One of the quickest ways of finding out what is in a solution is to send light through it and see how much comes back. UV-Vis spectrophotometry puts this simple idea into numbers: light in the ultraviolet and visible region is passed through the sample, and the intensity going in is compared with the intensity coming out.
The basic parts of the instrument are a light source, a device that selects the wavelength, a cuvette holding the sample and a detector that measures the emerging light. Before the measurement, the solvent has to be read on its own, that is, a blank reading has to be taken; the value obtained then reflects only the contribution of the dissolved substance.
The choice of cuvette also affects the result directly. Ordinary plastic and glass cuvettes absorb in the ultraviolet region themselves and so distort the reading at 280 nanometres; quartz cuvettes are used in this region. Clean cuvette windows, a solution free of air bubbles and an unobstructed light path are the basic conditions of the measurement.
What does absorbance depend on?
Absorbance varies with three things together: how readily the substance absorbs at that wavelength, the concentration in the solution and the length of the path the light travels through the sample. The linear relationship between these three is the basis of spectrophotometric measurement, and the path length is kept constant in standard cuvettes.
Linearity has a working range. In very concentrated solutions the light reaching the detector is reduced too far and the measurement loses its reliability; in very dilute solutions the signal is lost in the noise. The sample is therefore diluted so as to fall within the instrument's safe range, and the dilution factor is taken into account.
The instrument itself is also prepared before measurement. The lamp is expected to warm up, the baseline to be flat and the wavelength accuracy to be checked at regular intervals. If the baseline has drifted, the value read carries a contribution from the instrument rather than the sample.
Why 280 nanometres?
Three amino acids found in protein and peptide chains absorb markedly in the ultraviolet region: tryptophan, tyrosine and, more weakly, phenylalanine. The ring structure of these residues produces an absorbance peak at around 280 nanometres. The value read at this wavelength in a solution is therefore a practical indicator of the presence of protein.
The second reason for the choice is selectivity. The peptide bond itself absorbs very strongly at around 200 nanometres, but buffers and solvents absorb in this region too, so the measurement is noisy. 280 nanometres is a compromise point at which the protein signal can be read while the contribution from the medium remains relatively low.
| Wavelength | Absorbing structure | Practical note |
|---|---|---|
| ~200 nm | The peptide bond | Very sensitive, but the contribution from solvent and buffer is high |
| ~260 nm | Nucleic acids | Monitored as an indicator of nucleic acid contamination in the sample |
| ~280 nm | Tryptophan and tyrosine | The common choice in protein measurement |
| 320 nm and above | No marked absorbance is expected | A rise is taken as a sign of turbidity or aggregation |
Reading a second wavelength alongside strengthens the interpretation. Nucleic acids absorb strongly at around 260 nanometres; the ratio of the two values gives an idea of whether there is a nucleic acid contribution in the sample. In a peptide-dominated material such as venom, this ratio can be monitored as an auxiliary criterion showing how clean the sample preparation was.
What does the scan curve show?
If a broad range is scanned instead of reading at a single wavelength, the result comes out as a curve. The shape of this curve says more than a single figure: where exactly the peak sits, how smooth the region outside the peak is and whether there is a rise towards longer wavelengths all become visible. An unexpected rise at long wavelengths usually points to undissolved particles or aggregation.
In the scan carried out for our facility's reference batch, the range from 190 to 400 nanometres was read and the maximum peak was recorded at 280 nanometres. The measurement was made with a Shimadzu UV-1900i spectrophotometer at an independent laboratory outside the facility. The result is consistent with the sample consisting of components containing aromatic amino acids.
For the measurement to be reproducible, how the sample was prepared is recorded as well: the solvent, the dilution factor, the dissolution time and the measurement temperature. When the same sample is read at different dilutions, the values are expected to agree with the dilution factor; disagreement usually points to incomplete dissolution or aggregation, and the measurement is repeated.
Unlike most methods, a scan does not consume the sample. The solution in the cuvette can be recovered after the measurement and other analyses can continue on the same sample. With a material available in limited quantity, this is a practical advantage that places the method at the start of the analytical sequence.
For the same reason, a spectrophotometric reading is also used as the first check when a batch is received. If the expected peak is not in place, or the shape of the curve is out of the ordinary, sample preparation is reviewed before moving on to the later analyses.
What the method does not tell you
The value at 280 nanometres gives quick information about the presence of protein but no information about identity. Which proteins are present, how many components they fall into or what their molecular weights are cannot be deduced from this measurement. In addition, a peptide low in aromatic residues may give a weaker signal than its actual amount would suggest.
Spectrophotometry is therefore used not on its own but as the first link in a chain of analyses. The number of components is determined by separation with chromatography; the detail is described in the article on HPLC. Electrophoresis is used for the weight distribution. A numerical concentration requires calibration; building the standard curve is covered in the article on protein assay.
The greatest strength of the method is its speed: it provides a first assessment in a few minutes without consuming the sample. Our batch-level measurement results are published on the analysis page; values are measured afresh for every batch.
