UV-Vis Spectrophotometer

What is a UV-Vis Spectrophotometer?

Principals & History of UV-Vis Spectrophotometers

Ultraviolet-visible (UV-Vis) spectrophotometer is used to quantify and qualify samples by the means of UV and visible light (mainly 200 to 900 nm). The first mentioning of a spectroscope (predecessor of a spectrophotometer) dates back to 1814, when Joseph von Fraunhofer, the name patron of today’s world renowned Fraunhofer Gesellschaft, used his invention of this spectroscope to measure sunlight and discover the 574 dark fixed lines in the solar spectrum (Fraunhofer Lines). He also developed a diffraction grating in 1821 to separate the light from the sun, almost 40 years after the first manmade diffraction grating was invented by David Rittenhouse.

It took more than 100 years until the first commercial UV-Vis spectrophotometer to qualify and quantify samples by the means of ultra violet and visible light was introduced by Arnold O Beckman in 1941. The instrument utilized a quartz prism to separate light from a tungsten lamp into its absorption spectrum and a phototube, the predecessor of a modern photodiode to record the signal. To account for background influence from the lamp and the electronics a UV-Vis spectrophotometer measures the intensity of light transmitted through a sample and subtracts the described background automatically to provide precise readings that represent the determined properties of a sample.

Beckman Model DK1 Spectrophotometer

Science History Institute, CC BY-SA 3.0, via Wikimedia Commons

UV/Vis Spectrophotometer | Accuracy, Miniaturization and Speed

The basic optical principle of a modern UV-Vis spectrophotometer has not changed much; however, lamps, detectors, gratings, and other optical components have evolved significantly making modern instruments way more versatile, more robust, much smaller, and faster.

The latest NanoPhotometer® models of microvolume UV-Vis spectrophotometers feature the highest speed and smallest footprint in its class along with a maintenance-free mobile design allowing UV-Vis spectroscopy to be taken anywhere within the lab and out in the field. Up to 12 samples can be fully scanned and analyzed in a drop within a single 20-second run and without the need for expensive accessories or consumables; there are also models available that allow reading samples using a classical cuvette (quartz, optical glass or plastic).

The built-in web application server is another highlight of the NanoPhotometer®. It allows to control the instrument and access data from any computer (Windows or Mac), tablet or phone (Android and iOS). The NanoPhotometer® can also be integrated in any LIMS via REST API.

Prof. Beer introducing the NanoPhotometer® line of UV-Vis Spectrophotometers (with Lambert obviously being absolutely thrilled about the presentation)

Optical Setup of a Modern UV/Vis Spectrophotometer

Schematic drawing of the optical setup of a monochromatic UV/Vis spectrophotometer

Schematic drawing of the optical setup of a polychromatic UV/Vis spectrophotometer

In comparison to the first commercially available UV/Vis spectrophotometer, which has been a monochromatic scanner, the NanoPhotometer® represents a new class of UV-Vis spectrophotometer instruments with a polychromatic rather than a monochromatic optical setup.

The full spectrum of light produced by a Xenon Flash Lamp is sent through the sample and then separated after it passed using a polychromator. To record the signals, modern UV Vis spectrophotometers are utilizing a CCD or CMOS detector (linear camera), which allows to record high-resolution spectra over the entire wavelength range within a few seconds even.

Moreover, a major advantage of the NanoPhotometer® UV-Vis spectrophotometer instruments is that no regular service, maintenance or recalibration is required anymore to operate the instruments. This introduces great peace of mind for researchers since they can always trust their measurements over the entire lifetime of the instruments.

NanoPhotometer® UV/Vis spectrophotometer micro-volume head with compressed sample drop during reading

So how does a UV Vis Spectrophotometer Really Work?

Lambert will explain the technology by analyzing the beer that Prof. Beer is visibly enjoying.

The absorption of light is based on interaction of light with the electronic and vibrational state of a molecule.

Each type of molecule has an individual set of energy levels associated with its chemical bonds and therefore will absorb light of specific wavelengths showing unique spectral properties.

To run an experiment on the NanoPhotometer®, he only needs to swipe a small drop of beer and put it on the microvolume pedestal. Since daylight is considered white light (an overlay of the three basic colors red, green and blue – RGB) and beer is yellow, it can be expected that it absorbs light in the blue range and only lets pass green and red light (which when overlapped results in yellow to our eyes)… and voila: the scan confirms what we have anticipated perfectly!

UV-Vis Spectrum of Beer measured with the NanoPhotometer® NP80 using 2 µl of sample at 0.67 mm Pathlength – Lid 15 operating at 1/15 virtual dilution showing absorbance only in the blue range as anticipated based on the yellow color

So how to determine concentrations from the signals obtained: let’s take a look at the Beer’s law or also Beer-Lambert Law, which defines the absorbance as follows?

The Beer-Lambert Law

The Beer-Lambert law, also known as Beer’s Law, empirically relates the absorption of light to the properties of the sample. This law states that there is a logarithmic relationship between the transmission of light through a specific sample (T = I/Io with I = outgoing light and Io = incoming light), the molar extinction coefficient for a specific compound (ε), the concentration of the absorbing species in the material (c) and the distance the light travels (d).

We learn that the expected result depends on the molar extinction coefficient, the concentration of the sample and the pathlength.

Lambert showing the impact of Pathlength on a UV-Vis spectrophotometric scan – the longer the Pathlength, the higher the absorption will be, just what is expected from our visual experiment

Pathlength Matters

To show that the Pathlength is of the essence, Lambert again uses Prof. Beer’s favorite drink and fills it in a small glass instead of his liter stein mug (according to Lambert, he does not appreciate that at all).

Same beer, different color. So why is that: The liter mug is much wider in comparison to the elegant glass right next to it, hence the light is traveling longer through the beer and interacting more with the liter mug. The width of the glasses is equivalent to what UV Vis spectroscopists call Pathlength.

Side note from Lambert: Prof. Beer obviously prefers longer Pathlengths – for a good reason. The NanoPhotometer® UV-Vis spectrophotometer, on the other hand, is all about shorter Pathlengths to extend the dynamic range of the instruments by introducing a quasi-virtual dilution to the samples in comparison to a 1 cm reading in a standard cuvette spectrophotometer. Not sure how good the manual dilutions are by Prof. Beer given his preference to longer Pathlengths, so that virtual dilution comes in very handy.:-)

Important UV/Vis Spectrophotometer Features

Now that we have learned how a UV-Vis spectrophotometer is working in principle, we are ready to understand on what the NanoPhotometer® can do to improve the results in the lab.

The NanoPhotometer® is mainly used for nucleic acid (DNA, RNA, mRNA, Oligos with and without dye labels) and protein/antibody quantification and qualification, OD600 measurements and a lot of other applications like kinetics in a drop and scans of small molecules even in organic solvents. So let’s take a look at the specifications that are of relevance when planning your experiments (the specifications of the NanoPhotometer® are shown in parenthesis.)

Spectral range (200 to 900 nm)
Absorbance range (0 – 2.7 Abs)
Spectral resolution (better than 1.8 nm)
Pathlength (0.67 and 0.070 mm plus most cuvette sizes offered commercially – 1 cm and less)
Dynamic range for dsDNA:
N60, NP80: 1 – 16,500 ng/μl
N50: 5 – 7,500 ng/μl
N120: 2 – 8,000 ng/μl
Dynamic range for BSA:
N60, NP80: 0.03 – 478 mg/ml
N50: 0.15 – 217 mg/ml
N120: 0.06 – 230 mg/ml

NanoPhotometer®

N120

Microvolume High Throughput Spectrophotometer

• High-Performance Microvolume Spectrophotometer

• Only 1.7 sec per sample, 20 sec per run (12 samples)

• Full high-resolution scan from 200-900 nm for each sample

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NanoPhotometer®

NP80

All-in-One

Spectrophotometer

• All-in-One Spectrophotometer

• Micro-volume and cuvette capability

• Built-in vortexer

• Full high-resolution scan from 200-900 nm for each sample

• Temperature Controlled Cuvette Holder

• Starting at 0.3 µl sample volume

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NanoPhotometer®

N60*/N50

Microvolume

Spectrophotometer

• High-Performance Micro Volume Spectrophotometer

• Starting at 0.3 µl sample volume

• Full high-resolution scan from 200-900 nm for each sample*

• Built-in vortexer*

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NanoPhotometer®

C40

Cuvette

Spectrophotometer

• Standard and Microvolume cuvettes (quartz, glass and plastic)

• Additional Applications: OD600 (Cell, Bacterial, Yeast Density), Bradford, BCA

• Temperature Controlled Cuvette Holder

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