_j

Pesticide Analysis Using HPLC WithPDA/MS Detection

Joseph P.Romano,MatthewJ. Dilts, andYakov I. MakarovskyWatersCorporation34 Maple St., Milford,MA 01757, U.S.A.

Title of the presentationand authors.A new analytical method has been developed for thepreconcentration and analysis of pesticides using HPLCwith PDA/MS detection.

q• r_ • •

Objectives

P-Perform trace analysis of commonpesticides found in drinking water usingHPLC

• Utilize solid phase extraction (SPE) forsample preconcentration

• Demonstrate simultaneous PDA/MSdetection

The objectives of this work were:Perform trace HPLC analysis of seven common pesticidesfound in drinking water.Utilize solid phase extraction off-line for samplepreconcentration.And demonstrate simultaneous Photodiode Array(PDA)/Mass Spectrometer (MS) detection employing theWaters Integrity System.

• °

HPLC Analysis of PesticidesSystem: Waters DiscoverySystemColumn: Waters Nova-PakC18 150 x 2.0ramFluentA: 100raMAmmoniumAcetatepH 6.9

5 Buent B: 100% AcetonitrileLinearGradient: 95%A/5% B to 50% B, 15 minutes

Hold5 minutesthen to 100% B,4 22 minutes,hold8 minutes,back to

inilialcondilJons,5 minutes- Rowrate 0.35 ml/min

DeteclJon: PDAMaxPIot Display(200 nmto 450 nm)6 Amount: 0.5 ug on column

1.0o 2 3 7AU

1. 2,4-D2. Warfarin

_ 3. Dinoseb4. Atrazine

s. Carbaryt6. Diuro_7. Linuron

16 18 20 22 24 26 28Mnutes

Seven common pesticideswere selected for optimizationof the HPLC method. The chromatographic separation wasfirst developed using the Waters Discovery System.The optimized chromatographic conditions utilized thenarrow bore Waters Nova-Pak C18 column to reducesolvent consumption. A mobile phase consisting of anammonium acetate to acetonitrile binary linear gradient,created the best separation of the pesticides in less than 26minutes. In order to achieve the best sensitivity, the Waters996 PDA MaxPIot function was incorporated.

q,

Extracted Wavelengths2D Chromatograms at a specified wavelength

for Calibration and Quantitation

Absorbance!i@ 220nm

1AU

\!:

: i Absorbancei _ @ 254 nm

.... _.................. ....... • ...... ......... ..............

__ Tim Tim

The advanced capabilities of the Waters 996 PDA allow theuser to extract chromatograms at specified wavelengths inorder to obtain the best calibration and quantitationinformation.

Solid Phase Extraction

Recovery Results__.-;_:_.._:!_':_._._: _._.: _ _i_ ii;._;;i;_!_:_:_._;_._._::..';_!_@:_.::.i._.:_.!.i.::.i_:.:_:_:_:i:i:i:_:_:_:_:_:_:::::::_::_:_ii!:_ _:_::_._ _._._._._iiiiiiii_i_i!iii:_iii;iii;_iii_i_!_ii_i::_i_iiii!_ii_i_i_ii_iii;iiiii;_;;i_iiiiiiiii;iii;i_iiiiiiiiii;i_iii_!i;_!_ii;_!;ii;_;;;_;_i_!::;::i::!::!::i::_::i::i::i::ii;::iiiii_iii!iiiiiii

::.::-._:: :.":__:::i:_:_:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: :: :::::::: ::::::::: :::::: :_:!:!:: ::: :i:

_::::::::::_::::.'::-:.::::::::-':.::._:.-:::::._.'::_-'__::_.-::!_i._:::::_.':.:::_.:::3.'__.'_:!:_::_ :._':::_-._"_._:_':._'_:.-:.-:.-:.-::::-":-.':_._::::::::::::_::: :: ::::."::_ :._.':::.':_:::.':::::::."..':::.'::-:::::::::::¢-::::::::: :._::::::: :::::::::: :::::::::::::::: :::::

$&--_-._:_--+__45_:_.'_._.:$ $:__. $_'_._ _._::__'-':':;'::_:'__:_:::.__::."_::_::__$_:':__::__. :.-:.__:.:-_-:-_-__:::::::::::::::::::::::::::::::::::::::::::::::::':i:.;::::-:::::i:!:;:!:!:!::!::::;:!:!:.:!:

_:_: .............. ._:_'._._:_::_::::::::::_'_'_::_:_:_':_:... _:: _4_-;._:_"_:_::::_'.:-_:$::._:" . :::::::::::::::::::::::::::::::::::::::: ::::::.- :::::::::::::::::::::

_j .._$_....:._._._ ._._._: _ .._:._, _._'_:._;._,:.,_:.:._.=....,_:.._.._:.._,.._._,_,:_,,×+_..... .:_..×..:.:...:+:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:...................

:-;:_.t__ &._._._:_:__!::$!:_:_._i_ii_ i_ _:_:_i_._:_:_$i:i:i:i:¢i:i&_:_$:::::::::::::::::::::::::::::¢:::::::::::._..:_: ::::::::::::_:;:_:_:-_-:+.:_ ._':_.':':_&_ .:'-:'___. _'.:..'.:-.'..'-:-:_-:-:-_.':-':.:_:::_',_:_ _.:-:::-::::::::.':::.':_':"::_:_:::-.-.:..'-.':x'::::__.--::.'-:::::_:.:::::::::::'.-'::::::-:.':::::::::::::::::::::::.':::::::::::._::-:-:-:+:-:-:-:-:-:-:o._:._:-:-:-:-:-:-:-:-:-:-:::::-::::::::::::::::::::::::::::::::::::::::::::::::::

====================================================================_:,:::'.:::::::::::::::::.:::::::::.-::::_'_::::::::::::::::::::: :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: ::::::::::::;::::+_:.::::::::.:.:.:_.:.:._:.:¢.:.:+:.:..._,:_+:+:.:,..:.:.:.:.:_:.:.:.×_,:...._............ :;.:::.:_:. ::._..:.. . , ........... :......: .................... :::::::::::::::::::::::::::::::::::::::::::::::::::::: ::::::::::::::::::::. : . ::$:: ================================:: ;;::::: ::::::::::::::: ::: :.-.:+-.....:::--.--:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: ........ .......:.:....:.:.:+:+:+:.:.:.:.:.:.::

:::::_:_:::_:'-"i'_i:::::::::::::::::::::::::::::::::::::::::::::::::: :_:_¢:: ::_:::::: : :_::::::::::::::: :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::__:::_:::_:::_:_:_::::_:::::::::::::::_:_::::::::_::::::_::::_:::::::::::::::::::::::::::::::::::::::::::::::_::::::_::::_:::::_:::::_::::::::::::::::::::::::::::::_:::::::::::::::::::::::::::::::::::::::::::::::_:::::::::::::::::::::::::_:::_:::::::::::_:_:_:_:::_:_:_:_:_:_:_:_:_:_:_::;:::_::_:_::_::_:_::_:_::: ::

_;!;_N_!i;_;;!_;;i!;!;_i;_i_i;i_iiiiii_iiiiiii;ii;i_iii;;ii;;_!_;_;_;_N_;i_;i;_;!_i_ii_;_i_;_;_ii;i_i_ii_i_;;i;;_;_6_i_iii;_;_ii_!_!_;_;i!::_::_i::i::iii_i::_::_::_::9

The numberof replicates= 2EstimatedMDL< 0.1 ppbwithS/N=3Preconcentrationfactorof 1000X and 100 uL injection

An optimization study of the solid phase extraction (SPE) procedure for the7 pesticides produced the following recovery results. An economical wayto "batch" process many samples off-line is by using Waters Sep-Pakcartridges with the Sep-Pak cartridge vacuum manifold. Up to 24 samplescan be prepared simultaneously in this fashion. A variety of Sep-Pakcartridge chemistries are available for different applications. In this case,the tC18 Environmental Plus Sep-Pak cartridge was used for traceenrichment. Using 1 liter sample volumes a concentration factor of 1000Xwas obtained. Spiked samples containing 1,5, and 50 ug/I (ppb)respectively were concentrated and analyzed. A method detection limit(MDL) was estimated to be less than 0.1 ppb using the 1 ppb standard, a100 ul injection, and a signal to noise ratio of 3.

Pesticide Analysis by HPLCwith PDA/MS Detection

The compounds chosen for this study represent a variety ofpesticides used for specific actions and belong tocharacteristic families of compounds.The individual pesticides also possess unique electronimpact ionization spectra with primary and secondary ionsthat create a "finger print" for positive compoundidentification. In fact the EPA recommends monitoring boththe primary and secondary ions when using the MSdetector in Selected Ion Mode (SIM) for confirmation ofpositive ID.

,°

Mass Spectrum Index Plot300.00 2.84 9.643°_'-''-" 16.69 17.16 18.67 23.83 3OO.00

_ 250.0025O.00

20021___... 200218"._z200o0 ,,z_ 1_ ,,_.: ,oooon_1 1 14144 150.00

150.00 135_. 12_ /_'_--_' 11_1___.g_ g_ 100.00

O.2O 0.2O

0.15 0.15

intensity 0.10 0.10 Intensity

o_ LL oo_0.00 0.00

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00Mknutes

In keeping with the lookand feel as the 996 PDA detector,the mass spectrometer utilizes the same Millenniumtechnology to fully automate the data collection and resultsmanagement process, and provides complete control of allsystem operations from a single keyboard. Shown here is amass spectrum index plot of the pesticides. The massspectrum, over the preselected AMU range, is displayed foreach chromatographic peak.

° I

Pesticide Conversion for LC/MSo.55 System: Waters lntegdtySystem s 1. 2,4-D

Column: WatersNova-PakC18 15x?_Omm 2. Warlarino.50 Buem A: 10raMAmmoniumAcetate 3. Dinoseb

Buent B: 100%Acetonil'Ue 4. Atrazine

o.45. Unear Gradient: 85_',JV15%B,hold2 minutes, 5. Carbarytthen to 35% 17minutes, 6. Diuron0.40.hold3 minutesIhen to 10(WoB, 7. Linuron

0,_5. 22 minutes,hold3 Minutes,back 7to initialcondiUons,3 minutes

0.,t0. Row mrs 0.35 mPminAU Detec_on: Wsmrs996 PDA

0_s. MaxPIot Display 4(210 nmto 310 rim)

020. Amount: 300 ng on column 6

0.15. l 1

0.10. 21 3

o._, j . I__0.00 2:00 4:00 6:00 8:00 I0.00 I_-00 li.00 16.00 18.00 20.00 22.00 24.00

Mlnutes

The Waters Integrity System is the first HPLC to combinemass spectral and photodiode detection in a practicalbench top configuration providing positive compoundidentification and quantitation from a single injection. Amethod conversion for the analysis of these pesticides wasdeveloped on the Integrity System taking into considerationsolvent compatibility, narrow bore analytical columns, andlow flow rates. These conditions must be addressed inorder to achieve compatibility with a MS detector. Anexample of the chromatographic separation generated by

O the PDA detector using optimized conditions is shown here.

°

°

Total Ion Current Chromatogram0.8O

Detection: WatersThennaBeam 1. 2,4-DMassde_ctor 2. Warfarin

0.7o Scan: MaxRot 140 m/zto 320m/z 3. Atrazine

Smoothed: by 31 pointSavitsky-Golay 4. Carbaryl[] Source: 220 degreesC 5. Diuron

o.eo 6. Linuron5

0.50.

Intensity0.40'

0.30

3

o.ooJ,',-,'--_,. ---___..... _ .......... :-- __ ................o.oo zoo 4.00 6:oo e:oo 16.oo__oo_i.oo 16.oo_e.oo2ooo __oo2Loo

Mnutes

The total ion current produced by the Waters ThermaBeamMass detector in scan mode is displayed as achromatogram. Note that there is no loss of resolution orchange in peak shape of the pesticides.(Dinoseb was not detected)

Mass

Spectrum ofAtrazine

The mass spectral profile of each chromatographic peaksuch as atrazine, can be reviewed and stored in a libraryautomatically by Millennium software

Pesticide Analysis by HPLCwith PDA/MS Detection

ProtocolSpike 500 ng each into 500 mL (1 ppb)

-ISolid Phase Extraction 500 mL to 1 mL (500 ppb)

HPLC with 10 uL injection (5 ng on column)

In order to lower the detection limits for the pesticides usingPDA/MS detection, sample preconcentration using SPEwas performed. The sample prep protocol consisted ofspiking 500 mL of tapwater with 500 ng each of 2,4-D,warfarin, atrazine, carbaryl, and diuron. The 500 mL samplewas passed through a tC18 Environmental Plus Sep-Pakcartridge, then eluted with 5 mL of CH3CN/MEOH 70/30.The extract was blown down to 1 mL with nitrogen. Finally,10 uL was injected into the Integrity System.

_t_drllm r_eview _:_

__..__=,_.o,o_._,=_r._..,_ Positive

CompoundIdentification

UsingWiley

Library___._, __:_;_,_-.'-.'-:¢-.'___ _%_:_._'._.._:..'..____.__.:_ ___ _ _ __

__ _]_ti_._lE:_ _ _ _._:._.'..-,$.':.:.:.::.i_.:_z._i

Mass Spectral Malch Ruulls_PBMJ PerCent [ Spectrum lUbraryIMolecularl _ . _1

F, Icontami, I Name IName I weiaht I _ormua _111 gl I 10 ]1 3_5-Trlm_n_-Ta-dlamlnR6-JWtLEY_ :215 ICgH14CLN_I.... : ._- _:.; .-, . . . ,,,_._:..:.>. . _ _ - :-.:-_...:.:._:+. _0_'_._:::_• ..

i .11

The particle beam MS uses library-searchable electronimpact ionization spectra for compounds up to 1000 MWwith resolving power of 1 AMU. Using the integratedPalisade Probability-based matching (PBM) searchalgorithm, spectra are automatically searched against eitheran operator's own library of spectra or the optional WileyRegistry of mass spectral data. The Wiley registry isapproved for use by laboratories in the U.S. under theEnvironmental Protection Agency (EPA) ContractLaboratory Program (CLP). This library matching routineconfirmed the identification of atrazine.

Tapwater Spiked @ 1 ppb0.014 4

System: WatersIntegritySystem 1. 2,4-DColumn: WatersNova-Pak C18 15x2.0mm 2. Warfarin

o.o12 FluentA: 10raMAmmoniumAcetate 3. AtrazineFluentB: 100% Acetonitdle 4. CarbarylUnear Gradient:85%A/15% B, hold2 minutes, 5. Diuron

then to 35% 17minutes,o.olo hold3 minutes then to 100% B,

22 minutes,hold3 Minutes,back

o.006 to initialconditions,3 minutesRowrate 0.35 ml/min

AU Detection: Waters996 PDAMaxPIotDisplay 3

0.006 1 (210 nm to 310 nm)SamplePrep: SPE preconcent_'ation500X 5

0.004 2

0.002

0.000 '_

0.00 2.00 4.00 6.00 8.00 10.00 1_00 14.00 16.00 18.00 20.00 22.00Minutes

A chromatogram of the trace enriched tapwater samplespiked at 1 ppb is displayed here. The chromatographicseparation generated by the 996 PDA shows excellentsensitivity for the pesticides.

PDA Spectrum Index Plot of Tapwater340.00 340.00

320.00 320.00

300.00 300.00

nm 280.00 280.00 nm

260.00 _ 260.00

240.00 _._ 240,00

220.0O 220.00

0.006 , _ 0.0060.004 0,004

AU AU

0.002 ! 0.002

0.000 0.000

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00

Minutes

Using Millennium, the spectrum index plot of the tapwatersample generated from the 996 PDA detector is displayed.The spectral profile for each chromatographic peak can beautomatically compared to spectra stored in its library.

i

2,4-D Spectrum Index 3D Plot

Another way to look for trace impurities is by using thethree dimensional plot.

PDA Purity I Malch Results _

File Edil _tions Help-_ur,_...,,.. WarfarinRetention Time: 10.500 Minutes Peak: 2 of 5

_..u°..:,o._,,,o,o.,. Purity PlotPurity Angle: 1.611 Purity Threshold: 91.000

Peak Purl_ Passes Table

Going to the next peak, identified as peak 2 in the tapwatersample, its retention time corresponds to that of warfarinfrom the standard injection. First, a purity plot routine wasperformed. Again it passed the peak purity test.

MS Detection of Warfarin Using

o._,Selected Ion Model (SlM)0.0o8 Sample Concentration 1 ppb Tapwater I

PreconcentTatJon Factor:, 500X IB

Primary Ion: 265 ||0.007

0.006

Intensity

0.0O5

0.004

0.003

0.002 265

0.001

4.00 5.00 6.00 7_00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

Minutes

Positivecompound identificationwas confirmed by the MSdetection of warfarin in the tapwater at 1 ppb. UsingSelected Ion Mode and monitoring the primary andsecondary ion at 265 and 308 AMU respectively, excellentsignal was achieved confirming the signature "finger print"of warfarin and its presence in the sample.

PDA Identification of Wafarin

_ _._.._, ._._..._. ._ ..... ., " 1.. _ _._..... .. _ _._._._ _" _::_%_:_::_:_::_:_:_$_.:_:::_:_::::_:i_:_:::_:_:_:_:_:i:____ ::::::::::::::::::::::::::::_.:,.,._:; ._i!i!;ii!!i!!!ii)!)ili!!_!;!!)::i::)::i::i::!!i::!::;::;!)RBAMaldt I_esult=

MatchI Match ISpec'eum)LibraryIMatchl_ .I VWtnIMetchl I II !_e341911000lWarfarinIPesticides[- IYe_10_o0O011....... i 1 [

..... _ _._._. _:; . . . :-:..._._ ._ .... !$:-$!_._.....-_.,-.-.-_._-.:.:..-.:.:_.:.:.:.:.:.:

Comparing the spectrum to the storedpesticides libraryresults in a positive match for warfarin.

PDA Identification of Atrazine

___:""""""'_'_"_ """"_."_ P._._-;';';;;;:$_ :5:;;;$:.__; _;$"":_.;$::•".. _ _ ._.._._.;.:.:.:______$-;_"":.%<:_._._._;.';.-_........ _._-._y_:<:<-:-;:.'::;;;;:;._ -"_ .... _ .. :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::_... -: ........ .:.........__L=_,_..._..._.:-:-:-:-:-:-:-:-:-:-:-:-:+:-:-:-:.:.:........................................_.tr-_==___<;i_'i_:-_ __._ _;5.-;:;.-_;._::<'._¥_-_.il_-_._ _. _.'::'-_:':':':':':::::::::::._._._:_.::::::::::::::_:::::::::5::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: " _$] " ._" - !_ _-_:_1_._..._.:] : " =======================================================================================================:.__'_,:_-_._,_ .....................................................................................

PI_ Matc_t RLmBulI_

|Matchl Match Specbum Library Match i 'dWtn Match

[7,7o31 9!_ooo[_r-_ne IPestl¢lclesJ-]Yes|0_0oo0|- -[- J J /

Similar routineswere performed on peak 3 identified in thetapwater. PDA information identified the peak as atrazineusing the library matching function.

MS Detection of Atrazine Usingooo,,Selected Ion Mode (SIM)0.0070

2OO

O.0065 Sample ConcentTation 1 ppb TapwaterPmconcentration Factor: 500X

o.o06o Pdmary Ion: 200Secondary Ion: 215

0.0055Intec_sity

0.0050

0.0045 :,

0.0040

0.0035 "": :::"_:::..... : ..

/ ......:..:....:.................:i.:.:. _ ....0.0030 r

10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00

_n_es

Positive compound identification of atrazine was confirmedby MS detection. Note that up to 31 channels can beprogrammed and monitored simultaneously when usingSelected Ion Mode.

PDA Identification of Carbaryl

i:....................... : " _"_¢.... :'.::::i....... _ .<" "i'" _; _ _ _$_:_`_<i:::_<i:_:_<::_<_:::::::i:i;:i:::i:i:::.:_i:i:;:::::::i:i:i:::i:i:i:i:i:i:i:::iiiiiiiiii:i:i:i:i:i:i:i:i:i:i:!:_:i:!:_:_:_:

POA Match RBsults

I Match Match Spect_lm Ubra_f Match _ .I YWIn IMatch I I I IIoeal_o,,I_r--.o'd/Name [ Name I Fiac I/ I_M_I_-o_1 I I I 1

Peak 4 in the tapwater sample was also identified by PDAdetection and library matching as carbaryl.

F

0o,,MS Detection of Carbaryl Using

oo,° Selected Ion Mode (SIM)0.015

0.014 _

0.013

0.012 Sample Concentration 1 ppb Tapwater

Intensity Preco ncentralion Factor: SOOX II0.011 Primary Ion: 144 J J

O.OLO0.009

0.008

0.007 1440.006

12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00Minutes

MS detection of carbaryl using SIM mode provides positivecompound identification and confirmation of the presence ofcarbaryl at 1 ppb in the tapwater sample.

Conclusion

Successfultrace analysisof pesticidesusing Waters IntegritySystem

_-Sample preconcentrationusing tC18Sep-Pak cartridgesprovidesexcellentrecoveriesof commonpesticides

_- High resolutionPDAdetectionwithThermaBeam Massdetectioncombinesthe best in accuratecompoundquantitationwith the powerful identificationof compounds in one integratedsystem

Successful trace analysis of pesticides can be achievedusing the Waters Integrity System. Solid phase extractioncan provide excellent sensitivity and recoveries for commonpesticides. The combination of PDA detection with MSdetection provides conformational information for thequantitation and identification of unknown compounds.

PDAPurity/Match Results __File Edit _ptions Help

_""_"°" 2 4-D Purity PlotRetention ]]me: 4.133 Minutes Peak: 1 of 5 _1Peak Umits: 4.000 to 4.317 min

PurftyAngle: 7.425 PurityThreshold:91.000

PurityPNm TaMe

Millennium software contains a special peak purityalgorithm then uses vector analysis to mathematicallyconsolidate all these comparisons and determine peakpurity. Here the 2,4-D purity plot passes the peak puritytest.

PDA Identification of 2,4-D

ii__i___.._._..._._i__i_i_iiiiiiiiiii;i-.'...--_-.w--._-,-.--:: _.--_':._'_::-_-_::__::_;_:_;_;_'_:_:`;_::_:_:_._:_:_:_:_::::_:_:_:::K_::::_._:_:::_::::::_:_:_:_::_:_:_::_:::_::_::_:i.:._:i:::_:'::::::::':`:::!::i:i:::::::i:_:_::::!:!:!::_::i:i:_:_:i:!:!:i::i:i

,. PI)A Ma_ch Resu=s _i

After peak purity is established, a similar routine comparesthe peak spectrum to spectra in a library to locate a matchfor identification. In this case the spectrum taken from thepeak located on the shoulder of the spiked tapwater sampleis compared the UV spectra stored in a library calledpesticides. Note that a good match was found for 2,4-D.Also note that in the upper portion of the slide the twocorresponding spectra overlay perfectly confirmingpresence of 2,4-D in the tapwater.

PDA Spectrum Index Plot340.00 340.00

320.00 320.00

300.00 i 300.00

nm 280.00 280.00 nm

240.00 S 240.00

220.00 _ _,_ 220.000.20 0.20

0.15 j t 0.15

AU 0.10 0.10 AU

0.05 0.05

_" "[ oooo,oo:! J ' " "]'L" "

2.00 4.00 6.1111 8.1111 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

Minutes

The spectrum index plot is another way the 996 PDAdetector can display data. Here the UV spectrum, over thepreselected wavelength range, is displayed for eachchromatographic peak representing the individualpesticides.The spectral profile of each peak can beautomatically compared, by Millennium Software, to spectrastored in its library.

v

PDA Spectrum Index Plot of 2,4-D340.00 "_ - 340.00

320.00 _ 320.00300.00 _ 300.00

nm 280.00 _ 280.00 nm

260.00 _ 260.00240.00 240.00

220.00 _ 220.00

0.0060 0.0060

0.0050 _ _ 0.0050

AU 0.0040 0.0040 AU

0.0030 _ 0.0030

0.0020 00020

3.60 3.80 4.00 4.20 4,40 4.60 4.80

Mnutes

Taking a closer look at 2,4-D located on the shoulder of thesample matrix interference reveals a resolved peak.Located in the upper portion of the slide is the UV spectrumtaken at the peak apex of the chromatographic peak.Millennium compares the spectrum at the peak apex to allother spectra at each point along the peak.