Primesep A retains the hydrophilic zwitterion tricine by a combination of polar interactions and ion exchange. Tricine is not retained by traditional reversed-phase chromatography. The retention on Primesep A is adjustable by changing the concentration of TFA in the mobile phase. Excellent peak shape results with a mass spec compatible mobile phase of water, acetonitrile (MeCN, ACN) and trifluoracetic acid (TFA) with evaporative light scattering detection (ELSD).
Primesep mixed-mode columns are designed to separate basic, acidic, neutral and zwitter-ionic compounds. 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.
Taurine (2-aminoethanesulfonic acid) is organic zwitterionic compound. Taurine is a non-essential sulfur-containing amino acid that functions with glycine and gamma-aminobutyric acid as a neurotransmitter. Taurine is incorporated into one of the most abundant bile acids, chenodeoxycholic acid, where it serves to emulsify dietary lipids in the intestine, promoting digestion. It is used in food and pharmaceutical formulations. High polarity and zwitterionic nature of taurine complicates analysis of this compound by reverse-phase chromatography. Two methods for the analysis of taurine are developed on mixed-mode columns. Taurine is retained on Primesep A column by HILIC cation-exchange mechanism and on Primesep D column by HILIC anion-exchange mechanism. Method can be used for fast and effective quantitation of taurine in various products including energy drinks. Method requires ELSD or LC/MS detection due to lack of UV activity for taurine.
MOPS and MES are buffers used in biology and biochemistry. They consist of a morpholine ring and propanesulfonic acid. Both molecules are zwitter-ionic and very polar. No retention can be achieved on reversed-phase columns. MOPS and MES are analyzed in HILIC mode on a Primesep N HPLC column. Other biological buffers can be analyzed with this general method. Biological buffers are not UV active and can be monitored by ELSD, CAD or LC/MS.
Obelisc N mixed-mode columns provide different selectivity than other HILIC columns -- presence of ion-exchange mechanism contributes to a different selectivity. Depending on the pH of the mobile phase, ion-exchange mechanism can be enhanced or suppressed. Two amino acids (glycine and glycylglycine), two basic compounds (triethanolamine and TRIS) and two zwitter-ionic compounds (MOPS and MES) are separated by combination of HILIC and ion-exchange mechanisms. Compounds elution can be monitored by Evaporative Light-Scattering Detector (ELSD), Corona (CAD), LC/MS or other detection techniques.
GABA (neurotransmitter) and its isomers are polar zwitter-ionic compounds. Due to the position of amino-groups, all three compounds show different polar and basic properties. The isomers of aminobuturic acid are separated on an Obelisc N HILIC/cation-exchange column. Buffer concentration has a different effect on retention of alpha-, beta-, and gamma-aminobutyric acid. This general and robust method can be used for separation of other polar and ionizable compounds and isomers by mixed-mode chromatography.
HEPES, CAPS, MES and MOPS are zwitterionic organic chemical buffering agents. These are very polar compounds which are not retained by traditional reverse phase chromatography. These compounds are zwitterions in nature and can be separated by mixed-mode hydrophilic interaction chromatography on Obelisc N column. Retention is achieved by combination of HILIC and ion-exchange mechanisms. These buffering agents do not have UV active groups, but can be analyzed with ESLD, LC/MS, and CAD detection.
|Column||Primesep 100, 4.6x150 mm, 5 µm, 100A|
|Mobile Phase||MeCN/H2O - 10/90%|
|Buffer||TFA - 0.1%|
|Flow Rate||1.0 ml/min|
|Detection||UV, 215 nm, ELSD|
|Class of Compounds||Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements|
|Analyzing Compounds||Glutamic acid, GABA|
|Column||Obelisc N, 4.6x50 mm, 5 µm, 100A|
|Buffer||AmFm pH 3.0|
|Flow Rate||1.0 ml/min|
|Class of Compounds||Amino sulfonic acid, Hydrophilic, Ionizable, Vitamin, Supplements|