Category Archives: Trione

Troubleshooting – Trione

by David Mazawa

Ninhydrin reacts with primary amines and hydrindantin to form Ruhemann’s Purple which is detectable at 570nm. Ninhydrin reacts with secondary amines to form a yellow complex detectable at 440nm. The ninhydrin reaction is carried out at 130°C with a reactor volume of 500uL. The elevated temperature is required because at room temperature, the ninhydrin reaction is very slow and takes hours to go to completion.

Trione is a proprietary formulation containing ninhydrin, hydrindantin (reduced ninhydrin), a lithium acetate buffer, and sulfolane, a water-miscible organic solvent. The solvent is necessary to maintain the solubility of both the hydrindantin and the primary amine product, Ruhemann’s Purple. The buffer is required because the reaction is pH dependent. T100 and T100C (Case of 4 bottles of T100) are premixed and have a shelf life of 4 months at room temperature. T200 comes in 2 bottles and must be mixed together before use.

Trione will oxidize when exposed to air. Depending on the exposure, part or all of the hydrindantin will be oxidized to ninhydrin. Only primary amine development requires hydrindantin. Consequently, as the hydrindantin disappears, so will the primary amine signal. Secondary amines, such as Proline, will appear to be larger than normal because the signal for primary amines will so low.

The Trione aging process is different from Trione oxidation. The concentration of hydrindantin will increase as Trione ages. Consequently, the signal for primary amines will increase. The increase in hydrindantin can cause changes in reagent color and increase the risk of precipitation in the heated reactor. For this reason we have set the shelf life of T100 and T100C to 4 months at room temperature. The aging process begins as soon as T100 is bottled. The aging process for T200 is slower; however, once mixed together it will age at the same rate as T100. Unmixed T200 has a shelf life of 12 months at room temperature. Never top-off your old Trione with new Trione, as the old Trione acts as a catalyst and will make the new Trione age very fast. Always use a clean reagent bottle when using a new bottle of Trione.

Trione Oxidation

Hydrindantin decreases. Low primary amine signal.

Trione Aging

Hydrindantin increases. High primary amine signal. Increased risk for precipitation in the heated reactor.

ninhydrin reaction

Chromatography Quiz #16

Chromatography Quiz #15 Results

We would like to congratulate our grand prize winners of our last newsletter’s Amino Acids Analysis Chromatography Quiz: Tom Schneider from Suffolk County Water Authority, Jim Balk from DHHS Public Health Environmental Laboratory, and Narjes Ghafoori from LA County Environmental Toxicology Laboratory!

They have each won and will shortly be receiving: a $100 gift card for Best Buy, which can be used online or in the store!!!

We would like to thank all of you for your submissions!

The correct answer for the Amino Acids troubleshooting chromatogram: the baseline noise was caused by a dirty flow cell. As we have seen through several Chromatography Quizzes, baseline noise can have a variety of causes, both mechanical and chemical. In this chromatogram, the noise we are seeing is not regular or repeatable, steering us away from the HPLC pump, a trapped bubble, or the back-pressure regulator.

To learn more about baseline noise, please review Maria’s article entitled “The Art of Noise” ( from a previous newsletter.

Thank you!

Pickering Labs

Chromatography Quiz #16:

We are doing something a little bit different with this quiz! Identify what’s wrong with the picture below and win a prize! Simply email your answer as well as your full contact information to Rebecca at by July 1st, 2014 in order to win. You will receive email confirmation that your submission has been received. The answer to the quiz and winner congratulations will be published in the next issue (to be anonymous, please notify Rebecca in your submission).

A blast from the past: What is wrong with this picture?!

As you can probably tell from the photo, we’ve dug into Michael’s past for our quiz this quarter! Look carefully and see if you can figure out what’s wrong with this picture! If you have any questions, please feel free to email Rebecca at for further guidance.

Michael_Trione Ad

Amino Acid Analysis of Monoclonal Antibodies

Method Abstract 373, Amino Acid Analysis of Monoclonal Antibodies

The peptide and protein based pharmaceuticals are a rapidly expanding class of therapeutical agents that are used to treat a wide variety of health conditions, including cancer, metabolic and auto-immune diseases, HIV and more. Biologic drugs, such as monoclonal antibodies, are derived from living organisms and are usually very expensive. As many biologics are coming off of patents, the market is ready for cost-saving biogenerics. But all proteins, including monoclonal antibodies, have complex structures that determine their function. Differences in structure would alter biological activity leading to changes in safety and efficacy of the drug.

ICH Q6B is a guidance document that provides a set of internationally accepted specifications for biotechnological and biological products to support new marketing applications. It establishes the set of criteria to which a drug substance, drug product or material should conform to be considered acceptable for intended use.

aaa chromatograms from MA373Determining Amino Acid composition following hydrolysis is listed in ICH Q6B as a way to characterize the protein and to confirm its identity by comparing with Amino Acid composition deduced from the gene sequence of the desired product. Amino Acid Analysis data is also used to accurately determine the protein content.

The Amino Acids Analysis with post-column derivatization is a very sensitive, reproducible and rugged method and it has been a preferred approach for laboratories running biological samples, protein, peptides and foods analysis. Pickering Laboratories Inc. offers many Amino Acids Analysis products including post-column derivatization instruments, columns, eluants, reagents and standards. All products are designed to work together to deliver optimum results for any chosen sample.

Analytical conditions
Column: High-efficiency Sodium cation-exchange column, 4.6 x 110 mm, P/N 1154110T
Flow Rate: 0.6 mL/min
Mobile Phase: See method in Table 1

Post-Column Conditionsgradient table for AAA monoclonal AB
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

You can download this abstract, as well as our product catalog and other notes from our website:


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

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:

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

Pickering Laboratories in 2013

By Wendy Rasmussen

In recent years, we have released several new products and applications, and with still more on the horizon, it occurred to me that now would be a great time to summarize the Pickering of today – our mindset and our wide variety of products & applications.

Acai Berries
Acai Berries

No longer are we simply the “Post-Column Company”. We are the “Automated-Sample Antioxidants” company.  Think of us as the new “super fruit.” The Acai berry, or perhaps the new Chia Seeds (incidentally, we do have a post-column application for the identification & quantitation of  Antioxidants in a variety of matrices).

We are still very active, and we as a company plan to be here for many years to come. We are still the company founded on chemistry and a desire to to teach, to spread our technical expertise, to support our customers.

It’s been a few years now since Pickering began distributing and supporting our LCTech Product line.  The products have shown an ever increasing interest here in the US and in Canada (our official Sales Territory for this product line). We are very proud to offer these products and we hope we can develop it further in the future.

Historically, we have provided the back-end of an analysis (post-column derivatization). Nowadays, we can provide the front-end of analysis as well (the sample-cleanup).

In thinking about our product offerings, I realized that a simple list does not effectively show the scope of the products we have to offer in 2013 – primarily because we have a lot of overlap between products and product lines.  We’re not a vertical company in that regard.  I suppose one could say our product offerings are more circular in that many do not fall into a single distinct category.  I am a very visual person, and for me, a Venn diagram and our overall “product scale” really helped to understand and clarify our products:

Venn Diagram of Pickering Laboratories
Venn Diagram of Pickering Laboratories
General "Product Scale" for Pickering Offerings
General “Product Scale” for Pickering Offerings
For those of who like lists, you can find one Here, on Pickering’s website, and on LCTech’s Website

For any Questions, please feel free to contact us:

Pickering Laboratories, Inc.
Mountain View, California
Phone: (direct) 650-694-6700 or (toll-free) 800-654-3330


Image of Acai Berries:

Pickering Products

Mycotoxins: Clean-up Columns, ELISA Kits, Post-Column Derivatization Instruments & Methods, SPE manifolds (manual & automated) for the analysis of:

Ochratoxin A
Fumonisin FB1, FB2
Ergot Alkaloids

Post-Column Derivatization: Pinnacle PCX, Vector PCX, UVE Photochemical Reactor, Columns, Reagents, Eluants for the analysis of:

Mycotoxins (individual and multi-residue)
Amino Acids
Biogenic Amines
Paralytic Shellfish Toxin
Polyether Antibiotics
Hexavalent Chromium
Carbamate Pesticides
Glyphosate Herbicide

Sample Preparation (clean-up) Instruments:

FREESTYLE for SPE, GPC, online-concentration
FREESTYLE ThermELUTE for Aflatoxin analysis (direct inject onto HPLC)
AcceCLEAN for SPE (incl. Immunoaffinity columns)
EluVAC vacuum manifold for SPE (incl. Immunoaffinity columns)
GPC QUATTRO for manual GPC Cleanup
DECS System for the cleanup of samples for Dioxin analysis

Chemistry Products:

Artificial Perspiration
Artificial Saliva
High Purity Water

Stand-Alone OEM Syringe Pump:

For any applications requiring an inert flow path, volumes up to 70mL, and pressures up to 500psi

For More Information visit:

or email: