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!  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:


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


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 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

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).


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

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


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

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
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
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
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



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

New Air Barrier tubing

Air Barrier Tubing, PN 2103-0463

Dear valued customer,

SARAN tubing is oxygen-impermeable and is used for preventing oxidation of oxygen sensitive reagents commonly used in HPLC post-column analysis. To avoid future supplier issues, we have made a change to item part number 2103-0463. The new material is opaque white in color, impermeable to oxygen, and is effective at preventing oxidation of our post-column reagents. This material is a direct replacement for SARAN. We are keeping the part number the same, however, the new description will be: Air Barrier Tubing, 1/8” OD x 1/16” ID, 36in or 90cm per unit. You will be able to use the new air barrier tubing just like the old SARAN tubing.

Please contact Pickering Laboratories Support at (650)694-6700, (800)654-3330, or if you have any questions.

New description effective January 1, 2014:
PN 2103-0463, Air Barrier Tubing, 1/8” OD x 1/16” ID, 36in or 90cm per unit

David Mazawa
Technical Support Chemist
Pickering Laboratories, Inc.
1280 Space Park Way
Mountain View, CA 94043 USA
Phone: (650)694-6700 ext. 710
Fax: (650)968-0749

SARAN is a Registered Trademark of Dow Chemical Company

New! Product Testing Solutions

Pickering Laboratories Launches Product Testing Website

Building on the history of providing chemistry to the clinical and environmental market Pickering Laboratories has developed a line of Artificial Body Fluids for the Product Testing market.

Manufacturers of consumer and medical products test for compatibility with human fluids for safety as well as durability.  Multiple standard organizations specify the composition of these artificial body fluids for effective testing protocols. Pickering Laboratories developed the artificial perspiration, saliva and urine to these specifications for reliable and consistent product testing regimes.ld--perspiration-group

In 2009 Pickering Laboratories started offering artificial Perspiration at the request of Crime Science Incorporated a distributor of Law enforcement products. They wanted to provide an artificial perspiration product used as a control for finger print analysis called SwetCheck. Since then products as diverse as credit cards, textiles, eye glasses, guitar strings, electronics, cosmetics and more have requested the official specified solutions for their product testing needs. We continue to develop additional new solutions to requested standards

To better serve this market Pickering Laboratories is launching a website specifically for product testing solutions:

Product testing solutions are manufactured to official standards of ASTM, ACCTC, ISO, DIN as well as offering the most complete artificial perspiration available on the market today.

Visit Pickering at Pittcon 2014

We will be displaying our equipment at this year’s Pittcon in Chicago, Illinois from March 3-6, 2014. Be sure to stop by:  Booth 2955

We will have our Pinnacle PCX post-column derivatization instrument on display as well as our Mycotoxin Analysis Products. In addition, we will some new videos to highlight the latest in our Automated Sample Preparation instrument the FREESTYLE SPE system.

Wendy, David and Mike will be in Chicago. Please stop by. We would love to talk with you!

Freestyle Automated Sample Prep Instrument

The new FREESTYLE system is the most adaptable robotic equipment for sample preparation currently available on the market. With its nearly universally applicable system range, numerous processing steps can be elegantly automated. The FREESTYLE system takes on daily routine tasks in the laboratory, but also offers the user the unique opportunity to combine specific working steps that were previously carried out individually.

FREESTYLE SPE Module: The FREESTYLE SPE system is suitable for the fully automated processing of the most diverse column formats. The technology is unique:

    • One rack for different SPE columns: Different columns can be combined in any fashion. Adaptation is achieved through choosing the respective adapter. Different adapters are available for different sized columns.
    • Handling of both open as well as closed (e.g. IAC) columns is possible
    • Sample processing directly on the robotic arm under positive or negative pressure
    • Simple method set-up through pre-determined single steps, which can be arbitrarily combined and parametrized individually:
      • Conditioning
      • Emptying (e.g. let buffer drain off from immunoaffinity columns)
      • Loading: a variety of options for loading the sample onto the column
      • Washing
      • Drying the SPE cartridge with ambient air or nitrogen stream
      • Elution with various solvents, with multiple repetitions, and into different containers
    • Any combination of methods and/or columns within a sample list is possible
    • Various – almost unlimited – possibilities of using different containers for any type of sample or for filling of the eluate

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FREESTYLE EVAporation Module:
The FREESTYLE EVAporation system is suitable for the fully automated evaporation of many samples – if you wish overnight or over the weekend. The novel FREESTYLE technology:

    • allows for free end-volume selection of between 0.2 mL and 5.0 mL (automatic calibration),
    • individually for each sample,
    • combines the rotary evaporator technology (vacuum) with gentle blowing off with nitrogen (optional),
    • allows an automated solvent exchange liquid/liquid or to dryness (optional),
    • results in a concentrate, precisely bottled or dispensed in aliquots in free-selected glasses (e.g. closed GC vials).

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FREESTYLE GPC Module: The FREESTYLE GPC module facilitates the upgrade to a fully automated chromatographic sample preparation system (gel permeation chromatography), as is used for example for clean-up of food, animal feed and environmental samples. It complies in all areas with the requirements of general methods, e.g. DFG S19, EPA 3640A, AOAC 984.21, EN 1528 and more. The module consists of a preparative LC-double piston pump with all its advantages e.g. minimizing pulsation. With a flow rate of say 5 mL/min, the preparative pump incurs only a small load and is consequently particularly low maintenance.

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Be sure to visit for more details, configurator, and applications

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:


AOAC International Meeting

fountainThe AOAC International held their annual meeting in Chicago, Illinois from August 25-27, 2013.

Pickering Laboratories were on hand to share our latest methods and instruments.  We also gave an Exhibitor Presentation titled “An Automated Highly Sensitive Method for Aflatoxin B/G or Ochratoxin A Clean-up and Analysis – A Novel Approach in Mycotoxin Analysis”

The purpose of the talk was to showcase our newest method and instrument:

Building on the FREESTYLE Automated Sample Preparation Instrument, the new ThermELUTE module and columns enable rapid, sensitive analysis of Aflatoxin or Ochratoxin A. The ThermELUTE module uses a thermal denaturation technique to release jellybean4toxin from a new column format requiring only 20% of usual solvent volumes, and FREESTYLE quantitatively transfers the eluate into the HPLC. In combination with online injection, analytical sensitivity can be increased without manual manipulation. The increase in sensitivity provides a reduction of sample load, while maintaining excellent chromatographic results. The increase of sensitivity allow analysis of mycotoxin levels well below regulatory limits.

The ThermELUTE takes the separate steps of cleanup and analysis, and combines them into one automated system. Using a FREESTYLE robotic system equipped with the

FREESTYLE Automated Sample Preparation System

FREESTYLE Automated Sample Preparation System

ThermELUTE module, we can now clean up a sample for mycotoxins, and then directly inject the cleaned sample onto the HPLC column for analysis!

Automating Mycotoxin Analyses from Cleanup to Detection:

  • Results 3 times faster
  • 80% less solvent
  • Direct connect to HPLC

The brochure for ThermELUTE can be found HERE. To view the slides from our presentation, click HERE.


A Story with Bearing: Cholesterol

A Story with Bearing: Cholesterol

By Michael Pickering

Years ago our friend Peggy told us that her doctor had prescribed a diet devoid in cholesterol, as her blood test indicated worrisome numbers.  The doctor recommended that all of the usual suspects be excluded from her diet, such as egg yolks and butter, but it was all to no avail.  Regardless of how long she maintained the exclusive diet, her blood numbers did not budge.  So, she decided to experiment with her own dietary exclusions.  One of her first experiments was targeted at a food everyone who knows Peggy is well aware that she is addicted to – milk chocolate.  It was only then that her blood cholesterol numbers improved.

While it is true that one’s diet is an important factor in the level of cholesterol in one’s blood, the amount of cholesterol in one’s diet is not germane.  Unlike the essential amino acids and minerals which must be harvested from the diet, the cholesterol in our blood is synthesized inside our bodies from smaller synthons (many acetates, a popular biosynthetic mode, see flavonoids).

So the issue isn’t whether cholesterol is in one’s diet, but rather how cholesterol is behaving in one’s blood.

The key link between the importance of diet and the behavior of cholesterol in one’s blood is the amount and type of fat you ingest.  Highly saturated fats have the most negative impact on the solubility of cholesterol in the blood.

mink coat front

Blood chemistry is necessarily dominated by water soluble processes.  Magnesium, sodium, citrate, and all manner of water soluble nutrients must course around freely.  However, cholesterol is not water soluble, even though it must move as freely through our veins.  The body’s solution is to cloak the cholesterol with a hydrophobic interior (cozy coat) with a hydrophilic exterior (sort of like a 1960’s Bill Blass coat with the mink on the inside and the satin on the outside).mink coat back

These water taxis are called LDL and HDL: low density lipoproteins and high density lipoproteins.  Our body considers the HDL to be better than the LDL because, among other things, it’s easier to void.   Tom Scheve’s description of the reason for this in his article for Discovery Health, entitled “What’s the difference between LDL and HDL Cholesterol” eloquently expresses my own musings:

When the lipoprotein has more protein than cholesterol [HDL], it resembles a Ferrari, gunning through your body without stopping until the cholesterol arrives at your liver, where it’s converted into bile acids.  […]  When the lipoprotein has more cholesterol than protein [LDL], however, this makes for a rickety    ride, and that jalopy doesn’t get too far.  Cells have special receptors that bind tightly to these lipoproteins as they pass.  This LDL sputters down the road, careening off the arteries, running into things and leaving bits all over the place.  While the HDL Ferrari sees a pileup and nimbly speeds around it, the LDL jalopy crashes right into it, adding to the jumble of tangled fenders and tailpipes (or platelets and plaque).

The overall solubility of cholesterol in the blood is governed by a ternary phase diagram. ternary phase diagramMaintaining these three components in the proper ratio crates a zone of solubility in the triangle.  If the diet (the source of phospholipids and fats) biases the ratio out of the soluble zone, the cholesterol precipitates with the fenders and tailpipes.  And like all solids in a moving fluid, they deposit in the zones of slowest flow.  In a vascular system the slowest flow is in the arteries.

The lipids (fats) in our diet can be broadly sorted into two categories: 1) naturally occurring, and 2) man-made.  Obviously the naturally occurring fats and oils are derived from plants and animals.  The man-made fats are partially hydrogenated vegetable oils.  The saturation level determines the melting point and viscosity regardless of the source.  So highly unsaturated lipids like sesame oil have a low melting point and viscosity and so are inappropriate for frying, whereas poly-saturated lard and butter have a high melting point and viscosity, and are well suited for frying.  Similarly, an award-winning pie crust can be made with lard or butter, but not with unsaturated oil.  Partial hydrogenation thus controls the melting point of the fat and establishes its suitability for any particular application.

However, a side reaction also occurs during the hydrogenation: isomerization.  Natural unsaturation tends to be cis-configuration but hydrogenation isomerizes the bonds to trans-configuration.

cis trans fat structuresWhile the hydrogenation controls the melting point precisely (which is essential for processed foods), the resulting fat is not recognized by the body as food.

Our wild type diet is clearly designed around whole grains as the staple, a source uniquely rich in unsaturated (cis-) fats, phospholipids, and protein.  The goal is to manage the trace chemistry in our blood, the hydrophobic components.  So the lesson is: eat as little saturated (mostly animal) fat as you can tolerate, eat whole grains and exclude all partially hydrogenated vegetable oil.  Read the label!

So while the amount of cholesterol present in a milk chocolate bar (24 g) is comparable to that of a tablespoon of butter (30 g), the two had very different effects on Peggy’s blood work.  Cocoa fat is among the most saturated of the vegetable oils.  The melting point is so high that the bars are wax-like at room temperature.  The chocolate was in effect creating an excess of LDL jalopy wreckage in Peggy’s blood stream, by causing the LDL and HDL levels to get out of whack.

The cholesterol had no bearing.


Editor’s Note: Always consult a physician first. The views presented herein are strictly editorial in nature.

Chromatography Quiz No.14

Chromatography Quiz #13 Results

We would like to congratulate our grand prize winners of our last newsletter’s Carbamate Analysis Chromatography Quiz: Irene Taylor from Orange County Utilities, Jim Balk from DHHS Public Health Environmental Laboratory, Narjes Ghafoori from LA County Environmental Toxicology Laboratory, Helene Lachance from Shur-Gain Nutreco, and Jamie Palmer and Matthew Hartz from Underwriters Laboratories!!!

laser chess 1They have each won and will be receiving: a Laser Board Game from laser chess 2Sharper Image!  (This strategy-based board game is similar to chess and features an 80-square Egyptian themed grid.  The goal is to protect your game pieces while eliminating your opponent’s by bouncing eye-safe lasers at them through the maze of mirrors you’ve constructed.)

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

The correct answer for the modified Carbamate chromatogram: Reagent #2 (CB910, OPA, and Thiofluor) was partially oxidized.  The peak heights of Carbaryl and 1-Naphthol have significant diagnostic importance in regard to the OPA reagent system. Since 1-Naphthol naturally fluoresces, we can eliminate the fluorescence detector as the problem.  Though smaller peak sizes can reflect problems with Reagent #1, since the Carbaryl peak is full-sized, we can deduce that hydrolysis has occurred (Carbaryl naturally fluoresces after hydrolysis).

Particularly astute observation yields additional details about our troubleshooting chromatogram – slightly shifting retention times towards the end of the run could indicate a problem with HPLC hardware, such as a proportioning valve.

Thank you!

Pickering Labs

 Chromatography Quiz #14:

Complete the cross word puzzle below and win a prize!  Simply email your answer (copy of completed puzzle or list answers with clues) as well as your full contact information to Rebecca at by December 15, 2013 in order to win.  You will receive email confirmation that your submission has been received.  The answer to the puzzle and winner congratulations will be published in the next issue (to be anonymous, please notify Rebecca in submission).

Carbamate/Glyphosate Analysis: Cross Word Puzzle