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Laboratory Operations

Spectrophotometry & Photometry

Let’s pull back the curtain on what is arguably the most important single principle in all of automated chemistry: spectrophotometry. I know it’s a mouthful, but the concept is beautifully simple. Think about putting on a pair of sunglasses. The lenses have a certain color and darkness. When you look at the sun, they block some of the light from reaching your eyes. The darker the lenses, the more light they block. Spectrophotometry is the science of precisely measuring that effect

In the clinical laboratory, we have harnessed this simple idea into a powerful tool. We can’t see a glucose molecule or a cholesterol molecule, but we can run a chemical reaction that produces a colored product where the intensity of the color is directly proportional to the concentration of the analyte we want to measure. Spectrophotometry is the workhorse technology that allows us to turn color into a number, which we then report as a patient result

Beer’s Law: The Golden Rule of Spectrophotometry

This entire field is built on a fundamental rule called Beer’s Law (sometimes Beer-Lambert Law). This law is the mathematical statement that connects what we can see (color) to what we want to know (concentration). In its simplest form, it states that the amount of light absorbed by a solution is directly proportional to the concentration of the light-absorbing substance in that solution

Let’s break that down:

  • Absorbance (A): This is the measure of light that is “stopped” or absorbed by the solution. It is a logarithmic scale, so it doesn’t have units. The higher the absorbance, the darker the solution and the higher the concentration
  • Transmittance (%T): This is the flip side of absorbance. It’s the fraction of light that successfully passes through the solution and reaches the detector. A perfectly clear solution has 100% transmittance (and zero absorbance). An opaque solution has 0% transmittance. They are inversely related

Beer’s Law creates a linear relationship between absorbance and concentration. If you double the concentration of your analyte, you will double the absorbance of your solution. This is the magic that allows us to build the Standard Curves we discussed previously. We plot the known concentrations of our calibrators against their measured absorbance values, create a straight line, and then use that line to determine the concentration of our unknown patient samples

Anatomy of a Spectrophotometer

A spectrophotometer is simply a machine built to precisely measure absorbance. To do this, it needs several key components arranged in a specific order. Think of it as an assembly line for a beam of light

  1. Light Source It all starts with a steady, stable light. This is typically a tungsten lamp for visible light or a deuterium or mercury-arc lamp for ultraviolet (UV) light
  2. Monochromator This is the heart of the machine. “Mono” means one, and “chroma” means color. Its job is to isolate one specific, narrow wavelength (color) of light from the white light produced by the lamp. Why? Because a substance will absorb light most strongly at its peak absorbance wavelength, giving us the best sensitivity. Monochromators are often a prism or, more commonly, a diffraction grating (which acts like a tiny, mirrored rainbow-maker) that can be rotated to select the desired wavelength
  3. Exit Slit After the monochromator, a narrow slit allows only our chosen wavelength to pass through to the next stage
  4. Cuvette (Sample Cell) This is the small, optically clear container that holds our patient sample or QC material. It has a precisely known width, called the pathlength, which is critical because a wider cuvette would absorb more light even at the same concentration. In our instruments, this is a fixed, constant value (usually 1 cm)
  5. Detector After passing through the sample, the remaining light hits a detector. The most common type is a photomultiplier tube (PMT), which is incredibly sensitive. It converts the light energy it receives into a small electrical signal
  6. Readout Device This final component takes the electrical signal from the detector, amplifies it, and performs the logarithmic calculation to convert the signal into an absorbance value that is displayed for us or used to calculate the final concentration

Spectrophotometer vs. Photometer: A Key Distinction

While we often use the terms interchangeably in conversation, there is a technical difference that you should know

  • A Spectrophotometer uses a true monochromator (like a diffraction grating) that allows for the selection of very narrow, specific wavelengths. You could scan a sample across the entire spectrum if you wanted to
  • A Photometer is a simpler and more common instrument in many analyzers. Instead of a complex monochromator, it uses filters to isolate a broader band of wavelengths. A 600 nm filter, for example, might let light from 580-620 nm pass through. It is less precise but robust and perfect for routine clinical assays

Most of our big chemistry analyzers are technically sophisticated multi-wavelength photometers, but they operate on the exact same principles of Beer’s Law

Quality Assurance in Spectrophotometry

We must ensure our “ruler” is working correctly. Key checks for a spectrophotometer include:

  • Wavelength Accuracy: Is the monochromator pointing to the right wavelength? We check this using special materials like a holmium oxide filter, which has known, sharp absorbance peaks at specific wavelengths
  • Photometric Accuracy (Linearity): Does doubling the concentration truly double the absorbance? We check this by reading a set of special linearity filters with known absorbance values
  • Stray Light: Is any unwanted, un-monochromated light leaking through the system and hitting the detector? This can cause a falsely low absorbance reading, especially at high concentrations. We check this with special cutoff filters that should, in theory, block all light

Key Terms

  • Spectrophotometry: The quantitative measurement of the reflection or transmission properties of a material as a function of wavelength; the method of measuring how much a chemical substance absorbs light
  • Beer’s Law: The fundamental principle stating that the absorbance of a solution is directly proportional to the concentration of the analyte and the pathlength of the light through the sample
  • Absorbance: A logarithmic measure of the amount of light that is absorbed by (does not pass through) a solution. It is directly proportional to concentration
  • Transmittance: The fraction or percentage of incident light that passes through a solution. It is inversely and logarithmically related to absorbance
  • Wavelength: The distance between two successive peaks of a wave of light, which determines its color or energy. It is typically measured in nanometers (nm)
  • Monochromator: A device within a spectrophotometer (like a prism or diffraction grating) that isolates a specific, narrow band of wavelengths from a light source
  • Cuvette: The optically clear sample container with a fixed pathlength that holds the liquid being measured in a spectrophotometer