The Daigram Of Light Flow In A Spectrophotometer

The scientific and research lab has more instruments for interpreting the exact information from the biological specimens.  One of those instruments is the spectrophotometers which are designed for measuring the light wavelengths depending on the intensity of light.  These instruments characterize the color of samples or analytes used in the research lab. In this manner,  cent percent reflection happens which further transfers into transmittance of the light rays. Here, reflection happens with some of the reference points within the single and double beam spectrophotometers.  In the case of most of the spectrophotometers, they use color measurement techniques with a reference point in the instrument. This makes it a perfect reflecting diffuser to which the white standardization tile may be traced through the specimens.  

The output from a double beam UV spectrophotometer and single-beam spectrophotometer is a spectral curve. The curve obtained is like a fingerprint of the color of the biological sample. From these spectral curves results, tristimulus values and then other color metric values can be calculated in both single and double beam spectrophotometers.  These spectral curve results may also be used in the computer color matching technique which is connected to the spectrophotometers.  When it comes to spectrophotometers, it comes in many sizes, shapes, geometries, and types. But the most common is the two types of spectrophotometers  which are as follows :

  1. Single Beam Spectrophotometers
  2. Double Beam Spectrophotometers

Both of these beam spectrophotometers are used in measuring light from the analytes. The beam spectrophotometer usually uses ultraviolet rays, infrared rays, and visible rays   for the transmission process. However, the modern and recent spectrophotometers use a wide spectrum of electromagnetic wavelength, which includes ultraviolet rays,  x-rays, visible rays, infrared rays, microwave rays for the reflection and transmission process. 

Main Use Of An Single And Double Beam Spectrophotometers

The Use Of Collimator In A Spectrophotometer 

The main work of a single and double beam spectrophotometer is to measure the amount of light for a specific wavelength passing through the sample.  In the case of spectrophotometry,  the beams of light are absorbed by the analyte within a biological specimen. The samples in spectrophotometry can be gases,  liquids. The analytes are dissolved in a solvent in the process of spectrophotometry.  For the solvents used here, the solid pellets are mixed with a transparent matrix for being used as an analyte for spectrophotometry.  The disks for the double beam spectrophotometer are made by using a pellet press which has a disk suspended in the biological samples through which the light beams pass on.

Differences Between A Single And Double Beam Spectrophotometers

Following are the parameters of differences between the  single and double beam spectrophotometers:

1.  The Definition of Single And Double Beam Spectrophotometers

A single light beam spectrophotometer is defined as an analytical instrument that witnesses all the light rays coming from the light source passes through the specimen.  So here,  the measurements are taken as the intensity of light before and after the passing through the biological samples. The single-beam spectrophotometer is  compact in nature and optically simpler than any double beam spectrophotometer. Despite all these, they are less reproducible.  And also these optical instruments are less expensive.

A double beam spectrophotometer is usually used for the measurement of transmittance or reflectance of analytes in a sample.  The samples used here are transparent or opaque solids, like polished glass, or gases that can be used as solvents. It is seen that many types of biochemicals come colored, due to which they absorb visible light or ultraviolet light. The transmitted light can be measured by colorimetric procedures within the spectrophotometer. Even the colorless biochemicals used here can  be converted to colored compounds for the process. It is suitable for chromogenic color-forming reactions to result in compounds suitable for colorimetric analysis in a single and double beam spectrophotometer.

2.  The Number Of Light Beams

In a double-beam configuration spectrophotometer, the beam from the light source is split into two parts. While in the single beam spectrophotometer, there is only one beam that strikes from a light source.

The double beam spectrophotometer, the two beams of light has different parts to illuminate.  The first part illuminates the reference standard and the second part illuminates the whole of the samples. The light beams may be recombined before they reach a single monochromator attached to the spectrophotometer.  In some cases of spectrophotometry, two monochromators are used.  Here, in this case, the splitting of the light  beam is normally done in one of two manners:

  • First one is done statistically, with a partially-transmitting mirror or similar device with the actual instrument.
  • By attenuating the light beams using moving optical and mechanical devices attached to the double beam spectrophotometers.  

With such specifications, double beam spectrophotometers have become popular.

While in the single beam spectrophotometers, the light beam is single from a light source which work is to illuminate the specimen as well as the reference points.  The monochromator selects the wavelength of light used here.

3.  The Measured Things In Single and double beam spectrophotometers

In the case of a  single beam spectrophotometer, it measures the concentration of an analyte in a sample. This is done by measuring the amount of light absorbed by that analyte in the specimen.  Here, the Beer-Lambert Law comes into operation for measuring the light beams. According to this law, the concentration of an analyte is directly proportional to the absorbance factor.

When it comes to a double beam spectrophotometer, it can measure the absorbance of the biological sample. The reference beam of light can measure the absorption rate within the instrument.  Here, the sample can be compared with the reference point used.  So, it is said that absorption is the ratio between the sample beams used in spectrophotometry. The light beams are measured after passing through the sample and a reference beam of light. A double beam spectrophotometer has a monochromator that isolates the desired wavelengths from a light beam. These reference beams of light sand sample beams of light recombine before moving to the monochromator. Thus, the measurement is made. 

4. Nature Of Light Beams Used In Single And Double Beam Spectrophotometers

The light beam used in a single beam spectrophotometer is of single nonsplit type beam. This means that the light beams cannot be split apart throughout the spectrophotometry process. 

In the case of double-beam spectrophotometers, it uses a beam of light that splits down.  As a result of the splitting, the light beams are split into two fractions before passing through biological samples.

5. Measurements Done By An Single And Double Beam Spectrophotometers

The measurements done by a single-beam spectrophotometer are taken from a single beam of light. But the measurements made here are less reproducible as only a single light beam is used for the measurements. Because of this, the single beam spectrophotometers.

On the contrary, the double beam spectrophotometer’s measurements are highly reproducible. As it uses the electronic and mechanical effects on biological samples and reference beams at an equal space. So, because of this, the double beam spectrophotometer is used vastly over the single beam spectrophotometers. In this case, the beam of light affects references and samples in equal space. 


A spectrophotometer is an optical instrument that analyses the analytes of a sample by observing the capability to absorb light by them. There are two main types of spectrophotometers available in the market:  single beam and double beam spectrophotometer. The main difference between single beam and double beam spectrophotometer is: in a single-beam spectrophotometer, all the light rays pass through the biological sample. Whereas in a double beam spectrophotometer device, the light beam splits into two parts and only one part passes through the sample.

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