The Origins of Trione

By Mike Gottschalk

Michael Pickering got the idea for Trione on his first day as a research chemist at Durrum Instruments back in 1976. While they were showing him their Amino Acid analysis instrument they opened the refrigerator to reveal the eluents and reagents in their reservoirs. One solution was red. He asked, “What’s the red stuff?” The reply was that it was the ninhydrin reagent. He asked, “Why is it red, ninhydrin isn’t red!” The answer was no one knew. He said, show me the instrument later, take me to documents, “I want to see the formula for the ninhydrin.” Seeing the formula reveal that Stan Moore’s recipe included Dimethylsulfoxide (DMSO) as the essential water miscible organic solvent. While DMSO is a great solvent, it’s also reactive, and responsible for the secondary reactions that lead to the background red color.

Before join Durrum Instruments Michael had followed the work of George Olah in Carbonium ion chemistry. He’s the guy who developed “magic acid” which could protonate methane to make CH5+. His solvent of choice was sulfolane, a water miscible, non-reactive organic solvent.

The idea was born. However, Durrum Instruments had no interest in making consumables. The company where he next worked was Spectra Physics which also had no interest in making consumables. But on his starting interview with personal he was required to list his ideas that he thought were patentable. Number one was Trione.

Early on, he enlisted the help of Bertum Russel, a chemistry patent attorney to get the patent for Trione. Bertum’s conditions were “If you think you’re going to make money by licensing this I won’t waste your money, but if you are going to make it, I’ll get you a patent.”

That same year, Michael contacted Stan Moore at Rockefeller University to get his assessment after using Trione. Afterall, what better way to start a business than to have the endorsement of one of the researchers who developed the original ninhydrin. Stan said, “You should do well with this but I have post-docs making ninhydrin for me every day so I won’t need to buy it.”

Undeterred, Michael gave seminars and workshops thought the analytical community and soon acceptance by the analytical chemists made Trione the best in class ninhydrin for Amino Acid analysis it is today.

Pittcon 2014

This year’s Pittcon was held in Chicago, Illinois from March 3-6.

The overwhelming theme of this meeting was COLD. Winter was still very present in Chicago that week with temperatures dropping well below freezing.chicago 2014

But despite the cold, there was a good turnout and it was a successful show. As usual, we had on hand our Pinnacle PCX and our post-column applications. We also had our FREESTYLE Sample Cleanup system. This is always a great show for meeting distributors, suppliers, customers. Nowadays Pittcon is as much (if not more) about networking and meetings as it is about finding new equipment.

Beer sampler

The sampler at 18th Street Brewery

We usually try to find some adventure or other on the Sunday before the show starts, and this year was no exception. This year, we braved the cold and the snow to venture out to Gary, Indiana!  Before Pittcon, this editor had visited 45 of the 50 US States. Indiana was one of 5 remaining.

Waiting for the train under McCormick Place

Waiting for the train under McCormick Place

The requirements for the day were: 1) easy to get to without a car 2) be located outside the city limits of Chicago and 3) have a restaurant/bar close to the train station.  After some internet research, the 18th Street Brewery in Gary, IN seemed to fit the bill perfectly!

you are beautiful

We were told the Aquatorium is a fun place in summer

We took the South Shore Line from McCormick Place  out to Gary, where we trudged through a foot of snow to the 18th Street Brewery.  It turns out this is a wonderful brewery with good beer and friendly folks. And they even had a grill out back which was cooking up kielbasa sausage with kimchi. If you’re ever in Chicago and need a change of pace, or are in northern Indiana, it’s worth a stop.

All in all it was a good trip and a successful meeting. But I’m glad Pittcon 2015 will be in New Orleans…

Joint Western & Eastern Canada Workshop

Pickering Laboratories participated in the first joint Western & Eastern Canadian Trace Organic Workshop, held in Guelph, Ontario.   We shared a table with our Canadian distributor, Chromatographic Specialties. The meeting was well attended and there were many interesting talks. The purpose of the meeting is to share the latest methods and information about trace analysis — for a wide range of analytes from pesticides, to dioxins, to PCBs, flame retardants, and surfactants.

We presented two posters:

Cleanup and Determination of Aflatoxins in Peanuts and Peanut Butter using Immunoaffinity Cleanup with HPLC- Post-column UVE Photochemical Derivatization

and

Glyphosate analysis in soy beans, corn and sunflower seeds by HPLC with post-column derivatization and fluorescence detection

In addition to the two posters, we had the latest information on the FREESTYLE Sample Preparation system, and in particular the ThermELUTE module, for rapid and sensitive cleanup and analysis of Aflatoxins and Ochratoixn A.

In the News: Phthalates

By Michael Pickering

While reading the NY Times one morning in March, I came across an article titled “A Plastic Threat to Male Fertility” in the Science Times section. Having encountered phthalates in the past, I was curious to read further.

Federal researchers recently spent four years tracking 501 couples who were trying to have children. The goal of their study was to assess the impact of everyday chemicals on fertility. While both men and women were exposed to known toxins, men appeared much more likely to suffer fertility problems as a result.

“Anything you can think of that’s testosterone-dependent is likely to be affected.”   – Heather Patisaul, North Carolina State University

The gender disparity was most significant when it came to phthalates. This group of petrochemicals finds its way into many commercial products. Phthalates are among a group of compounds known as endocrine disruptors. As the endocrine system controls the production and distribution of hormones in the body, they have been implicated in a range of health problems. Unlike many of the study’s other industrial toxins that bio-accumulate, phthalates are metabolized within hours of ingestion. However, their pervasiveness in the environment means almost constant exposure, which increases their impact.

Among the myriad uses for phthalates are as plasticizers (rendering polymers flexible rather than brittle) and in cosmetics, where they improve smoothness of flow upon application.

Partial List:

  • Cosmetics
  • Plastics
  • Household products
  • PVC pipes
  • Hospital tubing
  • Medicine (pills, capsules)
  • Air filters, residential and commercial

While the evidence for an effect on male fertility is compelling, it is still difficult for researchers to gauge the full impact these prevalent phthalates are having. If you want to minimize your exposure, read labels, do not heat anything you intend to ingest in plastic, and bring your own glass to the keg party.

IN THE PAST: Phthalate Blues

Personally, my formal introduction to phthalates was when I quit teaching high school and went to grad school. My research involved isolating and characterizing secondary metabolites in the plant family Rutaceae. The process involved solvent extraction (soxhlet) and evaporation followed by gravitational silica column chromatography. We learned quickly that all the bulk silica, most of the solvents, and the most popular clear flexible tubing (Tygon) all contained di-octylphthalate (DOP), an oil. The air in the building was also loaded with DOP due to the filters. Fortunately, it was easy to keep track of DOP because of its blue fluorescence. The low polarity of the plasticizer made it easy to elute off of the columns with the slurry solvent. For our 20cm x 20cm x 1-2mm thick layer prep plates, we formed and dried them, and then placed them in the chromatography chamber with acetone. Upon re-drying, the plates viewed under UV light were flat white with a bright blue edge along the top.

Most of my targeted metabolites are fluorescent; yellow, green, pink, and blue to the eye. I once inadvertently isolated a non-chromophoric terpene because I was chasing an indigo blue fluorescent spot on the TLC. Upon elution from the plate, I got an oil. Mass spectrometric analysis revealed the terpene in a tableau of DOP fragments. The friend who ran the mass spec for me, Charlie, was in charge of a spectroscopy lab for a veterinary toxicology department at the time. He said he could recognize all the spectrographic manifestations of DOP from twenty paces.

My last encounter with DOP was several years later, in a Pickering customer’s lab. An extremely agitated gentleman had called to announce that he had “NO PEAKS!!!” So, our customer service chemist went for a visit, and I went along for the ride.

We arrived at 10:00am, and fortunately the system was running. He had a Carbamate post-column instrument paired with a water/methanol two-pump, four-piston binary gradient HPLC. The pistons were ganged in series. Our chemist engaged the customer while I observed the instrument. The fluorometer was so overloaded with signal that the PM tube was regularly turning off. There was no troubleshooting information available from the magnitude of the signal, but there was a very regular and periodic spike, implying a piston cycle.

Upon further observation of the HPLC, I noticed that both reservoirs’ contents had traveled up into the Nitrogen lines and was sloshing around in the Tygon tubing. With this back-flowed solution being sloshed back into the reservoir periodically, I realized the problem. Although Tygon tubing is phthalate-free today, it was loaded with DOP back then. The swamping fluorescence was in both reservoirs. The spike frequency was the last piston on the methanol pump, where the DOP concentration would be the highest.

The customer had his peaks, he just couldn’t see them for the bright lights.

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” (http://pickeringlabs-retentiontimes.com/?p=58) 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 rlsmith@pickeringlabs.com 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 rlsmith@pickeringlabs.com for further guidance.

Michael_Trione Ad

Chromatograpy Quiz No. 15

Chromatography Quiz #14 Results

We would like to congratulate our grand prize winners of our last newsletter’s Carbamate/Glyphosate Crossword Puzzle Chromatography Quiz: Jim Balk from DHHS Public Health Environmental Laboratory, Amanda Comando and Dan Raphaely from SCWA, June Black from the Pennsylvania Department of Environmental Protection, Narjes Ghafoori from LA County Environmental Toxicology Laboratory, Helene Lachance from Shur-Gain Nutreco, Holger Franz from Thermo Fisher Scientific, Matthew Hartz from Underwriters Laboratories, Hossein Hajipour from Texas Department of State Health Services, and Daniel Durham from Georgia Department of Natural Resources!!!

rum cake picThey have each won and will shortly be receiving: a Tortuga To Go Suitcase Gift Pack from tortugarumcakes.com!  This “vacation in a box” includes three rum cakes and an 8oz package of coffee, shipped directly from the Cayman Islands!

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

The correct answers for the Carbamate/Glyphosate crossword puzzle:

Across:

1. Pickering column protection system: GARD
6. Heated or Ambient: REACTOR
10. ChlorAC buffer for sample ________: PRESERVATION
11. Over-pressure ________ valve: RELIEF
12. Internal Standard: BDMC
13. Faulty check valves yield a noisy ________: BASELINE
14. Solution to late, broad Glyphosate and AMPA peaks: RESTORE

Down:

1. Glyphosate oxidized by hypochlorite: GLYCINE
2. Pickering Catalog number 3700-2000: THIOFLUOR
3. Reagent one: HYDROLYSIS
4. Fourth peak: METHOMYL
5. Glyphosate herbicide: ROUNDUP
7. _______ detection: FLUORESCENCE
8. o-phthaladehyde: OPA
9. Gradient, water and _______ : METHANOL

Thank you!
Pickering Labs

Chromatography Quiz #15:

Identify the error made when running the Amino Acids chromatogram below and win a prize!  Simply email your answer as well as your full contact information to Rebecca at rlsmith@pickeringlabs.com by May 1, 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 submission).

Amino Acid Analysis – Baseline Noise

Pinnacle PCX post-column instrument is being used, in a traditional HPLC setup as recommended by Pickering Labs.  The chromatogram is expanded to show the baseline noise.  The quiz question is: what is causing this problem?

Post-column conditions for amino acid analysis:
Reagent 1: Trione
Reactor 1: 130 °C, 0.5 mL
Reagent flow rate: 0.3 mL/min

Detection: UV-Vis Detector, 570 nm for primary amino acids, 440 nm for secondary amino acids

Problem Chromatogram:

problem chromatogram
Please use our website to view any reference Amino Acid chromatogram – the problem for this quiz is not application-specific, so any of our AAA methods could be applicable.  If you have any questions, please don’t hesitate to email Rebecca at rlsmith@pickeringlabs.com.

Analysis of Sugars in Feeds

by HPLC with Post-column Derivatization and Fluorescence Detection

The types and amounts of sugar in animal feeds are as important as the amount of protein, minerals and fats in the determination of nutritive value. We developed a simple and sensitive HPLC method for analyzing six sugars in animal feeds – Sucrose, Fructose, Glucose, Galactose, Maltose and Lactose. Post-column derivatization reagents convert reducing and non-reducing sugars into fluorescent derivatives, which greatly improves the sensitivity and selectivity of the detection.

The blends of feed examined varied from grains/vegetable products (live stock feeds) to meat/vegetable products (pet food).

Method

Standards Preparation:

Mix 2.5 g of feed sample with 50 mL of water. Heat using a water bath while constantly mixing for 1 hour at 65 °C. Centrifuge and filter through 0.45 um filter.

Analytical Conditions
Column: Carbohydrate column, 4.6×150 mm
Temperature: 30 ºC
Flow Rate: 1 mL/min
Mobile Phase: Acetonitrile/Water
Injection Volume: 10 uL – 50 uL

Post-Column Conditions
Post-Column System: Pinnacle PCX or Vector PCX
Reactor Volume: 1.4 mL
Temperature: 130 °C
Reagent 1: Guanidine hydrochloride 60 mM in 200 mM Boric acid adjusted to pH 11.5 with KOH
Reagent 2: 1.5 mM periodic acid adjusted to pH 11.5 with KOH
Flow Rate: 0.15 mL/min each reagent

Detection: FLD; λex: 325 nm, λem: 465 nm

HPLC GRADIENT
TIME (Min) WATER, % ACN, %
0.0 20 80
20.0  20  80
20.1  50  50
30.0  50  50
30.1  20  80

Calibration:

A quadratic calibration curve with correlation > 0.999 is observed for monosaccharides such as Fructose, Glucose and Galactose. A linear calibration curve with correlation > 0.999 is observed for disaccharides such as Maltose, Lactose and Sucrose. Examples of calibration curves presented in Fig. 1 and Fig. 2.M_237a

FRUCTOSE GLUCOSE GALACTOSE SUCROSE MALTOSE LACTOSE
FEED MATRIX 1
Content in feed, %  0.54 0.52 0.09 4.02 1.12 ND*
Spike Concentration, %  0.60 0.58 0.60 2.02 0.58 0.59
Recoveries, n=3, % 105 107 110 91 103 114
Spike Concentration, % 1.21 1.20 1.19 4.05 1.22 1.21
Recoveries, n=3, % 108 106 110 85 85 103
FEED MATRIX 2
Content in feed, % 0.23 0.46 ND* 3.21 0.59 ND*
Spike Concentration, % 0.42 0.57 0.56 2.50 0.57 0.58
Recoveries, n=3, % 98 101 104 106 106 107
Spike Concentration, %  0.81 1.14 1.12 4.80 1.11 1.15
Recoveries, n=3, % 103 101 101 102 102 106
FEED MATRIX 3
Content in feed, %  0.14 0.11 ND* 0.51 0.02 ND*
Spike Concentration, % 0.38 0.56 0.57 2.40 0.55 0.56
Recoveries, n=3, % 95 101 116 102 95 101
FEED MATRIX 4
Content in feed, %  0.17 0.13 ND* 1.35 0.21 ND*
Spike Concentration, % 0.41 0.57 0.58 2.43 0.56 0.56
Recoveries, n=3, % 92 97 116 101 95 102

*Not Detected

M_223b

M_223c

You can download the method abstract 233 here: MA223 sugars in feeds