Why is the blank cuvette needed in spectrophotometry?
When you use a spectrophotometer, you are not actually measuring the absolute absorption of the sample, but the sum of all light loss between the light source and the detector. Some of that light is simply not aimed directly at the detector, some of it is scattered by the cuvette, some is absorbed by the cuvette, some bounces off the detector instead of being absorbed by it, and of course some of the light is absorbed by your sample. You only care about the light lost by absorption of your sample, so you run a “blank” cuvette in the machine first (no sample), and the machine will internally save the absolute light value in its computer. Then when you put the sample in, the computer will automatically account for the light that was lost to all of the things that wasn’t in your sample.If you don’t have any microchips in your spectrophotometer (super-super-old-school), then what you have to do is measure the absorption of your blank (it won’t be zero) and then subtract the blank absorption from the measured sample absorption to calculate the actual absorption of your sample.
What is the functions of UV/Vis spectrophotometer?
It can also be used for other functions having to do with change in UV spectrum. We use it in our lab to track the folding and unfolding of proteins, since the two conformations have different absorbance maximums. We can tell the extent to which it folds/unfolds, and the time it takes to do so. Microbiologists use them to measure optical density (think of it as how cloudy a solution is) to track the growth of bacteria growing in liquid medium. The possibilities for good times are endless =)
Why is a spectrophotometer absorbance negative?
A measured absorbance may be negative if the absorbance of the reference used in the blank measurement in that given frequency is greater than that of your sample.Typical case is: you measure the absorbance of a substrate (absorbance 1), than that of sample + substrate (absorbance 2). You subtract absorbance 2 from absorbance 1 and the result is negative !! And you don`t understand, how can I add a sample and reduce the total absorbance in that frequency so that when I subtract from that of the substrate it gives negative?Simple => the substrate that you used as reference usually is not the same under your sample. It can be a little thicker and thus absorb more.
Why do we use quartz cuvettes in the spectrophotometer?
You don't always use quartz cuvette. It depends on the type of spectrophotometery you're trying to do.Typically quartz cuvette are used when the wavelength of the incident light is in the UV range because the quartz cuvette will not absorb the light. If you use the standard plastic cuvettes (although you can get UV safe plastic cuvettes) in the UV range the plastic will absorb some of the light so you won't get a correct reading.You will also use quartz cuvettes (or glass cuvettes if not in the UV range) if there is something in the solution, such as a solvent, that will react with a plastic cuvette and cause a change in its optical properties. (Some solvents will attack the plastic and make it go cloudy.)
What does negative numbers on the decibel scale mean?
I’m guessing you might be seeing this on a power amp, or on recording meters. In this case, zero dB represents the point at which clipping occurs. Clipping refers to the fact that you’ve reached the limits of the equipment, the peak, where your sinusoidal wave “clips” and becomes a square wave. It’s reached the top, and so flattens out. This is distortion. This is why you want to stay below zero dB. Square waves can also damage speakers.Say you have a 300 watt amplifier. When your outputting 300 watts, you’re at zero db. Reduce output to -3 dB, and you’re outputting 150 watts. Being a logarithmic scale, each 3 dB represents a halving, or doubling, of the power output.
Spectroscopy of ion versus neutral atom?
The frequencies/wavelengths of light that an atom/ion absorbs/emits (i.e. the spectrum) depends on the arrangement of its quantized energy levels and the probabilities of the electrons undergoing transitions between these levels, which is in turn affected by the electrostatic field experienced by the electrons. The electrons in atoms and ions obviously experience different degrees of attraction by the nuclei and repulsion by neighbouring electrons, so their energy levels are not going to be the same. In short, each atom/ion will show a unique set of energy levels and spectral frequencies/wavelengths that can be used to fingerprint its identity. There's no simple way to predict how the spectra differ unless you solve the Schrodinger equations for the atom/ion of interest.
Why am I getting a negative absorbance in FTIR spectroscopy?
It depends on the wavelength, but usually this is a case of a bad background scan. If your negative peaks are around 2300 wavenumber then this is due to a different amount of CO2 in your sample chamber compared to your background scan. Make sure the instrument is thorougly purged with CO2 free gas and the problem will go away.If it’s at a different wavelength, then you have a different problem but still likely due to something giving you some absorbance in your background scan.
Do data obtain from a spectrophotometer for absorbance of a solution have to be decimal or could it be a whole number like 53 or 46?
Juraj’s answer is definitely a good one. As (1) a fundamentally alternative approach, or (2) in the event there is no lab member with experience on that particular instrument (and the manual, from say 1989, is lost forever), I’ll offer the following:You can verify the instrument readings with a standard of known extinction coefficient/molar absorptivity. It sounds like you might be in a molec. bio. or biochem. lab. If that’s the case, you may have 4-nitrophenol or any of the free nucleobases or nucleosides (e.g. adenosine, gunaosine, etc) on hand. If not, I’m sure there is something laying around that would fit the bill.The aforementioned molecules have well-established extinction coefficients freely available in the literature (I think p-nitrophenol is in the ballpark of 18,000 1/M*cm at 405 nm, in aqueous media of high pH, for example). Make a stock solution you’d expect to give you 1 A.U. (based on Beer’s law) and see what the instrument tells you. In my experience, excellent agreement is obtained if (a) things are done right, and (b) the instrument is behaving. ‘Excellent’ as in you might expect an Abs of ~ 0.95 A.U or so. Remember to employ the appropriate blank and let us know how it goes!
Spectrometer. effects of fingerprints on cuvette?
a) Fingerprints will reduce the amount of light that can pass through the cuvette. As a result, transmittance will be lower and absorbance will be higher. b) From what I know about this experiment, you are measuring the absorbance of FeSCN^2+ in the solutions at equilibrium. But if your measured absorbance is erroneously high due to the fingerprints, then your calculated equilibrium concentration of FeSCN^2+ will end up being significantly higher than the actual concentration. Now, the amounts of Fe^3+ and FeSCN^2+ at equilibrium must add up to the original amount of Fe^3+ added to the solution. This means that your calculated equilibrium concentration of Fe^3+ will end up being erroneously low. Similarly, the amount of free and bound SCN^- must add up to the original amount of SCN^- added to the solution, so your calculated equilibrium concentration of SCN^- will also be erroneously low. c) The reaction of interest is: Fe^3+ + SCN^- <==> FeSCN^2+, for which the equilibrium constant expression is Kc = [FeSCN^2+]/([Fe^3+][SCN^-]). If we plug the bad data into this expression, the denominator will be lower and the numerator will be higher, so the end result is that we will get an erroneously high value for Kc.
Mass spectrometer question?
In case you are confused by the other answer: M/Z means Mass (M) divided by Charge (Z). So the m/z of 25Mg+ is 25/1=25. The m/z of 25Mg2+ is 25/2=12.5. The mass spectrometer works by firing ions into a pipe and having a magnetic field deflect, or make the ion fly in a different direction, so it reaches the detector at the end (the spectrometer has a right angle so it looks like a right angled pipe with magnets at the right angle to make the ions curve so they dont fly straight into the wall of the spectrometer). The spectrometer makes the ions curve into the detector by using magnets that pull the ion towards the walls of the spectrometer. One thing you have to know to answer this question is: the larger the ion's mass or the lower the charge, the stronger the magnetic field has to be to be able to deflect it. The smaller or higher the charge of the ion, the weaker the magnetic field has to be because the ion has a higher charge (in this question the mass of both ions is the same so ignore the mass) so it attracts to the magnets easier. Therefore you can see that 25Mg2+ has a higher charge than 25Mg+ so it's easier to deflect because it does most of the curving itself due to its charge. The 25Mg+ ion is harder to deflect so it requires a stronger magnetic field to deflect it.