Smart UV-VIS Spectrophotometry

Dr Ursula Tems & Dr Matt Quinn explain how simultaneously measuring standards, controls and unknown samples without any moving parts is giving a new meaning to analytical instrument reliability and reproducibility

In today’s lab, where efficiency and uncompromising quality is vital, managers need assurance that the instruments they purchase can meet this ever-increasing demand, and will improve workflows. This is particularly apparent in the pharmaceutical and biopharmaceutical industries, where quality at an optimal speed is crucial, data integrity is of utmost importance, and time to market is critical for success.

One way to increase sample throughput and save time is to buy more instruments. But the practical reality is not that simple: buying more instruments is a greater financial outlay while adding a large maintenance and validation burden.

Another approach to improve efficiency is to buy a faster instrument. Many UV-Vis spectrophotometers available today are fast – but efficiency and sample throughput are key features required to improve workflow. For example, the Cary 3500 enables a complete experiment to be performed in a single measurement because each sample is measured at the same time.

The Cary 3500 UV-Vis spectrophotometer was designed to improve the user workflow at the instrument, enabled by the modular instrument architecture that also allows system optimisation for the type of measurement performed. It contains a Xenon lamp that flashes at 250Hz (collecting 250 data points per second) and an ultra-fast monochromator. Together these allow data to be collected at rates up to 150,000nm/min, which means that a full spectrum can be collected and displayed on the screen in less than 1.0s. The speed gains are amplified by splitting the light from the single Xenon flash lamp and directing it to all eight cuvette positions simultaneously. Measurements are performed on every cuvette position at the exact same time. With air-cooled Peltier temperature control the system can configure to collect data from multiple samples at different temperatures simultaneously (Fig. 1). This simultaneity removes all unwanted variables from kinetics measurements and protein analysis.

Temperature ramping rate and sensitivity

Benjamin Tadgell and Eser M. Akinoglu from the ARC Centre of Excellence in Exciton Science School of Chemistry, University of Melbourne, Australia have been using the instrument to research how temperature affects the scattering properties of gold nanoparticles coated in PNIPAM polymer.

As temperature increases over the 30-40°C range, the polymer collapses and the particles aggregate. Wavelength scans at different temperatures, and thermal gradient scans are used to partially characterise these processes, along with measurements over a range of pH, salt concentrations and gold particle concentrations. The product’s temperature ramping function allows quick scanning of the effect of temperature on particle scattering at a given wavelength. Previously, there was no other way to do that than to manually take data points at different temperatures from wavelength spectra. The heating/cooling rates and rapid equilibration over all eight cell holders means that more experiments (over different concentrations, pH) can be achieved. The accurate results for high-absorbing samples eliminates dilutions and reduced errors.

Improving throughput and data accuracy

To understand how this speed improves sample throughput and data quality, let’s consider the following scenario. A common lab experiment is to create a calibration curve by sequentially measuring several standards then measuring a series of unknown samples to determine the relative concentrations of the analytes they contain. Duplicate analyses of the standards or unknowns might also be included. Most UV-Vis systems require that one solution (standard, “unknown” or duplicate) is measured at a time even when using a mechanical multicell accessory. It would take approximately 10 minutes to collect data across the wavelength range 350-800nm from seven standards and unknown samples. The Cary 3500 UV-Vis will collect the data from standards and unknowns, automatically generate the calibration curve and determine sample concentration data in a single measurement that takes less than five seconds. This not only saves time, it also helps improve data quality by eliminating errors or variability that can be introduced during the measurement process. This variability can be caused by human error, changing environmental factors or changes in the samples themselves – all detrimentally impact data quality and daily operational efficiency.

Case study

In another real-world example, Dr Mohammad Al Kobaisi from the School of Chemistry and Biotechnology, Swinburne University of Technology has experienced controlled stirring and wide range temperature control options with the Cary 3500, using UV-Vis to monitor processes in situ.

The stability of the signal allows for long-term kinetic processes too, opening up a range of experiments that were previously unable to do due to drift. The intuitive software and data management prevents the loss of results and ensures well organised projects and archives and expands the flexibility to control more parameters while conducting research experiments.

In conclusion, the experimental possibilities with new UV-Vis instruments are endless. They create new possibilities for quantitative and qualitative analysis. Simultaneous system design with integrated non-moving multicell holder provides productivity and reproducibility required in pharmaceutical and biopharmaceutical industries.

Dr Ursula Tems & Dr Matt Quinn are with Agilent

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