Liquid Chromatography - SUPELCO 110-147.pdf · Liquid Chromatography SUPELCO 112 Order:...

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oHPLC ApplicationsAmino Acids, Carbohydrates

Amino Acids, Dabsyl (HPLC)Column: SUPELCOSIL LC-DABS, 15cm x 4.6mm ID, 3µm particlesCat. No.: 59137

Mobile Phase: A = 0.025M KH2PO4, pH 7.0 with potassium hydroxideB = acetonitrile:methanol (70:30)

Flow Rate: 1.5mL/minDet.: VIS, 436nmInj.: 5µL, 50pmole each amino acid on-column

Carbohydrates (HPLC)Column: SUPELCOGEL Ca, 30cm x 7.8mm IDCat. No.: 59305-U

Mobile Phase: waterFlow Rate: 1.5mL/min

Temp.: 80°CDet.: RIInj.: 10µL, 2.5mg/mL each analyte in water

Dark Corn Syrup (HPLC)Column: SUPELCOGEL Ag, 30cm x 7.8mm IDCat. No.: 59315

Mobile Phase: waterFlow Rate: 0.5mL/min

Temp.: 85°CDet.: RIInj.: 10µL of 1g syrup/10mL DI water, filtered (0.20µm filter)

βββββ-Cyclodextrin, Hydrolyzed (HPLC)Column: Two TSKgel G-Oligo-PW, 30cm x 7.8mm ID, 6µm particlesCat. No.: 808031

Mobile Phase: distilled waterFlow Rate: 1mL/min

Temp.: 60°CDet.: refractive index

Gradient ProgramTime (min) %B

0 204 209 25

23 2524 3230 3237 4046 6054 7559 7560 20

712-

0135

1. o-Phospho-serine2. o-Phospho-threonine3. L-Aspartic acid4. o-Phospho-tyrosine5. Cysteic acid6. L-Glutamic acid7. Carboxymethyl

cysteine8. S-Sulfocysteine9. L-Asparagine

10. L-Glutamine11. L-Serine12. Methionine sulfoxide13. L-Threonine14. Glycine15. L-Alanine16. L-Arginine17. Methionine sulfone18. L-Proline19. L-Valine20. L-Methionine21. L-Isoleucine22. L-Leucine23. L-Tryptophan24. L-Phenylalanine25. Ammonia26. L-Cystine27. L-Lysine28. L-Histidine29. L-Tyrosine

0 10 20 30 40 50

2

3 45

6

7

8

9

10

13

1415

12

16

17

19

20

21

18

22

23

24

28292725

Min

26

1

11

795-

0016

1

2

34

5

6

78

0 5 10 20 2515Min

1. Maltotriose2. Maltose3. Glucose4. Xylose5. Arabinose6. Ribitol7. Arabitol8. Xylitol

794-

0029

1

5

6

4

32

0 5 10 15Min

1. DP62. DP53. DP44. DP35. DP26. Glucose

713-

0917

15 20Min

DP=1

DP=6

DP=4

DP=2

DP=3DP=7

DP=5

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HPLC ApplicationsCarbohydrates, Drugs

Chito Oligosaccharides (HPLC)Column: TSKgel G-Oligo-PW, 30cm x 7.8mm ID, 6µm particlesCat. No.: 808031

Eluent: distilled waterFlow Rate: 1.0mL/min

Temp.: 60°CDet.: RI

Carbohydrates (HPLC)Column: SUPELCOSIL LC-NH2, 25cm x 4.6mm ID, 5µm particlesCat. No.: 58338

Mobile Phase: acetonitrile:water (75:25)Flow Rate: 2mL/min

Det.: refractive indexInj.: 10µL, 150µg each sugar

Alkaloids (HPLC)Column: Discovery RP-AmideC16, 15cm x 4.6mm ID, 5µm particlesCat. No.: 505013

Mobile Phase: methanol:25mM KH2PO4, pH 3.0 (20:80)Flow Rate: 2mL/min

Temp.: 35°CDet.: UV, 254nmInj.: 10µL

Allegra Ingredients (HPLC)Column: Discovery Cyano, 15cm x 4.6mm ID, 5µm particlesCat. No.: 59356-U

Mobile Phase: acetonitrile:25mM KH2PO4, pH 7.0 (15:85)Flow Rate: 1mL/min

Temp.: 25°CDet.: UV, 254nm

794-

0325

1

2

34

5 1. Chitohexose2. Chitopentose3. Chitotetrose4. Chitotriose5. Chitobiose

15 20Min

713-

0908

32

4 5

0 4 8 12Min

1

1. Fructose2. Glucose3. Sucrose4. Maltose5. Lactose

G00

0235

1. Codeine2. Strychnine3. Quinidine4. Quinine5. Noscapine6. Papaverine

0 2 4 6 8 10 12Min

1

23

4

5

6

0 2 4 6 8 10Min

1. Methanol2. Pseudoephedrine3. Fexofenadine

G00

1279

1

2

3

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oHPLC ApplicationsDrugs

Analgesics (HPLC)Column: SUPELCOSIL LC-ABZ, 5cm x 4.6mm ID, 5µm particlesCat. No.: 59141

Mobile Phase: acetonitrile:25mM KH2PO4,pH 2.3 with phosphoricacid (20:80)

Flow Rate: 2mL/minDet.: UV, 230nmInj.: 5µL mobile phase, analyte quantities shown

Analgesics and Antihistamines in Dosage Form (HPLC)Column: Discovery RP-AmideC16, 15cm x 4.6mm ID, 5µm particlesCat. No.: 505013

Mobile Phase: A = acetonitrileB = 25mM KH2PO4, pH 2.3

Flow Rate: 2mL/minTemp.: ambient

Det.: UV, 214nmInj.: 10µL

Amides (HPLC)Column: Discovery RP-AmideC16, 15cm x 4.6mm ID, 5µm particlesCat. No.: 505013

Mobile Phase: 80:20 25mM Potassium Phosphate (pH 3.0):MeOHFlow Rate: 2.0mL/min

Temp.: 35°CDet.: UV at 254nmInj.: 10µL

Analgesic Drugs (HPLC)Columns: 15cm x 4.6mm ID, 5µm particlesCat. Nos.: 567516-U (HS F5), 504955 (C18)

Mobile Phase: (A) 10mM ammonium acetate, 0.1% formic acid; (B) MeOHFlow Rate: 1.5mL/min

Temp.: 35°CDet.: UV at 254nmInj.: 10µL

713-

0178

0 2Min

Dextromethorphan(100µg/mL)

Acetylsalicylic acid(50µg/mL)

G00

0785

0 4 6 8 102Min

2

3

4

1. Doxylamine2. Pseudoephedrine3. Codeine4. Acetaminophen5. Chlorpheniramine

5

1


0 902 906 70

10 70

0 2 4 6 8 10 12 14 16Min

1

2

3

4

5

61. Codeine2. Strychnine3. Quinidine4. Quinine5. Noscapine6. Papaverine

G00

0691

1. p-Aminophenol (100µg/mL)2. Acetaminophen (10µg/mL)3. Acetanilide (10µg/mL)4. Phenacetin (10µg/mL)

0 2 4 6 8 10 12 14Min

0 2 4 6 8 10 12 14Min

Discovery C18:Elutes at void volume

▼▼▼▼▼

Gradient ProgramTime (min) %A %B

0 90 103 90 10

10 50 5015 50 50

▼▼▼▼▼

4

3

1

2

4

3

1

2

G00

1527

G00

1526

Discovery HS F5:Good Retention

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HPLC ApplicationsDrugs

Aniline Homologs (HPLC)Coumn: Discovery C18, 15cm x 4.6mm ID, 5µm particles

Cat. No.: 504955Mobile Phase: MeOH:H2O (60:40)

Flow Rate: 1mL/minTemp.: 30°C

Det.: UV, 254nm

Antibiotics (Fluoroquinolones from Tablets) (HPLC)Columns: 15cm x 4.6mm ID, 5µm particlesCat. Nos.: 504955 (C18), 568522-U (HS C18)

Mobile Phase: (A) 25 mM potassium phosphate (pH 3.0)(B) CH3CN

Flow Rate: 1.0mL/minTemp.: 35°C


Anticonvulsants (HPLC)Column: Discovery C18, 15cm x 4.6mm ID, 5µm particlesCat. No.: 504955

Mobile Phase: acetonitrile:25mM KH2PO4, pH7.0 (40:60)Flow Rate: 1mL/min


0 2 4 6 8 10 12 14 16 18Min

1

2

3

4

56

1. Aniline2. N-Ethylaniline3. N-Propylaniline4. N,N-Dimethylaniline5. N-Butylaniline6. N,N-Diethylaniline

G00

0295

0 2 4 6 8 10 12 14Min

1

2 34

5

6

0 2 4 6 8 10 12 14Min

1

2 34

5

6

1. Levofloxacin2. Ciprofloxacin3. Lomefloxacin4. Sparfloxacin5. Grepafloxacin6. Trovafloxacin

Discovery HS C18

Discovery C18

G00

1425

G00

1424


0 90 1025 65 35

1. Clonazepam2. Clorazepate3. Diazepam

G00

0188

0 2 4 6 8 10Min

1

2

3

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1.0 2.0 3.0 4.0 5.0Min

1

2

3


Antihypertensive-Diuretic Combination (HPLC)Column: Discovery RP-AmideC16, 15cm x 4.6mm ID, 5µm particlesCat. No.: 505013

Mobile Phase: methanol: 25mM K 2HPO4, pH 7.0 (22:78)Flow Rate: 1mL/min


Anticonvulsants (HPLC)Columns: 15cm x 4.6mm ID, 5µm particlesCat. Nos.: 504955 (C18), 59353-U (C8)

Mobile Phase: 70:30 water:CH3CNFlow Rate: 2.0mL/min

Det.: UV, 254nmTemp.: 20°C

Inj.: 10µL

Antihistamines (HPLC)Column: Discovery C8, 15cm x 4.6mm ID, 5µm particlesCat. No.: 59353-U



Antihypertensive ACE Inhibitors (HPLC)Column: Discovery RP-AmideC16, 15cm x 4.6mm ID, 5µm particlesCat. No.: 505013

Mobile Phase: acetonitrile:25mM KH2PO4, pH 2.3 (33:67)Flow Rate: 0.6mL/min


1. Atenolol2. Amiloride3. Hydrochlorothiazide4. Caffeine

G00

0280

0 2 4 6 8 10Min

1

2

3

4

0 2 4 6 8 10 12 14 16 18Min

1

23

0 2 4 6 8 10 12 14 16 18Min

1

23

1. Clonazepam2. Clorazepate3. Diazepam

G00

0748

G00

0746

G00

1278

1. Maleic acid2. Doxylamine3. Chlorpheniramine4. Carbinoxamine5. Brompheniramine

0 2 4 6 8 10 12 14 16 18Min

1

3

2

4 5

1. Enalapril2. Lisinopril3. Captopril

G00

0279

Discovery C8

Discovery C18

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Anti-Inflammatory Drugs(Aminopryridine and Piroxicam) (HPLC)

Columns: Discovery C18, 15cm x 4.6mm ID, 5µm particlesCat. Nos.: 504955

Mobile Phase: A = 85:15, 10 mM potassium phosphate (pH 2.5): CH 3CNB = 85:15, 10 mM potassium phosphate (pH 6.8): CH 3CN

Flow Rate: 1.0mL/minDet.: UV, 220nmInj.: 5µL

Anti-Inflammatory Drugs and Diuretics in Serum (HPLC)Column: Hisep, 15cm x 4.6mm ID, 5µm particlesCat. No.: 58935

Mobile Phase: A = acetonitrile, B = 180mM ammonium acetate, pH 5.4Flow Rate: 2mL/min

Temp.: 30°CDet.: UV, 267nmInj.: 10µL horse serum containing 5µg/mL hydrochlorothiazide,

10µg/mL each other drug

Antihypertensive Drugs (HPLC)(Beta/Calcium Channel Blockers and ACE Inhibitor)

Column: Discovery RP-AmideC16, 15cm x 4.6mm ID, 5µm particlesCat. No.: 505013

Mobile Phase: methanol:25mM KH2PO4, pH 7.0 (40:60)Flow Rate: 1mL/min


Anti-Inflammatory Drugs, Non-Steroidal (HPLC)Column: Discovery RP-AmideC16, 15cm x 4.6mm ID, 5µm particlesCat. No.: 505013


Temp.: 30°CDet.: UV, 230nmInj.: 10µL, 1µg/mL each analyte

0 2 4 6Min

2

0 2 4 6 8 10 12 14Min

1

1. 2-Aminopyridine2. Piroxicam

2

G00

1658

G00

1657

2 4 6 8 10 12 14Min 85

-163

1

243

0

1. Hydrochlorothiazide2. Oxyphenbutazone3. Furosemide4. Phenylbutazone

Gradient ProgramTime (min) %A

0 205 30

15 20

G00

0112

1. Captopril2. Pindolol3. Desmethyldiltiazem4. Nifedipine5. Diltiazem6. Verapamil

0 2 4 6 8 10 12 14 16 18Min

1

2

3

4 5

6

1. Piroxicam2. Sulindac3. Tolmetin4. Ketoprofen5. Naprosyn6. Diflunisal7. Indomethacin8. Ibuprofen9. Diclofenac

10. Mefanamic acidG

0000

61

0 10 20Min

1

2

3

45 6

7

89

1

Mobile Phase B

Mobile Phase A

10

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Antipyretics/Analgesics/Antifungals (HPLC)Column: Discovery RP-AmideC16, 15cm x 4.6mm ID, 5µm particlesCat. No.: 505013

Mobile Phase: 0.1% TFA in (25:75) acetonitrile:waterFlow Rate: 1mL/min


Antitussive/Antihistamine/Antipyretic Mix (HPLC)Columns: 15cm x 4.6mm ID, 5µm particlesCat. Nos.: 505013 (RP-AmideC16), 504955 (C18)

Mobile Phase: (A) 25 mM Potassium Phosphate (pH 2.3)(B) CH3CN

Flow Rate: 2.0mL/minTemp.: ambient


Antioxidants (HPLC)Column: SUPELCOSIL LC-18, 15cm x 4.6mm ID, 5µm particlesCat. No.: 58230-U

Mobile Phase: A = 5% acetic acid in deionized waterB = acetonitrile:methanol (1:1)70% A/30% B to 100% B, linear gradient over 10 min, hold 10min

Flow Rate: 2.0mL/minDet.: UV, 280nmInj.: 10µL , 20µg/mL each antioxidant

(Cat. No. 47192)

G00

0088

1. Paracetamol2. Aspirin (acetylsalicylic acid)3. Sorbic acid4. Benzoic acid5. Salicylic acid

0 2 4 6 8 10 12 14Min

12

3

4

5

0 2 4 6 8 10Min

2

3

1

54

Elution order changes!

Discovery RP-AmideC16

Discovery C18

1. Acetaminophen2. Doxylamine3. Pseudoephedrine4. Codeine5. Chlorpheniramine

▼▼▼▼▼


0 90 102 90 104 70 308 70 30

10 50 50

0 10 20Min

▼▼▼▼▼

Elutionorderchanges!

23

1

4

G00

0787

G00

0785

795-

0438

2

4

5

7

9

8

3

1

0 2 4 6 8 10 1412Min

6

1. Propyl gallate2. THBP3. TBHQ4. NDGA5. BHA6. Ionox 1007. Octyl gallate8. BHT9. Dodecyl gallate

5

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Antiulcer Compounds (HPLC)Columns: 15cm x 4.6mm ID, 5µm particlesCat. Nos.: 505013 (RP-AmideC16), 504955 (C18),

569252-U (HS C18), 59353-U (C8)Mobile Phase: MeOH:25mM H3PO4/KOH, pH 3.0 (15:85)

Flow Rate: 0.5mL/minTemp.: ambient


Anxiolytics (HPLC)Column: Discovery C18,15cm x 4.6mm ID, 5µm particlesCat. No.: 504955


Temp: 20°CDet.: UV, 254nmInj.: 10µL

Anxiolytics (HPLC)Column: SUPELCOSIL LC-8, 15cm x 4.6mm ID, 5µm particlesCat. No.: 58220-U

Mobile Phase: methanol:acetonitrile:0.005M KH 2PO4 and 0.1M ammoniumacetate buffer (pH 6.0 with glacial acetic acid),26.5:16.5:57 (v/v/v)


Det.: UV, 245nmSample: 10µL

Discovery C18

Discovery RP-AmideC16 1. Nizatidine2. Ranitidine3. Famotidine4. Cimetidine

G00

1428

G00

1427

G00

1430

Discovery HS C18

797-

0662

1. Nordoxepin2. Oxazepam3. Chlordiazepoxide4. Desmethyldiazepam

0 10

1

2

3

4

Min

Nordoxepin is notoriously difficult tochromatograph, and the slight tailingis a good peak shape for this drug.

0 2 4 6 8 10 12 14Min

12

3

4

0 2 4 6 8 10 12 14Min

1 2

3

4

0 2 4 6 8 10 12 14Min

1 2

3

4

0 2 4 6 8 10 12 14Min

1 2

3

4

Discovery C8

G00

1429

1. Demoxepam2. Nitrazepam (int. std.)3. N-desmethyl Chlordiazepoxide4. Oxazepam5. Chlordiazepoxide6. Temazepam7. N-desmethyl Diazepam8. Diazepam

2 45 7

8

3

1

0 2 4 6 8 10 1412Min

6

713-

1390

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Barbiturates (HPLC)Column: Discovery C8, 15cm x 4.6mm ID, 5µm particlesCat. No.: 59353-U

Mobile Phase: 55:45 water:MeOHFlow Rate: 1.0mL/min

Det.: UV at 214nmTemp.: ambient

Inj.: 5µL

Barbiturates (HPLC)Column: Discovery RP-AmideC16, 15cm x 4.6mm ID, 5µm particlesCat. No.: 505013

Mobile Phase: methanol:acetonitrile, 60:40:water, 25:75Flow Rate: 2mL/min


Barbiturates (HPLC)Column: Discovery C18, 15cm x 4.6mm ID, 5µm particlesCat. No.: 504955

Mobile Phase: 55:45 water:MeOHFlow Rate: 1.0mL/min

Temp.: ambientDet.: UV, 214nmInj.: 10µL

Barbiturates (HPLC)Column: SUPELCOSIL LC-ABZ, 15cm x 4.6mm ID, 5µm particlesCat. No.: 59140-U

Mobile Phase: acetonitrile:25mM potassium phosphate buffer, pH 6.9 (25:75)Flow Rate: 1.5mL/min


1. Barbital2. Aprobarbital3. Phenobarbital4. Butabarbital5. Mephobarbital6. Pentobarbital7. Secobarbital

0 2 4 6 8 10 12 14 16 18Min

1

2

3

45

6 7

G00

0791

1. Barbital2. Phenobarbital3. Aprobarbital4. Butabarbital5. Mephobarbital6. Pentobarbital7. Secobarbital

G00

0193

0 10 20Min

13

2

4

6

5

7

0 10 20Min

1. Barbital2. Aprobarbital3. Phenobarbital4. Butabarbital5. Mephobarbital6. Pentobarbital7. Secobarbital

6

4

3

1

2

5

7G

0001

90

0 2 4 6 8 10Min

1. Methylprylon (2mg/mL)2. Butabarbital (50µg/mL)3. Butalbital (50µg/mL)4. Hexobarbital (50µg/mL)5. Mephobarbital (25µg/mL)

713-

0141

1

5

2

3

4

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Benzalkonium Chlorides (HPLC)Column: Discovery Cyano, 15cm x 4.6mm ID, 5µm particlesCat. No.: 59356-U

Mobile Phase: CH3CN:acetate buffer, pH 4.5, (60:40)Flow Rate: 1mL/min

Temp.: ambient temp.Det.: UV, 254nm

Sample: Undefined C8 to C18 Benzalkonium Chlorides

Catecholamines (HPLC)Column: Discovery C18, 15cm x 4.6mm ID, 5µm particlesCat. No.: 504955

Mobile Phase: acetonitrile:50mM KH2PO4, pH 3.0 with H 3PO4, 100mg/L EDTA,200mg/L 1-octane-sulfonic acid (5:95)



Cardiac Drugs (HPLC)Column: SUPELCOSIL ABZ+Plus, 15cm x 4.6mm ID, 5µm particlesCat. No.: 59196

Mobile Phase: A = acetonitrileB = 25mM KH2PO4 (pH 3.0)

Flow Rate: 2mL/minDet.: UV, 220nmInj.: 20µL methanol:water, 50:50 (30µg/µL pindolol and

diltiazem,100µg/mL dipyridamole, 50µg/mL other analytes)

Chlorzoxazone (HPLC)Column: Discovery HS PEG, 15cm x 4.6mm ID, 5µm particlesCat. No.: 567416-U

Mobile Phase: 70:30 20mM acetic acid in water(pH 4.5 with ammonium hydroxide):CH 3CN



G00

0726

0 2 4 6Min

797-

0659

1. Norepinephrine2. Epinephrine3. 3,4-Dihydroxybenzylamine4. Dopamine

Min

1

2

3

4

0 1079

4-06

38

Min

1. Procainamide2. Pindolol3. Oxprenolol4. Dipyridamole5. Diltiazem6. Verapamil7. Digoxin8. Lidoflazine9. Flunarizine

10. Nifedipine


0 9010 5012 5012.1 90

0 2 4 6 8 10

1098

7

6

4

3

1

2

5

0 10 20Min

1. 6-Hydroxychlorzoxazone2. Chlorzoxazone

12

G00

1674

CH2 NCH3

CH3

R

+

Cl-

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Cold Remedy Ingredients/Antihistamines (HPLC)Columns: 15cm x 4.6mm ID, 5µm particlesCat. Nos.: 505013 (RP-AmideC16), 59353-U (C8), 59356-U (Cyano)

Mobile Phase: acetonitrile:25mM KH2PO4 pH 7.0 (15:85)Flow Rate: 1mL/min


Cephalosporins (HPLC)Column: 15cm x 4.6mm ID, 5µm particles

Cat. Nos.: 59353-U (C8), 505013 (RP-AmideC16), 504955 (C18)Mobile Phase: methanol:25mM KH2PO4, pH 3.0, (20:80) for C18 and C8,

(10:90) for RP-Amide C16Flow Rate: 2mL/min


G00

0710

1. Pseudoephedrine2. Acetaminophen

Min

1

2

G00

0709

Discovery C8


2

1

0 2 4

0 2 4 6Min

0 2 4 6Min

1

2

3

1. Cefadroxil2. Cefaclor3. Cephalexin

G00

0696

0 2 4 6Min

1

2

3

0 2 4 6 8Min

1

23

Discovery C8


Discovery C18

G00

0746

G00

0747

0 2 4 6Min

2

1

Discovery Cyano

G00

0839

Liqu

idC

hrom

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raph

y

SUPELCO

121

Ord

er:

1.8

00

.32

5.3

01

0 T

ec

hn

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: 1

.80

0.3

59

.30

41

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Fluoxetine (Prozac) (HPLC)Columns: 15cm x 4.6mm ID, 5µm particlesCat. Nos.: 505013 (RP-AmideC16), 504955 (C18),

59353-U (C8), 59356-U (Cyano)Mobile Phase: CH3CN:25mM KH 2PO4, pH 7.0 (40:60)


Det.: UV, 214nmInj.: 1µL βββββ-Lactam Antibiotics (HPLC)

Column: 15cm x 4.6mm ID, 5µm particlesCat. Nos.: 59353-U (C8), 505013 (RP-AmideC16),

504955 (C18), 59356-U (Cyano)Mobile Phase: A = 0.01% TFA in water;

B = 0.01% TFA in CH3CNFlow Rate: 1.5mL/min


0 2 4 6Min

0 2 4 6Min

0 2 4 6Min

0 2 4 6Min

Discovery C18


Discovery C8

Discovery Cyano

G00

0902

G00

0900

G00

0901

G00

0899

G00

1180

1. Amoxicillin2. Ampicillin3. Piperacillin4. Penicillin G5. Penicillin V6. Cloxacillin


0 55 35

20 35

0 2 4 6 8 10 12 14Min

1

2

3

4 5

6

0 2 4 6 8 10 12 14 16Min

0 10 20Min

1

2

3

4 5 6

12

3

4

5

6

1 2

3 4 5

6

0 10 20Min

Discovery C8


Discovery C18

Discovery Cyano

G00

1178

G00

1179

G00

1181


0 520 25

Liqu

idC

hrom

atog

raph

y

SUPELCO

122

Ord

er:

1.8

00

.32

5.3

01

0 T

ec

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: 1

.80

0.3

59

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41

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Quinidine and Trace Impurity (HPLC)Columns: 15cm x 4.6mm ID, 5µm particlesCat. Nos.: 567516-U (HS F5), 504955 (C18)

Mobile Phase: 25 mM ammonium phosphate (pH 7.0):CH 3CNvarying ratios: (A) 35:65, (B) 70:30



5-Fluorouracil and Tegafur (HPLC)Column: SUPELCOGEL TPR-100, 15cm x 4.6mm ID, 5µm particlesCat. No.: 59154

Mobile Phase: acetonitrile:water (10:90)Flow Rate: 1.0mL/min


Methcathinone and Ephedrine (HPLC)Column: Discovery HS F5, 15cm x 4.6mm ID, 5µm particlesCat. No.: 567516-U

Mobile Phase: (30:70) 10mM Ammonium Acetate (pH 6.98): CH 3CNFlow Rate: 2.0mL/min

Temp.: 35°CDet.: Photodiode ArrayInj.: 5µL

0 10 20Min

0 10 20Min

1

(A) Discovery HS F5

(B) Discovery C181

1. Quinidine (50µg/mL)2. Impurity

Partiallyresolvedfrontshoulder

2

▼▼▼▼▼

2

▼▼▼▼▼

G00

1667

G00

16663 4 5 6 70 1 2

Min

Tegafur

5-Fluorouracil

96-0

095

HN

NH

O

FO

O

N

NHF

O O

G00

1673

1. Methcathinone (100µg/mL)2. (+/-) Ephedrine (200µg/mL)

Min0

2

1

876431 2 5

Liqu

idC

hrom

atog

raph

y

SUPELCO

123

Ord

er:

1.8

00

.32

5.3

01

0 T

ec

hn

ica

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ice

: 1

.80

0.3

59

.30

41

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Procainamides (HPLC)Column: Discovery HS F5, 15cm x 4.6mm ID, 5µm particlesCat. No.: 567516-U

Mobile Phase: (65:35) 25 mM ammonium phosphate (pH 7):CH 3CNFlow Rate: 1.0mL/min


Sample: 50µg/mL of each

Sinus Medications (HPLC)Column: Discovery RP-AmideC16, 15cm x 4.6mm ID, 5µm particlesCat. No.: 505013



Stimulants, Amphetamine (HPLC)Column: Suplex pKb-100, 15cm x 4.6mm ID, 5µm particlesCat. No.: 58932

Mobile Phase: acetonitrile:25mM K2HPO4, pH 7.0 with phosphoric acid (15:85)Flow Rate: 2mL/min

Det.: UV, 254nmInj.: 10µL mobile phase (50µg/mL each analyte)

Sulfa Drugs (HPLC)Column: Discovery C18,15cm x 4.6mm ID, 5µm particlesCat. No.: 504955

Mobile Phase: methanol:water with 1% CH 3COOH (20:80)Flow Rate: 1mL/min


0 2Min

1. 5-Fluorocytosine (to)2. N-Acetylprocainamide3. Procainamide

G00

1686

1

2

3

1. Pseudoephedrine2. Paracetamol3. Chlorpheniramine

G00

0087

85-8

2

Amphetamine

Methamphetamine

0 1 2 3 54Min

1

23 4

1. Sulfanilamide2. Sulfathiazole3. Sulfamerazine4. Sulfamethazine

797-

0655

4 60 2Min

0 2 4 6Min

1

2

3

Liqu

idC

hrom

atog

raph

y

SUPELCO

124

Ord

er:

1.8

00

.32

5.3

01

0 T

ec

hn

ica

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: 1

.80

0.3

59

.30

41

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Tricyclic Antidepressants (HPLC)Column: Discovery C8, 15cm x 4.6mm ID, 5µm particlesCat. No.: 59353-U

Mobile Phase: methanol:10mM K2HPO4, pH 7.0Flow Rate: 2mL/min


Tricyclic Antidepressants (HPLC)Column: SUPELCOSIL LC-PCN, 15cm x 4.6mm ID, 5µm particlesCat. No.: 58377

Mobile Phase: acetonitrile:methanol:0.01M dipotassium hydrogen phosphate,pH 7.0 with phosphoric acid (60:15:25)


Det.: UV, 215nmInj.: 50ng each drug

Tricyclic Antidepressants (HPLC)Column: Discovery RP-AmideC16, 15cm x 4.6mm ID, 5µm particlesCat. No.: 505013

Mobile Phase: methanol:25mM KH2PO4, pH 7, 60:40Flow Rate: 2mL/min


Tricyclic Antidepressants (HPLC)Column: Discovery Cyano, 15cm x 4.6mm ID, 5µm particlesCat. No.: 59356-U

Mobile Phase: acetonitrile:methanol:10mM K 2HPO4, pH 7.0, 60:20:20Flow Rate: 1mL/min


1. Desmethyl doxepin2. Protriptyline3. Desipramine4. Nortriptyline5. Doxepin6. Imipramine7. Amitriptyline8. Trimipramine

G00

0541

0 2 4 6 8 10 12 14 16Min

1

2

3

4

56

78

713-

0508

1. Trimipramine (int. std.)2. Doxepin3. Amitriptyline4. Imipramine5. Desmethyl doxepin6. Nortriptyline7. Desipramine8. Protriptyline (int. std.)

4 60 2Min

1

2 3

45

6

78

G00

0727

1. Desmethyl doxepin2. Protriptyline3. Desipramine4. Nortriptyline5. Doxepin6. Imipramine7. Amitriptyline8. Trimipramine

G00

0163

0 2 4 6 8 10 12Min

1

2

3

4

5

6

7

8

1. Trimipramine2. Doxepin3. Amitriptyline4. Imipramine5. Desmethyl doxepin6. Nortriptyline7. Desipramine8. Protriptyline

0 2 4 6Min

14

3

2

5

6

7

8

Liqu

idC

hrom

atog

raph

y

SUPELCO

125

Ord

er:

1.8

00

.32

5.3

01

0 T

ec

hn

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: 1

.80

0.3

59

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o

HPLC ApplicationsFatty Acids, Foods

Paraben Preservatives (HPLC)Columns: 15cm x 4.6mm ID, 5µm particles

conditions for RP-AmideC16, C18,and C8Cat. Nos.: 505013 (RP-AmideC16), 504955 (C18),

59353-U (C8)Mobile Phase: CH3CN:H2O, (40:60)



conditions for CyanoCat. No.: 59356-U (Cyano)

Mobile Phase: CH3CN:H2O, (30:70)Flow Rate: 1mL/min


Fatty Acids, Free (HPLC)Column: SUPELCOSIL LC-8, 15cm x 4.6mm ID, 3µm particlesCat. No.: 58983

Mobile Phase: acetonitrile:tetrahydrofuran: 0.1% phosphoric acid (50.4:21.6:28)Flow Rate: 1mL/min


Fatty Acid Methyl Esters (HPLC)Column: SUPELCOSIL LC-18, 25cm x 4.6mm ID, 5µm particlesCat. No.: 58298

Mobile Phase: acetonitrile:acetone (59.0:41.0, v/v)Flow Rate: 1.0mL/min

Det.: RISample: 10µL of 9% C8 to C18 saturated and unsaturated FAMEs in

mobile phase

0 10 20Min

1

2 3

4

0 2 4 6 8 10Min

1

23

4

1. Methyl paraben2. Ethyl paraben3. Propyl paraben4. Butyl paraben

0 2 4 6 8 10 12Min

1

2 3

4

0 2 4 6Min

1

2

3

4G

0012

35G

0012

34G

0012

36G

0012

37

Discovery Cyano

Discovery C8

Discovery C18


713-

0969

9

0 2 4 6 8 10 12Min

Ox Oxidation products1. 18:3c (2.65µg)2. 16:1c (0.53µg)3. 16:1t (10.6µg)4. 18:2c (0.53µg)5. 18:2t (2.65µg)6. 16:0 (15.8µg)7. 18:1c (10.6µg)8. 18:1t (10.6µg)9. 18:0 (10.6µg)

5

1

8

76

1. C82. C103. 12:0 + 18:34. 18:25. 14:06. 18:17. 16:08. 18:0

0 2 4 6Min

21

3

4

5

6

7

8

797-

0501

4

32Ox

Liqu

idC

hrom

atog

raph

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SUPELCO

126

Ord

er:

1.8

00

.32

5.3

01

0 T

ec

hn

ica

l S

erv

ice

: 1

.80

0.3

59

.30

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oHPLC ApplicationsFlavor, Mycotoxins, Nucleosides, Nucleotides

Flavones (HPLC)Column: Discovery HS PEG, 15cm x 4.6mm ID, 5µm particlesCat. No.: 567416-U

Mobile Phase: 45:55 0.1% formic acid in water :0.1% formic acid in MeOH

Flow Rate: 1.0mL/minTemp.: 30°C,


Aflatoxins (SPE/HPLC)Sample: cornmeal spiked with aflatoxins (30ppb G 2 and B2, 100ppb

G1 and B1) Blend 50g sample for 1 min in 100mLmethanol:water (8:2) and filter.

Extraction Tube: Supelclean LC-CN, 3mL, 0.5g packingCat. No.: 57013

Conditioning: 2mL 0.5% aqueous acetic acidSample Addition: 1mL filtered extract + 4mL 0.5% aqueous acetic acid

Washing: 500µL 20% THF in 0.5% aqueous acetic acid, then 2mLhexane, Dry packing under nitrogen. 3mL 25% THF inhexane, Dry packing 1 min under nitrogen.

Elution: 2 x 2mL 1% THF in methylene chlorideEvaporate eluate to dryness under nitrogen. Reconstitutewith 100µL of methanol, then dilute with 100µL of 0.5%aqueous acetic acid.

Column: SUPELCOSIL LC-18, 25cm x 4.6mm ID, 5µm particles (withguard column)

Cat. No.: 58298Mobile Phase: methanol:acetonitrile:water (22.5:22.5:55)

Flow Rate: 1.5mL/minDet.: VIS, 365nmInj.: 100µL

pBR322 DNA-Hae lll Digest (HPLC)Column: TSKgel DEAE-NPR, 3.5cm x 4.6mm ID,

2.5µm particles ( with 5mm DEAE-NPR guard column)Cat. No.: 813075

Mobile Phase: A = 1M NaCl/25mm Tris-HCl, pH 9.0B = 25mM Tris-HCl, pH 9.0

Flow Rate: 1mL/minTemp.: ambient

Det.: UV, 260nmInj.: 20µL of 50µg/mL HAE III digest of pBR322 DNA

Nucleotides (HPLC)Column: SUPELCOSIL LC-18-T, 15cm x 4.6mm ID, 3µm particlesCat. No.: 58970-U

Mobile Phase: A = 0.1M potassium phosphate buffer/4mM tetrabutylammoniumhydrogen sulfate, pH 6.0B = A:methanol (70:30), pH 7.2

Flow Rate: 1.5mL/minDet.: UV, 254nmInj.: 20µL deionized water (25-530µM each analyte)

1. Flavone2. 7-Hydroxyflavone3. Baicalein4. 5-Hydroxyflavone5. Chrysin6. Luteolin7. Myricetin8. Quercetin

0 2 4 6 8 10 12 14 16Min

7 86

3

5

4

2

1

G00

1665

0

713-

0874

1

2

4

3

862 4Min

10

1. Aflatoxin G22. Aflatoxin G13. Aflatoxin B24. Aflatoxin B1

1614121086420Min

1821

51

184

234 267

192

434

504

540

587

5764

8089104 123

124

213

794-

0036


0 250.5 455 50

20 6221 10024 10025 25

458

1

2

3

4

5

6

7

8

9

10

15

11

12

13

14

201612840Min 71

3-09

51


0 02.5 05 30

10 6013 10017 10018 0

1. HX2. XAN3. INO4. IMP5. NAD+

6. ADO7. AMP8. GDP

9. NADP+

10. ADP-rib11. GTP12. ADP13. NADH14. ATP15. NADPH

Liqu

idC

hrom

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SUPELCO

127

Ord

er:

1.8

00

.32

5.3

01

0 T

ec

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: 1

.80

0.3

59

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HPLC ApplicationsNucleosides, Nucleotides, Organic Acids

Single Species tRNAs (HPLC)Columns: SUPELCOSIL LC-18-S, 15cm x 4.6mm ID, 5µm particlesCat. No.: 58931

Mobile Phase: A = 0.01M KH2PO4 (pH 5.3):methanol, 97.5:2.5B = 0.01M KH2PO4 (pH 5.1):methanol, 80:20C = 0.01M KH2PO4 (pH 4.9):acetonitrile, 65:35

Equilibration: solvent A, 15 min at 1mL/minFlow Rate: 1mL/min

Temp.: 26°CDet.: UV, 210nm, 254nm, 280nmInj.: 50µL hydrolysate buffer (pH 7.8) containing 5µg bovine tRNA Leu

hydrolysate

Deoxyribonucleosides and Ribonucleosides (HPLC)Column: SUPELCOSIL LC-18-S, 15cm x 4.6mm ID, 5µm particlesCat. No.: 58931

Mobile Phase: 0.05M K2HPO4/KH2PO4, pH 4.0:methanolA = 97.5:2.5, B = 80:20


Det.: UV, 254nmInj.: nucleoside standards in water

Plant Growth Regulators (HPLC)Column: SUPELCOSIL LC-18-DB, 3.3cm x 4.6mm ID, 3µm particlesCat. No.: 58978

Mobile Phase: A = 1% acetic acid/1mM tetrabutylammonium phosphate inwater, pH 2.8B = acetonitrile


Det.: UV, 280nmInj.: compounds in 13:87 A:B

713-

0501

, 068

8

Figure provided by Dr. C.W. Gehrke and Mr. K.C. Kuo of the InterdisciplinaryChromatography-Mass Spectrometry Laboratories, University of Missouri(Columbia, MO, USA).

Gradient ProgramTime (min) %A %B %C

0 100 — —7.2 100 — —

12 90 10 —15 75 25 —19.2 40 60 —21.6 38 62 —27 — 100 —48 — — 10055 — — 100

0 10 3020Min

Detection at 280nm,50mAU

UCΨ

m1A

m5C

G

T

Um

Ψm

m1G

m2GA

m22Gm3C

C UΨ

hU

0 10


Min

Selective,Quantitative Analysis


Figure provided by Dr. C. W. Gehrke and Mr. K. C. Kuo, University of Missouri-Columbia, Experimental Station Chemical Laboratories, Columbia, MO USA.

0

m6dA

dA

A

dI

dGU

m3dC

m1dA

C

dU

hm5Um5dC

I

GdT

dC

5'-dA

15105

713-

0955Min


0 1003 100

12 015 019.2 40

713-

0852

1. 6-Benzylaminopurine riboside (445ng)2. Indole-3-acetic acid (222ng)3. Abscisic acid (222ng)4. α-Naphthalene acetamide (222ng)

I Abscisic acid impurity5. Colchicine (555ng)6. Indole-3-propanoic acid (222ng)7. p-Chlorophenoxy acetic acid (333.5ng)8. Indole-3-butyric acid (333.5ng)9. α-Naphthalene acetic acid (333.5ng)

10. β-Naphthalene acetic acid (333.5ng)11. 2,4-Dichlorophenoxy acetic acid (555ng)12. Indole-3-acetic acid ethyl ester (445ng)13. 2,4,5-Trichlorophenoxy acetic acid (555ng)


0 131.25 20

5.0-5.5 356.5-7.5 13

Kinetin(222ng)

310

13

10

8

7

I

63

2

15

4

1112

9

4 5 6Min

2

Liqu

idC

hrom

atog

raph

y

SUPELCO

128

Ord

er:

1.8

00

.32

5.3

01

0 T

ec

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ica

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: 1

.80

0.3

59

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oHPLC ApplicationsOrganic Acids

Organic Acids (HPLC)Column: Discovery C18, 15cm x 4.6mm ID, 5µm particlesCat. No.: 504955

Mobile Phase: methanol:water, 0.1% TFA (40:60)Flow Rate: 1mL/min

Temp.: 20°CDet.: UV, 254nmInj.: 10µL, 1mg/mL each analyte

ααααα-Hydroxy Aliphatic Acids (HPLC)Column: Discovery RP-AmideC16, 15cm x 4.6mm ID, 5µm particlesCat. No.: 505013

Mobile Phase: 25mM KH2PO4, pH 3.0Flow Rate: 1mL/min


Organic Acids (HPLC)Column: SUPELCOGEL C-610H, 30cm x 7.8mm IDCat. No.: 59320-U

Mobile Phase: 0.1% phosphoric acidFlow Rate: 0.5mL/min


Organic Acids (HPLC)Columns: 5cm x 4.6mm ID, 5µm particlesCat. Nos.: 504947 (C18), 568520-U (HS C18)

Mobile Phase: 60:40 0.1%TFA in water:MeOHFlow Rate: 2.0mL/min


797-

0657

1. Homovanillic acid2. Sorbic acid3. Salicylic acid4. p-Toluic acid

0 2 4 6

12

3

4

Min

1. Tartaric acid2. Malic acid3. Lactic acid4. Citric acid

G00

0624

0 2 4

12

3

4

Min

6

794-

0231

1

3

45 6

7

2

8

10 12Min

8 14

1. Oxalic (0.4µg)2. Citric (5µg)3. Tartaric (5µg)4. Malic (6.2µg)5. Succinic (12.5µg)6. Formic (7.5µg)7. Acetic (12.5µg)8. Fumaric (impurity)

1.0 2.0 3.0Min

1

2

34

1. Homovanillic acid (0.0625µg/mL)2. Sorbic acid (0.00625µg/mL)3. Salicylic acid (0.0625µg/mL)4. p-Toluic acid (0.00625µg/mL)

Discovery C18

Discovery HS C18

1.0 2.0 3.0 4.0Min

1

2

34

G00

1431

G00

1432

Liqu

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SUPELCO

129

Ord

er:

1.8

00

.32

5.3

01

0 T

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HPLC ApplicationsOrganic Acids, Peptides and Proteins

ααααα-Amylase, Crude (HPLC)Column: TSKgel Ether-5PW, 7.5cm x 7.5mm ID, 10µm particlesCat. No.: 808641

Mobile Phase: A = 0.1M phosphate buffer + 1.1M Na 2SO4, pH 7.0B = 0.1M phosphate bufferA to B over 60 min

Flow Rate: 1.0mL/minDet.: UV, 280nm

Hippuric and Methylhippuric Acids (HPLC)Column: SUPELCOSIL LC-18, 7.5cm x 4.6mm ID, 3µm particlesCat. No.: 58984

Mobile Phase: tetrahydrofuran:10mM KH 2PO4, pH 3.0 with phosphoric acid(3:97)

Flow Rate: 1.5mL/minDet.: UV, 260nmInj.: 10µL spiked urine

(300µg/mL HA, o-MHA; 150µg/mL m-MHA, p-MHA)

Amyloid ß-Protein Fragment 10-35 (HPLC)Columns: Discovery BIO Wide Pore C18, 25cm x 4.6mm ID, 5µm particlesCat. No.: 568223-U

Mobile Phase: (A) 0.1% TFA in water(B) 25:75, (0.1% TFA in water): (0.1% TFA in CH3CN)

Flow Rate: 1.3mL/minTemp: 30°C

Det.: UV, 215nmInj.: 10µL, 1mg/mL in water

Gradient: 35-55%B in 20 min, 55%B for 5 min, 35%B for 5 min

Angiotensins (HPLC)Column: Discovery BIO Wide Pore C18, 15cm x 4.6mm ID, 5µm particlesCat. No.: 568222-U

Mobile Phase: (A) 65:35, (10mM NH 4OAc, pH 7):(50% CH3CN in 20mMNH4OAc, pH 7)(B) 25:75, (10mM NH 4OAc, pH 7):(50% CH3CN in 20mMNH4OAc, pH 7)

Flow Rate: 1.0mL/minTemp: ambient

Det.: UV, 215nmInj.: 6µL (10µg each) in water

Gradient: 0-100%B in 12.5 min713-

0961

0Min10 20

713-

0961

20 4 6 8 10Min

3

5

4

2

1

1. Hippuric acid2. o-Methylhippuric acid3. p-Methylhippuric acid impurity4. p-Methylhippuric acid5. m-Methylhippuric acid

200

150

100

50

0

G00

15890 10 20 30

Min

0 2 4 6 8 10 12Min

1

23

G00

1585

Peptide Sequence1. Angiotensin II DRVYIHPF2. Angiotensin III DRVYIHPF3. Angiotensin I DRVYIHPFHL

Liqu

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SUPELCO

130

Ord

er:

1.8

00

.32

5.3

01

0 T

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59

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oHPLC ApplicationsPeptides and Proteins

Angiotensins (HPLC)Column: Discovery C18, 15cm x 4.6mm ID, 5µm particlesCat. No.: 504955

Mobile Phase: (A) 5mM (NH4)H2PO4/NH 4OH, pH 7.0(B) 5mM (NH 4)H2PO4/NH 4OH, pH 7.0: CH3CN (50:50)


Det.: UV, 220nmGradient: 30-60% B in 15 min

Angiotensins Capillary Separation (HPLC)Column: Discovery BIO Wide Pore C18, 10cm x 0.32mm ID,

3µm particlesCat. No.: 65527-U

Solution x: 10mM NH 4OAc/NH 4OH, pH 7.0Solution y: 50:50 (20mM NH 4OAc/NH 4OH, pH 7.0) : CH3CN

Mobile Phase A: x:y, 65:35Mobile Phase B: x:y, 25:75

Flow Rate: 6µL/minDet.: +ES, source 2.5kV, capillary 12V, 130° C

Temp.: ambientInj.: 50pmol

Angiotensins (HPLC)Column: Discovery BIO Wide Pore C8, 15cm x 4.6mm ID, 5µm particlesCat. No.: 568322-U

Mobile Phase: (A) 10mM NH4H2PO4/ NH 4OH, pH 7(B) 50:50, (20mM NH 4H2PO4/ NH 4OH, pH 7):CH3CN

Flow Rate: 1mL/minTemp: 30°C

Det.: UV, 215nmInj.: 6µL in water

Gradient: 30-60% B in 15 min

Antibiotics, Peptide (HPLC)Columns: 15cm x 4.6mm ID, 5µm particlesCat. Nos.: 505013 (RP-AmideC16), 59353-U (C8)

Mobile Phase: 25mM KH2PO4, pH 3:acetonitrile, 60:40Flow Rate: 1mL/min

Temp.: 35°CDet.: UV, 220nmInj.: 10µL 25mM KH 2PO4, pH 3 containing 50µg/mL each analyte

0 10 20Min

2

31

1.Angiotensin I2.Angiotensin II3.Angiotensin III

G00

1085

100

50

00 10 20

Rela

tive

Abun

danc

e

3.341046.6

8.36649.1

4.10931.5

III

III

G00

1718


0 2012.5 100

Min

G00

1503

0 10 20Min

1

2 3

1. Angiotensin II (1.67g/L) (DRVYIHPF)2. Angiotensin III (1.67g/L) D(RVYIHPF)3. Angiotensin I (1.67g/L) (DRVYIHPFHL)

G00

0920

0 2 4 6 8 10Min

1

2

1. Bacitracin A2. Virginiamycin M1

0 2 4 6 8 10Min

G00

0911

12


Discovery C8

Liqu

idC

hrom

atog

raph

y

SUPELCO

131

Ord

er:

1.8

00

.32

5.3

01

0 T

ec

hn

ica

l S

erv

ice

: 1

.80

0.3

59

.30

41

We

b:

ww

w.s

igm

a-a

ldri

ch

.co

m/s

up

elc

o

HPLC ApplicationsPeptides and Proteins

Apohemoglobin, Carboxymethylated, Tryptic Digest(HPLC)

Columns: 15cm x 4.6mm ID, 5µm particlesCat. Nos.: 568222-U (C18), 568322-U (C8), 568422-U (C5)

Mobile Phase: (A) 95:5, (0.1% TFA in H2O):(0.1% TFA in CH3CN); (B) 50:50,(0.1% TFA in H2O):(0.1% TFA in CH3CN);



Sample: carboxymethylated apohemoglobin tryptic digest in 50mMNH4HCO3

Gradient: 0-100% B in 65 min

Apohemoglobin (Horse), Carboxymethylated, TrypticDigest (HPLC)

Column: Discovery BIO Wide Pore C18, 15cm x 2.1mm ID,3µm particles

Cat. No.: 567202-UMobile Phase A: water/0.1% TFAMobile Phase B: 50:50, (water/0.1% TFA) : (CH3CN/0.1% TFA)



Sample: digest

Cytochrome c (HPLC)Column: Discovery BIO Wide Pore C18, 15cm x 4.6mm ID,


Mobile Phase: (A) 70:30, (0.1% TFA in water):(0.1% TFA in CH3CN);(B) 64:36, (0.1% TFA in water):(0.1% TFA in CH3CN)


Det.: UV, 220nmInj.: 12µL each at 0.8mg/mL in 0.1%TFA

Gradient: 0-100%B in 30 min

Carboxyamido-methylated BSA Tryptic Digest (HPLC)Columns: 25cm x 4.6mm ID, 5µm particlesCat. Nos.: 505013 (RP-AmideC16), 504955 (C18)

Mobile Phase: (A) 0.1% TFA, 1% CH3CN in H2O(B) 0.1% TFA, 40% CH3CN

Flow Rate: 1 mL/minDet.: UV, 215nm

Temp.: 35°CGradient: 0-100% B in 130 min

G00

1504

, 05,

06

Discovery BIO Wide Pore C5



Min0 20 40 60

0 20 40 60Min G

0017

19


0 100 050 0 100

0 10 20Min

1

1. Horse cytochrome c2. Rabbit cytochrome c3. Cow cytochrome c4. Pigeon cytochrome c5. Chicken cytochrome c6. Dog cytochrome c

6

4

31 2 5

G00

1581

0 20 40 60 80 100 120Min

0 20 40 60 80 100 120Min

G00

1087

G00

1086


Discovery C18

Liqu

idC

hrom

atog

raph

y

SUPELCO

132

Ord

er:

1.8

00

.32

5.3

01

0 T

ec

hn

ica

l S

erv

ice

: 1

.80

0.3

59

.30

41

We

b:

ww

w.s

igm

a-a

ldri

ch

.co

m/s

up

elc


Carboxymethylated Cytochrome c (Chicken) TrypticDigest (HPLC)


Cat. No.: 567202-UMobile Phase A: water/0.1% TFAMobile Phase B: 50:50, (water/0.1% TFA) : (MeCN/0.1% TFA)


Det.: 215nmInj.: 5µL

Sample: digest

Cytochrome c Tryptic Digest (HPLC)Columns: 15cm x 4.6mm ID, 5µm particlesCat. Nos.: 505013 (RP-AmideC16), 504955 (C18)

Mobile Phase: (A) 0.1% TFA, 3% n-propanol in H2O(B) 0.1% TFA, 3% n-propanol in CH3CN


Gradient: 1 - 37% B in 72 min

Cytochrome c Species Variants (HPLC)Column: ProtEx-SP, 5cm x 4.6mm ID, 5µm particlesCat. No.: 54740-U

Mobile Phase: A = 20mM Bis-Tris HCl, pH 7.0B = A + 0.5M NaCl24-69% B over 20 min

Flow Rate: 0.5mL/minDet.: UV, 280nmInj.: 10µg each variant

Desalting of Proteins (HPLC)Column: Supelguard LC-308, 2cm x 4.6mm ID, 5µm particles,

300Å poresCat. No.: 59511-U

Mobile Phase: A = 0.1% TFA in waterB = 0.09% TFA in acetonitrile

Flow Rate: 2mL/minDet.: UV, 220nmInj.: 40µL of 200µg BSA in 3M guanidine-HCl

0 20 40 60Min

G00

1721

Undigested protein


0 100 050 0 100

0 10 20 30 40 50Min

0 10 20 30 40 50Min

Discovery C18


G00

0308

G00

0307

Figure used with permission of Mitsubishi Chemical Co.

Cytochrome c Source1. Bovine2. Horse3. Rabbit

3

2

1

0 5Min10 15 20

796-

0477

1. Guanidine-HCl2. BSA

1

2

0 0.5 1.51.0 2.0 2.5 3.0Min

3.5


0 200.5 201.0 80

Liqu

idC

hrom

atog

raph

y

SUPELCO

133

Ord

er:

1.8

00

.32

5.3

01

0 T

ec

hn

ica

l S

erv

ice

: 1

.80

0.3

59

.30

41

We

b:

ww

w.s

igm

a-a

ldri

ch

.co

m/s

up

elc

o


Glutathione S-Transferase (HPLC)Column: TSKgel G3000SWXL, 30cm x 7.8mm ID, 5µm particlesCat. No.: 808541

Mobile Phase: 0.3M NaCl/0.05M NaH 2PO4, pH 7.0Flow Rate: 1mL/min

Temp.: 25°CDet.: UV, 220nmInj.: 100µL crude glutathione S-transferase preparation from guinea

pig liver (0.7mg)

Hemoglobin HbA 1c (HPLC)Column: Mono S HR5/5, 10µm particlesCat. No.: 54808

Mobile Phase: A = 0.01M malonate-NaOH, pH 5.7B = 0.3M LiCl in mobile phase A

Flow Rate: 2.0mL/minDet.: VIS, 405nmInj.: 50µL hemolysate

diluted 1:5

Ferredoxin NADP Reductase (HPLC)Column: TSKgel Phenyl-5PW column, 7.5cm x 7.5mm ID,

10µm particlesCat. No.: 807573

Mobile Phase: 60 min decreasing linear gradient from 1.5M to 0M ammoniumsulfate in 0.1M sodium phosphate buffer, pH 7


Det.: UV, 280nmInj.: 3mg (crude) or 0.2mg (standard) Purity Evaluation of Human Growth Hormone (hGH)

(HPLC)Column: ProtEx-DEAE, 5cm x 4.6mm ID, 5µm particlesCat. No.: 54742

Mobile Phase: A = 20mM Tris-HCl, pH 8.0B = A + 0.5M NaCl5-70% B over 30 min

Flow Rate: 0.5mL/minInj.: 10mg hGH

713-

0939

0 5 10 15

Standard

Active fraction (recovered activity = 89%)

Min79

4-04

12

A405nm

100

% fu

ll sc

ale HbA1c

%B

0 5Min

10

100

Reproduced with permission of Amersham Biosciences.

0

Standard

Crude Sample

713-

0922

0 30 60Min

90

FerredoxinNADP reductase

Heated hGH: 60°C, 5 hr.

Untreated hGH

Heated hGH: 70°C, 8 hr.

Figure used with permission of Mitsubishi Chemical Co.

0 5 10 15 20 25 30Min

hGH

Desamido hGH derivative

Desamido hGH derivatives

796-

0659

Liqu

idC

hrom

atog

raph

y

SUPELCO

134

Ord

er:

1.8

00

.32

5.3

01

0 T

ec

hn

ica

l S

erv

ice

: 1

.80

0.3

59

.30

41

We

b:

ww

w.s

igm

a-a

ldri

ch

.co

m/s

up

elc


Immunoglobulins, IgM (HPLC)Medium: Q Sepharose Fast FlowCat. No.: Q1126-100MLColumn: 30cm x 50mm ID

Mobile Phase: A = 0.02M NaH2PO4, pH 6.5B = A + 0.5M NaCl, pH 6.5

Flow Rate: 25cm/hrDet.: UV, 280nmInj.: culture supernatant

Insulin (HPLC)Column: Discovery BIO Wide Pore C8, 15cm x 4.6mm ID, 5µm particlesCat. No.: 568322-U

Mobile Phase: (A) 71:29, (0.1% TFA in H2O):(0.1% TFA in CH3CN);(B) 68:32, (0.1% TFA in H2O):(0.1% TFA in CH3CN)


Det.: UV, 215nmInj.: 50µL in 0.1% TFA

Gradient: 0-100% B in 30min

Immunoglobulins, IgG, Human (HPLC)Column: HiTrap Desalting, 5mLCat. No.: 54822

Mobile Phase: 0.2M sodium carbonate buffer, pH 8.5Flow Rate: syringe use

Det.: UV, 280nmInj.: 0.5mL unbound human IgG in 0.2M sodium carbonate buffer

after coupling to a HiTrap NHS 1mL column.Fraction volume 0.5mL

IGF-1, Insulin-like Growth Factor-1 (HPLC)Column: HiLoad 16/60 Superdex 75 prep grade, 34µm particlesCat. No.: 54802-U

Mobile Phase: 0.15M ammonium acetate, pH 6.0Flow Rate: 0.75mL/min

Det.: UV, 280nmInj.: IGF-1, ZZ fusion protein and

uncleaved material

A280nm

5.0

4.0

3.0

2.0

1.0

100

80

60

40

20Start ofelution

IgMcontainingfraction

200100 300 400Volume, mL

794-

0370


2 4 6 8 10G

0015

07

1. Bovine insulin (5µg)2. Human insulin (5µg)3. Porcine insulin (5µg)

23

1

Min

796-

0403

5 10 15 20

h-lgG

0.5

1.0

1.5

A280

N-Hydroxysuccinimide

Fraction number

2.0


Reproduced with permission of Amersham Biosciences. 794-

0420

A280nm

0.1

0.05

0 1.0 2.0Fraction 1 2 3 4

IGF-1

VOVC

Time (h)

Liqu

idC

hrom

atog

raph

y

SUPELCO

135

Ord

er:

1.8

00

.32

5.3

01

0 T

ec

hn

ica

l S

erv

ice

: 1

.80

0.3

59

.30

41

We

b:

ww

w.s

igm

a-a

ldri

ch

.co

m/s

up

elc

o


Insulin, Bovine (HPLC)Columns: Discovery C8, 5cm x 4.6mm ID, 5µm particlesCat. Nos.: 59353-U

Mobile Phase: 25mM KH2PO4, pH 3:acetonitrile, 60:40Mobile Phase: A = 50mM H2NaPO4, 50mM Na 2SO4, pH 3.0 (w/H 3PO4), 5%

acetonitrileB = 50mM H 2NaPO4, 50mM Na 2SO4, pH 3.0 (w/H 3PO4), 50%acetonitrile


Det.: UV, 220nm

Insulin (HPLC)Column: Discovery BIO Wide Pore C5, 15cm x 4.6mm ID, 5µm particlesCat. No.: 568422-U



Det.: UV, 215nmInj.: 5µL each at 1mg/mL in 0.1%TFA


Lactogen, Human Placental (HPLC)Column: Mono Q HR5/5, 10µm particlesCat. No.: 54807

Mobile Phase: A = 20mM bis-Tris, pH 6.0B = A + 1M NaCl

Flow Rate: 1.0mL/minDet.: UV, 280nmInj.: 2.5mL hPL fraction from size exclusion medium

0

0 10 20Min

A

B

Discovery C8

Discovery C8

Min G00

0765

10 20

1. Subunit A oxidized2. Holoenzyme3. Subunit B oxidized

1

2

3


0 33.318 73.3

Holoenzyme (Sigma I5500)reduced and carboxy-methylated

A. Subunit AB. Subunit B

G00

0766

0 2 4 6 8 10Min

1

2

3

1. Bovine insulin2. Human insulin3. Porcine insulin

G00

1580


0 010 025 1026 40


A280nm

0.1

hPL

0 20

NaCl

Min

794-

0421

Liqu

idC

hrom

atog

raph

y

SUPELCO

136

Ord

er:

1.8

00

.32

5.3

01

0 T

ec

hn

ica

l S

erv

ice

: 1

.80

0.3

59

.30

41

We

b:

ww

w.s

igm

a-a

ldri

ch

.co

m/s

up

elc


ß-Lactoglobulin (Cow), Carboxymethylated, TrypticDigest (HPLC)


Cat. No.: 567202-UMobile Phase A: water/0.1% TFAMobile Phase B: 50:50, (water/0.1% TFA) : (MeCN/0.1% TFA)


Det.: 215nmInj.: 5µL

Sample: digest

ß-Lactoglobulin Tryptic Digest, Capillary Separation(HPLC)


Cat. No.: 65519-UMobile Phase: (A) 0.1% TFA in water;

(B) 0.1% TFA in CH3CNFlow Rate: 14µL/min

Temp.: 30°CInj.: 500pmol (5µL) ß-Lactoglobulin tryptic digest in 50mM NH 4HCO3

Gradient: 5-40%B in 70 minMS conditions: +ESI mode Capillary Temp 130°C, Source Voltage 2.5KV, Capillary

Voltage 12V

Lipoxidase, Commercial (HPLC)Column: TSKgel Phenyl-5PW, 7.5cm x 7.5mm ID,

10µm particlesCat. No.: 807573

Mobile Phase: A = 0.1M phosphate buffer, pH 7.0 + 1.5M (NH 4)2SO4B = 0.1M phosphate buffer, pH 7.0linear gradient, 0% Bto 100% B over 60 min


Liver Extract, Bovine (HPLC)Column: Mono Q HR5/5, 10µm particlesCat. No.: 54807

Mobile Phase: A = 50mM diethanolamine, pH 8.8B = 50mM diethanolamine + 1.0M NaCl, pH 8.8

Flow Rate: 1.0mL/minDet.: UV, 280nmInj.: 100µL, 1.6mg protein

0 20 40 60Min


0 100 050 0 100

100

50

00 10 20 30 40

Min

Rela

tive

Abun

danc

e

12.36573.3

15.90458.9

21.42696.2

17.17337.3

18.46623.4

21.00419.4

22.80818.7

27.66859.2

24.54775.4

29.17555.2

30.49772.1

27.171044.6

34.37522.5

35.06522.6

36.66322.0

37.71522.6

G00

1591

794-

0320

Min0 30 60

794-

0376

NaC

l con

cent

ratio

n M

(----

-)

A28

0 (

)

0.5

0.4

0.3

0.2

0.1

1.0

0.25

0 5 10 15 20Volume, mL


Liqu

idC

hrom

atog

raph

y

SUPELCO

137

Ord

er:

1.8

00

.32

5.3

01

0 T

ec

hn

ica

l S

erv

ice

: 1

.80

0.3

59

.30

41

We

b:

ww

w.s

igm

a-a

ldri

ch

.co

m/s

up

elc

o


Monoclonal Antibody (HPLC)Column: HiTrap HIC cartridgesCat. No.: 54814

Mobile Phase: A = 50mM sodium phosphate, 1.0M ammonium phosphate,pH 7.0B = 50mM sodium phosphate, pH 7.0

Flow Rate: 1.0mL/minDet.: UV, 280nmInj.: 100µL anti-lac cl 507, 8.8mg/mL

Peptides (HPLC)Column: Discovery BIO Wide Pore C18, 5cm x 4.6mm ID, 5µm particlesCat. No.: 568220-U



Det.: UV, 220nmInj.: 5µL, 2.5µg each peptide (Sigma Peptide Mix, Cat. No. H 2016) in

mobile phase AGradient: 2-24%B in 22 min, 8 min at 100%A

Peptides (HPLC)Column: Discovery BIO Wide Pore C5, 15cm x 4.6mm ID, 5µm particlesCat. No.: 568422-U

Mobile Phase: (A) 0.1% PFPA (pentafluoropropionic acid) in water:CH3CN(81:19);(B) 0.1% PFPA in water:CH3CN (62:38)


Det.: UV, 215nmInj.: 10µL, ~0.25µg each peptide (Sigma Peptide Mix, Cat. No. P

2693) in mobile phase AGradient: 0-100%B in 19 min

Peptides, Basic, No TFA (HPLC)Columns: Discovery BIO Wide Pore C18, 15cm x 2.1mm ID,


Mobile Phase A: 25mM formic acid in waterMobile Phase B: 50:50 (25mM formic acid in water) : (20mM formic acid in

CH3CN)Flow Rate: 0.208mL/min


Inj.: 0.5µL (~0.25µg ea peptide)Sample: RP Peptide Ionic Interactions Standard, p/n RPS-I0020

(Alberta Peptide Institute)

796-

0397

- 04

01


HiTrap Phenyl Sepharose HighPerformance

HiTrap ButylSepharose 4 FastFlow

HiTrap OctylSepharose 4 FastFlow

HiTrap PhenylSepharose 6 Fast Flow(low sub)

HiTrap PhenylSepharose 6 Fast Flow(high sub)

10.0 20.0 30.0

0.00

0.10

0.30

0.20

0.40

0.50

Min

1. Gly-Tyr2. Val-Tyr-Val3. Met-Enkephalin4. Leu-Enkephalin5. Angiotensin II

G00

1562

, 63

Initial Injection

0 10 20Min

1

2

3

4 5

after 40,000columns volumes

1

2

3

4 5

G00

1492

, 93,

94

0 2 4 6 8 10 12 14 16 18Min

Peptide Sequence1. Arg8-vasopressin CFQNCPRG-amide; disulfide2. Bradykinin, fragment 1-5 RPPGF3. Oxytocin CYQNCPLG-amide; disulfide4. Met-enkephalin YGGFM5. Luteinizing hormone

releasing hormone PEHWSYGLRPG-amide6. Leu-enkephalin YGGFL7. Bradykinin RPPGFSPFR8. Bombesin PEQRLGNQWAVGHLM-amide9. Substance P RPKPQQFFGLM-amide

98764

312

5

0 10 20 30Min

020

40m

AU

215

nm

G00

1642

Peptide 1: ac-GGGLGGAGGLK-amidePeptide 2: ac-KYGLGGAGGLK-amidePeptide 3: ac-GGALKALKGLK-amidePeptide 4: ac-KYALKALKGLK-amide


0 1545 60

Liqu

idC

hrom

atog

raph

y

SUPELCO

138

Ord

er:

1.8

00

.32

5.3

01

0 T

ec

hn

ica

l S

erv

ice

: 1

.80

0.3

59

.30

41

We

b:

ww

w.s

igm

a-a

ldri

ch

.co

m/s

up

elc


Peptides, Alberta Mix, No TFA (HPLC)Column: Discovery BIO Wide Pore C18, 15cm x 2.1mm ID,


Mobile Phase A: 25mM formic acid in water,Mobile Phase B: 50:50 (25mM formic acid in water) : (20mM formic acid in

CH3CN)a,Flow Rate: 0.208mL/minb

Det.: +ESTemp.: ambient

Inj.: 1µLSample: RP Peptide Performance Standard, p/n RPS-P0010 (Alberta

Peptide Institute)

Peptides, Synthetic (HPLC)Columns: Discovery BIO Wide Pore C18, 15cm x 2.1mm ID, 5µm

particlesCat. No.: 568202-U

Mobile Phase A: 25mM formic acid in waterMobile Phase B: 50:50 (25mM formic acid in water):(20mM formic acid in

CH3CN)Flow Rate: 0.208 mL/min


Inj.: 0.5µL (~0.25µg ea peptide)Sample: RP Peptide Ionic Interactions Standard, p/n RPS-I0020 (Alberta

Peptide Institute)

Peptides, Synthetic (HPLC)TFA:


Cat. No.: 568222-UMobile Phase A: 80:20, (water/0.1%TFA) : (CH3CN/0.1%TFA)Mobile Phase B: 66:34, (water/0.1%TFA) : (CH3CN/0.1%TFA)

Flow Rate: 1.0mL/min;Det.: UV, 215nm

Temp.: 30°CInj.: 10µL Sigma Peptide Mix P2693 (~25µg ea. Peptide)

PFPA:Column: Discovery BIO Wide Pore C18, 15cm x 4.6mm ID,


Mobile Phase A: 81:19, (water/0.1%PFPA) : (CH3CN/0.1%PFPA)Mobile Phase B: 62:38, (water/0.1%PFPA) : (CH3CN/0.1%PFPA)


Temp.: 30°CInj.: 10µL Sigma Peptide Mix P2693 (~25µg ea. Peptide)

G00

1638

100

%

0

1

23

4

5

Peptide 1: ac-RGAGGLGLGK-amidePeptide 2: ac-RGGGGLGLGK-amidePeptide 3: ac-RGAGGLGLGK-amidePeptide 4: ac-RGVGGLGLGK-amidePeptide 5: ac-RGVVGLGLGK-amide


0 90 1020 50 50

0% TFA (25mM formic acid), 1µL injection

Min0 10 20

a) molarity of formic acid adjusted to provide minimum baseline driftb) linear velocity equal to 1mL/min on 4.6mm ID columns

G00

1642

0 2 4 6 8 10 12 14 16 18Min

G00

1593

0 2 4 6 8 10 12 14Min

1 23

4

5 67

89

Peak Peptide Amino Acid Sequence1. Arg8-vassopressin CYFQNCPRG-amide; disulfide2. Bradykinin, fragment 1-5 RPPGF3. Oxytocin CYIQNCPLG-amide; disulfide4. LHRH* pEHWSYGLRPG-amide **5. Met-enkephalin YGGFM6. Bradykinin RPPGFSPFR7. Leu-enkephalin YGGFL8. Bombesin pEQRLGNQWAVGHLM-amide **9. Substance P RPKPQQFFGLM-amide

* Luteinizing Hormone Releasing Hormone** pE is pyroglutamate


0 100 014 0 100

TFA mobile phase:

12

3

4

5

6 78

9

G00

1594


0 100 019 0 100

PFPA (penta-fluoropropionic acid) mobile phase:

0 10 20 30Min

Peptide 1: ac-GGGLGGAGGLK-amidePeptide 2: ac-KYGLGGAGGLK-amidePeptide 3: ac-GGALKALKGLK-amidePeptide 4: ac-KYALKALKGLK-amide


0 1545 60

Liqu

idC

hrom

atog

raph

y

SUPELCO

139

Ord

er:

1.8

00

.32

5.3

01

0 T

ec

hn

ica

l S

erv

ice

: 1

.80

0.3

59

.30

41

We

b:

ww

w.s

igm

a-a

ldri

ch

.co

m/s

up

elc

o


Peroxidase (HPLC)Column: TSKgel G3000SWXL, 30cm x 7.8mm ID, 5µm particlesCat. No.: 808541

Mobile Phase: 0.3M NaCl/0.05M phosphate buffer,pH 7.0


Det.: UV, 220nmInj.: 100µL crude peroxidase preparation from Japanese horseradish

(0.15mg) Proteins (HPLC)Columns: Discovery BIO Wide Pore C5, 15cm x 4.6mm ID, 5µm particlesCat. No. : 568422-U

Mobile Phase: 75:25, (0.1% TFA in water):(0.1% TFA in CH3CN);Flow Rate: 1.0mL/min

Temp: ambientDet.: UV, 220nmInj.: 12µL in 0.1%TFA


Proteins (HPLC)Column: TSKgel CM-5PW, 7.5cm x 7.5mm ID, 10µm particlesCat. No.: 813068

Mobile Phase: A = 0.1M NaH2PO4 buffer, pH 7.0B = 0.5M NaCl, A to B in 60 min


Proteins (HPLC)Column: ProtEx-DEAE or ProtEx-SP, 5cm x 4.6mm ID, 5µm particlesCat. No.: 54742 (ProtEx-DEAE) or 54740-U (ProtEx-SP)

Mobile Phase: Left: A = 20mM Tris-HCl, pH 8.0, B = A + 0.5M NaCl5-100% B over 15 minRight: A = 20mM sodium phosphate, pH 7.0,B = A + 0.5M NaCl5-100% B over 15 min

Flow Rate: 0.5mL/minDet.: UV, 280nmInj.: 10µg trypsin inhibitor, 5µg conalbumin, myoglobin or

10µg ribonuclease A, 5µg cytochrome c, a-chymotrypsinogen A

0 5 10 15

Recovered Activity = 98%

Min 713-

0940

0 10 20Min

0

200

400

600

1

2

3

4

5

6

G00

1488

Impurity inLysozyme

Impurity inMyoglobin

1. RNase (13.7kDa, 1mg/mL)2. Cytochrome c (12.4kDa, 1mg/mL)3. Lysozyme (14.3kDa, 1mg/mL)4. BSA (67.0kDa, 2.5mg/mL)5. Myoglobin (18.8kDa, 1mg/mL)6. Ovalbumin (45.3kDa, 3.5mg/mL)

713-

1002

1. Trypsinogen2. Ribonuclease A3. α-Chymotrypsinogen4. Cytochrome c5. Lysozyme 5

43

21

403020100Min

Abs

orba

nce

(280

nm)

1

2

3

151050Time (Min)

Abs

orba

nce

(280

nm)

020 2015105Time (Min)

1

2

3

ProtEx-SP1. Ribonuclease A2. α-Chymotrypsinogen A3. Cytochrome c

ProtEx-DEAE1. Myoglobin2. Conalbumin3. Trypsin inhibitor

Figures used with permission of Mitsubishi Chemical Co.

796-

0657

796-

0658

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SUPELCO

140

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Proteins (HPLC) Column A: TSKgel DEAE-NPR, 3.5cm x 4.6mm ID, 2.5µm particles

Cat. No.: 813075Column B: TSKgel DEAE-5PW, 7.5cm x 7.5mm ID, 10µm particles

Cat. No.: 807164Mobile Phase: A = 20mM Tris-HCl buffer, pH 8.0

B = 0.5M NaCl in ADet.: UV, 280nm

Proteins (HPLC)Column A: TSKgel Butyl-NPR, 3.5cm x 4.6mm ID, 2.5µm particles

Cat. No.: 814947Column B: TSKgel Phenyl-5PW, 7.5cm x 7.5mm ID, 10µm particles

Cat. No.: 807573Column C: TSKgel Ether-5PW, 7.5cm x 7.5mm ID, 10µm particles

Cat. No.: 808641Mobile Phase: A = 0.1M phosphate buffer, pH 7.0 plus (NH 4)2SO4, 2.3M to

0M in12 minB & C = 0.1M phosphate buffer, pH 7.0 plus (NH 4)2SO4, 1.8M to0M in 60 min

Flow Rate: 1mL/minDet.: UV, 280nm

713-

1020

, 713

-102

12

1

3020Min

DEAE-NPRA to B in 10 min

at 1.5mL/min

DEAE-5PWA to B in 60 min

at 1mL/min

1. Ovalbumin2. Trypsin inhibitor

2

1

65432Min

713-

1025

, 102

6, 1

027

A – Butyl-NPR

C – Ether-5PW

1. Myoglobin2. Ribonuclease3. Lysozyme4. α-Chymotrypsin5. β-Chymotrypsinogen

B – Phenyl-5PW

108642Min

0

5

4

3

2

1

4

5

3

2

1

5

4

3

2

1

60300Min

45150Min

30

1

Liqu

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SUPELCO

141

Ord

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00

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5.3

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0 T

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o

0 2 4 6 8 10 12Min


Substance P and Its Fragments (HPLC)Column: Discovery C18, 15cm x 4.6mm ID, 5µm particlesCat. No.: 504955

Mobile Phase: A = water + 0.1% TFAB = acetonitrile + 0.1% TFA


Det.: UV, 220nmInj.: Substance P in initial mobile phase

Trypsin (HPLC)Column: TSKgel Butyl-NPR, 3.5cm x 4.6mm ID, 2.5µm particlesCat. No.: 814947

Mobile Phase: 2M to 0M ammonium sulfate in 20mM Tris-HCI buffer, pH 7.5 in10 min

Flow Rate: 1.0mL/minDet.: UV, 280nmInj.: commercial trypsin sample

Proteins, Egg White (HPLC)Column: HiTrap Chelating, 1mLCat. No.: 54834

Mobile Phase: A = 0.2M NaH2PO4, 1M NaCl, pH 7.2B = 0.02M NaH2PO4, 1M NH 4Cl, pH 7.2


Det.: UV, 280nmInj.: 200µL egg white

Proteins, Milk (HPLC)Column: HiTrap SP, 1mLCat. No.: 54817

Mobile Phase: A = 50mM Na acetate, pH 6.0B = 50mM Na acetate/1.0M NaCl , pH 6.0

Flow Rate: 1mL/minDet.: UV, 280nmInj.: 0.5mL filtered casein-precipitated human milk (buffer exchanged

to mobile phase A)

G00

0322

1. Fragment 1-42. Fragment 1-73. Fragment 1-94. Fragment 8-115. Holopeptide6. Fragment 2-117. Fragment 7-11


0 56 29

11 34

6

4

3

1

2

5

7

0 2 4 106 8Min

α-Trypsin

β-Trypsin

713-

1204

794-

0425


1.0A280nm

0.5

0

5 2010 15 25 mL


0 08 0

24 10048 100


0493

Volume, mL

A280nm 100% B

40% B

10% B

Fract. 16Fract. 17

Fract. 24

0.5

0.4

0.3

0.2

0.1

0

5 10 302515 20

Fraction 2

Fraction 9

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oHPLC ApplicationsPeptides, Proteins, Pesticides

Pesticides and Herbicides (HPLC)Column: SUPELCOSIL LC-ABZ, 25cm x 4.6mm ID, 5µm particlesCat. No.: 59142

Mobile Phase: 10% to 90% acetonitrile in water at 0.5%/minFlow Rate: 1mL/min

Temp.: 40°CDet.: UV, 225nmInj.: 50µL water, spiked with analytes

Venom, White-Faced Hornet (HPLC)Column: Mono S HR5/5, 10µm particlesCat. No.: 54808

Mobile Phase: A = 50mM BICINE, pH 8.4B = A + 0.35M NaCl

Flow Rate: 1.0mL/minDet.: UV, 280nmInj.: 7mg venom in mobile phase A

Organo-Acid Pesticides (HPLC)Columns: 15cm x 4.6mm ID, 5µm particlesCat. Nos.: 504955 (C18), 59353-U (C8)

Mobile Phase: 60:40 25mM potassium phosphate, (pH 2.3):CH 3CNFlow Rate: 2.0mL/min


Inj.: 1µL

Urea Pesticides (HPLC)Column: Discovery Cyano, 15cm x 4.6mm ID, 5µm particlesCat. No.: 59356-U



794-

0033

Figures reproduced courtesy of Dr. Claude Corvi, Laboratories Cantonal deChimie, Geneva, Switzerland.

20 40 60 10080Min

8

11 12

13

141715

13

456

7

10

9218

16

19

U

20

0 120 140

1. Caffeine (100ng)2. Metamitron (50ng)3. Fenuron (50ng)4. Metoxuron (50ng)5. Simazine (5ng)6. Bromacil (50ng)7. Cyanazine (50ng)8. Atrazine (5ng)9. Carbaryl (5ng)

10. Isoproturon (50ng)11. Propham (50ng)

12. Propazine (50ng)13. Terbutylazine (5ng)14. Linuron (50ng)15. Propanil (50ng)16. Prometryn (50ng)17. Fenamiphos (50ng)18. Fenitrothion (50ng)19. Parathion (50ng)20. Permethrin (50ng)

U Unknown


0411

A280

50

%B100

0 10 3020Min

Phospholipase A

Phospholipase B

Hyaluronidase

Acid phosophatase

40

1. Solvent2. Dalapon3. 2,4-D4. 2,4-DB5. 2,4,5-T6. 2,4-D Methyl ester7. 2,4,5-T Methyl ester8. 2,4-DB Methyl ester

G00

1042

, 104

1

0 2 4 6 8 10 12 14Min

1

2

34

5

6 7

8

0 10 20Min

2

3 4

5

6 7

8

Discovery C18

Discovery C8

1.0 2.0 3.0Min

1. Fenuron2. Monuron3. Diuron4. Linuron

G00

1067

3

1

2

4

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143

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00

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HPLC ApplicationsPesticides

Carbamate and Urea Pesticides (HPLC)Column: SUPELCOSIL LC-8, 15cm x 4.6mm ID, 5µm particlesCat. No.: 58220-U

Mobile Phase: acetonitrile:water gradient, 18:82 to 65:35 in 9 min, hold 3 minFlow Rate: 2.0mL/min


Organophosphorus Pesticides (HPLC)Column: Discovery RP-AmideC16, 15cm x 4.6mm ID, 5µm particlesCat. No.: 505013

Mobile Phase: acetonitrile:water, 60:40Flow Rate: 1mL/ min


Urea/Carbamate/Triazine/An iline Pesticides (HPLC)Column: Discovery C18, 15cm x 4.6mm ID, 5µm particlesCat. No.: 504955

Mobile Phase: A = acetonitrileB = water



Organophosphorus Pesticides (HPLC)Column: Discovery Cyano, 15cm x 4.6mm ID, 5µm particlesCat. No.: 59356-U



1. Methomyl (80ng)2. Oxamyl (80ng)3. Fenuron (80ng)4. Monuron (80ng)5. Carbofuran (1000ng)6. Propoxur (800ng)7. Carbaryl (200ng)8. Fluometuron (80ng)

9. Diuron (80ng)10. Propham (80ng)11. Siduron (80ng)12. Linuron (80ng)13. Chlorpropham (80ng)14. Barban (80ng)15. Neburon (80ng)

794-

0803

1. Dichlorvos2. Guthion3. Methyl parathiion4. Ethoprophos5. Disulfoton6. Fenchlorvos7. Chlorpyrifos8. Prothiophos

G00

1031

0 10 20Min

10

9

8

76

4

3

1

2 5

11

12

1314

15

Min0 2 4 6 8 10 12

6

4

3

1

2

5

7

8

1. Acetone2. Oxamyl3. Methomyl4. Fenuron5. Aldicarb6. Simazine7. Monuron8. Cyanazin9. Carbofuran

10. Atrazine11. Carbaryl12. Diuron13. Propachlor14. Linuron15. Chlorpropham16. Barban

15

0 2 4 6 8 10 12 14 16 18Min

1

23

45

6

7

8

9

10

11

12

13

14

16

G00

0948


0 852 85

15 42.517 42.5

G00

1030

1. Dichlorvos2. Ethoprophos3. Guthion4. Methyl parathion5. Disulfoton6. Fenchlorvos7. Chlorpyrifos8. Prothiophos

87

6

5

0 10 20 30 40Min

1

42

3

Liqu

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SUPELCO

144

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oHPLC ApplicationsPhenols, Polymers, Oligomers

Phenols (HPLC)Column: SUPELCOSIL LC-8, 15cm x 4.6mm ID, 5µm particlesCat. No.: 58220-U

Mobile Phase: 1% acetic acid/methanol:1% acetic acid/water, 35:65 to 100:0(20 min), return to 35:65 (5 min)


Det.: UV, 280nm

Epoxy Resin, Epikote 1001 (HPLC)Column: TSKgel G3000HXL, 30cm x 7.8mm ID, 6µm particles*Cat. No.: 816136

Mobile Phase: tetrahydrofuranFlow Rate: 1mL/min

Det.: UV, 254nm*Two columns in series.

Polyethylene Glycol 200 (HPLC)Column: TSKgel G-OLIGO-PW, 30cm x 7.8mm ID, 6µm particles*Cat. No.: 808031

Mobile Phase: distilled waterFlow Rate: 1mL/min

Det.: refractive index*Two columns in series.

Nonionic Surfactants (HPLC)Column: SUPELCOSIL LC-Diol, 25cm x 4.6mm ID, 5µm particlesCat. No.: 58201

Mobile Phase: A = hexane:methylene chloride (95:5)B = hexane:methylene chloride:methanol (50:40:10)


Det.: UV, 280nmInj.: 10µL methylene chloride containing 20µg surfactant

1. Phenol2. 4-Nitrophenol3. 2,4-Dinitrophenol4. 2-Nitrophenol5. o-Chlorophenol6. 4,6-Dinitrophenol7. 2,4-Dimethylphenol8. 4-Chloro-3-methylphenol9. 2,4-Dichlorophenol

10. 2,4,6-Trichlorophenol11. Pentachlorophenol

713-

1031

109

8

7

6

4

3

1

2

511

Min0 2 4 6 8 10 12 14 16 18 20

713-

0783

5 10 15 20Min

713-

0778

n=2

n=3

n=4

15 20Min

n=9

n=8

n=7

n=6

n=5

713-

0782

, 106

8

0 4 8 12 16 20 24 0 12 16 20 24Min Min

20 Ethoxy Groups(Nonylphenol)

9-10 Ethoxy Groups(Octylphenol)

84


0 308 50

15 6025 8035 100

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o

HPLC ApplicationsPolynuclear Aromatic Hydrocarbons

Metabolites of 7,12-Dimethylbenzo(a)anthracene(HPLC)

Column: SUPELCOSIL LC-18, 25cm x 4.6mm ID, 5µm particlesCat. No.: 58298

Mobile Phase: methanol:water, 50:50 (10 min) to 100:0 at 2.5% /minFlow Rate: 0.8mL/min

Det.: fluorescenceInj.: rat liver, 9000 x g supernatant fraction from Aroclor-treated

animals

PAHs in Water (SPME/HPLC)Sample: 5µL PAH mix (Cat. No. 4-8743) in 5mL water

SPME Fiber: polydimethylsiloxane, 100µm filmCat. No.: 57301

Extraction: immersion, 30 min (rapid stirring)Desorption*: static, 200µL acetonitrile:water, 40:60, 2 min

Column: SUPELCOSIL LC-PAH, 15cm x 4.6mm ID, 5µm particlesCat. No.: 58318

Mobile Phase: acetonitrile:water gradient (see program)Flow Rate: 0-2 min: 0.2mL/min 2-45 min: 1.0mL/min

Det.: UV, 254nm*Requires SPME/HPLC interface.

Chelating Agent, DERT Test (HPLC)Column: SUPELCOGEL TPR-100, 15cm x 4.6mm ID, 5µm particlesCat. No.: 59154

Mobile Phase: ammonium acetate (25mM, pH 7):acetonitrile (55:45)Flow Rate: 1.0mL/min

Temp: ambientDet.: UV, 254nmInj.: 10µL

Figure provided by Drs. J. Milner and J. Grunau, University of Illinois,Urbana, Illinois, USA.

1. 2-OH-DMBA(t-diol)2. 7-OHM-12-MBA-(t-10,11-diol)3. 7-M-12-OHMBA-(t-8,9-diol)4. 7-OHM-12-MBA-(t-8,9-diol)5. 7-OHM-(t-3,4-diol)6. Mixed diols, incl. DMBA-

(t-8,9-diol & t-10, 11-diol)7. Position of 7,12-bis-OHMBA8. 7-OHM-12-MBA-2-ol9. 7-OHM-12-MBA-(3-ol)

10. 7-M-12-OHMNA-4-ol

11. 7-OHM-12-MBA-4-ol12. 7-M-12-OHMBA-(3-ol)13. 7-M-12-OHMBA-2-ol14. 7-OHM-12-MBA15. 7-M-12-OHMBA16. DMBA-2-ol17. DMBA-3-ol18. DMBA-4-ol19. DMBA

(parentheses = tentative identifications)

713-

1043

1

24

5

109

8

12

1315

17

6 11 19181614

37

0 4844403632282420161284Min

796-

0086

1. Naphthalene 1000ng/mL2. Acenaphthylene 2000ng/mL3. Acenaphthene 1000ng/mL4. Fluorene 200ng/mL5. Phenanthrene 100ng/mL6. Anthracene 100ng/mL7. Fluoranthene 200ng/mL8. Pyrene 100ng/mL9. Benzo(a)anthracene100ng/mL

10. Chrysene 100ng/mL11. Benzo(b)fluoranthene 200ng/mL12. Benzo(k)fluoranthene 100ng/mL13. Benzo(a)pyrene 100ng/mL14. Dibenzo(a,h)anthracene 200ng/mL15. Benzo(ghi)perylene 200ng/mL16. Indeno(1,2,3-cd)pyrene 100ng/mL

Gradient ProgramTime (min) % ACN

0 505 50

30 10045 100

Flow increased at 2.0 min

1

2

3

4

5

16

1514

13

12

11

10

9

8

7

6

0 10 20Min

30 40

796-

0042

1

2

10 432Min.

1. 2,7-Dihydroxynaphthalene (300µg/mL)2. 2,3-Dihydroxynaphthalene (1000µg/mL)

5 6 7 8

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oHPLC ApplicationsSemivolatiles, Toxins, Vitamins

Vitamins, Fat Soluble (D 2 and D 3) (HPLC)Column: Discovery HS C18, 15cm x 4.6mm ID, 3µm particlesCat. No.: 569252-U

Mobile Phase: 100% CH3CNFlow Rate: 0.8mL/min

Temp: 30°CDet.: UV, 290nmInj.: 10µL, 50µg/mL of each analyte

Vitamins, Fat Soluble (A and E) (HPLC)Column: 15cm x 4.6mm ID, 5µm particles

Cat. Nos.: 59353-U (C8), 504955 (C18)Mobile Phase: Discovery C18, MeOH:H2O (95:5),

Discovery C8 CH3CN:H2O (90:10)Flow Rate: 2mL/min

Temp: 30°CDet.: UV, 290nmInj.: 10µL

Bufalin Toxin (HPLC)Column: Discovery BIO Wide Pore C18, 25cm x 4.6mm ID,


Mobile Phase: A = 0.1% TFA in waterB = 0.1% TFA in acetonitrile:water (3:1)


Det.: UV, 298nmInj.: 10µL bufalin, 0.5mg/mL in acetonitrile

Benzidines by US EPA Method 605 (HPLC)Column: SUPELCOSIL LC-1, 15cm x 4.6mm ID, 5µm particlesCat. No.: 58210-U

Mobile Phase: 0.1M acetate buffer (pH 4.7):acetonitrile, 80:20Flow Rate: 1mL/min

Det.: UV, 254nmInj.: 10µL (0.5µg each analyte)

0 10 20Min

1. Ergocalciferol (Vitamin D2)2. Cholecalciferol (Vitamin D3)

G00

1419

0 2 4 6 8 10Min

0 2 4 6 8Min

1. Retinol acetate (Vitamin A acetate), 50µg/mL2. δ-Tocopherol, 165µg/mL3. γ-Tocopherol, 200µg/mL4. α-Tocopherol (Vitamin E), 292µg/mL5. α-Tocopherol acetate (Vitamin E acetate), 405µg/mL

43

1

2

5

431

2

5

Discovery C18

Discovery C8Gradient ProgramTime (min) %B

0 3515 8017 9019 9021 3529 35

G00

1722

794-

0800

0 42 6 8 10Min

Benzidine

3,3’-Dichlorobenzidine

2

1

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HPLC ApplicationsVitamins

Vitamins, Water Soluble (HPLC)Column: Discovery RP-Amide, 15cm x 4.6mm ID, 5µm particles

Cat. Nos.: 505013 (RP-AmideC16),Mobile Phase: MeOH:50mM KH2PO4 , pH 4.5 (30:70)


Column: Discovery C18 or C8, 15cm x 4.6mm ID, 5µm particlesCat. Nos.: 504955 (C18), 59353-U (C8)

Mobile Phase: 50mM KH2PO4, pH 4.5 to MeOH:50mM KH 2PO4, pH4.(30:70) in12 min (Discovery C8) or 8 min (Discovery C18)

Flow Rate: 1mL/minTemp.: ambient temp.


Vitamins, Fat Soluble (A and E) (HPLC)Column: SUPELCOSILLC-18, 25cm x 4.6mm ID, 5µm particlesCat. No.: 58298

Mobile Phase: methanol:deionized water (98:2)Flow Rate: 2mL/min

Det.: UV, 325nm (retinol, retinyl acetate) or 290nm (tocopherols,tocopherol acetate)

Inj.: 20µL methanol(0.2-1mg/mL each analyte)

Vitamins, Fat Soluble (A and E) (HPLC)Column: SUPELCOSIL LC-Si, 15cm x 4.6mm ID, 5µm particlesCat. No.: 58200-U

Mobile Phase: hexane:amyl alcohol (99.65:0.35)Flow Rate: 2mL/min


G00

0790

1. Ascorbic acid2. Nicotinic acid3. Thiamine4. Pyridoxine5. Nicotinamide6. Folic acid7. Riboflavin

0 2 4 6 8 10 12 14Min


0 08 30

7

6

4

3

1

2

5

Discovery C18

0 10 20Min

13

2

45

6

7

0 2 4 6 8 10 12Min

12

3

4

5

67


Discovery C8

G00

1171

G00

0789

712-

0679

0 2 4 6 8 10 12Min

3

4 5

61

2

1. Retinol2. Retinyl acetate3. δ-Tocopherol4. γ-Tocopherol5. α-Tocopherol6. Tocopherol acetate

Min

1. α-Tocopherol (0.3mg/mL)2. β-Tocopherol (0.4mg/mL)3. γ-Tocopherol (0.3mg/mL)4. δ-Tocopherol (1.3mg/mL)5. Ergocalciferol (1.0mg/mL)6. Retinol (0.7mg/mL)

713-

0881

1

4

5

6

0 5 10 15 20

2

3

Liquid Chromatography - SUPELCO 110-147.pdf · Liquid Chromatography SUPELCO 112 Order:...

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