Sweeteners

A. Natural Sweeteners

i. Lactose (Commercial)

The lactose is manufactured from the whey remaining after the manufacture of casein, cheese or chhana from milk. Lactose finds its use in dietary foods and pharmaceutical products.

Purity

Reagents

•  Acid mercuric nitrate solution: Dissolve purified mercury in twice its weight of nitric acid and dilute with 5 volumes of water.
•  Mercuric iodide solution: Dissolve 33.2 g of KI and 13.5 g mercuric chloride in 200 ml of glacial acetic acid and 640 ml of water.
•  Phosphotungstic acid solution: 5% (m/v)

 Procedure

Take two graduated flasks, one of 100 ml and the other of 200 ml capacity.Weigh accurately 65.8 g of the prepared sample into each flask. Add to each flask 20 ml of acid mercuric nitrate solution or 30 ml of mercuric iodide solution.To the 100 ml flask, add phopshotungstic acid solution and dilute to the mark with water. Shake both the flasks frequently during 15 min filter through dry filter paper and polarize.

Calculate percentage of lactose as follows:

Subtract reading of solution from 200 ml flask (using 400 mm tube) from reading of solution from 100 ml flask (using 200 mm tube). Multiply difference by 2.Subtract result from reading of solution from 100 ml flask; Divide result by 2.

Specific rotation

Accurately weigh 10 g of the sample and dissolve it in a beaker in about 60 ml of water. Cover with a watch glass and boil for 15 min. Allow to cool, add a few drops of dilute ammonium hydroxide of sp gravity 0.96 and make up to 100 ml in volumetric flask. Measure the optical rotation at 20°C.

Specific rotation, degrees =  (100*a* 100)/ l* c *w

Where,

a=observed optical rotation in degrees,

l= length in tube in decimeters,

c= weight, in g, of sample in 100 ml of water, and

w= lactose percent in the sample.

ii. Dextrose Monohydrate

Purity

Reagents

•  Fehling’s solution A: Aqueous solution of cupper sulphate (68.28 g CuSO4.5H2O/L).
•  Fehling’s solution B: Aqueous solution containing 346 g of sodium potassium tartrate, and 100 g sodium hydroxide per litre.
• Standard dextrose solution: Dissolve 2.5 g of pure dextrose dried at 70°C under reduced pressure, in distilled water to make 1000 ml.
•  Methylene blue indicator : 1% aqueous solution.

 Procedure

Weigh sample to 500 ml volumetric flask to give 0.25 g dextrose per 100 ml.With pipette transfer 5 ml of each Fehling’s solutions A & B into 250 ml conical flask. Add pumice stone and enough water suitable for titration. Put the conical flask on the heating device. Right from the start of heating add sample or standard dextrose solution amounting within 0.5 ml of the anticipated end point. Adjust the heating in such a way as to make the solution boil within 2.75±0.25 min and then make no more adjustment till the end of the test. After 2 min of boiling, add 2 drops of methylene blue solution and complete the titration drop by drop. Carry out the operation till the blue colour vanishes. Titration should be completed within 1.5-2.0 min after the addition of the indicator.

Dextrose, % by mass =125V/ VI M

Where,

V=volume, in ml, of standard dextrose solution,

V1= volume, in ml, of the test solution used, and

W= mass, in g, of the test portion.

iii. Refined Sugar

Reducing sugar

The complex formed between Cu++ ions and potassium tartrate is reduced by reducing sugars to Cu+ ions which is precipitated as Cu2O. The precipitated Cu2O is the determined by iodometric titration. The Cu2O is oxidized by an excess is back titrated with sodium thiosulphate. The reaction between the reducing sugars and Cu++ complex is not stoichiometric. The amount of Cu2O formed depends upon the prescribed conditions which therefore strictly followed.

Reagents

• Modified Ofner solution : Weigh out 7 g of copper sulphate pentahydrate, 10 g sodium carbonate, 10 g 300 g potassium sodium tartrate and 50 g disodium hydrogen phosphate in a 1000 ml flask. Dissolve in approximately 900 ml water.
•  Heat the solution for 2 h in a boiling water bath. Cool down to room temperature and fill up to the mark. Add approximately 10 g activated carbon and stir for 5- 10 min. Filter the solution.
• Potassium iodate solution (0.01667M)
•  Starch solution (1%)
•  Sodium thiosulphate solution (0.0333M)
•  Iodine solution (0.01667M)

 Procedure

Weigh 40 g sugar sample and make up with water to 200 ml (£ 25 mg invert sugar/ 50 ml).

Hot value : Mix 50 ml of the prepared solution with 50 ml Ofner solution. Add some pumice pieces to the mixture. Bring the mixture to the boil within 4-5 min.Boil for exactly 5 min. Cool the mixture down in a water bath with cold running water. After approximately 10 min the mixture should have reached room temperature. Add iodine solution until the colour of the mixture turns a typical iodine colour. This procedure dissolves the formed cuprous oxide with an excess of iodine. The surplus iodine should be so high that between 10-15 ml of sodium thiosulphate are consumed on back titration. Add 15 ml of 1M HCl by pouring it down the inner side of the flask so that the residual droplets are washed down into the solution. Cover the flask with a watch glass and move it gently for 2 min until the precipitate of cuprous oxide is completely dissolved. Titrate the sample with 0.0333M sodium thiosulphate. Add 1 ml of starch solution immediately before the end point is reached.

Cold value : Mix 50 ml of the prepared sample with 50 ml of the Ofner solution. Leave the mixture at room temperature for 10 min. Repeat the procedure as in hot value.

Blank value : Mix 50 ml of water with 50 ml of Ofner solution. Repeat the procedure as in hot value.

Reducing sugar, mg/kg =((A − B−C − D)×1000)/ S

Where,

A= calculated hot value; B= calculated cold value;

C= calculated blank value; D= sucrose correction, and

S= amount of sample in 50 ml of prepared solution.

Calculated cold/hot/blank value = (Vi×Fi-Vth×Fth)

Where,

 Vi & Vth = volume of iodine solution and thiosulphate solution used, respectively.

Fi & Fth = correction factors for iodine solution and thiosulphate solution,respectively.

iv. Sucrose

Weigh accurately about 5 g of the sample in a beaker, add about 50 ml of water and warm the mixture in a water bath at 50-60°C for about 5 min to dissolve the sucrose content of the sample. Cool and filter through a Whatman filter paper No.40. Collect the filtrate carefully in a 100 ml volumetric flask. Wash the beaker and the insoluble residue of starch in the filter paper carefully with water. Make up the volume of the filtrate to 100 ml.

Inversion : Take 10 ml of this solution in a conical flask and add 1.5 ml of conc HCl and about 10 ml of water. Heat the flask at 60-70°C for 10 min in a water bath. Cool immediately and transfer quantitatively the inverted solution to a volumetric flask and make up the volume to 100 ml.

Reducing Sugar : Pour the prepared solution into a 50 ml burette. Pipette 25 ml of Fehling’s solution into 250 ml conical flask and run in from the burette 15 ml of the prepared solution. Without further dilution, heat the contents of the flask over wire gauze, and boil. When it judged that nearly all the copper is reduced,add 1 ml of methylene blue indicator solution. Continue boiling the contents of the flask for 1-2 min from the commencement of ebullition and then add the prepared solution n small quantities (1 ml or less at a time), allowing the liquid to boil for about 10 sec between successive additions, till the blue colour of the indicator just disappears. Note the reading of the burette and calculate the volume of the prepared solution used up in titration.

Sucrose, % by mass =(0.95(Q−WR))/ W

Where,

Q = value in table corresponding to the titre,

R= percent of reducing sugar by mass, and

W= mass, in g, of the material taken for the test.

v. Honey

Specific Gravity

Clean and thoroughly dry the specific gravity bottle and weigh. Fill it up to the mark with freshly boiled and cooled distilled water which has been maintained at 27±1°C and weigh. Remove the water, dry the bottle again and fill it with the honey sample maintained at the same temperature. Weigh the bottle again.

Specific gravity at 27°C = Weight of honey in bottle/ Weight of water in bottle

Moisture

Determine the refractometer reading of honey at 20°C and calculate the percentage of moisture from the values given in table.

Fructose-Glucose Ratio

Pipette 50 ml of honey solution in a 250 ml flask. Add 40 ml of iodine solution and 25 ml of sodium hydroxide solution. Stopper the flask and keep in dark for 20 min. Acidify with 5 ml of sulphuric acid and titrate quickly the excess of iodine against standard sodium thiosulphate solution. Conduct a blank using 50 ml of water instead of honey solution.

Approx. glucose, % by mass (G) = ((B−S)× 0.004502×100)/ W

Where,

B = volume of sodium thiosulphate solution required for the blank,

S= volume of sodium thiosulphate solution required for the sample, and

W= mass, in g, of honey taken for test.

Approx. fructose, % by mass (F) = (Approx. reducing sugar, %-G)/ 0.925

True glucose, % by mass (D) =(Approx. reducing sugar, %-D)/ G-0.012F

True fructose, % by mass = 0.925

B. Artificial Sweetners

i. Aspartame

Aspartame is a non-nutritive sweetener, sugar substitute and flavour enhancer. It is 100-200 times sweeter than sucrose and has an acceptable dietary intake of 0-40 mg/kg body weight. It has been permitted for use under the PFA rules.

Purity

Weigh accurately about 150 mg of sample, previously dried at 105°C for 4 h. Dissolve in 35 ml of dimethylformamide. Add 5 drops of thymol blue and titrate with a micro burette to a dark blue end point with 0.1M lithium methoxide.Perform a blank determination and make any necessary correction. Each ml of 0.1M lithium methoxide is equivalent to 29.43 mg of C14H18N2O5.

Note : Sodium methoxide may be used for the titration.

5-Benzyl-3,6-Dioxo-2-Piperazine Acetic acid

Gas Chromatograph

GC equipped with FID containing a 1.83 m×4 mm I.D. glass column packed with 3 percent OV-1 on 80/100 mesh Supelcoport. Condition the column overnight at 250°C before readjustment and equilibration to the operating condition.

Operating Conditions

The operating parameters may vary depending upon the particular instrument used, but a suitable chromatogram may be obtained using the following conditions:

(a) Column temperature : 200°C

(b) Injector temperature : 200°C

(c) Detector temperature : 275°C

(d) Carrier gas-N2 flow rate : 75 ml/min

(e) H2 and air flow to burner : Optimized to give maximum sensitivity.

(f) Recorder : 1 mv full scale

Reagents

1. Silation reagent: Just before use, dilute 3 parts by volume of N,O-bistrimethylsilyl) acetamide with 2 parts of dimethylformamide.

2. Standard solution: Transfer 25 mg of 5-Benzyl-3,6-dioxo-2-piperazine acetic acid into a 50 ml volumetric flask, dissolve in methanol dilute to volume with methanol and mix. Pipette 10 ml of this solution into a 100 ml volumetric flask dilute volume with methanol and mix. Pipette 3 ml of second solution into a 2 dram vial with Teflon lined cap and evaporate to dryness on a steam bath. Add 1 ml of silation reagent to the residue, cap the vial tightly, shake and heat in an oven at 80°C for 30 min. Remove the vial from the oven, shake for 15 sec and cool to room temperature.

3. Sample preparation: Transfer about 10 mg of aspartame sample accurately weighed into a 2 dram vial with Teflon lined cap, add 1 ml of the silation reagent,cap tightly, shake and heat in an oven at 80°C for 30 min. remove the vial from the oven, shake for 15 sec and cool to room temperature.

Procedure

Inject a 3 ml portion of standard preparation into the GC obtain the chromatogram,measure the height of peak produced by 5-benzyl-3,6-dioxo-2-piperazxine acetic acid. Under the stated conditions, the elution time is about 7-9 min. Similarly inject a 3 ml portion of sample preparation, obtain the chromatogram, measure the height of the peak produced by the 5-benzyl-3,6-dioxo-2-piperazxine acetic acid contained in the sample.

ii. Sodium Saccharin

Sodium saccharin (sodium salt of 2,3-dihydro-oxobenzisulfonazole or 1,2-benzisothiazolin-3-one-1,1-dioxide sodium salt dihydrate is a non-nutritive sweetener. It is 500 times sweeter than sucrose. It is used in specifically notified food products under PFA rules as artificial sweetener within the prescribed limits.

Purity

Dissolve about 0.3 g of previously dried sample, accurately weighed in 20 ml glacial acetic acid. Add 2 drops of crystal violet-glacial acetic acid indicator and titrate with 0.1N perchloric acid. End point shall be when violet colour of solution change to green via blue. Perform a blank determination, make any necessary correction, Each ml of 0.1N perchloric acid is equivalent to 20.52 mg of sodium saccharin.

Toluenesulfonamides

Reagents

•  Solution A : 4 volumes of methanol plus 1 volume of acetone plus 0.5 percent (m/v) of the sample.
•  Solution B: 4 volumes of methanol plus 1 volume of acetone plus 0.005% (m/v) of 4-sulphamoylbenzoic acid.
•  Solution C : 4 volumes of methanol plus 1 volume of acetone plus 0.005% (m/v) of toluene-2-sulfonamide.

 Procedure

Prepare silica gel G plate (0.25 mm thick) and dry plates at 105-110°C for 1 h.Pour into the developing chamber sufficient quantity of mobile phase (100 volumes of chloroform+ 50 volumes of methanol+11.5 volume of strong ammonia solution)to form a layer about 15 mm deep. Close the tank for 1 h at 20-27°C. Using micropipette apply separately to the chromatoplates 2 ml each of solutions A, B and C.

Dry the spots and place the chromatoplates in the developing chamber at 20-27°C until the mobile phase has ascended to the 15 cm line. Remove the plates and dry them in current of warm air. Then heat at 105°C for 5 min. Spray the hot plates with the sodium hypochlorite solution (0.5% m/v). Dry in a current of cold air until sprayed area of the plate below the line of application give at most a faint blue colour with a drop of a mixture, prepared by dissolving 0.5% KI in starch mucilage containing 1% m/v of glacial acetic acid. Spray the plates with the same mixture. The spots in the chromatograms obtained with solution B and C should be more intense than any corresponding spots in the chromatogram obtained with solution A.

iii. Sorbitol Powder

Sorbitol powder (food grade) used as a food additive is permitted for use in certain foods under PFA rules.

Purity

Weigh accurately about 50 mg of sample and mix with 40 ml of sodium periodate solution and allow to stand for 1 h. Then add 1 g of potassium hydrogen carbonate,50 ml sodium arsenite solution and about 2 g of KI. Titrate against iodine solution using starch as indicator. Carry out a blank. The difference between the two titrations corresponds to the volume of sodium periodate taken up by the sorbitol powder. Each ml of 0.1N sodium periodate corresponds to 0.0018218 g of sorbitol.