Primesep mixed-mode columns are designed to separate basic, acidic, neutral and zwitter-ionic compound. Elution of these compounds is facilitated by acetonitrile and ions in the mobile phase. In all cases the retention time on mixed-mode columns is significantly longer than on traditional reverse-phase columns. Presence of cation-exchange mechanism in Primesep 100 column allows to achieve better retention and peak shape for analytes like phenylalanine and benzylamine. Various buffers can be used along with different detection techniques. This HPLC method can be adopted as a universal approach for analysis of basic, acidic and zwitter-ionic compounds in complex mixtures and formulations. Compounds can be monitored by UV, ELSD, CAD and LC/MS.

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 catecholamine neurotransmitters are amino-acid derivatives of tyrosine. DOPA, tyrosine, phenylalanine, norepinephrine, epinephrine, and dopamine and baseline are resolved on a Primesep 100 column with UV-transparent phosphate buffer. This method can be used for analysis of catecholamines and related impurities in various matrices. Peak order and retention time can be changed by changing the amount of ACN, buffer concentration and buffer pH. Various buffers can be used to accommodate desired detection technique. Primesep 100 is a reversed-phase cation-exchange mixed-mode column that can be used for analysis of polar neutral, polar ionizable, polar zwitter-ionic, hydrophobic neutral, and hydrophobic ionic compounds in the same run. Column can be operated in reverse-phase, cation-exchange, anion-exclusion, HILIC and mixed-modes depending on the mobile phase selection and nature of analytes. Column is compatible with LC/MS and does not require use of ion-pairing reagents.

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.

Amino acids can be retained and separated on a Primesep S2 column in HILIC/cation-exchange mode.

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