A spectrophotometer is used to determine the concentration of certain compounds, such as protein, in a solution. In general, a light shines through a cuvette filled with a sample. The amount of light that is absorbed by the sample is measured. Since compounds absorb light in different spectral ranges, the right wavelength must be set for the analysis. There are numerous ways to calculate the concentration of unknown samples through spectrophotometry, however using reference standards gives the best accuracy. Furthermore, standards also indicate if the spectrophotometer is malfunctioning or other problems with the analysis.
Things You'll Need
- Reference standards
- Unknown samples
- Excel, spreadsheet, or graphical software
Calculating through Straight Line Equations
Analyze reference standards at the beginning of the analysis. Standards are known concentrations and are used to calibrate equipment and check accuracy. All samples should fall within the working range of the standards. If not, then samples will need to be diluted or the concentration of standards increased.
Make a scatter chart or line graph with standard concentration and absorbency readings. The concentration will be on the y-axis, the absorbency on the x-axis. For example, the standards are 1 ppm, 2.5 ppm, and 5 ppm. The absorbency given was 1 ppm= .25, 2.5 ppm= .5, and 5 ppm= .75.
Format the trend line to display an equation on the graph. The equation will show the formula y=mx+b. For example, using the standards in step 2 the equation is y= .1224x + 0.1531. Most trend lines are intercepted at zero, but it depends on the analytical method and R-squared value.
Analyze unknown samples and record absorbency readings.
Use the equation (y=mx+b) to determine concentration of samples.
Understanding Straight Line Equation
Let "Y" equal the concentration. This is what will be solved for.
Let "X" equal the absorbency of the sample. This is the absorbency measured by the spectrophotometer.
Allow "" to equal the slope and "b" to equal the y-intercept. Both are given, however if the trend line was forced through 0, then "b" will be 0.
Solve for concentration. Using the example in step 3, substitute "x" as a given absorbency of .563. Therefore:
y= .1224(.563) + 0.1531
y (concentration)= .222011
Tips & Warnings
- The units of the concentration will be the same as the standards. For example, units are parts per million (ppm) or parts per billion (ppb).
- Reference standards and samples are easily contaminated.
- Always read all parts of assay or method before developing.
- Photo Credit spectre angulaire image by Unclesam from Fotolia.com
How to Calculate Concentration With a Spectrophotometer
How to Calculate Concentration With a Spectrophotometer. ... How to Calculate the Calculations for Spectrophotometers; Print this article; Things You'll Need. Pencil;
How to Create Calibration Curves
This example uses a spectrophotometer. Other People Are Reading. How to Make ... Use the graphing program to calculate the regression line...
How to Calculate Concentration Using Absorbance
Calculate the slope, m, of the line according to the formula m = (y1 - y2) / (x1 - x2). ... How...
How to Calculate RNA Concentration
Quantitate your RNA sample by measuring its absorbance of ultraviolet light (UV). A nano-drop spectrophotometer will use only one or two microliters...
How to Calculate HPLC Resolutions
What Are the Disadvantages of HPLC? How to Calculate Dead Time on the LC Column; How Does Paper Chromatography Work, and Why...
How to Use a Spectrophotometer
A spectrophotometer is a device used to measure light at a specific wavelength. ... How to Calculate the Calculations for Spectrophotometers.
How to Make a Graph in MS Excel
Microsoft Excel 2010 is well known for its spreadsheet capabilities and contains numerous built-in formulas to help you calculate everything from simple...
How to Calculate Concentration From Extinction Coefficient
In order to find the concentration ("c") of a chemical in solution using ... from its light transmittance. ... testing and the...
How to Do Spectrophotometer Analysis
Analytical chemists frequently use a spectrophotometer to estimate the ... How to Calculate Concentration Using ... The sample is fragmented ...