Applications:
Bufferless Ion Separation (BLIS™) Chromatography of Amino Acids (2)
Adding on to the previous HPLC separation of amino acids using Bufferless Ion Separation (BLIS) Chromatography; here we have additional amino acids separated on Primesep 200 column using only water and acetonitrile (MeCN, ACN) in the mobile phase. Primesep 200 is a reverse-phase (RP) column with weak acidic ion-pairing groups embedded. With no buffer present in the mobile phase, detection can be achieved with UV, mass spectrometry (MS), evaporative light scattering detection (ELSD).
Condition
Column |
Primesep 200, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN |
Buffer |
No |
Flow Rate |
1.0 ml/min |
Detection |
UV, 195 nm |
Description
Class of Compounds
|
Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid |
Analyzing Compounds |
Aspartic acid, Alanine, Valine, Methionine, Leucine |
AlanineAmino AcidsAspartic AcidLeucineMethionineValine
HPLC Separation of Amino Acids in Supplements Capsules

Glutamine, valine, isoleucine, and leucine are baseline resolved in a dietary supplement with a mobile phase of acetonitrile, water, and sulfuric acid with ultraviolet (UV) detection at 210 nm on a Primesep 100 column. Increasing the sulfuric acid concentration allows elution of arginine. Primesep 100 separates underivatized amino acids and amines by combining reversed-phase and ion-exchange mechanisms. The HPLC separation uses a mobile phase of water, acetonitrile (MeCN, ACN), and sulfuric acid with ultraviolet (UV) detection at 210 nm.
Condition
Column |
Primesep 100, 4.6x250 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O - 30/70% |
Buffer |
H2SO4 |
Flow Rate |
1.0 ml/min |
Detection |
UV, 210 nm |
Description
Class of Compounds
|
Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid |
Analyzing Compounds |
Arginine, Glutamine, L-Valine (Val/V), L-Isoleucine (Ile/I), L-Leucine (Leu/L) |
ArginineGlutamineIsoleucineLeucineValine
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 Lysine and Arginine from Other Amino Acids
Application Notes: Amino acids are polar ionic compounds which are not retained on reversed-phase column without ion-pairing reagent. In our application, lysine and arginine can be separated from other amino acids. Amino acids with a pH between 3 and 5 and with one basic and one acidic group become very polar. Therefore these amino acids don’t have strong ion-exchange interaction with Primesep C stationary phase. Amino acids with two amino groups still carry positive net charge and can interact with stationary phase by cations-exchange mechanism. pH variation of the mobile phase can be an effective tool to adjust selectivity of separation for zwitter-ionic, basic and acidic compounds. This method can be used for separation of mono-charged compounds from compounds having an extra charge.
Application Columns: Primesep C
Application compounds: Aspartic acid, Glutamic acid, Aspargine, Glycine, Proline, Alanine, Phenylalanine, Tyrosine, Leucine, Lysine, Arginine
Detection technique: UV, LC/MS, ELSD/CAD
Condition
Column |
Primesep C, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN - 15% |
Buffer |
AmAc pH 5.0- 15 mM |
Flow Rate |
1.0 ml/min |
Detection |
ELSD |
Description
Class of Compounds
|
Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements |
Analyzing Compounds |
Aspartic acid, Glutamic acid, Aspargine, Glycine, Proline, Alanine, Phenylalanine, Tyrosine, Leucine, Lysine, Arginine |
AlanineArginineAsparagineAspartic AcidGlutamic AcidGlycineLeucineLysinePhenylalanineProlineTyrosine
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 β‑Hydroxy-β-methylbutyric Acid, 2-Hydroxyisocaproic Acid and Leucine on Primesep 100 Column
Both β-hydroxy-β-methylbutyric acid (HMB) and 2-hydroxyisocaproic acid (HICA) are metabolites of the
essential dietary branched chain amino acid leucine.
Condition
Column |
Primesep 100, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN - 5%, 3 min hold, Gradient MeCN 5-30% 12 min |
Buffer |
H2SO4 - 0.05 % 3 min hold, GradientH2SO4 0.05-3% 12 min |
Flow Rate |
1.0 ml/min |
Detection |
UV, 210 nm |
Description
Class of Compounds
|
Acid, Hydrophilic, Ionizable, Normal human metabolite, Dietary supplement, Essential amino acid, Branched-chain amino acid (BCAA) |
Analyzing Compounds |
β‑Hydroxy-β-methylbutyric (HMB), 2-Hydroxyisocaproic Acid (HICA), Leucine |
2-Hydroxyisocaproic Acid (HICA)D-LeucineLeucineβ‑Hydroxy-β-methylbutyric Acid (HMB)
HPLC Separation of Allo-Isoleucine, Isoleucine, Leucine on Primesep 100 Column
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Condition
Column |
Primesep 100, 4.6x250 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O - 5/95% |
Buffer |
AmFM pH 3.0 - 30 mM |
Flow Rate |
1.0 ml/min |
Detection |
CAD (Corona) MS- compatible mobile phase |
Description
Class of Compounds
|
Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid |
Analyzing Compounds |
Allo-Isoleucine, Isoleucine, Leucine |
D-IsoleucineD-LeucineDL-IsoleucineIsoleucineL-AlloisoleucineL-IsoleucineLeucineallo-D-isoleucine
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