Uses of Spectrometers - Colorimetric Methods

Colorimetric method

Generally, there are several methods such as BCA, Bradford, and Lowry.

Lowry method: Based on the earliest Biuret reaction, and improved. Protein reacts with Cu2 to produce a blue reactant. However, compared with Biuret, Lowry method is more sensitive. The disadvantages are that several different reaction reagents need to be added in sequence; the reaction takes a long time; it is easily affected by non-protein substances; proteins containing EDTA, Triton x-100, ammonia sulfate and other substances are not suitable for this method.

BCA (Bicinchoninine acid assay) method: This is a newer and more sensitive protein test method. The protein to be analyzed reacts with Cu2 in an alkaline solution to produce Cu, which forms a chelate with BCA to form a purple compound with an absorption peak at 562nm. This compound has a strong linear relationship with protein concentration, and the compound formed after the reaction is very stable. Compared with Lowry method, it is simple to operate and has high sensitivity. However, similar to Lowry method, it is easily interfered by proteins and detergents.

Bradford method: The principle of this method is that the protein reacts with Coomassie Brilliant Blue to produce a colored compound with an absorption peak of 595nm. Its most significant features are good sensitivity, which is twice that of Lowry and BCA test methods; simpler operation and faster speed; only one reaction reagent is needed; the compound can be stable for 1 hour, which is convenient for results; and it is compatible with a series of reducing agents that interfere with Lowry and BCA reactions (such as DTT, mercaptoethanol). However, it is still sensitive to detergents. The main disadvantage is that different standards will lead to large differences in the results of the same sample and no comparability.

Some researchers who are new to colorimetric determination may be confused by the inconsistent results of various colorimetric methods. Which method should they believe? Because the reaction groups and color-developing groups of various methods are different, the sample concentrations obtained by using several methods at the same time for the same sample are incomparable. For example: Keller et al. tested the protein in human milk, and the concentrations measured by Lowry and BCA were significantly higher than Bradford, with significant differences. Even if the same sample is measured, the standard samples selected by the same colorimetric method are inconsistent, and the concentration after the test is inconsistent. For example, when Lowry is used to test the protein in the cell homogenate, BSA is used as the standard, with a concentration of 1.34mg/ml, and a globulin is used as the standard, with a concentration of 2.64mg/ml. Therefore, before choosing the colorimetric method, it is necessary to refer to the chemical composition of the sample to be tested and find a standard protein with a similar chemical composition as a standard. In addition, when the colorimetric method is used to quantify protein, a common problem is that the absorbance value of the sample is too low, resulting in a large gap between the measured sample concentration and the actual concentration. The key issue is that the color of the colorimetric cup, an important accessory of the 1011 spectrophotometer, has a certain half-life after the reaction, so each colorimetric method lists the reaction test time, and all samples (including standard samples) must be tested within this time. If the time is too long, the absorbance value obtained becomes smaller, and the converted concentration value decreases. In addition, the reaction temperature, solution pH value, etc. are important factors affecting the experiment. In addition, it is very important to use a plastic colorimetric method. Avoid using quartz or glass cuvettes, because the color after the reaction will stain the quartz or glass, resulting in inaccurate sample absorbance values.