Applications:
Amino Acids Analysis in Acid Gradient Condition

11 underivatized amino acids (aspartic acid, glutamic acid, alanine, valine, methionine, isoleucine, cysteine, phenylalanine, histidine, lysine, and arginine) are separated by a Primesep 100 HPLC column by reversed-phase and ion-exchange mechanisms with LC/MS compatible conditions without the use of ion-pair reagents. The HPLC separation uses a TFA (trifluoroacetic acid) gradient in a mobile phase of water acetonitrile (MeCN, ACN with evaporative light scattering detection (ELSD).
Condition
Column |
Primesep 100, 4.6x250 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O - 30/70% |
Buffer |
TFA , gradient 0.05-0.3 % , 25 min |
Flow Rate |
1.0 ml/min |
Detection |
ELSD |
Description
Class of Compounds
|
Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements |
Analyzing Compounds |
Gaspartic acid, Glutamic acid, Alanine, Valine, Methionine, Isoleucine, Cysteine, Phenylalanine, Histidine, Lysine, Arginine |
AlanineAmino AcidsArginineAspartic AcidCysteineGlutamic AcidHistidineIsoleucineLysineMethioninePhenylalanineValine
HPLC Separation of Creatine, Creatinine and Histidine with MS Conditions

Creatine, creatinine, and histidine are all important amino acids in the human body. All of these can be separated by liquid chromatography. The mobile phase contains acetonitrile (MeCN), water, and phormic acid. Smaller 3 µm particle columns are available for fast UPLC applications. This liquid chromatography method is scalable and can be used for isolation of impurities in preparative separation. It also suitable for pharmacokinetics.
Condition
Column |
Primesep 200, 3.0x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O - 20/80% |
Buffer |
Formic Acid - 0.1% |
Flow Rate |
0.5 ml/min |
Detection |
UV, 210 nm |
Description
Class of Compounds
|
Drug, Acid, Hydrophilic, Ionizable, Hormone |
Analyzing Compounds |
Creatine, Creatinine, Histidine |
CreatineCreatinineHistidine
HPLC Separation of Creatine, Creatinine and Histidine

Creatine is produced by the body and plays a major role in recycling ATP. It also stabilizes pH in certain tissues. Creatine can be analyzed by this reverse phase (RP) HPLC method with simple conditions. The mobile phase contains acetonitrile (MeCN), water, and TFA. For Mass-Spec (MS) compatible applications the phosphoric acid needs to be replaced with formic acid. Smaller 3 µm particle columns are available for fast UPLC applications. This liquid chromatography method is scalable and can be used for isolation of impurities in preparative separation. It also suitable for pharmacokinetics.
Condition
Column |
Primesep 200, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O - 1/90% |
Buffer |
TFA -0.1% |
Flow Rate |
1.0 ml/min |
Detection |
UV, 210 nm |
Description
Class of Compounds
|
Drug, Acid, Hydrophilic, Ionizable, Hormone |
Analyzing Compounds |
Creatine, Creatinine, Histidine |
CreatineCreatinineHistidine
HPLC Analysis of Polysorbate in Mixture with Amino Acids and Sugar

Polysorbates are a class of emulsifiers used in some pharmaceuticals and food preparation. They are often used in cosmetics to solubilize essential oils into water-based products. Polysorbates are oily liquids derived from PEG-ylated sorbitan (a derivative of sorbitol) esterified with fatty acids. Surfactants that are esters of plain (non-PEG-ylated) sorbitan with fatty acids are usually referred to by the name Span. It is often required to quantitate Polysorbate (Polysorbate/Tween 20, Polysorbate/Tween 40, Polysorbate 60/Tween 60 and Polysorbate 80) by HPLC in various formulations. Polysorbates exist in form of oligomers.
Condition
Column |
Primesep D, 3.2x50 mm, 5 µm, 100A |
Mobile Phase |
IPA |
Buffer |
Formic Acid |
Flow Rate |
0.5 ml/min |
Detection |
ELSD |
Description
Class of Compounds
|
Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid |
Analyzing Compounds |
Glycine, Histidine, Sucrose, Polysorbate 20 |
Amino AcidsGlycineHistidinePolysorbatePolysorbate 20Polysorbate 80SucroseTween 20Tween 80
HPLC Separation of Amino Acids in Zero Organic Mode on Primesep 200 column
Essential and non-essential amino acids can be retained and separated in zero-organic mode on Primesep mixed-mode HPLC columns. Zero-organic mode is required to monitor isotopes of carbon. Amino acids are retained by combination of reversed-phase and cation-exchange mechanisms. At lower pH, some of the amino acids are more hydrophobic. Buffer pH will affect ionization state of amino acids, and at higher pH (above 2.5), the amino acids will be less hydrophobic and retentive in zero-organic mode. Amino acids can be monitored by low UV. Method can be used in archeological research for analysis of various molecules where presence of organic component of the mobile phase interferes with analysis.
Condition
Column |
Primesep 200, 4.6x250 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O |
Buffer |
Na2HPO4 |
Flow Rate |
1.0 ml/min |
Detection |
UV 210 nm |
Description
Class of Compounds
|
Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements |
Analyzing Compounds |
Valine, Leucyne, Isoleycine, Tyrosine, Phenylalanine, Histidine, Lysine |
HistidineIsoleucineLeucineLysinePhenylalanineTyrosineValine
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-MethionineL-ThreonineLeucineMethioninePhenylalanineProlineTryptophanValine
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
HPLC Method For Analysis Of Histidine and Histidine Methyl Ester Dihydrochloride on Primesep 100 Column
Separation type: Liquid Chromatography Mixed-mode
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High Performance Liquid Chromatography (HPLC) Method for Analysis of Histidine and Histidine Methyl Ester Dihydrochloride
Histidine and Histidine Methyl Ester Dyhydrochloride are related compounds with interesting uses. Histidine is used in biosynthesis to create proteins, while histidine methyl ester dihydrochloride (a derivative of histidine) is used to create various other compounds.
This amino acid and its derivative can be detected in the low UV regime. Using a Primesep 100 mixed-mode column and a mobile phase consisting of water and acetonitrile (MeCN) with a sulfuric acid (H2SO4) buffer, Histidine and Histidine Methyl Ester Dyhydrochloride can be retained, separated, and analyzed. This analysis method can be UV detected at 200 nm.
Condition
Column |
Primesep 100, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O - 60/40% |
Buffer |
H2SO4 - 0.1% |
Flow Rate |
1.0 ml/min |
Detection |
UV, 200 nm |
Description
Class of Compounds
|
Amino acid |
Analyzing Compounds |
Histidine, Histidine Methyl Ester |
HistidineHistidine methyl esterL-Histidine hydrochloride monohydrate