Dopamine

For optimal results in HPLC analysis, it is recommended to measure absorbance at a wavelength that matches the absorption maximum of the compound(s) being analyzed. The UV spectrum shown can assist in selecting an appropriate wavelength for your analysis. Please note that certain mobile phases and buffers may block wavelengths below 230 nm, rendering absorbance measurement at these wavelengths ineffective. If detection below 230 nm is required, it is recommended to use acetonitrile and water as low UV-transparent mobile phases, with phosphoric acid and its salts, sulfuric acid, and TFA as buffers.
For some compounds, the UV-Vis Spectrum is affected by the pH of the mobile phase. The spectra presented here are measured with an acidic mobile phase that has a pH of 3 or lower.

HPLC Method for Separation of of Amines in Non Aqueous MP on BIST B+ by SIELC Technologies

Separation type: Bridge Ion Separation Technology, or BIST™ by SIELC Technologies

Dopamine is a key neurotransmitter and medical stimulant used to treat low blood pressure, low heart rate, and heart attacks. m-Xylylenediamine (MXDA) is a popular curing agent used on epoxy resins. 2,4,6-Tris(dimethylaminomethyl)phenol is another amine-based curing agent used on epoxy resins. Using SIELC’s newly introduced BIST method, these 3 amines can be retained on a positively-charged anion-exchange BIST B+ column. There are two keys to this retention method: 1) a multi-charged, negative buffer, such as Sulfuric acid (H2SO4), which acts as a bridge, linking the positively-charged analytes to the positively-charged column surface and 2) a mobile phase consisting mostly of less polar organic solvent (such as IPA) to minimize the formation of a solvation layer around the charged analytes. This method uses an entirely non-aqueous mobile phase to drive BIST retention. The gradient starts with a high concentration of the less polar IPA to generate the initial BIST retention and progresses to the more polar MeOH to elute the longer-retaining amines in a reasonable time. Using this new and unique analysis method, these 3 amines can be retained and UV detected at 210 nm.

High Performance Liquid Chromatography (HPLC) Method for Analysis of of Amines in Non Aqueous MP

Condition

Description

Separation type: Liquid Chromatography Mixed-mode

High Performance Liquid Chromatography (HPLC) Method for Analysis of Dopamine and Serotonin

Dopamine is a key neurotransmitter and medical stimulant used to treat low blood pressure, low heart rate, and heart attacks. Serotonin is another key neurotransmitter that has pharmaceutical applications – namely as the key ingredient in antidepressant and anti-anxiety medications.These two neurotransmitters can be retained, separated, and analyzed on a Primesep 200 mixed-mode column using an isocratic analytical method with a simple mobile phase of water, Acetonitrile (MeCN), and an Ammonium Formate (AmFm) buffer. This analysis method can be UV detected at 280 nm with high resolution and peak symmetry.

Separation type: Liquid Chromatography Mixed-mode

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Catecholamines are neurotransmitters that generate the ‘fight-or-flight’ responsein the body. The 3 main catecholamines are epinephrine (also known as adrenaline), norepinephrine, and dopamine, and they are produced by the adrenal glands.
These 3 catecholamines can be detected in the low UV regime. Using a Newcrom AH mixed-mode column and a mobile phase consisting of almost entirely water with either a phosphoric (H3PO4) acid or ammonium formate (AmFm) buffer, the catecholamines can be retained, separated, and measured. This analysis method can be UV detected at 275 nm with high resolution. The latter mobile phase (utilizing AmFm) is compatible with Mass Spectrometry.

FlipLC™ is an alternative method to avoid the interference of most of the contaminants by the use of an isolation column and a high pressure switching valve before the separation column. This method allows sample cleaning and analyte separation in one automated process. The isolation column and the separation column should have orthogonal retention characteristics to operate efficiently in this setup. Mixed-mode columns with reverse phase and ion-exchange characteristics were used in this analysis.

FlipLC™ is an alternative method to avoid the interference of most of the contaminants by the use of an isolation column and a high pressure switching valve before the separation column. This method allows sample cleaning and analyte separation in one automated process. The isolation column and the separation column should have orthogonal retention characteristics to operate efficiently in this setup. Mixed-mode columns with reverse phase and ion-exchange characteristics were used in this analysis.

Application Notes: Neurotransmitters DOPAC, homovanillic acid and dopamine were separated by mixed-mode chromatography on Primesep 100 and Primesep 200 HPLC columns. The method can be used for quantification of neurotransmitters with LC/MS compatible conditions. The compounds are retained by combination of reversed-phase, ion-exchange or ion-exclusion mechanisms. The retention time and selectivity of separation can be adjusted by variation of amount of acetonitrile, buffer pH and buffer concentration.

 
Application Columns: Primesep 100

Application compounds: Dopac, Homovanillic Acid, Dopamine

Detection technique: UV, LC/MS, ELSD/CAD

Application Notes: Neurotransmitters DOPAC, homovanillic acid and dopamine were separated by mixed-mode chromatography on Primesep 100 and Primesep 200 HPLC columns. The method can be used for quantification of neurotransmitters with LC/MS compatible conditions. The compounds are retained by combination of reversed-phase, ion-exchange or ion-exclusion mechanisms. The retention time and selectivity of separation can be adjusted by variation of amount of acetonitrile, buffer pH and buffer concentration.
Application Columns: Primesep 100, Primesep 200
Application compounds: Dopac, Homovanillic Acid, Dopamine
Detection technique: UV, LC/MS, ELSD/CAD

Application Notes: Dopamine is a naturally occurring neurotransmitter found in the brain. Dopamine is a well studied compound because dopamine is an important neurotransmitter known to regulate many functions from pain response to behavior disorders. According to USP methods, dopamine hydrochloride contains not less than 98% and not more than 102% dopamine hydrochloride calculated on the dried basis. The USP HPLC method for the separation of hydrocortisone was developed on Legacy L1 column according to the US Pharmacopeia methodology. L1 classification is assigned to reversed-phase HPLC column containing C18 ligand. Support for the material is spherical silica gel with particles size 3-10 um and pore size of 100-120A. Resolution between critical pairs corresponds to rules and specifications of USP.

Application Columns: Legacy L1 C18 HPLC column

Application compounds: Dopamine hydrochloride

Mobile phase: Water with 1% acetic and 5mM octanesulfonic acid/MeCN

Detection technique: UV

Reference: USP35: NF30

Dopamine and epinephrine were separated on a Primesep S2 HILIC mixed-mode column. Dopamine and epinephrine are retained by HILIC and cation-exchange mechanisms. Retention time is controlled by the amount of ACN, buffer and buffer pH. Method is compatible with LC/MS and can be used for analysis of polar metabolites in biofluids.

The catecholamine neurotransmitters are amino-acid derivatives of tyrosine. DOPA, tyrosine, phenylalanine, norepinephrine, epinephrine, and dopamine and baseline are resolved on a Primesep 100 column with UV-transparent phosphate buffer. This method can be used for analysis of catecholamines and related impurities in various matrices. Peak order and retention time can be changed by changing the amount of ACN, buffer concentration and buffer pH. Various buffers can be used to accommodate desired detection technique. Primesep 100 is a reversed-phase cation-exchange mixed-mode column that can be used for analysis of polar neutral, polar ionizable, polar zwitter-ionic, hydrophobic neutral, and hydrophobic ionic compounds in the same run. Column can be operated in reverse-phase, cation-exchange, anion-exclusion, HILIC and mixed-modes depending on the mobile phase selection and nature of analytes. Column is compatible with LC/MS and does not require use of ion-pairing reagents.

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).

The separation of amino acids, the building blocks of proteins, can be challenging to separate on a reverse-phase column due to their high polarity. Using a mixed-mode HPLC column, allows the separation of amino acids by cation-exchange and ion-exclusion mechanisms as well as hydrophobicity. Fine tuning of separation can be achieved with changes in organic concentration of the mobile phase as well as choice of buffer and pH.

Catecholamines are chemical compounds derived from the amino acid tyrosine containing catechol and amine groups. Some of them are biogenic amines. Retention of compounds of the catecholamine pathway is achieved on Obelisc N column. All polar compounds are well retained by combination of HILIC and ion-exchange mechanisms. Obelisc N columns produce very good peak shapes for all analytes. The method is very sensitive to amount of ACN, buffer and buffer pH. The retention time changes with variation of the main parameters. This method can be used for quantitation of biogenic amines and related compounds (homovanillic acid, dihydroxyphenyl acetic acid, serotonin, dopamine, epinephrine, hydroxytryptophan, epinephrine and DOPA) in urine, blood and other biological fluids. Further optimization of this HPLC method can be used during screening and validation. Amines and acids can be analyzed in the same run and retained by a combination of polar organic mode, cation-exchange and anion-exchange modes. Various buffers within specified pH can be employed (ammonium formate, ammonium acetate, sodium phosphate, etc.).

The catecholamine neurotransmitters are amino-acid derivatives of tyrosine. DOPA, tyrosine, phenylalanine, norepinephrine, epinephrine, and dopamine and baseline are resolved on a
Primesep 200 column with UV-transparent phosphate buffer. This method can be used for the analysis of catecholamines and related impurities in various matrices. The peak order and retention time can be changed by changing the amount of ACN, buffer concentration and buffer pH. Various buffers can be used to accommodate the desired detection technique. Primesep 200 is a reversed-phase cation-exchange mixed-mode column that can be used for analysis of polar neutral, polar ionizable, polar zwitterionic, hydrophobic neutral, and hydrophobic ionic compounds in the same run. Column can be operated in reverse-phase, cation-exchange, anion-exclusion, HILIC and mixed-modes depending on the mobile phase selection and the nature of the analytes. The column is compatible with LC/MS and does not require the use of ion-pairing reagents.

Dopamine, norphenylephrine and serotonin are separated on Primesep P HPLC column. Base line separation of these neurotransmitters can be achieved using mix-mode approach. Primesep P provides retention by reverse phase, pi-pi and cation-exchange mechanisms. Method can be used for fast quantitation of these compounds in various sample matrices. Detection technique ranges from UV to ELSD, LC/MS, RI and CAD. Primesep P column can be used with tetrahydrofuran or with 100% aqueous mobile phase to enhance pi-pi interaction.

Primesep 200 separates catecholamines in the catecholamine pathway in 10 minutes. Phenylalanine, tyrosine, DOPA, dopamine, norepinephrine, and epinephrine 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.

Primesep 200 separates catecholamines in less than 8 minutes. Tyrosine, dl-DOPA, dopamine, epinephrine 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.