Teacher Notes

Document Analysis

Student Laboratory Kit

Materials Included In Kit

Sodium hydroxide solution, NaOH, 0.1 M, 75 mL
Thymolphthalein indicator solution, 75 mL
Chromatography paper, 15 sheets
Chromatography pens, 6
Cotton-tipped applicators, 15
Filter paper, 15
Paper samples A–E, 15 sets
Pencils, #1, 12
Pencils, #2, 12
Pencils, #3, 12
Tape, cellophane, 1 roll
Wood splints, 90

Additional Materials Required

(for each lab group)
Beaker, 50- or 100-mL
Chromatography chambers, 6
Microscope or stereoscope
Notebook paper, several sheets
Spray bottle
UV lamp (shared)

Prelab Preparation

  1. Cut a sheet of paper B into small pieces and write a ransom note on each piece. These will be used in Part I of the lab.
  2. Run a chromatogram for one of the pens in Part IV of the lab prior to conducting the lab. This chromatogram will represent the pen used at a crime scene. Students’ results should reveal which pen was used to create your chromatogram.

Safety Precautions

Sodium hydroxide is a corrosive liquid. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Avoid exposure of eyes and skin to the disappearing ink chemicals. Do not allow students to place disappearing ink on their own clothing or on the clothing of others. Warn students about the dangers of viewing UV light. Wash hands upon completion of laboratory work. Please review current Safety Data Sheets for additional safety, handling and disposal information.


Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. The water from the chromatography can be disposed of according to Flinn Suggested Disposal Method #26b. The disappearing ink and any remaining solutions can be stored for future use or can be disposed of according to Flinn Suggested Disposal Method #10.

Teacher Tips

  • Enough materials are provided in this kit for 30 students working in pairs or for 15 groups of students. The entire lab will take two 50-minute class periods to complete.
  • Writing and developing disappearing ink messages works best on filter paper or chromatography paper. Some lower-grade filter papers tend to absorb the ink or developing solutions too fast and the resulting message may look more like an inkblot test rather than a written message. Index cards or plain paper, on the other hand, do not absorb the solutions as well and the solutions tend to simply “run off.”
  • Set up a special developing area for spraying the sodium hydroxide solution. This can be done over a glass tray or a plastic demonstration-style tray to contain the sprayed mist. Always spray in a direction away from others in the lab. Good housekeeping is important to clean up any residual basic solution from the work area after the demonstration is over.
  • If, after repeated use and exposure to air over time, the original disappearing ink solution loses its deep blue color, the color can be restored by the addition of a few drops of the 0.1 M sodium hydroxide solution.
  • Disappearing ink provides is an ideal activity for open-house days in the science lab. Have students prepare filter paper “cards” to hand out to parents and other visitors as they enter the classroom. The visitors can be directed to a special area where volunteers can develop their secret messages (e.g., Welcome, Chemistry is pHun, Chemists Love Solutions, ChemMystery). (Volunteers should wear goggles and gloves and should spray in a direction away from all visitors—the hood is a perfect place to set up developing trays.) The student volunteers are able to display their growing chemical knowledge to curious visitors by explaining the chemical basis of the mystery ink phenomenon.
  • Chromatography chambers can be 100-mL Erlenmeyer flasks, large test tubes or even jars. If you do not have enough chambers for Part IV, you can do one of three things:
    1. Have each student or group only use one pen and strip.
    2. Use an alternate chromatography chamber setup, as shown in Figure 3, using a large test tube. (A graduated cylinder may also be used.)
    3. You may try to place two strips in each flask. However, the strips cannot touch each other.
  • A terrific extension of this activity is to adjust the polarity of the solvent to try to achieve better separations. In fact, when a 50/50 mixture of water and acetone was tried, some very interesting results were achieved. The yellow ink, which previously appeared to contain only one pigment, separated into two yellow pigments, only one of which was fluorescent. The yellow and pink pigments in the red and brown inks actually reversed their order on the strips.
  • Good technique is required for the students to see well-separated compounds on their strips. Some of the common causes for poor separations or results are:
    1. Too much starting material is placed on the initial spot or band.
    2. Initial spot or band is too large.
    3. Initial spot or band is below the solvent level in the developing chamber.
  • Allowing enough time for the development of the strip is critical. The strip must be left in the chromatography chamber long enough for the solvent to be drawn up near the top of the strip. Do not stop the development until the solvent front nears the top of the strip. Underdevelopment will lead to incomplete separation.

Correlation to Next Generation Science Standards (NGSS)

Science & Engineering Practices

Asking questions and defining problems
Analyzing and interpreting data
Obtaining, evaluation, and communicating information

Disciplinary Core Ideas

MS-PS1.A: Structure and Properties of Matter
MS-PS1.B: Chemical Reactions
HS-PS1.A: Structure and Properties of Matter
HS-PS1.B: Chemical Reactions

Crosscutting Concepts

Cause and effect
Structure and function

Performance Expectations

MS-PS1-3: Gather and make sense of information to describe that synthetic materials come from natural resources and impact society.
MS-PS1-2: Analyze and interpret data on the properties of substances before and after the substances interact to determine if a chemical reaction has occurred.
HS-PS1-3: Plan and conduct an investigation to gather evidence to compare the structure of substances at the bulk scale to infer the strength of electrical forces between particles.

Sample Data

The apparent pigment colors we found in our six pens, using water as a solvent were: 

  1. Black: dark blue, light blue, orange, yellow
  2. Blue: dark blue, light blue
  3. Brown: dark purple, yellow‡, pink‡
  4. Green: light blue, yellow
  5. Red: yellow‡, pink‡, (under UV light, a fluorescent orange also appeared to be present)
  6. Yellow: yellow‡

Note: If the pigment fluoresced under UV light, it is marked with a ‡. The particular pens we used may or may not be the ones shipped with this kit. Manufacturers may change ink formulations without notice, or pens may be substituted depending on availability.

Student Pages

Document Analysis


Crime scene investigators use many kinds of evidence when trying to solve a crime. Often the evidence includes the analysis of written or printed documents. This may include analysis of the paper, the writing, the inks or other properties of the documents. Analyzing documents is an important aspect of forensic science and scientific analysis of documents often used as evidence in a court of law.


  • Forensic science
  • Microscopic observation
  • Chromatography
  • Acid–base indicators


The analysis of documents from a crime scene can take many different paths and can employ many different analysis techniques. The document experts of a crime lab will make use of chemistry, physics, microscopy, chromatography, photography, handwriting analysis and various other specialized techniques. Most of the work in the crime lab emphasizes comparison of materials and writing with known standards. This often allows the crime scene investigator to trace the materials back to a certain location or to a certain manufacturer. Documents at crime scenes can include wills, checks, handwritten notes, typed materials, printed materials, photographic materials and a variety of other paper documents. In addition to the actual documents, printing machines as well as an individual’s handwriting can also be analyzed. Analysis of tampered documents is often important in criminal investigations.

Paper, in its simplest terms, is a very thin layer of bonded fibers. Paper has been made from many different fibers throughout history, but today most paper fiber is cotton, linen, wood or some combination of these. Because the formula and mixture of fibers for all papers is slightly different, various papers have distinctive looks and structures. The structure of a paper can help to identify its source or authenticity. Some manufacturers place a translucent mark, called a watermark, on the paper. These are specific to a certain manufacturer and are changed periodically. Watermarks can reveal information about the date and origin of a document.

What is written on paper is often more revealing than the paper itself. Secret messages may be written in code or with “invisible” chemicals that can be revealed later. Inks, pens, and pencils all have distinctive “trails” that are left behind on the paper. All of these things are analyzed carefully by the document specialists on a crime scene investigation team.

One common laboratory technique used to analyze the makeup of materials, such as inks, lipsticks and other markings on paper is called paper chromatography. There are many different types of chroma tography but most work on the concept of absorbance. Two important components of any chromatography system are the absorbent and the eluent. A good absorbent is usually a solid material that will attract and absorb the materials to be separated. Paper, silica gel or alumina are all very good absorbents. The eluent is the solvent that carries the materials to be separated through the absorbent.

Chromatography works on the principle that the compounds to be separated are slightly soluble in the eluent and will spend some of the time in the eluent (or solvent) and some of the time on the absorbent. When the components of a mixture have different solubilities in the eluent, they can then be separated from one another. The polarity of the molecules to be separated and the polarity of the eluent are very important. Changing the polarity of the eluent will only slightly change the solubility of the molecules but will greatly change the degree to which they are held by the absorbent. This affinity for the eluent versus the absorbent is what separates the molecules in chromatography.

Paper chromatography is often used as a simple separation technique. In paper chromatography, the absorbent is the paper itself, while the eluent can be any number of solvents. The polarity of the eluent is very important in paper chromatography since a small change in polarity will dramatically increase or decrease the solu bility of some organic molecules. Many times, a mixture of a nonpolar solvent and a polar solvent is used to achieve an optimum polarity. When placed in a chromatography chamber as shown in Figure 1, the eluent moves up the paper strip, being drawn by capillary action. The organic molecules, which were “spotted” onto the paper chromatography strip, separate as they are carried with the eluent up the strip at different rates. Those molecules that have a polarity closest to the polarity of the eluent will be the most soluble, and will move up the strip the fastest.

The choice of the eluent or solvent is the most difficult task. Choosing the right polarity is critical because this determines the level of separation that will be achieved. Common solvents used in chromatography, in order of increasing polarity, are: petroleum ether or hexanes, cyclohexane, toluene, chloroform, ethyl ether, acetone, ethanol, methanol and water. Sometimes mixtures of solvents are used to achieve the desired degree of polarity. Many inks are actually mixtures made up of several basic pigments. Each of these pigments has a different molecular structure and, usually, a different polarity. Many of these pigments can be separated using paper chromatography.

Disappearing ink used in this lab is a mixture of thymolphthalein indicator, ethyl alcohol, sodium hydroxide solution, and water at pH 11. When the blue ink is applied to paper, the blue color quickly vanishes. The disappearance of the blue ink color in air is due to the effect of carbon dioxide (CO2), which reacts with moisture in the air to form carbonic acid (H2CO3)—the pH change is enough to push the basic form of the indicator (Ind) back to its colorless acidic form (HInd).
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Thymolphthalein is a weak organic acid that behaves as an acid–base indicator in the pH range 9.3 (colorless) to 10.5 (blue). It exists in two different forms—an acid form HInd, which is colorless, and a corresponding conjugate base form Ind, that is blue. The color transition range for an acid–base indicator depends on the strength of the weak acid HInd. The color change is due to the changing proportion of the indicator molecules in the acid or base form.


Sodium hydroxide solution, NaOH, 0.1 M, 3–5 mL
Thymolphthalein indicator solution, 3–5 mL
Beaker, 50- or 100-mL
Chromatography chambers, 6
Chromatography paper, 6 strips
Cotton-tipped applicator
Filter paper
Microscope (4X or stereoscope if available)
Paper, notebook, several sheets cut into fourths
Paper samples, 5 (A–E)
Pencils, 3 (#1, #2, #3)
Pens, 6
Spray bottle
Tape, 6 strips (½")
UV lamp
Wood splints, 6

Safety Precautions

Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Never look directly into a UV light source as eye damage can result. Wash hands with soap and water upon completion of laboratory work.


Part I. Paper Examination

  1. Obtain paper samples labeled A–E.
  2. Observe each paper type and record your observations of each paper on the Document Analysis Worksheet, Part I. Describe the color. Is it white, yellow-white, gray-white, blue-white, etc. Is it dull/shiny? Thick/thin? Smooth/rough?
  3. Hold the paper up to a light or window. Does it have a watermark? What does the watermark look like? Are any of the marks the same?
  4. Observe the papers under an ultraviolet light. (Use longwave ultraviolet light for best results). Shine the light on each paper and observe if the paper fluoresces or glows. What color is each paper under UV light? Warning: Do NOT look directly at a UV light; it may cause eye damage.
  5. Try to rank the papers by weight or thickness. Rank them from thinnest (1) to thickest (5). If a centigram balance (0.01) is available, each paper should be weighed.
  6. Use a microscope (on lowest power, or use a stereoscope, if available) to observe the fiber structure of each paper. Rank the fiber structure of each paper. Rank them from the smoothest (1) to the roughest (5).
  7. Record all observations on the Document Analysis Worksheet.
  8. Examine the ransom note provided by your instructor. Which paper type (A–E?) was used for writing the ransom note?
Part II. Pencils/Erasers
  1. Sharpen a #1, #2 and #3 pencil to the same degree.
  2. Draw a 2 cm-long mark from each pencil on a sheet of notebook paper side by side so that they are relatively close together. Try to apply the same pressure on the pencil when making the lines. Label the lines 1, 2 and 3, respectively.
  3. Examine each line using low power on a microscope. Focus on each line carefully and note its thickness, color intensity, sharpness, etc. Record your observations on Part II of the Document Analysis Worksheet.
  4. Have a partner secretly select one of the three pencils and write a short sentence on another small piece of paper. Compare the writing with the three original marks. Examine the sentence writing using a microscope. Can you determine which pencil your partner used to write the sentence?
  5. Have a partner make an “X” on a small piece of paper noting which line of the X was drawn first (bottom) and which line was drawn second (top). Use the microscope to determine which line is on top and which is on the bottom. (This technique could be important in trying to determine if one line has been written over the top of another.)
  6. Use a pencil to write your name on another small piece of paper. Press firmly as you write. Erase part of a letter. Examine the eraser mark under the microscope. Shine the UV light on the eraser mark. Are the erasures easily detected?
  7. Use a pencil to write a sentence on a piece of paper. Have a partner do the same. Erase a word in the sentence. Try to write the erased word again with the same pencil, being as careful to write right over the old writing as possible. (See if you are a good forger!) Swap the written sentences with your partner and use the microscope and UV light to track down the erased word. Were you able to detect the forgeries?
  8. Write a summary of your observations about erasing pencil from paper. Do this on the Document Analysis Worksheet, Part II.
Part III. Handwriting/Forgery
  1. Use a ballpoint pen to firmly write your signature onto a small piece of paper. Label this paper “O.”
  2. On a second sheet of paper write your signature again. Label this signature “A.”
  3. Find another person. Give them paper “O” and instruct them to place a piece of paper over the signature and attempt to trace it. Have them label this paper “B.”
  4. Find another person. Give them paper “A” and instruct them to forge the original signature by just looking at it and trying to make it look identical to “A.” Have them label this paper “C.”
  5. Give papers “O,” “A,” “B” and “C” to a fellow crime scene investigator. Using a microscope, can the investigator determine which signatures, A, B, C are forgeries and which is an original like “O”?
  6. Answer the question for Part III on the Document Analysis Worksheet.
Part IV. Inks/Chromatography
  1. From a chromatography sheet, cut six strips, each 13 cm long x 2 cm wide.
  2. Using a pencil, lightly draw a line across the width of each strip, 2 cm from one end (see Figure 2a).
  3. Cut off the bottom corners of each strip to create a point, as shown in figure 2b. Staple or tape the strip to a wooden splint, as shown in Figure 2c. Repeat for all six strips.
  4. Add 50 mL of water to six chromatography chambers.
  5. Use a chromatography pen, place a small dot on the center of the drawn line on one chromatography strip. Repeat for each pen on a separate chromatography strip. Using a pencil, write the color of the pen on the top of the strip or on the wooden splint.
  6. Slowly lower one chromatography strip into the chromatography chamber. The pointed end of the paper strip should just touch the solvent but the sample spot should remain above the solvent (the water). If the sample spot touches the solvent, it will simply dissolve in the solvent.
  7. Repeat step 6 for each chromatography strip.
  8. The solvent will be drawn up the chromatography strips by capillary action. As it is drawn up, it will carry the pigments in the ink samples up the strips at different rates depending on the characteristics of the individual components in the ink.
  9. When the solvent front is within 0.5–1.0 cm of the top of the chromatography strip, stop the run by removing the strip from the flask.
  10. It is a good idea to carefully mark the location of each of the pigment spots on the strips and the final solvent fronts, again using a pencil. This is done because some of the color and brightness of each of the spots may be lost over time. During this time, the residual water on the strips may continue to be drawn up the strip slightly by continued capillary action. If you have marked the location of the pigment spots in pencil, this is not a concern.
  11. If a UV light is available, shine it on each of the strips in a darkened room.
  12. Answer the Analysis Questions for Part IV of the Document Analysis Worksheet.
Part V. Secret Messages
  1. Use a cotton-tipped applicator to “write” a message with the thymolphthalein “disappearing ink” solution on a large piece of chromatography or filter paper. The color will fade from blue to colorless almost immediately.
  2. Allow the secret message to dry and disappear completely. If necessary, blow on the secret message to make the ink dry and disappear faster.
  3. Once the ink has disappeared it can be “developed” (made to reappear) by spraying the message with a mist of 0.1 M NaOH solution from a spray bottle. The blue color will reappear almost instantly and will usually last 3–5 minutes before it fades again.
  4. Answer the questions for Part V of the Document Analysis Worksheet.

Student Worksheet PDF


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