Colonne Application

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Colonnes HPLC KROMASILApplicationsAMINO ACIDSAmino acids, PTC derivatives18 amino acids as phenylthiocarbamyl (PTC) derivatives. (ref. 7)2= PTC-glutamic acid 9=PTC-proline 16=PTC-norleucine (I. S.) 1=PTC-aspartic acid 8=PTC-alanine 15=PTC-leucine

3=PTC-serine

10=PTC-tyrosine 17=PTC-phenylalanine

4=PTC-glycine

11=PTC-valine 18=PTC-tryptophan

5=PTC-histidine 16 14 15 13 17 18

12=PTC-methionine 19=PTC-lysine

4 7 1 3 2 5 6 8 10 9

11 12

19 6=PTC-arginine 13=PTC-cysteine

7=PTC-threonine0 5 10 15 20 25 30 35 (min.) 40

14=PTC-isoleucine

R=PTC derivative group

Phase: Column: Temperature: Eluent A: Eluent B:

Kromasil 100 , 5 m, C18 4.6 x 200 mm 38C 3% ACN in 0.1M sodium acetate ACN:water (80:20; v:v).

Gradient:

Flow rate: Detection:

Linear gradient elution. 0 min. 0% B, 13 min. 7% B, 23 min. 23% B, 29 min. 35% B, 35 min. 40% B, 40 min. 100% B, 45 min. 100% B, 47 min. 0% B 1 ml/min. UV 254 nm

Amino acids, Fmoc-derivativesef. 30)11 7 8 6 3=Fmoc-aspartic acid 9 5 2 4 1 3 12 14 15 17 16 18 6=Fmoc-glycine 14=Fmoc-phenylalanine 7=Fmoc-alanine 15=Fmoc-tryptophane 8=Fmoc-proline0 10 20 (min.) 30

20 21

1=Fmoc-arginine

9=Fmoc-tyrosine

16=Fmoc-cystine

19

2=Fmoc-serine

10=Fmoc-methionine

17=Fmoc-isoleucine

18=Fmoc-leucine 11=Fmoc-OH

4=Fmoc-glutamic acid 10 13 5=Fmoc-threonine

12=Fmoc-NH

2

19=Fmoc-histidine

13=Fmoc-valine

20=Fmoc-cysteine

21=Fmoc-lysine

R=Fmoc derivative group

Phase: Kromasil 100 , 5 m, C8 Column: 4 x 250 mm Temperature: 45C (75:15:10; v:v:v) ACN:THF (85:15; v:v). 0 2.5 min. 0% B, 2.5 6.6 min. 7% B, 6.6 8.3 Flow rate: Detection:

Eluent B: Gradient:

min. 14% B, 8.3 8.4 min. 21% B, 8.4 10 min. 21% B, 10 10.1 min. 17% B, 10.1 20 min. 19% B, 20 29 min. 55% B, 29 30 min. 100% B 1.5 ml/min. UV 263 nm

A.I.T FRANCE

Colonnes HPLC KROMASILApplicationsAMINO ACIDS

Amino acidsDetection of N-acetylaspartate and N-acetylglutamate in cerebral tissue extracts. (ref. 228) . (ref.1=aspartate 3=hypoxanthine 5=uric acid 8=N-acetylglutamate (NAG)

Amino acids, N-acetylatedSeparation of N-acetylated amino acids. (ref. 348)

1=N-acetylhistidine

2=N-acetylaspartate 2=glutamate 4=xanthine 6=inosine 9=adenosine 2 3=N-acetylglutamate

3

6

7=N-acetylaspartate (NAA) 9

3 1

1

2

4

5 8

7

0

10

20

30

(min.)

0

5

10

15

20

(min.)

25

Phase: Column: T Temperature: Eluent: Flow rate: Detection:

Kromasil 100 , 5 m, C18 4.6 x 250 mm 23C 2.8 mM tetrabutylammonium hydroxide, xide xide, 25 mM KH 2 PO 4 , 1.25% MeOH (pH 7) 1 ml/min. UV 210 nm

Phase: Column: Temperature: Eluent: Flow rate: Detection:

Kromasil 100 , 5 m, C18 4.6 x 250 mm 23C tetrabutylammonuim hydroxide 2.8 mM: KH 2 PO 4 25 mM and 1.25% MeOH, pH 7 1 ml/min. UV 210 nm

Amino acids, benzoylatedAnalysis of benzoylated amino acids. (ref. 51a)1=lysine (oxazol derivative) 5=glutamic acid (benzoylated) 9=isoleucine (benzoylated)

11

2=glycine (oxazol derivative)

6=phenylalanine (oxazol derivative)

10=tyrosine (benzoylated)

3=alanine (oxazol derivative) 3 1 2 4 5 6 7

7=valine (benzoylated)

11=cyclohexylalanine (benzoylated)

8 9 10 4=methionine (benzoylated) 8=leucine (oxazol derivative)

10

15

20

(min.)

Phase: Column: Eluent: Gradient: Flow rate: Detection:

Kromasil 100 , 5 m, C18 4 x 250 mm acetonitrile-water mixtures 70 95% ACN in 30 min. 1 ml/min. UV 274 nm

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Colonnes HPLC KROMASILApplicationsAMINO ACIDS

Aminosalicylic acidsDetermination of 5-aminosalicylic acid and 3-aminosalicylic acid. (ref. 279)1=5-aminosalicylic acid

Carnitines, aminoanthracene derivativesDetermination of L-carnitine, acetyl-L-carnitine and propionylL-carnitine in human plasma by HPLC with post-column derivatization with 1-aminoanthracene. (ref. 66)1=L-carnitine 1-aminoanthraceneamide 2=acetyl-L-carnitine 1-aminoanthraceneamide 3=methansulfonylL-carnitine 1-aminoanthraceneamide

2=3-aminosalicylic acid

1 1

4=propionyl-L-carnitine 1-aminoanthraceneamide R=1-aminoanthracene group

2

3

5=isobutyryl-L-carnitine 1-aminoanthraceneamide

2 40 1 2 3 4 5 6 (min.) 0 5 10 15 20

5(min.)

Phase: Column: Eluent: Flow rate: Detection:

Kromasil 100 , 5 m, C18 4.6 x 200 mm MeOH:phosphate buffer (35:65; v:v) 1 ml/min. UV 254 nm

Phase: Column: Eluent: Flow rate: Detection:

Kromasil 100 , 5 m, C18 4.6 x 250 mm ACN:ammonium acetate (0.1 M, pH 3.5) (30:70; v:v) 1.3 ml/min. spectrofluorimetric ( l ex 248 mn, l em 418 nm)

BoronophenylalanineDetermination of boronophenylalanine in biological samples after precolumn derivatization with o-phthalaldehyde (OPA). (ref. 231=OPA-aspartic acid 8=OPA-citrulline 15=OPA-valine

7)

2=OPA-glutamic acid

9=OPA-glycine

16=OPA-phenylalanine

3=OPA-asparagine

10=OPA-threonine

17=OPA-isoleucine

4=OPA-histidine 9 18 5 8 1 2 3 4 6 7 10 11 13 12 14 15 19 17 20 7=OPA-arginine 5=OPA-serine

11=OPA- g-aminobuturic acid (GABA)

18=OPA-leucine

12=OPA-alanine

19=OPA-ornitine

16 6=OPA-glutamine

13=OPA-tyrosine

20=OPA-lysine

14=OPA-p-boronophenylalanine

R=OPA derivative group

0

10

20

30

40

50

60

(min.)

70

Phase: Column: Temperature: Eluent A: Eluent B:

Kromasil 100 , 5 m, C18 4.6 x 250 mm 23C 50 mM CH 3 COONa (pH 7.4) : 50 mM NaHPO (pH 7.4) : MeOH : THF (48:48:2:2; v:v:v:v) MeOH:water (65:35; v:v).

Gradient:

4

Flow rate: Detection:

80% A in 3 min, 80% 70% A in 12 min, 70% 50% A in 15 min, 50% 45% A in 10 min, 45% 20% A in 10 min, 20% 15% A in 5 min, 15% 10% A in 3 min, 10% 0% A in 2 min, 0% A in 15 min. 1.2 ml/min. spectrofluorimetric ( ex 330 nm, em 430 nm)

A.I.T FRANCE

Colonnes HPLC KROMASILApplicationsDRUGS AND METABOLITESAmoxicillinMeasurement of amoxicillin in gastric tissue samples. (ref. 6)2

AmperozideSeparation of amperozide, derivate and metabolite. (ref. 45)3 1=amperozide's N-de-ethyl metabolite (II) 2

1=amoxicillin degradation derivative

2=ampicillin degradation derivative (I. S.)

2=amperozide (I) 1

1

3=amperozide's N-de-ethyl-N-butyl analogue (III)

0

2

4

6

8

(min.)

0

5

10

15

(min.)

Phase: Column: Temperature: Eluent: Flow rate: Detection:

Kromasil 100 , 5 m, C18 3.2 x 150 mm 40C MeOH-water (55:45; v:v) 0.4 ml/min. fluorescence ( l ex 365 nm, l

Phase: Column: Eluent: Flow rate: Detection:

em

445 nm)

Kromasil 100 , 5 m, C18 2.1 x 200 mm MeOH:ammonium phosphate buffer (pH 7.8) (78:22; v:v:) 0.2 ml/min. UV 265 nm

AnthocyanidinsSeparation of cyanidin from 3-Ob-glycosylated anthocyanidins. (ref. 347)1=petunidin-3-O-glycoside b4=malvidin-3-O-glycoside b7=cyanidin

5 1 6 2=delphinidin-3-O-glycoside b5=cyanidin-3-O-glycoside b-

3

2

4 7

3=pelargonidin-3-O-glycoside

b-

6=peonidin-3-O- b-glycoside

0

5

10

15

(min.)

20

Phase: Column: Temperature: Eluent A: Eluent B: Gradient:

Kromasil 100 , 5 m, C18 4.6 x 250 mm 23C HCOOH:water (1:10; v:v) HCOOH:water:MeOH (1:9:10; v:v:v) 0% 60% A in 5 min., 60% 45% A in 5 min., 45% 0% A in 6 min., 0% A in 10 min.

Flow rate: Detection:

1.2 ml/min. 520 nm

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Colonnes HPLC KROMASILApplicationsDRUGS AND METABOLITESAntibacterial drugsDetermination of metronidazole, clotrimazole and chlorhexidine acetate in Shuangzo effervescent tablets. (ref. 23)1=chlorhexidine 12 3

Antibacterial drugs, veterinarySimultaneous determination of sulfaquinoxaline, sulfamethazine and pyrimethamine. (ref. 246)1 = s ulfa m e t ha z ine 2=pyrimethamine 3 = s ulfa quinox a line

2=metronidazole

1

3=clotrimazole

3

2

0

20

(min.)

0

1

2

3

4

(min.)

5

Phase: Column: Eluent: Flow rate: Detection:

Kromasil 100 , 5 m, C18 4.6 x 250 mm MeOH:buffer (70:30; v:v) (NaAc 24.4 g, HAc 80 ml, (C4 H 9 )NBr 4.83 g in 1000 ml water, pH 3.6) 1 ml/min. UV 260 nm

Phase: Column: Eluent: Flow rate: Detection:

Kromasil 100 , 5 m, C18 4.6 x 150 mm 40 mM phosphate buffer (pH 3 containing 10 mM ClO 4 ) : ACN (65:35; v:v) 1.5 ml/min. UV 270 nm

Antibacterials, sulfa drugsDetermination of sulfamethoxypyridazine, sulfamethoxazole, sulfadimethoxine and associated compounds. (ref. 267)1 = t r i m e t ho pr i m 4=sulfadimethoxine 5 = b r om hex ine

Antibiotics and intermediatesDetermination of ceftriaxone, 7-aminocephalosporanic acid (7-ACA) and 7-amino-3-[[(2,5-dihydro-6-hydroxy-2-methyl-5-oxo-1,2,4trizin-3-yl)- thio]methyl]-cephalosporanic acid (7-ACT). (ref. 129)1=7-ACA 2

3

2=sulfamethoxypyridazine 4 2=7-ACT 1

3=sulfamethoxazole 3=ceftriaxone

2 3 1 5

0

5

10

(min.)

15

0

2

4

6

(min.)

8

Phase: Column: Eluent: Gradient: Flow rate: Detection:

Kromasil 100 , 5 m, C18 4.6 x 150 mm 10 mM citrate buffer (pH 3):MeOH 0 min. 31% MeOH, 4