[anonymous10012]

Hello,

Thank you for clicking on this post. I just bought a Spectronic 20 to do some quick and dirty enzyme activity assays. I want to make my own calibration check standards with things I have on hand. I thought that this would be VERY simple by just using Beer's law to calculate the theoretical absorbance at a concentration, making that concentration, and checking it on the spectrophotometer.

I found an academic lab experiment that discloses the molar absorption coeffients of food dyes and their wavelengths of maximum absorbance (Blue No 1, Red 40, and Yellow #5 are the standards I was planning to use) .

A=∈bc

Yellow # 5

ᵧmax = 425
∈=27,300  L/(cm ×mol)
b= 1 cm
c=0.1 mol/L


If I plug these values into the equation, all of the units cancel, but the numerical answer is rubbish. Can anyone help me to understand what I need to do here?

Thank you!

[Corribus]

Why do you think your numerical answer is rubbish? What numerical answer were you expecting?

[Babcock_Hall]

Offhand I would say that your value of the concentration is worth double checking.  How did you obtain it?

[anonymous10012]

The theoretical values are not consistent with the measured values. The spec 20 reads from 0-2 absorbance. If the sample was too concentrated to discern, I would have expected the instrument to max out. I took a few data points of serial dilutions and plotted them with the theoretical values using a pathlength of 1.2 cm because the cuvette is a test tube style. (picture attached)

I dont know if there is something I am missing between the relationship from beers law and how the instrument reads. Multiple sources note that absorbance of 1 means that 90% of the wavelength is being absorbed and reading of 2 means that 99.9% of the wavelength of light is being absorbed.

The point of this tartrazine test was to check the instrument to see if it works. It is looking to me like I need to return it. I want to use the spectrophotometer to measure guaiacol complexing with oxygen in an enzyme assay, so it's not really possible to make a standard curve and use the instrument in the shape it is in.

[anonymous10012]

Babcock,

I made the stock solution using an analytical balance then serial dilutions with volumetric flasks. I was not super precise when measuring the water in the stock solution. The end goal is to conduct semi-quantitative analysis. It should have been close. I just wonder if there is something to the percent and instrument output relationship. Can you read my other reply and see if it speaks to you?

Thank you!

[Corribus]

First, Beer's Law always fails at high concentrations. Your experimental results are consistent with this. Second, even great spectrophotometers costing tens of thousands of dollars struggle measuring accurate absorbances over OD values of 2, even though they claim to be good in some cases up to values of 5. An inexpensive one could struggle with OD values over 1. Even if they say it'll read 0-2, that doesn't mean it will read high values well - just that it'll return a number. Regardless of your instrument, you should almost never make a measurement with a value over 1. Quantum yield and extinction coefficient measurements are almost always done with OD value under 0.5, and frankly I don't trust them unless they are < 0.1. So any lack of agreement between theoretical and experimental absorbance values above OD = 1 are going to be suspect anyway. You are also seeing lack of agreement with low OD values. This is most likely due to either (a) inaccurate concentrations, (b) inaccurate extinction coefficient, (c) instrument calibration (both wavelength and intensity). Since (c) is what you're kind of trying to do, let's forget about that one for a second.

Thinking about (b), here's some things to keep in mind. The molar extinction coefficient is wavelength, solvent, and concentration dependent. They're also notoriously hard to measure well, and there are a lot of bad extinction coefficient values out there. Make sure you have a good one by checking around literature sources to see if the value you use is consistent with a lot of different reported values. (Also check and make sure you are using the same solvent the literature value was measured in, and make sure also that your solvent is good. For non-aqueous measurements, wet solvent can lead to big shifts in wavelength/intensity values, and impurities can lead to background absorption.)

Are your cuvettes good quality? If not, you could have light losses from your glass/plastic. (see additional note below)

I also suggest checking the linearity of your instrument by doing serial 20% dilutions starting with an OD approximately 1 and checking to make sure you get a nice line. This will give you an idea of whether your instrument is behaving well, irrespective of quantitative accuracy of a single point.

Regarding (a) - making accurate solutions is harder than most people think. But assuming you are making them well, some other things to consider: Where are you buying your dyes? If they are old or from poor quality sources, the effective concentration can be a lot lower than you think it will be. Trying your expt with a few different chromophores can help you determine if you've got a bad batch of one.

EDIT: I didn't check your actual numbers... but I noticed after reading again you said your path length is 1.2 cm and your cuvette is "test tube style". This would mean the cuvette is round, yes, with a diameter = 1.2 cm? Is the spectrometer specifically designed for a round cuvette? If the cuvette is round, keep in mind the actual pathlength won't be 1.2 unless the beam is really narrow. The beam will be experiencing all kinds of different path lengths,  most of them less than 1.2 cm, depending on where individual photons cross through the sample. This will make it really hard to apply Beer's Law, and your measured concentrations will probably be less than theoretical based on a path length of 1.2 cm. Plus you'll have all kinds of weird reflections could interfere with the measurement. There's a reason people use square cuvettes. If you're using a round tube as a cuvette, I strongly suggest you change to a square one, as this could be a significant portion of your problem. If you don't want to invest in quartz cuvettes, inexpensive plastic ones perform reasonably well for aqueous solutions in the visible region.

[anonymous10012]

Corribus,

All of that was very helpful. I am the only chemist at work, so there isn't really anyone to talk through this stuff with. I got impatient and started making dilutions to check the linearity (as you suggested)! When I purchased the spec 20 I forgot that it uses those stupid test tube cuvettes. I had already purchased some of the plastic square ones.

Dispite the light scattering you describe from the shape of the cuvette and the haphazard dilutions that I made when I was a little frustrated, the linearity looks pretty decent. There is an adapter that allows the standard quartz style cuvettes to be used in a spec 20, but there I go again trying to save money only to be disappointed.

I really only need a qualitative answer to the enzyme activity assay (does one root have more enzyme than another harvested under different conditions?). I have a GC-MS, but other than that, I'm kind of starting from the ground up with lab supplies. It is difficult to probe questions like these without blowing the budget or asking favors from professors at my alma mater. Your reply has helped me very much to reevaluate what I can do to potentially make the instrument work for what I need it to do.

Thank you so much for your time!

Best,

A

[Corribus]

No worries at all. I think we can be reasonably certain the round cuvette is your problem. But all isnt lost. When I said strongly recommend square cuvette, that's mostly for absolute measurement. Round cuvette is perfectly suitable for concentration as long as you accept that your measurements will have to be made relative to a standard concentration curve. The absorption value by itself doesnt mean anything. Not as bad as it sounds- most chemical analysis is done this way. This does put pressure on you to make sure your standard dilutions are good and, especially, free of systematic error. This is true of any analytic measurement of course. If you want to validate your method, have someone else make up a few blind samples and see how well you measure their concentrations. And do keep in mind that linearity will still disappear at high ODs for both instrumental and molecular reasons. Better to stick to OD values less than 1, diluting your samples if necessary.

As to not having chemists around, thankfully Chemical Forums is always just a click away. We are always happy to help you work thru a problem.