Primesep 100 separates a mixture of amino acids (tyrosine, phenylalanine), organic acids (benzoic acid, mandelic acid), amines (benzylamine, pyridine), and neutrals (benzonitrile, toluene) in one HPLC run by combining reversed-phase, cation-exchange, and polar interactions. The method is tunable and peak order can be changed significantly by adjusting acetonitrile and trifluoroacetic acid concentrations. The separation method uses a mobile phase mixture of water, acetonitrile (MeCN, ACN) and trifluoracetic acid (TFA) and compatible with UV, mass spec (LC/MS) and evaporative light scattering (ELSD) detection.
Lactic acid (milk acid) is natural organic acid that plays an important role in biochemical processes. It has carboxylic and hydroxy-groups which upon standing can react with each other and forms dimers, trimers and higher oligomers. Lactic acid is a polar molecule that produces no retention on reversed-phase column. Primesep B2 mixed-mode anion-exchange column is used for separation of lactic acid and its oligomers by combination of weak reversed-phase and anion-exchange mechanisms. Oligomers of lactic acid can be converted to lactic acid by hydrolysis. Primesep B2 column can be used for analysis of lactic acid alone and lactic acid in the mixture of oligomers. Lactates can be monitored by low UV, ELSD or LC/MS. In case of ELSD, CAD or LC/MS, phosphoric acid in the mobile phase needs to be replaced with buffer compatible with these detection techniques (ammonium formate or TFA).
Ascorbic, methylmalonic and succinic are weak organic acids. Retention of these three acids is achieved on Primesep N column in HILIC mode using acetonitrile/water and ammonium acetate. Compounds are monitored by ELSD. Method can be used for determination of ascorbic acid (Vitamin C), methylmalonic acid and succinic acid in various matrices. Other polar organic acids can be analyzed on this HILIC column.
Benzenesulfonic and toluenesulfonic acids are organic acids used in pharmaceutical industry as counter-ions for basic drugs. Both acids are hydrophilic in nature with strong acidic properties. Obtaining separation, retention and good peak shape with traditional reversed-phase columns is a challenging task. We developed robust mixed-mode approach for separation of benzenesulfonic acid and p-toluenesulfonic acid on a Primesep D reversed-phase anion-exchange column. Elution of these compounds is controlled by the amount of ions in the mobile phase and amount of acetonitrile. Both compounds are separated within 4 minutes and can be monitored by UV, ELSD, CAD or LC/MS. This column can be also used to analyze hydrophobic basic drugs by reversed-phase cation-exclusion mechanisms.
Organic and inorganic acids can be separated by reversed-phase anion-exchange Primesep D column. Succinic and methylmalonic acids were separated based on their hydrophobic and ionic properties. Method can be used for analysis of other acids by HPLC.
Pyridinecarboxylic acids exist as three isomers with different position of carboxylic acid relative to nitrogen in pyridine. Three isomers of pyridinecarboxylic acid (picolinic or 2-pyridinecarvoxylic acid, niacin or 3-pyridinecarboxylic acid, isonicotinic or 4-pyridinecarboxylic acid), along with pyridinedicarboxylic acid, are separated on a Primesep 100 column. Pyridinecarboxylic acids have a similar empirical formula, and are very similar in terms of hydrophobicity and ionic properties. Small differences in these properties are enough to achieve good separation on cation-exchange mixed-mode HPLC column like Primesep 100. Retention time for all compounds is controlled by the amount of acetonitrile and amount of ions in the mobile phase. Ions in the mobile phase can be created by organic and inorganic acids and corresponding salt buffers. Various detection techniques can be used for monitoring pyridinecarboxylic acids. Other ionizable isomers can be successfully separated on mixed-mode columns.
Primesep D separates organic acids such as fumaric, benzoic, phthalic, naphthoic, and maleic acids by a mixture of anion exchange and reversed phase. Retention times and elution order can be changed by adjusting the percentage of acetonitrile in the mobile. This can not be done by traditional ion-exchange and ion-exclusion chromatography. The HPLC separation uses a mobile phase of water, acetonitrile (MeCN, ACN) and trifluoroacetic acid (TFA) and UV detection at 250 nm.
Organic and inorganic acids and ions can be separated on a Primesep B4 column based on their ionic properties. Method can be used for quantitation of residual acids in various products and sample matrices. Trifluoracetic, hydrochloric, methanesulfonic, and nitric acids are separated using ACN-water-ammonium formate. Ions can be detected by ELSD, CAD or LC/MS.
Obelisc N column are used for separation of weak and strong organic acids in mixed-mode HILIC. Benzoic and naphthalenesulfonic acids are retained based on polar interaction mode and anion-exchange mode. Order of elution and retention pattern can be changed by modifying mobile phase. PH of the mobile phase changes ionization state of stationary phase and analytes. Fast quantitation method for benzoic and naphthalenesulfonic acid can be developed using UV, ELSD or LC/MS detection. HPLC Method can be used for mixture of organic and inorganic strong and weak acids.
Hydrophilic acids are separated on Obelisc N mixed-mode HILIC column. Seven carboxylic acids are separated based on their polarity and pKa values. Changes in ionization states of acids and stationary phase can be used to control elution order of organic and inorganic acids.
Organic and inorganic acids can be retained and separated on mixed-mode columns based on weak reversed-phase and weak/medium anion-exchange mechanisms. Amount of ACN, buffer concentration and buffer pH will affect retention time of organic and inorganic acids. Acids can be monitored by low UV, ELSD or LC/MS. Presence of ions is required to facilitate ion-exchange mechanism. Method can be used as a general approach for analysis of acidic hydrophilic and acidic hydrophobic compounds. Carboxylic acids along with inorganic acid can be retained and separated without ion-pairing reagent.
Organic acids were separated on a HILIC/cation-exchange column in HILIC/anion-exclusion mode. This column can be used for analysis of polar compounds in HILIC mode. If compounds are ionizable, additional mode of interaction can be added (cation-exchange or anion-exclusion).
Application Notes: maple syrup primarily consists of sucrose and other sugars, but also has small amounts of amino acids (arginine, threonine, proline), organic acids (malic and fumaric), vitamins (niacin and riboflavin) and traces of minerals (Na, K, Ca, Mn, Mg, Zn ions). One analysis performed on maple syrup is calcium ion determination. A Primesep 100 column was used in cation-exchange mode to isolate calcium ion from other components of the maple syrup. This method can be used for quantification of calcium ion by ELSD/CAD. Limit of detection depends on the type of ELSD/CAD detector used but usually reaches 20 ppm
Application Columns: Primesep 100
Application compounds: calcium ions, Maple Syrup
Detection technique: LC/MS, ELSD/CAD