Thursday 27 October 2016

Friday 14 October 2016

Spectrophotometer


Every chemical compound absorbs, transmits, or reflects light (electromagnetic radiation) over a certain range of wavelength. Spectrophotometry is a measurement of how much a chemical substance absorbs or transmits. Spectrophotometry is widely used for quantitative analysis in various areas (e.g., chemistry, physics, biology, biochemistry, material and chemical engineering, clinical applications, industrial applications, etc). Any application that deals with chemical substances or materials can use this technique. In biochemistry, for example, it is used to determine enzyme-catalyzed reactions. In clinical applications, it is used to examine blood or tissues for clinical diagnosis. There are also several variations of the spectrophotometry such as atomic absorption spectrophotometry and atomic emission spectrophotometry.

A spectrophotometer is an instrument that measures the amount of photons (the intensity of light) absorbed after it passes through sample solution. With the spectrophotometer, the amount of a known chemical substance (concentrations) can also be determined by measuring the intensity of light detected. Depending on the range of wavelength of light source, it can be classified into two different types:
  • UV-visible spectrophotometer: uses light over the ultraviolet range (185 - 400 nm) and visible range (400 - 700 nm) of electromagnetic radiation spectrum.
  • IR spectrophotometer: uses light over the infrared range (700 - 15000 nm) of electromagnetic radiation spectrum.
In visible spectrophotometry, the absorption or the transmission of a certain substance can be determined by the observed color. For instance, a solution sample that absorbs light over all visible ranges (i.e., transmits none of visible wavelengths) appears black in theory. On the other hand, if all visible wavelengths are transmitted (i.e., absorbs nothing), the solution sample appears white. If a solution sample absorbs red light (~700 nm), it appears green because green is the complementary color of red. Visible spectrophotometers, in practice, use a prism to narrow down a certain range of wavelength (to filter out other wavelengths) so that the particular beam of light is passed through a solution sample. 

Devices and mechanism
Figure 1 illustrates the basic structure of spectrophotometers. It consists of a light source, a collimator, a monochromator, a wavelength selector, a cuvette for sample solution, a photoelectric detector, and a digital display or a meter. Detailed mechanism is described below.  

A spectrophotometer, in general, consists of two devices; a spectrometer and a photometer. A spectrometer is a device that produces, typically disperses and measures light. A photometer indicates the photoelectric detector that measures the intensity of light.
  • Spectrometer: It produces a desired range of wavelength of light. First a collimator (lens) transmits a straight beam of light (photons) that passes through a monochromator (prism) to split it into several component wavelengths (spectrum). Then a wavelength selector (slit) transmits only the desired wavelengths, as shown in Figure 1.
  • Photometer: After the desired range of wavelength of light passes through the solution of a sample in cuvette, the photometer detects the amount of photons that is absorbed and then sends a signal to a galvanometer or a digital display, as illustrated in Figure 1.
Adams Chromatic Valence Color Space
Adams chromatic valence color spaces are a class of color spaces suggested by Elliot Quincy Adams

Two important Adams chromatic valence spaces are CIELUV and Hunter Lab.

Chromatic value/valence spaces are notable for incorporating the opponent process model, and the empirically-determined 2½ factor in the red/green vs. blue/yellow chromaticity components (such as in CIELAB).

In 1942, Adams suggested chromatic value color spaces. Chromatic value, or chromance, refers to the intensity of the opponent process responses, and is derived from Adams' theory of color vision.
A chromatic value space consists of three components:
·         {\displaystyle V_{Y},}VY the Munsell-Sloan-Godlove value function{\displaystyle V_{Y}^{2}=1.4742Y-0.004743Y^{2}}(VY)2 = 1.4742Y – 0.004743Y2
·         {\displaystyle V_{X}-V_{Y}}Vx – Vy,  the red-green chromaticity dimension, where {\displaystyle V_{X}}Vx is the value function applied to {\displaystyle (y_{n}/x_{n})X}(yn/xn)X  instead of Y
·         {\displaystyle V_{Z}-V_{Y}}VZ - VY, the blue-yellow chromaticity dimension, where {\displaystyle V_{Z}}VZ is the value function applied to {\displaystyle (y_{n}/z_{n})Z}(yn/zn)Z  instead of Y
A chromatic value diagram is a plot of {\displaystyle V_{X}-V_{Y}}VX - VY (horizontal axis) against {\displaystyle 0.4(V_{Z}-V_{Y})}0.4(VZ – VY) (vertical axis). The 2½ scale factor is intended to make radial distance from the white point correlate with the Munsell chroma along any one hue radius (i.e., to make the diagram perceptually uniform). For achromatic surfaces, {\displaystyle (y_{n}/x_{n})X=Y=(y_{n}/z_{n})Z}(yn/xn)X = Y = (yn/zn)Z  and hence {\displaystyle V_{X}-V_{Y}=0}{\displaystyle V_{Z}-V_{Y}=0}VX – VY = 0, VZ – VY = 0. In other words, the white point is at the origin.
Constant differences along the chroma dimension did not appear different by a corresponding amount, so Adams proposed a new class of spaces, which he termed chromaticvalence. These spaces have "nearly equal radial distances for equal changes in Munsell chroma".

Kubelka-Munk equations:

When light is fall on some sample, some part of the light reflected, some are absorbed and some are scattered. If the sample is having opacity more than 70% then as per Kubelka Munk (established in 1931) following is the relation between reflectance, scattering and absorption of light:
K/S= (1 – 0.01R)2/2(0.01R)
The mathematical basis for all color matching software is the Kubelka-Munk series of equations. These equations state that for opaque samples such as textile materials, the ratio of total light absorbed and scattered by a mixture of dyes is equal to the sum of the ratios of light absorbed and scattered by the dyes measured separately. Where absorption is defined as "K" and scattering is defined as "S", Kubelka-Munk states that _:
(K/S) mixture = (K/S) dye 1 + (K/S) dye 2 + (K/S) dye 3 + ...
K/S is not a readily measurable quantity, but it can be calculated from the reflectance of a sample -- "R" -- by the Kubelka-Munk equation that states
 K/S= (1-R)2/2R

K/S  value is proportional to dye  concentration in the substrate
K/S=kC

Where k is constant and C is concentration of dyes or colorant 

Delta E Differences and Tolerances.


The difference between two colour samples is often expressed as Delta E, also called  DE, or ΔE. 'Δ' is the Greek letter for 'D'. This can be used in quality control to show whether a dyed or printed sample, such as a colour swatch or proof, is in tolerance with a reference sample or industry standard. The difference between the L*, a* and b* values of the reference and sample will be shown as Delta E (ΔE). The resulting Delta E number will show how far apart visually the two samples are in the colour 'sphere'

 CIE L*C*h°
This is possibly a little easier to comprehend than the Lab colour space, with which it shares several features. It is more correctly known as  L*c*h*.  Essentially it is in the form of a sphere. There are three axes; L* , c* and .  

The L* axis represents Lightness. This is vertical; from 0, which has no lightness (i.e. absolute black), at the bottom; through 50 in the middle, to 100 which is maximum lightness (i.e. absolute white) at the top.

The c* axis represents Chroma or 'saturation'. This ranges from 0 at the centre of the circle, which is completely unsaturated (i.e. a neutral grey, black or white) to 100 or more at the edge of the circle for very high Chroma (saturation) or 'colour purity'.
The h* axis represents Hue. If we take a horizontal slice through the centre, cutting the 'sphere' ('apple') in half, we see a coloured circle. Around the edge of the circle we see every possible saturated colour, or Hue. This circular axis is known as  for Hue. The units are in the form of degrees (or angles), ranging from 0° (red) through 90° (yellow), 180° (green), 270° (blue) and back to  0°. 
The Lch colour model is very useful for retouching images in a colour managed workflow, using high-end editing applications. Lch is device-independent.

CIE XYZ:


The basic CIE colour space, or colour model, is based on a 'Standard Observer and 'Standard Illuminants' (D50, D65, etc.). This is a numerical model of colour sensitivity based on research commenced in the 1920s on a sample of people with normal colour vision. It is a 'universal colour space' representing the colour spectrum visible to the 'average human'. The light-sensitive retina at the back of the eye has three types of receptors near the centre, known as cones. They are sensitive to the three primaries, 'red, green and blue'. The CIE XYZ tristimulus values are assigned to the red, green and blue curves respectively. These approximate to the cones in the eye. The relative response of each is plotted on a diagram against the wavelength in nanometers. The eye also has rods, outside of the retina's centre, which are sensitive to low-wavelength light and which only operate at low levels of illumination. There are two axes: vertical and horizontal.

The vertical axis represents Relative Response 0 - 2.0 (shown here) or Reflective Intensity 0 - 120% (not shown).
The horizontal axis represents Wavelength in nanometers, usually from about 380 to about 720nm.
It should be emphasized that this is a 'device-independent' colour space in which each primary colour (X,Y,Z) is always constant, unlike  RGB which varies with every individual device (monitor, scanner, camera, etc.). XYZ is typically used to report the spectral response of a sample measured by a colorimeter or a spectrophotometer. A colorimeter may contain as few as three sensors, one each for red, green and blue, (or X, Y and Z), and will typically be used for display calibration and profiling. A spectrophotometer will report the entire spectral response at frequent intervals along the spectrum, say every 10 nanometres, and will typically be used to measure printed sheets to control a press or create an ICC profile.
While CIE XYZ is used to report colour from measuring instruments, it is not so useful for humans to describe colour. Another use is as the Profile Connection Space (PCS) within an ICC profile, where it may be used instead of CIE Lab.
You may notice that the Y ('green curve') covers the widest wavelength. This corresponds to the overall human visual response to all colours, or lightness. It is therefore also used to indicate luminance ('lightness').

CIE L*a*b*: In such case the vertical L* axis represents Lightness, ranging from 0-100.  The other (horizontal) axes are now represented by a* and b*. These are at right angles to each other and cross each other in the centre, which is neutral (grey, black or white). They are based on the principal that a colour cannot be both red and green, or blue and yellow. 
      The a* axis is green at one extremity (represented by -a), and red at the other (+a). The b* axis has blue at one end (-b), and yellow (+b) at the other. 
The centre of each axis is 0. A value of 0, or very low numbers of both a* and b* will describe a neutral or near neutral. In the case of paper, the white point in terms of a* and b* is usually carried through to the black, being gradually reduced towards '0'.
In theory there are no maximum values of a* and b*, but in practice they are usually numbered from -128 to +127 (256 levels).
The CIE Lab colour model encompasses the entire spectrum, including colours outside of human vision. CIE Lab is extensively used in many industries apart from printing and photography. Its uses include providing exact colour specifications for paint (including automotive, household, etc.), dyes (including textiles, plastics, etc.), printing ink and paper. 


CIE Color Systems

CIE Color Systems The CIE, or Commission Internationale de l’Eclairage (translated as the International Commission on Illumination), is the body responsible for international recommendations for photometry and colorimetry.

In 1931 the CIE standardized color order systems by specifying the light source (or illuminants), the observer and the methodology used to derive values for describing color.

The CIE system characterizes colour by a luminance Y and two colour coordinates x and y which specify the point on the chromatic diagram. This system offers more precision in colour measurement than do the Munsell system because the parameters are based on the spectral power distribution of the light emitted from a coloured object and is factored by sensitivity curves which have been measured for the human eye.

Based on the fact that the human eye has three different types of color sensitive cones, the response of the eye is best described in terms of three "tristimulus values". However, once this is accomplished, it is found that any color can be expressed in terms of the two color coordinates x and y.
The colors which can be matched by combining a given set of three primary colors (such as the blue, green, and red) are represented on the chromaticity diagram by a triangle joining the coordinates for the three colors.

The diagram given below represents the  mapping of human color perception in terms of two CIE parameters x and y. The spectral colors are distributed around the edge of the "color space" as shown, and that outline includes all of the perceived hues and provides a framework for investigating color.
The CIE Color Systems utilize three coordinates to locate a color in a color space. These color spaces include:

• CIE XYZ
• CIE L*a*b*
• CIE L*C*h°
Scales for Measuring Colour:
There are two important scales for measuring colour. These are:
      Munsell Scale
      CIE colour system

Munsell Scale:

In 1905, artist Albert H. Munsell originated a color ordering system — or color scale — which is still used today. The Munsell System of Color Notation is significant from a historical perspective because it’s based on human perception. Moreover, it was devised before instrumentation was available for measuring and specifying color. This system assigned numerical value to the three properties of the colour-Hue, Chroma and value. The Munsell color system match colors to a set of standard samples. The Munsell system divides hue into 100 equal divisions around a color circle and circle is distorted by assigning a unit of radial distance to each perceptable difference in saturation (called units of chroma). Since there are more perceptable differences for some hues, the figure will bulge outward to 18 values for some hues compared to only 10 for another. Perpendicular to the plane formed by hue and saturation is the brightness scale divided into a scale of "value" from 0 (black) to 10 (white). A point in the color space so defined is specified by hue, value, and chroma in the form H V/C.

The MUNSELL system is a collection of color samples for comparison, with adjacent samples based upon equal perceived differences in color.

Munsell saw that full chroma for individual hues might be achieved at very different places in the color sphere. For example, the fullest chroma for hue 5RP (red-purple) is achieved at 5/26.

Another color such as 10YR (yellowish yellow-red) has a much shorter chroma axis and reaches fullest chroma at 7/10 and 6/10:

Instrumentation for colour measurement:
As colour is perception, it cannot be directly measured; however we can measure and subsequently calculate certain factors which are responsible for producing this sensation of color. The quantification of the color properties of textile materials is of great economic value in industry and instruments are employed to some degree in almost every textile operation involved in textile coloration.

Color instrumentation has experienced a tremendous advancement in technology during the past few decades. The first devices for measuring color were absorptiometers which were used to determine by visual inspection whether two solutions were of equal color. This is very similar to holding two glass cylinders of dye solution up to a light and judging whether they are of equal strength and shade, except that the absorptiometer provided a method of adjusting the thickness or path width so that this change in width could be read from a scale. In measuring reflected light from opaque materials such as textiles, the first instruments were reflectometers developed around 1915-1920. 
Perception of colour: It involves a series of events which are interdisciplinary in nature. Perception of colours includes source of light, object that is illuminated and eye and brain that perceive the colour.
                                                   Object
Metamerism: It is a phenomenon observed when two specimens appears to have the similar colour under one set of viewing conditions, but different under another. The change in viewing conditions refers to change in source, observer or geometry of the observation.
Types of metamerism
         illuminant metamerism
         observer metamerism
         geometry metamerism
Illuminant metamerism
When the colour of two specimens matches under one illuminant but not with another illuminant, it is said to be illuminant metamerism.


Observer metamerism
When colour of two objects appears to match to one observer but not to the other, it is said to be observer metamerism. In this case observer may not be colour blind but the spectral sensitivity of colour receptors of one observer may be slightly different than the other.
Geometry metamerism
Two objects which match in one arrangement of illumination, sample and observer may mismatch by altering the positions. 

MEASUREMENT OF COLOUR
Colour is perception and sensation experienced caused by light reflected from or transmitted through objects. If you have some coloured object, it means it has certain reflectance characteristics—the patterns of light wavelengths that are reflected and absorbed—that are physical properties of object. However, colour is our subjective perception of the wavelengths of light that end up bouncing off the object and onto our retina. Photoreceptors in the retina begin the process by selectively responding to different wavelengths. A single type of photoreceptor alone cannot accomplish color vision since it cannot distinguish between enough wavelengths. We overcome this difficulty by using three different types of cone photoreceptors to code for color, a concept known as trichromacy. One of the key observations in developing the trichromatic theory of color vision was that lights can be added together to form mixtures that look identical to other, single light wavelengths. Another activity illustrates additive and other forms of color mixing.
Human Visual System

The human visual system consists of two functional parts, the eye and (part of the) brain. The brain does all of the complex image processing, while the eye functions as the biological equivalent of a camera.


What our eyes perceive of a scene is determined by the light rays emitted or reflected from that scene. When these light rays are strong enough (have enough energy), and are within the right range of the electromagnetic spectrum (about 300 to 700 nm), the healthy eye will react to such a ray by sending an electric signal to the brain through the optic nerve. When a light ray hits the eye, it will first pass through the cornea, then subsequently through the aqueous humor, the iris, the lens, and the vitreous humor before finally reaching the retina. The cornea is a transparent protective layer, which acts as a lens and refracts the light. The iris forms a round aperture that can vary in size and so determines the amount of light that can pass through. Under dark circumstances the iris is wide open, letting through as much light as possible. In normal daylight, the iris constricts to a small hole. The lens can vary its shape to focus the perceived image onto the retina. In the retina, the light rays are detected and converted to electrical signals by photoreceptors. The eye has two types of photoreceptors: rods and cones, named after their approximate shape. The rods are abundant, about 100 million in a human eye, and spread evenly about the retina, except at the fovea, where there are almost none. The fovea is the area of the retina where our vision is sharpest. There are much fewer cones, about 6 to 7 million, which are mainly located around the fovea, but can be found in a low density in the entire retina. No photoreceptors are found at the point where the optic nerve attaches to the eye (the so-called blind spot), so we cannot perceive anything there. Since rods are more responsive to light than cones we can identify three types of vision, depending on the amount of light that reaches the eye. Under dark circumstances, practically only the rods are active. Since rods cannot discriminate colors, we perceive only shades of grey. We call this scotopic or night vision. Under daylight circumstances, the cones are most active, and we experience photopic or day vision. In dimly lighted circumstances there is an intermediate stage where both rods and cones are active called mesopic vision. We are able to distinguish colors because there are three distinct types of cones, each sensitive to a different band of the electromagnetic spectrum.