UJI KUANTITATIF KARBOHIDRAT

Summary

This video script details a step-by-step laboratory procedure for measuring reducing sugars in samples. The process begins with weighing the sample and performing extractions, followed by using a centrifuge and preparing standard curves for glucose concentrations. The key part of the procedure involves using a spectrophotometer to analyze the absorbance of the sample, from which the concentration of reducing sugars is calculated. Additionally, the script explains how to handle reagents, pipettes, and the importance of precision in measurements, culminating in the calculation of the sugar concentration using linear regression equations.

Takeaways

• 😀 The sample is first weighed and then mixed with a buffer for extraction, followed by pipetting 10 mL of liquid for processing.
• 😀 Centrifugation is used to separate the mixture, isolating the supernatant, which contains the reducing sugars (like glucose).
• 😀 A standard curve with varying glucose concentrations (0-200 g/L) is prepared to facilitate comparison with the sample absorbance.
• 😀 The absorbance of both the sample and standards is measured using a spectrophotometer at 540 nm to determine sugar concentration.
• 😀 The standard curve is created by plotting absorbance versus concentration of glucose, helping to quantify reducing sugars in the sample.
• 😀 Reducing sugar concentration is calculated using the regression equation derived from the standard curve and the sample's absorbance.
• 😀 The formula to calculate reducing sugar concentration includes the dilution factor and the sample weight, ensuring accuracy in results.
• 😀 Aquades (distilled water) is used throughout to ensure that the solution is free from impurities, which could affect the analysis.
• 😀 The procedure emphasizes the importance of precise pipetting and handling to avoid errors and ensure accurate spectrophotometric readings.
• 😀 The final percentage of reducing sugars in the sample is calculated and averaged from the measurements taken for each sample.
• 😀 A common mistake in the procedure is incorrect absorbance readings, where the absorbance should increase with concentration, not decrease.

Q & A

What is the first step in the procedure described in the transcript?

The first step is to weigh the sample (e.g., corn) and place it into a Falcon tube for extraction.

How is the extraction buffer added to the sample?

The extraction buffer is added using a pipette. 10 mL of the buffer is transferred into the Falcon tube containing the sample.

What is the purpose of the centrifugation step in the process?

Centrifugation is used to separate the components of the sample. The heavier particles are forced to the bottom of the tube, and the supernatant is collected for further analysis.

What is the role of the spectrophotometer in the procedure?

The spectrophotometer is used to measure the absorbance of the samples at a wavelength of 540 nm. The absorbance values are used to create a standard curve and calculate the concentration of reducing sugars.

Why is a standard curve necessary in this experiment?

A standard curve is necessary to correlate the absorbance values to known concentrations of glucose. This allows the concentration of reducing sugars in the samples to be calculated accurately.

What is the significance of using aquades (distilled water) in this experiment?

Aquades is used because it is free from impurities and minerals that could interfere with the accuracy of the experiment. It ensures that only the intended substances are present in the solutions.

What temperature is required for the reaction in the procedure?

The reaction is heated at 100°C in a water bath to facilitate the chemical reaction that leads to the formation of a colored product, which is essential for absorbance measurement.

What happens if the standard curve does not show a straight line?

If the standard curve does not form a straight line, it indicates a problem with the pipetting or sample preparation, suggesting that the experiment was not performed correctly.

How is the concentration of reducing sugars calculated from the absorbance values?

The concentration of reducing sugars is calculated using the regression equation derived from the standard curve. The absorbance values of the samples are used to determine their corresponding concentrations.

What formula is used to calculate the percentage of reducing sugar in the sample?

The formula for calculating the percentage of reducing sugar is: Reducing Sugar (%) = (PPM from curve × Volume of Extract) / (Weight of Sample × Dilution Factor) × 100.