Quantitative Pipetting and Spectrophotometry

In this exercise we will use a spectrophotometer to monitor our pipetting technique.

Spectrophotometers are instruments that measure the wavelength and amount of light that are absorbed by molecules in solution. According to the Beer-Bouger Law there is a relationship between a solution's concentration of suspended particles or dissolved molecules and the solution's ability to transmit light.

The terms percent transmittance and absorbance are used to describe spectrophotometric analysis. Percent transmittance (T) is the ratio the amount of light passing through the sample to the amount of light applied to the sample multiplied by 100.:

% T = [(light passing through the sample) / (light shined on sample) ]X 100

Absorbance (A) or Optical Density (O.D.) is directly proportional to concentration of solute and it represents the logarithm to the base 10 of the reciprocal of the transmittance. O.D. can be derived from % T according to the following formula:

O.D. = 2 - log₁₀ of % Transmittance

***Example: If the % T of one of your dilutions is 69.5 then:

O.D. = 2 - log of 69.5

= 2 - 1.8420

= 0.158

*** Remember that the mantissa, the number behind the decimal, is derived from a log table. You can determine the characteristic, the number in front of the decimal, by converting the number you are trying to get the log of into scientific notation then use the exponent on the 10 as the characteristic of the logarithm.

For example, the characteristic of 253 is 2 since 253 = 2.53 x 10².

The characteristic of 2.53 is 0 since 2.53 = 2.53 x 10 ⁰. The mantissa in both cases would be 4031. Of course you can always use a calculator.

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EXPERIMENT

In this exercise you will practice your pipetting technique by accurately pipetting a pink, cobalt chloride solution into a series of test tubes containing accurately measured amounts of distilled water. In this manner you will be gradually and predictably diluting the dye concentration in the water.

This type of dilution technique is called a serial dilution and variations of this technique are widely used in microbiological and immunological examinations of many types of specimens. (For example see figure 6.15 on page 172 of Tortora et. al.)

A spectrophotometer will allow you to measure the precision of your technique. Since the intensity of the color as well as the Optical Density of the solution is directly proportional to the concentration of the cobalt chloride in that solution, and since you are precisely measuring each dilution in the dilution series; then you should be able to predict the O.D. of each dilution in the series if you know the O.D. of the initial, undiluted solution.

*** Example:

The original solution had an O.D. of 1.6.

1.) If you took 1.0 ml of the original solution and mixed it with 1.0 ml distilled water, you would now have 2.0 ml of solution and this diluted solution should have and OD of 0.8 since you performed a 1 to 2 dilution.

Another way of saying this is that you have divided the solute concentration in half by performing this dilution.

2.) If you took 0.1 ml of the original solution and mixed it with 0.9 ml dH₂O, you would now have 1.0 ml solution and the diluted solution should have an O.D. of 0.16 since you performed a 1 to 10 dilution (1 part original plus 9 parts dH₂O equals 10 parts total). You have 1/10th the original solute concentration in the dilute solution.

MATERIALS

Spectronic 20 set at 510 nm and warmed up for 20 minutes

Acidic solution of 1% cobalt chloride in water

13 x 100 test tubes without side markings

1 ml and 5 ml pipettes

250 ml beakers with water

pipette pumps

METHODS

Before you do these experiments, please watch the quicktime videos or realvideos on spectrophotometry at this site!

Choose one of the following options to view a real video tutorial film

28.8 K modem           28.8 K modem large screen

56 K modem              56 K modem large screen 

T1 connection             T1 connection large screen 

Or watch the tutorial as a Quicktime movie (16 MB) - Click Here

 

EXPERIMENT 1:

• Set up a rack with 4 small test tubes. Number them 1, 2, 3, 4 with small numbers up near the top of the tube.

   

  • Pipette 3.0 ml water into tube 1
  • 2.7 ml water into tube 3
  • 2.7 ml water into tube 4.
  • Pipette 3.0 ml cobalt chloride into tube 2.

• Pipette 0.3 ml from tube 2 into tube 3. Pipette up and down several times to mix and then pipette 0.3 ml from tube 3 into tube 4. Pipette up and down several times to mix.

• Wipe off the tubes with Kim Wipes and measure %T and the O.D. of each tube. Tube 1 is the "blank" therefore use it to Zero the instrument as demonstrated in the videos.

EXPERIMENT 2

• Set up a rack with 6 small test tubes. Number them 1, 2, 3, 4, 5, 6 with small markings up near the top of the tube.

• Pipette 3.0 ml water into tubes 1, 3, 4, 5, and 6. Do not pipette any water into tube 2.

• Pipette 6.0 ml cobalt chloride solution into tube 2.

• Pipette 3.0 ml color solution from tube 2 into tube 3. Pipette carefully up and down several times to mix.
• Pipette 3.0 ml from tube 3 into tube 4, mix again and continue in a similar manner thru to tube 6.

• Wipe off the tubes with Kim Wipes and measure %T and O.D. of each tube.
• Tube 1 is the "blank" therefore use it to Zero the instrument as demonstrated in the videos.

ANALYSIS

Construct a table with the following data:

Exp # 1

Exp # 2