Spectrophotometry: Videos & Practice Problems

Spectrophotometry is a technique used to measure the absorbance of light by a molecule, which can help determine the concentration of that molecule in a solution. This method is particularly useful in biochemistry for quantifying proteins. The key instrument used in this process is the spectrophotometer, which measures light absorbance values to ascertain the concentration of the absorbing solute.

The relationship between light absorbance and solute concentration is described by Beer's Law, also known as the Lambert-Beer Law. This law can be expressed mathematically as:

\[A = \epsilon \cdot c \cdot l\]

In this equation, A represents the absorbance of the solute, c is the concentration of the solute, l is the path length of the light through the sample (typically measured in centimeters), and \(\epsilon\) is the molar extinction coefficient, which is a specific property of the solute that indicates how much light is absorbed.

The process begins with a light source emitting white light, which is then directed through a diffractor that separates the light into its various wavelengths. The selected wavelength of light is referred to as the incident light, denoted as \(I_0\). When this light passes through the protein sample, some of it is absorbed, while the remainder is transmitted, referred to as transmitted light and denoted as I. The intensity of the transmitted light is always less than that of the incident light due to the absorption by the protein.

Several factors influence the amount of light absorbed by the sample. The concentration of the protein is crucial; a higher concentration results in a cloudier solution, leading to greater light absorption. Additionally, the length of the container through which the light passes affects absorption; a longer path length allows more light to be absorbed. Lastly, the extinction coefficient is unique to each solute and determines how effectively a solute can absorb light.

Beer's Law can be rearranged to express the relationship between absorbance and the ratio of incident to transmitted light:

\[A = \log\left(\frac{I_0}{I}\right)\]

Understanding these components of Beer's Law is essential for solving problems related to spectrophotometry. Depending on the information provided in a problem, different parts of Beer's Law may be utilized. This foundational knowledge will be built upon in future lessons, particularly regarding the extinction coefficient.

In the study of spectrophotometry, understanding the extinction coefficient, often denoted by the Greek letter epsilon (ε), is crucial. This coefficient quantifies how strongly a chemical absorbs light at a specific wavelength, represented by the Greek letter lambda (λ). The relationship between the extinction coefficient and absorbance is direct: a higher molar extinction coefficient indicates greater absorbance. It's important to note that the molar extinction coefficient varies with different wavelengths, making the choice of wavelength critical when applying Beer's Law.

The units for the molar extinction coefficient are typically expressed as inverse molarity (M^-1) and inverse centimeters (cm^-1). This means that when solving problems using Beer's Law, ensuring that all units are consistent is essential, and unit conversions may be necessary.

Graphical representations often illustrate the extinction coefficient across various wavelengths, with the y-axis showing the extinction coefficient and the x-axis displaying the wavelength in nanometers (nm). Such graphs reveal how the extinction coefficient changes with wavelength, further emphasizing that both the extinction coefficient and absorbance are wavelength-dependent. This highlights the importance of selecting the appropriate wavelength for accurate analysis, particularly in applications like protein analysis, which will be explored in subsequent lessons.

In the study of spectrophotometry, the Lambert-Beer law plays a crucial role in understanding how light interacts with different substances. An absorbance spectrum is a graphical representation that plots absorbance values on the y-axis against the wavelength of light on the x-axis. This spectrum allows for the analysis of how various molecules absorb light at specific wavelengths.

For instance, in an absorbance spectrum, two distinct types of molecules can be represented: proteins and nucleic acids. The protein molecule is typically shown with a blue curve, while the nucleic acid is represented by a red curve. Notably, these two molecules absorb light at different wavelengths; nucleic acids exhibit strong absorbance around 260 nanometers, whereas proteins show significant absorbance at 280 nanometers.

The strong absorbance of proteins at 280 nanometers is primarily attributed to the presence of two specific amino acids: tryptophan and tyrosine. Tryptophan has a strong absorbance peak at this wavelength, while tyrosine exhibits a weaker absorbance. This characteristic property is essential for biochemists, as it allows them to quantify protein concentrations effectively. Since proteins are generally large and composed of numerous amino acids, the likelihood of containing tryptophan or tyrosine is high, making 280 nanometers a reliable wavelength for protein quantification.

However, it is important to note that not all amino acids absorb light at characteristic wavelengths. The remaining 18 amino acids do not have significant absorbance at specific wavelengths, which limits their use in protein quantification. Therefore, if a protein lacks tryptophan or tyrosine, alternative methods must be employed to determine its concentration.

In summary, the absorbance spectrum is a vital tool in biochemistry, particularly for analyzing proteins through their absorbance characteristics at specific wavelengths, especially 280 nanometers due to the unique properties of tryptophan and tyrosine.