The Osazone test is a type of biochemical test which is used to detect reducing sugars. This test can distinguish between different types of reducing sugars by the appearance time of the complex. Osazone test can also be called Phenyl hydrozine test depending on the reagent used.

Purpose

• To detect reducing sugars.
• To distinguish between reducing and non-reducing sugars.
• To differentiate different reducing sugars from each other.

Principle of Osazone Test 

Osazones are the characteristic derivatives of carbohydrates that result from the reaction of reducing sugars with Phenylhydrazine. These osazone derivatives are crystallized. This made it possible to identify the carbohydrate type with ease.

This test does not work for sugars that lack free anomeric hydroxyl groups (non-reducing sugars). While glucose, fructose, and mannose produce similar types of osazone (glucosazone), The melting point, crystal shape, and time of crystallization are all different for each osazone crystal.

Requirements

Reagent

• Osazone mixture: 0.5 g of phenylhydrazine hydrochloride and 0.1 g of sodium acetate.
• Glacial acetic acid
• Test sample

Apparatus

• Test tube
• Test tube holder
• Dropper
• Pipette
• Stand 
• Spirit or gas lamp
• Light Microscope
• Slides
• Coverslip
• Solution to be tested

Procedure

1. In a small container, take 5 ml of the solution.
2. To the above-mentioned test tube, add 3 pinches of osazone mixture.
3. Mix thoroughly
4. Place the flame on the test and boil for five minutes
5. Look out for yellow crystals. If they are not formed, boil them.
6. Check every 5 minutes to ensure yellow crystals are not missed.
7. After the crystals have formed, let the solution cool at room temperature.
8. Take the crystals out and prepare slide    
9. Under microscope, examine the slide

Observations

Osazone crystals formed when examined under the microscope can take on different shapes depending upon the type of carbohydrate.

Results

The following inferences are drawn from the shapes of osazone crystals.

1. Needle shaped/ broom-stick crystals are formed in 5 minutes indicate glucose
2. Needle shaped/ broom-stick crystals are formed in 2 minutes indicate fructose
3. Cotton ball-shaped crystals are formed by lactose in 30 minutes
4. Sunflower shaped crystals are formed by maltose in 30 to 40 minutes

Carbohydrate	Time of formation (min)	Crystalline structure
Fructose	2	Needle shape
Glucose	5	Needle or broomstick shape
Galactose	20	Thorny ball or rhombic plate shape
Maltose	30-45	Sunflower/ petal shaped
Lactose	30-45	puff shaped powder or cotton balls

Advantages of Osazone Test

• This test distinguishes between reducing and non-reducing sugars. It is also the cheapest, easiest to do, and most importantly, interprets well.
• This test can be used to identify sugars in plant tissues.
• Different reducing sugars form different osazone compounds. It is an effective way to seperate them.

Limitations of Osazone Test

• Reducing sugars must be present at greater quantities in the sample for identification.
• If heated for longer than 30 minutes, it can give false-positive results for sucrose (a non-reducing sugar).
• If more than one reducing sugars are in the sample, detection is impossible.

References

• Tiwari A. (2015). Practical Biochemistry. LAP Lambert Academic Publishing.
• Shah, Tejas. (2016). Utility of Osazone Test to Identify Sugars. Journal of Medical Science And clinical Research. 04. 14361-14365. 10.18535/jmscr/v4i12.14.
• Mester, L.; El Khadem, H.; Horton, D. (1970). “Structure of saccharide osazones.” Journal of the Chemical Society C: Organic (18): 2567. doi:10.1039/J39700002567.
• https://www.topperlearning.com/answer/what-is-osazone-test-please-explain-with-the-help-of-example%20/d1yd9w11
• https://pubs.acs.org/doi/10.1021/ja01971a011
• http://ecoursesonline.iasri.res.in/mod/page/view.php?id=53489
• https://idswater.com/2019/12/08/what-is-osazone-formation-and-its-significance/
• https://pdfcoffee.com/osazone-test-pdf-pdf-free.html
• https://aklectures.com/lecture/reactions-and-disaccharides/osazone-formation-from-d-glucose
• https://www.jaypeedigital.com/book/9788184482591/chapter/ch2
• https://alevelbiology.co.uk/notes/tests-for-carbohydrates/#64-osazone-test