Aspartic Acid

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High Performance Liquid Chromatography (HPLC) Method for Analysis of Non-Essential Amino Acids on Primesep 100 by SIELC Technologies

High Performance Liquid Chromatography (HPLC) Method for Analyses of Non-Essential Amino Acids

Amino acids are the building blocks of proteins. Based on their dietary requirement, they are classified into essential and non-essential amino acids. Essential amino acids cannot be synthesized by the human body in sufficient quantities and must be obtained from the diet. Non-essential amino acids, on the other hand, can be synthesized by the body and are not dependent on dietary intake.

It’s worth noting that while these amino acids are considered “non-essential” for adults under normal circumstances because the body can synthesize them, there are situations where some may become “conditionally essential.” This means that under certain conditions like illness, stress, or trauma, the body might not produce them in sufficient quantities, and dietary intake becomes necessary. Arginine, for instance, is considered conditionally essential, especially during periods of rapid growth, illness, or trauma.

Amino acids can be retained, separeted and analyzed on a Primesep 100 mixed-mode stationary phase column using an isocratic analytical method with a simple mobile phase of water, Acetonitrile (MeCN), and a phosphoric acid (H3PO4) as a buffer. This analysis method can be detected in the UV regime at 200 nm.

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

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.

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.

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.

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).

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).