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Amino Acid Analysis, Chromatography Quiz

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.

post-column derivatization

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

 

 

 

Uncategorized

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 support@pickeringlabs.com 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

Regards,
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

artificial perspiration, product testing, saliva, sweat

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: www.pickeringtestsolutions.com

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.

pickering laboratories, Pittcon

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!
fountain
clean-up, freestyle, GPC, immunoaffinity cleanup, lctech, mycotoxin, spe

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 www.freestyle-robotic.com for more details, configurator, and applications

Amino Acid Analysis, HPLC, pickering laboratories, pinnacle pcx, post-column derivatization, Trione

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.

METHOD
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: www.pickeringlabs.com

 

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