Today’s compact spectrometers deliver SNR performance, optical resolution and acquisition speed comparable to more expensive and less flexible spectrometers. Here we explore the absorbance linearity of the Ocean HR2 spectrometer.
Ocean HR2 offers high resolution performance (<1.0 nm FWHM), rapid acquisition speed (1 µs integration time) and great signal to noise ratio (SNR) performance (380:1) in a single instrument. These characteristics make Ocean HR2 an excellent option for applications including protein sample concentration measurements, which we demonstrate here.
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Absorbance of Bovine Serum Albumin
To evaluate the HR2, we measured nearly 30 different concentrations of 503 mg bovine serum albumin (BSA) protein samples in distilled water. BSA is often used as a protein concentration standard in biochemical applications.
Our setup comprised an Ocean HR2 spectrometer (190-880 nm), a deuterium-tungsten halogen light source with attenuator, a pair of 400 µm optical fibers, and a quartz cuvette in a cuvette holder. OceanView operating software and OceanDirect, a device driver platform, completed the system.
To ensure best measurement results, we first warmed up the spectrometer and light source for 30 minutes. Light source output can change slightly until the source is in thermal equilibrium, affecting measurements.
Also, as part of the sampling process, we never removed the quartz cuvette from the cuvette holder, as doing so can introduce errors. Instead, we made our dilutions in the cuvette, using a disposable pipet to remove some of each sample, then mixing in the DI water, and repeating the process for each sample.
Our measurements focused on the BSA absorbance peak at 280 nm (Figure 1), which mapped to absorbance linearity of 0.999, up to 2.5 AU (Figure 2). This level of performance would be beneficial in quantifying other types of protein concentration, as well as for biotechnology applications including quality assurance of pharmaceutical formulations.
High Speed Averaging Mode to Improve SNR
High Speed Averaging Mode is a hardware-accelerated signal averaging function accessible via the OceanDirect device driver that improves SNR performance in Ocean Insight spectrometers. With better SNR comes higher quality spectra and more accurate results.
SNR is a function of several factors, some more easily managed than others. For example, signal averaging to improve SNR can be carried out in operating software on the host computer but can take longer to process than what may be acceptable for your application. HSAM overcomes that limitation by enabling many more spectral averages over a given time period, yielding a much higher SNR per unit time. This can be important for time-critical or real-time applications, where decisions must be made very quickly and with high accuracy.
The advantage of HSAM can be quantified easily. For example, the standard operating software can process 10,000 averages in about 2 seconds. But because HSAM performs the averages on the hardware, the OceanDirect device driver can process 10,000 averages in 200 ms – 10x faster.
Summary
From determining concentration levels of proteins to identifying contaminants in pharmaceutical materials, the Ocean HR2 spectrometer delivers research-grade spectrometer performance in an instrument that works equally well in the lab or on the line, as part of a standalone setup, or as a key subsystem or component within another device.