Phenylalanine

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.

Coresep 100 PURCHASE LEARN ABOUT Coresep 100

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

Mixed-mode stationary phases were designed to retain and separate compounds with drastic difference in properties using one column and method. Carboxylic acids, amino acids and sugars are poorly retained in reversed-phase chromatography. Derivatization procedures, ion-pairing reagents and HILIC columns can be used tp analyze these complex mixtures. The use of ion-pairing reagent makes MS detection problematic due to volatility issues of IP and signal suppression in MS. HILIC mixed-mode columns, like Amaze TH, have polar ionizable groups on the surface which help significantly increase retention time of polar ionizable analytes like amines, amino acids, carboxylic acids and sugars. We have developed a method for separation and retention of succinic acid, phenylalanine, sucrose, glycine, aspartic acid and raffinose. Isocratic conditions provide separation within 15 minutes with good peak shape and retention control. Retention time on Amaze TH column is controlled by amount of ACN, buffer concentration, buffer ph and buffer nature. Method can be used for other amines, amino acids, carboxylic acids and sugars where fast MS-compatible separation is required.

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Conditions of Experiment

Column:	Amaze TH
Separation Modes:	HILIC, cation-exchange, anion-exchange
Column Dimenstions:	4.6 x 100 mm 5 um, 100A
Mobile Phase:	80% ACN with 10 mM AmAc pH 4.8
Detection:	ELSD
Sample:	0.3 mg/ml
Injection:	3 uL
Flow rate:	1 ml/min

Analytes

Class of compounds:	Amino acid, Organic acid, Sugar, Supplement
Nature of compounds:	Acidic, Hydrophilic, Neutral, Polar, Zwitterionic
Compounds:	Succinic acid, Phenylalanine, Sucrose, Glycine, Aspartic acid, Raffinose

Four neurotransmitters - pseudoephedrine, norephedrine, phenylephrine and norphenylephrine - were separated in hydrogen-bonding and cation-exchange mode. Pseudoephedrine is sympathomimetic drug used either as a single ingredient or part of the composition of OTC or Rx medications. Phenylephrine is a hydrophilic basic drug which is used as decongestant in OTC medication composition, such as Sudafed, Benadryl, Advil, Nyquil, etc. Norphenylephrine is an adrenergic agent used as sympathomimetic drug. Norephedrine is naturally occurring endogenous trace amine that plays a role as neurotransmitter in the brain, and is used as decongestant in many OTC compositions as well as an appetite suppressant. All four drugs are hydrophilic and basic in nature, and have no retention on traditional reversed-phase column without an ion-pairing reagent. Method provides an efficient and robust separation for all drugs. The method can be used in analysis of neurotransmitters in biofluids and other sample matrices. The method is compatible with all detection techniques: MS, RI, UV, ELSD and CAD.

Amaze HD PURCHASE LEARN ABOUT Amaze HD

Conditions of Experiment

Column:	Amaze HD
Separation Modes:	hydrogen-bonding and cation-exchange
Column Dimenstions:	3.2 x 50 mm, 3 um
Mobile Phase:	MeCN/MeOH (95/5) with 0.5% HCOOH and 0.05% AmFm
Detection:	UV 275 nm
Sample:	0.3 mg/ml
Injection:	1 uL
Flow rate:	1.0 mL/min

Analytes

Class of compounds:	Amino acid, Aromatic base, Catecholamine, Drug, Neurotransmitter, Stimulant, Supplement
Nature of compounds:	Basic, Hydrophilic, Polar, Zwitterionic
Compounds:	Pseudoephedrine, Norepinephrine, Phenylalanine, Norphenylephrine

Amino acids are very polar compounds which are used as a building blocks in pharmaceutical industry. They are widely used as supplements and food additives. Depending on the pH of the mobile phase they can exist in acidic, basic or zwitter-ionic form. At pH below 3, amino acids are basic in nature and have the highest hydrophobicity. Within pH of 3 to 7 amino acids are in zwitter-ionic form where they are the most hydrophilic. There is no mechanism of retention on reversed-phase column and amino acids are not retained. Bufferless ion-separation (BLIS) was introduced by SIELC as a way to retain and analyze amino acids in reversed-phase cation-exchange mode. This mode allows to analyze amino acids as zwitter-ions without any ions/buffers in the mobile phase. The method was adopted for analysis of amino acids on core-shell mixed-mode columns. A similar approach on a reversed-phase core-shell column results in distorted peaks and no separation or significant retention to achieve baseline separation.

Coresep 100 PURCHASE LEARN ABOUT Coresep 100

Conditions of Experiment

Column:	Coresep 100
Separation Modes:	reversed-phase and cation-exchange
Column Dimenstions:	3.2 x 50 mm, 2.7 um, 90A
Mobile Phase:	20% ACN with no additives
Detection:	UV 205 nm, ELSD
Sample:	0.3 mg/ml
Injection:	1 uL
Flow rate:	1 ml/min

Analytes

Class of compounds:	Amino acid, Supplement
Nature of compounds:	Hydrophilic, Polar, Zwitterionic
Compounds:	Methionine, Tyrosine, Phenylalanine

Catecholamines (neurotransmitters) are derivatives of the amino acid tyrosine. DOPA, tyrosine, phenylalanine, norepinephrine, epinephrine, and dopamine. Fast and efficient method with base-line separation was achieved on Coresep 100 column. All compounds are separated by combination of reversed-phase and cation-exchange mechanisms. Peak order and retention time can be changed by switching from TFA to ammonium formate in the mobile phase, by adjusting mobile phase composition and by changing pH. The method is fully compatible with mass spectroscopy and can be used for fast analysis of neurotransmitters in biofluids. regular. Method can be used a replacement for analysis of neurotransmitters by UPLC with ion-pairing reagents.

Coresep 100 PURCHASE LEARN ABOUT Coresep 100

Conditions of Experiment

Column:	Coresep 100
Separation Modes:	reversed-phase and cation-exchange
Column Dimenstions:	3.2 x 100 mm, 2.7 um, 90A
Mobile Phase:	ACN gradient from 5% to 10% in 5 min, buffer gradient - Ammonium formate pH 2.9 from 5 to 25 mM in 5 min
Detection:	UV 270 nm, ELSD
Sample:	0.3 mg/ml
Injection:	1 uL
Flow rate:	1 ml/min

Analytes

Class of compounds:	Amino acid, Aromatic base, Catecholamine, Drug, Neurotransmitter, Supplement
Nature of compounds:	Basic, Hydrophilic, Polar, Zwitterionic
Compounds:	Dopamine, Tyrosine, Phenylalanine, Norepinephrine, Epinephrine, DOPA

Amino acids serve as building blocks, drugs and nutrients. The usual approach for analysis of amino acids include reversed-phase chromatography with an ion-pairing reagent, derivatization, and hydrophilic interaction chromatography (HILIC). Three amino acids (phenylalanine, tyrosine, and DOPA) were retained and separated in underivatized form in HILIC cation-exchange mode of core-shell mixed-mode HPLC column. This method is fast and reliable and provides base-line separation for all amino acids in the application. Method can be adopted for analysis of other amino acids.

Coresep S PURCHASE LEARN ABOUT Coresep S

Conditions of Experiment

Column:	Coresep S
Separation Modes:	HILIC and cation-exchange
Column Dimenstions:	3.2 x 100 mm, 2.7 um, 90A
Mobile Phase:	85 % ACN with 15 mM AmAc pH 5
Detection:	UV 270, ELSD
Sample:	0.3 mg/ml
Injection:	3 uL
Flow rate:	0.5-2 ml/min

Analytes

Class of compounds:	Amino acid, Catecholamine, Drug, Neurotransmitter, Supplement
Nature of compounds:	Basic, Hydrophilic, Polar, Zwitterionic
Compounds:	Phenylalanine, Tyrosine, DOPA