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General Description

Agilent Technologies has developed a number of key high performance liquid chromatography (HPLC) applications forthe food market. This guide offers an overview of food sampleanalysis using HPLC, recommended starting conditions forapplication development, and information on Agilent literatureconcerning the various applications.

The first section in this document, the Application Overview,provides excerpts from Agilent publications that relate toHPLC applications for 13 major food groups. These highlightsinclude detailed chromatographic conditions, chromatogramsof standards, real-life samples, and information about themethods. To review a publication referenced in this guide, turnto the Application Reference Index for the publication’snumber; entering this number at the Agilent Web site will giveyou access to the complete text.

The System Configurations section of this guide providesdescriptions of the types of HPLC solvent delivery systems and detectors. The information in the Food Quick ReferenceGuide repeats the information provided in the previously mentioned sections; however, this section organizes the information by food group. Basic Principles of Liquid Chromatography offers an overview of separation modes,column dimensions and materials, sample preparation techniques, solid phase extraction, and method translation.

Please note that the described instruments are recommendations only.

Food Solutions with HPLC

Solutions Guide

November 2006

2

Application Overview 3

System ConfigurationsCarbohydrates, sugars, sugar alcohols 64Dyes, colorants, pigments 64Fats and oils 64Flavors, sweeteners, organic acids 64Herbal supplements, natural products, plant hormones 64Preservatives 64Proteins, peptides, amino acids 65Regulated/hazardous drug substances 65Regulated/hazardous miscellaneous substances 65Regulated/hazardous natural toxin substances 65Regulated/hazardous pesticides/herbicide substances 66Fat-soluble vitamins 66Water-soluble vitamins 66

Food Quick Reference GuideCarbohydrates, sugars, sugar alcohols 67Dyes, colorants, pigments 67Fats and oils 67Flavors, sweeteners, organic acids 67Herbal supplements, natural products, plant hormones 67Preservatives 68Proteins, peptides, amino acids 68Regulated/hazardous drug substances 68Regulated/hazardous natural toxin substances 68Regulated/hazardous pesticides/herbicide substances 69Fat-soluble vitamins 69Water-soluble vitamins 69

Basic Principles of Liquid ChromatographyBasic Principles of Liquid Chromatography 70Concepts of the Rapid Resolution Systems and Methods 73Method Translation 74Sample Preparation Techniques 76

Application Reference IndexCarbohydrates, sugars, sugar alcohols 78Dyes, colorants, pigments 78Fats and oils 79Flavors, sweeteners, organic acids 79Herbal supplements, natural products, plant hormones 80Preservatives 82Proteins, peptides, amino acids 82Regulated/hazardous drug substances 83Regulated/hazardous miscellaneous substances 85Regulated/hazardous natural toxin substances 85Regulated/hazardous pesticides/herbicide substances 86Fat-soluble vitamins 88Mixed vitamins 89Water-soluble vitamins 89Mixed publications 90Packaging 90

Table of Contents

3

Application Overview

HPLC is increasingly applied to the analysis of foodsamples for additives and contaminants (see Table 1). The method enables complex mixtures tobe separated into individual compounds and to beidentified and quantified by suitable detectors anddata handling systems. Separation and detectionoccurs at ambient temperature or slightly above andtherefore the method is also well suited to com-pounds of low thermal stability. Although HPLC

Applications

Group Major analytes Matrix Page

Carbohydrates, sugars, sugar alcohols Carbohydrates 5Sugar alcohols Beverage 6Dextran 7 Starch 8Carbohydrates Lemonade 9

Dyes, colorants, pigments Sudan dyes Food 10FDC food dyes, paraben 11Cyanidins Cabbage 12

Fats and oils Phospholipids Soybean 14Triglycerides 15Triglycerides Edible oil 16Triglycerides and their hydroperoxides Edible oil 17

Flavors, sweeteners, organic acids Flavors, sweeteners, preservatives Soft drinks 18Organic acids Foods 19Flavoring agents Mouthwash 20Semivolatile flavors 21Aspartame, degradants Cola 22

Herbal supplements, natural products, plant hormones Xanthine metabolites 23Glycyrrhizin Licorice root 24Xanthines Tea, chocolate 25Ginsenosides part 1 Root 26Anthocyanins Blueberry 27Anthocyanins 28Flavonoids, catechins 29

Preservatives Flavors, sweeteners, preservatives Soft drinks 30Paraben, phenoxyethanol 31

Proteins, peptides, amino acids Proteins Wheat 32Proteins Wheat 33BSA (bovine serum albumin) digest, peptide 34AAA amino acid 35

Table 1 Food Compounds Typically Analyzed by HPLC

detectors in general are not as sensitive as gas chromatography (GC) detectors, HPLC is a sensi-tive method, mainly due to the possibility ofinjecting large sample amounts up to 1–2 mL perinjection. This, and the nondestructive nature ofmany of the detection techniques, also enables thecollection of fractions for further analysis, whichis especially important for the analysis of proteinsand synthesis environments. Modern samplepreparation techniques, such as solid phaseextraction (SPE) and supercritical fluid extrac-tion (SFE), also enable high sensitivity HPLCanalysis in the in the parts per trillion(ppt) range.

4

Group Major analytes Matrix Page

Regulated/hazardous drug substances Drugs Water 36Nitrofurans Poultry, shrimp 37Fluoroquinolones Beef kidney 38Chloramphenicol Shrimp, honey 39Sulfa drugs Meat 40Sulfonamides 41

Regulated/hazardous miscellaneous substances HMF hydroxymethylfurfural Bread, cereal, 42yogurt

Acrylamide Drinking water 43Chromium speciation 44Perchlorate Water, vegetables 45Arsenobetaine Fish 46

Regulated/hazardous natural toxin substances DSP algal toxins Shellfish 47Mycotoxin, fumonisin Corn 49Aflatoxins Various 50

Regulated/hazardous pesticides/herbicide substances 44 pesticides Vegetables, fruit 51Acid herbicides Water 52Postharvest fungicides Citrus 54Chloronicotinyl insecticides Vegetables, fruit 55Phenylurea, triazine herbicides Water 56

Fat-soluble vitamins Retinol isomers 58Fat-soluble vitamins 59Vitamin D3 Poultry feed 60Fat-soluble vitamins A,D, E 61

Water-soluble vitamins Water-soluble vitamins 62Water-soluble vitamins Cat food 63

Table 1 Food Compounds Typically Analyzed by HPLC (Continued)

The above information highlights only a fraction of the available Agilent application notes. Refer to the Application Reference Index (pages 78 to 90) at the back of this guide for amore comprehensive listing of available solutions. All of these application notes can be downloaded from the Agilent Web site www.agilent.com/chem.

5

0 2 4 6 8 10 12 14 16 18

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28

1

23

4

5 6

1

23

4

56

1 Fructose2 Glucose3 Saccharose4 Palatinose5 Trehalulose6 Isomaltose

Major analytes

Carbohydrates

Matrix

Standard

Reference

Robert Ricker,”High-Performance Carbohydrate Analysis,”Agilent Technologies, publication 5988-6351EN,www.agilent.com/chem

ConditionsColumn ZORBAX NH2

250 mm × 4.6 mm(Agilent p/n 880952-708)

Mobile phase ACN : H2O, as indicated

Flow rate 1 mL/min

Detector Refractive index (RID)

Carbohydrates, Sugars, Sugar Alcohols

System Summary

LC System

Isocratic

Detection

RID

Columns

ZORBAX NH2, 250 mm × 4.6 mm

Column part number

880952-708 or 840300-908

6

Major Analytes

Sugar alcohols

Matrix

Beverage

Reference

Hiroki Kumagai, “Application of liquidchromatography/mass spectrometry to the analysis ofsugars and sugar alcohol,” Agilent Technologies, publication 5988-4236EN, www.agilent.com/chem

Abundance120000

80000

40000

0

1 2 3 4 5 6 7 8 9

Time (min)

Coffee Sucrose

Time (min)

Abundance140000

100000

60000

20000

1 2 3 4 5 6 7 8 9

Orange juice

Glucose

Fructose

Time (min)

Abundance

300000

200000

100000

0

1 2 3 4 5 6 7 8 9

Tea Sucrose


System Summary

LC System

Isocratic

Detection

MSD negAPCI

Columns

150 mm × 2 mm Asahipak NH2-50 2D

Column part number

843300-908

ConditionsColumn 150 × 2 mm Asahipak NH2-50 2D

Mobile phase Acetonitrile/Water (75/25)

Flow rate 0.2 mL/min

Column temperature 40 °C

Injection volume 10 µL

Post column addition Acetonitrile/CHCl3 (50/50) at 0.2 mL/min

Detector MSD Quadrupole Ionization APCI (negative)Scan range m/z 100–500Vaporizer 400 °CNebulizer pressure 40 psiFragmentor 20 VCorona current 30 ADrying gas 13 L/min, 350 °CSIM (m/z) negativeSorbitol 217, 219Glucose, fructose 215, 217Xylitol 187, 189Sucrose 377,379

Confirmation MS spectral information and RT

7

Major Analytes

Dextran

Matrix

Standard

Reference

Heinz Goetz, “Quality control of dextran,” Agilent Technologiespublication 5988-0118EN, www.agilent.com/chem

Time [min]

Dextran

Impurities

7.5 10 12.5 15 17.5 20 22.5

2000

4000

0

14000

8000

6000

10000

12000

Norm.


0.25

0.75

1.25

Molar mass [g/mol]104 105 106

W [

log

M]

rid1A

Mn: 9.0685e4 g/molMw: 1.8436e5 g/molMz: 3.8872e5 g/molMv: 1.8436e5 mL/gD: 2.0330e0[n]: 0.000000Vp: 1.4450e1 mLMp: 9.2727e4 g/molA: 2.0790e4 mL*V1.0e4 D 1.679e-1 %2.8e5 D 8.0762e1 %7.8e5 D 1.0000e2 %0.0e D 0.000000 %0.0e D 0.000000 %

Report subsets - additional informationon user-selected parts of chromatogram

Molecular weight distribution

Molecular weight data

ConditionsSample preparation Sample was dissolved in the mobile phase

(concentration 0.1 %) and filtered

Column PL aquagel-OH MXA, 7.5 mm × 300 mm, 8 µm (Agilent p/n 79911GF-MXA) in series with PL aquagel-OH 30A, 7.5 mm × 300 mm, 8 µm (Agilent p/n 79911GF-083)

Mobile phase Water

Flow rate 1 mL/min

Column compartment 25 °Ctemperature



Polymer standards Polyethylene oxide EasyCal standards in vials for calibration (Agilent p/n 5064-8280)

Software ChemStation Plus with SEC data analysis software

Figure 1. Analysis of dextran sample.

Figure 2. Typical GPC report.

System Summary

LC System

Isocratic

Detection

RID

Columns

PL aquagel-OH MXA, 7.5 mm × 30 mm, 8 µm

PL aquagel-OH 30A, 7.5 mm × 30 mm, 8 µm

Column part number

79911GF-MXA and 79911GF-803

8

10 11 12 13 14 15 16 17 18

0

1000

2000

3000

4000

5000

6000

Starch afterincorrectprocessing

Originalstarch

I

II

nRIU

Time [min]

Starch Mn Mw

Original * *Degradation I 1000 4700Degradation II 910 6100

Glucose

* Not calculated because of steric exclusion


Major Analytes

Starch

Matrix

Standard

Reference

Heinz Goetz and Peter Kilz, “Process Control of Starch,”Agilent Technologies publication 5988-0117EN, www.agilent.com/chem

ConditionsSample preparation Sample was dissolved in 1 mL eluent at

20 °C (concentration 0.1 % w/w). Dextran standards from Polymer Standards Services (PSS) were used for narrow standard calibration.

Column PSS Suprema 100 + 1000 in series,8 mm × 300 mm, 10 µm

Mobile phase 0.1 M sodium nitrate

Flow rate 1 mL/min

Column compartment 25 ° Ctemperature



Software ChemStation Plus with SEC data analysis software

Figure 1. Overlay of chromatograms of original starch after incorrect processing.

System Summary

LC System

Isocratic

Detection

RID

Columns

Aq. GPC PSS Suprema 100 + 1000, 2 of 8 mm × 300 mm, 10 µm

Column part number

See application note

9

Norm

120

100

80

140

160

180

15 20105Time [min]

Standard

Raf

fin

ose

Gal

acto

seLact

ose

Glu

cose

Fru

ctos

e

CellbioseMaltose Sucrose

Standard

Corn extract


Norm

200

400

600

800

Standard

Lemonade

Raffinose

Citric acid? Lactose

Glucose

Galactose

Fructose

15105Time [min]

Figure 1 Analysis of carbohydrates in lemonade.

Figure 2 Analysis of carbohydrates in corn extract.

ConditionsColumn 300 × 7.8 mm Bio-Rad HPXP, 9 µmMobile phase Water H2SO4

Column compartment 80 ºCtemperatureFlow rate 0.7 mL/minDetector Refractive index (RID)

Sample preparation Samples were directly injected.

HPLC method performanceLimit of detection < 80 ng with S/N = 2

Repeatability of RT RT over 10 runs < 0.05 %areas over 10 runs 2 %

Major Analytes

Carbohydrates

Matrix

Lemonade

Reference

Angelika Gratzfeld-Huesgen, “Analysis of Carbohydrates in Lemonade using HPLC,” Agilent Technologies publication5966-0637EN, www.agilent.com/chem

System Summary

LC System

Isocratic

Detection

RID

Columns

BioRad HPXP

Column part number

Contact manufacturer

10

Dyes, Colorants, Pigments

Major Analytes

Sudan dyes

Matrix

Food

Reference

Yanyan Fang and Michael Zumwalt,“Using TOF for Screen-ing and Quantitation of Sudan Red Colorants in Food,” Agilent Technologies publication 5989-4736EN, www.agilent.com/chem

ReproducibilitySudan Red I Sudan Red II

Standard RSD Accuracy RSD Accuracy(ppm) (%) (%, avg) (%) (%, avg)

0.2 6.04 97.31 5.76 97.61

0.4 6.98 101.95 5.64 100.27

0.8 4.61 104.75 6.12 103.78

1.6 5.17 102.53 5.99 105.92

2 6.12 96.77 4.74 94.72

Figure 2. TIC showing the four peaks of the Sudan Red dyes.

Experimental

Instrument

Agilent 1100 Series LC/MSD TOF with Agilent 1100 binary pump andwell plate autosampler

LC ConditionsColumn ZORBAX XDB C18, 2.1 mm × 50 mm, 1.8 µm

Agilent p/n: 922700-902

Mobile phases A: H2O with 5 mM NH4OAcB: Acetonitrile

Gradient 0–3 min 95% B to 98% B3–5 min 98% B

Post time 3 min


MS ConditionsIonization ESI, Positive

Gas temp 350 °C

Drying gas 8 L/min

Nebulizer pressure 45 psi

Capillary V (+ve) 4000 V

System Summary

LC System

Binary gradient

Detection

MS TOF posESI

Columns

ZORBAX XDB-C18, 2.1 × 50 mm, 1.8 µm

Column part number

922700-902

11


Major Analytes

FDC food dyes, paraben

Matrix

Ricker

Reference

Robert Ricker, “FD&C Colors,” Agilent Technologies, publication 5988-6355EN, www.agilent.com/chem

ConditionsColumn ZORBAX XDB-C18,

50 mm × 4.6 mm, 3.5 µm, (Agilent p/n 935967-902)

Mobile phase A: 0.1% TFA with triethylamine (TEA) B: Methanol

Gradient 17% B to 100 % B in 4 min

Flow rate 1 mL/min

Column temperature Ambient

Detector DAD, detection wavelength 254 nm

min1 2 3 4 5

mAu800

600

400

200

0

0

ZORBAX Eclipse XDB-C18

1

2

34 5

1. Yellow #5 C16H9N4Na3O9S2 MW=5342. Red #40 C18H14N2Na2O8S2 MW=4963. Blue #1 C37H34N2Na2O9S3 MW=7604. Propylparaben C10H12O3 MW=1805. Red #3 C20H4I4Na2O5 MW=878

System Summary

LC System

Quaternary gradient

Detection

DAD

Columns

ZORBAX XDB-C18, 50 mm × 4.6 mm, 3.5 µm

Column part number

935967-902

12


Major Analytes

Cyanidins

Matrix

Cabbage

Reference

Masahiko Takino, “Analysis of Natural Food Colorants byElectrospray and Atmospheric Pressure Chemical IonizationLC/MS,” Agilent Technologies publication 5968-2979E,www.agilent.com/chem

LC Conditions Column 250 × 2.1 mm Inertsil

ODS3, 5 µm

Mobile phase A = 1% formic acidB = Acetonitrile/water

Gradient Start with 5% BAt 30 min 50% B


Column temp 40 °C

Injection vol 10 µL

MS Conditions Source ESI

Ion mode Positive

Vcap Voltage 4000 V

Nebulizer 50 psig

Drying gas flow 10 L/min

Drying gas temp 350 °C

Corona 4 µA

Vaporizer temp 350 °C

Scan range 100–1200 amu

Step size 40.1

Peak width 0.15 min

Time filter On

Fragmentor 200 V

Continued

Figure 2. Total and extracted ion chromatograms of red cabbage colorant.

System Summary

LC System

Binary gradient

Detection

MSD posESI APCI

Columns

Inertsil ODS3, 2.1 mm × 250 mm, 5 µm

Column part number

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

935967-902

13

Figure 2. The structure of major pigments in red cabbage colorant.

Figure 3. Mass spectra of major pigments in red cabbage colorant.

Conditions

Mobile phase A = 95:2.5:2.5% hexane-isopropanol-methanolB = 40:60% isopropanol-methanolBoth solvents contain 10-mM ammonium acetate

14

Fats and Oils

Major Analytes

Phospholipids

Matrix

Soybean

Reference

Cliff Woodward and Ron Majors, “The HPLC PreparativeScale-Up of Soybean Phospholipids,” Agilent Technologiespublication 5989-2848EN, www.agilent.com/chem

Phospholipid Abbreviation

Phosphatidylethanolamine PE

Phosphatidylserine PS

Phosphatidylinositol PI

Phosphatidylcholine PC

min5 10 15 20 25 30 35

mV

0

250

500

750

1000

1250

1500

1750

2000

25 µg

50 µg 100 µg

200 µg

500 µg

PE PS PI

Z1

Z2

PC

min0 5 10 15 20 25 30 35

mV

0

250

500

750

1000

1250

1500

1750

2000

PE PS PI

20 mg

10 mg

4 mg2 mg

1 mg

Z1 Z2

PC

Figure 6. Agilent prep 21.2 × 150 mm, 10-µm – Mixture of soy phospholopidstandards, – Loadability.

Figure 3. Agilent prep 4.6 × 150 mm, 10-µm – Mixture of soy phospholopid standards, – Loadability.

System Summary

LC System

Prep/35900E

Detection

ELSD (ESA), some MSD

Columns

Prep SIL, 4.6 mm × 150 mm, 10 µm

21.2 mm × 150 mm, 10 µm

Column part number


15

Fats and Oils

Major Analytes

Triglycerides

Matrix

Standard

Reference

Hiroki Kumagai, “Analysis of Triglycerides by Liquid Chromatography/Mass Spectrometry,” Agilent Technologies, publication 5988-4235EN,www.agilent.com/chem

ConditionsColumn Develosil ODS OG-3, 75 mm × 4.6 mm

Mobile phase Acetone/Water (98/2)

Flow rate 1 mL/min



Gradient At 0 min 20% Aat 30 min 100% A

Detector MSD Quadrupole, SLIonization APCI (positive)Scan range m/z 100–1000Vaporizer 400 °CNebulizer pressure 50 psiFragmentor 160 VDrying gas 4 L/min, 350 °C

Confirmation MS spectral information and RT

40000

35000

30000

25000

20000

15000

10000

5000

Abundance

1.00 2.00 3.00 4.00 5.00 6.00 min

1

2

34 5 6

1. Trilaurin2. Trilinolein3. Trimyristin4. Triolein5. Tripalmitin6. Tristearin

Figure 1. TIC of triglyceride standards, each at 0.2 ppm.

Figure 2. Mass spectra of individual triglyceride standards, each at 0.2 ppm.

90

50

Tristearin 607.5

90

50211

495.5 Trimyristin

90

50

Tripalmitin551.5

90

50

Trilinolein

305

599.5 879.8 (MH)+

90

50

Triolein 603.5

881

150 350 550 750 150 350 550 750

150 350 550 750

150 350 550 750

150 350 550 750

150 350 550 750

90

50

Trilaurin439.5

183305

System Summary

LC System

Isocratic

Detection

MSD posAPCI

Columns

Develosil ODS OG-3, 4.6 mm × 75 mm

Column part number


866953-902

16

Fats and Oils

Major Analytes

Triglycerides

Matrix

Edible oil

Reference

Doug McIntyre, “The Analysis of Triglycerides in Edible Oilsby APCI LC/MS,” Agilent Technologies, publication 5968-0878E, www.agilent.com/chem

Chromatographic ConditionsColumn 200 mm × 2.1 mm Hypersil MOS, 5 µm

Mobile phase A = 60:40 water:isopropanol + 25 mM ammonium formate

B = 10:10:80 water:isopropanol:n-butanol + 25 mM ammonium formate

Gradient Start with 30% B at 1.5 min 30% Bat 25 min 60% B at 28 min 100% B


Column temp 50 °C

Injection vol 1.5 µL

MS ConditionsSource APCI

Ion mode Positive

Vcap 4000 V

Nebulizer 50 psig

Drying gas flow 4 l/min

Drying gas temp 325 ¡C

Corona 4 µA

Vaporizer 300 ¡C

Scan range 300–1100 m/z

Stepsize 0.1 m/z

Peakwidth 0.3 min

Time filter On

Fragmentor 80 V

16.00

10000

20000

30000

40000

50000

60000

70000

80000

90000

100000

Abundance

Trielaidin(C18:1,[trans]-9)

Tristearin(C18:0)

Triolein(C18:1,[cis]-9)

Trilinolein(C18:2,[cis,cis]-9,12)

min23.0022.0021.0020.0019.0017.00 18.00

650600550500450400350 700

650600550500450400350 700

2000

4000

6000

8000

2000

4000

6000

8000

439 467502

684.65

411

439

467

495

684.65

Fragmentor = 150 volts

Fragmentor = 80 volts

loss of C14 fatty acid

loss of C12 fatty acid

Figure 1. TIC showing separation obtained for four C18 triglyceride standards.Note that cis and trans isomers are well resolved.

Figure 1. The effect of raising the fragmentor voltage on a mixed triglyceride incoconut oil. The fragment masses and ratios are consistant with twoC12 fatty acids and one C14 fatty acid.

System Summary

LC System

Binary gradient

Detection

MSD posAPCI

Columns

Hypersil MOS, 2.1 mm × 200 mm, 5 µm

Column part number


830990-902

17

Fats and Oils

Major Analytes

Triglycerides and their hydroperoxides

Matrix

Edible oil

Reference

Rainer Schuster, “Analysis of Unsaturated Triglyceridesusing HPLC,” Agilent Technologies, publication 5966-0744E, www.agilent.com/chem

ConditionsColumn 200 × 2.1 mm Hypersil MOS, 5 µmMobile phase A = water

B = ACN/methyl-tert.butylether (9:1)Gradient At 0 min 87% B; at 25 min 100% BPost time 4 minFlow rate 0.8 mL/minColumn compartment 60 ºCInjection vol 1 µL standardUV absorbance 200 nm and 215 nm to detect

triglycerides, 240 nm to detect hydroperoxides, and 280 nm to detect tocopherols and decomposed triglycerides (fatty acids with three conjugated double bonds)

Sample preparation Samples were dissolved in tetrahydrofuran (THF).

HPLC method performanceLimit of detection for >10 µgsaturated triglycerides

Repeatability of RT over 10 runs <0.7 %areas over 10 runs <6 %

Time [min]

140

Abs

orba

nce

[sc

aled

]

120

100

80

60

40

20

0

5 10 15 20 25

215 nm

LLL

OLL

OO

L

PLL

OO

O

SO

O

240 nm

* Hydroperoxides

*

* *

*

*

*

215 nm

280 nm

215 nm

280 nm

LLL

OO

O

OO

L

OO

L

LLO

LLO

LLO

OO

O

SO

OSO

O

Olive oil

Good quality Poor quality

mAU

0

13.0Time [min]

23.0 13.0Time [min]

23.0

5

10

15

20

mAU

0

5

10

15

20

Figure 1. Triglyceride pattern of aged sunflower oil. The increased response at240 nm indicates hydroperoxides.

Figure 2. Analysis of olive oil. The response at 280 nm indicates a conjugateddouble bond and therefore poor oil quality.

Sunflower oil

System Summary

LC System

Quaternary gradient

Detection

DAD 3channel

Columns

Hypersil MOS, 2.1 mm × 200 mm, 5 µm

Column part number


830990-902

18

Flavors, Sweeteners, Organic Acids

Major Analytes

Flavor, sweetener, preservative

Matrix

Soft drinks

Reference

Michael Woodman, “Rapid Screening and Analysis of Components in Nonalcoholic Drinks,” Agilent Technologies,publication 5989-5178EN, www.agilent.com/chemFlo

Conditions

SystemAgilent 1200 Series Rapid Resolution LC, consisting of:

G1379B micro degasser

G1312B binary pump SL

G1367C HiP ALS autosampler SL, with thermostatic

temperature control

G1316B thermostatted column compartment SL

G1315C UV/VIS diode array detector SL, flow cell as indicated in

individual chromatograms

ChemStation 32-bit version B.02.01

ColumnsAgilent ZORBAX SB-C18, 4.6 mm × 250 mm, 5 µmAgilent ZORBAX SB-C18, 3.0 mm × 50 mm, 1.8 µm

Mobile phase conditionsOrganic solvent Acetonitrile (ACN) or ACN containing

0.1% formic acid

Aqueous solvent 20 mm phosphoric acid in Milli-Q water, with ammonium hydroxide, or Milli-Q water containing 0.1% formic acid

Gradient conditionsGradient slope 2.8% per column volume

See individual chromatograms for

flow rate and gradient time.

Samples1. Standard mixture of sodium saccharin, caffeine,

aspartame, vanillin, benzoic acid, sorbic acid,

benzaldehyde, all 50 µg/mL in 1/1 methanol/water

2. Various soft drinks, decarbonated where applicable

min0 2 4 6 8 10 12 14 16 18

mAU

0

100

200

300

400

500

600

1.90

1

4.38

9 -

Sac

char

in

5.64

1

6.70

1 -

Caf

fein

e

8.10

4 -

Asp

arta

me

9.95

5 -

Van

illin

12.

053

- B

enzo

ate

12.4

99 -

Sor

bate

15.5

12 -

Ben

zald

ehyd

e

min0.5 1 1.5 2 2.5 3 3.5 4

mAU

-50

0

50

100

150

200

0.79

3 -

Sac

char

in

1.25

4 -

un

k 1.83

2 -

Caf

fein

e

2.69

7 -

Asp

arta

me

2.95

2 -

Van

illin

3.45

5 -

Ben

zoat

e

3.68

1 -

Sor

bate

4.27

0 -

Ben

zald

ehyd

e

Figure 1. Gradient separation of soft drink additives on 4.6 mm ×× 250 mm, 5-µm ZORBAX SB-C18.

Figure 2. Gradient separation of soft drink additives on 3.0 mm ×× 50 mm, 1.8-µm ZORBAX SB-C18.

System Summary

LC System

1200SL

Detection

DAD

Columns

ZORBAX SB-C18, 4.6 mm × 250 mm, 5 µm

3 mm × 50 mm, 1.8 µm (600 bar)

Column part numbers

880975-902 (5 µm)

827975-302 (1.8 µm)

19


Major Analytes

Organic acids

Matrix

Foods

Reference

M.A. van Straten, H.A. Claessens, and A. Dams, “Analysis of Organic Acids in Aqueous Samples,” Agilent Technologies, publication 5989-1265EN,www.agilent.com/chem

2 4 6

mAU

0

10

20

30

40

50

60

1.5

12 1

.660

1.8

57 -

Tar

tari

c ac

id 2

.152

- M

alic

aci

d 2

.232

- L

acti

c ac

id 2

.413

- A

ceti

c ac

id

2.9

29 -

Cit

ric

acid

3.1

89 -

Su

ccin

ic a

cid

4.5

02 -

Fu

mar

ic a

cid

Time [min]1 2 3 4 5 6

mAU

0

20

40

60

80

100

120

Mal

ic a

cid

Asc

orbi

c ac

id

Cit

ric

acid

Su

ccin

ic a

cid

Fu

mar

ic a

cid

ConditionsColumn ZORBAX SB-Aq, 4.6 mm × 150 mm × 5 µm Mobile phase 20 mM aqueous phosphate buffer/

acetonitrile = 99/1 (v/v)Flow rate 1.0 mL/minInjection volume 1 µLColumn temperature 25 °CDetection UV-DAD detection wavelength/window

210/8 nm, reference wavelength 360/80 nmSample preparation Filtration over 0.45-µm filter

Figure 2. Standard mixture 1.

Figure 4. Orange juice.

System Summary

LC System

Quaternary gradient

Detection

DAD

Columns

ZORBAX SB-Aq, 4.6 mm × 150 mm, 5 µm

Column part number

883975-914

20


Major Analytes

Flavoring agents

Matrix

Mouthwash

Reference

Robert Ricker, “Flavoring Agents,” Agilent Technologies,publication 5988-6353EN, www.agilent.com/chem

ConditionsColumn ZORBAX SB-Phenyl,

50 mm × 2.1 mm, 5 µm (Agilent p/n 860975-912)

Mobile phase 0.3% TFA:acetonitrile/water (65:35)




0.6

0.5

0.4

0.3

0.2

0.1

0.0

0 1 2 3 4 5 6 7 8 9 10

Minutes

1

2

3

4

5

6

7

Cool Mint Listerine sample1. Unknown2. Benzoic acid3. Methyl salicylate4. Carvone5. Unknown6. Thymol7. Anethole

System Summary

LC System

Binary gradient

Detection

DAD

Columns

ZORBAX SB-Phenyl, 2.1 mm × 50 mm, 5 µm

Column part number

860975-912

21


Major Analytes

Semivolatile flavors

Matrix

Standard

Reference

Robert Ricker,"Aromatics,” Agilent Technologies, publication 5988-6352EN, www.agilent.com/chem

1

2

3

4

5

6

0.14

0.12

0.10

0.08

0.06

0.04

0.02

0.00

0 5 10 15 20

Minutes

Aromatic sample1. Acetophenone2. Cinnamaldehyde3. Eugenol4. Cinnamaldehyde impurity5. Ethyl cinnamate6. p-Cymene

ConditionsColumn ZORBAX XDB-Phenyl,

150 mm × 4.6 mm, 3.5 µm (Agilent p/n 963967-912)

Mobile phase Water : Methanol (40:60)

Flow rate 1 mL/min



System Summary

LC System

Isocratic

Detection

DAD

Columns

ZORBAX XDB-Phenyl, 4.6 mm × 150 mm, 3.5 µm

Column part number

963967-912

22

System Summary

LC System

Isocratic

Detection

DAD

Columns

ZORBAX SB-C18, 4.6 mm × 75 mm, 3.5 µm

Column part number

866953-902


Major Analytes

Aspartame, degradants

Matrix

Cola

Reference

Robert Ricker, “Aspartame: Metabolites and Applications,”Agilent Technologies, publication 5988-6349EN,www.agilent.com/chem

ConditionsColumn ZORBAX SB-C18,

75 mm × 4.6 mm, 3.5 µm, (Agilent p/n 866953-902)

Mobile phase 0.1% TFA:acetonitrile/water (85:15)

Flow rate 1 mL/min




1

2Aspartame and its metabolites1. Phenylalanine piperazineacetic acid2. 5-benzyl-3, 6-dioxo-2-piperazimeacetic acid3. Aspartic acid-phanylalanine dipeptide4. Aspartame

Diet Coke1. Caffeine2. Aspartame3. Unknown

Equal sweetener1. Aspartame

3 4

1

2

3

1

0 1 2 3 4 5 6 7 8 9 10Minutes

23

Herbal Supplements, Natural Products, Plant Hormones

Major Analytes

Xanthine metabolites

Matrix

Standard

Reference

John W. Henderson Jr. and William J. Long, ”Superior PeakShape of Xanthines and Metabolites Separated by EclipsePlus C18,” Agilent Technologies, publication 5989-5237EN, www.agilent.com/chem

21 3

45

6Eclipse Plus

mAU

04080

0 2 4 6 8 min

Competitor GmAU

04080

0 2 4 6 8 min

Competitor SmAU

04080

0 2 4 6 8 min

0 2 4 6 8

mAU

04080 Competitor L

min

0 2 4 60

4080

Competitor X

min

mAU

ConditionsColumn Eclipse Plus C18,

4.6 mm × 150 mm, 5 µm, (Agilent p/n 959993-902)

Mobile phase A: 25 mM PhosphateB: ACN (90:10)

Flow rate 1 mL/min


Detector DAD

Analyte elution order:

1. 1-Methylxanthine2. Theobromine3. Theophylline4. b-hydroxyethyltheophylline5. 3-Propylxanthine6. Caffeine

Figure 1. Separation of xanthines on various C18 columns.

System Summary

LC System

Isocratic

Detection

DAD

Columns

Eclipse Plus C18, 4.6 mm × 150 mm, 5 µm

Column part number

959993-902

24

System Summary

LC System

1200SL

Detection

DAD

Columns

ZORBAX SB-C18 RRHT, 4.6 mm × 150 mm, 1.8 µm

Column part number

829975-902


Major Analytes

Glycyrrhizin

Matrix

Licorice root

Reference

Bernard Permar and Ronald E. Majors, “The High-Resolu-tion Reversed-Phase HPLC Separation of Licorice RootExtracts Using Long Rapid Resolution HT 1.8-µm Columns,”Agilent Technologies, publication 5989-4907EN, www.agilent.com/chem

HPLC conditionsInstrument Agilent 1200SL Series Rapid Resolution

System

Detector Multiple wavelength detector (MWD), 254 nm/100 BW, 450 nm reference

Mobile phase A = 1% Acetic acid in waterB = 1% Acetic acid in acetonitrile

Gradient conditions for ZORBAX SB-C18 columnsConventional 4.6 mm × 250 mm, 5 µm

5% to 100% B in 50 minutes

RRHT 4.6 mm × 150 mm, 1.8 µm5% to 100% B in 30 minutes

Flow 1.0 mL/min

Temperature Ambient

GA

min25 30 35 40 45

mAU

0

10

20

30

40

50

60

70

22.0

16

Analysis of licorice root extract B using a ZORBAX SB-C18 4.6 × 250-mm column, 5 µm

GA

min16 18 20 22 24 26 28 30

mAU

20

30

40

50

60

70

80

90

Analysis of licorice root extract B using a ZORBAX SB-C18 RRHT 4.6 × 150-mm column

O

COOH

O

O

COOH

O

COOH

Figure 1. Structure of glycyrrhizinic acid.

25


Major Analytes

Xanthines

Matrix

Tea, chocolate

Reference

John W. Henderson and Ronald E. Majors, “High Through-put Separation of Xanthines by Rapid Resolution HPLC,”Agilent Technologies, publication 5989-4857EN,www.agilent.com/chem

SB-CN 1.8 µm1,2

43

min0.5 1 1.5 2 2.5

mAU

0

50

100

ZORBAX Bonus RP 100 mm 3.5 µm14

2,3

min0.5 1 1.5 2 2.5

mAU

02040

ZORBAX StableBond (SB)-C18 1.8 µm

min0.5 1 1.5 2 2.5

mAU

02040

12 3 4

mAU

0102030

12

SB-Phenyl 1.8 µm*

min0.5 1 1.5 2 2.5

3 4

Compounds:1 1-methylxanthine2 1,3-dimethyluric acid3 3,7-dimethylxanthine4 1,7-dimethylxanthine* Preproduction batch

System Summary

LC System

1200SL

Detection

DAD 2-µL cell

Columns

RRHT, 1.8 µm various

Column part number

Various

ConditionsColumns ZORBAX SB-C18, 4.6 × 50 mm, 1.8 µm

SB-Phenyl, 4.6 × 50 mm, 1.8 µmZORBAX Bonus-RP, 4.6 × 50 mm, 1.8 µmBonus-RP, 4.6 × 100 mm, 3.5 µm

Mobile phase A = 0.2% FAB = ACN w 0.2% FA

Isocratic composition 98% A 2% B (v/v)


Injection volume 2, 4, 6 µL, respectively

Detector DAD, 254 nm

Flowcell 3 µL, 2-mm flow path

Figure 2. ZORBAX stationary phase selectivity comparisons for zanthines.

26

Methods:The Agilent 1200 Series binary pump SL was operated under the following conditions: Solvent A Water + 0.1 % TFA Solvent B ACN + 0.1 % TFA Flow: 0.5 mL/minGradient 0 min 5 % B, 1 min 5 % B,

60 min 85 % B, 61 min 95 % B, 70 min 95 % B

Stop time 70 min Post time 15 min

System Summary

LC System

1200SL

Detection

MS TOF posESI

Columns

SB-C18, 2.1 mm × 150 mm, 1.8 µm

Column part number

820700-902


Major Analytes

Ginsenosides part 1

Matrix

Root

Reference

Bernard Permar and Ronald E. Majors, “Analysis of a Com-plex Natural Product Extract From Ginseng – Part 1: Struc-ture Elucidation of Ginsenosides by Rapid ResolutionLC–ESI TOF with Accurate Mass Measurement,” AgilentTechnologies, publication 5989-4506EN,www.agilent.com/chem

Thermostat

Column compartment

DAD SL

Degasser

650 mm x 0.17 mm capillary 320 mm x 0.12 mm capillary

2-L UV flow cell

Low delayvolume heatexchanger

High-performance autosampler SL

Binary pump SL

1.8-mm SB C18column

To MS

19.0 21.0 23.0 25.0 27.0 29.0 31.0 33.0 35.0Time, min

0.0

1.0e6

2.0e6

3.0e6

4.0e6

5.0e6

6.0e6

7.0e6

8.0e6

9.0e6In

ten

sity

cps

Rb1

Rc

Rb2

Rd

Rf

F11

Re

Figure 3. RR-LC-TOF basepeak chromatogram of the area containing the maincomponents of the Asian ginseng root extract.

Measured Calculated Formula Mass accuracy Mass accuracymass mass [mDa] [ppm]

1109.6129 1109.6108 C54H93O23 2.10 –1.901091.6012 1091.6002 C54H91O22 1.00 –0.91785.5047 785.5051 C42H73O13 –0.40 0.53767.4950 767.4946 C42H71O12 0.40 –0.58749.4854 749.4840 C42H69O11 –1.40 1.88425.3784 425.3783 C30H49O 0.10 –0.14407.3679 407.3678 C30H47 0.10 –0.30343.1248 343.1240 C12H23O11 0.80 2.23325.1136 325.1135 C12H21O10 0.10 –0.39

Table 1. Empirical Formulas and Achieved Mass Accuracies for the StructureElucidation of Ginsenoside

Figure 1. Agilent 1200 Series binary LC system for MS using a low delay volumeconfiguration.

27

Delphinidin-3-galactoside

Delphinidin-3-glucoside

5 10 15 20 25 30 35 40 min

5 10 15 20 25 30 35 40 min

5 10 15 20 25 30 35 40 min

Agilent Prep-C1821.2 × 250 mm, 10 µ

Agilent Prep-C184.6 × 250 mm, 5 µ

Fraction 1 rechromatographed

Fraction 2 rechromatographed

Agilent Prep-C18 4.6 × 250 mm, 5 µ

Threshold setting

Slope setting

Actual peaks collected are between the dashed vertical lines

Figure 2. Fraction collection and rechromatography to demonstrate purity.

System Summary

LC System

Prep

Detection

DAD

Columns

Prep-C18, 21.2 mm × 250 mm, 10 µm, and 4.6 mm × 250 mm, 5 µm

Column part numbers

410910-102 and 440905-902


Major Analytes

Anthocyanins

Matrix

Blueberry

Reference

Cliff Woodward, “Scale-Up of Anthocyanin Separations andRe-Analysis of Collected Fractions on an Agilent Prep-C18Column,” Agilent Technologies, publication 5989-0591EN,www.agilent.com/chem

10 min20 30 40 50 60 70 80

Agilent Prep-C18, 21.2 × 250 mm, 10 µm(p/n 410910-102)

1A

10 min20 30 40 50 60 70 80

Agilent Prep-C18, 4.6 × 250 mm, 5 µm(p/n 440905-902)

1B

Conditions1A

Column Agilent Prep-C18, 21.2 × 250 mm, 10 µmTemperature AmbientDAD wavelength 525 nmInjection 2000 µLSample Blueberry extract, 46.1 mg/mL total

dissolved solids (~5 mg/mL anthocyanins)Flow 21.2 mL/min

1B

Column Agilent Prep-C18, 4.6 × 250 mm, 5 µTemperature AmbientDAD wavelength 525 nmInjection 100 µLSample Blueberry extract, 46.1 mg/mL total

dissolved solids (~5 mg/mL anthocyanins)Flow 1.0 mL/min

Figure 1. Scalability of Agilent Prep columns.

Mobile phase

A = 0.1% TFA in water

B = 0.1% TFA in methanol

Gradient timetable

Time (min) % Solvent B0.00 23.0

35.00 26.085.00 53.5

See application note for details on resulting fraction purity.

28


Major Analytes

Anthocyanins

Matrix

Standard

Reference

Robert Ricker, “High-Efficieny and High-Speed Separation ofNatural Anthocyanins,” Agilent Technologies, publication5988-6362EN, www.agilent.com/chem

ConditionsColumn ZORBAX SB-C18,

250 mm × 4.6 mm, 5 µm, (Agilent p/n 880975-902)

ZORBAX SB-C18, 150 mm × 4.6 mm, 3.5 µm, (Agilent p/n 863953-902)

ZORBAX SB-C18, 75 mm × 4.6 mm, 3.5 µm, (Agilent p/n 866953-902)

Mobile phase A: 3% phosphoric acid, B: methanolwater

Gradient 23% B to 60% B in 97 min23% B to 60% B in 58 min23% B to 60% B in 29 min

Flow rate 1 mL/min




0

Time (min)

10 20 30 40 50 60 70

0

Time (min)

5 10 15 20 25 30 4035

0

Time (min)

5 10 15 20

4.6 mm × 250 mm, 5 µmTime Percent B

0 23%

35 26%

97 60%

4.6 mm × 150 mm, 3.5 µmTime Percent B

0 23%

21 26%

58.2 60%

4.6 mm × 75 mm, 3.5 µmTime Percent B

0 23%

10.5 26%

29.1 60%

System Summary

LC System

Quaternary or binary gradient module

Detection

DAD

Columns

ZORBAX SB-C18, 4.6 mm, 5 µm and 3.5 µm

Column part number


29

20

15

10

5

0

0

15

43

2

5 10 15

50

25

0

0

1

Unknown

43

2

5 10 15

Minutes Minutes

Green tea extractCatechin mixture

1. Epigallocatechin

2. Epicatechin

3. Epigallocatechin

gallate

4. Catechol

5. Epicatechin

gallate


Major Analytes

Flavonoids, catechins

Matrix

Standard

Reference

Robert Ricker, “Epigallocatechin 3-O-gallate extract fromgreen tea,” Agilent Technologies, publication 5988-6357EN, www.agilent.com/chem

ConditionsColumn ZORBAX SB-C8, 4.6 mm, 150 mm × 3.5 µm

(Agilent p/n 863953-906)

Mobile phase A 0.1% trifluoroacetic acid in water 75%, Mobile phase B Methanol 25%

Flow rate 1 mL/min

Oven temperature 40 °C



System Summary

LC System


Detection

DAD

Columns


Column part number

863953-906

30

Preservatives

Major Analytes

Flavors, sweeteners, preservatives

Matrix

Soft drinks

Reference

Michael Woodman, “Rapid Screening and Analysis of Components in Nonalcoholic Drinks,” Agilent Technologies,publication 5989-5178EN, www.agilent.com/chem

ConditionsSystem

Agilent 1200 Series Rapid Resolution LC, consisting of:G1379B micro degasserG1312B binary pump SLG1367C HiP ALS autosampler SL, with thermostatic temperature control G1316B thermostatted column compartment SL G1315C UV/VIS diode array detector SL, flow cell as indicated in individual chromatogramsChemStation 32-bit version B.02.01

Columns Agilent ZORBAX SB-C18, 4.6 mm × 250 mm, 5 µmAgilent ZORBAX SB-C18, 3.0 mm × 50 mm, 1.8 µm

Mobile phase conditions

Organic solvent Acetonitrile (ACN) or ACN containing 0.1% formic acid

Aqueous solvent 20 mm phosphoric acid in Milli-Q water, withammonium hydroxide, or Milli-Q water containing 0.1% formic acid

Gradient conditions

Gradient slope 2.8% per column volume See individual chromatograms for flow rate and gradient time.

Samples

1. Standard mixture of sodium saccharin, caffeine, aspartame, vanillin,benzoic acid, sorbic acid, and benzaldehyde, all 50 µg/mL in 1/1 methanol/water

2. Various soft drinks, decarbonated where applicable

min0 2 4 6 8 10 12 14 16 18

mAU

0

100

200

300

400

500

600

1.90

1

4.38

9 -

Sac

char

in

5.64

1

6.70

1 -

Caf

fein

e

8.10

4 -

Asp

arta

me

9.95

5 -

Van

illin

12.

053

- B

enzo

ate

12.4

99 -

Sor

bate

15.5

12 -

Ben

zald

ehyd

e

min0.5 1 1.5 2 2.5 3 3.5 4

mAU

-50

0

50

100

150

200

0.79

3 -

Sac

char

in

1.25

4 -

un

k 1.83

2 -

Caf

fein

e

2.69

7 -

Asp

arta

me

2.95

2 -

Van

illin

3.45

5 -

Ben

zoat

e

3.68

1 -

Sor

bate

4.27

0 -

Ben

zald

ehyd

e

Figure 1. Gradient separation of soft drink additives on 4.6 ×× 250 mm, 5-µm ZORBAX SB-C18.

Figure 2. Gradient separation of soft drink additives on 3.0 ×× 50 mm, 1.8-µm ZORBAX SB-C18.

System Summary

LC System

1200SL

Detection

DAD

Columns

ZORBAX SB-C18, 4.6 mm × 250 mm, 5 µm;

3 mm × 50 mm, 1.8 µm (600 bar)

Column part numbers

827975-302 (1.8 µm), 880975-902 (5 µm)

31

Table 1. Structures and Concentrations of Preservative Compounds

Preservatives

Major Analytes

Paraben, phenoxyethanol

Matrix

Standard

Reference

Coral Barbas, Javier Rupérez, Andre Dams, and Ronald E. Majors, “Separation of Paraben Preservatives byReversed-Phase HPLC,” Agilent Technologies, publication5989-3635EN, www.agilent.com/chem

OCH2

CH2OH

COCH3

O

OH

COC2H5

O

OH

CO

CH2

O

OH

CH2

CH3

COCH2

O

OH

CHCH3

CH3

CO C4H9

O

OH

2PX: 2-Phenoxyethanol

(1.4 mg/mL)

MEP: Methylparaben

(0.30 mg/mL)

ETP: Ethylparaben

(0.07 mg/mL)

PRP: Propylparaben

(0.04 mg/mL)

IBP: Isobutylparaben

(0.04 mg/mL)

BTP: Butylparaben

(0.08 mg/mL)

ConditionsColumn ZORBAX Eclipse XDB-C18 Rapid

Resolution, 4.6 mm × 150 mm, 3.5 µm

Mobile phase Solvent A: WaterSolvent B: Methanol

Gradient Time % MeOH0 385 386 6016 6017 6220 38

Flow rate 0.8 mL/minTemperature 40 °CDetector UV 254 nmInjection volume 5 µL

1

2

3

45

6

1 MEP2 2PX3 ETP4 PRP5 IBP6 BTP

Minutes

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0

mA

U

0

200

400

600

800

Figure 1. Separation of preservatives by reversed-phase HPLC.

System Summary

LC System


Detection

DAD

Columns

ZORBAX XDB-C18, 4.6 mm × 150 mm, 3.5 µm

Column part number

963967-902

32

Proteins, Peptides, Amino Acids

Major Analytes

Proteins

Matrix

Wheat

Reference

Robert Ricker, “Stationary-Phase Selectivity Comparison:High-Molecular-Weight, Alkylated, Wheat Glutenin, ProteinSubunits,” Agilent Technologies, publication 5988-6358EN,www.agilent.com/chem

ZORBAX 300 SB-C8Gradient: 23%-48% ACN

ZORBAX 300 SB-CNGradient: 23%-48% ACN

A

B

2*

910

2*9

10

7*

7*

5

50.21

Ab

sorb

ance

, 210

nm

, mv

x10

3A

bso

rban

ce, 2

10 n

m, m

v x1

03

0.15

0.09

0.03

0.50

0.35

0.20

0.05

0 20

Time (minutes)

40 60

0 20

Time (minutes)

40 60

ω-gliadins

ω-gliadins

ConditionsColumn ZORBAX 300 SB-CN, 300 SB-C8

4.6 mm × 150 mm(Agilent p/n 883995-906, 883995-905)

Mobile phase As above with 0.1% TFA, water

Gradient 60 minFlow rate 1.0 mL/minTemperature 50 °CDetector DAD

System Summary

LC System


Detection

DAD

Columns

ZORBAX 300 SB-CN, 300 SB-C8

Column part numbers

883995-906, 883995-905

33

0 20 40 60 80 100

0 20 40 60 80 100

70°

50°

A-F

H

I

J

K-P

G


Major Analytes

Proteins

Matrix

Wheat

Reference

Robert Ricker, “Effect of temperature on separation of awheat protein,” Agilent Technologies, publication 5988-6348EN, www.agilent.com/chem

ConditionsColumn ZORBAX 300 SB-C8, 150 mm × 4.6 mm,

(Agilent p/n 883995-906)

Mobile phase A: 0.1% trifluoroacetic acid in water B: 0.1% TFA in acetonitrile

Flow rate 1 mL/min

Gradient At 0 min 23% B at 120 min 48% B

Oven temperature 50 and 70 °C



System Summary

LC System


Detection

DAD

Columns

ZORBAX 300 SB-C8, 4.6 mm × 150 mm, 5 µm

Column part number

883995-906

34


Major Analytes

BSA (bovine serum albumin) digest, peptide

Matrix

Standard

Reference

Robert D. Ricker and Cliff Woodward, “Decreasing AnalysisTime Using Poroshell 300 SB-C18 in Analysis of a ProteinDigest,” Agilent Technologies, publication 5988-6081EN,www.agilent.com/chem

mAU

0

100

200 1 mL/min0-100%B12 min

Poroshell 300 SB-C182.1 × 75 mm, 5 µmPart number: 660750-902

6 min

ConditionsColumn 300 SB-C18 (as indicated above)

Mobile phase A = 95% H2O, 5% ACN, 0.1% TFAB = 5% H2O, 95% ACN, 0.07% TFA

Flow As above

Piston stroke 20 µL

Detection UV: 215 nm

Temperature 70 °C

Agilent 1100 well plate autosampler with delay volume reduction

Injection volume 20 µL (0.22 µg/1 µL)

Sample BSA Tryptic digest (15 hours, 70 pmol)

System Summary

LC System

Binary gradient

Detection

DAD

Columns

Poroshell 300SB-C18, 2.1 mm × 75 mm, 5 µm

ZORBAX 300SB-C18, 2.1 mm × 150 mm, 5 µm

Column part number

660750-902 and 883750-902

0

mAU

0

100

200 0.208 mL/min0-100%B120 min

Zorbax 300 SB-C182.1 × 150 mm, 5 µmPart number: 883750-902

60 min

Aligned

35


Major Analytes

AAA amino acid

Matrix

Standard

Reference

John W. Henderson, Robert D. Ricker, Brian A. Bidling-meyer,and Cliff Woodward, “Rapid, Accurate, Sensitive, andReproducible HPLC Analysis of Amino Acids,” AgilentTechnologies, publication 5980-1193EN,www.agilent.com/chem

mAU

0

5

10

min0 4 6 8 10 12

mAU

0

5

10

15

2

1 2

3

4

56

78

9

10 11 1221

2019

1817

16

1514

13

22

23

2421

A

B

338 nm

262 nm

mAU

0

4

8

min0 5 10 15 20 25

mAU

0

4

8

12

3

4

5678

9

1011 12 1314

1517

16

1819

20

3

21

12 4

56

78

9

1110 12 1314

15

1617

1819

20

21

5 µm, Zorbax Eclipse-AAA

3.5 µm Zorbax Eclipse-AAA

Figure 1. Routine Analysis, High-Throughput Separation of 24 Amino AcidsUsing the Eclipse-AAA Protocol. The column dimensions are 4.6 × 75 mm, 3.5 µm. See Table 1 for peak identification. Detection: A. 338 nm (OPA amino acids), B. 262 nm (FMOC-amino acids).

Figure 2. High-Resolution Analysis of 21 Amino Acids on the 5-µm and 3.5-µm ZORBAX Eclipse-AAA Column. Column dimensionsare 4.6 × 150 mm. See Table 1 for peak identification. Detection: 338 nm (OPA aminoacids).

Mobile PhaseMobile phase A 40 mM Na2HPO4 [5.5 g NaH2PO4,

monohydrate + 1 liter water, adjust pH withNaOH solution (10 N)]

Mobile phase B ACN:MeOH:water (45:45:10, v/v/v)

It is convenient to make mobile phase A as a 10X stock solution withno pH adjustment. The solution can be kept for several weeks and canbe diluted and titrated, as needed. All mobile-phase solvents should beHPLC grade

Pump SettingsFlow 2 mL/min

Stoptime 14 min (75-mm column) or 26 min (150-mm column)

Post time Off

Gradients For 75-mm column length

Time (min) % B

0 01 09.8 5710 10012 10012.5 014 0

System Summary

LC System


Detection

FLD/OPA-FMOC

Columns

Eclipse-AAA, 4.6 mm × 75 mm and 4.6 mm × 150 mm, 3.5 µm;

3 mm × 150 mm, 3.5 µm; and 4.6 mm × 150 mm, 5 µm

Column part number


36

Regulated/Hazardous Drug Substances

Major Analytes

Drugs

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

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 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2

×106

Hydrochlorothiazide

Aspirin

Enalaprilat Furosemide

Ketoprofen

Clofibric acidNaproxen

Diclofenacsodium salt

IbuprofenIbuprofen - d3

Gemfibrozil

Triclocarban

Figure 1. Negative ion mode TIC of 11 pharmaceuticals.

ConditionsColumn ZORBAX Extend-C-18, RRHT,

2.1 mm × 100 mm, 1.8 µm

Column temperature: 40 °C

Mobile phases A 0.1% formic acid in water, add NH4OH buffer Mobile phases B Acetonitrile (ACN)

Flow rate: 0.3 mL/min

Gradient: Time %B0 015 10020 10021.5 0

System Summary

LC System

1200SL

Detection

MSMS QQQ pos/negESI

Columns

Extend-C18, 2.1 mm × 100 mm, 1.8 µm

Column part number

728700-902

37


Major Analytes

Nitrofurans

Matrix

Poultry, shrimp

Reference

Bernhard Wüst, Christian Sauber, and Hans (J.) A. van Rhijn“Quantitation of Nitrofuran Metabolites in Shrimp and Poul-try by LC/MS/MS Using the Agilent LC/MSD Trap XCT,” Agilent Technologies, publication 5989-0738EN,www.agilent.com/chem NBA-d4 AOZ

x104

0.0

0.4

0.8

1.2

NBA-AOZ

x104

0.00

0.50

1.00

1.50

NBA-AHD

0

2000

4000

6000

NBA-d5 AMOZ

x104

0

2

46

NBA-AMOZ

0

2

4x104

Intensity

NBA-SEM

x104

0

2

4

2 4 6 8 10 12 Time [min]

LC/MS/MS Method DetailsHPLC Agilent 1100


Column ZORBAX XDB-C8, 2.1 mm × 50 mm, 3.5 µm

Mobile phases A: Water + 0.1% acetic acidB: Acetonitrile + 0.1% acetic acid

Gradient 0–14 min: 10% A - 45% A; 14–16 min: 45% A - 90% A

Injection 50 µL out of 500 µL

MS 1100 LCMSD XCT Ion TrapIonization mode Positive ESI



Drying gas 350 °C temperature

Skimmer 20 V

Capillary exit 55 V

Trap drive 55

ICC On

Figure 5. Representative chromatogram of a positive shrimp sample at a levelof 0.25 µg/kg.

System Summary

LC System

Binary gradient

Detection

MSn Trap XCT posESI

Columns

ZORBAX XDB-C8, 2.1 mm × 50 mm, 3.5 µm

Column part number

971700-906

HPLC Method performance

Overall amount RSD 4.3%

Overall recovery 100%

38


Major Analytes

Fluoroquinolones

Matrix

Beef kidney

Reference

Ralph Hindle and Chin-Kai Meng, “The Analysis of Fluoroquinolones in Beef Kidney Using HPLC ElectrosprayMass Spectrometry,” Agilent Technologies, publication5989-0596EN, www.agilent.com/chem

HPLCColumn ZORBAX Eclipse XDB-C8,

150 mm × 4.6 mm, 5 µm (P/N 993967-906)

Solvent A 0.1% formic acid in water

Solvent B 0.1% formic acid in acetonitrile

Gradient t0 = 20% Bt1 = 20% Bt8 = 90% Bt15 = 90% BPost time = 2.0 min



Column temp 30 °C

MSDSource Electrospray ionization (ESI)

(positive ion mode)

Ion dwell time 14 ions at 40 ms each

Fragmentation Varies by ion



Drying gas temperature 350 °C

Capillary voltage 4000 V

min5.5 6 6.5 7 7.5 8 8.5 9

min5.5 6 6.5 7 7.5 8 8.5 9

5.484 - NOR

5.485 - NORq1

5.482 - NORq2

0

4000

8000

0

4000

8000

12000

12000

0

4000

8000

12000

0

4000

8000

12000

0

4000

8000

12000

0

4000

8000

12000

5.530 - NOR

5.531- NORq1

5.532 - NORq2

320

302

276

320

302

276

System Summary

LC System

Binary gradient

Detection

MSD posESI

Columns

ZORBAX XDB-C8, 4.6 mm × 150 mm, 5 µm

Column part number

993967-902

Beef kidney blank

Norfloxacin (IS)

330 = [M + H]+

302 = [M – H2O + H]+

276 = [M – CO2 + H]+

39


Major Analytes

Chloramphenicol

Matrix

Shrimp, honey

Reference

G. Vanhoenacker, F. Davis, P. Sandra, “Detection, Confirma-tion, and Quantitation of Chloramphenicol in Honey andShrimp at Regulatory Levels Using Quadrupole and Ion TrapLC/MS,” Agilent Technologies, Publication 5988-9920EN,www.agilent.com/chem

1000

0

2000

4 5Time (min)

1000

0

176

194

249

257

160 240

176 194

249

257

160 240 320 m/z

180199 254

262

m/z160 240 320

CAP: EIC 176, 194, 249, 257 m/z(2.5 pg/µL)

CAP-d5: EIC 180, 199, 254, 262(5 pg/µL)

2.5 pg/µL CAP

0.2 pg/µL CAP(LOD)

5 pg/µL d5-CAP6

Figure 2. Analysis of a standard solution containing 2.5 pg/mL CAP and 5 pg/mL CAP-d5 (IS) on the LC/MSD Trap together with the corre-sponding MS/MS spectra and the MS/MS spectrum resulting froman analysis of a standard solution containing 0.2 pg/mL CAP.

30000

60000

7500

15000

Time (min)

Time (min)

4 5 6 70

6000

12000

0

15000

4 5 6 7

15000

0

QU

AR

UP

OLE

TR

AP

TIC

EIC 262, 326, 328 m/z (CAP-d5)

EIC 257, 321, 323 m/z (CAP)

EIC 176, 194, 249, 257 m/z (CAP)

EIC 180, 199, 254, 262 m/z (CAP-d5)

Figure 4. Analysis of a blank honey sample containing 1 ppb CAP-d5.

System Summary

LC System

Binary gradient

Detection

MSD and MSn Ion Trap negESI

Columns

ZORBAX Eclipse XDB-C18, 4.6 mm × 150 mm, 5 µm

Column part number

993967-902

ConditionsMobile phase 10 mM ammonium acetate in water

(solvent A) Methanol/acetonitrile 1/9 (solvent B)

40


Major Analytes

Sulfa drugs

Matrix

Meat

Reference

Hiroki Kumagai and Adebayo Onigbinde, “Analysis of Residual Synthetic Antibacterials in Meat by HPLC,” Agilent Technologies, publication 5988-7135EN, www.agilent.com/chem

ConditionsColumn RP-18 Purospher, 250 mm × 4 mm, 5 µm

(Agilent p/n 79925PU-584)

Mobile phase A: 0.7% phosphoric acid B: Acetonitrile

Gradient at 0 min 5% Bat 10 min 65% Bat 40 min 65% Bat 45 min 65% B

Post time 7 min

Flow rate 1 mL/min



Detector DAD detection wavelengths at 338/10 nm, 264/8 nm and 360/8,reference off

Figure 1. Chromatogram of standard solution, 2 ug/mL each analyte.

Figure 2. Chromatogram of extract of bovine muscle.

System Summary

LC System


Detection

DAD

Columns

RP-18 Purospher, 250 mm × 4 mm, 5 µm

Column part number

79925PU-584

20

10

0

0

Absorbance [mAU]

5 10 15 20

FZD

DFZ

25 30 35 Time (min)

20

10

0

0

Absorbance [mAU]

5 10 15 20 25 30 35 Time (min)

1 SMr2 PYM3 TCP4 SDD5 FZD

6 SMMX7 DFZ8 SDMX9 SQX10 OXA

1

34

2

at 224 nm

at 360 nm

56 7 8 9

10

4

2

0

0

Absorbance [mAU]

5 10 15 20

SDD

25 30 35 Time (min)

4

2

0

0

Absorbance [mAU]

5 10 15 20 25 30 35 Time (min)

at 224 nm

at 360 nm

41

Conditions

Mobile phase A = 0.1 % formic acid in water

B = 0.1 % formic acid in acetonitrile


Major Analytes

Sulfonamides

Matrix

Standard

Reference

Rainer Schuster and Christine Miller, “Analysis of sulfonamides in the low pg range by capillary LC usingdiode array and mass spectrometric detection,” AgilentTechnologies, publication 5980-2499EN,www.agilent.com/chem

[min]

Abs

orba

nce

[m

AU

]

-1

0

0 2 4 6 8 10

1

2

3

4 1

2

3

4

5 6 7

1 Sulfanilamide2 Sulfacetamide

3 Sulfadiazine4 Sulfapyridine

5 Sulfamethazine6 Sulfamerazine

7 Sufameter

100 pg

10 pg

min2 4 6 8 10

0

1000

2000

3000

4000

5000

2

1

34

5

6

7

8

Abu

nda

nce

10 pg

1 pg

min2 4 6 8

2200

2500

600

2200

400

1600 Sulfameter m/z 281.1

Sulfamethazine m/z 279.1

Sulfacetamidem/z 215.1

A

B

1 Sulfaguanidine2 Sulfanilamide3 Sulfacetamide4 Sulfadiazine

5 Sulfapyridine6 Sulfamerazine7 Sulfamethazine7 Sufameter

Figure 2. Analysis of the standard mix with 10 pg and 1 pg with MSD and SIM – total ion chromatogram from SIM (A) and extracted ion chromatogram from SIM (B).

Figure 1. Analysis of a mixture of 7 sulfonamides with diode array at 10 pg and100 pg absolute with 100-nL injection volume.

System Summary

LC System

CapLC

Detection

DAD MSD posESI

Columns


Column part number

5064-8262

42

Regulated/Hazardous Miscellanous Substances

Major Analytes

HMF hydroxymethylfurfural

Matrix

Bread, cereal, yogurt

Reference

Vural Gökmen, Hamide Z. Senyuva, “Rapid Determination of Hydroxymethylfurfural in Foods UsingLiquid Chromatography-Mass Spectrometry,” Agilent Technologies, publication 5989-5403EN,www.agilent.com/chem

1600

1400

1000

1200

800

600

400

200

2.5 5 7.5 10 12.5 min

m/z 127

OOHO

HMF

7000

6000

4000

5000

3000

2000

1000

2.5 5 7.5 10 12.5 min

m/z 127

OOHO

HMF

Figure 4. EIC of a bread sample (HMF concentration is 17.5 µg/g).

Figure 3. EIC of a fruited yogurt sample (HMF concentration is 0.2 µg/g).

Conditions

LC/MSFlow rate 0.2 mL/min

Gradient ZORBAX Bonus RP, 100 mm × 2.1 mm, 3.5 µm

Mobile phase 0.01 mM acetic acid in 0.2% aqueoussolution of formic acid

Injection 20 µL out of 1000 µL

MS conditionsIonization mode Positive APCI



Drying gas 325 °C temperature

Vaporizer 425 °Ctemperature

Skimmer 20 V

Capillary voltage 4 kV

Fragmentor voltage 55 eV

Dwell time 439 ms

System Summary

LC System

Binary gradient G1312A or G1312B

Detection

MSD posAPCI

Columns

Bonus-RP, 2.1 mm × 100 mm, 3.5 µm

Column part number

861768-901

43

System Summary

LC System

Dual binary w/6-port valve for autoSPE

Detection

MS TOF posESI

Columns

ZORBAX SB-C18, 2.1 mm × 150 mm, 5 µm

Column part number

883700-922


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 5988-2884EN, www.agilent.com/chem

1

4

2

3

6

5

1

4

2

3

6

5

LC/MSD TOF

Sample extraction

Second pump

Analytical column

Analytical pump

1100 Auto sampler 1100 Column compartment

WasteExtraction pre-column

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

Acry 1 µg/L - Acrylamide d3 (Unknown) 75.0 - 75.0 amu amu - Acry 1 µg wiffArea: 2.21e+002 counts Height: 1.12e+002 cps RT: 1.95 min

5000

4000

3000

Inte

nsi

ty, c

ps2000

1000

01.00.0 2.0 3.0

Time, min

4.0 5.0

6000

5000

4000

3000

Inte

nsi

ty, c

ps

2000

1000

01.00.0 2.0 3.0

Time, min

4.0 5.0

0.41 1.071.42

1.93

2.383.50 4.24 4.99

0.29 1.02 1.46 1.952.18

2.543.18

4.06 4.18 4.89 5.31

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

ConditionsElution solvents A: 97% HPLC water (0.01% formic acid)

B: 3% CH3CN (0.01% formic acid)

44

System Summary

LC System

Metrohm 818 pump, Agilent 7500 ISIS sampler

Detection

IC-ICPMS

Columns

Agilent Cr, 4.6 mm × 30 mm, 5 µm

Column part number

G3268A


Major Analytes

Chromium speciation

Matrix

Metrohm

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

Valve 1 ISIS

Drain

ICP-MSNebulizer

Metrohm818 IC Pump

Sample

Column(G3268A)

Pump 1ISIS

0.05~1.0 mL

Integrated Sample Introduction System(ISIS)

Autosampler (ASX500)

Mobile phase

0.00 1.00 2.00 3.00 4.00 5.00

2000

3000

4000

5000

6000

7000

8000

9000

Cr(III) 0.1 µg/L

Retention Time / min

Abu

nda

nce

/ c

oun

ts Cr(VI) 0.1 µg/L

Abu

nda

nce

/C

oun

ts

0.00 1.00 2.00 3.00 4.00 5.00 6.00

2000

3000

4000

5000

6000

7000

8000

9000

10000

11000

12000

13000

14000

RT/min

Original Water B

Cr(III): 0.05 µg/L

Cr(VI): 0.24 µg/L

Water B + 1 µg/L each Cr(III),Cr(VI)

Cr(III): 1.10 µg/L

Cr(VI): 1.27 µg/L

Figure 1. IC-ICP-MS configuration used for Cr speciation.

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

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

ConditionsCr column Agilent part number G3268A,

30 mm × 4.6-mm id

Mobile phase 5 mM EDTA (Na2), adjust pH by NaOH



Injection volume 50~500 µL

Sample preparation

Reaction 40 °Ctemperature

Incubation time 3 h

EDTA concentration 5~15 mM, adjust pH by NaOH

45

System Summary

LC System

Metrohm IC

Detection

MSD negESI

Columns

See description of metrohm column on this page

HPLC Method Performance

Overall amount RSD 4.3%

Overall recovery 101.5%


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 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"




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, water


Agilent 1100 MSDTune mode Negative mode “auto-tune”

Vcap 1400 V

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

Nebulizer pressure 20 psig

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-

(4 mm × 100 mm) p/n 6.1006.510

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

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.

46

System Summary

LC System

Isocratic G1310A

Detection

ICP-MS

Columns

See Hamilton decribed at right

2.00 4.00 6.00 8.00 10.00 12.00 14.00

0

2000

4000

6000

8000

10000

12000

14000

Time (min)

Abu

nda

nce

(co

un

ts)

AsB

As(III)DMA

MMAA


Major Analytes

Arsenobetaine

Matrix

Fish

Reference

Raimund Wahlen, “Fast and accurate determination ofarsenobetaine (AsB) in fish tissue using HPLC-ICP-MS,”Agilent Technologies, publication 5988-9893EN,www.agilent.com.chem

ConditionsColumn Hamilton PRP X-100

Mobile phase A: 2.2-mM NH4HCO3,B: 2.5-mM tartaric acid, 1% methanol

Flow rate 1 mL/min



Gradient At 0 min 20% Aat 30 min 100% A

Detector HPLC-ICP-MSRF power 1430–1550RF matching 1.89–1.92 VSampling depth 4.0–4.8 mmCarrier gas flow 0.89–0.93 L/minMake up gas flow 0.10–0.14 L/minOptional gas oxygen at 5%Spray chamber temperature 0 °CCones platinumIsotopes monitored 75As, 103Rh, 77Se, 53CrInjector diameter 2.4 mmNebulizer 100 L/min PFA2 interface pumps

Confirmation MS spectral information and RTs

Figure 1. Chromatography A: 2.2-mM NH4HCO3 2.5-mM tartaric acid, 1% MeOH, Hamilton PRP X-100 column. Concentration of standard ~ 5 ng/g as As.

47

Regulated/Hazardous Natural Toxin Substances

Major Analytes

DSP algal toxins

Matrix

Shellfish

Reference

Norbert Helle, Sebastian Lippemeier, and Jürgen Wendt, “Identification and Isolation of DSP-Toxins Using a Combined LC/MS-System for Analytical and Semiprepara-tive Work,” Agilent Technologies, publication 5989-2912EN, www.agilent.com.chem

m/z650 675 700 725 750 775 800 825 850

m/z650 675 700 725 750 775 800 825 850

0

20

40

60

80

100 Max: 611673

Okadaic acid (OA)positive

0

5

10

15

20

25

30 Max: 177930

804.4

805.4

Okadaic acid (OA)negative

751.4

733.4 753.4

752.4

769.4

770.4

771.4787.4

827.4

828.4

829.4

17.0 17.2 17.4 17.6

Okadaic acid (OA) positive

Okadaic acid (OA) negative

Figure 4. LC/MS analysis of OA.

Continued

System Summary

LC System

Quaternary gradient

Detection

MSD pos/negESI

Columns

ZORBAX SB-C18, 3 mm × 150 mm, 5 µm;

9.4 mm × 50 mm, 5 µm semiprep

Column part number

883975-302 and 846975-202

48

LC/MS Method Details – AnalyticalLC Conditions

Instrument Agilent 1100 HPLC (Quaternary pump)Column ZORBAX SB-C18, 3.0 mm × 150 mm, 5 µm Mobile phase A Water (0.1% Formic acid)

B MethanolGradient 20% B at 0 min

20% B at 5 min80% B at 20 min

Stop time 28 min; Post time 4 min Flow rate 0.6 mL/min

Injection vol 10 µL

MS Conditions

Instrument Agilent LC/MSDSource Positive/negative switching ESIDrying gas flow rate 12 L/minNebulizer 60 psigDrying gas temp 350 °C Vcap 3000 V (positive and negative)

LC/MS Method Details – SemipreparativeLC Conditions

Instrument 1 Agilent 1100 HPLC (quaternary pump)Column ZORBAX SB-C18, 9.4 mm × 50 mm, 5 µm Mobile phase A water (0.1% formic acid)

B methanol

Gradient 20% B at 0 min20% B at 5 min80% B at 20 min

Stop time 28 minPost time 4 min Flow rate 7.0 mL/min Injection vol 100 µL (250 µL using multiple draw mode)

Instrument 2 Agilent 1100 HPLC (isocratic pump) formakeup flowFlow rate 0.8 mL/min (50% H2O + 50% MeOH +

0.1% formic acid)Active splitter Split ratio 271:1

MS Conditions

Instrument Agilent LC/MSDSource Negative ESIDrying gas flow 12 L/minNebulizer 60 psigDrying gas temp 350 °C Vcap 3000 V (positive)

MSD Fraction Collection Setup

FC Mode Use method target mass; Adducts: (M–H)–

1467 µg/kgOkadaic acid

mV

mV

1467 µg/kgOkadaic acid

HPLC/Fluorescence

LC-MS, API pos.

2 6 10 14 18 min

0 10 20 30 40 min

Figure 5. Conparative analysis of DSP toxins in shellfish.

49


Major Analytes

Mycotoxin, fumonisin

Matrix

Corn

Reference

Friedrich Mandel, “Analysis of Fumonisin Mycotoxins,” Agilent Technologies, publication 5968-2124E, www.agilent.com.chem

System Summary

LC System

Binary gradient

Detection

MSD posESI

Columns


Column part number

993700-902

1 2 3 4 5

Retention Time (min)

6 7 8 9 min

1.251

3.281

6.274

7.690

800000

Abundance Fumonisin B1 Fumonisin B3 Fumonisin B2

400000

0

200 300 400 500 600 700 800

m/z

722.5

706.5

706.5

0

100000

200000

0

100000

200000

0

100000

200000

Fumonisin B1

Fumonisin B3

Fumonisin B2

Figure 3. Chromatographic separation of fumonisin analogues at 25 ng.

Figure 2. Mass spectra for fumonisin analogues.

Chromatographic ConditionsColumn ZORBAX Eclipse XDB, C18,

2.1 mm × 150, 5 µm

Mobile phase A = 5 mM ammonium acetate in waterB = acetonitrile

Gradient Start with 33% Bat 8 min 60% Bat 9 min 33% B

Flow rate 250 µL/min

Injection vol 5 µl

Column temp 40 °C

Diode-array detector Signal 220, 4 nm; reference 550,100 nm

MS ConditionsSource ESIIon mode PositiveVcap 4000 VNebulizer 30 psigDrying gas flow 10 l/minDrying gas temp 350 °CScan range 120-820 amuStep size 0.1Peak width 0.15 minTime filter OnFragmentor Variable 230 V (100-680)

100 V (680-800)

50


Major Analytes

Aflatoxins

Matrix

Various

Reference

Norbert Helle, Sebastian Lippemeier, and Jürgen Wendt, “Identification and Isolation of DSP-Toxins Using a Combined LC/MS-System for Analytical and Semiprepara-tive Work,” Agilent Technologies, publication 00060329.pdf, www.agilent.com.chem

System Summary

LC System

Agilent/Gerstel onlineSPE

Detection

MSD posESI

Columns

Phenomenex MAX RP, 2.1 mm × 250 mm, 5 µm

Column part number

Suggest ZORBAX SB-C18, 2.1 mm × 150 mm, 3.5 µm, 830990-902

clean-up

MOVE -moves vial to the spe unit

MOVE -moves affinity cartridge to the spe unit

ADD -transfers defined volume to the cartridge

WASH -washes the cartridge with 20 ml water

SHIFT -transports the spe unit from waste to vial

ADD -elution of the sample with methanol

derivatization

MOVE -moves the sample vial to agitator tray

ADD -adds derivatization solution (bromine/CHCl3)

MIX -mixing of the reagents for 2 min (derivatization)

MOVE -moves the sample vial to the injection tray

INJECT

Method:

1. Sample Preparation and Derivatization

The clean-up and derivatization steps of the aflatoxins were performed on-line using a Gerstel MPS-3 autosampler with its new spe-capabilities. Twouser defined injector programs were automatically executed one after theother.

Figure 3. Program for automatic spe clean-up and derivatization.

2. LC/MS Method

The autosampler was integrated in an Agilent 1100 LC/MSD system, con-sisting of a binary pump, thermostatted column compartment, diode arraydetector and a LC/MSD (single quadrupole). The LC/MSD was used withelectrospray ionization in positive ion mode. Chromatographic separationswere performed on a Phenomenex Max RP (250*2.1 mm, 5 µm), using aflow rate of 0.3 mL/min in gradient mode (Eluent A: 0.1% formic acid,water; Eluent B: acetonitrile, water). Complete system control (includingthe autosampler) and data evaluation were carried out using the AgilentChemStaion (Rev.A10.03)

O

O

O

OO

O

Br

O

O

O

O

O

O

O

OCH3

H3C

H3C

CH3

Br

O

Figure 4. Monobrominated aflatoxins.

2 4 6

B1/G2

B2

G1

G1

brom

B1

brom

8 10 12 14

Figure 6. TICs of aflatoxins before (top) and after (bottom) derivatization.

51

Regulated/Hazardous Pesticides/Herbicide Substances

Major Analytes

44 pesticides

Matrix

Vegetables, fruit

Reference

Masahiko Takino and Toshitsugu Tanaka, “Determination of 44 Pesticides in Foodstuffs by LC/MS/MS,” Agilent Technologies, publication 5989-5459EN, www.agilent.com.chem

LC ConditionsInstrument Agilent 1100 HPLCColumn ZORBAX Extend C18,

100 mm × 2.1 mm, 1.8 µm (p/n 728700-902)

Column temp 40 °C Mobile phase A = 0.1% formic acid + 5 mM ammonium

formate in waterB = Acetonitrile

Gradient: 10% B at 0 min, 80% B at 30 minFlow rate 0.2 mL/min Injection vol 5 µL

MS ConditionsInstrument Agilent 6410 QQQSource Positive ESIDrying gas flow 10 L/minNebulizer 50 psigDrying gas temp 350 °C Vcap 4000 VScan m/z 100 to 550 Fragmentor Variable 100 VMRM ions Shown in Tables 1 and 2Collision energy Shown in Tables 1 and 2

x107

0.4

0.8

1.2

1

2

3

4

5

6

7,8

9

10

11

12

13

14

15,16

17

1819, 20

33

32

31

29,30

27, 28

2625

24

23

2221

x106

0.5

2.5

4.5

6.5

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31

1

2

3 4

5

6

7, 89 10

11

12

1314

15, 16

18

19, 20

33

32

31

29, 30

27, 2826

25

24

23

22

21

17

Abundance versus acquisition time (min)

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31

Abundance versus acquisition time (min)

(A)

(B)

Figure 1. TIC of 33 pesticides standard in full scan mode (A) and product ionscan mode (B) at 1 µg/mL.

System Summary

LC System

Binary gradient

Detection

MSMS QQQ posESI

Columns

Extend-C18, 2.1 mm × 100 mm, 1.8 um

Column part number

728700-902

52


Major Analytes

Acid herbicides

Matrix

Water

Reference

Michael Woodman, “Rapid Analysis of Herbicides by RapidResolution 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

- P

iclo

ram

13.

303

14.

436

- C

hlo

ram

ben

17.

653

17.

845

- D

icam

ba

18.

895

- B

enta

zon

19.

480

- 2,

4-D

20.

238

21.

104

- D

ich

lorp

rop

23.

095

- 2,

4,5-

TP

24.

678

- A

cifl

uor

fen

29.

609

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

ConditionsColumn EPA Method 555 with ZORBAX SB-C18

columns and fast DAD detectorZORBAX SB-C18, 4.6 mm × 250 mm, 5 µm

Column temp 25 °CGradient 25 mM H3PO4, ACN, 10% to 90% ACN

in 30 minGradient slope 7.8% ACN/column volume Analysis flow rate 1 mL/min Group A compounds 1 µL of 100 µg/mLTotal analysis time 60 minDetection UV 230 nm, 10-mm 13-µL flow cell, filter

2 seconds (default)

System Summary

LC System

AutoSPE/1200SL with dual binary pumps and 6-port valve

Detection

DAD

Columns

ZORBAX SB-C18, 5, 3.5, and 1.8 µm

Column part number


Continued

ConditionsEPA Method 555 with ZORBAX SB-C18 columns and fast DADdetector ZORBAX SB-C18, 2.1 mm × 80 mm, 3.5 µm Column temp 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

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

2.2

06

2.3

10

2.3

85

2.5

77

2.6

26

2.7

23

2.8

30

3.0

25

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

53

Experimental Conditions

SystemAgilent 1200SL Series Rapid Resolution LC consisting of:

G1379B micro degasser

G1312B binary pump SL

G1312A binary pump with solvent selection valve option, or

G1354A quaternary pump

G1367C HiP ALS autosampler SL, and

G2258A dual loop prep autosampler 5 mL

G1316B thermostatted column compartment SL with 6- or 10-port

2-position switching valve

G1315C UV/VIS diode array detector (DAD) SL, flow cell as indicated in

individual chromatograms

ChemStation 32-bit version B.02.01

Columns

Agilent ZORBAX SB-C18, 4.6 × 250 mm, 5 µm

Agilent ZORBAX SB-C18, 3.0 × 150 mm, 3.5 µm

Agilent ZORBAX SB-C18, 2.1 × 80 mm, 1.8 µm

Agilent ZORBAX SB-Aq, 4.6 × 12.5 mm, 5 µm

Mobile phase conditions

Organic solvent Acetonitrile

Aqueous solvent 25 mm phosphoric acid in Milli-Q water

Gradient conditions

Gradient slope 7.8 or 2.3% per column volume, as indicated. See

individual chromatograms for flow rate and time

Sample

EPA 555 Group A chlorinated phenoxy acid herbicides (picloram,

chloramben, dicamba, bentazon, 2,4-D, dichlorprop, and 2,4,5-TP,

acifluorfen), 100 µg/mL in methanol or diluted to 20 ng/L (20 ppb)

in reagent water acidified with 25 mm phosphoric acid.

54


Major Analytes

Postharvest fungicides

Matrix

Citrus

Reference

E. Michael Thurman, Imma Ferrer, Michael Woodman, andJerry Zweigenbaum, “Analysis of Post-Harvest Fungicides and Their Metabolites in Citrus Fruits and Juices by Time-of-Flight and Ion Trap LC/MS,” Agilent Technologies,publication 5989-2728EN, www.agilent.com.chem

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time, min

0.0

4.0e6

8.0e6

1.2e7

1.6e7

2.0e7

2.4e7

2.8e7

3.2e7

3.6e7

3.9e7

Inte

nsi

ty, c

ps

17.9

16.7

7.6

21.521.0

20.426.4

26.024.8

14.92.4

25.4

18.2 22.72.1

23.92.7

2

Imazalil

Thiabendazole

Imazalil degradate

Figure 2. LC/MSD TOF total ion chromatogram (TIC) of orange peel, note thetwo large peaks in the chromatogram are imazalil and thiabendazole.

System Summary

LC System

Binary gradient

Detection

MS TOF, MSn Ion Trap, both posESI

Columns


Column part number

993967-906

LC/TOF MS MethodLC pumps Agilent 1100 binary pumps, injection volume

50 µL with standard Agilent 1100 ALS

Column ZORBAX Eclipse XDB-C8, 4.6 mm × 150 mm, 5 µm, product number 993967-906

Mobile phases A = acetonitrile and B = 0.1% formic acid in water

Gradient 5 minutes isocratic at 10% A, followed by a linear gradient to 100% A in 30 minutes at a flow rate of 0.6 mL/min

Instrument Agilent LC/MSD TOF time-of-flight mass spectrometer with dual electrospray source, positive ESI, capillary 4000 V

Nebulizer 40 psig

Drying gas 9 L/min, 300 °C

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

Reference masses m/z 121.0509 and 922.0098

Resolution 9500±500 at m/z 922.0098

Mass range 50–1000 m/z

Flow rate Reference A Sprayer 2 is at a constant flow rate during the run

55


Major Analytes

Chloronicotinyl insecticides

Matrix

Vegetables, fruit

Reference

Imma Ferrer, E. Michael Thurman, Agilent Contact: JerryZweigenbaum, “Determination of Chloronicotinyl Insecticides in Salad Vegetables by LC/MSD TOF andLC/MSD Trap,” Agilent Technologies, publication 5989-1842EN, www.agilent.com.chem

2.429.0

3.6

24.1

30.331.028.627.1 29.425.2

20.912.821.8

11.0 23.5 26.014.88.3 22.5

19.110.2 18.02.1 20.59.1

19.56.89.87.45.16.0 14.5

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32

Time, min

0.0

2.0e6

4.0e6

6.0e6

8.0e6

1.0e7

1.2e7

1.4e7

1.6e7

1.8e7

Inte

nsi

ty, c

ps

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32

Time, min

0.0

2.0e5

4.0e5

6.0e5

8.0e5

1.0e6

1.2e6

1.4e6

1.6e6

Inte

nsi

ty, c

ps

Imidacloprid

Acetamiprid

Thiacloprid

ImidaclopridAcetamiprid

Thiacloprid

Figure 3. LC/MSD TOF analysis of the three chloronicotinyl insecticides at 0.5 mg/kg in a fortified tomato matrix. The total and extracted ionchromatograms are shown in upper and lower panels, respectively.

N

NH N

CH3

CH3

Cl

Cl

Cl

N

CH

N

N

NH N

CH3

CH3

N

Acetamiprid[M+H]+ = m/z 223.0745 theoretical

m/z 223.0749 measured

Theoretical neutral loss of 97.0640 uMeasured neutral loss of 97.0644 u

m/z 126.0105 theoretical

126.0105 measured

Figure 5a. Structure and fragmentation pathways for acetamiprid usingLC/MSD Trap mass spectrometer and LC/MSD TOF. Only the m/z 126 ion was seen with LC/MSD TOF as a fragment ion.

Agilent LC/MSD TOFLC pump Agilent 1100 binary

Autosampler Agilent 1100 standard ALS


Column ZORBAX Eclipse XDB C-84.6 mm × 150 mm × 5 µm p/n 993967-906

Mobile phase A: acetonitrile B: 0.1% formic acid in water

Gradient 15% to 100% A over 30 minutes at 0.6 mL/min

System Summary

LC System

Binary gradient

Detection

MS TOF, MSn Ion Trap, both posESI

Columns

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

Column part number

993967-906

56


Major Analytes

Phenylurea, triazine herbicides

Matrix

Water

Reference

Neil Cullum and Pete Stone, “Determination of Phenyl Ureaand 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

System Summary

LC System

Dual binary w/6-port valve for autoSPE

Detection

MSD pos/negAPCI

Columns

ZORBAX Eclipse XDB-C8, 2.1 mm × 50 mm, 3.5 µm

Column part number

971700-906

3

8

4

5 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

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

Figure 1. Flow path diagram.

HPLC ConditionsColumn ZORBAX Eclipse XDB-C8

50 mm × 2.1 mm, 3.5 µm @ 40 °CFlow rate 0.5 mL/min

Precolumn ZORBAX SB C18 Rapid Resolution30 mm × 2.1 mm, 3.5 µm @ 20 °C


Mobile phase A = 0.001% formic acid in waterB = methanol

Pump program Pump 1 (Analytical column)

Time Mobile Mobile Flow rate(min) phase phase (mL/min)

A B

Initial 90 10 0.54.5 90 0 0.5

17.5 30 70 0.517.6 90 10 0.522.0 90 10 0.5

Pump 2 (Precolumn)

Time Mobile Mobile Flow rate(min) phase phase (mL/min)

A B

Initial 90 10 0.55.0 90 10 0.55.1 90 10 0.1

18.0 90 10 0.118.1 90 10 0.5

Column switching valve Time Valve Temp (L) Temp (R)(min) Position (°C) (°C)

Initial 1 20.0 40.03.0 2 20.0 40.0

20.0 1 20.0 40.0

Continued

57

ResultsFigures 2, 3, and 4 show an extracted ion chromatogram from spiked tap water containing atrazine, isoproturon, anddiuron, respectively, at a concentration of 0.1 µg/L. Figure 5also shows an extracted ion chromatogram, again from aspiked tap water containing prometryn and terbutryn at aconcentration of 0.01 µg/L (5 pg on-column).

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 5. Spiked tap water containing 0.01 µg/L of prometryn and terbutryn.

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

Minute14 15 16 17 18 19 20 21

0

2000

4000

6000

8000

10000

12000

14000

MSD1 242, EIC=241.7:242.7 (VAL3_4\PT3_4024.D) API-ES, Pos, SIM, Frag: 70 (TT)

16.5

98

Prometryn

16.8

13

Terbutryn

Validation of the method was done on 11 sample batches.Standards were prepared at three levels: 0.01, 0.10, and 0.40 µg/L. The borehole raw water was spiked at twolevels: 0.01 and 0.10 µg/L. The potable tap water (whichwas derived from a surface water source) was also spikedat two levels: 0.01 and 0.10 µg/L. All samples were analyzed in duplicate in each batch in a random order.

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

58

Fat-Soluble Vitamins

Major Analytes

Retinol isomers

Matrix

Standard

Reference

Robert Ricker, “Separation of Retinol (Vitamin A) Isomers:Comparison of Mobile-Phase Composition,” Agilent Technologies, publication 5988-6361EN, www.agilent.com.chem

System Summary

LC System

Isocratic

Detection

DAD

Columns

ZORBAX Sil, 4.6 mm × 250 mm, 5 µm

Column part number

880952-701

11,13-di-cis-13-cis-

9, 11, 13-tri-cis-9, 13-di-cis-

11-cis-7, 11-di-cis-

9-cis-7, 9-di-cis-

7, 13-di-cis-

A = Retinol Isomers

AT all trans retinolB-F = Retinol Isomers

A

B

C

D

E

F

6 8 10 12 14

Time (min)

16 18 20 22 24

11, 1

313

13

13

9, 1

1, 1

39

, 11,

13

9, 1

1, 1

3

9, 1

3

9, 1

3

9, 1

3

7, 1

1

7, 1

1

7, 1

37,

11

11

11

119

9

9

7, 9

AT

AT

AT

13

9, 1

1, 1

39, 1

3

7, 1

1

11

9

AT

7, 1

3 13

9, 1

1, 1

3

9, 1

3 7, 9

11 9

AT

7, 1

313

9, 1

1, 1

39, 1

3

11

9A

T

ConditionsColumn ZORBAX Sil, 4.6 mm × 250 mm

Agilent p/n 880952-701

Mobile phase Dioxane, (MTBE), hexane

Injection volume 20 µL, 25 °C

Courtesy of Dr. G. Nöll Physiologisches Inst. – Justus Liebig Uni.Giessen

59


Major Analytes

Fat-soluble vitamins

Matrix

Standard

Reference

Robert Ricker and Thomas Schellenberger, "Fat-Soluble Vitamins on ZORBAX XDB-C8," Agilent Technologies, publication 5988-6359EN, www.agilent.com/chem

System Summary

LC System

Isocratic

Detection

DAD

Columns


Column part number

993967-906

0 5 10 15 20 25 30

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 5 10 15 20 25 30

0.22

0.20

0.18

0.16

0.14

0.12

0.10

0.08

0.06

0.04

0.02

0.00

Minutes Minutes

Ambient 50 °C

1

2

3

4

5

6

7

1

2

3

4

5

6

7

1. Retinol2. Retinol acetate3. Vitamin D34. γ-Tocopherol5. α-Tocopherol6. Tocopherol acetate7. Retinol palmitate

ConditionsColumn ZORBAX XDB-C8, 150 mm × 4.6 mm, 5 µm

(Agilent p/n 993967-906)

Mobile phase A: water 5%, B: methanol 95%

Flow rate 1 mL/min

Oven temperature 50 °C


60


Major Analytes

Vitamin D3

Matrix

Poultry feed

Reference

Linda L. Lopez and Paul C. Goodley, “Rapid determination of Vitamin D3 in poultry feed supplements,” Agilent Technologies, publication 5968-9408E,www.agilent.com/chem

ConditionsMobile phase 0.05% acetic acid : methanol (50:50)



Detector MSD ion trapIonization APCI (positive)Nebulizer pressure 40 psiDrying gas 10 L/min, 350 °CSkim 1 15.0 VCap exit offset 60.0 VAverages 2ICC onMax acc.time 200 msTarget 50000

Confirmation MS spectral information and RTs

System Summary

LC System

Binary gradient

Detection

MSn Ion Trap posAPCI

Columns

Flow injection – no column

Column part number

NA

158.6

185.0

213.0

255.2

271.1

325.5

Pure standardVitamin D3MS3

m/z 385 → 367 →10-µL loop injection

349.2367.6

185.0213.1

255.1

285.4311.2 353.2

241.3

241.2

271.1

285.3

325.5

227.2

227.2

158.5

0

20

40

60

80

100

150 200 250 300 350 400 500m/z

% R

elativ

e Ab

unda

nce

0

20

40

60

80

100

150 200 250 300 350 400 500m/z

% R

elativ

e Ab

unda

nce Poultry feed extract

MS3

m/z 385 → 367 →10-µL loop injection

Comparison of production spectra from a pure standard ofvitamin D3 and an enriched poultry feed extract

61


Major Analytes

Fat-soluble vitamins A, D, E

Matrix

Standard

Reference

Udo Huber, “Analysis of Fat-Soluble Vitamins by HPLC,”Agilent Technologies, publication 5968-2970E,www.agilent.com/chem

System Summary

LC System

Quaternary gradient

Detection

DAD

Columns

ZORBAX Eclipse XDB-C18, 4.6 mm × 75 mm, 3.5 µm

Column part number

7995118-344

ConditionsColumn ZORBAX Eclipse XDB-C18,

4.6 mm × 75 mm, 3.5 µmMobile phase A = water,

B = methanolFlow rate 1.0 ml/minGradient At 0 min 90 % Bat 15 min 100 % Bat 20 min

100 % BColumn wash At 21 min 90 % BUV detector Variable wavelength detector 210 nm,

standard cellColumn compartment 20 °Ctemperature

1 Retinol (A)2 Cholecalciferol (D

3)

3 α-Tocopherol (E)

1

2

3

Time [min]

0 2.5 5 7.5 10 12.5 15 17.5 20

Abso

rban

ce, [

mAU

]

200

0

400

600

800

Figure 1. Analysis of three fat-soluble vitamins.

HPLC PerformanceCompound LOD for Precision of RT Precision of Area

S/N = 2 (RSD of 10 runs) (RSD of 10 runs)(mg/L)* (1000 mg/L)* (1000 mg/L)*

Retinol 4.0 0.10 0.14

Cholecalciferol 2.5 0.04 0.16

a-Tocopherol 2.0 0.04 0.20

* Injection volume: 5 µL

62

Water-Soluble Vitamins

Major Analytes

Water-soluble vitamins

Matrix

Standard

Reference

Robert Ricker, “Separation of water-soluble vitamins usingreversed phase chromatography,” Agilent Technologies,publication 5988-6365EN, www.agilent.com/chem

ConditionsColumn ZORBAX SB-C8, 150 mm × 4.6 mm,

(Agilent p/n 883975-906)

Mobile phase A: 50-mM sodium phosphate:methanol(90 : 10)B: 50-mM sodium phosphate:methanol (10 : 90)

Gradient At 0 min 0% BAt 18 min 70% B

Flow rate 1 mL/min




System Summary

LC System


Detection

DAD

Columns

ZORBAX SB-C8, 150 mm × 4.6 mm, 5 µm

Column part number

883975-906

0

1 3

4

2

5

6

7

8

2.5 5 7.5 10 12.5 min

1. B1 - Thiamine2. Vitamin C3. B3 - Niacin4. B6 - Pyridoxine5. Pantothenic acid6. Folic acid7. B12 - Cyanocobalamin8. B2 - Riboflavin

63


Major Analytes

Water-soluble vitamins

Matrix

Cat food

Reference

Robert Ricker and Thomas Schellenberger, “Rapid analysisof water-soluble vitamins with extraction from cat food,” Agilent Technologies, publication 5988-5761EN, www.agilent.com/chem 0 2 4 6 8 10 12 min

0

5

20

15

10

Riboflavin B2

Thiamin B1

B6

Nicotinic acid

Folicacid

4

Pantothenicacid

UV = 200nm

ConditionsColumn ZORBAX SB-C18, 75 mm × 4.6 mm, 3.5 µm

(Agilent p/n 866953-902)

Mobile phase A: 1-L water, 2.5-g hexane sulfonate, 2.5-mL acetic acid, 4-g NaH2PO4, 50-L triethylamineB: 600-mL water, 2.5-g hexane sulfonic acid, 2.5-mL acetic acid, 50-L triethylamine 400-mL acetonitrile

Gradient At 0 min 0% Bat 3.6 min 0% Bat 6 min 20% Bat 13.5 min 55% Bat 14.4 min 90% Bat 16.5 min 90% Bat 16.8 min 0% Bat 20 min 0% B





System Summary

LC System


Detection

DAD

Columns

See description at right

64

System Configurations

Application Area – Carbohydrates, Sugars, Sugar Alcohols

LC solvent PublicationMajor analytes delivery system* Detection** Notes number Page

Carbohydrates Isocratic G1310A RID 5988-6351EN 5Sugar alcohols Isocratic G1310A MSD negAPCI 5988-4236EN 6Dextran Isocratic G1310A RID 5988-0118EN 7Starch Isocratic G1310A RID 5988-0117EN 8Carbohydrates Isocratic G1310A RID 5966-0637EN 9

Application Area – Dyes, Colorants, Pigments

Sudan dyes Binary gradient 5989-4736EN 10G1312A or G1312B MS TOF posESI

FDC food dyes, paraben Quaternary gradient 5988-6355EN 11G1354A DAD

Cyanidins Binary gradient 5968-2979E 12G1312A or G1312B MSD posESI APCI

Application Area – Fats and Oils

Phospholipids Prep/35900E ELSD (ESA), 5989-2848EN 14some MSD

Triglycerides Isocratic G1310A MSD posAPCI 5988-4235EN 15Triglycerides Binary gradient MSD posAPCI 5968-0878E 16

G1312A or G1312BTriglycerides and Quaternary gradient DAD 3channel 5966-0744E 17their hydroperoxides G1354A

Application Area – Flavors, Sweeteners, Organic Acids

Flavors, sweeteners, 1200SL DAD 5989-5178EN 18preservativesOrganic acids Quaternary gradient DAD 5989-1265EN 19

G1354A DADFlavoring agents Binary gradient DAD 5988-6353EN 20

G1312A or G1312BSemivolatile flavors Isocratic G1310A DAD 5988-6352EN 21Aspartame, degradants Isocratic G1310A DAD 5988-6349EN 22

Application Area – Herbal Supplements, Natural Products, Plant Hormones

Xanthine metabolites Isocratic G1310A DAD 5989-5237EN 23Glycyrrhizin 1200SL DAD 5989-4907EN 24Xanthines 1200SL DAD 2-µL cell 5989-4857EN 25Ginsenosides part 1 1200SL MS TOF posESI 5989-4506EN 26Anthocyanins Prep DAD 5989-0591EN 27Anthocyanins Quaternary or binary DAD 5988-6362EN 28

gradient moduleFlavonoids, catechins Quaternary or binary DAD 5988-6357EN 29

gradient module

Application Area – Preservatives

Flavors, sweeteners, 1200SL DAD 5989-5178EN 30preservatives

Paraben, phenoxyethanol Quaternary or binary DAD 5989-3635EN 31gradient module

65

Application Area – Proteins, Peptides, Amino Acids


Proteins Quaternary or binary DAD Compare various 5988-6358EN 32gradient module bonded phases

Proteins Quaternary or binary DAD Column temperature vs. 5988-6348EN 33gradient module separation quality and

selectivity

BSA (bovine serum Binary gradient DAD Fast 5988-6081EN 34albumin) digest, peptide G1312A or G1312BAAA amino acid Quaternary or binary FLD/OPA-FMOC Precolumn 5980-1193EN 35

gradient module derivatization

Application Area – Regulated/Hazardous Drug Substances

Drugs 1200SL MSMS QQQ Offline SPE 5989-5319EN 36pos/negESI

Nitrofurans Binary gradient MSn Trap XCT 5989-0738EN 37G1312A or G1312B posESI

Fluoroquinolones Binary gradient MSD posESI 5989-0596EN 38G1312A or G1312B

Chloramphenicol Binary gradient MSD and MSn 5988-9920EN 39G1312A or G1312B Ion Trap negESI

Sulfa drugs Quaternary or binary DAD 5988-7135EN 40gradient module

Sulfonamides CapLC DAD MSD posESI 5980-2499EN 41

Application Area – Regulated/Hazardous Miscellaneous Substances

HMF hydroxymethylfurfural Binary gradient MSD posAPCI 5989-5403EN 42G1312A or G1312B

Acrylamide Dual binary w/6-port MS TOF posESI 5988-2884EN 43valve for autoSPE

Chromium speciation Metrohm 818 pump, IC-ICPMS Column normally bundled 5989-2841EN 44Agilent 7500 ISIS with ICP-MS. Contact yoursampler agent/representative

for detailsPerchlorate Metrohm IC MSD negESI 5989-0816EN 45Arsenobetaine Isocratic G1310A ICP-MS 5988-9893EN 46

Application Area – Regulated/Hazardous Natural Toxin Substances

DSP algal toxins Quaternary gradient MSD pos/negESI 5989-2912EN 47G1354A

Mycotoxin, fumonisin Binary gradient MSD posESI 5968-2124E 49G1312A or G1312B

Aflatoxins Agilent/Gerstel MSD posESI Bromination of B1 and 00060329.pdf 50onlineSPE G1 prior to separation

System Configurations (Continued)

66

Application Area – Regulated/Hazardous Pesticides/Herbicide Substances


44 pesticides Binary gradient MSMS QQQ SPE cleanup 5989-5459EN 51G1312A or G1312B posESI

Acid herbicides AutoSPE/1200SL with DAD 5989-5176EN 52dual binary pumps and 6-port valve

Postharvest fungicides Binary gradient MS TOF, MSn Ion QuECHERS 5989-2728EN 54G1312A or G1312B Trap, both posESI Sample prep

Chloronicotinyl insecticides Binary gradient MS TOF, MSn Ion 5989-1842EN 55G1312A or G1312B Trap, both posESI

Phenylurea, triazine Dual binary w/6-port MSD pos/negAPCI 5989-0813EN 56herbicides valve for autoSPE

Application Area – Vitamins, Fat-Soluble

Retinol isomers Isocratic G1310A DAD 5988-6361EN 58Fat-soluble vitamins Isocratic G1310A DAD 5988-6359EN 59Vitamin D3 Binary gradient MSn Ion Trap 5968-9408E 60

G1312A or G1312B posAPCIFat-soluble vitamins A, D, E Quaternary gradient DAD 5968-2970E 61

G1354A

Application Area – Vitamins, Water-Soluble

Water-soluble vitamins Quaternary or binary DAD 5988-6365EN 62gradient module

Water-soluble vitamins Quaternary or binary DAD Titriplex-V hot extraction 5988-5761EN 63gradient module

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- and 4.6-mm id columns. For veryfast 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 multiwavelength monitoring is required. If not, you may substitute amultiwavelength or variable-wavelength detector. Choose standard flow cells for most 3- and 4.6-mm id column applications. For 2.1-mm applications, especially with 1.8-µmparticle 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 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 if this is required.

67

Food Quick Reference Guide


LiteraturePage Major Analytes Matrix LC system Detection Mobile phase reference

5 Carbohydrates Isocratic RID ACN/water 5988-6351EN6 Sugar alcohols Beverage Isocratic MSD negAPCI ACN/water 5988-4236EN7 Dextran Isocratic RID Water 5988-0118EN8 Starch Isocratic RID 0.1M NaNO3 5988-0117EN9 Carbohydrates Lemonade Isocratic RID Water H2SO4 5966-0637EN


10 Sudan dyes Food Binary gradient MS TOF posESI ACN/water 5989-4736ENAmmOAc

11 FDC food dyes, Ricker Quaternary DAD MeOH/water 5988-6355ENparaben gradient TFA/TEA

12 Cyanidins Cabbage Binary gradient MSD posESI ACN/water 5968-2979EAPCI formic acid

Fats and Oils

14 Phospholipids Soybean Prep/35900E ELSD (ESA), MeOH, IPA, 5989-2848ENsome MSD hexane, AmmOAc,

water15 Triglycerides Isocratic MSD posAPCI Acetone/water 5988-4235EN16 Triglycerides Edible oil Binary gradient MSD posAPCI IPA, n-ButOH, 5968-0878E

water,amm. form.

17 Triglycerides Edible oil Quaternary DAD 3channel ACN/MTBE/ 5966-0744ENand their gradient waterhydroperoxides


18 Flavors, sweeteners, Soft drinks 1200SL DAD ACN/water 5989-5178ENpreservatives Amm. HPO4

formic

19 Organic acids Foods Quaternary DAD ACN/water 5989-1265ENgradient 20 mM phosphate

20 Flavoring agents Mouthwash Binary gradient DAD ACN/water TFA 5988-6353EN21 Semivolatile flavors Isocratic DAD MeOH/water 5988-6352EN22 Aspartame, degradants Cola Isocratic DAD ACN/water TFA 5988-6349EN


23 Xanthine metabolites Isocratic DAD ACN/water 5989-5237EN25 mM phosphate

24 Glycyrrhizin Licorice root 1200SL DAD ACN/water acetic 5989-4907EN25 Xanthines Tea, chocolate 1200SL DAD 2-µLcell ACN/water formic 5989-4857EN26 Ginsenosides part 1 Root 1200SL MS TOF posESI ACN/water TFA 5989-4506EN27 Anthocyanins Blueberry Prep DAD MeOH/water TFA 5989-0591EN28 Anthocyanins Gradient DAD MeOH/water 5988-6362EN

phosphoric acid29 Flavonoids, catechins Gradient DAD MeOH/water TFA 5988-6357EN

68

Preservatives


30 Flavors, sweeteners, Soft drinks 1200SL DAD ACN/water 5989-5178EN preservatives AmmHPO4

31 Paraben, phenoxyethanol Gradient DAD MeOH/water 5989-3635EN


32 Proteins Wheat Gradient DAD ACN/water TFA 5988-6358EN33 Proteins Wheat Gradient DAD ACN/water TFA 5988-6348EN34 BSA (bovine serum Binary DAD ACN/water TFA 5988-6081EN

albumin) digest, peptide gradient35 AAA amino acid Gradient FLD/OPA-FMOC ACN,MeOH, 5980-1193EN

water, Na2HPO4, NaOH


36 Drugs Water 1200SL MSMS QQQ ACN/water 5989-5319ENpos/negESI amm. form

37 Nitrofurans Poultry, shrimp Binary MSn Trap XCT ACN/water 5989-0738ENgradient posESI acetic

38 Fluoroquinolones Beef kidney Binary MSD posESI ACN/water 5989-0596ENgradient formic

39 Chloramphenicol Shrimp, honey Binary MSD and MSn MeOH/ACN/ 5988-9920ENgradient Ion Trap negESI water AmmOAc

40 Sulfa drugs Meat Gradient DAD ACN/water 5988-7135ENH3PO4

41 Sulfonamides CapLC DAD MSD posESI ACN/water 5980-2499ENformic

Regulated/Hazardous Miscellaneous Substances

42 HMF Bread, cereal, Binary gradient MSD posAPCI Water acetic 5989-5403ENhydroxymethylfurfural yogurt G1312A or G1312B formic

43 Acrylamide Drinking water Dual binary MS TOF posESI ACN/water 5988-2884ENw/6-port valve formicfor autoSPE

44 Chromium speciation Metrohm Metrohm 818 pump, IC-ICPMS water Na2EDTA 5989-2481ENAgilent 7500 ISIS NaOHsampler

45 Perchlorate Water, vegetables Metrohm IC MSD negESI MeOH/water 5989-0816EN30 mm NaOH

46 Arsenobetaine Fish Isocratic G1310A ICP-MS AmmHCO3/ 5988-9893ENtartaric, methanol


47 DSP algal toxins Shellfish Quaternary MSD pos/negESI MeOH/water 5989-2912ENgradient formic

49 Mycotoxin, fumonisin Corn Binary MSD posESI ACN/water 5968-2124Egradient AmmOAc

50 Aflatoxins Various Agilent/Gerstel MSD posESI ACN/water 00060329.pdf onlineSPE formic

Food Quick Reference Guide (Continued)

69



51 44 pesticides Vegetables, fruit Binary MSMS QQQ ACN/water, 5989-5459ENgradient posESI amm. form.

52 Acid herbicides Water AutoSPE/1200SL DAD ACN/water 5989-5176ENwith dual binary pumps H3PO4

and 6-port valve54 Postharvest fungicides Citrus Binary MS TOF, MSn Ion ACN/water 5989-2728EN

gradient Trap both posESI formic55 Chloronicotinyl Vegetables, fruit Binary MS TOF, MSn ACN/water 5989-1842EN

insecticides gradient Ion Trap, both formicposESI

56 Phenylurea, triazine Water Dual binary w/ MSD MeOH/water 5989-0813ENherbicides 6-port valve for pos/negAPCI formic

autoSPE


58 Retinol isomers Isocratic DAD Dioxane, 5988-6361EN(MTBE), hexane

59 Fat-soluble vitamins Isocratic DAD MeOH/water 5988-6359EN60 Vitamin D3 Poultry feed Binary MSn Ion Trap MeOH/water 5968-9408E

gradient posAPCI acetic61 Fat-soluble Quaternary DAD MeOH/water 5968-2970E

vitamins A,D, E gradient


62 Water-soluble vitamins Gradient DAD MeOH/water 5988-6365ENphosphate

63 Water-soluble vitamins Cat food Gradient DAD ACN/water, 5988-5761ENacetic, phosphate,TEA, hexanesulfonate

Food Quick Reference Guide (Continued)

70

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.

71

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

HPLC Separation Modes

The 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

72

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 Materials

A 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

73

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 Methods

The 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 all

HPLC and STM-LC operational modes on one system andfacilitates the use of existing and newly developed HPLCmethods without the need for revalidation or extensivereconfiguration.

Optimizing the Instrument Setup for Different Column IDs

When 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 ordecreased particle size will increase the resolving power of

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

74

the 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 Volume

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

Summary

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

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.

Method Translation

It 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 formodernization 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.

75

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 fast

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 thantypically 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,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).

that 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 columnsrequires 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 changein 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:

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

Volumecolumn1

Volumecolumn2Inj. vol.col. 1 ×

(eq. 3)#Column volumes

(End% – Start%)% Gradient slope =

(eq. 1)= Flowcol. 2

Diam.column1

Diam.column2Flowcol. 1

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 linearvelocity, 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 observation

76

min0 2 4 6 8 10 12 14 16 18

mAU

0

100

200

300

400

500

600

1.90

1

4.38

9 -

Sac

char

in

5.64

1

6.70

1 -

Caf

fein

e

8.10

4 -

Asp

arta

me

9.95

5 -

Van

illin

12.

053

- B

enzo

ate

12.4

99 -

Sor

bate

15.5

12 -

Ben

zald

ehyd

e

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

-25

0

25

50

75

100

125

150

175

0.2

97 -

Sac

char

in

0.4

52 -

un

k.

0.6

24 -

Caf

fein

e

0.8

49 -

Asp

arta

me

0.9

33 -

Van

illin

1.0

71 -

Ben

zoat

e

1.1

36 -

Sor

bate

1.3

20 -

Ben

zald

ehyd

e

Conditions

ZORBAX 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)

gradients 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 higherresolution 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 Techniques

Proper 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.

Filtration

Sample 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. (add pub notedescribing available products here)

Extraction

Liquid/liquid extraction techniques, with or without pHmodification of the aqueous phase, have long been used toselectively extract general classes of compounds based on

77

solubility. 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, or ionexchange-it may be possible to process individual samplesmanually or use automation for offline processing of largernumbers of samples. (add pub note(s)/links for applica-tions and products here)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 samplepreparation 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 been

applied 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 Preparation

The 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

78


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

5988-6351EN page 5Carbohydrates Isocratic RID ZORBAX NH2 880952-708 or

4.6 mm × 250 mm, 5 µm 840300-908 ACN/water

5988-4236EN page 6Sugar alcohols Beverage Isocratic MSD negAPCI Asahipak NH2-50 2D Substitute

2 mm × 150 mm 843300-908 ACN/water

5988-0118EN page 7

Dextran Isocratic RID PL aquagel-OH MXA 79911GF-MXA Water7.5 mm × 30 mm, 8 µm and 79911GF-083PL aquagel-OH 30A, 7.5 mm × 30 mm, 8 µm

5988-0117EN page 8Starch Isocratic RID Aq. GPC PSS Suprema See app. note 0.1M NaNO3

100 + 1000, 2 of 8 mm × 300 mm, 10 µm

5966-0637EN page 9Carbohydrates Lemonade Isocratic RID BioRad HPX-87 Contact mfr. Water H2SO4

5968-5144ENSugars, sugar Isocratic RID ZORBAX Carbohydrate NH2 840300-908, ACN/water 1alcohols 4.6 mm × 250 mm, 843300-908

4.6 mm × 150 mm, 5 µm

5965-9797Sugars ECD PAD IEX NA Water, NaOH 2


5989-4736EN page 10Sudan dyes Food Bin. grad MS TOF ZORBAX XDB-C18 922700-902 ACN/water

posESI 2.1 mm × 50 mm, 1.8 µm AmmOAc

5988-6355EN page 11FDC food dyes, Quat. grad DAD ZORBAX XDB-C18 935967-902 ACN/water paraben 4.6 mm × 50 mm, 3.5 µm TFA/TEA

5968-2979E page 12Cyanidins Cabbage Bin. grad MSD posESI Inertsil ODS3 Suggest SB-C18 ACN/water

APCI 2.1 mm × 250 mm, 5 µm 2.1 mm × 150 mm, formic acid3.5 µm

5980-0914FDC food dyes, Henderson DAD ZORBAX XDB-C18 935967-902 MeOH/water paraben 4.6 mm × 75 mm, 3.5 µm TEA TFA

5965-9042Dyes, disperse Plastics DAD Hypersil BDS ACN/water and soluble 3 mm × 125 mm, 3 um TBAHS, H2SO4

Application Reference Index

The table below lists Agilent Technologies applications forthe food industry. In this guide, the page number where youcan review the most current application overview is listedafter the publication number. The complete publication forall application overviews listed below can be viewed on theAgilent Web site, www.agilent.com/chem.

79

5964-3559Dyes DAD Hypersil

Fats and Oils


5989-2848EN page 14Phospholipids Soybean Prep/35900E ELSD (ESA), Prep SIL See app. note MeOH, IPA,

some MSD 4.6 mm × 150 mm, 10 µm hexane, salt,21.2 mm × 150 mm, 10 µm water, CHCl3

5988-4235EN page 15Triglycerides Isocratic MSD Develosil ODS OG-3 Suggest SB-C18, Acetone/water

posAPCI 4.6 mm × 75 mm 4.6 mm × 75 mm, 3.5 µm866953-902

5968-0878E page 16Triglycerides Edible oil Bin. grad MSD Hypersil MOS Suggest SB-C18 IPA,n-ButOH,

posAPCI 2.1 mm × 200 mm, 5 µm 2.1 mm × 150 mm, water, 3.5 µm amm. formate830990-902

5966-0744E page 17Triglycerides Edible oil Quat. grad DAD Hypersil MOS Suggest SB-C18 ACN/MTBE/and their 3 channel 2.1 mm × 200 mm, 5 µm 2.1 mm × 150 mm, waterhydroperoxides 3.5 µm

830990-902

5966-0635ESaponified to Food DAD, Hypersil MOS ACN, THF, 3fatty acids derivatized 2.1 mm × 200 mm, 5 µm water

5966-0634ETriglycerides Olive, Isocratic RID Hypersil MOS ACN/acetone

rapeseed oil 2.1 mm × 200 mm, 5 µm

5954-6269E obsoleteTriglycerides Vegetable oil Old HP DAD Hypersil MOS ACN, MTBE,

2.1 mm × 200 mm, 3 µm water


5989-5178EN page 18Flavor, sweetener, Soft drinks 1200SL DAD ZORBAX SB-C18 827975-302 ACN/water preservative 4.6 mm × 250 mm, 5 µm, (1.8 µm) Amm, HPO4

3 mm × 50 mm 1.8 µm 880975-902(600 bar) (5 µm)

5989-1265EN page 19

Organic acids Foods Quat. grad DAD ZORBAX SB-Aq 883975-914 ACN/water 4.6 mm × 150 mm, 5 µm 20 mM phosphate

5988-6353EN page 20Flavoring agents Mouthwash Bin. grad DAD ZORBAX SB-Phenyl 860975-912 ACN/water TFA

2.1 mm × 50 mm, 5 µm

5988-6352EN page 21Semivolatile flavors Isocratic DAD ZORBAX XDB-Phenyl 963967-912 MeOH/water

4.6 mm × 150 mm, 3.5 µm

5988-6349EN page 22Aspartame, Cola Isocratic DAD ZORBAX SB-C18degradants 4.6 mm × 75 mm, 3.5 µm 866953-902 ACN/water TFA

Dyes, Colorants, Pigments (Continued)


80

5966-0741EAPM aspartame DAD/FLD w/ Hypersil ODS MeOH/water 4

OPA deriv. 2.1 mm × 200 mm, 5 µm NaOAc

5966-0631EBitter naringenin Citrus DAD Hypersil BDS ACN/water hesperidin 4 mm × 125 mm, 5 µm H2SO4

5966-0630EVanillin Impurities DAD Hypersil BDS ACN/water

4 mm ×100 mm, 3 µm H2SO4

5966-0627EOrganic acids Wine DAD BioRad HPX-87 Water, H2SO4


5989-5237EN page 23Xanthine metabolites Isocratic DAD Eclipse Plus C18 959993-902 ACN/water

4.6 mm × 150 mm, 5 µm 25 mm phosphate

5989-4907EN page 24

Glycyrrhizin Licorice root 1200SL DAD ZORBAX SB-C18 RRHT, 829975-902 ACN/water4.6 mm × 150 mm, 1.8 µm acetic

5989-4857EN page 25Xanthines Tea, chocolate 1200SL DAD RRHT 1.8 µm Various ACN/water

2 µL cell various formic

5989-4506EN page 26Ginsenosides Root 1200SL MS TOF SB-C18 RRHT, 820700-902 ACN/water Part 1 posESI 2.1 mm × 150 mm,1.8 µm TFA

5989-0591EN page 27Anthocyanins Blueberry Prep DAD Prep-C18, 410910-102 MeOH/water

21.2 mm × 250 mm, 10 µm 440905-902 TFA4.6 mm × 250 mm, 5 µm

5988-6362EN page 28Anthocyanins Gradient DAD ZORBAX SB-C18 See app. note MeOH/water

4.6 mm, 5 µm, and 3.5 µm phosphoric acid

5988-6357EN page 29Flavonoids, Gradient DAD ZORBAX SB-C8 863953-906 MeOH/watercatechins 4.6 mm × 150 mm, 3.5 µm TFA

5989-5493EN

Ginsenosides Ginseng 1200SL MS Ion Trap, SB-C18 820700-902 ACN/water TFATOF both 2.1 mm × 150 mm 1.8 µmposESI, and DAD

5989-4705ENGinsenosides Root 1200SL MSn Ion Trap SB-C18 820700-902 ACN/water TFAPart 2 posESI 2.1mm × 150 mm 1.8 µm

5989-0389EN

Ephedrine isomers Ephedrine MSD posESI SB-Aq 871700-914 Water formic2.1 mm × 50 mm, 3.5 µm

5989-0261ENErgot alkaloids Fungal extract Nano LC, MSn Ion Trap Nano, unspecified Unspecified

semiprep LC posESI

Flavors, Sweeteners, Organic acids (Continued)


81

5988-7136EN Alkaloids Goldenseal DAD ZORBAX XDB-C18 963967-902 ACN/water berberine, etc. 4.6 mm × 150 mm, 3.5 µm TEA AmmOAc

5988-6285ENPlant growth DAD SB-C8 866953-906 ACN/water hormones 4.6 mm × 75 mm, 5 µm TFA

5988-5749ENFormononetin Red clover Quat. grad DAD/FLD SB-C18 883975-302 ACN/water

3 mm × 150 mm, 5 µm HOAc

5988-2399ENPhytoestrogens, Red clover Quat. grad DAD/FLD SB-C18 880975-902 ACN/water glucosides, 4.6 mm × 250 mm, 5 µm H3PO4

isoflavonoids

5988-0637ENHydroxyanthro- Rhubarb Prep DAD SB-C18 883975-302 ACN/water quinones emodin, 3 mm × 150 mm, 5 µm, HOAcrhein 21.2 mm × 150 mm, 5 µm

5980-2081EEphedrines, Mahaung, CE CEMS 75 µm fused silica Various Boratealkaloids mahonia, capillaryberberine, otherstetrandrine,fangchinoline,chlorogenic acid,etc.

5968-8869EGinsenosides, Ginseng MSn Ion Trap SB-C18 863954-302 ACN/water 5saponins posESI 2.1 mm × 50 mm, 3.5 µm acetic

5968-6252Rhein, emodin Rhubarb DAD SB-C18 883975-302 ACN/water laxative 3 mm × 150 mm, 5 µm, 846975-202 AmmOAc, H2SO4

9.4 mm × 150 mm, 5 µm

5968-2975EAtropine Atropa DAD SB-C8 866953-906 ACN/water hyoscyamine Belladonna 4.6 mm × 75 mm, 3.5 µm KH2PO4

5968-2974EQuinine quinidine Cinchona DAD Purospher RP18 ACN/water

(Cortex 4 mm × 125 mm, 5 µm KH2PO4

Cinchonae) bark

5968-2973EQuercetin, Gingko DAD Hypersil ODS MeOH/water kaempferol, (Gingko biloba) 4 mm × 125 mm, 5 µm H3PO4

isorhamnetin

5968-2972ERhein, emodin Rhubarb DAD Hypersil ODS ACN/water

(Rheum 4 mm × 125 mm, 5 µm AmmOAcpalmatum)

5968-2884EEphedrine Ma Huang, DAD SB-C8 866953-906 ACN/water norephedrine Ephedra sinica 4.6 mm × 75 mm, 3.5 µm KH2PO4

stimulant stapf

Herbal Supplements, Natural Products, Plant Hormones (Continued)


82

5968-2882EProtocatechuic acid, Dan Shen extr. DAD SB-C8 866953-906 ACN/water aldehyde, various 4.6 mm × 75 mm, 3.5 µm KH2PO4

tanshinones

5966-2591ENPlant hormones DAD SB-C8 866953-906 ACN/water TFA

4.6 mm × 75 mm, 3.5 µm

5965-9802ECatechins Tea ECD Hypersil BDS Recommend MeOH/water,

4 mm × 250 mm, 5 µm XDB-C18 or NaNO3, H2SO4

cartridge column SB-C18chemistry

Preservatives

5989-5178EN page 30Flavors, sweeteners, Soft drinks 1200SL DAD ZORBAX SB-C18 827975-302 ACN/water preservatives 4.6 mm × 250 mm, 5 µm, (1.8 µm) and AmmHPO4

3 mm × 50 mm, 1.8 µm 880975-902 (600 bar) (5 µm)

5989-3635EN page 31Paraben, Gradient DAD ZORBAX XDB-C18 963967-902 MeOH/waterphenoxyethanol 4.6 mm × 150 mm, 3.5 µm

5988-6356ENParaben Food DAD SB-C18 833975-902 ACN/water

4.6 mm × 30 mm, 3.5 µm H3PO4

cartridge

5966-0629EPreservative Wine dressing DAD Hypersil BDS ACN/water

4 mm × 125 mm, 5 µm H2SO4

5966-0628EAntioxidants Gum DAD Hypersil BDS ACN/water

4 mm × 100 mm, 3 µm H2SO4


5988-6358EN page 32Proteins Wheat Gradient DAD ZORBAX 300 SB-CN, 883995-906, ACN/water TFA 6

300 SB-C8 883995-905

5988-6348EN page 33Proteins Wheat Gradient DAD ZORBAX 300 SB-C8 883995-906 ACN/water TFA 7

4.6 mm × 150 mm, 5 µm

5988-6081EN page 34BSA (bovine serum Bin. grad DAD Poroshell 300 SB-C18 660750-902, ACN/water TFA Fastalbumin) digest, 2.1 mm × 75 mm, 5 µm 883750-902peptide ZORBAX 300 SB-C18

2.1 mm × 150 mm, 5 µm

5980-1193EN page 35AAA amino acid Gradient FLD/ Eclipse-AAA See app. note ACN, MeOH, water 8

OPA-FMOC 4.6 mm × 75 mm, 3.5 µm Na2HPO4, NaOH4.6 mm × 150 mm, 3.5 µm3 mm × 150 mm, 3.5 µm 4.6 mm × 150 mm, 5 µm

Herbal Supplements, Natural Products, Plant Hormones (Continued)


83

5989-0015ENProteins DAD 300 SB-x, Poroshell SB-x Various ACN/water TFA

5988-7930ENProtein, peptides MSn Ion Trap MALDI NA NA

AP/MALDI

5988-0897ENPeptide MSn Ion Trap 300 SB-C18 5064-8291 ACN/water phosphorylation with library, 0.3 mm × 150 mm, 5 µm formic

DAD

5980-2155Proteins CapLC MSn Ion 180 µm 300A C18 Unknown ACN/water

Trap posESI formic

5966-0746AAA amino acid Beer FLD OPA/ Hypersil ODS ACN, THF, water,

FMOC 2.1 mm × 200 mm, 5 µm NaOAc


5989-5319EN page 36Drugs Water 1200SL MSMS QQQ Extend-C18 728700-902 ACN/water Offline

pos/negESI 2.1 mm × 100 mm, 1.8 µm AmmForm SPE

5989-0738EN page 37Nitrofurans Poultry, shrimp Bin. grad MSn Trap XCT ZORBAX XDB-C8 971700-906 ACN/water

posESI 2.1 mm × 50 mm, 3.5 µm acetic

5989-0596EN page 38Fluoroquinolones Beef kidney Bin. grad MSD posESI ZORBAX XDB-C8 993967-906 ACN/water

4.6 mm × 150 mm, 5 µm formic

5988-9920EN page 39Chloramphenicol Shrimp, honey Bin. grad MSD and ZORBAX XDB-C18 993967-902 MeOH/ACN/

MSn Ion Trap 4.6 mm × 150 mm, 5 µm water AmmOAcnegESI

5988-7135EN page 40Sulfa drugs Meat Gradient DAD RP-18 Purospher 79925PU-584 ACN/water

4 mm × 250 mm 5 µm H3PO4

5980-2499EN page 41Sulfonamides CapLC DAD MSD ZORBAX SB-C18 5064-8262 ACN/water

posESI 0.5 mm × 150 mm, 3.5 µm formic

5989-4858ENEstrogens River water, MS TOF Luna Phenyl Hexyl Various Propanol/ 10

sewage, treated negAPPI 2.0 mm × 150 mm 3 µm See app. note cyclohexanesewage effluent w/guard

2.0 mm × 4 mm, 3 µm

5989-1302EN PosterNitrofuran Poultry, shrimp MS TOF ZORBAX XCB-C18 971700-902 ACN/water 11

posESI 2.1 mm × 50 mm 3.5 µm acetic

5989-0182ENSulfonamides Pork MSD posAPCI ZORBAX XDB-C8 993967-906 ACN/water 12

4.6 mm × 150 mm, 5 µm formic

Proteins, Peptides, Amino Acids (Continued)


84

5988-8999ENChloramphenicol Fish MSD negAPPI, ZORBAX XDB-C18 993967-302 MeOH/water 13

DAD 3 mm × 150 mm, 5 µm AmmOAc

5988-8903ENNitrofuran Poultry MSD posESI, Inertsil ODS3 ACN/water 14

DAD 2.1 mm × 150 mm, 5 µm formic

5988-6926ENSteroids Water MSn Ion Trap ZORBAX XDB-C18 971700-902 ACN/water 15

pos/negAPCI, 2.1 mm × 50 mm, 3.5 µm AmmOAcposAPPI, DAD

5966-1619Tetracyclines Meat, food DAD Hypersil BDS Recommend ACN/water 16

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

5965-9794Antibiotics, Meat DAD Purospher RP18 Recommend ACN/water sulfas 4 mm × 250 mm, 5 µm SB-C18 chemistry H3PO4

5989-5505ENArsenic species Urine, water LC-ICPMS ICPMS G3288-80000 G3288-80000 plus EtOH, water,

Arsenic column, G3154-65002 phosphate4.6 mm × 250 mm (Guard Column) EDTA, NaOAc,

NaNO3

5989-3572ENMethylmercury, Water, LC/ICP-MS ICPMS ZORBAX XDB-C18 960967-902 MeOH/water mercury, synthetic 2.1 mm × 50 mm, 5 µm AmmOAc ethylmercury seawater, soil 2-mercaptoethanol

5989-2883ENAcid herbicides, Water MSD negESI Extend-C18 763750-902 ACN/water 17steroids 2.1 mm × 150 mm, 3.5 µm formic NH3 TEA

5989-2324ENHerbicide Bialaphos MSn Ion Trap, Infusion NA NA 18antibiotic, MS TOF, both peptide bialaphos, posESIbilanaphos

5989-1270ENVarious including Food Various Various Various Various 19pesticides, drugs

5989-0814ENAmitrol Groundwater MSD posAPCI SB-C18 863954-302 MeOH/water 20

3 mm × 150 mm, 3.5 µm AmmOAc

5989-0181ENEPA 3640A Vegetable oil, GPC DAD Various Organic GPC See app. note Various 21Standard mix broccoli,

animal fat

5989-5403EN page 42HMF hydroxy- Bread, cereal, Bin. grad MSD Bonus-RP 861768-901 Water acetic Offline methylfurfural yogurt posAPCI 2.1 mm × 100 mm, 3.5 µm formic SPE

5988-2884EN page 43Acrylamide Drinking water Dual binary w/ MS TOF ZORBAX SB-C18 883700-922 ACN/water

6-port valve for posESI 2.1 mm × 150 mm, 5 µm formicautoSPE

Regulated/Hazardous Drug Substances (Continued)


85

5989-2481EN page 44Chromium Metrohm Metrohm 818 IC-ICPMS Agilent Cr G3268A Water Na2 EDTA 22speciation pump, Agilent 4.6 mm × 30 mm, 5 µm NaOH

7500 ISISsampler

5989-0816EN page 45Perchlorate Water, Metrohm IC MSD negESI MetroSep See app. note MeOH/water

vegetables ASUPP-5 30 mm NaOH4 mm × 100 mm

5988-9893EN page 46Arsenobetaine Fish Isocratic ICP-MS Hamilton PRP X-100 Contact AmmHCO3/

G1310A manufacturer tartaric

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

9.4 mm × 250 mm HNO3

5968-3049Bromate, iodate Ozone-treated Yokagawa IC ICP-MS or DAD IC See app. note Water, carbonate/ 23

water post col. deriv. bicarbonate

5966-0633Anions Water Isocratic DAD, indirect Contact author for ACN/water NaOH 24

UV reversed phase column plus UV modand moble phase details


5989-2912EN page 47DSP algal toxins Shellfish Quat. grad MSD pos/ ZORBAX SB-C18 883975-302 MeOH/water

negESI 3 mm × 150 mm, 5 µm, and formic9.4 mm × 50 mm, 5 µm 846975-202semiprep

5968-2124E page 49Mycotoxin, Corn Bin. grad MSD posESI ZORBAX XDB-C18 993700-902 ACN/water fumonisin 2.1 mm × 150 mm, 5 µm AmmOAc

00060329.pdf page 50Aflatoxins Various Agilent/ MSD posESI Phenomenex MAX RP Suggest SB-C18 ACN/water 25

Gerstel 2.1 mm × 250 mm, 5 µm 2.1 mm × 150 mm, formiconlineSPE 3.5 µm

830990-902

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

4.6 mm × 150 mm, 3.5 µm water

5968-3796E

Anatoxin A, (Algae) drinking MSD posESI Inertsil ODS3 Recommend ACN/water 26alkaloid water 2.1 mm × 150 mm, 5 µm SB-C18 or AmmOAcneurotoxin XDB-C18

chemistry

5968-2123EMicrocystins Fresh (surface) MSD posESI Mytisil ODS Recommend ACN/water

water 2.1 mm × 100 mm, 5 µm SB-C18 chemistry formic

Regulated/Miscellaneous Substances


86

5966-0632Aflatoxins, Various DAD/FLD Various Various Various 27mycotoxins, patulin, etc

5952-5852 obsoleteMycotoxins Old HP MS Vydac 201HSB Recommend ACN/water

Thermospray 4.6 mm × 150 mm, 5 µm SB-C8 or XDB-C8 AmmOAc(obsolete) chemistry

5952-5852Various fungal Various 5988A MS Various Various Various Old HP toxins Thermospray note

(obsolete)

5091-8692Mycotoxins Various DAD w/ Various See app. note Various

Library, FLD

Regulated/Hazardous Pesticide/Herbicide Substances

5989-5459EN page 5144 pesticides Vegetables, Bin. grad MSMS QQQ Extend-C18 728700-902 ACN/water SPE

fruit posESI 2.1 mm × 100 mm, 1.8 µm AmmForm cleanup

5989-5176EN page 52Acid herbicides Water AutoSPE/1200SL DAD ZORBAX SB-C18 See app. note ACN/water

with dual binary 5-, 3.5-, and H3PO4

pumps and 1.8-µm columns6-port valve

5989-2728EN page 54Postharvest Citrus Bin. grad MS TOF, MSn ZORBAX XDB-C8 993967-906 ACN/water 28fungicides Ion Trap, both 4.6 mm × 150 mm, 5 µm formic

posESI

5989-1842EN page 55Chloronicotinyl Vegetables, Bin. grad MS TOF, MSn ZORBAX XDB-C8 993967-906 ACN/waterinsecticides fruit Ion Trap, both 4.6 mm × 150 mm, 5 µm formic

posESI

5989-0813EN page 56Phenylurea, triazine Water Dual binary w/ MSD pos/ ZORBAX XDB-C8 971700-906 MeOH/water herbicides 6-port valve negAPCI 2.1 mm × 50 mm, 3.5 µm formic

for autoSPE

5989-5469EN100 pesticides Vegetables, MSMS QQQ ZORBAX XDB-C8 993967-906 ACN/water

fruit posESI 4.6 mm × 150 mm, 5 µm formic

5989-5320ENPesticides Water 1200SL MSMS QQQ Extend-C18 728700-902 ACN/water 29

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

5989-3573ENTerbuthylazine Olive oil MS TOF, MSn ZORBAX XDB-C8, 993967-906 ACN/water 30

Ion Trap both 4.6 mm × 150 mm, 5 µm formicposESI

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

fruit Ion Trap both 4.6 mm × 150 mm, 5 µm formicposESI

Regulated/Hazardous Natural Toxin Substances (Continued)


87

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

Ion Trap both 4.6 mm × 150 mm, 5 µm formicposESI

5989-1688ENPesticides Drinking MSD posESI Hypersil BDS ACN,MeOH,

water 2 mm × 100 mm, 3 µm water AmmOAc

5989-0930ENVarious including DAD Eclipse CN, C18 Various MeOH/waterpesticides

5989-0371ENPesticides Water MSD pos/ ZORBAX XDB-C18 930990-902 ACN/water 31Imazapyr negESI, DAD 2.1 mm × 150 mm, 3.5 µm formic

5989-0184ENAmitrol herbicide Water MSD ZORBAX SB-C18 863954-302 MeOH/water 32

posAPCI 3 mm × 150 mm, 3.5 µm AmmOAc

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

5988-8595ENHerbicides Water MSD pos/ ZORBAX XDB-C8 971700-906 MeOH/water phenylurea negESI 2.1 mm × 50 mm, 3.5 µm formictriazine

5988-8449ENPesticides DAD ZORBAX SB-C18 880975-302 ACN/water

3 mm × 250 mm, 5 µm NaOAc

5988-7351ENParaquat diquat Water Dual binary w/ MSD Extend-C18 763750-902 ACN/water 33

6-port valve for posESI 2.1 mm × 150 mm, 3.5 µm TDFHA autoSPE (tetradecafluoro-

heptanoic acid)

5988-7220ENParaquat diquat Water Dual binary w/ MSD posESI Extend-C18 763750-902 ACN/water 34

6-port valve for 2.1 mm × 150 mm, 3.5 µm TDFHA autoSPE (tetradecafluoro-

heptanoic acid)

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

5988-6635ENPesticides MSD ESI, ZORBAX XDB-C8 935967-906 ACN, MeOH, 35phenylureas, APCI, APPI 4.6 mm × 50 mm, 3.5 µm aceticcarbamates

5988-5882ENAcid herbicides, Water MSD negESI ZORBAX XDB-C18 930990-902 ACN/water 36propyzamide 2.1 mm × 150 mm, 3.5 µm formic

5988-4981ENGlyphosate, Water MSD posESI ZORBAX XDB-C8 946975-906 ACN/water 37AMPA 4.6 mm × 50 mm, 5 µm AmmOAc

Regulated/Hazardous Pesticides/Herbicides Substances (Continued)


88

5988-4708ENCarbamates Vegetable MSD posESI ZORBAX XDB-C18 960967-902, ACN/water

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

5988-4233ENSimazine, MSD posESI Inertsil ODS3 MeOH/water 38thiobencarb, 2.1 mm × 250 mm, 5 µm AmmOActhiuram

5988-3844ENGlycosylated Plant, hostas MSD negESI 300SB-C18 883750-902 ACN/water 39flavonoids 2.1 mm × 150 mm, 5 µm AmmOAc

5988-3774ENOrganophosphate MSD posESI, ZORBAX SB-C18 871700-902 ACN/water 40pesticides DAD 2.1 mm × 50 mm, 3.5 µm AmmOAc

5988-3649ENSulfonylureas, Surface water Cohesive MSn Ion Trap Metasil Basic ACN/water 41

posESI 2.1 mm × 100 mm, 5 µm acetic AmmOAc

5980-0561ERodenticides Sausage, dog MSD pos/neg ZORBAX XDB-C18 993700-902 MeOH/water 42difenacoum, stomach of ESI, APCI, 2.1 mm × 150 mm, 5 µm AmmOAccoumatetralyl, contents DADcoumafuryl

5980-0332ECarbaryl Complex food MSn Ion Trap ZORBAX XDB-C8 990967-906 ACN/water 43

homogenate posESI 4.6 mm × 250 mm, 5 µm acetic AmmOAc

5966-0743Glyphosate FLD post-col. IEX 44

deriv.

5966-0742Triazines, Salad spices DAD Hypersil BDS Recommend ACN/waterphenylureas, 3 mm × 100 mm, 3 µm XDB-C18 methabenzthiazuron, chemistrydiquat, paraquat, mercaptobenzothiazole

5954-7852E obsoleteCarbamates Old HP FLD w/post- C18 MeOH/water 45

col. deriv. 4.6 mm × 250 mm, 5 µm

5091-3621Glyphosate Drinking water LC/Pickering FLD w/ SAX-300 79919QA-754 Water, phosphate 46

precol. or 4.6 mm × 100 mmpostcol. deriv.


5988-6361EN page 58Retinol isomers Isocratic DAD ZORBAX Sil 880952-701 Dioxane,

4.6 mm × 250 mm, 5 µm tBME, hexane

5988-6359EN page 59Fat-soluble vitamins Isocratic DAD ZORBAX XDB-C8 993967-906 MeOH/water

4.6 mm × 150 mm, 5 µm

5968-9408E page 60Vitamin D3 Poultry feed Bin. grad MSn Ion Trap Flow inject – no column NA MeOH/water

posAPCI acetic

Regulated/Hazardous Pesticides/Herbicides Substances (Continued)


89

5968-2970 page 61Fat-soluble Quat. grad DAD ZORBAX XDB-C18 7995118-344 MeOH/watervitamins A,D, E 4.6 mm × 75 mm, 3.5 µm

5988-6354ENFat-soluble VWD with SB-C18 883975-902 MeOH/ACNvitamins wavelength 4.6 mm × 150 mm, 5 µm, XDB-C18

switching XDB-C18 4.6 mm × 150 mm, 4.6 mm × 150 mm, 5 µm 5 µm

5988-6350ENFat-soluble vitamins VWD with ODS 884950-543, MeOH/ACNA,D and E wavelength 4.6 mm × 250 mm, 5 um, 883952-702isomers, K1 switching ODS classic

4.6 mm × 150 mm, 5 µm

5980-1390Ergosterol Soil DAD Rx-C18 866967-902 MeOH/waterpre-Vitamin D 4.6 mm × 75 mm, 3.5 µm

5966-0745Fat-soluble DAD, Hypersil MOS Suggest ACN/watervitamins multiwave- 2.1 mm × 100 mm, 5 µm 2.1 mm × 150 mm,

length 3.5 µm Zorbax Sil

5966-0641Tocopherol Margarine DAD/FLD Hypersil SI Suggest IPA/hexaneisomers, 2.1 mm × 100 mm, 5 µm 2.1 mm × 150 mm, Vitamin E 3.5 µm Zorbax Sil

Mixed Vitamins

5091-3194Vitamins A,B,C,E Various ECD Lichrospher, Hypersil Various


5988-6365EN page 62Water-soluble Gradient DAD ZORBAX SB-C8 883975-906 MeOH/water vitamins 4.6 mm × 150 mm, 5 µm phosphate

5988-5761EN page 63Water-soluble Cat food Gradient DAD ZORBAX SB-C18 866953-902 ACN/water acetic 47vitamins 4.6 mm × 75 mm, 3.5 µm acetic phosphoric

TEA hexane sulfonate

5988-6364ENWater-soluble Tablets DAD SB-C18 880975-902 MeOH/water vitamins 4.6 mm × 250 mm, 5 µm, acetic hexane

USP23, L1 sulfonate

5988-6363ENWater-soluble DAD SB-C8 866953-906 MeOH/water vitamins 4.6 mm × 75 mm, 3.5 µm phosphoric hexane

sulfonate

5968-2971Water-soluble Chilled ALS DAD SB-C18 866953-902 ACN/water vitamins 4.6 mm × 75 mm, 3.5 µm phosphate

Fat-Soluble Vitamins (Continued)


90

5966-0639Water-soluble Tablets DAD Hypersil BDS ACN/watervitamins 4 mm × 100 mm, 3 µm H2SO4

Mixed Publications

5989-5177ENMethod transfer 1200SL DAD SB-C18 See app. note ACN/water example acid 5-, 3.5- and 1.8-µm H3PO4

herbicides columns

5989-4934ENSteroids, alkaloid DAD Various Eclipse RP Various Various

5989-4721ENSunscreen, DAD Various RP Various Various 48phenones,analgesics

5989-4086ENLuteoskyrin Various Various Eclipse RP Various Various

5989-1947ENFood solution guide Various Various Various Various 49

5988-4931ENPesticides, DAD Various Various Various 50peptide, APM (aspartame), etc.

5988-1786ENVarious DAD ZORBAX general Various Various 51

5968-9346EApplication FLD Various Various Various 52overview of FLD

5968-8569pdfPeptides, DAD ZORBAX misc. Various Various 53aspartame, NSAIDs 5 and 3.5 µm

Packaging

5988-8610ENAntioxidants Polymer MSD pos/ ZORBAX XDB-C8, 935967-906 ACN,MeOH,

negAPCI, DAD 4.6 mm × 50 mm, 3.5 µm THF, water

5967-6102EBisphenol Food MSD posESI Hypersil ODS ACN/water A esters 2.1 mm × 200 mm, 5 µm AmmOAc

Water-Soluble Vitamins (Continued)


91

Notes for the Application Reference Index1 12 pp intro

2 Post-column addition of NaOH solution

3 Bromophenacyl bromide derivatizing agent

4 Precolumn deriv. w/ OPA/mercaptoethanol

5 MS and MSMS, some infusion, some chromatography

6 Compare various bonded phases

7 Column temperature vs. separation quality and selectivity

8 Precolumn derivatization

9 In Japanese; for English version contact Woodman, Agilent.

10 Offline automated SPE procedure, normal phase cleanup (SB-CN 4.6 mm × 50 mm 5 µm and guard with propanol/cyclohexane), GPC cleanup (PLgel 50A in MeCl2), postcolumn addition of reference mass solution

11 Precolumn offline derivatization

12 Detailed sample prep including offline SPE

13 Postcolumn dopant acetone

14 Offline precolumn derivatization, detailed sample prep

15 Large volume direct injection with good comparison of strong and weak diluents on peak shape

16 Detailed sample prep provided

17 Ion suppression problems studied, postcolumn addition used

18 Elaborate structural elucidation via Trap MSMS and TOF accurate mass analysis

19 Food safety primer_LC, GC, ICP

20 Derivatization hexylchloroformate, SPE trace enrichment

21 Loading guidelines, various solvents

22 Column normally bundled with ICP-MS. Contact your agent/representative for details

23 Submitted to : Journal of Chromatography A, 789, 259-265 (1997)

24 UV modifier, probably aromatic acid like 4-OH-benzoic or trimesic; some applns have used cetylpyridinium chloride for indirect or displacement UV of anions.

25 Bromination of B1 and G1 prior to separation

26 Online derivatization with FMOC; good details

27 Detailed sample preps and references in this review article

28 QuECHERS sample prep

29 Offline SPE trace enrichment

30 Liquid/liquid extraction and SPE

31 Ion suppression problems studied, postcolumn addition used, “fully automated offline SPE”

32 Derivatization, SPE trace enrichment

33 Novel ion pairing reagent

34 Novel ion pairing reagent

35 Post-column addition of APPI dopant

36 “Fully automated offline SPE”

37 Offline precolumn derivatization FMOC, post-column addition dilute formic acid

38 QuECHERS sample prep

39 Post-column addition of TEF in IPA

40 Compares DAD to MSN-SIM results

41 Tubulent flow trace enrichment

42 Detailed sample prep, compares DAD to MSD, and neg vs. pos and ESI vs. APCI

43 Complex and detail sample prep.

44 Pickering post-column system with Agilent LC/FLD

45 Pickering system postcolumn base hydrolysis followed by OPA

46 Pickering post column system with Agilent LC/FLD

47 Titriplex-V hot extraction

48 RRHT standalone brochure

49 Former rev (2004) of Food Solution Guide

50 Zorbax SB LC columns

51 Zorbax applications guide various markets

52 Applications book various markets

53 Zorbax Rapid Resolution Columns, various applications

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. 2006

Printed in the USADecember 15, 20065989-5674EN

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