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Absorbance

AbsorbanceThe technique of absorbance is as old as the first alchemists. They sought to identify and understand their elixirs by looking at the color and opacity of solutions as different reagents were mixed, heated, and stirred.

 

Today it remains the most widely used spectroscopic technique for studying liquids and gases due to its simplicity, accuracy, and ease of use. An absorbance spectrum can be used as a qualitative tool to identify or “fingerprint” substances, or as a quantitative tool to measure the concentration of a molecule in solution.

 

The most common image of an absorbance measurement is a solution in a cuvette, measured in transmission with a dual-beam spectrometer – the classic introductory chemistry lab experiment. In practice, however, absorbance measurements can take many forms. They work equally well for gases as for liquids, and have found their way into consumer products and industrial applications alike. Samples no longer need to fit into the standard 1 cm pathlength cuvette, as flow cells, dip probes, micropipetters, folded gas cells, and micro-cuvettes allow the sampling optic to be customized to the sample.

 

Modular spectroscopy has provided infinitely more flexibility to choose the wavelength range and resolution needed, and to move between sampling optics quickly and easily for measurements in the lab or field. With our wide range of spectrometers, light sources, and accessories, we can help you to create a flexible system to measure a wide range of solutions and concentrations. Are you ready to think outside the cuvette? Read on.

 

 

Advantages

  • Non-destructive: Unless the sample is photo-sensitive, the measurement can be repeated without altering the sample. Can be performed in-situ or within process flows.
  • Quantitative: Allows determination of solution concentration or the extinction coefficient of a substance.
  • Accurate: Can quantify absorbance to within 0.001 absorbance units when implemented properly.

Applications

Other Common Applications

  • Kinetics: reaction monitoring, endpoint detection, protein and DNA thermodynamics, enzyme kinetics, on-line thermal cycling of biological particles
  • Quality & process control: pharmaceutical and textile manufacturing, particle size analysis, ethylene production, polymer processing
  • Chemical analysis: fluorophore characterization, phenol determination, column liquid chromatography, trace detection of metals
  • Research: analysis of freshwater and marine environments, characterization of liquid crystals, study of eye tissues, photostability studies of compounds in various environments
  • Environmental monitoring: SO2 detection as a predictor of volcanic activity, fenceline monitoring near chemical plants, airborne pollution monitoring in cities, prediction of red tide events, soil contamination analysis, ozone monitoring
  • Food testing: analysis of composition in dairy products, determining solids content in fruit, predicting odor and flavor suitability in wines
  • Biomedical: reading microtiter plates and labs-on-a-chip, analysis of nucleic acids and proteins, clinical and in-vitro blood diagnostics

What Is Absorbance?

When light is incident on a sample in a cuvette, it can be transmitted, absorbed, or scattered. This is often written as T + A + S = 1. Transmission is the light that passes through the sample without interacting with it. Light that encounters a molecule or particle can be either absorbed or scattered. Elastic scattering occurs when the interaction changes the direction of light, but not its wavelength or energy.

T + A + S = 1

When an absorption measurement is made, however, it is assumed that scatter is zero, in which case all light not transmitted to the detector is absorbed by the sample, i.e., T + A = 1.  This is true for the ideal case of an infinitely dilute solution of infinitely small particles in a transparent solvent. Luckily, it is also reasonably accurate in practice for a wider range of absorbing substances, solvents and concentrations. Absorbance occurs when the light encountering the molecule in the solvent matches the frequency of molecular vibrations or transitions in electronic energy-level states within the molecule. The chance of this happening is dependent on the cross section of the molecule for a particular energy level transition, and determines how absorptive a molecule is in solution. The more concentrated the solution, the greater the chance that a photon travelling through the solution will be absorbed. In fact, the probability of absorption increases linearly with both the pathlength and concentration of the solution, a relationship which has been quantified in the Beer-Lambert Law, also known as Beer’s Law.

What Is Beer’s Law?

Beer’s Law (also called the Beer-Lambert law) says that the absorbance of a solution will depend directly on the concentration of the absorbing molecules and the pathlength traveled by light through the solution. Beers Law where

  • A(λ) is the absorption of the solution as a function of wavelength            
  • ε(λ) is the molar absorptivity or extinction coefficient of the absorbing molecule as a function of wavelength (in L/mol·cm)
  • c is the concentration of the solution (in mol/L)
  • l is the pathlength traveled by light through the solution (in cm)

But how can we determine the amount of light absorbed? By measuring the transmission through the sample. Provided the sample has low scatter (as with a relatively dilute, clean solution), almost all of the light not absorbed will be transmitted. Transmission is the ratio of incident intensity, I0 to transmitted intensity, I, and will decrease with increasing path length or concentration.

Transmission as a function of concentration By taking the negative log10 of each side of this equation, we get a linear absorbance equation that is useful for calculations from measurements. Absorbance as a function of concentration This explains why absorbance is a dimensionless number that scales with concentration on a log scale. A perfectly transparent sample (T = 100%) will have an absorbance value of zero, while a perfectly opaque sample (T = 0%) will have an absorbance value of infinity. When units are specified, absorbance is usually described in terms of absorbance units (AU) or optical density (OD). The linearity of absorbance makes it conveniently additive.

For example, if one sample has an absorbance of 0.5 AU and another has an absorbance of 0.3 AU, then putting both samples in the light path in tandem will yield an absorbance of 0.8 AU. Similarly, if two different substances are present in the same sample, then the total absorbance will equal the sum of their individual absorbance values at that wavelength. It is important to keep in mind that many factors can affect the validity of Beer’s Law. Before using it to calculate the concentration of a solution or extinction coefficient of a substance, it is best to validate the relationship by measuring a set of standard solutions and plotting a calibration curve. The sweet spot for measuring absorbance with best accuracy is between 0.5 and 1.0 absorbance units, so aim to work in this range when choosing the pathlength of your sample cell, and create a calibration curve using concentrations across this range.

Application Note:

Technical Notes:

Featured Products for Absorbance in Gases:

Absorbance of Gases

Maya2000 Pro This high-resolution spectrometer is configured for UV absorbance in the sample setup. We use Grating #H7 set for 200-300 nm, with a 5 µm slit as entrance aperture and a detector collection lens for increased sensitivity.
D-2000 Deuterium source produces continuous output from ~215-400 nm
CUV-UV-10 Cuvette holder for 10-cm pathlength cuvettes
CV-Q-100 Cylindrical quartz cell with Teflon stopper; has volume of 28.2 mL
QP455-025-XSR-BX Pair of extreme solarization-resistant patch cord assemblies, 455 µm diameter, 0.25 m length
OceanView Spectroscopy software

Featured Products for Absorbance in Solutions:

UV-Vis Absorbance of Solutions

USB4000-UV-VIS General-purpose spectrometer is preconfigured for 200-850 nm and has a 25 µm slit and order-sorting filter
DH2000-BAL Balanced deuterium tungsten halogen light source provides illumination from 215-2000 nm
CUV-UV This sturdy cuvette holder accepts 1 cm pathlength cuvettes
CV-Q-10 A quartz cuvette is recommended for UV applications in particular
QP450-2-XSR Two 450 µm extreme solarization-resistant optical fibers will transmit and receive light in this setup
Absorbance Standards (optional) NIST-traceable photometric absorbance standards for 200-450 nm (STAN-ABS-UV) and 400-900 nm (STAN-ABS-VIS) ranges
OceanView Spectroscopy software

What light source should I use for illumination?

This will really depend on the molecule or substance you want to measure. Chromophores are substances that absorb visible light, giving them a colored appearance. These can be conveniently measured with visible light. Not all molecules, however, absorb in the visible.

Transitions between different electronic energy states often require high-energy, shorter-wavelength light in the ultraviolet or visible. Vibrational energy level transitions, on the other hand, tend to be at longer wavelengths in the near-infrared and infrared (hence the popularity of IR and Raman spectroscopy for chemical identification).

The electronic transitions that give rise to absorption spectra are almost always overlaid with vibrational and rotational states of the same molecule, making the peaks typically broad. Though this makes absorption spectroscopy less useful than other molecular techniques for identifying compounds, it does work well for absorption measurements using Beer’s Law, as a range of wavelengths can be monitored using more boxcar averaging for best possible signal-to-noise.

Use of a light source that covers only the wavelength range of interest will help reduce stray light, which is particularly important for higher absorbance readings.

Visible light sources: The broad, smoothly varying output of a tungsten halogen light source is ideal for absorption at visible wavelengths. The LS-1 is the most economical, and comes in long-lifetime and rack mounted versions. The HL-2000 has very similar spectral output, and has additional shuttered and high power versions. The bluLoop is an LED-based light source with four different bulbs to yield balanced spectral output over the visible range and greater intensity at blue wavelengths.

UV-visible light sources: A deuterium and tungsten based light source has a broad, smoothly varying spectrum and stable output from 190 to 2500 nm, making it well suited to most absorbance measurements. Since its output comes from two different bulbs, the UV and visible portions of the spectrum can be used separately to reduce stray light and optimize signal to noise. The DH-2000 comes in a shuttered version for light-sensitive samples, and in a balanced version where the strong deuterium emission line at 655 nm is attenuated.

A xenon light source would not usually be recommended for absorbance measurements, as it has a jagged, pulsed spectrum, making averaging and boxcar smoothing absolutely necessary to get good quality measurements. It may be considered for UV absorbance measurements in the field, since it runs on 12 V DC power. Though higher in cost and power consumption, the DT-MINI-2-GS is just as portable, with a much smoother spectrum. It also features the ability to switch the visible portion off to focus on UV wavelengths and reduce stray light.

NIR light sources: Both the LS-1 and HL-2000 have output into the NIR, out to about 2400 nm. Even though intensity decreases at the longer wavelengths, this effect is compensated by higher sensitivity of the detectors in our NIR spectrometers at those wavelengths. The Vivo NIR light source is also a tungsten halogen light source, and uses four spatially separated bulbs to avoid overheating the sample. The Cool Red is a silicon nitride light source, with output from 1 to 5 μm, and a shutter capable of rapid modulation.

What is the best sampling optic for my measurement?

The most common way to measure the absorbance of a solution is in a standard 1 cm pathlength cuvette, whether it be a disposable plastic UV-VIS cuvette or a high-quality quartz cuvette. This is typically a good pathlength unless the sample happens to be very high or very low concentration. If extinction coefficient is being measured, it is very important to use a high-quality quartz cuvette for which the pathlength is accurately known.

Cuvettes can be housed in a standard CUV-UV cuvette holder with two fiber inputs for absorbance only, a CUV-ALL-UV with four fiber inputs for conversion to fluorescence and scattering measurements, or in a CUV-FL-DA direct attach version with three fiber inputs for doing the same while mounted directly to the light source. All have spring-loaded ball plungers for precise, repeatable positioning of the cuvette, and a filter slot for blocking light during dark measurements and/or additional filtering.

The CUV-QPOD four-way cuvette holder has additional Peltier-cooler temperature regulation from -30°C to +105°C and a magnetic stirrer, ideal for absorbance and scattering measurements of temperature-sensitive samples. Typical applications include protein and DNA thermodynamics, fluorophore characterization, enzyme kinetics and thermal cycling of DNA for PCR-based amplification.

If a 1 cm pathlength cuvette isn’t the right solution, the best sampling optic can be narrowed down using a few key questions:

  • What is the optical density of the sample? The OD of the sample directly affects the pathlength needed for measurement. The more optically dense the sample, the shorter the pathlength needs to be. SpecVette™ cuvettes range in pathlength from 250 to 1000 μm, and require only a few microliters of sample solution. On the other hand, low OD solutions require a longer pathlength, such as a 10 cm cuvette holder or a custom cuvette in a variable pathlength cuvette holder. For extremely dilute solutions, an LPC longpass flow cell with 50 to 500 cm pathlength may be required.
  • What is the sample volume? The amount of sample to be measured also affects the choice of sampling accessory. SpecVette™ micro-cuvettes use very little sample solution and measure short pathlengths. A SpectroPipetter, in contrast, draws just 2 µl of sample solution into a long, narrow tip to probe a full 1 cm of pathlength.
  • Is the sample a liquid or a gas? Gases can pose a problem, as their greatly reduced density requires either a highly absorptive sample or extremely long pathlength. Depending on the sample, a 10 cm pathlength may be sufficient, achieved using a CUV-UV-10 or variable pathlength cuvette holder with cuvette, or simply by placing an adjustable collimating lens and cuvette holder directly in the gas to be measured.
  • Where will the measurement be made? If the measurement needs to be made in situ, like in a process stream or within a reaction chamber, a transmission dip probe may be useful.  It looks like a reflection probe, but with a mirror at the end of a spacer to reflect light back into the probe after traversing the sample solution twice. These are available with different fiber diameters (T300-RT, T200-RT, TP300) and in different materials for use in a range of chemical solutions and environments, including industrial process applications (TI300).
  • Does the sample require continuous flow? Sampling accessories like flow cells can be coupled with pumps, tubing and fittings to stream sample fluids for absorbance and other measurements. These include Z-cells like the FIA-Z-SMA and FIA-1000-Z, cross flow cells like the PRO-CFC, and chromatography and capillary electrophoresis cells (CUV-CCE), as well as micro-volume cells (LPC, PRO-MFC), ultra short path flow cells (FIA-USP), long path process flow cells (PRO-FC-LP), and adjustable-pathlength end-of-column flow cells (PRO-FC-BIO). These flow cells are available in materials like PEEK, plexiglas, Teflon, stainless steel, ultem, and fused silica to provide multiple options for chemical and environmental compatibility. They are supported by flow injection analysis systems and kits (FIA-LAB-2500, FIA-SIA-LOV, FIA-1000-Z).

What spectrometer should I use for detection?

The spectrometer in an absorbance system needs to match the wavelength range of interest, and have the right sensitivity for the sampling optic being used.  A preconfigured UV-VIS or VIS-NIR spectrometer works well for most cuvette-based measurements, but a more sensitive spectrometer may be needed for measurements in low volume cells or with fiber optics probes.

An extended range USB2000+XR1 or USB4000-XR1 spectrometer can be a good choice for absorbance, as it features ~2.0 nm (FWHM) resolution over a 200 – 1025 nm range, covering UV through NIR quite handily, with an enhanced sensitivity (-ES) option available. The HR2000+CG or HR4000CG-UV-NIR spectrometers extend slightly further into the NIR, with ~1.0 nm and ~0.75 nm resolution, respectively, albeit at higher cost.

For enhanced sensitivity at UV or NIR wavelengths, consider the Maya2000 Pro spectrometer line, which features back-thinned 2D FFT-CCD detectors. When stray light must be minimized for maximum accuracy or low absorbance solutions, however, a Torus or QE65 Pro spectrometer may be a better option.

The Torus spectrometer covers 360 – 825 nm with ≤1.6 nm resolution (FWHM), and uses a concave, variable line spacing grating to minimize stray light (0.015% at 400 nm) and ensure high throughput.  It also has excellent thermal stability, making it well-suited to extended kinetics measurements and precision measurements of optically dense solutions.

The QE Pro delivers similar high thermal stability, high sensitivity, and low stray light (0.4% at 435 nm) using an optimized optical design and a 2D back-thinned, FFT, TE cooled detector for low noise and dark signal.  It can be configured with a range of gratings to achieve 0.14 – 7.7 nm resolution (FWHM), and is also available as a preconfigured unit for absorption (QE Pro-ABS) that covers 200 – 950 nm.  One advantage of this high-performance unit is that it features a replaceable slit design for maximum flexibility.

Are there any integrated systems I can use?

Yes! Just keep in mind that they tend to use uncollimated light, so not all light incident on the sample travels the same pathlength. This makes them suitable for relative absorbance measurements where concentration is to be determined from a calibration curve, but not for absolute measurements of pathlength or extinction coefficient.

The USB-ISS-VIS and USB-ISS-UV/VIS are light sources with integrated 1 cm pathlength cuvette holder that attach directly to a USB2000+ or USB4000, covering 350 – 1000 nm and 200 – 850 nm, respectively. They are also available as fully integrated systems with a matching USB4000 spectrometer as the CHEMUSB4-VIS-NIR and CHEMUSB4-UV-VIS. These can be attractive for educational labs since they eliminate the risk of broken fibers entirely. Even the light source draws its power from the USB in the case of the visible unit.

Why do I need a reference measurement?

In an absorbance experiment, light is attenuated not only by the solution, but also by the solvent and reflections from every interface in the light path. Instead of attempting to calculate or measure these separately, it is easier define the incident light, I0, as the light passing through a reference solution, also called a “blank” or “baseline”.

In the case of a cuvette-based measurement, the appropriate reference would be the cuvette filled with just the solvent. Ensure that no dust or fingerprints are on the transmitting surfaces, as these will add scatter and absorbance signatures of their own. If the sample is in a buffer solution, the reference must be the buffer solution with no sample present.

In dual beam spectrophotometers, the reference solution would be placed in a reference slot for direct comparison for the duration of the experiment. When using an Ocean Optics spectrometer, you simply store a reference spectrum and then replace the cuvette with the sample to be measured. The software takes care of subtracting the reference spectrum, as well as applying any other correction factors that are selected.

It is a good idea to look at the reference solution periodically as a gauge of system drift. Drift can be caused by lamp intensity variations, changes in temperature, or even changes in the reference solution (depending on the chemistry). It takes very little time to take a new dark and a new reference measurement, so it is good practice to do so frequently while making measurements.

Do I need an absorbance standard?

If you’re not sure, then probably not. Absorbance standards are used to verify the accuracy and consistency of response of an absorbance system relative to NIST-traceable standards. This is required as part of quality control procedures in biomedical and pharmaceutical industries, as well as the food and beverage, petrochemical, semiconductor, and pulp and paper industries. Neutral density filters can be used for the same purpose, but do not assess factors like staff ability to prepare stock solutions, liquid and cuvette handling procedures, or the quality of cuvettes being used.

Our STAN-ABS liquid absorbance standard kits are submicron-sized, non-surface charged, solid polymer spheres in ultrapure water. They are provided with absorbance charts for each solution, collected using NIST-calibrated instruments.

Why is stray light important?

Stray light refers to any light that reaches the detector via scattering. This can include light that did not travel through the sample, or photons of the wrong wavelength hitting a pixel. Stray light has many sources, including ambient light that leaks into the instrument, light that bypasses the sample (like light that gets wave-guided through a cuvette wall), higher-order diffraction from the grating, and scattering from optical surfaces inside the spectrometer.

Even when a spectrophotometer is designed to minimize stray light effects, there is a physical limit imposed by the zero-order scattering of the grating. Fortunately the stray light of an instrument can be measured and a correction applied in software.

Stray light (Is) always appears as additional signal in both the sample (I) and reference (I0) measurement in the absorbance equation:

Absorbance equation

When the absorbance equals zero, like when the reference solution is inserted as the sample, the stray light terms cancel out. As absorbance increases, (I) decreases and stray light begins to affect the absorbance value, reducing it from the true value. At very low light levels, the stray light, Is, approaches or exceeds the transmitted light, I, and becomes the dominant term. At this point, it becomes the limiting factor for the absorbance that can be measured.

When the stray light of a system is quantified, it is usually expressed as a percentage of the reference value. Our spectrometer models with the lowest stray light are the Torus (0.015% at 400 nm) and the QE Pro (0.4% at 435 nm, 0.08% at 600 nm).

As shown in the figure below, absorbance readings in the 0.5 – 1.0 range are not significantly affected by stray light, but at higher absorbance readings the effects become important.

Absorbance Error

Since stray light is a property of both the spectrometer and of the light source used, it must be characterized at the system level. One method is to measure absorbance using a highly concentrated sample. For example, if the concentrated sample is known to have an absorbance near 5, then the light reaching the detector would be reduced by a factor of 10,000. Any light measured above this level could be considered stray light. A longpass filter can also be used, as these are designed to block by 5 or more orders of magnitude at the shorter wavelengths.

When using an Ocean Optics spectrometer, the stray light value can be entered into the software and then scaled and subtracted from all other readings. Note that stray light varies with wavelength. Near infrared wavelengths often contribute more than others to stray light, so using a shortpass filter prior to the spectrometer may help to reduce the amount of stray light reaching the spectrometer detector.

Reducing stray light can also be as simple as turning off the tungsten halogen light source when working at wavelengths covered by the deuterium portion of the light source, or using a bandpass filter to limit the light source to illumination over only the wavelength range of interest.

How do I use Beer's Law to calculate concentration or extinction coefficient?

Although it can be tempting to use Beer’s Law in combination with a known extinction coefficient and the pathlength of the cuvette to directly calculate concentration, it is not the most accurate method. It is better to first validate the linearity of Beer’s Law for your sample and system. This can also help to remove errors that are particular to the experiment, the equipment, or the particular set of samples being studied.

First, carefully prepare a series of standard solutions for which the concentrations are known, and then measure their absorption as a function of wavelength. For most compounds, there is typically at least one wavelength where the sample absorption peaks. Choose one of these wavelengths to monitor and create your calibration curve. Looking at Beer’s Law:

Beers Law where   A(λ) is the absorption of the solution as a function of wavelength            

            ε(λ) is the extinction coefficient as a function of wavelength (in L/mol·cm)            

            c is the concentration of the solution (in mol/L)            

            l is the pathlength travelled by light through the solution (in cm)

This equation tells us that plotting absorbance as a function of concentration for the wavelength of interest should yield a straight line intercepting zero, for which the slope is equal to ε(λ)l. If the system truly follows Beer’s Law, then the plot will be linear and pass through zero. Linear regression can be used to calculate the best straight line and determine the slope, or it can be calculated automatically in our software using the Beer’s Law wizard.

As an example, the graph below shows the calibration curve for calcium dipicolinic acid in deionized water. Measurements were taken at 270 nm, the wavelength of maximum absorbance for this sample. The reference was pure deionized water. Calcium Dipicolonic Acid plot Using this graph, the concentrations of unknown samples can easily be calculated using the slope of the line. Similarly, the extinction coefficient can be determined if the other parameters are known. For the most accurate results, it is best to use ten or more data points evenly distributed over the desired interpolation range. Four points does not a calibration make! Beer's Law slope equation Using a calibration curve conveniently eliminates the need to know the extinction coefficient or exact pathlength. This is handy, because very few chemicals come with a molar absorptivity curve for the exact solvent being used. It also allows the use of a sample holder of unknown pathlength, provided it is used consistently.

If the calibration curve turns out to be non-linear, it can still be used by fitting a polynomial function to the data. Interpolations have the potential to be just as accurate as those from a linear calibration curve, as accuracy is dependent on the quality of the curve fit, not the shape of the line.

Deviations from linearity may indicate chemistry that is occurring within the sample, including, but not limited to, other species and changing equilibrium conditions. So before a nonlinear calibration curve is used, it is a good idea to verify the result using a fresh set of standard solutions.

Why isn't my calibration curve linear?

All calibrations will be non-linear if a wide enough range of concentrations are used due to stray light and of limitations in the instrument. For example, the effect of stray light on measurements is shown in the figure below.

Absorbance Linearity vs Stray Light

The detector measures the numbers of photons striking the detector during the integration time. Those photons arrive randomly, and the random variation in how many photons strike the detector is equal to the square root of the number of photons (an effect called shot noise). At high absorbance values, the number of photons reaching the detector is low and the signal-to-noise is relatively poor.  Less of the detector’s dynamic range is being used, and the shot noise is a much larger fraction of the total signal.

At low absorbance values, there are plenty of photons and higher signal-to-noise. However, the uncertainty in measuring the percent transmission dominates the error in the measured absorbance value.  In other words, the spectrometer is not as sensitive to small changes in absorbance (concentration) when absorbance is already low.

The ideal range to work in is from about 0.5 – 1.0 absorbance units, where the sensitivity to changes in absorbance is maximized and good signal-to-noise can still be achieved. By adjusting the concentration of the samples through dilution, by choosing cuvettes of different pathlength, or both, the absorbance values measured can be adjusted to be in this range to get the most accurate results.

What's the difference between relative and absolute absorbance?

In order to apply Beer’s Law directly using the pathlength of the cuvette, the light passing through the cuvette must be collimated. If the illumination is diffuse, there will be a variety of paths taken through the cuvette, resulting in an average pathlength for the combined cuvette and system that differs from the specified length of the cuvette being used.

Uncollimated light through a sample

Uncollimated light through a sample

Collimated light through a sample

Collimated light through a sample

If diffuse illumination is combined with collimated detection (using a lens for light gathering), the result is a relative absorbance measurement. That is, the absorbance reading for a specific sample will depend on the specific instrument used. Our direct-attach and integrated cuvette holders provide diffuse illumination to the cuvette via light focused directly from a bulb. Transmission dip probes and flow cells are also relative absorbance sampling optics.

Collimated illumination combined with collimated detection results in absolute absorbance measurements, which are independent of the instrument used to make the measurement. Any of our cuvette holders which use a fiber and collimating lens combination provide collimated illumination, as the light exiting the fiber does so with a well-defined range of angles, allowing proper collimation. Be aware, however, that making adjustments to the position of the collimating lens in these cuvette holders will affect collimation of the incident light.

Relative absorbance systems work very well for determining concentration when used with a calibration curve. They are not suitable, however, for determining extinction coefficients, as the exact optical pathlength is not known. Absolute absorbance sampling optics:

  • CUV-ALL (1 cm cuvettes)
  • CUV-UV (1 cm cuvettes)
  • CUV-UV-10 (10 cm cuvettes)
  • CUV-VAR (1 – 10 cm pathlength)
  • CUV-TLC-50F (1 cm cuvettes)

Relative absorbance sampling optics:

  • ISS-2  (1 cm cuvettes)
  • ISS-UV-VIS (1 cm cuvettes)
  • CUV-FL-DA (1 cm cuvettes)
  • USB-ISS-VIS (1 cm cuvettes)
  • USB-ISS-UV (1 cm cuvettes)
  • USB-ISS-T (12 mm round test tubes)
  • All dip probes
  • All flow cells

How do I take the best dark measurement?

When taking a dark measurement, it is best to block the light at the light source if possible. Turning the light source off and then on again will throw the light source out of thermal equilibrium and require a new reference measurement. Alternatively, many cuvette holders have a filter slot where the light can be blocked. Just be sure to use a piece of metal or another object that is guaranteed to be 100% opaque. Paper, even cardboard, can be deceptively transmitting, and it only takes a very low level of light to affect a measurement.

How do I take the best reference measurement?

To take the best reference measurement, first allow the light source to come to thermal equilibrium (this can take up to 30 minutes in some cases). Ensure that all surfaces of the cuvette are clean and clear of fingerprints, dust, and dirt. Fill the cuvette with the exact solvent or buffer solution to be used for the sample, and check for bubbles. It is especially important to check for bubbles if using a transmission dip probe or flow cell, as they are prone to this source of error, particularly at the shorter path lengths.

Optimize integration time so that maximum signal is at ~80% of full scale, and use the highest number of averages tolerable. Keep boxcar width to approximately the same value as the pixel resolution of the spectrometer, otherwise it can affect the spectral resolution.

Once the reference has been acquired, go into transmission mode and take a look at the resulting spectrum. If a good quality reference spectrum has been stored, the transmission of the reference solution should be at 100%, with some noise around this value. Wavelength regions with more noise indicate the wavelengths at which accuracy will be the least for the sample measurement (typically the shortest and longest wavelengths). OceanView Reference in Transmission Mode Screenshot

How do I make repeatable measurements?

In any measurement that is made relative to a reference, the enemy is instability. Temperature changes or heating in the light source can cause the reference or dark measurement to change slightly, affecting all data. Here’s how to get the most accurate, repeatable measurements:

  • Remember that transmission may vary from cuvette to cuvette. It will also vary with the orientation of the cuvette in the sample holder if the cuvette does not have exactly the same pathlength in all directions. For best accuracy, always use the same cuvette. If this isn’t feasible, make sure cuvettes are oriented the same way every time they are placed in the cuvette holder.
  • Warm up the light source for the recommended amount of time (up to 30 minutes in some cases). Output will continue to change very slightly until the light source is in thermal equilibrium, affecting measurements.
  • Take frequent dark and reference measurements to re-establish an accurate baseline.
  • Set the integration time so that the reference spectrum peaks at 80% to 90% of the full scale of counts. This takes advantage of the full dynamic range of the spectrometer, improving signal to noise (S:N).
  • Never turn off the light source or disconnect fibers to take a dark measurement. Instead, block the light source at its origin, or at the filter slot in the cuvette holder. Make sure the object used will block 100% of the light; metal works well.
  • Set averages as high as tolerable to improve signal-to-noise. The S:N improves with the square root to the number of averages taken.  For example, setting averages to 9 improves the S:N by a factor of 3.
  • Increasing the boxcar value can also smooth out noise in the spectrum. This is a moving average with wavelength, so a boxcar value of 2 will average an additional 2 pixels on each side (5 in total) and assign that average to the center pixel.  If the boxcar value is set too high, it will begin to blur the spectral shape, so use this feature carefully. To smooth data without affecting resolution, set the boxcar value equal to the pixel resolution of your spectrometer.
  • Use the nonlinearity and stray light correction options. The stray light correction factor can be determined using high pass filters inline between the sampling optic and the spectrometer. Stray light is often in the NIR, which means it could be reduced through use of a shortpass filter prior to the spectrometer.

How do I measure very low concentrations or low absorbance?

At low absorbance values, the uncertainty in measuring the percent transmission may be as large as the attenuation created by the sample. This makes it very hard to get an accurate absorbance reading for dilute or low attenuation samples. First, ensure that the wavelength of peak attenuation for the sample is being monitored. Then, increase the pathlength of the sample cell until the absorbance readings are in the sweet spot of 0.5 – 1.0 absorbance units. Alternatively, concentrate the sample prior to measurement or use less solvent for sample preparation.

If the amount of sample available is limited or precious, try using our SpectroPipetter. It uses only 2 μL of sample, while still achieving a 1 cm pathlength. Our longpass flow cells can provide 50 – 500 cm of pathlength using just 125 – 1250 μL of sample at a time (additional volume will be required for the injection process).

How do I measure very high concentrations or high absorbance?

At high absorbance values, the signal reaching the detector is very low, and signal-to-noise is limited by the shot noise of the detector. The effect of stray light also increases at higher absorbance values, reducing the perceived absorbance from its true value (see section on stray light for more information).

Using a spectrometer that has been specifically designed for low stray light can improve the accuracy of high absorbance measurements considerably. The stray light of a system is usually expressed as a percentage of the reference value. Our spectrometer models with the lowest stray light are the Torus (0.015% at 400 nm) and the QE Pro (0.4% at 435 nm, 0.08% at 600 nm).

Since stray light can’t be eliminated completely, the best defense is to measure it. Stray light is instrument-dependent, so once measured for a specific system, it can be corrected in the software. This also improves the accuracy of high absorbance measurements significantly.

Stray light correction is highly recommended for any measurements above 2 absorbance units, and can even improve accuracy for lower measurements. Near infrared wavelengths often contribute more than others to stray light, so using a shortpass filter prior to the spectrometer may help to reduce the amount of stray light reaching the spectrometer detector.

When possible, it is best to reduce the pathlength of the system or dilute the sample to bring measurements into the 0.5 – 1.0 absorbance unit range. Our SpecVette™ cuvettes come in pathlengths from 250 μm to 1 mm, and can be mounted in a universal adaptor for use in any 1 cm cuvette holder. We also offer ultrashort path flow cells, including adjustable pathlength micro flow cells for process applications.

Is there an easy way to make kinetics measurements in the software?

Yes! The strip chart feature allows you to select the spectrometer, spectrum type (absorbance vs. reflectance, etc.), total time for data to be collected, and wavelength to be monitored (single wavelength or a range). This can then be easily imported into Excel for analysis.

Should I correct for electrical dark?

Think carefully about whether you want to turn on electrical dark correction. Dark current levels tend to vary slightly over time due to temperature variations, and the electrical dark correction option will keep the offset dark levels stable for a longer period of time. This correction does add noise, however, as it is a subtraction calculation. It would not be advisable for very low absorbance measurements.

So consider your environment, absorbance value and the amount of temperature variation you typically see. If it’s minimal, the dark correction probably won’t add value, but it can be a benefit in unstable temperatures.

Should I use the non-linearity correction?

The non-linearity correction compensates for the non-linearity effects of the detector, which occur above 80% of the saturation level. Non-linearity correction improves the accuracy of the spectral shape, but also adds more noise to your original signal.

The error is somewhat subtle and usually smaller than the experimental errors associated with handling liquid reagents. Compared to detector readings at around mid-scale, readings at the top and bottom end of the scale will show a drop of 2 – 8% (in counts). The effect on the absorbance reading will depend on the value of the reference and the sample in relation to this raw count. With the non-linearity correction turned on this error becomes undetectably small.

Ocean MZ5 ATR-MIR Spectrometer

Ocean MZ5 ATR-MIR Spectrometer

Rapid, Accurate Mid-Infrared Analysis
OCEAN HDX Spectrometers

OCEAN HDX Spectrometers

High Definition Optics in Small Bench Design
BUNDLE-QEPRO-ABS

BUNDLE-QEPRO-ABS

High-sensitivity Spectroscopic System for Absorbance
Education Kits for Science

Education Kits for Science

Spectroscopy Experiment Kit for Teaching STEM Labs
Flame Spectrometer

Flame Spectrometer

High Thermal Stability, Interchangeable Slits
FLAME-CHEM Spectrometer Systems

FLAME-CHEM Spectrometer Systems

Compact Spectrophotometers for STEM Teachers
JAZ Spectrometer

JAZ Spectrometer

Handheld Spectrometer for UV-Vis Measurements
SpecLine – Analysis Software

SpecLine – Analysis Software

Software for Compound Identification
BUNDLE-FLAME-ABS

BUNDLE-FLAME-ABS

Application-Ready System for Absorbance
USB2000+ (Custom)

USB2000+ (Custom)

Custom Configured Spectrometer for Setup Flexibility
STS Developers Kit

STS Developers Kit

Connect, Code, Create with New Sensing Tools
QE Pro (Custom)

QE Pro (Custom)

High-sensitivity Spectrometer for Low Light Level Applications
Maya2000 Pro (Custom)

Maya2000 Pro (Custom)

High Sensitivity Spectrometer
NIRQuest512

NIRQuest512

Small-footprint Spectrometer for Near-Infrared Measurements
STS-UV

STS-UV

UV Spectral Analysis in a Tiny Footprint
USB4000 (Custom)

USB4000 (Custom)

Custom Configured for Maximum Flexibility
NIRQuest512-2.5

NIRQuest512-2.5

Small-footprint Spectrometer for Near-Infrared Measurements
HR4000 (Custom)

HR4000 (Custom)

High Resolution Spectrometer for Maximum Flexibility
Maya2000 Pro-NIR

Maya2000 Pro-NIR

High-sensitivity Spectrometer for Raman and NIR Applications
STS-VIS

STS-VIS

Vis Spectral Analysis in a Tiny Footprint
QE Pro-ABS

QE Pro-ABS

High-sensitivity Spectrometer for Absorbance
STS-NIR

STS-NIR

NIR Spectral Analysis in a Tiny Footprint
USB2000+UV-VIS

USB2000+UV-VIS

Application-ready Spectrometer for the UV-VIS
EMBED Spectrometer

EMBED Spectrometer

Robust, Stable Spectrometer for OEM Applications
NIRQuest512-1.9

NIRQuest512-1.9

Small-footprint Spectrometer for Near-Infrared Measurements
USB2000+XR1

USB2000+XR1

Extended Range Spectrometer for UV-NIR applications
NIRQuest512-2.2

NIRQuest512-2.2

Small-footprint Spectrometer for Near-Infrared Measurements
USB-TC Temperature Controller

USB-TC Temperature Controller

Increase Thermal Wavelength Stability
USB2000+UV-VIS-ES

USB2000+UV-VIS-ES

Application-ready Spectrometer for the UV-VIS with Enhanced Sensitivity