chromatography 1

Chromatography of Photosynthetic Pigments Study Notes

Lab Overview

• Purpose Statement: The objective of this lab is to utilize paper chromatography to separate and characterize the photosynthetic pigments present in spinach leaves (Spinacia oleracea). This method will allow us to visualize the different pigments and their absorption properties.

• Hypothesis Development:
    - Predict the separation order of pigments based on polarity:
      - More polar pigments (like xanthophylls) will move slower compared to non-polar pigments (like β-carotene) on the chromatogram.
    - Predict light wavelengths reflected by pigments:
      - Based on color, hypothesize wavelengths that will be reflected by each pigment as the absorbed wavelengths correspond inversely to reflected wavelengths.

Background Information

• Chromatography Definition:
    - Chromatography is a technique used for separating molecules in a mixture based on their distribution between a stationary phase and a mobile phase.

• Principles of Paper Chromatography:
    - Separation is based on the differential solubility of pigments in the mobile phase (organic solvent) and stationary phase (water in filter paper).
    - Movement of Pigments: More soluble pigments travel further up the paper with the solvent. Those more soluble in water will show less movement and remain closer to the application origin.
    - Rf Value Calculation:
      - Defined as:
        $Rf = \frac{\text{distance traveled by the solute}}{\text{distance traveled by the solvent}}$
      - Interpretation: Values close to 1 indicate higher solubility in the organic solvent.

• Spectrophotometry Overview:
    - A spectrophotometer quantifies the amount of light absorbed by a sample at specific wavelengths, allowing for the characterization of pigments after extraction via chromatography.

Procedures

Preparation of Chromatography Jar

1. Solvent Preparation:
      - Mix 9 volumes of petroleum ether with 1 volume of acetone.
      - Pour the solvent to a depth of 1-2 cm in the chromatography jar.
      - Cover the jar for 30 minutes for saturation with solvent vapor (to be done in a fume hood).

Preparation of Photosynthetic Pigment Extract

1. Prepare an ice bath and chill mortar and acetone.

2. Select and prepare spinach leaves:
      - Remove large veins and weigh out 3.0 grams of leaf tissue.
      - Chop tissue finely using scissors and add to ice-cold mortar with 15 ml of chilled acetone and sand.
      - Grind for 1 minute until a homogenous mixture is achieved.

3. Transfer the mixture into a 50-ml conical tube and shake; allow to settle on ice for 10 minutes.

4. Extract a small volume of the dark green liquid into a beaker for chromatography.

Preparation of Chromatography Paper

1. Obtain and handle Whatman 3MM chromatography paper only by edges with gloves.

2. Draw a pencil line 1.5 cm from the bottom of the paper, ensuring it is above solvent level in the jar.

3. Apply 10 streaks of the pigment extract along the pencil line using a capillary tube, allowing each to dry before the next.
      - Maintain line thickness at 6-7 mm maximum.

Chromatography Process

1. Bend the top of the chromatography paper over the chromatography jar.

2. Allow chromatogram development for approximately 10-15 minutes, monitoring solvent front without timing.

3. Remove the paper just before solvent reaches the end, marking the solvent front immediately.

4. Allow the chromatogram to dry, marking each pigment band's position consistently.

5. In the lab notebook, document band colors, distance traveled from origin, and solvent front distance.

6. Calculate and record Rf values for each pigment in a data table.
       - Use the Rf formula:
         $Rf = \frac{\text{distance pigment traveled}}{\text{distance solvent front traveled}}$

Elution of Pigments and Spectrophotometric Analysis

1. Turn on the spectrophotometer and allow self-tests to complete.

2. Label small test tubes for each pigment (Chl b, Chl a, xanthophylls, β-carotene, blank).

3. Cut pigment bands from chromatogram and place in respective labeled tubes (lutein and violaxanthin in xanthophyll tube).

4. Add 4.0 ml acetone to all tubes and seal with parafilm, allowing elution for 5 minutes.

5. Remove paper and seal tubes after elution time.

6. Set up spectrophotometer:
      - Select Spec 200E Modern Interface and switch to Scan mode.
      - Set measurement mode to absorbance (ABS), wavelengths to 350 nm and 750 nm as low and high respectively.

7. Zero the spectrophotometer using 4 ml acetone in the blank tube.
      - Wait for the device to display performing auto zero.

8. Scan each pigment tube, recording absorbance at specified wavelengths from Table 2 in the lab notebook.

9. If necessary, re-blank before each new spectrophotometric scan.

Data Tables

• Table 1: Rf Values for Spinach Pigments
    | Color or Pigment | Distance from Origin (mm) | Rf value |
    |--------------------------|---------------------------|----------|
    | Solvent | NA | NA |
    | β-carotene | | |
    | Lutein | | |
    | Violaxanthin | | |
    | Chlorophyll a (Chl a) | | |
    | Chlorophyll b (Chl b) | | |

• Table 2: Absorbance Readings for Photosynthetic Pigments
    | Wavelength (nm) | Chl b | Chl a | Xanthophylls | β-carotene |
    |------------------|-------|-------|--------------|------------|
    | 400 | | | | |
    | 410 | | | | |
    | 420 | | | | |
    | 430 | | | | |
    | 440 | | | | |
    | 450 | | | | |
    | 460 | | | | |
    | 470 | | | | |
    | 480 | | | | |
    | 490 | | | | |
    | 500 | | | | |
    | 520 | | | | |
    | 540 | | | | |
    | 560 | | | | |
    | 580 | | | | |
    | 600 | | | | |
    | 620 | | | | |
    | 640 | | | | |
    | 645 | | | | |
    | 660 | | | | |
    | 663 | | | | |
    | 680 | | | | |
    | 700 | | | | |

References

• Bregman, A. (2002). Laboratory Investigations in Cell and Molecular Biology, Fourth Edition. Wiley.