Summary
Amino acid is a general name for those organic compounds with amino and carboxy. According to the different positions of amino and carbocy, they can be classified into alpha-, beta-, gamma-, S-, omega- amino acid. Among these, alpha- amino acid is the basic unit of proteins. So to analyze amino acid is getting more and more important for researchers and scientists.
This article is going to introduce some analyzing methods of amino acid in Creative Proteomics. And then you can get clear what you can get from it.
Before this introduction, we have to explain some common definitions of all related substances.
1.Pre-column derivatization
In order to increase the detection sensitivity and separation selectivity of amino acid analysis, the method of derivatized amino acids is always utilized. The pre-column derivatization is to make sample derivatized before separated by the analytical column. And then separating derivative through column.
2.Gradient elution
3.Baseline
4.Chromatographic peak
5.Peak area
6.Retention time
7.Standard sample
8.Standard sample calibration curve
Methods and theory
Using phenyl isothiocyanate (PITC) as a pre-column derivatization agent. PITC occurs quantitative derivatization with amino of amino acid molecule to generate PTC-AA, and then the excessive PITC can be removed through a vacuum drying.
While the compounds generated by PTC-AA are moving relatively with the mobile phase in the column, the mixed amino acid derivatives are allocated in two phases repeatedly. Those components can be separated according to the different partition coefficient.
Reagents and materials
1. HAA detection reagent
Mobile phase
Solution A: 19.0g NaAc.3H2O, 0.5ml of triethylamine and 200μl 0.0% EDTA dissolved in 1000ml ultrapure water. Adjusting PH to 6.40 with glacial acetic acid aqueous solution and then filtering it with o.45u membrane filter. Degassing 940ml this liquid and 60ml of acetonitrile under ultrasonic for 20s and then filling it with nitrogen.
Solution B: mixing 600ml acetonitrile, 400ml ultrapure water and 200μl 0.0% EDTA, and then degassing with ultrasonic under vacuum for 20s.
2.Dissolving 1g phenol in 6 mol/L HCl 100 ml or preparing 200μl 6mol / L HCl and 0.5mg phenol (crystals) in empty sample tube.
3.Dry reagent: ethanol: water: triethylamine = 2:2:1
4.Derived reagents: phenyl isothiocyanate: ethanol: triethylamine: water = 1:7:1:1
5.Sample dilution: dissolving 710mg Na2HPO4 with 1000ml clean aqueous (10% phosphoric acid: acetonitrile (95:5) phosphoric acid in acetonitrile solution adjusted pH = 7.40] backup.)
FAA detecting reagent
1.Mobile phase
Solution A: dissolving 9.54g NaAc·3H2O in 1000ml ultra-pure water and filtering it with 0.45μm membrane. And then mixing this solution 975ml with 25 ml of acetonitrile, 250μl 10mmol/L EDTA and then degassing with ultrasonic under vacuum for 20s.
Solution B: mixing 450ml acetonitrile, 150ml methanol and 400ml ultra pure water and degassing with ultrasonic under vacuum for 30s.
2.Dry reagent: methanol :1.0mol / L sodium acetate: triethylamine = 2:2:1 4.2.3
3.Derivatization reagent: phenyl isothiocyanate: methanol: triethylamine: water = 1:7:1:1
These are the main substances that used by Creative Proteomics to meet your amino acid analysis standards. You can find more information on its website.
Amino acid is a general name for those organic compounds with amino and carboxy. According to the different positions of amino and carbocy, they can be classified into alpha-, beta-, gamma-, S-, omega- amino acid. Among these, alpha- amino acid is the basic unit of proteins. So to analyze amino acid is getting more and more important for researchers and scientists.
This article is going to introduce some analyzing methods of amino acid in Creative Proteomics. And then you can get clear what you can get from it.
Before this introduction, we have to explain some common definitions of all related substances.
1.Pre-column derivatization
In order to increase the detection sensitivity and separation selectivity of amino acid analysis, the method of derivatized amino acids is always utilized. The pre-column derivatization is to make sample derivatized before separated by the analytical column. And then separating derivative through column.
2.Gradient elution
3.Baseline
4.Chromatographic peak
5.Peak area
6.Retention time
7.Standard sample
8.Standard sample calibration curve
Methods and theory
Using phenyl isothiocyanate (PITC) as a pre-column derivatization agent. PITC occurs quantitative derivatization with amino of amino acid molecule to generate PTC-AA, and then the excessive PITC can be removed through a vacuum drying.
While the compounds generated by PTC-AA are moving relatively with the mobile phase in the column, the mixed amino acid derivatives are allocated in two phases repeatedly. Those components can be separated according to the different partition coefficient.
Reagents and materials
1. HAA detection reagent
Mobile phase
Solution A: 19.0g NaAc.3H2O, 0.5ml of triethylamine and 200μl 0.0% EDTA dissolved in 1000ml ultrapure water. Adjusting PH to 6.40 with glacial acetic acid aqueous solution and then filtering it with o.45u membrane filter. Degassing 940ml this liquid and 60ml of acetonitrile under ultrasonic for 20s and then filling it with nitrogen.
Solution B: mixing 600ml acetonitrile, 400ml ultrapure water and 200μl 0.0% EDTA, and then degassing with ultrasonic under vacuum for 20s.
2.Dissolving 1g phenol in 6 mol/L HCl 100 ml or preparing 200μl 6mol / L HCl and 0.5mg phenol (crystals) in empty sample tube.
3.Dry reagent: ethanol: water: triethylamine = 2:2:1
4.Derived reagents: phenyl isothiocyanate: ethanol: triethylamine: water = 1:7:1:1
5.Sample dilution: dissolving 710mg Na2HPO4 with 1000ml clean aqueous (10% phosphoric acid: acetonitrile (95:5) phosphoric acid in acetonitrile solution adjusted pH = 7.40] backup.)
FAA detecting reagent
1.Mobile phase
Solution A: dissolving 9.54g NaAc·3H2O in 1000ml ultra-pure water and filtering it with 0.45μm membrane. And then mixing this solution 975ml with 25 ml of acetonitrile, 250μl 10mmol/L EDTA and then degassing with ultrasonic under vacuum for 20s.
Solution B: mixing 450ml acetonitrile, 150ml methanol and 400ml ultra pure water and degassing with ultrasonic under vacuum for 30s.
2.Dry reagent: methanol :1.0mol / L sodium acetate: triethylamine = 2:2:1 4.2.3
3.Derivatization reagent: phenyl isothiocyanate: methanol: triethylamine: water = 1:7:1:1
These are the main substances that used by Creative Proteomics to meet your amino acid analysis standards. You can find more information on its website.