CH 150: Introduction to Biochemistry

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Introduction: Pipetting and Beer's Law

The objectives of this experiment are as follows:

 1. to learn the operation of the spectrophotometers in the laboratory;

2. to learn how to use the micro-pipettes

2. to determine the visible absorbance spectra of a colored solution;

3. to determine the relationship between concentration and absorbance by producing a standard curve;

4. to calculate extinction coefficients and determine the concentration of an unknown solution.

Introduction:

            UV-Visible light spectroscopy is an analytical method used to characterize a light-absorbing material (chromophore) in a sample.  Based on their unique molecular structures, all materials absorb energy in the electromagnetic spectrum.  Some materials absorb energy in the visible light range, and thus appear colored to the eye.  The wavelength at which given amount of a material absorbs light is the absorbance maximum, or ”l_max”.   The extent to which a material absorbs light at any wavelength,  but especially at the l_max, can be used to identify the material and its concentration in solution.

         The extent to which a material absorbs light of a given wavelength is dependent on the concentration of the chromophore in solution.  The relationship is linear up to a concentration threshold, beyond which the relationship is no longer linear.  If the absorbance of light at a specific wavelength is measured for samples containing increasing concentrations of a chromophore, one will find a direct linear relation between the two, with the absorbance increasing as concentration increases, as shown in the figure below.

           A = b c E

where A = absorbance

            b = optical path length (cm)

            c = concentration (M)

E = extinction coefficient (M^-1 cm^-1)

 Assuming a 1 cm pathlength cuvette, the slope of the plot above is the extinction coefficient (E). While E is normally calculated and reported using l_max, extinction coefficients can be calculated using any wavelength at which a compound absorbs light.

Experimental Procedure:

Become familiar with the various controls on the spectrophotometers in the lab.

         You will be given a stock solution containing 0.05 mM  (5X10^-5 M) DNP-glutamate in 0.1 M Tris-Cl. Prepare 4 mL of 25 mM  (2.5 X 10^–5 M) DNP-glutamate using 0.1 M Tris-Cl buffer.

 Describe how you accomplished this:____________________________

____________________________________________________________

___________________________________________________________

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A.   l_max Determination

 mM DNP-glutamate solution (solution 1). Put the "blank" cuvette into the cuvette holder, set the wavelength to 350 nm, and set the absorbance to read 0.000.  Remove the blank and insert the cuvette containing the DNP-glutamate solution; measure and record the absorbance value.  Repeat this procedure, increasing the wavelength in 10 nm intervals, to 500 nm, blanking the absorbance and reading the absorbance of the DNP-glutamate solution at each interval.  The wavelength at which the absorbance is maximum is the l_max.

• Using the data from Part A, plot absorbance (y-axis) versus wavelength (x-axis) for DNP-glutamate. Determine the l _max for your sample.

                    l _max __________

B.   Reproducibility of Absorbance Readings vs Reproducibility of Pipetting

       Measure the absorbance of the 0.05 mM DNP-glutamate solution five (5) times at l_max. Place 4 mL of the sample in one cuvette, and read that sample 5 times (use Tris-Cl as the blank). Calculate the mean, standard deviation, and relative standard deviation for these five values using the following equations:

where å x_I  is the sum of the individual values and n is the number of observations.

  relative standard deviation (RSD) = (SD/X_m) *100%

             Dilute 0.5 mM (5X10^-4 M) DNP-glutamate solution by a factor of 10.  Such a dilution is a 1 to 10 (1:10) dilution.  The concentration of the diluted solution is 0.05 mM.  Prepare five (5) replicates of this dilution.  Determine the absorbance of each replicate at the l_max for DNP-glutamate. Calculate the mean, standard deviation, and relative standard deviation (RSD) for these 5 replicates.

How does your ability to prepare replicate samples compare with your ability to replicate absorbance readings of a single sample?_______________

__________________________________________________________

__________________________________________________________

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C.   Determination of an Unknown solution of DNP-glutamate

         Using the 0.05 mM solution of DNP-glutamate in 0.10 M Tris-Cl buffer, pH 8.0, make 5 mL solutions containing 0.01, 0.02, 0.03, 0.04, and 0.05 mM DNP-glutamate solution.  Use 0.10 M Tris-Cl buffer, pH 8.0 as the diluent.  Measure the absorbance of these solutions at the l_max for DNP-glutamate by placing 5 mL of each dilution per cuvette.  Use the 0.10 M Tris-Cl buffer, pH 8.0 buffer alone for a blank.

• Plot absorbance (y-axis) versus DNP-glutamate concentration (mM) at l_max. Plot the data using only data points (do not connect them with a line).   Add a linear trendline. Be sure to print the equation of the line of the plot and also include the correlation coefficient as well. 

• Calculate the Extinction Coeffiecient.

                E =      _____________ mM^-1cm^-1

_____________  mM^-1cm^-1

                        ^{_______________} M^-1cm^-1

        Dilute your DNP-glutamate unknown with 0.10 M Tris-Cl buffer, pH 8.0 until its absorbance at l_max  is within the linear range of the standard curve.  Measure the absorbance of this diluted solution.  Calculate the concentration (mM) of DNP-glutamate in the original unknown solution (undiluted solution) using the equation from the linear trendline from the standard curve.

 Unknown letter_______________

Concentration of DNP-glutamate in unknown ________________________