[orgo814]

I'm trying to troubleshoot here.. What are some of the reasons my standard curve could have come out bad? My r^2 was terrible at 0.3. My cuvettes may have been contaminated since they've been used before although I cleaned them. Could that be cause?

[Arkcon]

That's certainly a possible reason.  However, that is really a horrible r².  I suspect many things -- could you have calculated it wrong, and of course there be another serious fault.  Can you give a a tale table of values, and possibly a chart, because really, how do you call this a line.  Maybe you need a higher order polynomial for your data, if that's permissible.

[Corribus]

Telling us what technique/experiment you're doing could also be helpful.

[orgo814]

I redid it and got a 0.85 by changing the cuvettes I was using but that still is not good enough

[orgo814]

The concentrations are: 1650, 825, 412.5, 206.25 (ug/mL)
The absorbances I got through UV-Vis were: 0.090, 0.078, 0.08, 0.073

I redid it using 20 mL standards instead of 10 mL. My r^2 ended up worse..
Absorbances: 0.194, 0.172, 0.012, 0.005

I always struggle once I put the 412.5 solution in. I am using 15 uL of each of the solutions (concentrations above) with 1 mL of a coumassie blue reagent I made up.

[Corribus]

The R² isn't your primary problem. I'm not sure exactly what you're doing, but if you double the concentration, you should double your absorbance - as long as you're in the linear range. You're not even close. In fact, between 412.5 and 825 ug/mL, your absorbance is going down. This is basic Beer's Law.

Are you doing a single read measurement, or are you acquiring the entire spectrum? If the former, are you sure you're reading the right wavelength (i.e., where there's a peak)? I would suggest you acquire an entire spectrum to be sure.

[Arkcon]

Here's a quick and dirty chart.  Sorry I didn't take the time to label axes, or the series.  You should be able to tell by the numbers, but pink is your "20 ml" sample, and blue is your "10 ml" sample.  I'm sorry you don't like the "10 ml" sample, it gives a pretty straight line -- although not a useful one, as absorbance doesn't change much as a function of concentration.

I'm wondering if you can come up with some conclusions, looking at the data thi way.

[orgo814]

I've done beers law before and know how to make standards well... The only thing I can think of is my pipettes weren't calibrated correctly or my coumassie blue went bad. This is so frustrating! :/

[Corribus]

The latter can be ruled out (mostly) by taking an entire spectrum of a dilute solution of your coumassie blue and comparing it to literature. Are the peaks in the right place? Are there any extra ones that shouldn't be there? Do your extinction coefficients roughly match. UV-Vis is fairly tolerant of impurities.

I think your pipettes would have to be pretty terrible in order to be off by so much.

What wavelength are you measuring at?

[Corribus]

It seems the extinction coefficient at 595 nm at pH 7 is around 43,000. This should help you estimate what your absorbance should be at the concentrations you are using.

[orgo814]

I've been working at 595

[orgo814]

I'm just completely confused at what could cause this

[Corribus]

Based on an extinction coefficient of 43,000 M-1 cm-1, which seems about right, what do you expect your absorbance to be for a 206 ug/mL solution?

[orgo814]

I know beers law is A = EBC, b being path length (1 cm). How would i convert the concentration to fit this formula

[orgo814]

I may have solved my problem. Did research and the coumassie I had was the reddish kind with max absorbance at 470 and I was doing mine at 595.