A plasma reaction converts A (Molecular weight = 200) to B and uses NAD as a co-substrate and produces NADH as a by product (1 mole of NADH is produced per every 1 mole of A converted to B). The amount of NADH can be determined by monitoring its’ absorbance at 340nm in a spectrophotometer.

Question

A plasma reaction converts A (Molecular weight = 200) to B and uses NAD as a co-substrate and produces NADH as a by product (1 mole of NADH is produced per every 1 mole of A converted to B). The amount of NADH can be determined by monitoring its’ absorbance at 340nm in a spectrophotometer.

1ml of plasma is taken and diluted with 4 ml of reaction buffer with the enzyme required to convert A to B inhibited. 1ml of this sample is added to a cuvette and the inhibition is then stopped by adding 2 ml of a “inhibition prevention” buffer and the natural conversion of A to B allowed to occur. This conversion is completed in 10 minutes after which time the change in absorbance at 340nm is recorded.
The change in absorbance is 0.82 absorbance units over the 10 minutes. The molar extinction coefficient of NADH at 340nm is 6220.
What is the concentration of compound A in micrograms/100mL?

Answer 1

Two dillutions and assume volumes are additive

C1*V1=C2*V2

1ml was mixed with 4ml giving a total volume of 5ml.

C1*1ml=X*5ml

1ml of X was mixed with 2ml to give a total of 3ml.

X*1ml=X2*3ml

Absorbance was taken of X2:

Absorbance (A)=l*M*E

E=molar absorbtivity=6220
M=Molarity (moles/L)
l=length of cuvette (1)

Solve for M:

M=0.82/(6220*1)

Let M=X2

You know X2, so solve for X:

X=(X2*3ml)/1ml

You know X, so solve for C1:

C1=(X*5ml)/1ml

C1=mole/L

So convert moles to grams using molecular weight (200g/mole):

C1*200g/mole=g/L

And convert L to ml:

1L=1000ml

g/L*(1L/1000ml)=g/ml

Answer with the appropriate number of significant figures

Answer 2

Additional information for reply:

The info given will give g/ml. Convert g to ug and do not convert L to ml. Divide answer by 10 to get units desired.

Answer 3

To determine the concentration of compound A in micrograms/100mL, we need to calculate the amount of NADH produced in the reaction first.

Given information:
- Change in absorbance = 0.82 absorbance units
- Molar extinction coefficient of NADH at 340nm = 6220
- Volume of the reaction mixture = 1 ml (after dilution)
- Conversion time = 10 minutes

We can use the Beer-Lambert Law, which relates the concentration of a substance to its absorbance:

A = ε * c * l

Where:
A = Absorbance
ε = Molar extinction coefficient
c = Concentration
l = Path length (usually 1 cm in spectrophotometers)

Rearranging the equation to solve for concentration (c):

c = A / (ε * l)

Now, let's calculate the concentration of NADH produced:

c(NADH) = 0.82 / (6220 * 1)

c(NADH) = 0.0001319 moles

Since 1 mole of NADH is produced for every 1 mole of compound A converted to B, the molar ratio is 1:1. Therefore, the concentration of compound A is also 0.0001319 moles.

To convert moles to grams, we need to know the molecular weight of compound A. The molecular weight of compound A is given as 200.

Mass(A) = moles(A) * molecular weight(A)

Mass(A) = 0.0001319 moles * 200 g/mol

Mass(A) = 0.02638 g

To convert grams to micrograms (μg), we multiply by 1000:

Mass(A) = 0.02638 g * 1000 μg/g

Mass(A) = 26.38 μg

Finally, to express the concentration of compound A per 100 mL:

Concentration(A) = (Mass(A) / Volume) * 100

Concentration(A) = (26.38 μg / 1 mL) * 100

Concentration(A) = 2638 μg/100 mL

Therefore, the concentration of compound A in micrograms per 100 mL is 2638 μg/100 mL.