Tryptophan and Tyrosine, which are aromatic molecules, are amino acids that have a major role in detecting proteins in native fluorescence. 5-HIAA and Mandelic Acid (HMMA) are bigger molecules. 5-HIAA is a muscle stimulant and the primary metabolite of serotonin, a hormone derived from the amino acid tryptophan. HMMA is part of the family of fruit acids, also called alpha-hydroxy acid (AHA) which is used in the treatment of infections or in cosmetics for its ability to treat a number of skin imperfections such as wrinkles, pigment spots or acne. This application note shows the separation of these four molecules with a 275 nm LED induced fluorescence detector.
Method of separation
Key features of the method | • Hydrodynamic injection of sample : 50 mBar during 10 s, • Separation: 16 kV during 17 min. • Temperature: 25°C • Buffer: CAPS 10 mM, Sodium |
Instruments | • Capillary Electrophoresis: Agilent Technologies 7100 CE • Detector: ZETALIF LED 275 • Fused Silica Capillary: 75 μm ID • Total Length = 65 cm / Effective Length = 44 cm |
Limit of Detection (LOD)
Molecules | Minimum Concentration Tested (nM) | S/N Ratio | LOD (nM) |
Tryptophan | 50 | 23.6 | 6 |
Tyrosine | 500 | 10.7 | 140 |
HIAA | 50 | 30.7 | 5 |
HMMA | 500 | 28.1 | 50 |
Conclusion:
The 275 nm LED offers a sensitivity comaparable to the 266 nm laser for the detection of molecules in native fluorescence.
Unlike the pulsed laser, it makes it possible to obtain linear calibration curves.