Transmittance relates the intensity of light that enters a sample to the intensity of light that exits the sample. The reduction in light is due to the absorbance of light by the sample.
Absorbance is the amount of light absorbed by the sample. Absorbance increases as concentration increases and transmittance decreases. Calculated by: A=-logT. Can be related to concentration using Beer's law.
A spectrophotometer is used to quantitatively determine how a sample is interacting with a specific wavelength in light. For more info, follow link:
Serial Dilutions are used to make several "standards" of known concentration from the same stock concentration. This allows for a calibration curve to be made. The dilution formula is used to determine the concentrations of the standard solutions.
Blank ing the spectrophotometer allows the machine to reset its 100% T value. A blank is made by adding everything in your sample EXCEPT what you are trying to test (in our case, phosphate). Note that whatever solvent you put your sample in, you must use for your blanking measurements.
The Color Wheel is used to determine what wavelength of light is most optimum for obtaining the highest absorbance values is a sample. For example, if a sample transmits red light (appears red), then that sample is absorbing light the most in the green region (the color directly across from red in the color wheel), and a wavelength between 480nm and 560 nm should be used.
Beer's law relates absorbance to concentration. A=EbC where A is absorbance, Epsilon is the molar extinction coefficient, b is the path length, and C is the concentration. A calibration curve must be made if epsilon is unknown
Calibration Curves are made by graphing the concentration of standard solutions on the x-axis and their corresponding absorbance on the y-axis. By inserting a line of best fit, a y=mx+b equation can be obtained (where y is A, m is Eb, and x is C). This y=mx+b equation can be used to find the concentration of solutions by plugging in the absorbance obtained for the sample into the equation.
