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While the same scan functioncan be used for all T4 CDDs, as they do not co-elute,different scan functions must be used in sequence forthe determination of a specific T4 CDD and its co-elutinglabeled isotopomer.A total ion number target of 35 000 counts was setfor the AGC algorithm; with a filament emission currentof 50 µA, the maximum ionization time employed was20 ms.
The supplementary alternating voltages applied tothe end-cap electrodes in dipolar fashion are referredto as waveforms; these waveforms are employed forion isolation, ion excitation, and axial modulation. Apreisolation waveform was imposed during ionization(A) and prolonged after the cessation of ionizationduring period B. The pre-isolation waveform consistedof multiple frequencies covering the range 3.7 – 513.5 kHzwith a 1 kHz notch corresponding to the secular frequencyof the molecular ions to be isolated. For the example ofa T4 CDD scan function shown in Figure 16, the notchis centered at 174.5 kHz in order to isolate both m/z322ab100%257Intensity194315331 341310 320 330 340 350309229285166 176279209297337160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350m/zFigure 17 Mass spectra of T4 CDD: (a) mass spectrum showing the isolated [M]Cž and [M C 2]Cž ions of the molecular ion cluster;(b) product ion mass spectrum obtained by CID of the two isolated ion species shown in part (a).
Note the complete disappearanceof the isolated molecular ions.19QUADRUPOLE ION TRAP MASS SPECTROMETERthe duration of the scan function can be reduced toonly 50 ms; in this case, the number of mass spectralfiles can be increased to five per second, each basedon four microscans. The mass spectra obtained fromthe application of each scan function within an ionpreparation file were merged together to form a singlemerged mass spectrum. The significance of this mergingprocedure is described below.11.3 Tandem Mass Spectrometry Determination ofEluting and Co-eluting CompoundsThe total ion chromatogram shown in Figure 18 wasobtained by GC/MS/MS of 1 µL of solution containing200 pg of each of a T4 CDF, a 13 C12 -T4 CDF, a T4 CDDand a 37 Cl4 -T4 CDD and 100 pg of each of two 13 C12 T4 CDDs.
The peak indicated with an asterisk in Figure 18is an impurity present in the sample. This chromatogramis described more properly as a merged total ionchromatogram since it is a display of the total ion count ofeach merged mass spectrum obtained as described above.Let us consider the first three peaks of interest whichwere observed in this merged total ion chromatogram,that is, those peaks labeled 1, 2 and 3. Peak 1 is dueto a T4 CDF and its co-eluting 13 C12 -labeled isotopomer(a component of the solution injected); peak 2 is duesolely to a 13 C12 -T4 CDD while peak 3 is a compositepeak of a native T4 CDD and its 13 C12 - and 37 Cl4 -labeledisotopomers.For the T4 CDF of molecular weight is 304, themolecular ion MCž of m/z 304 and the [M C 2]Cž ofm/z 306 were isolated simultaneously then dissociatedcollisionally using MFI in accordance with a specificscan function.
The signal intensities of fragment ionsformed by the loss of COClž or CO37 Clž to yield m/z3100%1Total ion signal320 and m/z 322, [M]Cž and [M C 2]Cž , respectively,having qz values of 0.454 and 0.451, respectively. Theamplitude of the pre-isolation waveform was 20 V.0 p/for all scan functions; the function of the pre-isolationwaveform was to eject all ions except those within amass range of ca.
10 Da about the selected ones. Fineisolation was achieved by ramping the rf amplitude untilthe LMCO was just less than m/z 320 at which point ionsof lower mass/charge ratios were ejected; ejection of ionsof higher mass/charge ratio was facilitated by concurrentapplication of axial modulation with an amplitude of3 V.0 p/ . The rf amplitude was modulated moderatelyover a small amplitude range in order to avoid ejection ofthe selected ions.
The ejection of ions with m/z > 322 (C)was achieved by applying a broad band waveform havingan amplitude of 30 V.0 p/ and lasting for 5 ms.Once isolation of the selected ion species m/z 320 andm/z 322 for T4 CDD as illustrated in Figure 17(a) wascompleted, the rf amplitude was reduced to obtain a qzvalue of 0.4 for m/z 322; for T4 CDD, a qz value of 0.4 form/z 322 corresponds to a LMCO value of m/z 142.
Thebandwidth of the waveform employed to carry out CIDusing MFI was composed of 13 frequency componentsspaced at intervals of 0.5 kHz so as to cover a 6 kHz bandof frequencies. The total bandwidth is almost double therequired bandwidth since the Toolkit software locks thecenter of the MFI band to the axial secular frequency ofthe previously selected ion species; only the width of theMFI band can be varied. The MFI waveform amplitudeswere 2.55 V.0 p/ for T4 CDF and 2.45 V.0 p/ for T4 CDD.The voltage amplitude of each component of the MFIwaveform is equal to the amplitude of the waveformdivided by the number of frequency components. Therf amplitude was modulated so as to cover completelythe frequency range of the 13 frequency componentsof MFI.Following CID by MFI for 10 ms, the analytical massrange of 165 – 350 Da was scanned for product ions fromT4 CDF and T4 CDD so as to monitor all major productions save that resulting from chlorine atom loss.
A massspectrum of the product ions from dioxin is shown inFigure 17(b): m/z 257 and m/z 194 are due to the lossesof COClž or 2COClž , respectively, from MCž (m/z 320).The analytical ramp was scanned at 5555 Da s 1 ; axialmodulation was carried out with an amplitude of 3 V.0 p/and at a frequency of 485 kHz. The electron multiplierwas biased at a voltage of ca. 1800 V to provide an ionsignal gain of 105 .Each scan function used had a duration of ca. 125 mssuch that each acquisition point or mass spectral filewas generated from four microscans; thus two massspectra were accumulated each second.
It should be notedthat when a small mass range only is monitored duringthe analytical rf ramp, say for two fragment ions only,223.2724.10*24.9425.7726.60Time (min)Figure 18 Merged total ion chromatogram of six dioxins andfurans and their labeled isotopomers. Each point expresses thesum of all of the relevant fragment ion peaks.20MASS SPECTROMETRYIntensity of selected ions241+24314689252+254268+270165822,3,7,8-T4CDF13C12-,2,3,7,8-T4CDF13C12-1,2,3,4-T4CDD6418886313257+259158872632299823.2724.10C12-2,3,7,8-T 4CDD2,3,7,8-T4CDD37Cl4- 2,3,7,8-T4CDD24.9425.7726.60Time (min)Figure 19 Selected ion chromatogram showing that the signal area of each of six compounds can be determined using GC/MS/MS.241 and m/z 243 were recorded, summed and shown inFigure 19. For MS/MS of the co-eluting 13 C12 -T4 CDF, thesecond scan function comes into play for the ionizationand simultaneous isolation of both MCž of m/z 316 and[M C 2]Cž of m/z 318; the isolated ion species were thensubjected to CID with MFI.
The signal intensities offragment ions formed by the loss of COClž or CO37 Clžto yield m/z 252 and m/z 254 were recorded and summedas shown in Figure 19. Throughout the period when thenative and labeled T4 CDFs are co-eluting, the first twoscan functions are used alternately.Now let us consider peak 2 of Figure 18. A third scanfunction is required for the MS/MS determination of thelabeled 13 C12 -T4 CDD to control the ionization, isolation,and CID of both the molecular ion MCž of m/z 332 and the[M C 2]Cž of m/z 334.
The signal intensities of fragmentions formed by the loss of COClž or CO37 Clž to yield m/z268 and m/z 270 were summed and recorded in Figure 19.Peak 3 of Figure 18 is composed of the fragment ioncounts from three compounds. The scan function usedin peak 2 was used again for the MS/MS determinationof a T4 CDD together with two additional scan functionsfor the co-eluting 13 C12 - and 37 Cl4 -T4 CDD isotopomers.For T4 CDD of molecular weight 320, the molecularions MCž , of m/z 320, and [M C 2]Cž , of m/z 322, wereisolated simultaneously then were dissociated as before;the resulting signal intensities of the fragment ions of m/z257 and m/z 259, as shown in Figure 17, were summedand recorded in Figure 19. From the MS/MS of the13C12 -T4 CDD, the signal intensities of the fragment ionsof m/z 268 and m/z 270 were summed and recorded inFigure 19.
For the MS/MS of 37 Cl4 -T4 CDD of molecularweight 328, the molecular ion MCž of m/z 328 was isolatedthen dissociated as before and the signal intensity of thefragment ions arising from the loss of CO37 Clž to yieldm/z 263 was recorded in Figure 19.The signal areas of each of the six separated compoundsare shown in Figure 19 as selected ion chromatograms;the signal intensities of fragment ions due to loss of COClžor CO37 Clž are plotted here.
In order to perform MS/MSon the total of six different tetrachlorinated dioxins andfurans which constituted the first three peaks of Figure 18,a total of five different scan functions was required. Inthis example, the enormous power of MS/MS is evidentin the determination of three co-eluting compounds.In the above discussion, only a single fragmentationchannel was considered, that of the loss of COClž orCO37 Clž . While COClž loss is the major fragmentationchannel, it is not the sole loss channel.
For the dioxins,minor losses of Clž and 2COClž are observed while forthe furans, minor losses of Clž , COCl2 and COClž3 areobserved. The duration of the analytical rf ramp wasreduced by selecting a small mass range that excluded thefragment ions of low intensity.11.4 Tandem Mass Spectrometric OperationThe above sequence of ion isolation and CID can berepeated many times in a process known as (MS)n . Whilethis process is not illustrated here, multiple stages of massselective operation are used frequently for ion structuredetermination where the quadrupole ion trap is used21QUADRUPOLE ION TRAP MASS SPECTROMETERin combination with external ionization sources, such asESI.
Here, multiply charged molecules having massesof thousands of daltons can be stored in an ion trapand subjected to (MS)n so as to follow the stepwise iondissociation for the elucidation of ion structure.ClClClClCongener 77ClClClCl12 CHEMICAL IONIZATION ANDION/MOLECULE REACTIONSClCongener 110Scheme 1 PCB congeners 77(3,30 ,4,40 ,-tetrachlorobiphenyl)and 110(2,3,30 ,40 ,6-pentachlorobiphenyl).In the quadrupole ion trap, several types of reactionscan and do occur simultaneously and spontaneouslyonce EI of a compound has occurred. Ion/moleculereactions involving charge transfer, proton transfer, andclustering occur sequentially in a type of thermodynamic‘‘waterfall’’ which results in the formation of stable evenelectron ions of greater mass/charge ratio than that of themolecular ion.
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