Biochemistry Practical 1: Blood Analysis

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Questions and Answers

What is the concentration of the saline solution used for sample preparation?

1.0% NaCl

0.5% NaCl

0.8% NaCl (correct)

0.25% NaCl

How should a cuvette be handled to avoid obstructing the reading?

Handle it via the bottom part

Keep it upright at all times

Avoid touching the light path (correct)

Touch any part of the cuvette

What is the main reason for using glass containers for solution storage?

To save plastics (correct)

They are lighter than plastic

They are more affordable

They can be thrown away easily

What is the approximate volume requirement for a cuvette?

Only that the light path is submerged Signup and view all the answers

What kind of graph is generated when using a spectrophotometer?

Absorption spectrum Signup and view all the answers

What is the characteristic of the peak wavelengths in the absorption spectrum of a molecule such as haemoglobin?

They signify the signature of the molecule Signup and view all the answers

What should you do to save on micropipette tips while filling a cuvette?

Pour the liquid in until approximately 2/3 full Signup and view all the answers

Which peak in the absorption spectrum of haemoglobin should be ignored according to the guidelines?

The last peak indicated by the machine Signup and view all the answers

What does the abbreviation ABS represent in the context of the calibration curve equation?

Absorbance Signup and view all the answers

In calculating the original concentration of hemoglobin in mg/mL, what must be taken into account?

Dilution factor Signup and view all the answers

If the final absorbance (ABS) is recorded as 6.6087, what is the next step to determine the concentration of hemoglobin?

Substitute the value into the calibration curve equation Signup and view all the answers

Which unit is used for expressing original concentration in the calibration curve?

mg/mL Signup and view all the answers

What does "[Hb]\

The concentration of hemoglobin in mg/mL Signup and view all the answers

What is the equation that represents the calibration curve based on the provided data?

ABS = 6.6087[Hb] Signup and view all the answers

How is the trend line for the data plotted on the scatter graph customized?

It includes the origin where concentration is zero. Signup and view all the answers

What is the primary purpose of using the spectrophotometer in this experiment?

To measure the absorbance of the hemoglobin solutions. Signup and view all the answers

Which concentration of hemoglobin would likely produce the measured absorbance of 1.008?

0.20 mg/mL Signup and view all the answers

In constructing a scatter graph in Excel, what is a necessary step after plotting the data?

Adding a trend line that fits the data. Signup and view all the answers

What should be checked for accuracy when plotting the calibration curve?

The title must describe the relationship clearly. Signup and view all the answers

If a hemoglobin solution has an absorbance of 1.36, what would be its concentration based on the calibration curve equation?

Approximately 0.25 mg/mL Signup and view all the answers

What is a common mistake when drawing a trend line by hand on graph paper?

Using a line that curves to fit data instead of being straight. Signup and view all the answers

What is the molecular weight of Mr?

64,500 g/mol Signup and view all the answers

Which of the following statements is true about hemoglobin concentration?

Hb in mg/ml can be converted directly to g/l. Signup and view all the answers

How can Hb concentration in mol/l be calculated?

"[Hb] (g/l) / 64500" Signup and view all the answers

What is the correct unit for a concentration that is 2.14 μM?

μM Signup and view all the answers

What is the relationship defined by the calibration curve?

A linear relationship between concentration and absorbance. Signup and view all the answers

What does the broad peak at 273 nm in the Hb spectrum correspond to?

Absorption by different amino acids Signup and view all the answers

In the calibration curve's equation Y = a x + b, what does 'b' represent?

The value of Y when x is zero Signup and view all the answers

If the slope 'a' is determined in the calibration curve, what does it indicate?

The sensitivity of the measurement Signup and view all the answers

Which peak in the fake blood spectrum correlates with yellow colorant?

327 nm Signup and view all the answers

What does the term 'intercept' mean in the context of a linear equation?

The point where the line crosses the y-axis Signup and view all the answers

What characteristic does the broad peak at 514 nm indicate in the fake blood spectrum?

Absorption of red colorant Signup and view all the answers

What will the spectrum look like if 'Hb in saline' is used as a blank reference instead of saline alone?

It will resemble the fake blood spectrum Signup and view all the answers

What is the significance of the peaks found around 259 nm and 327 nm in the context of fake blood?

They correspond to the absorption of colorants Signup and view all the answers

What determines the color seen in a solution according to the absorption properties?

The color seen is complementary to the color absorbed Signup and view all the answers

The sharp peak at 406 nm in the Hb spectrum is attributed to which component?

Haem group in hemoglobin Signup and view all the answers

What is the effect of having a peak around 430 nm in analyzing fake blood?

It indicates a possible hidden lower peak Signup and view all the answers

Study Notes

Practical 1: Is this blood real?

Aims: Identify and quantify biological molecules (hemoglobin, proteins, and DNA) using spectrophotometry
Intended Learning Outcomes (ILOs): Students will be able to:
- Perform calculations to prepare solutions and dilutions
- Create calibration curves (by hand or Excel)
- Calculate solution concentrations and convert units
- Use spectrophotometers to measure absorption spectra
- Identify molecules in solution via absorption spectra
- Create standard curves using known hemoglobin concentrations for determining unknown concentrations
Specific techniques:
- Use micropipettes accurately (automatic)
- Determine absorption spectra and measure absorbance at specific wavelengths
- Identify molecules in solution based on their absorption spectra
- Plot a standard curve of absorbance versus hemoglobin concentration
Equipment: Micropipette/automatic pipettor, universal tube (30 mL), bijou bottle (7 mL), cuvette, cuvette rack, sample in cuvette, spectrophotometer, etc.

Useful Data-Handling Modules

Measurements and units
Moles and concentrations
Accuracy and precision
Graphs and trendlines
Functions and equations

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Description

This quiz focuses on the practical aspects of identifying and quantifying biological molecules such as hemoglobin, proteins, and DNA through spectrophotometry. Students will learn to prepare solutions, create calibration curves, and measure absorption spectra to determine concentrations of unknowns. Get ready to apply your knowledge of laboratory techniques and analytical skills!