Category Archives: Amino Acid Analysis

Chromatography Quiz No. 4

Chromatography Quiz #3 Results
Congratulations to the grand prize winners of our last newsletter’s Glyphosate Chromatography Quiz: Jim Balk from Nebraska DHHS Public Health Environmental Laboratory, Matthew Hartz and Keena Njoroge from Underwriters Laboratories, and Narjes Ghafoori from the County of Los Angeles Environmental Toxicology Laboratory! They have won, and will shortly be receiving from Apple.com, a new iPod Shuffle! Additionally, for this third quiz all of our participants will be receiving a $10 gift card from iTunes. Thank you all for your submissions!

The correct answer for the modified Glyphosate chromatogram: the Glyphosate ‘doublet’ is caused by injecting a sample at basic pH. An improperly buffered sample extract at a large injection volume will not mix with the mobile phase sufficiently to create the acidic pH necessary for Glyphosate to be at the proper single charge state, impacting the interaction between Glyphosate and the active
sites on the column resin. Adding a couple drops of Restore TM(pH 1.3) to the sample before injection will eliminate the ‘doublet’ and return proper peak shape.
Thank you!
Pickering Labs

Chromatography Quiz #4:
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 September 30th in order to win. You will receive email confirmation when your submission is received, and the troubleshooting answer and winner congratulations will be published in the next issue (to be anonymous, please notify Rebecca in submission).

Amino Acid Analysis of Physiological Fluids
Pickering Standard: 011006P Native Sample Standard 0.25 µmole/mL, 10 µL injection
Pickering Column: 0354100T High Efficiency Lithium Cation-exchange Column, 4.0 x 100 mm
Pickering Guard Column: 0352020, 2.0 X 20 mm

Normal Operating Conditions: (for reference only, condition changes may be reflected in chromatogram)

Column Temperature: 37 °C
Flow rate: 0.35 mL/min

Eluent Gradient:

TIME….Li275%…….Li750%…….RG003%
0…………100……………0………………0

12……….100……………0………………0
48………..65…………….35…………….0
90………..0………………100…………..0

95………..0………………100…………..0

120………0……………….94……………6

122………0……………….94……………6

122.1…..100……………0………………0

140……..100……………0………………0



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


You can find an example of a good chromatogram on our website. Either click on the link, or go to www.pickeringlabs.com

New! GARD: Column Protection System

A new Column protection system for Cation-exchange HPLC applications will be unveiled exclusively at Pittcon this year.

The new GARD™ manufactured by Pickering Laboratories is a substantial improvement over the standard packed guards. The new GARD™ adds little pressure, is invisible to the chromatography, and has substantially more capacity for strongly retained compounds that can foul the analytical column.

The new GARD Column Protection System significantly prolongs column life without band spreading or added pressure. We will have a poster at Pittcon demonstrating, by means of a performance comparison for Amino Acid Analysis, that the use of a GARD will protect the analytical column more effectively than traditional guard cartridges, is more cost-effective for the laboratory, is easy to change, and most importantly has zero band spreading.

Chromatography Quiz No. 2

Congratulations to the winners of our last newsletter’s Chromatography Quiz: Matthew Hartz, Jamie Palmer, and Keena Njoroge from Underwriters Laboratories, Sudheer Reddy from Chemtex, and Becky Canela from Environmental Laboratory Services!

They’ve each won, and will shortly be receiving from Gifttree.com, two dozen irresistible cookies in five flavors: White Chocolate Hazelnut, Snickerdoodle, Peanut Butter, Oatmeal Raisin, and Chocolate Chip.

The correct answer for the modified Carbamates chromatogram: we reversed the two reagents. The OPA reagent was pumped in the Reagent One position, and the Hydrolysis reagent was pumped in the Reagent Two position. Thus 1-Naphthol, which is naturally fluorescent, appears full-sized. The other Carbamate peaks have different sizes due to their varying rate of hydrolysis – the high pH of the OPA reagent will allow for some but not complete hydrolysis prior to detection.

Chromatography Quiz: Amino Acid Analysis

Identify the error made when running the Amino Acids chromatogram below and win a prize! Simply email your answer and your full contact information to Rebecca at rlsmith@pickeringlabs.com by March 1st in order to win. The troubleshooting answer and winner congratulations will be published in the next issue (to be anonymous, please notify Rebecca in submission).

Amino Acid Analysis of Physiological Fluids:

Pickering Standard: 011006P Native Sample Standard 0.25 µmole/mL, 10 µL injection

Pickering Column: 0354100T High Efficiency Lithium Cation-exchange Column, 4.0 x 100 mm

Normal Operating Conditions: (for reference only, condition changes may be reflected in chromatogram)

Column Temperature: 36 °C

Flow rate: 0.35 mL/min

Eluent Gradient:

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, 570nm for primary amino acids, 440nm for secondary amino acids
Hint: Assume in this case that both Guard and Analytical column are good. To see a standard Amino Acid chromatogram, click here

New Product Lines from Pickering, October 2009

Mycotoxin product line
We are now distributing Mycotoxin Immunoaffinity products for Ochratoxin and Aflatoxin. The performance and batch-to-batch reproducibility of the columns is exceptional and far exceeds that of other manufacturers. The columns can be used for any matrix, from wine and juice, to nuts and grains, to herbs and spices. Contact Sales for more information.

GPC Sample Clean up line
We have a new GPC Sample Clean-up product line! We have both automated and manual GPC cleanup systems and we also have systems that include concentration & solvent exchange, or just GPC. Sample cleanup using GPC is especially useful for fatty matrices, but also perfect for vegetable matter and spices, as well as soil & waste water.

New faster AAA columns
We now have a Lithium amino acid run which will separate 45 amino acids in 70 minutes for Physiologic fluids, an a new 30-min Sodium amino acid run which will separate the 20 amino acids commonly found in protein hydrolysate samples. These columns are for use with our Pinnacle PCX.

Histamine Product Line
Our newly launched Histamine product line consists of Dip-sticks and Elisa kits as well as Post-column derivatization for fast and in-situ testing as well as quick, reproducible, and sensitive methods for follow-up confirmation. Contact Pickering Laboratories at 1-800-654-3330 or sales@pickeringlabs.com for more information!

About Post-Column derivatization analysis for HPLC – Part Three

Detector Considerations

Refractive Index Sensitivity

RI sensitivity applies only to UV-vis detectors. There are two sources of RI noise in post-column applications:

– RI discontinuities due to imperfect mixing.
– RI discontinuities due to temperature gradients in the eluant/reagent stream as it leaves a heated reactor.

In either case, when such inhomogeneities enter the flowcell, they bend light into the wall or off the photomultiplier tube, causing detector noise. The noise usually correlates with the piston cycles of the pumps, thus limiting the detector to low-sensitivity applications.
Most flowcells in modern UV-vis detectors are designed to minimize the effects of RI.

In order to minimize the temperature-related RI effects mentioned above, some manufacturers have a capillary heat exchanger at the flowcell entrance. In some instances this heat exchanger has an internal diameter of 0.12 mm (0.005 inches), which can result in post-column pressures in excess of 42 bar (600 psi). Since this can exceed the pressure rating of a heated reactor made with fluorocarbon tubing, this small-diameter heat-exchanger tube may need to be replaced with a 0.25 mm (0.010 inch) i.d. tube.

Detector Pressure Ratings

When the eluant-reagent stream from the heated reactor reaches the detector, it can release dissolved gas as it cools. The Pickering Laboratories derivatization instruments place a back-pressure of 7 bar (100 psi) on the detector flowcell in order to prevent the formation of bubbles.

– suppress boiling in the reactor
– prevent outgassing in the detector flowcell.

The back-pressure regulator can be factory-adjusted to accommodate flowcells with a lower back-pressure rating, depending upon the reactor temperature. but a setting lower than 3.2 5 bar (70 psi) is not recommended for reactor temperatures over 100° C.

Operating an HPLC system with a post-column derivatization system can be as routine as regular LC. The benefits from this LC/post-column combination include minimal sample pre-treatment, greatly improved sensitivity, and enhanced selectivity for compounds that would normally be much more difficult to detect.

About Post-Column derivatization analysis for HPLC – Part Two

Chemical Requirements

The chemical requirements for post-column derivatization are generic.

  • Stability of Reagent: The minimum reagent stability sufficient for routine work is one day. This means that the yield and signal-to-noise ratio for a given sample must remain constant for at least 8 hours.
  • Completeness of Reaction: The analytical separation is complete when the reagent is mixed with the column effluent. Therefore, in order to minimize band spreading, it is important to keep the volume small between the mixing tee and the detector. If the reaction is slow (in excess of one minute), an elevated temperature can be used to decrease the reaction time.
  • Reproducibility: Unless the system consistently produces the same signal for the same sample, quantitation is impossible. Because the reaction is occurring ³on the fly² as the combined column and reagent stream flows toward the detector, the reproducibility is linked to the flow-rate precision of the pumps and to the temperature. Accordingly, even an incomplete reaction will be as repeatable as the retention time for any given species. The completeness of the reaction, then, is not strictly necessary for reproducibility, but it is important for maximum sensitivity.
  • Minimal Detector Response of Reagents: The color or background fluorescence of the reagent (or its by-products) represents a continuous noise source. Because the reagent is present in excess relative to the analyte, the analyte’s signal could be obliterated by the reagent’s strong background signal. Pickering’s Chromatographic Grade® eluants and reagents are guaranteed to produce the absolute minimum possible detector background signal in post-column applications.
  • Solubility: All species must remain in solution, including the combined components of the eluants and the reagent(s), as well as the newly formed derivative(s). Precipitates can block capillary tubes, burst reactors and foul detector flowcells.

Ninhydrin chemistry provides a good example of multiple solubility considerations. Ninhydrin reagents contain a lithium acetate buffer, ninhydrin, hydrindantin, and a water-miscible organic solvent. The organic solvent is necessary to maintain both the hydrindantin and the new purple chromophore (derivative) in solution. Also the presence of lithium ion in the formula precludes the use of eluants containing phosphate, because lithium phosphate is insoluble and would precipitate at the point of mixing.

About Post-Column derivatization analysis for HPLC – Part One

Chromatography is a science of separations. High Performance Liquid Chromatography (HPLC) like other forms of chromatography, is used to separate complex mixtures into their components. There are many flavors of HPLC, but what they have in common is that the separation takes place in solution. Having separated a mixture, you need to see the components. The most popular detectors use either UV/VIS light absorption, or fluorescence. Unfortunately, many substances are difficult to detect. Moreover, you want to see the components of interest without distraction from the background.

Post-column derivatization, also known as post-column reaction, renders visible certain compounds that are normally invisible. This trick is accomplished after the separation by performing a chemical reaction on the substances that gives them an easily-detectable physical property. Typically you use a reaction that produces a strong color or makes a fluorescent product. You can increase the sensitivity of detection by several orders of magnitude in favorable cases. Most reagents are selective for a particular class of substances, so analytes of that class are more easily seen against a complex background. So, post-column derivatization is used to increase sensitivity and selectivity in HPLC analysis.

The post-column reaction system mixes the stream of eluant from the HPLC column with a stream of reagent solution. The mixture usually flows through a reactor to allow enough time for the chemical reactions to complete. If the reaction is slow, the reactor may be heated to speed things up. Some reactions need two or more reagents added in sequence. Finally the mixed streams pass into the detector, typically UV/VIS absorbance or fluorescence. Of course a practical system requires metering pumps, pulse-dampeners, thermostats, and safety systems to give reliable results.

Examples of the chemistry and hardware are given in the catalog and user’s manuals.