Dilution and Spectrophotometry

Dilution and Spectrophotometry

Introduction

In this laboratory session, you will learn the principles of dilution, serial dilutions, and determination of concentrations by use of the spectrophotometer. You will also brainstorm on their practical applications. These practical skills are needed for the completion of many exercises throughout the semester, as well as actual cases in your field of practice. A spectrophotometer, to be precise, is an immensely useful instrument and one of the most significant technological developments in the field of laboratory medicine. It operates by a simple mechanism that involves shining a beam of light that is filtered to give only a specific wavelength through a test sample in a cuvette and measuring the absorbance of the sample.

Objectives

The following are the main objectives of the practical:

1. To state the basic mechanics and principles of a spectrophotometer.
2. To perform normal and serial dilutions of a sample.
3. To determine the absorbance and subsequent concentration of the sample.

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Background Information

Diverse mechanisms are usable in spectrometry. These include the absorbance and emission spectrophotometry. Whereas the absorbance spectrophotometer measures the fraction of incident light that is absorbed by a sample, the latter measures the amount of incident energy that is emitted. In this case, the absorbance spectrophotometer is used.

Figure 1: Block diagram of a spectrophotometer

The picture above is a representation of the parts of a spectrophotometer. The source of light generates the incident light, which is then passed through a wavelength selector (a monochromator in this case), which selects only a single wavelength of light to pass through the sample. The photoresistor then converts the light that passes through the example into an electronic signal that then is amplified and measured by the meter.

Principle

The spectrophotometer operates on the Beer-Lambert’s law. The law describes the relationship between the absorbance of a sample and its concentration. According to the law, the absorbance of a sample is directly proportional to the strength of the same sample.

Materials/Equipment:

1. Deionized water
2. Stock Cibracon blue dye (20 mg/dL)
3. Spectrophotometer
4. Micropipettes (2-20uL, 20-200uL,and 100-1000 uL)
5. Serological pipettes (2ml, 5ml, and 10 mL)
6. Kim wipes
7. Parafilm
8. Tubes

Method

1. Dilutions

The principle of dilution is that with each addition of the solvent, the solute is further dispersed, and the concentration reduces. The percentage or fraction representing the new concentration denotes the amount of solute out of the total volume of solution. For example, a 1/3 dilution implies that 1 part of the solute is dissolved in 2 parts of the solvent.

1. Normal Dilutions
2. To make a 1/3 dilution, pipette 2ml of the deionized water, wipe the outside of the pipette, and dispense into a test tube. Now pipette 1ml of the stock solution, clean the outside of the pipette tip and dispense entirely into the test tube. Mix thoroughly.
3. While following the above example perform 1/6, 1/10, 1/15, and 1/25 while ensuring the volume does not exceed 5ml.
4. Serial Dilutions
5. To make a serial dilution of factor 2, dispense 0.5ml of the deionized into a test tube. Pipette 0.5ml of the stock solution, wipe the outside of the pipette, and dispense into the same test tube. Mix thoroughly.
6. Now, by the same method described above, and using the prepared dilution above as the stock solution, repeat the process four times. The final dilution will be 1/32
7. Determining Absorbance

To determine the absorbance of the sample:

1. Switch on the spectrophotometer and allow it to adjust to room temperature
2. Wash the machine by following the manufacturer’s instruction
3. Carry out the measurements against a reagent blank (use deionized water) as indicated in the user manual
4. Record results in the table below

1. Determining Concentration

Two main methods do the determination of the concentration. Firstly a calibration curve may be prepared using absorbance values obtained for the standard solution and used to determine the concentration of the unknown. Secondly, the absorbance values may be compared with the standard using the formula below.

(OD of test /OD of STD) X Conc. of the stand. =Concentration of the test sample.