Proteins are large biological molecules comprised of one or multiple amino acids. Proteins in solution absorb ultraviolet light with an absorbance maximum at 280nm. The primary reason for this is due to the aromatic amino acids, e.g. phenylalanine, tryptophan, tyrosine and histidine.
For post chromatography column detection, UV absorption is used to detect the onset and end of proteins as they elute the column, in addition to the total volume eluted from the column. Liquid buffer and solvent gradients in the mobile phase are utilized in order to elute the sample components from the chromatography column. UV absorption is the preferred technique as the mobile phase often has no absorption at 280nm allowing a precise control of the chromatographic separation irrespective of the continuous phase composition.
It is also possible to measure the purity of biomolecules by ratio of absorbance at 280nm and 260nm. The A260/A280 ratio provides an indication of protein contamination in nucleic acid isolates and less commonly, nucleic acid contamination in protein isolates.
Benefits of in-line UV measurement:
- Strong UV absorbance
- Highly concentrated to trace detection
- Chromatography control
- Concentration monitoring
- Real time in-line measurement
A Kemtrak DCP007-UV LED photomter is the recommended instrument to accurately measure protein concentration. Traditional UV photometers use powerful mercury vapor UV lamps that subject the sample to broad spectrum UV radiation and heat generated by the lamp. The resulting light that has passed through the sample then passes through a UV filter and is detected by a photodiode. The hot broad-spectrum UV radiation used on these traditional photometers destroys the sample being analyzed resulting in unknown and potentially hazardous breakdown products.
The Kemtrak DCP007-UV LED photometer uses a state of the art high performance UV LED light source and robust fiber optics. The sample is subjected to ultra-low power cold light of the exact wavelength required for the analysis, thousands of times less powerful than that used on traditional photometers. The Kemtrak UV LED light source is also modulated and can be configured to measure during very short pulses, further reducing the exposure of the sample to harmful UV radiation.
Measurement cells are available in intrinsically safe hygienic TriClamp flanges to custom manifolds with less than 1 µL internal volume for fast and accurate concentration measurement of proteins.
Contact a Kemtrak sales representative for more information.