A critical part of any chromatography process is identifying your product. A number of different sensors can be used in the flow path, such as conductivity and pH, but the most common is UV absorption. It is paramount to the process that these sensors perform reliably and consistently. We will describe the performance of the built-in UV detector in ÄKTA process™ CFG chromatography system.
Multiple wavelength monitoring
While many compounds can be identified by monitoring a single UV or visible light wavelength, sometimes it is useful or even necessary to look at alternate or multiple wavelengths to distinguish otherwise similar compounds from one another or to monitor impurities. This capability can also be used to collect multiple compounds in a single run. ÄKTA process™ systems include the UV-90 detector which can monitor up to three user-selectable wavelengths simultaneously (between 190 and 700 nm). The UV-90 is used to monitor the post-column eluent and can measure both absorption unit (AU) and optical density (OD). The UV signals are captured in UNICORN™ control software in three channels (UV1, UV2, and UV3) that are logged as part of the chromatogram result file from each run. The desired wavelength(s) can be set for ÄKTA process™ system using the interactive Process Picture or the UNICORN™ instruction Monitors-Wavelength.
Comparison of UV-90 to UV-900 and U9-M monitors
The UV-90 has several improved features compared to its predecessor UV-900 monitor used in earlier generation ÄKTA™ systems and has a proven design based on the U9-M multi-wavelength monitor used in our lab-scale ÄKTA™ systems. The UV-90 contains updated lamp driver electronics, more robust mechanical design, and communicates through EtherNet/IP. It also has built-in tools for calibration and wavelength accuracy check and works with pre-calibrated flow cells. These features are described below in more detail.
Flow cell path length
One aspect which influences sensitivity and range is the optical path length of the flow cell. A longer flow cell path length is more sensitive but saturates more easily with high-absorbing sample or buffer. A shorter flow cell path length is useful for higher concentration of sample, at the expense of sensitivity. To accommodate your needs, we offer the choice between flow cell path lengths of 1 mm, 2 mm, and 5 mm as standard to give optimum performance for each process. The difference is demonstrated in Figure 1, showing measurements from a 1 mm flow cell and a 5 mm flow cell. In both cases the full 0 to 2 AU range of the detector is used. The 5 mm flow cell path length has higher resolution but saturates at 4 AU/cm while the 1 mm flow cell path length has a useful range up to 20 AU/cm.
Fig 1. Comparison of measured absorption versus reference optical density for 1 mm and 5 mm flow cell path lengths.
Flow cells
Figures 2 and 3 are images of the flow cells for each system size.
Fig 2. Black PEEK flow cell for 1" system size.
Fig 3. Black PEEK flow cell for 6 mm, 1/2", 3/8", and 10 mm system sizes.
Dual channel support
The AKTA process™ CFG chromatography system comes standard with one channel and one flow cell. The UV-90 also supports two channels (ordered as a customization) to use two flow cells in series, combining their benefits for increased dynamic range.
Calibrated flow cells from production
In order to measure absorption quantitatively, there must be consistency between instruments and between replacement parts. Normally, calibration fluids, that is, liquid mixtures with known absorptions, are used to calculate flow cell path length. With the UV-90, the flow cell length is measured as part of the production process, with the optical path length etched on each flow cell. The nominal and actual flow cell path lengths are entered manually into UNICORN™ control software when a new or replacement flow cell is fitted to the system. These values are automatically used in calculations to ensure normalized performance results regardless of which instrument or flow cell was used for the separation.
Figure 4 below shows a comparison between the UV-90 and Optek AF46. The optical density of a reference liquid is measured and plotted versus reference measurements by spectrophotometer (PerkinElmer Lambda 1050+). Linear fit slope and R2 (calculated with intercept at 0) and p-values for each curve are listed in Table 1.
Fig 4. Optical density measured using the UV-90 and Optek AF46 versus reference liquids. Experimental work was performed from December 2023 to May 2024, and data is held at Cytiva Uppsala, Sweden. Fitted slope and R2 values are listed in Table 1.
Table 1. Slope and R2 for linear fits for each optical density measurement in Figure 4 were calculated using the Pearson correlation function giving statistically significant data (p < 0.01).
| Instrument | Slope | R2 |
| Cytiva UV-90 1 | 0.9887 | 1 |
| Cytiva UV-90 2 | 0.9965 | 1 |
| Cytiva UV-90 3 | 1.0108 | 1 |
| Optek AF46 1 | 1.0108 | 1 |
| Optek AF46 2 | 1.0198 | 1 |
| Optek AF46 3 | 1.0236 | 1 |
Securing accurate performance
Lamp intensity check
Lamp intensity will affect the signal-to-noise ratio. The UV-90 has a light intensity check that is measured in the reference channel at 229.6 nm. The result is presented as a percentage value where the monitor is fully functional down to 0% (the lower limit for accurate measurement). At 20% it is recommended to plan for lamp replacement. The typical lifetime of the lamp is more than 20 000 h. The lamp can be turned off to save on lamp hours.
One-button linear calibration
To ensure that the instrument and sensor are working correctly and within specification it is necessary to occasionally perform calibration. A novel feature of the UV-90 is the inclusion of four built-in neutral density filters for fast, easy, one-button calibration from software (Fig 5). This allows the users to calibrate the flow cell and monitor as often as they like with virtually no down-time, training, or specialist skill set. The risk for operator error is minimized using this automatic calibration. Each filter is measured several times in different positions to account for any variations. The four absorption values are fitted to a straight line and the deviation from this line is presented as a percentage. A typical value is below 0.5%. The 10 most recent calibrations are stored and accessible from the control software.
Fig 5. Calibration unit on UV-90. Front and rear views.
The filters in the built-in calibration unit are requalified at each preventative maintenance (PM) visit, using a calibration tool including four neutral density filters and a stray-light filter (Fig 6).
Fig 6. Calibration kit used by field service engineers (FSE) at preventative maintenance (PM) visits. The kit includes a reference filter with certificates and optical fiber for connection.
Wavelength check
The UV-90 wavelength control is based on a proven design with a motor-controlled grating. The natural peaks from the xenon lamp at 229.6 nm, 459.3 nm, and 541.9 nm are identified at each startup of the lamp. If a check of the wavelength accuracy is desired, for example, after a run, there is a function in UNICORN™ control software that will verify that these peaks are correctly identified. Typically, the deviation is less than 1 nm. The wavelength resolution is 0.33 nm.
Conclusion
UV performance is critical to accurately monitoring the purity of your product during the downstream purification process. ÄKTA process™ CFG chromatography system was designed with improved components including the innovative UV-90 multi-wavelength UV monitor. The UV-90 contains updated lamp driver electronics, more robust mechanical design, built-in tools for calibration and wavelength accuracy check, works with pre-calibrated flow cells for direct measurement in OD, and communication using EtherNet/IP. For more information about ÄKTA process™ CFG system, visit the product page.
RELATED CONTENT
Video: view how to perform UV linearity calibration for ÄKTA process chromatography system
Efficient chromatography buffer prep with inline dilution
ÄKTA process™ flow accuracy and gradient performance
Cleaning components/external surfaces of ÄKTA process systems
CY44035-16Aug24-AN