Absorbance and Concentration Question

In summary, the protein oxyhemoglobin has specific molar absorbance coefficients at two different wavelengths, 276 nm and 415 nm. Using the equation A=\epsilonbc, where A is absorbance, \epsilon is the molar absorbance coefficient, b is the length of light path through the cell, and c is concentration, the concentration at 415 nm can be calculated based on the given absorbance value. It is also possible to calculate the absorbance at 276 nm by substituting the concentration and molar absorbance coefficient at this wavelength. The path length, b, is assumed to be 1 cm unless otherwise stated.
  • #1
fuzzy
6
0

Homework Statement


The protein oxyhemoglobin has the following molar absorbance coefficients:
[tex]\epsilon[/tex]276 nm = 3.44 x10[tex]^{4}[/tex]M[tex]^{-1}[/tex]cm[tex]^{-1}[/tex]
[tex]\epsilon[/tex]415 nm = 1.25 x 10[tex]^{5}[/tex]M[tex]^{-1}[/tex]cm[tex]^{-1}[/tex]
If a solutioin of oxyhemoglobin has an absorbance at 415nm of 0.234,
a) What is the concentration at 415nm?
b) What is the absorbance at 276 nm?

Homework Equations


A=[tex]\epsilon[/tex]bc, where A is absorbance, [tex]\epsilon[/tex] is the molar absorbance coefficient in M[tex]^{-1}[/tex]cm[tex]^{-1}[/tex], b is the length of light path through the cell in cm, and c is concentration in mol/L.

(I don't know if this one is needed...)
[tex]\frac{As}{Au}[/tex]=[tex]\frac{Cs}{Cu}[/tex]
where As and Au are absorbance of the standard and unknown and Cs and Cu are concentration of the standard and unknown.


The Attempt at a Solution


Can someone give me a hint at where to start here? I can't see how to do the problem without at least 1 of the concentration values or a light path length. :confused: Do I assume that the concentrations remain the same at 276 and 415?
 
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  • #2
Assume that the path length, b, is the same for both samples and is 1 cm. You are given the absorbance, the extinction coefficient and you are asked to solve for 'c'. Seems pretty straightforward.

The second part asks you to substitute the concentration and molar extinction coefficient at a different wavelength and determine the absorbance at that wavelength... again, fairly straightforward.

Do I assume that the concentrations remain the same at 276 and 415?

Will the concentration change when you shine a different color light on it?
 
  • #3
Check your textbook about how to handle quantitative absorbance measurements. You probably need A=e*C, where A is absorbance, e is your epsilon, C is concentration; but I could be mistaken. Each wavelength has it own A=e*C for the oxyhemoglobin.

... Yes, you DO assume the concentration remains the same at both wavelengths.

For part (a), subsituting the values given for the 415 nm,
0.234 = 1.25*10^5 * C,
Find C.

As I say, this stuff is a bit murky now, so recheck your textbook.
 
  • #4
Thank you for the replies.

Assume that the path length, b, is the same for both samples and is 1 cm.
So this is always the case if it's not otherwise stated? Because in other problems it was given as 0.5cm, so I was confused a bit.

Other than that I think I get it now :D
So I get
a)
c=[tex]\frac{A}{\epsilon}[/tex]

c=[tex]\frac{0.234}{1.25x10^{5}}[/tex]

c=1.87x10[tex]^{-6}[/tex]M
This isn't too low for a concentration?

b)
A=[tex]\epsilon[/tex]c
A=(3.44x10[tex]^{4}[/tex])(1.87x10[tex]^{-6}[/tex])
A=0.064
 
  • #5
fuzzy said:
...So this is always the case (path length = 1 cm)if it's not otherwise stated? Because in other problems it was given as 0.5cm, so I was confused a bit.

No, it is not always the case. If the path length information is absent (an oversight in my opinion), you can assume anything. Assuming 1 cm. (a standard photometric cell dimension) must be part of your answer.

Other than that I think I get it now :D
So I get
a)
c=[tex]\frac{A}{\epsilon}[/tex]

c=[tex]\frac{0.234}{1.25x10^{5}}[/tex]

c=1.87x10[tex]^{-6}[/tex]M
This isn't too low for a concentration?

It is fairly low but you can express it as 1.87 ppm as well. ppm levels are determined routinely.

b)
A=[tex]\epsilon[/tex]c
A=(3.44x10[tex]^{4}[/tex])(1.87x10[tex]^{-6}[/tex])
A=0.064

Looks good.
 

1. What is absorbance and how is it related to concentration?

Absorbance is a measure of how much light is absorbed by a substance. It is related to concentration through the Beer-Lambert Law, which states that absorbance is directly proportional to the concentration of a substance in a solution.

2. How is absorbance measured?

Absorbance is measured using a spectrophotometer, which measures the amount of light absorbed by a sample at a specific wavelength.

3. What is the relationship between absorbance and transmittance?

Absorbance and transmittance are inversely related. As absorbance increases, transmittance decreases, and vice versa. This is because absorbance measures how much light is absorbed, while transmittance measures how much light passes through a substance.

4. How can absorbance be used to determine concentration?

Using the Beer-Lambert Law, absorbance can be used to calculate the concentration of a substance in a solution. By measuring the absorbance of a known concentration of a substance and then measuring the absorbance of an unknown concentration, the concentration of the unknown can be determined.

5. What factors can affect the accuracy of absorbance measurements?

The accuracy of absorbance measurements can be affected by factors such as the quality of the spectrophotometer, the wavelength of light used, the path length of the sample, and the presence of interfering substances in the solution. It is important to carefully control these factors in order to obtain accurate absorbance measurements.

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