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Calculation of Whey Protein Fraction in Milk-Based Infant Formula

A method for the calculation of whey protein fraction in milk-based formula products was developed and validated by a group of researchers from Abbott Nutrition, Covance Laboratories and AOAC International. The method is based on calculating the ratio of (Asx + Ala)/(Pro+Phe) determined from the amino acid profile of the hydrolyzed sample.

The method was approved as AOAC Official First Action Method 2012.07 and is appropriate for accessing compliance with whey content requirements of GB 10765-2010 (National Food Safety Standard Infant Formula)*. It is applicable to finished products containing both intact and partially hydrolyzed whey proteins.

According to this method, the Amino Acid Profile of hydrolyzed samples is determined using cation-exchange chromatography with post-column derivatization and UV/Vis detection. The researchers used Pickering Laboratories post-column derivatization system, our patented Trione Ninhydrin reagent and Pickering Sodium analytical column and buffers.

The method performance was evaluated using non-fat dry milk and products with whey levels from 32% to 63%, including NIST Infant Formula Standard Reference Material. The repeatability ranged between 0.3 and 2.5 %, while intermediate precision were between 2.6 and 3.4%. Average recoveries ranged from 97 to 100%.

The unmatched accuracy and precision of amino acid analysis by HPLC with post-column derivatization allowed the researches to develop a robust mathematical model for calculating whey protein fraction that can be successfully applied to a wide range of products. This proved once again that post-column derivatization analysis of amino acids is still a method of choice for laboratories that require highly accurate results.

Pickering Laboratories offers a total solution for amino acids analysis, including post-column derivatization systems, reagents, columns, buffers and standards.

The reference to the published study is below. The First Action Official Method 2012.07 can also be found online at http://www.eoma.aoac.org/.

Calculation of Whey Protein Fraction in Milk-Based Infant Formula: First Action 2012.07

Authors: Wesley Jacobs, Paul Johns, Phillip Haselberger, Joseph J. Thompson, Darryl Sullivan, Steve Baugh.

Source: Journal of AOAC International, Volume 96, No. 3, 2013, pp. 502-507

 

(*) this is a Chinese Standard

Amino Acid Analysis of Cell Culture Media

We have a new Method Abstract! MA371, Amino Acid Analysis of Cell Culture Media

Cell cultures are widely used to produce biopharmaceuticals and other biologically active compounds. The composition of the cell culture media affects the yield and structure of the desired products and must be carefully optimized. Cell culture media is typically composed of mixtures of amino acids, vitamins, carbohydrates, inorganic salts as well as different peptides, proteins and other compounds. As the cells grow, they consume nutrients and release target biopharmaceuticals as well as waste products.

Amino Acids serve as the building blocks of proteins, as well as intermediates in many metabolic pathways. Amino Acids are typically added to cell culture media to provide nutritional requirements for the cells. Monitoring and adjusting Amino Acid composition is an essential part of optimizing the manufacturing process to ensure high quality and optimum yield of the final product.

Amino Acid Analysis using cation-exchange chromatography with post-column Ninhydrin derivatization allows for easy determination of Amino Acid concentrations in many complex matrices, including cell culture media. The post-column method is very sensitive, reproducible and rugged. It has been and continues to be a method of choice for laboratories running biological samples, protein, peptides and foods analysis. Most chemical compounds present in the media do not interfere with analysis, so the majority of samples only need diluting with citric buffer and filtering before analysis. If serum is added to the media, then the proteins need to be precipitated using either Seraprep™ solution or ultrafiltration.

Pickering Laboratories, Inc. offers the complete solution for Amino Acid Analysis, including post-column derivatization instruments, columns, eluants, reagents and standards. The Pinnacle PCX derivatization system has a programmable column oven to allow for shorter run times and easy method optimization.

YChromatograms of Cell Culture Mediaou can download this application note, and many others from our website: www.pickeringlabs.com

METHOD
Analytical conditions
Column: High-efficiency Lithium cation-exchange column, 4.6 x 75 mm, Catalog Number 0354675T
Flow Rate: 0.55 mL/min
Mobile Phase: See method in Table 1

Post-Column Conditions
Post-column System: Pinnacle PCX
Reactor Volume: 0.5 mL
Reactor Temperature: 130 °C
Flow Rate: 0.3 mL/min
Detection: UV/VIS 570 nm for primary amino acids, 440 nm for secondary amino acids
Injection Volume: 10-50 uL

gradient table for AAA

Polyether Antibiotics in Animal Feed

Our updated Method Abstract 264 describes the analysis of Monensin, Salinomycin, Narasin, & Lasalocid in Animal Feeds.

Polyether Antibiotics are commonly used for preventing coccidiosis and other infections in poultry and for improving feed efficiency for beef cattle and swine. The use of Polyether Antibiotics is strictly regulated, with only specific ionophores approved for use in feeds intended for different animals.

Analysis of Polyether Antibiotics by HPLC with post-column derivatization and UV/Vis detection has been proven to successfully identify and quantify Monensin, Narasin and Salinomycin in medicated feeds, supplements and premixes as well as to determine trace contamination levels in non-medicated feeds [1, 2].

Post-column derivatization of Polyether Antibiotics is done using highly acidic Vanillin or DMAB reagents. The Pinnacle PCX derivatization system (Pickering Laboratories, Inc.) has an inert flow path and automated system wash capabilities that make it uniquely suitable for handling corrosive reagents. The two-pump system is recommended to extend reagent stability, but the single-pump system for this application is also available.

Adding a Fluorescence detector to the instrumentation allows for using the same extraction procedure and HPLC conditions to also determine Lasalocid, which doesn’t require post-column derivatization.

Chromatograms of Polyether AntibioticsMETHOD
Sample Preparation
To 25 g of finely ground feed sample, add 100 mL of extraction solution (90% Methanol – 10% water). Shake for 1 hour at high speed using a mechanical shaker. Let the solids settle and filter an aliquot of the extract for injection. Dilute with extraction solution if needed to fit the calibration curve. Use a 2.5 g portion when testing premixes.

Analytical conditions
Analytical Column: Polyether Column, C18, 4.6 x 250 mm, Catalog No 2381750
Temperature: 40 ºC
Flow Rate: 0.7 mL/min
Mobile Phase: 90% Methanol, 10% of 5% Acetic Acid solution in water, isocratic
Injection volume: 20 μL

Post-Column Conditions
Post-column System: Pinnacle PCX
Reactor Volume: 1.4 mL
Reactor Temperature: 90 °C
Reagent 1: Concentrated Sulfuric acid / Methanol (4:96 v/v)
Reagent 2: 60 g of Vanillin in 950 mL of Methanol
Reagent Flow Rate: 0.3 mL/min
Detection: UV/VIS 570 nm (for Lasalocid – FLD, Ex. 322 nm, Em. 370 nm)

Conclusion
Analysis of Polyether Antibiotics by HPLC with Post-column derivatization is a robust and sensitive method that utilizes standard equipment and could easily be adopted by testing laboratories. It allows for testing of different ionophores at wide range of concentrations,
including at trace levels. Using Pinnacle PCX post-column derivatization system, factory configured for the analysis, guarantees stable and reproducible results.

The full application note as well as our complete Product Catalog can be downloaded from our website: www.pickeringlabs.com

table for polyether antibiotics

NEMC 2013

Pickering Laboratories participated in the National Environmental Monitoring Conference in San Antonio, Texas from August 4-8.

niosita low res
Old San Antonio

Rebecca and Wendy were on-hand to share Pickering’s products with the environmental laboratories involved with the conference.  On display were the new FREESTYLE sample clean-up system and the Pinnacle PCX.

Some highlights of the conference included a new product by InnovaPrep, which will rapidly concentrate pathogens in liquid samples, and the 2-D GCMS analysis of POPs in human milk. There were also several interesting discussions about pesticide extraction and analysis as well as the use of Hydrogen as a carrier gas in GC (instead of Helium, for which there is currently a shortage).

The meeting was held at the Hyatt in downtown San Antonio on the Riverwalk. This area of town is full of history and interesting architecture. We were also located directly across the plaza from the Alamo.

alamo low res
Remember the Alamo

2013 Pickering Laboratories North American Catalog

Our latest Catalog for North America is now available!

Click on the link to download the latest product Catalog from Pickering Laboratories, Inc.

Pickering Catalog: http://pickeringlabs-retentiontimes.com/wp-content/uploads/2013/08/2013_PickeringLabs_Catalog_NorthAmerica_LowRes.pdf

Inside our catalog you will find a complete product listing for all instruments and consumables we currently have available. From Sample Cleanup to Post-Column Derivatization, to artificial Perspiration and Saliva, we hope you find something you will find useful!

Iodine Clock

As part of our new blogging format, we’d like to share a video with you.
We have a new favorite YouTube channel:  It’s the Periodic Table of Videos, and it’s produced by the University of Nottingham in the UK and features The Prof. We hope you enjoy this, and their other videos about general chemistry:

Iodine Clock

If only all chemistry classes could be so much fun…

 

Wicking – Don’t Sweat It

A Study of the Effect of Perspiration, Laundering, and Abrasion on Polyester Performance Fabric

By Tony Kedzierski

Abstract:

The purpose of this project was to determine if abrasion, perspiration, and laundering had an effect on the wicking ability of performance fabric.  Wicking is the ability of fabric to move sweat away from the skin.  It keeps athletes cool and dry during competition, helping them regulate their body temperature.   Wicking depends on a fabric’s capillary action.  Capillary tubes are areas found within the weave of material.  Larger tubes will wick faster; narrow tubes will wick slowly.  Longer tubes will wick farther; shorter tubes will wick a shorter distance.

A Vertical Wicking Apparatus was used to test these variables.  The distance distilled water traveled up each sample was measured.  Averages were compared to results of testing on untreated control samples.  All three variables wicked farther than control samples.  Viewed under a microscope, abraded fabric showed capillary tubes had gotten wider and narrower.  Fibers had been broken, blocking tubes and making flow uneven or impossible.  Dried perspiration left mineral build-up that slowed capillary flow.  Surfactants in the laundry detergent reduced surface tension of the water, allowing it to spread further.  Perhaps a finish had been applied to control samples, causing them to wick less than treated samples.

Knowing what variables affect the wicking ability of athletic wear can help athletes better prepare for competition.  Abrasion will cause performance fabric to behave differently each time it is worn as weave and capillary tubes change.  Proper washing to remove sweat is equally important. Understanding wicking properties of performance fabric gives athletes an edge.

Conclusions: Control Sample

The original hypothesis stated that, over time, exposure to abrasion would negatively affect polyester performance fabric’s ability to wick.  The hypothesis was based on the understanding and importance of capillary flow in a fabric’s ability to move liquid along its surface.  Because capillary flow must happen in capillary tubes that provide a continuous and uninterrupted flow of liquid, any break in the flow, such as those caused by abrasion of the fabric, might slow or halt the flow.  It was also assumed that as the fabric was abraded, the weave of the fabric would stretch and loosen, creating larger capillary tubes that would wick moisture quickly but at a shorter distance.  The data collected, however, did not support the hypothesis.  In fact, the opposite occurred.

After 30 minutes, the control samples wicked the water an average of 8.1 centimeters.  The abraded samples wicked an average of 10.2 centimeters after 30 minutes.  This is a difference of 2.1 centimeters.  While polyester is made to be highly resistant to abrasion, it is not completely resistant to wear that might come from normal use, such as laundering and wearing while playing sports.  There was visible evidence of wear on the abraded fabric strips.  The edges were frayed and the fabric no longer felt smooth like the other samples tested.  Despite the noticeable wear on the tested fabric, wicking was not impacted.  In fact, abrasion resulted in a 26% increase in the wicking ability of the samples tested.  Because wicking continued to occur between the 10 and 30 minute intervals, perhaps 30 minutes was not a long enough testing time for the fabric to wick to its peak distance and maximize its capillary effect.

Like abrasion, perspiration had a positive effect on the performance fabric’s ability to wick when compared to the control samples.  After 30 minutes, the control samples averaged 8.1 centimeters of wicking distance. The perspiration samples wicked, on average, 10.3 centimeters of wicking distance.  This is a difference of 2.2 centimeters, or an increase of 27% wicking capability.  As the perspiration dried on the fabric strips,Perspiration Sample the water portion of the perspiration evaporated leaving the mineral solids on the fabric. There was even visible evidence of the build-up of solids on the fabric because there was a dark line at the top of the fabric’s wicking peak.  The mineral left on the fabric might have affected the wicking capability by clogging or narrowing the capillary tubes in the fabric.

Like abrasion and perspiration, laundering improved the polyester ‘s ability to wick.  The control samples averaged a wicking distance of 8.1 centimeters. The laundering samples averaged a wicking distance of 18.8 centimeters after 30 minutes. This is an improvement of 10.7 centimeters, or 132% increase of wicking capability.  Perhaps, surfactants, surface active agents, in the detergent caused the wicking ability to improve.  Surfactants reduce surface tension of waterLaundering Sample and allow it to spread further across material during laundering.  These surfactants might have helped the distilled water move further along the surface of the fabric during testing.

After the 15th trial, treated fabric samples were examined under a 130X microscope camera.  The pictures of the abraded fabric showed that some of the capillary tubes became narrow while others were larger.  This variation in the weave creates inconsistency in the wicking rate across the fabric.  In addition, abrasion creates differences in wicking performance each time the fabric is worn, as rubbing will occur with regular wear and laundering.  Abrasion SampleThe pictures also showed fraying and breaking of fibers in the abraded fabric.  This would also contribute to uneven wicking.  The pictures of the samples soaked in perspiration showed a build-up of minerals on the fabric that could clog the capillary tubes, giving the appearance of a dull white crust on the material.  The pictures of the laundered samples showed little difference when compared to the control sample.

It is unclear why the untreated control samples wicked less than the samples treated with perspiration, abrasion, and laundering.   One possible explanation is that there was a finish applied to the performance fabric before it was shipped out to stores.  A finish is something that can be applied to fabric during or after manufacture to enhance the way it feels, looks, or performs.  These finishes can create a fabric that more easily releases soil, is waterproof, fire retardant, odor-resistant, and even softer.  Because there was no labeling of the test fabric indicating that it was any more than a wicking fabric, was the finish one that was applied so it had a better appearance in retail stores?  Did the abrasion rub a finish off that was applied after manufacture?  Did the perspiration dilute the finish?  Did the laundering wash it off?  Finally, had more time been added to the wicking trials in this study, would the results have been different?  Would extra time have allowed the untreated control, perspiration, laundering, and abrasion samples to maximize their capillary effect and reach an equal wicking distance?

 

Editor’s Note:
The above experiment design, text and photos were copied (with permission) verbatim from a report received from Tony. We were so excited that Tony won so many awards with his project, that we just had to share with our readers.  Pickering Laboratories supplied only the Aritificial Persipration; Tony is not related to Pickering in any way. In April, 2013, Tony sent us the following letter:

—————————————————————————————————————-

Dear Ms. George,

My name is Tony Kedzierski.  Several months ago you very generously sent me two bottles of  your artificial perspiration for use in my science fair project, WICKING:  Don’t Sweat It:  A Study of the Effect of Perspiration, Laundering, and Abrasion on Polyester Performance Fabric.  I have completed my project and have attached my results and conclusions for you to look at.  Through this project I learned a lot about the effects of everyday wear and use on performance fabric, as well as the importance of capillarity on this fabric’s ability to wick and keep an athlete dry and comfortable.

In our school district’s science fair (Sci://Tech Exposition), I placed 2nd in the 7th grade
chemistry division.  At the regional science fair (Science and Engineering Fair of Houston) I placed first in the junior division for 7th and 8th graders.  In addition, I received the Naval
Science Award from the Office of Naval Research, the Most Outstanding Exhibit in Materials Science Award from The ASM Materials Education Foundation, and the Award of Excellence from The Krishen Foundation for Arts and Sciences.  Last week, I went to San Antonio, Texas for the Exxon Mobil Texas Science and Engineering Fair.  There I placed first in the chemistry division and was nominated to participate in the Broadcom MASTERS national science, technology, engineering, and math competition for 6th, 7th, and 8th graders.

While International Science and Engineering Fair rules did not permit me to acknowledge
Pickering Laboratories on my science fair display board, I was able to mention Pickering
Laboratories Artificial Eccrine Sweat in my materials list/research plan and in the protocol
papers submitted prior to each science fair.  Without your generous contribution I do not think my project would have been possible, so I thank you so much for supporting me.

Sincerely,
Tony Kedzierski
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We’re so glad we could help! Congratulations, Tony! And Best of Luck for the future!

Guaranteed Chemistry