What Is Spectrophotometer and How to Use it?

A spectrophotometer is an analytical instrument that measures the intensity of light as a function of its wavelength. A common tool in physics, spectrophotometers are commonly used to measure light absorption. There are two major measurement classes of spectrophotometers–single beam and double beam spectrophotometers.

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There are a few major differences between the single beam and the double beam spectrophotometers. A single beam spectrophotometer measures absolute light intensity, while the double beam spectrophotometer measures the ratio of light intensities on two separate light paths–the reference standard and the sample.

Although double beam spectrophotometers were popular in the early days of spectroscopy, it is now thought that the single beam spectrophotometer is more advantageous. This is because it becomes difficult to recombine the light beam prior to reaching the monochromator with the double beam spectrophotometers.

The monochromator is a device for selecting light from a narrow band of wavelengths. The monochromator splits the light into its component wavelengths, so that only the light from the desired wavelengths reach the sample. One example of a monochromator is a prism.

The single beam spectrophotometers also have a larger dynamic range than the double beam spectrophotometers.The oldest form of spectroscopy is ultraviolet/visible spectroscopy.

An ultraviolet/visible spectrophotometer works by placing a sample in the spectrophotometer and shining ultraviolet and/or visible light through. Measurements are made depending on how much light was absorbed by the sample.


The basic components of a spectrophotometer are a light source, a wavelength monochromatic, focusing devices, a cuvette, a photo detector, and a display device. Light passes through a monochromatic that separates the light into its component wavelengths.

Slits then isolate a band pass (the segment of the spectrum isolated by the monochromatic) of the wavelength needed for measurement, improving its purity.

Next, the light passes through the sample, and a portion of the radiant energy is absorbed; the amount depends on the nature and concentration of the sample.

The portion of the band pass that is not absorbed is transmitted to a photo detector, which converts light energy to electrical energy that can be registered on a meter or digital readout.

All spectrophotometer instruments designed to measure the absorption of radiant energy have the basic components as follows

  1. A stable source of radiant energy (Light).
  2. A wavelength selector to isolate a desired wavelength from the source (filter or monochromator)
  3. Transparent container (cuvette) for the sample and the blank.
  4. A radiation detector (phototube) to convert the radiant energy received to a measurable signal.
  5. A readout device that displays the signal from the detector.

Components of a spectrophotometer

The energy source is to provide a stable source of light radiation, whereas the wavelength selector permits separation of radiation of the desired wavelength from other radiation. Light radiation passes through a glass container with sample. The detector measures the energy after it has passed through the sample.

The readout device calculates the amount of light absorbed by the sample displays the signal from the detector as absorbance or transmission.

The spectrophotometers which are used for such measurements may vary from simple and relatively inexpensive colorimeters to highly sophisticated and expensiveinstruments that automatically scan the ability of a solution to absorb radiation over a wide range of wavelengths and record the results of these measurements.

One instrument cannot be used to measure absorbance at all wavelengths because a given energy source and energy detector is suitable for use over only a limited range of wavelengths.

Spectrophotometers are instruments equipped with monochromators that permit the continuous variation and selection of waveiength.The sample containers, cells or cuvettes, must be fabricated from material that is transparent to radiation in the spectral region of interest.

Types Of Spectrophotometer

There are two major classes of devices: single beam and double beam. A double beam spectrophotometer compares the light intensity between two light paths, one path containing a reference sample and the other the test sample.

A single beam spectrophotometer measures the relative light intensity of the beam before and after a test sample is inserted. Although comparison measurements from double beam instruments are easier and more stable, single beam instruments can have a larger dynamic range and are optically simpler and more compact.

Additionally, some specialized instruments, such as spectrophotometer built onto microscopes or telescopes, are single beam instruments due to practicality.

Single Beam Spectrophotometer

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A single beam spectrophotometer has one light path that passes from the light source through the monochromator system and sample cuvette and then to the detector.A blank is used to set the instrument to 100%T(0 A),then the samples are read.

Double Beam Spectrophotometer

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A Double beam spectrophotometer has two light paths,both originating from the same light source.One path is for the sample and other for the blank or reference.The beam from the source strikes a vibrating or rotating mirror that alternate directs light through the reference cell and the sample cell. Light passing through each cell is sent to the detector.

How to Use a Spectrophotometer

Flinn Spectrophotometer

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  1. Plug in and power on the spectrophotometer. Run the machine for five to 10 minutes to allow it to warm up.
  2. Find the wavelength knob beside the sample compartment and rotate it to set the wavelength.
  3. Turn the filter wheel to select the corresponding filter. Use violet for wavelengths between 300 and 375 nm, blue for wavelengths between 375 and 520 nm, yellow for wavelengths between 520 and 740 nm, and red for wavelengths of 740 to more than 900 nm.
  4. Press the mode button located at the front of the spectrophotometer to select the mode that displays Percent Transmittance and Absorbance simultaneously.
  5. Open the sample chamber to ensure it is empty, then close it. Turn the left front dial to set Percent Transmittance to 0 percent.
  6. Don gloves and clean a cuvette with a lab wipe to ensure cleanliness and reduce the risk of erroneous results. Insert the cuvette filled three-quarters of the way with solvent into the sample chamber and close the door.
  7. Rotate the right front dial until it reads 100 percent T.
  8. Remove the solvent cuvette and replace it with a sample cuvette. Close the sample chamber.
  9. View the meter to determine the reading and record it in your records.

Spectrophotometer 20/20D

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  1. Plug in and turn on the spectrophotometer. Allow it to warm up for 15 minutes. This is necessary for the machine to perform properly.
  2. Adjust the wavelength knob located beside the sample compartment to the desired wavelength.
  3. Check the sample compartment to ensure it is empty and closed. Adjust the zero control knob located at the left front of the spectrophotometer by rotating it until it reads 0.
  4. Don gloves and wipe a blank cuvette with a lab wipe. Insert the cuvette into the sample compartment and close the door.
  5. Adjust the light control knob located at the right front of the spectrophotometer by rotating it until it reads 100. Remove the blank cuvette and insert a sample cuvette. Record the value shown on the meter.

Spectrophotometer Educator

  1. Plug in and turn on the spectrophotometer. Allow it to warm up for 15 minutes.
  2. Press the Percent T/A selector to select Percent Transmittance or Percent Absorbance mode. Locate the wavelength dial beside the sample chamber and set it to the desired wavelength.
  3. Don gloves and wipe a cuvette with a lab wipe to clean it and remove any fingerprints. Insert a clean, solvent-filled cuvette into the sample chamber and close the door.
  4. Adjust the right front dial to read 100 percent if in Percent Transmittance mode or 0 if in Absorbance mode.
  5. Replace the solvent-filled cuvette with a sample cuvette. Record the value shown by the spectrophotometer.

Spectronic 401

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  1. Check the sample chamber to ensure it is empty and that both the chamber and test tube access doors are not open. Press the power button located on the back of the spectrophotometer to turn on the machine. Wait 10 minutes to allow the machine to warm up.
  2. Type the desired wavelength on the keypad and press the “go to” key.
  3. Don gloves and wipe the blank solution cuvette carefully with a lab wipe to remove any residue or fingerprints. Insert the blank solution cuvette into the sample chamber, aligning the clear face of the cuvette to face the front of the machine.
  4. Locate the “auto zero” button on the spectrophotometer keypad and press it.
  5. Replace the blank solution cuvette with a sample cuvette that has been cleaned with a lab wipe. Record the absorbance displayed on the screen.

Uses of a Spectrophotometer

Spectrophotometers are directly used to measure light intensity at different wavelengths, and this can be represented as percent of incident light transmitted or absorbed. Using this information and comparing it to other data obtained or known, spectroscopy can be used as a tool.

One example is comparing spectra to determine concentrations of a solute in solution. This can be done by recording transmittance/absorbance at a specific wavelength (a wavelength that the solute absorbs) and known concentration.

Then analysis of a solution of unknown concentration can be compared to the known data, and be interpolation the concentration can be calculated. This can even be done with solutions containing multiple solutes, however is it most accurate when the different solutes absorb different wavelengths.

Spectrometers that do not have a light source, but generate spectra based on the incoming light can be used in a similar way to identify light sources. The spectra graph obtained from an unknown light source (or mixture of sources) can be compared to a database of graphs for different known light sources to identify the unknown light source.

Another application of the spectrophotometer is to determine the equilibrium constant of a reaction involving ions, which takes place in aqueous solution. Starting a solution containing only one reactant, the spectrum is measured. Then small, measured amount of the other reactant is added and after each addition, the spectrum is measured again.

This method works best if there is a known wavelength that the product absorbs. Then, as more products are formed from adding more reactant, more light will be absorbed. When the solution becomes saturated and the reaction reaches net equilibrium, the increase in light absorption will level out, indication equilibrium.

Application case: Pharmaceutical uses of bench top spectrophotometers for R&D and batch control.

Pharmaceutical companies must monitor and control production of the small molecules they manufacture and they often rely on spectrophotometers for many of these procedures.

It’s no surprise that the ingredients used in making pharmaceuticals are highly regulated, requiring a series of tests and quality control steps to ensure that patients are receiving safe and correct dosages of sometimes dangerous compounds. Manufacturers take these controls seriously, but the toll they can take on a pharmaceutical plant’s output can be high.

Thankfully, technology in particular, bench top spectrophotometers are helping pharmaceutical makers find a way to improve testing and save valuable time and money.

Pharmaceutical companies use bench top spectrophotometers for color measurement in a variety of applications. One use is to ensure that the color is consistent in the dosage. Pills, for example, need to have unique colors and shapes so they are not mistaken for other medicines.

Bench top spectrophotometers are used in the measurement of solids, liquids, powders, pastes and creams. The CM-5 spectrophotometer is in widespread use in pharmaceutical companies due to its versatility in sample measuring.

Many pharmaceutical labs also use bench top spectrophotometers to evaluate the effect of different dosages of ingredients in a medication. Assays are created on a sectioned plate, and each section is administered with a different amount of the active product ingredient (API). A dye that activates when cells in the assay incorporate the ingredient is also administered to each section of the plate.

Using the bench top spectrophotometer, scientists can easily measure numerically the effect the ingredient has on a cell by measuring the color of the dye in each section.

Since pharmaceutical labs have so many uses for bench top spectrophotometers, these instruments must be versatile, accurate and easy to use in a variety of ways. The Konica Minolta CM-5 spectrophotometer uses 3 easy steps to measure color in laboratory samples. Basically the user turns it on, employs a wizard to adjust the settings, positions the sample to be measured and presses a button to start.

All the color data, spectral graphs and colorimetric plots, are displayed on its LCD screen, so that the user does not have to use a separate computer to see the data. It has two ways of positioning the object to be measured. There is a top port for measuring pills, granules, and pastes, and a large transmittance chamber with no sides, to measure liquids, films or plates up to 60mm thick.

Results can be evaluated in terms of industry specific color scales such as Gardner, Iodine, Hazen (APHA), European Pharmacopoeia, and US Pharmacopeia. The instrument also has the ability to interface with PC software which can be FDA 21 CFR Part 11 compliant.

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