Nylander’s Test for Carbohydrates Principle, Procedure, Result

Nylander’s Test sometimes also called Bismuth Reduction test, is used for detecting reducing sugars like glucose / fructose etc.

The principle is based on reduction of bismuth oxynitrate (BiONO₃) or bismuth subnitrate into metallic bismuth, in alkaline medium.

In this reaction, reducing sugar which have free aldehyde or ketone group, they reduce bismuth compound into black colored bismuth metal.

The reagent used, called Nylander’s reagent, it is mixture of bismuth nitrate (Bi(NO₃)₃), potassium sodium tartrate (Rochelle salt) and potassium hydroxide (KOH).

The potassium sodium tartrate helps to keep bismuth ions in solution and prevail the formation of insoluble bismuth hydroxide.

The sample is boiled with reagent, and if reducing sugar is present, a black / dark brown precipitate of metallic bismuth forms.

Reaction occur by bismuth tartrate complex, which goes by several steps before final reduction happen.

Positive result show black coloration; negative stays colorless or slightly yellowish, sometimes greyish also.

It was first given by Emil Nylander (1835–1907), Swedish chemist from University of Lund.

Mainly used for urine sugar detection (for diagnosing diabetes mellitus), also it detect sugar concentration up to near about 0.08–0.1%, though less reliable under 0.3%.

Principle of Nylander’s Test 

• The principle of Nylander’s Test is based on reduction reaction occur between reducing sugar and bismuth salt in alkaline medium.
• In this test, sugars that having free aldehyde or ketone group act as reducing agent, they reduce bismuth oxynitrate / subnitrate into black metallic bismuth (Bi).
• The reaction happen only under alkaline condition, because it activate the glucose molecule – converting it into reactive enolate form.
• Nylander’s reagent consist of bismuth nitrate (Bi(NO₃)₃), potassium sodium tartrate (Rochelle salt) and potassium hydroxide (KOH), sometimes NaOH also used.
• The tartrate plays important role, it makes complex with bismuth ion and stop formation of insoluble bismuth hydroxide, which otherwise formed easily in strong alkali.
• During heating, bismuth tartrate complex is reduced by sugar molecules, forming metallic bismuth that appear as black or dark brown precipitate.
• Thus the black coloration indicates the presence of reducing sugars or reducing functional groups in the sample solution.

Requirements for Nylander’s Test

• A sample solution which suspected to contain reducing sugars like glucose / fructose is required.
• The main reagent needed called Nylander’s Reagent, it is freshly prepared before use mostly.
• Nylander’s reagent contain three main components –
  • Bismuth nitrate / Bismuth subnitrate – reduced by the sugar to form metallic bismuth.
  • Potassium sodium tartrate (Rochelle salt) – help to keep bismuth ions in solution, by forming tartrate complex, also stop formation of insoluble bismuth hydroxide.
  • Potassium hydroxide (KOH) or sometimes Sodium hydroxide (NaOH) – gives alkaline condition needed for activation of sugar molecule.
• Test tube, test tube stand, and pipette (for accurate mixing) are required in lab setup.
• A heat source like water bath or spirit lamp is essential, since the reaction proceed only by heating.
• The mixture usually boiled for around 2–5 min, until color / black precipitate appear.
• All glassware must be clean and dry before using, otherwise reaction may not show proper result.

Step-by-Step Procedure of Nylander’s Test

1. About 5 mL of sample solution (like urine / glucose solution) is taken in clean test tube.
2. Around 5–8 drops of Nylander’s reagent is added carefully into same tube. Sometimes 0.5 cc of reagent added to 5 cc of 2 % glucose sol., this vary by method used.
3. The mixture is shaken / mixed gently to make uniform solution.
4. Then test tube is placed in boiling water bath for about 3 min, sometimes heating continued up to 5 min depending on result visibility.
5. After heating, tube is removed and allowed to cool down slowly to room temperature.
6. The color change is observed carefully — appearance of black / dark brown precipitate means positive result for reducing sugar.
7. If coloration come only while cooling, that’s not confirmed positive; sometimes reagent darkens slightly by long heating.
8. When no color / no precipitate appear, it is taken as negative result.
9. The tube must be cleaned right after, because black bismuth deposit stick on glass walls easily.

Nylander’s Test Result

Nylander’s Test result gives qualitative idea about presence of reducing sugars in sample.

When reducing sugar present, a positive reaction is observed.

Positive Result show clear color change from light yellow → brown → dark brown / black coloration.

Formation of black precipitate of metallic bismuth (Bi) at bottom of tube confirm the positive test.

This happen because bismuth subnitrate / oxynitrate get reduced by the sugar molecule in alkaline condition.

The shades sometimes differ – glucose give dark olive brown, fructose may give olive green, while lactose produce light yellow tone, etc.

Historical data mention color names like chocolate coloration, dark amber, light amber for weaker solutions (0.5%–0.005%).

Negative Result – no change in color, the solution stay clear or slightly cloudy, no black deposit seen.

It means reducing sugar absent or concentration below detection limit (about 0.08–0.1%, sometimes not reliable under 0.3%).

Sucrose, starch, and other non-reducing sugars usually give negative reaction.

The timing also matter – color should appear during boiling or soon after; if it come later (on cooling), that’s not considered positive.

So, dark bismuth precipitate act like signal / alarm showing reducing activity present in tested sample.

Uses of Nylander’s Test

• Nylander’s Test mainly used for detection of reducing sugars like glucose / fructose in solution.
• It’s applied in qualitative analysis of carbohydrates, showing presence of reducing functional groups.
• Often used in organic chemistry labs for identifying aldehyde containing compounds.
• Urine testing for reducing sugars is major use, especially for clinical diagnosis of diabetes mellitus.
• It serve as optimum assay for glucose detection in urine, sensitive for small concentration also.
• In food industry, it used for measuring sugar content in food items / beverages etc., important for sweetness and flavor control.
• Quality control units in industries apply this test where residual reducing sugars can affect taste, texture, shelf life.
• Research and teaching labs use it as standard test to demonstrate carbohydrate reduction and redox chemistry to students.
• In agriculture field, it help to check sugar levels in fruits & produce, to estimate ripeness and quality.
• Sometimes also employed as screening test before using more advanced carbohydrate analysis methods.

Advantages of Nylander’s Test

• Nylander’s Test is regarded as one of the most sensitive assays for glucose detection in urine samples.
• It can detect very small concentration of reducing sugars, even about 0.08–0.1%, which make it highly reliable.
• The appearance of black metallic bismuth precipitate give clear visual proof, so result interpretation is simple & direct.
• It work effectively for trace detection of reducing carbohydrates, even when present in minute amount.
• The bismuth–tartrate complex principle maintain stability of reagent, allowing bismuth ion to stay soluble in strong alkaline medium.
• The test show high precision & specificity, since only reducing sugars yield black coloration.
• Historically it was said that when result found negative, no further sugar test required — showing strong reliability.
• The reaction product (metallic bismuth) itself is distinct, making identification easy without extra apparatus.
• It’s simple, cost-effective, and valuable for biochemical and clinical laboratory works.
• Overall, it serve as dependable qualitative tool for routine detection of reducing sugars in different types of sample.

Limitations of Nylander’s Test

• Nylander’s Test is non–specific, it gives positive result with any reducing substance, not only glucose.
• Reaction respond to reducing functional groups, mainly aldehydes, also to sugars like fructose and lactose, so confusion may occur.
• Historically it was concluded that test become of no value when dextrose concentration below about 0.3 %.
• The sensitivity for dextrose detection is lower when compared with other carbohydrate assays like Fehling’s Test.
• When performed with urine, sample must be protein-free, because proteins interfere with color & precipitate formation.
• Using excess urine (more than ~15 drops) sometimes gives dirty green color, which is not characteristic of positive reaction.
• Color interpretation can be subjective — sometimes amber yellow seen even when sugar present, causing false negative reading.
• The test require heating to start reaction; without heat, reduction not proceed, so it cannot be used at room temp.
• The dark coloration must appear during boiling stage; if it appear later (during cooling), it’s not considered valid evidence for sugar.
• Hence, the Nylander’s Test though simple & cheap, still limited by subjectivity, low sensitivity, and non-specificity.

Precautions

• While interpreting Nylander’s Test, make sure dark color or black precipitate must appear during boiling, not when fluid cooling.
• If color appear only after cooling, that not confirm presence of reducing sugar.
• When testing urine with dextrose, not more than 15 drops should be added, else reaction turn dirty green and misleading.
• The urine sample should be free from proteins, because proteins interfere by forming unwanted turbidity.
• Always wear protective gloves, lab coat, eye & face protection when handling Nylander’s Reagent.
• Reagent is corrosive / caustic, it can cause skin burn or eye injury, so handle carefully.
• Avoid any contact with eyes, skin, or clothes, if contact happen, wash immediately with plenty water.
• Do not inhale vapors, dust or mist from the reagent; work in well ventilated area.
• After handling, wash hands and exposed skin properly, before eating or leaving lab.
• In case of ingestion, rinse mouth – but do not induce vomiting.
• Store reagent in tightly closed container, in cool dry ventilated place away from incompatible stuff.
• Keep it away from sunlight and acids / organic materials / peroxides / chlorinated solvents / metals like aluminum, tin, zinc, phosphorus.
• Avoid long air exposure, since reagent absorbs CO₂ and lose strength.
• Always store reagent locked & labeled, separated from food items or chemicals that react with alkali.