Using a spectrophotometer

Summary

This video provides a comprehensive guide to using a spectrophotometer to measure the absorbance of samples, such as algae or bacteria, to estimate their concentration. Viewers will learn how to prepare the blank and sample, calibrate the device, and take accurate readings using a cuvette. The video also explains how to interpret absorbance values with the help of a standard curve, which relates absorbance to cell concentration. This informative tutorial is ideal for beginners in laboratory analysis, highlighting the necessary tools, techniques, and procedures for effective spectrophotometry.

Takeaways

• 😀 The spectrophotometer measures how much light is absorbed by a liquid sample, helping to determine the concentration of substances like algae or bacteria.
• 😀 A blank sample is essential for calibration to ensure accurate absorbance readings.
• 😀 When there are no cells in the sample, the absorbance value is zero because all light passes through.
• 😀 The addition of cells (like algae or bacteria) increases the absorbance as some light is absorbed by the cells.
• 😀 The more cells added to the sample, the higher the absorbance value.
• 😀 The clear side of the cuvette must always face the light source for accurate readings.
• 😀 A standard curve is used to correlate absorbance values with the concentration of cells per milliliter.
• 😀 The spectrophotometer needs to be set to the correct wavelength for the analysis.
• 😀 Always use a new pipette tip when handling a fresh sample to avoid contamination.
• 😀 After measuring the blank sample, press the 0% ABS button to calibrate the spectrophotometer to a zero absorbance value.
• 😀 Once the absorbance value is measured for the test sample, it can be used to estimate the concentration of microorganisms in the sample.

Q & A

What is the primary purpose of using a spectrophotometer in this procedure?

The primary purpose of using a spectrophotometer is to measure the amount of light absorbed by a liquid sample, allowing us to quantify the concentration of cells, such as algae or bacteria, in the sample.

What happens when there are no cells in the sample being measured?

If there are no cells in the sample, all the light will pass through, resulting in an absorbance value of zero.

How does the presence of cells like algae or bacteria affect the absorbance value?

When cells like algae or bacteria are present, they absorb some of the light, causing the absorbance value to increase. The more cells in the sample, the higher the absorbance value will be.

What materials are needed for this spectrophotometric analysis?

The materials needed are a spectrophotometer, a blank sample, a test sample, pipetters, tips, and clean cuvettes.

What is the correct orientation for inserting the cuvette into the spectrophotometer?

The clear side of the cuvette should always face the light source when it is inserted into the spectrophotometer.

What is the purpose of setting the spectrophotometer to 0% absorbance with the blank sample?

Setting the spectrophotometer to 0% absorbance with the blank sample calibrates the instrument, ensuring that any light absorbed by the sample itself is accounted for, thus establishing a baseline for the test sample measurements.

What is the significance of the 'standard curve' in this procedure?

The standard curve allows for the interpretation of the absorbance values by providing a known relationship between the number of cells per milliliter and the corresponding absorbance. This enables the estimation of cell concentration in unknown samples based on their absorbance value.

Why is it important to use a new pipette tip when transferring the blank sample?

Using a new pipette tip ensures that there is no contamination between different samples, which could alter the accuracy of the absorbance readings.

What should be done after measuring the absorbance of a sample in the spectrophotometer?

After measuring the absorbance of a sample, the previous sample should be removed from the spectrophotometer to avoid contamination, and the absorbance value should be recorded.

What types of organisms can be quantified using this spectrophotometric method?

This spectrophotometric method can be used to quantify unicellular organisms such as algae, bacteria, and other similar microorganisms.