# Spectrophotometer concentration and standard curve

### How to calculate unknown concentration from standard curve in excel

Interpreting a Standard Curve A spectrophotometer measures light quantity. The original solution was diluted to prepare these unknowns, however, so you now need to work backwards and calculate the concentration of the original solution based on the dilution factor. Take the equation for the best-fit line from your spreadsheet program and solve it for y by subtracting b from both sides and dividing both sides by m. Photo Credits. To calculate the concentration, you need to compare your reading with readings for standards of known concentration. There are various methods which can be used to measure the glucose concentration but in this experiment a highly specific enzymatic method using the GOD-PAP assay glucose oxidase peroxidase aminophenazone phenol schedule coventry, In other words, unknown 1 will contain 0. Spectrophotometry is a process that uses a sample solution to determine how much light a chemical substance absorbs by measuring the intensity of a beam of light passing through the solution. Cap each of the unknowns with parafilm, and carefully invert to mix. The first portion of the experiment seemed to be a practice 10 fold serial dilution using the chemical bromophenol blue, and was treated as a quantitative experiment. Consult the user's manual for your spreadsheet program for details about how to use the linear regression feature. Try watching the two videos under the Resources section to familiarize yourself with the procedure. An orange which is an excellent source of vitamin C gets its sweetness from natural sugars which are sucrose, glucose and fructose livestrong.

While you can estimate concentration of an unknown from just looking at the graph, a more accurate way to determine concentration to actually use the equation of the line which fits to your data points. For the time being, use the following five concentrations, but remember that you will need to modify these when performing your own experiment: Standard 1: 0.

The wavelength will depend on the type of chemical in your experiment.

Choose five concentrations for your standards. To estimate the concentration of an unknown solution of the analyte, we read the absorbance, and then use the standard curve :.

Add your results together, and divide them by the number of results.

You have now calculated the concentration of the chemical of interest in three of your diluted unknowns. If you input the formula, your spreadsheet program will do the calculation for you. Notice that as concentration increases, absorbance increases as well.

### How to make a standard curve absorbance vs concentration

Calibrate your spectrophotometer. A colorimetric indicator was added to the standards, and a graph was formed. This will give you an average. The wavelength will depend on the type of chemical in your experiment. Using the spreadsheet program, fit a linear equation to this graph. Lift the edge of your finger slightly to let a little solution flow out of the pipet, until you reach your desired volume. If all five fall in the same range as the standards, you can use all five instead, but you need to use at least three.

A line of best fit is then drawn through the points. To create the standard curve, we plot a line graph of Absorbance Y axis vs Concentration X Axis for each of the standards. Using the spreadsheet program, fit a linear equation to this graph.

But in order for the ratio to be accurate the system must of reached equilibrium and temperature must have stayed constant. You want the standard concentrations to be separated from each other by about the same interval -- e.

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