[Blue Humour]

Hi Everyone,

I have made up some samples of known concentrations of Toluene in Methanol as should be seen from the graph attached.

The purpose of this was to help me identify the concentrations of Toluene from some UV data I have generated.

Is there any way I can calculate, rather than estimate (by seeing which UV conc. curve is closest), the concentration from an unknown conc. UV graph?

Cheers.

[Blue Humour]

I've not worked with UV much before and I'm really not sure where to start...

[Arkcon]

Is there any way I can calculate, rather than estimate (by seeing which UV conc. curve is closest), the concentration from an unknown conc. UV graph?

Yes.  You have built a standard curve, and now you can fit the absorbance values vs concentrations to a line, and compute the formula of that line.  Then you can reverse the calculations to determine the concentration at any point on the line.  To the accuracy of the line, which you will also know.

You won't be using curve data, but instead the value at one point on the curve.  the best one will be near, but not at the top of the curve, and you have two (or more) curve apexes to select from.  No one is, on the face of it, better or worse than any other.

Learning to build a standard curve and fit the results to a line with linear regression are not easy concepts to communicate one posting at a time, so I hope you have access to a university instructor, or some other expert.  Or can check out the general topic online, or in a library book.  Possibly a book dedicated to using Excel for scientific applications.

[Blue Humour]

Okay, I understand the concept of what you have explained and I cannot see any difficulties you suggest may be present?

For future reference, is there a way to calculate the equation of a line like this without actually preparing 'known conc.' solutions? If all I know is that I am going to be diluting Toluene in Methanol is there any way of magic-ing the equation with clever tricks? I'm guessing there must be? Or perhaps there is a library with all of these absorbance/conc. lines?

[Corribus]

For future reference, is there a way to calculate the equation of a line like this without actually preparing 'known conc.' solutions?

In principle, you can determine concentration simply without having an empirical concentration curve if you know the extinction coefficient, then using Beer-Lambert Law. This is pretty standard practice in research laboratories. This presupposes you know the extinction coefficient for your analyte, if course. Sometimes you can look this up or find values in the literature, if it is a common analyte. However the extinction coefficient is dependent on solvent choice, and the true observed absorption can also be dependent on other external factors like temperature and instrument function (and using the extinction coefficient with Beer's Law subjects you to the limitations of Beer's Law), so for analytical applications of high precision and accuracy, building your own concentration curve may be preferable even if the extinction coefficient is known.

[Blue Humour]

Okay, I have taken the absorptions at the 250nm and made the graph attached. It is not a straight line, is this a problem?

[mjc123]

For a start, your data points are not spaced along the x axis in proportion to the x coordinate (concentration). 10, 21, 42, 168 etc. are not equally spaced! You need to plot a scatter (X-Y) chart in Excel. When you do that, your line should (I hope) be linear at lower concentrations, but it will still level off at high concentrations because the absorption saturates - too little light is transmitted to measure accurately. For linear calibration you want to be working with absorbances of ideally <1 AU. If your samples are concentrated you may need to dilute them, determine the concentration of the diluted sample, and back-calculate the concentration of the original sample.

[Blue Humour]

For a start, your data points are not spaced along the x axis in proportion to the x coordinate (concentration). 10, 21, 42, 168 etc. are not equally spaced! You need to plot a scatter (X-Y) chart in Excel. When you do that, your line should (I hope) be linear at lower concentrations, but it will still level off at high concentrations because the absorption saturates - too little light is transmitted to measure accurately. For linear calibration you want to be working with absorbances of ideally <1 AU. If your samples are concentrated you may need to dilute them, determine the concentration of the diluted sample, and back-calculate the concentration of the original sample.

Thanks for your feedback. I noticed that terrible error too, what was I thinking!?

I have already scanned and disposed of the samples with high concentration, so I guess I will have to estimate.

I have two questions:
1. Are there intruments available which would help me determine concentrations as large as I am without diluting?

2. Why does the first peak appear to shift in wavelength the more concentrated the Toluene becomes? Would I not be able to use these peaks to create my Abs. vs. Conc. line?

[Corribus]

1. Different UV-Vis instruments have different OD ranges. Some go up to 3 and some go up to 5. There might be some that go up even higher. Depending on your application, though, I would recommend against pushing up against the top of this range. Absorption is a logarithmic scale, so an OD of 5 means a transmission of the incident light of only 0.001%. As a rule of thumb for measuring extinction coefficients, I like to keep ODs below 1. At high concentrations you also subject your experiment to various inner filter effects and other limitations of Beer's Law, where the observed absorbance is no longer linear with respect to concentration. Which leads us to:

2. A (usually red) shift in the absorbance wavelength at high concentrations is often observed for flat, aromatic molecules. This is because the molecules stack up and aggregate as they approach their solubility limit (some like pyrene stack up even far below their solubility limit). You will especially see pronounced effects in the corresponding fluorescence experiments. Needless to say, such effects will greatly impact the linearity of a concentration curve, which is why it's a good idea to acquire spectra over a full wavelength range rather than just analyzing a single wavelength, at least until you know you are not experiencing any spectral shape changes over the concentration range of interest.