The analysis of the amorphous fraction is a fundamental task in the production of polypropylene (PP). It provides a measurement of the small amounts of undesirable low tacticity and low molar mass fractions in homopolymer resins, which have significant influence on the polymer properties and processing. The incorporation of amorphous material is usually done within the same production plant at a second stage of the polymerization process by adding ethylene to the continuous reaction, resulting in the formation of amorphous ethylene-propylene copolymer. The continuous measurement of the overall amount of this rubbery fraction is essential for plant control in order to obtain the desired specifications and performance of the polymer resin. 

Historically, the amorphous or soluble content has been measured by solubility in xylene using manual wet chemistry methods that are time-consuming and involve large amounts of solvent. CRYSTEX QC is an innovative approach that automates the analysis of PP samples in quality control (QC) plant or processing laboratories. In addition, CRYSTEX QC quantifies ethylene incorporation and intrinsic viscosity in the original sample and in both the amorphous and crystalline fractions. 

This traditional method is based on a crystallization process in which the crystalline matrix is segregated from the amorphous or soluble fraction by cooling a xylene solution according to a specified method with fixed crystallization temperature and time (1–2). The process is described as follows: 

"A weighed amount of polymer is dissolved in hot xylene, then cooled under controlled conditions down to 25 °C, which results in the precipitation of the insoluble fraction. The soluble matter remains in the xylene. The suspension is filtered and an aliquot of the solution is then evaporated, dried, and the residue weighed. The ratio of the corresponding weights is the basis for the final calculation of the soluble fraction."

This procedure has been extensively used in the industry and has proven to be of great value to the manufacturing of PP. However, the determination of the xylene solubles with this approach demands significant manpower, solvent consumption, and an analysis time of around 5–6 h. The ethylene content of the soluble fraction is measured separately by FTIR on a pressed film; and measurement of intrinsic viscosity demands re-dissolution of the dried fraction and injection into a viscometer.   

CRYSTEX QC is a fully automated instrument that quantifies the amorphous fraction while delivering improvements in time, safety and precision. The instrument is based on a proprietary Temperature Rising Elution Fractionation (TREF) column where a small aliquot of homogeneous polymer solution is crystallized, in di- or tri-chlorobenzene, on a support under reproducible and well-controlled conditions, therefore, delivering very reproducible results. Solvent selection is not so important as the intention to separate two different polymer phases quite apart in crystallinity (3) and it has been proven by previous CRYSTEX methodology (4–5) 

Figure 1: CRYSTEX QC for process and QC laboratories of polypropylene manufacturing plants.			

The amorphous fraction is measured with a sensitive infrared (IR) detector that delivers equivalent values to the xylene solubles measurement but obtained with outstanding precision. The IR detector also measures the concentration of the amorphous and crystalline fractions, as well as their ethylene incorporation. The integration of a capillary viscometer provides an automated measurement of intrinsic viscosity of the whole sample and of both the amorphous and crystalline fractions.

Figure 2: Schematic diagram of CRYSTEX QC.

The schematics of the system are presented in Figure 2. All the analyst is required to do is put an approximate amount of sample in a disposable bottle, place it in the stirred-heated plate, and lower a handle to pierce the septum of the bottle with a needle. The automated process is as follows: 

 a) Filling the disposable bottle with 100 mL of solvent preheated at 160 °C. Initiate stirring in the heated plate. 

b) An aliquot of the solution is pumped through the TREF column at 160 °C into the detectors to measure concentration, content of ethylene, and intrinsic viscosity of the whole sample (first eluted peak in Figure 3).  

c) A new aliquot of the solution is injected into the middle of the column at 160 °C, the flow is stopped, and the column temperature is reduced down to controlled ambient temperature to precipitate the crystalline fraction. The column is then flushed to elute the soluble fraction (second peak in Figure 3). The ratio of this peak area by the one of the whole sample provides the soluble percentage in the sample. As the soluble material passes through the IR and viscometer detectors, ethylene content and intrinsic viscosity of this fraction are also measured.  

d) Temperature is raised rapidly up to 160 °C with stop flow to re-dissolve the crystalline fraction and after a pre-set time, this fraction is eluted through both IR and viscometer detectors. 

e) The column is rinsed, and the instrument is ready for a new sample analysis. 

Figure 3: Elution of the whole sample and PP fractions in a TREF column.

Reproducibility is remarkable for the soluble fraction determination. Table 1 shows data obtained from seven replicate analyses using 4 g of sample for a set of three PP products. Average standard deviations are shown for each type of measurement. 

Table 1: Soluble fraction, ethylene content, and intrinsic viscosity of three different PP products. 			

No additional experimental effort is required since the data are collected by the IR and viscometer detectors during the automated analysis and a measurement of the ethylene content and intrinsic viscosity of all polymer parts is provided. 

A new method that measures the soluble fraction in PP resins has been developed based on a TREF crystallization approach. The analytical process is fully-automated with no need for weighing, filtration, or solvent handling. 

The total time of analysis is 2 h, and samples are analyzed one at a time in disposable bottles. Total volume consumption is as low as 180 mL for a 2 g sample analysis. Analysis of 4 g of resin in the form of powder can be done for enhanced sample representativeness. 

In addition to the soluble fraction, the new system measures ethylene content and intrinsic viscosity simultaneously in both amorphous and crystalline fractions, and in the whole sample. 

(2) ISO standard 6427, (1992), ISO standard 16152, (2005).

 (John Wiley & Sons, 2000) pp. 8074–8094.   

(4) L. Romero, B. Monrabal, and A. Ortín, Pittcon 2001 USA. 

(5) A. Ortín, B. Monrabal, and M. D. Romero, 

Gustave Eiffel 8, Paterna, Valencia E-46980 Spain

Tel: +34 96 131 81 20  fax +34 96 131 81 22

Articles

Polymer

Soluble Fraction Analysis in Polypropylene for Quality Control Laboratories

Peptide mapping by reversed-phase chromatography is the mainstay technique in biotherapeutic analysis, delivering the comprehensive characterization of biopharmaceutical products. When interfaced with a mass spectrometer (MS), it can deliver the identification of proteins and its variants, determine post-translational modifications (PTMs) and locations, and confirm protein sequences. 

However, peptide mapping represents a significant chromatographic challenge because of the inherent complexity of protein digests. As a result, many organizations struggle with developing robust and reliable peptide maps. In general, their peptide maps have suffered from low sensitivity, poor peak shapes, and very long separation times to achieve the desired resolution. 

The new 2.7 um AdvanceBio Peptide Mapping column has been introduced to fulfill a critical segment in biotherapeutic characterizations, for generating both rapid and highly efficient peptide maps at low liquid chromatography (LC) system pressures. Using superficially porous chromatographic media, the AdvanceBio Peptide Mapping columns achieve substantial improvements in peptide mapping during very fast run times, while still maintaining high efficiency peak performance. 

Figure 1: Fast HPLC optimizations of mAb tryptic digest. Mobile phase A: water (0.1% FA) Mobile phase B: 90% acetonitrile (0.1% FA), Injection: 15 ÂµL, Temperature: 40 Â°C, DAD: 215 nm (a): 75 min. separation on a 2.1 Ã 150 mm AdvanceBio Peptide Mapping column generating 59 peptide peaks.  (b): 14 min. separation on a 2.1 Ã 100 mm AdvanceBio Peptide Mapping column generating 57 peptide peaks.			

Rapid mAb Tryptic Digest Peptide Mapping by HPLC–UV 

To highlight this performance, the AdvanceBio Peptide Mapping column was used for the rapid LC–MS analysis of a monoclonal antibody (mAb) tryptic digest. Traditionally, shallow gradients of 2 h  or longer are employed for mapping mAb peptide maps to achieve the desired baseline performance. The longer run times ensure that optimum resolution is achieved and that critical PTM peak information is not sacrificed during the shorter run. However, the AdvanceBio Peptide Mapping column has the flexibility to increase analysis speed without sacrificing separation performance. The UV–LC AdvanceBio column separations in Figure 1 demonstrate this flexibility. The top chromatogram provides an example of a more common peptide map generated during 75 min on a 2.1 × 150 mm column at a standard flow rate (relative to column internal diameter) of 0.2 mL/min. The separation provides excellent baseline resolution with 59 mAb tryptic digest peaks resolved over the entire gradient profile. In the bottom chromatogram comparison, a 2.1 × 100 mm AdvanceBio column was used with a very steep gradient at a high flow rate of 0.6 mL/min but at a fraction of the runtime in under 14 min. In this separation, the mAb tryptic peak count has remained relatively unchanged (57 peaks) while baseline resolution, peak shapes, sensitivity, and selectivity have not been compromised. In addition, the column back pressure remained well under 500 bar making this rapid and highly efficient separation achievable on traditional high perfomance liquid chromatography (HPLC) instrumentation. 

Delivering UHPLC-like Performance Without the High Back Pressure Constraints

Peptide mapping by ultrahigh-pressure liquid chromatography (UHPLC) can provide the basis for a more detailed characterization for protein biotherapeutics taking advantage of smaller particles packed in shorter column lengths to achieve high efficiency maps during faster run times. However, the pressure requirement for these separations becomes increasingly high, thereby preventing operation on traditional 400 and 600 bar HPLC instruments and severely limiting its broader application. The new 2.7 um AdvanceBio Peptide Mapping column can generate UHPLC-like mapping performance while providing the flexibility to use with UHPLC or HPLC instrumentation. 

Figure 2: (a) AdvanceBio Peptide Mapping column and (b) UHPLC peptide mapping column total ion chromatograms (TIC) for a mAb tryptic digest.			

Figure 2 displays the reversed-phase LC–MS separation performance between an AdvanceBio Peptide Mapping column and a competitor UHPLC peptide mapping column. The total ion chromatogram (TIC) comparisons clearly detail very comparable resolving performance and indicate the ability of the AdvanceBio peptide columns to adequately and efficiently resolve a mAb tryptic digest to deliver comparable performance to the UHPLC–MS separation at HPLC traditional pressure (433 bar). 

Figure 3: Extracted compound chromatograms (ECC) for a mAb tryptic digest. (a) AdvanceBio Peptide Mapping column, 433 bar yielding 99.63% sequence coverage. (b) UHPLC peptide mapping column, 700 bar yielding 99.0% sequence coverage.			

In the extracted compound chromatogram (ECC) (Figure 3) the AdvanceBio Peptide Mapping HPLC–MS analysis delivered excellent sequence coverage performance in comparison to the UHPLC–MS column analysis. Sequence coverage for the AdvanceBio column was 99.63%, while the UHPLC column delivered 99.0%. The flexibility to obtain UHPLC-like results on the AdvanceBio peptide mapping column, without the pressure constraints of UHPLC, can make this column a more attractive and universal choice for rapid and efficient characterization of biotherapeutics. 

The AdvanceBio Peptide Mapping column reduced the analysis time for reversed-phase LC–MS peptide mapping of a monoclonal antibody (mAb) tryptic digest. In combination with optimized short gradient conditions, this application describes methods for generating rapid (14 min) and highly efficient mAb peptide maps requiring only low LC pressure operation (<450 bar). Alternatively, a UHPLC peptide mapping column was compared for rapid mapping performance with the AdvanceBio Peptide Mapping column. In this evaluation, the AdvanceBio Peptide Mapping column delivered well-resolved peptide peaks across the entire gradient profile comparatively and resulted in high sequence coverage of the mAb. However, the AdvanceBio Peptide Mapping separations were generated at low system pressures, highlighting important flexibility for operating on either HPLC or UHPLC instrumentation. 

2850 Centerville Road, Wilmington, Delaware 19808, USA

Pharmaceutical/Drug Discovery

Increasing Speed for Biocharacterizations: Peptide Mapping ofMonoclonal Antibodies with an Agilent AdvanceBio Peptide Mapping Column

According to the recent IPCC report (1), climate change as a result of global warming is estimated to proceed at a faster rate than previously assumed. Consequently, easy and efficient monitoring of the greenhouse gases methane (CH

O) is becoming an increasingly important issue. The analytical challenges that surround this approach are the varying concentration ranges of the components in ambient air, ranging from high ppm levels for carbon dioxide, low ppm to ppb levels for methane, and trace levels below 100 ppb for nitrous oxide. So far, a rather complex GC system including two or three detectors was needed to fulfill all requirements. With the new Barrier Ionization Discharge Detector (BID), this approach can be simplified to a gas chromatography (GC) system with one detector and one column only.  

The BID detector belongs to the family of helium ionization detectors. It is a universal detector able to measure all types of organic and inorganic compounds except for helium and neon. Because of the novel plasma technology, the BID detector combines the high detection sensitivity of the helium ionization technique with robustness and long-term stability.  

Gas injection was carried out using a two-position six-port VALCO valve with a 1 mL sample loop. Chromatographic separation was performed on a 2 m Restek ShinCarbon ST micropacked column with a 1 mm inner diameter and 1/16" outer diameter. The column was selected because of its sample capacity, and therefore its ability to obtain sufficient separation of methane from the ambient air matrix peak, even with a 1 mL injection volume. Figure 1 shows the chromatogram of an ambient air sample taken in the vicinity of a larger city. This is why carbon monoxide is also present in the chromatogram. 

Figure 1: Ambient air chromatogram sampled at the southern edge of the city of Duisburg (Germany). Results are shown in Table 1. 			

The separation of matrix air requires a temperature programme starting at 30 °C, then hold for 5 min, and a temperature increase at a rate of 10 °C/min to 120 °C. Helium with a purity of 6.0 or better was used as carrier gas and discharge gas for the BID detector. The carrier gas flow over the ShinCarbon ST column was 14 mL/min. The BID was operated at 250 °C with a discharge gas flow of 80 mL/min. 

Table 1: Quantitation results from the chromatogram in Figure 1. 			

Quantitation was based on a 500 ppb gas standard of methane, carbon dioxide, and nitrous oxide in helium (Figure 2). Despite the low concentration, good reproducibility results of RSD 1.1% for methane, 1.4% for carbon dioxide, and 1.1% for nitrous oxide were obtained. The achieved detection limits for the respective compounds are shown in Table 2 and calculated at a signal-to-noise ratio of 3.3. 

Figure 2: Standard chromatogram of 500 ppb methane, carbon dioxide, and nitrous oxide in helium. 			

The results show that detection limits below 100 ppb can be easily obtained using the BID detector. With the ShinCarbon ST, sufficient separation of methane from matrix air is achieved. If carbon monoxide is present in the sample, it can be separated from methane as well (resolution R = 0.92). More important is the good separation of carbon dioxide, even in high concentrations, from trace concentration of nitrous oxide (resolution R>4). In conclusion, GC systems with BID detectors offer a new and easy solution for greenhouse gas trace measurements with a simple one-column, one-detector configuration. 

Table 2: Detection limits calculated at a signal-to-noise ratio of 3.3. 			

(1) 5th IPCC Report Final Draft (accepted): 

http://www.climatechange2013.org/report/review-drafts/

				Albert-Hahn-Str. 6–10, D-47269 Duisburg, Germany				

				Tel: +49 203 76 87 0  Fax +49 203 76 66 25 

Environmental

Trace Analysis of Greenhouse Gases Using a Barrier Ionization Discharge Detector

2-methylimidazole (2-MeI) and 4-methylimidazole (4-MeI) are by-products from the manufacture of caramel colour additives used in beverages, soy sauces, baked foods, and so on. The International Agency of Research of Cancer classified these two compounds as "possibly carcinogenic to humans", and proposed the no significant risk level (NSRL) to be 29 µg/day for 4-MeI, while California listed 4-MeI as a probable carcinogen and proposed a 16 µg/day NSRL. This application note presents a simple, fast, and cost-effective method to determine 2-MeI and 4-MeI in beverages simultaneously. Beverage samples were degassed, diluted 10 times with acetonitrile (MeCN), and filtered through a solid-phase extraction (SPE) cartridge with 200 mg FASt sorbent, onto which the undesired matrix components, such as sugars and organic acids were retained, while the analytes were passed through and collected. A new HILIC HPLC method was developed to separate the 2-MeI and 4-MeI isomers, which underwent the same MS–MS transitions. Baseline separation was achieved in an 8-min run. 

Pour the beverage samples into 500-mL beakers, and degas the samples by stirring at high speed for 2 h. 

a. Transfer 0.1 mL of the degassed samples into test tubes or vials. Add 0.9 mL of MeCN and 100 ng/mL imidazole as internal standard (IS). Vortex for 10 s.  

b. Attach the FASt cartridges (CSFAS203) to a glass block or positive pressure manifold, insert test tubes or 2-mL auto-sampler vials into the manifold. 

c. Transfer the entire diluted samples into the cartridges, apply low vacuum or positive pressure, and collect the filtrates.  

d. The samples are ready for LC–MS–MS analysis. 

: Thermo Scientific Ultimate 3000®  LC coupled to Vantage MS/MS system 

: Thermo Scientific Accucore HILIC, 100 × 2.1 mm (2.6 µm), with 10 mm guard column  

: 8-min isocratic with 5% of 50 mM ammonium formate in water and 95% of MeCN  

Retention times and MS–MS transitions: 

a. IS: 1.96 min; 69.07→42.01; 69.07→28.08 

b. 4-MeI: 3.18 min; 83.08→56.05; 83.08→42.00  

c. 2-MeI: 5.72 min; 83.07→42.04; 83.07→56.05  

This efficient and reliable FASt procedure has been applied to the analysis of 12 real samples, including one colourless naturallyflavoured soda, one root beer, one sweet tea, and nine colas of the same brand obtained from nine different states in the USA (AZ, CO, LA, MD, MS, OR, TN, TX, and WA). 2-MeI was not detected in any of the samples, while 4-MeI was detected in 11 of the samples. The only exception was the colourless soda, which was therfore used to prepare matrix matched calibration standards and spiked samples. The nine cola samples from the different states were found to contain 4-MeI at varying levels; the lowest result of about 100 ng/mL was detected in the samples from WA and OR, while the highest of 840 ng/mL was from AZ. 

 Table 1: Accuracy and precision data.			

A simple, fast, and cost-effective method has been developed for the simultaneous quantification of 2-MeI and 4-MeI in beverage samples utilizing a filter and shoot procedure. Excellent recovery (103.6% and 102.9% for 4-MeI and 2-MeI, respectively) with RSD less than 5% was obtained. An 8-min HILIC HPLC method was successfully developed for the baseline separation of the 2-MeI and 4-MeI isomers with identical MS–MS transitions. 

					2731 Bartram Road, Bristol, Pennsylvania19007, USA  				

Food and Beverage

Simultaneous Quantification of 2- and 4-Methylimidazoles in Beverages Using a Simple Filter and Shoot (FASt) Procedure

The presence of polymerized triacylglycerides or chlorophyll degradation products in olive oil indicates that the oil has been refined or subject to other thermal treatment processes. In this study, several olive oils were analyzed using the Agilent 1260 Infinity LC with refractive index and fluorescence detection.

Virgin olive oil can be created only by mild, cold pressing of olives (

). In addition to the determination of stigmastadienes (1), the quantification of polymerized triacylglycerides and chlorophyll degradation products in olive oil is another important factor to determine whether thermal treatment was used. 

Table 1: Polymerized triacylglycerides. 			

During different refining steps, the oil components are chemically altered (2). Polymerized triacylglycerides are normally not present in crude vegetable oils. During the extraction processes, the chlorophyll a and b pigments are converted into the more stable pheophytins (3), which in turn undergoes slow decarboxymethylation to give pyropheophytin. 

Table 2: Chlorophyll degradation products. 			

In contrast to virgin olive oil, the partly refined olive oil chromatogram showed dimerized triacylglycerides (Figure 1a). In addition, a higher percentage of chlorophyll a degradation products are found in partly refined olive oils (Figure 1b). 

Figure 1: Detection of (a) polymerized triacylglycerides and (b) chlorophyll degradation products in partly refined olive oil. 			

 were detected in all six virgin olive oils. In addition, in fresh virgin olive oils, only a minor amount of pyropheophytin was detectable, but as it develops slowly during storage, higher amounts were detected in several longer stored virgin olive oils as well as in the partly refined olive oil.   

(1) Quality Analysis of Virgin Olive Oils – Part 1, Agilent Application Note, Publication Number 5991-1894EN, 2013. 

European Journal of Lipid Science and Technology

				5301 Stevens Creek Blvd., Santa Clara, California 95051, USA 			

Thermal Treatment Analysis — Determination of Polymerized Triglycerides and Chlorophyll Degradation Products

Acrylamide has been found to occur in many cooked carbohydrate-rich foods and is of concern as a possible carcinogen. The supported liquid extraction (SLE) method described in this application note achieves high recoveries of acrylamide in fried potato chips (crisps). The method is sensitive enough to potentially measure levels as low as 10 ppb in a popular brand and flavour and has also been tested in flavoured varieties that were both machine and hand fried.

 ISOLUTE® SLE+ 1 mL Sample Volume Columns, part number 820-0140-C 

 Add 10 mL water to 1 g crushed sample (previously spiked with 

 acrylamide internal standard), and mix for 1 h. Centrifuge and remove a 0.65 mL aliquot of the aqueous layer, taking care not to include any of the thin upper oil layer. 

 Load the pre-treated sample (0.65 mL) onto the ISOLUTE SLE+ column. Apply a pulse of vacuum or positive pressure to initiate flow. Allow the sample to absorb for 5 min. 

 Elute with ethyl acetate–tetrahydrofuran, (1:1, v/v, 2 × 2.5 mL) and allow to flow under gravity into tubes containing 2 µL ethylene glycol. Apply vacuum or positive pressure to elute any remaining extraction solvent. 

 Dry the volatile constituents of the eluate in a stream of air or nitrogen. Reconstitute in water (200 µL).  

 Phenomenex Hydro 4 µm 50 mm × 2 mm C18 column with a C18 guard cartridge and on-line filter 

 Initial 100% A, hold for 0.6 min; Linear ramp to 100% B over 0.25 min (0.85 min), hold 1.65 min (2.5 min); Linear ramp to 100% A in 0.01 min (2.51 min), hold 2.49 min (5 min) 

Table 1: Positive ion mode â MRM parameters. 			

Ions were selected on the basis of achieving maximum sensitivity using multiple reaction monitoring. 

Figure 1: Chromatogram of untreated potato chip (crisp) extract showing level of incurred acrylamide. 			

A method has been developed which measures acrylamide from a challenging matrix at highly sensitive levels. Despite acrylamide being a relatively polar molecule, excellent separation was demonstrated between this and matrix interferences on the ISOLUTE SLE+ material. The method delivers good recovery (90%), low %RSD (<10%), and is significantly easier, quicker, and more cost-effective to perform than SPE-based procedures, making it ideal for routine analysis of fried and baked foods. 

Tel: +46 18 56 59 00  fax: +46 18 59 19 22 

The Application Notebook-12-02-2013

Soluble Fraction Analysis in Polypropylene for Quality Control Laboratories

Increasing Speed for Biocharacterizations: Peptide Mapping ofMonoclonal Antibodies with an Agilent AdvanceBio Peptide Mapping Column

Trace Analysis of Greenhouse Gases Using a Barrier Ionization Discharge Detector

Simultaneous Quantification of 2- and 4-Methylimidazoles in Beverages Using a Simple Filter and Shoot (FASt) Procedure

Thermal Treatment Analysis — Determination of Polymerized Triglycerides and Chlorophyll Degradation Products

Extraction of Acrylamide from Fried Potato Chips (Crisps) Using ISOLUTE® SLE+ Prior to LC–MS–MS Analysis