User:Michaelmazzeo25/sandbox

The purposes of editing the spectrophotometry page on the wikipedia page is in order to add more relevant information to the page which will help in future understanding and research of the topic. In addition the page does not have an experimental application section which I believe would be relevant in terms of understanding the content. Therefore I am adding several minor sentences and details to the Applications in biochemistry section and creating an experimental application section referring to the experiment run in lab involving B-galactosidase.

Applications in biochemistry

Spectrophotometry is an important technique used in many biochemical experiments that involve DNA, RNA, and protein isolation, enzyme kinetics and biochemical analyses. A brief explanation of the procedure of spectrophotometry includes comparing the absorbency of a blank sample, that usually contains the same reacting enzyme, to a sample that contains a colored compound. This coloring can be accomplished by either a dye such as Coomasie Brilliant Blue G-250 dye or by an enzymatic reaction as seen between β-galactosidase and ONPG (turns sample yellow). The spectrophotometer is used to measure colored compounds in the visible region of light (between 350 nm and 800 nm)[9], thus it can be used to find more information about the substance being studied. In biochemical experiments, a chemical and/or physical property is chosen and the procedure that is used is specific to that property in order to derive both quantitative and qualitative data about the sample, such as the quantity, purity, enzyme activity, etc. Spectrophotometry can be used for a number of techniques such as determining optimal wavelength absorbance of samples, determining optimal pH for absorbance of samples, determining concentrations of unknown samples, and determining the pKa of various samples. Spectrophotometry is also a helpful procedure for protein purification[10] and can also be used as a method to create optical assays of a compound. Spectrophotometric data can also be used in conjunction with the Beer-Lambert Equation, A= -log10T=εcl=OD, in order to determine various relationships between transmittance and concentration, and absorbance and concentration. Because a spectrophotometer measures the wavelength of a compound through its color, a dye binding substance can be added so that it can undergo a color change and be measured. [11] Spectrophotometers have been developed and improved over decades and have been widely used among chemists. Additionally, Spectrophotometers are specialized to measure either UV or Visible light wavelength absorbance values. It is considered to be a highly accurate instrument that is also very sensitive and therefore extremely precise, especially in determining color change.[12] This method is also convenient for use in laboratory experiments because it is an inexpensive and relatively simple process.

Experimental Application:

As described in the applications section, spectrophotometry can be used in both qualitative and quantitative analysis of DNA, RNA, and proteins. One experiment that can demonstrate the various uses that visible spectrophotometry can have is the separation of  β-galactosidase from a mixture of various proteins. Largely spectrophotometry is best used to help quantify the amount of purification your sample has undergone relative to total protein concentration. By running an affinity chromatography you can isolate B-Galactosidase and this can be tested by reacting collected samples with ONPG and determining if the sample turns yellow. [cite textbook] Following this testing the sample at 420nm for specific interaction with ONPG and at 595 for a Bradford Assay the amount of purification can be assessed quantitatively. [cite textbook] In addition to this spectrophotometry can be used in tandem with other techniques such as SDS-Page electrophoresis in order to purify and isolate various protein samples.