scsynthsis
Rising Star
Beers Law. Live By It.
This IS My introductory essay.
If you read all it of you you not only will learn how to use a spectrophotometer but I even tell you where to buy one for VERY reasonable price.
Foreword: Endlessness' analytical methods thread on this forum is a pretty decent summary of some viable analytical methods & apparatus, albeit most(if not all..) of these techniques and theories involved (not to mention equipment) seem to be well beyond the scope of the average person on the forum.
This is my attempt to elaborate on UV-Vis. Beer-Lambert Plots. Serial Dilutions. BASIC Color Change Chemistry. Analytical Methodology.Applied Linear Regression.
- This post should be moved to Endlessness' Analytical Methods Post -
I thought it was funny that someone would ask a chemist to generalize something as complex as color changing reactions, and then ask about which peak on a UV-Vis standard to target in a workup..
However, if i were asked to explain color changing chemistry to some budding "scientists", phenolphthalein is a relatively simple example of pH related functional group cleavage relating directly to color change.
Phenolphthalein - Wikipedia
Perhaps they will catch the planar positioning of the benzene ring and relative differences in functional groups relative to color. However, I dont see the utility for explaining the mechanism any further, it involves free radicals in an organic setting...that wouldn't really serve to strengthen the understanding of the color differences of phenolphthalein in relation only to the structural differences as different pH values. basically..lets keep it simple! these aren't O-chem grad students cramming for cumes.
Multiple Peaks in UV-Vis can mean different things depending on the context. ie A solution containing more than one unknown can in theory have more than one peak in UV-Vis. But! A solution containing a chemically pure standard can also have more than peak given the chemical structures.
Go look at an Acetone molecule, Yes! effing right now! no whining.
(3D - http://upload.wikimedia.org/wikipedia/commons/8/8d/Acetone-3D-balls.png)
(2D - File:Acetone-CRC-MW-ED-dimensions-2D.png - Wikipedia)
Where do you think the UV Energy goes when it interacts with this molecule?
(Answer: Carbonyl group. Explanation: Now this one's up to you to wrestle with.. food for thought!)
On the topic of UV-Vis:
That ergot alkaloid paper posted here is actually a really amazing piece of history.. however it isnt clear enough.
People wont know what e is or even know how to approach doing a beer's law plot.
So I wanted to make some clarifications.
UV Vis is a great introduction to analytical methods.
As long as a chemical standard is available UV Vis is an excellent analytical method!
(older spectrometers need both a chemical standard and known maximum absorption wavelength ie Spectronic-20, because they scan at only one wavelength.)
How a typical procedure would go is to take your analyte "sample", weigh it on at the least a miligram scale.(.000g) Next dissolve in selected non-polar solvent.
(methanol and petroleum ether both would work well in regards to this forum.)
Which ever solvent you used to dissolve your analyte, will be used for "background corrections". Background corrections or calibrations are different for different machines.
For example:
On a Perkin Elmer Lambda 35 UV-Vis. Spectrophotometer, background correction is performed by placing only the solvent into a cuvette and then into the machine's "sample" cuvette slot. (the other cuvette slot is the reference cuvette would be filled with your chosen solvent as well, as a background reference while scanning) - the PE Lambda 35 is an expensive machine only available to scientists or maybe theres a few wealthy eccentric people out there that actually own one in their home? unlikely.
On a Milton Roy Spectronic - 20. Calibration is done by flipping a switch and dialing in knobs for Transmission/Absorption & Lamp power. it is easy and literally explained on the machine itself in the form of a sticker (usually, unless removed).
However the spectronic 20 is only useful IF you know your Analytes peak absorption wavelength AND have a known chemically pure standard to use in a serial dilution. (Spectronic 20's are great and affordable for common folk!)
http://www.ebay.com/itm/Bausch-Lomb-Spectronic-20-Spectrophotometer-Used-/390363850735#vi-content - 60.00USD i think? used - needs cover for sample port, im not sure i would trust it.. that lamp could be tired..
350.00USD looks pristine, and a huge bargain might i add.. secksy chrome'd knobs and all.
After background steps are done with a PE-Lambda 35 You can scan from 190nm-1100nm, This is done to determine your initial analyte concentration and location of highest peak. IF Absorption is over 1.0 shadowing effects in the linear optical path will correlate to inaccurate data, in normal speak - for accurate data you must have absorption values at least under 1.1, to achieve this we dilute our sample solution. until we find a happy concentration under 1.1 (stick as close to 1.0 - 1.1 as possible for the high end concentration range - it is necessary for linear beer's law plots)
After we have our most concentrated sample (x,y) = (75% Sample, 1.1 A) we will do a series of dilutions on our Standard and make up 5-6 min. data points for the different concentrations and absorption values.
(std , standard = standard known concentration chemically pure standard in soln)
ie. 100% Standard, diluted 1:1.5 = 75% standard. 100% standard, 1:2 = 50%std, 50% std, 1:2 = 25% std, 25%std 1:2 = 12.5% std. And so on.. the end factor (not including 1:1.5 example) is 1:8 (2*2*2).
After you have your concentration and absorption plotted , use a least squares linear regression to fit a line to your data. correlation co-efficient for accurate linear beer plots must be over .990!
Now use your linear model and solve X (conc.) for the Analyte absorption value.
ie
1.1 = (mx + b , Linear equation), solve for x (we know x = 75% conc.)
DONT forget to multiply back by 1/3 to represent 100% Analyte and not only 75% of it!
In my example i use %conc. but concentration can be expressed as Molarity(mol/L) or unit mass per unit volume , mg/ml,dg/dl,etc.. concentration is a broad term.
I prefer to use Molarity because its easy to convert to anything. Couple that with excel or a TI-89 and entire X lists can go from M to mg/ml in a few steps.
et, Voila! Beer would be so proud!
let me know what you guys think. If you want to know more about a different analytical technique let me know.. Believe it or not uv.vis is the simplest, when compared to NMR,HPLC,LC/MS,GC/MS,GC,FT-IR, AA,MS, etc..
Honestly though, I feel like beyond simple curiosity all of these techniques and methods mentioned above will not be utilized. Why not just determine melting point of your sample in a thiele tube and compare to literature known standards... simple technique worked just fine for cocaine in the 80's.
oh by the way,
I am scsynthesis.
Somewhere in Bay Area - California.
This IS My introductory essay.
If you read all it of you you not only will learn how to use a spectrophotometer but I even tell you where to buy one for VERY reasonable price.
Foreword: Endlessness' analytical methods thread on this forum is a pretty decent summary of some viable analytical methods & apparatus, albeit most(if not all..) of these techniques and theories involved (not to mention equipment) seem to be well beyond the scope of the average person on the forum.
This is my attempt to elaborate on UV-Vis. Beer-Lambert Plots. Serial Dilutions. BASIC Color Change Chemistry. Analytical Methodology.Applied Linear Regression.
- This post should be moved to Endlessness' Analytical Methods Post -
I thought it was funny that someone would ask a chemist to generalize something as complex as color changing reactions, and then ask about which peak on a UV-Vis standard to target in a workup..
However, if i were asked to explain color changing chemistry to some budding "scientists", phenolphthalein is a relatively simple example of pH related functional group cleavage relating directly to color change.
Phenolphthalein - Wikipedia
Perhaps they will catch the planar positioning of the benzene ring and relative differences in functional groups relative to color. However, I dont see the utility for explaining the mechanism any further, it involves free radicals in an organic setting...that wouldn't really serve to strengthen the understanding of the color differences of phenolphthalein in relation only to the structural differences as different pH values. basically..lets keep it simple! these aren't O-chem grad students cramming for cumes.
Multiple Peaks in UV-Vis can mean different things depending on the context. ie A solution containing more than one unknown can in theory have more than one peak in UV-Vis. But! A solution containing a chemically pure standard can also have more than peak given the chemical structures.
Go look at an Acetone molecule, Yes! effing right now! no whining.
(3D - http://upload.wikimedia.org/wikipedia/commons/8/8d/Acetone-3D-balls.png)
(2D - File:Acetone-CRC-MW-ED-dimensions-2D.png - Wikipedia)
Where do you think the UV Energy goes when it interacts with this molecule?
(Answer: Carbonyl group. Explanation: Now this one's up to you to wrestle with.. food for thought!)
On the topic of UV-Vis:
That ergot alkaloid paper posted here is actually a really amazing piece of history.. however it isnt clear enough.
People wont know what e is or even know how to approach doing a beer's law plot.
So I wanted to make some clarifications.
UV Vis is a great introduction to analytical methods.
As long as a chemical standard is available UV Vis is an excellent analytical method!
(older spectrometers need both a chemical standard and known maximum absorption wavelength ie Spectronic-20, because they scan at only one wavelength.)
How a typical procedure would go is to take your analyte "sample", weigh it on at the least a miligram scale.(.000g) Next dissolve in selected non-polar solvent.
(methanol and petroleum ether both would work well in regards to this forum.)
Which ever solvent you used to dissolve your analyte, will be used for "background corrections". Background corrections or calibrations are different for different machines.
For example:
On a Perkin Elmer Lambda 35 UV-Vis. Spectrophotometer, background correction is performed by placing only the solvent into a cuvette and then into the machine's "sample" cuvette slot. (the other cuvette slot is the reference cuvette would be filled with your chosen solvent as well, as a background reference while scanning) - the PE Lambda 35 is an expensive machine only available to scientists or maybe theres a few wealthy eccentric people out there that actually own one in their home? unlikely.
On a Milton Roy Spectronic - 20. Calibration is done by flipping a switch and dialing in knobs for Transmission/Absorption & Lamp power. it is easy and literally explained on the machine itself in the form of a sticker (usually, unless removed).
However the spectronic 20 is only useful IF you know your Analytes peak absorption wavelength AND have a known chemically pure standard to use in a serial dilution. (Spectronic 20's are great and affordable for common folk!)
http://www.ebay.com/itm/Bausch-Lomb-Spectronic-20-Spectrophotometer-Used-/390363850735#vi-content - 60.00USD i think? used - needs cover for sample port, im not sure i would trust it.. that lamp could be tired..
350.00USD looks pristine, and a huge bargain might i add.. secksy chrome'd knobs and all.
After background steps are done with a PE-Lambda 35 You can scan from 190nm-1100nm, This is done to determine your initial analyte concentration and location of highest peak. IF Absorption is over 1.0 shadowing effects in the linear optical path will correlate to inaccurate data, in normal speak - for accurate data you must have absorption values at least under 1.1, to achieve this we dilute our sample solution. until we find a happy concentration under 1.1 (stick as close to 1.0 - 1.1 as possible for the high end concentration range - it is necessary for linear beer's law plots)
After we have our most concentrated sample (x,y) = (75% Sample, 1.1 A) we will do a series of dilutions on our Standard and make up 5-6 min. data points for the different concentrations and absorption values.
(std , standard = standard known concentration chemically pure standard in soln)
ie. 100% Standard, diluted 1:1.5 = 75% standard. 100% standard, 1:2 = 50%std, 50% std, 1:2 = 25% std, 25%std 1:2 = 12.5% std. And so on.. the end factor (not including 1:1.5 example) is 1:8 (2*2*2).
After you have your concentration and absorption plotted , use a least squares linear regression to fit a line to your data. correlation co-efficient for accurate linear beer plots must be over .990!
Now use your linear model and solve X (conc.) for the Analyte absorption value.
ie
1.1 = (mx + b , Linear equation), solve for x (we know x = 75% conc.)
DONT forget to multiply back by 1/3 to represent 100% Analyte and not only 75% of it!
In my example i use %conc. but concentration can be expressed as Molarity(mol/L) or unit mass per unit volume , mg/ml,dg/dl,etc.. concentration is a broad term.
I prefer to use Molarity because its easy to convert to anything. Couple that with excel or a TI-89 and entire X lists can go from M to mg/ml in a few steps.
et, Voila! Beer would be so proud!
let me know what you guys think. If you want to know more about a different analytical technique let me know.. Believe it or not uv.vis is the simplest, when compared to NMR,HPLC,LC/MS,GC/MS,GC,FT-IR, AA,MS, etc..
Honestly though, I feel like beyond simple curiosity all of these techniques and methods mentioned above will not be utilized. Why not just determine melting point of your sample in a thiele tube and compare to literature known standards... simple technique worked just fine for cocaine in the 80's.
oh by the way,
I am scsynthesis.
Somewhere in Bay Area - California.
