	Amino acids have significant biological importance as they are the building blocks of life, 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 × 150 mm), a polymer based column for HILIC analysis. From each sample, 5 µL was injected into the system. The column temperature was kept at 30 °C 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) 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 with 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, 1017
	tel. (212) 370-0033 x109, fax (212) 370-4566
	Website: 

Medical/Biological

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

	Adenosine triphosphate (ATP) is the universal energy currency in biological systems. ATP is synthesized from adenosine mono and di-phosphate substrates. The relative ratio of ATP, ADP, and AMP is an important indicator of metabolic standing: the energy charge

	is considered a quantitative measure of energy status, and serves as a barometer of sorts, as aberrant change in EC is associated with onset of numerous pathological states. Measurement of adenosine nucleotides, therefore, represents an important and insightful diagnostic.

	In the present study, an ion pair reversed-phase HPLC method was developed to separate ATP, ADP, and AMP from like-phosphorylated guanosine metabolic products (such as GTP, GDP, GMP) enabling quantification of these nucleotides from tissue extracts.


	4970 Energy Way, Reno, NV 89502
	tel. (775) 858-3000, (800) 648-5950
	Website: 

Determination of Adenine and Guanine Nucleotides in Tissue Extracts

	Cannabinoids are a class of terpenophenolic compounds that are associated with the pharmacological activity of cannabis. Cannabinoids exist in the plant mainly as carboxylic acids and are converted to neutral analogs by light and heat while in storage or during the preparation of edible products.

	A new HPLC method with post-column derivatization was developed to analyze cannabinoids in cannabis plants as well as in cannabis-containing edible products. This post-column method is based on the reaction with Fast Blue Salt reagent under basic conditions and implements a simple sample extraction with acidified water–acetonitrile followed by QuEChERS sample clean-up. The method is suitable for analysis of the major neutral cannabinoids such as THC, CBD, CBN, and CBG as well cannabinoid acids THCA-A and CBDA with high sensitivity and selectivity of detection.

	The following cannabinoids were analyzed: delta-9 tetrahydrocannabinol (THC), cannabinol (CBN), cannabidiol (CBD), cannabigerol (CBG), cannabichromene (CBC), delta-9 tetrahydrocannabinolic acid A (THCA-A), and cannabidiolic acid (CBDA).


	1280 Space Park Way, Mountain View, CA 94043
	tel. (800) 654-3330, (650) 694-6700
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Food & Beverage

Analysis of Cannabinoids Using HPLC with Post-Column Derivatization

	Ephedra alkaloids are phenethylamines that occur naturally in plants, including the herb Ma Huang used in traditional Chinese medicine. The aim of this study was to develop a multi-analyte procedure for the extraction, cleanup, and quantification of the ephedra alkaloids in functional foods and natural products. High capacity strong cation-exchange SPE cartridges were used for the isolation of the phenethylamines from dietary supplements. HPLC separation, including separation of the stereoisomers, was carried out using a UCT Selectra® PFPP column prior to detection by LC–MS/MS.

	Weigh 1 ± 0.1 g of dietary supplement in question into a 15 mL polypropylene centrifuge tube. Add 10 mL of 1% formic acid to each sample. Shake or vortex sample for 15 min to fully extract the ephedra alkaloids*. Ensure samples are fully dissolved. Centrifuge the sample for 10 min at ≥3000 × g and 4 °C.

	* For this study, a SPEX® SamplePrep® GenoGrinder® was used to pulverize the tablets.

	1. Add 2 × 4 mL of methanol to CUBCX1HL56 SPE cartridge.

	Note: Do not let the cartridge go dry otherwise repeat steps 1 through 3.

	1. Load supernatant from sample pretreatment step.

	2. Allow sample to percolate through the cartridge or apply a vacuum if necessary (adjust vacuum for flow of 1–3 mL per min).

	1. Add 2 × 4 mL of 0.1% formic acid and slowly draw through.

	2. Add 2 × 4 mL methanol and slowly draw through.

	3. Dry under vacuum for ≈30 s to remove excess solvent.

	1. Elute the ephedra alkaloids using 8 mL of methanol containing 2% ammonium hydroxide.

	2. Evaporate off the methanol solvent at 40 °C under a gentle stream of nitrogen until it reaches a volume of ≈1 mL.

	3. Add 1 mL of aqueous mobile phase (10 mM ammonium acetate).

	4. Evaporate off any remaining methanol.


	2731 Bartram Rd, Bristol, PA 19007
	tel: (800) 385-3153
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Determination of Ephedra Alkaloids and Synephrine in Dietary Supplements via Strong Cation-Exchange SPE and LC–MS/MS Detection

	Pesticides have gone far in increasing food supply, important in a world expecting to have 9.8 billion people by 2050. However, the mechanisms that make pesticides effective in eliminating pests can also cause harm to humans and animals. News stories continue to point to contamination through spills and exposure through the respiratory system. This work demonstrates the use of SPE disks on wastewater samples using automation to provide control and consistency in all the steps in the extraction process, including conditioning the disk, loading the water sample, rinsing the sample bottle, and eluting the sample in solvent. Since the use of a halogen-specific detector precludes use of a chlorinated solvent for the sample, the method specifies solvent exchange from methylene chloride into hexane before the analysis step. When SPE is used, the extraction and elution steps are separate, so extraction onto the SPE disk is one step and then elution can be considered separately. Since the final extract should be in hexane, it is possible to elute the disk directly into hexane, eliminating the elution with methylene chloride and subsequent solvent exchange step. Both elution with methylene chloride and elution with hexane are demonstrated in the full note.

	The results are very good for a complex matrix such as wastewater influent, shown in the table, meeting the goals in all cases for spike recovery. In the case of trans-chlordane, however, the agreement between the spike and spike duplicate is outside the acceptable range. There was some interference with the chromatography and the spike was cleaned with copper. The spike duplicate appeared cleaner and was not treated with copper. This issue was not observed with synthetic wastewater and was unexpected. The particulate matter in the spike and spike duplicate were different in amount and color. This could have contributed to a matrix effect which influenced the recovery of compounds more in one sample than the other, in spite of the copper treatment.

	Elution with hexane can save time and effort required in solvent exchange required for an ECD detector, improving the workflow in a busy laboratory. Analysis of a medium-sized wastewater treatment plant influent wastewater showed good results using the hexane elution procedure, further demonstrating the ability of automated disk SPE to improve the laboratory workflow while maintaining method performance.

	(1) W.R. Jones and Z.A, Grosser, "Analysis of Wastewater Samples Using Disk Solid Phase Extraction (SPE) Following US EPA Method 608.3, AN1151707_01," available from 


	16 Northwestern Dr., Salem, NH 03079
	tel. (603) 893-3663, fax (603) 893-4994
	Website: 

Environmental

Analysis of Wastewater Samples Using Disk Solid-Phase Extraction (SPE) Following US EPA Method 608.3

Raptor C18 SPP 5 µm core-shell silica particle columns offer excellent resolution for fluorochemicals with short total cycle times. For even faster analysis, 2.7 µm core-shell particles are available.

Unique, robust Raptor C18 column design increases instrument uptime.

	Perfluorinated alkyl acids are man-made fluorochemicals used as surface-active agents in the manufacture of a variety of products, such as firefighting foams, coating additives, textiles, and cleaning products. They have been detected in the environment globally and are used in very large quantities around the world. These fluorochemicals are extremely persistent and resistant to typical environmental degradation processes. As a result, they are widely distributed across the higher trophic levels and are found in soil, air, groundwater, municipal refuse, and landfill leachates. The toxicity, mobility, and bioaccumulation potential of perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), in particular, pose potential adverse effects for the environment and human health.

	Perfluorinated alkyl acid analysis can be challenging because these compounds are chemically different from most other environmental contaminants. They are difficult to quantify because some are more volatile than others, and they also tend to be more hydrophilic and somewhat reactive. In addition, fluorochemicals are present in polytetrafluoroethylene (PTFE) materials, so excluding the use of any PTFE labware throughout the sampling and analytical processes (including HPLC solvent inlet tubing) is essential for accurate analysis. Typically, perfluorinated alkyl acids are analyzed by LC–MS/MS methods, such as EPA Method 537, but long analysis times can significantly limit sample throughput.

	As written, the EPA 537 requires a 27-min cycle per sample, but the method does allow flexibility in the column used as long as there is sufficient resolution for the MS dwell time for all compounds in a specific retention time window. In Figure 1, all target perfluorinated alkyl acids were analyzed on a Raptor C18 column in under 8 min with a total cycle time of 10 min-resulting in an approximately three-fold faster analysis than the EPA method. While this analysis is significantly faster, there is no sacrifice in peak resolution or selectivity, meaning all fluorochemicals are easily identified and they elute as highly symmetrical peaks that can be accurately integrated and quantified by MS/MS. If PFOA and PFOS are the only target fluorochemicals, the analysis can be further optimized, which results in a fast, <2-min separation with a total cycle time of just 4.5 min, as shown in Figure 2.

	Whether labs conducting perfluorinated alkyl acid analysis by LC use longer target analyte lists or focus just on PFOA and PFOS, the excellent peak shapes and separations achieved here result in consistent, accurate quantification with much shorter analysis times. By switching to a Raptor C18 column, labs can process more samples per hour while still meeting fluorochemical method requirements.


	110 Benner Circle, Bellefonte, PA 16823
	tel. (800) 356-1688, fax (814) 353-1309
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Rapid Perfluorinated Alkyl Acid Analysis by LC–MS/MS Increases Sample Throughput

	The continued interest in persistent organic pollutants (POPs), such as polychlorinated dibenzo-p-dioxins (PCDDs), furans (PCDFs), biphenyls (PCBs), and PBDEs, has led to a variety of automated systems for the cleanup of complex sample matrices. This has resulted in development of our fully automated "Power Prep" sample cleanup instead of manual preparative open column chromatography.

	To meet demands for a lower cost method that requires little financial investment, we combined the features of the "PowerPrep" system-accurate, fast, reliable, with short turnaround times, and low background using FMS pre-packaged columns-with a relatively simple semi-automated approach. An important feature of the semi-automated technique is that a minimum amount of solvent is used and no dichloromethane is used. This is important since many laboratories around the world are phasing out this solvent. This manual method is ideal for those laboratories that want high quality sample processing without much financial investment.

		Thermo Trace GC Ultra with Thermo Quantum XLS Triple Quad GC/MS

		The FMS EZPrep123 System operates under vacuum for fast elution

		Samples are loaded onto columns in hexane and the silica column is eluted with hexane

		Carbon and alumina columns are eluted with toluene for collection

		Two fractions are collected: Fraction 1 with PCDD/Fs and coplanary- PCBs and Fraction 2 with mono- and di-ortho PCBs

		Low solvent volume of collected Fractions reduces time required for sample concentration

		No re-use of tubing, syringes, parts, and glassware

		No electronics and mechanical parts to fail

		No service contract or maintenance to worry about

		No repetitive motions and minimal cleaning of reusable parts

	Excellent recoveries are seen with the new semi-automated method using the FMS EZPrep123 System, as can be seen in Tables I and II. Because the system is mostly composed of disposable parts, the risk of cross-contamination is very low. Note that no dichloromethane is used. The system can be set up at no cost and is a cheaper alternative to the fully automated cleanup equipment, and processing times are much shorter than other manual procedures. The certified columns and simple, versatile system guarantee same morning or afternoon POPs analysis.


	580 Pleasant Street, Watertown, MA 02472
	tel. (617) 393-2396, fax: (617) 393-0194
	Website: 

Fast, Low Solvent, DCM-Free, Sample Cleanup for Persistent Organic Pollutants Analysis

This study demonstrates the excellent performance of Markes International's CIA Advantage–Kori–UNITY-xr pre-concentration system for confident GC–MS canister analysis of "air toxics" in humid environments, in accordance with US EPA Method TO-15.

	US EPA Method TO-15 involves sampling of ambient air using canisters followed by thermal desorption pre-concentration, and GC–MS or GC–FID analysis. It is widely used by commercial laboratories for analysis of volatile organic compounds (VOCs) in urban and industrial settings, especially in the USA and Asia. However, traditional canister pre-concentration technologies are challenged by the ever-greater range of analytes and concentrations of interest, and by the range of temperatures and humidities at sampling locations. This study describes the performance of a new analytical system able to handle samples at high humidities, while remaining compliant with Method TO-15.

-xr canister auto-sampler with a Kori-xr™ water condenser and UNITY-xr™ thermal desorber, coupled to a GC–MS system. The entire system uses electrical trap cooling, eliminating the cost and inconvenience associated with liquid cryogen. Full analytical conditions are available in Markes
	International's Application Note 081 (

Figure 1 shows a typical analysis of the 10 ppbv TO-15 standard at 100% relative humidity (RH), and Figure 2 shows extracted-ion chromatograms (EICs) for five components spanning the volatility range. Note the excellent peak shape, especially for lighter VOCs, and the high degree of similarity between the three humidity levels, indicating the effectiveness of the Kori-xr water-removal module.

At all three humidity levels, excellent linearities with respect to sample volume and concentration were obtained-Figure 3 shows examples of the former for the 100% RH sample, for the set of nine compounds spanning the volatility range.

These were calculated on the basis of seven replicate samples at 0.1 ppbv. To comply with Method TO-15, MDLs are required to be ≤0.5 ppbv. Our calculations gave a mean MDL of 14 pptv, with values for 54 out of the 65 compounds being at 20 pptv or below. Values ranged from 4 pptv for dichlorotetrafluoroethane, tetrahydrofuran, 1,1,1-trichloroethane and 1,2,4-trichlorobenzene to 45 pptv for ethanol, with a solitary "outlier" at 95 pptv for propene. These values are all very much lower than the requirement of ≤0.5 ppbv.

Method TO-15 requires that the calculated RSDs for the RRFs for each compound in the calibration table must be less than 30%, with at most two exceptions up to a limit of 40%. Results were well within the requirements of Method TO-15, with values at the 50%, 75%, and 100% RH levels being 7.9%, 9.0%, and 8.5% respectively.

–Kori–UNITY-xr pre-concentration system described allows confident GC–MS canister analysis of "air toxics" in humid environments, in accordance with US EPA Method TO-15. Key features of the results are the excellent chromatographic performance for the analysis of a 65-component TO-15 mix (from propene to naphthalene), at 50%, 75%, and 100% relative humidity. Performance was well within the requirements of Method TO-15, with method detection limits as low as 4 pptv.

	Crucially, this performance has been achieved for even the most volatile of components in the TO-15 mix, due to the ability of the Kori-xr module to efficiently and selectively remove water from humid air streams, without compromising the analysis of very volatile organic compounds or polar species.

For the complete study, including a full table of results, download Markes International's Application Note 081


	Gwaun Elai Medi-Science Campus, Llantrisant, Wales, UK
	tel. +44 (0)1443 230935, 

Innovative Cryogen-Free Ambient Air Monitoring in Compliance with US EPA Method TO-15

Typically, silica-based size-exclusion columns require high concentrations of non-volatile salts to facilitate timely elution, and require dedicated columns for GPC or GFC applications. Addressing these issues, our new size-exclusion chromatography silica-based column utilizes volatile salts making it compatible with MS

, CAD, or ELSD; provides excellent results in aqueous and/or organic mobile phases; and offers the mechanical stability of silica-based columns.

	Size exclusion chromatography is an established method to separate a mixture based on the size of a variety of large molecules, such as proteins and synthetic polymers. Polymer-based columns are commonly used today because of their chemical inertness, but they are mechanically unstable (vulnerable to swelling in certain solvents or compression under high pressure). Silica-based columns have better mechanical durability, but they require high concentrations of non-volatile salts and suffer from exposed silanols.

	Here we highlight a new technology, the Intrada SEC, which utilizes a wide-pore (300 Å), silica-based particle with a diol-substituted stationary phase. This new technology enables excellent resolution in aqueous and/or organic mobile phases (GFC and GPC), the use of volatile salts, and compatibility with MS detection.

	Separations were run using Intrada SEC columns (250 × 4.6 mm, length × ID). Mobile phase conditions in figure inset.

	Figure 1 shows gel filtration chromatography (GFC) of recombinant human serum albumin (rHSA) using an aqueous mobile phase with volatile salts and mass spec detection.

	This chromatogram highlights the high resolution capability that Intrada SEC offers. This is the only silica-based SEC column designed specifically to use mobile phase conditions compatible with MS detection. This dramatically improves the resolution and quantification capabilities of SEC as shown here where the size of the peak of interest was characterized down to the single Dalton.

	Figure 2 shows a synthetic polymer separation with gel permeation chromatography (GPC), using a purely organic mobile phase. This highlights the wide range of mobile phase options for Intrada SEC, in that a single column type is compatible in either GFC or GPC conditions.

	Intrada SEC is truly a breakthrough for silica-diol SEC technology. It delivers outstanding performance in simple aqueous mobile phase with volatile salts making these methods MS

, CAD, or ELSD compatible. Additional benefit of using Intrada SEC is that it provides excellent separation over a wide range of mobile phases, from aqueous to organic, in either GFC or GPC conditions. Finally, Intrada SEC offers the mechanical stability of silica, expanding the range of acceptable solvents without worrying about particle swelling or compression upon high pressures. We feel that this makes this column a much better option over standard polymer-based SEC columns.


	1104 NW Overton St., Portland, OR, 97210
	tel. (888) 456-HPLC, (215) 775-8902, fax (501) 646-3497
	Website: 

Pharmaceutical/Drug Discovery

Intrada SEC: GFC/GPC Silica-Diol SEC Column Designed to Work with Volatile Salts and Is MS-Compatible

Modelling of peptide and protein separations is investigated using commercially available software (ACD/LC Simulator). A model that accounts for the changing structure and retention of proteins with temperature is proposed, delivering similar retention accuracy as found with simulation of small molecule separations.

	Proteins and peptides are analytes of increasing interest within the pharmaceutical industry. While retention modelling has been successfully used for optimization of analytical scale separations of small molecules for approximately 30 years, it has not been expanded to accurate modelling of protein separations.

	In this paper we describe the methods used to create retention models that allow accurate simulation of protein separations in commercially available software.

	Isocratic retention models were used in combination with numerical solutions to calculate retention times for gradient conditions (1).

	Six proteins with MW ~25,000 Da were chromatographed and modelled by reversed phase chromatography (RPC) and ion exchange chromatography (IEC).

	ACD/LC Simulator (2) was used for gradient modelling using the following equations:

	For small molecules, modelling gradient shape and temperature simultaneously has been found to be very effective, however, this was found to be insufficient for proteins and a second order temperature model was applied.

	As heat is applied a protein unfolds and refolds, and it is proposed that these changes lead to changes in protein retention with temperature.

	Combined gradient and temperature models were evaluated in RPC and IEC mode by fitting the models based in nine experiments (3 gradient times x 3 temperatures). Predictions were then made for four additional combinations of gradient time and temperature.

	For a simple linear gradient, the accuracy of prediction was found to be systematic and greater than 98% for retention time (ΔtR <2%) and greater than 80% for peak width (Δw <20%). The impact of 20% peak width error is not significant for optimization purposes, and is similar for proteins and small molecules. Predictions for more complex, multi-step gradients showed similar accuracy.

	RPC and IEC gradient chromatography, at different temperatures, can be modeled with the same accuracy for proteins as for small molecules. A second order temperature model is likely needed due to the unfolding of proteins at higher temperatures. Modeling of other types of separations, including HILIC and HIC as previously described by Snyder and colleagues (1), should also be possible.

High-Performance Gradient Elution: The Practical Application of the Linear-Solvent-Strength Model

	(2) ACD/LC Simulator, Advanced Chemistry Development, Inc., Toronto, ON., Canada, 


	8 King Street East, Suite 107, Toronto, Ontario, Canada M5C 1B5
	tel. 1(800) 304-3988, email 

Retention Modelling of Peptide and Protein Separations

Coordination compounds tend to exhibit poor peak shape when run on traditional stainless steel HPLC columns due to interactions with the metals used in column housings and frits. PEEK-lined columns and frits help minimize these issues by providing an inert environment for these types of analytes. This work highlights improvements in peak shape obtained for two coordination compounds (Hinokitiol and 8-Quinolinol) when run on YMC's new Triart C18 PEEK-lined columns, as compared to a standard stainless steel column.

	Stock solutions of 1 mg/mL Hinokitiol and 8-Quinolinol were made using 50:50: H

O:ACN as the diluent. A standard solution (0.1 mg/mL) of both compounds was subsequently made using the same diluent.

	This application began by first evaluating a separation of Hinokitiol and 8-Quinolinol using a standard stainless steel column. The column used for this part of the experiment was a 150 × 4.6 mm, 120 Å, 3 µm, YMC-Triart C18 column (part number: TA12S03-1546WT). The two coordination compounds exhibited low response and poor peak shape with excessive tailing as seen in Figure 1.

	The analysis was then repeated on a 150 × 4.6mm, 120 Å, 3 µm, YMC-Triart C18 PEEK-lined column (part number: TA12S03-1546PTP). The improved performance can be seen in Figure 2.

	In order to eliminate as many variables as possible, the procedure was run under the same conditions, same day, using the same mobile phases, samples, and instrument. Tabulated results of the different analyses can be seen in Table I.

	In summary, the results indicate that YMC-Triart C18 PEEK-lined columns and frits outperform traditional stainless steel columns when used for analyzing troublesome coordination compounds such as 8-Quinolinol and Hinokitiol. These columns are also useful for other coordination compounds that contain phosphate groups in their structure such as ADP, ATP, and AMP. YMC-Triart PEEK-lined columns are available in all YMC-Triart stationary phases including C18, C18 ExRS, C8, Phenyl, PFP, and Diol-HILIC for extremely polar compounds. In addition to 3 µm and 5 µm particle sizes, these phases are also offered in 1.9 µm for high-throughput UHPLC analysis.


	941 Marcon Blvd., Suite 201, Allentown, PA 18109
	tel. (610) 266-8650, fax (610) 266-8652
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Use of YMC-Triart C18 PEEK-Lined Columns for Improved Peak Shape of Coordination Compounds

	Liposomes are often used as nanocarriers to encapsulate in their cores hydrophobic or highly toxic drugs, and deliver these drugs safely to target tissue. During drug nanocarrier research and development as well as production quality control, it is of great importance to monitor liposome size distributions accurately while also verifying drug encapsulation. FFF-MALS-DLS, consisting of field-flow fractionation (FFF) combined with multi-angle light scattering (MALS) and dynamic light scattering (DLS), is a powerful tool for characterizing the size, concentration and structure of large nanoparticle ensembles.

	Encapsulation might cause changes in liposomal dimensions, but that is not always the case and other effects could cause such changes. Therefore, a more sophisticated analysis is warranted than mere size. Here, we report the analyses of two liposome samples, one empty and one filled with drug, by means of an Eclipse® FFF system followed by a DAWN® MALS detector with embedded WyattQELS™ DLS module. The WyattQELS detector was placed at approximately 143° in order to extend the rh measurement to well over 60 nm. The FFF separation method was optimized with the aid of Wyatt's proprietary FFF simulation software. ASTRA software was used to collect and analyze the light scattering data to determine size and concentration (number density).

	FFF separates particles according to hydrodynamic radius. Thanks to upstream separation, quantitative size distributions by FFF-MALS-DLS provide far more resolution and quantification than does batch (unfractionated) DLS. Online DLS directly measures the hydrodynamic radius rh sequentially for each eluting size fraction, while MALS measures simultaneously the root-mean square radius 

 are plotted against elution time in Figure 1. The results from duplicate runs demonstrate clean separation and excellent reproducibility of the FFF-MALS-DLS method. Figure 1a shows that the 

 values for both empty and filled liposomes are well-overlaid, suggesting that drug loading does not impact hydrodynamic size. However, as shown in Figure 1b, 

 values for these two liposomes do not overlay, which indicates different internal structures.

; ρ is the slope of the linear fit. The values of ρ for these two populations correlate precisely to empty and filled liposomal structures. In addition to size and structure, FFF-MALS can determine quantitative size distributions (number density vs. size) of size-fractionated nanoparticles if the refractive index of the constituent material is known. For lipids this is quite straightforward and Figure 2b provides the quantitative nanoparticle concentration analysis.

	For liposomes or other nanoparticles, FFF-MALS-QELS provides an easily adaptable yet powerful characterization tool to obtain information on particle size, size distribution, particle count, as well as structure-all without making assumptions about the particles or their composition. FFF-MALS-DLS instrumentation is essential for robust drug nanocarrier development and quality control.


	6330 Hollister Avenue, Santa Barbara, CA 93117
	tel. (805) 681-9009, fax (805) 681-0123
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Liposome Size, Concentration, and Structural Characterization by FFF-MA LS-DLS

	Industrial polymer samples may contain fillers, gels/crosslinked material or are additivated with small-particle size carbon black as light stabilizer. In those cases, a filtration step is required prior to a liquid chromatography (LC) analysis to protect the life of the columns, and/or fouling the detector cells.

	There are new instruments that may incorporate an in-line filter system prior to injection with a back flush mode, which can be effective for most standard samples contaminated with small amounts of residues (1). However, when samples contain significant amount of crosslinked material or inorganic residues, an external filtration apparatus is advised, and it is a must when samples contain carbon black of very small-particle size due to the difficulties of removing it with standard filtration elements. Samples produced in batch reactors or pilot plants with reduced catalyst efficiency may also contain significant catalyst residues, which also need to be removed with an external filtration step.

	The most difficult case is the removal of fine carbon black particles in polyolefins, usually analyzed by high temperature gel permeation chromatography (GPC), which is quite often used with a light scattering detector (LS). The presence of very small particles may damage frits or column packing, and the particles capable of passing through the column to the detector cell will result in a noisy baseline when using a LS detector. Thus, an efficient external filtration system is required in the analysis of these types of samples.

	An external filtration instrument capable of handling polymer solutions of up to 180 °C has been designed. It consists of two small modules:

	1) An external dissolution oven (EDO) with a shaking motion for dissolution of polymer samples that require high temperature to dissolve. Samples are handled in vials of 10 and 20 mL volume and up to six samples can be dissolved simultaneously, Figure 1a.

	2) An external filtration system (EFS) with an oven and a vacuum system controlled by a microprocessor to filtrate one solution sample at a time, Figure 1b.

	The filtration element is an aluminum cartridge capable of handling polymer solutions of up to 180 °C. It is filled with an inert, high surface packing and has top and bottom needles. The in-depth filtration cartridge has a clear advantage over a classical surface filtration element in terms of capacity and filtration speed, and it is the only alternative for completely removing fine particles such as carbon black.

	The EFS oven has been designed to maintain at the desired temperature, the vial with the solution to be filtrated, the filtration cartridge, and the filtrated solution collecting via, Figure 1c.

	1) An empty vial, used to collect the filtered solution, is inserted in the bottom part of the EFS oven, Figure 1c.

	2) A new filtration cartridge is placed at the center of the EFS oven and the bottom needle pierces the empty collecting vial.

	3) The vial containing the polymer sample solution (typically with a concentration of 1 or 2 mg/mL) is taken out of the EDO unit at the dissolution temperature, placed in the top part of the EFS oven and then pierced by the top needle of the filtration cartridge element, Figures 1b and 1c.

	4) The start button initiates a pre-established filtration method. The aluminum cartridge is preheated in less than 1 min and the vacuum pump is immediately turned ON to bring the initial polymer solution through the cartridge, and into the empty collecting vial, Figure 1c. Typically, a 20 mL vial is used to dissolve the sample in 10 mL of solvent. Roughly 8 mL of the filtered solution are collected in a 10 mL vial. The remaining 2 mL are kept at the bottom of the dissolution vial.

	The whole process takes less than 3 min and the vial with the filtered solution can then be taken to the analytical equipment (GPC, HPLC, TREF...) for subsequent analysis.

	The design of the filtration process prevents any possible cross-contamination because the polymer solution goes from the initial dissolution vial, through a single-use cartridge, and into the collecting vial, as shown in Figure 1c. The dissolution vial, now almost empty, and the used cartridge are disposed properly, with no instrument cleaning required.

	The filtration process of the most difficult samples can be performed in a short time (less than 3 min). Figure 2a shows the initial solution of a polyolefin containing 40% of carbon black and then the transparent solution obtained after going through the filtration cartridge, with the polymer precipitated to the upper layer when cooled down. Figure 2b shows the vial with the remaining solution volume and the used cartridge for disposal. All types of inorganic particles and gels are easily removed in a short time.

	Two different polyolefins were chosen to investigate if part of the polymer would be retained in the inert packing of the filter cartridge. For that purpose, the two samples were analyzed by GPC and the results of the original samples were compared to those after the solutions were filtered in the EFS.

	Calculated recoveries of the samples filtered are close to 100%, indicating that no irreversible adsorption in the filter packing is taking place.

	A linear low density polyethylene (LLDPE) of MI 1 and density of 0.920, was analyzed by GPC in triplicate, with and without filtration. The molar mass distributions obtained show a perfect match as seen in Figure 3, indicating that neither degradation nor selective fractionation of this polymer happened in the filtration process.

	The level of antioxidant (BHT) in the original solution remained constant after going through the filtration process; thus, ensuring that the same antioxidant level remains in the final analytical process.

	An isotactic homopolymer polypropylene sample of 250,000 molar mass was analyzed by GPC (triplicate analysis), with and without EFS filtration, as in the previous case but at a concentration of 1 mg/mL. The sample preparation incorporated a vial nitrogen purge to prevent thermal degradation in the dissolution step (2).

	The molar mass distributions obtained are presented in Figure 4 and indicate that EFS filtration does not modify significantly the microstructure of the polypropylene samples (3).

	A fast and effective filtration apparatus has been developed for polymer solutions. It is prepared to handle polymers with large content of solids that demand high dissolution temperatures.

	The filtration process is automated and proceeds in a closed environment in less than 3 min. The user is not required to handle solvents, and the single-use cartridge design prevents any possible cross-contamination between samples.

	The most difficult samples, such as polyolefins with a high content of carbon black, are easily filtered, resulting in a transparent solution.

	No selective precipitation or fractionation of the sample takes place and the dissolved additives like BHT are not removed in the filtration process.

	(1) B. Monrabal and W. Yau, "Engineering advances in High Temperature GPC Instrumentation," The Column, 

Polyolefin Characterization: Recent Advances in Separation Techniques,

203–251, Springer Berlin Heidelberg, (2013).

	(3) T. González and B. Monrabal, to be presented at ICPC 2018, Houston, Texas.


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Polymer

Sample Preparation in Liquid Chromatography Filtration of Polymer Solutions and Carbon Black Removal Prior to a GPC Analysis

	"Click" chemistry is a class of efficient and selective reactions that is characterized by quantitative yields, tolerance to a broad range of functional groups, facile experimental set-up, and minimal synthetic work-up. This class of reactions combines particularly well with controlled radical polymerization methods, such as atom transfer radical polymerization (ATRP), and the two techniques have been prolifically employed for the synthesis of a wide variety of novel polymeric materials, including (multi) block co-polymers, stars, brushes, and gels. This note describes the analysis of multisegmented block copolymers prepared by click-coupling of block copolymers synthesized by ATRP.

	A triblock copolymer consisting of polystyrene (PS) and poly(ethylene oxide) (PEO) was synthesized via ATRP of styrene from a difunctional PEO macroinitiator. The bromine end groups were substituted with azide by reaction with NaN

 in DMF, and the material (N3-PS-PEO-PS-N3) was coupled with propargyl ether to form a multisegmented block copolymer ((PS-PEO- PS)x).

	Due to the presence of multiple blocks within a single polymer chain, conventional size-exclusion chromatography (SEC) could not be used to accurately analyze the molecular weight of the starting materials or products. A triple detector system was employed in conjunction with SEC (TD-SEC), utilizing the following TD-SEC demonstrated that the click coupling was successful (Figure 2), and a product of higher molecular weight (

 = 33,100 g/mol) was obtained with a broader molecular weight distribution.

	A combination of click chemistry with ATRP was utilized to synthesize multisegmented block copolymers. Absolute molecular weight characterization via TD-SEC demonstrated a degree of polymerization of 5–7, which includes up to 21 separate blocks in a single polymer chain.

	TD-SEC analysis combining multi-angle light scattering, differential viscometry and differential refractometry provides additional characterization capabilities not mentioned here, including the determination of Mark-Houwink-Sakurada coefficients as well as conformation and branching ratio.

Analyzing Multisegmented Block Copolymers by SEC-MALS-IV

The unique selectivity, pH, and temperature stability of the ZirChrom®-CARB phase allows baseline resolution of acrylamide and acrylic acid using a 4-min gradient method.

	Small polar molecules such as acrylamide and acrylic acid (see Figure 1) can be difficult if not impossible to separate on conventional reversed-phase bonded silica type phases using HPLC. Acrylamide historically has been analyzed using sample derivitization coupled with gas chromatography (1). The distinctive selectivity and increased retentiveness of the ZirChrom®-CARB phase, compared to bonded silica or polymeric phases, enables the baseline resolution of these small polar molecules.

	A mixture of acrylamide monomer and acrylic acid was separated using ZirChrom®-CARB.

	This method is an excellent example of how the enlarged zirconia

	method development "tool box" can be used to overcome the toughest separation challenges. Using temperature, extreme pH, and the unique surface chemistry of ZirChrom®-CARB a fast separation of acrylamide and acrylic acid was achieved using a 4-min gradient (see Figure 2).

	This method can be tailored to your specific application needs. ZirChrom method developers can help you optimize and transfer this method to your site. Please contact ZirChrom technical support at 1-866-STABLE1 or 

	ZirChrom phases offer unique selectivity for ionic compounds, high efficiency, and excellent chemical and thermal stability.

	(1) E. Tareke et al., "Acrylamide: A Cooking Carcinogen?" 


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The Application Notebook-09-02-2017