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Development of an HRGC-HRGC-LRMS system: Instrument design and performance data
Chemosphere, Vol.20, No.6, pp 635-645, Printed in Great Britain
1990
0045-6535/90 $3.00 + .OO Pergamon Press plc
DEVELOPMENT OF AN HRGC-HRGC-LRMS SYSTEM: INSTRUMENT DESIGN AND PERFORMANCE DATA
L.L.Lamparski,* T.l.Nestrldg* D.Ianson* and G.Wilson~ *The Dow Chemical Company, Michigan Applied Science & Technology Laboratories, Analytical Sciences, 1602 Building, Midland, Michigan 48674, USA. *Analytical Controls, Incorporated, 3448 Progress Drive, Bensalem, Pennsylvania 19020. q'he Hewlett Packard Company, 39550 Orchard Hill Place, Novi, Michigan 48050, USA.
ABSTRACT Analytical Controls, Inc., can supply a Hewlen Packard Model 5890 high resolution gas chromatograph that is appropriately configured to accomplish fully automated, single-oven, 2-dimensional gas chromatography. As a portion of an extended research program dedicated to improving the capabilities of conventional instrumentation, we have developed and assembled the necessary components to permit the linkage of snch a unit to a Hewlen Packard Model 5987A high resolution gas chromatograph-low resolution mass spectrometer (HRGC-LRM$). The results of this project have produced an instrument that can routinely conduct fully automated, dual-oven, 2-dimensional HRGC separations in conjunction with LRM$ identification and detection for solutes elutingfrom the secondary analytical gas chromatograph. In this paper we will endeavor to describe this new analytical instrument and the techniques required to assemble it from commercially available resources. Preliminary evaluation and testing of the operational chamctm~tics of the unit indicate that it may be a valuable tool for measuring compounds at trace concentrations in extremely complex matrtce~ KEYWORDS Multi-dimensional capillary gas chromatography; multi-dimensional capillary gas chromatography-low resolution mass spectrometry (HRGC-HRGC-LRMS); high resolution gas chromatography-low resolution mass spectrometry (HRGC-LRMS); Deans switch; trace analyses; chlorinated dibenzo-p-dioxins and dibenzofurans (CDDs/CDFs). INTRODUCTION Analytical procedures for the measurement of chlorinated dibenzo-p-dioxins and dibenzofurans (CDDs/CDFs) in complex environmental matrices at extremely low concentration levels (e.g., parts per trillion = PIT, or parts per quadrillion = PPQ) rely upon one of two basic techniques to accomplish such determinations. Either an extensive sample preparation method is employed to impart analytical reliability,'-2 or highly sophisticated and expensive instrumentation such as high resolution gas chromatography-high resolution mass spectrometry (HRGC-H1LMS).S It is easy to surmise that each of these basic techniques suffers inherent disadvantages from the general analytical perspective. Extensive sample cleanup, including such procedures as high performance liquid chromatography (HPLC) fractionation to isolate specific analytes prior to instrumental measurement, can be a labor-intensive process which may severely limit the number of samples that can be examined in a given time period. Alternatively the sophisticated instrumental approach using HRGC-HRMS,while potentially resolving the problem of analytical speed by reducing the need for complicated cleanups, requires substantially more expensive equipment and can often preclude many laboratories from performing such analyses. In
635
636
response to these potential difficulties surrounding trace analyses we have developed, constructed and tested an instrument which permits combining the s e p a r a t i o n p o w e r of a m u l t i dimensional capillary gas chromatograph with the selectivity, ruggedness and operational ease of a low resolution mass spectrometer. This new hybrid instrument is a dual-oven, dual-column, 2-dimensional HRGC-HRGC-LRMS.
Figure 1. Operational conflguration for HRGC-HRGC-LRMS unit in the authors' laboratory; components for MS data system are not shown but are situated to the right of the HP-5890 preparatory gas chromatograph. The autosampler is shown unattached to provide an unobstructed view of the heated transfer line.
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The s u b s t a n t i a l power of multi° dimensional gas chromatography for the separation of complex mixtures has been reported in the literature for at least twenty years.~s However, it has been the recent d e v e l o p m e n t of c o m p u t e r controlled switching devices that has allowed the evolution of easy to operate multi-dimensional gas chromatography. '-8 Typically these systems use a Deans-type pressure switch to perform the heart-cntting, cryogenic trapping and desorption operations necessary to achieve such muiti-dimensional chromatographic separations. One disadvantage to many of these commercially available instruments is that they are primarily designed for use in a single-oven gas chromatographic system. Such configuration may result in operating constraints for the unit with regard to the types of capillary columns that can be used because of the requirement to maintain operational temperature compatibility. In addition, these instruments are generally not configured for use in a research-type mass spectrometer suitable for performing trace analyses at the PPT or PPQ concentration level The coupling of multi-dimensional gas chromatography to mass spectrometers is not a new phenomenon from the viewpoint of research applications. Ligon and May have described its implementation with HRMS instrumentation in 1984, 9-~°and Claude and Tabacchi devised a triple quadrupole mass spectrometer unit (GCGC-MS-MS) in 1988. ~ Neither of these groups reported the ability to perform measurements at PPT concentration levels that are routinely necessary for typical environmental monitoring of CDDs/CDFs. However, Ligon a n d May d i d d e m o n s t r a t e the c a p a b i l i t y to measure 20 PPB (parts p e r billion) of 2,3,7,8tetrachlorodibenzofuran (2378-TCDF) in transformer oil using their HRMS unit. In this paper we report the coupling of a commercially available, ACI-modified, Hewlett Packard Model 5890 (HP-5890) capillary gas chromatograph to a conventional Hewlett Packard Model 5987A (HP-5987A) HRGCLRMS instrument to produce a fully automated, computer-controlled, dual-oven HRGC-HRGC-LRMS unit that is capable of measuring PPT concentrations of specific CDDs/CDFs in complex environmental samples on a routine basis. Details of specific instrument components, their assembly and control, as well as resultant trace analytical performance data will be described. INSTRUMENT DESCRIFHON Integral components of the HRGC-HRGC-LRMS system to be described are illustrated in their typical operating configuration in Figure 1. The HP-5890 gas chromatograph (far right in Figure 1) contains the preparatory capillary column (precolumn) and all of the valving components associated with the Deans switch to control the gas flows to both capillary columns in the system. This gas chromatograph is equipped with a flame ionization detector (FIE)) and an electron capture detector (ECD), as well as a Hewletr Packard Model 7673A Autosampler. Either detector can be used to monitor the effluent fzom the precolumn or, under stand-alone operation as a single-oven unit, from the analytical capillary column.'
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General system control, including the valving sequence, autosampler control and the HP-5890 instrument parameters, is accomplished by the ACI Controller and its associated software operating on a Howlett Packard Model 3396 (HP-3396) Integrator. These programs are written in Basic and are stored on a 3.5 inch floppy disc in a Hewiett Packard Model 9122 dual disc drive unit. Data from the precolumn analyses of injected samples are also stored on floppy disc. Hgure 2. Simplifiedflowpath for HRGC-HRGC-LRMSinstrument.
CryogenicTrap
NI Switch
~
Transfer Line
HP-5890 Preparatory Gas Chromatograph
Butt Connector
1
The p r e c o l u m n effluent c a n be cryogenically trapped (liquid carbon dioxide) a n d transferred to the analytical column through the heated transfer line that connects the HI)5890 to the HP-5880 gas chromatographs. This transfer unit consists of an outer, electrically heated section of Vs-inch copper tubing with an inner, concentric piece of 0.20 m m ID fused silica tubing. For this application, the inner fused silica line is a portion of a HP-5 capillary column (df = 0.11 gm) obtained from The Hewiett Packard Company (Avondale, PAL Other types of deactivated and/or coated fused silica t u b i n g c a n be used in the transfer line d e p e n d i n g upon the chromatographic characteristics of the desired solutes to be examined. The temperature of the heated transfer line is controlled to +5 ° C over its entire length (-125 cm) by three independently heated zones regulated by the B-injection port of the HP-5890 and the two detector heaters of the HP5880. In this manner, the transfer line t e m p e r a t u r e can be regulated for o p t i m u m transfer of the analyte between the two gas chromatographs.
HP-~
The HP-5987A HRGC-LRMS system, including the HP-5880, operate in essentially normal mode except for the elimination of the need to use the HP-5880 injection port since the carrier gas is s u p p l e d to the analytical column through the transfer line from the preparatory gas chromatograph. We have found that the analytical column can be easily and reliably connected to the transfer tubing via a Supelco Capillary Column Butt Connector TM (Supelco, Inc., Bellefonte, PA) with no observable loss in chromatographic performance. This configuration permits changing the analytical column without removing the transfer tubing thereby simplifying the mechanics of the operation.
Analytical Gas Chromatograph
A schematic representation of the carrier gas flowpath between the preparatory and analytical gas chromatographs is shown in Figure 2. This figure is intended to show the basic configuration of the precolumn, Deans switch, transfer line and the analytical column. The configuration of Analytical Controls' Deans switch is provided in Figure 3. The nomenclature for this figure is: NV is a needle valve that is manually set to provide controlled restriction to achieve system flow balancing; V is a solenoid actuated valve that controls flowpath switching; the flow controller is used to provide a constant carrier gas flow to the precolumn; and the pressure regulator provides constant pressure carrier gas to the analytical column and directs the effluent from the precolumn to either the FID or ECD or to the cryogenic trap. There are four primary operations initiated by the Deans switch as used in our present application. These functions are: (1) precolumn separation, (2) analyte trapping (3) reinjection into the analytical column, and
638
Figure 3-1.
P r e c o l u m n Separation. Solute separation occurs on the precolumn operating under constant flow conditions. The effluent is directed to the FID or ECD by constant pressure carrier gas switched from V3 which continues to maintain constant pressure on the analytical column. Ir~or
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Figure 3-2. Ana/yte Trapping. Valve V9 is opened a//owing l/qu/d COt to cool the cryogenic trap and V3 is switched to divert the precolumn effluent through the trap. Excess carrier gas from this operation is vented through V4 while maintaining constant pressure on the analytical column.
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Figure 3-4. Precolumn Backflush. As the analyte
discontinued by closing 1/9 and the trap is then resistively heated to desorb the analyte onto the analytical column which is operating under constant carrier gas pressure. At this time data acquisition on the HP-5987A begins.
separation proceeds, 1/2 is opened and V1 is diverted to allow reverse carrier gas f l o w through the precolumn in a pressure-controlled manner to backflush residual solutes from the precolumn thereby shortening the total analysis time.
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(4) backflush of the precolumn. The cartier gas and solute flowpaths for each of these functions are illustrated in the four windows of Figure 3. In addition to these operations, other possibilities such as precooling the cryogenic trap and a precolumn stop-flow mode are described elsewhere.7 Custom Interface Circuit An important facet of developing the instrument involved synchronizing the desorption of the analyte from the cryogenic trap with the initiation of an analysis run on the HP-5987A unit via a compatible electronic channel. Accomplishment of this task necessitated the production of a specialized circuit to serve as an interface (I/F). In essence, the purpose of this I/F was to connect an HP-5890 gas chromatograph that was modified by Analytical Controls, by equipping with an ACI Controller, to an HP-5987A HRGC-LRMS system in the functions of Remote Enable and Remote Start signals. The resultant circuit design, shown in Figure 4, is for an original application and is not necessarily approved byThe Hewlett Packard Company. The specific goals of this design are: (1) pass a Start signal from the HP-5890 HRGC to the HP-5987A HRGC-LRMS system upon injection into the former instrument; [2) pass the NotReady status from the HRGC-LRMS system to the HP-5890 to prevent automaticinjectionofsubsequentsamplesbeforethe HRGC-LRMS is ready; (3) allconnectionsto theinstntment systems should be simple, external additions and not hard wired to the major components to simplify potential troubleshooting; (4) maintain electrical isolation between the two systems at the signal level; and (5) provide a reliable and potentially long term solution for system operation. The Start signal in the HP-5890 HRGC is generated by a microprocessor command that doses a relay contact for 50 milliseconds. Normally this closure would be sufficient for other instruments to sense, but in this case the
Hgure 4. Circuit diagram for the custom interface (I/F) linking the HP-5890 and I-1P-5987A instruments. HP-SS87A Remote Start 9
Connector to HP.5987A
(Rudy) BNC Custom I/F Box
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Start signal was to be connected into a de-bounced push button circuit on the I-lP-5987A Remote Start module via the existing coaxial connector. The de-bounced nature of the receiving circuit ignores the 50 millisecond relay closure form the HP-5890 HRGC thereby necessitating a pulse widening circuit. A low power 555 timer was used to lengthen the pulse to -700 milliseconds and its output was also a relay to maintain isolation. The power supply for the 555 timer and the relay is a large capacitor which is charged through a diode from a 5 volt puUup source within the ACI Controller, that is, it steals power from the sensing circuit of the ACI device without loading it significantly. The Remote Enab/e signal from the HRGC-LRMS system is derived from the LED circuit used to indicate system readiness on the Remote Start module. By tapping into the main connector of the module, this I/F senses and inverts the HRGC-LRMS system ready signal and closes a relay contact while a Not Ready condition exists. The I/F itself is housed in a plastic box that fits neatly under the cover of the HP-5890 HRGC and has the ACI/HP5890 remote start cable passing through it where connections are made to its four wires. The circuit design, as illustrated in Figure 4, functions properly but suffers one minor drawback. When the Start signal is passed to the HRGC-LRMS system, it responds by turning off its Ready LED on the Remote Start module. The I/F then passes this not ready condition back to the HP-5890 HRGC which illuminates its red Not Ready LED and puts the message "EXT DEVICE NOT READY" on the HP-5890 display. The running of the overall system is not affected by this status and the run continues normally with the curious message remaining. INSTRUMENT OP~P,ATION Operation and testing of the HRGC-HRGC-LRMS unit for the determination of 2,3,7,8-tetrachlorodibenzo-pdioxin (2378-TCDD) and 2378-TCDF at trace level concentrations is the subject of this section, however, it is important to understand that these conditions can easily be adapted for any analytes that are amenable to typical HRGC-LRMS examination. The gas chromatographic conditions used for the precolumn chromatograph and the HRGC-LRMS analytical chromatography are presented in Table 1. We have found that the combination of the two fused silica capillary columns described in Table 1 is broadly suitable for the determination of many CDDs/CDFs (chlorinated dibenzo-p-dioxins and dibenzofurans) in a variety of environmental sample matrices. The DB-5 precolumn provides reasonable chromatographic durability and is substantially resistant to fouling caused by the injection of dirty extracts. Its maximum allowable operating temperature permits routine temperature programming up to -300 ° C for removal of low volatility solutes without significant loss of general chromatographic efficiency. The Lee SB-Smectic column has been demonstrated to yield an isomer specific separation for 2378-TCDD. However, elution of 2378-TCDD typically requires operation of the column near the maximum allowable temperature for this liquid crystal stationary phase and therefore reduces its applicabilit Table 1. HRGC-HRGC-LRMS instrumental and gas chromatographic operating conditions for the identification
and measurement of 2378-TCDD and 2378-~_,DF analytes. Preparatory HRGC Conditions
Analytical HRGC Conditions
Column: 0.32 mm ID x 30 m DB-5 (dr -- 0.25 prn), J &W Retention Gap: 0.53 mm ID x 1 m DB-5 (dr = 0.25 pro), J &W CarderGa¢" Helium at 2.5 cm'/minute Initial Temperature: 135° C Initial Time: 1.0 minute ProgramRote: 20" C/minute RuM Temteratura: 275 ° C Final Time: 5.0 minute leJecttenSize: 2.0 pL, splitless injection technique Injection Porl Tamlmretura: 250 ° C MonitorDetecter:. ECD at 300° C
Column: 0.20 mm ID x 25 m SB-Smectic (dr = 0.15 pro), Lee Scientific
CryogenicTrappingTemperature: -10" C Analyte RelnlecUon Temperature: 260° C Analyte Trap Window: 9.40 to 10.10 minutes Anelyte DesorMtenT/me: 1.0 minute Precolurml TemlmraturedurlnDDnorption: 270° C PrecolumnBactfflL~h Time: 10.10 minutes TransferUnn Temperetera: 250° C
(Lee Scientific, Salt Lake City, Utah USA)
CarrlerSu: Helium at30 PSI Initial Temlem~m: 170° C Initial T/me: 1.0 minute Initial TamperetumProgramRata: 30° C/minute TomlmmtumLevel?: 220° C Temterehn Level? HuM Time: 0.0 minutes Temmratem Pmgrem Rate 2." 5.0° C/minute
Finl Te~retem: 270 ° C RaM TemWerateraHold Time: 2.4 minutes TotelRua Time: 15.0 minutes GC-MS IMerlace Temperetum: 250° C MS ~u~e Temleratum: 300° C ten MassesMonitored: "rCDD = 320, 322, 324, 334 TCDF = 304,306,308,318 Dwell TimefurMonltemd Al~w$: 200 milliseconds each
6~i
to normal HRGC-LRMS analyses due to fouling of the column by low volatility solutes. Within the perspective of the described HRGC-HRGC-LRMS application, this column deficiency is largely eliminated via the precolumn separation which provides a heart-cut of the residue containing only solutes of similar volatility to that of 2378TCDD. Additionally, the combination of vastly different stationary phase selectivities exhibited by DB-5 and SB-Smectic columns provides a reduction in the potential for analyte interferences caused by solute co-elution from the analytical column. The advent of new liquid phases demonstrating special selectivity for CDDs/CDFs can be immediately employed in this HRGC-HRGC-LRMS system to further increase chromatographic selectivity. As illustrated in Figure 1, the I-IBGCHRGC-LRMS unit is d e s i g n e d to operate in conjunction with an HP7673A Autosampler which provides the capability of processing up to 100 samples in an unattended fashion once the i n s t r u m e n t a l o p e r a t i n g parameters have been established. The operational conditions specified for the determination of 2378-TCDD and 2378-TCDF in Table I lead to an HRGC-HRGC-LRMS system control sequence that is presented in the format of a Gandt chart in Figure 5. In this chart, the t i m e w i n d o w s during which an operation is being performed are highlighted by a solid black bar. The windows that an instrument is in the ready mode, waiting for a signal from another component, are highlighted in grey. During the r e m a i n i n g time, the components are in a standby mode or are equilibrating for the next step in the sequence.
Figure 5. HRGC-HRGC-LRMS system components operating sequence for the determination o f 2378- TCDD and 2378- TUDF per instrumental parameters given in Table 1. See text for description o f events and component idenu'fications.
(min)
Prep GC Cryo valve Cryotrap Trap heat Backflush Foreflush Ovencod
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To determine the operations that are LRMS taking place at a given time in the control sequence, read down the i n= chart along the vertical time lines Ready defined at the top. O p e r a t i n g components will have a black bar in the row to the right o f the name. Components ready to begin an operation, pending the completion of another event, are shown in grey. At the start of the analysis the six main components of the system (Prep Computer = ACI Controller, Autoinjector = HP-7673A, Prep GC = I-IP-5890, Analytical Computer = HP-5987A data system, Analytical GC = HP-5880, and the LRMS) are all in a ready state and are synchronized at the time of the start signal which is coincident with sample injection into the I-IP-5890 gas chromatograph. The Prep Computer, Autoinjector and the Analytical Computer temporarily suspend operation while the Prep GC performs the precolumn separation. At this time the Analytical GC and LRMS function for a short time in a d u m m y run. This run is necessitated by the fact that the start signal from the Autoinjector initiates the HRGC-LRMS analysis and then places the HRGC-LRMS system in a not ready mode. in order for analyte desorption to occur after the precolumn separation is completed, the Prep Computer must receive a ready signal f ~ m all of the external devices attached to the system. We have adopted the d u m m y run as a work-around for this complication. Initiating the dummy run allows the HRGC-LRMS to equilibrate during the time that the precolumn separation is progressing so that at the time of analyte desorption (15.2 minutes per Figure 5), the HRGC-LRMS is in the ready state and can accept the remote start signal to begin the final analytical separation. Analyte desorption at 15.2 minutes run-time is the point of maximum activity in the system. The Prep GC begins the second portion of its analysis sequence by initiating procedures to bacldlush the precolumn, closing
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the cryogenic valve to discontinue liquid COz flow to the trap, heating the trap, and adjusting the carrier gas flow paths to correspond with those depicted In Figure 3-3. At 16.2 minutes run-time the precolumn is put into the bacldlush mode and low volatility solutes are vented through valve V2 as indicated in Figure 3-4. With analyte desorption, the focus of the operational sequence switches to the Analytical GC (HP-5880) and the LRMS. The analytical separation proceeds for 15 minutes with data acquisition occurring over the last 11 minutes of the window. The analytes, 2378-TCDD and 2378oTCDE typically elute from the analytical column in the region from -11.5 to 12.0 minutes. After completion of the analytical separation, all system components return to their starting conditions and equilibrate prior to beginning analysis of the next sample. Under the chromatographic conditions presented in Table 1, the total analysis time for a sample is 37.2 minutes. HRGC-HRC~-I.RMS INSTRUMENT VALIDATION Prior to actual e m p l o y m e n t of the instrument for the determination of 2378-TCDD and 2378-TCDF in environmental samples, it was necessary to demonstrate the operating characteristics of the system with regard to general reliability. In this section we will describe the results of experiments to measure the reproducibility of analysis, linearity of response, analyte carry-over after m e a s u r e m e n t of c o n c e n t r a t e d solutions, and the sensitivity of the instrument compared to operation in the HBGC-LRMS mode.
Table 2. Results from HRGC-HRGC-LRMS
instrumental reproducibility test for the measurement of 40 pg 2378- TCDD injected. mh322 ~/z 324
n (number of runs) ~o (averageresponse)
10
10
7 784
3 774
To test the r e p r o d u c i b i l i t y of the HRGC-HRGC-LRMS instrument, a series of ten replicate Injections of a 20 PPB o ~ (relatives(andard deviation) 5.5 % 3.8 % (part per billion = pg/pL) reference standard of 2378-TCDD were accomplished over an -8 hour period. The system was operated in the automated mode with injections from the HP-7673A Autosampler. The results from this experiment are shown in Table 2. As indicated therein, the reproducibility of the system over an -8 hour time period is certainly acceptable for most trace analytical applications. There was some evidence of LRMS instrumental drift over the course of the experiment, and therefore it is anticipated that the reproducibility for analysis sets requiring shorter operational periods may demonstrate somewhat improved results. The linearity of 2378-TCDD response versus concentration is plotted in the two graphs in Figure 6. In this experiment a series of four calibration standards of 2378-TCDD and 2378-TCDF were examined over a period of -30 hours. These standards (at concentrations ranging from 2 to 1000 pglpL) were each analyzed in triplicate and the resuits averaged to yield the data in the graphs. The first graph shows the response linearity over the entire range of the test (4 to 2000 pg injected), and as indicated, acceptable linearity was obtained for this concentration range. However, the highest concentration level examined shows a slightly elevated response which in turn causes a slight skewing of the calculated response line away form the true response at the lower concentrations. This deviation is quite small and can be ignored for most typical applications. For analyses in which greater accuracy is required, the second graph in Figure 6 illustrates the response linearity obtainable when the analyte concentration is limited to the range from 2 to 200 pg injected. In this case the response for both TCDD ions monitored shows excellent llnearity and an acceptable value for the y-intercept. It should be noted that the data reported in Figure 6 are for 2378-TCDD, however, they are virtually identical to that obtained for 2378-TCDF with respect to each concentration range examined. The ability to suitably clean the chromatographic system between sample injections, to avoid carry-over from high concentration to following low concentration samples, is of crucial importance to the analyst if he or she is to avoid generating fa/se posit/oe results. We examined this system characteristic by analyzing a concentrated reference~standard of 2378-1L-DD and 2378-TCDF followed by a nonane solvent blank containing neither of the analytes. These tests were done in the manual injection mode. In the initial experiment a 500 PPB solution of [13Clz]-2378-TCDD and P3CI2]-2378-TCDF was injected into the HRGC-HRGC-LRMS system and the analytical run completed. Next a nonane solvent blank was injected and again the analytical run completed. Based upon the LRMS output for the latter run, neither analyte could be detected at a level of 0.1%. The entire experiment was then repeated using a much higher concentration of the analytes; 5000 PPB each. Since this concentration level was greater than the highest concentration reference standard employed during the linearity studies, and
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Hgure 6. HRGC-HRGC-LRMS de~ctor response dam for 2378-TCDD over two different operating ranges.
Detector Response
ExtendedConcentration Range /
Detector Response
NormalConcentration Range
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is therefore outside the limits bounding acceptably linear response for sample analyses, it is an example of the worst case scenario. The nonane solvent blank injection was accomplished after routine cleaning of the syringe, and the analytes carry-over was found to be -0.015% for each species. The syringe washing procedure was repeated and the precolumn GC injector was cleaned by making three consecutive injections of 2.0 gL benzene with the column temperature adjusted to 270 ° C isothermal. Following system reequilibration, the solvent blank analysis was again repeated and no detectable response for either analyte was obtained at a limit of detection in the range of -0.005%. This experiment demonstrates that it is possible for analyte carry-over to cause an apparent false positive response, however, the extent of this problem does not appear to be any greater than that for typical HRGC-LRMS instrumentation (i.e., apparently there is no carry-over that is peculiar to the Deans switch or the transfer line associated with the HRGC-HRGC-LRMS unit). Routine cleaning of both the injection syringe and the GC injector between sample analyses, as is common practice in the author's laboratory, can virtually eliminate common sample cross contamination effects attributable to analyte carry-over. Global instrument sensitivity for HRGC-HRGC-LRMS, as compared to the same detector operating in the HRGC-LRMS mode, is a function of the efficiency of the carrier gas plumbing system related to the Deans switch and also the efficiency at which the particular analyte(s) can be trapped and then subsequently desorbed onto the analytical column. Based upon two experiments that will be described, it was found that there is very little difference in instrumental sensitivity between HRGC-HRGC-LRMS and HRGC-LRMS. The efficiency of trapping and desorption of 2378-TCDD and 2378-TCDF is illustrated by an experiment in which the analytes were kept in the cold trap for an extended period of time prior to desorption. In this case, holding the analytes in the cold trap for 75 minutes resulted in <10% decrease in response when compared to that observed for a comparable standard that was desorbed after only 5.5 minutes in the trap. Of interest here is the fact that both of these tests were conducted using 40 pg of each native analste and 1000 pg of each P3C~=]-labeled internal standard. Such levels are not atypical of those encountered in general environmental analyses. A comparison of instrument sensitivity while operating in HRGC-LRMS and HRGC-HRGC-LRMS modes can be derived from the chromatograms in Figure 7. Although the analyte retention times are different, because the HRGC-LRMS run was accomplished on a DB-5 capillary column operating under conditions which permitted elution of 2378-TCDD in 6.6 minutes and the HRGC-HRGC-LRMS run used the conditions given in Table 1 where an elution time of 11.4 minutes is observed, it is still evident from the perspective of signal to noise ratio
644
FiguR 7. Comparison of instrumental sensitiuity for 2378-TCDD uia HRGC-LRMS and HRGC.HRGCLRMS techniques. Note comparable peak shapes, peak widths and signal to noise ratios for these runs.
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-
|
-
i
-
(minutes)
that the sensitivities for these measurements are comparable. It is important to mention that this level of instrumental performance depends upon the proper adjustment of gas flows within the Deans switch, however, such adjustments are not complicated to perform and can easily be accomplished in -2 hours when the capillary columns are being installed. Routine readjustment of the initial settings are typically not necessary. We have found that it is even possible to replace a column with one of the same dimensions and not have to make any adjustments in the flow or pressure settings for the Deans switch. CONCLUSIONS An HRGC-HRGC-LRMS instrument that is based entirely upon Hewlett Packard equipment has been developed, assembled and tested. It is capable of automated operation in an unattended mode for up to 100 sample analyses. It is based upon a dual-oven design wherein the preparatory column and the analytical column can be operated independently of one another thereby eliminating compatibility problems. Testing of the system, especially those components of the Deans switch and the cryogenic trap which contact solute species, with trace concentrations of 2378-TCDD and 2378-TCDF has indicated that it is reasonably inert and capable of transmission rates equivalent to those obtained in conventional HRGC-LRMS equipment. Because of these characteristics, the new HRGC-HRGC-LRMS instrument described offers analyte sensitivities that are essentially equivalent to those of typical HRGC-LRMS units for reference standards examined in the absence of matrix constituents. However, considering the negative impact that complicated matrix components can have upon the analytical detection limits for a given analyte, the HRGC-HRGC-LRMS unit is anticipated to possess superior usable sensitivity under typical trace analysis monitoring conditions. This contemplation will be~the subject of future reports concerning the application of HRGC-HRGC-LRMS to real wor/d samples. REFERENCES 1.
L.L. Lamparski and T. J. Nestrick, "AnalyticalMethodology of The Dew Chemical Company for the Determi-
2.
nation of Selected Chlorinated Dlbenzo-p-dioxins and Dibenzofurans in Stack Gas Effluent Matrices. Part I: Full Method for Determination of Tetra- through Octa-Chlorinated Congener Group Total Concentrations and "2378"-Substituted Isomers," Chemo~phere 19, 1165-1177 (1989). T. ]. Nestrick and L. L. Lamparski, 'Pma/ytical Methodology of The Dew Chemical Company for the Determination of Selecwd Chlorinated Dibenzo-p-dioxins and Dibenzofurans in Stack Gas Effluent Matrices. Part ll: Isomer Method for Determination of Tetra- through Octa-Chlorinated Congener Group "2378"-Substituted Isomer Concentrations," Chemosphere 19, 1179-1185 (1989).
645
3.
4.
"Method 1613: Tetra- through Octa-chlorinated Dioxins and Furans by Isotope Dilution High Resolution Gas Chromatograph-High Resolution Mass Spectrometry," United States Environmental Protection Agenc~ Office of Water Regulations and Standards, Washington, DC, July 1989. D.R. Deans, Chromatographia 1, 18 (1968).
5. 6.
G. Schomburg, H. HusmanandEWeeke, J.ofChrornatogr. 112, 205-217 (1975). W. Blass, K. Riegner and H. Hulpke, J. of Chromatogr. 172, 67-75 (1975).
7.
Analytical Controls, Inc., Product Bulletin #AC3020, Bensalem, PA (1968).
8.
V.G. vanI-Iese and D. Grant, American Laboratory 20, No. 12, 26-33 (1988).
9.
W.V.LigonandR. J. May, J.ofChromatogr. 294, 77-86 (1984).
10. W.V. Ligon and R. J. May, J. of Chromatogr. 294, 87-98 (1984). 11. S.G. Claude and R. Tabacchi, J. of HR & CC 11, 187-190 (1968). (Received in Germany 14 February 1990; accepted iO April 1990)
1990
0045-6535/90 $3.00 + .OO Pergamon Press plc
DEVELOPMENT OF AN HRGC-HRGC-LRMS SYSTEM: INSTRUMENT DESIGN AND PERFORMANCE DATA
L.L.Lamparski,* T.l.Nestrldg* D.Ianson* and G.Wilson~ *The Dow Chemical Company, Michigan Applied Science & Technology Laboratories, Analytical Sciences, 1602 Building, Midland, Michigan 48674, USA. *Analytical Controls, Incorporated, 3448 Progress Drive, Bensalem, Pennsylvania 19020. q'he Hewlett Packard Company, 39550 Orchard Hill Place, Novi, Michigan 48050, USA.
ABSTRACT Analytical Controls, Inc., can supply a Hewlen Packard Model 5890 high resolution gas chromatograph that is appropriately configured to accomplish fully automated, single-oven, 2-dimensional gas chromatography. As a portion of an extended research program dedicated to improving the capabilities of conventional instrumentation, we have developed and assembled the necessary components to permit the linkage of snch a unit to a Hewlen Packard Model 5987A high resolution gas chromatograph-low resolution mass spectrometer (HRGC-LRM$). The results of this project have produced an instrument that can routinely conduct fully automated, dual-oven, 2-dimensional HRGC separations in conjunction with LRM$ identification and detection for solutes elutingfrom the secondary analytical gas chromatograph. In this paper we will endeavor to describe this new analytical instrument and the techniques required to assemble it from commercially available resources. Preliminary evaluation and testing of the operational chamctm~tics of the unit indicate that it may be a valuable tool for measuring compounds at trace concentrations in extremely complex matrtce~ KEYWORDS Multi-dimensional capillary gas chromatography; multi-dimensional capillary gas chromatography-low resolution mass spectrometry (HRGC-HRGC-LRMS); high resolution gas chromatography-low resolution mass spectrometry (HRGC-LRMS); Deans switch; trace analyses; chlorinated dibenzo-p-dioxins and dibenzofurans (CDDs/CDFs). INTRODUCTION Analytical procedures for the measurement of chlorinated dibenzo-p-dioxins and dibenzofurans (CDDs/CDFs) in complex environmental matrices at extremely low concentration levels (e.g., parts per trillion = PIT, or parts per quadrillion = PPQ) rely upon one of two basic techniques to accomplish such determinations. Either an extensive sample preparation method is employed to impart analytical reliability,'-2 or highly sophisticated and expensive instrumentation such as high resolution gas chromatography-high resolution mass spectrometry (HRGC-H1LMS).S It is easy to surmise that each of these basic techniques suffers inherent disadvantages from the general analytical perspective. Extensive sample cleanup, including such procedures as high performance liquid chromatography (HPLC) fractionation to isolate specific analytes prior to instrumental measurement, can be a labor-intensive process which may severely limit the number of samples that can be examined in a given time period. Alternatively the sophisticated instrumental approach using HRGC-HRMS,while potentially resolving the problem of analytical speed by reducing the need for complicated cleanups, requires substantially more expensive equipment and can often preclude many laboratories from performing such analyses. In
635
636
response to these potential difficulties surrounding trace analyses we have developed, constructed and tested an instrument which permits combining the s e p a r a t i o n p o w e r of a m u l t i dimensional capillary gas chromatograph with the selectivity, ruggedness and operational ease of a low resolution mass spectrometer. This new hybrid instrument is a dual-oven, dual-column, 2-dimensional HRGC-HRGC-LRMS.
Figure 1. Operational conflguration for HRGC-HRGC-LRMS unit in the authors' laboratory; components for MS data system are not shown but are situated to the right of the HP-5890 preparatory gas chromatograph. The autosampler is shown unattached to provide an unobstructed view of the heated transfer line.
I
Ill
The s u b s t a n t i a l power of multi° dimensional gas chromatography for the separation of complex mixtures has been reported in the literature for at least twenty years.~s However, it has been the recent d e v e l o p m e n t of c o m p u t e r controlled switching devices that has allowed the evolution of easy to operate multi-dimensional gas chromatography. '-8 Typically these systems use a Deans-type pressure switch to perform the heart-cntting, cryogenic trapping and desorption operations necessary to achieve such muiti-dimensional chromatographic separations. One disadvantage to many of these commercially available instruments is that they are primarily designed for use in a single-oven gas chromatographic system. Such configuration may result in operating constraints for the unit with regard to the types of capillary columns that can be used because of the requirement to maintain operational temperature compatibility. In addition, these instruments are generally not configured for use in a research-type mass spectrometer suitable for performing trace analyses at the PPT or PPQ concentration level The coupling of multi-dimensional gas chromatography to mass spectrometers is not a new phenomenon from the viewpoint of research applications. Ligon and May have described its implementation with HRMS instrumentation in 1984, 9-~°and Claude and Tabacchi devised a triple quadrupole mass spectrometer unit (GCGC-MS-MS) in 1988. ~ Neither of these groups reported the ability to perform measurements at PPT concentration levels that are routinely necessary for typical environmental monitoring of CDDs/CDFs. However, Ligon a n d May d i d d e m o n s t r a t e the c a p a b i l i t y to measure 20 PPB (parts p e r billion) of 2,3,7,8tetrachlorodibenzofuran (2378-TCDF) in transformer oil using their HRMS unit. In this paper we report the coupling of a commercially available, ACI-modified, Hewlett Packard Model 5890 (HP-5890) capillary gas chromatograph to a conventional Hewlett Packard Model 5987A (HP-5987A) HRGCLRMS instrument to produce a fully automated, computer-controlled, dual-oven HRGC-HRGC-LRMS unit that is capable of measuring PPT concentrations of specific CDDs/CDFs in complex environmental samples on a routine basis. Details of specific instrument components, their assembly and control, as well as resultant trace analytical performance data will be described. INSTRUMENT DESCRIFHON Integral components of the HRGC-HRGC-LRMS system to be described are illustrated in their typical operating configuration in Figure 1. The HP-5890 gas chromatograph (far right in Figure 1) contains the preparatory capillary column (precolumn) and all of the valving components associated with the Deans switch to control the gas flows to both capillary columns in the system. This gas chromatograph is equipped with a flame ionization detector (FIE)) and an electron capture detector (ECD), as well as a Hewletr Packard Model 7673A Autosampler. Either detector can be used to monitor the effluent fzom the precolumn or, under stand-alone operation as a single-oven unit, from the analytical capillary column.'
637
General system control, including the valving sequence, autosampler control and the HP-5890 instrument parameters, is accomplished by the ACI Controller and its associated software operating on a Howlett Packard Model 3396 (HP-3396) Integrator. These programs are written in Basic and are stored on a 3.5 inch floppy disc in a Hewiett Packard Model 9122 dual disc drive unit. Data from the precolumn analyses of injected samples are also stored on floppy disc. Hgure 2. Simplifiedflowpath for HRGC-HRGC-LRMSinstrument.
CryogenicTrap
NI Switch
~
Transfer Line
HP-5890 Preparatory Gas Chromatograph
Butt Connector
1
The p r e c o l u m n effluent c a n be cryogenically trapped (liquid carbon dioxide) a n d transferred to the analytical column through the heated transfer line that connects the HI)5890 to the HP-5880 gas chromatographs. This transfer unit consists of an outer, electrically heated section of Vs-inch copper tubing with an inner, concentric piece of 0.20 m m ID fused silica tubing. For this application, the inner fused silica line is a portion of a HP-5 capillary column (df = 0.11 gm) obtained from The Hewiett Packard Company (Avondale, PAL Other types of deactivated and/or coated fused silica t u b i n g c a n be used in the transfer line d e p e n d i n g upon the chromatographic characteristics of the desired solutes to be examined. The temperature of the heated transfer line is controlled to +5 ° C over its entire length (-125 cm) by three independently heated zones regulated by the B-injection port of the HP-5890 and the two detector heaters of the HP5880. In this manner, the transfer line t e m p e r a t u r e can be regulated for o p t i m u m transfer of the analyte between the two gas chromatographs.
HP-~
The HP-5987A HRGC-LRMS system, including the HP-5880, operate in essentially normal mode except for the elimination of the need to use the HP-5880 injection port since the carrier gas is s u p p l e d to the analytical column through the transfer line from the preparatory gas chromatograph. We have found that the analytical column can be easily and reliably connected to the transfer tubing via a Supelco Capillary Column Butt Connector TM (Supelco, Inc., Bellefonte, PA) with no observable loss in chromatographic performance. This configuration permits changing the analytical column without removing the transfer tubing thereby simplifying the mechanics of the operation.
Analytical Gas Chromatograph
A schematic representation of the carrier gas flowpath between the preparatory and analytical gas chromatographs is shown in Figure 2. This figure is intended to show the basic configuration of the precolumn, Deans switch, transfer line and the analytical column. The configuration of Analytical Controls' Deans switch is provided in Figure 3. The nomenclature for this figure is: NV is a needle valve that is manually set to provide controlled restriction to achieve system flow balancing; V is a solenoid actuated valve that controls flowpath switching; the flow controller is used to provide a constant carrier gas flow to the precolumn; and the pressure regulator provides constant pressure carrier gas to the analytical column and directs the effluent from the precolumn to either the FID or ECD or to the cryogenic trap. There are four primary operations initiated by the Deans switch as used in our present application. These functions are: (1) precolumn separation, (2) analyte trapping (3) reinjection into the analytical column, and
638
Figure 3-1.
P r e c o l u m n Separation. Solute separation occurs on the precolumn operating under constant flow conditions. The effluent is directed to the FID or ECD by constant pressure carrier gas switched from V3 which continues to maintain constant pressure on the analytical column. Ir~or
V1
J'd ~ 1 1
Figure 3-2. Ana/yte Trapping. Valve V9 is opened a//owing l/qu/d COt to cool the cryogenic trap and V3 is switched to divert the precolumn effluent through the trap. Excess carrier gas from this operation is vented through V4 while maintaining constant pressure on the analytical column.
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Figure 3-3. Analyte lt~lnjection. Coolant flow is
Figure 3-4. Precolumn Backflush. As the analyte
discontinued by closing 1/9 and the trap is then resistively heated to desorb the analyte onto the analytical column which is operating under constant carrier gas pressure. At this time data acquisition on the HP-5987A begins.
separation proceeds, 1/2 is opened and V1 is diverted to allow reverse carrier gas f l o w through the precolumn in a pressure-controlled manner to backflush residual solutes from the precolumn thereby shortening the total analysis time.
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639
(4) backflush of the precolumn. The cartier gas and solute flowpaths for each of these functions are illustrated in the four windows of Figure 3. In addition to these operations, other possibilities such as precooling the cryogenic trap and a precolumn stop-flow mode are described elsewhere.7 Custom Interface Circuit An important facet of developing the instrument involved synchronizing the desorption of the analyte from the cryogenic trap with the initiation of an analysis run on the HP-5987A unit via a compatible electronic channel. Accomplishment of this task necessitated the production of a specialized circuit to serve as an interface (I/F). In essence, the purpose of this I/F was to connect an HP-5890 gas chromatograph that was modified by Analytical Controls, by equipping with an ACI Controller, to an HP-5987A HRGC-LRMS system in the functions of Remote Enable and Remote Start signals. The resultant circuit design, shown in Figure 4, is for an original application and is not necessarily approved byThe Hewlett Packard Company. The specific goals of this design are: (1) pass a Start signal from the HP-5890 HRGC to the HP-5987A HRGC-LRMS system upon injection into the former instrument; [2) pass the NotReady status from the HRGC-LRMS system to the HP-5890 to prevent automaticinjectionofsubsequentsamplesbeforethe HRGC-LRMS is ready; (3) allconnectionsto theinstntment systems should be simple, external additions and not hard wired to the major components to simplify potential troubleshooting; (4) maintain electrical isolation between the two systems at the signal level; and (5) provide a reliable and potentially long term solution for system operation. The Start signal in the HP-5890 HRGC is generated by a microprocessor command that doses a relay contact for 50 milliseconds. Normally this closure would be sufficient for other instruments to sense, but in this case the
Hgure 4. Circuit diagram for the custom interface (I/F) linking the HP-5890 and I-1P-5987A instruments. HP-SS87A Remote Start 9
Connector to HP.5987A
(Rudy) BNC Custom I/F Box
HP-5890 Remote Connector
Remote EnableI/P Pulldown= Not Reod~
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.
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640
Start signal was to be connected into a de-bounced push button circuit on the I-lP-5987A Remote Start module via the existing coaxial connector. The de-bounced nature of the receiving circuit ignores the 50 millisecond relay closure form the HP-5890 HRGC thereby necessitating a pulse widening circuit. A low power 555 timer was used to lengthen the pulse to -700 milliseconds and its output was also a relay to maintain isolation. The power supply for the 555 timer and the relay is a large capacitor which is charged through a diode from a 5 volt puUup source within the ACI Controller, that is, it steals power from the sensing circuit of the ACI device without loading it significantly. The Remote Enab/e signal from the HRGC-LRMS system is derived from the LED circuit used to indicate system readiness on the Remote Start module. By tapping into the main connector of the module, this I/F senses and inverts the HRGC-LRMS system ready signal and closes a relay contact while a Not Ready condition exists. The I/F itself is housed in a plastic box that fits neatly under the cover of the HP-5890 HRGC and has the ACI/HP5890 remote start cable passing through it where connections are made to its four wires. The circuit design, as illustrated in Figure 4, functions properly but suffers one minor drawback. When the Start signal is passed to the HRGC-LRMS system, it responds by turning off its Ready LED on the Remote Start module. The I/F then passes this not ready condition back to the HP-5890 HRGC which illuminates its red Not Ready LED and puts the message "EXT DEVICE NOT READY" on the HP-5890 display. The running of the overall system is not affected by this status and the run continues normally with the curious message remaining. INSTRUMENT OP~P,ATION Operation and testing of the HRGC-HRGC-LRMS unit for the determination of 2,3,7,8-tetrachlorodibenzo-pdioxin (2378-TCDD) and 2378-TCDF at trace level concentrations is the subject of this section, however, it is important to understand that these conditions can easily be adapted for any analytes that are amenable to typical HRGC-LRMS examination. The gas chromatographic conditions used for the precolumn chromatograph and the HRGC-LRMS analytical chromatography are presented in Table 1. We have found that the combination of the two fused silica capillary columns described in Table 1 is broadly suitable for the determination of many CDDs/CDFs (chlorinated dibenzo-p-dioxins and dibenzofurans) in a variety of environmental sample matrices. The DB-5 precolumn provides reasonable chromatographic durability and is substantially resistant to fouling caused by the injection of dirty extracts. Its maximum allowable operating temperature permits routine temperature programming up to -300 ° C for removal of low volatility solutes without significant loss of general chromatographic efficiency. The Lee SB-Smectic column has been demonstrated to yield an isomer specific separation for 2378-TCDD. However, elution of 2378-TCDD typically requires operation of the column near the maximum allowable temperature for this liquid crystal stationary phase and therefore reduces its applicabilit Table 1. HRGC-HRGC-LRMS instrumental and gas chromatographic operating conditions for the identification
and measurement of 2378-TCDD and 2378-~_,DF analytes. Preparatory HRGC Conditions
Analytical HRGC Conditions
Column: 0.32 mm ID x 30 m DB-5 (dr -- 0.25 prn), J &W Retention Gap: 0.53 mm ID x 1 m DB-5 (dr = 0.25 pro), J &W CarderGa¢" Helium at 2.5 cm'/minute Initial Temperature: 135° C Initial Time: 1.0 minute ProgramRote: 20" C/minute RuM Temteratura: 275 ° C Final Time: 5.0 minute leJecttenSize: 2.0 pL, splitless injection technique Injection Porl Tamlmretura: 250 ° C MonitorDetecter:. ECD at 300° C
Column: 0.20 mm ID x 25 m SB-Smectic (dr = 0.15 pro), Lee Scientific
CryogenicTrappingTemperature: -10" C Analyte RelnlecUon Temperature: 260° C Analyte Trap Window: 9.40 to 10.10 minutes Anelyte DesorMtenT/me: 1.0 minute Precolurml TemlmraturedurlnDDnorption: 270° C PrecolumnBactfflL~h Time: 10.10 minutes TransferUnn Temperetera: 250° C
(Lee Scientific, Salt Lake City, Utah USA)
CarrlerSu: Helium at30 PSI Initial Temlem~m: 170° C Initial T/me: 1.0 minute Initial TamperetumProgramRata: 30° C/minute TomlmmtumLevel?: 220° C Temterehn Level? HuM Time: 0.0 minutes Temmratem Pmgrem Rate 2." 5.0° C/minute
Finl Te~retem: 270 ° C RaM TemWerateraHold Time: 2.4 minutes TotelRua Time: 15.0 minutes GC-MS IMerlace Temperetum: 250° C MS ~u~e Temleratum: 300° C ten MassesMonitored: "rCDD = 320, 322, 324, 334 TCDF = 304,306,308,318 Dwell TimefurMonltemd Al~w$: 200 milliseconds each
6~i
to normal HRGC-LRMS analyses due to fouling of the column by low volatility solutes. Within the perspective of the described HRGC-HRGC-LRMS application, this column deficiency is largely eliminated via the precolumn separation which provides a heart-cut of the residue containing only solutes of similar volatility to that of 2378TCDD. Additionally, the combination of vastly different stationary phase selectivities exhibited by DB-5 and SB-Smectic columns provides a reduction in the potential for analyte interferences caused by solute co-elution from the analytical column. The advent of new liquid phases demonstrating special selectivity for CDDs/CDFs can be immediately employed in this HRGC-HRGC-LRMS system to further increase chromatographic selectivity. As illustrated in Figure 1, the I-IBGCHRGC-LRMS unit is d e s i g n e d to operate in conjunction with an HP7673A Autosampler which provides the capability of processing up to 100 samples in an unattended fashion once the i n s t r u m e n t a l o p e r a t i n g parameters have been established. The operational conditions specified for the determination of 2378-TCDD and 2378-TCDF in Table I lead to an HRGC-HRGC-LRMS system control sequence that is presented in the format of a Gandt chart in Figure 5. In this chart, the t i m e w i n d o w s during which an operation is being performed are highlighted by a solid black bar. The windows that an instrument is in the ready mode, waiting for a signal from another component, are highlighted in grey. During the r e m a i n i n g time, the components are in a standby mode or are equilibrating for the next step in the sequence.
Figure 5. HRGC-HRGC-LRMS system components operating sequence for the determination o f 2378- TCDD and 2378- TUDF per instrumental parameters given in Table 1. See text for description o f events and component idenu'fications.
(min)
Prep GC Cryo valve Cryotrap Trap heat Backflush Foreflush Ovencod
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To determine the operations that are LRMS taking place at a given time in the control sequence, read down the i n= chart along the vertical time lines Ready defined at the top. O p e r a t i n g components will have a black bar in the row to the right o f the name. Components ready to begin an operation, pending the completion of another event, are shown in grey. At the start of the analysis the six main components of the system (Prep Computer = ACI Controller, Autoinjector = HP-7673A, Prep GC = I-IP-5890, Analytical Computer = HP-5987A data system, Analytical GC = HP-5880, and the LRMS) are all in a ready state and are synchronized at the time of the start signal which is coincident with sample injection into the I-IP-5890 gas chromatograph. The Prep Computer, Autoinjector and the Analytical Computer temporarily suspend operation while the Prep GC performs the precolumn separation. At this time the Analytical GC and LRMS function for a short time in a d u m m y run. This run is necessitated by the fact that the start signal from the Autoinjector initiates the HRGC-LRMS analysis and then places the HRGC-LRMS system in a not ready mode. in order for analyte desorption to occur after the precolumn separation is completed, the Prep Computer must receive a ready signal f ~ m all of the external devices attached to the system. We have adopted the d u m m y run as a work-around for this complication. Initiating the dummy run allows the HRGC-LRMS to equilibrate during the time that the precolumn separation is progressing so that at the time of analyte desorption (15.2 minutes per Figure 5), the HRGC-LRMS is in the ready state and can accept the remote start signal to begin the final analytical separation. Analyte desorption at 15.2 minutes run-time is the point of maximum activity in the system. The Prep GC begins the second portion of its analysis sequence by initiating procedures to bacldlush the precolumn, closing
642
the cryogenic valve to discontinue liquid COz flow to the trap, heating the trap, and adjusting the carrier gas flow paths to correspond with those depicted In Figure 3-3. At 16.2 minutes run-time the precolumn is put into the bacldlush mode and low volatility solutes are vented through valve V2 as indicated in Figure 3-4. With analyte desorption, the focus of the operational sequence switches to the Analytical GC (HP-5880) and the LRMS. The analytical separation proceeds for 15 minutes with data acquisition occurring over the last 11 minutes of the window. The analytes, 2378-TCDD and 2378oTCDE typically elute from the analytical column in the region from -11.5 to 12.0 minutes. After completion of the analytical separation, all system components return to their starting conditions and equilibrate prior to beginning analysis of the next sample. Under the chromatographic conditions presented in Table 1, the total analysis time for a sample is 37.2 minutes. HRGC-HRC~-I.RMS INSTRUMENT VALIDATION Prior to actual e m p l o y m e n t of the instrument for the determination of 2378-TCDD and 2378-TCDF in environmental samples, it was necessary to demonstrate the operating characteristics of the system with regard to general reliability. In this section we will describe the results of experiments to measure the reproducibility of analysis, linearity of response, analyte carry-over after m e a s u r e m e n t of c o n c e n t r a t e d solutions, and the sensitivity of the instrument compared to operation in the HBGC-LRMS mode.
Table 2. Results from HRGC-HRGC-LRMS
instrumental reproducibility test for the measurement of 40 pg 2378- TCDD injected. mh322 ~/z 324
n (number of runs) ~o (averageresponse)
10
10
7 784
3 774
To test the r e p r o d u c i b i l i t y of the HRGC-HRGC-LRMS instrument, a series of ten replicate Injections of a 20 PPB o ~ (relatives(andard deviation) 5.5 % 3.8 % (part per billion = pg/pL) reference standard of 2378-TCDD were accomplished over an -8 hour period. The system was operated in the automated mode with injections from the HP-7673A Autosampler. The results from this experiment are shown in Table 2. As indicated therein, the reproducibility of the system over an -8 hour time period is certainly acceptable for most trace analytical applications. There was some evidence of LRMS instrumental drift over the course of the experiment, and therefore it is anticipated that the reproducibility for analysis sets requiring shorter operational periods may demonstrate somewhat improved results. The linearity of 2378-TCDD response versus concentration is plotted in the two graphs in Figure 6. In this experiment a series of four calibration standards of 2378-TCDD and 2378-TCDF were examined over a period of -30 hours. These standards (at concentrations ranging from 2 to 1000 pglpL) were each analyzed in triplicate and the resuits averaged to yield the data in the graphs. The first graph shows the response linearity over the entire range of the test (4 to 2000 pg injected), and as indicated, acceptable linearity was obtained for this concentration range. However, the highest concentration level examined shows a slightly elevated response which in turn causes a slight skewing of the calculated response line away form the true response at the lower concentrations. This deviation is quite small and can be ignored for most typical applications. For analyses in which greater accuracy is required, the second graph in Figure 6 illustrates the response linearity obtainable when the analyte concentration is limited to the range from 2 to 200 pg injected. In this case the response for both TCDD ions monitored shows excellent llnearity and an acceptable value for the y-intercept. It should be noted that the data reported in Figure 6 are for 2378-TCDD, however, they are virtually identical to that obtained for 2378-TCDF with respect to each concentration range examined. The ability to suitably clean the chromatographic system between sample injections, to avoid carry-over from high concentration to following low concentration samples, is of crucial importance to the analyst if he or she is to avoid generating fa/se posit/oe results. We examined this system characteristic by analyzing a concentrated reference~standard of 2378-1L-DD and 2378-TCDF followed by a nonane solvent blank containing neither of the analytes. These tests were done in the manual injection mode. In the initial experiment a 500 PPB solution of [13Clz]-2378-TCDD and P3CI2]-2378-TCDF was injected into the HRGC-HRGC-LRMS system and the analytical run completed. Next a nonane solvent blank was injected and again the analytical run completed. Based upon the LRMS output for the latter run, neither analyte could be detected at a level of 0.1%. The entire experiment was then repeated using a much higher concentration of the analytes; 5000 PPB each. Since this concentration level was greater than the highest concentration reference standard employed during the linearity studies, and
643
Hgure 6. HRGC-HRGC-LRMS de~ctor response dam for 2378-TCDD over two different operating ranges.
Detector Response
ExtendedConcentration Range /
Detector Response
NormalConcentration Range
/
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500 000
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400 000 300000
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Picograms2378-TCDDinjected
200
is therefore outside the limits bounding acceptably linear response for sample analyses, it is an example of the worst case scenario. The nonane solvent blank injection was accomplished after routine cleaning of the syringe, and the analytes carry-over was found to be -0.015% for each species. The syringe washing procedure was repeated and the precolumn GC injector was cleaned by making three consecutive injections of 2.0 gL benzene with the column temperature adjusted to 270 ° C isothermal. Following system reequilibration, the solvent blank analysis was again repeated and no detectable response for either analyte was obtained at a limit of detection in the range of -0.005%. This experiment demonstrates that it is possible for analyte carry-over to cause an apparent false positive response, however, the extent of this problem does not appear to be any greater than that for typical HRGC-LRMS instrumentation (i.e., apparently there is no carry-over that is peculiar to the Deans switch or the transfer line associated with the HRGC-HRGC-LRMS unit). Routine cleaning of both the injection syringe and the GC injector between sample analyses, as is common practice in the author's laboratory, can virtually eliminate common sample cross contamination effects attributable to analyte carry-over. Global instrument sensitivity for HRGC-HRGC-LRMS, as compared to the same detector operating in the HRGC-LRMS mode, is a function of the efficiency of the carrier gas plumbing system related to the Deans switch and also the efficiency at which the particular analyte(s) can be trapped and then subsequently desorbed onto the analytical column. Based upon two experiments that will be described, it was found that there is very little difference in instrumental sensitivity between HRGC-HRGC-LRMS and HRGC-LRMS. The efficiency of trapping and desorption of 2378-TCDD and 2378-TCDF is illustrated by an experiment in which the analytes were kept in the cold trap for an extended period of time prior to desorption. In this case, holding the analytes in the cold trap for 75 minutes resulted in <10% decrease in response when compared to that observed for a comparable standard that was desorbed after only 5.5 minutes in the trap. Of interest here is the fact that both of these tests were conducted using 40 pg of each native analste and 1000 pg of each P3C~=]-labeled internal standard. Such levels are not atypical of those encountered in general environmental analyses. A comparison of instrument sensitivity while operating in HRGC-LRMS and HRGC-HRGC-LRMS modes can be derived from the chromatograms in Figure 7. Although the analyte retention times are different, because the HRGC-LRMS run was accomplished on a DB-5 capillary column operating under conditions which permitted elution of 2378-TCDD in 6.6 minutes and the HRGC-HRGC-LRMS run used the conditions given in Table 1 where an elution time of 11.4 minutes is observed, it is still evident from the perspective of signal to noise ratio
644
FiguR 7. Comparison of instrumental sensitiuity for 2378-TCDD uia HRGC-LRMS and HRGC.HRGCLRMS techniques. Note comparable peak shapes, peak widths and signal to noise ratios for these runs.
100%"HRGC-LRMS
I
l ~_Ab......~40pg2378-TCDD
Ion Intensity "h322
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5.0 100%
7.0
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40pg2378-TCDD
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(minutes)
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-
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-
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(minutes)
that the sensitivities for these measurements are comparable. It is important to mention that this level of instrumental performance depends upon the proper adjustment of gas flows within the Deans switch, however, such adjustments are not complicated to perform and can easily be accomplished in -2 hours when the capillary columns are being installed. Routine readjustment of the initial settings are typically not necessary. We have found that it is even possible to replace a column with one of the same dimensions and not have to make any adjustments in the flow or pressure settings for the Deans switch. CONCLUSIONS An HRGC-HRGC-LRMS instrument that is based entirely upon Hewlett Packard equipment has been developed, assembled and tested. It is capable of automated operation in an unattended mode for up to 100 sample analyses. It is based upon a dual-oven design wherein the preparatory column and the analytical column can be operated independently of one another thereby eliminating compatibility problems. Testing of the system, especially those components of the Deans switch and the cryogenic trap which contact solute species, with trace concentrations of 2378-TCDD and 2378-TCDF has indicated that it is reasonably inert and capable of transmission rates equivalent to those obtained in conventional HRGC-LRMS equipment. Because of these characteristics, the new HRGC-HRGC-LRMS instrument described offers analyte sensitivities that are essentially equivalent to those of typical HRGC-LRMS units for reference standards examined in the absence of matrix constituents. However, considering the negative impact that complicated matrix components can have upon the analytical detection limits for a given analyte, the HRGC-HRGC-LRMS unit is anticipated to possess superior usable sensitivity under typical trace analysis monitoring conditions. This contemplation will be~the subject of future reports concerning the application of HRGC-HRGC-LRMS to real wor/d samples. REFERENCES 1.
L.L. Lamparski and T. J. Nestrick, "AnalyticalMethodology of The Dew Chemical Company for the Determi-
2.
nation of Selected Chlorinated Dlbenzo-p-dioxins and Dibenzofurans in Stack Gas Effluent Matrices. Part I: Full Method for Determination of Tetra- through Octa-Chlorinated Congener Group Total Concentrations and "2378"-Substituted Isomers," Chemo~phere 19, 1165-1177 (1989). T. ]. Nestrick and L. L. Lamparski, 'Pma/ytical Methodology of The Dew Chemical Company for the Determination of Selecwd Chlorinated Dibenzo-p-dioxins and Dibenzofurans in Stack Gas Effluent Matrices. Part ll: Isomer Method for Determination of Tetra- through Octa-Chlorinated Congener Group "2378"-Substituted Isomer Concentrations," Chemosphere 19, 1179-1185 (1989).
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3.
4.
"Method 1613: Tetra- through Octa-chlorinated Dioxins and Furans by Isotope Dilution High Resolution Gas Chromatograph-High Resolution Mass Spectrometry," United States Environmental Protection Agenc~ Office of Water Regulations and Standards, Washington, DC, July 1989. D.R. Deans, Chromatographia 1, 18 (1968).
5. 6.
G. Schomburg, H. HusmanandEWeeke, J.ofChrornatogr. 112, 205-217 (1975). W. Blass, K. Riegner and H. Hulpke, J. of Chromatogr. 172, 67-75 (1975).
7.
Analytical Controls, Inc., Product Bulletin #AC3020, Bensalem, PA (1968).
8.
V.G. vanI-Iese and D. Grant, American Laboratory 20, No. 12, 26-33 (1988).
9.
W.V.LigonandR. J. May, J.ofChromatogr. 294, 77-86 (1984).
10. W.V. Ligon and R. J. May, J. of Chromatogr. 294, 87-98 (1984). 11. S.G. Claude and R. Tabacchi, J. of HR & CC 11, 187-190 (1968). (Received in Germany 14 February 1990; accepted iO April 1990)