Analysis of Individual Sugars in a Mixture by HPLC using Refractive Index Detector
A HPLC method with refractive index detector (RID) was developed for sugar determination in fruit juice and fermented drinks. The method enables the determination of the three main sugars of fruit juice (fructose, glucose and sucrose), lactose for the milk product and maltose. The method ensures adequate sensitivity, quantitative recoveries, does not suffer from matrix interferences and shows good repeatability.
It was used for the analysis of 3 (2 mango & 1 apple) fruit juice samples, 1 carbonated soft drinks and one Flavoured Chilled Milk along with one whisky and one beer of different origin and the results were found to be in agreement with those reported in Nutritional label of the respective product. The method is simple and suitable for a routine quality control of fruit juice composition and represents an alternative to gas chromatographic methods, which require more complex purification and derivatization steps.
Food carbohydrates are characterized by a wide range of chemical reactivity and molecular size. Because carbohydrates do not possess chromophores or fluorophores, they cannot be detected with UV-visible or fluorescence techniques. Nowadays, however, refractive index detection can be used to detect concentrations in the low parts per million (ppm) range and above.
The mandate of nutrition labeling by the Food and Drug Administration (FDA) in 1993 has made sugar analysis (mono and disaccharides) a necessity for all of the food industry. Previous reports on sugar analysis of various food products were based on the total sugar or sucrose, with little emphasis on the other individual sugars. Colorimetric and iodometric methods (Somogyi 1952, Ting 1956) were unable to quantitate individual sugars. Gas-liquid chromatography (GLC) has been successful in determining individual sugars, but it requires a derivatization procedure (Li et al 1985). Recently, high-performance liquid chromatography (HPLC) has become the preferred method for quantitating simple sugars in a variety of food products.
Sample preparation varies depending on the food product in question. High-fat samples, such as chocolates, require defatting before analysis. High-fat samples have been pretreated by various methods, including boiling water (AACC 1983), a Carrez reagent (Bugner and Feinberg 1992), or a petroleum ether wash (AOAC 1990). Extraction methods for other food products include boiling in 80% ethanol (Picha 1985), 50% alcohol in an 80-850C water bath (Zygmunt 1982), or a water extraction (AACC 1983). Li et al (1985) extracted samples using hexane followed by water or 80% ethanol. The objectives of our study were to develop an uncomplicated and rapid HPLC method for quantitating individual sugars, particularly in various fermented products.
i. Sample or samples.
ii. 0.28 M Copper sulphate solution.
iii. 1 N sodium hydroxide
iv. Acid-washed Celite (Merck).
v. Injection solvent : 50% (v/v) HPLC-grade acetonitrile in water.
vi. Mobile phase : 78:22 (v/v) acetonitrile/10mM Sodium Phosphate
vii. Syringe with PTFE filter.
viii. High Performance Liquid Chromatograph (HPLC; PerkinElmer) with :
- Isocratic LC pump 200
- 200a series refractive index (RI) detector
A. Sample Preparation –
i. 10g – 20g of each sample was taken in 200 ml beaker, 40 ml deionized water was added and stirred for 1 hr. (approx.) on a magnetic stirrer.
ii. 10 ml of 0.28 M copper sulfate solution was added to each of the samples while stirring.
iii. pH of the samples were adjusted to 6.4 using 1 N sodium hydroxide and a pH meter.
iv. The samples were carefully transferred to 100 ml volumetric flask and volume was made-up to the mark with deionized water.
v. The solutions were filtered through Whatmann 41 filter paper overlaid with approx. 0.5g acid-washed celite.
vi. 2 ml of aliquot were taken from each of the solution into 4 dram vials, dried in heating block at 50 oC (approx.).
vii. 2 ml injection solvent (50% acetonitrile) were added to each of the vials, shaken until no residue was found on the wall of the vials.
viii. The resultant solutions were filtered using Syringe filter and injected into HPLC.
B. Preparation of calibration standards –
ix. For each reference sugar, a set of calibration standards using stock and working reference standard solutions. These 5 sets of calibration standards (i.e., fructose, glucose, sucrose, lactose and maltose) each at concentrations of 0.1%, 0.5%, 1.0%, 1.5% and 2.0%.
x. HPLC – The system (PerkinElmer) consisted of: model Series 200 pump, Series 200a Refractive Index Detector (PerkinElmer) and associated a compatible computer. The aminopropyl-bonded phase column (Pinnacle II Amino 3mm 150×4.6 mm) was operated at 40oC. The mobile phase was an isocratic acetonitrile and 10mM Sodium Phosphate (monobasic) solution (78:22). Sugar standards were dried at 60 0C in a vacuum oven overnight and dissolved in 50% Acetonitrile (Injection solvent). Injection volume was 20 ml. Sugar concentration was calculated based on peak area measurements.
Results & Discussion:–
Samples were procured from market of six brands along with whisky and beer. Sugars were extracted from the samples as per the described process and analyzed by HPLC using Refractive Index Detector. The obtained results are summarized below (Table 1) which shows a good correlation with the declared value in the respective product.
From the respective chromatogram it is clear that the fermented drinks contain trace amount of sugar.
The HPLC method for analyzing fruit juice, soft drinks, milk products and fermented products is a composite of methods previously applied to various food products, with modifications. Utilizing an isocratic solvent system improved detection limits. Little sugar was present in the fermented samples. Glucose decreased faster than fructose in yeasted products. Lactose values did not vary.
Contributed by: Mrs. Nandita Das & Mr. Bamdev Sinha