The Monochromator and Its Role in the Spectrograph

Spectrographs are important tools for studying many biological and chemical processes and substances, from pigments to plant growth and nucleic acids to pharmaceuticals, providing information about analytes based on their optical properties. A spectrograph is essentially a machine that separates light into particular wavelengths that are then detected and recorded immediately using an array detector,¹such as a charge coupled device (CCD).²The first processing step in a spectrograph is separating the incoming light, which is performed by a monochromator. This article describes what a monochromator is and how it works, the different types of monochromators, what monochromators are used for and their role in the spectrograph.

A monochromator is a device that separates different wavelengths of light from a given light source. The main components typically include an entrance slit, mirrors and a light disperser.³A prism or grating is often used as the light disperser.³Most monochromators have an exit slit where the separated light leaves the device, but monochromators used in spectrographs have an array detector, usually a CCD, replacing the exit slit to capture data from a range of wavelengths at once, resulting in real‑time data.²

When light rays travel in a parallel direction, it is often called collimated light, and when they are in this form, the rays can be controlled.³In a monochromator, the incoming light rays are usually collimated using mirrors, dispersed and aimed towards the detector. There are different ways to implement the setup, which are beyond the scope of this article, but here we will focus on the most common form, known as a Czerny‑Turner arrangement (Figure 1).

In a Czerny‑Turner arrangement, there are two concave mirrors and a diffraction grating. Light rays enter the monochromator's entrance slit (Figure 1B) and travel to the first concave mirror (Figure 1C), which aligns the rays so they are traveling in parallel. The parallel light rays then reach a diffraction grating, which then bends different wavelengths of light at different angles (Figure 1D). The light then reaches a second concave mirror that focuses different wavelengths of light at different points (Figure 1E). Rotating the grating (Figure 1D) controls the range of light wavelengths that will then subsequently pass to the CCD detector (Figure 1F).³

Figure 1: A schematic diagram of a Czerny-Turner monochromator indicating the component parts and the path of the light from the light source through to detection.Credit: Technology Networks.

Optical instruments that are designed to make absorbance measurements in colorimetric analyses commonly have a single monochromator. However, having multiple monochromators in one instrument is becoming more prevalent to achieve more accurate high absorbance measurements. A double monochromator setup is often used in higher quality spectrophotometers where two monochromators are operating in tandem, arranged in series (Figure 2A). Using a double monochromator arrangement improves resolution,^6-8but results in a weaker light intensity arriving at the detector. The light leaving the first monochromator feeds into the second monochromator to help remove light with undesirable wavelengths, often called stray light.³

Some stray light will always be expected and may originate from undesired diffraction or scattering of light inside the monochromator or from the environment if the optical device is not completely sealed. Stray light is problematic when measuring very high absorbance values or very faint fluorescence emissions from samples as a large portion of the light reaching the detector will be stray light.³

A regular single monochromator can be modified into a double⁶or multiple monochromator.^7–9构造简洁,单色仪这样的that the light is reflected back to the light disperser using a pair of mirrors at right angles to each other before reaching the detector.^6,7,9If the light is dispersed twice, similar to passing light through a monochromator twice, then it behaves just like a double monochromator. The same method has been implemented to effectively mimic a quadruple monochromator where light is passed through the light disperser four times, resulting in even better resolution and less stray light.⁷

从技术上讲,一个四单色仪可以有ght pass through the same monochromator four times,⁷behaving similarly to passing light through four monochromators arranged in series, but this is not what the term typically means. Quadruple monochromators normally refer to a setup in which two double monochromators are utilized and are popular for fluorescence studies (Figure 2B). In this arrangement, a double monochromator is first used to select a narrow range of wavelengths to excite a fluorescent sample and another double monochromator is used to select the emitted wavelength range of light. Reducing stray light in fluorescence studies is imperative due to the weak light intensity emitted from a fluorescent sample competing with the light intensity from stray light.

Sometimes, a double monochromator may be referred to as a two-monochromator system, but a two-monochromator system is usually not a double monochromator. For example, an optical instrument called a fluorometer that measures fluorescence has two monochromators: one for selecting the excitation or absorption wavelength, and another separately for selecting the emission wavelengths (Figure 2C).

Figure 2: (A), (B) and (C) each represent a different spectrograph design. Schematics of a double monochromator arrangement (A) known to increase resolution and significantly reduce stray light when compared to using a single monochromator. ^{7 , 8} A typical quadruple monochromator configuration (B) consists of a pair of double monochromators, often used for high resolution fluorescence studies and a common two monochromator configuration used in fluorescence analysis (C). The colored arrows conceptually illustrate the separation of light, beginning with the rainbow arrow from the light source, followed by the extraction of specific wavelengths after each passing through a monochromator. Note that MC indicated in this figure is an abbreviation for a monochromator. The assumption is that a sample is being examined using a spectrograph design where (A) is typical for absorbance measurements and designs (B) and (C) are used for fluorescence.Credit: Technology Networks.

Different types of monochromators can be categorized based on how light is dispersed inside them, with prisms and gratings being the most common.

Prism monochromators

As the name suggests, a prism monochromator uses a prism to disperse light. The prism relies on a difference in the index of refraction, which means that the speed of light varies as it travels through a prism material. Consequently, the colors of white light will be split accordingly. For example, blue light is slowed and bent more than red light in a glass prism.

A prism may sometimes inherently have certain material disadvantages. For example, prism materials made of sodium chloride or potassium chloride can be used for analysis in the infrared spectrum,¹⁰but the prism properties of both degrade over time due to absorption of water from the air. A sapphire prism for infrared analysis¹⁰would be a better substitute but is generally expensive. As another example, glass prisms are not used for examining wavelengths in the ultraviolet (UV) range because glass absorbs UV light.¹⁰Instead, a quartz prism is necessary to examine the UV spectrum.¹⁰

Prism monochromators are generally preferred when working in the UV range, but in practice most monochromators today use gratings.

Grating monochromators including the Czerny-Turner monochromator

Gratings are currently by far the most commonly used dispersing element in a monochromator. Several gratings can be included in a monochromator that give differing selectivity across a range of wavelengths in a single instrument. This is useful as each usually works best for a specific wavelength range and certain applications.³

A grating is an optical component that has regular, evenly spaced grooves, usually with a reflective coating placed on top of another material. Light reaching a grating may be reflected at different angles, with each groove acting as a source of light when the light reflects.³A blazed grating, also called an echelette grating, is a specific specialized type of grating designed to operate best at a specific wavelength of light.Gratings used for examining the infrared range (longer wavelengths) have much wider spacing between grooves, whereas those for the UV range (shorter wavelengths) have much tighter spacing between grooves.

The reflected light rays dispersed from a grating can interfere constructively when they are in phase, interfere destructively when out of phase or somewhere in between.³Reflected light that interferes destructively decreases light intensity as expected, but adisadvantage inherent in all gratings is interference that leads toorder overlap. For instance, light with a wavelength of 300 nm in the second order will overlap the light with a wavelength of 600 nm in the first order, thus a CCD detector would recognize both wavelengths at the same time. Order overlap is undesirable and can be removed by adding filters, a second monochromator connected in series or an additional grating.

The majority of gratings in monochromators are made as replicas of a high quality original grating, also called a master grating.^11,12Instead of producing master gratings directly for use in monochromators, creating copies of a master grating using a mold significantly lowers the production cost and eases the large-scale production of gratings.

High-quality gratings have minimal surface imperfections, which can cause stray light,¹³and consequently provide high resolution. Traditionally, high-resolution diffraction gratings were ruled, but gratings made holographically generate less stray light.¹³When discussingresolution in the context of diffraction grating,a higher or better resolution refers to instances where the difference in wavelengths of light that can be distinguished becomes smaller, meaning that more details become visible.³The resolution improves when more grooves are present in a grating.³For example, low‑cost projects may use a compact disk (CD) or a digital versatile disk (DVD) as a diffraction grating, with the DVD having more grooves than a CD and therefore providing better resolution.¹⁴

Decreasing the groove spacing on a grating is known to increase both the dispersion (how easily the wavelengths of light will separate and spread) and resolution.³In a monochromator, smooth rotation of the grating is expected to yield a linear change in wavelength.The dispersion of light is linear when using a grating, which is a large advantage when compared to a prism because a prism has non‑linear light dispersion. This in turn simplifies the data processing, analysis and interpretation.

Overall, grating monochromators, and the Czerny-Turner monochromator design in particular, are most commonly used. The basic function of the Czerny-Turner monochromator is explained in three minutes by M. Leandro Heien in this video.¹⁵

A summary of the advantages and disadvantages when compared to a prism monochromator are presented in Table 1.

Table 1:A comparison of the advantages and disadvantages of using a reflective diffraction grating and a prism to disperse light in a monochromator. Entries inboldare more advantageous.

	Reflective diffraction grating	Prism
Dispersion method	Diffraction	Refraction
Transmission efficiency	Lower efficiency overall (due to dispersion of light into several directions)	High efficiency
Low light intensity situations	Worse	Better
Dispersion	Linear	Non-linear
Spectral resolution	Better overall	Worse overall but better in UV
Temperature dependence on dispersion	Low (deforms)	High (changing refraction index)
Order overlap	Yes	None
Stray light	More	Less
Weight	Lighter	Heavier
Overall cost	Less	More

Monochromators are used to control the wavelength of light when needed, such as in spectroscopic analysis techniques. Examples that commonly employ monochromators include:

• Ultraviolet‑visible (UV-Vis) spectroscopy
• Atomic absorption spectroscopy
• Fluorescence spectroscopy
• Raman spectroscopy
• Circular dichroism spectroscopy

Monochromators are probably most often used in UV‑Visabsorptionspectroscopy, where sample types including proteins,pharmaceuticals, beverages, DNA and RNA are analyzed.¹⁶

It is best to keep in mind the particular application when deciding whether a prism or a diffraction grating should be used. If a high signal‑to‑noise ratio with high sensitivity is required in a low‑light situation, then a prism monochromator should be used. Highly efficient light transmission generally yields high sensitivity; prisms usually have a higher efficiency than diffraction gratings.¹⁷This is important for situations involving low intensity light experiments, such as measuring the absorbance of deeply colored samples using visible spectrophotometry or faintly fluorescing samples using fluorometry.

In the vast majority of modern spectrographs, diffraction gratings are present in monochromators to disperse light. There are specific uses for rapid absorbance measurements, particularly with applications in analyzing DNA, RNA, pharmaceuticals and dyes.¹⁶These measurements are often done using a microplate reader that can take hundreds or thousands of rapid measurements using a double monochromator spectrograph. Additionally, microplate readers can also take rapid, high-resolution fluorescence measurements using a quadruple monochromator spectrograph. When several rapid spectrophotometric measurements or analyses are required, the spectrograph is the first choice.

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