Starch contains large homopolymers of linear amylose (AMY) and branched amylopectin (AMP). Its functional properties are influenced by the ratio and molar masses of its macromolecular constituents, which vary with source, crop year, and climate.

With average radii in the hundreds of nanometers, and molecular weights ranging into the hundreds of millions, starch polymeric components cannot be separated by GPC. However, field-flow fractionation coupled to multi-angle light scattering (FFF-MALS) is suitable for separation and characterization of polymers and nanoparticles from 1 to 1000 nm, and hence is applicable to starch analysis. FFF performs non-shearing separation by hydrodynamic size, and MALS analyzes absolute molar mass and size regardless of conformation or retention properties.

This note demonstrates the application of FFF-MALS to separating AMY and AMP in order to calculate their mass ratio, determine molar mass distributions, the average molecular weights of AMY and AMP, and the 

Separation was performed by an Eclipse® FFF system with short channel, a 350 μm spacer, and a 10 kDa cutoff regenerated cellulose membrane. Detection was accomplished with DAWN® 18-angle MALS and Optilab® refractive index (RI) detectors. The channel flow was maintained at 1.0 mL/min and the cross-flow was varied linearly from 1.0 to 0.1 mL/min for 10 min, then switched to 0.0 mL/min.

Figure 1 shows that FFF-RI fractograms separate into distinct peaks for AMY and AMP. The molar mass distributions determined for these samples ranged from 10 kDa to 1 GDa, though there is evidence for smaller starch components as well.

Table I summarizes the results of the analyses. Integration of the respective peak areas enabled calculation of the AMY:AMP ratios, in excellent agreement with the nominal values. The values for 

 fall within the generally accepted limits found in the literature. Polymer conformation is assessed by comparing 

 with molar mass. Conformational plots for the AMP component (not shown) indicate a log-log slope of 0.39–0.41 for all starches measured, verifying their branched nature. Species-specific differences in the overall density of the amylopectins are apparent in the vertical offsets of the conformation plots.

Starch characterization by size-exclusion chromatography can be limited due to shear degradation or column adsorption of the higher mass fractions of AMP, and SEC columns cannot cover the entire size range. Due to branching, the usual linear calibration standards would not have been applicable. The open-channel separation of FFF eliminates these limitations and enables recovery of intact AMP. The molar masses and mass ratios of AMY:AMP were accurately determined over five orders of magnitude. Only FFF-MALS fully addresses the multiple analytical challenges presented by starch.

Articles

Starch Amylose and Amylopectin Molar Mass and Size Distributions by FFF-MALS

The Empore EZ-Trace (Figure 1) is a manual, vacuum-controlled extraction workstation designed to perform up to four extractions simultaneously to improve efficiency in a clean and reproducible manner. The Empore EZ-Trace workstation is universally compatible with all Empore 47/90 mm disks and cartridges. The unique independent channel design ensures that each extraction is precisely controlled, preventing cross-contamination with the other channels. High-throughput flow path switches provide a unique, environmentally friendly extraction by collecting organic and aqueous wastes in separate reservoirs. This is demonstrated using an example application of EPA Method 525.3, which is designed for identification and quantification of semivolatile organic compounds (SVOCs) such as PCBs, PAHs, and pesticides. Under this method, SVOCs are extracted from three reagent water samples having different concentrations and one sample of ground water, with four replicate measurements required for each sample. Methods such as EPA 525.3 require ways to improve efficiency when numerous extractions are needed.

Four extractions were done simultaneously using the Empore EZ-Trace system (catalog #: 8000) by following EPA 525.3 extraction procedures to extract SVOCs onto an Empore SDB-XC 47mm disk (catalog #: 2240). The extract was then evaporated, diluted, and then analyzed with a Shimadzu GC-2010 instrument using a Restek Rxi-5Sil-MS system (30 m x 0.25 mm x 0.25 μm) column for separation and MS QP2010 for detection. In this method, 125 analytical calibration standards, three surrogate compounds, and four internal standards are efficiently separated (Figure 2). The recoveries and concentrations of 125 analytes were determined from four replicate measurements using a five-point calibration curve. For all compounds except for two, the acceptable range of recoveries is 70–130%. The accepted range for the other two compounds is 60–130%. The combined recovery results are summarized in Table I. For each sample, the average recovery is >89.9% with <7.4% RSD. These results demonstrate how the Empore EZ-Trace provides a clean, efficient, and reproducible extraction.

Application Notes: Environmental

This application note demonstrates how the Empore EZ-Trace SPE system is used to perform multiple extractions simultaneously while achieving clean and reproducible results using EPA Method 525.3 as an example application.

Using the Empore EZ-Trace SPE Workstation to Perform Efficient and Reproducible Extractions of SVOCs

Biotherapeutics are generally larger molecules such as peptides, proteins and monoclonal antibodies with monomer molecular weight ranging from 3,000 to 150,000 Da. The drug must remain free from impurities such as fragment, dimer, and other higher order aggregates because they may cause severe immunogenic response. This becomes even more important particularly if the biotherapeutic protein is thermally susceptible. Historically, size exclusion is the preferred mode of chromatography for separation and characterization of such applications. Here we report the online detection of absolute molecular weight of two recombinant protein samples using a 2 μm size exclusion chromatography (SEC) column directly connected to the LenS

 Multi-Angle Light Scattering (MALS) detector.

Sample 1 – Recombinant proteins (~90 kDa) at 1.72 mg/mL in mobile phase
Sample 2 – Recombinant proteins (~90 kDa) at 3.64 mg/mL in mobile phase

The samples were stored at -20 °C and thawed to 8 °C just before analysis. The concentration was adjusted by diluting the sample in mobile phase pre-chilled at 8 °C.

Instrument: ThermoFisher Ultimate® 3000 UHPLC and Chromeleon® software

Column: TSKgel UP-SW2000, 2 μm, 4.6 mm ID × 30 cm 

Mobile Phase: BupH modified Dulbecco’s phosphate buffer prepared in light scattering grade water and filtered through a 0.1 μm PES membrane (The buffer was prepared from saline packs as per directions contained in Thermo Scientific– catalog # 28374, lot TL275790)

Detectors: UltiMate 3000 VWD variable wavelength detectors @ 280 nm wavelength and Tosoh LenS

 MALS detector (positioned in series: Column → UV → MALS)

Column Oven Temp.: 25°C
Autosampler temp.: 8 °C
Injection Volume: Sample 1: 25 μL, Sample 2: 10 μL

The multi-detector setup was calibrated using a freshly prepared bovine serum albumin (BSA) solution. Figure 1 shows the overlay trace of UV and MALS detectors. The one-step calibration procedure in the SECview

 software adjusted the dead volume between the detectors and corrected for the band-broadening effect caused by the in-series detector configuration while determining the detectors’ calibration constants and offsets.

Figures 2 and 3 illustrate the UV detector overlays for two consecutive injections of Sample 1 and Sample 2, respectively. Zoomed-in figures (inset) show the excellent separation resolution between the monomer and the aggregates obtained from TSKgel UP-SW2000 column.

Figures 4 and 5 demonstrate the molecular weight (green) profiles for Sample 1 and Sample 2, respectively. The concentrations of the aggregate contents differ in the two samples.

Looking closer at the UV trace, it appears that the monomer peaks in the samples illustrate a slight shoulder on the higher retention region, suggesting a bimodal shape. Further analysis using the molecular weight trace by the MALS detector reveals two separate populations of molecular weights. Figures 6 and 7 zoom in on the monomer peaks and demonstrate the two molecular weight plateaus, 1a and 1b, in both samples. Considering the sensitive nature of these recombinant proteins to the ambient conditions, the shoulder peak (1b) suggests the beginning of temperature-induced modifications, which varies in extent in both samples.

Tables I and II list the results of the chromatography analysis, including molecular weight and percent content, for the identified aggregate peaks.

This study shows that the molecular weight species including monomer and multiple aggregate levels present in the recombinant protein therapeutics could be determined and quantified using a SEC-MALS configuration. The TSKgel UP-SW2000 demonstrates excellent separation of the higher order aggregates from the monomer in both Sample 1 and Sample 2, as well as the slightly higher molecular weight temperature-induced variants that are almost co-eluted with the monomer. The column yields reproducible results, allowing accurate and precise chromatograms to be analyzed using the LenS3 MALS detector. The LenS3 accompanied by the SECview software also produces reproducible, accurate results in terms of MW determination for all peaks, in addition to area calculations, even at extremely low concentrations or in the presence of the aggregates.

TSKgel and Tosoh Bioscience are registered trademarks of Tosoh Corporation.
LenS is a trademark of Tosoh Bioscience LLC.
SECview is a registered trademark of Tosoh Bioscience LLC in the USA and EU and of Tosoh Corporation in Japan. UltiMate and Chromeleon are registered trademarks of the Dionex Corporation.

3604 Horizon Drive, Suite 100, King of Prussia, PA 19406 

Pharma/Drug Discovery

Characterization of Recombinant Protein Biotherapeutic Using TSKgel® UP-SW2000 Size Exclusion Chromatography Column In-Line with LenS3TM MALS Detector

Amino acids have significant biological importance, as they are the building blocks of life, and they are also used in nutritional supplements, fertilizers, and food technology. Industrial amino acid use includes the production of drugs, biodegradable plastics, and even chiral catalysts. With the wide variety of uses, there are many ways to classify amino acids, including polarity, pH, size, hydrophobicity, side chain group type, and so on. However, the ideal quantitative analysis of amino acids combines speed and sensitivity with reliability of the analytical technique.

Many columns cannot offer a single method to analyze the 20 building block amino acids. Shodex introduces the new HILICpak VC-50 2D column, which can offer speed, sensitivity, and reproducibility. This column is packed with polyvinyl alcohol material with carboxyl functional groups. When used according to the protocol described below, the column enables the user to analyze all 20 amino acids using a single method. A single run can be completed in 25 min with good resolution. In addition to analyzing the essential amino acids, the VC-50 2D has been shown to separate amino sugars.

Each amino acid was dissolved in water and diluted to 0.1 μg/mL. The analysis of all 20 amino acids was successfully accomplished with the Shodex HILICpak VC-50 2D (2.0 mm ID x 150 mm), a polymer-based column for HILIC analysis. A 5 μL measure of each sample was injected into the system. The column temperature was kept at 30 oC and a flow rate of 0.2 mL/min. Eluent conditions were H

CN = 30/20/50 to 0/90/10 (0 to 5 min), 0/90/10 (5 to 30 min) in a lower pressure linear gradient. The HPLC system was coupled with an ESI-MS SIM detector.

The Shodex team has successfully developed a method for detecting 20 amino acids using a single method on the HILICpak VC-50 2D under positive MS mode. From the data shown above, isoleucine and leucine have some overlap. Through some modifications to the method, this analysis can result in a better separation. The Shodex HILICpak VC-50 2D is an ideal column for amino acid analysis.

420 Lexington Avenue Suite 2335A, New York, NY, 10170 

Tel. (212) 370-0033 x109, Fax: (212) 370-4566 

Application Note - Shodex HILICpak VC-50 2D for All Essential Amino Acids Detection

Since the discovery of aflatoxins in 1960, many areas of human food sources have shown considerable propagation of the fungal infection. Although the majority of food samples are cereals, the toxins can be found in a variety of medias, including corn, peanuts, legumes, livestock, milk, and even cannabis, to name a few. The aflatoxins mentioned above are part of approximately 40 secondary metabolites derived from the fungus, 

 infection can be found in both the pre-harvest and the post-harvest analysis of crops. Analogously, the effects from these metabolites can be either acute or chronic, and have been found to be teratogenic, mutagenic, carcinogenic, immunotoxic, or hepatotoxic in nature. Due to the nature of these attributes, the toxins have been implicated in liver cancer, Reyes syndrome, cirrhosis, and chronic gastritis. As such, the FDA has imposed strict guidelines on the amounts of acceptable aflatoxins present in food, <20 ppb, with even stricter guidelines for milk, at <0.5 ppb.

In this application note, we have developed an HPLC method that isolates five different aflatoxins with the aid of Hamilton’s HxSil-C18 (3 μm) column. The fast isocratic method is completed in under 6 min, and provides excellent resolution between the various aflatoxins. The excellent sensitivity shown in the method affords the analyst a robust, accurate, and affordable product.

Food and Beverage

Application Note from Hamilton - Separation of Aflatoxins on HxSil C18

Per- and polyfluoroalkyl substances (PFAS) are a diverse group of synthetic organofluorine compounds that have been widely used in industrial applications and consumer products, such as nonstick cookware, food packaging, fire-fighting foams, carpeting, apparels, and metal plating. PFAS are persistent in the environment and bio accumulative in humans and wildlife, and said to cause reproductive and developmental toxicity. This application note summarizes a validated procedure developed by the US Food and Drug Administration (FDA) for the measurement of 16 PFAS in food matrices using a QuEChERS sample preparation approach and LC–MS/MS analysis.

The edible portion of the food sample is collected and homogenized using an IKA tube mill with a disposable 100 mL polypropylene grinding chamber (an alternative homogenizer may also be used for this step). Samples are ground at 5000 rpm for approximately 2 min. The minimum sample size for analysis is 5 g.

(1) Add amount of sample and LC–MS grade water based on Table II and commodity type to a 50 mL polypropylene (PP) centrifuge tube.

(2) Add 10 μL of 1 μg/mL isotopically labeled surrogate standard solution to the sample.

(3) Add 10 mL acetonitrile and 150 μL formic acid to the 50 mL PP conical centrifuge tube.

(5) Add QuEChERS salt packet and shake for 5 min.

Glas-Col® digital pulse vortexer at 1500 rpm with pulse set to 70

(6) Centrifuge the samples at 10000 rcf for 5 min.

(1) Transfer supernatant to the 15 mL dSPE tube

(3) Centrifuge the samples at 10000 rcf for 5 min.

(4) Filter 5 mL of the extract with a 0.2 μm nylon syringe filter and transfer to a 15 mL conical centrifuge tube.

(5) Add internal standard: For samples that do not require nitrogen concentration: 

Add 5 μL of 1 μg/mL d5-N-EtFOSAA internal standard solution to the 5 mL extract to give a final concentration of 1 ng/mL. Surrogates will also have a final concentration of 1ng/mL in the final extract.

For samples that require nitrogen concentration: 

Concentrate to near dryness with nitrogen and reconstitute to 0.5 mL with methanol.

Add 5 μL of the 1 μg/mL d5-N-EtFOSAA internal standard solution to give a final concentration of 10 ng/mL in solution. Surrogates will also have a final concentration of 10 ng/mL in the final extract.


(7) Transfer sample to a polypropylene autosampler vial for analysis by LC–MS/MS.

A level 2 single laboratory validation was conducted under the Guidelines for the Validation of Chemical Methods for the FDA FVM Program 2nd Ed. A total of four different types of foods and beverages were evaluated. These include produce, milk, fish, and bread. The method was validated at six concentrations (0.05, 0.15, 0.5, 1.5, 2, 5 ng/mL) in four food matrices. Acceptable recovery ranges for these compounds based on the FDA guidelines for the validation of chemical methods is 40–120% for concentrations spiked at 1 ng/mL. All compounds were within the acceptable range, except for 11Cl-PF3OUdS in bread samples which were on the lower side at 26–42% recovery.