Hydroxyproline seems to be the most promising amino acid used in carbon dating when isolated from bone collagen. Separation of amino acids is challenging, especially without the use of ions or inorganic buffers that can interfere with Mass spectrometer (MS) or contaminate the sample with modern carbon. Amino acids are also not retained in reverse-phase chromatography. The ideal solution would be using water only to separate the amino acids. This would allow a direct coupling to MS. We were able to separate hydroxyproline from proline and other simple amino acids like glycine and alanine in HPLC on Newcrom AH column using water only as a mobile phase. Using water also allowed UV detection at 205 nm which can’t be done if using a buffer based on acetic or formic acid.
See more information on radiocarbon dating here.
The same method can be modified to get symmetrical peaks and higher efficiency if a mobile phase with ionic modifier such as formic acid is used.

HPLC Method for Asparagine, L-Cysteine, Cysteine, Proline, Valine, D-Valine, Methionine, L-Methionine, Isoleucine, D-Isoleucine, DL-Isoleucine, D-Leucine, Phenylalanine, Tryptophan, Histidine, Arginine, Amino Acids, Leucine, L-Threonine on Primesep 100 by SIELC Technologies

High Performance Liquid Chromatography (HPLC) Method for Analysis of Asparagine, L-Cysteine, Cysteine, Proline, Valine, D-Valine, Methionine, L-Methionine, Isoleucine, D-Isoleucine, DL-Isoleucine, D-Leucine, Phenylalanine, Tryptophan, Histidine, Arginine, Amino Acids, Leucine, L-Threonine.

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 sulfuric acid (H2SO4) as a buffer. This analysis method can be detected in the UV regime at 200 nm.

HPLC Method for L-Glutamine, Glycine, Cysteine, L-Cysteine, Proline, Tyrosine, Arginine, Amino Acids on Primesep 100 by SIELC Technologies

High Performance Liquid Chromatography (HPLC) Method for Analysis of L-Glutamine, Glycine, Cysteine, L-Cysteine, Proline, Tyrosine, Arginine, Amino Acids.

Glutamine is an amino acid with the chemical formula C₅H₁₀N₂O₃. It is a building block for protein, but it also supports the immune system, gut health, and detoxification. It is usually found in meat, fish, eggs, dairy, and whole grains; though some consider it the most abundant amino acid in the body. People also take glutamine as treatment for sickle cell disease, healing of burns, recovery after surgery, and injuries.

L-Glycine is an important amino acid compounds widely used in pharmaceutical, biochemical, and peptide research. It has he chemical formula C₂H₅NO₂. It is water-soluble and plays a critical role in protein synthesis, peptide modification, and metabolic studies. It can be found in meat., eggs. and bones.

L-Cysteine is an amino acid with the chemical formula C₅H₁₀N₂O₃. It is primarily a building block for protein, but it also has antioxidant effects. On occasion, ot os used to support people dealing with cancer, diabetes, and hangover, but there is yet to be substantial evidence that it works. Poultry, egg, beef, and whole grains are rich sources of the amino acid.

L-Proline is an amino acid with the chemical formula C₅H₉NO₂. It is naturally produced in the body, but it is also recommended to be consumed through the diet. It can be found in meat, fish, and dairy. It is used for skin healing and treatment of other skin conditions as it is an essential component of collagen. It is important for upkeep of joints and tendons, as well as heart muscles.

L-Tyrosine is an amino acid with the chemical formula C₉H₁₁NO₃. It is naturally used in the body to create neurotransmitters like dopamine and norepinephrine. While it is produced in the body naturally, it can also be consumed in beef, pork, fish, dairy products, and beans. As a supplement, it is also used to treat Phenylketonuria (PKU).

L-Arginine is an amino acid with the chemical formula C₉H₁₁NO₃. It is a precursor for nitric acid, making it crucial for blood flow and general cardiovascular health. It can help lower blood pressure, help with chest pain, and improve symptoms of Peripheral arterial disease (PAD).

L-Glutamine, Glycine, Cysteine, L-Cysteine, Proline, Tyrosine, Arginine, Amino Acids can be retained and analyzed using the Primesep 100 stationary phase column. The analysis utilizes an isocratic method with a simple mobile phase consisting of water and acetonitrile (MeCN) with a sulfuric acid buffer. Detection is performed using UV.

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

Polar amino acids are very often used as components of vitamin and supplement composition. Analysis of such complex composition is a challenging task. In this application, 5 amino acids (asparagine, glutamic acid, proline and arginine) and two preservatives (methyl paraben and propyl paraben) are separated on a Primesep 100 reversed-phase cation-exchange column with LC/MS compatible mobile phase. Method does not require ion-pairing reagent in the mobile phase. Compounds are monitored by ELSD and UV. Method is validated for quantitation of underivatized amino acids in complex mixtures. The method is simple and robust and can be used for analysis of various vitamin formulations.

Primesep 100 separates the components of a Swiss cheese extract by HPLC using cation exchange and reversed phase as retention mechanisms. The amino acids, glutamic acid and proline, as well as glycine betaine were resolved in less than 10 minutes. A mobile phase gradient of water, acetonitrile (MeCN, ACN,) and phosphoric acid (H3PO4) and ultraviolet (UV) detection was used.