110
HPLC Applications
Amino Acids, Carbohydrates
20
20
25
25
32
32
40
60
75
75
20
17
24
10
22
21
7
1
18
14
19
16
11 15 23
12
13
5
3 4
2 6
0
10
20
30
40
50
Min
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
1.
2.
3.
4.
5.
6.
6
5
DP6
DP5
DP4
DP3
DP2
Glucose
4
3
2
0
5
10
15
Min
Dark Corn Syrup (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
SUPELCOGEL Ag, 30cm x 7.8mm ID
59315
water
0.5mL/min
85°C
RI
10µL of 1g syrup/10mL DI water, filtered (0.20µm filter)
Amino Acids, Dabsyl (HPLC)
Column: SUPELCOSIL LC-DABS, 15cm x 4.6mm ID, 3µm particles
Cat. No.: 59137
Mobile Phase: A = 0.025M KH2 PO4, pH 7.0 with potassium hydroxide
B = acetonitrile:methanol (70:30)
Flow Rate: 1.5mL/min
Det.: VIS, 436nm
Inj.: 5µL, 50pmole each amino acid on-column
DP=1
DP=2
DP=7
2
DP=3
DP=4
3
5
7
4
8
6
1.
2.
3.
4.
5.
6.
7.
8.
Maltotriose
Maltose
Glucose
Xylose
Arabinose
Ribitol
Arabitol
Xylitol
DP=5
DP=6
15
0
5
10
15
20
25
Min
Carbohydrates (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
SUPELCOGEL Ca, 30cm x 7.8mm ID
59305-U
water
1.5mL/min
80°C
RI
10µL, 2.5mg/mL each analyte in water
Min
20
β -Cyclodextrin, Hydrolyzed (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Two TSKgel G-Oligo-PW, 30cm x 7.8mm ID, 6µm particles
808031
distilled water
1mL/min
60°C
refractive index
713-0917
1
795-0016
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
8
SUPELCO
28
20 25 26 27 29
9
1
794-0029
Liquid
Chromatography
0
4
9
23
24
30
37
46
54
59
60
o-Phospho-serine
o-Phospho-threonine
L-Aspartic acid
o-Phospho-tyrosine
Cysteic acid
L-Glutamic acid
Carboxymethyl
cysteine
S-Sulfocysteine
L-Asparagine
L-Glutamine
L-Serine
Methionine sulfoxide
L-Threonine
Glycine
L-Alanine
L-Arginine
Methionine sulfone
L-Proline
L-Valine
L-Methionine
L-Isoleucine
L-Leucine
L-Tryptophan
L-Phenylalanine
Ammonia
L-Cystine
L-Lysine
L-Histidine
L-Tyrosine
712-0135
1.
2.
3.
4.
5.
6.
7.
Gradient Program
Time (min) %B
111
HPLC Applications
Carbohydrates, Drugs
4
1.
2.
3.
4.
5.
5
2
Chitohexose
Chitopentose
Chitotetrose
Chitotriose
Chitobiose
4
2
1
6
1.
2.
3.
4.
5.
6.
Codeine
Strychnine
Quinidine
Quinine
Noscapine
Papaverine
Liquid
Chromatography
1
3
3
Min
20
4
TSKgel G-Oligo-PW, 30cm x 7.8mm ID, 6µm particles
808031
distilled water
1.0mL/min
60°C
RI
6
8
Min
10
G000235
2
12
Alkaloids (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
Chito Oligosaccharides (HPLC)
Column:
Cat. No.:
Eluent:
Flow Rate:
Temp.:
Det.:
0
Discovery RP-AmideC16, 15cm x 4.6mm ID, 5µm particles
505013
methanol:25mM KH 2PO4, pH 3.0 (20:80)
2mL/min
35°C
UV, 254nm
10µL
2
1.
2.
3.
4.
5.
1
Fructose
Glucose
Sucrose
Maltose
Lactose
1. Methanol
2. Pseudoephedrine
3. Fexofenadine
3
1
2
3
4
8
12
Min
Carbohydrates (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Det.:
Inj.:
SUPELCOSIL LC-NH2, 25cm x 4.6mm ID, 5µm particles
58338
acetonitrile:water (75:25)
2mL/min
refractive index
10µL, 150µg each sugar
0
2
4
6
Min
8
10
Allegra Ingredients (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Discovery Cyano, 15cm x 4.6mm ID, 5µm particles
59356-U
acetonitrile:25mM KH 2PO4 , pH 7.0 (15:85)
1mL/min
25°C
UV, 254nm
G001279
0
713-0908
4 5
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
15
794-0325
5
SUPELCO
112
HPLC Applications
Drugs
Liquid
Chromatography
3
1.
2.
3.
4.
5.
6.
6
2
4
Codeine
Strychnine
Quinidine
Quinine
Noscapine
Papaverine
1.
2.
3.
4.
5.
4
Doxylamine
Pseudoephedrine
Codeine
Acetaminophen
Chlorpheniramine
Gradient Program
Time (min) %B
0
2
6
10
90
90
70
70
1
5
2
4
6
8
Min
10
12
14
16
G000691
0
2
1
3
Discovery RP-AmideC16, 15cm x 4.6mm ID, 5µm particles
505013
80:20 25mM Potassium Phosphate (pH 3.0):MeOH
2.0mL/min
35°C
UV at 254nm
10µL
0
2
6
4
8
Analgesics and Antihistamines in Dosage Form (HPLC)
Column: Discovery RP-AmideC16, 15cm x 4.6mm ID, 5µm particles
Cat. No.: 505013
Mobile Phase: A = acetonitrile
B = 25mM KH 2 PO4, pH 2.3
Flow Rate: 2mL/min
Temp.: ambient
Det.: UV, 214nm
Inj.: 10µL
Dextromethorphan
(100µg/mL)
Acetylsalicylic acid
(50µg/mL)
Gradient Program
Time (min)
%A
%B
0
3
10
15
0
Min
2
10
Min
90
90
50
50
1.
2.
3.
4.
10
10
50
50
Discovery HS F5:
Good Retention
713-0178
p-Aminophenol (100µg/mL)
Acetaminophen (10µg/mL)
Acetanilide (10µg/mL)
Phenacetin (10µg/mL)
4
2
3
▼
1
Column: SUPELCOSIL LC-ABZ, 5cm x 4.6mm ID, 5µm particles
Cat. No.: 59141
Mobile Phase: acetonitrile:25mM KH 2PO4,
pH 2.3 with phosphoric
acid (20:80)
Flow Rate: 2mL/min
Det.: UV, 230nm
Inj.: 5µL mobile phase, analyte quantities shown
0
2
4
6
8
Min
10
12
14
G001526
Analgesics (HPLC)
4
Discovery C18:
Elutes at void volume
3
2
▼
1
0
2
4
6
8
Min
10
12
14
Analgesic Drugs (HPLC)
Columns:
Cat. Nos.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
15cm x 4.6mm ID, 5µm particles
567516-U (HS F5), 504955 (C18)
(A) 10mM ammonium acetate, 0.1% formic acid; (B) MeOH
1.5mL/min
35°C
UV at 254nm
10µL
G001527
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
SUPELCO
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
G000785
5
Amides (HPLC)
113
HPLC Applications
Drugs
1.
2.
3.
4.
5.
6.
1
Aniline
N-Ethylaniline
N-Propylaniline
N,N-Dimethylaniline
N-Butylaniline
N,N-Diethylaniline
0
25
90
65
1.
2.
3.
4.
5.
6.
10
35
Levofloxacin
Ciprofloxacin
Lomefloxacin
Sparfloxacin
Grepafloxacin
Trovafloxacin
Liquid
Chromatography
Gradient Program
Time (min)
%A
%B
3
1
2
2 3
Discovery C18
4
5
4
6
5
0
2
4
6
8
10
12
14
16
18
G000295
6
Coumn:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Discovery C18, 15cm x 4.6mm ID, 5µm particles
504955
MeOH:H2O (60:40)
1mL/min
30°C
UV, 254nm
0
2
4
6
8
10
12
14
Min
1
2 3
Discovery HS C18
5
4
6
1
1. Clonazepam
2. Clorazepate
3. Diazepam
2
0
2
4
6
8
Min
Anticonvulsants (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
Discovery C18, 15cm x 4.6mm ID, 5µm particles
504955
acetonitrile:25mM KH 2PO4 , pH7.0 (40:60)
1mL/min
40°C
UV, 254nm
10µL
10
G000188
0
2
4
6
8
Min
10
12
14
Antibiotics (Fluoroquinolones from Tablets) (HPLC)
Columns: 15cm x 4.6mm ID, 5µm particles
Cat. Nos.: 504955 (C18), 568522-U (HS C18)
Mobile Phase: (A) 25 mM potassium phosphate (pH 3.0)
(B) CH3CN
Flow Rate: 1.0mL/min
Temp.: 35°C
Det.: UV, 220nm
Inj.: 10µL
G001425
3
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
Aniline Homologs (HPLC)
G001424
Min
SUPELCO
114
HPLC Applications
Drugs
Liquid
Chromatography
1
2
1. Clonazepam
2. Clorazepate
3. Diazepam
Discovery C18
1. Enalapril
2. Lisinopril
3. Captopril
1
3
2
2
4
6
8
10
Min
1
12
14
16
G000746
0
18
1.0
2.0
3.0
4.0
5.0
Min
Antihypertensive ACE Inhibitors (HPLC)
Discovery C8
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
2
2
4
6
8
10
Min
12
14
16
Discovery RP-AmideC16, 15cm x 4.6mm ID, 5µm particles
505013
acetonitrile:25mM KH 2PO4, pH 2.3 (33:67)
0.6mL/min
35°C
UV, 214nm
3µL
G000748
0
18
3
Anticonvulsants (HPLC)
Columns:
Cat. Nos.:
Mobile Phase:
Flow Rate:
Det.:
Temp.:
Inj.:
1.
2.
3.
4.
2
15cm x 4.6mm ID, 5µm particles
504955 (C18), 59353-U (C8)
70:30 water:CH3CN
2.0mL/min
UV, 254nm
20°C
10µL
Atenolol
Amiloride
Hydrochlorothiazide
Caffeine
4
1
2
Maleic acid
Doxylamine
Chlorpheniramine
Carbinoxamine
Brompheniramine
0
3
4
2
4
6
8
10
Min
5
Antihypertensive-Diuretic Combination (HPLC)
0
2
4
6
8
10
12
14
16
Min
Antihistamines (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
Discovery C8, 15cm x 4.6mm ID, 5µm particles
59353-U
acetonitrile:25mM KH 2PO4 , pH 2.3 (8:92)
2mL/min
25°C
UV, 254nm
2µL
18
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
Discovery RP-AmideC16, 15cm x 4.6mm ID, 5µm particles
505013
methanol: 25mM K 2HPO4 , pH 7.0 (22:78)
1mL/min
30°C
UV, 254nm
10µL
G000280
1.
2.
3.
4.
5.
1
G001278
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
3
SUPELCO
G000279
3
115
HPLC Applications
Drugs
2
1
Captopril
Pindolol
Desmethyldiltiazem
Nifedipine
Diltiazem
Verapamil
1.
2.
3.
4.
Gradient Program
Time (min) %A
6
1
3
2
4
6
0
5
15
G000112
4 5
0
8
10
Hydrochlorothiazide
Oxyphenbutazone
Furosemide
Phenylbutazone
Liquid
Chromatography
1.
2.
3.
4.
5.
6.
12
14
16
2
3
20
30
20
4
18
Min
Discovery RP-AmideC16, 15cm x 4.6mm ID, 5µm particles
505013
methanol:25mM KH 2PO4, pH 7.0 (40:60)
1mL/min
35°C
UV, 214nm
5µL
0
4
6
8
10
12
14
Min
Anti-Inflammatory Drugs and Diuretics in Serum (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
2
1. 2-Aminopyridine
2. Piroxicam
2
Mobile Phase A
Hisep, 15cm x 4.6mm ID, 5µm particles
58935
A = acetonitrile, B = 180mM ammonium acetate, pH 5.4
2mL/min
30°C
UV, 267nm
10µL horse serum containing 5µg/mL hydrochlorothiazide,
10µg/mL each other drug
1
G001657
2
0
2
4
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
1
6
5
Min
6
4
2
7
Mobile Phase B
3
Piroxicam
Sulindac
Tolmetin
Ketoprofen
Naprosyn
Diflunisal
Indomethacin
Ibuprofen
Diclofenac
Mefanamic acid
9
8
0
2
4
6
8
10
12
14
Min
Anti-Inflammatory Drugs
(Aminopryridine and Piroxicam) (HPLC)
Columns: Discovery C18, 15cm x 4.6mm ID, 5µm particles
Cat. Nos.: 504955
Mobile Phase: A = 85:15, 10 mM potassium phosphate (pH 2.5): CH 3CN
B = 85:15, 10 mM potassium phosphate (pH 6.8): CH 3 CN
Flow Rate: 1.0mL/min
Det.: UV, 220nm
Inj.: 5µL
G001658
0
10
20
Min
Anti-Inflammatory Drugs, Non-Steroidal (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
Discovery RP-AmideC16, 15cm x 4.6mm ID, 5µm particles
505013
acetonitrile:25mM KH 2PO4 , pH 3.0 (40:60)
1mL/min
30°C
UV, 230nm
10µL, 1µg/mL each analyte
G000061
10
1
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
85-163
Antihypertensive Drugs (HPLC)
(Beta/Calcium Channel Blockers and ACE Inhibitor)
SUPELCO
116
HPLC Applications
Drugs
2
Liquid
Chromatography
7
9
1
1.
2.
3.
4.
5.
6.
7.
8.
9.
Propyl gallate
THBP
TBHQ
NDGA
BHA
Ionox 100
Octyl gallate
BHT
Dodecyl gallate
0
2
4
8
10
90
90
70
70
50
5
1.
2.
3.
4.
5.
10
10
30
30
50
Acetaminophen
Doxylamine
Pseudoephedrine
Codeine
Chlorpheniramine
Discovery RP-AmideC16
1
4
3
8
6
6
8
10
12
14
2
4
Antioxidants (HPLC)
4
1.
2.
3.
4.
5.
1
2
3
0
2
4
6
8
Min
Elution
order
changes!
1
Discovery C18
▼
Paracetamol
Aspirin (acetylsalicylic acid)
Sorbic acid
Benzoic acid
Salicylic acid
3
2
4
0
5
10
20
Min
Antitussive/Antihistamine/Antipyretic Mix (HPLC)
5
0
2
4
6
8
10
12
14
Min
Antipyretics/Analgesics/Antifungals (HPLC)
Column :
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
Discovery RP-AmideC16, 15cm x 4.6mm ID, 5µm particles
505013
0.1% TFA in (25:75) acetonitrile:water
1mL/min
30°C
UV, 254nm
10µL
10
G000785
5
Column: SUPELCOSIL LC-18, 15cm x 4.6mm ID, 5µm particles
Cat. No.: 58230-U
Mobile Phase: A = 5% acetic acid in deionized water
B = acetonitrile:methanol (1:1)
70% A/30% B to 100% B, linear gradient over 10 min, hold 10
min
Flow Rate: 2.0mL/min
Det.: UV, 280nm
Inj.: 10µL , 20µg/mL each antioxidant
(Cat. No. 47192)
G000088
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
Min
3
Columns: 15cm x 4.6mm ID, 5µm particles
Cat. Nos.: 505013 (RP-AmideC16), 504955 (C18)
Mobile Phase: (A) 25 mM Potassium Phosphate (pH 2.3)
(B) CH3CN
Flow Rate: 2.0mL/min
Temp.: ambient
Det.: UV, 214nm
Inj.: 10µL
G000787
4
795-0438
2
Elution order changes!
▼
0
SUPELCO
Gradient Program
Time (min)
%A
%B
117
HPLC Applications
Drugs
1.
2.
3.
4.
3
Nordoxepin is notoriously difficult to
chromatograph, and the slight tailing
is a good peak shape for this drug.
Nizatidine
Ranitidine
Famotidine
Cimetidine
1.
2.
3.
4.
3
4
2
1
Liquid
Chromatography
Discovery RP-AmideC16
Nordoxepin
Oxazepam
Chlordiazepoxide
Desmethyldiazepam
2
4
6
8
Min
10
Discovery C18
12
14
4
0
3
1
2
2
4
6
8
Min
10
Min
Anxiolytics (HPLC)
G001428
0
1
10
12
14
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp:
Det.:
Inj.:
Discovery C18,15cm x 4.6mm ID, 5µm particles
504955
acetonitrile:25mM KH 2PO4 , pH 7.0 (30:70)
1mL/min
20°C
UV, 254nm
10µL
4
Discovery HS C18
1
1.
2.
3.
4.
5.
6.
7.
8.
3
1
2
0
2
4
6
8
Min
10
12
14
G001430
3
5
2
Demoxepam
Nitrazepam (int. std.)
N-desmethyl Chlordiazepoxide
Oxazepam
Chlordiazepoxide
Temazepam
N-desmethyl Diazepam
Diazepam
7
4
4
Discovery C8
6
8
3
2
0
2
4
6
8
Min
10
12
Antiulcer Compounds (HPLC)
Columns: 15cm x 4.6mm ID, 5µm particles
Cat. Nos.: 505013 (RP-AmideC16), 504955 (C18),
569252-U (HS C18), 59353-U (C8)
Mobile Phase: MeOH:25mM H 3 PO4/KOH, pH 3.0 (15:85)
Flow Rate: 0.5mL/min
Temp.: ambient
Det.: UV, 215nm
Inj.: 3µL
14
G001429
0
2
4
6
8
10
12
14
Min
Anxiolytics (HPLC)
Column: SUPELCOSIL LC-8, 15cm x 4.6mm ID, 5µm particles
Cat. No.: 58220-U
Mobile Phase: methanol:acetonitrile:0.005M KH 2 PO4 and 0.1M ammonium
acetate buffer (pH 6.0 with glacial acetic acid),
26.5:16.5:57 (v/v/v)
Flow Rate: 2mL/min
Temp.: 30°C
Det.: UV, 245nm
Sample: 10µL
713-1390
1
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
2
797-0662
0
G001427
4
SUPELCO
118
HPLC Applications
Drugs
Liquid
Chromatography
3
1
4
2
1.
2.
3.
4.
5.
Methylprylon (2mg/mL)
Butabarbital (50µg/mL)
Butalbital (50µg/mL)
Hexobarbital (50µg/mL)
Mephobarbital (25µg/mL)
1.
2.
3.
4.
5.
6.
7.
5
Barbital
Phenobarbital
Aprobarbital
Butabarbital
Mephobarbital
Pentobarbital
Secobarbital
2
4
5
4
6
8
10
Barbiturates (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Det.:
Inj.:
0
SUPELCOSIL LC-ABZ, 15cm x 4.6mm ID, 5µm particles
59140-U
acetonitrile:25mM potassium phosphate buffer, pH 6.9 (25:75)
1.5mL/min
UV, 254nm
25µL
1
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
Barbital
Aprobarbital
Phenobarbital
Butabarbital
Mephobarbital
Pentobarbital
Secobarbital
Discovery RP-AmideC16, 15cm x 4.6mm ID, 5µm particles
505013
methanol:acetonitrile, 60:40:water, 25:75
2mL/min
30°C
UV, 214nm
2µL
2
3
1.
2.
3.
4.
5.
6.
7.
1
4
5
3
6
20
Barbiturates (HPLC)
2
1.
2.
3.
4.
5.
6.
7.
10
Min
Barbital
Aprobarbital
Phenobarbital
Butabarbital
Mephobarbital
Pentobarbital
Secobarbital
4
7
5
2
4
6
8
10
12
14
Min
Barbiturates (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Det.:
Temp.:
Inj.:
Discovery C8, 15cm x 4.6mm ID, 5µm particles
59353-U
55:45 water:MeOH
1.0mL/min
UV at 214nm
ambient
5µL
16
18
6
0
10
Min
Barbiturates (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
Discovery C18, 15cm x 4.6mm ID, 5µm particles
504955
55:45 water:MeOH
1.0mL/min
ambient
UV, 214nm
10µL
7
20
G000190
0
G000791
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
Min
7
G000193
2
6
713-0141
0
SUPELCO
3
1
119
HPLC Applications
Drugs
+
1.
2.
3.
4.
2
CH 2 N R
CH 3
Norepinephrine
Epinephrine
3,4-Dihydroxybenzylamine
Dopamine
Liquid
Chromatography
1
CH 3
Cl-
3
4
6
Min
0
Benzalkonium Chlorides (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Sample:
10
Min
Discovery Cyano, 15cm x 4.6mm ID, 5µm particles
59356-U
CH3CN:acetate buffer, pH 4.5, (60:40)
1mL/min
ambient temp.
UV, 254nm
Undefined C8 to C18 Benzalkonium Chlorides
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
5
1
Catecholamines (HPLC)
Column: Discovery C18, 15cm x 4.6mm ID, 5µm particles
Cat. No.: 504955
Mobile Phase: acetonitrile:50mM KH 2 PO4, pH 3.0 with H 3 PO4, 100mg/L EDTA,
200mg/L 1-octane-sulfonic acid (5:95)
Flow Rate: 1mL/min
Temp.: 20°C
Det.: UV, 254nm
Inj.: 10µL
Procainamide
Pindolol
Oxprenolol
Dipyridamole
Diltiazem
Verapamil
Digoxin
Lidoflazine
Flunarizine
Nifedipine
1
2
2
1. 6-Hydroxychlorzoxazone
2. Chlorzoxazone
Gradient Program
Time (min) %B
0
10
12
12.1
3
6
4
4
10
7
6
8
10
Min
Cardiac Drugs (HPLC)
Column: SUPELCOSIL ABZ+Plus, 15cm x 4.6mm ID, 5µm particles
Cat. No.: 59196
Mobile Phase: A = acetonitrile
B = 25mM KH 2PO4 (pH 3.0)
Flow Rate: 2mL/min
Det.: UV, 220nm
Inj.: 20µL methanol:water, 50:50 (30µg/µL pindolol and
diltiazem,100µg/mL dipyridamole, 50µg/mL other analytes)
0
10
Min
Chlorzoxazone (HPLC)
Column: Discovery HS PEG, 15cm x 4.6mm ID, 5µm particles
Cat. No.: 567416-U
Mobile Phase: 70:30 20mM acetic acid in water
(pH 4.5 with ammonium hydroxide):CH 3 CN
Flow Rate: 1.0mL/min
Temp.: 30°C
Det.: UV, 285nm
Inj.: 10µL
20
G001674
2
9
794-0638
0
8
90
50
50
90
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
2
797-0659
0
G000726
4
SUPELCO
120
HPLC Applications
Drugs
1
2
Liquid
Chromatography
Discovery C8
1. Cefadroxil
2. Cefaclor
3. Cephalexin
Discovery RP-AmideC16
2
3
4
6
0
Min
2
4
Min
1
1
Discovery RP-AmideC16
Discovery C8
2
2
4
Min
6
8
2
0
1
2
4
6
G000709
0
G000747
3
Min
Discovery C18
1
Discovery Cyano
2
3
2
4
6
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 KH 2PO4 , pH 3.0, (20:80) for C18 and C8,
(10:90) for RP-Amide C16
Flow Rate: 2mL/min
Temp.: 20°C
Det.: UV, 254nm
Inj.: 1µL
2
0
2
4
6
Min
Cold Remedy Ingredients/Antihistamines (HPLC)
Columns:
Cat. Nos.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
15cm x 4.6mm ID, 5µm particles
505013 (RP-AmideC16), 59353-U (C8), 59356-U (Cyano)
acetonitrile:25mM KH 2PO4 pH 7.0 (15:85)
1mL/min
20°C
UV, 214nm
1µL
G000839
0
G000746
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
G000710
2
G000696
1
0
SUPELCO
1. Pseudoephedrine
2. Acetaminophen
121
HPLC Applications
Drugs
1.
2.
3.
4.
5.
6.
Discovery C8
3
2
6
4
1
Amoxicillin
Ampicillin
Piperacillin
Penicillin G
Penicillin V
Cloxacillin
Gradient Program
Time (min) %B
5
6
0
2
4
6
8
10
12
14
Min
Discovery C18
2
Discovery RP-AmideC16
3
1
0
2
4
6
6
5
G000900
4
G001178
Min
Discovery C8
0
10
Min
20
3
Discovery C18
4
2
6
0
2
4
6
G000901
1
5
Min
0
2
4
6
8
10
12
14
G001179
Discovery Cyano
16
Min
1
2
4
Min
6
6
G000899
0
Discovery Cyano
2
3
Gradient Program
Time (min) %B
4 5
0
20
5
25
Columns: 15cm x 4.6mm ID, 5µm particles
Cat. Nos.: 505013 (RP-AmideC16), 504955 (C18),
59353-U (C8), 59356-U (Cyano)
Mobile Phase: CH3CN:25mM KH 2PO4 , pH 7.0 (40:60)
Flow Rate: 2mL/min
Temp.: 30°C
Det.: UV, 214nm
Inj.: 1µL
0
10
20
Min
β -Lactam Antibiotics (HPLC)
Column: 15cm x 4.6mm ID, 5µm particles
Cat. 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 CH3CN
Flow Rate: 1.5mL/min
Temp.: 30°C
Det.: UV, 254nm
G001181
Fluoxetine (Prozac) (HPLC)
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
4
Min
G001180
2
5
35
35
G000902
0
5
20
0
Liquid
Chromatography
Discovery RP-AmideC16
SUPELCO
122
HPLC Applications
Drugs
5-Fluorouracil
Liquid
Chromatography
H
N
O
NH
F
(A) Discovery HS F5
O
O
F
NH
Tegafur
O
N
O
4
3
5
6
▼
96-0095
2
1
7
Min
0
10
Min
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
SUPELCOGEL TPR-100, 15cm x 4.6mm ID, 5µm particles
59154
acetonitrile:water (10:90)
1.0mL/min
30°C
UV, 270nm
1
20
(B) Discovery C18
1. Methcathinone (100µg/mL)
2. (+/-) Ephedrine (200µg/mL)
Partially
resolved
front
shoulder
1
▼
2
0
10
Min
Quinidine and Trace Impurity (HPLC)
0
1
2
3
4
Min
5
6
7
8
Methcathinone and Ephedrine (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
Discovery HS F5, 15cm x 4.6mm ID, 5µm particles
567516-U
(30:70) 10mM Ammonium Acetate (pH 6.98): CH 3 CN
2.0mL/min
35°C
Photodiode Array
5µL
Columns: 15cm x 4.6mm ID, 5µm particles
Cat. Nos.: 567516-U (HS F5), 504955 (C18)
Mobile Phase: 25 mM ammonium phosphate (pH 7.0):CH 3CN
varying ratios: (A) 35:65, (B) 70:30
Flow Rate: 1.0mL/min
Temp.: 30°C
Det.: UV, 235nm
Inj.: 10µL
20
G001667
2
G001673
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
5-Fluorouracil and Tegafur (HPLC)
G001666
2
0
SUPELCO
1. Quinidine (50µg/mL)
2. Impurity
1
123
HPLC Applications
Drugs
1
Amphetamine
3
2
Liquid
Chromatography
1. 5-Fluorocytosine (t o)
2. N-Acetylprocainamide
3. Procainamide
Procainamides (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
Sample:
0
Discovery HS F5, 15cm x 4.6mm ID, 5µm particles
567516-U
(65:35) 25 mM ammonium phosphate (pH 7):CH 3CN
1.0mL/min
30°C
UV, 280nm
5µL
50µg/mL of each
2
3
4
5
Min
Stimulants, Amphetamine (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Det.:
Inj.:
Suplex pKb-100, 15cm x 4.6mm ID, 5µm particles
58932
acetonitrile:25mM K 2HPO4 , pH 7.0 with phosphoric acid (15:85)
2mL/min
UV, 254nm
10µL mobile phase (50µg/mL each analyte)
1. Pseudoephedrine
2. Paracetamol
3. Chlorpheniramine
1
1
1
1.
2.
3.
4.
3
Sulfanilamide
Sulfathiazole
Sulfamerazine
Sulfamethazine
2
3
2
4
6
Min
Sinus Medications (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
Discovery RP-AmideC16, 15cm x 4.6mm ID, 5µm particles
505013
acetonitrile:50mM KH 2PO4 , pH 3.0 (25:75)
1mL/min
30°C
UV, 220nm
10µL
0
2
4
6
Min
Sulfa Drugs (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
Discovery C18,15cm x 4.6mm ID, 5µm particles
504955
methanol:water with 1% CH 3COOH (20:80)
1mL/min
20°C
UV, 254nm
10µL
797-0655
0
4
G000087
2
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
2
Min
85-82
0
G001686
Methamphetamine
SUPELCO
124
HPLC Applications
Drugs
1.
2.
3.
4.
5.
6.
7.
8.
Liquid
Chromatography
1
3
4
Desmethyl doxepin
Protriptyline
Desipramine
Nortriptyline
Doxepin
Imipramine
Amitriptyline
Trimipramine
1
2
4
3
Trimipramine
Doxepin
Amitriptyline
Imipramine
Desmethyl doxepin
Nortriptyline
Desipramine
Protriptyline
5
7
6
5
2
6
7
8
6
8
Min
10
12
14
16
0
2
4
G000727
4
G000541
2
8
6
Min
Tricyclic Antidepressants (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Tricyclic Antidepressants (HPLC)
Discovery C8, 15cm x 4.6mm ID, 5µm particles
59353-U
methanol:10mM K 2 HPO4, pH 7.0
2mL/min
40°C
UV, 254nm
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Discovery Cyano, 15cm x 4.6mm ID, 5µm particles
59356-U
acetonitrile:methanol:10mM K 2HPO4, pH 7.0, 60:20:20
1mL/min
35°C
UV, 254nm
2
2
1
1.
2.
3.
4.
5.
6.
7.
8.
3
4
5
Trimipramine (int. std.)
Doxepin
Amitriptyline
Imipramine
Desmethyl doxepin
Nortriptyline
Desipramine
Protriptyline (int. std.)
1.
2.
3.
4.
5.
6.
7.
8.
1
3
4
6
Desmethyl doxepin
Protriptyline
Desipramine
Nortriptyline
Doxepin
Imipramine
Amitriptyline
Trimipramine
5
7
8
7
6
0
2
Min
4
6
Tricyclic Antidepressants (HPLC)
Column: SUPELCOSIL LC-PCN, 15cm x 4.6mm ID, 5µm particles
Cat. No.: 58377
Mobile Phase: acetonitrile:methanol:0.01M dipotassium hydrogen phosphate,
pH 7.0 with phosphoric acid (60:15:25)
Flow Rate: 2mL/min
Temp.: 30°C
Det.: UV, 215nm
Inj.: 50ng each drug
0
2
4
6
Min
8
10
12
Tricyclic Antidepressants (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
Discovery RP-AmideC16, 15cm x 4.6mm ID, 5µm particles
505013
methanol:25mM KH 2PO4 , pH 7, 60:40
2mL/min
40°C
UV, 230nm
10µL
G000163
8
713-0508
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
0
SUPELCO
1.
2.
3.
4.
5.
6.
7.
8.
125
HPLC Applications
Fatty Acids, Foods
1
4
1.
2.
3.
4.
1
Oxidation products
18:3c (2.65µg)
16:1c (0.53µg)
16:1t (10.6µg)
18:2c (0.53µg)
18:2t (2.65µg)
16:0 (15.8µg)
18:1c (10.6µg)
18:1t (10.6µg)
18:0 (10.6µg)
Methyl paraben
Ethyl paraben
Propyl paraben
Butyl paraben
Discovery RP-AmideC16
4
3
2
3
2
Ox
0
8
10
Min
6 7
20
G001235
Ox
1.
2.
3.
4.
5.
6.
7.
8.
9.
Liquid
Chromatography
5
1
9
4
6
Min
8
10
12
4
3
2
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
SUPELCOSIL LC-8, 15cm x 4.6mm ID, 3µm particles
58983
acetonitrile:tetrahydrofuran: 0.1% phosphoric acid (50.4:21.6:28)
1mL/min
35°C
UV, 215nm
10µL
0
2
4
6
Min
8
10
1
12
G001234
Fatty Acids, Free (HPLC)
Discovery C8
4
3
3
C8
C10
12:0 + 18:3
18:2
14:0
18:1
16:0
18:0
2
0
2
4
6
Min
8
G001236
1.
2.
3.
4.
5.
6.
7.
8.
10
4
5
1
Discovery Cyano
6
3
2
4
2
8
0
2
Min
4
6
Fatty Acid Methyl Esters (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Det.:
Sample:
SUPELCOSIL LC-18, 25cm x 4.6mm ID, 5µm particles
58298
acetonitrile:acetone (59.0:41.0, v/v)
1.0mL/min
RI
10µL of 9% C8 to C18 saturated and unsaturated FAMEs in
mobile phase
797-0501
0
2
4
Min
6
Paraben Preservatives (HPLC)
Columns: 15cm x 4.6mm ID, 5µm particles
conditions for RP-AmideC16, C18,and C8
Cat. Nos.: 505013 (RP-AmideC16), 504955 (C18),
59353-U (C8)
Mobile Phase: CH3CN:H 2O, (40:60)
Flow Rate: 1mL/min
Temp.: 20°C
Det.: UV, 254nm
Inj.: 10µL
conditions for Cyano
Cat. No.: 59356-U (Cyano)
Mobile Phase: CH3CN:H 2O, (30:70)
Flow Rate: 1mL/min
Temp.: 20°C
Det.: UV, 254nm
Inj.: 10µL
G001237
7
1
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
2
713-0969
0
Discovery C18
SUPELCO
126
HPLC Applications
Flavor, Mycotoxins, Nucleosides, Nucleotides
Liquid
Chromatography
1
1.
2.
3.
4.
5.
6.
7.
8.
2
540
Flavone
7-Hydroxyflavone
Baicalein
5-Hydroxyflavone
Chrysin
Luteolin
Myricetin
Quercetin
Gradient Program
Time (min)
%A
0
0.5
5
20
21
24
25
5
4
2
4
6
8
Min
10
12
14
16
G001665
0
8
7
587
458
434
234 267
213
184 192
104 123
124
89
57
80
64
51
6
3
504
25
45
50
62
100
100
25
18
21
SUPELCO
1.
2.
3.
4.
4
0
4
6
8
Min
10
12
14
16
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 9.0
B = 25mM Tris-HCl, pH 9.0
Flow Rate: 1mL/min
Temp.: ambient
Det.: UV, 260nm
Inj.: 20µL of 50µg/mL HAE III digest of pBR322 DNA
Aflatoxin G 2
Aflatoxin G 1
Aflatoxin B2
Aflatoxin B1
1.
2.
3.
4.
5.
6.
7.
8.
Gradient Program
Time (min) %B
2
1
2
794-0036
Column: Discovery HS PEG, 15cm x 4.6mm ID, 5µm particles
Cat. No.: 567416-U
Mobile Phase: 45:55 0.1% formic acid in water :
0.1% formic acid in MeOH
Flow Rate: 1.0mL/min
Temp.: 30°C,
Det.: UV, 254nm
Inj.: 10µL
0
2.5
5
10
13
17
18
3
0
0
30
60
100
100
0
5
HX
XAN
INO
IMP
NAD +
ADO
AMP
GDP
9.
10.
11.
12.
13.
14.
15.
NADP+
ADP-rib
GTP
ADP
NADH
ATP
NADPH
11
14
9
13
6
1
0
2
4
6
Min
8
10
3
15
7
12
2
713-0874
8
4
10
Aflatoxins (SPE/HPLC)
Sample: cornmeal spiked with aflatoxins (30ppb G 2 and B2 , 100ppb
G1 and B1 ) Blend 50g sample for 1 min in 100mL
methanol:water (8:2) and filter.
Extraction Tube: Supelclean LC-CN, 3mL, 0.5g packing
Cat. No.: 57013
Conditioning: 2mL 0.5% aqueous acetic acid
Sample Addition: 1mL filtered extract + 4mL 0.5% aqueous acetic acid
Washing: 500µL 20% THF in 0.5% aqueous acetic acid, then 2mL
hexane, Dry packing under nitrogen. 3mL 25% THF in
hexane, Dry packing 1 min under nitrogen.
Elution: 2 x 2mL 1% THF in methylene chloride
Evaporate eluate to dryness under nitrogen. Reconstitute
with 100µL of methanol, then dilute with 100µL of 0.5%
aqueous acetic acid.
Column: SUPELCOSIL LC-18, 25cm x 4.6mm ID, 5µm particles (with
guard column)
Cat. No.: 58298
Mobile Phase: methanol:acetonitrile:water (22.5:22.5:55)
Flow Rate: 1.5mL/min
Det.: VIS, 365nm
Inj.: 100µL
0
4
8
12
Min
16
20
713-0951
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
Flavones (HPLC)
Nucleotides (HPLC)
Column: SUPELCOSIL LC-18-T, 15cm x 4.6mm ID, 3µm particles
Cat. No.: 58970-U
Mobile Phase: A = 0.1M potassium phosphate buffer/4mM tetrabutylammonium
hydrogen sulfate, pH 6.0
B = A:methanol (70:30), pH 7.2
Flow Rate: 1.5mL/min
Det.: UV, 254nm
Inj.: 20µL deionized water (25-530µM each analyte)
127
HPLC Applications
Nucleosides, Nucleotides, Organic Acids
dC
0
7.2
12
15
19.2
21.6
27
48
55
100
100
90
75
40
38
—
—
—
%C
—
—
10
25
60
62
100
—
—
Gradient Program
Time (min) %A
hm5U
m5dC
C
dA
U
—
—
—
—
—
—
—
100
100
G dG
dU
Selective,
Quantitative Analysis
0
3
12
15
19.2
dT
A
I
dI
Liquid
Chromatography
Gradient Program
Time (min)
%A
%B
100
100
0
0
40
m3dC
m1dA
m6dA
Detection at 210nm,
250mAU
ΨC U
Ψ
A
G
C
U
5'-dA
2
2
m G
Um
m3C
hU
15
Figure provided by Dr. C. W. Gehrke and Mr. K. C. Kuo, University of MissouriColumbia, Experimental Station Chemical Laboratories, Columbia, MO USA.
Deoxyribonucleosides and Ribonucleosides (HPLC)
0
T
Ψm
10
10
Min
Detection at 254nm,
50mAU
Detection at 280nm,
50mAU
20
Min
30
Figure provided by Dr. C.W. Gehrke and Mr. K.C. Kuo of the Interdisciplinary
Chromatography-Mass Spectrometry Laboratories, University of Missouri
(Columbia, MO, USA).
713-0501, 0688
m5C
Column: SUPELCOSIL LC-18-S, 15cm x 4.6mm ID, 5µm particles
Cat. No.: 58931
Mobile Phase: 0.05M K2HPO4/KH 2PO4, pH 4.0:methanol
A = 97.5:2.5, B = 80:20
Flow Rate: 1.0mL/min
Temp.: 30°C
Det.: UV, 254nm
Inj.: nucleoside standards in water
1.
2.
3.
4.
I
5.
6.
7.
8.
9.
10.
11.
12.
13.
Single Species tRNAs (HPLC)
Columns: SUPELCOSIL LC-18-S, 15cm x 4.6mm ID, 5µm particles
Cat. No.: 58931
Mobile Phase: A = 0.01M KH 2 PO4 (pH 5.3):methanol, 97.5:2.5
B = 0.01M KH2PO4 (pH 5.1):methanol, 80:20
C = 0.01M KH 2 PO4 (pH 4.9):acetonitrile, 65:35
Equilibration: solvent A, 15 min at 1mL/min
Flow Rate: 1mL/min
Temp.: 26°C
Det.: UV, 210nm, 254nm, 280nm
Inj.: 50µL hydrolysate buffer (pH 7.8) containing 5µg bovine tRNA
hydrolysate
6-Benzylaminopurine riboside (445ng)
Indole-3-acetic acid (222ng)
Abscisic acid (222ng)
α-Naphthalene acetamide (222ng)
Abscisic acid impurity
Colchicine (555ng)
Indole-3-propanoic acid (222ng)
p-Chlorophenoxy acetic acid (333.5ng)
Indole-3-butyric acid (333.5ng)
α-Naphthalene acetic acid (333.5ng)
β-Naphthalene acetic acid (333.5ng)
2,4-Dichlorophenoxy acetic acid (555ng)
Indole-3-acetic acid ethyl ester (445ng)
2,4,5-Trichlorophenoxy acetic acid (555ng)
Kinetin
(222ng)
Leu
6
3
Gradient Program
Time (min) %B
2
4
0
1.25
5.0-5.5
6.5-7.5
10
1
5
8
9
13
20
35
13
I
7
12
11
0
1
2
3
4
13
5
6
Min
Plant Growth Regulators (HPLC)
Column: SUPELCOSIL LC-18-DB, 3.3cm x 4.6mm ID, 3µm particles
Cat. No.: 58978
Mobile Phase: A = 1% acetic acid/1mM tetrabutylammonium phosphate in
water, pH 2.8
B = acetonitrile
Flow Rate: 2mL/min
Temp.: 35°C
Det.: UV, 280nm
Inj.: compounds in 13:87 A:B
713-0852
m1A
0
10
Min
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
5
0
m1G
713-0955
m2G
SUPELCO
128
HPLC Applications
Organic Acids
4
1.
2.
3.
4.
5.
6.
7.
8.
Homovanillic acid
Sorbic acid
Salicylic acid
p-Toluic acid
2
4
4
6
5
7
3
1
2
6
Min
8
Organic Acids (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
Discovery C18, 15cm x 4.6mm ID, 5µm particles
504955
methanol:water, 0.1% TFA (40:60)
1mL/min
20°C
UV, 254nm
10µL, 1mg/mL each analyte
10
Min
12
14
Organic Acids (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Det.:
Inj.:
2
1.
2.
3.
4.
8
6
794-0231
0
Oxalic (0.4µg)
Citric (5µg)
Tartaric (5µg)
Malic (6.2µg)
Succinic (12.5µg)
Formic (7.5µg)
Acetic (12.5µg)
Fumaric (impurity)
797-0657
3
Homovanillic acid (0.0625µg/mL)
Sorbic acid (0.00625µg/mL)
Salicylic acid (0.0625µg/mL)
p-Toluic acid (0.00625µg/mL)
SUPELCOGEL C-610H, 30cm x 7.8mm ID
59320-U
0.1% phosphoric acid
0.5mL/min
UV, 210nm
5µL
1
2
Discovery C18
4
1
3
1.
2.
3.
4.
1.0
2.0
Min
2
3.0
G001431
4
Tartaric acid
Malic acid
Lactic acid
Citric acid
3
Discovery HS C18
0
1
4
1.0
2.0
3.0
4.0
Min
Organic Acids (HPLC)
Columns:
Cat. Nos.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
5cm x 4.6mm ID, 5µm particles
504947 (C18), 568520-U (HS C18)
60:40 0.1%TFA in water:MeOH
2.0mL/min
20°C
UV, 254nm
10µL
2
Min
4
α -Hydroxy Aliphatic Acids (HPLC)
3
G001432
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
SUPELCO
1
1.
2.
3.
4.
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
Discovery RP-AmideC16, 15cm x 4.6mm ID, 5µm particles
505013
25mM KH2 PO4, pH 3.0
1mL/min
30°C
UV, 220nm
10µL
G000624
Liquid
Chromatography
2
129
HPLC Applications
Organic Acids, Peptides and Proteins
Hippuric acid
o-Methylhippuric acid
p-Methylhippuric acid impurity
p-Methylhippuric acid
m-Methylhippuric acid
200
Liquid
Chromatography
1.
2.
3.
4.
5.
1
150
100
50
0
10
20
Min
G001589
0
4
30
2
0
2
4
6
Min
8
713-0961
3
10
Hippuric and Methylhippuric Acids (HPLC)
Column: SUPELCOSIL LC-18, 7.5cm x 4.6mm ID, 3µm particles
Cat. No.: 58984
Mobile Phase: tetrahydrofuran:10mM KH 2PO4 , pH 3.0 with phosphoric acid
(3:97)
Flow Rate: 1.5mL/min
Det.: UV, 260nm
Inj.: 10µL spiked urine
(300µg/mL HA, o-MHA; 150µg/mL m-MHA, p-MHA)
Columns: Discovery BIO Wide Pore C18, 25cm x 4.6mm ID, 5µm particles
Cat. 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/min
Temp: 30°C
Det.: UV, 215nm
Inj.: 10µL, 1mg/mL in water
Gradient: 35-55%B in 20 min, 55%B for 5 min, 35%B for 5 min
Peptide
1. Angiotensin II
2. Angiotensin III
3. Angiotensin I
1
Sequence
DRVYIHPF
DRVYIHPF
DRVYIHPFHL
3
G001585
2
0
2
4
6
8
10
12
Min
0
10
Min
20
α -Amylase, Crude (HPLC)
Column: TSKgel Ether-5PW, 7.5cm x 7.5mm ID, 10µm particles
Cat. No.: 808641
Mobile Phase: A = 0.1M phosphate buffer + 1.1M Na 2 SO4, pH 7.0
B = 0.1M phosphate buffer
A to B over 60 min
Flow Rate: 1.0mL/min
Det.: UV, 280nm
713-0961
Angiotensins (HPLC)
Column: Discovery BIO Wide Pore C18, 15cm x 4.6mm ID, 5µm particles
Cat. No.: 568222-U
Mobile Phase: (A) 65:35, (10mM NH 4 OAc, pH 7):(50% CH 3 CN in 20mM
NH 4OAc, pH 7)
(B) 25:75, (10mM NH 4OAc, pH 7):(50% CH 3CN in 20mM
NH 4OAc, pH 7)
Flow Rate: 1.0mL/min
Temp: ambient
Det.: UV, 215nm
Inj.: 6µL (10µg each) in water
Gradient: 0-100%B in 12.5 min
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
Amyloid ß-Protein Fragment 10-35 (HPLC)
5
SUPELCO
130
HPLC Applications
Peptides and Proteins
1.Angiotensin I
2.Angiotensin II
3.Angiotensin III
Liquid
Chromatography
3
1
1. Angiotensin II (1.67g/L)
2. Angiotensin III (1.67g/L)
3. Angiotensin I (1.67g/L)
1
20
Min
G001085
10
Angiotensins (HPLC)
Column: Discovery C18, 15cm x 4.6mm ID, 5µm particles
Cat. No.: 504955
Mobile Phase: (A) 5mM (NH 4)H2PO4/NH 4OH, pH 7.0
(B) 5mM (NH 4)H2PO4/NH 4 OH, pH 7.0: CH 3 CN (50:50)
Flow Rate: 1mL/min
Temp.: 35°C
Det.: UV, 220nm
Gradient: 30-60% B in 15 min
10
Min
20
Angiotensins (HPLC)
Column: Discovery BIO Wide Pore C8, 15cm x 4.6mm ID, 5µm particles
Cat. No.: 568322-U
Mobile Phase: (A) 10mM NH 4H2 PO4/ NH 4 OH, pH 7
(B) 50:50, (20mM NH 4 H2PO4/ NH 4 OH, pH 7):CH 3 CN
Flow Rate: 1mL/min
Temp: 30°C
Det.: UV, 215nm
Inj.: 6µL in water
Gradient: 30-60% B in 15 min
1
3.34
1046.6
100
II
I
1. Bacitracin A
2. Virginiamycin M1
Discovery RP-AmideC16
8.36
649.1
Gradient Program
Time (min) %B
2
20
100
G000911
0
12.5
Relative Abundance
4.10
931.5
III
50
0
2
4
6
8
10
8
10
Min
1
2
Discovery C8
0
0
10
Min
20
G001718
Angiotensins Capillary Separation (HPLC)
Column: Discovery BIO Wide Pore C18, 10cm x 0.32mm ID,
3µm particles
Cat. No.: 65527-U
Solution x: 10mM NH 4OAc/NH 4OH, pH 7.0
Solution y: 50:50 (20mM NH 4OAc/NH 4 OH, pH 7.0) : CH 3 CN
Mobile Phase A: x:y, 65:35
Mobile Phase B: x:y, 25:75
Flow Rate: 6µL/min
Det.: +ES, source 2.5kV, capillary 12V, 130° C
Temp.: ambient
Inj.: 50pmol
0
2
4
6
Min
Antibiotics, Peptide (HPLC)
Columns:
Cat. Nos.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
15cm x 4.6mm ID, 5µm particles
505013 (RP-AmideC16), 59353-U (C8)
25mM KH2PO4, pH 3:acetonitrile, 60:40
1mL/min
35°C
UV, 220nm
10µL 25mM KH 2 PO4, pH 3 containing 50µg/mL each analyte
G000920
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
0
SUPELCO
3
2
0
(DRVYIHPF)
D(RVYIHPF)
(DRVYIHPFHL)
G001503
2
131
HPLC Applications
Peptides and Proteins
Discovery BIO Wide Pore C18
Liquid
Chromatography
Discovery RP-AmideC16
G001087
Discovery BIO Wide Pore C8
Discovery C18
40
60
0
Apohemoglobin, Carboxymethylated, Tryptic Digest
(HPLC)
20
40
60
Min
80
100
1.
2.
3.
4.
5.
6.
0
100
1
20
40
Min
4
2
3
120
0
0
60
Apohemoglobin (Horse), Carboxymethylated, Tryptic
Digest (HPLC)
Column: Discovery BIO Wide Pore C18, 15cm x 2.1mm ID,
3µm particles
Cat. No.: 567202-U
Mobile Phase A: water/0.1% TFA
Mobile Phase B: 50:50, (water/0.1% TFA) : (CH3 CN/0.1% TFA)
Flow Rate: 0.208mL/min
Temp: 35°C
Det.: UV, 215nm
Inj.: 5µL
Sample: digest
80
1
G001719
0
60
Min
100
120
Columns: 25cm x 4.6mm ID, 5µm particles
Cat. Nos.: 505013 (RP-AmideC16), 504955 (C18)
Mobile Phase: (A) 0.1% TFA, 1% CH3CN in H 2O
(B) 0.1% TFA, 40% CH3CN
Flow Rate: 1 mL/min
Det.: UV, 215nm
Temp.: 35°C
Gradient: 0-100% B in 130 min
Gradient Program
Time (min)
%A
%B
100
0
40
Carboxyamido-methylated BSA Tryptic Digest (HPLC)
Columns: 15cm x 4.6mm ID, 5µm particles
Cat. 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 H2 O):(0.1% TFA in CH3 CN);
Flow Rate: 1mL/min
Temp: 30°C
Det.: UV, 215nm
Inj.: 50µL
Sample: carboxymethylated apohemoglobin tryptic digest in 50mM
NH 4HCO3
Gradient: 0-100% B in 65 min
0
50
20
Horse cytochrome c
Rabbit cytochrome c
Cow cytochrome c
Pigeon cytochrome c
Chicken cytochrome c
Dog cytochrome c
5
6
10
Min
Cytochrome c (HPLC)
Column: Discovery BIO Wide Pore C18, 15cm x 4.6mm ID,
5µm particles
Cat. No.: 568222-U
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 CH3 CN)
Flow Rate: 1.0mL/min
Temp: ambient
Det.: UV, 220nm
Inj.: 12µL each at 0.8mg/mL in 0.1%TFA
Gradient: 0-100%B in 30 min
20
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
Min
G001086
20
G001581
0
G001504, 05, 06
Discovery BIO Wide Pore C5
SUPELCO
132
HPLC Applications
Peptides and Proteins
3
Liquid
Chromatography
Discovery RP-AmideC16
Cytochrome c Source
1. Bovine
2. Horse
3. Rabbit
2
G000308
1
0
10
20
30
Min
40
50
Discovery C18
SUPELCO
10
Min
15
20
796-0477
5
Figure used with permission of Mitsubishi Chemical Co.
0
10
20
30
40
50
Min
G000307
Cytochrome c Species Variants (HPLC)
Column: ProtEx-SP, 5cm x 4.6mm ID, 5µm particles
Cat. No.: 54740-U
Mobile Phase: A = 20mM Bis-Tris HCl, pH 7.0
B = A + 0.5M NaCl
24-69% B over 20 min
Flow Rate: 0.5mL/min
Det.: UV, 280nm
Inj.: 10µg each variant
Cytochrome c Tryptic Digest (HPLC)
Columns: 15cm x 4.6mm ID, 5µm particles
Cat. 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 CH 3CN
Flow Rate: 1mL/min
Temp.: 35°C
Gradient: 1 - 37% B in 72 min
Gradient Program
Time (min) %B
0
0.5
1.0
2
20
20
80
1. Guanidine-HCl
2. BSA
Gradient Program
Time (min)
%A
%B
0
50
100
0
Undigested protein
0
100
1
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Min
Desalting of Proteins (HPLC)
0
20
40
Min
60
Carboxymethylated Cytochrome c (Chicken) Tryptic
Digest (HPLC)
Column: Discovery BIO Wide Pore C18, 15cm x 2.1mm ID,
3µm particles
Cat. No.: 567202-U
Mobile Phase A: water/0.1% TFA
Mobile Phase B: 50:50, (water/0.1% TFA) : (MeCN/0.1% TFA)
Flow Rate: 0.208mL/min
Temp: 35°C
Det.: 215nm
Inj.: 5µL
Sample: digest
G001721
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
0
Column: Supelguard LC-308, 2cm x 4.6mm ID, 5µm particles,
300Å pores
Cat. No.: 59511-U
Mobile Phase: A = 0.1% TFA in water
B = 0.09% TFA in acetonitrile
Flow Rate: 2mL/min
Det.: UV, 220nm
Inj.: 40µL of 200µg BSA in 3M guanidine-HCl
133
HPLC Applications
Peptides and Proteins
hGH
Ferredoxin
NADP reductase
Liquid
Chromatography
Untreated hGH
Crude Sample
Standard
Desamido hGH derivative
0
30
60
90
Min
713-0922
Heated hGH: 60°C, 5 hr.
Heated hGH: 70°C, 8 hr.
Desamido hGH derivatives
0
25
30
Column: ProtEx-DEAE, 5cm x 4.6mm ID, 5µm particles
Cat. No.: 54742
Mobile Phase: A = 20mM Tris-HCl, pH 8.0
B = A + 0.5M NaCl
5-70% B over 30 min
Flow Rate: 0.5mL/min
Inj.: 10mg hGH
A405nm
100
TSKgel G3000SWXL, 30cm x 7.8mm ID, 5µm particles
808541
0.3M NaCl/0.05M NaH 2PO4, pH 7.0
1mL/min
25°C
UV, 220nm
100µL crude glutathione S-transferase preparation from guinea
pig liver (0.7mg)
HbA1c
%B
100
0
0
5
Min
10
Reproduced with permission of Amersham Biosciences.
Hemoglobin HbA
1c
(HPLC)
Column: Mono S HR5/5, 10µm particles
Cat. No.: 54808
Mobile Phase: A = 0.01M malonate-NaOH, pH 5.7
B = 0.3M LiCl in mobile phase A
Flow Rate: 2.0mL/min
Det.: VIS, 405nm
Inj.: 50µL hemolysate
diluted 1:5
794-0412
Glutathione S-Transferase (HPLC)
% full scale
15
Min
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
20
713-0939
10
15
Min
Purity Evaluation of Human Growth Hormone (hGH)
(HPLC)
Standard
5
10
Figure used with permission of Mitsubishi Chemical Co.
Active fraction (recovered activity = 89%)
0
5
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
Column: TSKgel Phenyl-5PW column, 7.5cm x 7.5mm ID,
10µm particles
Cat. No.: 807573
Mobile Phase: 60 min decreasing linear gradient from 1.5M to 0M ammonium
sulfate in 0.1M sodium phosphate buffer, pH 7
Flow Rate: 0.5mL/min
Temp.: 25°C
Det.: UV, 280nm
Inj.: 3mg (crude) or 0.2mg (standard)
796-0659
Ferredoxin NADP Reductase (HPLC)
SUPELCO
134
HPLC Applications
Peptides and Proteins
A280nm
Liquid
Chromatography
A280nm
5.0
100
4.0
80
3.0
60
0.1
0.05
IGF-1
Fraction 1 2 3 4
2.0
Time (h)
Reproduced with permission of Amersham Biosciences.
1.0
40
IgM
containing
fraction
Start of
elution
100
SUPELCO
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Det.:
Inj.:
HiLoad 16/60 Superdex 75 prep grade, 34µm particles
54802-U
0.15M ammonium acetate, pH 6.0
0.75mL/min
UV, 280nm
IGF-1, ZZ fusion protein and
uncleaved material
200
Volume, mL
300
400
Reproduced with permission of Amersham Biosciences.
Immunoglobulins, IgM (HPLC)
Medium:
Cat. No.:
Column:
Mobile Phase:
Q Sepharose Fast Flow
Q1126-100ML
30cm x 50mm ID
A = 0.02M NaH2PO4, pH 6.5
B = A + 0.5M NaCl, pH 6.5
Flow Rate: 25cm/hr
Det.: UV, 280nm
Inj.: culture supernatant
A280
2.0
N-Hydroxysuccinimide
1
1.5
2
3
1. Bovine insulin (5µg)
2. Human insulin (5µg)
3. Porcine insulin (5µg)
1.0
h-lgG
2
5
10
20
15
Fraction number
Reproduced with permission of Amersham Biosciences.
Immunoglobulins, IgG, Human (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Det.:
Inj.:
HiTrap Desalting, 5mL
54822
0.2M sodium carbonate buffer, pH 8.5
syringe use
UV, 280nm
0.5mL unbound human IgG in 0.2M sodium carbonate buffer
after coupling to a HiTrap NHS 1mL column.
Fraction volume 0.5mL
4
6
Min
8
10
Insulin (HPLC)
Column: Discovery BIO Wide Pore C8, 15cm x 4.6mm ID, 5µm particles
Cat. No.: 568322-U
Mobile Phase: (A) 71:29, (0.1% TFA in H2 O):(0.1% TFA in CH3CN);
(B) 68:32, (0.1% TFA in H2O):(0.1% TFA in CH3CN)
Flow Rate: 1mL/min
Temp: 30°C
Det.: UV, 215nm
Inj.: 50µL in 0.1% TFA
Gradient: 0-100% B in 30min
G001507
0.5
796-0403
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
IGF-1, Insulin-like Growth Factor-1 (HPLC)
20
794-0370
1.0
794-0420
0
2.0
VC
VO
135
HPLC Applications
Peptides and Proteins
2
1
Discovery C8
1. Bovine insulin
2. Human insulin
3. Porcine insulin
2
3
Liquid
Chromatography
1. Subunit A oxidized
2. Holoenzyme
3. Subunit B oxidized
3
Gradient Program
Time (min) %B
0
18
33.3
73.3
0
2
4
6
8
G001580
1
10
Min
20
B
Holoenzyme (Sigma I5500)
reduced and carboxy-methylated
A. Subunit A
B. Subunit B
Discovery C8
Column: Discovery BIO Wide Pore C5, 15cm x 4.6mm ID, 5µm particles
Cat. No.: 568422-U
Mobile Phase: (A) 71:29, (0.1% TFA in water):(0.1% TFA in CH3CN);
(B) 68:32, (0.1% TFA in water):(0.1% TFA in CH3 CN)
Flow Rate: 1.0mL/min
Temp: ambient
Det.: UV, 215nm
Inj.: 5µL each at 1mg/mL in 0.1%TFA
Gradient: 0-100%B in 30 min
A280nm
Gradient Program
Time (min) %B
0.1
A
hPL
10
20
Min
Insulin, Bovine (HPLC)
Discovery C8, 5cm x 4.6mm ID, 5µm particles
59353-U
25mM KH2 PO4, pH 3:acetonitrile, 60:40
A = 50mM H2 NaPO4 , 50mM Na 2SO4, pH 3.0 (w/H 3PO4), 5%
acetonitrile
B = 50mM H 2 NaPO4 , 50mM Na 2 SO4 , pH 3.0 (w/H 3PO4), 50%
acetonitrile
Flow Rate: 1mL/min
Temp: 35°C
Det.: UV, 220nm
0
0
10
40
G000766
0
0
10
25
26
NaCl
Columns:
Cat. Nos.:
Mobile Phase:
Mobile Phase:
0
20
Min
Reproduced with permission of Amersham Biosciences.
Lactogen, Human Placental (HPLC)
Column: Mono Q HR5/5, 10µm particles
Cat. No.: 54807
Mobile Phase: A = 20mM bis-Tris, pH 6.0
B = A + 1M NaCl
Flow Rate: 1.0mL/min
Det.: UV, 280nm
Inj.: 2.5mL hPL fraction from size exclusion medium
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
10
Min
794-0421
0
G000765
Insulin (HPLC)
SUPELCO
136
HPLC Applications
Peptides and Proteins
100
0
0
100
0
SUPELCO
40
Min
Lipoxidase, Commercial (HPLC)
60
Column: TSKgel Phenyl-5PW, 7.5cm x 7.5mm ID,
10µm particles
Cat. No.: 807573
Mobile Phase: A = 0.1M phosphate buffer, pH 7.0 + 1.5M (NH 4)2 SO4
B = 0.1M phosphate buffer, pH 7.0
linear gradient, 0% B
to 100% B over 60 min
Flow Rate: 0.5mL/min
Det.: UV, 280nm
ß-Lactoglobulin (Cow), Carboxymethylated, Tryptic
Digest (HPLC)
A280 (
0.5
27.66
859.2
21.42 22.80
21.00 696.2 818.7
419.4
0.2
15.90
458.9
29.17
27.17 555.2
1044.6
30.49
772.1 35.06
522.6
24.54
34.37
775.4
522.5
12.36
573.3
0.25
0.1
36.66
322.0
37.71
522.6
0
0
10
20
Min
30
40
G001591
50
0.4
0.3
18.46
623.4
17.17
337.3
1.0
)
Column: Discovery BIO Wide Pore C18, 15cm x 2.1mm ID,
3µm particles
Cat. No.: 567202-U
Mobile Phase A: water/0.1% TFA
Mobile Phase B: 50:50, (water/0.1% TFA) : (MeCN/0.1% TFA)
Flow Rate: 0.208mL/min
Temp.: 35°C
Det.: 215nm
Inj.: 5µL
Sample: digest
100
60
NaCl concentration M (-----)
20
Relative Abundance
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
0
30
Min
794-0320
0
50
ß-Lactoglobulin Tryptic Digest, Capillary Separation
(HPLC)
Column: Discovery BIO Wide Pore C18, 15cm x 0.5mm ID,
5µm particles
Cat. No.: 65519-U
Mobile Phase: (A) 0.1% TFA in water;
(B) 0.1% TFA in CH3 CN
Flow Rate: 14µL/min
Temp.: 30°C
Inj.: 500pmol (5µL) ß-Lactoglobulin tryptic digest in 50mM NH 4HCO3
Gradient: 5-40%B in 70 min
MS conditions: +ESI mode Capillary Temp 130°C, Source Voltage 2.5KV, Capillary
Voltage 12V
0
5
10
15
Volume, mL
20
Reproduced with permission of Amersham Biosciences.
Liver Extract, Bovine (HPLC)
Column: Mono Q HR5/5, 10µm particles
Cat. No.: 54807
Mobile Phase: A = 50mM diethanolamine, pH 8.8
B = 50mM diethanolamine + 1.0M NaCl, pH 8.8
Flow Rate: 1.0mL/min
Det.: UV, 280nm
Inj.: 100µL, 1.6mg protein
794-0376
Liquid
Chromatography
Gradient Program
Time (min)
%A
%B
137
HPLC Applications
Peptides and Proteins
0.50
HiTrap Phenyl Sepharose High
Performance
0.40
6.
7.
8.
9.
HiTrap Butyl
Sepharose 4 Fast
Flow
Peptide
Arg 8 -vasopressin
Bradykinin, fragment 1-5
Oxytocin
Met-enkephalin
Luteinizing hormone
releasing hormone
Leu-enkephalin
Bradykinin
Bombesin
Substance P
Sequence
CFQNCPRG-amide; disulfide
RPPGF
CYQNCPLG-amide; disulfide
YGGFM
Liquid
Chromatography
1.
2.
3.
4.
5.
PEHWSYGLRPG-amide
YGGFL
RPPGFSPFR
PEQRLGNQWAVGHLM-amide
RPKPQQFFGLM-amide
0.30
5
0.20
HiTrap Octyl
Sepharose 4 Fast
Flow
HiTrap Phenyl
Sepharose 6 Fast Flow
(low sub)
HiTrap Phenyl
Sepharose 6 Fast Flow
(high sub)
4
1
0.10
6
8
7
9
3
10.0
20.0
Min
30.0
Reproduced with permission of Amersham Biosciences.
0
2
4
6
8
10
Min
12
14
16
18
Peptides (HPLC)
Monoclonal Antibody (HPLC)
Column: Discovery BIO Wide Pore C5, 15cm x 4.6mm ID, 5µm particles
Cat. No.: 568422-U
Mobile Phase: (A) 0.1% PFPA (pentafluoropropionic acid) in water:CH3CN
(81:19);
(B) 0.1% PFPA in water:CH3 CN (62:38)
Flow Rate: 1.0mL/min
Temp: 30°C
Det.: UV, 215nm
Inj.: 10µL, ~0.25µg each peptide (Sigma Peptide Mix, Cat. No. P
2693) in mobile phase A
Gradient: 0-100%B in 19 min
Column: HiTrap HIC cartridges
Cat. No.: 54814
Mobile Phase: A = 50mM sodium phosphate, 1.0M ammonium phosphate,
pH 7.0
B = 50mM sodium phosphate, pH 7.0
Flow Rate: 1.0mL/min
Det.: UV, 280nm
Inj.: 100µL anti-lac cl 507, 8.8mg/mL
1
1.
2.
3.
4.
5.
1
2
2
Gly-Tyr
Val-Tyr-Val
Met-Enkephalin
Leu-Enkephalin
Angiotensin II
Peptide 1:
Peptide 2:
Peptide 3:
Peptide 4:
ac-GGGLGGAGGLK-amide
ac-KYGLGGAGGLK-amide
ac-GGALKALKGLK-amide
ac-KYALKALKGLK-amide
3
3
20
15
60
0
10
Min
G001562, 63
Initial Injection
0
45
Peptides (HPLC)
Column: Discovery BIO Wide Pore C18, 5cm x 4.6mm ID, 5µm particles
Cat. No.: 568220-U
Mobile Phase: (A) 5:95, (0.5% TFA in water):(0.5% TFA in CH3CN);
(B) 25:75, (0.5% TFA in water):(0.5% TFA in CH3 CN)
Flow Rate: 2.0mL/min
Temp: 70°C
Det.: UV, 220nm
Inj.: 5µL, 2.5µg each peptide (Sigma Peptide Mix, Cat. No. H 2016) in
mobile phase A
Gradient: 2-24%B in 22 min, 8 min at 100%A
0
10
20
30
Min
Peptides, Basic, No TFA (HPLC)
Columns: Discovery BIO Wide Pore C18, 15cm x 2.1mm ID,
5µm particles
Cat. No.: 568202-U
Mobile Phase A: 25mM formic acid in water
Mobile Phase B: 50:50 (25mM formic acid in water) : (20mM formic acid in
CH3CN)
Flow Rate: 0.208mL/min
Det.: UV, 215nm
Temp.: 35°C
Inj.: 0.5µL (~0.25µg ea peptide)
Sample: RP Peptide Ionic Interactions Standard, p/n RPS-I0020
(Alberta Peptide Institute)
G001642
after 40,000
columns volumes
0
Gradient Program
Time (min) %B
40
5
5
mAU 215nm
20
4
4
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
0.00
G001492, 93, 94
796-0397 - 0401
2
SUPELCO
138
HPLC Applications
Peptides and Proteins
Peptide 1:
Peptide 2:
Peptide 3:
Peptide 4:
Peptide 5:
Liquid
Chromatography
Gradient Program
Time (min)
%A
%B
90
50
10
50
TFA mobile phase:
Peak Peptide
Amino Acid Sequence
1. Arg 8 -vassopressin
CYFQNCPRG-amide; disulfide
2. Bradykinin, fragment 1-5 RPPGF
3. Oxytocin
CYIQNCPLG-amide; disulfide
4. LHRH*
pEHWSYGLRPG-amide **
5. Met-enkephalin
YGGFM
6. Bradykinin
RPPGFSPFR
7. Leu-enkephalin
YGGFL
8. Bombesin
pEQRLGNQWAVGHLM-amide **
9. Substance P
RPKPQQFFGLM-amide
* Luteinizing Hormone Releasing Hormone
** pE is pyroglutamate
0% TFA (25mM formic acid), 1µL injection
5
100
4
3
2
Gradient Program
Time (min)
%A
%B
4
1
0
10
Min
20
5 6
1 2
100
0
0
100
7
8
3
9
Peptides, Alberta Mix, No TFA (HPLC)
Column: Discovery BIO Wide Pore C18, 15cm x 2.1mm ID,
3µm particles
Cat. No.: 567202-U
Mobile Phase A: 25mM formic acid in water,
Mobile Phase B: 50:50 (25mM formic acid in water) : (20mM formic acid in
CH3 CN) a,
Flow Rate: 0.208mL/min b
Det.: +ES
Temp.: ambient
Inj.: 1µL
Sample: RP Peptide Performance Standard, p/n RPS-P0010 (Alberta
Peptide Institute)
0
2
4
6
5
ac-GGGLGGAGGLK-amide
ac-KYGLGGAGGLK-amide
ac-GGALKALKGLK-amide
ac-KYALKALKGLK-amide
0
20
Min
30
2
4
0
100
6 7
8
3
6
100
0
8
10
Min
9
12
14
16
Peptides, Synthetic (HPLC)
15
60
G001642
10
14
Gradient Program
Time (min)
%A
%B
4
1
Gradient Program
Time (min) %B
0
12
0
19
2
0
45
10
PFPA (penta-fluoropropionic acid) mobile phase:
a) molarity of formic acid adjusted to provide minimum baseline drift
b) linear velocity equal to 1mL/min on 4.6mm ID columns
Peptide 1:
Peptide 2:
Peptide 3:
Peptide 4:
8
Min
G001593
0
G001638
0
14
Peptides, Synthetic (HPLC)
Columns: Discovery BIO Wide Pore C18, 15cm x 2.1mm ID, 5µm
particles
Cat. No.: 568202-U
Mobile Phase A: 25mM formic acid in water
Mobile Phase B: 50:50 (25mM formic acid in water):(20mM formic acid in
CH3 CN)
Flow Rate: 0.208 mL/min
Det.: UV, 215nm
Temp.: 35°C
Inj.: 0.5µL (~0.25µg ea peptide)
Sample: RP Peptide Ionic Interactions Standard, p/n RPS-I0020 (Alberta
Peptide Institute)
TFA:
Column: Discovery BIO Wide Pore C18, 15cm x 4.6mm ID,
5µm particles
Cat. No.: 568222-U
Mobile Phase A: 80:20, (water/0.1%TFA) : (CH3 CN/0.1%TFA)
Mobile Phase B: 66:34, (water/0.1%TFA) : (CH3 CN/0.1%TFA)
Flow Rate: 1.0mL/min;
Det.: UV, 215nm
Temp.: 30°C
Inj.: 10µL Sigma Peptide Mix P2693 (~25µg ea. Peptide)
PFPA:
Column: Discovery BIO Wide Pore C18, 15cm x 4.6mm ID,
5µm particles
Cat. No.: 568222-U
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)
Flow Rate: 1.0mL/min
Det.: UV, 215nm
Temp.: 30°C
Inj.: 10µL Sigma Peptide Mix P2693 (~25µg ea. Peptide)
18
G001594
%
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
SUPELCO
0
20
ac-RGAGGLGLGK-amide
ac-RGGGGLGLGK-amide
ac-RGAGGLGLGK-amide
ac-RGVGGLGLGK-amide
ac-RGVVGLGLGK-amide
139
HPLC Applications
Peptides and Proteins
Recovered Activity = 98%
RNase (13.7kDa, 1mg/mL)
Cytochrome c (12.4kDa, 1mg/mL)
Lysozyme (14.3kDa, 1mg/mL)
BSA (67.0kDa, 2.5mg/mL)
Myoglobin (18.8kDa, 1mg/mL)
Ovalbumin (45.3kDa, 3.5mg/mL)
Liquid
Chromatography
1.
2.
3.
4.
5.
6.
3
5
10
15
Min
4
2
5
1
Impurity in
Lysozyme
400
6
Impurity in
Myoglobin
200
Column: TSKgel G3000SWXL, 30cm x 7.8mm ID, 5µm particles
Cat. No.: 808541
Mobile Phase: 0.3M NaCl/0.05M phosphate buffer,
pH 7.0
Flow Rate: 1mL/min
Temp.: 25°C
Det.: UV, 220nm
Inj.: 100µL crude peroxidase preparation from Japanese horseradish
(0.15mg)
1
Absorbance (280nm)
ProtEx-DEAE
1. Myoglobin
2. Conalbumin
3. Trypsin inhibitor
0
0
10
Min
20
Proteins (HPLC)
Columns:
Cat. No. :
Mobile Phase:
Flow Rate:
Temp:
Det.:
Inj.:
Gradient:
Discovery BIO Wide Pore C5, 15cm x 4.6mm ID, 5µm particles
568422-U
75:25, (0.1% TFA in water):(0.1% TFA in CH3 CN);
1.0mL/min
ambient
UV, 220nm
12µL in 0.1%TFA
0-100%B in 25 min
2
1.
2.
3.
4.
5.
3
Trypsinogen
Ribonuclease A
α-Chymotrypsinogen
Cytochrome c
Lysozyme
0
5
10
Time (Min)
4
5
2
796-0657
1
3
15
ProtEx-SP
1. Ribonuclease A
2. α-Chymotrypsinogen A
3. Cytochrome c
Absorbance (280nm)
10
20
Min
30
Proteins (HPLC)
Column: TSKgel CM-5PW, 7.5cm x 7.5mm ID, 10µm particles
Cat. No.: 813068
Mobile Phase: A = 0.1M NaH2PO4 buffer, pH 7.0
B = 0.5M NaCl, A to B in 60 min
Flow Rate: 1.0mL/min
Det.: UV, 280nm
1
0
5
10
Time (Min)
15
20
Figures used with permission of Mitsubishi Chemical Co.
Proteins (HPLC)
Column: ProtEx-DEAE or ProtEx-SP, 5cm x 4.6mm ID, 5µm particles
Cat. No.: 54742 (ProtEx-DEAE) or 54740-U (ProtEx-SP)
Mobile Phase: Left: A = 20mM Tris-HCl, pH 8.0, B = A + 0.5M NaCl
5-100% B over 15 min
Right: A = 20mM sodium phosphate, pH 7.0,
B = A + 0.5M NaCl
5-100% B over 15 min
Flow Rate: 0.5mL/min
Det.: UV, 280nm
Inj.: 10µg trypsin inhibitor, 5µg conalbumin, myoglobin or
10µg ribonuclease A, 5µg cytochrome c, a-chymotrypsinogen A
796-0658
20
0
40
713-1002
3
2
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
Peroxidase (HPLC)
G001488
0
713-0940
600
SUPELCO
140
HPLC Applications
Peptides and Proteins
A – Butyl-NPR
Liquid
Chromatography
DEAE-NPR
A to B in 10 min
at 1.5mL/min
2
1. Ovalbumin
2. Trypsin inhibitor
5
3
1
1
4
2
0
2
3
1.
2.
3.
4.
5.
4
Min
5
2
4
Min
6
6
8
Myoglobin
Ribonuclease
Lysozyme
α-Chymotrypsin
β-Chymotrypsinogen
10
SUPELCO
5
2
DEAE-5PW
A to B in 60 min
at 1mL/min
3
1
2
20
Min
30
713-1020, 713-1021
0
4
30
Min
60
1
1
C – Ether-5PW
Proteins (HPLC)
Column A:
Cat. No.:
Column B:
Cat. No.:
Mobile Phase:
TSKgel DEAE-NPR, 3.5cm x 4.6mm ID, 2.5µm particles
813075
TSKgel DEAE-5PW, 7.5cm x 7.5mm ID, 10µm particles
807164
A = 20mM Tris-HCl buffer, pH 8.0
B = 0.5M NaCl in A
Det.: UV, 280nm
5
3
2
4
0
15
30
Min
45
713-1025, 1026, 1027
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
B – Phenyl-5PW
1
Proteins (HPLC)
Column A:
Cat. No.:
Column B:
Cat. No.:
Column C:
Cat. No.:
Mobile Phase:
TSKgel Butyl-NPR, 3.5cm x 4.6mm ID, 2.5µm particles
814947
TSKgel Phenyl-5PW, 7.5cm x 7.5mm ID, 10µm particles
807573
TSKgel Ether-5PW, 7.5cm x 7.5mm ID, 10µm particles
808641
A = 0.1M phosphate buffer, pH 7.0 plus (NH 4)2 SO4, 2.3M to
0M in12 min
B & C = 0.1M phosphate buffer, pH 7.0 plus (NH 4) 2SO4 , 1.8M to
0M in 60 min
Flow Rate: 1mL/min
Det.: UV, 280nm
141
HPLC Applications
Peptides and Proteins
1.0
Gradient Program
Time (min) %B
Gradient Program
Time (min) %B
0
8
24
48
0
6
11
0
0
100
100
1.
2.
3.
4.
5.
6.
7.
3
5
29
34
5
4
2
Fragment 1-4
Fragment 1-7
Fragment 1-9
Fragment 8-11
Holopeptide
Fragment 2-11
Fragment 7-11
Liquid
Chromatography
A280nm
6
1
7
2
4
6
Min
8
10
12
Substance P and Its Fragments (HPLC)
5
10
15
20
25
mL
Reproduced with permission of Amersham Biosciences.
794-0425
0
Proteins, Egg White (HPLC)
Column: HiTrap Chelating, 1mL
Cat. No.: 54834
Mobile Phase: A = 0.2M NaH2PO4 , 1M NaCl, pH 7.2
B = 0.02M NaH 2 PO4, 1M NH 4Cl, pH 7.2
Flow Rate: 0.5mL/min
Temp.: 40°C
Det.: UV, 280nm
Inj.: 200µL egg white
Column: Discovery C18, 15cm x 4.6mm ID, 5µm particles
Cat. No.: 504955
Mobile Phase: A = water + 0.1% TFA
B = acetonitrile + 0.1% TFA
Flow Rate: 1mL/min
Temp.: 35°C
Det.: UV, 220nm
Inj.: Substance P in initial mobile phase
α-Trypsin
Fraction 2
A280nm
100% B
β-Trypsin
0.5
0.4
0.2
0
40% B
2
4
Min
6
8
10
Fract. 24
0.1
Trypsin (HPLC)
Fract. 16
Fract. 17
Fraction 9
0
5
10
15
20
Volume, mL
25
Reproduced with permission of Amersham Biosciences.
30
795-0493
10% B
Proteins, Milk (HPLC)
Column: HiTrap SP, 1mL
Cat. No.: 54817
Mobile Phase: A = 50mM Na acetate, pH 6.0
B = 50mM Na acetate/1.0M NaCl , pH 6.0
Flow Rate: 1mL/min
Det.: UV, 280nm
Inj.: 0.5mL filtered casein-precipitated human milk (buffer exchanged
to mobile phase A)
Column: TSKgel Butyl-NPR, 3.5cm x 4.6mm ID, 2.5µm particles
Cat. No.: 814947
Mobile Phase: 2M to 0M ammonium sulfate in 20mM Tris-HCI buffer, pH 7.5 in
10 min
Flow Rate: 1.0mL/min
Det.: UV, 280nm
Inj.: commercial trypsin sample
713-1204
0.3
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
0
G000322
0.5
SUPELCO
142
HPLC Applications
Peptides, Proteins, Pesticides
A280
3
%B
100
Liquid
Chromatography
Phospholipase A
Phospholipase B
1.
2.
3.
4.
Hyaluronidase
Acid phosophatase
4
50
1
30
40
Reproduced with permission of Amersham Biosciences.
Venom, White-Faced Hornet (HPLC)
Column: Mono S HR5/5, 10µm particles
Cat. No.: 54808
Mobile Phase: A = 50mM BICINE, pH 8.4
B = A + 0.35M NaCl
Flow Rate: 1.0mL/min
Det.: UV, 280nm
Inj.: 7mg venom in mobile phase A
2
Discovery C18
3
1.
2.
3.
4.
5.
6.
7.
8.
4
6
7
2.0
3.0
Min
Urea Pesticides (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Solvent
Dalapon
2,4-D
2,4-DB
2,4,5-T
2,4-D Methyl ester
2,4,5-T Methyl ester
2,4-DB Methyl ester
Discovery Cyano, 15cm x 4.6mm ID, 5µm particles
59356-U
60:40 water:CH3CN
2.0mL/min
20°C
UV, 214nm
1µL
Caffeine (100ng)
Metamitron (50ng)
Fenuron (50ng)
Metoxuron (50ng)
Simazine (5ng)
Bromacil (50ng)
Cyanazine (50ng)
Atrazine (5ng)
Carbaryl (5ng)
Isoproturon (50ng)
Propham (50ng)
12.
13.
14.
15.
16.
17.
18.
19.
20.
U
Propazine (50ng)
Terbutylazine (5ng)
Linuron (50ng)
Propanil (50ng)
Prometryn (50ng)
Fenamiphos (50ng)
Fenitrothion (50ng)
Parathion (50ng)
Permethrin (50ng)
Unknown
16
5
8
U
9
2
18
10
Min
20
10
2
3
Discovery C8
4 6
5 7
13
1
8
14
11 12
19
15
17
20
4
3
1
0
0
7
6
4
8
6
8
Min
10
12
14
Organo-Acid Pesticides (HPLC)
Columns:
Cat. Nos.:
Mobile Phase:
Flow Rate:
Det.:
Temp.:
Inj.:
40
60
80
100
120
Min
5
2
20
15cm x 4.6mm ID, 5µm particles
504955 (C18), 59353-U (C8)
60:40 25mM potassium phosphate, (pH 2.3):CH 3CN
2.0mL/min
UV, 214nm
20°C
1µL
Figures reproduced courtesy of Dr. Claude Corvi, Laboratories Cantonal de
Chimie, Geneva, Switzerland.
Pesticides and Herbicides (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
SUPELCOSIL LC-ABZ, 25cm x 4.6mm ID, 5µm particles
59142
10% to 90% acetonitrile in water at 0.5%/min
1mL/min
40°C
UV, 225nm
50µL water, spiked with analytes
794-0033
0
G001042, 1041
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
20
Min
794-0411
1.0
10
G001067
2
0
SUPELCO
Fenuron
Monuron
Diuron
Linuron
140
143
HPLC Applications
Pesticides
Methomyl (80ng)
Oxamyl (80ng)
Fenuron (80ng)
Monuron (80ng)
Carbofuran (1000ng)
Propoxur (800ng)
Carbaryl (200ng)
Fluometuron (80ng)
9.
10.
11.
12.
13.
14.
15.
Diuron (80ng)
Propham (80ng)
Siduron (80ng)
Linuron (80ng)
Chlorpropham (80ng)
Barban (80ng)
Neburon (80ng)
1
1.
2.
3.
4.
5.
6.
7.
8.
6
12
Dichlorvos
Ethoprophos
Guthion
Methyl parathion
Disulfoton
Fenchlorvos
Chlorpyrifos
Prothiophos
Liquid
Chromatography
1.
2.
3.
4.
5.
6.
7.
8.
4
2
13
5
3
3
14
7
5
11
15
8
2
4
10
2
4
6
Min
8
10
12
794-0803
0
0
Carbamate and Urea Pesticides (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
8
7
SUPELCOSIL LC-8, 15cm x 4.6mm ID, 5µm particles
58220-U
acetonitrile:water gradient, 18:82 to 65:35 in 9 min, hold 3 min
2.0mL/min
35°C
UV, 240nm
10µL
10
20
Min
30
40
Organophosphorus Pesticides (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
Discovery Cyano, 15cm x 4.6mm ID, 5µm particles
59356-U
75:25 water:CH3CN
2.0mL/min
20°C
UV, 214nm
1µL
6
Gradient Program
Time (min) %B
85
85
42.5
42.5
11
12
14
6
3
16
10
7
2
9
5
4
Acetone
Oxamyl
Methomyl
Fenuron
Aldicarb
Simazine
Monuron
Cyanazin
Carbofuran
Atrazine
Carbaryl
Diuron
Propachlor
Linuron
Chlorpropham
Barban
1
2
5
3
4
13
8
8
1
7
2
4
6
8
10
Min
Urea/Carbamate/Triazine/An
12
14
16
18
iline Pesticides (HPLC)
Column: Discovery C18, 15cm x 4.6mm ID, 5µm particles
Cat. No.: 504955
Mobile Phase: A = acetonitrile
B = water
Flow Rate: 2mL/min
Temp.: 20°C
Det.: UV, 214nm
Inj.: 1µL
G000948
0
0
Dichlorvos
Guthion
Methyl parathiion
Ethoprophos
Disulfoton
Fenchlorvos
Chlorpyrifos
Prothiophos
10
20
Min
Organophosphorus Pesticides (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp.:
Det.:
Inj.:
Discovery RP-AmideC16, 15cm x 4.6mm ID, 5µm particles
505013
acetonitrile:water, 60:40
1mL/ min
20°C
UV, 214nm
1µL
G001031
0
2
15
17
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
15
1.
2.
3.
4.
5.
6.
7.
8.
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
9
G001030
6
1
SUPELCO
144
HPLC Applications
Phenols, Polymers, Oligomers
Liquid
Chromatography
6
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
23
n=5
n=3
n=6
4
8
n=7
1
4
6
8
10
Min
12
n=9
14
16
18
20
713-1031
2
n=2
n=8
9
Phenols (HPLC)
15
713-0778
7
20
Min
Polyethylene Glycol 200 (HPLC)
Column: SUPELCOSIL LC-8, 15cm x 4.6mm ID, 5µm particles
Cat. 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)
Flow Rate: 1.5mL/min
Temp.: 30°C
Det.: UV, 280nm
Column: TSKgel G-OLIGO-PW, 30cm x 7.8mm ID, 6µm particles*
Cat. No.: 808031
Mobile Phase: distilled water
Flow Rate: 1mL/min
Det.: refractive index
*Two columns in series.
9-10 Ethoxy Groups
(Octylphenol)
20 Ethoxy Groups
(Nonylphenol)
Gradient Program
Time (min) %B
0
5
10
Min
15
20
Epoxy Resin, Epikote 1001 (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Det.:
TSKgel G3000H XL, 30cm x 7.8mm ID, 6µm particles*
816136
tetrahydrofuran
1mL/min
UV, 254nm
*Two columns in series.
4
8
12
Min
16
20
24
30
50
60
80
100
0
4
8
12
Min
16
20
Nonionic Surfactants (HPLC)
Column: SUPELCOSIL LC-Diol, 25cm x 4.6mm ID, 5µm particles
Cat. No.: 58201
Mobile Phase: A = hexane:methylene chloride (95:5)
B = hexane:methylene chloride:methanol (50:40:10)
Flow Rate: 1mL/min
Temp.: 35°C
Det.: UV, 280nm
Inj.: 10µL methylene chloride containing 20µg surfactant
24
713-0782, 1068
0
8
15
25
35
713-0783
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
0
11
10
5
SUPELCO
n=4
Phenol
4-Nitrophenol
2,4-Dinitrophenol
2-Nitrophenol
o-Chlorophenol
4,6-Dinitrophenol
2,4-Dimethylphenol
4-Chloro-3-methylphenol
2,4-Dichlorophenol
2,4,6-Trichlorophenol
Pentachlorophenol
145
HPLC Applications
Polynuclear Aromatic Hydrocarbons
1.
2.
3.
4.
5.
6.
Gradient Program
Time (min)
% ACN
0
50
5
50
30
100
45
100
Flow increased at 2.0 min
7.
8.
9.
10.
2-OH-DMBA(t-diol)
7-OHM-12-MBA-(t-10,11-diol)
7-M-12-OHMBA-(t-8,9-diol)
7-OHM-12-MBA-(t-8,9-diol)
7-OHM-(t-3,4-diol)
Mixed diols, incl. DMBA(t-8,9-diol & t-10, 11-diol)
Position of 7,12-bis-OHMBA
7-OHM-12-MBA-2-ol
7-OHM-12-MBA-(3-ol)
7-M-12-OHMNA-4-ol
11. 7-OHM-12-MBA-4-ol
12. 7-M-12-OHMBA-(3-ol)
13. 7-M-12-OHMBA-2-ol
14. 7-OHM-12-MBA
15. 7-M-12-OHMBA
16. DMBA-2-ol
17. DMBA-3-ol
18. DMBA-4-ol
19. DMBA
(parentheses = tentative identifications)
6
19
14 16 18
11
1
8
11
6
5
10
9
5
12
4
4
2
2
10
13
3
17
15
7
7
9
13
0
4
8
12
20
16
28
24
32
36
40
44
48
3
8
12
713-1043
Min
1
Figure provided by Drs. J. Milner and J. Grunau, University of Illinois,
Urbana, Illinois, USA.
16
10
20
Min
30
40
PAHs in Water (SPME/HPLC)
Sample:
SPME Fiber:
Cat. No.:
Extraction:
Desorption*:
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Det.:
5µL PAH mix (Cat. No. 4-8743) in 5mL water
polydimethylsiloxane, 100µm film
57301
immersion, 30 min (rapid stirring)
static, 200µL acetonitrile:water, 40:60, 2 min
SUPELCOSIL LC-PAH, 15cm x 4.6mm ID, 5µm particles
58318
acetonitrile:water gradient (see program)
0-2 min: 0.2mL/min 2-45 min: 1.0mL/min
UV, 254nm
796-0086
14 15
0
Liquid
Chromatography
Naphthalene 1000ng/mL
Acenaphthylene 2000ng/mL
Acenaphthene 1000ng/mL
Fluorene 200ng/mL
Phenanthrene 100ng/mL
Anthracene 100ng/mL
Fluoranthene 200ng/mL
Pyrene100ng/mL
Benzo(a)anthracene100ng/mL
Chrysene100ng/mL
Benzo(b)fluoranthene 200ng/mL
Benzo(k)fluoranthene 100ng/mL
Benzo(a)pyrene100ng/mL
Dibenzo(a,h)anthracene 200ng/mL
Benzo(ghi)perylene 200ng/mL
Indeno(1,2,3-cd)pyrene 100ng/mL
Metabolites of 7,12-Dimethylbenzo(a)anthracene
(HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Det.:
Inj.:
SUPELCOSIL LC-18, 25cm x 4.6mm ID, 5µm particles
58298
methanol:water, 50:50 (10 min) to 100:0 at 2.5% /min
0.8mL/min
fluorescence
rat liver, 9000 x g supernatant fraction from Aroclor-treated
animals
1. 2,7-Dihydroxynaphthalene (300µg/mL)
2. 2,3-Dihydroxynaphthalene (1000µg/mL)
2
*Requires SPME/HPLC interface.
0
1
2
3
4
Min.
5
6
7
Chelating Agent, DERT Test (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp:
Det.:
Inj.:
SUPELCOGEL TPR-100, 15cm x 4.6mm ID, 5µm particles
59154
ammonium acetate (25mM, pH 7):acetonitrile (55:45)
1.0mL/min
ambient
UV, 254nm
10µL
8
796-0042
1
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
SUPELCO
146
HPLC Applications
Semivolatiles, Toxins, Vitamins
2
Liquid
Chromatography
1. Ergocalciferol (Vitamin D2)
2. Cholecalciferol (Vitamin D 3 )
0
10
20
Min
0
2
4
6
8
10
Min
794-0800
Vitamins, Fat Soluble (D
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Temp:
Det.:
Inj.:
2
and D 3) (HPLC)
Discovery HS C18, 15cm x 4.6mm ID, 3µm particles
569252-U
100% CH3 CN
0.8mL/min
30°C
UV, 290nm
10µL, 50µg/mL of each analyte
Benzidines by US EPA Method 605 (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Det.:
Inj.:
SUPELCOSIL LC-1, 15cm x 4.6mm ID, 5µm particles
58210-U
0.1M acetate buffer (pH 4.7):acetonitrile, 80:20
1mL/min
UV, 254nm
10µL (0.5µg each analyte)
1.
2.
3.
4.
5.
Retinol acetate (Vitamin A acetate), 50µg/mL
δ-Tocopherol, 165µg/mL
γ-Tocopherol, 200µg/mL
α-Tocopherol (Vitamin E), 292µg/mL
α-Tocopherol acetate (Vitamin E acetate), 405µg/mL
4
3
Gradient Program
Time (min) %B
0
15
17
19
21
29
2
Discovery C8
1
35
80
90
90
35
35
5
G001722
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
SUPELCO
3,3’-Dichlorobenzidine
1
0
2
4
6
8
Min
1
Bufalin Toxin (HPLC)
3
4
2
Column: Discovery BIO Wide Pore C18, 25cm x 4.6mm ID,
5µm particles
Cat. No.: 568223-U
Mobile Phase: A = 0.1% TFA in water
B = 0.1% TFA in acetonitrile:water (3:1)
Flow Rate: 1.3mL/min
Temp: ambient
Det.: UV, 298nm
Inj.: 10µL bufalin, 0.5mg/mL in acetonitrile
Discovery C18
5
0
2
4
6
Min
8
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:H2 O (90:10)
Flow Rate: 2mL/min
Temp: 30°C
Det.: UV, 290nm
Inj.: 10µL
10
G001419
Benzidine
147
HPLC Applications
Vitamins
Retinol
Retinyl acetate
δ-Tocopherol
γ-Tocopherol
α-Tocopherol
Tocopherol acetate
1.
2.
3.
4.
5.
6.
7.
Gradient Program
Time (min) %B
0
8
3
1
0
30
Ascorbic acid
Nicotinic acid
Thiamine
Pyridoxine
Nicotinamide
Folic acid
Riboflavin
2
Liquid
Chromatography
1.
2.
3.
4.
5.
6.
7
Discovery RP-AmideC16
5
1
6
4
6
3
0
10
Min
5
4
20
G001171
2
2
4
6
Min
8
10
12
Discovery C18
Vitamins, Fat Soluble (A and E) (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Det.:
SUPELCOSILLC-18, 25cm x 4.6mm ID, 5µm particles
58298
methanol:deionized water (98:2)
2mL/min
UV, 325nm (retinol, retinyl acetate) or 290nm (tocopherols,
tocopherol acetate)
Inj.: 20µL methanol
(0.2-1mg/mL each analyte)
α-Tocopherol (0.3mg/mL)
β-Tocopherol (0.4mg/mL)
γ-Tocopherol (0.3mg/mL)
δ-Tocopherol (1.3mg/mL)
Ergocalciferol (1.0mg/mL)
Retinol (0.7mg/mL)
6
2
7
4
6
0
2
4
6
8
10
12
14
G000790
1.
2.
3.
4.
5.
6.
5
1
Min
5
3
Discovery C8
1
1
5
2
4
2
6
7
4
0
2
4
6
8
10
12
0
5
10
Min
15
Vitamins, Fat Soluble (A and E) (HPLC)
Column:
Cat. No.:
Mobile Phase:
Flow Rate:
Det.:
Inj.:
SUPELCOSIL LC-Si, 15cm x 4.6mm ID, 5µm particles
58200-U
hexane:amyl alcohol (99.65:0.35)
2mL/min
UV, 280nm
20µL
20
713-0881
Min
G000789
3
Vitamins, Water Soluble (HPLC)
Column:
Cat. Nos.:
Mobile Phase:
Temp.:
Det.:
Inj.:
Discovery RP-Amide, 15cm x 4.6mm ID, 5µm particles
505013 (RP-AmideC16),
MeOH:50mM KH2PO4 , pH 4.5 (30:70)
35°C
UV, 220nm
10µL
Column: Discovery C18 or C8, 15cm x 4.6mm ID, 5µm particles
Cat. Nos.: 504955 (C18), 59353-U (C8)
Mobile Phase: 50mM KH2PO4 , pH 4.5 to MeOH:50mM KH 2 PO4, pH4.(30:70) in
12 min (Discovery C8) or 8 min (Discovery C18)
Flow Rate: 1mL/min
Temp.: ambient temp.
Det.: UV, 254nm
Inj.: 1µL
O r d e r : 1 . 8 0 0 . 3 2 5 . 3 0 1 0 Te c h n i c a l S e r v i c e : 1 . 8 0 0 . 3 5 9 . 3 0 4 1 We b : w w w. s i g m a - a l d r i c h . c o m / s u p e l c o
0
712-0679
3
SUPELCO