AP Chemistry · Handsworth Secondary 2026–27 · Unit 3

Lab · Spinach & Ink Chromatography

Dr. Ras Mulinta
Handsworth Secondary
IMF · Polarity · Separation

Spinach leaves are green, but the green hides a mixture of photopigments: two chlorophylls plus the yellow and orange accessory pigments underneath. In this short activity you let a polar solvent climb a strip of paper and watch the pigments separate, because each one clings to the polar paper versus the moving solvent with a different strength, and that difference is set by polarity and intermolecular forces. You run a black-ink dot beside it to prove the ink is a mixture too, then put a number on each separation with the retention factor, Rf.

CED 3.1 (Intermolecular Forces) & 3.9 (Separation by Chromatography) · Unit 3.  ~1 block, hands-on ·  FORMATIVE-CATEGORY activity: not a graded formal report. Hand in the data table + analysis answers.

Name:Block:Date:Partner (setup only):

Background CED 3.1 & 3.9

Paper chromatography separates a mixture using two "phases." The stationary phase is the chromatography paper, cellulose, rich in –OH groups, so it is polar and holds a thin layer of bound water. The mobile phase is the solvent that wicks up the paper: here a 70:30 isopropyl alcohol : water mixture, also polar. As the solvent rises past a spot of mixture, each component is pulled two ways at once.

Like dissolves like, the IMF tug-of-war: a component that bonds strongly to the polar paper (hydrogen bonding, dipole–dipole) is held back and travels a short distance. A component that is nonpolar has only weak London dispersion forces with the cellulose, so it would rather ride along with the moving solvent and travels far. Same mixture, same paper, same solvent, the separation comes entirely from each molecule's own polarity and the IMF it can form.
Retention factor Rf: a unitless ratio that fingerprints each component:
Rf = (distance travelled by the component) ÷ (distance travelled by the solvent front)
Both distances are measured from the start line. Rf is always between 0 and 1: a low Rf means the component stuck to the polar paper (more polar); a high Rf means it ran with the solvent (less polar / more nonpolar).
The spinach pigments, most polar to least polar: chlorophyll b (an extra polar –CHO aldehyde group → most hydrogen bonding to the paper → travels least, lowest Rf) · chlorophyll a (–CH3 in place of that aldehyde → slightly less polar → travels a bit farther) · xanthophylls (yellow; oxygen-containing carotenoids, moderately polar) · carotenes (orange; pure hydrocarbons, only London forces → essentially no attraction to the polar paper → ride the solvent front, highest Rf). So on this polar paper with a polar mobile phase you expect the band order, from the start line upward: chlorophyll b, chlorophyll a, xanthophyll, carotene.

Why two phases of green? The bright green of chlorophyll masks the yellow/orange accessory pigments in a living leaf; chromatography pulls them apart so you can see the carotenes and xanthophylls that the eye normally never separates from the chlorophyll.

The ink dot: a "black" marker is usually a blend of coloured dyes. Running it beside the spinach shows the same physics on a man-made mixture, the dyes separate by their own polarity into distinct bands, proving black ink is a mixture, not a pure substance.

Safety read before you start

Isopropyl alcohol is flammable. The mobile phase is 70% isopropyl alcohol. Keep it away from any open flame or spark, no Bunsen burners or hot plates anywhere in the room during this lab. Work in a well-ventilated area and keep the watchglass on the beaker to limit vapour.
Eyes and skin. Splash goggles on the whole period. Isopropyl alcohol dries and irritates skin; wipe spills and wash your hands after handling. Avoid breathing the vapour for long stretches.
Sharp / glass. Handle the scissors and the thin glass capillary/pipet carefully. Tell Dr. Mulinta if a capillary breaks; do not pick up glass with bare fingers.
Disposal: the small volume of used solvent goes in the labelled organic-solvent waste container, not the sink. Used paper strips go in the regular bin once dry.

Materials & Equipment low-gear setup

Everything here is everyday stock plus the pre-made spinach mixture. There is no instrument to share, every group runs its own two strips.

Per group: one clean, dry 600 (or 400) mL beaker · wooden splint · two strips of chromatography paper (~30 cm region) · two small pieces of tape · scissors · pencil · ruler · clean dry 100 mL beaker · medicine dropper · 25 mL graduated cylinder · watchglass (lid) · fine-point black Sharpie · a thin glass capillary tube / fine pipet · paper towel.
Shared at the bench: bottle of isopropyl alcohol · bottle of distilled water · the labelled bottle of pre-made green spinach mixture · organic-solvent waste container.

Mrs. Kathy (lab tech) pre-sets: the pre-made green spinach pigment mixture (extracted ahead of time and kept cool/dark), bottles of isopropyl alcohol and distilled water at each bench, the cut chromatography paper, capillary tubes, and the organic-solvent waste container. No flames are set out for this lab. You only measure and mix the 70:30 mobile phase yourself.

Procedure ~1 block · talk theory while it runs

Step 1: Prepare the stationary phase

  1. Take the clean, dry 600 (or 400) mL beaker, the wooden splint, two pieces of chromatography paper, two small pieces of tape, and scissors.
  2. Tape the two paper strips so they hang from the wooden splint. They must hang freely, the paper must not rest on the bottom of the beaker when the splint sits across the rim.
  3. Lay each strip flat. With a pencil (never pen), draw a straight start line across each strip exactly 2 cm from the bottom edge.

Step 2: Prepare the mobile phase (70:30 IPA : water)

  1. In the 25 mL graduated cylinder, measure 14.0 mL isopropyl alcohol and 6.0 mL distilled water = 20.0 mL total (a 70:30 isopropyl-alcohol-to-water mixture).
  2. Pour into the clean 100 mL beaker and swirl to mix.
  3. Transfer just enough of the mixture into the large beaker so it is no more than 1 cm deep. Cover the large beaker with the watchglass to slow evaporation while you prepare the samples.

Step 3: Prepare the samples (keep dots small)

  1. On the start line of the first strip, make one small dot with the fine-point black Sharpie (this is the ink sample).
  2. On the start line of the second strip, use the glass capillary / thin pipet to place a very small dot of the pre-made green spinach mixture (get about 10 mL of the pre-made mixture for your group).
  3. Let the spinach dot dry (blow gently), then dot the same spot again, repeat for three applications total to concentrate the pigment. A small, dark, tight dot separates far better than a big smeared one.

Step 4: Run the separation

  1. Lower both strips into the large beaker so the splint rests across the rim and the bottoms dip into the mobile phase. Check that the solvent level is below the start line: the start line must sit above the solvent, or your samples will wash off.
  2. Re-cover with the watchglass. Let the solvent climb for about 15–20 minutes, or until the solvent front is near the top of the strips. Do not bump the bench. (Use this time to discuss the chromatography theory questions.)

Step 5: Mark, measure, calculate Rf

  1. Lift both strips out and lay them on a paper towel. Immediately, with a pencil, mark (i) the solvent front and (ii) the centre of each distinct coloured band before they fade or spread.
  2. With the ruler, measure from the start line to the solvent front, and from the start line to the centre of each band. Record in centimetres.
  3. For each band, calculate Rf = (distance band travelled) ÷ (distance solvent front travelled). Record in the data table.

Data Tables fill in lab

Spinach strip   solvent-front distance is the same denominator for every band.

Band (colour → pigment)Distance from start line (cm)Rf
Solvent front__________1.00
Orange → carotene____________________
Yellow → xanthophyll____________________
Blue-green → chlorophyll a____________________
Yellow-green → chlorophyll b____________________

Black-ink strip   record one row per dye band you see (you may not see all four).

Dye band (colour)Distance from start line (cm)Rf
Solvent front__________1.00
______________________________
______________________________
______________________________

Analysis Questions show the Rf math · reason from IMF

Answer on this sheet or your own paper. For full marks, show each Rf calculation and justify your ranking with polarity / intermolecular-force reasoning, not just colour.

  1. Rf values. Using your measured distances, calculate Rf for every spinach band. Show the division for at least one band and fill the rest into the table.
  2. Rank by Rf. List the four spinach pigments from highest Rf to lowest. Which travelled farthest, and which barely left the start line?
  3. Identify the phases. Name the stationary phase and the mobile phase in this experiment, and state whether each is polar or nonpolar.
  4. Polarity from Rf. The stationary phase (cellulose paper) is polar. Use that fact plus your Rf ranking to order the pigments from most polar to least polar. Explain in one sentence why a high Rf means a less polar molecule on this paper.
  5. IMF reasoning. Carotene is a pure hydrocarbon; chlorophyll b carries polar groups including an aldehyde. Using intermolecular forces, explain why carotene races to the solvent front while chlorophyll b stays near the start line. Name the specific IMF each pigment can (or cannot) form with the cellulose –OH groups.
  6. Predict a change. If you re-ran the spinach using a more nonpolar mobile phase (say, mostly hexane), predict what happens to the carotene Rf and to the chlorophyll Rf. Justify with "like dissolves like."
  7. The ink. How many dye bands did the black Sharpie separate into? Is "black" ink a pure substance or a mixture? What does the spread of Rf values tell you about the relative polarities of the dyes?
  8. Technique. Explain, with a reason tied to the chemistry, why each of these matters: (a) drawing the start line in pencil, not pen; (b) keeping the solvent level below the start line; (c) making the sample dot small and concentrated.
  9. Connect to Unit 3. In one or two sentences, tie this separation back to the idea of solubility and "like dissolves like" from CED 3.10, why is chromatography really just a controlled competition of intermolecular forces?
  10. Error analysis, measuring a band. Identify one measurement slip that would make a pigment's Rf read too high and one that would make it read too low. For each, explain the direction through Rf = (band distance) ÷ (solvent-front distance).
  11. Error analysis, the solvent front. Now identify one error in how you locate or mark the solvent front that would push every Rf too high and one that would push every Rf too low. Reason through the same ratio.
AP Chemistry · Unit 3, Spinach & Ink Chromatography (Intermolecular Forces & Properties) · Dr. Ras Mulinta · Handsworth Secondary 2026–27. Formative-category activity (~1 block), not a graded formal report. Procedure adapted from a proven school version (the spinach pigment mixture is pre-extracted by the lab tech); pegged to the College Board AP Chemistry CED (3.1 Intermolecular Forces; 3.9 Separation of Solutions and Mixtures: Chromatography; 3.10 Solubility) and the AP recommended lab "Separation by Chromatography."