- Spectrophotometry
Lab Goals
Using the standard curve, Estimate the Protein concentration of unknowns

Lab: Introduction - We have learned how Spectrophotometry can be used to measure the absolute or relative concentration of a characterized substance in solution. We have also constructed a standard concentration curve from known concentrations. Be sure and have the standard concentration curve you constructed. Next, we will use that standard curve to determine the unknown protein concentration of two solutions:

Sample 1 and Sample 2

Procedures
This part of the lab requires that you have constructed a standard curve and kept your data.

Determine and record the absorbance and transmittance values at 595 nm, successively, for the different unknown solutions (Sample 1 and Sample 2). Using the graph area below, recreate your standard curve, then clearly indicate the absorbance values of your unknowns.

The unknown protein concentrations can be determined from the standard curve by drawing a horizontal line on the graph parallel to the X-axis and through the point on the Y-axis which corresponds to the absorbance. This line will intersect the standard curve; at this intersection, a vertical line is dropped to the X-axis and the concentration read from the X-axis.

example Graph

Two factors are important in determining unknown or relative concentrations from a standard curve. The absorption value of the unknowns should be within your standard curve values (higher than the lowest & lower than the highest) and your standard curve should have a nice linear correlation (the values should lie close to your best fit line.

Now, using your standard curve, determine the protein concentration of Sample 1 and Sample 2.

See another Example

Questions to answer:

Name___________________Section__________________Lab 1

(1 pt. for each Protein Conc., 0.5 pts. for each Absorbance value):

Enter data from today Protein Concentration Absorbance
Sample 1  
Sample 2  


Plot your standard curve using your data from the previous page, include the "best-fit" line and clearly indicate (plot) your unknown values as Absorbance vs mg/ml (you should now be able to determine the protein concentration of the two unknows).

Print this page with the graph and your data to turn in. (Click the graph to open a new graph page that will print without being split.) The graph must be drawn by hand and is worth 2.5 pts:

spec2a.swf

Ho Print this page, along with the graph page, and you are done. (Don't forget the extra blank copies for use in class.) me