**Determination of DNA and Protein Content Using Ultraviolet Spectrophotometry**
This experiment focuses on determining the protein content in a solution using ultraviolet (UV) spectrophotometry. The method relies on the absorption properties of aromatic amino acids such as tyrosine and tryptophan, which are commonly found in proteins. These residues have conjugated double bonds that absorb UV light at specific wavelengths, with the maximum absorption typically occurring around 280 nm. This makes UV spectrophotometry an efficient and widely used technique for protein quantification.
The principle behind this method is based on the Lambert-Beer Law, which states that the absorbance (A) of a solution is directly proportional to its concentration (c), given a constant path length (b) and molar absorptivity (ε). Mathematically, A = εbc. By measuring the absorbance at the wavelength of maximum absorption (λmax), we can determine the concentration of the protein in the sample.
In this experiment, we use a UV-1700 spectrophotometer, which consists of several key components: a light source, a monochromator, an absorption cell, a detector, and a signal display. For UV measurements, a xenon lamp is used along with quartz cuvettes, while visible light measurements typically use a tungsten lamp and glass cuvettes.
The procedure involves preparing a standard curve by measuring the absorbance of various concentrations of a known protein solution at 280 nm. Once the standard curve is established, the absorbance of the unknown protein sample is measured under the same conditions. The concentration of the unknown sample is then determined by comparing its absorbance to the standard curve.
The experimental steps include:
1. **Instrument Setup**: Turn on the spectrophotometer, start the software, and calibrate the instrument.
2. **Blank Measurement**: Measure the absorbance of a blank solution (0.9% NaCl) to zero the baseline.
3. **Absorption Curve**: Prepare a diluted protein solution and measure its absorbance across a range of wavelengths (190–400 nm).
4. **Standard Curve Preparation**: Prepare a series of standard protein solutions with known concentrations and measure their absorbance at 280 nm.
5. **Sample Measurement**: Measure the absorbance of the unknown protein solution at 280 nm and repeat the measurement in triplicate.
6. **Data Analysis**: Plot the standard curve and calculate the concentration of the unknown sample using linear regression.
From the data obtained, the standard curve equation was found to be A = 0.6208C + 0.0186 with a high correlation coefficient (R² = 0.9998). The average absorbance of the sample was 0.674, leading to a calculated concentration of approximately 1.056 mg/mL. After adjusting for dilution, the final concentration of the protein solution was determined to be 3.520 mg/mL.
This method is simple, fast, and highly sensitive, making it ideal for routine protein analysis in biochemical and molecular biology laboratories. It also allows for non-destructive measurements and minimizes interference from other substances, provided the solution is properly prepared.
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