**Data Collection**

*Estimating Concentration form Absorbance*

__Lambert-Beer Law__

Lambert-Beer
Law is used to determine concentration [c] from Absorbance [A], if the molar
extinction coefficient [e] of a compound and the distance light passes through
the solution are known. The formula is:

A = ecl

Thus, concentration can be calculated by: c=A/el

Example: the molar extinction coefficient for betacyanin is approximately 38,000 L/mole-cm, the path length for the Spectronic 20 spectrophotometer is 1.2cm. If the Absorbance of beet betacyanin extract is 1.0, then

c = 1.0 A /38,000 L/mole-cm X 1.2cm

c = 1.0 A /45,600 L/mole (use this value for your data)

c = 2.193 x10 eM^{-5}

or 22 microM (mM)

ShortCut to finding betacyanin concentration inmM

c = A_{530}/0.0456

For any given solute, the amount of light absorbed is proportional to the concentration of the solute in the solution.

[ ] = Concentration Units: 1 mole/l = 1M, molar

__Making
solutions of known concentration using the mass balance principle
__In order to construct a standard curve, it is necessary to make a
series of reference solutions of known concentration. Usually such solutions are
prepared from a stock solution using the formula:

**C1V1 = C2 V2**

where,

C1 = the concentration of the stock
solution V1 = the volume of stock solution used

C2 = final concentration of diluted stock
solution V2 = final volume of diluted stock
solution

If one knows the stock concentration C1 and the final concentration of diluted stock solution C2 and the final volume of diluted stock solution V2, then one solves for V1. Remember that V2 is greater than V1, so to bring V1 to the final volume =
V2, a
volume of solution usually distilled water, dH_{2}O, must be added to dilute
the stock solution to a lower concentration.

Thus, V2 = V1 + VdH20. The VdH20 can be calculated by subtraction,

V2 -V1 = VdH2O.

Try these problems to test your understanding of mass-balance [Review Mass Balance]

__Constructing
a table of dilutions__

Constructing a table of dilutions for all volumes and concentrations will help in
preparing your standard curve. Try this technique to form the following solutions.

**Example**

The stock solution is 80% ethanol, EtOH (C1), the diluted stock solutions are 50% (C2) and
10% (C2) EtOH, each has a final volume (V2)of 10 mL.

Table of Dilution for a 100mM betacyanin stock solution

C1 |
V1 |
C2 |
V2 |
VdH |

80% |
40% |
10 mL |
||

80% |
10% |
10 mL |

__To calculate V1 and VdH _{2}O to make 50__

1. Solve for V1

V1=C2/C1*V2, thus V1= 40% / 80% * 10mL

V1= **0.5** * 10mL = 5mL of 80% EtOH (C1)

2. Solve for VdH_{2}O to adjust the final volume to 10mL by
subtracting: V2-V1 = VdH_{2}O.

10mL - 5mL = 5mL dH_{2}O.

Thus, a 40% EtOH solution can be made by adding 5mL of
80% EtOH to 5mL of distilled water. Does this make sense? Sure a 40% solution is
half, or **0.5**, less concentrated than a 80% solution, so
combining equal volumes of 80% EtOH and dH_{2}O should result in a
solution half as concentrated.

__To calculate V1 and VdH _{2}O to 10mM
betacyanin __.

1. Solve for V1

V1=C2/C1*V2, thus V1= 10% / 80% * 10mL

V1= **0.125** * 10mL = 1.25mL of C1

2. Solve for VdH_{2}O to adjust the final volume to 10mL by
subtracting: V2-V1 = VdH_{2}O.

10mL - 1.25mL = 8.75mL dH_{2}O.

Thus, a 105 EtOH solution can be made by adding 1.25mL of 80% EtOH to 8.75mL of distilled water.

Constructing a dilution table of all volumes and concentrations will help in preparing your ethanol concentrations.

C1 stock conc. EtOH |
V1 |
C2 |
V2 desired final vol. for expt. |
VdH2O vol. of water to add for V2. |