Applications:
Separation of Diacid: Ion Exclusion mode

Primesep 100 separates a mixture of dicarboxylic acids in ion-exclusion mode with a mobile phase of water, acetonitrile (MeCN, ACN), and sulfuric acid (H2SO4) with UV detection at 210 nm. Baseline resolution of fumaric, maleic, malic, and succinic acids is obtained in less than 8 minutes. The separation combines ion-exclusion and reversed-phase mechanisms in one method.
Dicarboxylic AcidsFumaric AcidMaleic AcidMalic AcidSuccinic Acid
Separation of Diacid Hydrophobic and Ion Exclusion Modes

Primesep 200 retains and separates the organic diacids (malic, succinic, fumaric, and maleic) by a combination hydrophobic, reversed-phase interactions and ion exclusion. The separation uses a mobile phase of water, acetonitrile (MeCN, ACN) and trifluoracetic acid (TFA) with UV detection at 210 nm.
Dicarboxylic AcidsFumaric AcidMaleic AcidMalic AcidSuccinic Acid
Separation of Diacid Hydrophobic and Ion Exchange Modes

Primesep B combines a hydrophobic, reversed-phase mechanism with ion exchange to separate the diacids, fumaric, benzoic, phthalic, naphthoic, and maleic acids. Changing the acetonitrile content of the mobile phase reverses the peak order for naphthoic and maleic acids. Primesep B combines reversed-phase and anion-exchange mechanism with a mobile phase of water, acetonitrile (MeCN, ACN) and trifluoracetic acid (TFA) and UV detection at 250 nm.
Condition
Column |
Primesep B, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O |
Buffer |
TFA |
Flow Rate |
1.0 ml/min |
Detection |
UV, 250 nm |
Description
Class of Compounds
|
Acid, Hydrophilic, Ionizable |
Analyzing Compounds |
Fumaric acid, Benzoic acid, Phthalic acid, Naphthoic acid, Maleic acid, ) |
Benzoic AcidDicarboxylic AcidsFumaric AcidMaleic AcidNaphthoic AcidPhthalic Acid
HPLC Separation of Organics Acids

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.
Condition
Column |
Primesep D, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O |
Buffer |
AmFm |
Flow Rate |
1.0 ml/min |
Detection |
UV 250 nm |
Description
Class of Compounds
|
Acid, Hydrophilic, Ionizable |
Analyzing Compounds |
Fumaric Acid, Benzoic Acid, Phthalic Acid, Maleic Acid, Naphtoic Acid |
Benzoic AcidFumaric AcidMaleic AcidNaphthoic AcidOrganic AcidsPhthalic Acid
HILIC Separation of Carboxylic 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.
Fumaric AcidHydroxybenzoic AcidMalic AcidMandelic AcidMethylmalonic AcidOrganic AcidsSuccinic AcidTartaric Acid
HPLC Analysis of Basic Drugs and Acidic Counter-Ions by Mixed-Mode Chromatography

The majority of drugs in the pharmaceutical industry are administered in salt form. The presence of two counter-ions very often necessitates the use of two methods. The nature of these counterparts in drugs can be an inorganic cation and organic acid, inorganic anion and organic base, and organic cation and organic anion. Furthermore, the properties of the molecules will result in a differing stoichiometry. The task of simultaneous quantitation of counter-ions can be achieved by using mixed-mode columns. The general approach for analysis is based on properties of corresponding counter-ions. Hydrophobic basic drugs, like dextromethorphan, verapamil, trimipramine, and corresponding acidic counter-ions (chloride, chlorate, bromide, bromate, perchlorate, maleate, fumarate,tartrate, succinate, phosphate, citrate, benzosulfonate, toleuensulfonate) can be separated and quantitated in the same run on reversed-phase anion-exchange column. Basic hydrophobic drugs are retained by the reversed-phase mechanism, and counter-ions are retained by the reversed-phase and anion-exchange mechanism. Some polar counter-ions are retained only by the anion-exchange mechanism. Retention time and selectivity of HPLC separation of drugs and counter-ions can be achieved by changing the amount of acetonitrile and the amount of ions in the mobile phase. The detection technique depends on the properties of the counter-ions. In case of low or no UV activity, ELSD can be employed if the counter-ion forms a non-volatile salt with the mobile phase additive (ammonium formate). This HPLC method can be used for simultaneous quantitation of other basic drugs and counter-ions. The presence of two mechanisms of retention allows control over retention times of drug and counter-ion independently, and even allows a change of order of elution when necessary.
Condition
Column |
Primesep D , 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O |
Buffer |
AmFm pH 3.0 |
Flow Rate |
1.0 ml/min |
Detection |
ELSD, UV 270 |
Description
Class of Compounds |
Ions, Hydrophilic, Hydrophobic, Base, Acids, Ionizable |
Analyzing Compounds |
Sodium Chloride, Sodium chloride, Sodium Chlorate, Sodium bromide, Sodium bromate, Perchloric Acid, Maleic Acid, Fumaric Acid, Tartaric Acid, Succinic Acid, Phosphoric Acid, Citric acid, Benzosulfonic acid, Dextromethorphan, Verapamil, Trimipramine |
Benzenesulfonic AcidBromideChlorateChlorideCitric AcidDextromethorphanFumaric AcidMaleic AcidOrganic AcidsPerchloratePhosphoric AcidPyrilamineSuccinic AcidTartaric AcidVerapamilp-Toluenesulfonic Acid (PTSA)
Analysis of Compounds of TCA Cycle on SHARC 1 Column

This is an alternative approach for the separation of the TCA cycle intermediates that was developed using novel hydrogen-bonding HPLC column. The organic nature of the mobile phase helps obtain a highly sensitive LC/MS compatible method.
Citric AcidFumaric AcidIsocitric AcidMaleic AcidMalic AcidSuccinic Acid
Separation of Compounds in TCA Cycle on Primesep D Column
The citric acid cycle (tricarboxylic acid cycle, Krebs cycle) is a key process in the metabolic pathway by which all aerobic organisms generate energy. Compounds generated during TCA are hydrophilic, acidic compounds. Some of the hydrophilic acids are very strong while others are relatively weaker. All compounds have very low hydrophobicity and do not retain by the reversed-phase mechanism on the C18 or C8 HPLC columns. Citric acid and related products were separated in reversed-phase, anion-exchange mode on the Primesep D mixed-mode column. All hydrophilic, acidic compounds are well separated and demonstrate good shape. This robust method can be used for analysis of components of the TCA cycle and other highly hydrophilic compounds. This method is fully compatible with LC/MS and prep chromatography.
Condition
Column |
Primesep D, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O |
Buffer |
AmFm, pH 3.0 |
Flow Rate |
1.0 ml/min |
Detection |
ELSD |
Description
Class of Compounds
|
Acid, Hydrophilic, Ionizable |
Analyzing Compounds |
Sodium, Succinic acid, Malic acid, Isocitric acid, Citric acid, Fumaric acid, Maleic acid |
Citric AcidFumaric AcidIsocitric AcidMaleic AcidMalic AcidSodiumSuccinic Acid
HPLC Separation of Small Organic Acids on Newcrom B Column
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Condition
Column |
Newcrom B, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O - 5/95% |
Buffer |
AmAc pH 5.0, Formic Acid |
Flow Rate |
1.0 ml/min |
Detection |
CAD (Corona) MS- compatible mobile phase |
Description
Class of Compounds |
Acid, Hydrophilic, Ionizable |
Analyzing Compounds |
Glycolic acid, Succinic Acid, Methylmalonic acid, Malic Acid, Maleic Acid, Citric Acid, Tartaric Acid, Fumaric Acid, Malonic Acid |
Citric AcidFumaric AcidGluconic acidGlycolic acid sodium saltMaleic AcidMalic AcidMalonic AcidMethylmalonic AcidSuccinic AcidTartaric Aciddl-Tartaric acid
HPLC Separation of Methylmalonic, Fumaric, Maleic acids on Newcrom B Column
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Condition
Column |
Newcrom B, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeOH/H2O - 5/93% |
Buffer |
Formic Acid - 2% |
Flow Rate |
1.0 ml/min |
Detection |
CAD (Corona) (MS-compatible mobile phase) |
Description
Class of Compounds |
Acid, Hydrophilic, Ionizable |
Analyzing Compounds |
Methylmalonic acid, Fumaric acid, Maleic acid |
Fumaric AcidMaleic AcidMethylmalonic Acid
HPLC Separation of Sorbic, Fumaric, Malonic Acids on Newcrom BH Column
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Acids with different strengths can be retained and elution order controlled by the use of a mixed-mode column, which has an ion-exchange component in addition to hydrophobic retention. In this example, the Newcrom BH mixed-mode column was used to separate fumaric, malonic and sorbic acids in HPLC. Sorbic acid was mostly retained by hydrophobicity, while fumaric and malonic acids were also retained by the adjustment strength of the mobile phase
Condition
Column |
Newcrom BH, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O - 20/80% |
Buffer |
H3PO4 |
Flow Rate |
1.0 ml/min |
Detection |
UV 200nm |
Description
Class of Compounds |
Acid |
Analyzing Compounds |
Sorbic Acid, Levulinic Acid |
Fumaric AcidMalonic AcidSorbic acid