Absorbance Testing

Absorbance testing is a technique that measures the amount of light absorbed by a sample at a specific wavelength. Dissolved analytes such as molecules or ions have distinct arrangements of atoms connected by chemical bonds. When photons of light interact with these bonds, ground-state electrons can be promoted to an excited state at a higher energy level. The relative amounts of electron excitement vary between different bond types, and therefore the absorbance of light for each molecule of interest is unique based on concentration. Absorbance can be used to quantify dissolved solute types, including nucleic acids, proteins, and others, as well as their concentrations [1]. The calculated absorbance of samples can be used to identify solutions and to determine concentration to provide a base level of reproducibility in future formulations, as each molecule has a unique wavelength of absorbance that is directly correlated with its chemical structure.

History of Absorbance Testing

Absorbance tests have been developed and used over centuries of scientific research. An important figure in its history was Louis Pasteur, a French chemist and microbiologist who made significant contributions to the fields of vaccination, fermentation, and microbiology [2]. He developed several methods for measuring the concentration of substances in solutions using absorbance measurements. One of his most well-known methods was known as "the colorimetric method", which involved adding a known volume of a solution containing an indicator (a substance that changes color depending on the pH) to another solution containing the unknown substance. The absorbance of both solutions was measured at different wavelengths using a spectrophotometer, and then compared to calculate the concentration ratio. This procedure established the groundwork for current absorbance testing and spectrophotometers used within the biotechnology industry today.

Absorption-image-1

Figure 1: An atom of an analyte: Photons of light at variable wavelengths are absorbed by the electrons of analyte atoms, causing them to shift from a grounded energy state to an excited energy state.

Importance of Absorbance Testing for Quality Assurance

Absorbance testing as a Quality Control measurement offers an analysis of a product's composition over selected wavelengths. Each wavelength chosen is specific to the molecule type present in each sample, as the absorption spectrum of each molecule is directly correlated with its structure and stability. Common wavelengths often used as composition indicators include but are not limited to:

    • 260 nm: This wavelength can often be used for UV absorption of nucleic acids, as they contain nucleotides that absorb light at this wavelength. DNA or RNA solutions can often be used as standards for this wavelength.
    • 280 nm: This wavelength can often be used for UV absorption of proteins, as they contain large amounts of tryptophan and tyrosine residues that absorb strongly at this wavelength. Bovine Serum Albumin (BSA), or human/rabbit IgG (immunoglobulin) solutions can be used as standards for this wavelength.
    • 320 nm: This wavelength can often be used for UV absorption of acidic p-nitrophenol. Uncontrolled p-nitrophenol is a toxic organic pollutant that can contaminate chemicals of interest. Not only does it affect product quality, but it can also have adverse effects on human health. P-nitrophenol is also known as a substrate for enzymatic assays, as it reacts with enzymes known as alkaline phosphatases. Detecting p-nitrophenol levels may help to understand the levels of these enzymes in a product of interest [3].
    • 340 nm: This wavelength can be used to measure the availability of Nicotinamide Adenine Dinucleotide (NAD), a coenzyme to many cellular enzymes, at specific time points in a chemical reaction. NAD absorption is highest at this wavelength. This absorption value can then demonstrate the specific activity of the enzyme associated with NAD in a sample.

Absorbance Testing at Boston BioProducts

At Boston BioProducts, we utilize a spectrophotometer that employs a wavelength range from 200-999 nm and measures absorption through absorption spectroscopy. To do this, the device shines a beam of light of a specific wavelength at a product sample, and then at that wavelength records the relative amount of light that passes through the sample, using a detector.

Light-Absorption-1

Figure 2: The mechanism of absorbance technology. To calculate absorbance the spectrophotometer shines a beam of light through a monochromator, which narrows the beam down to a select wavelength. The wavelength of light then passes through the sample, and any remaining light is detected by an internal detector. Using this derived data, absorbance can then be calculated.

To accurately calculate absorbance, the spectrophotometer uses the collected data and inputs it into the Beer-Lambert Law, written as follows:

A = log10I0
I

= ∈cL

 

Where:

A = Absorbance
I0 = Incident Intensity
I = Transmitted Intensity
= Molar Absorptivity
c = Concentration
L = Path length of light

The Beer-Lambert Equation relates the absorption a light by a substance (A) to its molar absorptivity (∈) concentration (c), incident and transmitted light intensities (I0 and I), and path length (L), allowing for the accurate measurement of absorption of light by an analyte. For an accurate measurement, the solvent used to dissolve the analyte should also be used as a testing standard.

Considerations and Limitations of Absorbance Testing

Formulation Accuracy The accuracy and reproducibility of absorbance testing is directly associated with the saturation of formulations. All solutes used must be fully saturated in solution prior to sample use.
Sampling Technique The accuracy of results depends on proper sample collection techniques. Sampling should occur in a clean, well-regulated environment.
Temperature and pH of Samples The temperature and pH of samples can alter the chemical composition of the expected analyte if not carefully monitored, potentially affecting the absorbance spectra of the molecule and absorbance readings.
Absorbance Range Beer's law typically requires an absorbance between 0.05 and 1.0 for accurate measurements. If the solution concentration is too high, the solution may be too concentrated, and light may not pass through the sample. If the solution is too diluted, the sample may be affected by noise or stray light.
Wavelength used The wavelength used for absorbance testing should match the absorbance spectrum of the analyte. Different compounds have different spectra, and the wavelength used should be chosen carefully.

In addition to Absorption Testing, Boston BioProducts provides a comprehensive set of QC tests for custom reagents. Learn more about custom reagent development services.

References: