NAME: _______________________________

Biology 244 Standard Curve Activity

As the concentration of a substance increases, its absorbance will also increase. The exact relationship
between absorbance and concentration for a particular substance is determined by creating a standard
curve. To create a standard curve, first measure the absorbances of a series of samples with known
concentrations using a spectrophotometer (this data is provided for you below). Then, graph each data
point with concentration on the x-axis and absorbance on the y-axis. Create a line of best-fit. Excel
instruction are on the last page. Now you can use interpolation to estimate the concentration of an
unknown sample by measuring its absorbance (absorbance values also provided below). Using the
y=mx+b doesn’t always give answers that make sense for this worksheet; therefore, check your answer
on the graph and if needed just look at the graph and make a best guess estimation of where the
unknown falls. Report your answers on the second page.

1. Examine the following absorbance values for known concentrations of bovine serum albumin:

Concentration (g/ml) 1.5 1.0 0.75 0.5 0.25 0.125 0.025

Absorbance 5.4 4.1 3.3 2.3 1.5 0.8 0.4
Draw or use EXCEL to create a standard curve with the data. Cut and paste your figure in the space
below. Include all labels including axes, the equation for the line, and the squared correlation
coefficient (r2).

Answer questions 2 and 3 on the following page.

2. Here are duplicate absorbances for unknowns #1 and #2 run in the same assay as in question 1:

Unknown #1: 3.1

Unknown #1: 3.7

Unknown #2: 0.9

Unknown #2 1.2

Use the mean of the unknowns to estimate the protein concentration using the standard curve you
created. If y=mx+b doesn’t give an answer that makes sense (check it against the graph) just chart the
unknown and make a best guess estimate of the concentration.

Estimate the concentration of protein in Unknown #1: __________________

Estimate the concentration of protein in Unknown #2:___________________

3. What was your R-squared value? ________________________

What does this tell you about the strength of your linear regression?
EXCEL Instructions:
Head column A as concentration (units) and list all data underneath.
Head column B as absorbance and list the data provided underneath.
Highlight both columns, including the heading.
Go to insert:

Under the charts section select scatter plot .

You should get a graph with concentration on the x-axis and absorbance on the y-axis.
Beside the graph, select the + symbol and under chart elements select axis titles and trend line.

On the graph, select the axis titles to label them (make sure to include units). Also change the title.
Go back to the chart elements (see photo above), click on trendline, and select more options.

At the bottom, select to display the equation and R squared value.

Select the R-squared and equation on the chart to move them to a good location on the graph.