What is the origin of the shift (up) in the baseline of the UV-VIS spectrum as noticed from 300 nm to 800 nm in the screenshot attached? I'm measuring phenobarbital in 0.2 NaOH against 0.2 NaOH blank. I have tried turning off fluorescent lights, CRT monitors, and capping the cuvette while measuring the sample on my HP 8453 chemstation.
Mr. Assaf,
The picture is, due to, solvent effect. It is not associated with the baseline.
Bojidarka B. Ivanova,
please check the attached picture for the same samples under the same experimental conditions.
All of the scans shown appear to be the same. Only their measured concentration appears to be different. If you normalize the scan plots, they will probably overlay. The rise in low UV baseline may be due to heating (temp) or because the samples were taken at different times (baseline values change over time). You can compensate for this.
Bojidarka B. Ivanova , Can you elaborate more on the solvent effect that you think is occuring?
William Letter, Im well aware that these are different concentrations. Why isn't the baseline (>400nm) not superimposable?
The compound absorbs in the low UV/VIS region. As you raise the concentration, you increase the absorbance. The baseline changes each time you measure it. You can compensate for this using the instrument.
You stated that the spectrum shown was from "300 nm to 800 nm". That is mostly in the visible region of the spectrum. Depending on lamp types used, the output may be in the UV, visible, IR or some combination of them. Your spectrum shows the visible and UV regions. Most UV lamps output into the VIS region (often to >600 nm) which we assume yours does, as you stated it went to 800 nm. So if you turn OFF the VIS lamp, you will still see a spectrum all the way up to 600 nm (maybe higher, but it should sharply decay after that as noise takes over) as this represents the normal output of a deuterium lamp. Please refer to the operator's manual of your spectrophotometer for more info).
"As you raise the concentration, you increase the absorbance." Sound familiar? Think about it...
Phenobarbital has a lamdba max ~ 256 nm. You should be monitoring at that wavelength (~10 nm bandwidth). You should see very little increase in baseline within the VIS region (confirmed by your scan). You should also see an increase in ALL UV signals in the UV region as sample concentration increases (also confirmed by your scan). Just about everything absorbs in the UV region (including the NaOH). The more there is, the more absorbance you will see. Please read up (or get some initial training in) the use of a spectrophotometer to better understand this.
With your argument ( "As you raise the concentration, you increase the absorbance." Sound familiar? ), we should be observing an incremental baseline with increasing concentrations. I am well aware how UV spectroscopy work, I just dont think your explanation is correct.
You do not understand or maybe you have some of the terminology confused. No argument was made, just stated a simple fact. Let's look at your spectral plot and review some basics and terminology too.
The parts of your sample which absorb go up with increasing concentration. BTW: The "baseline" is the entire 'X' axis from 190 to 800 nm (or whatever the scan range is. You did not provide any scale so we are not sure). The areas of the baseline which are flat are areas where your sample does not absorb light. Those areas should not increase as the sample concentration increases UNLESS the solution the sample is dissolved in is also absorbing some light too). Any observed baseline changes may be due to sampling time changes and can be compensated through the instrument (or software).
Look at the spectra that you provided. Do you see how the lowest end of the UV signal keeps increasing as the sample concentration also increases? You kept the pathlength of the flow cell the same. The only thing that was changed was the concentration. You increased the concentration. So the sample wavelengths which absorb also show increases too (higher AU). That is exactly what your spectral plot shows with the overlaid signals. Any diode leakage (Dark Current) would be subtracted out from each reading so no net change would be observed run-to-run unless a lot of time has passed. This is a training issue and as you are new user and student, please discuss this with your teacher. This is basic spectrophotometry.
My question says specifically on the region 300 nm - 800 nm. Yes it's obvious that at 254 nm the AU increases with concentration. Turns out that the machine had excessive dark current on the photodiode array and this "shift" is not observed anymore. Thank you for your contribution.
Hi Zaid Zaid Assaf I would like to know how you remove the shift. I think I am facing the same problems as you faced previously. I attached my UV-Vis spectrum below. My experiment is about photocatalytic degradation of Rhodamine B. As the reaction time gets longer, more and more rhodamine b is degraded, so its concentration gets lower and the intensity of its absorbance peak gets smaller. But there is a shift of baseline. I am curious about the phenomenon and think it is similar to yours.
Did you remove your shift by eliminating the excessive dark current on the photodiode array of the machine? I don't understand well. Can you explain further? Thanks!
Mengqi , Dark current was and still is not the main issue here.
In your case, as the process continues (Rhodamine degradation), the spectra changes and that means the absorbance at different wavelengths changes too (more of the broken down sample moves towards the lower UV end). That "shift" you see is normal over time. You do not want to remove it, but instead normalize the signals to account for it. With software normalized signals, you can use the total areas or just the peaks for comparison. The 'normalize signal' feature can often be found in your spectrophotometer software application. It will allow you to overlay multiple signals and adjust them to have the same relative baseline. This should be covered in the software operating manual for your instrument.
PS: When you have a new question on RG or any web forum, please do not hijack another user's post. Instead, create a brand new post and be sure to include the make, model and type of spectrophotometer you are using (plus the software) along with the settings, sample solution used and signal details. This type of basic information is needed to offer useful suggestions.
Hi William William Letter, thanks for your quick reply and very helpful tips on my question and how to use RG efficiently! I am a brand new user of RG haha.
I will check the name of the UV-Vis spectrometer and check if the software I used has the function of overlaying multiple signals. I will update my progress then.
The spectrophotometer I used is Agilent Cary 5000 UV-Vis-NIR. But it seems that the software can't adjust the baseline of multiple signals to the relative same position. I would like to know if there is any software recommended to do the analysis.
Baseline drift is a common problem in UV-vis spectroscopy - see https://ibsen.com/resources/detector-resources/subtracting-dark-spectra/ for a useful outline of the issue. I am glad that you were able to pinpoint the source of error in your experiment, Zaid Assaf. Increases in dark current (e.g. due to heating) seem to be a frequent offender, though loss of minor absorbing species (e.g. consumption of acid during a reaction) or changes in the solvent environment (e.g. in gradient chromatography) could also contribute.
My advice to William Letter is to read questions carefully before jumping to conclusions regarding the poster's knowledge and experience level. Condescending comments like "This is basic spectrophotometry" are not constructive and may deter other researchers from seeking assistance online. In this instance, you mistook the poster's issue with baseline drift as a misunderstanding of the Beer-Lambert law, despite repeated clarification. I hope that you will take greater care in your future replies.
Christopher D Jones , the issue was indeed the overheating of the photodiode array with time. Multiple instrument logs showed that the temperatures exceeded the alarm limit and those were correlated to samples with deviating baselines. I hope that my explanation was not too confusing in the original post as I have taken great care to highlight that the issue is with the baseline not linearity around the 255 nm region. In my case those were standard solutions prepared identically and don't exhibit any form of degradation or hydrolysis. I can't confidently say the same for Duan's situation. I have attached a spectrograph for the same solutions after installing a fan on the back of the detector. The baselines are identical.
Thank you for taking greater care in reading the original post.
- Badges
- Science topic