Overview of the ATR Pro Penta X

The ATR method is widely used in FTIR spectroscopy for measuring solutions without the need for sample preparation. The current, mainstream single-reflection ATR requires a sample concentration on the order of several percent, making it difficult to measure many biological samples that are often in low concentrations of less than 0.1%.

Figure 1. ATR PRO Penta X

The newly developed ATR Pro Penta X (Figure 1), a highly sensitive multiple-reflection ATR, can measure low-concentration samples of 0.1% or less using only a small amount of sample (several microliters) by adopting a uniquely designed 14-reflection Ge prism (Figure 2).*

* Since proteins adsorb on Ge, the spectral intensity does not change below a certain concentration.

* Strong acids and strong alkalis cannot be measured as they damage the crystal.

Time Reflective Ge Prism
Figure 2. Time Reflective Ge Prism

Here, we introduce JASCO’s ATR PRO PENTA as a useful tool in the biochemistry field for samples such as lipids, sugars, and proteins.

Quantitation of low-concentration solutions

The spectra of 0.5%, 0.1%, and 0.05% sucrose aqueous solutions are shown in Figure 3. The spectra in Figure 3 are the resulting difference spectra from subtracting the spectrum of water from the spectrum of the sucrose aqueous solution. The calibration curve for the concentration of sucrose aqueous solutions shows a very high linearity (Figure 4), suggesting that the ATR PRO PENTA is sufficient for the quantification of low-concentration solutions that were previously untestable.

Difference spectrum of each sucrose aqueous solution Figure 4.Sucrose content metering (1056 cm-1 peak height)
Figure 3. Difference spectrum of each sucrose aqueous solution*

* The quantitation limit of sucrose aqueous solution in this experiment was 0.042%, and the detection limit was 0.014%.

Difference spectrum of each sucrose aqueous solution
Figure 4. Calibration curve for the concentration of sucrose aqueous solutions

Secondary structure analysis of IR proteins using extremely low-concentration samples

IR-SSE (IR protein secondary structure analysis) was performed using the difference spectrum of 0.01% lysozyme aqueous solution (Figure 5). Even at a low concentration of 0.01%, the peaks of amide I and amide II are clearly separated, and the peak shape is clear, so amide I information can be used for secondary structural analysis. Detection of lysozyme at a concentration of 0.01% was possible, and SSE was performed based on the Amide I band in the difference spectrum. Figure 6 shows the resulting concentration values. The results of the secondary structure analysis of the difference spectra measured using a heavy aqueous solution were reasonably consistent with those obtained by Sarver and his team [1].

[1] Sarver, R. W., Krueger, W.C., 1991. Anal. Biochem., 194, 89-100.

Measurement results and difference spectra of 0.01% lysozyme aqueous solution with ATR PRO PENTA
Figure 5. Measurement results and difference spectrum of 0.01% lysozyme aqueous solution with ATR PRO PENTA
SSE spectra of 0.01% lysozyme heavy aqueous solution
Figure 6. SSE spectra of 0.01% lysozyme-heavy aqueous solution

SSE quantitation

Structure Percentage (%)

Orthogonal Similarity Assessment of Monoclonal Antibodies Using CD, FTIR, and Raman

In this webinar we outline the study of monoclonal antibody structure using qHOS and various spectroscopic techniques.

The biopharmaceutical market has been expanding dramatically over the past decade, particularly for therapeutic antibodies. As innovative antibodies are developed there is a strong push to develop biosimilars with identical higher order structure (HOS). The HOS of biopharmaceuticals directly impacts their efficacy and safety, which means verifying HOS is of great importance. Current FDA guidelines emphasize the use of orthogonal quantitative methods in the comparison of reference products with biosimilars. For proteins, such as antibodies, circular dichroism, Fourier transform Infrared, and Raman spectroscopy are excellent tools for structural determination. To quantitate spectral difference for these spectroscopic techniques, Jasco has developed qHOS, a powerful spectral assessment tool.