Environmental Solutions with HPLC and Related … Environmental Solutions Guide...General...

General Description

Through our own laboratories and in collaboration with exter-nal sources, Agilent Technologies has developed a largenumber of HPLC, LC/MS, GC, GC/MS, CE, and ICP-MS applica-tions for the environmental market. This guide gives a con-densed overview of the application solutions for this market,with particular emphasis on LC, LC/MS, and selected LC-ICP-MSapplications. Liquid phase and SPE sample preparation tech-niques used prior to some GC or GC/MS analyses are alsoincluded.

This guide illustrates the varied solutions by showing high-lights of the extensive Agilent application collection. Thisguide provides information on:

• Various analyte classes and matrices

• Chromatographic conditions

• Reference chromatograms

• Spectral conditions (where applicable)

• Spectral data (where applicable)

• Instrument and column parameters

For more detailed information about the systems or condi-tions, refer to the configuration table at the end of this guideor to the cited application. Applications that are not includedin the abstract section can be found in the application index,which references more than 100 additional Agilent applicationsfor environmental analyses.

Please note that the described instruments are recommendations only. Please contact your local Agilentsupplier to refine a solution based on your specific method and performance requirements.

Environmental Solutions with HPLC andRelated Technologies

Solutions Guide

May 2007

2

Table of Contents

Applications Overview 3Agilent Hardware and ChemStation Software 5Non-Agilent Hardware, Consumables, and Documentation 5

Other Resources 5

Featured Applications 6System ConfigurationsRegulated/Hazardous Drug Substances 60Regulated/ Hazardous Miscellaneous Substances 60Regulated/Hazardous Natural Toxin Substances 60Regulated/Hazardous Pesticide/Herbicide Substances 61Sample Preparation Techniques 61

Quick Reference GuideRegulated/Hazardous Drug Substances 62Regulated/ Hazardous Miscellaneous Substances 62Regulated/Hazardous Natural Toxin Substances 62Regulated/Hazardous Pesticide/Herbicide Substances 63Sample Preparation Techniques 63

Basic Principles of Liquid ChromatographyBasic Principles of Liquid Chromatography 64Instrumentation for HPLC 64HPLC Separation Modes 65Column Dimensions and Materials 66Concepts of the Rapid Resolution Systems and Methods 67Method Translation 68Sample Preparation Techniques 70

Application Reference IndexRegulated/Hazardous Drug Substances 72Regulated/ Hazardous Miscellaneous Substances 73Regulated/Hazardous Natural Toxin Substances 77Regulated/Hazardous Pesticide/Herbicide Substances 77Sample Preparation Techniques 82Cosmetics, Personal Care Products 82Dyes, Colorants, Pigments 82Fine Chemicals 82Forensics 83Industrial Miscellaneous 83Mixed Publications 83Packaging 84Petrochemicals, Crude Oil 84Polymer Additives 84Polymers, Organic Soluble 84Special Techniques 84

3

Applications Overview

Much like food and industrial samples, the diver-sity of organic molecules encountered in environ-mental and food safety settings is substantial andrequires careful thought prior to executing a sepa-ration scheme. With knowledge of the analyte andmatrix properties and a general search of availableliterature, it is possible to design a separation planthat has a high probability of success.

Solubility is one of our most important parametersin liquid chromatography, regardless of the separa-tion mechanism. It is tied to molecular weight,carbon content, and the number and type of func-tional groups (polar elements such as oxygen,nitrogen, sulfur, and phosphorous that are presentin polar or ionizable functionalities). Solubilitiesrange from purely hydrocarbon solvents to bufferedaqueous solutions. The structural differencesbetween the analytes can be organized in the fol-lowing categories:

• Small molecules soluble in • Organic or water-miscible organics, differing

by solubility due to organic carbon contentand the absence, presence, and type of polarand/or ionizable functional groups

• Organic or water-miscible organics, differingprimarily by ionizable functional groups variably present on all analytes

• Nonpolar to moderately polar organics, differing primarily by organic carbon contentand absence, presence, and type of polar functional groups

• Large molecules (homogeneous or heterogeneouspolymers) soluble in• Organic or water-miscible organics, differing

by size due to molecular weight and/or structural conformation

• Organic solvents exclusively, differing by sizedue to molecular weight and/or structuralconformation

With macromolecules we need to answer one spe-cific question: Are we trying to fully resolve eachmolecule in the mixture or do we simply wish tohave an answer that expresses the general or statis-tical molecular weight distribution?

Full resolution of macromolecules in a mixture islimited by several parameters, including the hetero-geneity among the available species and the degreeof similarity of the monomeric building blocks. If

the building blocks are largely identical, as is truefor homopolymers and copolymers, it is unlikelythat a complete separation can be achieved by anymeans with more than 50 or so monomeric units.The difference from one analyte to the next simplybecomes too small to be distinguished.

If the sample is significantly heterogeneous, asmight be said of peptides or small proteins, then itis quite possible to distinguish two analytes evenwhen they have the identical molecular weight andidentical molecular size. The separation mode,however, would not be based on size. It would bebased on any mechanism that can distinguish theanalytes on the basis of chemical properties of thesubunits.

With a good definition of solubility, functionalgroups, and molecular weight we can make goodchoices about the mode of separation that is leastlikely to fail. We want to choose a mechanism thatcan retain and elute all the species of interest, becompatible with the dilution or dissolution sol-vents, and can distinguish the analytes by how theyare different. Because considerable overlap existsin the interpretation of these data, it is alwaysuseful to survey the literature for the specific ana-lytes or for analyte classes that have reasonablesimilarities to the analytes in question.

Four major classes of separation are available:

• Molecular weight (actually molecular size in solu-tion) separation

• Ion exchange separation, for which all analytesmust possess an ionizable group

• Normal phase separation, which is predomi-nantly used for small molecules differing in theirpolar functional groups and soluble in nonpolarto moderately polar solvents

• Reversed phase separation, which separates bysolubility or partition coefficients with water orpH-modified water in conjunction with water-miscible solvents

If the analytes have ionizable functional groups,this behavior will need to be controlled, preferablyby ion suppression, with buffered or pH-modifiedmobile phases that ensure that the ionizable groupsdo not cause the analytes to have poor retentiondue to ion exclusion from the nonpolar and neutralreversed phase surface.

4

Many people will lean toward reversed phase sepa-ration over the other mechanisms. This is justifi-able, because about 75% of all HPLC separationsare performed under reversed phase conditions.That does not mean that it is the best choice, how-ever, and you should carefully survey the literatureto support your choice before precious laboratorytime and materials are consumed strictly on themajority rule that reversed phase enjoys.

Another consideration is the ability of the newanalysis to fit into the general laboratory workflow.If every application in the lab is run underreversed phase conditions, a normal phase separa-tion added to the method list might require a dedi-cated instrument or demand a strict cleaningprocedure to ensure that residual polar solventsused in reversed phase separations do not deacti-vate the polar silica surface of the normal phaseseparation. Although the normal phase separationmight be optimal, users in that situation often pushthe separation onto a reversed phase system toexpedite getting the application started up andshut down on shared LC systems.

For molecular weight applications using refractiveindex detection on a simple isocratic system, a ded-icated instrument is often the most productivechoice because of the relatively long equilibrationand stabilization times that are required before ausable baseline can be obtained.

Finally, a detection scheme must be selected.UV/VIS spectrophotometry holds the majority of allapplications but only when the analytes have usefulchromophores, such as aromatic rings, double-bondcarbonyls, and other oxygen-containing functionalgroups. Fluorescence is present in only a small per-centage of UV-absorbing molecules unless a deriva-tization step is used to create or enhancefluorescence behavior. Refractive index is often

used in molecular weight separations due to its relative lack of bias for chemical structure. It isrestricted to isocratic separations, however, and islately often replaced by evaporative light-scatteringdetectors, which dry away the mobile phase as partof the detection process.

More obscure detectors include conductivity, elec-trochemical reduction/oxidation, chirality (sophis-ticated polarimeters with relatively small flowcells), radioactivity, and so on. Recently, mass spec-trometry has been introduced into HPLC separa-tions and, while expensive and somewhat limitingwith respect to mobile phase contents (every bit ofthe mobile phase must be volatile), it can not onlydetect many diverse molecules, but it can also yieldimportant structural data used to support identityconfirmation or unknown identification.

About the Instrumentation

State-of-the-art HPLC equipment, including auto-matic samplers, injectors, analytical conditionscontrolled by microprocessors, and data evalua-tions performed by Agilent ChemStation softwarecan automate HPLC separations.

Important requirements for automation are:

• Excellent precision of the LC system

• Data evaluation with report printouts

• The ability to store chromatograms and results

• The ability to detect leaks and other errors forsafety reasons

Automation not only increases sample throughput,it also yields precise results by eliminating humanerrors. Following is an overview of samples typi-cally analyzed with HPLC, which are shown in thisguide.

5

Agilent Hardware and ChemStation Software

HPLC hardware and Agilent ChemStation softwareare ordered using standard product and optionnumbers. This leaves all flexibility to configure thesystem according to local regulations and company-specific requirements.

Non-Agilent Hardware, Consumables,and Documentation

For some applications, hardware or consumablesmust be ordered from the external vendor. In thesecases, please contact your local Agilent supplier foradvice how your supplier and vendor can worktogether to ensure smooth ordering and delivery ofthe product.

A Special Note About ZORBAX Rapid ResolutionHigh Throughput (RRHT) Columns

As of 2007, a much wider range of products is avail-able in the newer 600 bar column hardware, whichuses the same sub-2-micron particle technology asthe original 400 bar RRHT columns. All 400 barproducts are still available; however, we encourageyou to use the newer 600 bar columns whereverpossible.

Other Resources

Environmental methods and food safety methodsoverlap a great deal in respect to analytes of inter-est and the difficulties of sample preparation forcomplex matrices with low analyte levels. Weencourage you to search the Agilent Web site(www.agilent.com/chem) for specific techniques,matrices, and analytes, and to review these Agilentsolution guides for additional information:

• Food Solutions with HPLC: includes mainly LCand LC/MS methods, with some CE and ICP-MSmethods (Agilent publication 5989-5674EN)

• Food Safety Primer, Applications in Mass Spec-trometry: includes LC/MS, ICP-MS, and GC/MSmethods (Agilent publication 5989-1270EN)

• HPLC Solutions for the Hydrocarbon ProcessingIndustry: encompasses many industrial and con-sumer applications for LC, LC/MS, CE, and ICP-MS methods (Agilent publication 5989-5847EN)

6

ApplicationsGroup Major analytes Matrix Page

Regulated/hazardous drug substances Chloramphenicol Honey, shrimp, chicken 7Pharmaceuticals Water 9Estrogens, steroids River water, sewage, treated 10

sewage effluentVeterinary pharmaceuticals Surface water, soil, 12

sediment

Regulated/hazardous miscellaneous Crude oil Soil 13substances Arsenic speciation Urine, water 15

VX, Sarin, hydrolysis products Soil 16Disperse dyes, azo dyes Water 18Methylmercury, mercury, ethylmercury Water, synthetic seawater, 19

soilAcrylamide Drinking water 21Chromium speciation 22Nitroaromatics explosives Soil 24Herbicide, antibiotic, peptide, bialaphos, 26bilanaphosPerchlorate Water, vegetables 28Organotin compounds Sediments 29Nitroaromatics explosives 31DNPH aldehydes Air, Brazil 32Inorganic fluoride Water 33Anions, metals Plating bath 34Inorganic anions, miscellaneous Pulping liquor 35Bromate, iodate Ozone-treated water 36HGA hydrocarbons, aromatics Oil, fuels 37Polynuclear aromatic hydrocarbons (PAHs) Soil (SFC extraction) 38

Regulated/hazardous natural toxin substances Anatoxin A, alkaloid neurotoxin Drinking water 39Microcystins, algal toxins Fresh (surface) water 40

Regulated/hazardous pesticide/herbicide 600 pesticides screening Fruit, vegetable 41substances Chlorophenoxy acid herbicides Soil 42

Chlorophenoxy acid herbicides Water 44Various, including trimethoprim Sediments 45Phenylurea, triazine herbicides Water 46Herbicides phenylurea, triazine Water 48Paraquat, Diquat, amitrole, chlormequat Water 50Phenylureas, carbamate pesticides 52Glyphosate, AMPA Water 53Sulfonylurea herbicides Surface water 55

Sample preparation techniques Various in water sources Water 57EPA 3640A standard mix Vegetable oil, broccoli, 58

animal fat

Table 1 Featured Applications

7

×101

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Abundance vs. Acquisition Time (min)0.2 0.6 1 1.4 1.8 2.2 2.6 3 3.4 3.8 4.2

0.1 ppt CAP

ConditionsHPLC

Column ZORBAX SB-C18, 2.1 × 50 mm, 1.8 µm (p/n 827700-902)

Flow rate 0.4 mL/minMobile phase A: water

B: methanolGradient 0-5 min, 30~70% B

5-6 min, 70~100% B8 min, 100% B

Post time 4 minTemperature 45 °CInjection 5 µL

MS Source Settings

Source ESIIon polarity NegativeDrying gas 350 °CtemperatureDrying gas flow rate 10 L/minNebulizer 45 psiVcap 3500 VFragmentor 100 VCollision energy 10 V for m/z 257(qualifier ion)

15 V for m/z 152 (quantitation ion)

System Summary

LC System1200SL

DetectionMS QQQ negESI

ColumnsZORBAX SB-C18, 2.1 mm × 50 mm, 1.8 µm

Column part number827700-902

Figure 9. MRM chromatogram of 0.1 ppt CAP in solvent with injection volume of 5 µL.

Continued

Regulated/Hazardous Drug Substances

Major analytes

Chloramphenicol

Matrix

Honey, shrimp, chicken

Reference

Yanyan Fang, Jerry Zweigenbaum and Zhuwei Wang,”Detection, Confirmation, and Quantification of Chloram-phenicol in Honey, Shrimp and Chicken Using the Agilent6410 LC/MS Triple Quadrupole,” Agilent Technologies publication 5989-5975EN, www.agilent.com/chem

8

Table 4. Integrated Areas of the Quantitation Ion and Qualifier Ion and Their Associated Internal Standard Ion

Chloramphenicol d5-chloramphenicolquantitative Qualifier quantitative Qualifier ion (321–152) ion (321–257) Ratio ion (326–157) ion (326–262) Ratio

1 350 165 47.1 262 121 50.4

2 346 157 45.2 258 114 55.3

3 346 5 44.6 259 118 49.4

4 313 164 52.3 267 127 47.6

5 301 154 49.5 261 121 46.4

6 313 168 53.6 253 124 49.0

7 320 160 50.1 228 111 48.6

8 326 145 44.5 225 113 50.4

9 317 141 44.5 241 117 48.6

10 290 135 46.6 226 107 47.1

11 300 138 46.2 253 90 45.7

12 281 136 48.4 240 90 47.6

13 303 143 47.3 220 101 45.9

14 290 140 48.3 214 107 49.8

15 261 131 50.3 217 101 46.6

RSD 8.11% 8.30% 5.91% 7.67% 9.99% 4.83%

9

1

2

3

4

5

Abundance vs. acquisition time (min)

3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.84.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2

×106

Hydrochlorothiazide

AspirinEnalaprilat

Furosemide

Ketoprofen

Clofibric acidNaproxen

Diclofenacsodium salt

IbuprofenIbuprofen-d3 Gemfibrozil

Triclocarban

System Summary

1200SL

DetectionMS QQQ pos/negESI

ColumnsZORBAX Extend-C18, 2.1 mm × 100 mm, 1.8 µm


Figure 1. Negative ion mode TIC of 11 pharmaceuticals.

´101

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6


1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13

Metformin

Acetaminophen

Salbutamol

Cimetidine1,7-dimethylxanthine

CotinineCodeine

CaffeineTrimethoprim

Thiabendazole

Sulfamethoxazole

Azithromycin

Diphenhydramine Carbamazepine

Diltiazem

Fluoxetine

Dehydronifedipine

Warfarin

Miconazolenitrate salt

5 pg on column

Figure 4. Overlaid MRM chromatograms of the 19 pharmaceuticals in positive ion mode.


Major Analytes

Pharmaceuticals

Matrix

Water

Reference

Chin-Kai Meng, Stephen L. Werner and Edward T. Furlong,“Determination of Pharmaceuticals in Water by SPE andLC/MS/MS in Both Positive and Negative Ion Modes,” Agilent Technologies publication 5989-5319EN, www.agilent.com/chem

10

An elaborate and effective three-step sample preparation,including SPE, normal phase, and GPC, is described inthe application note.

System Summary

LC SystemBinary gradient

DetectionMS TOF negAPPI

ColumnsPhenyl hexyl

Column part numberVarious, see application note

LC/MS-TOF Conditions

Source APPI operated in negative ionization mode

Dry gas temp 350 °C

Dry gas flow rate 8.0 L/min

Aporiser temp 475 °C

Nebulizer pressure 40 psig

Vcap 3000 V

Fragmentor 240 V

Skimmer 60 V

Scan mode 100–800 amu, transients/scan 20000, scan/sec 0.67

Reference mass 100 mg/L solutions, purine 2 mL/L, solutions reserpine 1 mL/L

Analytical HPLC ConditionsColumn Luna Phenyl Hexyl,

2.0 mm × 150 mm, 3.0 µm, at 60 °C

Guard column Luna Phenyl Propyl, 2.0 mm × 4.0 mm, 3.0 µm

Mobile phase A HPLC grade waterMobile phase B Methanol:acetone (95:5)

(Acetone required as the dopant)

Gradient Time (min) A (%) B (%)Initial to 0.50 95 50.50 to 1.00 80 201.00 to 12.0 60 4012.0 to 14 20 8014.5 95 5

Flow rate 0.3 mL/min

Injection volume 100 µL

Continued


Major Analytes

Estrogens, steroids

Matrix

River water, sewage, treated sewage effluent

Reference

Neil Cullum and Paul Zavitsanos, “Determination of Steroid Estrogens in River Water, Final Effluents and Crude Sewage by Validated APPI-LC/MS-TOF Method,”Agilent Technologies publication 5989-4858EN, www.agilent.com/chem

11

Figure 2. Chromatograms for the 2 ng/L standards.

Estrone std = 2 ng/L

Estradiol std = 2 ng/L

Ethinyl estradiol std = 2 ng/L

Figure 3. Calibration curve for estrone. It shows that the method is nearly linear over the range 0 to 25 ng/L.

12

Table 1. Target Compounds and Their Required Analytical LODs

System Summary

LC SystemQuaternary gradient

DetectionMSD, MSn Ion Trap

ColumnsZORBAX SB-C18, 2.1 mm × 150 mm, 3.5 µm


900850800750700650600550m/z

8

16

24

32

40

48

56

64

72

80

88

96

Rel

ativ

e In

tens

ity

(%)

505.0

565.1

623.4 709.0790.3

830.3

876.2

910.3

Figure 7. MS/MS spectrum of eprinomectin (SL Trap), negative ion APCI.

Soil and sediment WaterCompound LOD (mg/kg) LOD (µg/L)

Doramectin 5 0.001

Eprinomectin NA 0.004

Ivermectin 5 0.0002

Moxidectin NA 0.0003

NA Not applicable

LC ConditionsInstrument Agilent 1100 LC comprising degasser,

quaternary pump, automatic liquid sampler (ALS), column and column oven compartment.

Column ZORBAX 80 SB-C18, 2.1 × 150 mm, 3 µm (p/n 830990-902) with Eclipse XDB-C8 narrow bore guard column 2.1 × 12.5 mm, 5 µm (p/n 821125-926)

Column temp 60 °C

Mobile phase A = 5 mM ammonium formate in water (pH 5.5)B = Methanol

Isocratic 80% B


Injection volume 100 µL

MS Conditions

Instruments Agilent LC/MSD SL

Source Positive APCI

Drying gas flow 5 L/min

Nebulizer 60 psig

Vaporizer temp 300 °C

Drying gas temp 250 °C

Vcap 2500 V

MSD1 915, EIC=914.5:915.5 (C:\HPCHEM\DATA\1\06JUL04\CAL1_E.D) APCI, Pos, SIM, Frag: 130 (TT)

Eprinomectin

010002000

Moxidectin

MSD1 623, EIC=622.3:623.3 (C:\HPCHEM\1\DATA\06JUL04\CAL1_E.D) APCI, Pos, SIM, Frag: 130 (TT)

200004000060000

Doramectin

MSD1 917, EIC=916.5:917.5 (C:\HPCHEM\1\DATA\06JUL04\CAL1_E.D) APCI, Pos, SIM, Frag: 130 (TT)

0100020003000

IvermectinMSD1 893, EIC=892.5:893.5 (C:\HPCHEM\1\DATA\06JUL04\CAL1_E.D) APCI, Pos, SIM, Frag: 130 (TT)

min4 6 8 10 12 140

20004000

Figure 2. EICs for the low level standards.


Major Analytes

Veterinary pharmaceuticals

Matrix

Surface water, soil, sediment

Reference

Anthony Gravell, “Validated Method for the Determinationof Veterinary Medicines in the Environment Using the Agilent LC-MSD Quad and Ion Trap,” Agilent Technologies publication 5989-2980EN, www.agilent.com/chem

13

Regulated/Hazardous Miscellaneous Substances

Major Analytes

Crude oil

Matrix

Soil

Reference

Wei Luan and Chuanhong Tu, and Michael Woodman,“Evaluation of Total Petroleum Hydrocarbon in Soil Using LCwith Fraction Collection and GC/MS Fraction Evaluation,”Agilent Technologies publication 5989-6012EN, www.agilent.com/chem

System Summary

LC SystemBinary gradient with fraction collector

DetectionDAD RID

ColumnsZORBAX NH2, 4.6 mm × 250 mm, 5 µm


Figure 2. Chromatography of soil sample extract and fractions collected in different vials.

Experimental

Instrumentation and Conditions

Agilent 1200 Series LC, consisting of:G1379B Micro vacuum degasserG1312B Binary pump SLG1367C High-performance autosampler SLG1316B Thermostatted column compartment SL

with 55-column switching optionG1315C UV/VIS diode array detector SLG1364C Fraction collector (analytical scale)ChemStation 32-bit version B.02.01-SR1

Agilent 6890GC with 5973 MSD, consisting of:G1540N 6890N network GC system with options:

201 MSD interfaceG3243A 5975B inert MSD/DS perf turbo EI bundleG3397A Ion gauge/controller for use with

5975 MSDG2913A 7683B autoinjector module G2614A 7683 autosampler tray moduleG2589A 5973 inert mass selective detectorMSD Chemstation version D.02.00 with NIST 05 MS Library version 2.0d

The LC and GC/MS operating conditions are listed in Table 1.

min0 2 4 6 8 10 12 14

mAU

0

500

1000

1500

2000

2500

1-P1-A

-01

1-P1-A

-02

1-P1-A

-03

1-P1-A

-04

1-P1-A

-05

1-P1-A

-06

1-P1-A

-07

1-P1-A

-08

1-P1-A

-09

1-P1-

B-01

1-P1-

B-02

1-P1-

B-03

1-P1-

B-04

1-P1-

B-05

1-P1-

B-06

1-P1-

B-07

1-P1-

B-08

1-P1-

B-09

1-P1-

C-01

1-P1-

C-02

1-P1-

C-03

1-P1-

C-04

1-P1-

C-05

Continued

14

4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

Time

Abu

ndan

ce

120

165

20 40 60 80 100 140 160 180 200 220 240 260 2800

20004000

6000

8000

Abu

ndan

ceA

bund

ance

Scan 1190 (7.885 min): SOIL17.D\data.ms192

1659482 207139 15263 115 17750 281127 253

20 40 60 80 100 120 140 160 180 200 220 240 260 2800

2000

4000

6000

8000 #51411: Phenanthrene, 2-methyl-192

9663 139 15239 8227 51 115 178127

Tri-aromatics

Figure 3. GC/MS total ion chromatography of typical fractions including paraffins and tri-aromatics.

15

0

2

4

6

8

10

12

14

0 2 4 6

Ret

entio

n tim

e/m

in

AB*DMAAs(III)

MMAAs(V)Cl

Conc. of NaNO3/mM

2.0 4.0 6.0 8.0 10.0 12.0 14.00

4000

8000

12000

16000

20000

24000

28000

32000

36000

40000

Sig

nal/

coun

ts

Retention time/min

AB*DMA

As(III)

MMA

As(V)Cl (m/z 75)

*Arsenobetaine, while well separated from the four anionic species, elutes with the void volume and may coelute with other neutral or cationic species if present.

Figure 1. Optimization of concentration of sodium nitrate.

ConditionsColumn G3288-80000 (4.6 × 250 mm),

G3154-65002 (Guard Column)

Mobile phase 2.0 mM PBS/0.2 mM EDTA/10 mM CH3COONa/3.0 mM NaNO3/1% EtOH pH 11.00 adjusted with NaOH


Injection volume 5 to 100 µL

RF power 1550 W

Sample depth 9.0 mm

Spray chamber temp 2 °C

Carrier gas 0.7 L/min

Makeup gas 0.42 L/min

Nebulizer MICRO MIST

System Summary

LC SystemIsocratic

DetectionICP-MS

ColumnsArsenic column, 4.6 mm × 250 mm, 3.5 µm

Column part numberG3288-80000 plus G3154-65002 (Guard column)


Major Analytes

Arsenic speciation

Matrix

Urine, water

Reference

Tetsushi Sakai and Steve Wilbur, “Routine Analysis of ToxicArsenic Species in Urine Using HPLC with ICP-MS,” Agilent Technologies publication 5989-5505EN, www.agilent.com/chem

16

0 200 400 600 800200

250

300

350

400

450

500

550

600

Res

pons

e (C

PS)

Time (sec)

0 200 400 600 800100

150

200

250

300

350

400

450

500

550

600

Res

pons

e (C

PS)

Time (sec)

Spiked Top Soil

31P

31P

50 ppb

A

B

PCH3 OH

OOCH2CH3

PCH3 OH

OOCH(CH3)2

PCH3 OH

OOH

Figure 2 Separation of MPA, EMPA, and IMPA in a standard mixture (A) andspiked topsoil (B).

Conditions

ICP-MS parametersForward power 1500 W (with shielded torch)Plasma gas flow rate 15.6 L/minAuxiliary gas flow rate 1.0 L/minCarrier gas flow rate 1.20 L/minNebulizer Glass expansion micro-

concentricSpray chamber ≈2 °C (Scott double channel)Sampling depth 6 mmSampling and skimmer cones NickelDwell time 0.1 sIsotopes monitored (m/z) 31P and 47PO+

Octopole reaction system He (Flow optimized prior to experiment)

HPLC parameters

Instrument Agilent 1100 HPLCFlow rate 0.5 mL/minInjection volume 100 µL

50 mM Ammonium acetate; 2% Methanol

Buffer 5 mM Myristyltrimethyl-ammonium bromide pH 4.85

Column Alltima C8 (3.2 × 150 mm) 5 µm

System Summary


DetectionDAD, ICP-MS

ColumnsAlltima C8, 3.2 mm × 150 mm, 5 µm

Column part numberContact manufacturer

Continued


Major Analytes

VX, Sarin, hydrolysis products

Matrix

Soil

Reference

Douglas D. Richardson, Baki B.M. Sadi, and Joseph A.Caruso, “Ultra-Trace Analysis of Organophosphorus Chemi-cal Warfare Agent Degradation Products by HPLC-ICP-MS,” Agilent Technologies publication 5989-5346EN, www.agilent.com/chem

17

Degradation productAgent liquid LD50 Chemical warfare Degradation oral-human LDLO

Chemical warfare agent (mg kg–1)* degradation products product pKa (mg kg–1)

0.14 2.16

24 2.24 143–428**

See above pKa1 = 2.41pKa2 = 7.54

Table 1. Chemical Warfare Agents and Degradation Products

EMPA

OCH2CH3P

O

H3C OH

VX

OCH2CH3P

O

H3C S CH2 CH2 NCH(CH 3)2

CH(CH 3)2

Sarin (GB)

P

O

H3C F

OCH(CH3)2

IMPA

OCH(CH3)2P

O

H3C OH

MPA

P

O

H3C OH

OH

* Vapor form LD50 values range from ~0.09–2 mg-min/m3 (Agent MSDS)

**Cerilliant MSDS

VX

OCH2CH3P

O

H3C S CH2 CH2 NCH(CH 3)2

CH(CH 3)2

Sarin (GB)

P

O

H3C F

OCH(CH3)2

18

LC/MS Method DetailsLC Conditions

Instrument Agilent 1100 HPLCColumn ZORBAX XDB-C8,

50 mm × 2.1 mm × 1.8 µm (p/n 922700-932)

Column temp 55 °C Mobile phase A = 5-mM ammonium acetate in water

B = AcetonitrileGradient 40% B at 0 min

60% B at 6 min85% B at 8.5 min95% B at 10.0 min

Flow rate 0.8 mL/min Injection volume 1 µL

MS Conditions

Instrument Agilent LC/MSD TOFSource Positive ESIDrying gas flow 10 L/minNebulizer 40 psigDrying gas temp 300 °C Vcap 4000 V (negative)Scan m/z 200–1500 References masses Purine (m/z 121.0509) and HP-0921

(m/z 922.0098)Fragmentor 120 V for (M+H)+ identification and

quantitation, and 320 V for fragmentationResolution 10,000 at m/z 922.0098

TIC of +TOF MS: from 050224008.wiff, Baseline Subtracted

Max. 1.4e7 cps.

3.1 3.3 3.5 3.7 3.9 4.1 4.3 4.5 4.7 4.9 5.1 5.3Time, min

0.01.0e62.0e63.0e64.0e65.0e66.0e67.0e68.0e69.0e61.0e71.1e71.2e71.3e7

Inte

nsity

, cps

4.02

Figure 1. TIC showing the Disperse Red 1 dye compound peak.

System Summary


DetectionMS TOF posESI

ColumnsZORBAX Eclipse XDB-C8, 2.1 mm × 50 mm, 1.8 µm


Table 2. Linearity and LOD for Each Disperse Dye Analyzed Using an EIC of 0.5 amu Width

LOD Disperse dye R2 (ng on-column)

1 Disperse Blue 1 0.9976 0.332 Disperse Blue 3 0.9992 0.423 Disperse Blue 7 0.9982 1.344 Disperse Blue 26 0.9984 0.875 Disperse Blue 35 0.9875 1.126 Disperse Blue 102 0.9921 1.227 Disperse Blue 106 0.9997 1.028 Disperse Blue 124 0.9967 1.019 Disperse Orange 1 0.9982 0.4610 Disperse Orange 3 0.9983 0.0811 Disperse Orange 37/76 0.9976 0.3612 Disperse Red 1 0.99996 0.1613 Disperse Red 11 0.9982 0.2614 Disperse Red 17 0.9986 0.2515 Disperse Yellow 1 0.9932 1.1816 Disperse Yellow 3 0.9992 0.2817 Disperse Yellow 9 0.9995 0.5218 Disperse Yellow 49 0.9999 0.4419 Disperse Yellow 39 0.9990 2.0120 Disperse Orange 11 0.9987 0.17


Major Analytes

Disperse dyes, azo dyes

Matrix

Water

Reference

Yanyan Fang, Ping Li, and Michael Zumwalt, “Determination of EU-Banned Disperse Dyes by LC/MSD TOF,” AgilentTechnologies publication 5989-3859EN,www.agilent.com/chem

19

Hg2+

3.24

MeHg2.55

EtHg6.53

3000

2800

2400

2000

Abu

ndan

ce

1600

1200

800

400

01.000.00 2.00 3.00 4.00

Time [min]5.00 6.00 7.00 8.00

Figure 6. HPLC-ICP-MS TIC for soil sample, S-A-03, extracted by 7.6% HCl,spiked with 0.9-ng (as Hg) mixed Hg species standard. MeHg 2.55 min, Hg2+ 3.24 min and EtHg 6.53 min, 100-µL loop.

Figure 1. Agilent 1100 HPLC schematic.

Q-pole

Turbopump

Turbopump

Rotarypump

ICP Torch

Argon gas controller

(ICP-MS not shown to scale)

Agilent 1100 HPLC

Agilent 7500 ICP-MS

Eluent bottles

Degasser

Pump

Automatedsample trayand injector

Columncompartment

Nebulizer/Spraychamber

Continued


Major Analytes

Methylmercury, mercury, ethylmercury

Matrix

Water, synthetic seawater, soil

Reference

Dengyun Chen, Miao Jing and Xiaoru Wang, “Determina-tion of Methyl Mercury in Water and Soil by HPLC-ICP-MS,”Agilent Technologies publication 5965-3572EN, www.agilent.com/chem

20

System Summary

LC SystemLC/ICP-MS

DetectionICP-MS

ColumnsZORBAX Eclipse XDB-C18, 2.1 mm × 50 mm, 5 µm


Table 5. Spike Recoveries of Hg Species in Soil Samples by HPLC-ICP-MS

True Value Measured Recovery Sample Hg-Species (pg) value (pg) (%)

S-BLK-1 MeHg NA 2 NAHg2+ 61 63 103EtHg NA + NA

S-BLK-2 MeHg NA 9 NAHg2+ 61 65 107EtHg NA + NA

S-A-03 MeHg 90 85 95Hg2+ 151 185 122EtHg 90 82 91

S-A-04 MeHg 90 80 89Hg2+ 151 181 120EtHg 90 75 83

S1-1 MeHg 36 34 94Hg2+ 97 88 91EtHg 36 28 77

S1-2 MeHg 36 37 104Hg2+ 97 105 108EtHg 36 35 97

S1-3 MeHg 36 41 113Hg2+ 97 98 101EtHg 36 43 120

NA Not applicable+ No measurements made

21

6000

Acry 1 µg/L - Acrylamide (Unknown) 72.0 - 72.0 amu amu - Acry 1 µg wiffArea: 1.92e+004 counts Height: 4.09e+003 cps RT: 1.93 min

5000

4000

3000

Inte

nsity

, cps

2000

1000

01.00.0 2.0 3.0

Time, min4.0 5.0

0.41 1.071.42

1.93

2.383.50 4.24 4.99

69.5

1.00e4

+TOF MS: 1.941 min from pt 5.wiff Agilent, substracted (1.889 min and 2.09 to 2,29 min)

9500.00

8500.00

7500.00

6500.00

5500.00

Inte

nsity

, cou

nts

4500.00

3500.00

2500.00

1500.00

500.00

70.0 70.5 71.0 71.5 72.0m/z, amu

72.5 73.0 73.5 74.0 74.5

72.0460

69.512070.0240

70.516473.0142

Figure 5. Extracted ion profile of acrylamide at 1 µg/L..

Figure 4. Example of an acrylamide mono-isotopic molecular ion mass of(M+H)+ at 72.0460 from a single injection.

System Summary

LC SystemDual binary w/ 6-port valve for autoSPE


ColumnsZORBAX SB-C18, 2.1 mm × 150 mm, 5 µm


3.02.82.62.42.22.01.81.6

Ana

lyte

are

a/IS

are

a

1.41.21.00.80.60.40.20.0

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4Analyte con./IS conc.

1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0

Table 1. Optimized Precolumn Extraction

HPLC separation Agilent 1100: binary pump 2, column oven

Column ZORBAX SB-C18, 2.1 mm × 150 mm, 5 µm

Column temperature 40 °CAnalytical flow 0.5 mL/minElution solvents A: 97% HPLC water (0.01% formic acid)

B: 3% CH3CN (0.01% formic acid)Backflush OffAnalysis start 0.6 minAnalysis end 3.2 min

Analytical detection Agilent MSD TOFSource ESIIon polarity PositiveDrying gas flow 12 mL/minDrying gas temperature 300 °CNebulizer pressure 50 psiFragmentor 150 VOctopole RF 100 VMass range 50 to 1000 m/zReference ions 121.05087 and 922.00979

Table 2. Optimized Analytical Conditions

Figure 7. Linearity plot for acrylamide/acrylamide-d3 response.

Apparatus Agilent 1100 series binary pump, autosampler, 6-port switching valve, and column oven

Extraction column ZORBAX SB-C18, 4.6 mm × 12.5 mm, 5 µm

Precolumn temperature 20 °CInjection volume 400 µLLoading flow 0.5 mL/minLoading solvent 100% HPLC water Backflush start 0.6 min (via the analytical column)Backflush stop 3.2 min (via the waste)

Major Analytes

Acrylamide

Matrix

Drinking water

Reference

Sophie Men, Johanne Beausse, Jean-Francois Garnier,“Determination of Acrylamide in Raw and Drinking Waters,”Agilent Technologies publication 5989-2884EN, www.agilent.com/chem


22

0.00 1.00 2.00 3.00 4.00 5.00 6.00

2000

3000

4000

5000

6000

7000

8000

9000

RT/min

Original Water A

Cr(III): 0.005 µg/L

Cr(VI): 0.055 µg/L

Water A + 0.1 µg/L each Cr(III), Cr(VI)

Cr(III): 0.105 µg/L

Cr(VI): 0.153 µg/L

Na 7.3 mg/L

Ca 91.0 mg/L

Mg 19.9 mg/L

K 4.9 mg/L

Abu

ndan

ce/C

ount

s

0.00 1.00 2.00 3.00 4.00 5.002000

3000

4000

5000

6000

7000

8000

9000

Cr(III) 0.1 µg/L

Retention Time / min

Abu

ndan

ce/

coun

ts Cr(VI) 0.1 µg/L

Figure 5. Major element composition (mg/L) and chromatogram for spikedand unspiked mineral water A.

Figure 2. Separation and detection of Cr(III) and Cr(VI) at a concentration of0.1 µg/L each species.

System Summary

LC SystemIC Metrohm 818 pump, Agilent 7500 ISIS sampler

DetectionICP-MS

ColumnsAgilent Cr, 4.6 mm × 30 mm

Column part numberG3268A

Table 1. Chromatographic Conditions for Cr Speciation

Cr column Agilent part number G3268A, 30 mm × 4.6-mm id

Mobile phase 5 mM EDTA (2Na), pH 7 adjust by NaOH

Flow rate 1.2 mL/minColumn temperature AmbientInjection volume 50~500 µL

Sample preparationReaction temperature 40 °CIncubation time 3 hEDTA concentration 5~15 mM pH 7 adjust by NaOH

Continued


Major Analytes

Chromium speciation

Matrix

Standard

Reference

Tetsushi Sakai, Ed McCurdy, and Steve Wilbur, “Ion Chromatography (IC) ICP-MS for Chromium Speciation inNatural Samples,” Agilent Technologies publication 5989-2481EN, www.agilent.com/chem

23

Table 3. Stability of RT and Peak Area for Multiple 500 µL Injections of 0.5 µg/L Each Cr Species

RT/min Peak height/counts Peak area/counts

Number Cr(III)-EDTA Cr(VI) Cr(III)-EDTA Cr(VI) Cr(III)-EDTA Cr(VI)

1 0.969 2.338 23514 18437 5331427 4621752

2 0.969 2.338 22642 18784 5280683 4758462

3 0.969 2.338 22832 18615 5220349 4742259

4 0.952 2.338 24104 19944 5470760 4800723

5 0.969 2.372 22797 19203 5287094 4726640

6 0.969 2.405 23830 19328 5498172 4760285

7 0.985 2.338 23971 19479 5481984 4824934

8 0.969 2.338 23393 19675 5474510 4883193

9 0.969 2.355 23070 20097 5355106 4892160

10 0.969 2.372 23826 19896 5428247 4886400

Avg 0.97 2.38 23398 19346 5382833 4789681

STD 0.008 0.014 534.45 581.88 100413.18 85782.42

RSD% 0.80 0.57 2.28 3.01 1.87 1.79

24

System Summary


DetectionMS TOF negAPCI

ColumnsZORBAX Extend-C18, 4.6 mm × 250 mm, 5 µm



Major Analytes

Nitroaromatics explosives

Matrix

Soil

Reference

Russell Kinghorn, Courtney Milner and Jerry Zweigenbaum,“Analysis of Trace Residues of Explosive Materials by Time-of-Flight LC/MS,” Agilent Technologies publication5989-2449EN, www.agilent.com/chem

Continued

# Name Abbreviation CAS no. Molecular formula

1 Hexamethylenetriperoxidediamine HMTD NA C6H12N2O6

2 Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine HMX 2691-41-0 C4H8N8O8

3 Hexahydro-1,3,5-trinitro-1,3,5-triazine RDX 121-82-4 C3H6N6O6

4 1,3,5-triamino-2,4,6-trinitrobenzene TATB 3058-38-6 C6H6N6O6

5 Ethylene glycol dinitrate EGDN 628-96-6 C2H4N2O6

6 1,3,5-Trinitrobenzene 1,3,5-TNB 99-35-4 C6H3N3O6

7 1,3-Dinitrobenzene 1,3-DNB 99-65-0 C6H4N2O4

8 Methyl-2,4,6-trinitrophenylnitramine Tetryl 479-45-8 C7H5N5O8

9 4-amino-2,6-dinitrotoluene 4A-DNT 1946-51-0 C7H7N3O4

10 Nitrobenzene NB 98-95-3 C6H5NO2

11 Nitroglycerin NG 55-63-0 C3H5N3O9

12 2-amino-4,6-dinitrotoluene 2A-DNT 355-72-78-2 C7H7N3O4

13 2,4,6-Trinitrotoluene TNT 118-96-7 C7H5N3O6

14 2,6-Dinitrotoluene 2,6-DNT 606-20-2 C7H6N2O4

15 2,4-Dinitrotoluene 2,4-DNT 121-14-2 C7H6N2O4

16 Hexanitrostilbene HNS 19138-90-0 C14H6N6O12

17 2-Nitrotoluene 2-NT 88-72-2 C7H7NO2


19 Pentaerythritol tetranitrate PETN 78-11-5 C5H8N4O12


21 Triacetone triperoxide TATP NA C9H18O6

22 Carbamite Carbamite NA C17H20N2O

Table 1. Names, Abbreviations and Molecular Formulae of Explosives Studied

25

3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5Time, min

10 µg/L RDX

10 µg/L HNS

10 µg/L TNT

10 µg/L 1,3,5-TNB

Time, min

Time, min

0

40

80

120

160

200

240

280

320

360

400

440

480

4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.00

100

200

300

400

500

600

700

800

900

1000

5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.00

100

200

300

400

500

600

700

800

Inte

nsity

, cps

Inte

nsity

, cps

Inte

nsity

, cps

Inte

nsity

, cps

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0Time, min

0

100

200

300

400

500

600

700

800

Figure 6. Representative chromatographic responses for four of the explosive compounds at the 10 µg/L concentration.

LC Conditions

Solvents Methanol and waterFlow rate 0.9 mL/min

Gradient Time [min] % Methanol % Water0 60 401 60 4015 92 816 100 018 100 019 60 40

Post time 5 minutesTotal run time 24 minutesInjection volume 10 µL, with needle washColumn temperature 40 °CColumn ZORBAX Extend-C18

4.6 mm × 250 mm, 5 µmp/n 770450-902

MS Detection conditionsIonization APCIGas temperature 350 °CVaporizer temperature 325 °CDrying gas flow 5 L/minNebulizer pressure 40 psigPCI corona current 4 µAPCI capillary voltage 4000 VNCI corona current 10 µANCI capillary voltage 1500 VScan m/z range 70–1000Fragmentor voltage 100 VStorage mode ProfileSkimmer 60 VOct RF 250 V

26


Major Analytes

Herbicide, antibiotic, peptide, bialaphos, bilanaphos

Matrix

Bialaphos

Reference

Sheher Mohsin and Patrick D. Perkins, “Characterization of the Tripeptide Antibiotic Bialaphos Using MSn IonTrap and Accurate Mass TOF Data,” Agilent Technologies publication 5989-2324EN, www.agilent.com/chem

System Summary

LC SystemInfusion

DetectionMSn Ion Trap, MS TOF, both posESI

ColumnsNone

Column part numberNA

Figure 2. AutoAssignment of MS/MS spectrum of precursor ion at m/z 324.1 of bialaphos. Over 95% of the spectral abundance was assigned to fragments automatically. The lines labeled with ** are discussed in the text.

320300280260240220200180160140120100m/z

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e In

tens

ity (%

)

99136

137162 190

207

235

270

288

306

No. Fragment Formula m/z Calc.

1 1,3-21 C11H21N3O5P 306

2 1-20 C11H21N3O5P 306

3 1-6,8-21(-H) C11H19N2O6P 306

4 1-12,14-21(-2H) C10H17N3O6P 306

5 1-17,19-21(-2H) C10H17N3O6P 306

6 1,3-20(-2H) [H+] C11H19N3O4P 288

7 1-15(+H) C8H17N2O4P 236

8 1-12,14-16(+H) C7H15N3O4P 236

9 1-15 C8H16N2O4P 235

10 1-12,14-16 C7H14N3O4P 235

11 1-6,8-15(-2H) [H+] C8H13NO4P 218

12 1-13 C7H16N2O3P 207

13 1-6,8-13(-2H) [H+] C7H13NO3P 190

**14 1-10(-2H) C5H9NO3P 162

**15 1-6,8-11(-H) C5H9NO3P 162

16 1-8(-H) [H+] C4H11NO2P 136

19 O20

17NH16

18

HO21

14

O15

12

NH11

13

9

O10

6

NH27

5

4P1

O3

OH2

8

Continued

27

LC/MSD Trap ConditionsSample introduction Infusion, 6 µL/minIonization mode Positive ESIDrying gas flow 5 L/minNebulizer 15 psigDrying gas temperature 300 °C Vcap –3500 VSkim 1 40 V Capillary exit 136.1 Trap drive 43.5Scan mode UltrascanScan range m/z 70–400Averages 1ICC OnMax. accumulation time 200 msTarget 200,000Rolling, averages On, 3Automatic MS(5)

No. of precursors 1SPS OnMS(n) averages 3Fragmentation ampl. 1.0 Active exclusion OffSmart Frag On, 30%–200%Isolation width 4 m/z

Data Analysis Conditions for ACD/Labs MS Manager Suite MSAssignment options

Maximum number of 10,000fragments generated at each stepFragmentation steps 4

Table 1. Suggested empirical formulae from TOF data for bialaphos ions using a 3 ppm error tolerance. In most cases, the best andonly match confirms the AutoAssignments of the data obtained by ion trap MS/MS (compare with Figure 2). The differencefor m/z 162 is discussed in the text.

Proposed empirical formula Calculated m/z Experimental m/z Error (mDa) Error (ppm)

C11 H22 N3 O6 Na P 346.1138 346.1145 0.7 2.0C11 H23 N3 O6 P 324.1319 324.1324 0.5 1.5C8 H16 N2 O4 P 235.0842 235.0844 0.2 0.9C7 H16 N2 O3 P 207.0893 207.0892 –0.1 –0.5C6 H13 N O2 P 162.0678 162.0675 –0.3 –1.9C4 H11 N O2 P 136.0522 136.0525 0.3 2.2

**C5 H9 N O3 P 162.0315 162.0675 36.0 222.4

** If the empirical formula from the AutoAssignment of the ion trap data was correct

LC/MSD TOF Conditions

LC ConditionsColumn None, flow injection analysisMobile phase A = 0.1% formic acid in water

B = 0.1% formic acid in ACN Gradient Isocratic, 50% BFlow rate 0.25 mL/minInjection volume 1 µL

MS ConditionsIonization mode Positive ESIDrying gas flow 9.5 L/minNebulizer 40 psigDrying gas temperature 300 °C Vcap –3000 VSkim 1 60 VFragmentor 175, 225, 255 V (programmed)Scan range m/z 100–1000Averages 10,000 transients/scanReference masses m/z 121.0509, 922.0098

(internal reference)

28


System Summary

LC System

Metrohm IC

Detection

MSD negESI

Columns

MetroSep ASUPP-5 4 mm × 100 mm

Column part number

See application note

min2 4 6 8 10 12 14 16

8000

10000

12000

14000

16000

18000

MSD1 TIC, MS File (ICDATA~1\ICBLK1D\IC000045.D) API-ES, Neg, SIM, Frag: 140, "neg sim" MSD1 TIC, MS File (ICDATA~1\ICBLK1D\IC000046.D) API-ES, Neg, SIM, Frag: 140, "neg sim" MSD1 TIC, MS File (ICDATA~1\ICBLK1D\IC000047.D) API-ES, Neg, SIM, Frag: 140, "neg sim" MSD1 TIC, MS File (ICDATA~1\ICBLK1D\IC000048.D) API-ES, Neg, SIM, Frag: 140, "neg sim"

No interferents

200 ppm Cl_, CO3_2, SO4

_2

500 ppm Cl_, CO3_2, SO4

_2

1000 ppm Cl_, CO3_2, SO4

_2

Table 1. Operating Parameters

Metrohm Advanced ICInjection loop size 100 µL

Column MetroSep ASUPP-5 (4 mm × 100 mm)

Eluent 3/7 v/v MeOH/30 mm NaOH


Agilent 1100 MSDTune mode Negative mode “auto-tune”

Vcap 1400 V

Drying gas flow and 9 L/min @ 320 °Ctemperature


Fragmentor 140 V

Dwell time m/z 99 1 s

Dwell time m/z 101 1 s

Table 2. Metrohm-Peak Ion Chromatograph Parameters and Setup

HardwareMetrohm Advanced IC consists of Metrohm 788 Autosampler, 830 Interface with ICNet 2.3 software, 833 Suppressor Module,819 Advanced IC Detector, 820 IC Separation Center, 818 IC serialdual-piston pump

SetupColumn Metrohm ASUPP-5 – 100

(4 mm × 100 mm) p/n 6.1006.510

Eluent 3/7 v/v MeOH/30 mM NaOH

Regenerant solution 5/95 v/v MeOH/60 mM HNO3

Rinse solution 5/95 v/v MeOH/H2O


Suppressor regenerantand rinse flow rate 0.5 mL/min

Connection to synchronized start MSD and IC by MSD-com portand events on 820 IC separation center.

Figure 5. Synthetic matrix spikes overlaid with 1-ppb perchlorate standard.

Major Analytes

Perchlorate

Matrix

Water, vegetables

Reference

Johnson Mathew, Jay Gandhi, Joe Hedrick, “The Analysisof Perchlorate by Ion Chromatography/Mass Spectrometry,”Agilent Technologies publication 5989-0816EN,www.agilent.com/chem

Range for calibration standards0.1 ppb to 5 ppb (ClO4

_)

Amount (ng/mL)0 1

Area

0

50000

100000

150000

200000

Perchlorate 99, MSD1 101

Correlation: 0.99874

Rel. Res% (4): _3.377

Area = 44472.7433*Amt -1931.1962

2 3 54

Figure 4. Calibration data (m/z 101).

29


System Summary

LC System

LC and GC

Detection

ICP-MS

Columns


Column part number


1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.000

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

22000

24000

Time

Abu

ndan

ce

Ion 120.00 (119.70 to 120.70): msd1.ms

Ion 117.00 (116.70 to 117.70): msd1.msDBT

TPhT

TBT

Figure 2. HPLC-ICP-MS chromatogram.

Major Analytes

Organotin compounds

Matrix

Sediments

Reference

Raimund Wahlen, “A Comparison of GC-ICP-MS and HPLC-ICP-MS for the Analysis of Organotin Compounds,” AgilentTechnologies publication 5988-6697EN,www.agilent.com/chem

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.000

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

600000

650000

Time

a)

b)

c)

MBT

DBT

TBT

TeBT

TPhTDPhT

MPhT

Figure 1. Sensitivity increase on a 20 ng mL-1 mixed standard by using a) noadditional gas, b) 5% O2, and c) 5% N2.

HPLC-ICP-MS GC-ICP-MSInterface cones Platinum Platinum

Plasma gas flow 14.5−14.9 L min-1 14.5−14.9 L min-1

Carrier gas flow 0.65−0.75 L min-1 0.80−0.85 L min-1

Make-up gas flow 0.15−0.25 L min-1 Not used

RF power 1350−1550 W 1100−1200 W

Sampling depth 4−7 mm 6.5−7.5 mm

Integration time per mass 300 ms 100 ms

Isotopes monitored 120Sn 120Sn117Sn 118Sn103Rh 117Sn

Other parameters ICP torch injector diameter: 1.5 mm 5% N2 or O2 added to enhancePeltier cooled spray chamber at -5 °C sensitivity5% O2 added post-nebulization ShieldTorch fittedShieldTorch fitted

Table 1. ICP-MS Parameters Used

Continued

LC of sedimentextract

GC of standards

30

Table 3. TBT Data for Sediment Extracts

HPLC-ICP-MS Standard GC-ICP-MS Standard(ng/g as Sn) uncertainty k = 1 (ng/g as Sn) uncertainty k = 1

Sample n = 4 (ng/g as Sn) n = 4 (ng/g as Sn)

1 827 19 853 122 805 38 846 133 845 9 838 8

Mean 826 22 846 11

Expanded uncertainty (k = 2) ±87 ±39

31


System Summary

LC System

Binary gradient

Detection

DAD

Columns

ZORBAX SB-C18, SB-CN, 2.1 mm × 150 mm, 5 µm

Column part number

883700-883700-922

Major Analytes

Nitroaromatics explosives

Matrix

Standard

Reference

Robert Ricker, “Qualitative and Quantitative Analysis of Explosives and Related Compounds Using Polar and Non-polar HPLC Columns,” Agilent Technologies publication5988-6345EN, www.agilent.com/chem

-0.002

0.000

10.00 20.00 30.00Minutes

Courtesy of Th. Renner, Hess. Landesanstalt f. Umwelt, Dez. III/2, P.O. Box, 65022 Wiesbaden, Germany

1

2 3

4

5

6

7

8

9

10

11+12

13

1415

16

17

18

121

2 3

4

5

6

7

8

10

11

16

17

18

19

13+9

14+15

10.00 20.00 30.00Minutes

40.00

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

0.022

0.024

0.026

0.028

0.030

0.032

0.034

0.036

0.038

0.042 0.36

0.34

0.32

0.30

0.28

0.26

0.24

0.22

0.20

0.18

0.16

0.14

0.12

0.10

0.08

0.06

0.04

0.02

0.00

0.040ZORBAX SB-C18 ZORBAX SB-CN

Conditions

Column ZORBAX SB-C18, SB-CN (2.1 mm × 150 mm) (Agilent P/N: 883700-905, 883700-922)

Mobile Phase A: ACN + 5% H2O + 5 mM CF3COONH4

B: H2O + 5% ACN + 5 mM CF3COONH4

pH 2.7 (CF3COOH)

Inject 10 µL of 19 nitromethanes in ACN:H2O (20:80), 5 mM CF3COONH4;

Flow rate 0.23 mL/min, 18°C, Detection UV (210, 240, 360 nm)

32

mAU

0

10

20

30

40

50

60

70

80 DAD1 A, Sig = 360,40 Ref = 540,40

MSD1 TIC, APCI, Neg, Scan

0 5 10 15 20 25 30 35 min

0

100000

200000

300000

400000

500000

600000

700000

0 5 10 15 20 25 30 35 min

C1

C2

C3K+ACR

C3CR

BZC4 TOL

C5

C6MEK + MTH

209

223235 + 237

237249

285251

299265

279249 + 251

Chromatographic ConditionsColumn Nucleosil 100-5 C18 HD,

3 mm × 250 mm, 5 µm Guard column Phenomenex Security Guard C18,

3 mm id × 4 mmMobile phase A = water

B = acetonitrileGradient Start with 49% B

at 26 min 49% Bat 40 min 100% B

Post-time 5 minutesFlow rate 1.0 mL/minColumn temp 38 °CInjection vol 20 µLDiode-array Signal: 360, 40; 385, 40; 430, 40 nm detector Reference: 540, 40 nmMS ConditionsSource APCIIonization mode NegativeVcap 1500 VCorona current 10 µANebulizer 60 psigDrying gas flow 4 L/minDrying gas temp 350°CVaporizer temp 500°C Scan 125–600 amuThreshold 150 countsGain 5Step size 0.1 amu Peak width 0.1 minTime filter OnFragmentor 50 V

Figure 1. Liquid chromatography analysis of a mixture of the DNPH derivativesof 13 carbonyls by ultraviolet absorption at 360 nm (diode array detec-tor, top) and by atmospheric pressure negative chemical ionizationmass spectrometry (total ion current, bottom): C1, formaldehyde; C2, acetaldehyde; C3K, acetone; ACR, acrolein; C3, propanal; CR, croton-aldehyde; MEK, 2-butanone; MTH, methacrolein; C4, butanal; BZ, benzaldehyde; C5, pentanal; TOL, m-tolualdehyde; C6, hexanal.

System Summary


DetectionDAD MSD negAPCI

ColumnsNucleosil C18, 3 mm × 250 mm, 5 µm

Column part numberSee application note

Major Analytes

DNPH aldehydes

Matrix

Air, Brazil

Reference

John M. Hughes et al, “LC/DAD/MS Analysis of Carbonyl(2,4-Dinitrophenyl)hydrazones,” Agilent Technologies publication 5968-8850E, www.agilent.com/chem


Sample Collection and Preparation

Carbonyl-DNPH standards were synthesized in our labora-tory as described previously. [2, 3] Carbonyls were pur-chased from commercial suppliers (Aldrich Chemical Co.,Lancaster Synthesis, Wiley Organics, Fluka Chemical Corp.)or were prepared as described in previous work. [2, 3]

Air samples were collected by drawing air at 1 liter/minutethrough C18 Sep-Pak cartridges (Waters Corporation)impregnated with (2,4-dinitrophenyl)hydrazine/phosphoricacid. [4] Collected carbonyl compounds were derivatized to(2,4-dinitrophenyl)hydrazones on the cartridge, and werethen eluted with 2 mL of acetonitrile. The eluate was ana-lyzed directly by LC/MS. The sample can be concentratedfor the analysis of the higher molecular weight carbonyls,which are present at lower levels in ambient air.

33

1.000.00 2.00 3.00 4.00 5.00 6.000

10000

20000

30000

40000

50000

60000

70000

80000

7.00

Time (min)

Cou

nts

AlF+2

Al+3

Table 3 Results Obtained for Fluoride in Water Samples Using ICP-MS

Figure 3. Typical calibration curve of fluoride.

System Summary

LC SystemIC

DetectionICP-MS

ColumnsDionex IonPac

Column part numberHPIC-CG2

Major Analytes

Inorganic fluoride

Matrix

Water

Reference

Maria Montes Bayon, “Indirect Determination of FluorideTraces in Natural Waters by Ion Chromatography and ICP-MS Detection,” Agilent Technologies publication 5968-8232E, www.agilent.com/chem


Conc. found Conc. SpikedWater sample (n=3) ICP-MS found FISE amount Recovery(dilution factor) (ng/g–1) (ng/g–1) (ng/g–1) (%)

Fontecelta (200) 8050±80 7700 4300 104

Font-Vella (10) 182±2 – 205 97.8

Tap-water (10) 161±1 – 210 90

*Sea-water (100) 1030±60 1080 1080 97.5

Figure 2. Chromatogram corresponding to 20 ng/g–1 F–.

y = 38515x + 128019R2 = 0,9986

0,E+00

5,E+05

1,E+06

2,E+06

2,E+06

3,E+06

0 10 20 30 40 50 60Conc. fluoride (ppb)

Pea

k a

rea

34

-10.0

Abs

orba

nce

[mA

U]

-12.5

-15.0

-17.5

-20.0

-22.5

-25.0

-27.5

-30.0

-32.5

3.5

1

2

3

4

5

6

7

8

9

10

4 4.5 5 5.5

Time [min]

6 6.5 7 7.5

1 Sulfate2 Formate3 Malate4 Hypophosphite5 EDTA

6 Phosphite7 Acetate8 Copper9 Nickel10 Lactate

ConditionsSample Electroless nickel- and copper-plating

bath, 1:500 diluted with waterInjection 8 s at 50 mbarCapillary Fused silica capillary

Total length 80.5 cmEffective length 72 cmInternal diameter 50 µm

Buffer Agilent Plating Bath Analysis BufferVoltage –25 kVTemperature 20 °CDetection Signal 350/20 nm

Reference 275/10 nm

System Summary

LC SystemCE

DetectionDAD

ColumnsSee application note


Major Analytes

Anions, metals

Matrix

Plating bath

Reference

Tomoyoshi Soga and Maria Serwe, “Monitoring of Electroless Plating Baths by Capillary Electrophoresis,” Agilent Technologies publication 5968-5761E, www.agilent.com/chem


Figure 1. Analysis of electroless nickel- or copper-plating baths.

Electroless plating is mainly used for the plating of non-metals, for example, ceramics and plastics, and allows theplating of complex shaped parts with a uniform film-thick-ness. In addition to metal cations, the bath solutions con-tain additives such as reducing agents (which drive theplating reaction) and organic acids (as buffering and/ormetal complexing agents). Inorganic anions are also pre-sent as counter-ions of the plating metals. These ions caneasily be monitored using capillary electrophoresis (CE)with indirect UV detection.

35

SO42-

SO42-

Cl-

Cl-

S2O32-

S2O32-

SO32-

SO32-

S2-

S2-

Green liquor

Time [min]

0.5 1 1.5 2 2.5 3

Absorbance[mAU]

0

5

10

15

-5

-10

White liquor

ConditionsSample Green and white Kraft liquorInjection 1 sec at 50 mbar sample, dip inlet in

water2 sec at 50 mbar buffer

Capillary Effective length 24.5 cmTotal length 33 cmInternal diameter 50 µm

Buffer 2.25 mM pyromellitic acid6.5 mM sodium hydroxide0.75 mM hexamethonium hydroxide1.6 mM triethanolamine, pH 11.2

Voltage –18 kVTemperature 35 °CDetection Signal 350/50 nm

Reference 235/10 nmPreconditioning 2 min flush with 0.1 N NaOH

4.2 min flush with buffer, at 1 bar each

System Summary

LC SystemCE

DetectionDAD

ColumnsSee application note


Major Analytes

Inorganic anions, miscellaneous

Matrix

Pulping liquor

Reference

Maria Serwe, “Analysis of Sulfur Anions in Kraft LiquorsUsing Capillary Electrophoresis,” Agilent Technologies publication 5968-3306EN, www.agilent.com/chem


Figure 1. Analysis of green and white Kraft liquors.

The proper function of Kraft recovery furnaces is a veryimportant part of the chemical recovery process at a Kraftpulp mill. In the recovery furnace, sulfur anions such as sul-fate in the black liquor, are converted to sulfide anions inthe green liquor. Sulfide anions in the pulping reactions pro-duce stronger paper. If the concentration is too low, thepaper produced loses its strength. If the sulfide concentra-tion is very high, polluting emissions from the Kraft recoveryfurnaces are increased. Wet chemical tests and ion chro-matography (IC) have traditionally been used to analyzeprocess liquors. Capillary electrophoresis (CE) is a worthyreplacement for IC due to less buffer consumption, lessmaintenance, and considerably shorter run times.

36

RF power 1300 WRf reflected power < 1 WPlasma gas 16.0 L/minAuxillary gas 1.00 L/minCarrier gas flow 1.06 L/minSampling depth 7.5 mmMass 79 amu (Br), 127 amu (I)Integration time 0.5 sNumber of scans 1

0 2.5 5 7.5 10

0 2.5 5Retention time/min

7.5 10

79:Br

127:I

2000BrO3

_

IO3

_

I_

Br_

ICP-

MS

sign

al/c

ount

s 1000

0

5000

2500

0

Figure 1. Chromatograms of halogen anion standards by IC-ICP-MS. Peaks:BO3 (10 µg/L), Br (10 µg/L), IO3 (1 µg/L), and I (2 µg/L). Experimental conditions: column, Excelpak ICS-A23; mobile phase, 0.03 mol/L (NH4)2CO3; flow rate, 1.0 mL/min; column temperature, 40 °C; injection volume, 0.5 mL.


Major Analytes

Bromate, iodate

Matrix

Ozone-treated water

Reference

Michiko Yamanaka, “Specific Determination of Bromate andIodate in Ozonized Water by Ion Chromatography with TwoDetection Methods: Post-Column Derivatization and ICP-MS Detection,” Agilent Technologies publication 5968-3049EN, www.agilent.com/chem

System Summary

LC SystemYokagawa IC

DetectionICP-MS or DAD post-column derivatization

ColumnsIC, see application note

Column part numbersSee application note

79:Br

I_

IO3

_

BrO3

_

ArK+

Br_

0 2.5 5 7.5 10

0 2.5 5Retention time/min

7.5 10

127:I

2000

ICP-

MS

sign

al/c

ount

s 1000

0

5000

2500

0

Figure 4. Chromatograms of ozonized water (sample E). Peaks: BO3 (1.87 µg/L),Br (5.73 µg/L), IO3 (5.45 µg/L), and I (0.05 µg/L).Experimental conditions are same as those given in Figure 1. Unit of theconcentrations is µg/L as species.

Determined concentrations[µg/L as species]

IC-ICP-MS Post-columnSamples BrO3

– Br– IO3– I– BrO3

– IO3–

A. Raw water 0.26 28.9 0.44 0.63 0.29 0.09B. Ozonized sample A 13.0 17.8 3.57 3.56 15.7 4.13C. Raw water 1.64 59.1 1.26 2.97 1.65 0.60D. Ozonized sample C 1.88 38.5 5.66 0.14 2.31 4.98E. Ozonized water 1.87 5.73 5.45 0.05 1.85 4.77

Table 3. Comparison of determined concentrations of halogen anions in rawand ozonized water.Experimental conditions are same as those given in Figure 1 and Table 2.

Table 1. Operational Conditions of ICP-MS

Ion ChromatographyColumn Excelpak ICS-A13 × 2Mobile phase 5 × 10–3 mol/L Na2CO3/1 × 10–3 mol/L

NaHCO3, 1.0 mL/minColumn temperature 40 °CInjection volume 0.1 mL

Reagent PreparationReagent 5 mg/L NaNO2 in 0.5 mol/L NaBr,

1.0 mL/minPreparation reagent 0.75 mol/L H2SO4, 1.0 mL/minCation hollow fiber 5 m

Post-column DerivatizationReaction coil 3 m × 0.5 mm idReaction temperature 40 °CDetection UV-268 nm

Table 2. Operating Conditions of Post-column Derivation

37

0 2 4 6 8

Time [min]

10 12 14

500

400

300

200

100

1

2

3

4

1 Cyclohexane2 0-Xylene3 1-Methylnapthalene4 Phenanthrene

Switch ofbackflushvalve

Norm.

ConditionsColumn 200 × 4.6 mm AP NH2, 5 µmMobile Phase HeptaneFlow Rate 0.8 mL/minInjection Vol 2 µLOven Temp 20 ºCBackflush on 5 min refractive index detector Sample preparation 1 g diesel sample diluted to 10 mL

with heptane


System Summary

LC SystemIsocratic with switching valve

DetectionRID

Columns4.6 mm × 200 mm AP NH2, 5 µm

Column part numberRecommend 4.6 mm × 250 mm ZORBAX NH2

Major Analytes

HGA hydrocarbons, aromatics

Matrix

Oil, fuels

Reference

Angelika Gratzfeld-Huesgen, “Analysis of Aromatic Hydro-carbons in Middle Distillates with HPLC Using IP StandardMethod 391/95,” Agilent Technologies publication 5965-9044E, www.agilent.com/chem

Figure 3. Repeatability – overlay of 5 runs.

43

2

1




o-Xylene, ADC1 AArea = 2044.21111*Amt+11.755183Rel. Res%(4): 2.997

0

4 3

2

1

0

43

2

1

0 2 4

Amount [g/100 mL]Amount [g/100 mL]

Amount [g/100 mL]

8000

6000

4000

2000

0

1-Methylnapthalene, ADC1 AArea = 3565.76887*Amt -0.0649756Rel. Res%(4): 4.967

Phenanthrene, ADC1 AArea = 4851.34241*Amt -6.1479635Rel. Res%(4): 4.967

Area

14000

10000

6000

2000

0

Area

2000

1500

1000

500

0

Area

Figure 2. Linearity.

38

Time [min]

mAU

0

5

10

15

20

25

30

35

2 18 °C Column temp.

25 °C Column temp.

30 °C Column temp.

13

5

4

6

978

10

11

1214

13

15

16

50 10 15 20 25 30

ConditionsColumn 250 mm × 2.1 mm PAH column, 5 mm

(Agilent part no. 79918PAH-582) Buffer A: waterBuffer B: acetonitrile

Temperature 18 °C, 25 °C, 30 °C; see figure


Gradient 50% B to 60% in 3 min

to 90% in 14.5 min

to 95% in 22.5 min

Detector Sample wavelength 270 nm, bandwidth 40 nm

Samples See Table 1

System Summary


DetectionDAD/FLD

ColumnsPAH 2.1 mm × 250 mm, 5 µm

Column part number79918PAH-582


Major Analytes

Polynuclear aromatic hydrocarbons (PAHs)

Matrix

Soil (SFC extract)

Reference

Angelika Gratzfeld-Hüsgen and Rainer Schuster, “ImprovedData Quality in the Automated HPLC Analysis of PNAs(PAHs),” Agilent Technologies publication 5964-3540, www.agilent.com/chem

Figure 3 Separation of DIN/EPA standards at different column temperatures.

Table 1. Precision of Retention Times at 18 °C Column Temperature,with Ambient Temperature of 25 °C

Results

Impact of column temperature on separation

We investigated the impact of column temperature on theseparation at three different temperatures: 30, 25 and 18 °C.The 16 EPA compounds could be separated at all tempera-tures (see Figure 3); however, a temperature of 18 °C hadseveral advantages:

• The resolution between critical compound pairs such asbenzo(ghi)perylene and indeno(1,2,3-cd)pyrene wasbetter. This allowed trouble-free switching of excitationand emission wavelengths when using a programmable fluorescence detector.

• Additional interfering compounds such as anthrachinoncould be separated from the PNAs. Figure 3 clearlyshows the improved separation of PNAs far below 30 °C.

Peak RSD tR % RSD areanumber Name of compound (15 runs) (15 runs)

1 Naphthalene 0.12 1.412 Anthrachinon 0.05 3.703 Acenaphthylene 0.10 3.514 Acenaphthene 0.09 3.715 Fluorene 0.08 1.766 Phenanthrene 0.06 1.727 Anthracene 0.05 1.428 Fluoranthene 0.05 1.409 Pyrene 0.05 1.6210 Benzo(a)anthracene 0.05 1.5911 Chrysene 0.06 1.6012 Benzo(b)fluoranthene 0.07 1.9013 Benzo(k)fluoranthene 0.08 1.9614 Benzo(a)pyrene 0,08 1.7615 Dibenzo(a,h)anthracene 0.09 1.6216 Benzo(ghi)perylene 0.09 1.9917 Indeno(123-cd)pyrene 0.11 2.50

39

System Summary


Detection

MSD posESI with FMOC derivatization

Columns

Inertsil ODS3 2.1 mm × 150 mm, 5 µm

Column part numberRecommend SB-C18 or XDB-C18 chemistry

Regulated/Hazardous Natural Toxin Substances

Major Analytes

Anatoxin A, alkaloid neurotoxin

Matrix

Drinking water

Reference

Masahiko Takino, “Analysis of Anatoxin-a in Drinking Waterby Automated On-Line Derivatization Electrospray LC/MS,”Agilent Technologies publication 5968-3796E, www.agilent.com/chem

Figure 1. Chemical reaction of anatoxin-a with FMOC.

Figure 2. Total ion chromatogram and mass spectrum of derivatized anatoxin-a (1 µg/ml).

+

OH

N –HCI20

ON

FMOC Anatoxin-a

CH2OCOCI CH2OCO

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

80000

0

160000

240000

320000

400000

Minute

388 (M+H)210

160 220

100

50

5280 340

m/z400 460 520

179

410 (M+Na)

40

CH3CH3

NH

Z

OCH3 CH3

CH3 CH3

O

NH

X

NH

O

N CH2

NH

O

CH3

CH3

COOHH

H R1

R2

COOH

H3C

H

System Summary


DetectionMSD posESI

ColumnsMytisil ODS 2.1 mm × 100, 5 µm

Column part numberRecommend SB-C18 chemistry

Regulated/Hazardous Natural Toxin Substances

Major Analytes

Microcystins, algal toxins

Matrix

Fresh (surface) water

Reference

Masahiko Takino and Yutaka Kyono, “LC/MS Analysis ofMicrocystins in Fresh Water by Electrospray Ionization,” Agilent Technologies publication 5968-2123E, www.agilent.com/chem

Figure 1. Structure of microcystins.

200000

Abundance

1 Microcystin-RR2 Microcystin-YR3 Microcystin-LR

12

3

100000

5.00 7.00 9.00 11.00 13.00Retention time [min]

15.00 17.00 19.00

Figure 2. Total ion chromatogram of microcystin standards at 1 ng/µL.

Chromatographic ConditionsGuard column Mytisil ODS (10 mm × 2.1mm 5 µm)Column 10 × mm 2.1 mm Mytisil ODS, 5 µmMobile phase A = 0.2% formic acid in water

B = acetonitrileGradient conditions Start with 10% B

at 20 min 55% B Flow rate 0.6 mL/minInjection volume 100 µL

MS ConditionsSource ESIIon mode PositiveVcap 4000 VNebulizer 50 psigDrying gas flow 10 L/minDrying gas temp 350 °CScan range 100–1200 amuSIM target ions 520, 1045, 995Fragmentor Variable 120 V (520); 160 V (995, 1045)Step size 0.1 amuPeak width 0.15 min

X Z R1 R2 MW

Microcystin-RR Arg Arg CH3 CH3 1037Microcystin-YR Tyr Arg CH3 CH3 1044Microcystin-LR Leu Arg CH3 CH3 994

41

System Summary



ColumnZORBAX Eclipse XDB-C8, 4.6 mm × 150 mm, 5 µm


Major analytes

600 pesticides screening

Matrix

Fruit, vegetable

Reference

E. Michael Thurman, Imma Ferrer and Jerry A. Zweigenbaum,“Automated Screening of 600 Pesticides in Food by LC/TOFMS Using a Molecular-Feature Database Search,” AgilentTechnologies publication 5989-5496ENwww.agilent.com/chem

Regulated/Hazardous Pesticide/Herbicide Substances

LC/MS TOF Methods• LC pumps: Agilent 1100 binary pumps, injection volume

50 µL with standard Agilent 1100 ALS

• Column: ZORBAX Eclipse XDB-C8, 4.6 × 150 mm, 5 µm(p/n 993967-906)

• Mobile Phase A = 0.1% formic acid in water, and B = acetonitrile, gradient began with 5 minutes isocraticat 10% B followed by a linear gradient to 100% B in 30 minutes at a flow rate of 0.6 mL/min

• Agilent 6210 LC/MS TOF with dual spray electrospray source

• Positive ESI, Capillary 4000 V

• Nebulizer 40 psig, drying gas 9 L/min, gas temp 300 °C

• Fragmentor 190 V, skimmer 60 V, Oct DC1 37.5 V, OCT RF V 250 V

• Reference Masses: mass range (m/z) 121.0509 and922.0098, resolution: 9500 ± 500 at m/z 922.0098, m/z 50 to 1,000, Reference A Sprayer 2 is constant flow rate during the run

S

O

CH3

P

H3CO

SH

HN

OCH3

C5H13NO3PS2+

Exact mass: 230.0069

Figure 1. Dimethoate mass spectrum showing low intensity of MH+ ion and importance ofusing characteristic fragment ions to lower LODs on some compounds of lowintensity, specifically, m/z 124.9819 ion.

Figure 2. Black pepper sample showing complexity of the sample ~3000 accurate masspeaks were detected in this sample at signal to noise of 10:1 or greater.

42

×106

0

1

2

3

4

5


3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8

-EIC (161.0 m/z) MRM (247.0 m/z); from mont_neg_8000.d

Clopyralid

Picloram

Dicamba

MCPA

2, 4-D

MCPP

2, 4-DB2, 4-DP

Triclopyr

InstrumentationLC 1200 LC

Column ZORBAX Extend-C18, RRHT,2.1 mm × 100 mm, 1.8 µm

Column temperature 60 °CMobile phases A: 0.04% Glacial acetic acid in water

B: Acetonitrile (ACN)Flow rate: 0.3 mL/minInjection volume: 1.0 µL

Gradient Time, min. %B0 01 402 523 604 1008 1009 0

MS G6410A QQQIonization ESI (–)Mass range 120 to 400 amu Capillary 3500 VNebulizer pressure 40 psiDrying gas flow 9 L/min Gas temperature 200 °CSkimmer 35 V

MRM parameters are listed in Table 2

System Summary


DetectionMS QQQ negESI

ColumnZORBAX Extend C18, 2.1 mm × 100 mm, 1.8 µm


Major analytes

Chlorophenoxy acid herbicides

Matrix

Soil

Reference

Chin-Kai Meng, “Determination of Chlorinated Acid Herbicides in Soil by LC/MS/MS,” Agilent Technologiespublication 5989-5246EN, www.agilent.com/chem


Figure 1. Overlaid MRM results from the nine selected herbicides.

Continued

43

×102

×102

×102

×102

×102

×103

×103

×103

×103

13

Abundance vs. Acquisition Time (min)

3.4 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 6

-EIC (146.0 m/z) MRM (190.0 m/z); from mont_neg_10.d Smooth (1)3.522

0.5

1.5


0

1

-EIC (175.0 m/z) MRM (219.0 m/z); from mont_neg_10.d Smooth (1)

4.379

0

4


13


0.5

1.5-EIC (196.0 m/z) MRM (254.0 m/z); from mont_neg_10.d Smooth (1) 5.327

2

6 -EIC (161.0 m/z) MRM (233.0 m/z); from mont_neg_10.d Smooth (1) 5.481

12

-EIC (141.0 m/z) MRM (213.0 m/z); from mont_neg_10.d Smooth (1) 5.497

0.51

-EIC (161.0 m/z) MRM (247.0 m/z); from mont_neg_10.d Smooth (1) 5.670

Clopyralid, 7.4 pg

Picloram, 2.3 pg

Dicamba, 10.3 pg

2,4 -D, 2.2 pg

MCPA, 6.9 pg

Triclopyr, 1.6 pg

2,4 -DP, 1.8 pg

MCPP, 3.4 pg

2,4 -DB, 8.6 pg

Figure 2. MRM results.

44

System Summary

LC System1200SL dual binary with 6-port valve for autoSPE

DetectionDAD

ColumnZORBAX SB-C18, 5-, 3.5-, and 1.8-µm columns


Major analytes

Chlorophenoxy acid herbicides

Matrix

Water

Reference

Michael Woodman, “Rapid Analysis of Herbicides by Rapid Resolution LC with Online Trace Enrichment,” Agilent Technologies publication 5989-5176EN,www.agilent.com/chem


min10 12 14 16 18 20 22 24 26 28

mAU

0

20

40

60

80

100

120

12.

679

- Pic

lora

m

13.

303

14.

436

- Chl

oram

ben

17.

653

17.8

45 -

Dic

amba

18.

895

- Ben

tazo

n

19.

480

- 2,4

-D 2

0.23

8

21.

104

- Dic

hlor

prop

23.

095

- 2,4

,5-T

P

24.

678

- A

ciflu

orfe

n

29.

609

min1.25 1.5 1.75 2 2.25 2.5 2.75 3 3.25

mAU

0

100

200

300

400

500

600

700

1.1

21

1.5

32

1.6

43 1

.672

2.0

48

2.1

61 2

.206

2.3

10 2

.385

2.5

77 2

.626

2.7

23

2.8

30

3.0

25

ConditionsEPA Method 555 with ZORBAX SB-C18 columns and fast DADdetector ZORBAX SB-C18 4.6 mm × 250 mm, 5 µmColumn temperature 25 °CGradient 25 mM H3PO4, ACN,

10% to 90% ACN in 30 min7.8% ACN/column volume

Analysis flow rate 1 mL/min Group A compound 1 µL of 100 µg/mLTotal analysis time 60 minDetection UV 230 nm, 10-mm 13-µL flow cell,

filter 2 seconds (default)

Figure 2. Gradient separation of herbicides on 4.6 mm × 250 mm, 5 µm ZORBAX SB-C18.

ConditionsEPA Method 555 with ZORBAX SB-C18 columns and fast DADdetectorColumn ZORBAX SB-C18,

2.1 mm × 80 mm, 50 mm, and 30 mm; 1.8 µm

Column temperature 50 °C Gradient 25 mM H3PO4/ACN, 10% to 90% ACN

in 2.7 min7.8% ACN/column volume

Analysis flow rate 0.72 mL/minDetection UV 230 nm, 3-mm 2-µL flow cell,

filter 0.2 secondsSample Aged 1 µL 100 µg/mLTotal analysis time 6 min

Figure 5. High-speed gradient separation of herbicides on 2.1 × 80 mm, 1.8-µmZORBAX SB-C18.

45

System Summary


DetectionMS TOF, MSn ion trap both posESI

ColumnZORBAX Eclipse XDB-C18, 2.1 mm × 50 mm, 3.5 µm


Major analytes

Various, including trimethoprim

Matrix

Sediments

Reference

Paul Zavitsanos, “Finding and Confirming Identification ofUnknowns in Sediment Samples by LC/TOF and LC/TrapMS,” Agilent Technologies publication 5989-0815EN, www.agilent.com/chem


0.0

1.0 2.0 3.0 4.0 5.0

Time (min)

6.0 7.0 8.0 9.0 10.0 11.0

1.0e6

2.0e6

3.0e6

4.0e6

5.0e6

6.0e6

7.0e6

8.0e6

9.0e6

0.0

1.0 2.0 3.0 4.0 5.0

Time (min)

6.0 7.0 8.0 9.0 10.0 11.0

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

0.45

2.11

Trimethoprim

0.360.45

0.77

1.04 1.52

4.17

4.57

5.08

5.66

6.20 6.68 7.078.48

9.72 9.98 10.60

2.70 3.11 3.70

4.084.32

4.79

5.27

5.66

6.04

6.23

6.45

7.09

7.447.95

8.08

8.388.59

9.209.83

10.2010.62

Inte

nsity

(cps

)In

tens

ity (c

ps)

2A

2B

Figure 2. The TIC (2A) and EIC (2B) of the sediment extract. Ion extraction was set for 291.12 to 291.16 m/z.

LC ConditionsMobile phases A = 10 mM ammonium formate, @ pH 4.5

B = acetonitrile (ACN)

Gradient 5% B to 90% B over 7 min, hold 2 min at

90% B, and return to 5% B by 10 min


Column ZORBAX Eclipse XDB C-18,

2.1 mm × 50 mm, 3.5 µm

MS Conditions

Instrument Agilent LC/TOF in positive ion

ESI and Agilent LC/Ion TRAP XCT in positive

ion ESI, depending on experiment

Nebulizer pressure 55 psi

Vcap 3500 V

Sample injection 2 to 20 µL depending on sample

Samples A 20-drug standard

Sediment sample: A methanol/water solu-

tion derived from a lake sediment, extracted

by the USGS

46

System Summary

LC SystemDual binary with 6-port valve for autoSPE

DetectionMSD pos/neg APCI

ColumnZORBAX XDB-C8, 2.1 mm × 50 mm, 3.5 µm


Major analytes

Phenylurea, triazine herbicides

Matrix

Water

Reference

Neil Cullum and Pete Stone, “Determination of Phenyl Urea and Triazine Herbicides in Potable and Groundwater by LC/MS Using API-ESI Selective Ion Monitoring andDirect Large-Volume Aqueous Injection,” Agilent Technologies publication 5989-0813EN, www.agilent.com/chem


3

8

45 1

6 10

2

7 9

12

54

63

Right heat exchanger

Detector

Analytical column

Left heat exchanger

Precolumn

Column compartment

Autosampler valve

Pump 1

Pump 2

Waste

Waste

Metering head

Needle seat + 400 µL loop

Figure 1. Flow path diagram.

Flow Path DiagramThe flow path through the column compartment 10-portvalve and the wellplate 6-port autosampler valve is shownin Figure 1.

Minute12 12.5 13 13.5 14 14.5 15 15.5 16 16.5

0

5000

10000

15000

20000

25000

30000

35000

40000 1

4.11

5

Atrazine

MSD1 216, EIC=215.7:216.7 (VAL3_4\PT3_4026.D) API-ES, Pos, SIM, Frag: 70 (TT)

Figure 2. Spiked tap water containing 0.1 µg/L of atrazine.

MS ConditionsIonization mode Positive/Negative API-ES

Drying gas flow 13.0 L/min


Drying gas temperature 350 °C

Vcap voltage 3000V (Positive); 2500 (Negative)

Selected Ion Monitoring [SIM] Table parameters

[Positive ion mode]

Table 2. Mass Spectrometer Conditions

Continued

47

Table 3. Method Performance Data

Borehole raw water Potable treated water

Compound %Recovery %RSD %Recovery %RSD LOD (µg/L)

Metamitron 101.0 4.9 101.5 3.8 0.0039

Chloridazon 99.4 6.7 98.4 4.1 0.0055

Monuron 102.1 4.7 98.4 4.9 0.0038

Carbetamide 100.6 6.4 96.4 6.2 0.0054

Simazine 101.7 5.1 98.2 4.0 0.0049

Cyanazine 99.5 4.2 99.3 5.3 0.0073

Chlortoluron 99.2 5.3 99.3 5.3 0.0044

Atrazine 99.1 3.4 97.0 3.3 0.0020

Diuron 100.8 4.6 97.7 3.8 0.0042

Isoproturon 100.8 4.3 98.5 4.9 0.0037

Linuron 102.5 5.1 99.9 3.1 0.0075

Propazine 101.4 5.0 100.5 4.3 0.0035

Terbuthylazine 100.5 6.3 99.5 3.8 0.0038

Trietazine 102.5 5.4 100.5 4.3 0.0043

Prometryn 102.0 4.8 101.1 4.5 0.0045

Terbutryn 102.3 6.0 100.8 4.0 0.0054

48

System Summary


DetectionMSD pos/neg ESI



Major analytes

Herbicides phenylurea, triazine

Matrix

Water

Reference

Neil Cullum and Paul Stephens, “Validated Method for theDetermination of Phenylurea and Triazine Herbicides inPotable and Groundwater by LC/MS Using Selective IonMonitoring,” Agilent Technologies publication 5988-8595EN,www.agilent.com/chem


min4 6 8 10 12 14 16 180

100000

200000

300000

400000MSD1 TIC, MS File (T:\AGILEN~2\922259~1\DATA\PT1\SAMPLE03.D) API-ES, Pos, SIM, Frag: 70 (TT)

12.9

02

1 23 4

5 + 6 7

89

12 13

14

15 + 16

Figure 1. Chromatogram for the low level standard in positive ion mode.

MS ConditionsIonization mode Positive/negative API-ESDrying gas flow 13.0 L/minNebulizer pressure 40 psigDrying gas 350 °CtemperatureVcap voltage 3000 V (positive), 2500 V (negative)

The selected ion monitoring (SIM) ions and fragmentor voltageslisted in the SIM table parameters of Table 1 were all optimizedusing Flow Injection Analysis (FIA). Ten mg/L standard solutionsof each herbicide were injected using scan mode 150 – 400 amuand the fragmentor voltage was ramped from 70 to 150 V in stepsof 5 V.

Continued

LC ConditionsColumn ZORBAX Eclipse XDB-C8

50 mm long × 2.1 mm id, 3.5 µm particles, 40 °C

Flow rate 0.5 mL/minInjection volume 25 µLMobile phase A = 0.001% formic acid in water

B = Methanol

A BGradient program Initial 90% 10%

2.0 min 90% 10%15.0 min 30% 70%15.1 min 90% 10%22.0 min 90% 10%

49

Groundwater sample Potable water sampleCompound Recovery % RSD % Recovery % RSD % LOD µg/L

Atrazine 83.4 5.1 83.3 5.6 0.00146Carbetamide 92.0 7.5 88.5 7.8 0.00544Chloridazon 94.8 5.8 94.0 4.6 0.00293Chlortoluron 89.5 3.9 89.5 4.6 0.00219Cyanazine 95.1 4.3 96.3 4.7 0.00352Diuron 92.7 4.6 94.3 4.5 0.00348Isoproturon 93.0 2.6 93.2 3.3 0.00209Linuron 89.4 4.9 91.0 4.6 0.00330Metamitron 102.4 2.8 102.8 3.1 0.00257Monuron 96.6 4.6 97.3 4.8 0.00221Prometryn 81.8 4.6 82.6 4.4 0.00208Propazine 85.8 5.8 85.6 6.2 0.00218Simazine 91.0 4.7 91.1 4.8 0.00179Terbuthylazine 78.1 8.3 80.2 6.8 0.00397Terbutryn 81.9 4.4 83.6 4.6 0.00268Trietazine 79.1 4.7 78.7 4.8 0.00179

Table 2. Results

50

System Summary

LC SystemDual binary with 6-port valve for autoSPE

DetectionMSD posESI

ColumnZORBAX Extend-C18, 2.1 mm × 150 mm, 3.5 µm


Major analytes

Paraquat, Diquat, amitrole, chlormequat

Matrix

Water

Reference

Neil Cullum and Paul Stephens, “Rapid Screening Methodfor the Analysis of Paraquat and Diquat by LC/MSD UsingSelective Ion Monitoring and Large Volume Injection,” Agilent Technologies publication 5988-7220EN, www.agilent.com/chem


N+

N+

CH3CH3

N+

N+

CH3 C 2 Br_

2 CI_

H3

Paraquat Diquat

Figure 3. Total ion chromatogram of mixed standard, 10 µg/L, 250 µL injection, 2.5 ng on column.

LC ConditionsColumn ZORBAX Extend-C18

150 mm × 2.1 mm id, 3.5 µm, 60 °CFlow rate 0.45 mL/minInjection volume 250 µLMobile phase Isocratic elution

A: 5 mM TDFHA in water (75%)B: Acetonitrile (ACN) (25%)

MS ConditionsIonization mode API-ES, positiveDrying gas flow 13.0 L/minNebulizer gas 30 psigpressureDrying gas 350 °CtemperatureVcap voltage 3500 V

min2 4 6 8 10 12 14

0

100000

200000

300000

400000

500000

MSD1 TIC, MS File (E:\QUATS\QUATS\TAP00001.D) API-ES, Pos, SM, Frag: Var

2.88

6

3.64

6

13.6

57

AmitroleParaquat/Diquat

Chlormequat

10 µg/L mixed standard

250 µL injection

2.5 ng on column

Continued

51

m/z100 150 200

0

50000

100000

150000

200000

250000

300000

350000

*MSD1 SPC, time=16.609:16.796 of E:\QUATS\INDIV\PARAQ000.D API-ES, Pos, Scan, Frag: Var

Max: 374848

171.

1

185.

118

6.1

Figure 2. Mass spectrum of Paraquat.

52

System Summary


DetectionMSD ESI, APCI, APPI



Major analytes

Phenylureas, carbamate pesticides

Matrix

Standard

Reference

Chin-Kai Meng, “Analyzing Phenyl Ureas and CarbamatePesticides Using ESI-, APPI-, and APCI-LC/MSD,” Agilent Technologies publication 5988-6635EN, www.agilent.com/chem


min2 4 6 8 10 12 140

20000400006000080000

100000120000140000

min2 4 6 8 10 12 140

500000

1000000

1500000

2000000

APPI without post-column acetone

APPI with post-column acetone

Carbofuran

Carbamate

Mexacarbate Diuron

Phenyl urea

Monuron

ESI

APPI

APCI

Figure 3. TICs show the effect of adding post-column acetone to enhance ana-lyte signal in APPI. Methanol was used as solvent B for both TICs.

Figure 10. The S/N of four analytes from the three ionization modes.

ESI APCI APPI

Column ZORBAX Eclipse XDB-C8, 4.6 × 50 mm, 3.5 µm (p/n 935967-906)Column temperature 30 °C 30 °C 30 °CColumn flow rate 1 mL/min 1 mL/min 1 mL/minSolvent A H2O, 0.1% acetic acid H2O H2O

or as specifiedSolvent B Acetonitrile, Acetonitrile Methanol

0.1% acetic acidor as specified

Post-column n/a n/a 40 µL/min acetoneadded as dopant

Solvent gradient B: 10% at 0 min, 30% B: 30% at 0 min, 40% at 4 min, 70% at 13 min, at 4 min, 80% at 10 min, 80% at 16 min or as specified in the figure80% at 16 min

Injection volume 2 µL 2 µL 2 µLDrying gas flow 12 L/min 11 L/min 11 L/minDrying gas temperature 350 °C 275 °C 275 °CFragmentor voltage 60 V 110 V 110 VVcap 3500 V 4500 V 4500 VNebulizer pressure 60 psi 35 psi 35 psiVaporizer n/a 225 °C 225 °CStep size 0.1 0.1 0.1Peak width (min) 0.15 0.15 0.15Time filter Off Off OffScan (m/z) 150−800 115−500 115−500Polarity Positive Positive Positive

Table 1. LC/MS Conditions

53

Continued

System Summary


DetectionMSD posESI

ColumnZORBAX Eclipse XDB-C8, 4.6 mm × 50 mm, 5 µm


Major analytes

Glyphosate, AMPA

Matrix

Water

Reference

Paul Zavitsanos, Chin-Kai Meng, Lorna Grey, Bick Nguyen,and Paul Yang, “Analysis of Glyphosate and AminomethylPhosphonic Acid by Liquid Chromatography/Mass Spec-trometry,” Agilent Technologies publication 5988-4981EN,www.agilent.com/chem


LC ConditionsColumn ZORBAX Eclipse XDB-C8,

4.6 mm × 50 mm, 5 µm, 40 °C

Precolumn pump Agilent 1100 binary

Mobile phase A: 50 mM ammonium acetate, aqueous

Mobile phase B: acetonitrile, 0 to 95% in 5 min, hold 3 min

Precolumn flow rate 0.7 mL/min

Sample size injected 1 µL

Post-column pump Agilent 1100 isocratic.Flow 0.3 mL/min of 0.6% formic acid

Binary pump with diode array detector (DAD) and well-plate sampler

Instrument: Agilent 1100 LC/MS with electrosprayionization (ESI) in Positive Ion ModeDrying gas 12 L/min, 350 °C

Nebulizer gas 60 psi

Vcap 3500 V

Fragmentor 100 V for both scan and SIM runs

SIM ions 334 and 392 m/z

Scan range 120 to 1000 m/z

CH2

N H

OHO

CH2

P

OH OH

O

OCl

O

CH2

N

OHO

CH2

P

OH OH

O

O

O

O

NH2CH2

P

OH OH

O

OCl

O

NH

CH2

P

OH OH

O

O

+ + HCl

+ HCl+

Glyphosate (MW = 169)

AMPA (MW = 111) FMOC (MW = 259) AMPA-FMOC (MW = 333)

FMOC (MW = 259) Gly-FMOC (MW = 391)

Figure 1. Derivatization reactions for glyphosate and AMPA with FMOC.

54

m/z300 400 500 600 700 800

0

5

10

15

20

25

30

35

[2M

+H

]+[M+

H]+

[M+

NH

4]+

334.

0

667.

0

351.

0

668.

1

335.

0

352.

0

m/z300 400 500 600 700 800

0

20

40

60

80 [M+

H]+

[2M

+H

]+

392.

039

3.0

783.

1

AMPA

Glyphosate

Figure 2. Mass spectra of target molecules.

55

Continued

System Summary

LC SystemDual binary with Cohesive 2300 valve for autoSPE

Detection

MSn Ion Trap posESI

Column

Metasil Basic, 2.1 mm × 100 mm, 5 µm

Column part numberRecommend ZORBAX Eclipse XDB-C18 chemistry

Major analytes

Sulfonylurea herbicides

Matrix

Surface water

Reference

Robert D. Voyksner and Jennifer A. Townsend, “Applicationof Online Solid-Phase Extraction Ion Trap Mass Spectrome-try in Environmental Matrices,” Agilent Technologies publi-cation 5988-3649EN, www.agilent.com/chem


Time (minutes)10 15 20 25 30 35 40 45

mA

U

0

2

4

6

8

12 3 4 5

678

9

10

1112 13

Time (minutes)10 20 30 40 50

0

25000

50000

75000

100000

125000

150000

175000

200000

225000

1

2 34

56

7

8

9

10

11

12

13

LC/UV of Marsh Water Sample Single-Quadrupole LC/MS of Marsh Water Sample

Figure 3. Chromatographic results for LC/UV (A), LC/MS (B).

0m/z

20

40

60

80

100 186.0

213.0

317.0

369.0

414.9143.1160.1

Abund.

S

O O

OC

O

C O

NH NH

N

N

OCH3

Cl

CH2 CH3

MS/MS of [M+H]+ ion at m/z 415

Chlorimuron ethylM.W. = 414

100 150 200 250 300 350 400 450

Figure 5. LC/ion trap MS/MS mass spectrum of chlorimuron ethyl obtainedfrom a 1 ng/ml spike of sulfonylureas in marsh water. The CID massspectrum was generated from the [M+H]+ ion.

56

[M+H]+ MS/MS Fragment Solvent Standard Marsh WaterCompound (m/z) Ions (m/z) % Recovery % RSD % Recovery % RSD

Flumetsulam 326 129*, 192, 262 92 5.9 58 6.7Oxasulfuron 407 150*, 210, 284, 258 112 9.4 95 8.0Thifensulfuron methyl 388 141, 167* 104 13.0 62 14.0Metsulfuron methyl 382 141, 167* 114 32.0 81 23.0Sulfometuron methyl 365 106, 150*, 199, 333 124 18.0 85 7.2Triasulfuron 402 141, 167*, 359 108 7.5 87 7.6Chlorsulfuron 358 167* 73 19.0 55 9.7Ethametsulfuron methyl 411 142, 168, 196* 110 2.8 97 7.3Sulfosulfuron 471 165, 211*, 265 100 5.9 88 6.1Bensulfuron methyl 411 119, 149*, 182, 213 116 5.2 102 7.1Prosulfuron 420 141*, 167 110 2.0 78 3.3Chlorimuron ethyl 415 186*, 213, 369 101 12.0 74 6.7Triflusulfuron methyl 493 238, 264*, 460 98 3.8 95 3.6

*Ion used for quantitation

Table 1. SPE LC/MS/MS Results (LOQ = 200 pg/mL)

57

System Summary

LC System1200 AESOP

Detection

Open

Column

Open

Column part numberOpen

Major analytes

Various in water sources

Matrix

Water

Reference

“Agilent 1200 Series Sample Enrichment System – ARobust, Fully Automated and Sensitive Solution to MeetToday’s EU Environmental Requirements,” Agilent Technologies publication 5989-5692EN,www.agilent.com/chem

Sample Preparation Techniques

Basic system forneutral compounds only

Basic system forneutral and acid compounds

Enhanced system forneutral and acid compoundswith capacity extension

58

System Summary

LC SystemIsocratic

Detection

DAD

Column

Various organic GPC


Major analytes

EPA 3640A standard mix

Matrix

Vegetable oil, broccoli, animal fat

Reference

M. Woodman, “Optimizing Sample Loading in AutomatedSize Exclusion Chromatography Sample Preparation forSmall Molecule Analysis from Complex Matrices,” AgilentTechnologies publication 5989-0181EN,www.agilent.com/chem

Sample Preparation Techniques

min8 10 12 14 16 18

Norm.

0

200

400

600

800

1000

1200

DAD1 A, Sig=254,4 Ref=off (C:\HPCHEM\2\DATA\CLNUP3\G1CGD103.D)

Collection zone

Corn oil

DEHP

Methoxychlor

Perylene

Sulfur

Figure 1. Chromatogram of a calibration mixture using SEC method EPA 3610A.

min8 10 12 14 16 18

Norm.

0

200

400

600

800

1000

1200



Extractable solids: ~25% w/w50 g sample = ~12 g solids

Corn oil

DEHP

Methoxychlor

Perylene

Sulfur

High extractablemass in meats

Figure 4. Chromatographic comparison of spiked post-GPC sausage extract and EPA test mix.

Continued

59

min5 10 15 20

min5 10 15 20

min5 10 15 20

min5 10 15 20

mAU

0

100

200

300

400

500

600

700

DAD1 A, Sig=254,4 Ref=off (CLNUP5\PL100_03.D)

Variable volumes from 100–600 µL used 6.2-mg load

mAU

0

200

400

600

800

1000


12.5-mg load

mAU

0

200

400

600

800

1000

1200

DAD1 A, Sig=254,4 Ref=off (CLNUP5\JO100_05.D)

25-mg load

mAU

0

200

400

600

800

1000

1200

1400

DAD1 A, Sig=254,4 Ref=off (CLNUP5\JO100_06.D)

37.5-mg load

min5 10 15 20

min5 10 15 20

min5 10 15 20

min5 10 15 20

mAU

0

200

400

600

800

1000


mAU

0

200

400

600

800

1000

DAD1 A, Sig=254,4 Ref=off (CLNUP5\PL1C_083.D)

mAU

0

500

1000

1500

2000


mAU

0

500

1000

1500

2000


Figure 5. LC chromatograms of EPA test mix, as a function of sample loading. Figure 6. LC comparison of EPA test mix using different elution solvents. Fromthe top: DCM, THF, MTBE, and EA/isooctane mix.

60

System Configurations

Application Area – Regulated/Hazardous Drug SubstancesLC solvent Publication

Major analytes delivery system* Detection** Notes number Page

Chloramphenicol 1200SL MS QQQ neg ESI 5989-5975EN 7Pharmaceuticals 1200SL MS QQQ pos/neg ESI 1 5989-5319EN 9Estrogens, steroids Binary gradient MS TOF neg APPI 2 5989-4858EN 10Veterinary pharmaceuticals Quaternary gradient MSD, MSn Ion Trap 3 5989-2980EN 12

Application Area – Regulated/Hazardous Miscellaneous Substances

Crude oil Binary gradient with frac. coll. DAD RID 4 5989-6012EN 13Arsenic speciation Isocratic ICP-MS 5989-5505EN 15VX, Sarin, hydrolysis product Binary gradient DAD, ICP-MS 5989-5346EN 16Disperse dyes, azo dyes Binary gradient MS TOF pos ESI 5989-3859EN 18Methylmercury, mercury, LC/ICP-MS ICP-MS 5989-3572EN 19ethylmercuryAcrylamide Dual binary with 6-port valve MS TOF pos ESI 5989-2884EN 22

for autoSPEChromium speciation IC Metrohm 818 pump, ICP-MS 5 5989-2481EN 24

Agilent 7500 ISIS samplerNitroaromatics explosives Binary gradient MS TOF neg APCI 6 5989-2449EN 24Herbicide, antibiotic, peptide, Infusion MSn Ion Trap, MS TOF, 7 5989-2324EN 26bialaphos, bilanaphos both pos ESIPerchlorate Metrohm IC MSD neg ESI 5989-0816EN 28Organotin compounds LC and GC ICP-MS 5988-6697EN 29Nitroaromatics explosives Binary gradient DAD 5988-6345EN 31DNPH aldehydes Binary gradient DAD MSD neg APCI 8 5968-8850E 32Inorganic fluoride IC ICP-MS 5968-8232E 33Anions, metals CE DAD 5968-5761E 34Inorganic anions, CE DAD 5968-3306EN 35miscellaneousBromate, iodate Yokagawa IC ICP-MS or DAD post- 9 5968-3049 36

column derivatizedHGA hydrocarbons, Isocratic with RID 10 5965-9044E 37aromatics switching valvePolynuclear aromatic Binary gradient DAD/FLD 5964-3540 38hydrocarbons (PAHs)

Application Area-Regulated/Hazardous Natural Toxin Substances

Anatoxin A, alkaloid Binary gradient MSD pos ESI with 11 5968-3796E 39neurotoxin FMOC derivatizedMicrocystins, algal toxins Binary gradient MSD pos ESI 5968-2123E 40

1 Offline SPE2 Offline automated SPE procedure, normal phase cleanup (ZORBAX Extend SB-CN 4.6 × 50 mm 5 µm and guard with propanol/cyclohexane),

GPC cleanup (PLgel 50A in MeCl2), post-column addition of reference mass solution3 ASE prep for soils, SPE trace enrichment for water4 Requires fraction collector, note includes GC/MS fraction analysis5 Column normally bundled with ICP-MS; contact agent/representative for details6 Post-column reference mass addition7 Elaborate structural elucidation via Ion Trap MSMS and TOF accurate mass analysis8 DNPH cartridge for direct air sample collection9 Submitted to Journal of Chromatography A, 789, 259-265 (1997)10 Classic hydrocarbon group analysis per IP391 guidelines11 Online derivatization with FMOC

61

System Configurations (Continued)

* Selection of the injector, including accessory thermostat module, should be based on budget and additional user requirements. Consult your Agilent representative or agent.

Where isocratic is shown, choose an isocratic, binary, or quaternary pump module. Binary and quaternary gradient modules offer greater flexibility. Isocratic pumps are field-upgradeable to quaternary gradient pumps.

Where binary gradient is shown, quaternary gradients may often be substituted when using flow rates 1 mL/min or higher and with most 3 mm and 4.6 mm id columns. For very fast gradients with steep gradient slopes, as in a high throughput analysis environment, the binary gradient module would be more suitable.

** Where DAD is shown, review the original application note carefully to determine if spectral content or multi-wavelength monitoring is required. If not, you may substitute amultiwavelength or variable wavelength detector. Choose standard flow cells for most 4.6 and 3 mm id column applications. For 2.1 mm applications, especially with 1.8 µm particle sizes, consider smaller volume flow cells to minimize extracolumn dispersion.

MS detectors require the user to specify the ionization source (that is, APCI, ESI, multimode ESI/APCI, AP-MALDI, APPI/APCI, dual-spray ESI [TOF], ChipCube ESI, and othersources) for standard, nano, or capillary LC operations.

Not all MS detector models permit concurrent positive/negative polarity switching. Review the original application note carefully and consider future needs to determine ifthis is required.

Application Area-Regulated/Hazardous Pesticide/Herbicide Substances

LC solvent PublicationMajor analytes delivery system* Detection** Notes number Page600 pesticides screening Binary gradient MS TOF pos ESI 12 5989-5496EN 41Chlorophenoxy acid Binary gradient MS QQQ neg ESI 5989-5246EN 42herbicidesChlorophenoxy acid 1200SL dual binary with 6-port DAD 5989-5176EN 44herbicides valve for autoSPEVarious, including Binary gradient MS TOF, MSn Ion Trap, 5989-0815EN 45trimethoprim both pos ESIPhenylurea, triazine Dual binary with 6-port valve MSD pos/neg APCI 5989-0813EN 46herbicides for autoSPEHerbicides phenylurea, Binary gradient MSD pos/neg ESI 5988-8595EN 48triazineParaquat, Diquat, amitrole, Dual binary with 6-port valve MSD pos ESI 13 5988-7220EN 50chlormequat for autoSPEPhenylureas, carbamate Binary gradient MSD ESI, APCI, APPI 14 5988-6635EN 52pesticides Glyphosate, AMPA Binary gradient MSD pos ESI 15 5988-4981EN 53Sulfonylurea herbicides Dual binary with cohesive 2300 MSn Ion Trap pos ESI 16 5988-3649EN 55

valve for autoSPE

Application Area – Sample Preparation TechniquesVarious in water sources 1200 AESOP Open 17 5989-5692EN 57EPA 3640A standard mix Isocratic DAD 18 5989-0181EN 58

12 QuEChERS prep, Molecular Feature Database search13 Novel ion pairing reagent14 Post-column addition of APPI dopant requires extra pump or large syringe pump15 Offline precolumn derivatization FMOC, post-column addition dilute formic acid16 Turbulent flow trace enrichment17 AutoSPE system with special ChemStation macro program18 Loading guidelines, various solvents

62

Application Area – Regulated/Hazardous Drug Substances

LiteraturePage Major Analytes Matrix LC system Detection Mobile phase reference

7 Chloramphenicol Honey, shrimp, 1200SL MS QQQ neg ESI MeOH/water 5989-5975ENchicken

9 Pharmaceuticals Water 1200SL MS QQQ ACN/water 5989-5319ENpos/neg ESI ammonium formate

10 Estrogens, steroids River water, sewage, Binary gradient MS TOF neg APPI Propanol/cyclohexane 5989-4858EN treated sewageeffluent

12 Veterinary pharmaceuticals Surface water, soil, Quaternary gradient MSD, MSn Ion Trap MeOH/water 5989-2980EN sediment ammonium formate

Application Area-Regulated/Hazardous Miscellaneous Substances

13 Crude oil Soil Binary gradient with DAD RID Hexane 5989-6012ENfrac. coll.

15 Arsenic speciation Urine, water Isocratic ICP-MS EtOH, water, phosphate, 5989-5505EN EDTA, NaOAc, NaNO3

16 VX, Sarin, hydrolysis Soil Binary gradient DAD, ICP-MS MeOH/water 5989-5346ENproducts AmmOAc

Myristyltrimethyl-ammonium bromide

18 Disperse dyes, azo dyes Water Binary gradient MS TOF pos ESI ACN/water AmmOAc 5989-3859EN

19 Methylmercury, mercury, Water, synthetic LC/ICP-MS ICP-MS MeOH/water 5989-3572EN ethylmercury seawater, soil AmmOAc

2-mercaptoethanol

21 Acrylamide Drinking water Dual binary with 6-port MS TOF pos ESI ACN/water formic 5989-2884ENvalve for autoSPE

22 Chromium speciation IC Metrohm 818 pump, ICP-MS Water Na2 EDTA 5989-2481EN Agilent 7500 ISIS NaOHsampler

24 Nitroaromatics explosives Soil Binary gradient MS TOF neg APCI MeOH/water 5989-2449EN

26 Herbicide antibiotic peptide, Bialaphos Infusion MSn Ion Trap, MS TOF, Not applicable 5989-2324EN bialaphos, bilanaphos both pos ESI

28 Perchlorate Water, vegetables Metrohm IC MSD neg ESI MeOH/water 5989-0816EN30 mm, NaOH

29 Organotin compounds Sediments LC and GC ICP-MS See application note 5988-6697EN

31 Nitroaromatics explosives Binary gradient DAD ACN/water TFA 5988-6345ENammonia

32 DNPH aldehydes Air, Brazil Binary gradient DAD MSD neg APCI ACN/water 5968-8850E

33 Inorganic fluoride Water IC ICP-MS Water, nitric acid 5968-8232E

34 Anions, metals Plating bath CE DAD See application note 5968-5761E35 Inorganic anions, Pulping liquor CE DAD See application note 5968-3306EN

miscellaneous

36 Bromate, iodate water Ozone-treated Yokagawa IC ICP-MS or DAD Water, carbonate/ 5968-3049 water post-column bicarbonate

derivatized

37 HGA hydrocarbons, Oil, fuels Isocratic with RID Heptane 5965-9044Earomatics switching valve

38 Polynuclear aromatic Soil (SFC extr) Binary gradient DAD/FLD ACN/water 5964-3540hydrocarbons (PAHs)

Application Area-Regulated/Hazardous Natural Toxin Substances

39 Anatoxin A, alkaloid Drinking water Binary gradient MSD pos ESI with ACN/water 5968-3796Eneurotoxin FMOC derivatized AmmOAc

40 Microcystins, algal toxins Fresh (surface) Binary gradient MSD pos ESI ACN/water formic 5968-2123Ewater

Quick Reference Guide

63

41 600 pesticides screening Fruit, vegetable Binary gradient MS TOF pos ESI ACN/water formic 5989-5496EN

42 Chlorophenoxy acid Soil Binary gradient MS QQQ neg ESI ACN/water HOAc 5989-5246ENherbicides

44 Chlorophenoxy acid Water 1200SL dual binary with DAD ACN/water H3PO4 5989-5176ENherbicides 6-port valve for autoSPE

45 Various, including Sediments Binary gradient MS TOF, MSn Ion ACN/water 5989-0815ENtrimethoprim Trap, both pos ESI ammonium formate

46 Phenylurea, triazine Water Dual binary with 6-port MSD pos/neg APCI MeOH/water formic 5989-0813ENherbicides valve for autoSPE

48 Herbicides phenylurea, Water Binary gradient MSD pos/neg ESI MeOH/water formic 5988-8595ENtriazine

50 Paraquat, Diquat, amitrole, Water Dual binary with MSD pos ESI ACN/water 5988-7220ENchlormequat 6-port valve tetradecafluoro-

for autoSPE heptanoic acid (TDFHA)

52 Phenylureas, carbamate Binary gradient MSD ESI, APCI, ACN, MeOH, acetic 5988-6635ENpesticides APPI

53 Glyphosate, AMPA Water Binary gradient MSD pos ESI ACN/water AmmOAc 5988-4981EN

55 Sulfonylurea herbicides Surface water Dual binary with MSn Ion Trap ACN/water acetic 5988-3649ENCohesive 2300 valve pos ESI AmmOAcfor autoSPE

Application Area-Sample Preparation Techniques

57 Various in water sources Water 1200 AESOP Open Open 5989-5692EN

58 EPA 3640A standard mix Vegetable oil, Isocratic DAD Various 5989-0181ENbroccoli, animal fat

Quick Reference Guide (Continued)

LiteraturePage Major Analytes Matrix LC system Detection Mobile phase reference


64

Basic Principles of Liquid Chromatography

Classical liquid chromatography (LC) was first used in 1903by the Russian scientist Mikhail Tswett (1872-1919) to sep-arate plant pigments. In his initial publications, Tswettcalled the new technique “chromatography” because theresult of the analysis was “written in color” along thelength of the adsorbent column.

Chromatography is a separation technique that places (orinjects) a small amount of liquid sample into a tube, knownas a column, that is packed with porous particles; this iscalled the stationary phase. The sample’s individual compo-nents are transported down the packed column by a liquidthat is moved by gravity; this is called the mobile phase.

The sample’s components are separated by the columnpacking through various chemical and/or physical interac-tions between their molecules and the packing particlesand are moved through the column bed by the flowing sol-vent. The separated components are collected at thecolumn exit and identified by an external measurementtechnique, such as spectrophotometry, which measures theintensity of the color, by gravimetric analysis, or by anothertechnique that can measure the amount of each separatedcomponent. The modern form of column liquid chromatog-raphy is now referred to as “flash chromatography.”

High-performance liquid chromatography (HPLC) is a sepa-ration technique that injects a small amount of liquidsample into a column packed with 1- to 10-micron diameterparticles; this is the stationary phase. Individual samplecomponents are moved along the column by a liquid thatforced through the column by high pressure delivered by apump; this is the mobile phase.

The competitive interaction of the stationary and mobilephases on the individual analytes and the various chemicaland/or physical interactions between their molecules andthe packing particles work together to separate the samplecomponents from one another. When the separated compo-nents exit the column, a detector measures the amount ofeach component. The detector’s graphical recorded outputdetector is called a “liquid chromatogram.”

HPLC is used for one of three reasons: qualitative analysis,quantitative analysis, and to prepare pure compounds. Qual-itative analysis is used to identify the individual compoundsin a sample. The most common parameter for identifying acompound is its retention time, the time it takes to elutefrom the column after injection. Depending on the detectorused, identification may also be based on chemical struc-ture, molecular weight, or some other molecular property.

The second reason for using HPLC is for quantitative analy-sis; that is, to determine what compounds are in a sampleand to measure the amount (or concentration) of each one.There are two primary ways to interpret a chromatogram orperform quantitation. The first is to measure the height of achromatographic peak from the baseline; the second is todetermine the peak area. To quantitatively assess the com-pound, a sample or standard with a known amount of thecompound of interest is injected under identical operatingconditions, and its peak height or peak area is measured.This measurement is compared to the response of the sameanalyte in a subsequently analyzed sample mixture.

The final reason HPLC is used is to prepare a pure com-pound. A pure substance can be prepared for later use (forexample, organic synthesis, identification, clinical studies,or toxicology studies) by collecting the chromatographicpeaks at the detector’s exit and concentrating the com-pound (also called the analyte) by removing the solvent.This methodology is called preparative chromatography.

Instrumentation for HPLC Although in principle LC and HPLC work the same way, thespeed, efficiency, sensitivity, and ease of operation makeHPLC vastly superior. The main components of an HPLCsystem are described below.

65

it to determine the sample components' elution time (quali-tative analysis) and the sample's amount (quantitativeanalysis).

HPLC Separation ModesThe four main types of columns offer separations based ona variety of analyte properties. The main examples or modesof separation are reversed phase; ion exchange; normalphase; and gel permeation, gel filtration, and size exclusionchromatography.

Reversed Phase (C18, ODS, C8, C4, CN, RP, and bondedphase, among others). Reversed phase separation is primar-ily used for compounds that are somewhat organic or aque-ous soluble and that differ in solubility with respect toorganic/aqueous mixtures.

This mode of separation is by far the most widely usedtechnique in LC today, due to column durability, the compat-ibility of mobile phases with typical sample matrices, andthe availability of versatile separation mechanisms. Thecolumns used for reversed phase separation are usuallypacked with a porous silica-based material that is covalentlybonded with linear alkyl chains like octyldimethylsilane (a C8 column) or octydecyldimethylsilane (a C18 column, or ODS).

Mobile phases are generally mixtures of water-misciblereagents and solvents. Methanol (MeOH), acetonitrile (ACNor MeCN), and tetrahydrofuran (THF) are the commonorganic solvents. Water, phosphate buffers, acetate buffers,and ion pairing reagents (such as alkylsulfonic acids or qua-ternary alkyl ammonium compounds) are commonly used.

Halide salts or corrosive acids (for example, hydrochloricacids, perchloric acid, and even sulfuric acid) are oftenavoided because of possible corrosive effects on the instru-ment's stainless steel parts. Proper care and use of theinstrument allows almost all types of reagents to be used;however, the use of these corrosive reagents can createmaintenance problems.

Ion Exchange (IEX, cation exchange [CX], anion exchange[AX]). Ion exchange separations are most commonly usedfor organic carboxylic or sulfonic acids, sugars, proteins,classical amino acid techniques using post-column derivati-zation, DNA/RNA-related and oligonucleotides, inorganicanions and cations, and some small organic amines.

Pump

As part of the mobile phase, the HPLC's pump forces aliquid through the liquid chromatograph at a specific flowrate, expressed in milliliters per minute (mL/min). Normalflow rates are between 0.2 and 2 mL/min; however, theycan be lower for capillary and nanoseparations or higher forpurification separations. Typical pumps can reach pressuresbetween 6,000 and 9,000 psi (400 and 600 bar). During thechromatographic experiment, a pump can deliver a constant(isocratic) or dynamic (gradient) mobile phase composition.

Injector

The injector, which must withstand the system’s high pres-sures, introduces the liquid sample into the flow stream ofthe mobile phase without unduly interrupting or disturbingthe system's flow or pressure. Typical sample volumes are 1 to 20 microliters (µL). The injector must be able to with-stand the system's high pressures. An autosampler is anautomatic injector for when there are many samples to ana-lyze or when greater precision is required and manual injection is not practical.

Column

Considered the heart of the chromatograph, the column’sstationary phase separates the sample’s components ofinterest by using various physical or chemical parameters.The typically small particles (< 10-micron), densely packedinside the column, are what cause both the separation ofthe sample's components and the incidental high backpres-sure at normal flow rates. The pump must push hard tomove the mobile phase through the column, and this resis-tance creates high pressure within the chromatograph.

Detector

The detector can see the individual molecules come out(elute) of the column, normally in dilute solution within themobile phase. Modes of detection include UV/VIS, fluores-cence; differential refractive index; evaporative light scatter-ing; conductivity; electrochemistry in various forms,including oxidative or reductive measurements; and massspectrometry. The detector measures the amount of theanalyte and may provide orthogonal information (spectraldata) relative to identification or confirmation so that thechemist can quantitatively analyze the sample’s compo-nents. The signal that the detector provides to a recorder orcomputer results in a liquid chromatogram.

Computer

Frequently called the data system, the computer usuallycontrols all HPLC instrument’s system modules andacquires and stores the detector signal. In the final step, thecomputer processes the signal from the detector and uses

66

These columns are made of a silica or cross-linked styrene-divinyl benzene or methacrylate polymer base material witha charged site (anionic or cationic) covalently grafted ontothe surface. The solvents used with this mode are predomi-nantly buffers with small amounts of organic solvent some-times added for improved solubility or selectivity (that is, toimprove separation). Ion exchange is often used in the gra-dient separation mode (complex mixtures especially DNA-related, proteins, amino acids).

Normal Phase (adsorption and silica). The normal phase isthe least commonly used technique today, representing lessthan 5% of total applications worldwide. The base materialfor these columns is silica; it is usually not derivatized,except sometimes cyanopropyl or aminopropyl groups areadded. Normal phase is used almost exclusively with non-polar solvents that are typically not water miscible. The pre-ferred separations for this mode include preparativechromatography (because of easy solvent removal fromfractions), some fat-soluble vitamins like vitamins A and E,some synthesis products, and some polymer additives.

Gel Permeation, Gel Filtration, and Size Exclusion Chromatography (GPC, GFC, and SEC, respectively). All ofthese modes imply separations based on the size of a mole-cule in a particular solution, in this case the mobile phase.Columns may be based on silica or polymeric materials,with or without the addition of covalently bound functionalgroups, and are generally larger and more expensive thanother columns for analytical separations.

GPC is commonly used for synthetic or natural polymersand GFC is used for biomolecules like proteins or large pep-tides. SEC describes the mechanism of separation mostaccurately. SEC separations are generally easy, require littlemobile phase development, and are inherently isocratic, forgood detectability. These columns are from three to 10 times larger than for other separation modes, yet theseparation takes place relatively quickly because thecolumns do not (or generally should not) interact chemicallywith the sample components; thus, the sample componentstravel at the same or faster speed than the mobile phase.Separation times are from 10 to 60 minutes, depending onthe range of molecular sizes in the sample. (Molecularweight is an analogous term that can be used in most situations.)

Column Dimensions and MaterialsA modern column for HPLC is normally constructed of astainless steel tube with a highly polished interior and endcaps with integral or removable porous frits. The frit poros-ity is designed to allow solvent to flow through while preventing particles from entering and packing material

particles from escaping. PEEK, TEFLON™, other inert poly-mers, glass, and other inert materials have also been used.With proper handling and reasonable attention to chemicalcompatibility and sample preparation, columns oftendeliver thousands of usable injections before their performance begins to fail.

The column dimensions and particle size of the packingmaterial are as important as the separation mode. Typicalanalytical columns vary from about 1.0 to 6.0 mm internaldiameter (id) and from as short as 10 to 300 mm long.

Small-diameter columns consume small volumes of solvent, may allow enhanced detection sensitivity, andoperate at flow rates that are compatible with mass spec-trometers (MS) and evaporative light-scattering detectors(ELSD). The small bed volume, however, makes the columnsusceptible to resolution losses due to extracolumn disper-sion or band spreading. Special tubing, fittings, and flowcells may need to be used to minimize the extracolumnvolume, maximizing the usable efficiency, which is alwayslower than the column's theoretical efficiency.

In contrast, larger diameter columns operate at relativelyhigh flow rates (1.5 to 3 mL/min, typically), have a relativelylarge bed volume, and consume significantly more mobilephase. Flow splitting may be required when these columnsare used with MS- or ELSD-type detectors. Large-diametercolumns enable larger injection volumes and samplecapacity. It also may be possible to use these analyticalsize columns for low milligram scale purification. The largerbed volume yields comparatively small extracolumn dispersion effects.

In addition, very short columns make extremely fast separa-tions possible, although lower efficiency (that is, resolvingpower) will be observed. Larger diameter long columnsdeliver the highest resolution but require more time andsolvent to perform the same separations. For further dis-cussion about converting methods from one column dimen-sion to another, with appropriate adjustments in flow rate,gradient, or run times, see “Method Translation” below.

Particle size is another important physical variable thatshould be considered carefully. We have long known thatsmaller particle sizes and narrower particle size distribu-tions allow higher efficiency, which, in turn, contribute togreater resolution in the separation. The increase in effi-ciency is inversely proportional to the particle size change,so halving the particle size doubles the efficiency, whichincreases actual resolution by about 1.4 (the square root of2, per the standard resolution equation). Operating pres-sure, though, shows inverse but exponential increases;thus, halving the particle size is theoretically expected toincrease pressure by 4 times. For these reasons, we try to

67

balance the resolution requirements against the negativeimpact that increased pressure may have on system reliabil-ity, column lifetime, and possibly increased analysis time incases where maximum pressures are reached and desiredoperating flow rates must be reduced.

Concepts of the Rapid Resolution Systems and MethodsThe Agilent 1200 Series Rapid Resolution LC concept hasbeen developed to allow increased speed and resolution ofchromatographic analyses while keeping system pressureat a minimum. The system provides faster analyses andhigher resolution than conventional LC, which beneficiallyallows higher sample throughput and higher data quality. Itoptimizes performance while minimizing the risks that ultra-high pressure might impose on instrument reliability andlongevity.

In general, shorter analyses and increased resolution andsensitivity may be reached by optimizing the column,column thermostatting, and gradient delay volume and byreducing the extracolumn dispersion volume. These stepsensure the best possible system performance for high-speed and high-resolution separations while offering theextra benefits of solvent reduction and increased sensitivity.The user may enjoy a substantial improvement in the over-all chromatographic process, especially if higher operatingpressures are available and compatible with the columnpacking materials.

A new high-performance pump with flow rates from 0.05 to5 mL/min and up to 600 bar pressure was developed for thenew Agilent system. The system also features a high-per-formance degasser, a 600 bar low-dispersion autosampler,and new UV and MS detectors. It can be optimized for high-est speed and resolution in both LC/UV and LC/MS appli-cations and can run any traditional LC method, making thenew Agilent system very flexible. Flow rates from 0.05 to 5 mL/min ensure flexibility from semi-micro to semi-prepar-ative operation on the single platform. It accommodates allHPLC and STM-LC operational modes on one system and

facilitates the use of existing and newly developed HPLCmethods without the need for revalidation or extensivereconfiguration.

Optimizing the Instrument Setup for DifferentColumn IDsWhen performing high-throughput sample analyses, themajor focus is on having short run times. The usual way todo this is to use very short columns to achieve high-column-volume-per-minute flow rates. In the flexible RRLCsystem concept, one can choose 2.1-mm, 3.0-mm, or 4.6-mm inside diameter (id) columns, with slightly differentinstrument configurations recommended for each. It is gen-erally important to have the lowest possible extracolumnvolume when using 2.1 mm id STM columns.

When compared to conventional system configurationsusing 4.6 mm, 5-um columns, the major difference is fromthe autosampler onward-the point where the sample entersthe flow path and is subject to peak dispersion. The firststep is to change from 0.17-mm id capillaries to smaller0.12-mm id capillaries, a 50% reduction in extracolumnvolume. Further means of reducing the extracolumn volumeinclude a specially designed low-dispersion heat-exchangerin the thermostatted column compartment and small-volume flow cells with a specially designed inlet and outletflow path for improved flush-out behavior. In general, allcapillaries are kept as short as possible, and the use of connecting unions is minimized.

It has often been said that the column is “the heart of thesystem.” Indeed, proper column selection, based on a rangeof user requirements, is a critical step in the method-devel-opment or method-improvement process. The variable para-meters are length, id, and particle size. Increased length or

Figure 2. Single stack configuration with MS detector, example.

68

decreased particle size will increase the resolving power ofthe column. Increasing the column length will result in aproportional increase in the operating pressure, solventconsumption, and analysis time. Reducing the particle sizewill result in an exponential increase in the pressure withminimal effect on solvent consumption or analysis time,providing that the pressure requirement does not exceedthe system maximum or user preference. Decreasing thecolumn id is a common approach to reducing solvent con-sumption with minimal effect on resolution and analysistime. However, efficiency may diminish slightly due to extra-column effects, and care must be taken to minimize thisdetrimental parameter.

The 2.1-mm id column is very popular in LC/MS methodsbecause the typical flow rate is ideal for the most popularionization sources. The 3-mm id column offers a balancebetween the more demanding system requirements of a 2.1 mm id column and the high solvent consumption of a 4.6 mm id column. The nearly twofold increase in bedvolume, over 2.1 mm columns, allows larger volume UV flowcells with longer paths to be used without loss of resolu-tion, which can improve sensitivity. Depending on the ion-ization source and flow rate, a flow split before an MSdetector might be required.

The most commonly used columns are the 4.6-mm idcolumn. They typically have the most available stationaryphases, tolerate extracolumn dispersion reasonably well,and allow flow cells with long paths to be used, often givingthe best sensitivity on a purely signal-to-noise basis. How-ever, 4.6 mm id columns have the highest solvent consump-tion per analysis. In reality, the smaller volume columns willinvariably give higher sensitivity when the same samplemass is injected under comparable analysis conditions.

Gradient Delay VolumeThe Agilent binary pump SL can be optimized to favorlowest delay volume or maximum solvent mixing perfor-mance. Two flow paths are available, and only two fittingsneed to be moved. If the pump’s internal volume is quitelarge, the time until the gradient reaches the column andmakes the compounds move along the stationary phase willbe long. When operating with very small column volumes, itwill require longer run times to compensate for this delay.For larger columns or long, shallow gradients (typical ofpeptide and other macromolecule separations), this is not acritical parameter. In general, one should consider howmany column volumes of delay, not absolute volume, will bepresent and compare it proportionately to the total numberof column volumes in the gradient analysis.

Like all Agilent samplers, the Agilent 1200 high-perfor-mance ALS sampler SL is also designed to allow a bypassmode (advanced delay volume reduction) to be assumedafter the sample aliquot is completely beyond the injectorplumbing. This gives the user more flexibility in controlling

the small but sometimes significant delay volume associ-ated with sending the gradient through the sample loop.

SummarySystem flexibility is typically ranked as highly as systemperformance and reliability. The RRLC system was devel-oped with all of these parameters in mind, to offer dynamicperformance without compromising precision, resolution, orspeed. Using the available tools for method design and con-version (see other sections in this solutions guide and atwww.agilent.com/chem), the user can preemptively designa method around a particular column and then preselect theoptimum system configuration to ensure the best possibleoverall performance. Users with a wide variety of liquidchromatographic tasks can select one or more RRLC sys-

400 µL mixerDisconnectonly here

Standard delayvolume(600-800 µL)

Purgevalve

600 bar damper

Pressuresensor

Flow path

Pressure-sensor

Mixing-T

Damper

400 µL mixer

Purge valve

400 µL mixerDisconnectonly here

Low delayvolume(120 µL)

Purgevalve

600 bar damper

BA

BA

Pressuresensor

Flow path

Pressure-sensor

Mixing-T

Purge valve

Figure 2. How to change between standard and low delay volume configurationof the binary pump SL.

tems to operate a wide range of methods without worryingabout the system limitations inherent in traditional generalpurpose instruments or narrowly focused specialty systems.

Method TranslationIt is sometimes advantageous or necessary to change aseparation's overall scale when adapting an existingmethod for a new purpose. This might include increasingthe mass capacity (scaling an analytical separation forpurification), increasing the sensitivity (reducing the columnsize to improve detectability by increasing the average peakconcentration eluting to the detector), or increasingthroughput. In every case, following simple mathematicalguidelines will ensure that the method is scaled appropri-ately and will deliver the required capacity, sensitivity, reso-lution, and throughput according to your requirements.

Analysis methods developed on older columns packed withlarge 5- or 10-µm particles are often good candidates for

69

requires that the gradient slope be preserved. While manypublications have referred to gradient slope in terms of percent change per minute, it is more useful to express it aspercent change per column volume. In this way, the change

modernization by simply replacing these large columns withsmaller ones packed with smaller particle sizes. This canreduce analysis time and solvent consumption, improvesensitivity, and enable greater compatibility with massspectrometer ionization sources.

Simplistically, a 250-mm long column that contains 5-µmparticles can be replaced by a 150-mm long column packedwith 3-µm particles. If the ratio of length (L) to particle size(dp) is equal, the two columns are considered to have equalresolving power. Solvent consumption is reduced by L1/L2so a 250 mm column length separation converted to a 150mm length results in about a 1.6-fold reduction in solventusage per analysis. If an equal mass of analyte can then besuccessfully injected, the sensitivity should also increaseby 1.6-fold due to reduced dilution of the peak as it travelsthrough a smaller column of equal efficiency.

Liquid chromatography/mass spectrometry ionizationsources, especially the electrospray ionization mode, havedemonstrated greater sensitivity at lower flow rates than

typically used in normal liquid chromatography/ultraviolet(LC/UV) optical detection methods, so it may also beadvantageous to reduce the internal diameter of a columnto allow timely analysis at lower flow rates. The relationshipof flow rate between different column diameters is shownin Equation 1.

The combined effect of reduced column length and diame-ter contributes to a reduction in solvent consumption and,

velocity, as commonly defined by van Deemter [1] andothers. The second parameter is the often overlooked effectof extracolumn dispersion on the column's observed orempirical efficiency.

Although Van Deemter observed and mathematicallyexpressed the relationship of column efficiency to a varietyof parameters, we are most interested in his observationthat in a well-packed HPLC column there is an optimumlinear velocity for any given particle size and that the opti-mum linear velocity increases as the particle sizedecreases. The practical application is that a reduction inparticle size, as discussed earlier, can often be further opti-mized by increasing the linear velocity, resulting in a furtherreduction in analysis time. This increased elution speed willdecrease absolute peak width and may require an increasein data acquisition rates and reduction in signal filteringparameters to ensure that the chromatographic separationis accurately recorded in the acquisition data file.

The second important consideration is the often-overlookedeffect of extracolumn dispersion on the observed or empiri-cal efficiency of the column. As column volume is reduced,peak elution volumes are proportionately reduced. If smallerparticle sizes are also used, there is a further reduction inthe expected peak volume. The liquid chromatograph, andparticularly the areas where the analytes will traverse, is acollection of various connecting capillaries and fittings thatwill cause a measurable amount of band spreading. Fromthe injector to the detector flow cell, the cumulative disper-sion that occurs degrades the column performance andresults in observed efficiencies that can be far below thevalues that would be estimated by purely theoretical means.It is fairly typical to see a measured dispersion of 20 to 100 µL in an HPLC system. This has a disproportionateeffect on the smallest columns and smallest particle sizes,both of which are expected to yield the smallest possiblepeak volumes. Care must be taken to minimize the extracol-umn volume and, where practical, reduce the number ofconnecting fittings and the volume of injection valves anddetector flow cells.

For gradient elution separations, where the mobile phasecomposition increases through the initial part of the analy-sis until the analytes of interest have been eluted from thecolumn, successful method conversion to smaller columns

(eq. 2)= Inj. vol.col. 2Volumecolumn1

Volumecolumn2Inj. vol.col. 1 ×

(eq. 3)#Column volumes

(End% – Start%)% Gradient slope =

(eq. 1)= Flowcol. 2Diam.column1

Diam.column2Flowcol. 1

2

×

again assuming the same analyte mass can be injected intothe smaller column, a proportional increase in peakresponse. The injection mass is normally scaled to the sizeof the column, though, and a proportional injection volumewould be calculated from the ratio of the void volumes ofthe two columns multiplied by the injection volume on theoriginal column (see Equation 2).

For isocratic separations, the above conditions will normallyresult in a successful conversion of the method with little orno change in overall resolution. Several other parameterscan be considered to improve the method conversion’s out-come. The first parameter is the column efficiency relativeto flow rate, or, more correctly, efficiency relative to linear

70

min0 2 4 6 8 10 12 14 16 18

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ConditionsZORBAX SB-C18 4.6 mm × 250 mm, 5 µmColumn temp 30 °CGradient 20 mM H3PO4 pH 3.65 with ammonium

hydroxide, 10% to 50% ACN in 25 min

Gradient slope 2.8% ACN/column volume Analysis flow rate 1.41 mL/min Sample Standards 50 µg/mL each in

methanol/water 1/1, 15-µL injection

Total analysis time 37.5 minDetection UV 230 nm, 10-mm 13-µL flow cell, filter

2 seconds (default)

(Datafile SDADDS000006.D)

min0 0.2 0.4 0.6 0.8 1 1.2 1.4

mAU

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50

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ConditionsZORBAX SB-C18, 3.0 mm × 50 mm, 1.8 µm Column temp 45 °C Gradient 20 mM H3PO4 pH 3.65 with ammonium

hydroxide, 10% to 50% ACN in 1.5 minGradient slope 2.8% ACN/column volumeAnalysis flow rate 2.0 mL/minSample Standards 50 µg/mL each in

methanol/water 1/1, 2.5-µL injectionTotal analysis time 3 min Detection UV 210 nm, 6-mm 5-µL flow cell with

0.12-mm id inlet heat exchanger, filter 0.2 seconds

(Datafile SDADDS_3MM31029.D)

in column volume during method conversion can be used toaccurately render the new gradient condition. If we think ofeach line of a gradient table as a segment, we can expressthe gradient by the following equation:

Note that using percent change per column volume ratherthan percent change per minute enables users to controlgradient slope by altering gradient time and/or gradientflow rate. A large value for gradient slope yields very fastgradients with minimal resolution, while lower gradientslopes produce higher resolution at the expense ofincreased solvent consumption and somewhat reducedsensitivity. Longer analysis time may also result unless thegradient slope is reduced by increasing the flow rate (withinacceptable operating pressure ranges) rather than byincreasing the gradient time. Resolution increases withshallow gradients because the effective capacity factor, k*,is increased. Much like in isocratic separations, where thecapacity term is called k', a higher value directly increasesresolution. The effect is quite dramatic up to a k value ofabout 5 to 10, after which little improvement is observed. Inthe subsequent examples, we will see the results associ-ated with the calculations discussed above.

Careful analysis of the existing gradient conditions, coupledwith an awareness of the need to accurately calculate newflow and gradient conditions can lead to an easy and reli-able conversion of existing methods to new faster or higher

resolution conditions. In addition, awareness of extracol-umn dispersion, especially with small- and high-resolutioncolumns, will ensure good column efficiency, which is critical to a successful translation of the method.

Further reading can be found in application notes 5989-2908EN, 5989-4721EN, 5989-5176EN, 5989-5177EN,5989-5178EN, RRLC system brochure 5989-4330EN, and5989-5200EN.

Sample Preparation TechniquesProper sample preparation is a key component of success-ful HPLC analysis. From techniques as simple as dissolutionor dilution to complex, multistep matrix interference-remov-ing procedures, the choices are abundant and diverse. Thekey goal of good sample preparation is to prepare thesample in an injectable form that is compatible with theoperating conditions of the intended analysis. Miscibilityand pH compatibility are primary elements at this step. Fur-ther goals include removing unwanted matrix componentsthat may complicate or lengthen the analysis or reduce theuseful life of the separation column or other system components.

71

FiltrationSample filtration is the most fundamental procedure, pro-tecting the instrument and column from insoluble particu-late materials. Selective precipitation of some matrixcomponents, followed by filtration or centrifugation, mayalso be advised if some matrix components might precipi-tate on contact with the mobile phase or adsorb strongly orirreversibly to the column packing material.

ExtractionLiquid/liquid extraction techniques, with or without pHmodification of the aqueous phase, have long been used toselectively extract general classes of compounds based onsolubility. While this is effective for a limited number ofsamples, it may be cumbersome and too labor-intensive forprocessing large numbers of samples unless appropriateautomation methods (such as automatic pipetting andshaking) are applied in the scale-up phase. Depending onthe matrix complexity, these extraction techniques may beineffective at removing critical interferences and may besubject to troublesome emulsion formation that limitsspeed and reduces recovery and cleanup efficiency.

SPE, solid/liquid extraction, may be a convenient way tomore extensively fractionate a sample mixture. It is a simpleand generally small form of open-column liquid chromatog-raphy. By using one or more of the chromatographicprocesses–reversed phase, adsorption/normal phase, orion exchange–it may be possible to process individual sam-ples manually or use automation for offline processing oflarger numbers of samples.

http://www.chem.agilent.com/ecommerce/product/p2_cas_main.aspx?orhttp://www.chem.agilent.com/ecommerce/product/p2_cas_level2.aspx?groupid=1003&anchor=Sample%20Prepara-tion%20Supplies&bgc=7bba00

Typical SPE formats include syringe barrels of various sizes,low profile SPE devices resembling membrane filtration car-tridges, simple polymeric cartridges and mini-cartridges thatmay be attached to disposable syringes, and 96 wellmicrotiter plate compatible devices.

In some cases, it is possible to move the SPE proceduredirectly to the liquid chromatograph as an integral part ofthe analysis. When the sample matrix is not highly loadedwith particulates or dissolved matrix components, onlineSPE techniques usually use reusable media packed in guardcolumn hardware. The fractionation is controlled through aswitching valve and additional pump to deliver sample

preparation and SPE column regeneration reagents independently of the main analysis hardware and column.This has been widely demonstrated for trace enrichmentassociated with drinking water analysis and has also beenapplied to more complex matrices where total mass loadingis appropriate for online sample processing. For more information, access publications 5989-0813EN and 5988-9917EN.

Gel Permeation Chromatography (GPC)GPC has also been used as an effective high-performancesample cleanup technique. Because the separation mecha-nism is one of size rather than chemistry, it is a useful wayto remove lipids, proteins, and other larger molecules fromsamples containing broad classes of small molecule ana-lytes. Nowhere has this been more frequently used than inthe analysis of various molecule classes from environmen-tal samples, especially sludge, soil, and other solid matri-ces, which are first thoroughly extracted to obtain thesoluble analytes and a variety of larger molecules inherentin the matrix. Additional discussion is found in applicationnotes 5989-5401EN and 5989-0181EN.

Ideal Sample PreparationThe ideal sample preparation removes unwanted or interfer-ing matrix components, shortens and simplifies the analy-sis, reduces overall sample analysis time, lowers theper-sample cost, extends column life, and improves theoverall performance of the analysis to which the purifiedsample is finally subjected. A wide selection of publishedtechniques from peer-reviewed journals and suppliers ofsample preparation products is available to help you adaptexisting techniques and materials to your specific samplepreparation requirements.

An appropriate combination of HPLC components, a suit-able column chemistry and mobile phase chemistry, andeffective sample preparation are the keys to developing andusing a robust and reliable analysis or purification methodthat can deliver good results time after time and with a vari-ety of operators at the controls. To view the various hard-ware and chemical products and references available fromAgilent Technologies, refer to our general product catalog orvisit us online at www.agilent.com/chem. Once registered,you can shop for chromatographic components, consum-ables, and supplies; download technical references; reviewFAQs; or contact us for further technical information.

Credits for material used here: Ron Majors and Mike Woodman, Agilent Technologies

72

Application Area-Regulated/Hazardous Drug Substances

Major Analytes Matrix LC System Detection Column(s) Col. P/N Mobile Phase Notes

5989-5975EN (page 7)Chloramphenicol Honey, shrimp, 1200SL MS QQQ ZORBAX SB-C18, 827700-902 MeOH/water

chicken neg ESI 2.1 mm × 50 mm, 1.8 µm

5989-5319EN (page 9)Pharmaceuticals Water 1200SL MS QQQ ZORBAX Extend-C18, 728700-902 ACN/water 1

pos/neg ESI 2.1 mm × 100 mm, 1.8 µm ammonium formate

5989-4858EN (page 10)Estrogens, steroids River water, Binary MS TOF Luna Phenyl Hexyl, See application Propanol/ 2

sewage, treated gradient neg APPI 2.0 mm × 150 mm, 3 µm, note cyclohexanesewage effluent with guard, Luna

Phenyl Propyl,2.0 mm × 4 mm, 3 µm

5989-2980EN (page 12)Veterinary Surface water, Quaternary MSD, MSn ZORBAX SB-C18, 830990-902 MeOH/water 3pharmaceuticals soil, sediment gradient Ion Trap 2.1 mm × 150 mm, 3.5 µm, ammonium

formate

5989-5808ENNitrofuran Fish, tilapia LC MS MS QQQ ZORBAX Extend-C18, Contact author ACN/water metabolites pos ESI 2.1 mm × 150 mm, 3 µm formic

5989-5807ENMalachite green, Food LC MS MS QQQ ZORBAX Extend-C18, Contact author ACN/water leucomalachite pos ESI 2.1 mm × 150 mm, 5 µm AmmOAcgreen

5989-1302EN (poster)Nitrofuran Poultry, shrimp Binary MS TOF ZORBAX Eclipse XDB-C18, 971700-902 ACN/water 4

gradient pos ESI 2.1 mm × 50 mm, 3.5 µm acetic

5989-0738ENNitrofuran Poultry, shrimp Binary MSn Trap XCT ZORBAX Eclipse XDB-C8, 971700-906 ACN/water

gradient pos ESI 2.1 mm × 50 mm, 3.5 µm acetic

5989-0596ENFluoroquinolones Beef kidney Binary MSD pos ESI ZORBAX Eclipse XDB-C8, 993967-906 ACN/water

gradient 4.6 mm × 150 mm, 5 µm formic

5989-0182ENSulfonamides Pork Binary MSD pos APCI ZORBAX Eclipse XDB-C8, 993967-906 ACN/water 5

gradient 4.6 mm × 150 mm, 5 µm formic

72

Application Reference Index

The table below lists Agilent Technologies applications forthe hydrocarbon processing and polymer markets. In thisguide, the page number where you can review the most current application overview is listed after the publicationnumber. The complete publication for all applicationoverviews listed below can be viewed on the Agilent Website, www.agilent.com/chem.

1 Offline SPE2 Offline automated SPE procedure, normal phase cleanup (ZORBAX SB-CN, 4.6 x 50 mm, 5 µm, and guard with propanol/cyclohexane), GPC cleanup (PLgel 50A in MeCl2),

post-column addition of reference mass solution3 ASE prep for soils, SPE trace enrich for water4 Precolumn offline derivatization5 Detailed sample prep, including offline SPE

73

Application Area-Regulated/Hazardous Drug Substances (Continued)


5988-9920ENChloramphenicol Shrimp, honey Binary MSD and ZORBAX Eclipse XDB-C18, 993967-902 MeOH/ACN/

gradient MSn Ion 4.6 mm × 150 mm, 5 µm water AmmOAcTrap neg ESI

5988-8999ENChloramphenicol Fish Binary MSD neg APPI, ZORBAX Eclipse XDB-C18, 993967-302 MeOH/water 6

gradient DAD 3 mm × 150 mm, 5 µm AmmOAc

5988-8903ENNitrofurans Poultry Binary MSD pos Inertsil ODS3, Recommend ACN/water 7

gradient ESI, DAD 2.1 mm × 150 mm, 5 µm ZORBAX SB-C18 or formicEclipse XDB-C18 chemistry

5988-7135ENSulfa drugs Meat Gradient DAD RP18 Purospher, 79925PU-584 ACN/water H3PO4

4 mm × 250 mm, 5 µm

5988-6926ENSteroids Water Binary MSn Ion Trap ZORBAX Eclipse XDB-C18, 971700-902 ACN/water 8

gradient pos/neg APCI, 2.1 mm × 50 mm, 3.5 µm AmmOAcpos APPI, DAD

5980-2499ENSulfonamides Cap LC DAD MSD ZORBAX SB-C18, 5064-8262 ACN/water formic

pos ESI 0.5 mm × 150 mm, 3.5 µm

5968-8185ECisplatin platinum IC ICP-MS See application note See application See applicationcompounds note note

5966-1619Tetracyclines Meat, food LC DAD Hypersil BDS, Recommend ACN/water 9

4 mm × 100 mm, 3 µm ZORBAX SB-C18 H2SO4

chemistry

5965-9794EAntibiotics, sulfas Meat Binary DAD Purospher RP18, Recommend ACN/water

gradient 4 mm × 250 mm, 5 µm ZORBAX SB-C18 H3PO4

chemistry

Application Area-Regulated/Hazardous Miscellaneous Substances

5989-6012EN (page 13)Crude oil Soil Binary DAD RID ZORBAX NH2, 880952-708 Hexane 10

gradient with 4.6 mm × 250 mm, 5 µmfrac. coll.

5989-5505EN (page 15)Arsenic speciation Urine, water Isocratic ICP-MS G3288-80000 Arsenic G3288-80000 + EtOH, water, EDTA,

column, 4.6 mm × 250 mm G3154-65002 phosphate,(Guard Column) NaOAc, NaNO3

5989-5346EN (page 16)VX, Sarin, hydrolysis Soil Binary DAD, ICP-MS Alltima C8, Contact MeOH/water products gradient 3.2 mm × 150 mm, 5 µm manufacturer AmmOAc Myristyl-

trimethyl-ammoniumbromide

5989-3859EN (page 18)Disperse dyes, Water Binary MS TOF pos ESI ZORBAX Eclipse XDB-C8, 922700-932 ACN/water azo dyes gradient 2.1 mm × 50 mm, 1.8 µm AmmOAc

6 Post-column dopant acetone, isocratic G1310A pump, or large syringe pump required7 Offline precolumn derivatization, detailed sample prep8 Large volume direct injection with good comparison of strong and weak diluents on peak shape9 Detailed sample prep provided10 Requires fraction collector, note includes GC/MS fraction analysis

74

Application Area-Regulated/Hazardous Miscellaneous Substances (Continued)


5989-3572EN (page 19)Methylmercury, Water, synthetic LC/ICP-MS ICP-MS ZORBAX Eclipse XDB-C18, 960967-902 MeOH/water mercury, ethyl- seawater, soil 2.1 mm × 50 mm, 5 µm AmmOAc mercury 2-mercapto-

ethanol

5989-2884EN (page 21)Acrylamide Drinking water Dual binary MS TOF pos ESI ZORBAX SB-C18, 883700-922 ACN/water

with 6-port 2.1 mm × 150 mm, 5 µm formicvalve for autoSPE

5989-2481EN (page 22)Chromium speciation IC Metrohm ICP-MS Agilent Cr, 4.6 mm × 30 mm G3268A Water Na2 EDTA 11

818 pump, NaOHAgilent 7500ISIS sampler

5989-2449EN (page 24)Nitroaromatics Soil Binary MS TOF ZORBAX Extend-C18, 770450-902 MeOH/water 12explosives gradient neg APCI 4.6 mm × 250 mm, 5 µm

5989-2324EN (page 26)Herbicide, antibiotic, Bialaphos Infusion MSn Ion Trap, Infusion Not applicable Not applicable 13peptide, bialaphos, MS TOF, both bilanaphos pos ESI

5989-0816EN (page 28)Perchlorate Water, Metrohm IC MSD neg ESI MetroSep ASUPP-5, See application MeOH/water

vegetables 4 mm × 100 mm note 30 mm, NaOH

5988-6697EN (page 29)Organotin Sediments LC and GC ICP-MS See application See application See applicationcompounds note note note

5988-6345EN (page 31)Nitroaromatics Binary DAD ZORBAX SB-C18, SB-CN, 883700-922, ACN/water TFA explosives gradient 2.1 mm × 150 mm, 5 µm 883700-905 ammonia

5968-8850E (page 32)DNPH aldehydes Air, Brazil Binary DAD MSD Nucleosil C18, See application ACN/water 14

gradient neg APCI 3 mm × 250 mm, 5 µm note

5968-8232E (page 33)Inorganic fluoride Water IC ICP-MS Dionex IonPac HPIC-CG2 Water,

nitric acid

5968-5761E (page 34)Anions, metals Plating bath CE DAD See application note See application See application

note note

5968-3306EN (page 35)Inorganic anions, Pulping liquor CE DAD See application note See application See applicationmiscellaneous note note

5968-3049 (page 36)Bromate, iodate Ozone-treated Yokagawa ICP-MS or IC; see application note See application Water, carbonate/ 15

water IC DAD post-column note bicarbonatederivatized

11 Column normally bundled with ICP-MS; contact your agent/representative for details12 Post-column reference mass addition13 Elaborate structural elucidation via trap MSMS and TOF accurate mass analysis14 DNPH cartridge for direct air sample collection15 Submitted to Journal of Chromatography A, 789, 259-265 (1997)

75



5965-9044E (page 37)HGA hydrocarbons, Oil, fuels Isocratic RID 4.6 mm × 200 mm, Recommend Heptane 16aromatics with AP NH2, 5 µm ZORBAX NH2,

switching 4.6 x 250 mmvalve

5964-3540 (page 38)PAH aromatic Soil (supercritical Binary DAD/FLD PAH 2.1 mm × 250 mm, 79918PAH-582 ACN/waterhydrocarbons fluid chromato- gradient 5 µm

graphy [SFC] extract)

5989-5403ENHMF hydroxymethyl- Bread, cereal, Binary MSD pos APCI Bonus-RP 861768-901 Water acetic 17furfural yogurt gradient 2.1 mm × 100 mm, 3.5 µm formic

5989-4736ENSudan dyes Food Binary MS TOF pos ESI ZORBAX Eclipse XDB-C18, 922700-902 ACN/water

gradient 2.1 mm × 50 mm, 1.8 µm AmmOAc

5989-4565CHCNAldehydes, ketones Air LC DAD with DNPH HC-C18 Agilent China only ACN/water 18

derivatized

5989-2883ENChlorophenoxy acid Water Binary MSD neg ESI ZORBAX Extend-C18, 763750-902 ACN/water formic 19herbicides, steroids gradient 2.1 mm × 150 mm, 3.5 µm NH3 TEA

5989-1270ENVarious, including Food Binary Various Various Various Various 20pesticides, drugs gradient

5989-0814EN (obsolete)Various drugs, Groundwater Binary MS TOF pos ESI ZORBAX Eclipse XDB-C18, 971700-902 ACN/water 21pesticides gradient 2.1 mm × 50 mm, 3.5 µm ammonium

formate

5989-0264PAH by GC GC See application See application note See application Not applicable

note note

5989-0027ENOrganomercury Food Infusion ICP-MS None Not applicable Not applicable

5988-9893ENArsenobetaine Fish Isocratic ICP-MS Hamilton PRP X-100 Contact AmmHCO3/

manufacturer tartaric

5988-6346ENCarbonyls Binary DAD with DNPH ZORBAX ODS, 884950-543 ACN/water(12 analytes) gradient derivatized 4.6 mm × 250 mm, 5 µm

5988-6344ENCarbazole, catechol Binary DAD ZORBAX 300 SB-C18, 883995-902 ACN/water metabolites gradient 4.6 mm × 150 mm, 5 µm TEA TFA

5988-6342ENExplosives Soil Isocratic DAD ZORBAX SB-C18, 880975-302 MeOH/water(15 analytes) 3 mm × 250 mm, 5 µm

16 Classic hydrocarbon group analysis per IP391 guidelines17 Offline SPE18 Column available only in China market19 Ion suppression problems studied, post-column addition used20 Food safety primer, LC, GC, ICP21 Contact author; publication unavailable online

76



5988-4056ENPolynuclear aromatic Water GC See application See application note See application Not applicablehydrocarbons (PAHs) note note

5988-4055ENPolychlorinated Water GC See application See application note See application Not applicable 22biphenyls (PCBs) note note

5988-3161ENBromate Drinking water LC ICP-MS IC Dionex PA-100, Contact Water, AmmNO3, 22

9.4 mm × 250 mm manufacturer HNO3

5980-1477 (obsolete)Benzidines phenols LC ECD DC, PAD Various Various Various

5980-0262ENArsenic speciation Drinking water Isocratic ICP-MS G1354A/101,G1354A/102 See application Water, phosphate,

note EDTA

5968-8660EInorganic anions Wastewater CE DAD indirect UV 75 µm Not applicable Chromate electrolyte

5968-7929EAromatic amines Leather CE MSD pos ESI 50 µm Not applicable Water acetate

5968-5731EArsenate, cyanide, Food CE DAD 50 µm Not applicable See application anions, organic acids note

5968-3050EArsenic compounds Rat urine IC ICP-MS IC; see application See application See application

note note note

5967-5598DNPH aldehydes Air, Japan Isocratic DAD with DNPH ZORBAX Eclipse XCB-C18, 990967-902 ACN/water 23

derivatized 4.6 mm × 250 mm, 5 µm

5966-0633Inorganic anions Water Isocratic DAD, indirectUV Contact author for reversed ACN/water NaOH 24

phase column and mobile + UV modphase details

5965-9796DNPH aldehydes Air, Brazil Isocratic DAD with DNPH Inertsil ODS Recommend ACN/water 23

derivatized 4.6 mm × 250 mm ZORBAX SB-C18 or Eclipse XDB-C18chemistry

5091-7626Explosives Soil HP 1090 DAD Hypersil BDS Recommend MeOH/waternitroaromatics 4 mm × 100 mm, 3 µm ZORBAX SB-C18

chemistry

5091-7260PAH aromatic Soil, meat HP 1090 DAD/FLD PAH See application ACN/waterhydrocarbons note

5091-1815 (obsolete)Anions LC ECD See application See application See application

note note note

22 SPE trace enrichment23 DNPH cartridge for direct air sample collection24 UV modifier, probably aromatic acid like 4-OH-benzoic or trimesic; some applications have used cetylpyridinium chloride for indirect or displacement UV of anions

77

Application Area-Hazardous Natural Toxin Substances


5968-3796E (page 39)Anatoxin A, alkaloid Drinking water Binary MSD pos ESI Inertsil ODS3 Recommend ACN/water 25neurotoxin gradient with FMOC 2.1 mm × 150 mm, 5 µm ZORBAX SB-C18 or AmmOAc

derivatized Eclipse XDB-C18 chemistry

5968-2123E (page 40)Microcystins, Fresh (surface) Binary MSD pos ESI Mytisil ODS Recommend ACN/water formicalgal toxins water gradient 2.1 mm × 100 mm, 5 µm ZORBAX SB-C18

chemistry

5989-3634ENAflatoxins Isocratic DAD ZORBAX Eclipse XDB-C18, 963967-902 MeOH, ACN,

4.6 mm × 150 mm, 3.5 µm water

5989-2912ENDSP algal toxins Shellfish Quaternary MSD pos/ ZORBAX SB-C18, 883975-302 and MeOH/water

gradient neg ESI 3 mm × 150 mm, 5 µm, 846975-202 formic9.4 mm × 50 mm, 5 µm,semiprep

5968-2124EMycotoxin, fumonisin Corn Binary MSD pos ESI ZORBAX Eclipse XDB-C18, 993700-902 ACN/water

gradient 2.1 mm × 150 mm, 5 µm AmmOAc

5966-0632Aflatoxins, Various LC DAD/FLD Various Various Various 26mycotoxins, patulin

5952-5852 (obsolete)Mycotoxins Old MS Vydac 201HSB Recommend ACN/water

HP 5988A Thermospray 4.6 mm × 150 mm, 5 µm ZORBAX SB-C8 or AmmOAc(obsolete) Eclipse XDB-C8

chemistry

5091-8692Mycotoxins Various LC DAD with Various See application Various

Library, FLD note

00060329 (poster) HPLC 2006Aflatoxins Various Ag/Gerstel MSD pos ESI Phen. MAX RP Suggest ACN/water 27

online SPE 2.1 mm × 250 mm, 5 µm ZORBAX SB-C18, formic2.1 mm × 150 mm, 3 and 5 µm (830990-902)


5989-5496EN (page 41)600 pesticides Fruit, vegetable Binary MS TOF pos ESI ZORBAX Eclipse XDB-C8, 993967-906 ACN/water formic 28screening gradient 4.6 mm × 150 mm, 5 µm

5989-5246EN (page 42)Chlorophenoxy Soil Binary MS QQQ neg ESI ZORBAX Extend-C18, 728700-902 ACN/water HOAcacid herbicides gradient 2.1 mm × 100 mm, 1.8 µm

5989-5176EN (page 44)Chlorophenoxy Water 1200SL dual DAD ZORBAX SB-C18, 5-, 3.5-, See application ACN/water H3PO4

acid herbicides binary with and 1.8-µm, columns note6-port valve for autoSPE

25 Online derivatization with FMOC; good details26 Detailed sample preps and references in this review article27 Bromination of B1 and G1 prior to separation28 QuEChERS prep, molecular feature database search

78

5989-0815EN (page 45)Various, including Sediments Binary MS TOF, MSn ZORBAX Eclipse XDB-C18, 971700-902 ACN/water trimethoprim gradient Ion Trap, 2.1 mm × 50 mm, ammonium

both pos ESI 3.5 µm formate

5989-0813EN (page 46)Phenylurea, Water Dual binary MSD pos/ ZORBAX Eclipse XDB-C8, 971700-906 MeOH/water triazine herbicides with 6-port neg APCI 2.1 mm × 50 mm, 3.5 µm formic

valve for autoSPE

5988-8595EN (page 48)Herbicides Water Binary MSD pos/ ZORBAX Eclipse XDB-C8, 971700-906 MeOH/water phenylurea, triazine gradient neg ESI 2.1 mm × 50 mm, 3.5 µm formic

5988-7220EN (page 50)Paraquat, Diquat, Water Dual binary MSD pos ESI ZORBAX Extend-C18, 763750-902 ACN/water 29amitrole, chlormequat with 6-port 2.1 mm × 150 mm, 3.5 µm tetradeca-

valve for fluoroheptanoicautoSPE acid (TDFHA)

5988-6635EN (page 52)Phenylureas, Binary MSD ESI, APCI, ZORBAX Eclipse XDB-C8, 935967-906 ACN, MeOH, 30carbamate pesticides gradient APPI 4.6 mm × 50 mm, 3.5 µm acetic

5988-4981EN (page 53)Glyphosate, AMPA Water Binary MSD pos ESI ZORBAX Eclipse XDB-C8, 946975-906 ACN/water 31

gradient 4.6 mm × 50 mm, 5 µm AmmOAc

5988-3649EN (page 55)Sulfonylurea Surface water Dual binary MSn Ion Trap Metasil Basic Recommend herbicides with Cohesive pos ESI 2.1 mm × 100 mm, 5 µm ZORBAX Eclipse ACN/water acetic 32

2300 valve XDB-C18 AmmOAcfor autoSPE chemistry

5989-5469EN100 pesticides Fruit, vegetable Binary MS QQQ ZORBAX Eclipse XDB-C8, 993967-906 ACN/water

gradient pos ESI 4.6 mm × 150 mm, 5 µm formic

5989-5459EN44 pesticides Fruit, vegetable Binary MS QQQ ZORBAX Extend-C18, 728700-902 ACN/water 33

gradient pos ESI 2.1 mm × 100 mm, 1.8 µm ammonium formate

5989-5320ENPesticides Water 1200 MS QQQ pos/ ZORBAX Extend-C18, 728700-902 ACN/water 34

neg ESI 2.1 mm × 100 mm, 1.8 µm acetic formic AmmOH

5989-5177ENChlorophenoxy 1200SL DAD ZORBAX SB-C18, See application ACN/water H3PO4

acid herbicides 5-, 3.5- and 1.8-µm notecolumns

5989-3573ENTerbuthylazine Olive oil Unspecified MS TOF, MSn ZORBAX Eclipse XDB-C8, 993967-906 ACN/water 35

Ion Trap, 4.6 mm × 150 mm, 5 µm formicboth pos ESI

Application Area-Regulated/Hazardous Pesticide/Herbicide Substances (Continued)


29 Novel ion pairing reagent30 Post-column addition of APPI dopant requires extra pump or large syringe pump31 Offline precolumn derivatization FMOC; post-column addition of dilute formic acid32 Turbulent flow trace enrichment33 SPE cleanup34 Offline SPE trace enrichment35 Liquid/liquid extraction and SPE

79

5989-3198ENHerbicides, Binary MSD APCI/ESI ZORBAX Eclipse XDB-C18, 930990-902 MeOH/water 36pesticides, insecticides gradient multimode 2.1 mm × 150 mm, 3.5 µm AmmOAc(35 analytes)

5989-2728ENPost-harvest Citrus Binary MS TOF, ZORBAX Eclipse XDB-C8, 993967-906 ACN/water 37fungicides MSn Ion 4.6 mm × 150 mm, 5 µm formic

Trap, bothpos ESI

5989-2209ENFungicides Vegetables, fruit Binary MS TOF, MSn ZORBAX Eclipse XDB-C8, 993967-906 ACN/water

gradient Ion Trap, both 4.6 mm × 150 mm, 5 µm formicpos ESI

5989-1924ENPesticides Vegetables Binary MS TOF, MSn ZORBAX Eclipse XDB-C8, 993967-906 ACN/water

gradient Ion Trap, both 4.6 mm × 150 mm, 5 µm formicpos ESI

5989-1842ENChloronicotinyl Vegetables, Binary MS TOF, MSn ZORBAX Eclipse XDB-C8, 993967-906 ACN/water insecticides fruit gradient Ion Trap, both 4.6 mm × 150 mm, 5 µm formic

pos ESI

5989-1688ENPesticides Surface and Binary MSD pos ESI Hypersil BDS Recommend ACN,MeOH, 22

drinking water gradient 2 mm × 100 mm, 3 µm ZORBAX SB-C18 water AmmOAcchemistry

5989-0930ENVarious, including Isocratic DAD ZORBAX Eclipse XDB-CN 993967-905, MeOH/waterurea pesticides or XDB-C18, 4.6 mm × 993967-902

150 mm, 5 µm

5989-0414ENPesticides LC, GC, Various Various Various Various 38

ICP-MS

5989-0371ENPesticides, imazapyr Water Binary MSD pos/ ZORBAX Eclipse XDB-C18, 930990-902 ACN/water 39

gradient neg ESI, DAD 2.1 mm × 150 mm, 3.5 µm formic

5989-0184ENAmitrol herbicide Water Binary MSD posAPCI ZORBAX SB-C18, 863954-302 MeOH/water 40

gradient 3 mm × 150 mm, 3.5 µm AmmOAc

5988-8692ENPesticides, Binary DAD 3 mm, RP columns Various Variousantibacterials gradient

5988-8449ENPesticides Drinking water Binary DAD ZORBAX SB-C18, 880975-302 ACN/water 41

gradient 3 mm × 250 mm, 5 µm NaOAc

5988-7220ENParaquat, Diquat Water Dual binary MSD pos ESI ZORBAX Extend-C18, 763750-902 ACN/water 42

with 6-port 2.1 mm × 150 mm, 3.5 µm TDFHAvalve for autoSPE



36 Published in LC/GC Applications Notebook, June 200537 QuECHERS sample prep38 General capabilities demonstrated39 Ion suppression problems studied, post-column addition used, "fully automated offline SPE"40 Derivatization with hexylchloroformate, SPE trace enrichment41 Separation Times, Vol 16, No.1, 200342 Novel ion pairing reagent

80

5988-7071ENVarious mixtures LC, GC, Various See application See application See application 43

ICP-MS note note note

5988-6686ENTriforine fungicide Gradient DAD ZORBAX Eclipse XDB-C8, 993967-906 ACN/water isomers 4.6 mm × 150 mm, 5 µm formic

5988-6347ENPesticides Isocratic DAD ZORBAX Eclipse XDB-C18, 931975-932 MeOH/water

cartridge 4.6 mm × 30 mm, 3.5 µm

5988-6341ENPesticides Water Binary DAD ZORBAX SB-C18, 880975-302 ACN/water (28 analytes) gradient 3 mm × 250 mm, 5 µm acetate

5988-6340ENHerbicides Isocratic DAD ZORBAX SB-C8, CN, See application ACN/water 44

phenyl note

5988-6289ENChlorophenoxy Binary DAD ZORBAX SB-C18, 880975-902 ACN/water acid herbicides gradient 4.6 mm × 250 mm, 5 µm phosphate

5988-6288ENTriazine herbicides Binary DAD ZORBAX SB-C8, 866953-906, ACN/water TFA

gradient 4.6 mm × 150 mm, 5 µm, 883975-9064.6 mm × 75 mm, 3.5 µm

5988-6287ENSulfonylurea Water Binary DAD ZORBAX SB-C18, 880975-302 ACN/water pesticides gradient 3 mm × 250 mm, 5 µm acetic

5988-6286ENTriazine herbicides Isocratic DAD ZORBAX SB-C18, 883975-902 ACN/water 45

4.6 mm × 150 mm, 5 µm acetate

5988-6284ENTriazine herbicides Isocratic DAD ZORBAX SB-C18, 883975-902 MeOH/water 46

4.6 mm × 150 mm, 5 µm acetate

5988-5882ENChlorophenoxy acid Water Binary DAD, MSD ZORBAX Eclipse XDB-C18, 930990-902 ACN/water 47herbicides, gradient neg ESI 2.1 mm × 150 mm, 3.5 µm formicpropyzamide

5988-4708ENCarbamates Vegetable, Binary MSD pos ESI ZORBAX Eclipse XDB-C18, 993700-902, ACN/water

broccoli gradient 2.1 mm × 150 mm, 5 µm; 993967-902 AmmOAc4.6 x 150 mm, 5 µm

5988-4233ENSimazine, Binary MSD pos ESI Inertsil ODS3 Recommend MeOH/water thiobencarb, thiuram gradient 2.1 mm × 250 mm, 5 µm ZORBAX SB-C18 or AmmOAc

Eclipse XDB-C18 chemistry

5988-4057ENChlorinated Water GC See note See application note See application Not applicable 22pesticides note



43 Separately showing GC, LC, and ICP-MS44 Examples of bonded phase selectivity differences45 50 °C versus 90 °C46 40 °C versus 90 °C47 "Fully automated offline SPE"

81

5988-3774ENOrganophosphate Binary MSD pos ESI, ZORBAX SB-C18, 871700-902 ACN/water 48pesticides gradient DAD 2.1 mm × 50 mm, 3.5 µm AmmOAc

5988-1664ENParaquat, Diquat CE MSD pos ESI PVA coated 75 µm id See application MeOH/water

note AmmOAc

5980-0561ERodenticides Sausage, Binary MSD pos/ ZORBAX Eclipse XDB-C18, 993700-902 MeOH/water 49difenacoum, dog stomach gradient neg ESI, APCI, 2.1 mm × 150 mm, 5 µm AmmOAccoumatetralyl, contents DADcoumafuryl

5980-0332ECarbaryl Complex food MSn Ion Trap ZORBAX Eclipse XDB-C8, 990967-906 ACN/water acetic 50

homogenate pos ESI 4.6 mm × 250 mm, 5 µm AmmOAc

5966-2629EHerbicides Isocratic DAD ZORBAX SB-C18, 883975-902 MeOH/water temperature effects 4.6 mm × 150 mm, 5 µm NaOAc

5966-2625EN (old HP)Sulfonylurea pesticides Binary DAD ZORBAX SB-C18, 880975-302 ACN/water

gradient 3 mm × 250 mm, 5 µm acetic

5966-1876Insecticide, aldicarb, Isocratic DAD Hypersil ODS Recommend ACN/waterbendiocarb 2.1 mm × 100 mm, 5 µm ZORBAX Eclipse

XDB-C18 chemistry

5966-1875Paraquat, Diquat Isocratic DAD Hypersil ODS Recommend Water, TEA, hexane sulfonate 2.1 mm × 100 mm, 5 µm ZORBAX SB-C18 H3PO4,

chemistry hexanesulfonate

5966-0743Glyphosate Water Binary FLD post-column Pickering IEX 8 µm Contact Water, phosphate 51

gradient derivatized manufacturer

5966-0742Triazines, Salad spices LC DAD Hypersil BDS Recommend ACN/waterphenylureas, 3 mm × 100 mm, 3 µm ZORBAX Eclipse methabenzthiazuron, XDB-C18 chemistryDiquat, Paraquat, mercaptobenzothiazole

5954-7852E (obsolete)Carbamates Old HP FLD with post- ZORBAX C18, Recommend MeOH/water 52

column 4.6 mm × 250 mm, 5 µm ZORBAX Eclipse derivatized XDB-C18 chemistry

5952-2229 (obsolete)Pesticides Water Old HP DAD Hypersil ODS Various ACN/water 53

5091-3621Glyphosate Drinking water LC/ FLD with SAX-300 79919QA-754 Water, phosphate 54

Pickering precolumn or 4.6 mm × 100 mmpost-column derivatized



48 Compares DAD to MSD-SIM results49 Detailed sample prep, compares DAD to MSD, and negative versus positive and ESI versus APCI50 Complex and detailed sample prep51 Pickering post-column system with Agilent LC/FLD52 Pickering system post-column base hydrolysis followed by OPA53 Many sample prep SPE details54 Pickering post-column system with Agilent LC/FLD

82

5091-0302 (obsolete)Bentazone, phenoxy Water LC DAD Hypersil ODS Recommend ACN/water acid herbicides 4 mm × 100 mm, 5 µm ZORBAX Eclipse phosphate

XDB-C18, 3 mm × acetate150 mm, 5 µm

Application Area-Sample Preparation Techniques

5989-5692EN (page 57)Various in water Water 1200 AESOP Open Open Open Open 55sources

5989-0181EN (page 58)EPA 3640A Vegetable oil, Isocratic DAD Various organic See application Various 56standard mix broccoli, animal GPC note

fat

5966-1873GPC cleanup Soil, sediment Isocratic DAD Per EPA See application MeCl2 57

note

5091-6241 (obsolete)GPC cleanup Various LC UV DNPH GPC See application MeCl2 58

derivatized note

5965-9057Sudan red dye Diesel Binary DAD Hypersil BDS Recommend ACN/water

gradient 4 mm × 100 mm, 3 µm ZORBAX SB-C18 H2SO4

chemistry

Application Area-Cosmetics, Personal Care Products

5989-3633ENPyrethroid insecticides, Shampoo, lotion Unspecified DAD ZORBAX Eclipse XDB-C18, 963967-902 MeOH/waterpermethrin, piperonyl 4.6 mm × 150 mm, 3.5 µmbutoxide

5966-1877Tenside nonylphenol Binary DAD Hypersil ODS Recommend ACN/water surfactant gradient 2.1 mm × 250 mm, 5 µm ZORBAX Eclipse KH2PO4

XDB-C18 chemistry

Application Area-Dyes, Colorants, Pigments

5965-9042Dyes, disperse and Plastics LC DAD Hypersil BDS Recommend ACN/water soluble 3 mm × 125 mm, 3 µm ZORBAX SB-C18 TBAHS, H2SO4

chemistry

Application Area-Fine Chemicals

5989-1042ENWarfarin and related Tablets Isocratic DAD ZORBAX Eclipse XDB-CN 993967-905 ACN/water 59Compound A 4.6 mm × 150 mm, 5 µm acetic

5988-6085ENFungicide triforine, Binary MSD pos ESI ZORBAX Eclipse XDB-C8, 993967-906 ACN/water degradants gradient 4.6 mm × 150 mm, 5 µm formic



55 AutoSPE system with special ChemStation macro program56 Loading guidelines, various solvents57 1-mm path flow cell58 See 5989-0181EN59 References USP27-NF22

83

5968-9463ECE for the CE DAD See application note See application See application determination of note noteforensic anions (arsenite, azide) in adulterated foods and beverages

Application Area-Industrial Miscellaneous

5988-6343ENSubstituted anilines Binary DAD ZORBAX SB-C18, 880975-902 ACN/water

gradient 3 mm × 250 mm, 5 µm KOAc

5988-6339ENSubstituted phenols Binary DAD ZORBAX SB-C18, 883975-902 ACN/water

gradient 4.6 mm × 150 mm, 5 µm H3PO4

Application Area-Mixed Publications

5989-5847ENHPLC Solutions for Various Various Various Various Variousthe Hydrocarbon Processing Industry

5989-4934ENSteroids, alkaloids, Binary DAD Various ZORBAX Various Varioussulfas gradient Eclipse RP

5989-4894ENReserpine, alprazolam 1200 MS QQQ Unknown Contact Unspecified

manufacturer

5989-4721ENSunscreen 1200 DAD Various RP Various Various 60Padimate-O

5989-4086ENLuteoskyrin LC Various Various ZORBAX Various Various

Eclipse RP

5989-1243ENTrace metals High ionic matrix Infusion ICP-MS None Not applicable Not applicable

5989-0510ENVarious Various LC with Various Various Various Various 61

valving

5988-8631ENToxic chemicals, GC, LC, ICP Various Various Various Variouspesticides

5988-6441ENOSHA LC methods Various Gradient Various Various ZORBAX See application See application 62

note note

5988-4931ENPesticides, peptide, LC DAD Various Various Various 63aspartame (APM)

5988-3294Various, including Food LC Various Various Various Various 64pesticides, drugs

Application Area-Forensics


60 RRHT standalone brochure61 Use of online valving for autoSPE62 OSHA Methods for Analysis Using ZORBAX HPLC Columns63 ZORBAX SB LC columns64 Food safety primer, LC, GC, ICP

84

5988-2266 LC Various Various Various Various 65

5988-1786ENVarious LC DAD ZORBAX general Various Various 66

5968-9346EApp. overview of FLD LC FLD Various Various Various 67

5968-8569Peptides, aspartame, LC DAD Various ZORBAX, Various Various 68NSAIDs 5 and 3.5 µm

5968-6726EN LC Various See application note See application See application 69note note

5965-9032PAH herbicides CE DAD Various packed Various ACN/water TRIS

CEC columns

Application Area-Packaging

5989-5898ENEpoxies, phenolic 1200SL MS TOF pos ESI ZORBAX Eclipse XDB-C18, 961753-902 ACN/water 70polymers 2.1 mm × 100 mm, 3.5 µm

Application Area-Petrochemicals, Crude Oil

5956-4167GPC Crude oil LC RID See application note See application See application

note note

Application Area-Polymer Additives

5965-9056Brenzcatechol Antiox LC DAD See application note See application See applicationantioxidant note note

5965-9041Antioxidants, LC DAD See application note See application See applicationUV stabilizers note note

Application Area-Polymers, Organic Soluble

5965-5231ENBisphenol A Imps 1200SL DAD Various ZORBAX Eclipse Various ACN/MeOH/

XDB C18 water formic

5967-6102EBisphenol A esters Food LC MSD pos ESI Hypersil ODS Recommend ACN/water

2.1 mm × 200 mm, 5 µm ZORBAX AmmOAcSB-C18 chemistry

5965-9043Bisphenol A Polycarbonate LC Fluor See application note See application See application

note note

Application Area-Special Techniques

5988-5445ENNIST-compatible Infusion MSD Not applicable Not applicable Not applicable 71library

Application Area-Mixed Publications (Continued)


65 Sample prep solutions66 ZORBAX Applications Guide; various markets67 Applications book; various markets68 ZORBAX Rapid Resolution columns; various applications69 Agilent Environmental Solution Guide, 200070 Leaches into food products from can coatings71 Build API libraries with MSD and search NIST database

www.agilent.com/chem

Agilent shall not be liable for errors contained herein or for incidental or consequentialdamages in connection with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this publication are subject to changewithout notice.

© Agilent Technologies, Inc. 2007

Printed in the USAMay 23, 20075989-5851EN

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