into the TID. Varying the nitrogen flow between 0 and 30 mL/min revealed that optimal signal-to-noise ( S I N ) ratios were found at a flow of 3-5 mL/min. It was one of our aims to keep the GC-TID system intact and to allow rapid switching from the LC-TID to the GC-TID unit and vice versa. Therefore, the evaporation interface, which was placed in the GC-TID detector block, was coupled to the outlet of the GC column and the TID inlet via a homemade T-piece. The temperature of the GC oven was kept at 275 “C for the LC-TID experiments. The present setup has the advantage that it allows an easy daily control of the TID performance by analysis of a test mixture via the GC-TID unit. Another attractive feature of the setup is the possibility of repeating the injection of test solutes by GC-which ensures constant and known chromatographic behavior-while simultaneously introducing LC effluent vapor into the TID. This allows one to study the influence of LC parameters such as mobile-phase composition and flow rate and TID parameters such as hydrogen, nitrogen, and air flow rates on TID sensitivity and selectivity, and to compare the potential of LC-TID and GC-TID. In all optimization studies (next three sections), the “mixed” approach was used; Le., test solutes were injected via the GC system, while the LC mobile phase was introduced via the evaporation interface.
1635
Influence of Gas Flows and Bead Heating Current on Sensitivity. Experiments were started using the TID settings recommended for GC, i.e., an air flow of 50 mL/min, a H2flow of 5 mL/min, and a background current, ih, of 20 PA. Two test solutes were selected, viz. triethyl thiophosphate (TETP) and Nfl-dimethylaniline (Nfl-DMA); they were injected via the GC system, and no LC effluent was directed to the detector. Under the specified conditions, the TID showed excellent sensitivity toward both solutes, and the detection limits roughly agreed with the values quoted by the manufacturer (cf. Methods section). When, however, mobile-phase vapor was introduced into the TID via the LC system, the sensitivity decreased dramatically. The loss in sensitivity was distinctly more pronounced with the nitrogen- as compared with the phosphorus-containing analyte. This is probably caused by an increase of the temperature of the plasma surrounding the bead. The effect is similar to that in GC-TID when the hydrogen flow is increased (5). In such a case, the temperature of the plasma surrounding the rubidium bead is too high for optimal nitrogen detection; the elevated temperature has a negligible effect on the phosphorus response. All further work was carried out with phosphorus-containing test solutes. In order to reduce the number of parameters that can influence the TID performance (12), the Nz gas flow through the interface (3 mL/min; for optimization, see previous section) and the N2gas flow through the GC column (30 mL/min) were kept constant. In addition, all experiments were done with methanol-water (80:20) at a flow rate of 20 pL/min as the eluent, and neither the detector geometry nor the position of the rubidium bead was varied. The remaining parameters that can be varied were the Hzand air flow through the detector and the electrical bead heating current, ib. With i b and the air flow constant, it was observed that an increase of the Hzflow caused a distinct increase in ih and a smaller increase of the TETP response and the noise level. The optimal SIN ratio was found at Hzflows of less than 5 mL/min. Varying the air flow from 20 to 300 mL/min caused a rapid increase of ih. A maximum was reached at about 240 mL/min, with a subsequent slight decrease in the 240-300 mL/min region. A similar type of curve was found by us in GC-TID, where the maximum ih occurred at an air flow of 25 mL/min. The shift of the maximum background current to much higher air flows in LC-TID may well be caused by the necessary combustion of methanol. The decrease of ibc after the maximum is probably due to the fact that, gradually, the amount of oxygen present becomes excessive so that the (additional) air flow only serves to cool the rubidium bead and to dilute the fuel gas mixture. Finally, S I N measurements revealed that optimum sensitivity with our setup occurred at an air flow of 180-300 mL/min. The third parameter to be varied was the bead heating current, ib. It turned out that varying this parameter is the best means to change ibc. Because, conventionally, ib is indeed used to obtain a certain ih value during a series of experiments and, also, to keep the ih value constant when changes occur due to variations in other parameters, Figure 2 depicts the dependence of signal and noise on ih rather than ib. In agreement with data reported on GC-TID (13, 14), background noise and signal intensity increased with an increase in ib and, consequently, ih. The maximum S I N ratio occurs at rather low ibe values of around 2 PA. Based on the experimental data we decided to use a low ih setting in order to obtain a maximum SIN ratio. This will also help to enhance the lifetime of the rubidium bead. We also decided to work at constant i b This is recommended in the literature (13)to obtain constant sensitivity and good reproducibility over long periods of time and helps to compensate for degradation in bead performance during the usage.
1636
ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986
-
-%-
3300
m
.-ul
i
3250
v)
X
50 40
30
20
a, C
6
150
10
F W 2. Dependence of ndse,response for TETP, and dgna~to-noise ratio on the background current ( i k ) in LC-TID. CondRions are as follows: LC effluent, methanol-water (80:20) at 20 ,uL/min; GC injections of 24 ng of TETP and a 3% OV-101 on 80-100 mesh Chromosorb WHP column; T,,, 300 O C ; To,, 120 O C ; T,, 300 O C ; gas flows, H2, 5 mL/min; air, 200 mL/min; N, carrier gas, 30 mL/min; N, interface, 3 mL/min.
It is interesting to add here that the rubidium bead used in the present project has not shown noticeable degradation in performance during 8 months of intensive use. The lifetime of a rubidium bead in GC-TID is typically 3-6 months. Obviously, the use of a lower background current in LC-TID as compared to GC-TID is highly beneficial. The performance of a final series of experiments at ibc= 1.6 PA, in which the H2and air flow were varied, revealed optimal settings of 2.5-5 mL/min and 200-300 mL/min, respectively, when methanol-water (805%)vapor was introduced. These results are in agreement with those of McGuffin and Novotny (9)who found that the S I N ratio was improved at low H2 and at high air flows. Further it is interesting to note that changing the air flow had a larger effect on response (and noise) than changing the H2flow. Influence of Eluent Composition and Flow Rate on Sensitivity. The introduction of mobile-phase vapor into the TID considerably influenced the ih and, consequently, also the detector response and noise. For ih,the actual change strongly depended on the composition and flow rate of the LC eluent. For methanol-water mixtures with a total flow of up to 60 pL/min, the general situation was as follows. With 100% methanol, ib increased exponentially with the flow rate. In the presence of an increasing proportion of water in the eluent, the increase of ibc gradually became less pronounced, and at high water contents of over 70%, ibc even started to decrease with increasing flow rate. As explained in the previous section, for good performance of the LC-TID unit, it was necessary to work at a low and constant value of ibc (about 2 PA). The dependence of the response for TETP, at a constant ibc of 1.6 PA, on the flow rate (methanol-water (80:ZO)) and on eluent composition (at a flow rate of 20 pL/min) is shown in Figures 3 and 4,respectively. Figure 3 shows that the TID response dramatically decreased when going from "no eluent" flow to a flow rate of l pL/min. Simultaneously, the noise level increased by a factor of 10. A further 50% reduction of the response occurred between 1and 5 pL/min, and again between 5 and 20 pL/min. In the range of 1-30 hL/min, the noise level was fairly constant. Unfortunately, the noise in the present system increased rather substantially for flow rates of over 40 pL/min, and was so excessive at 60 pL/min that detector operation became
0
10
20
30
40
50
60
(pl rnin-') Flgure 3. Dependence of the response for TETP on the flow rate of methanol-water (80:20): i , = 1.6 PA. Further condltions are as in Figure 2. Flow-rate
Flgure 4. Dependence of the response for TETP and the noise on the
composition of the methand-water mixtures used as eluent; flow rate, 20 pL/min; i,, = 1.6 PA. Further conditions are as in Figure 2. virtually impossible. We should add that the inner diameter and length of the LC-TID interface were optimized for flows of about 20 pL/min. Probably, therefore, the high noise level at elevated flow rates was a t least partly caused by unsatisfactory interface performance. In practice, with narrow-bore LC columns of 0.7 mm i.d., one typically uses flow rates of between 20 and 60 pL/min. Because the TID sensitivity was higher at lower flow rates, 20 pL/min was used in our LC analyses. Figure 4 indicates that the detector sensitivity for T E T P increases with increasing water content of the eluent. Under these conditions, the detector noise also showed a distinct, but minor, increase. Consequently, best SIN ratios were found at high percentages of water. This contrasts with the results previously reported (9) for the dual-flame TID in which an increase of the water content in the mobile phase decreased the nebulization efficiency and caused a substantial increase in the background noise level, such that only 15-20'70 of water could be handled at a total eluent flow rate of 2 pL/min. Selectivity of LC-TID. The importance of the TID for GC is due to its sensitivity and selectivity for N- and P-con-
ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986
1
-
5
10
I
I
I
1637
/
0
tR(min)
Figure 5. Reversed-phase LC-TID chromatogram of six phosphorus-containing compounds in the system LiChrosorb RP-181 25 20 15 10 5 C methanol-water (80:20): flow rate, 20 wL1min; attenuation, X8; ibC c- tR(min) = 1.6 PA. Gas flows are as in Figure 2. Solutes used include (1) triethyl phosphate, (2) diethyl chlorothiophosphate, (3) triethyl thioFlgure 6. Reversed-phase LC-UV and LC-TID chromatograms of the phosphate, (4) fenitrothion, (5) parathion, and (6)trCn-butyl phosphate. extract of an onion spiked with 3.6 ppm diazinon in the system LiInjected amounts were 20-50 ngkompound. Chrosorb RP-18/methanoi-water (80:20): flow rate, 20 pL/min; UV detection at 254 nrn, attenuation, X 0.08;TID; i , = 1.6 PA, attenutaining compounds. Therefore, we estimated the selectivity ation, X 4. Gas flows are as in Figure 2. of our TID with and without the introduction of LC eluents 45-180 pg of phosphorus injected or, alternatively, to an avvia GC injections of n-hexadecane and TETP. The selectivity erage mass flux of 3-6 pg of P/s. The values quoted here may in GC-TID was determined to be 5 X 105 g of C/g of P, which be compared with a detection limit of about 0.01 pg of P/s coincides well with the manufacturer’s specifications. When measured by us for T E T P in GC-TID and quoted in the 20 wL/min methanol-water (80:20) was introduced into the manufacturer’s manual for tri-n-butyl phosphate As for detector the selectivity was found to be 1 X lo5 g of C/g of LC-TID, a detection limit of 14 pg of P / s has been reported P. The influence of the flame gas flows on the selectivity was (9) for trimethyl phosphate a t S I N = 8 and at a flow rate of also tested, revealing that the effect of the Hzflow is negligible, 1.3 pL/min of pure methanol. while selectivity for phosphorus was optimal a t an air flow With regard to the interface used in this study, a comabove 150 mL/min. Reducing the air flow below 100 mL/min parison of the peak areas of the six rather volatile test solutes resulted in a strong increase in the signal for n-hexadecane injected via the GC and the LC system revealed no difference and, therefore, a decrease in selectivity. in response between the two modes with methanol-water Variation of the mobile-phase composition to include higher mobile phases having up to 50% water, except for tri-n-butyl water percentages resulted in a slight decrease in selectivity. phosphate. For this compound, LC injection caused a response At high water percentages of over 90% the response for n20% lower than did GC injection. In other words, evaporation hexadecane increased strongly, and therefore, the decrease of the test solutes proceeded satisfactorily. In order to measure in selectivity became significant, reaching a value of about 1 the contribution of the interface to band broadening, a flow x IO4 g of C/g of P at 100% water. injection setup was used, and second moments (Mz) were As was mentioned previously the response for nitrogen is calculated (15,16)with TETP as the test solute. An Mz value much lower than that for phosphorus. The difference in of 5.4 s2 (relative standard deviation = 4%;n = 4) was found sensitivity was determined to be about lo3g of N/g of P using for the interface plus the detector. This corresponds to a NJV-DMA as a model compound. The response for the volumetric variance, 62,of 0.6 pL2 at an eluent flow rate of sulfur-containing compound dimethylsulfoxide was also 20 pL/min. It is a fully acceptable result, which is comparable measured and was found to be only slightly higher than that to that observed for several other detectors used in narrowfor carbon. bore LC (11, 17). LC-TID Studies. For actual LC-TID experiments, the Application. In order to demonstrate the applicability of same system was used without any alteration (i.e., with the our LC-TID system for environmental analyses, an onion was GC carrier gas still connected); a reversed-phase narrow-bore spiked with diazinon a t the parts-per-million level and anacolumn was installed to achieve LC separations, and test lyzed by reversed-phase LC-UV and LC-TID. Onions are solutes were injected into the LC system. The test mixture known to contain large amounts of sulfur-containing comconsisted of six P-containing compounds, viz., two organopounds. Pretreatment involved the following steps: (I) 50 phosphorus pesticides, two phosphate esters, and two thiog of onion was mixed with 50 mL of dichloromethane in a phosphate esters. Methanol-water (80:20) at a flow rate of Waring blender; after centrifugation the organic layer was 20 rL/min was used as the eluent. A typical chromatogram removed, and the residue was washed with 20 mL of diis shown in Figure 5. The analytical data for this system were chloromethane. (11) The organic phases were combined, rather satisfactory. Repeatability, at a level of 20-40 ng of washed with an equal volume of dimineralized water, and injected compounds was acceptable having a relative standard dried over anhydrous magnesium sulfate. (111)The solution deviation of 2.5-5% ( n = 10). A calibration curve, plotted was evaporated to 0.2 mL; 1.5 ml of methanol was added; and, for TETP, was found to be linear (P = 0.998) from 85 pg to finally, the solution was evaported to 1 mL. 300 ng of injected phosphorus; that is, the linear dynamic range was 3.5 orders of magnitude. The minimum detectable The recovery of diazinon was found to be about 70% in the 0.4-4 ppm range, with a detection limit of 0.1 ppm in the onion quantity (in nanograms of injected amount, and for S I N = sample. This corresponds to 2 ng of diazinon injected. Figure 3) varied from 0.3 ng for triethyl phosphate (k’ = 0.8) to 1.3 ng for tri-n-butyl phosphate ( k ’ = 4.2). This corresponds to 6 demonstrates the improved selectivity of LC-TID over
1638
Anal. Chem. 1986, 58, 1638-1643
LC-WV (A = 254 nm) analysis.
LITERATURE CITED
CONCLUSIONS An inexpensive evaporation interface, which can easily be
mounted in commercially available GC-TID equipment, has been used for coupling LC with TID. This system has been constructed in such a way that one can easily switch from LC to GC and vice versa. Compared to other equipment for LC-TID reported in the literature @-IO), the present device appears to be rather simple and more flexible in terms of range of application. Also, no special maintenance problems have been encountered thus far, although the LC-TID has been in uninterrupted use for over 8 months now. Future studies will focus on the evaluation of the potential and the limitations of the present system by using more polar and less volatile compounds, and of the usefulness of further miniaturization of the LC-TID system. The latter aspect will, however, require redesign or adaptation of the currently used instrumentation.
ACKNOWLEDGMENT We wish to thank J. C. Gluckman for her comments on this paper.
Desiderio, D. M.; Fridland, G. H. J. Liq. Chromatogr. 1984, 7, 317. Brlnkman, U. A. Th.; Geerdink, R. 6.; De Kok, A. J. Chromatogr. 1984, 297, 195. Maris, F. A.; Geerdink, R . 6.; Brinkman, U. A. Th. J. Chromatogr. 1985, 328, 93. Koib, 6.; Bischoff, J. J. Chromatogr. Sci. 1974, 72,625. Kolb, 6.; Auer, M.; Posporsii, P. J. Chromatogr. Sci. 1977, 15, 53. Slais, K.; Krejci, M. J. Chromatogr. 1874, 9 7 , 181. COmptOn, B. J.; Purdy, W. C. J. Chromatogr. 1979, 769, 39. McGuffin, V. L.; Novotny, M. J. Chromatogr. 1981, 278, 179. McGuffin, V. L.: Novotny, M. Anal. Chem. 1983, 55,2296. Gluckman, J. C.;Novotny, M. J. Chromatogr. 1984, 374, 103. Apffel, J. A.; Brinkman, U. A. Th.; Frei, R. W.; Evers, E. A. I. M. Anal. Chem. 1983, 55, 2280. Braznikov, V. V.; Gur'ev, M. V.: Sakodynsky, K. I . Cbromatogr. Rev. 1970, 72, 1. Lubkowitz, J. A.; Glajck, J. C.; Semonian, B. P.; Rogers, L. B. J. Chromatogr. 1977, 733, 37. Semonian, B. P.; Lubkowitz, J. A.: Rogers, L. B. J. Chrornatogr. 1978, 757, 1. Foley, J. P.; Dorsey, J. G. Anal. Chem. 1983, 55, 730. Maris, F. A.; Van der Vliet, A.; Geerdink, R. B.; Brinkman, U. A. Th. J . Chromatoor. 1985. 347. 75. (17) Jansen, HT; Brinkman, U'. A. Th.; Frei, R. W. Chromatographia 1985, 20, 453.
RECEIVED for review October 28, 1985. Accepted February 13, 1986.
High-Resafution Reversed-Phase Liquid Chromatography System for the Analysis of Complex Solutions of Primary and Secondary Amino Acids S t e f h Einarsson, S t a f f a n Folestad,* BjBm Josefason, and Soren Lagerkvist
Department of Analytical and Marine Chemistry, Chalmers University of Technology and University of Goteborg, S-412 96 Goteborg, Sweden
Packed fused &a columns have been employed for the separatknof 34 amho addsammtmiyfornd h phyrkkolcal fMds. The amlno ackls, dorlvatlzed with S-tluorenyhnethyl cMoroformate (FMOGCI) were separated wlth qadlent elutlon, uslng a conventlonal pump system and a shnple flow spHnlyl arrangement. A standard spectrofluorometer was wred for on-dumn detection. Good preckion was obtained In the separatlom, with a relatlve standard deviatlon of ret& Unns bekw 0.5%. The method was appUed to urlne sampb, whlch could be prepared In advance and then stored at room temperature until analysls. A mean recovery of standards added to wlne was 102% ( n = 12), and the mean relatlve standard devlatbn of 20 amlno acMs In urlne (peak helght) run overnight was 6.6% ( n = 10).
The need for rapid and versatile high-resolution chromatographic techniques in life sciences has long been recognized ( I ) . Reversed-phase liquid chromatography has developed into a powerful technique for separation of a large variety of substances. In combination with selective precolumn fluorescence labeling the technique has shown to be useful for amino acid analysis. The advantages are shorter analysis time and increase in sensitivity compared to the classical ion exchange chromatography (2,3).The most commonly used reagents are DNS-C1(4,5) and o-phthaldialdehyde (OPA)/ mercaptoethanol(6, 7),which has gained wide popularity in recent years. OPA reacts only with primary amino acids, but 0003-2700/86/0358- 1638$01.50/0
the formed derivatives are unstable. This has promoted development of automated derivatization coupled to the injection, for reproducible results (8, 9). The reagent has been applied to a variety of samples including physiological fluids (10, 11). The recently proposed fluorescent amino acid reagent 9fluorenylmethyl chloroformate (FMOC-Cl) has the advantage of reacting rapidly under mild conditions with both primary and secondary amino acids to yield stable derivatives (12,13). Thus a sample can be immediately stabilized by derivatization and thereafter stored for subsequent (automated) analysis. The reagent has been applied to protein hydrolysates, cerebrospinal fluids, and serum samples (12, 13). A notable trend in LC is the increased interest in columns of reduced internal diameters known collectively as microcolumns (14). Efficient packed columns have been shown to be readily prepared from fused silica capillaries (15-18). Demonstrated advantages are high separation efficiencies, high mass sensitivity, low-cost column material, and reduced solvent consumption. However, the number of biochemical applications is limited so far due to a lack of commercially available equipment. This paper describes a way to achieve high-resolution separations of FMOC amino acids using packed fused silica capillary columns. The UV transparency of the column is used for on-column fluorescence detection. The instrumental setup is based on conventional modules, and thus the equipment is commonly available. 0 1986 American Chemical Society
