Paper chromatography separates a mixture into its individual components using nothing more than a strip of filter paper, a solvent, and a container. You apply a small spot of the mixture near the bottom of the paper, dip the paper’s edge into a solvent, and wait while capillary action draws the liquid upward through the fibers. Different substances in the mixture travel at different speeds, producing a trail of separated spots you can see and measure. The whole process takes about 30 to 90 minutes depending on the solvent, and the materials cost almost nothing.
What You Need
The beauty of paper chromatography is that the supply list is short. Here is what to gather before you start:
- Filter paper: Standard laboratory filter paper works best. Whatman No. 1 is the traditional choice for school and lab work. In a pinch, white coffee filters can substitute for casual experiments, though they give less consistent results because their fiber density is less uniform.
- Solvent: Water works for separating water-soluble dyes like food coloring or certain ink pigments. For mixtures that do not dissolve well in water, rubbing alcohol (isopropanol) or a mixture of water and alcohol is common. The solvent you choose matters enormously, and matching the right solvent to your mixture is half the technique.
- Container: A tall glass jar, a beaker, or even a clean drinking glass. It needs to be tall enough that the paper can hang or stand inside without the top folding over, and it needs a lid or cover (plastic wrap works) to keep the inside saturated with solvent vapor.
- Pencil: A regular graphite pencil for drawing your baseline and labeling. Never use a pen, because ink from the pen will dissolve in the solvent and contaminate your results.
- Capillary tube or toothpick: For applying tiny, concentrated spots of your sample onto the paper.
- Ruler: For measuring distances after the run.
- Samples: Whatever you want to separate. Black marker ink, food coloring, leaf extract, or a set of known dyes for comparison.
Preparing the Paper and Applying Your Samples
Cut your filter paper into a strip that fits inside your container with a little room to spare on each side. The paper should not touch the walls of the container while it is developing, because contact with the glass creates uneven solvent flow and distorts separation. A strip about 2 to 3 centimeters wide and 15 to 20 centimeters long is a good starting size for most jars.
Using your pencil, draw a light horizontal line across the strip about 2 centimeters up from the bottom edge. This is your origin line, the baseline where you will place your sample spots. The line must be drawn in pencil because graphite does not dissolve in common solvents. If you are running several samples side by side, mark small dots or letters along the line to label each spot’s position, spacing them at least 1.5 centimeters apart so the spots do not merge as they travel upward.
Now apply your samples. Dip a toothpick or capillary tube into the mixture and touch it gently to the pencil line, making the smallest spot you can. Smaller, more concentrated spots give sharper separation. If the spot looks faint, let it dry completely and then apply a second dot directly on top of the first. Repeating this two or three times builds concentration without spreading the spot too wide. A spot that starts out large and blobby will produce smeared, overlapping bands that are difficult to read.
Running the Chromatogram
Pour your chosen solvent into the bottom of the container to a depth of about 1 centimeter. The critical rule here is that the solvent level must sit below your origin line. If the solvent touches the sample spots directly, the samples will dissolve off the paper into the pool of liquid instead of traveling upward through the fibers. Getting this depth right is the single most common make-or-break step for beginners.
Place the paper strip into the container so its bottom edge sits in the solvent. You can tape or clip the top of the strip to a pencil laid across the mouth of the jar, letting the strip hang straight down into the liquid. Alternatively, you can lean the strip against the inside wall at a slight angle, though hanging produces more even results. Once the paper is in position, cover the container with a lid, a watch glass, or a layer of plastic wrap.
Covering the container is not optional. The enclosed atmosphere fills with solvent vapor, and this saturated environment prevents the solvent from evaporating unevenly off the surface of the paper as it climbs. Without a cover, the edges of the strip dry faster than the center, the solvent front becomes uneven, and the spots warp sideways. Letting the covered container sit undisturbed for a few minutes before inserting the paper, so the air inside can become saturated with vapor, improves results even further.
Once the paper is in place, do not move, jostle, or peek inside the container repeatedly. The solvent will creep upward through the paper by capillary action. Research on liquid penetration into filter paper shows that the height of ascent is roughly proportional to the square root of time in the early stages, then gradually slows as the solvent climbs higher and gravity pulls back against the capillary force.1Bulletin of the Chemical Society of Japan. Penetration of Liquids into Filter Paper Used in Paper Chromatography In practical terms, this means the solvent front moves quickly at first and then slows down. You will typically wait 20 to 60 minutes for the front to climb most of the way up the strip, depending on the solvent and the paper.
When the solvent front is about 1 to 2 centimeters from the top of the strip, remove the paper from the container. Immediately mark the highest point the solvent reached with a pencil line. This is called the solvent front, and you need to know its exact position for your measurements. Let the strip dry completely on a flat surface or hang it up with a clip.
How to Read Your Results
Once the strip is dry, you should see your original sample spot replaced by a series of separated spots or bands, each a different color or shade. Each spot represents a different component of the mixture. If you started with black marker ink, for instance, you might see bands of blue, red, and yellow spread along the strip.
The key measurement in paper chromatography is the Rf value, short for “retention factor” or “retardation factor.” It is simply the distance a particular spot traveled divided by the distance the solvent front traveled, both measured from the origin line. Measure from the center of each spot to the pencil baseline, and from the solvent front line to the baseline. Divide the spot distance by the solvent front distance. The result is always a number between 0 and 1. A substance that did not move at all has an Rf of 0. A substance that traveled as far as the solvent front has an Rf of 1.
Rf values are useful because they are relatively consistent for a given substance under the same conditions: same paper, same solvent, same temperature. That means you can compare your unknown spot to a known reference substance run on the same strip. If a mystery spot has an Rf of 0.65 and your reference dye also lands at 0.65, they are likely the same compound. Running a known reference alongside your unknown on the same strip is always better than comparing to Rf values from a textbook, because even small differences in temperature, paper brand, or solvent composition can shift the numbers.
Why the Separation Happens
Paper chromatography is classified as a partition technique, one of the core separation methods in chromatography alongside ion exchange, surface adsorption, and size exclusion.2Europe PMC. Separation techniques: Chromatography In partition chromatography, the mixture’s components distribute themselves between two liquid phases. The filter paper’s cellulose fibers hold a thin layer of water trapped within and between them. This bound water acts as the stationary phase. The solvent you pour into the jar is the mobile phase, and it flows upward through the paper carrying dissolved sample molecules with it.
As the mobile phase moves, each substance in your sample constantly shifts back and forth between the moving solvent and the water trapped in the paper. Components that are more attracted to the water layer spend more time sitting still and travel a shorter distance. Components that dissolve more readily in the mobile solvent get carried further up the strip. The outcome is that different substances end up at different heights, each one reflecting its own balance of affinity for the two phases. That balance is what the Rf value captures in a single number.
Common Mistakes and How to Fix Them
Beginners tend to hit the same problems. Knowing them in advance saves a lot of wasted paper and frustration.
- Spot too large: If your starting dot is wide and wet, the separated bands will overlap and smear. Apply smaller amounts and let each layer dry before adding the next.
- Solvent too high: If the liquid in the jar reaches the origin line, your sample washes off the paper into the pool. Pour out some solvent or use a taller strip with the baseline drawn higher.
- Paper touching the wall: Contact between the strip and the jar wall creates a shortcut for the solvent, pulling it up faster along the edge and distorting the spots. Hang the strip so it sits freely in the center of the container.
- No cover on the jar: Evaporation causes the solvent front to climb unevenly or stall out entirely. Always seal the container.
- Using pen for the baseline: Pen ink dissolves in most solvents, adding unwanted spots and confusing your results. Always use pencil.
- Moving the jar: Vibrations and tilting ripple the solvent front, producing wavy, uneven separation. Set the jar on a stable surface and walk away.
If your spots are visible but poorly separated, meaning they overlap or barely spread apart, the problem is usually your choice of solvent. A solvent that is too similar to the stationary water phase will not differentiate the components well. Switching solvents or adjusting the ratio in a mixed solvent system often produces dramatically better separation.
Choosing the Right Solvent
Solvent selection is the single most influential variable in paper chromatography, and there is no universal best answer. The right solvent depends entirely on the chemistry of whatever you are trying to separate. Water works well for water-soluble dyes like food coloring because the different dye molecules have slightly different polarities, and water can exploit those differences. For plant pigments like chlorophyll and carotenoids, water will not dissolve them at all, and you need something less polar like a mixture of petroleum ether and acetone.
A common school experiment uses a solvent of roughly one part isopropanol to two parts water with a small splash of table salt dissolved in the water. This mixture works reasonably well for separating many types of ink. Adjusting the ratio lets you fine-tune how far and how fast different components travel. If everything races to the top with the solvent front, the mobile phase is too strong and you should dilute it with more water. If nothing moves at all, the mobile phase is too weak and you need a higher proportion of the organic component.
The trial-and-error nature of solvent selection can feel frustrating, but it is genuinely how chromatographers work. Even in professional labs, optimizing the mobile phase for a new separation involves running multiple trials with different solvent compositions.
Experiments Worth Trying
Paper chromatography is one of those rare techniques that is simple enough for a kitchen table and powerful enough for a research lab. Here are a few classic experiments that demonstrate different aspects of the technique.
Separating black marker ink is the most common starting point. Different brands of black markers use different blends of colored dyes, and chromatography reveals those hidden colors beautifully. Try several brands on the same strip for an easy comparison. You will often find that one “black” is really a mix of blue, red, and yellow, while another contains only blue and purple.
Food coloring provides another accessible experiment with a twist: commercial food dyes are sometimes single compounds and sometimes mixtures. Green food coloring, for example, is typically a blend of a blue dye and a yellow dye, and chromatography splits them apart neatly. Running the individual colors alongside the green confirms the identity of each band.
Leaf pigment separation is a step up in complexity. Grind fresh leaves in a mortar with a small amount of rubbing alcohol to extract the pigments, then spot the extract onto the paper. With a suitable solvent, you can separate chlorophyll a (blue-green), chlorophyll b (yellow-green), xanthophylls (yellow), and carotenoids (orange). This experiment shows the range of pigment molecules hiding behind the uniform green of a leaf.
Detecting Invisible Spots
Not all substances are colored. Amino acids, sugars, and many other compounds produce invisible spots on the paper. To make them visible, you need a detection method applied after the run is complete.
Ultraviolet light is the simplest approach. Some compounds absorb UV and appear as dark spots against a fluorescent background when viewed under a UV lamp. For amino acids, a chemical spray called ninhydrin is the classic choice: the paper is lightly misted with a ninhydrin solution and then heated gently, and amino acid spots turn purple. Sugars can be revealed by spraying with a solution of aniline and phosphoric acid and heating until colored spots appear.
These detection steps are standard in educational and research settings, though they involve chemicals that require more care than the basic dye experiments. For a home setup, sticking with colored samples avoids the need for detection reagents entirely.
Beyond the Basic Strip
The strip method described above is one-dimensional ascending chromatography, the simplest configuration. Two other setups are worth knowing about if you want to push the technique further.
Descending chromatography reverses the direction of flow. The paper hangs from a trough of solvent at the top of the chamber, and the mobile phase flows downward under gravity in addition to capillary action. Because gravity assists the flow rather than opposing it, the solvent moves faster and the run covers a longer distance, sometimes improving separation for mixtures whose components have very similar Rf values.
Two-dimensional chromatography tackles complex mixtures that a single run cannot resolve. You spot the sample in one corner of a square sheet of filter paper and run the chromatogram in one direction with one solvent. After drying, you rotate the sheet 90 degrees and run it again in the perpendicular direction with a different solvent. Components that co-migrated in the first solvent may separate in the second, spreading the spots across a two-dimensional grid instead of a single line. This is useful for separating complex biological extracts like amino acid mixtures from protein digests.
Modern Twists on an Old Technique
Paper chromatography dates to the 1940s, but it has not been frozen in time. Researchers have found ways to pair the simplicity of paper with modern digital analysis. One recent method uses a smartphone camera to photograph spots on paper, then feeds the image into free software that reads the color intensity of each spot. By analyzing the red, green, and blue color channels of the image and plotting them against known standard concentrations, a simple paper experiment becomes a quantitative measurement tool, with selected spot areas typically around 3,500 to 5,000 square pixels providing enough data for a reliable calibration curve.3MethodsX. A smartphone-based method for extraction-free antioxidant capacity analysis of emulsions on paper by the DPPH assay
This kind of approach is appealing for fieldwork and low-resource settings where expensive instruments are not available. It also makes paper chromatography more than a qualitative “which colors are present” exercise. With a phone, a piece of filter paper, and a free image analysis program, you can estimate how much of a substance is in your sample, not just confirm that it is there. For student projects, the combination of a hands-on chemistry technique with smartphone data analysis bridges two worlds in a way that tends to hold people’s attention far longer than the paper strip alone.