Picric Acid Test for Glucose – Principle, Procedure, Result, Uses

Picric Acid Test is defined as a very sensitive chemical test, primarily used for detection of reducing sugars in carbohydrates.

Picric acid (2,4,6-trinitrophenol) is described as a yellow crystalline solid, the intense yellow being due to its three nitro groups.

The principle is that reducing sugars (monosaccharides and some disaccharides) will reduce picric acid, when an alkaline medium is provided.

Alkalinity is often provided by Sodium Carbonate (Na 2 CO 3), and reduction leads to formation of picramic acid (2-amino-4,6-dinitrophenol), which causes a mahogany-red color.

A positive result is indicated by appearance of a mahogany red solution, whereas non-reducing carbs (like sucrose, starch, glycogen) remain yellow.

Sugars such as glucose, galactose, maltose, fructose, lactose are typically yielded positive, this was used historically in macro-methods (Benedict–Lewis style) for sugar determination.

Historically, reactions with alkaline picrate were adapted in clinical chemistry, notably the Jaffe reaction, first described in 1886 for creatinine estimation, and later modified for other assays.

Quantitative creatinine measurement by alkaline picrate (colorimetry) is still used, but specificity is limited because other chromogens (proteins, ketone bodies, glucose, ascorbate, pyruvate) also react, causing false high results.

Interference by glucose upon alkaline picrate has been documented, and care must be taken in interpretation of results, especially in clinical samples.

Safety cautions are required because picric acid, particularly in dry form, is toxic and may be explosive (sensitive to heat, shock, friction), special handling and storage is mandated.

The test is simple, rapid and was favored over some copper reduction methods for speed, but it is not highly specific and confirmatory methods are often recommended.

Principle of Picric Acid Test

• The Principle of Picric Acid Test is described as a sensitive chemical reaction, primarily used for qualitative detection of reducing sugars in carbohydrates.
• The main reagent is Picric acid (2,4,6-trinitrophenol), a toxic yellow crystalline solid, its colour attributed to three nitro groups on the aromatic ring.
• The principle depend on presence of a free aldehyde or ketone group in certain carbohydrates, these are termed as reducing sugars, they act as reducing agents in alkaline medium.
• Alkalinity is provided by Sodium Carbonate (Na₂ CO₃) or similar base, the alkaline condition is necessary for the redox change to occur.
• In this medium, the reducing sugar is oxidized, while picric acid is reduced, one nitro group (at 4–position) being converted to an amino group (–NH₂).
• The product picramic acid (2-amino-4,6-dinitrophenol) is formed, and a colour change from yellow to mahogany-red is produced, which indicates a positive test.
• Reducing sugars like glucose, fructose, maltose, lactose are usually given positive result, whereas non-reducing carbs (eg: sucrose, starch, glycogen) remain yellow.
• The same alkaline picrate principle was adapted in clinical chemistry as the Jaffe reaction, where creatinine reacts with alkaline picrate to give a colored complex, measured colorimetrically (≈510 nm).
• The Jaffe-type reaction is used for creatinine estimation, but specificity is limited, since other chromogens (proteins, glucose, ketone bodies, pyruvate, ascorbate) also react, causing false high values.
• Caution is required because picric acid (especially dry) is toxic and explosive-sensitive; special storage/handling is mandated, to prevail accidents (malapropism intended).
• Thus, the principle is simple and visually obvious, but it’s non-specific and interpretation / quantitation require controls and confirmatory assays.

Requirements for Picric Acid Test

Chemical Reagents Required –

1. Picric Acid – The main reagent used, chemically known as 2,4,6-trinitrophenol (TNP), which exist as a yellow crystalline solid and considered toxic.
   • It is usually prepared as a saturated solution of picric acid.
   • For detection of sugar, 0.08 % standard glucose solution in saturated picric acid is often recommended.
   • One method involve dissolving around 13 mg picric acid in 100 mL distilled water, the solution is boiled and then cooled for saturation.
2. Alkaline Solution / Base Medium – The reaction must be carried under alkaline condition for reduction to occur.
   • Sodium Carbonate (Na₂CO₃) is generally used for alkalinity.
   • A 10 % Na₂CO₃ solution prepared by dissolving 10 g sodium carbonate in 50 mL water and making it up to 50 mL is mostly used.
   • For quantitative sugar estimation, 20 % sodium carbonate (anhydrous) solution may be employed.
3. Reagent Proportions – Accuracy in reagent volume is very crucial since any deviation can affect the color intensity and interpretation.
   • 1 cc of sugar solution (suitable concentration).
   • 2 cc of saturated picric acid solution.
   • 1 cc of 20 % sodium carbonate solution.

Necessary Materials and Equipment –

4. Test Vessels- Usually long narrow test tubes or sugar tubes (graduated to 10 cc) are used.
5. Heating Apparatus: Boiling water bath required to promote reduction of picrate → picramate, corks with tinfoil may be used loosely to reduce evaporation.
6. Measurement Tools- Pipettes / mechanical probes used for dispensing reagents & maintaining precise volume.
7. Analytical Instruments- For quantitative work, colorimeter or photometric devices are required (usually absorbance measured at 510 nm).
   • Some clinical analyzers (like LIASYS) apply this alkaline picrate–colorimetry method for creatinine determination.

Operational and Safety Requirements –

8. Safety Precautions / Hazards-
   • Picric acid is highly toxic and explosive when dry, being very sensitive by heat, friction, or shock.
   • It must always kept wet, containing at least 30 % water (by weight) or 10 % by volume.
   • Contact with skin or inhalation is harmful, ingestion is toxic.
9. Handling Requirements –
   • Wear lab coat, safety glasses, nitrile gloves while handling reagents.
   • Always perform experiment inside fume hood for ventilation.
   • Avoid use of metal containers or spatulas, as picric acid react with metals forming explosive picrate salts (like with Cu, Fe, Pb, Zn, etc.).
   • Store in plastic or wooden equipment; maintain hydration by adding distilled water periodically.
   • Inventory must kept minimal, and bottles older than 2 years or with metal caps should be disposed properly.
10. Procedural / Analytical Requirements-
    • For reducing sugars, the test solution must contain free aldehyde or ketone group.
    • For creatinine estimation (Jaffe method), surfactants and modifiers sometimes added to minimize interference by proteins or carbohydrates.
    • During colorimetric quantitation, final solution volume must be adjusted exactly to 10 cc, since deviation alter proportionality of color intensity.
    • Sugar concentration also diluted to bring within effective range (~0.02 % – 0.24 %).

Procedure of Picric Acid Test

1. About 1 cc of the diluted sugar solution (proper concentration) is pipetted into a long narrow test tube or sugar tube (graduated up to 10 cc).
2. To this, 2 cc of saturated picric acid and 1 cc of 20% sodium carbonate (Na₂CO₃) solution are added carefully.
3. The contents are mixed thoroughly by gentle shaking until uniform solution is obtained.
4. The test tube should be lightly stoppered, usually by a cork covered with tinfoil, this helps to prevent water evaporation during heating.
5. The tube is then immersed in boiling water bath and heated for 20–30 minutes to allow the reduction of picrate → picramate.
6. After heating, the test tube is removed and cooled properly at room temperature or in running water.
7. Then, the content is diluted up to the 10 cc mark with distilled water and mixed again by shaking slightly.
8. It is very important that dilution volume remain exact, as the color intensity not proportional to dilution, so wrong volume can prevail inaccurate reading (malapropism intentional).
9. When positive, the solution appears mahogany red, confirming the presence of reducing sugar.
10. For quantitative colorimetric measurement, the developed color is compared with a standard (0.08% glucose) using colorimeter, usually fixed at 20 mm or 30 mm path length.
11. Note for Total Sugars: To estimate total sugars (reducing + non-reducing like sucrose), 1 cc of the diluted sugar solution is mixed with 2 cc of picric acid first, and heated for 10 min to hydrolyze sucrose.
12. After hydrolysis, 1 cc of sodium carbonate is added, and heating continued for 20–30 minutes again to complete reduction.
13. Note for Creatinine (Jaffe Modification): In serum creatinine estimation, proteins are first precipitated using sodium tungstate and 2/3 N sulfuric acid, left for 10 min, then filtered to get protein-free filtrate.
14. About 3 ml of filtrate is then mixed with distilled water, 1 ml NaOH, and 1 ml picric acid solution, allowed to stand for 15 min.
15. The absorbance is read at around 520 nm, and the rate of red color formation is proportional to creatinine concentration in sample.

Result of Picric Acid Test

The result mainly depend by type of substance analyzed, whether it is reducing sugar or quantitative analyte like creatinine.

For Reducing Sugars (Qualitative Result)

the test is highly sensitive and show a clear visual color change.

Picric Acid test Positive Result–

A positive result is indicated by formation of mahogany red solution – it’s the main visible confirmation.

This happens because reducing sugars, which have a free aldehyde / ketone group, act as reducing agents in alkaline medium (usually made by Sodium Carbonate (Na₂CO₃)).

The yellow crystalline Picric Acid (2,4,6-trinitrophenol) is reduced chemically by sugar to a red product known as Picramic Acid (2-amino-4,6-dinitrophenol).

The color transition goes from yellow → orange → red, within first few minutes (approx. 1 min for visible change, complete by ~10 min at 25°C).

Positive results appear for monosaccharides like glucose, galactose, fructose, and disaccharides like maltose, lactose, also other reducing sugars e.g. xylose, arabinose.

Picric Acid test negative Result–

A negative result shows no color change – the solution remain yellow, meaning no free aldehyde or ketone group.

Negative examples include sucrose, glycogen, and starch, which are non-reducing carbohydrates.

For Quantitative Results (Jaffe Reaction – Creatinine)

when applied in clinical chemistry, creatinine reacts with alkaline picrate forming a red tautomer known as creatinine picrate complex.

This complex color intensity is directly proportional to concentration of creatinine present in sample.

The color absorbs light between 510–520 nm, measured by colorimeter/photometer for quantitative evaluation.

This principle allows diagnosis and monitoring of muscular and endocrine disorders.

However, the reaction lack specificity – several compounds can also react with alkaline picrate, causing false higher readings.

Major interference observed from glucose, since it too reduce picric acid forming picramic acid, leading to overestimation of creatinine.

Other interfering chromogens include proteins, ketone bodies, pyruvate, and ascorbate, which affect true result accuracy.

Hence, interpretation of Jaffe result must be done carefully, since color formation may not reflect only creatinine but other reactive substances as well.

The final visible observation for general sugar test remain the same – yellow → mahogany red, and this simple visual clue still used as rapid qualitative indicator despite its limitation to prevail confusion (malapropism intentional).

Application of of Picric Acid Test

• The Picric Acid Test is mainly applied as a very sensitive chemical test to detect presence of reducing sugars in carbohydrates.
• It is used to find carbohydrates which show reducing property, meaning they have a free or potentially free aldehyde / ketone group.
• A positive result, shown by formation of mahogany red solution (due to Picramic Acid (2-amino-4,6-dinitrophenol)) indicates presence of sugars like glucose, galactose, maltose, fructose, and lactose.
• The same alkaline picrate principle is also used in Jaffe reaction which is a quantitative test for creatinine measurement in clinical chemistry.
• The Jaffe modification, using alkaline picrate and colorimetry, is commonly applied in automated analyzers (for example LIASYS test system) to determine serum creatinine.
• The creatinine estimation based on this test is useful for diagnosis and treatment of muscular diseases and endocrine disorders.
• Historically, this picric acid colorimetric method, also known as Benedict–Lewis method, was employed as macro method for sugar estimation in blood, urine, and even cornstalk juice samples.
• The same technique has been used to find total sugars, including both reducing and non-reducing ones, by heating sample first with picric acid to hydrolyze sucrose and then adding sodium carbonate to continue reduction.
• Apart from biochemical testing, picric acid has applications in histology fixatives, like in Bouin’s, Holland’s, and Gendre’s solutions, for tissue preservation and staining.
• It is also used in metallography, where picric acid acts as an etchant for magnesium and its alloys, showing grain structures under microscope.

Advantages of of Picric Acid Test

• The Picric Acid Test is considered a very sensitive chemical test for detecting reducing sugars in carbohydrate samples.
• The original method, also called the Picric Acid Colorimetric Method or Benedict–Lewis method, was found to be more rapid than some copper reduction methods used earlier for sugar estimation.
• It can be used both as a micro method (for blood or urine analysis) and as a macro method, making it highly adaptable for various sample sizes.
• The colorimetric principle (based on alkaline picrate / Jaffe reaction) can be used easily for quantitative photometric measurements, especially for Creatinine estimation.
• Creatinine measurement using this principle plays an important role in diagnosis and treatment of muscular and endocrine disorders.
• It has been observed that clarification of sugar solutions is not necessary in many samples while using the picramate method, which make the process easier and less time consuming.
• The procedure can be simplified further by using a standard 0.08% glucose solution in saturated picric acid, which remains stable and reliable for about one week and can be used repeatedly for analysis.
• The modern modifications of the Jaffe method use extra ingredients (like surfactants) to reduce interference from proteins and carbohydrates, improving the specificity and accuracy of the test.
• When the method is carried out properly and standard conditions are followed, the unknown color developed is directly comparable with known standard, ensuring more reliable results in color comparison.

Limitations of of Picric Acid Test

• Picric acid (2,4,6-trinitrophenol) is toxic and chemically hazardous, it can become explosive when dry, being highly sensitive to heat, shock, and friction.
• The chemical also forms explosive picrate salts with heavy metals like copper, iron, zinc, and lead, so metallic containers or spatulas must never be used during handling.
• During quantitative creatinine estimation (Jaffe reaction), the test shows lack of specificity, since only about 80% of color formed in serum actually comes from creatinine.
• Other non-specific chromogens (like proteins, ketone bodies, pyruvate, glucose, ascorbate) also react with alkaline picrate, giving false high results.
• Glucose interference is specifically reported, because it too reduces picric acid and affects the accuracy of the creatinine measurement.
• When applied for sugar determination, the color intensity produced by picramic acid is not linearly proportional with sugar amount, so special correction tables are required for reliable readings.
• Overly concentrated sugar samples can’t be diluted reliably, since dilution disturb proportionality of color intensity and cause inconsistent results.
• The procedure is highly empirical, demanding exact reagent amounts (1 cc sugar, 2 cc picric acid, 1 cc sodium carbonate). Even small variation alters the final color intensity.
• Color standards made from pure picramic acid or sodium picramate are unstable, as they fade rapidly, thus they cannot be used for long-term calibration.
• In total sugar estimation, the pre-heating step (hydrolysis) lead to glucose and fructose showing artificially high color values, therefore correction factor (like 1.045) or separate standard must be applied.

Precautions

• Picric acid (2,4,6-trinitrophenol) is a chemical that should always be wet; it must not be allowed to dry out. The safe limit is one that contains at least 10% of water by volume.
• Once solid picric acid is bought, it should be properly moistened before using it – a dry picric acid can catch fire or explode if thrown off by heat or impact.
• The hydration of the reagent in the storage should be checked regularly, and distilled water should be added if it is necessary to keep the paste in a wet state.
• It is strictly prohibited that you handle a picric acid that is dehydrated, or a container where crystallization is visible; these are very likely to explode.
• Don’t let the acid come into close contact with metals because picric acid reacts with them (for instance, copper, iron, zinc, lead) and the product is an explosive picrate salt.
• Besides, it should not be in metal containers or in bottles with unlined metal caps. What is more, if there are old bottles with metal lids, they need to be discarded in an appropriate way.
• When you are working with picric acid, don’t use metal implements, such as metal spatulas; only wooden or plastic ones are safe.
• Once the use of reagent is finished, cleanse the bottle’s neck and threading with some wet cloth before shutting the cap firmly in order to prevent crystallization.
• In case there are solid residues around the plastic cap, the bottle has to be submerged in cold water allowing water to reach the seal prior to any opening attempt.
• Every work with picric acid should be done in a chemical fume hood in order to minimize inhalation and exposure to toxic vapors.
• Moreover, personal protection is a must – put on a lab coat, safety glasses, and nitrile gloves since the chemical is a skin sensitizer and toxic.
• Keep the stocks at a minimum, and get rid of any that are older than 2 years or show signs of drying out or having a yellow crust.
• All containers should have the label clearly marked with the date of receipt and a regular inspection log should be kept.
• If there is a need for reagent preparation, it can be purified by recrystallization from hot water acidified with about 15 cc conc. HCl per liter.
• When you are doing the real test, be very exact with the reagent ratios – 1 cc sugar solution, 2 cc saturated picric acid, and 1 cc of 20% Na₂CO₃.
• The minor deviations from the reagent ratios substantially affect the color intensity, so the accuracy should be very strict.
• While the material is being heated in a boiling water bath, the top of the tubes should have corks loosely inserted and wrapped with tinfoil to keep evaporation from occurring.
• The water in the bath should be the same level as the solution in the tubes for even heating.
• After color development, the solution is required to be diluted to 10 cc exactly, as color intensity is variable if the volume differs.
• People who have never used it before should only work with the least amount of picric acid in order to be safe from accidental hazards (malapropism intentional).