# Path length and absorbance relationship advice

### electromagnetism - Light's wavelength and pathlength - Physics Stack Exchange

If you use absorbances, you get the nice Beer-Lambert law, which states the concentration and path length are proportional to absorbance. (With this knowledge, it is possible to use this relation for transmittance: T Absorbance is linearly proportional to concentration (c) of substance and to thickness of layer (l), which is the length of optical path or width of cuvette ( usually 1 cm). . Safety advice For work with solutions of malachite green and indigotin wear. Module 2. Determination of an Unknown NADPH Concentration Using Absorbance Spectrophotometry you have values for absorbance, path length, and the extinction coefficient, you can calculate the Common Mistakes and Some Advice.

It is specific for every individual substance and wave length.

**Beer's Law Laboratory**

With this knowledge, it is possible to use this relation for transmittance: Absorbance is linearly proportional to concentration c of substance and to thickness of absorbing layer lwhich is the length of optical path or width of cuvette usually 1 cm. Spectroscopic properties in visible VIS and ultraviolet UV are studied in spectroscopy and photometry. They use light the UV area wavelength nmvisible areas wavelength nm and adjacent infrared areas wavelength nm - 1 mm.

This can be done using two different methods: All samples have different concentrations but are placed in the same cuvettes and the same wave length is used to measure them. The resulting dependence should be in ideal case linear. However, this method is only viable for simple matrix such as fresh water. Thereafter the concentration of the substance in the sample is increased by the defined standard addition and corresponding absorbance of the solution is measured.

This method is used in complex matrix such as wastewater, where application of calibration curve method would be quite difficult. The formula of the linear regression equation is based on the Lambert-Beer law. It contains two variables x, y and two regression coefficients a, b. Both variables can be easily replaced with the magnitude from the graph of the calibration curve which expresses the concentration of the absorbance on the concentration which means that x means concentration c and y absorbance A.

Regression coefficient a expresses possible systematic error of measurement or deviations caused by dirt on cuvette or similar.

Concentration is in both expressions thus, we can say that coefficient of regression b is equal to multiplication of molar extinct coefficient and width of cuvette: This element expresses slope of a line in regression equation. Devices which measure in one or more specifically defined wavelengths of monochromatic lights are called photometers. More technically advanced devices where you can set or measure any wavelength of monochromatic light are called spectrophotometers.

### Spectrophotometry Practical - WikiLectures

They measure intensity of light after going through sample I and put it in ratio with intensity of light before going through sample I0.

Based on mentioned relations it is possible to determine transmittance, absorbance, and sample concentration. The range of wavelengths depends on the slot — it can be changed or permanently preset from the factory. The wider the slot is the more intense the resulting light coming out of the machine but this causes less specific measurements.

## Beer-Lambert Law

Increased time results in increased accuracy of the measurement, except for photosensitive substances i. Disadvantages of longer integration time are also longer measurement times, which is very important when processing a larger number of samples, when using larger number of wavelengths i.

Double-beam spectrophotometers use one beam for measuring the sample, and the other for blank solvent without the substance. When using the single-beam device we must first measure attributes of blank and then measure the sample.

Modern spectrophotometers are fully automatized and controllable via computers. They are used for measuring absorbing specters wider spectrum of wavelengths or quantitative measurement for one or more wavelengths.

They can also be used for measuring kinetics of simple, for example enzymatic reactions. In practice the validity of Lamber-Beer law is limited by dispersion of light because of tiny impurities in the sample; phosphorescence or fluorescence of the sample; small amount of light passing through highly concentrated solutions; changes of values of absorbing coefficients; shifting chemical equilibrium caused by high concentration of substances in the sample.

Their ability to measure concentrations of metabolically important substances in body fluids, such as blood, cerebrospinal fluid, urine and amniotic fluid among others, is crucial for correct diagnostic findings and continuous monitoring of patients. Equally early identified small deficiencies of certain essential substances might help preventing correlated health problems.

In intensive medicine the use of spectrophotometric analyses is more frequent, due to the fact that patients in unstable states are more prone to drastic changes in the amount of different substances in, for example, their blood.

## Spectrophotometry Practical

The official derivation relies on calculus, but you can get a sense of things by thinking of the solution not as a single thing, but as a succession of "slabs": Take a single thin slab of a colored material. This absorbs a certain amount of light at a given frequency. But it doesn't absorb a fixed amount of light. Instead, each photon that passes through has a certain probability of encountering an absorbant molecule and being absorbed.

The more light, the more absorbed. But what happens if you start varying the width of the material. Or equivalently, if you stack several of such slabs together.

The second slab would only "see" half the original light, and would then absorb half of that light. So the amount being transmitted out the back of the second slab would be only 0.

Note the necessity of converting from percentages to fractions to do the calculation here. Okay, but how about varying concentration?