Determination of Lactose In Milk by Lane-Eynon Method

What is Lane-Eynon Method?

• The Lane-Eynon method is a volumetric technique used for quantifying the amount of reducing sugars in a solution. This analytical procedure involves a titration process with Fehling’s solution, which serves as the reagent to detect and quantify the presence of reducing sugars based on a redox reaction. Fehling’s solution is a blue alkaline liquid that contains copper(II) sulfate mixed with sodium potassium tartrate (Rochelle salt) in an alkaline medium. The Rochelle salt acts to stabilize the copper ions, keeping them in solution.
• In the presence of reducing sugars, the copper(II) ions in the Fehling’s solution are reduced to copper(I) oxide, which precipitates out of solution as a red insoluble solid. This change is indicative of the reduction process and is used to determine the endpoint of the titration. The point at which the blue solution turns to a brick-red color, indicating the complete reduction of copper(II) ions and thus the presence of reducing sugars, is carefully noted.
• The method requires the determination of a ‘Fehling factor,’ which is essentially a calibration step to ascertain the amount of a standard reducing sugar solution needed to completely reduce a specific volume of Fehling’s solution. This factor is then used to calculate the concentration of reducing sugars in the sample being tested.
• The Lane-Eynon method is particularly useful in the food industry and in laboratories where the sugar content of various substances needs to be analyzed. It can be applied to simple reducing sugars directly, and with additional steps involving hydrolysis, it can also be used to estimate the amounts of more complex sugars like sucrose and starch, which are non-reducing but can be converted into reducing sugars under certain conditions.

Principle of Lane-Eynon Method

The Lane-Eynon method is a titrimetric procedure that quantifies reducing sugars in a solution through the utilization of Fehling’s solution as the primary reagent. This method operates on the fundamental chemistry of reduction-oxidation reactions, where Fehling’s solution, composed of copper sulfate and an alkaline solution of sodium potassium tartrate (Rochelle salt), acts as the oxidizing agent. The Rochelle salt serves a critical function by stabilizing the copper ions in solution, forming a complex with cupric hydroxide that prevents it from precipitating out.

When a reducing sugar is present in the solution, it interacts with the copper complex in Fehling’s solution, reducing the copper(II) ions to copper(I) oxide. This reduction process results in the formation of a red, insoluble precipitate of cuprous oxide, indicating the presence of reducing sugars. To accurately determine the endpoint of this redox reaction, an oxidation-reduction indicator, such as methylene blue, is employed. This indicator changes color to signal that all the copper(II) ions have been reduced, marking the completion of the reaction.

Before the quantitative analysis of reducing sugars can be conducted using this method, it’s essential to establish the ‘Fehling factor’. This factor represents the specific amount of a standard reducing sugar, typically invert sugar, required to fully reduce a defined volume of Fehling’s solution. This calibration step is crucial for ensuring the accuracy of subsequent measurements.

The Lane-Eynon technique is not limited to simple reducing sugars; it can also be applied to substances like sucrose and starch. Sucrose, a non-reducing sugar, can be transformed into reducing sugars through mild acid hydrolysis, allowing for its quantification using this method. Similarly, starch can be broken down into glucose, a reducing sugar, using strong acids, making it possible to measure starch content indirectly through the Lane-Eynon method.

This titration method is valuable in various fields, particularly in food chemistry and industry, for analyzing sugar content in products and ingredients, providing a reliable means of assessing the quality and composition of food items.

Requirements for Lane-Eynon Method

1. Fehling’s Solution A: This is one of the two parts that constitute Fehling’s solution. It is prepared by dissolving 34.639 grams of copper(II) sulfate pentahydrate (CuSO4·5H2O) in distilled water, with the volume made up to 500 milliliters. The solution should then be filtered to remove any insoluble impurities, ensuring clarity and purity for accurate titration results.
2. Fehling’s Solution B: The second component of Fehling’s solution involves dissolving 173 grams of Rochelle salt (potassium sodium tartrate, KNaC4H4O6·4H2O) and 50 grams of sodium hydroxide (NaOH) in distilled water. This mixture is then diluted to 500 milliliters. It’s essential to allow this solution to stand for at least two days before filtering to ensure all constituents are fully dissolved and any precipitates are settled.
3. Methylene Blue: This is used as an oxidation-reduction indicator in the titration process. A 1% solution of methylene blue is prepared in water, which will indicate the end point of the titration by changing color when all the copper(II) ions have been reduced.
4. Sample for Analysis: In this context, a milk sample is specified, but the Lane-Eynon method can be applied to various samples containing reducing sugars. The sample must be prepared appropriately, often requiring dilution, to fall within the method’s quantifiable range.
5. Standard Reducing Sugar: Pure lactose is used as a standard for calibration in this setup. The standard substance is essential for determining the Fehling factor, which is a critical step in quantifying the reducing sugar in the sample.
6. Heating Source: A spirit lamp with wire gauze is mentioned as the heating element to provide the necessary energy for the reaction. The wire gauze helps distribute the heat evenly, preventing direct flames from impacting the reaction vessel, which could lead to uneven heating or localized overheating.

Procedure

1. Preparation of Reagent Mixture: Begin by accurately measuring 5 or 12.5 milliliters of Fehling’s solution A and an equal volume of Fehling’s solution B. Combine both solutions in a 250-milliliter flask. This mixture serves as the reactive medium for the reducing sugars.
2. Standard Solution Preparation: Create a standard lactose solution, with its concentration adjusted so that the volume needed to completely react with the copper in the Fehling’s solution falls between 15 and 50 milliliters. The exact volume required for complete reduction provides the basis for calculating the concentration of reducing sugars in the sample.
3. Initial Titration: Add nearly the entire calculated volume of the lactose solution to the Fehling’s mixture, leaving a small amount (about 0.5 to 1.0 milliliters) for fine adjustment later in the process.
4. Heating: Heat the mixture to boiling, using a spirit lamp and wire gauze to distribute the heat evenly. Maintain boiling for approximately 2 minutes, adjusting the flame to prevent vigorous bubbling that could disrupt the titration.
5. Indicator Addition and Completion of Titration: While maintaining the heat, introduce 2 to 5 drops of a 1% methylene blue solution. This serves as an indicator for the end of the reaction. Continue the titration by adding the remaining lactose solution in small increments until the blue color disappears, indicating the complete reduction of copper ions. This should be achieved within a total boiling time of around 3 minutes.
6. Calculation of Factor: Determine the total volume of lactose solution used (titre) and multiply by the known concentration of lactose in the standard solution to calculate the ‘factor,’ which correlates to the amount of reducing sugar present.
7. Adjustment and Standardization: Compare the calculated factor with standard values to apply any necessary corrections. This adjustment accounts for variations in reagent composition or procedural deviations. For results within a 1% margin of error, this step may be skipped if the analytical procedure and reagent specifications are strictly followed.
8. Incremental Titration for Unknown Samples: If the sugar concentration in a sample is not predetermined, use an incremental titration approach. Mix a known volume of the sample with Fehling’s solution and heat to boiling. Gradually add the sample in small quantities until a very faint blue hue persists.
9. Final Titration and Factor Application: Add the indicator and finalize the titration with dropwise addition of the sample until the indicator’s color is neutralized. Refer to standardized tables to correlate the titre to the sugar concentration, adjusting with the previously determined correction factor.

Results of Lane-Eynon Method

Upon completing a Lane-Eynon titration, the analysis shifts towards interpreting the results to determine the lactose content in the tested sample. The fundamental outcome of this method is a percentage, representing the amount of lactose relative to the sample’s total volume or weight.

To compute the percentage of lactose, the following formula is used:

Lactose (%)= (Factor/tire) ×100

In this equation:

• Factor is a predetermined value representing the amount of lactose that corresponds to the complete reduction of the Fehling’s solution used in the titration. It’s calculated based on a standard lactose solution.
• Titre is the volume of the sample that was required to reach the end point of the titration, at which the blue color of the methylene blue indicator disappears, signaling the complete reduction of all copper(II) ions in the Fehling’s solution.

To elucidate the percentage of lactose present in a sample, the calculated factor is divided by the titre value obtained during the titration. The result is then multiplied by 100 to convert it into a percentage. This final value is the percentage concentration of lactose in the sample.

For example, if the factor determined for a standard lactose solution is 0.5 mg/mL and the titre volume is 25 mL, the percentage of lactose in the sample would be calculated as follows:

Lactose (%)=(0.5/25​)×100=2%

This indicates that the sample contains 2% lactose by volume or weight, depending on the initial sample preparation.

Interpreting the results requires precision and an understanding of the titration process. Any deviation in the standardization of the factor or errors in measuring the titre can significantly impact the accuracy of the lactose percentage calculated. Therefore, meticulous technique and careful calculations are crucial in the Lane-Eynon method to obtain reliable and valid results.

Factor for Fehlin solution to be used in correction with the Lane-Eynon general volumetric method.