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 Polar Compounds
The separation of amino acids, the building blocks of proteins, can be challenging to separate on a reverse-phase column due to their high polarity. Using a mixed-mode HPLC column, allows the separation of amino acids by cation-exchange and ion-exclusion mechanisms as well as hydrophobicity. Fine tuning of separation can be achieved with changes in organic concentration of the mobile phase as well as choice of buffer and pH.
Condition
Column |
Primesep 200, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O |
Buffer |
AcOH, AmFm |
Flow Rate |
1.0 ml/min |
Detection |
ELSD |
Description
Class of Compounds
|
Drug, Acid, Hydrophilic, Ionizable, Hormone |
Analyzing Compounds |
Aspartic acid, Glutaric acid, Glycine, Hydroxytriptophan, GABA, Norepinephrine, Dopamine |
Aspartic AcidDopamineGlutamic AcidNorepinephrinegamma-Aminobutyric Acid (GABA)
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
Effect of both pH and Organic Content on a Separation of Sugars, Amino Acids, and Carboxylic Acids
In mixed-mode HILIC chromatography, selectivity of separation can be adjusted by amount of acetonitrile, amount of buffer and buffer pH. Buffer concentration and pH will affect retention of ionizable compounds to a different degree. Retention of neutral compounds can be adjusted by the amount of acetonitrile. Carboxylic acid, three amino acids and two sugars are separated by combination of HILIC and ion-exchange mechanisms. Compounds can be monitored by ELSD, Corona (CAD), LC/MS or low UV. UV-transparent mobile phase /buffer is required for UV monitoring of this mixed-mode separation. This HPLC method can be adopted as general approach for analysis of sugars, amino acids and carboxylic acids.
Condition
Column |
Obelisc N, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O |
Buffer |
AmAc |
Flow Rate |
1.0 ml/min |
Detection |
ELSD |
Description
Class of Compounds
|
Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid |
Analyzing Compounds |
Succinic Acid, Phenylalanine, Sucrose, Glycine, Aspartic Acid, Raffinose |
Aspartic AcidPhenylalanineSuccinic Acid
HPLC Separation of Aspartic Acid and Asparagine on Obelisc R Column
Two amino acids, aspartic acid and asparagine, are separated on an Obelisc R column by combination of reverse-phase and ion-exchange mechanism. Method can be used for analysis of these and other underivatized amino acids by HPLC with UV, ELSD, CAD and LC/MS detection.
Condition
Column |
Obelisc R, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O |
Buffer |
AmFm, AmAc |
Flow Rate |
1.0 ml/min |
Detection |
ELSD |
Description
Class of Compounds
|
Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid |
Analyzing Compounds |
Asparagine, Aspartic Acid |
AsparagineAspartic Acid
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 Non-Essential Amino Acids
Condition
Column |
Primesep 100, 4.6x250 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O - 20/80% |
Buffer |
H3PO4 - 0.1% |
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-Aspartic Acid (Asp/D), L-Serine (Ser/S), L-Glutamic Acid Glu/E), L-Alanine Ala/A) |
Amino AcidsAspartic AcidD-AlanineDL-AlanineGLU (L-Glutamic acid)Glutamic AcidSerine
HPLC Separation of Amino Acids on Newcrom AH Column
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Condition
Column |
Newcrom AH, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN - 50% |
Buffer |
Formic Acid - 0.1% |
Flow Rate |
1.0 ml/min |
Detection |
CAD |
Description
Class of Compounds
|
Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid |
Analyzing Compounds |
Aspartic acid (Asp/D), Glutamine (Gln/Q), Glutamic acid (Glu/E), Glycine (Gly/G) |
Aspartic AcidGlutamic AcidGlycineL-Glutamine