# Homework Help: Spectrophotometry of an indicator

1. Aug 12, 2009

### pyridine

1. The problem statement, all variables and given/known data
OK, so if your indicator has 2 clearly different wavelengths of absorption for acidic and basic forms, you can measure the absorbance of buffered samples at a wavelength which only absorbs, for instance, the acidic form. Then you can use beer's law to find the concentration of the absorbing form A=elc (e=molar absorptivity, l= path length (1cm) and c=concentration of the thing which is absorbing). Then you can find by difference the concentration of the non absorbing form and then the pKa with the Henderson Hasselbalch equation.

BUT!

I want to know, how you find the concentration of the absorbing and non absorbing forms if there isn't a wavelength which only one absorbs. How would you figure out the ratio of acid/base???

2. Relevant equations
Beer's Law: A=elc

Henderson Hasselbalch equation: pKa = pH+ log(acid/base)

3. The attempt at a solution

I know Beer's Law would then be A(total)=A(base)+A(acid), and hence A(total)= ebc(base) +ebc(acid) but that's as far as i know.

Someone please help! I'm so close and this lab report has taken me nearly 2 weeks to do :(
1. The problem statement, all variables and given/known data

2. Relevant equations

3. The attempt at a solution

2. Aug 13, 2009

### symbolipoint

You want separate scans of wavelengths for the acid form and for the alkaline form of this indicator. Look for nonoverlapping wavelengths, significantly separated, in which both forms of the indicator absorb. When you measure your sample solution, you must use the two separate wavelengths. This way, you can have two equations; one for one wavelength for the acid form, and the other for the wavelength for the alkaline form. See your sample may be expected to have the two, differently colored forms of the indicator present, the concentration of each being unknown; but you then have two equations - each based on the absorbance at different wavelengths. I am not sure if I misunderstood your question, but I say that you want two different wavelengths which are not close to eachother, and that one wavelength absorbs significantly the acidic form and the other wavelength is absorbed significantly by the alkaline form.

3. Aug 13, 2009

### Staff: Mentor

Would it help if you will know sum of concentrations of both forms?

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4. Aug 13, 2009

### pyridine

Symbolipoint: Even if you scan an acid wavelength and a basic wavelength like you said, if the wavelengths overlap, all of the absorption wouldn't be related to a single form. Do you think that'd matter?

Borek: You'd know the sum of the concentrations of both, it's just the total concentration of your sample.

5. Aug 13, 2009

### pyridine

Awright....
I've inched further forward. I know the total concentration and the total absorbance. The total absorbance is made up of x concentration of base and y concentration of acid. What would be a way to work out the ratio of acid/base, if you know the absorbance of a purely acid sample as well as a purely base sample at the same wavelength?

6. Aug 13, 2009

### chemisttree

I find the http://en.wikipedia.org/wiki/Isosbestic_point" [Broken] to me most useful for these problems. You will need the molar absorptivity at the isosbestic wavelength for it to be useful. After that you can calculate the ratio of both the protonated and deprotonated species (in the case of an acid/base indicator).

Last edited by a moderator: May 4, 2017
7. Aug 13, 2009

### Staff: Mentor

Doesn't it give you two equations in two unknowns?

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8. Aug 13, 2009

### symbolipoint

I still believe that the member named 'pyridine' needs two different wavelengths. Borek, what am I missing?

9. Aug 13, 2009

### Staff: Mentor

I have already proved today (in other life) that I am an idiot, so I don't trust myself too seriously, but

[HA] + [A-] = Ctotal

kHA[HA] + kA[A-] = Atotal

If kHA<>kA there are two independent equations in two unknowns. Note that the second equation is for one measurement, at one wavelength.

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10. Aug 13, 2009

### pyridine

2 equations, 2 unknowns..of course. Thanks!!

11. Aug 13, 2009

### symbolipoint

If you adjust the pH to the isosbestic pH and then choose the wavelength for this isosbestic point, you could then directly measure the total concentration of the indicator compound. This would require an isosbestic point to exist for this compound. Pyridine and Borek, is that what you wanted to do?

12. Aug 14, 2009

### Staff: Mentor

I suppose total concentration was already given.

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