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
Neutral, Base, and Acid on Primesep A Column

Primesep A separates acids, bases, and neutrals in one injection. Maleic acid, benzonitrile, and benzylamine are baseline resolved by a combination of reversed-phase, ion-exchange, and ion-exclusion mechanisms. Excellent peak shape results with a mass spec compatible mobile phase of water, acetonitrile (MeCN, ACN) and trifluoracetic acid (TFA) with UV detection at 210 nm.
BenzonitrileBenzylamineMaleic Acid
Neutral, Base, and Acid on Primesep 100 Column

Primesep 100 retains and separates an acid, base, and neutral in one HPLC injection. Maleic acid, benzylamine, and benzonitrile are resolved by ion-exclusion, ion-exhange and reversed-phase modes. The separation uses a mobile phase of water, acetonitrile (MeCN, ACN) and trifluoracetic acid (TFA) with UV detection at 210 nm.
BenzonitrileBenzylamineMaleic Acid
Neutral, Base, and Acid on Primesep 200 Column

Primesep 200 separates acids, bases, and neutrals in one injection. Maleic acid, benzonitrile, and benzylamine are baseline resolved by a combination of reversed-phase, ion-exchange, and ion-exclusion mechanisms. Excellent peak shape results with a mass spec compatible mobile phase of water, acetonitrile (MeCN, ACN) and trifluoracetic acid (TFA) with UV detection at 210 nm.
BenzonitrileBenzylamineMaleic Acid
Neutral, Base, and Acid on Primesep B Column

Primesep B separates acids, bases, and neutrals in one injection. Maleic acid, benzonitrile, and benzylamine are baseline resolved by a combination of reversed-phase, ion-exchange, and ion-exclusion mechanisms. Excellent peak shape results with a mass spec compatible mobile phase of water, acetonitrile (MeCN, ACN) and trifluoracetic acid (TFA) with UV detection at 210 nm.
BenzonitrileBenzylamineMaleic Acid
HPLC Analysis of Polar Basic and Acetic Compounds on Primesep AB Column
Mixture of polar acidic and basic compounds is separated on a Primesep AB mixed-mode HPLC column. Dopamine and tyrosine are retained by combination of reversed-phase and cation-exchange mechanisms. Maleic acid is retained by anion-exchange mechanism, and benzoic acid is retained by reversed-phase mechanism. Primesep AB is a trimodal column with a C12 hydrophobic chain and cation-exchange and anion exchange groups on the surface. Method utilizes UV detection but can be used with other detection techniques (ELSD, LC/MS, Corona).
Condition
Column |
Primesep AB, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O |
Buffer |
TFA - 0.1% |
Flow Rate |
1.0 ml/min |
Detection |
UV, 210 nm |
Description
Class of Compounds
|
Drug, Acid, Monocarboxylic acid, Hydrophilic, Ionizable, Hormone |
Analyzing Compounds |
Tyrosine, Dopamine, Maleic Acid, Benzoic Acid |
Benzoic AcidDopamineMaleic AcidTyrosine
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
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)
Separation of Maleic and Succinic Acid on Primesep B2 column
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.
Condition
Column |
Primesep B2, 4.6x50 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O - 10/90% |
Buffer |
AmFm pH 3.0 |
Flow Rate |
1.0 ml/min |
Detection |
ELSD, UV 230nm |
Description
Class of Compounds |
Acid, Hydrophilic, Ionizable |
Analyzing Compounds |
Succinic Acid, Maleic Acid |
Maleic AcidOrganic AcidsSuccinic Acid
HPLC Separation of Maleic Acid and Pentaerythritol
Application Notes: Maleic acid is a small hydrophilic acid, which is poorly retained in reversed-phase chromatography. Pentaerythritol is hydrophilic neutral molecule. A miixed-mode HILIC/anion-exchange method was developed on Obelisc N HPLC column. The retention time for maleic acid can be controlled by amount of ACN, buffer pH, and buffer concentration. Pentaerythritol retention time is only controlled by the amount of acetonitrile. The method is LC/MS and ELSD compatible.
Application Columns: Obelisc N
Application compounds: Maleic Acid, Pentaerythritol
Detection technique: LC/MS, ELSD/CAD
Condition
Column |
Obelisc N, 3.2x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O - 95/5% |
Buffer |
AmFm pH 3.0- 5 mM |
Flow Rate |
0.5 ml/min |
Detection |
ELSD |
Description
Class of Compounds
|
Acid, Hydrophilic, Ionizable |
Analyzing Compounds |
Maleic Acid, Pentaerythritol |
Maleic AcidPentaerythritol
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 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