Category Archives: amino acids

Chromatography Quiz #9

Chromatography Quiz #8 Results
We would like to congratulate the grand prize winners of our last newsletter’s Carbamates Analysis Chromatography Quiz: Mary Benzinger from North Coast Laboratories, and Keena Njoroge and Matthew Hartz from Underwriters Laboratories!!!
They have won, and will shortly be receiving: Wine Hourglasses courtesy of Uncommongoods.com! Additionally, all participants will be receiving a $20 gift card from Jamba Juice!  Again, we would like to thank all of you for your submissions.  

The correct answer for the modified Carbamates chromatogram: the noisy baseline is due to the HPLC pump having excessively large pulsations (or pressure variations).  Post-column systems containing reciprocating pumps can also cause noisy pulsations in the baseline, and the frequency of the sine waves should be evaluated to determine which pump is causing the noise.

This problem is most often caused by one of the following: bad pump seals, bad check valves, scratched or damaged pump pistons, faulty pulse dampener, or problems with connections to the pump. 

This problem can also be caused by dirty reagents or eluents, however in this case the pulsations would be accompanied by the elevation of the background signal. 

We had a lot of excellent responses to this quiz! 

Thank you! 
Pickering Labs

Chromatography Quiz #9:

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.comby May 15th in order to win.  You will receive email confirmation that your submission has been received.  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 Fluid

Pickering Standard: 1700-0180 Native Sample Standard, acidics and neutrals, in 0.1 N HCl, 2.5 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:

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
An example of a good chromatogram can be found here: Standard Lithium Chromatogram

Pickering Products

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

Aflatoxins
Ochratoxin A
DON
Nivalenol
Fumonisin FB1, FB2
Zearalenone
Ergot Alkaloids

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

Antioxidants
Mycotoxins (individual and multi-residue)
Amino Acids
Biogenic Amines
Paralytic Shellfish Toxin
Polyether Antibiotics
Hexavalent Chromium
Vitamins
Sugars
Nitrate/Nitrite
Bromate
Carbamate Pesticides
Glyphosate Herbicide
NDELA
Formaldehyde

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:

www.pickeringlabs.com

or email: sales@pickeringlabs.com

RESULTS: Chromatography Quiz Number 2 (Amino Acids)

Congratulations to the Grand Prize Winner of our Newsletter’s Amino Acid Chromatography Quiz: Mary Barnes from Marin General Hospital!

She has won, and will shortly be receiving from Amazon.com, a new Kindle Wireless Reading Device and leather book cover!

The correct answer for the modified Amino Acid chromatogram: we did not allow the instrument proper equilibration. The column was equilibrated using a mixture of Li275 and Li750 buffers, and the higher starting pH caused peaks in the beginning of the chromatogram to come too early and co-elute. A similar chromatogram can also be caused by a higher pH of the sample or first buffer or an insufficient equilibration time between runs.

Also, we are extending our deadline for the Chromatography Quiz Number 3 (Glyphosate) until July 15th – so please get your entries in! Rebecca will accept submissions to rlsmith@pickeringlabs.com and confirm via email that she has received your answer.

Thank you and Good Luck!
The Pickering Labs Team

(Kindle image copyright Amazon.com)

The Art of Noise, by Maria Ofitserova, Senior Research Chemist

Baseline noise is a common and often frustrating problem in HPLC analysis. It makes integration difficult and adversely affects reproducibility and sensitivity of analysis. The most common sources of baseline noise are from pumps and bubbles in the lines or detectors. Not all noisy baselines can be easily explained but understanding common noise patterns will help you to determine what part of your system has a problem.

The Sine Wave
To observe the noise pattern you need to find a portion of the chromatogram that does not have any peaks and zoom in to look at about 5-10 min of the baseline. Baseline noise caused by reciprocating HPLC or post-column pump looks like a fairly regular sine wave. This kind of noise is usually due to old/poorly installed seals or bad piston. Ups and downs in the baseline follow flow/pressure variations as the piston moves. The period of the sine wave is different for different pumps. Measure the interval (in seconds) between the maximums of two waves to determine which pump is causing the noise. Most HPLC pumps have an interval of 6 -13 seconds. Pickering PCX5200 and Vector PCX reagent pumps have 2 sec and 4 sec intervals respectively. Pinnacle PCX contains a syringe pump which moves the piston in a single stroke hence it does not produce sine wave noise.

Most HPLC software programs record the column pressure during the analysis. It is very helpful to look at the trace to check if pressure variations have a similar pattern to your baseline noise. Pinnacle PCX users can also take advantage of log files collected by the Pickering software. Reagent pump pressure recorded in the log file helps Pickering technical support to evaluate the performance of the post-column system and determine if the syringe pump needs maintenance.

Bubbles
Baseline noise caused by bubbles consists of random spikes of varying amplitudes. Bubbles can occur in solvent lines or in the detector flow cell and are often caused by solutions outgassing. To prevent this from happening use a properly working degasser and install a backpressure regulator on the detector waste line to prevent boiling and outgassing in the heated reactor.

Detector Noise
Detector noise is always present and can be visible even on a “good” baseline if you zoom in deep enough. It is random and looks about the same throughout the chromatogram. An old detector lamp, dirt in a flow cell or problems with electronics can greatly increase noise level. If detector noise is suspected make sure the flow cell is clean and check the lamp hours. Built-in detector tests are also useful in assessing detector performance.

Shooting in the Dark
A common mistake people make when troubleshooting baseline noise in post-column applications is turning off the post-column reagent pump. Noise in the baseline is essentially variations in signal so it is proportional to background signal. Common eluants don’t fluoresce or absorb light in the visible range so when eluants alone go through the detector there is no signal and hence no noise. Post-column reagents, on the other hand, are often either colored or have background fluorescence so elevated noise caused by any part of HPLC system becomes visible. Turning off the reagent pump is akin to turning off the detector lamp and taking a shot in the dark – the noise is still there but we just can’t see it.

Let Us Help
When contacting Pickering support about elevated baseline noise please be ready to fax or e-mail your chromatogram and zoomed in portion of the baseline. For Pinnacle PCX users sending the log files will also help us to find the problem. You can email support@pickeringlabs.com or send a fax to 650-968-0749.

Pickering Laboratories rolls out up-grade to Pinnacle PCX: New Sigma Series, by Mike Gottschalk


The Pinnacle PCX Delta Series post-column derivatization instrument was first introduced in January 2005 to replace the PCX 5200 instrument. The Pinnacle PCX introduction brought new technologies to post-column systems including programmable temperature gradient column oven, inert flow path, reactor coil cartridge switching system, computer controlled software among others.

With the inclusion of the column temperature gradient feature, our amino acid analysis time for hydrolysates was reduced by half from 60 minutes to 30 minutes. In addition to improved analysis speed the ability to change reactor volumes easily made the Pinnacle PCX ideal for method development and application switching.

The development team at Pickering has been working behind the scenes to improve and expand the advantages of the Pinnacle PCX. Now with the confluence of several new features and improvements a complete series up-grade is occurring to the Sigma Series.


Notable improvement highlights:

  • Fully ROHS compliant – the European Union directive to eliminate toxic compounds in electronic equipment.
  • Power cooling – additional fans and air ducts have been developed to speed airflow in the column oven for faster cooling.
  • USB connection to PC – in addition to the Ethernet and relay connections USB has been added.
  • Pinnacle PCX Software version 1.0.0.7 includes 4 day log files for over the weekend log files, timer algorithm that runs independent of the system clock in the PC.
  • PEEK Front end on the pumps to prevent corrosion.
  • Injected composite parts – Column oven door and instrument base are 50 % lighter – saving on shipping costs.

Best of all the work flow of all methods are unaffected and migration of existing methods to the new Sigma series is seamless.

Pickering continually improves the components and manufacturability of all our products to provide the best analytical tools in the industry.