Applications:
HPLC Separation of Amino Acids, Bases, Acids, and Neutrals on Obelisc R

Separating basic, acidic and zwitterionic compounds in one run in reverse-phase HPLC can be very challenging. The methods might require the use of ion-pairing reagents and complex gradients that can make MS-compatibility difficult. Obelisc R column which has both positive and negative ion-pairs embedded in the stationary phase allows for fine tuning and separation of a wide range of compounds with different ionic properties. Acids, bases, amino acids and neutral compounds were separated isocratically in one run using a simple MS-compatible mobile phase of acetonitrile (ACN) and water with Ammonium Acetate (AmAc) buffer. Can also be UV detected at 250nm.
Condition
Column |
Obelisc R, 4.6x250 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O - 35/65% |
Buffer |
AmAc 10 mM pH 4.0 |
Flow Rate |
1.0 ml/min |
Detection |
UV, 250 nm |
Description
Class of Compounds
|
Drug, Acid, Bases, Neutral, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid |
Analyzing Compounds |
Amino acids |
2,6-LutidineBenzoic AcidBenzonitrileBenzylaminePhenolPhenylalaninePyridineTolueneTryptophan
HPLC Separation of Amino Acids on Obelisc R Column
Closely related compounds like amino acids can be separated on an Obelisc R column by various buffers depending on the amount of baseline separation required. By choosing different buffers, the separation between compounds can be adjusted based on application needs, especially those that require low organic concentration in the mobile phase. UV detection at 250nm.
Condition
Column |
Obelisc R, 4.6x250 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O - 5/95% |
Buffer |
AmFm |
Flow Rate |
1.0 ml/min |
Detection |
UV, 250 nm |
Description
Class of Compounds
|
Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid |
Analyzing Compounds |
Amino acids |
3-Aminobenzoic AcidAspartamePhenylalanineTryptophan
HPLC Separation of Phenylalanine, Glucosamine, and Tryptophan on Mixed-Mode Column
The Separation of Phenylalanine, Glucosamine and Tryptophan demonstrates how the small changes to the organic and/or buffer concentrations in the mobile phase affect the retention of compounds on a mixed-mode column by hydrophobic, ion-exchange and ion-exclusion mechanisms in reverse-phase HPLC chromatography. Evaporative Light Scattering Detector (ELSD) used
Condition
Column |
Primesep 100, 2.1x100 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O |
Buffer |
AmFm |
Flow Rate |
0.25 ml/min |
Detection |
ELSD |
Description
Class of Compounds
|
Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid |
Analyzing Compounds |
Phenylalanine, Glucosamine, Tryptophan |
GlucosaminePhenylalanineTryptophan
Generic Screening Method for Complex Mixtures
Condition
Column |
Primesep 200, 4.6*150 mm 5 µm, 100A |
Mobile Phase |
MeCN/H2O |
Buffer |
H2SO4 |
Flow Rate |
1.0 ml/min |
Detection |
UV, 215 nm |
Description
Class of Compounds
|
Drug, Acid, Hydrophilic, Ionizable, Hormone |
Analyzing Compounds |
Uracil, Epinephrine, DOPA, 2,6-Lutidine, Benzylamine, Hydroxytrypthophan, Homovanillic acid, Phenol, Tryptophan , 2,3-DHBA, Benzoic acid, Methylparaben, Ethylparaben, Toluene, Amitriptyline |
2,3-Dihydroxybenzoic Acid2,6-LutidineAmitriptylineBenzoic AcidBenzylamineDOPA (3,4-dihydroxy-L-phenylalanine)EpinephrineEthylparabenHomovanillic AcidHydroxytryptophanMethylparabenPhenolTolueneTryptophanUracil
HPLC Separation of Mixture of Essential Amino Acids on Primesep 100 Column
Condition
Column |
Primesep 100, 4.6x250 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O - 25/75% |
Buffer |
Gradient H2SO4 0.05-0.2% 25 min |
Flow Rate |
1.0 ml/min |
Detection |
UV, 200 nm |
Description
Class of Compounds
|
Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid |
Analyzing Compounds |
L-Threonine (Thr/T), L-Valine (Val/V), DL-Methionine (Met/M), L-Isoleucine (Ile/I), L-Leucine (Leu/L), L-Phenylalanine (Phe/F), L-Histidine (His/H), L- Tryptophan (Trp/W) |
Amino AcidsD-IsoleucineD-LeucineD-ValineDL-IsoleucineHistidineIsoleucineL-Histidine hydrochloride monohydrateL-IsoleucineL-MethionineL-ThreonineMethioninePhenylalanineTryptophanValine
HPLC Separation of Mixture of 12 Amino Acids on Primesep 100 Column
Condition
Column |
Primesep 100, 4.6x250 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O - 35/65% |
Buffer |
H2SO4 0.05% 12 min hold, gradient 0.05-0.20, 13 min |
Flow Rate |
1.0 ml/min |
Detection |
UV, 200 nm |
Description
Class of Compounds
|
Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid |
Analyzing Compounds |
L-Asparagine (Asn/N), L-Threonine (Thr/T), L-Cysteine (Cys/C), L-Proline (Pro/P), L-Valine (Val/V), DL-Methionine (Met/M), L-Isoleucine (Ile/I), L-Leucine (Leu/L), L-Phenylalanine (Phe/F), L- Tryptophan (Trp/W), L-Histidine (His/H), L-Arginine (Arg/R) |
Amino AcidsArginineAsparagineCysteineD-IsoleucineD-LeucineD-ValineDL-IsoleucineHistidineIsoleucineL-CysteineL-MethionineLeucineMethioninePhenylalanineProlineTryptophanValine
HPLC Separation of Biogenic Amines on Primesep 100 Column

Tryptophan is an essential amino acid used in the synthesis of proteins. Tryptamine is structurally similar to tryptophan. Serotonin is a neurotransmitter derived from tryptophan. Dimethyltryptamine and 5-MeO-DMT are psychedelic substances derived from tryptamine. All the compounds have similar structures and can present difficulties to separation in reverse-phase HPLC. They can be separated using the Primesep 100 mixed-mode column in gradient analysis with acetonitrile (ACN) and water mobile phase with ammonium formate (AmFm) buffer, making the method MS-compatible. The amines can also be UV detected at 280nm.
Condition
Column |
Primesep 100, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
Gradient MeCN - 40-50%, 15 min |
Buffer |
Gradient AmFm pH 3.0- 20-80 mM, 15 min |
Flow Rate |
1.0 ml/min |
Detection |
UV 280 nm, MS-compatible mobile phase |
Description
Class of Compounds
|
Drug, Hydrophilic, Supplements, Monoamine, Neurotransmitter |
Analyzing Compounds |
Serotonin, Tryptophan, N,N-dimethyltryptophan, N,N-Dimethyl-5-methoxytryptamine, Tryptamine, N,N-dimethyltryptamine |
N,N-Dimethyl-5-methoxytryptamineN,N-DimethyltryptamineN,N-dimethyltryptophanSerotoninTryptamineTryptophan
HPLC Separation of Mixture of Nine Essential Amino acids and Arginine on Newcrom AH Column
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Essential amino acids cannot be made by the body. As a result, they must come from food.
The 9 essential amino acids are: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine.
Condition
Column |
Newcrom AH, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN - 5% |
Buffer |
Gradient Formic Acid - 3-9%, 20 min |
Flow Rate |
1.0 ml/min |
Detection |
CAD |
Description
Class of Compounds
|
Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid |
Analyzing Compounds |
L-Threonine (Thr/T), L-Valine (Val/V), DL-Methionine (Met/M), L-Isoleucine (Ile/I), L-Leucine (Leu/L), L-Phenylalanine (Phe/F), L-Histidine (His/H), Lysine (Lys/K)
, L- Tryptophan (Trp/W), L-Arginine (Arg/R) |
ArginineHistidineIsoleucineL-ThreonineLeucineLysineMethioninePhenylalanineTryptophanValine