Benzoic acid

Caffeine, potassium sorbate, and sodium benzoate are common additives used in the production of soft drinks, energy drinks, and various food products. Accurate quantification of these compounds is crucial for quality control and regulatory compliance in the beverage industry. High-Performance Liquid Chromatography (HPLC) provides a robust method for the analysis of caffeine, potassium sorbate, and sodium benzoate mixtures. In this study, we describe the HPLC analysis of this mixture utilizing an Amaze HA mixed-mode column. Caffeine is a naturally occurring alkaloid found in coffee beans, tea leaves, and cacao pods. It is a white, crystalline powder with a bitter taste and acts as a stimulant, enhancing alertness and reducing fatigue. Potassium sorbate is a potassium salt of sorbic acid, a compound naturally found in some berries. It is widely used as a preservative in food and beverage products due to its ability to inhibit the growth of molds, yeasts, and bacteria. Sodium benzoate is the sodium salt of benzoic acid, a common food preservative found naturally in fruits. It is utilized in the food and beverage industry to prevent microbial growth and extend shelf life. The Amaze HA mixed-mode column features a unique stationary phase that combines hydrophobic, anion-exchange and cation-exclusion properties. The stationary phase consists of hydrophobic alkyl chains with embedded hydroxyl and amine groups, providing both reverse-phase and ion interactions. This mixed-mode chromatography enables enhanced selectivity and resolution of analytes, making it suitable for the analysis of polar and non-polar compounds, such as caffeine, potassium sorbate, and sodium benzoate. The Amaze HA column offers exceptional versatility and robustness, making it well-suited for various applications in the food and beverage industry. The HPLC analysis of a mixture of caffeine, potassium sorbate, and sodium benzoate on the Amaze HA mixed-mode column provides an effective method for the quantitative determination of these compounds commonly used in soft drinks, energy drinks, and other food products. This approach offers high sensitivity, selectivity, and reproducibility, making it suitable for quality control and regulatory compliance. Moreover, the unique design and superior performance of the Amaze HA mixed-mode column ensure efficient separation and analysis of complex mixtures, further enhancing its utility in beverage analysis and research endeavors.

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Conditions of Experiment

Column:	Amaze HA
Separation Modes:	reversed-phase, Anion-exchange
Column Dimenstions:	3x100 mm, 3 um, 100 A
Mobile Phase:	ACN/water/ammonium formate
Detection:	UV 255 nm, ELSD
Sample:	various-various mg/ml
Injection:	3 uL
Flow rate:	0.6 ml/min

Analytes

Class of compounds:	Aromatic acid, Inorganic cation, Organic acid, Preservative, Stimulant, Xanthine
Nature of compounds:	Acidic, Basic, Hydrophilic, Neutral, Polar
Compounds:	Sodium ion, Potassium ion, Caffeine, Sorbic acid, Benzoic acid

This method details the High-Performance Liquid Chromatography (HPLC) analysis of six preservatives: benzyl alcohol, dehydroacetic acid, sorbic acid, methylparaben, benzoic acid, and ethylparaben. The separation is conducted on an Amaze HA Mixed-Mode Column, utilizing both reversed-phase and anion-exchange modes for comprehensive separation. Benzyl Alcohol: A commonly used preservative with antimicrobial properties, benzyl alcohol is a water-miscible compound with a phenyl group. It is often employed in pharmaceutical formulations and cosmetic products. Dehydroacetic Acid: Another antimicrobial preservative, dehydroacetic acid is water-soluble and contains carboxylic acid functional groups. It is frequently utilized in the preservation of food, cosmetics, and personal care products. Sorbic Acid: Widely employed as a preservative in food and beverages, sorbic acid is water-soluble and possesses a carboxylic acid group. It inhibits the growth of mold, yeast, and fungi in various formulations. Methylparaben: Methylparaben is an ester of p-hydroxybenzoic acid, serving as a preservative in pharmaceuticals, cosmetics, and food products. It is soluble in alcohol and exhibits antimicrobial activity against a range of microorganisms. Benzoic Acid: A naturally occurring compound with antimicrobial properties, benzoic acid is soluble in water and commonly used as a preservative in acidic food products, beverages, and personal care items. Ethylparaben: Ethylparaben is an ester of p-hydroxybenzoic acid, similar to methylparaben, and is utilized as a preservative in various consumer products. It exhibits solubility in alcohol and serves to extend the shelf life of formulations. Amaze HA Mixed-Mode Column: This column features a mixed-mode stationary phase with both hydrophobic and ionic interaction capabilities. In reversed-phase mode, hydrophobic interactions predominate, facilitating separation based on differences in hydrophobicity. In anion-exchange mode, ionic interactions occur, allowing for separation based on differences in charge. By employing this method, comprehensive analysis and quantification of the six preservatives can be achieved, making it valuable for quality control and regulatory compliance in various industries.

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Conditions of Experiment

Column:	Amaze HA
Separation Modes:	reversed-phase, Anion-exchange
Column Dimenstions:	3x100 mm, 3 um, 100 A
Mobile Phase:	ACN/water/ammonium formate pH 3.7
Detection:	235 nm
Sample:	various-0.2 mg/ml
Injection:	3 uL
Flow rate:	0.6 ml/min

Analytes

Class of compounds:	Aromatic acid, Aromatic compound, Organic acid, Organic compound, Preservative
Nature of compounds:	Acidic, Hydrophilic, Hydrophobic, Neutral
Compounds:	Benzyl alcohol, Dehydroacetic acid, Sorbic acid, Methylparaben, Benzoic acid, Ethylparaben

Comparison run on Amaze PFP, Amaze AR, Amaze SPF, and Heritage C20 columns.

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Conditions of Experiment

Column:	Amaze SPF
Separation Modes:	reversed-phase, pi-pi
Column Dimenstions:	4.6x150 mm, 5 μm, 100A
Mobile Phase:	35% ACN with 20 mM AmAc pH 4.5
Detection:	UV 235 nm
Sample:	1 mg/ml
Injection:	3 μL
Flow rate:	1 ml/min

Analytes

Class of compounds:	Amines, Aromatic acid, Aromatic base, Aromatic compound, Organic acid, Organic compound, Preservative
Nature of compounds:	Acidic, Basic, Hydrophilic, Hydrophobic, Neutral, Polar
Compounds:	4-Aminophenol, Mandelic acid, 4-Hydroxybenzoic acid, Phenol, Benzoic acid, Methylparaben

Comparison run on Amaze PFP, Amaze AR, Amaze SPF, and Heritage C20 columns.

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Conditions of Experiment

Column:	Amaze AR
Separation Modes:	reversed-phase, pi-pi
Column Dimenstions:	4.6x150 mm, 5 μm, 100A
Mobile Phase:	28% ACN with 20 mM AmAc pH 4.5
Detection:	UV 255 nm
Sample:	1 mg/ml
Injection:	3 μL
Flow rate:	1 ml/min

Analytes

Class of compounds:	Amino acid, Aromatic acid, Aromatic compound, Drug, Isomer, Organic acid, Preservative, Stimulant, Xanthine
Nature of compounds:	Acidic, Hydrophilic, Hydrophobic, Neutral, Polar, Zwitterionic
Compounds:	Caffeine, 4-Aminobenzoic acid, Aspirin, 4-Hydroxybenzoic acid, Sorbic acid, Benzoic acid

Comparison run on Amaze PFP, Amaze AR, Amaze SPF, and Heritage C18 columns.

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Conditions of Experiment

Column:	Amaze PFP
Separation Modes:	reversed-phase, pi-pi
Column Dimenstions:	4.6x150 mm, 5 μm, 100A
Mobile Phase:	50% ACN with 20 mM AmFm pH 3
Detection:	UV 255 nm
Sample:	1 mg/ml
Injection:	3 μL
Flow rate:	1 ml/min

Analytes

Class of compounds:	Aromatic acid, Aromatic compound, Organic acid, Organic compound, Organic solvent
Nature of compounds:	Acidic, Hydrophilic, Hydrophobic, Neutral, Polar
Compounds:	Benzyl alcohol, Phenol, Benzoic acid, Toluene, Benzenesulfonic acid

Seven compounds with different properties were used to study an effect of different acids in the mobile phase on retention of neutral, acidic, basic and zwitterionic compounds on core-shell mixed-mode Coresep 100 column. Retention of neutral compounds (propylparaben and benzoic acid to some extent) are not affected by change of the acid. Basic compounds (norphenylephrine, pyridine, 2-aminopyridine) respond the most to change of the strength of acidic additive. Several folds increase in retention time was observed when sulfuric acid was replaced with formic acid in the mobile phase. Effect of acid on retention time of zwitterionic amino acids was less pronounced but still provided excellent retention and peak shape for zwitterionic compounds (5-aminosalicylic acid, phenylalanine). This methods and Coresep 100 column can be used to retain and separate other acidic, basic, neutral and zwitterionic compounds without the use of ion-pairing reagent in the mobile phase. Various detection techniques can be used based on the properties of the mobile phase and analytes. Methods are compatible with mall major detection techniques 9UV, ELSD, CAD, RI, MS). Core-shell mixed-mode columns provide great combination of unique selectivity, speed and efficiency.

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Conditions of Experiment

Column:	Coresep 100
Separation Modes:	reversed-phase, cation-exchange
Column Dimenstions:	4.6 x 150 mm, 2.7 um, 90A
Mobile Phase:	30% ACN with various acids
Detection:	UV 255 nm
Sample:	0.3 mg/ml
Injection:	3 uL
Flow rate:	1 mL/min

Analytes

Class of compounds:	Amines, Amino acid, Aromatic acid, Aromatic base, Aromatic compound, Catecholamine, Drug, Isomer, Neurotransmitter, Organic acid, Preservative, Pyridine, Supplement
Nature of compounds:	Acidic, Basic, Hydrophilic, Hydrophobic, Neutral, Polar, Zwitterionic
Compounds:	5-Aminosalicylic acid, Norphenylephrine, Phenylalanine, Pyridine, Benzoic acid, 2-Aminopyridine, Propylparaben

Core-shell mixed-mode columns offer unique selectivity which comes from the presence of two mechanisms - reversed-phase and ion-exchange. The alternative selectivity of mixed-mode columns can be used when order of elution or resolution between compounds needs improvement. Even for neutral compounds a different selectivity can be obtained. A method for common components of headache medications (acetaminophen, caffeine, and benzoate) was developed on our Coresep 100 column. Efficiency and peak shape are comparable to leading brand core-shell reversed-phase columns.

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Conditions of Experiment

Column:	Coresep 100
Separation Modes:	reversed-phase
Column Dimenstions:	3.2 x 50 mm, 2.7 um, 90A
Mobile Phase:	20% ACN with 0.1% TFA
Detection:	UV 250 nm, ELSD
Sample:	0.3 mg/ml
Injection:	1 uL
Flow rate:	1 ml/min

Analytes

Class of compounds:	Aromatic acid, Aromatic compound, Drug, Organic acid, Stimulant, Xanthine
Nature of compounds:	Acidic, Hydrophilic, Neutral
Compounds:	Acetaminophen, Caffeine, Benzoic acid

Mixture of hydrophilic acidic compounds are separated in reversed-phase anion-exchange mode on core-shell mixed-mode column. Method is robust, reliable and fast and can be used for analysis of underivatized acids without ion-pairing reagent.

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Conditions of Experiment

Column:	Coresep SB
Separation Modes:	reversed-phase and anion-exchange
Column Dimenstions:	3.2 x 100 mm, 2.7 um, 90A
Mobile Phase:	50% ACN with 50 mM sodium phosphate (monobasic) pH 3
Detection:	UV 230 nm
Sample:	0.3 mg/ml
Injection:	3 uL
Flow rate:	0.5 mg/ml

Analytes

Class of compounds:	Aromatic acid, Organic acid
Nature of compounds:	Acidic, Hydrophilic, Polar
Compounds:	Benzoic acid, Maleic acid, Fumaric acid