Use Of Uv Visible Spectroscopy Biology Essay

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Rate studies kinetics involve the measurements of the change in the concentration of a participant/reactant in the reaction as a function of time. Spectroscopic rate measurements involve the measurements of the fall and rise in absorption of the solution at a particular wavelength. This measurement provides us the information about the change in concentration of either of the reactants or products.

UV visible spectroscopy refers to the absorption spectroscopy in the UV visible spectral region. This means it uses light in the UV visible and adjacent infrared regions .In this region of the electromagnetic spectrum, molecules undergo electronic transitions.

A general method for estimating reaction rate constants of chemical reactions using ultraviolet-visible (UV-visible) spectroscopy is presented. The only requirement is that some of the chemical components involved be spectroscopic ally active. The method uses the combination of spectroscopic measurements and techniques from numerical mathematics. Therefore, the method can be used in cases where a large spectral overlap of the individual reacting absorbing species is present. No knowledge about molar absorbance of individual reacting absorbing species is required for quantification. The results obtained were also excellent.

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HOW UV VISIBLE SPECTROSCOPY WORKS

When light either visible or ultra violet is absorbed by valence electrons these electrons are promoted from their normal ground states to higher energy states. the energies of the orbital's involved in the electronic transitions have fixed values. Because energy is quantized, it seems safe to assume that absorption peaks in a UV-Visible spectrum will be sharp peaks. However, rarely, the spectrum has sharp peaks. This is because there are also vibrational and rotational energy levels available to absorbing materials.

TRANSMISSION, ADSORPTION

When a incident beam of radiation hits a transparent solution held in a sample cell, and if the solution contains chromophores, which absorb photons from the incident beam (by absorbing they can promote electrons to a higher orbital or state), then the Intensity of the transmitted radiation will smaller than the intensity of the incident beam. The percentage of the light transmitted is known as the transmittance (T), which is thus defined

as:

T = I / Io

where I is the light intensity after it passes through the sample and Io is the initial light intensity.

By taking the negative logarithm of this quantity then it is known as the absorbance:

A = -log T = - log (I / Io).

U V SPECTROSCOPY IN CHEMICAL KINETICS

UV spectroscopy can be used to study the kinetics of the rate of reaction. In order to determine the kinetics of a reaction, the change in concentration of either a reactant or product with time is measured. As absorbance is directly proportional to concentration, UV spectrophotometry can be used to follow the course of reaction. The method is based upon the fact that one of the reactants or products exhibiting suitable absorption in the ultraviolet region is not overlapped by absorption due to other species present. The method can be employed to study such rate which must be relatively slow.

UV SPECTROPHOTOMETER

The UV spectrophotometer is used to study fast reaction by following a step flow method. In this method, two solutions are entering through two sides.These are allowed to pass through reaction chamber and flow of the mixed solution is stopped by the piston.The absorbance of any species which absorbs the UV region is measured with the UV spectrophotometer. With suitable adjustments of flow rates, reaction with half lives down to milliseconds can be studied.

In the stop flow method, photomultiplier is used as a detector whose output is displayed on the screen with a time base.

UV spectroscopy can characterize the type of compounds which absorb UV radiation. These compounds are with unbounded electrons. In order to record UV absorption spectrum, the practice is to measure the amount of radiation absorbed at various wavelength .A spectrophotometers is a device used to measure the intensities and wavelengths at those particular intensities.

SPECTROSCOPIC TECHNIQUES

The spectroscopic technique form the largest and important techniques used in chemistry. This technique provides a wide range of qualitative and quantitative information. All spectroscopic techniques are more or less dependent on the emission or absorption of electromagnetic radiation characteristic of certain energy changes within an atomic or molecular system. The energy changes are associated with the complex series of discrete or quantized energy levels in which atoms and molecules are assumed to exist.

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The ways in which measurements of radiation frequency are made experimentally and energy

Levels deduced from these comprise the practice of spectroscopy.

OTHER APPLICATIONS OF UV SPECTROSCOPY

1. Qualitative Analysis

2. Detection of Impurities

3. Quantitative Analysis

4. Molecular Weight Determination

5. Chemical Kinetics

6. Charge Transfer Transition

LAMBART'S LAW

When a beam of monochromatic radiation passes through a homogenous absorption medium, the

rate of decrease of intensity of radiation with thickness of absorbing medium is proportional to

the intensity of incident radiation.

Mathematically the law can be expressed as

-dI = KI

dx

where

I= intensity of radiation after passing through a thickness x, of the medium.

dI = infinitesimally small decrease in the intensity of radiation on passing through thickness, dx of the medium.

-dI/dx = rate of decrease of intensity of radiation with thickness of the absorbing material

KINETIC STUDY OF THE REACTION

As we know that the concentration of the reactants is the main factor that directly influences the rate of reaction. Kinetic studies have established that the mechanism of the initial reaction is not a simple change. To determine the order of a reaction, experimenters can measure the rate of reaction at a variety of reactant concentrations to see how the rate changes. For example if the concentration of reactant is doubled, while all other concentrations remain constant, the reaction rate will also double, since the reactant corresponds to first order.

A general method for estimating reaction rate constants of chemical reactions using ultraviolet-visible (UV-visible) spectroscopy is presented. The only requirement is that some of the chemical components involved be spectroscopic ally active. The method uses the combination of spectroscopic measurements and techniques from numerical mathematics and chemo metrics. Therefore, the method can be used in cases where a large spectral overlap of the individual reacting absorbing species is present. No knowledge about molar absorbances of individual reacting absorbing species is required for quantification.

During a reaction, as by changing the concentrations of reactants one at a time, the rate of reaction may vary in different proportions relative to the change in concentration. So each different reaction's rate may change in a different proportion to a specific reactant. The product formed during the reaction will change in absorbency and have different absorbance.

As the reaction proceeds, the change in the absorbance of the product solution formed during the reaction determines the initial reaction rate of the reaction. By using a colorimeter, to measure the change in absorbency of solution products, over time, reaction rates of different concentrated reactions can be determined and can be compared to each other.

E.g.:

FeCl3 + 3KI=>3KCl + FeI2

If ferric chloride and potassium iodide were reacted at various molarities, the change in ferric chloride will likely bring a larger change in rate of reaction.

As during the course of reaction, the concentration of reactants goes on decreasing, so by measuring the absorbance at different concentrations, we can find the rate of reaction.

The procedure is like:

.Prepare the solutions of KI and FeCl3.Set up and calibrate the colorimeter. As the reaction mixture is put into the calorimeter to measure the absorbance during the course of reaction, the absorbance is noted at different time intervals and for different concentrations of the reactants. Hence a graph is plotted . With maximum three trials of reactions, execute a linear regression on each of the graphs to obtain a slope representing each reaction. The slope of the reaction denotes the initial reaction rate.

Reaction rates of various concentrations of Iron (III) Chloride and Potassium Iodide

As from the above graph, the average rate of reaction is calculated by knowing the slope of the

Graph from the first and the last point in each function. Also instantaneous rate of reaction is

Determined by finding the slope of the graph between any two points say between two data

points like 0 to 10 seconds.

EXAMPLE 2:

Consider the reaction

2I- + 2Fe3+ (aq) èI- + 2Fe2+

The concentration of the reactants I- and Fe3+will be varied accordingly .and to measure the rate

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Of reaction, we will measure the effect of the changes in solution absorbencies using a visible

Spectrophotometer. After measuring the absorbance at different intervals of time, plot average

absorbance on y axis and time on x axis and We will get a straight line graph. Calculate the slope

of graph.From the slope and concentration data, results can be extracted.

The slope of (absorbance vs. time) curve in proportional to the rate of reaction.

We will also determine the rate constants To determine the reaction order with respect to reactants in the reaction, we will measure absorbance vs. time. We first need to relate the integrated rate law, in terms of concentrations, to the measured absorbance values. We will use the relationship between concentration and absorbance of Beer's Law:

A = c L

The absorbance of light at a given wavelength is defined as the sum of the absorbance of the different reactants or products (substances) in solution. By combining this according to the Beer's Law and according to the stoichiometry of the reaction, it can be shown that

Where A0 is the initial absorbance, at time=0, and A is the final absorbance, at infinite time. also c is the concentration of reactant at time t and c0 is the initial concentration of reactant .By substituting the above expression into the integrated rate law gives

This above equation gives us the result that how to use the absorbance vs. time data to determine the rate constant for a particular reaction. When we draw a plot of ln [A - A / A0 - A] vs. time we will have a straight line with slope -k, for a first order or pseudo first order reaction, if we want to determine the reaction rate constants successfully, our measurements will need to include the initial absorbance, A0, and the final absorbance, A.As if we are using pseudo first order conditions , we can easily determine the pseudo first order rate constant k' from the plot.

RESULT:

The rate of reaction and specific rate constant k, can be calculated :

Rate of reaction = change in concentration/time taken

Bt we measure that absorbance/ time as slope

and absorbance is directly proportional to concentration. So by calculating the slope we can easily determine the rate as well as order of the overall reaction.