News from LabRulezLCMS Library - Week 11, 2025

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This week we bring you application notes by Agilent Technologies, Knauer, Shimadzu, Thermo Fisher Scientific and Waters Corporation!

1. Agilent Technologies: Determination of Asphaltenes in Crude Oil

Using the Agilent 1260 Infinity III LC system with ELSD

• Application note
• Full PDF for download

The Agilent 1260 Infinity III LC system with Agilent 1260 Infinity III evaporative light scattering detector was used to evaluate heptane-insoluble asphaltenes in crude oil. The separation of maltenes and asphaltenes is enabled using Agilent InfinityLab Quick Change inline filters for HPLC. The test solution was injected into a flow of heptane under normal phase HPLC conditions using an inline filter (see Figure 1). The precipitation of asphaltenes is induced as soon as the sample enters the flow of the heptane between the autosampler and inline filter. Precipitated asphaltenes are retained by the inline filter (4.6 mm ID, 0.5 µm porosity) while maltenes pass through to the detector. When the mobile phase is flashed from heptane to a solvent with a high solvent power (e.g., toluene), the blend redissolves the asphaltenes. The concentration of asphaltenes is quantified using evaporative light scattering detection. This method is fast, repeatable, and reproducible, with excellent sensitivity and detection limits.

Conclusion 

An improved analytical method was developed to determine asphaltene content in crude oils and petroleum samples. The inline filtration method takes just under 10 minutes and has a repeatability equivalent to or better than conventional gravimetric methods. Among other advantages, filters are inexpensive and can be easily replaced, as they are commercially produced and available, and their characteristics are less variable than handmade columns. The Agilent 1260 Infinity III LC system with Agilent 1260 Infinity III ELSD was used to evaluate heptane-insoluble asphaltenes in crude oil. This solution is fast, more repeatable, and reproducible with better sensitivity and detection limits than traditional gravimetric methods.

2. Knauer: Determining molecular weights for P(D,L)LA in THF – an internal validation

• Application Note
• Full PDF for download

Plastics are one of the most widely used and most versatile materials of the 21st century. However, the majority of the world’s annual production of approximately 370 million tons of plastics is neither biodegradable nor produced from renewable raw materials.(1) In contrast, poly lactic acid (PLA) is a promising plastic that has sustainable properties. The physical properties of plastics are mostly determined by their molar mass distribution, with the parameters Mn, Mw and the PD being the most important. GPC is the method of choice for the determination of these parameters. When no absolute method is available, the two most frequently used calibrations are conventional and universal calibration.

For the conventional calibration, calibration standards of the polymer to be analysed are required or, at the very least, standards of a chemically similar plastic are required. For universal calibration, first published by Benoit(2), a universal calibration curve is created using the Mark-Houwink relationship, so that a wide variety of polymers can be analysed, for example with a narrow polystyrene (PS) calibration. In 1998, S. Mori published the results of a multistage round-robin test in which PS standards were analysed by GPC using conventional calibration (CC) with PS under various framework conditions.(3) The relative interlaboratory standard deviation (%RSD) for Mn of more than 20% was reduced by setting strict framework conditions for the analysis, such as the injection volume, sample concentration, and sample size.

Results and Discussion 

Initially, a calibration was made using PS. The two largest and lowest molar masses were excluded due to their peak shape and overlap with the flow marker. Additionally, two standards had the same molar mass, resulting in a 14-point universal calibration with a 5th degree fit function (1 230 Da – 729 500 Da). The overlay of the calibration function and the chromatograms is shown in Fig. 1. For universal calibration with PS in THF, the following Mark-Houwink parameters were used: K = 14·105 dl/g, α=0.70.(5) For PLA K = 17.4·105 dl/g and α = 0.74 were used.(6)

Conclusion

The investigations have shown that the determination of the Mn and Mw of low molecular weight P(D,L)LA is easily possible using a KNAUER HPLC system combined with a gel permeation chromatography (GPC)-column. The intralaboratory validation in THF proves that the developed method is robust within the limits shown here with respect to changes in temperature, concentration, flow rate and injection volume. Furthermore, the repeatability of the molar mass determination as well as the intermediate precision of the sample preparation was verified. It was shown that the CC fails for this column/solvent/polymer combination, whereas the UC provides good results. The conventional calibration fails because the different hydrodynamic radii of PLA and PS in THF are not taken into account. The flow rate has a very strong influence on the molar mass determination and must be as constant as possible. Slight fluctuations affect the retention volume. In order to take fluctuations into account, a flow marker, in this case BHT, was used and is recommend as mandatory for all GPC/SEC applications.

3. Shimadzu: Fermentation Monitoring of Yeast Using Size Exclusion-ligand Exchange (Na-type) Column

• Application Note
• Full PDF for download

User Benefits:

• Oligosaccharides, monosaccharides, and sugar alcohols of less than trisaccharide can be separated.
• There is no need to prepare complicated mobile phase since just water can be used as mobile phase for HPLC analysis.
• The variations of saccharides and ethanol in the fermentation process caused by microbes can be monitored.

Quantitative determinations of saccharides are performed in various fields, including food products. Especially in the production of alcoholic beverages and biofuels, where ethanol is produced from saccharides through microbial fermentation, it is important to perform quantitative analysis and monitoring of saccharides accurately for process design and quality control purposes. Shim-packTM SUR-Na ligand exchange chromatography column provides the combination of two separation modes of size exclusion and sodium-type ligand exchange to analyze saccharides with high separation performance. There is no need for mobile phase preparation since only water is used as the mobile phase, resulting in easy execution ofsaccharide analysis.

Analysis of standard mixture

Fig. 1 shows the flow path diagram of this system setup. Shimpack SUR-Na requires water as the mobile phase to separate saccharides, it allows to employ the simple system configuration of the Nexera lite isocratic setup connected with RID-20A differential refractive index detector. Table 1 shows the analytical conditions. A retention index was created by analyzing twenty standard saccharides, involving oligosaccharides, monosaccharides, and sugar alcohols of less than trisaccharide.

Conclusion

In this article, the retention index and the calibration curves for saccharides and sugar alcohols were created, and as an application, quantitative monitoring of fermentation process was performed based on HPLC analyses of the yeast fermentation culture. Since Shim-pack SUR-Na can easily analyze a variety of saccharides, it is expected to be used in a wide range of application fields, including the energy and food industries.

4. Thermo Fisher Scientific: Direct injection of drinking water for the analysis of 54 PFAS compounds by LC-MS/MS aligned with current and evolving global regulations

• Application Note
• Full PDF for download

Application benefits

• A simple, reproducible, and robust sample preparation based on the dilution of the sample with mobile phase B followed by direct injection for reducing labor, errors, and potential contamination issues compared to standard methods requiring preconcentration with solid phase extraction. 
• A complete PFAS workflow solution using direct injection of 100 µL of water samples that meets challenging regulatory detection and reporting limits within the EU and UK. 
• Dedicated instrumental setup and appropriate sample handling procedures have been established to overcome the main challenges of this application which are sensitivity, contamination, and carryover handling.

Per- and polyfluoroalkyl substances are a large, diverse group of synthetic compounds containing chains of linked carbon and fluorine atoms. Due to the ubiquitous use of PFAS, environmental exposure can come from numerous sources, including household dust, food, and drinking water,¹ and has been linked to a variety of health effects² . To evaluate the health risk, an expanding list of governmental regulatory agencies have established validated methods stating the quantitative levels for individual or collective PFAS. 

In Europe, the main law regulating the quality of drinking water is the drinking water directive (2020/2184/EU) that sets two thresholds for PFAS as follows: 0.1 µg/L for the sum of a group of 20 PFAS, and 0.5 µg/L for the PFAS total, which means the totality of per- and polyfluoroalkyl substances. In the case of natural waters, they are regulated under the Water Framework Directive (WFD, 2000/60/EC), the Environmental Quality Standards Directive (EQSD, 2008/105/EC), and the Groundwater Directive (GWD, 2006/118/EC). Table 1 summarizes some of the European3 and national4-7 regulations for analysis of PFAS in drinking water.

Experimental 

Instrumental method 

A chromatographic method of 23 minutes was used for the analysis of 54 PFAS in drinking water samples using a Thermo Scientific™ Vanquish™ Flex Binary UHPLC system, coupled to a Thermo Scientific™ TSQ Altis™ Plus triple quadrupole mass spectrometer equipped with a HESI ionization probe. To handle the 100 µL injection volume, a strong solvent loop was added to the fluidics between the autosampler and analytical column. To prevent the issues of solubility of long chain PFAS, a user-defined injection program was included in the method to shake the samples before each injection. Considering the low limits of detection required in the different European regulations, a Thermo Scientific™ Acclaim™ 120 C18, 50 x 2.1 mm, 2.2 µm column (P/N 068981) was used as a delay column to prevent potential contamination coming from solvent and system. The analytical separation was performed with an Acclaim 120 C18, 150 x 2.1 mm, 3 µm analytical column (P/N 059130) heated at 40 °C. Gradient elution was performed with water (phase A) and methanol containing 2 mM ammonium acetate and 0.1% of acetic acid (phase B) at a flow rate of 400 µL/min. 

Acquisition was performed using selected reaction monitoring (SRM) in negative mode. The spray voltage was set at 500 V, sheath gas was set to 40 arb, auxiliary gas was set to 10 arb, and ion transfer tube and vaporizer temperatures were set to 200 °C and 300 °C, respectively. Table 2 summarizes the monitored SRM transitions.

Data analysis 

All LC-MS/MS data were acquired and processed using the Chromeleon Chromatography Data System (CDS), version 7.3.2. A *.cmbx file is included with the workflow that contains all the optimized SRM transitions for the 54 native and associated labeled PFAS, with software view settings for easy data review and templates to allow laboratories to generate reports for a given regulation.

Conclusions

The method presented in this work achieves high-level sensitivity when performing direct injection analysis of 54 PFAS compounds in the low ng/L range in drinking water using the TSQ Altis Plus mass spectrometer. This workflow allows laboratories to overcome the challenges associated with this analysis, i.e., contamination and solubility, with the use of practical tools such as adapted consumables, a delay Acclaim short LC column, specific fluidics, and a defined injection program performed for each injected sample. The ease of use and robustness of the method are based on a fixed configuration including a SOP with detailed hardware and consumables, a complete acquisition and processing method with customized view settings and reports, and all data handling performed with Chromeleon CDS 7.3.2. Additionally, the benefit of direct injection allows laboratories to improve sample throughput in the lab due to significantly less sample preparation being required compared to traditional SPE clean-up workflows.

5. Waters Corporation: Using a 2.1 mm ID Narrow Bore GTxResolve™ Premier™ SEC Column to Reduce Sample Consumption During Size Exclusion Analyses

• Application Note
• Full PDF for download

In the cell and gene therapy industry, it is size-exclusion chromatography (SEC) that has become an essential method for detecting both product-related and process-related impurities. Product-related impurities include aggregates or degraded forms of the therapeutic molecules that can impact efficacy or safety. Process-related impurities, such as residual host cell proteins, DNA, or buffer components, may also compromise product quality if not thoroughly removed. SEC efficiently separates these impurities from the desired product, allowing an analyst to collect a precise purity profile. One of the important impurities to check for is molecular aggregates. SEC, when operated under native conditions, is one of the most suitable techniques for aggregate quantitation. Beyond impurity detection, SEC is valuable for concentration determination, as it can quantify the therapeutic biomolecule based on its peak area, offering a multiattribute measurement to the purity assessment. This combined functionality makes SEC an indispensable tool for rigorous process development and quality control testing of cell and gene therapy drugs. The availability of new column technology in 2.1 mm ID narrow bore hardware means that more SEC runs can be applied to facilitate the development of drug substances. Narrow bore SEC columns make it possible to complete SEC-UV/FLR characterization without consuming as much precious lead candidate material.

Experimental 

The Solution 

Size exclusion chromatography separates biomolecules according to their hydrodynamic size and shape in solution. This technique is an entropy driven process, but it can be impacted by non-specific secondary interactions between analytes and the column packing material. Most SEC column manufacturers have relied on metal column hardware that requires significant method optimization through use of mobile phase additives including the addition of increasing concentrations of salt. To address this problem, Waters has developed MaxPeak Premier hydrophilic high-performance surface (HPS) column hardware to minimize non-specific electrostatic interactions. This technology makes use of vapor deposited surfaces comprised of an organic and inorganic (carbon and silica) composition that shield analytes from interacting with the surfaces of metallic flow path components. 

Traditionally, SEC columns are used in 4.6 or 7.8 mm ID dimensions to overcome the negative impact of large extra-column volume and dispersion effects imposed by HPLC instruments. However, narrower bore SEC columns continue to be requested to help reduce sample consumption, especially when drug substances are limited during discovery and pre-clinical project work. Fortunately, Waters can provide optimal packing into 2.1 mm columns constructed with MaxPeak HPS hardware. GTxResolve Premier SEC columns can be purchased as custom manufactured parts using Waters p/n: 186011284 by reaching out to a Waters customer representative. Use of narrower bore columns reduce sample consumption while maintaining high data quality of wider bore columns, though it should be noted that 2.1 mm ID should only be performed on a low dispersion UHPLC instrument. Moreover, care should be given to optimize pre- and post-column dispersion effects as much as possible. Upon request, MaxPeak Premier 2.1 mm ID Columns can be prepared with both diol bonded 2.5 µm BEH™ 450 Å particles and bridged ethylene polyethylene oxide (BE-PEO) modified 3 µm SEC 1000 Å particles. This application note demonstrates the performance of a 2.1 x 150 mm GTxResolve Premier BEH SEC 450 Å 2.5 µm Column compared to a commercially available 2.1 x 150 mm SEC titanium hardware column containing 3 µm 700 Å particles.

Conclusion

In this application, we demonstrate the generation of high quality chromatography from a GTxResolve Premier BEH SEC 450 Å 2.5 µm 2.1 x 150 mm Column. With small considerations made to pre- and post-column accessories, the fidelity of peaks can be effectively maintained. Although the performance of the separation is not as precisely optimized as a 4.6 x 150 mm column but the slight loss in resolving power is compensated for by a sizable reduction in sample consumption and a corresponding increase in senstivity. A Waters custom made, narrow bore column showed higher resolution of biomolecules when compared to a commercially available alternative (a 2.1 x 150 mm 700 Å 3 µm titanium hardware column). 

The Waters custom made column also exhibited little or no salt dependency while characterizing biotherapeutic moleclules. That feature was further confirmed upon use of the column to analyze a AAV2 sample where high quality information was obtained with a 1x strength dPBS buffer. In contrast, the alternative column (Column P) showed decreased resolution and high salt dependence. Although the use of narrow bore (2.1 mm ID) columns requires extra system considerations, it is our hope that their availability will hasten the development of gene therapies by giving project teams a new option to preserve more drug material for critical in vitro and in vivo studies.