Defination of Colorimetry

Colorimetry is defined as "the measurement of color"

and a colorimetric method is defined as "any technique used to evaluate an unknown color in reference to known colors". In a colorimetric chemical test the intensity of the color from the reaction must be proportional to the concentration of the substance being tested. The most basic colorimetric method involves the reacted test sample is visually compared to a known color standard.

 The colour intensity can be measured by:

Colorimeter Spectrophotometer

Colorimeters
The colorimeter is an apparatus that allows the

absorbance of a solution at a particular frequency (color) of visual light to be determined. Colorimeters hence make it possible to ascertain the concentration of a known solute, since it is proportional to the absorbance.

Working principle
Pass a colored light beam through an optical filter,

which transmits only one particular color or band of wavelengths of light to the colorimeter's photo detector where it is measured. The difference in the amount of monochromatic light transmitted through a colorless sample (blank) and the amount of monochromatic light transmitted through a test sample is a measurement of the amount of monochromatic light absorbed by the sample.

Spectrophotometers
A spectrophotometer is a photometer (a device for

measuring light intensity) that can measure intensity as a function of the color, or more specifically, the wavelength of light. The most common application of spectrophotometers is the measurement of light absorption.

Working principle
Either a tungsten or xenon flash lamp as the source of

white light. The white light passes through an entrance slit and is focused on a ruled grating. The grating causes the light to be dispersed into its various component wavelengths. The monochromator design allows the user to select which specific wavelength of interest will be passed through the exit slit and into the sample. The use of mirrors and additional filters prevents light of undesired wavelengths (diffraction of higher order, stray light) from making it to the sample. A photo detector measures the amount of light, which passes through the sample.

Differences
A colorimeter measures the absorbance of a particular wavelength by a solution. It is usually used to determine the concentration of a known solute in a known solvent through the application of the Beer-Lambert law (absorbance is proportional to concentration). A spectrophotometer is related to a colorimeter but may be used to scan across a spectrum of wavelengths. It also measures slightly different properties. A spectrophotometer usually measures transmittance (which is related to absorbance) or reflectance of solutions, transparent or opaque solids, such as polished glass, or gases. Hence a spectrophotometer is of broader application than a colorimeter. It can perform the same functions as a colorimeter, but it can also do more.
a colorimeter can only use one wavelenght which has to be in the visible range

only. a spectrophotometer on the other hand can not only function like a colorimeter but also take a spectrum ie the abosorbance/transmittance of a substance across the entire wave spectrum especially if it is a UV-vis or UV-vis-IR (uv visible infra red).

Visible Spectrum
visible spectrum that portion of the range of

wavelengths of electromagnetic vibrations (from extreme red, 770 to extreme violet, 390 nm) which is capable of stimulating specialized sense organs and is perceptible as light. This part is visible to human eye.

Visible spectrum and colorimetry

Principle
Colorimeters rely on the principle that the

absorbance of a substance is proportional to its concentration i.e., a more concentrated solution gives a higher absorbance reading. This is quantified by Beer law.

Beers Law
The Beer-Lambert law or simply Beer's law is the linear

relationship between absorbance and concentration of an absorber of electromagnetic radiation. It is generally written as:

Reasons behind colorimetric analysis

as to allow for a practical assay. We may have to employ one or more reagents to produce colored compounds in proportion to the concentration of unknown What we have is a shift to absorption at a higher wavelength in addition of a reagent. This shift to higher wavelength is associated with a greater degree of delocalization.

 The more delocalization there is, the smaller the gap

between the highest energy pi bonding orbital and the lowest energy pi anti-bonding orbital. The smaller the energy jump, the higher the wavelength of the light absorbed

Colour region

Wavelength(nm)

380 435
435 500 500 520 520 565 565 590 590 625 625 740

Delocalization
Electrons belonging to certain molecules are not attached to a

particular atom or bond in that molecule. These electrons are said to be "delocalized" because they do not have a specific location. Delocalization gives molecules resonance stability, and based on the resonance stability, we can determine the range of absorption of ultraviolet and visible light. of a molecule in the light spectrum. delocalization is a stabilizing force because it spreads energy over a larger area rather than keeping it confined to a small area. Since electrons are charges, the presence of delocalized electrons brings extra stability to a system compared to a similar system where electrons are localized. The stabilizing effect of charge and electron delocalization is known as resonance energy. the more stable the molecule the lower its potential energy and higher its wavelength.

DELOCALIZATION SETUP
It is generally enhanced by presence of a conjugated

system. Other arrangements include: (a) The presence of a positive charge next to a pi bond. The positive charge can be on one of the atoms that make up the pi bond, or on an adjacent atom. (b) The presence of a positive charge next to an atom bearing lone pairs of electrons. (c) The presence of a pi bond next to an atom bearing lone pairs of electrons.

Ways to setup
Often one or more reagents are added to standard and the sample solution as colours are not produced by itself. It would be useful to have a number of standards that span the full range of likely concentrations of our unknown. That's where a standard curve comes into it. We prepare a series of standards of known concentration of X, ranging from low to high concentration. We run the assay and plot absorbance versus concentration for each standard. Using this standard curve we can read the concentration for an unknown given its absorbance reading.

Standard curve
A standard curve is a quantitative research tool, a method of

plotting assay data that is used to determine the concentration of a substance, particularly proteins and DNA. In practice, a series of standard solutions with know concentration of analyte are prepared. The absorbances of the standard solutions are measured and used to prepare a calibration curve, which is a graph showing how the experimental observable (the absorbance in this case) varies with the concentration. For this experiment, the points on the calibration curve should yield a straight line (Beer's Law). The slope and intercept of that line provide a relationship between absorbance and concentration: A = slope c + intercept

 A normal standard curve is as follow:

absorbance

Sample preparation
It helps to have a reasonable estimate of ranges of

concentrations of sample that one can expect. Even with such an estimate it is good to prepare samples with a range of dilutions, in case a sample is so concentrated that its absorbance readings are out of range.

 The sample absorbance is measured and its

corresponding concentration is find out from the graph.

Control
When we run an assay we must ensure that only the substance we are

assaying is responsible for absorbance of light in the wavelength range of interest. All conditions under which standards and unknowns are prepared should be kept identical. If solutes in the sample buffers affect absorbance, then we have a problem. We won't obtain accurate results if we vary the volumes in which we prepare and assay standards and unknowns. The timing of reading absorbance, temperature at which we keep the materials, and all other physical factors should be kept the same. Because it is not always practical to use identical buffers for all unknowns and standards, we need only ensure that none of the components of any of the buffers has a significant effect on absorbance.

 Several factors may influence the experimenter's choice of

one colorimetric or photometric assay over another. Among these are sensitivity, relative volumes of reagent and sample required, selectivity for the compound of interest and against potentially interfering substances, linearity and range of the assay, variation in sensitivity (i.e., in this context, variations in sensitivity to different proteins) and whether the sample is destroyed by the assay.

Complementary colors
Colours directly opposite each other on the colour

wheel are said to be complementary colours Mixing together two complementary colours of light will give you white light.

Importance
Complementary colours is important to find put the

wavelength of maximum absorbance. Normally the wavelength of maximum absorbance is the wavelength at which the complementary colour of a particular coloured samples maximum absorbance takes place. for example if a sample is blue in colour then its correct wavelength will be 565-590, wavelength where yellow is absoprbed maximum.