KNAUER HPLC Applications Journal - December 2006quimica.udea.edu.co/.../knauer_applications_journal_2007.pdf · This Applications Journal was ... DNPH derivates of Formaldehyde

HPLC Applications

KN06

0309

Applications Journal V7801

V 12/2006

Contents

ENVIRONMENTAL APPLICATIONS 2

FOOD APPLICATIONS 24

PHARMACEUTICAL APPLICATIONS 55

CHIRAL APPLICATIONS 85

SUBSTANCE AND KEYWORD INDEX 110

This Applications Journal was designed to provide you with detailed information on the chromatographic conditions used with a variety of HPLC applications. With our extensive line of HPLC products and expert technical service we are positioned to fulfill all of your separation needs.

We invite requests for support with collaborative studies and method validation projects in cooperation with our application laboratory. We look forward to discussing with you how we can support your applications with our HPLC columns and HPLC equipment.

For more information on these products and services please do not hesitate to contact us.

KNAUER · ASI · Advanced Scientific Instruments HPLC · SMB · Osmometry Wissenschaftliche Gerätebau Dr. Ing. Herbert Knauer GmbH Hegauer Weg 38, D-14163 Berlin, Germany Telephone: +49 (0)30 80 97 27-0 Telefax: +49 (0)30 80150 10 [email protected] http://www.knauer.net

Environmental Applications

Page

Determination of multiple additives in cosmetics 3

Determination of aldehydes and ketones as DNPH derivates 4

Ion pair chromatography of alkyl sulphates and alkyl sulphosuccinates 5

Anion determination according to U.S. EPA Method 300.0 / 317.0 and 326.0 with suppressed conductivity 6

Determination of anions with Ion pair chromatography and indirect UV detection 7

Determination of carbamate insecticides by HPLC with post-column derivatization 8

Determination of monovalent cations 9

Determination of divalent cations 10

Separation of explosives 11

Determination of ketones as DNPH derivates 12

Analysis of nitro polycyclic aromatic hydrocarbons 13

Biomonitoring of hydroxypyrene 14

Separation of ortho-, nitro- para nitroaniline 15

Separation of parabens 16

Chromatographic determination of pesticides 17

Determination of priority pollutant phenols 18

Separation of PAHs on a 2 mm ID column 19

Ion pair chromatography of alkyl sulphates and alkyl sulphosuccinates 20

Determination of non-ionic surfactants using HPLC 21

Analysis of surfactants in toothpastes by HPLC 22

Determination of aromatic hydrocarbon types in middle distillates acc. to prEN 12916:2006 23

V 12/2006 3

1 Determination of multiple additives in cosmetics

Method Matrix HPLC cosmetics

Column: Eurospher 100-5 C18, 250 x 4 mm ID Order No. 25DE181ESJ Phase: Eurospher 100-5 C18

Eluent: Water - Methanol (65:35), adjusted to pH 2.5 with phosphoric acid Gradient: 26 min linear gradient to Methanol - Phosphoric acid (1000:1)

12 min final elution Flow rate: 1.0 ml/min Temperature:

Conditions:

Volume: 20 µl (50 µg/ml of each component) Detection: UV at variable wavelengths (205 nm, 230 nm, 255 nm, 285 nm) Substances: Benzyl nicotinate, Butyl p-hydroxybenzoate, 2,6-Di-tert.-butyl-4-methylphenol, 4-

Isopropyl-3-methylphenol, Methyl p-hydroxybenzoate, Monoammonium glycyrrhizinate, Pantothenylethylether, Pyridoxine dioctanoate, Salicylic acid, Stearyl glycyrrhetinate, Trichlorocarbanilide, Tocopheryl acetate

Keywords: Additives Chromatogram:

1. Pantothenyl ethyl 2. Methyl p-

hydroxybenzoate 3. Salicylic acid 4. Benzyl nicotinate 5. 4-Isopropyl-3-

methylphenol 6. Butyl p-

hydroxybenzoate 7. Monoammonium

glycyrrhizinate 8. Trichlorocarbanilide 9. 2,6-Di-tert.-butyl-4-

methylphenol 10. Pyridoxine dioctanoate 11. Tocopheryl acetate 12. Stearyl glycyrrhetinate

0 10 20 30 40

1

2

3

45

6

7 8 9

10

11

12

min

4 Environmental Applications

2 Determination of aldehydes and ketones as DNPH derivates

Method Matrix HPLC natural gas

Collection on a DNPH coated solid phase cartridge, elution with methanol


Eluent: Methanol - Water (84:16, v/v) Gradient: isocratic Flow rate: 0.7 ml/min Temperature: 30 °C

Conditions:

Volume: 10 µl Detection: UV at 365 nm Substances: DNPH derivates of Formaldehyde, Acetaldehyde, Acrolein, Propionaldehyde,

Acetone, Butyraldehyde, Methacrolein, Methyl ethyl ketone, Pentanone, Cyclohexanone

Keywords: Aldehydes, Carbonyl compounds, Ketones Chromatogram: 1. DNPH 2. Formaldehyde 3. Acetaldehyde 4. Acrolein 5. Propionaldehyde 6. Acetone 7. n-Butyraldehyde and

Methacrolein 8. Methyl ethyl ketone 9. 2-Pentanone and

Cyclohexanon

0 5 10

1

2

36

45

7

8

9

15 min

V 12/2006 5

3 Ion pair chromatography of alkyl sulphates and alkyl sulphosuccinates

Method Matrix HPLC


Eluent: 0.01 M aqueous Tetrabutylammonium sulphate Methanol (23:77, v/v), adjusted to pH 3.0 with 0.01 M HCl

Gradient: isocratic Flow rate: 2.0 ml/min Temperature: ambient

Conditions:

Volume: 50 µl Detection: Refractive Index Substances: Octyl hydrogen sulphate, C10 Sulphosuccinate,

C12 Sulphosuccinate, C14 Sulphosuccinate Keywords: Surfactants Chromatogram: 1. Octyl hydrogen sulphate 2. C10 Sulphosuccinate 3. C12 Sulphosuccinate 4. C14 Sulphosuccinate

0 2 4 6 8

1

2

3

4

min


4 Anion determination according to U.S. EPA Method 300.0 / 317.0 and 326.0 with suppressed conductivity

Method Matrix HPLC

Column: Novosep A-2 Anion (5 µ), 250 x 4 mm ID Order No. B92 Phase: Novosep A-2 Anion

Eluent: 3.6 mM Sodium Carbonate (Na2CO3) Gradient: isocratic Flow rate: 0.8 ml/min Temperature: 45 °C

Conditions:

Volume: 50 µl (Chromatogram for a mixture of anions with concentration of 0.25 ppm)

Detection: suppressed Conductivity (Alltech Model 650), Range 0.5 µS Substances: Fluoride, Bromate, Chloride, Nitrite, Bromide, Nitrate, Phosphate, Sulfate Keywords: Anions Chromatogram: 1. Fluoride 2. n.n. 3. Bromate 4. Chloride 5. Nitrite 6. Bromide 7. Nitrate 8. Phosphate 9. Sulfate

0 5 10 15 20 25 min

1

2

3

4

56

7

8

9

V 12/2006 7

5 Determination of anions with Ion pair chromatography and indirect UV detection

Method Matrix HPLC Column: Eurospher 100-5 C18, 250 x 4.6 mm ID Order No. 25EE181ESJ Phase: Eurospher 100-5 C18

Eluent: 0.11 % Acetyl trimethylammonium p-toluenesulphonate + 0.002 M p-Toluenesulphonate in Acetonitrile - Water (39:61), adjusted to pH 5.5 - 6 with Ammonium hydroxide

Temperature: ambient Flow rate: 1.5 ml/min

Conditions:

Volume: Detection: UV at 236 nm and 254 nm,

Chromatograms for a mixture of anions (1µg each, S.P. = system peak) Substances: Cl

-, ClO3

-, SO4

2-, NO3

-, I

-, IO3

-, SCN

-, Br

-

Keywords: Anions Chromatogram: 1. IO3

-

2. Cl-

3. Br-

4. NO3-

5. ClO3-

6. I-

7. SCN- 8. SO4

2-

1

2

3

4

5

6

7

8

1

2

34

5

6

78

236 nm 254 nm


6 Determination of carbamate insecticides by HPLC with post-column derivatization

Method Matrix HPLC Column: Eurospher 100-5 C18, 250 x 4.6 mm ID Order No. 25EE181ESJ Phase: Eurospher 100-5 C18

Eluents: A: water, B: methanol Gradient: 0 – 0.5 isocratic, 82% A

0.5 – 29 linear, 82 – 30% A 29 – 31 isocratic, 100% B, column rinsing 31 – 36 isocratic, 82% A, re-equalibration

Flow rate: 1.0 ml/min Temperature: 42 °C

Conditions:

Volume: 10 µl Derivatization: 1st Reactor, 500 µl 100°C, first derivatization step with NaOH 2nd Reactor, 150 µl Ambient, derivatization with OPA and 2-mercaptoethanol Detection: Post-column derivatization with OPA, two step reaction

Fluorescence: excitation 330 nm, emission 465 nm Substances: Aldicarb, Aldicarb sulphoxide, Aldicarb sulphone, BDMC, Carbaryl, Carbofuran, 3-

Hydroxycarbofuran, Methiocarb, Methomyl, 1-Naphtol, Oxamyl, Propoxur Keywords: Carbamates, Insecticides Chromatogram:

1. Aldicarb sulphoxide 2. Aldicarb sulphone 3. Oxamyl 4. Methomyl 5. 3-Hydroxycarbofuran 6. Aldicarb 7. Propoxur 8. Carbofuran 9. Carbaryl

10. 1- Naphtol 11. Methiocarb 12. BDMC

0 10 20

12

3

4

56

7

8

9

10

1112

V 12/2006 9

7 Determination of monovalent cations

Method Matrix HPLC

Column: PRP-X 200, 150 x 4.1 mm with Precolumn Order No. B89 Phase: PRP-X 200, cation exchange resin

Eluent: 4 mM Nitric Acid in 30 % MeOH Gradient: isocratic Flow rate: 1.7 ml/min Temperature: 35°C

Conditions:

Volume: 20 µl Detection: Conductivity (negative Polarity) Substances: Ammonium, Lithium, Potassium, Sodium Keywords: Cations, monovalent Chromatogram: 1. Lithium (5.0 ppm) 2. Sodium (10.0 ppm) 3. Ammonium (10.0 ppm) 4. Potassium (10.0 ppm)

0 2 3 4 5 min

1

2 3

4


8 Determination of divalent cations

Method Matrix HPLC

Column: PRP-X 200, 150 x 4.1 mm with Precolumn Order No. B89 Phase: PRP-X 200, cation exchange resin

Eluent: 1.5 mM Ethylene diammonium dichloride Gradient: isocratic Flow rate: 2 ml/min Temperature: 35°C

Conditions:

Volume: 10 µl Detection: Conductivity (negative Polarity) Substances: Barium, Calcium, Magnesium, Strontium Keywords: Cations, divalent Chromatogram: 1. Magnesium (20.0 ppm) 2. Calcium (20.0 ppm) 3. Strontium (20.0 ppm) 4. Barium (20.0 ppm)

1

2

34

0 2 5 8 min

V 12/2006 11

9 Separation of explosives

Method Matrix HPLC Column: Ultra Sep ES EX, 250 x 3 mm ID Order No. I0034

Eluent: A) Methanol B) Water Gradient: linear, 25-60 % Methanol in 40 min,

5 min hold and equilibrate column 5 min with 25% A for next injection


Conditions:

Volume: Detection: UV at 235 nm Substances: 2-Amino-4,6-dinitrotoluene, 4-Amino-2,6-dinitrotoluene, 2-Amino-4-nitrotoluene, 2-

Amino-6-nitrotoluene, 1,3-Dinitrobenzene, 2,4-Dinitrotoluene, 2,6-Dinitrotoluene, Hexogen, Nitrobenzene, 2-Nitrotoluene, 4-Nitrotoluene, 3-Nitrotoluene, Octogen, Tetryl

Keywords: Explosives, EPA 8330 Chromatogram:

0 5 10 15

1

2

3

4 5

6

7

8

9

10

11

12

13

14

1516

20 25 30 35 min

1. Octogen 2. Hexogen 3. 1,3,5-Trinitrobenzene 4. 2-Amino-6-nitrotoluene 5. 2-Amino-4-nitrotoluene 6. 1,3-Dinitrobenzene 7. Tetryl 8. Nitrobenzene 9. 2,4,6-Trinitroluene

10. 4-Amino-2,6-dinitrotoluene 11. 2-Amino-4,6-dinitrotoluene 12. 2,6-Dinitrotoluene 13. 2,4-Dinitrotoluene 14. 2-Nitrotoluene 15. 4-Nitrotoluene 16. 3-Nitrotoluene


10 Determination of ketones as DNPH derivates

Method Matrix HPLC Column: ProntoSIL 120-3 C18 AQ, 250 x 3 mm ID Order No. 15DF080PSJ Phase: ProntoSIL 120-3 C18 AQ

Eluent: A) Water B) Acetonitrile Gradient: 10 – 100 % B in 18 min Temperature: 30 °C

Conditions:

Flow rate: 0.35 ml/min Volume: 2 µl Detection: UV at 270 nm Substances: DNPH derivates of Formaldehyde, Acetaldehyde, Acrolein, Propionaldehyde,

Acetone, Butyraldehyde, Methacrolein, Methyl ethyl ketone, Pentanone, Cyclohexanone

Keywords: Aldehydes, Carbonyl compounds, Ketones Chromatogram:

0 5 10 15 min

1

2

3

4

5 6 78

911

10

1. Tetramethylketone 2. 2-Pentanone 3. 2-Hexanone 4. 2-Heptanone 5. 2-Octanone 6. 2-Nonanone 7. 2-Decanone 8. 2-Undecanone 9. 2-Dodecanone 10. 2-Tridecanone 11. 2-Hexadecanone

V 12/2006 13

11 Analysis of nitro polycyclic aromatic hydrocarbons

Method Matrix HPLC Column: ProntoSIL 120-3 Phenyl, 125 x 4 mm ID Order No. 12DF050PSG Phase: ProntoSIL 120-3 Phenyl

Eluent: A) Methanol / Water (60:40, v/v) B) Methanol Gradient: 100 % A for 13 min

0 – 100% B in 29 min Temperature: 25 °C

Conditions:

Flow rate: 0.8 ml/min Volume: 2 µl Detection: Fluorescence (wavelength program) Substances: 2-Nitro-Naphthalene, 2-Nitro-Fluorene, 9-Nitro-Phenanthrene,

1-Nitro-Pyrene, 6-Nitro-Chrysene, 6-Nitro-Benzo(a)pyrene Keywords: Nitro-PAH Chromatogram:

0 10 20 30 min

1

2

3

45

6

1. Nitro-Naphthalene 2. Nitro-Fluorene 3. Nitro-Phenanthrene 4. Nitro-Pyrene 5. Nitro-Chrysene 6. Nitro-Benzo(a)pyrene


12 Biomonitoring of hydroxypyrene

Method Matrix HPLC

Column: Eurospher 100-5 C18, 250 x 4 mm Order No. 25DE181ESJ Phase: Eurospher 100-5 C18

Eluent: A) ACN B) H2O Gradient: linear 60% A in 28 min to 100% A, 5 min hold, equilibrate column 10 min

with 60% A for next injection Flow rate: 1 ml/min Temperature: 30°C

Conditions:

Volume: 10 µl Detection: Fluorescence: excitation 242 nm, emission 388 nm Substances: Hydroxypyrene, Biomarker of PAH contamination Keywords: Hydroxypyrene Chromatogram:

1. Hydroxypyrene

0 5 10 15 20

1

V 12/2006 15

13 Separation of ortho-, nitro- para nitroaniline

Method Matrix HPLC

Column: Eurospher 100-5 Si, 120 x 4 mm Order No. 11DE000ESJ Phase: Eurospher 100-5 Si

Eluent: A) Heptan B) Ethanol Gradient: isocratic, 85 % A / 15 % B Flow rate: 1 ml/min Temperature: 30°C

Conditions:

Volume: 10 µl Detection: UV at 225 nm Substances: 2-Nitroaniline, 3-Nitroaniline, 4-Nitroaniline Keywords: Nitroaniline Chromatogram:

1. 2-Nitroaniline

2. 3-Nitroaniline

3. 4-Nitroaniline

0 2 4 6 8 min

1

2

3


14 Separation of parabens

Method Matrix HPLC Column: ProntoSIL 120-3 Phenyl, 125 x 4 mm ID Order No. 12DF050PSG Phase: ProntoSIL 120-3 Phenyl

Eluent: 20 mM Potassium Hydrogen Phosphate (KHPO4)/Acetonitrile (50 : 50 v/v) Gradient: isocratic Flow rate: 1 ml/min Temperature: 40 °C

Conditions:

Volume: 10 µl Detection: UV at 254 nm Substances: Methylparabene, Ethylparabene, Propylparabene, Butylparabene Keywords: Parabenes Chromatogram:

1

2

34

1 3 5 min

1. Methylparabene 2. Ethylparabene 3. Propylparabene 4. Butylparabene

V 12/2006 17

15 Chromatographic determination of pesticides

Method Matrix HPLC Column: UltraSep ES PEST, 250 x 3 mm Order No. I0025 Phase: UltraSep ES PEST

Eluent: ACN - Water Gradient: from 20 to 26 % ACN within 5 min,

26 to 30 % ACN within 19 min 30 to 60 % within 12 min

Temperature: 30 °C Flow rate: 0.9 ml/min

Conditions:

Volume: 10 µl Detection: UV at 215 nm and 230 nm Substances: Atrazine, Chlortoluron, Cyanazin, Desethylatrazine, Diuron, Hexazinon,

Isoproturon, Linuron, Metoxuron, Methabenzthiazuron, Metolachlor, Metobromuron, Metazachlor, Monolinuron, Sebuthylazin, Simazin, Terbuthylazine

Keywords: Pesticides Chromatogram:

5 10 15 20 3025 min

1

2

3

4

5

67

89

10

1112

13

14

15

1617

1. Desethylatrazin 2. Hexazinon 3. Metoxuron 4. Simazin 5. Cyanazin 6. Methabenzthiazuron 7. Atrazine 8. Chlortoluron 9. Monolinuron

10. Isoproturon 11. Metobromuron 12. Diuron 13. Metazachlor 14. Sebuthylazin 15. T erbuthylazine 16. Linuron 17. Metolachlor


16 Determination of priority pollutant phenols

Method Matrix HPLC


Eluent: A) MeOH plus 1 % acetic acid B) Water plus 1 % acetic acid

Gradient: 30 % A in 9 min to 40 % A 40 % A in 7 min to 100 % A, 6 min hold

Flow rate: 1 ml/min Temperature: 40 °C

Conditions:

Volume: 5 µl Detection: UV at 280 nm Substances: 2-Chlorophenol, 4-Chloro-3-methylphenol, 2,4-Dichlorophenol,

2,3-Dimethylphenol, 2,4-Dinitrophenol, 2-Methyl-4,6-dinitrophenol, 2-Nitrophenol, 4-Nitrophenol, Pentachlorphenol, Phenol, 2,4,6-Trichlorphenol

Keywords: Phenols Chromatogram:

0 5 10 15 20

1

2

34

5

6

7

8

9

10

11

min

1. Phenol 2. 4-Nitrophenol 3. 2,4-Dinitrophenol 4. 2-Chlorophenol 5. 2-Nitrophenol 6. 2,3-Dimethylphenol 7. 2-Methyl-4,6-dinitrophenol 8. 4-Chloro-3-methylphenol 9. 2,4-Dichlorphenol

10. 2,4,6-Trichlorphenol 11. Pentachlorphenol

V 12/2006 19

17 Separation of PAHs on a 2 mm ID column

Method Matrix HPLC

Column: Eurospher 100-3 C18, 250 x 2 mm ID Order No. 25BE181ESG

Eluent: A) MeOH - Water (80:20) B) ACN – THF (93:7) Gradient: linear 0-100 % B in 15 min, 5 min hold;

equilibrate column 10 min with 100% A for next injection Temperature: 30 °C Flow rate: 0.2 ml/min

Conditions:

Volume: 5 µl Detection: Fluorescence, wavelength program (excitation / emission):

1. 275/350 nm, 2. 260/390 nm, 3. 260/420 nm, 4. 290/430 nm, 5. 300/500 nm UV at 254 nm

Substances:

Naphthalene, Acenaphthylene, Acenaphthene, Fluorene, Phenanthrene, Anthracene, Fluoranthene, Pyrene, Benzo(a)anthracene, Chrysene, Benzo(b)fluoranthene, Benzo(k)fluoranthene, Benzo(a)pyrene, Dibenzo(a,h)anthracene, Benzo(g,h,i)perylene, Indeno(1,2,3-cd)pyren

Keywords: 16 PAH´s according to EPA (550.1 / 610 / 8310) Chromatogram:

12

3

4

56

7

8

9

10

11

12

13

14

15

16

0 10 20 min

1. Naphthalene 2. Acenaphthylene 3. Acenaphthene 4. Fluorene 5. Phenanthrene 6. Anthracene 7. Fluoranthene 8. Pyrene 9. Benzo(a)anthracene

10. Chrysene 11. Benzo(b)fluoranthene 12. Benzo(k)fluoranthene 13. Benzo(a)pyrene 14. Dibenzo(a,h)anthracene 15. Benzo(g,h,i)perylene 16. Indeno(1,2,3-cd)pyrene


18 Ion pair chromatography of alkyl sulphates and alkyl sulphosuccinates

Method Matrix HPLC


Eluent: 0.01 M aqueous Tetrabutylammonium sulphate Methanol (23:77, v/v), adjusted to pH 3.0 with 0.01 M HCl


Conditions:

Volume: 50µl Detection: Refractive Index Substances: Octyl hydrogen sulphate, C10 Sulphosuccinate,

C12 Sulphosuccinate, C14 Sulphosuccinate Keywords: Surfactants Chromatogram:

0 2 4 6 8

1

2

3

4

min

1. Octyl hydrogen sulphate 2. C10 Sulphosuccinate 3. C12 Sulphosuccinate 4. C14 Sulphosuccinate

V 12/2006 21

19 Determination of non-ionic surfactants using HPLC

Method Matrix HPLC


Eluent: MeOH - Water (90:10 v/v) Temperature: 25 °C Flow rate: 1.5 ml/min

Conditions:

Volume: 20 µl Detection: Refractive Index Substances: Polyethylene glycol, Polyoxyethylene sorbitane monolaurate, Nonylphenol

polyglycol ether, Laurylalcohol, Laurylalcohol polyglycol ether, Oleic acid diethanolamide, Oleic amide polyglycol ether, Myristylalcohol, Myristyl alcohol polyglycol ether, Palmitic acid polyglycol ester, Polyoxyethylene glycerol monostearate, Stearic acid polyglycol ester

Keywords: Surfactants Chromatogram:

0 5 10 15 min

1

2

3

4

5

6

78

9

10

1112

1. Polyethylene glycols 2. Polyoxyethylene sorbitane monolaurate 3. Nonylphenol polyglycol ether 4. Lauryl alcohol 5. Lauryl alcohol polyglycol ether 6. Oleic acid diethanolamide 7. Oleic amide polyglycol ether 8. Myristyl alcohol 9. Myristyl alcohol polyglycol ether

10. Palmitic acid polyglycol ester 11. Polyoxyethylene glycerol monostearate 12. Stearic acid polyglycol ester.


20 Analysis of surfactants in toothpastes by HPLC

Method Matrix HPLC toothpaste


Eluent: MeOH - Water (90:10 v/v) + 0.25 M NaClO4 Gradient: isocratic Flow rate: 1.5 ml/min Temperature: 25 °C

Conditions:

Volume: 20 µl Detection: Refractive Index Substances: Sodium soap (C8 - C18),

Sulpho fatty acid-α-methyl esters (C10 - C18) Keywords: Surfactants Chromatogram:

5 10 15 min 5 10 15

1 2

3

4

5

6

71

2

3

4 5

Sodium soaps: 1. (C8) 2. (C10) 3. (C12) 4. (C14) 5. (C16) 6. (C18 =) 7. (C18) α-sulpho fatty acid methyl esters: 1. (C10) 2. (C12) 3. (C14) 4. (C16) 5. (C18)

V 12/2006 23

0 5 10 15 min

Cyclohexane

MAH 20.14 % [m/m]

DAH 2.33 % [m/m]

T+AH 0.11 % [m/m]

Total aromatic hydrocarbons: 22.59 % [m/m]

12

13

11

1

21 Determination of aromatic hydrocarbon types in middle distillates acc. to prEN 12916:2006

Method Matrix diesel fuel HPLC Sample preparation: dilution with n-heptane

Column: Ultrasep ES AP 5 µm, 250 x 4 mm ID Order No. 25DE450USJ

Phase: Ultrasep ES AP 5 µm

Eluent: n-Heptane (dried with molecular sieve) Gradient: isocratic Flow rate: 0.5 ml/min Temperature: 25 °C

Conditions:

Volume: 10 µl Detection: RI Substances: MAH: phenyldodecane; 1,2 dimethylbenzene; hexamethylbenzene

DAH: naphthalene, fluorene T-AH: dibenzothiphene; phenanthrene, 9-methylanthracene

Keywords: MAH (mono-aromatic compounds); DAH (di-aromatic compounds);

T+AH (tri+-aromatic compounds) Chromatogram:

0 5 10 15 min

1

2

3

4

5

67

0 5 10 15 min

8

9

10

Retention time standard Calibration standard

Retention time standard 1. Cyclohexane 2. Phenyldodecane 3. 1,2 Dimethylbenzene 4. Hexamethylbenzene 5. Naphthalene 6. Dibenzothiophene 7. 9-Methylanthracene

Calibration standard 8. 1,2 Dimethylbenzene 9. Fluorene

10. Phenanthrene

Sample 11. MAH 20.14% [m/m] 12. DAH 2.33% [m/m] 13. T+AH 0.11% [m/m] Total aromatic hydrocarbens: 22.59 % [m/m]

Sample

Food Applications

Page

Separation of additives in soft drinks 25

Determination of aflatoxins by HPLC with post column derivatization 26

Simultaneous determination of alcohols, carbohydrates and organic acids I 27

Determination of alcohols, carbohydrates and organic acids II 28

Separation of amines in wine with precolumn derivatization (DANSYL) 29

HPLC determination of amines in wine with precolumn derivatization (OPA) 30

Determination of amino acids in beer with precolumn derivatization (OPA) 31

Inorganic and organic anions in beer 32

Anthocyanins in red and rose wine 33

Determination of biogenic amines in food products by HPLC in combination with ninhydrin derivatization 34

Separation of β-carotene isomers 35

Determination of diketopiperazine in soft drinks 36

Analysis of flavonoids in fruit juice 37

Stilbene and flavonoids in red wine samples 38

Analysis of flavonoid glycosides 39

Separation of methylfurfural and hydroxyfurfural in beer 40

Determination of mono- and disaccharides 41

Determination of deoxynivalenol (DON) 42

Determination of nivalenol (NIV) and deoxynivalenol (DON) with postcolumn derivatisation 43

Analysis of fusarium mycotoxines 44

Ion chromatography for determination of NO2- and NO3- in beverages 45

Determination of organic acids 46

Determination of organic acids II 47

Determination of patulin in apple juice 48

Separation of preservatives 49

Determination of various sterols 50

Separation of water soluble vitamins 51

Separation of tocopherols and vitamin D2 52

Separation of fat soluble vitamins 53

Fat soluble vitamins by HPLC with electrochemical detection 54

V 12/2006 25

1 Separation of additives in soft drinks

Method Matrix HPLC

Column: ProntoSIL 120-5 C8 SH, 150 x 4 mm ID Order No. 15DF080PSJ Phase: ProntoSIL 120-5 C8 SH

Eluent: 20 mM KH2PO4 (pH 3) / Acetonitrile 5:1 Gradient: isocratic Flow rate: 1.2 ml/min Temperature: 30 °C

Conditions:

Volume: 10 µl Detection: UV at 220 nm Substances: Acesulfam K, Ascorbic acid, Aspartame, Benzoic acid, Caffeine, Saccharin, Sorbic

acid, Quinine, Vanilin Keywords: Sweeteners, Additives Chromatogram:

10 min0 5

1

3

4

5

6 7

8

9

2

1. Ascorbic acid 2. Acesulfam K 3. Saccharin 4. Caffeine 5. Aspartame 6. Quinine 7. Vanilin 8. Sorbic acid 9. Benzoic acid

26 Food Applications

2 Determination of aflatoxins by HPLC with post column derivatization

Method Matrix HPLC food

Column: Nucleosil 120-5 C18, 250 x 4 mm ID Order No. 25DF181ESJ Phase: Nucleosil 120-5 C18

Eluent: Acetonitrile - Methanol - Water (23.5 : 29.5 : 47) Gradient: isocratic Flow rate: 0.7 ml/min Temperature: ambient

Conditions:

Volume: 10 µl, 10 ng/ml each Detection:

postcolumn derivatization with 1.3 mM bromine solution at a flow rate of 0.18 ml/min, fluorescence: excitation 375 nm, emission 455 nm

Substances: Aflatoxin B1, Aflatoxin B2, Aflatoxin G1, Aflatoxin G2 Keywords: Aflatoxins Chromatogram:

0 5 10 min

1

2

3

4

1. Aflatoxin G2 2. Aflatoxin G1 3. Aflatoxin B2 4. Aflatoxin B1

V 12/2006 27

3 Simultaneous determination of alcohols, carbohydrates and organic acids I

Method Matrix HPLC beverage Column: Eurokat H 10 µm, 300 x 8 mm ID Order No. 30GX340EKN Phase: Eurokat H 10 µm

Eluent: 0.01 M Sulfuric acid Gradient: isocratic Flow rate: 0.4 ml/min Temperature: 80 °C

Conditions:

Volume: 10 µl Detection: Refractive Index Substances: Maltose, Glucose, Succinic acid, Lactic acid, Formic acid, Acetic acid, Methanol,

Ethanol Keywords: Carbohydrates, Organic acids, Alcohols Chromatogram:

0 10 20

3

2

1

4

5

67

8

9

30 min

1. Higher carbohydrates 2. Maltose 3. Glucose 4. Succinic acid 5. Lactic acid 6. Formic acid 7. Acetic acid 8. Methanol 9. Ethanol


4 Determination of alcohols, carbohydrates and organic acids II

Method Matrix HPLC

Column: Eurokat H 10 µm, 300 x 8 mm ID Order No. 30GX340EKN Phase: Eurokat H 10 µm

Eluent: 0.00125 N Sulfuric acid (pH 2) Gradient: isocratic Flow rate: 0.4 ml/min Temperature: 90 °C

Conditions:

Volume: 10 µl Detection: Refractive Index Substances: Acetic acid, Butandiol, Citric acid, Ethanol, Fructose, Glucose, Lactic acid, Malic acid,

Succinic acid, Tartaric acid Keywords: Organic acids Chromatogram:

0 5 10 15 20 25 30 35

10

9

876

5

4

3

1

2

1. Citric acid 2. Tartaric acid 3. Glucose 4. Malic acid 5. Fructose 6. Sucinic acid 7. Lactic acid 8. Acetic acid 9. Butandiol

10. Ethanol

V 12/2006 29

5 Separation of amines in wine with precolumn derivatization (DANSYL)

Method Matrix HPLC wine

Column: Eurospher 100-5 C18, 250 x 4.6 mm ID Order No. 25EE181ESJ Phase: Eurospher 100-5 C18

Eluent: ACN - Water 30 :70 % Gradient: over 40 min to 60% ACN and 15% Ethanol in water, 10 min

hold Flow rate: 1 ml/min Temperature: ambient

Conditions:

Volume: 20 µl Detection: Fluorescence: excitation 338 nm, emission 455 nm Substances: Ethanolamine, Ammonia, Methylamine, Serotonin, Ethylamine, Morpholine,

Isopropylamine, Dimethylamine, Propylamine, Pyrrolidine, Pyrrolidone, Octopamine, Indole, Butylamine, Diethylamine, Phenethylamine, Methylbutylamine, Isopentylamine, Pentylamine, Piperidine, Diaminopropane, Diaminobutane, Hexylamine, Diaminopentane, Histamine

Keywords: Amines Chromatogram:

0 10 20 30 40 50 min

25

22

1

2

3

4

5

6

9 17,181615

14

1923,24

10-13

7,8

20,21

1. Ethanolamine, 2. Ammonia, 3. Methylamine, 4. Serotonin, 5. Ethylamine, 6. Morpholine, 7. Isopropylamine, 8. Dimethylamine, 9. Propylamine

10. Pyrrolidine, 11. 2-Pyrrolidone, 12. Octopamine, 13. Indole 14. Butylamine, 15. Diethylamine, 16. Phenethylamine, 17. 2-Methylbutylamine, 18. Isopentylamine, 19. Pentylamine, 20. Piperidine, 21. 1,3-Diaminopropane, 22. 1,4-Diaminobutane, 23. Hexylamine, 24. 1,5-Diaminopentane, 25. Histamine


6 HPLC determination of amines in wine with precolumn derivatization (OPA)



Eluent: 0.08 M Acetic acid – Acetonitrile 70:30 (v/v) 3 min, Gradient: in 6.5 min to 65 % Acetonitrile, in 11 min to 72 % Acetonitrile,

in 1 min to 80 % Acetonitrile, 3 min hold Flow rate: 1 ml/min Temperature: ambient

Conditions:

Volume: 20 µl Detection: Fluorescence: ex 230 nm, em 440 nm Substances: Cadaverine, Heptylamine, Histamine, Isoamylamine, Pentyl-i-amine, Phenethylamine,

Putrescine Keywords: Amines Chromatogram:

0 5 10 15 20 min

1

2 3

456

7

1. Histamine 2. Tyramine 3. Putrescine 4. Phenethylamine 5. Cadaverine 6. Isoamylamine 7. internal Standard IS

V 12/2006 31

7 Determination of amino acids in beer with precolumn derivatization (OPA)

Method Matrix HPLC beer

Column: OPA column B801, 300 x 4 mm ID Order No. B801 Phase: OPA Column

Eluent: A: 0.05 M Acetate – Methanol 81:19 (v/v) pH 7.2 B: 0.05 M Acetate – Methanol 25:75 (v/v)

Gradient: 28 min 100 % A, in 7 min to 70 % A, in 15 min to 100 % B (5 min hold), In 5 min 100 % A

Flow rate: 1 ml/min Temperature: 40 °C

Conditions:

Volume: 10 µl Detection: Fluorescence: excitation 330 nm, emission 450 nm Substances: Aspartic acid, Glutamic acid, Asparagin, Serine, Glutamin, Histidine, Glycine,

Threonine, Arginin, Alanine, Tyrosine, Methionine, Valine, Phenylalanine, Isoleucine, Leucine, Lysine

Keywords: Amines Chromatogram:

0 10 20 30 40 50 60 min

1

2

3 45

6

7

8

9

10

11

12

13

14

15

16

17

1. ASP 2. GLU 3. ASN 4. SER 5. GLN 6. HIS 7. GLY 8. THR 9. ARG

10. ALA 11. TYR 12. MET 13. VAL 14. PHE 15. ILE 16. LEU 17. LYS


8 Inorganic and organic anions in beer


Column: Allsep Anion, 100 x 4.6 mm ID Order No. B90

Phase: Allsep Anion

Eluent: 0.85 mM Sodium Hydrogen Carbonate (NaHCO3) Gradient: isocratic Flow rate: 1.2 ml/min Temperature: 35 °C

Conditions:

Volume: 100 µl Detection: suppressed Conductivity (Alltech Model 650), Range 10 µS Substances: Chloride, Nitrate, Phosphate, Sulfate, Oxalate Keywords: Anions, inorganic and organic Chromatogram:

5 10 15 min

1

2

3

4

5

Chromatogram of beer sample (dilution 1:10) 1. Chloride 2. Nitrate 3. Phosphate 4. Sulfate 5. Oxalate

V 12/2006 33

9 Anthocyanins in red and rose wine


Column: ProntoSIL 120-3 C18 SH, 250 x 4 mm ID Order No. 25DF180PSG Phase: ProntoSIL 120-3 C18 SH

Eluent: A: Water/Formic Acid/Acetonitrile 87:10:3 B: Water/Formic Acid/Acetonitrile (pH 1.3)

Gradient: 0 min 94 % A 6 % B, 15 min 70 % A 30 % B, 30 min 50 % A 50 % B, 35 min 40 % A 60 % B, 41 min 94 % A 6 % B (2 min hold)


Conditions:

Volume: 20 µl Detection: UV at 518 nm Substances: Group 1: nonacylated anthocyanidin-3-glycosides

Group 2: acylated anthocyanidin-3-glycosides Group 3: coumarylated anthocyanidin-3-glycosides

Keywords: Anthocyanins Chromatogram:

0 5 10 15 20

13

4

5

67 8 92

25 30 35 40 45 min

Group 1 1. Delphinidol-3-glucosid 2. Cyanidol-3-glucosid 3. Petunidol-3-glucosid 4. Peonidol-3-glucosid 5. Malvidol-3-glucosid

Group 2 6. Peonidol-3-acetylglucosid 7. Malvidol-3-acetylglucosid

Group 3 8. Peonidol-3-coumarylglucosid 9. Malvidol-3-coumarylglucosid


10 Determination of biogenic amines in food products by HPLC in combination with ninhydrin derivatization

Method Matrix HPLC Food


Eluent: 16 g Ninhydrin and 1.2 g Hydrindantin, dissolved in 322 ml Dimethyl sulphoxide (DMSO) by sonication, plus 350 ml 2.8 M Sodium acetate buffer pH 5.0, mixed with a solution of 2 g Sodium dodecyl sulphate in 618 ml DMSO - 710 ml Water

Gradient: isocratic Flow rate: 1.0 ml/min Temperature: ambient

Conditions:

Volume: Detection: UV-VIS at 546 nm Substances: Tyramine, Histamine, Putrescine, Cadaverine, Tryptamine, Phenylethylamine Keywords: Amines, Histamine Chromatogram:

0 10 20 30 40 50 60 70 min

1

2

3

4

56

1. Tyramine 2. Histamine 3. Putrescine 4. Cadaverine 5. Tryptamine 6. Phenylethylamine

V 12/2006 35

11 Separation of β-carotene isomers

Method Matrix HPLC

Column: ProntoSIL 200-3 C30, 250 x 4.6 mm Order No. 25EH300PSG Phase: ProntoSIL 200-3 C30

Eluent: MeOH / TBME 80:20 (v/v) Gradient: isocratic Flow rate: 1.4 ml/min Temperature: 25 °C

Conditions:

Volume: 5 µl Detection: UV at 450 nm Substances: β-Carotene Keywords: Vitamins, fat soluble Chromatogram:

0 5 10 15 20 25 min

1

2

1. all-trans β-Carotene 2. 9-cis β-Carotene


12 Determination of diketopiperazine in soft drinks

Method Matrix HPLC soft drink


Eluent: 10 mM KH2PO4 - ACN (85:15, v/v), adjusted to pH 4.0 Gradient: isocratic Flow rate: 0.7 ml/min Temperature: 30 °C

Conditions:

Volume: 10 µl Detection: UV at 210 nm Substances: Diketopiperazine, Caffeine, Aspartame, Benzoate Keywords: Additives, Sweeteners Chromatogram:

0 10 20 30 min

1

2 34

1. DKP = diketopiperazine 2. Caf = caffeine 3. APM = aspartame 4. BA = Na benzoate

V 12/2006 37

13 Analysis of flavonoids in fruit juice

Method Matrix HPLC Beverage (orange juice sample)


Eluent: 5 mM Ammonium acetate / ACN 75:25 (pH 4,45; adjusted with conc. acetic acid)

Gradient: isocratic Flow rate: 1.0 ml/min Temperature: 40 °C

Conditions:

Volume: 5 µl Detection: UV at 285 nm Substances: Naringin, Hesperidin Keywords: Flavonoids Chromatogram:

0 5 9 min

1

2

1. Naringin (28.03 ppm) 2. Hesperidin (79.25 ppm)


14 Stilbene and flavonoids in red wine samples



Eluent: A: MeOH / H2O 50:50 (pH 3 with TFA) B: MeOH / H2O 80:20 (pH 3 with TFA) C: MeOH / H2O 20:80 (pH 3 with TFA)

Gradient: 0 – 5 min 90 % A 10 % C � 100 % A 5 – 8 min 100 % A � 70 % A 30 % B 8 - 12 min 70 % A 30 % B � 20 % A 80 % B 12 - 14 min 20 % A 80 % B� 100 % B 14 - 16 min 100 % B isocratic)


Conditions:

Volume: 20 µl Detection:

UV at different wavelength (Resveratrol: 305 nm, Myrcetine: 254/365nm, Quercetine: 365 nm, Caempferol: 265/365 nm)

Substances: trans-Resveratrol, cis-Resveratrol, Myrcetine, Quercetine, Caempferol Keywords: Stilbene, Flavonoids Chromatogram:

0 5 10 15

1

23

4

5

20 min

1. trans Resveratrol 2. Myrcetine 3. cis Resveratrol 4. Quercetine 5. Caempferol

V 12/2006 39

15 Analysis of flavonoid glycosides

Method Matrix HPLC


Eluent: ACN / H2O, adjusted to pH 2.5; with Phosphate buffer Gradient: 17 - 23 % ACN over 35 min Flow rate: 1.0 ml/min Temperature: 25 °C

Conditions:

Volume: Detection: UV at 280 nm Substances: Eriocitrin, Eriodictyol 7-O-glucoside, Luteolin 7-O-rutinoside, Narirutin, Hesperidin,

Isorhoifolin, Rosmarinic acid, Diosmin Keywords: Flavonoid glycosides Chromatogram:

0 5 10 15 20 25 30 min

24

3

5

6

7

8x3

x4x1x2

1

1. Eriocitrin 2. Eriodictyol 7-O-glucoside 3. Luteolin 7-O-rutinoside 4. Narirutin 5. Hesperidin 6. Isorhoifolin 7. Rosmarinic acid 8. Diosmin

X1-4 unknown


16 Separation of methylfurfural and hydroxyfurfural in beer



Eluent: H2O / ACN 90 : 10 (v/v) Gradient: isocratic Flow rate: 1.0 ml/min Temperature: 30°C

Conditions:

Volume: 20 µl Detection: UV at 277 nm Substances: Furfural, Hydroxymethylfurfural (HMF) Keywords: HMF Chromatogram:

0 5 10 min

HMF

FU

RF

UR

AL

0 5 10 min

HMF

FURFURAL

V 12/2006 41

17 Determination of mono- and disaccharides

Method Matrix HPLC

Column: Eurospher 100-5 NH2, 125 x 4 mm ID Order No. 12DE190ESJ Phase: Eurospher 100-5 NH2

Eluent: ACN / Water 75 / 25 v/v Gradient: Flow rate: 1.0 ml/min Temperature: 25 °C

Conditions:

Volume: 5 µl Detection: Refractive Index Substances: Fructose, Glucose, Lactose, Maltose, Saccharose Keywords: Saccharides Chromatogram:

0 5 10 15 min

1

23

4 5

1. Fructose 2. Glucose 3. Saccharose 4. Maltose 5. Lactose


18 Determination of deoxynivalenol (DON)

Method Matrix HPLC

Column: Eurospher 100-5 C18, 150 x 4 mm ID Order No. 15EE181ESJ Phase: Eurospher 100-5 C18

Eluent: Water - Acetonitrile (90:10) Gradient: isocratic Flow rate: 0.6 ml/min Temperature: 30 °C

Conditions:

Volume: 20 µl Detection: UV at 218 nm Substances: Deoxynivalenol (DON) Keywords: Mycotoxins, Trichotecenes Chromatogram:

0 5

0 5

10 min

10 min

1

a.)

b.)

a) blank wheat sample b) spiked wheat sample

V 12/2006 43

19 Determination of nivalenol (NIV) and deoxynivalenol (DON) with postcolumn derivatisation

Method Matrix HPLC fungus

Column: ProntoSIL 120-5 C18 AQ, 250 x 3 mm ID Order No. 25CF184PSJ Phase: ProntoSIL 120-5 C18 AQ

Eluent: A) ACN B) 0.01 M acetic acid Gradient: 0-16 min 10 % A

16-22 min 90 % A 22-40 min 10 % A

Flow rate: 0.4 ml/min

Conditions:

Temperature: 25 °C Volume: up to 200 µl

2 channel postcolumn derivatization, flow rate of 0.25 ml/min, temperature of 115 °C Reactor Coil 1 : 1.2 ml, 0.15 M sodium hydroxide Reactor Coil 2 : 1.6 ml, 0.03 M methyl acetoacetate and 2 M ammonium acetate

Detection: Fluorescence: ex at 360 nm, em at 470 nm Substances: Deoxynivalenol (DON), Nivalenol (NIV) Keywords: Mycotoxins, Trichothecens Chromatogram:

0

1

2

5 10 15 min

1. Nivalenol (NIV) 2. Deoxynivalenol (DON)


20 Analysis of fusarium mycotoxines

Method Matrix HPLC


Eluent: A) ACN B) Water/ACN (90:10, v/v) Gradient:

0 – 28 min 8 % A 28 – 32 min 20 % A 32 – 36 min 100 % A 36 – 40 min 99 % A 40 – 45 min 8 % A

Flow rate: 0 – 7 min 0.65 ml/min 7 – 40 min 0.75 ml/min 40 – 45 min 0.65 ml/min

Temperature: 25 °C

Conditions:

Volume: 10 µl Detection: DAD at 220 and 230 nm Substances: Nivalenol, Deoxynivalenol (DON), 15-o-Acetyl-4-deoxynivalenol, 3-

Acetyldeoxynivalenol Keywords: Fusarium mycotoxines Chromatogram:

0 5 10 15 min

1

2

3 4

1. Nivalenol (2.8 ppm) 2. Deoxynivalenol (2.9 ppm) 3. 15-o-Acetyl-4-deoxynivalenol (3.0 ppm) 4. 3-Acetyldeoxynivalenol (2.9 ppm)

V 12/2006 45

21 Ion chromatography for determination of NO2- and NO3- in beverages

Method Matrix HPLC

Column: Novosep A-2 Anion, 250 x 4 mm ID Order No. B92 Phase: Novosep A-2 Anion

Eluent: 3.6 mM Sodium carbonate (Na2CO3) Gradient: isocratic Flow rate: 0.4 ml/min

Conditions:

Temperature: 45 °C Volume: 5 µl, Chromatogram for a mixture of anions with concentration of 1 ppm Detection: suppressed Conductivity (Alltech Model 650), Range 1 µS Substances: Chloride (Cl

-), Nitrite (NO2

-), Nitrate (NO3

-)

Keywords: Anions Chromatogram:

1

23

0 5 10 15 20 min

1. Chloride 1 ppm 2. Nitrite 1 ppm 3. Nitrate 1 ppm


22 Determination of organic acids

Method Matrix HPLC

Column: ProntoSIL 120-3 C18 AQ, 300 x 3 mm ID Order No. 30CF184PSG Phase: ProntoSIL 120-3 C18 AQ

Eluent: 5 mM Li2SO4 / H2SO4 (pH 2.81) Gradient: isocratic Flow rate: 0.56 ml/min

Conditions:

Temperature: 20 °C Volume: 10 µl Detection: Refractive Index Substances: Acetic acid, Ascorbic acid, Citric acid, Fumaric acid, Lactic acid, Malic acid, Malonic

acid, Oxalic acid, Quinic acid, Shikimic acid, Succinic acid, Tartaric acid Keywords: Organic acids Chromatogram:

5 10 min

1

2

3

4

5 6

7

8

9

10

11

12 13

0

1. Oxalic acid 2. not identified 3. Tartaric acid 4. Quinic acid 5. Malic acid 6. Malonic acid 7. Shikimic acid 8. Lactic acid 9. Ascorbic acid

10. Acetic acid 11. Citric acid 12. Fumaric acid 13. Succinic acid

V 12/2006 47

23 Determination of organic acids II

Method Matrix HPLC

Column: Eurokat H, 300 x 8 mm ID Order No. 30GX340EKN Phase: Eurokat H (polymer phase)

Eluent: 0.01 N sulfuric acid Gradient: isocratic Flow rate: 0.4 ml/min Temperature: 75 °C

Conditions:

Volume: 20 µl Detection: Refractive Index Substances: Malic acid, Lactic acid, Quinic acid, Shikimic acid Keywords: Organic acids Chromatogram:

1

2

3

4

0 10 20 30 min

1. 1 Malic acid 2. Quinic acid 3. Shikimic acid 4. Lactic acid


24 Determination of patulin in apple juice

Method Matrix HPLC Apple juice Sample preparation:

extraction of apple juice with ethyl acetate (3x), extraction of the combined phases with Na2CO3 solution, extraction of the separated aqueous phase with ethyl acetate, addition of glacial acetic acid, reduction of ethyl acetate, redissolution of the dry residue in mobile phase


Eluent: ACN - Water (3 : 97, v/v) Gradient: isocratic Flow rate: 1 ml/min Temperature: 25 °C

Conditions:

Volume: 10 µl Detection: UV (DAD) at 275 nm Substances: Patulin Keywords: Mycotoxins Chromatogram:

0 5 10 15 min

Patolin

O

O

O

H

H

H

H

H

OH

Patulin

V 12/2006 49

25 Separation of preservatives

Method Matrix HPLC


Eluent: A: Buffer / Methanol (50:20), B: Buffer / Methanol (50:70) Buffer: 0.4 ml formic acid, 0.8 ml NH3 (25 %) filled with water to 1l

Gradient: 0 – 15 min: 100 % A to 100 % B 15 – 20 min: 100 % B


Conditions:

Volume: 20 µl Detection: UV at 235 nm and 255 nm Substances: Benzoic acicd, 4-Hydroxy-benzoic acid, p-Hydroxy methylbenzoate, p-Hydroxy

ethylbenzoate, p-Hydroxy propylbenzoate, p-Hydroxy butylbenzoate, 2-Methoxy benzoic acid, Sorbic acid

Keywords: Chromatogram:

0 5 10 15 20 min

1

2

3

4

56 7

8

1. 4-Hydroxy benzoic acid 2. 2-Methoxy benoic acid 3. Benzoic acid 4. Sorbic acid 5. p-Hydroxy methylbenzoate 6. p-Hydroxy ethylbenzoate 7. p-Hydroxy propylbenzoate 8. p-Hydroxy butylbenzoate

255 nm / 235 nm


26 Determination of various sterols

Method Matrix HPLC


Eluent: A: Water B: Acetonitrile/Methanol 99:1 (v/v) Gradient: Isocratic 10% A and 90% B Flow rate: 1.0 ml/min Temperature: 25 °C

Conditions:

Volume: 2 µl Detection: UV at 205 nm Substances: Ergosterol, Lanosterol, Cholesterol, Campesterol, Stigmasterol, Sitosterol Keywords: Steroids, steroles Chromatogram:

0 5 10 15 min

1

2

3

45

6

1. Ergosterol 2. Lanosterol 3. Cholesterol 4. Campesterol 5. Stigmasterol 6. Sitosterol

V 12/2006 51

27 Separation of water soluble vitamins

Method Matrix HPLC

Column: ProntoSIL 120-3-C18 AQ, 250 x 3 mm Order No. 25CF184PSG Phase: ProntoSIL 120-3-C18 AQ

Eluent: A) 50 mM H3PO4 in Water B) Acetonitrile Gradient: 0 – 3 min 100 % A

3 – 6.66 min 1000 – 70 % A 6.66 - min 70 % A, 7 min isocratic


Conditions:

Volume: 5 µl Detection: UV at 230 nm Substances: Pyridoxamine, Thiamine (Vit. B1), Ascorbic acid (Vit. C), Nicotinic acid (Niacin)

Nicotinamide (Vit. B3), Pyridoxale, Pyridoxine (Vit. B6), Folic acid, Cyanocobalamine (Vit. B12), Riboflavine (Vit. B2)

Keywords: Vitamins, water soluble vitamins Chromatogram:

0 5 10 min

12 3

4

5

6

78

9

10

1. Pyridoxamine 2. Thiamine (Vit. B1) 3. Ascorbic acid (Vit. C) 4. Nicotinic acid 5. Nicotinamide (Vit. B3) 6. Pyridoxale 7. Pyridoxine (Vit. B6) 8. Folic acid, 9. Cyanocobalamine (Vit. B12)

10. Riboflavine (Vit. B2)


28 Separation of tocopherols and vitamin D2

Method Matrix HPLC

Column: Eurospher 100-5 Si, 150 x 4 mm ID Order No. 15DE000ESJ Phase: Eurospher 100-5 Si

Eluent: Hexan / 2-Butanol (1000:4 v/v) Gradient: isocratic Flow rate: 2 ml/min Temperature: 25°C

Conditions:

Volume: 20 µl Detection: UV at 280 nm Substances: Tocopherols (alpha, beta, gamma, delta), Vitamin D2 Keywords: Vitamins, fat soluble Chromatogram:

0 5 10 15 20 25 min

1

23

45

6

1. alpha-Tocopherol 2. beta-Tocopherol 3. gamma-tocopherol 4. delta-Tocopherol 5. Vitamin D2 6. trans-Retinol

V 12/2006 53

29 Separation of fat soluble vitamins

Method Matrix HPLC

Column: ProntoSIL 120-3-C18 SH, 250 x 3 mm ID Order No. 25CF180PSG Phase: ProntoSIL 120-3-C18 SH

Eluent: Methanol Gradient: isocratic Flow rate: 1 ml/min Temperature: ambient

Conditions:

Volume: 5 µl (50 – 300 ppm each substance) Detection: Evaporated Light Scattering Detector (T: 33 °C) / UV at 280 nm Substances: Vitamin A acetate, Vitamin D3, Vitamin K1, Vitamin A alcohol, Vitamin D2, Vitamin E Keywords: Vitamins, fat soluble Chromatogram:

0 5 10 15 20 min

1 2

3 4

6

5

1. Vitamin A acetate 2. Vitamin D3 3. Vitamin K1 4. Vitamin A alcohol 5. Vitamin D2 6. Vitamin E

54

30 Fat soluble vitamins by HPLC with electrochemical detection

Method Matrix HPLC carrot juice

Column: Eurospher 100-5 C18, 250 x 4.6 mm ID Order No. 25EE181ESJ Sample Preparation:

vitamins in carrot juice, extracted with methanol

Phase: Eurospher 100-5 C18

Eluent: Methanol with 2 g/l LiClO4 and 1 g/l Acetic acid Gradient: isocratic Flow rate: 1.5 ml/min Temperature: 25°C

Conditions:

Volume: 10 µl (20 ng of each standard) Detection:

electrochemical, glassy carbon working electrode, left +1000 mV, right +800 mV

Substances: Vitamin A acetate, α-Tocopherol, β-Carotene Keywords: Vitamins Chromatogram:

0 5 10

2

3

1

15 min

1. Vitamin A acetate 2. α-Tocopherol 3. β-Carotene

Pharmaceutical Applications

Page

Quality control mixture of acetyl cystein 56

Separation and determination of opium alkaloids 57

HPLC method for the determination of amineptine and its main metabolite in human plasma 58

Determination of amino acids with OPA 59

Separation of aniline derivatives with electrochemical detection 60

Separation of anilines and sulfonamides by HPLC with electrochemical detection 61

Separation of trimethoprim and sulfamethoxazol 62

Determination of anticonvulsant drugs and methyl xanthine derivatives in serum 63

Separation of antiepileptics in serum 64

Separation of antirheumatic drugs 65

Separation of catecholamines 66

Clinical analysis of chloramphenicol 67

Determination of citrate and oxalate in human urine 68

Determination of guaifenesin, codein and pseudoefedrin in cough syrup 69

Isocratic separation of the digoxin and digitoxin series of glycosides 70

Separation of Narcotics 71

Analysis of β-lactam antibiotic (Moxalactam) 72

Analysis of bisphosphonate pamidronate disodium by precolumn derivatization and fluorescence detection 73

Determination of paracetamol and by-products 74

Separation of penicillin antibiotics 75

Separation of peptides 76

Fast separation of proteins 77

Separation of sedativa 78

Separation of steroids 79

Separation of steroids II 80

Separation of sulfa drugs 81

Separation of sulfa drugs II 82

Separation of tricyclic antidepressant drugs 83

Separation of D,L-tryptophan and aromatic acids 84

56 Pharmaceutical Applications

1 Quality control mixture of acetyl cystein

Method Matrix HPLC


Eluent: 1.6 g Heptansulfonic acid in 960 ml H2O (pH with H3PO4 85%) and 40 ml MeOH

Gradient: isocratic Flow rate: 0.6 ml/min Temperature: 25°C

Conditions:

Volume: 10 µl Detection: UV at 240 nm (Ascorbic acid); UV at 216 nm Substances: Ascorbic acid, Citric acid, Adipic acid, Acetyl cystein (ACC), Cystein Keywords: Organic acids, Cystein, ACC Chromatogram:

0 10 20 30 min

1

23

45

1. Ascorbic acid 2. Citric acid 3. Adipic acid 4. Acetyl cystein 5. Cystein

V 12/2006 57

2 Separation and determination of opium alkaloids

Method Matrix HPLC opium

Column: Eurospher 100-5 C18, 250 x 4mm ID Order No. 25DE181ESJ Phase: Eurospher 100-5 C18

Eluent: 1% Ammonium acetate pH 5.8 - Acetonitrile (65:35 v/v) Gradient: isocratic Flow rate: 1.5 ml/min Temperature: 25 °C

Conditions:

Volume: 20 µl Detection: UV at 254 nm Substances: Morphine, Codeine, Cryptopine, Thebaine, Papaverine, Narcotine Keywords: Alkaloids Chromatogram:

0 5 10 15 min

1

2

3

4

5

6

1. Morphine 2. Codeine 3. Cryptopine 4. Thebaine 5. Papaverine 6. Narcotine


3 HPLC method for the determination of amineptine and its main metabolite in human plasma

Method Matrix HPLC plasma


Eluent: Aqueous phase containing 1.2 g/l heptane sulphonate adjusted to pH 3.0 with H3PO4 - Acetonitrile (62:38, v/v)

Gradient: isocratic Flow rate: 1 ml/min Temperature: 30 °C

Conditions:

Volume: 20 µl Detection: UV at 220 nm Substances: Amineptine Keywords: Drugs, Antidepressant drugs Chromatogram:

505 1005 10

32

1

32

1

0 10 min

Chromatograms of extracts from a) blank plasma b) plasma spiked with:

1. 250 ng/ml metabolite 2. 250 ng/ml amineptine 3. 500 ng/ml internal standard

c) a patient plasma sample

obtained 45 min after taking a 100 mg oral dose of amineptine, spiked with

1. 230 ng/ml metabolite 2. 180 ng/ml amineptine 3. 500 ng/ml internal standard

a b c

V 12/2006 59

4 Determination of amino acids with OPA

Method Matrix HPLC

Column: ProntoSIL 120-3-C18 H, 250 x 3.0 mm ID Order No. 25CF185PSG Phase: ProntoSIL 120-3-C18 H Sample Preparation:

Amino acid standard with OPA precolumn derivatization

Eluent: A: 20 mM CH3COONa in H2O / ACN, (97:3 v/v)

B: 20 mM CH3COONa in H2O / ACN, (50:50 v/v) Gradient: 5 – 28 % B, 0 – 144 s

28 – 45 % B, 145 – 560 s 45 – 82 % B, 561 – 896 s 82 – 90 % B, 897 – 1215 s


Conditions:


Fluorescence: excitation 330 nm emission 450 nm UV at 330 nm

Substances: Aspartic acid, Glutamic acid, Serine, Glutamin, Histidine, Glycine, Threonine, Arginine,

Alanine, Tyrosine, Methionine, Valine, Phenylalanine, Isoleucine, Leucine, Lysine Keywords: Amino acids Chromatogram:

0 5 10 15 20 min

1

23

4

5

6

7

89

1011

1213

1415

16

1. Asp 2. Glu 3. Ser 4. Gly 5. Thr 6. His 7. Ala 8. Arg 9. Tyr

10. Val 11. Cys 12. Met 13. Ile 14. Phe 15. Leu 16. Lys


5 Separation of aniline derivatives with electrochemical detection

Method Matrix HPLC


Eluent: Methanol - Water (1:1) with 2 g/l KNO3 and 0.05 g/l H2SO4 Gradient: isocratic Flow rate: 0.7 ml/min Temperature: ambient

Conditions:

Volume: 20 µl Detection: Amperometric detection, glassy carbon 1000 mV / 800 mV, 100 nA Substances: Phenylene diamine, Chloroaniline Keywords: Amines Chromatogram:

0 4 8 min

1

2

1. p-Phenylene diamine (20 ng) 2. 3-Chloroaniline (25 ng)

V 12/2006 61

6 Separation of anilines and sulfonamides by HPLC with electrochemical detection

Method Matrix HPLC


Eluent: Methanol - Water (1:1) with 2 g/l KNO3 and 0.05 g/l sulfuric acid Gradient: Flow rate: 0.7 ml/min Temperature: 25 °C

Conditions:


Amperometric detection, glassy carbon electrode, range 100 nA +1000 mV for o-Toluidine / 1200 mV for sulfonamides,

Substances: Toluidine-o, Sulfanilamide, Sulfamethoxydiazine Keywords: Drugs, Antibiotics Chromatogram:

0 4 8 min 0 4 8 min

1

2

3

1. o-Toluidine 2. Sulfanilamide 3. Sulfmethoxydiazone

Upot = 1000 mV Upot = 1200 mV


7 Separation of trimethoprim and sulfamethoxazol

Method Matrix HPLC

Column: ProntoSIL 120-5 C18 SH, 250 x 4 mm ID Order No. 25DF180PSJ Phase: ProntoSIL 120-5 C18 SH

Eluent: Methanol / Water with 1% formic acid 15:85 (v/v) Gradient: isocratic Flow rate: 1.0 ml/min Temperature: 30°C

Conditions:

Volume: 10 µl Detection: UV at 220 nm Substances: Trimethoprim, Sulfamethoxazol Keywords: Drugs, antibiotic Chromatogram:

1

2

0 5 10 15 20 min

1 Trimethoprim 2. Sulfamethoxazol

V 12/2006 63

8 Determination of anticonvulsant drugs and methyl xanthine derivatives in serum

Method Matrix HPLC serum Sample preparation:

Precolumn loading and washing with Eluent A


Eluent: A: 14 mM NaH2PO4 - 6mM Na2HPO4

B: Eluent A : Acetonitrile : Methanol (6.5 : 1.5 : 2, v/v) Gradient: isocratic Flow rate: 0.8 ml/min Temperature: 25 °C

Conditions:

Volume: 20 µl Detection: UV at 230 nm Substances: Primidone, Phenobarbital, Phenytoin, Carbamazepine Keywords: Drugs, Anticonvulsant drugs Chromatogram:

0 10 20 30 min

12 3 4

1. Primidone (10 µg/ml) 2. Phenobarbital (10 µg/ml 3. Phenytoin (20 µg/ml) 4. Carbamazepine (5 µg/ml)


9 Separation of antiepileptics in serum

Method Matrix HPLC

Column: Eurospher 100-5-C18, 100 x 4.0 mm ID Order No. 10DE181ESJ Phase: Eurospher 100-5-C18

Eluent: Recipe eluent: antiepileptics in serum (Order no. 15010) Gradient: isocratic Flow rate: 1 ml/min Temperature: 55 °C

Conditions:

Volume: 10 µl Detection: UV at 205 nm Substances: PEMA, Ethosuximide, Primidone, Carbamazepine-diol, 10-OH-Carbamazepine,

Phenobarbital, Carbamazepine-epoxide, Oxcarbazepine, DPH, Carbamazepine Keywords: Antiepileptics, Drugs Chromatogram:

0 10 20 30 40 min

1

2

3

4

5

6

7

8

9

10 11

1. PEMA 2. Ethosuximide 3. Primidone 4. Carbamazepine-diol 5. 10-OH-Carbamazepine 6. Phenobarbital 7. Carbamazepine-epoxide 8. Oxcarbazepine 9. Internal Standard (IS)

10. DPH 11. Carbamazepine

V 12/2006 65

10 Separation of antirheumatic drugs

Method Matrix HPLC


Eluent: Methanol - Water (70:30, v/v), 0.1 % acetic acid with 6 g/l ammonium acetate and 1.5 g/l sulfuric acid


Conditions:

Volume: 20 µl Detection: UV at 254 nm Substances: Camphor, Ethylenglycolmonosalicylate, Nicotinic acid methyl ester, Nicotinic acid

benzyl ester Keywords: Antirheumatic drugs Chromatogram:

0 5 10 min

1

2

3

4

1. Camphor 2. Nicotinic acid methyl ester 3. Nicotinic acid benzyl ester 4. Ethylenglycolmonosalicylate


11 Separation of catecholamines

Method Matrix HPLC


Eluent: prepared standard eluents “Urinary Catecholamines” Gradient: isocratic Flow rate: 1.4 ml/min Temperature: 40 °C

Conditions:

Volume: 10 µl Detection: Amperometric detection, Cell potential 0.55 V, 10 nA/V, glassy carbon electrode Substances: Barbital, Luminal, Prominal, Revinal Keywords: Catecholamines Chromatogram:

0 10 20 30 min

1

2

3

4

1. Noradrenaline 2. Adrenaline 3. Dihydroxybenzylamine (IS) 4. Dopamine

V 12/2006 67

12 Clinical analysis of chloramphenicol

Method Matrix HPLC serum

Column: Eurospher 100-5 C18, 100 x 2mm ID Order No. 10BE181ESJ Phase: Eurospher 100-5 C18

Eluent: 0.1 N Acetate buffer (pH 6.0) - Acetonitrile (8:2, v/v) Gradient: isocratic Flow rate: 0.2 ml/min Temperature: ambient

Conditions:

Volume: 0.5 µl Detection: UV at 254 nm Substances: Chloramphenicol Keywords: Drugs, Antibacterial drugs Chromatogram:

1

2

1

2

0 5 10 0 5 10 15 min

(a) 10 mg/l Chloramphenicol (b) 25mg/l Chloramphenicol 1. Chloramphenicol 2. Internal Standard

(a) (b)


13 Determination of citrate and oxalate in human urine

Method Matrix HPLC urine

Column: Novosep A-2 Anion (5µ), 250 x 4 mm ID Order No. B92 Phase: Novosep A-2 Anion

Eluent: 15 mM Sodium Carbonate (Na2CO3) Gradient: isocratic Flow rate: 1.0ml/min Temperature: 50 °C

Conditions:

Volume: 5 µl, urine sample 1:20 (blue) Detection: suppressed Conductivity (Alltech Model 650), Range 5 µS Substances: Citrate, Oxalate Keywords: Organic Anions Chromatogram:

0 5 10 15 20 min

1

2

3

45

1. Chloride 2. Phosphate 3. Sulfate 4. Oxalate 5. Citrate

V 12/2006 69

14 Determination of guaifenesin, codein and pseudoefedrin in cough syrup

Method Matrix HPLC cough syrup

Column: ProntoSIL 120-3 C18 SH, 250 x 4 mm ID Order No. 25EK185PSJ 25EK185PSJ Phase: ProntoSIL 120-3 C18 SH

Eluent: Water/Methanol 70:30 with 0.1 % TFA (pH 1.87) Gradient: isocratic Flow rate: 0.8 ml/min Temperature: 40 °C

Conditions:

Volume: 5 µl Detection: UV at 210 nm Substances: Codein, Guaifenesin, Pseudoefedrin Keywords: Codein, Guaifenesin, Pseudoefedrin Chromatogram:

0 5 10 15 20 min

1

2

3

n.i.

1. Codein 2. Pseudoefedrin 3. Guaifenesin


15 Isocratic separation of the digoxin and digitoxin series of glycosides

Method Matrix HPLC

Column: Eurospher 100-5 C18, 250 x 4mm ID Order No. 25DE181ESJ Phase: Eurospher 100-5 C18

Eluent: Acetonitrile - Methanol - Water (30:30:40) Gradient: isocratic Flow rate: 1ml/min Temperature: 30 °C

Conditions:

Volume: 10 µl Detection: UV at 220 nm Substances: Digoxigenin, Digoxigenin monodigitoxoside, Digoxigenin bisdigitoxoside, Digoxin,

Digitoxigenin, Digitoxigenin monodigitoxoside, Digitoxigenin bisdigitoxoside, Digitoxin Keywords: Drugs, Glycosides, Digitalis Glycosides Chromatogram:

0 5 10 15

8

7

6

5

4

3

2

1

20 min

1. Digoxigenin 2. Digoxigenin monodigitoxoside 3. Digoxigenin bisdigitoxoside 4. Digoxin 5. Digitoxigenin 6. Digitoxigenin monodigitoxoside 7. Digitoxigenin bisdigitoxoside 8. Digitoxin

V 12/2006 71

16 Separation of Narcotics

Method Matrix HPLC

Column: Eurospher 100-3 C18, 150 x 4 mm ID Order No. 15DE181ESG Phase: Eurospher 100-3 C18

Eluent: Water - ACN (45:55, v/v) Gradient: isocratic Flow rate: 1 ml/min, Temperature: 40 °C

Conditions:

Volume: 2 µl Detection: UV at 230 nm Substances: Veronal, Luminal, Prominal, Revonal, Thiogenal Keywords: Drugs, Narcotics Chromatogram:

0 5 10 min

12 3

4

1. Barbital 2. Luminal 3. Prominal 4. Revinal


17 Analysis of β-lactam antibiotic (Moxalactam)

Method Matrix HPLC


Eluent: Methanol - 0.05 M Kalium phosphate (5:95) adjusted to pH 6.5 Gradient: isocratic Flow rate: 2.0 ml/min Temperature: 20°C

Conditions:

Volume: 10 µl Detection: UV at 254 nm Substances: Moxalactam, Methyl-1H-tetrazole-5-thiol Keywords: Drugs, Antibiotics Chromatogram:

0 10 20 30 min

1

2

3

1. 1-Methyl-1H-tetrazole-5-thiol 2. R-Moxalactam 3. S-Moxalactam

V 12/2006 73

18 Analysis of bisphosphonate pamidronate disodium by precolumn derivatization and fluorescence detection

Method Matrix HPLC urine


Eluent: A) 1 mM aqueous Na2EDTA, adjusted to pH 6.5 with 1 M NaOH B) Methanol (97:3, v/v)

Gradient: isocratic Flow rate: 1 ml/min Temperature: ambient

Conditions:

Volume: 20µl Detection: Fluorescence: excitation 395 nm, emission 480 nm

a. drug-free human urine b. drug-free dog urine c. urine from a dog receiving a single intravenous injection of 5 mg of pamidronate

disodium in 0.15 M saline Substances: Pamidronate disodium,

Disodium 3-amino-1-hydroxypropylidenebisphosphonate pentahydrate (IS) Keywords: Drugs Chromatogram:

0 5 10 0 5 1015 min 15 min 0 5 10 15 min

1 1 1

2

3

1. Injection Peak 2. Pamidronate 3. Internal Standard (IS)

a) b) c)


19 Determination of paracetamol and by-products

Method Matrix HPLC

Column: ProntoSIL 120-5 C8 ace-EPS, 250 x 3 mm ID Order No. 25CF08APSJ Phase: ProntoSIL 120-5 C8 ace-EPS

Eluent: A: ACN B: H2O (pH 2.75 with H3PO4) Gradient: 0 – 0.65 min 10 % A

0.65 – 6.4 min 10 � 60 % A 6.4 – 10.4 min 60 % A 10.4 - 12 min 10 % A

Flow rate: 1.3 ml/min Temperature: 50°C

Conditions:

Volume: 5µl Detection: UV at 245 nm Substances: Paracetamol, N-(2-hydroxyphenyl)acetamide, N-(4-hydroxyphenyl)propanamide,

N-phenylacetamide, Chloracetanilide, 1-(4-hydroxyphenyl)ethanone oxime, 1-(2-hydroxyphenyl)ethanone, 4-Aminophenol, 4-Nitrophenol

Keywords: Paracetamol Chromatogram:

0 2.5 5 7.5 min

1

2

3

4

56 7

8

9

1. 4-Aminophenol 2. Paracetamol 3. N-(2-hydroxyphenyl)acetamide 4. N-(4-hydroxyphenyl)propanamide 5. N-phenylacetamide 6. 1-(4-hydroxyphenyl)ethanone oxime 7. 1-(2-hydroxyphenyl)ethanone 8. Chloracetanilide 9. 4-Nitrophenol

V 12/2006 75

20 Separation of penicillin antibiotics

Method Matrix HPLC

Column: Eurospher 100-5 C18, 250 x 4 mm ID Order No. 25DE181ESJ 25DE181ESJ Phase: Eurospher 100-5 C18

Eluent: ACN / H2O (0.1 % TFA) 50 : 50 (v/v) Gradient: isocratic Flow rate: 1.8 ml/min Temperature: 25 °C

Conditions:

Volume: 1µl Detection: UV at 254 nm Substances: Ampicillin, Penicillin G, Penicillin V, Nafcillin, Dicloxacillin Keywords: Drugs, Antibiotics, Penicillines Chromatogram:

0 5 10 min

1

2

3

4

5

1. Ampicillin 2. Penicillin G 3. Penicillin V 4. Nafcillin 5. Dicloxacillin


21 Separation of peptides

Method Matrix HPLC

Column: ProntoSIL 300-5 C18 SH, 250 x 4.6 mm ID Order No. 25EK185PSJ Phase: ProntoSIL 300-5 C18 SH

Eluent: A) H2O / 0.1 % TFA B) ACN / H2O (70 / 30, v/v) + 0.1 % TFA Gradient: 20-100% B in 30 min Flow rate: 1.5 ml/min Temperature: 30 °C

Conditions:

Volume: 5 µl Detection: UV at 220 nm Substances: Angiotensin I und II, Bradykinin, Eledoisin, Neurotensin, Oxytocin Keywords: Peptides Chromatogram:

0 5 10 15 20 min

1

2

3

4

5

1. Oxytocin 2. Bradykinin 3. Angiotensin II 4. Eledoisin 5. Neurotensin 6. Angiotensin I

V 12/2006 77

22 Fast separation of proteins

Method Matrix HPLC

Column: ProntoSIL 300-3 C18 SH, 75 x 4.6 mm ID Order No. 0746K185PS030 (*B) Phase: ProntoSIL 300-3 C18 SH

Eluent: A) H2O / 0.1 % TFA B) ACN / H2O (95 : 5 v/v) + 0.1 % TFA Gradient: 0-25 s 29 - 48% B

25-65 s 48 - 100% B Flow rate: 4.0 ml/min Temperature: 30 °C

Conditions:

Volume: 5 µl Detection: UV at 220 nm Substances: BSA, Insulin bovine, Lysozyme, Myoglobin, Ovalbumin, Ribonuclease A Keywords: Proteins Chromatogram:

1

2

3

4

5

6

7

0 1 2 min

1. Ribonuclease A 2. Insulin bovine 3. Lysozyme 4. BSA 5. Myoglobin 6. Ovalbumin 7. Not identified


23 Separation of sedativa

Method Matrix HPLC

Column: Eurospher 100-5 C18, 250 x 4.6 mm Order No. 25EE181ESJ Phase: Eurospher 100-5 C18

Eluent: A) Methanol B) Water (60/40 v/v) Gradient: isocratic Flow rate: 0.5 ml/min Temperature: ambient

Conditions:

Volume: 10 µl Detection: UV at 220 nm Substances: Barbital, Luminal, Prominal, Revonal, Thiogenal Keywords: Sedativa Chromatogram:

0 5 10 15 20 min

1

2

3

4

5

1. Barbital 2. Luminal 3. Prominal 4. Revonal 5. Thiogenal

V 12/2006 79

24 Separation of steroids

Method Matrix HPLC


Eluent: A) Methanol B) Water Gradient: 0 - 35 min 50 % � 95 % A

35 – 45 min 95 % � 50 % A Flow rate: 1ml/min Temperature: 30 °C

Conditions:

Volume: 10 µl Detection: UV at 240 nm Substances: Cortisone, Corticosterone, Desoxycorticosterone, Testosterone, Norgestrel,

Progesterone, Keywords: Steroids Chromatogram:

1

2

3

4

5 6

0 10 20 30 min

1. Cortisone 2. Corticosterone 3. Desoxycorticosterone 4. Testosterone 5. Norgestrel 6. Progesterone


25 Separation of steroids II

Method Matrix HPLC

Column: ProntoSIL 120A-5 C8 SH, 125 x 4 mm ID Order No. 12DF08PSJ Phase: ProntoSIL 120A-5 C8 SH

Eluent: A) H2O B) MeOH Gradient: 80% A (1 min hold), 65% A in 10 min, 65% A (3 min),

40 % A in 6 min, 40 % A 4 min hold Flow rate: 1 ml/min Temperature: 30°C

Conditions:

Volume: 10 µl Detection: UV at 230 nm Substances: Esteriol, Prednisolone, Cortisone, Testosterone, Methyl-6α-hydroxy-11β-progesterone,

Methyl-6α-hydroxy-17α-progesterone, Methyl-6α-hydroxy-17α-progesterone acetate, Estradiol, Estrone, Progesterone

Keywords: Steroids Chromatogram:

0 5 10 15 20 min

1

2

34

5

6

7

8

9

10

1. Esteriol 2. Prednisolone 3. Cortisone 4. Testosterone

5. Methyl-6α-hydroxy-11β-progesterone 6. Estradiol

7. Methyl-6α-hydroxy-17α-progesterone

8. Methyl-6α-hydroxy-17α-progesterone acetate 9. Estrone

10. Progesterone

V 12/2006 81

26 Separation of sulfa drugs

Method Matrix HPLC

Column: ProntoSIL 120-5 C18 ace-EPS, 150 x 4.6 mm ID Order No. 15CE181ESG Phase: ProntoSIL 120-5 C18 ace-EPS

Eluent: Methanol - Water – Acetic Acid (20:79:1, v/v/v) with 6 g/l ammonium acetate and 1.5 g/l sulfuric acid

Gradient: isocratic Flow rate: 1 ml/min Temperature: 25 °C

Conditions:

Volume: 3 µl Detection: UV at 254 nm Substances: Sulfanilamide, Sulfadiazine, Sulfathiazole, Sulfamerazine, Sulfamethazine Keywords: Sulfa drugs Chromatogram:

0 5 10 min

1

2

3

4 5

1. Sulfanilamide 2. Sulfadiazine 3. Sulfathiazole 4. Sulfamerazine 5. Sulfamethazine


27 Separation of sulfa drugs II

Method Matrix HPLC

Column: Eurospher 100-3 C18, 150 x 3 mm ID Order No. 15CE181ESG Phase: Eurospher 100-3 C18

Eluent: A: ACN B: 0.2 g NaH2PO4 in 1000 ml H2O (pH 4 with H3PO4)

Gradient: 22 % A 0 – 3.5 min 30 % A 3.5 – 4.5 min 90 % A 4.5 – 6 min, 2 min hold

Flow rate: 0 - 3.5 min 0.2 ml/min, 3.5 - 10 min 0.4 ml/min Temperature: 40 °C

Conditions:

Volume: 10 µl Detection: UV at 265 nm Substances: Sulfadiazine, Sulfamethiazole, Sulfamethoxypyridazine, Sulfamethoxazole,

Sulfadimethoxine Keywords: Sulfa drugs, antibiotics Chromatogram:

1 5 10 min

1

2 3

4

5

1. Sulfadiazine 2. Sulfamethiazole 3. Sulfamethoxypyridazine 4. Sulfamethoxazole 5. Sulfadimethoxine

V 12/2006 83

28 Separation of tricyclic antidepressant drugs

Method Matrix HPLC

Column: ProntoSIL 120-3-C18 AQ, 60 x 4.0 mm ID Order No. 06DF184PSG Phase: ProntoSIL 120-3-C18 AQ

Eluent: ACN / MeOH / 10 mM Phosphate Buffer (62:13:25, v/v/v) Gradient: isocratic Flow rate: 0.7 ml/min Temperature: 20 °C

Conditions:

Volume: 5 µl Detection: UV at 254 nm Substances: Amitriptyline, Doxepin, Imipramine, Nortiptyline, Trimipramine Keywords: Antidepressant, Drugs Chromatogram:

0 5 10 15 min

1

2

3

45

1. Doxepin 2. Imipramin 3. Nortriptyline 4. Amitriptyline 5. Trimipramine

84

29 Separation of D,L-tryptophan and aromatic acids

Method Matrix HPLC


Eluent: Methanol - Water (30:70, v/v) with 6 g/l ammonium acetate and 1.5 g/l sulphuric acid


Conditions:

Volume: 10 µl (40 ng each) Detection: Amperometric detection, +1000 mV / 800 mV, range 100 nA Substances: Dihydroxyphenylacetic acid, Tryptophan, Homovanillic acid Keywords: Biogenic amines, aromatic acids Chromatogram:

0 5 10 15 20 min

1

2

3

1. 3,4-Dihydroxyphenylacetic acid, 2. D,L-tryptophan, 3. Homovanillic acid

Chiral Applications

Page

Chiral separation of abscisic acid in reversed phase mode 86

Chiral separation of 3-amino-2(2-naphtyl)-1-phenyl-propanol in normal phase mode 87

Chiral separation of atenolol in normal phase mode 88

Chiral separation of atropine in normal phase mode 89

Chiral separation of 1-aza-[6]-helicene in normal phase mode 90

Chiral separation of 1-benzene-2-naphthene-3-amino propan(1)ol in normal phase mode 91

Chiral separation of benzoin 92

Chiral separation of 4-bromphenyl-glycinamid in normal phase mode 93

Chiral separation of carbinoxamine in normal phase mode 94

Chiral separation of dilactid in normal phase mode 95

Chiral separation of 1,2-diphenyl-2-(tosylamino)ethanone in normal phase mode96

Chiral separation of etozolin in polar organic mode 97

Chiral separation of flavanone in normal phase mode 98

Chiral separation of hexahydro-6,6-dimethyl-2-tosylpyrrolo[1,2-e]imidazol-3-one in normal phase mode 99

Chiral separation of hydroxyzine in normal phase mode 100

Chiral separation of n-methylephedrin in normal phase mode 101

Chiral separation of metoprolol in normal phase mode 102

Chiral separation of 1-(naphthalene-6-yl)ethane-1,2-diol in normal phase mode103

Chiral separation of 1-phenylethane-1,2-diol in normal phase mode 104

Chiral separation of 1-phenylpropane-1,2-diol in normal phase mode 105

Chiral separation of pindolol in reversed phase mode 106

Chiral separation of pindolol in polar organic mode 107

Chiral separation of propranolol in reversed phase mode 108

Chiral separation of propranolol in normal phase mode 109

86 Chiral Applications

1 Chiral separation of abscisic acid in reversed phase mode

Method Matrix HPLC

Column: Eurocel 01 5 µm, 250 x 4.6 mm ID Order No. 25DM370ECJ Phase: Eurocel 01

Eluent: Methanol / Water 50:50 + 0.1 TFA Gradient: isocratic Flow rate: 1 ml/min Temperature:

Conditions:

Volume: 5 µl Detection: UV at 230 nm Substances: Abscisic acid Keywords: Plant hormone Chromatogram:

0 5 10 15 20 min

k´1 = 2,67

k´2 = 3,19

α = 1.19

V 12/2006 87

2 Chiral separation of 3-amino-2(2-naphtyl)-1-phenyl-propanol in normal phase mode

Method Matrix HPLC


Eluent: Heptane/ Isopropanol 90:10 + 0.1 TFA Gradient: isocratic Flow rate: 1.0 ml/min Temperature:

Conditions:

Volume: 10 µl Detection: UV at 230 nm Substances: 3-Amino-2(2-naphtyl)-1-phenylpropanol Keywords: Chromatogram:

0 5 10 15 20 min

k´1 = 2,46

k´2 = 3,13

α = 1.27

OH

NH2


3 Chiral separation of atenolol in normal phase mode

Method Matrix HPLC

Column: Eurocel 01 5µm, 250 x 4.6 mm Order No. 25DM370ECJ Phase: Eurocel 01

Eluent: n-Heptane / IPA / EtOA (80:20:0.1) Gradient: isocratic Flow rate: 1 ml/min Temperature: 25°C

Conditions:

Volume: Detection: UV at 230 nm Substances: Atenolol (D,L) Keywords: Chromatogram:

0 10 20 30 min

k´1 = 6.53

k´2 = 8.17

α = 1.25

V 12/2006 89

4 Chiral separation of atropine in normal phase mode

Method Matrix HPLC


Eluent: n-Heptane / IPA / ETOA (80:20:0.1) Gradient: isocratic Flow rate: 1 ml/min Temperature: 25°C

Conditions:

Volume: Detection: UV at 218 nm Substances: Atropine Keywords: Chromatogram:

0 5 10 15 20 min

k´1 = 2.35

k´2 = 2.79

α = 1.18


5 Chiral separation of 1-aza-[6]-helicene in normal phase mode

Method Matrix HPLC


Eluent: Heptane/ Isopropanol 3:1 Gradient: isocratic Flow rate: 0.8 ml/min Temperature: ambient

Conditions:

Volume: 20 µl Detection: UV at 254 nm Substances: 1-aza-[6]helicene Keywords: Chromatogram:

0 5 10 15 min

k´1 = 1.28

k´2 = 1.59

α = 1.24

V 12/2006 91

6 Chiral separation of 1-benzene-2-naphthene-3-amino propan(1)ol in normal phase mode

Method Matrix HPLC Sample preparation:

Column: Eurocel 01 5 µm 250 x 4.6 mm ID Order No. 25EM370ECJ

Phase: Eurocel 01

Eluent: Heptane/ Isopropanol 90:10 + 0.1 TFA Gradient: isocratic Flow rate: 1 ml/min Temperature: ambient

Conditions:

Volume: 10 µl Detection: UV at 230 nm Substances: 1-Benzene-2-naphthene-3-amino propan(1)ol Keywords: Chromatogram:

OH

NH2

0 10 20 30 min

k´1 = 2,46

k´2 = 3,13

α = 1.27


7 Chiral separation of benzoin



Phase: Eurocel 01

Eluent: n-Hexane / IPA 90:10 Gradient: isocratic Flow rate: 1 ml/min Temperature: ambient

Conditions:

Volume: 10 µl Detection: UV at 254 nm Substances: Benzoin Keywords: Benzoin Chromatogram:

O

OH

0 5 10 15 20 min

k1´ = 7.18 k2´ = 11.33 α = 1.58

V 12/2006 93

8 Chiral separation of 4-bromphenyl-glycinamid in normal phase mode

Method Matrix HPLC


Eluent: n-Heptane / IPA / DEA (90:10:0.1) Gradient: isocratic Flow rate: 1 ml/min Temperature: ambient

Conditions:

Volume: 10 µl Detection: UV at 230 nm Substances: 4-Bromphenyl-glycinamid Keywords: Chromatogram:

0 5 10 15 min

k´1 = 3,09

k´2 = 4,54

α = 1.47

Br

NH2

O NH2


9 Chiral separation of carbinoxamine in normal phase mode

Method Matrix HPLC


Eluent: n-Heptane / IPA / DEA (90:10:0.1) Gradient: isocratic Flow rate: 1 ml/min Temperature: 25°C

Conditions:

Volume: Detection: UV at 230 nm Substances: Carbinoxamine (D,L) Keywords: Chromatogram:

0 5 10 min

k´1 = 1.69

k´2 = 1.94

α = 1.14

V 12/2006 95

10 Chiral separation of dilactid in normal phase mode

Method Matrix HPLC


Eluent: n-Hexane / Ethanol (90:10) Gradient: isocratic Flow rate: 1 ml/min Temperature: 25°C

Conditions:

Volume: Detection: UV at 220 nm Substances: Dilactid (D-, L-, meso) Keywords: Chromatogram:

0 5 10 15 min

k´1 = 2.01

k´2 = 2.22

α = 1.10


11 Chiral separation of 1,2-diphenyl-2-(tosylamino)ethanone in normal phase mode



Phase: Eurocel 01

Eluent: n-Hexane / IPA 90:10 Gradient: isocratic Flow rate: 1 ml/min Temperature: 25°C

Conditions:

Volume: 10 µl Detection: UV at 254 nm Substances: 1,2-diphenyl-2-(tosylamino)ethanone Keywords: Chromatogram:

OHN

Tos

1,2-diphenyl-2-(tosylamino)ethanone

0 10 20 30 40 min

k´1 = 7.66

k´2 = 8.66

α = 1.13

V 12/2006 97

12 Chiral separation of etozolin in polar organic mode

Method Matrix HPLC


Eluent: MeOH Gradient: isocratic Flow rate: 1 ml/min Temperature: 25°C

Conditions:

Volume: Detection: UV Substances: Etozolin (D,L) Keywords: Chromatogram:

0 3 6 9

k´1 = 0.68

k´2 = 1.05

α = 1.54


13 Chiral separation of flavanone in normal phase mode

Method Matrix HPLC


Eluent: n-Heptane / IPA (9:1) Gradient: isocratic Flow rate: 1 ml/min Temperature: 25°C

Conditions:

Volume: Detection: UV at 254 nm Substances: Flavanone (D,L) Keywords: Chromatogram:

0 3 6 9 12 15 min

k´1 = 1.41

k´2 = 1.83

α = 1.30

V 12/2006 99

14 Chiral separation of hexahydro-6,6-dimethyl-2-tosylpyrrolo[1,2-e]imidazol-3-one in normal phase mode



Phase: Eurocel 01

Eluent: n-Hexan / IPA (75:25) Gradient: isocratic Flow rate: 0.5 ml/min Temperature: 25°C

Conditions:

Volume: Detection: UV at 220 nm Substances: hexahydro-6,6-dimethyl-2-tosylpyrrolo[1,2-e]imidazol-3-one Keywords: Chromatogram:

NN

O

Tos

hexahydro-6,6-dimethyl-2-tosylpyrrolo[1,2-e]imidazol-3-one

0 10 20 30 40 50 min

k´1 = 3.92

k´2 = 4.92

α = 1.25


15 Chiral separation of hydroxyzine in normal phase mode

Method Matrix HPLC



Conditions:

Volume: Detection: UV at 230 nm Substances: Hydroxyzine (D,L) Keywords: Chromatogram:

0 5 10 15 min

k´1 = 1.88

k´2 = 2.15

α = 1.14

V 12/2006 101

16 Chiral separation of n-methylephedrin in normal phase mode

Method Matrix HPLC



Conditions:

Volume: Detection: UV at 210 nm Substances: N-Methylephedrin (D,L) Keywords: Chromatogram:

0 5 10 15 min

k´1 = 0.88

k´2 = 1.25

α = 1.42


17 Chiral separation of metoprolol in normal phase mode

Method Matrix HPLC


Eluent: n-Heptane / Ethanol / DEA (90:10:0.1) Gradient: isocratic Flow rate: 1 ml/min Temperature: 25°C

Conditions:

Volume: Detection: UV at 230 nm Substances: Metoprolol (D,L) Keywords: Chromatogram:

0 5 10 min

k´1 = 1.34

k´2 = 1.96

α = 1.46

V 12/2006 103

18 Chiral separation of 1-(naphthalene-6-yl)ethane-1,2-diol in normal phase mode

Method Matrix HPLC


Eluent: n-Hexan / IPA (90:10) Gradient: isocratic Flow rate: 1 ml/min Temperature: 25°C

Conditions:

Volume: Detection: UV at 254 nm Substances: 1-(Naphthalene-6-yl)ethane-1,2-diol Keywords: Chromatogram:

OHOH

1-(naphthalen-6-yl)ethane-1,2-diol

0 10 20 30 min

k´1 = 7.98

k´2 = 6.63

α = 1.20


19 Chiral separation of 1-phenylethane-1,2-diol in normal phase mode



Phase: Eurocel 01


Conditions:

Volume: Detection: UV at 254 nm Substances: 1-phenylethane-1,2-diol Keywords: Chromatogram:

OH

OH

1-phenylethane-1,2-diol

0 10 20 30 40 min

k´1 = 3.65

k´2 = 3.98

α = 1.09

V 12/2006 105

20 Chiral separation of 1-phenylpropane-1,2-diol in normal phase mode



Phase: Eurocel 01


Conditions:

Volume: Detection: UV at 254 nm Substances: 1-phenylpropane-1,2-diol Keywords: Chromatogram:

OH

OH

1-phenylpropane-1,2-diol

0 10 20 30 min

k´1 = 2.76

k´2 = 2.93

α = 1.06


21 Chiral separation of pindolol in reversed phase mode

Method Matrix HPLC

Column: Eurocel 01 5µm, 125 x 4 mm Order No. 12DM370ECJ Phase: Eurocel 01

Eluent: A) 20 mM Sodium borate buffer B) Acetonitrile

Gradient: 60 : 40 (A:B) isocratic Flow rate: 1 ml/min Temperature: 25°C

Conditions:

Volume: Detection: UV at 263 nm Substances: Pindolol (D,L) Keywords: Chromatogram:

0 3 6 9 min

k´1 = 1.43

k´2 = 3.68

α = 2.57

V 12/2006 107

22 Chiral separation of pindolol in polar organic mode

Method Matrix HPLC


Eluent: Acetonitrile / EtOA (99.9:0.1) Gradient: isocratic Flow rate: 1 ml/min Temperature: 25°C

Conditions:

Volume: Detection: UV at 263 nm Substances: Pindolol (D,L) Keywords: Chromatogram:

0 3 6 9 12 min

k´1 = 0.6

k´2 = 1.37

α = 2.28


23 Chiral separation of propranolol in reversed phase mode

Method Matrix HPLC

Column: Eurocel 01 5µm, 125 x 4 mm Order No. 12DM370ECJ Phase: Eurocel 01

Eluent: A) Water , B) Acetonitrile, 68 : 32 (A:B) with 0.1% DEA Gradient: isocratic Flow rate: 1 ml/min Temperature: 25°C

Conditions:

Volume: Detection: UV at 230 nm Substances: Propranolol (D,L) Keywords: Chromatogram:

0 10 20 30 min

k´1 = 12.8

k´2 = 14.32

α = 1.12

V 12/2006 109

24 Chiral separation of propranolol in normal phase mode

Method Matrix HPLC

Column: Eurocel 01 5µm, 250 x 4.6 mm Order No. 25EM370ECJ Phase: Eurocel 01

Eluent: n-Heptane / IPA / EtOA (80:20:0.1) Gradient: isocratic Flow rate: 1 ml/min Temperature: 25°C

Conditions:

Volume: Detection: UV at 230 nm Substances: Propranolol (D,L) Keywords: Chromatogram:

0 5 10 15 min

k´1 = 1.25

k´2 = 2.49

α = 1.99

110 Substance and Keyword Index

Substance and Keyword Index

1-(naphthalene-6-yl)ethane-1,2-diol ......................103 1,2 Dimethylbenzene ...............................................23 1,2-Diphenyl-2-(tosylamino)ethanone......................96 10-OH-Carbamazepine............................................64 1-Aza-[6]helicene .....................................................90 1-Benzene-2-naphthene-3-amino propan(1)ol ........91 1-Phenylethane-1,2-diol.........................................104 1-Phenylpropane-1,2-diol.......................................105 2,6-Di-tert.-butyl-4-methylphenol ...............................3 3-Amino-2(2-naphtyl)-1-phenylpropanol ..................87 4-Isopropyl-3-methylphenol .......................................3 6-Nitro-Benzo(a)pyrene ...........................................13 9-Methylanthracene .................................................23 Abscisic acid ............................................................86 Acenaphthene..........................................................19 Acenaphthylene .......................................................19 Acesulfam K.............................................................25 Acetaldehyde .......................................................4, 12 Acetic acid................................................... 27, 28, 46 Acetone ................................................................4, 12 Acetyl cystein ...........................................................56 Acetyldeoxynivalenol ...............................................44 Acids

aromatic................................................................84 organic..................................................... 46, 47, 56

Acrolein ................................................................4, 12 Additives........................................................ 3, 25, 36 Adipic acid................................................................56 Aflatoxin ...................................................................26 Alanine .............................................................. 31, 59 Alcohols....................................................................27 Aldehydes ............................................................4, 12 Aldicarb ......................................................................8

sulphone .................................................................8 sulphoxide ..............................................................8

Alkaloids...................................................................57 Amineptine ...............................................................58 Amines ............................................ 29, 30, 31, 34, 60 Amino acids..............................................................59 Amino-dinitrotoluene ................................................11 Amino-nitrotoluene...................................................11 Aminophenol ............................................................74 Amitriptyline..............................................................83 Ammonia ..................................................................29 Ammonium.................................................................9 Ampicillin ..................................................................75 Angiotensin ..............................................................76 Anions

inorganic ................................................ 6, 7, 32, 45 organic........................................................... 32, 68

Anthocyanidin-3-glycosides .................................... 33 Anthocyanins........................................................... 33 Anthracene.............................................................. 19 Antibiotics........................................ 61, 62, 72, 75, 82 Anticonvulsants ....................................................... 63 Antidepressant ........................................................ 83 Antidepressants ...................................................... 58 Antiepileptics ........................................................... 64 Antirheumatics ........................................................ 65 Arginin ..................................................................... 31 Arginine ................................................................... 59 aromatic acids ......................................................... 84 Ascorbic acid......................................... 25, 46, 51, 56 Asparagin ................................................................ 31 Aspartame......................................................... 25, 36 Aspartic acid...................................................... 31, 59 Atenolol ................................................................... 88 Atrazine ................................................................... 17 Atropine................................................................... 89 Barbital .............................................................. 66, 78 Barium..................................................................... 10 BDMC........................................................................ 8 Benzo ...................................................................... 19 Benzo(a)pyrene....................................................... 19 Benzo(b)fluoranthene ............................................. 19 Benzo(g,h,i)perylene............................................... 19 Benzo(k)fluoranthene.............................................. 19 Benzoate ................................................................. 36 Benzoic acid...................................................... 25, 49 Benzoin ................................................................... 92 Benzyl nicotinate ....................................................... 3 Biogenic amines...................................................... 84 Bradykinin ............................................................... 76 Bromate..................................................................... 6 Bromide................................................................. 6, 7 Bromphenyl-glycinamid........................................... 93 BSA ......................................................................... 77 Butandiol ................................................................. 28 Butyl p-hydroxybenzoate .......................................... 3 Butylamine .............................................................. 29 Butylparabene ......................................................... 16 Butyraldehyde ..................................................... 4, 12 Cadaverine........................................................ 30, 34 Caempferol.............................................................. 38 Caffeine............................................................. 25, 36 Calcium ................................................................... 10 Campesterol............................................................ 50 Camphor ................................................................. 65 Carbamates............................................................... 8 Carbamazepine................................................. 63, 64

V 12/2006 111

Carbamazepine-diol.................................................64 Carbamazepine-epoxide..........................................64 Carbaryl .....................................................................8 Carbinoxamine.........................................................94 Carbofuran.................................................................8 Carbohydrates .........................................................27 Carbonyl compounds...........................................4, 12 Carotene-β .........................................................35, 54 Catecholamines .......................................................66 Cations

divalent .................................................................10 monovalent.............................................................9

Chloracetanilide .......................................................74 Chloramphenicol......................................................67 Chlorate .....................................................................7 Chloride......................................................6, 7, 32, 45 Chloroaniline............................................................60 Chloromethylphenol.................................................18 Chlorophenol ...........................................................18 Chlortoluron .............................................................17 Cholesterol...............................................................50 Chrysene .................................................................19 cis-Resveratrol.........................................................38 Citrate ......................................................................68 Citric acid .....................................................28, 46, 56 Codein......................................................................69 Codeine....................................................................57 Corticosterone .........................................................79 Cortisone ...........................................................79, 80 Coumarylated anthocyanidin-3-glycosides..............33 Cryptopine ...............................................................57 Cyanazin ..................................................................17 Cyanidol-3-glucosid .................................................33 Cyanocobalamine ....................................................51 Cyclohexanone ....................................................4, 12 Cystein .....................................................................56 Deoxynivalenol ............................................42, 43, 44 Desethylatrazine ......................................................17 Desoxycorticosterone ..............................................79 Diaminobutane.........................................................29 Diaminopentane.......................................................29 Diaminopropane ......................................................29 di-aromatic compounds ...........................................23 Dibenzo(a,h)anthracene ..........................................19 Dibenzothiphene......................................................23 Dichlorophenol.........................................................18 Dicloxacillin ..............................................................75 Diethylamine ............................................................29 Digitalis Glycosides..................................................70 Digitoxigenin ............................................................70 Digitoxigenin bisdigitoxoside ...................................70 Digitoxigenin monodigitoxoside ...............................70 Digitoxin ...................................................................70 Digoxigenin ..............................................................70 Digoxigenin bisdigitoxoside .....................................70 Digoxigenin monodigitoxoside.................................70 Digoxin .....................................................................70 Dihydroxyphenylacetic acid .....................................84

Diketopiperazine ......................................................36 Dilactid .....................................................................95 Dimethylamine .........................................................29 Dimethylphenol ........................................................18 Dinitrobenzene.........................................................11 Dinitrophenol............................................................18 Dinitrotoluene...........................................................11 Diosmin ....................................................................39 Di-tert.-butylmethylphenol..........................................3 Diuron ......................................................................17 Doxepin....................................................................83 DPH .........................................................................64 Drugs .......................................................................73

Antibacterial .........................................................67 Antibiotics ...........................................61, 62, 72, 75 Anticonvulsant ......................................................63 Antidepressant ...............................................58, 83 Antiepileptics ........................................................64 Antirheumatics .....................................................65 Glycosides............................................................70 Narcotics ..............................................................71

Eledoisin ..................................................................76 Elphinidol-3-glucosid................................................33 Ergosterol.................................................................50 Eriocitrin ...................................................................39 Eriodictyol 7-O-glucoside.........................................39 Esteriol .....................................................................80 Estradiol ...................................................................80 Estrone.....................................................................80 Ethanol...............................................................27, 28 Ethanolamine...........................................................29 Ethosuximide ...........................................................64 Ethylamine ...............................................................29 Ethylenglycolmonosalicylate....................................65 Ethylparabene..........................................................16 Etozolin ....................................................................97 Explosives................................................................11 Flavonoid glycosides ...............................................39 Flavonoids..........................................................37, 38 Flavonone ................................................................98 Fluoranthene............................................................19 Fluorene.............................................................19, 23 Fluoride ......................................................................6 Folic acid..................................................................51 Formaldehyde......................................................4, 12 Formic acid ..............................................................27 Fructose.............................................................28, 41 Fumaric acid ............................................................46 Furfural.....................................................................40 Fusarium mycotoxines.............................................44 Glucose........................................................27, 28, 41 Glutamic acid .....................................................31, 59 Glutamin.............................................................31, 59 Glycine ...............................................................31, 59 Glycosides ...............................................................70 Guaifenesin..............................................................69 Heptylamine.............................................................30 Hesperidin..........................................................37, 39


Hexahydro-6,6-dimethyl-2-tosylpyrrolo[1,2-e]imidazol-3-one...................................................99

Hexamethylbenzene ................................................23 Hexazinon ................................................................17 Hexogen...................................................................11 Hexylamine ..............................................................29 Histamine .................................................... 29, 30, 34 Histidine ............................................................ 31, 59 HMF .........................................................................40 Homovanillic acid .....................................................84 Hydroxy-benzoic acid...............................................49 Hydroxycarbofuran.....................................................8 Hydroxymethylfurfural (HMF)...................................40 Hydroxyphenyl ethanone .........................................74 Hydroxyphenyl ethanone oxime...............................74 Hydroxypyrene.........................................................14 Hydroxyzine ...........................................................100 Imipramine ...............................................................83 Indeno(1,2,3-cd)pyren..............................................19 Indole .......................................................................29 Insecticides ................................................................8 Insulin bovine ...........................................................77 Iodate .........................................................................7 Iodide .........................................................................7 Isoamylamine...........................................................30 Isoleucine .......................................................... 31, 59 Isopentylamine.........................................................29 Isopropylamine.........................................................29 Isopropylmethylphenol ...............................................3 Isoproturon...............................................................17 Isorhoifolin................................................................39 Ketones ................................................................4, 12 Lactic acid ............................................. 27, 28, 46, 47 Lactose.....................................................................41 Lanosterol ................................................................50 Laurylalcohol ............................................................21

polyglycol ether ....................................................21 Leucine.............................................................. 31, 59 Linuron .....................................................................17 Lithium........................................................................9 Luminal........................................................ 66, 71, 78 Luteolin 7-O-rutinoside.............................................39 Lysine................................................................ 31, 59 Lysozyme.................................................................77 Magnesium...............................................................10 Malic acid .................................................... 28, 46, 47 Malonic acid .............................................................46 Maltose.............................................................. 27, 41 Malvidol-3-glucosid ..................................................33 Metazachlor..............................................................17 Methabenzthiazuron ................................................17 Methacrolein.........................................................4, 12 Methanol ..................................................................27 Methiocarb .................................................................8 Methionine......................................................... 31, 59 Methomyl....................................................................8 Methoxy benzoic acid ..............................................49 Methyl ethyl ketone ..............................................4, 12

Methyl p-hydroxybenzoate........................................ 3 Methyl-1H-tetrazole-5-thiol...................................... 72

Methyl-6α-hydroxy-11β-progesterone .................... 80

Methyl-6α-hydroxy-17α-progesterone .................... 80

Methyl-6α-hydroxy-17α-progesterone acetate ....... 80 Methylamine............................................................ 29 Methylbutylamine .................................................... 29 Methyldinitrophenol ................................................. 18 Methylparabene ...................................................... 16 Metobromuron......................................................... 17 Metolachlor.............................................................. 17 Metoprolol ............................................................. 102 Metoxuron ............................................................... 17 Monoammonium glycyrrhizinate ............................... 3 mono-aromatic compounds .................................... 23 Monolinuron ............................................................ 17 Morphine ................................................................. 57 Morpholine .............................................................. 29 Moxalactam............................................................. 72 Mycotoxins .................................................. 42, 43, 48 Myoglobin................................................................ 77 Myrcetine................................................................. 38 Myristylalcohol......................................................... 21 Myristylalcohol polyglycol ether .............................. 21 N-(2-hydroxyphenyl)acetamide............................... 74 N-(4-hydroxyphenyl)propanamide .......................... 74 Nafcillin.................................................................... 75 Naphthalene...................................................... 19, 23 Naphtol...................................................................... 8 Narcotics ................................................................. 71 Narcotine................................................................. 57 Naringin................................................................... 37 Narirutin................................................................... 39 Neurotensin............................................................. 76 Nicotinamide ........................................................... 51 Nicotinic acid ........................................................... 51 Nicotinic acid benzyl ester ...................................... 65 Nicotinic acid methyl ester ...................................... 65 Nitrate........................................................ 6, 7, 32, 45 Nitrite................................................................... 6, 45 Nitroaniline .............................................................. 15 Nitrobenzene........................................................... 11 Nitro-Benzo(a)pyrene.............................................. 13 Nitro-Chrysene ........................................................ 13 Nitro-Fluorene ......................................................... 13 Nitro-Naphthalene................................................... 13 Nitro-PAH ................................................................ 13 Nitro-Phenanthrene................................................. 13 Nitrophenol........................................................ 18, 74 Nitro-Pyrene ............................................................ 13 Nitrotoluene............................................................. 11 Nivalenol ........................................................... 43, 44 N-Methylephedrin.................................................. 101 Nonylphenol polyglycol ether .................................. 21 Norgestrel................................................................ 79 Nortiptyline .............................................................. 83 N-phenylacetamide ................................................. 74 Octogen................................................................... 11

V 12/2006 113

Octopamine .............................................................29 Octyl hydrogen sulphate ......................................5, 20 Oleic acid diethanolamide........................................21 Oleic amide polyglycol ether....................................21 Organic acids.....................................................27, 28 Ovalbumin................................................................77 Oxalate...............................................................32, 68 Oxalic acid ...............................................................46 Oxamyl.......................................................................8 Oxcarbazepine.........................................................64 Oxytocin ...................................................................76 PAH..........................................................................19 Palmitic acid polyglycol ester...................................21 Pamidronate disodium.............................................73 Pantothenylethylether ................................................3 Papaverine...............................................................57 Parabenes ...............................................................16 Paracetamol.............................................................74 Patulin ......................................................................48 PEMA.......................................................................64 Penicillin...................................................................75 Pentachlorphenol.....................................................18 Pentanone ...........................................................4, 12 Pentylamine .............................................................29 Pentyl-i-amine..........................................................30 Peonidol-3-glucosid .................................................33 Peptides...................................................................76 Pesticides ................................................................17 Petunidol-3-glucosid ................................................33 Phenanthrene ....................................................19, 23 Phenethylamine .................................................29, 30 Phenobarbital.....................................................63, 64 Phenol......................................................................18 Phenols ....................................................................18 Phenylalanine ....................................................31, 59 Phenyldodecane ......................................................23 Phenylene diamine ..................................................60 Phenylethylamine ....................................................34 Phenytoin.................................................................63 Phosphate............................................................6, 32 p-Hydroxy butylbenzoate .........................................49 p-Hydroxy ethylbenzoate .........................................49 p-Hydroxy methylbenzoate ......................................49 p-Hydroxy propylbenzoate.......................................49 Pindolol ..........................................................106, 107 Piperidine.................................................................29 Plant hormone .........................................................86 Polyethylene glycol ..................................................21 Polyoxyethylene

glycerol monostearate..........................................21 sorbitane monolaurate .........................................21

Potassium ..................................................................9 Prednisolone............................................................80 Primidone...........................................................63, 64 Progesterone .....................................................79, 80 Prominal.......................................................66, 71, 78 Propionaldehyde..................................................4, 12 Propoxur ....................................................................8

Propranolol.....................................................108, 109 Propylamine.............................................................29 Propylparabene .......................................................16 Proteins....................................................................77 Pseudoefedrin..........................................................69 Putrescine ..........................................................30, 34 Pyrene......................................................................19 Pyridoxale ................................................................51 Pyridoxamine ...........................................................51 Pyridoxine ................................................................51 Pyridoxine dioctanoate ..............................................3 Pyrrolidine ................................................................29 Pyrrolidone...............................................................29 Quercetine ...............................................................38 Quinic acid .........................................................46, 47 Quinine.....................................................................25 Revinal .....................................................................66 Revonal..............................................................71, 78 Rhodanide..................................................................7 Riboflavine ...............................................................51 Ribonuclease A........................................................77 Rosmarinic acid .......................................................39 Saccharides .............................................................41 Saccharin .................................................................25 Saccharose..............................................................41 Salicylic acid ..............................................................3 Sebuthylazin ............................................................17 Sedativa ...................................................................78 Serine.................................................................31, 59 Serotonin..................................................................29 Shikimic acid......................................................46, 47 Simazin ....................................................................17 Sitosterol ..................................................................50 Sodium.......................................................................9 Sodium soap (C8 - C18) ..........................................22 Sorbic acid .........................................................25, 49 Stearic acid polyglycol ester ....................................21 Stearyl glycyrrhetinate ...............................................3 Steroids........................................................50, 79, 80 Steroles....................................................................50 Stigmasterol .............................................................50 Stilbene ....................................................................38 Strontium..................................................................10 Succinic acid ................................................27, 28, 46 Sulfa drugs.........................................................81, 82 Sulfadiazine .......................................................81, 82 Sulfadimethoxine .....................................................82 Sulfamerazine..........................................................81 Sulfamethazine ........................................................81 Sulfamethiazole .......................................................82 Sulfamethoxazol ......................................................62 Sulfamethoxazole ....................................................82 Sulfamethoxydiazine................................................61 Sulfamethoxypyridazine...........................................82 Sulfanilamide .....................................................61, 81 Sulfate..............................................................6, 7, 32 Sulfathiazole ............................................................81 Sulpho fatty acid-α-methyl esters ............................22


Sulphosuccinate...................................................5, 20 Surfactants .............................................. 5, 20, 21, 22 Sweeteners ....................................................... 25, 36 Tartaric acid ...................................................... 28, 46 Terbuthylazine .........................................................17 Testosterone ..................................................... 79, 80 Tetryl ........................................................................11 Thebaine ..................................................................57 Thiamine ..................................................................51 Thiogenal .......................................................... 71, 78 Threonine .......................................................... 31, 59 Tocopherols .............................................................52 Tocopherol-α ............................................................54 Tocopheryl acetate ....................................................3 Toluidine-o ...............................................................61 trans-Resveratrol .....................................................38 tri-aromatic compounds ...........................................23 Trichlorocarbanilide....................................................3 Trichlorphenol ..........................................................18

Trichotecenes.......................................................... 42 Trichothecens.......................................................... 43 Trimethoprim ........................................................... 62 Trimipramine ........................................................... 83 Tryptamine .............................................................. 34 Tryptophan .............................................................. 84 Tyramine ................................................................. 34 Tyrosine ............................................................ 31, 59 Valine ................................................................ 31, 59 Vanilin ..................................................................... 25 Veronal.................................................................... 71 Vitamin A acetate .............................................. 53, 54 Vitamin A alcohol .................................................... 53 Vitamin D2......................................................... 52, 53 Vitamin D3............................................................... 53 Vitamin E................................................................. 53 Vitamin K1............................................................... 53 Vitamins .......................................... 35, 51, 52, 53, 54

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KNAUER HPLC Applications Journal - December 2006quimica.udea.edu.co/.../knauer_applications_journal_2007.pdf · This Applications Journal was ... DNPH derivates of Formaldehyde