Like we measure DNA at 260nm and protein in 280nm.So, here my question is that, How a specific nanometer measure a molecules? .I just wanted to know its mechanism or reason of using a specific nano-meter towards measuring a molecules correctly.
1. One of the most important issues in scientific studies is being able to know how much of a reagent one actually has in an experiment. Certain reagents can be weighed out before they are dissolved in clearly measured volumes of solvent. Other reagents, for a variety of reasons, need to be measured while they are fully dissolved. This is usually the case for DNA, RNA or protein. Because of this, a way is sought to determine objectively what the concentration of a particular protein (for example) is in a solution.
2. All molecules absorb energy from their environment. The simplest form of absorption is in the form of heat, or kinetic energy. When energy is absorbed it affects the behavior of the molecule in a variety of ways, potentially also including the shifting of some electrons to higher energy levels. When this happens some amount of light energy of specific wavelength is absorbed. The particular wavelengths thus affected are largely determined by the properties of the absorbing molecules. Many times we view entire absorbance spectra to see the characteristic absorbance patterns. Very few molecules have only one absorbance peak, because there are usually a variety of chemical bonds whose electrons can be involved. We usually look for the particular distinctive peaks that will be characteristic of what we are trying to measure. Hence the specific wavelengths for the absorbance measurements.
3. Most proteins include at least 1 Tryptophan (Trp), an amino acid with a characteristic absorbance peak at 280nm, in aqueous solution. Needless to say, trying to measure a concentration of something in the presence of impurities is likely at least inaccurate.
4. DNA, in aqueous solution, absorbs around 250-260nm. Unfortunately, so do Phenylalanine (Phe) and Tyrosine (Tyr). Thus, trying to determine DNA levels by measuring absorbance at 260nm would be reasonable only if the proteins (containing Phe or Tyr) and other impurities were already separated away.
5. There is a specific relationship for each particular molecule, represented by the Beer-Lambert law: A=εcl, where these represent absorbance, molar extinction coefficient, concentration and path length, respectively. The determination of protein extinction coefficients is described by von Hippel (1989) Anal.Biochem.182(2):319-326. Cuvettes are usually chosen with path lengths of 1 cm. Based on this, the concentration of a particular protein can be determined by simply measuring absorbance of the solution and dividing it by the extinction coefficient.
In basic terms, spectrophotometers work by passing light through a sample. Some molecules are able to absorb light at certain wavelengths. Using your example, DNA absorbs light at 260nm..thus, when you pass light at 260nm through the sample, DNA absorbs some of it and the machine will detect how much light "isnt" reaching the detector (due to absorbtion) and translate that into a value. Basic description but you get the idea.
1. One of the most important issues in scientific studies is being able to know how much of a reagent one actually has in an experiment. Certain reagents can be weighed out before they are dissolved in clearly measured volumes of solvent. Other reagents, for a variety of reasons, need to be measured while they are fully dissolved. This is usually the case for DNA, RNA or protein. Because of this, a way is sought to determine objectively what the concentration of a particular protein (for example) is in a solution.
2. All molecules absorb energy from their environment. The simplest form of absorption is in the form of heat, or kinetic energy. When energy is absorbed it affects the behavior of the molecule in a variety of ways, potentially also including the shifting of some electrons to higher energy levels. When this happens some amount of light energy of specific wavelength is absorbed. The particular wavelengths thus affected are largely determined by the properties of the absorbing molecules. Many times we view entire absorbance spectra to see the characteristic absorbance patterns. Very few molecules have only one absorbance peak, because there are usually a variety of chemical bonds whose electrons can be involved. We usually look for the particular distinctive peaks that will be characteristic of what we are trying to measure. Hence the specific wavelengths for the absorbance measurements.
3. Most proteins include at least 1 Tryptophan (Trp), an amino acid with a characteristic absorbance peak at 280nm, in aqueous solution. Needless to say, trying to measure a concentration of something in the presence of impurities is likely at least inaccurate.
4. DNA, in aqueous solution, absorbs around 250-260nm. Unfortunately, so do Phenylalanine (Phe) and Tyrosine (Tyr). Thus, trying to determine DNA levels by measuring absorbance at 260nm would be reasonable only if the proteins (containing Phe or Tyr) and other impurities were already separated away.
5. There is a specific relationship for each particular molecule, represented by the Beer-Lambert law: A=εcl, where these represent absorbance, molar extinction coefficient, concentration and path length, respectively. The determination of protein extinction coefficients is described by von Hippel (1989) Anal.Biochem.182(2):319-326. Cuvettes are usually chosen with path lengths of 1 cm. Based on this, the concentration of a particular protein can be determined by simply measuring absorbance of the solution and dividing it by the extinction coefficient.
Another good resource on this:
http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/beers1.htm
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