UV-Vis Frequently Asked Questions - Resolution and Stray Light
What is resolution?
Resolution is the ability to resolve spectral features and bands into their separate components. As such, it is an important experimental parameter. If the resolution is too low, spectral information will be lost, preventing correct identification and characterization of the sample. If the resolution is too high, total measurement time can be longer than necessary. What makes resolution “too low” or “too high” depends upon the particular application, and what information is desired from the experiment. In a UV/Vis spectrum there is spectral resolution that is controlled by the instrument’s slit setting as well as data point resolution defined by the data point collection interval.
First, we need to understand the characteristics of a well resolved absorbance peak generated by a typical spectrophotometer. At left we see a diagram of a single fully resolved absorbance peak. It shows the property of the full peak width at half maximum or FWHM. The FWHM property is an expression of the extent of function given by the difference between the two extreme values of the wavelength (X-axis) at which the absorbance (Y-axis) is equal to half of its maximum value. In other words, it is the width of a spectrum curve measured between those points on the Y-axis which are half the maximum amplitude.
Let’s now consider spectral resolution which is controlled by the slit function (bandwidth) of a spectrophotometer’s monochromator. The slit parameter is usually expressed in terms of wavelength (nanometers). It is important to note that the slit value is not the actual size of the physical slit width in the instrument. The slit width is converted to a wavelength value that allows us to relate the slit width parameter to the FWHM of a spectral peak. The general rule of thumb is that the instrument’s slit width should be at least 5 times less than the FWHM value. The FWHM minimum for most molecules in solution is 60 nm or higher; therefore, a slit width of 6 nm or lower will adequately resolve these peaks. The exceptions to this rule are dissolved organometallic complexes or rare earth compounds, which can have FWHM values of 10 nm or lower. Also, solid samples measured on an integrating sphere could be in this range as well. As seen at bottom right, gases are line absorption materials and have very narrow bands (< 0.01 nm).
How does resolution (slit width) influence spectral peak height and shape?
Besides spectral resolution, the slit also controls the amount of light energy incident on the sample. Therefore, the spectral noise level will be significantly affected by changes in the slit setting. The larger the slit setting, the lower the noise in the spectra. The smaller the slit setting, the higher the noise in the spectra.
Above are spectra that show the results of a lack of resolution. The black spectrum is fully resolved with a slit width five times lower than the FWHM. The red spectrum has a slit width equal to the FWHM. Note how the unresolved spectrum has photometric values lower than their “real values”. In addition, the unresolved spectrum has broader peaks with less separation between overlapping bands. Having an instrument with a large fixed slit width could cause inaccurate absorbance measurements on materials where narrow bands are a possibility.
What is stray light?
Stray light, along with detector sensitivity, work together to define the upper absorbance limit in dispersive UV/Visible/NIR spectrophotometers. However, the stray light value is the primary specification that limits the maximum sample absorbance possible for any instrument. Stray light is defined as any light that reaches the detector which is outside the spectral region isolated by the monochromator. High stray light frequently leads to deviations from the Beer-Lambert Law, with subsequent inaccuracies in sample photometric values. In theory stray light dictates the dynamic range for most spectrophotometers. In general, the lower the stray light specification, the more expensive the instrument.
Stray light in an instrument is defined as light in the instrument that is not of the wavelength set on the monochromator. For example, if the monochromator is set to 600 nm, then any light other than 600 nm is stray light. Stray light can originate from anywhere either inside (optics) or outside (light leaks) the instrument. Spectrophotometers that are well designed should not have stray light from either leaks or errant internal reflections. Typically, the primary source of stray light is from the diffraction grating component of the monochromator (above). There are flaws in the regular lines etched on the grating as a result of the manufacturing process and are the cause of most instrumental stray light. Modern holographic gratings produced by photo-lithographic processes have much lower stray light than the less expensive mechanically “ruled” gratings. To reduce stray light even more, two gratings can be used in series to achieve upper absorbance limits exceeding 6. By having the light reflect off of two gratings in series the stray light is significantly reduce from that of a single grating instrument.
Stray light, by convention, is measured in percent transmission. This is not very useful when most users tend to think in absorbance, at least as far as dynamic range is concerned. As one can see from the conversion equation on the right, absorbance is a simple -log transformation of the transmission value. The chart enables us to visualize the transmission/absorbance relationship over many orders of magnitude. From this chart we can easily see that a spectrophotometer with a stray light specification of 0.01 %T will not be able to measure any sample over 4.0 absorbance units. In addition to setting the highest absorbance, the stray light introduces a photometric error that increases in significance as the sample %T value becomes lower and approaches the stray light value.
How does stray light influence high absorbance measurements?
“Real verses “Measured” Absorbance - The plot above shows how stray light not only sets the upper absorbance level, but also is responsible for deviations from Beer’s Law. The black dotted line is the theoretical Beer’s Law relationship without any stray light affects; whereas, the solid red line has incorporated an instrumental stray light component of 0.001 %T. Note that the linear Beer’s Law relationship is maintained up to an absorbance of about 4. After a short region of increasing decline, the measured absorbances plateau as they approach the stray light values for the instrument. Stray light photometric artifacts always yield lower than actual absorbance values.
The percent error from Beer’s Law linearity due to the consequences of stray light is graphed above. The value for the stray light in this plot is the same as the previous page, 0.001 %T. Note that the error does not become significant (i.e. 1%) until around 4 absorbance, One percent is typically the linearity cutoff, since above 1% the error increases dramatically with small increases in absorbance.
How does stray light result in varied absorbance ranges on different instruments?
Pictured here are plots for various instruments having different stray light specifications. As you can see, the shapes of the plots are similar with only the Beer’s Law linearity cutoff point and the stray light plateau levels being different. The utility of this type of plot is that it defines the dynamic range envelope of any instrument based on its stray light specification.
How is 8 Plus Absorbance Even Possible?
So, what happens to stray light when we place any sample in the instrument for measurement? If the sample has any absorbance it has the ability to block the stray light in that absorbance wavelength range. Thus, the sample becomes its own stray light filter (blocker). So, samples, depending on how broad in wavelength their absorbance is, can frequently allow high absorbance measurements that appear to exceed the stray light specification of the instrument.
These are other unique samples that have a high absorbance level across an extend wavelength range very much like a high absorbance neutral density filter. These samples have the unique property of acting as their own stray light filter over the entire wavelength range. These samples are the darlings of instrument vendors to show 10 absorbance or even higher. Be warned, not many samples have this type of absorbance profile. So, if your sample has several absorbance peaks and returns to a low absorbance baseline in between, don’t expect to be measuring 8 plus absorbance values.
How does noise change between fixed slit and servo slit mode?
As can be seen at left, the amount of radiant energy in a typical spectrophotometer varies greatly as a function of wavelength. The amount of noise in a UV/Vis/NIR spectrum is directly related to these energy levels. The ability to measure high absorbance values is critically dependent on the amount of noise close to the 0 %T axis. High absorbance measurements will be more difficult to obtain in low energy wavelength regions of the instrument. Key instrumental parameters associated with the control of spectrum noise are slit width, detector gain, and integration (response) time.
One common methodology for improving noise through the use of instrumental parameters is to servo the slit as a function of wavelength (at right). This technique has the advantage of maintaining a constant energy and noise level throughout the NIR wavelength range. The servo slit mode maintains 70% energy through almost the entire NIR region, with the exception of the long wavelength area above 2900 nm. The energy declines here because the slit is open to its maximum value of 20 nm. Once the slit has opened to this maximum size, the energy will start to decline.
How does noise influence high absorbance measurements?
Spectrophotometers are unable to measure sample absorbance directly. They can only directly measure transmittance of a sample. Absorbance is a post data acquisition processing step. This fact is critical in understanding high absorbance spectra. High absorbance samples should always be measured in %T mode, this is so that one can directly see what is happening to the signal in the instrument. The spectrum above is a 7 plus absorbance blocking filter. Note the %T values for the blocking regions. The values are so close to zero %T that some of the values are negative due to instrumental noise. This causes a slight data handling problem for the absorbance processing step.
Above is a graphical representation of the relationship between percent transmission and absorbance values. The logarithmic nature of absorbance is apparent. On the bottom is the Beer’s Law conversion from percent transmission to absorbance. Remember those negative %T values due to noise for a highly absorbing sample? They represent a real problem. What is the logarithm of a negative number? The Log function Y = Logb (X) is the inverse of the exponential function X = bY. Since the base b is positive (b>0), the base b raised to the power of Y must be positive (bY>0) for any real value of y; therefore, the number x must be positive (x>0) as well. So, the real base b logarithm of a negative number must be undefined.
Since spectra cannot have “holes” in the wavelength axis due to undefined absorbance values, something must be done. The only solution is to “substitute” a real value that is on the same order of magnitude as the noise data. While this data can serve as a “placeholder”, it is inserting an unmeasured, artifactual value into the spectrum.
One methodology that can be employed to eliminate the negative %T values is to minimally smooth the original percent transmission spectrum. This smoothing “averages” the data above the 0 %T threshold; thereby, eliminating the negative values in the native spectrum. This result can be seen in the spectra on the right obtained from that 7 plus absorbance blocking filter. The original raw data, red spectrum, is subject to a 5-point smoothing which results in the blue spectrum where all %T values are positive. The resulting absorbance transformation would then be free from any artifactual data due to “undefined” absorbance from negative %T numbers. Since the correct minimal level of smoothing is basically a “trial and error” procedure, this type of methodology is difficult to automate properly in instrument software.