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Plant Detectives Manual: a research-led approach for teaching plant science

Activity 5: Qualitative analyses of photosynthetic pigments by high-performance liquid chromatography (HPLC) — optional

5.1) Introduction and objectives

High-performance liquid chromatography (HPLC) is a technique used to separate, identify and quantify complex mixtures of compounds based on their solubility in a solvent compared to a stationary phase (Fig. 7). The principle is similar to thin-layer chromatography, but instead of running on the surface of a stationary plate, the compounds are separated on a column packed with a stationary phase, and solvent is pumped through the column at high pressure. HPLC provides better resolution of complex mixes than TLC, it is more quantitative, it can be coupled to an absorbance and fluorescence spectrophotometer and it produces reproducible results.

 

In our case, the total sample extract from wild type and mutant plants will be run through a narrow column at high pressure. The pigments are separated via hydrophobic interactions with the column matrix and detected by measuring absorbance. Their retention time, or the time a specific pigment takes to reach the detector, is used to identify each pigment.

 

The main objectives of Activity 5 are to:

  1. isolate total pigments from both wild type and mutant plants
  2. resolve different pigments by using HPLC
  3. identify pigments by looking at the HPLC chromatogram and assess differences of the pigment composition between wild type and mutant samples.

 

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5.2) Materials

  1. micropipettes of 200–1000 microlitres (µl) and tips
  2. 1.5 millilitre (ml) Eppendorf tubes
  3. microcentrifuge (one per class or one per group)
  4. sterile distilled water
  5. vortex (one per class or one per group)
  6. plastic pistil for grinding tissue
  7. liquid nitrogen (–197 °C!!!)
  8. 60% (v/v) acetone: 40% (v/v) ethyl acetate
  9. HPLC vials and liners

 

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Figure 7. Analyses of plant pigments using HPLC

A) Schematic diagram of setup for HPLC. Two solvents (usually one polar and one slightly less polar) are mixed in a pump at certain ratios changing over time. The solvent mixture is pumped onto a column packed with stationary phase. The mixture to be separated is introduced onto the column and the solvents will separate the compounds, leading to distinct retention times (the time it takes for each compound to be eluted from the column). B) Each compound flows through an absorbance or fluorescence spectrophotometer and produces a typical absorbance or fluorescence spectra that can be used to identify each compound compared to a known standard. Each peak of this chromatogram from an Arabidopsis leaf sample represents a different pigment. The retention time of each peak is specific for individual pigments and the area under it is proportional to its abundance. Fig. 7A is modified from Linde (The Linde Group)3.

5.3) Procedure

5.3.1) Total pigment extraction for HPLC analysis

Using the same plants as per Activity 3, use one set of plants (wild type and mutant) to extract plant pigments for HPLC analyses. This method is adapted from the one described in (Förster et al. 2009).

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Materials

Method

Eppendorf tubes

  1. Harvest three x 30 milligrams of leaf material from your wild type and mutant plant.

Liquid nitrogen

  1. Freeze the material immediately by dipping the sample in liquid nitrogen. IMPORTANT: wear goggles and gloves to avoid burning your skin. Liquid nitrogen boils at −196 °C (very cold!) and causes rapid freezing when in contact with living tissue.

Pistil for grinding

  1. Grind the frozen tissue into a paste.

Acetone/ethyl acetate, pipette, tips

  1. Add 500 µl of filtered 60% (v/v) acetone: 40% (v/v) ethyl acetate to your ground tissue samples.

Vortex

  1. Vortex for ten seconds.

H2O, vortex, microcentrifuge

  1. Add 400 µl of H2O to each sample.

Vortex

  1. Vortex for ten seconds.

Centrifuge

  1. Centrifuge for five minutes at 16,000 g (depending on rotor, approx. 13,000 revolutions per minute (rpm))

HPLC vials

  1. Label HPLC vials and place the bottom inserts in.

Eppendorf tubes

  1. Recover approximately 200 µl of the upper phase containing the pigments into another labelled tube. It is no problem if some of the lower phase is also carried over.

Microcentrifuge

  1. Spin the pigment at 13,000 rpm for three minutes.

 

  1. Transfer 100 µl to the corresponding HPLC vial bottom insert.

5.3.2) HPLC run

This step will be performed by your demonstrator using an HPLC system. This method is adapted from the one described in (Förster et al. 2009).

 

You will be invited for a tour and explanation of the technique.

Materials

Method

 

  1. The demonstrator will load 10 µl of the extract onto the HPLC.

Waters Spherisorb 5 micrometre (µm) ODS2 column for reverse-phase HPLC (Agilent Technologies HP1100 series); acetonitrile: water: triethylamine, 90:10:0.1, v/v; ethyl acetate

  1. Separate pigments using a linear gradient decreasing solvent A (acetonitrile: water: triethylamine, 90:10:0.1, v/v) from 100% to 33% (v/v) while increasing solvent B (ethyl acetate) from 0% to 67% (v/v) over 31 minutes, followed by a four-minute elution with 100% (v/v) solvent B at a flow rate of 1 mL min–1.

 

  1. Your instructors will give you the raw data and explain how to interpret it. See also Fig. 7 for an example.

5.4) Expected outcomes

  1. Graph Abs vs retention time.
  2. Identify peaks using the example provided in Fig. 7B. Specifically, look at potential differences in peak composition (retention time) between wild type and mutant samples.

 


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