Lysine

Retaining polar ionizable and non-ionizable compounds is a challenging task in chromatography. This is due to the lack of retention and poor peak shape occuring due to secondary interactions on reversed-phase and HILIC columns. We have developed a method for the separation of glucose and lysine on the mixed-mode Amaze TH column. Glucose is a monosaccharide which is polar (hydrophilic) and non-ionizable. Lysine is one of the essential amino acids, it is polar and zwitterionic with two basic and one carboxylic groups. Glucose is retained separated by pure HILIC mechanism and lysine is retained by combination of HILIC and cation-exchange mechanisms. The retention time is controlled by the amount of ACN, buffer pH and buffer concentrations. Buffer pH affects the ionization state of lysine and the ionization state of the stationary phase. This method is robust. The Amaze TH column can be used to retain and separate amino acids, sugars, amino sugars, organic and inorganic acids as well as polar neutral compounds like sugars. The column is compatible with major detection technique (ELSD, CAD, RI, UV and mass spec)

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Conditions of Experiment

Column:	Amaze TH
Separation Modes:	HILIC, cation-exchnage
Column Dimenstions:	4.6x50 mm, 3 um, 100A
Mobile Phase:	65% ACN with 175 mM AmAc pH 5
Detection:	ELSD 45°C, RI-compatible
Sample:	1 mg/ml
Injection:	3 uL
Flow rate:	1 ml/min

Analytes

Class of compounds:	Amino acid, Sugar, Supplement
Nature of compounds:	Hydrophilic, Neutral, Polar, Zwitterionic
Compounds:	Glucose, Lysine

Arginine is a semi-essential or conditionally essential amino acid which is zwitterionic in nature. It contains two carboxylic acid fragments and two primary amino groups. Meat and dairy products as well as grains, beans and nuts are a source for this amino acid. Arginine plays an important role in cell division, wound healing, various immune functions and the release of hormones. It is an important compound for the regulation of blood pressure. Like any other amino acid, it is not retained well in reversed-phase chromatography without the use of ion-pairing reagents which, in general, are not compatible with the mass spectrometry that is usually required in bio and chemical studies. Arginine and related impurities are retained by combination of weak reversed-phase and cation-exchange mechanisms. At a lower pH (below 3), the compound is more basic and retentive on this Coresep 100 mixed-mode reversed-phase cation-exchange column. The retention time for all compounds is adjusted by playing with acetonitrile, buffer pH and buffer concentration. Various buffers and acids can be used. The selection of the components of the mobile phase depends on the properties of analytes and the detection technique. The Coresep 100 column can be used for retention and separation of underivatized amino acids without ion-pairing reagent. In case of complex sample matrices like biofluids, additional sample preparation is required for developing a robust and reproducible method for analysis of underivatized amino acids

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Conditions of Experiment

Column:	Coresep 100
Separation Modes:	reversed-phase, cation-exchange
Column Dimenstions:	4.6 x 150 mm, 2.7 um, 90A
Mobile Phase:	ACN gradient from 2% to 20%, H2SO4 from 0.02% to 0.2% in 12 minutes
Detection:	UV 210 nm
Sample:	0.3 mg/ml
Injection:	10 uL
Flow rate:	1.0 mL/min

Analytes

Class of compounds:	Amino acid, Aromatic acid, Supplement
Nature of compounds:	Acidic, Hydrophilic, Polar, Zwitterionic
Compounds:	Glutamic acid, Citrulline, Ornithine, Lysine, Arginine

Histidine is one of the essential amino acids which is zwitterionic in nature. It contains one carboxylic acid fragment and two basic groups, one weak basic group in the imidazole ring and a primary amine in the side chain of the ring. Histidine is a precursor for histamine, a nitrogen containing a compound necessary for implantation. Histidine can be converted to 3-methylhistidine which serves as a biomarker for muscle damage. Like any other amino acids, histidine is not retained well in reversed-phase chromatography without the use of ion-pairing reagents, which are not compatible with mass spectrometry usually required in bio and chemical studies. When histidine is retained on a traditional RP column, it produces a poor peak shape due to residual secondary interactions between basic groups of histidine and residual acidic silanols on the surface of silica gel. Histidine and related impurities are retained by a combination of weak reversed-phase and cation-exchange mechanisms. At a lower pH (below 3), the compound is more basic and retentive on this Coresep 100 mixed-mode reversed-phase cation-exchange column. The retention time for all compounds is adjusted by playing with acetonitrile, buffer pH and buffer concentration. Various buffers and acids can be used. The selection of components of the mobile phase depends on the properties of analytes and the detection technique. The Coresep 100 column can be used for the retention and separation of underivatized amino acids without ion-pairing reagent. In case of complex sample matrices like biofluids, additional sample preparation is required for developing a robust and reproducible method for analysis of underivatized amino acids

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Conditions of Experiment

Column:	Coresep 100
Separation Modes:	reversed-phase, cation-exchange
Column Dimenstions:	4.6 x 150 mm, 2.7 um, 90A
Mobile Phase:	ACN from 2% to 20%, Na2HPO4 pH 6 from 10 mM to 50 mM in 12 min
Detection:	UV 210 nm
Sample:	0.3 mg/ml
Injection:	10 uL
Flow rate:	1 mL/min

Analytes

Class of compounds:	Amino acid, Supplement
Nature of compounds:	Hydrophilic, Polar, Zwitterionic
Compounds:	Alanine, Histidine, Lysine, Arginine