Peter J. Schoenmakers

End-group-selective size-exclusion chromatography (esSEC) coupled with mass spectrometry (MS) is presented as an analytical approach for the separation and characterization of acid-terminated and non-acid-terminated poly(lactic acid) (PLA) and poly(lactide-co-glycolide) (PLGA). The method employs a gradient normal-phase liquid chromatography system with basic (triethylamine) and acidic (formic acid) additives in ethyl acetate-based mobile phases, enabling selective retention of acid-terminated polymers, while non-acid-terminated species elute according to hydrodynamic volume, i.e., their effective size in solution. Following their release from the stationary phase, the acid-terminated polymers also undergo separation according to hydrodynamic volume.The separation involves adsorption of acid-terminated polymers under basic conditions, followed by their release upon introduction of an acidic mobile phase and subsequent size-based separation. Gradient steepness strongly influenced the elution behavior of acid-terminated polymers. Shallow gradients resulted in broader elution profiles reflecting prolonged adsorption–desorption, whereas steeper gradients produced sharper, more symmetrical peaks.The method can be directly coupled to MS, enabling molecular-level characterization of end-group structures and chemical composition across the chromatographic separation.The proposed esSEC–MS method provides a novel strategy for end-group-resolved polymer analysis. Its applicability is demonstrated by monitoring the formation of cyclic structures by intramolecular transesterification and the degradation of an ester-terminated PLGA copolymer over time.

End-group-selective size-exclusion chromatography (esSEC) coupled with mass spectrometry (MS) is presented as an analytical approach for the separation and characterization of acid-terminated and non-acid-terminated poly(lactic acid) (PLA) and poly(lactide-co-glycolide) (PLGA). The method employs a gradient normal-phase liquid chromatography system with basic (triethylamine) and acidic (formic acid) additives in ethyl acetate-based mobile phases, enabling selective retention of acid-terminated polymers, while non-acid-terminated species elute according to hydrodynamic volume, i.e., their effective size in solution. Following their release from the stationary phase, the acid-terminated polymers also undergo separation according to hydrodynamic volume. The separation involves adsorption of acid-terminated polymers under basic conditions, followed by their release upon introduction of an acidic mobile phase and subsequent size-based separation. Gradient steepness strongly influenced the elution behavior of acid-terminated polymers. Shallow gradients resulted in broader elution profiles reflecting prolonged adsorption–desorption, whereas steeper gradients produced sharper, more symmetrical peaks. The method can be directly coupled to MS, enabling molecular-level characterization of end-group structures and chemical composition across the chromatographic separation. The proposed esSEC–MS method provides a novel strategy for end-group-resolved polymer analysis. Its applicability is demonstrated by monitoring the formation of cyclic structures by intramolecular transesterification and the degradation of an ester-terminated PLGA copolymer over time.

We introduce a novel analytical strategy for characterizing PLGA's monomer sequence and block-length distribution (BLD). Our approach couples controlled chemical degradation with RPLC-HRMS analysis. By employing two degradation strategies (0.5 vs 3 hrs), we characterize the microstructure of the PLGA co-polymer in terms of the dimer alternating structures (0.5 hr) and the BLD (3 hrs). This method allows for precise monitoring of the randomization caused by transesterification reactions. The BLD, characterized using a specialized algorithm, accurately reflects the polymer's blockiness without underestimation due to dimer-alternating structures, which is a common limitation of other methods like 13C NMR. We demonstrate the method's utility by showing that heterogeneities in block structure significantly influence PLGA's solubility. This new strategy provides critical insights into the relationship between monomer sequence and polymer properties, supporting the development of sequence-controlled PLGA for a wide range of applications.

There is a pervasive need for analytical separations in many fields of science and in many different industries. Analytical measurements are vital to understand processes and materials, to develop or improve products and to protect the health of individuals and entire ecosystems. Separations are needed in many situations because most samples are (complex) mixtures. Thus, scientists in academia and industry need analytical separations. This book teaches (almost) all the important aspects of analytical separation science to students and practicing analysts at various points in their careers. For those new to separation science, it features basic modules in which fundamental concepts and analytical practice are described. Master modules provide more in-depth treatments intended for graduate students. Advanced modules bring the reader to the current frontiers of separation science and encourage further reading. Emphasis is on the main analytical separation techniques of gas chromatography, liquid chromatography and capillary electrophoresis, but due attention is paid to a number of other methods and to sample preparation. Knowledge on statistics and chemometrics is provided, so that separation scientists can obtain relevant information from analytical data. Finally, the book describes how a sound understanding can be used to successfully develop optimal separation methods.

The characterization of the microstructure of in vivo degradable polyesters is gaining increased interest thanks to their high-performance applications, such as drug delivery systems. The design of such material requires a high level of understanding of the critical material attributes of the polyesters, such as molecular-weight distribution (MWD), chemical-composition distribution (CCD), and end-groups (functionality-type distribution, FTD). Size-exclusion chromatography (SEC) hyphenated with mass spectrometry (MS) is an effective method for analyzing the microstructure of polymers. While the MWD can be determined by size-exclusion chromatography hyphenated with ultraviolet spectrometry and refractive index, the CCD and FTD can be determined by SEC-MS. However, previous applications of SEC-MS have not assessed if polymer fragmentation can occur during the analysis process. In order to correctly interpret CCD and FTD, it is important to establish whether SEC-MS methods can be applied to biodegradable polymers and to recognize if fragmentation processes occur. In this study, we investigate whether SEC-MS methods can be applied to PLGA biodegradable polyesters. The research demonstrates that the choice of alkali metal salt used during ionization can influence the stability of PLGA during SEC-MS analysis. CsI was found to minimize fragmentations during ESI-MS, simplifying the MS spectra and allowing isomeric PLGA structures to be distinguished. The resulting method facilitates FTD and CCD determination. Additionally, when combined with selective degradation, the described method can provide insights into the "blockiness" of the polymer and support the development of sequence-controlled PLGA synthesis.

The characterization of the microstructure of in vivo degradable polyesters is gaining increased interest thanks to their high-performance applications, such as drug delivery systems. The design of such material requires a high level of understanding of the critical material attributes of the polyesters, such as molecular-weight distribution (MWD), chemical-composition distribution (CCD), and end-groups (functionality-type distribution, FTD). Size-exclusion chromatography (SEC) hyphenated with mass spectrometry (MS) yields SEC-MS, which is an effective method for analyzing the microstructure of polymers. While the MWD can be determined by size-exclusion chromatography hyphenated with ultraviolet spectrometry and refractive index (SEC-UV/RI), the CCD and FTD can be determined by SEC-MS. However, previous applications of SEC-MS have failed to address the risk of polymer fragmentation during the analysis process. It is crucial to establish whether SEC-MS methods can be applied to biodegradable polymers and to recognize if fragmentation processes occurred during the SEC separation or during the ESI-MS process. In this study, we investigate whether SEC-MS methods can be applied to PLGA biodegradable polyesters. The research demonstrates that the choice of alkali metal salt used during ionization strongly influences the stability of PLGA during SEC-MS analysis. CsI was found to minimize fragmentations during ESI-MS, simplifying the MS spectra and allowing isomeric PLGA structures to be distinguished. The resulting method facilitates end-group and CCD determination. Additionally, when combined with selective degradation, the described method can provide insights in the “blockiness” of the polymer and support the development of sequence-controlled PLGA synthesis.

Multidimensional chromatography offers enhanced chromatographic resolution and peak capacity, which are crucial for analyzing complex samples. This study presents a novel comprehensive online multidimensional chromatography method for the lipidomic analysis of biological samples, combining lipid class and lipid species separation approaches. The method combines optimized reversed-phase ultrahigh-performance liquid chromatography (RP-UHPLC) in the first dimension, utilizing a 150 mm long C18 column, with ultrahigh-performance supercritical fluid chromatography (UHPSFC) in the second dimension, using a 10 mm long silica column, both with sub-2 μm particles. A key advantage of employing UHPSFC in the second dimension is its ability to perform ultrafast analysis using gradient elution with a sampling time of 0.55 min. This approach offers a significant increase in the peak capacity. Compared to our routinely used 1D methods, the peak capacity of the 4D system is 10 times higher than RP-UHPLC and 18 times higher than UHPSFC. The entire chromatographic system is coupled with a high-resolution quadrupole-time-of-flight (QTOF) mass analyzer using electrospray ionization (ESI) in both full-scan and tandem mass spectrometry (MS/MS) and with positive- and negative-ion polarities, enabling the detailed characterization of the lipidome. The confident identification of lipid species is achieved through characteristic ions in both polarity modes, information from MS elevated energy (MS^E) and fast data-dependent analysis scans, and mass accuracy below 5 ppm. This analytical method has been used to characterize the lipidomic profile of the total lipid extract from human plasma, which has led to the identification of 298 lipid species from 16 lipid subclasses.