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Spotlight on Absorbance: a closer look at the conventional detection method for BET

Caitlin East

Key Account Manager @ Lonza | Biomedical Engineering, Process Improvement

发布日期: 2021年5月26日

Our visible existence is defined by the way light interacts with matter. Visible light is the part of the electromagnetic spectrum that we can see with the human eye. Objects don't inherently possess "color" on an atomic level, they interact with light, whether it be from an artificial source or the sun, and portions of the spectrum are either absorbed or reflected; our eyes are able to see the reflected light and perceive it as color. The clover appears green because it contains chlorophyll, which absorbs the red portion of the electromagnetic spectrum and reflects back the remaining green wavelengths. Likewise, the straps of my sandals are absorbing the red portion of the spectrum, reflecting the green, but my nail polish is absorbing the green and reflecting the red back into our eyes.

The implications of this phenomena are significant. Color allows us to distinguish visually a difference in materials. We can also see relative concentration of something based on color intensity. As the concentration of a material increases, the total amount of energy absorbed, and resulting intensity of the complementary wavelength reflected back, increases with intensity.

Qualitatively, we process color intensity and relate it to concentration. I could hand you a glass of lemonade, and if it is too sweet, you would intuitively grab the less intensely colored glass next to it and find it more palatable.

Pierre Bouguer, first documented the beverage-potency-concentration relationship while looking deeply into his glass of wine while on vacation. Johann Heinrich Lambert referenced Bouguer's observations, noting that the loss of intensity, the attenuation, is related to path length and initial light intensity . Combine this with August Beer's observation, that the light transmitted is constant when the product of concentration and path length is constant , and we have the three observations central to our quantitative measurements of absorption to come.

The Beer-Lambert law mathematically describes the relationship between attenuation of a signal (light) to the measurable properties of the medium the signal is passing through.

Absorbance (A) is defined as the log of the ratio of incident intensity to transmitted intensity.

For instances where the incident and transmitted intensity are equal, the ratio is 1, and absorbance is 0.

Lambert's observation, that absorbance is related to path length A ∝ l, combined with Beer's observation, A ∝ c (concentration), yields A ∝ cl. This becomes a formula when we insert the constant ε, the molar extinction coefficient, relating the concentration, c, and path length, l. In practice, the path length is fixed - typically a cuvette or microwell - so the remaining variables are only A and c.

The modern microbiology lab has a device to quantify absorbance - the spectrophotometer. A theoretical spectrophotometer contains these basic components:

a light source (typically a halogen bulb, more on this later)
wavelength selector - a monochromator or filter to take the broadband source light and isolate the wavelength of interest
a cuvette or similar container for the sample of interest
a detector (PMT or CCD)

Modern spectrophotometers have additional components, but they operate in essentially the same way.

The light source is energized - the type of light source varies on application. Halogen lamps are sufficient when the wavelength of interest is between 300 nm to 1000nm, however they have a limited lifespan and frequently need to be replaced.
The energized filament generates light which radiates into a filter, the light from the source passes through the filter, which allows only the wavelength of that filter. For more advanced systems using a monochromator, the source light it is diffracted into the corresponding wavelengths. Once diffracted, the light passes through an adjustable monochromator, which allows the end-user to select the wavelength desired for the measurement.
This incident light (incident intensity from our formula for absorbance) strikes the sample in the container - cuvette or microwell - and passes through.
The outgoing light, with reduced intensity if we've chosen an appropriate wavelength, strikes our detector. The detector converts the incoming light into an electrical signal that can be measured as current. The electrical signal is used to generate an output from the instrument, giving us an absorbance measurement to work with.

For microbiologists, absorbance has been the gold standard for kinetic endotoxin detection assays. Both the turbidimetric and chromogenic assays rely on this technology.

Kinetic chromogenic assays monitor for an increase in absorbance units over a period of time. Once the reaction has proceeded to completion, or yields an increase in 0.200 absorbance units, the reaction time is recorded. A log-log linear correlation of the reaction time of each standard with its corresponding known endotoxin concentration needs to be performed manually or with software, and if the correlation coefficient meets the requirements, the resulting standard curve can be used to determine the endotoxin concentration of samples.

Instrumentation for this method is cost effective with fairly low maintenance. This technology has been widely available for decades, making it an obvious choice for rolling out a methodology that would be affordable and easy to implement in its infancy. However, as with all good things, there are disadvantages that need to be addressed.

Limitations due to the sample arise out of the assumption that the sample is pure and the wavelength being measured is solely absorbed by the component of interest. If a sample contains multiple absorbing components, the total absorbance is the sum of the individual components. As illustrated in the clover picture, multiple materials can absorb at the same wavelength (clover and sandal straps). When the sample itself absorbs at a similar wavelength, it is impossible to discriminate what proportion of the absorbance is due to the sample and what is due to the assay itself. If the sample being tested already absorbs at time zero, the detector will likely be unable to detect a significant enough change in absorbance over the time period to determine the endotoxin concentration.

Along the same lines, the sample is assumed to have a consistent path length with no obstructions. One of the most common issues with the assay is path length disruptions due to bubbles. Bubbles increase the effective pathlength and refract the light, altering the measured transmitted light by the detector.

There are limitations due to instrumentation itself, as well. Beer's law makes the assumption that a single wavelength is reaching the sample. Stray light that reaches the detector is a small value, but it becomes more significant at higher concentrations, as the light reaching the detector is smaller. The detectors in absorbance spectrophotometers are not very sensitive at low concentrations - incident light is approximately the same as transmitted light - and the BET method demonstrates that by extending the read time to permit a detectable change in absorbance units to occur.

Limitations aside, this method is sufficient and acceptable for use in detecting endotoxin. Absorbance spectrometry has afforded the industry an economical option, at the time of its introduction, that allowed for a more precise method of quantitatively detecting levels of endotoxin in samples. Conventional methodology is adequate and fulfills the requirements of the assay, but newer detection methods provide work arounds to the limitations discussed.

References and further reading