February Spectra of the Month: Amino Acids

February Spectra of the Month: Amino Acids
 
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Avantes US Engineers reproduced a series of experiments conducted with samples of tryptophan, tyrosine, and bovine serum albumin recently to demonstrate our protein detection capabilities. The team used conventional excitation with a continuous wave broadband optical tungsten/halogen light source (AvaLight-DHc). Absorption measurements were recorded correlating changes in concentration to changes in absorption. Fluorescence was not measured during this experiment.

 

While tryptophan and tyrosine can be excited at similar wavelengths, they have different quantum yields produce distinctly different fluorescence signals in their pure form, but tryptophan and tyrosine are both present in Bovine Serum Albumin (BSA) with the effect that the fluorescence signal of tyrosine is quenched by the tryptophan through the process of resonance energy transfer.

The team running this experiment prepared three different concentrations for each of the three sample analytes: 1mg/ml, 0.5mg/ml, and 0.25mg/ml, and took broadband absorption spectra profiles for each.


All Samples with Varying Concentrations

AvaSpec Mini
All Samples
at 0.25 mg/ml
concentration
AvaSpec Mini
All Samples
at 0.50 mg/ml
concentration
AvaSpec-NIR
All Samples
at 1.00 mg/ml
concentration

By Sample at Each Concentrations

AvaSpec Mini
Tryptophan
AvaSpec Mini
Tyrosine
AvaSpec-NIR
BSA Albumin

 

Measurement System

  • AvaSpec-ULS3648-UA-25
  • AvaLight DHc
  • Inline Cuvette holder with hood (CUV-UV/VIS)
  • Fiber Optic Cables
  • Short path-length (1mm) cuvette

 

Absorption and Fluorescence Wavelengths for Common Amino Acids

(reference data from original experiment)

 

Absorption

Fluorescence

Amino Acids

Concentrations

Wavelength

Absorptivity

Wavelength

Quantum Yield

Tryptophan

1.0 mg/ml

0.5 mg/ml

0.25mg/ml

280 nm

5,600

348 nm

0.20

Tyrosine

1.0 mg/ml

0.5 mg/ml

0.25mg/ml

274 nm

1,400

303 nm

0.14

Phenylalanine

(BSA)

1.0 mg/ml

0.5 mg/ml

0.25mg/ml

257 nm

200

282 nm

0.04

 


 

References

Held, P. "Quantitation of peptides and amino acids with a Synergy™ HT using UV fluorescence." B.-T. Instruments, Winooski, VT (2003).

 

 Download the Complete Dataset

The data set includes absorbance and reference minus dark measurements collected with the AvaSpec-ULS3648.