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Amino Acid Analysis, Chromatography Quiz

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 email Rebecca Smith 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

Post-column conditions for amino acid analysis
You can find an example of a good chromatogram page on our website.  

amino acids, Chromatography Quiz

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 Kindle Wireless Reading Devicehas 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 at 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)

Chromatography Quiz, glyphosate

Chromatography Quiz Number 3

Reminder: Please resubmit your answers to last quarter’s Amino Acid Chromatography Quiz by June 1st to be eligible for a prize. You can find that quiz located on our Blog at Chromatography Quiz #2.

Chromatography Quiz #3

Identify the error made when running the Glyphosate chromatogram below and win a prize! Simply email your answer and your full contact information to Rebecca at rlsmith@pickeringlabs.com by July 1st 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).

Glyphosate Analysis for US EPA Method 547
Normal Operating Conditions: (for reference only, changes may be reflected in chromatogram)

Customer’s Glyphosate Standard: 25 ppb, 100 µL injection

Pickering Column: 1954150 Cation-exchange Column for Glyphosate Analysis, 4 x 150 mm

Column Temperature: 55 °C
Flow rate: 0.4 mL/min

Eluent Gradient:
Glyphosate Analysis for US EPA Method 547

Post-column conditions for pesticide analysis:
Reagent 1: Oxidizing reagent – 100uL of 5% Sodium Hypochlorite in 950mL of GA116
Reagent 2: 100 mg of OPA, 2 g Thiofluor™ in 950 mL of GA104

Reactor 1: 36 °C, 0.5 mL

Reactor 2: ambient. 0.1 mL
Reagent flow rates: 0.3 mL/min

Detection: Fluorometer ex 330 nm, em 465 nm

Post-column conditions for pesticide analysis

 
Uncategorized

Please Re-submit Your Answers to Amino Acid Quiz (No. 2)

Due to an unfortunate set of circumstances, all submitted answers to the previous Chromatography Quiz on Amino Acid Analysis have been lost. We sincerely apologize for the inconvenience to everyone who submitted an answer, and ask for all entries to be resubmitted. As we genuinely regret our technical difficulties, Rebecca will be sending prizes to all submissions, and grand prizes to everyone with the correct answer. Please resend your email, or resubmit your answer to rlsmith@pickeringlabs.com. Rebecca will send you confirmation via email that she has received your submission. All entries will be reviewed on June 11th, and prizes will be sent at that time. You can review the quiz at: http://pickeringlabs.blogspot.com/2010/01/chromatography-quiz-no-2.html. Again, our apologies, and if you have any questions please contact Wendy or Rebecca at 650-694-6700.

bacteria, food myth, plastic, wood cutting board

Persistent Food Myth No. 2: Wood Bad, Plastic Good, by Michael Pickering

In the early 1970’s, the USDA recommended that meat handlers such as butchers and restaurants replace wood cutting boards with plastic. The recommendation was heard as a categorical “MUST”. The assumption was that plastic cutting surfaces were less likely to transfer pathogens. No scientific evidence existed to support their opinion. Years later, researches decided to eliminate the information void by challenging the plastic and wood surfaces with E. coli O157:77, Salmonella, et al. Results indicated that new plastic and wood surfaces were comparable in cleaning and disinfecting properties.

It was discovered that bacteria were not recoverable from wood surfaces a short time after application. Bacteria did migrate into the interior of the wood, however they 1) did no multiply, 2) were not transferable, and 3) eventually died. The same bacterial concentrations on the plastic were transferable, but both surfaces were easily cleaned. Blade damaged plastic surfaces are very different.

Here is the rub: Blade damaged plastic surfaces are very different. First, plastic suffers much greater damage than wood under similar use. More importantly, damaged plastic surfaces proved difficult or impossible to decontaminate while the marred wood behaved much like new wood.

The plastic smoothness factor has reach the street according to my friend Brad Daley, Partner and General Manager at one of my favorite haunts, Cascal, a popular tapas restaurant and bar in Mountain View. He said the Health Department inspectors tell him when the plastic cutting surfaces need sanding. If the surface is visually scarred or discolored, they are told to replace or sand them; no other specifications. I’ll bet there’s an assumption hiding there. Another dangling end is that plastic surfaces vary greatly in chemical properties compared to close-grained wood, and only a few polymers have been tested.

In 1999, the USDA Meat & Poultry inspection manual (official regulations) and the US FDA Food Code (recommendations for restaurants, etc.) permitted the use of close-grained hardwoods, like maple, for cutting boards. Neither has a recommendation for the type of polymer that is acceptable nor do they specify how the plastic surfaces must be maintained. That’s a Gilda Radner “Never mind” to me.

allergies, glutamate, msg

Persistent Food Myths Part 1: MSG, The Last Sentence, by Michael Pickering

The amino acids important to our well-being are a small set divided into two groups: essential and non-essential. The essential amino acids typically must be supplied in the diet. The normal body makes the non-essential amino acids from molecules harvested from the diet. Glutamic acid can be synthesized in the body and so falls into the non-essential group. It has two carboxylate groups and under physiological conditions travels as the half salt, aka, glutamate and hence the mono- in monosodium glutamate (MSG). Besides its key role in protein and energy metabolism, Glutamate serves as a neurotransmitter in the brain.

One would be hard pressed to find anything edible containing no MSG. It is so ubiquitous that it is one of the most intensely studied food ingredients. Human breast milk contains 10x more MSG than cow’s milk and nursing infants recognize its taste.

MSG has been valued in world cuisine for more than 2000 years. The unique taste was first described in print in 1866 and proposed as a distinct taste in 1908. In 2000, the L-Glutamate taste receptor was identified: taste mGluR4. This fifth taste receptor is named “umami.” When stimulated, the umami signal to the brain is extremely positive. It is variously translated from the Japanese as “yummy,” “delicious” and “wow.” Of the long list of translations, I prefer “savory.” The list of positives regarding MSG in one’s diet would be wearisome to enumerate.

So why in 1968, when Robert Ho Man Kwok, M.D. coined the phrase “Chinese restaurant syndrome” (CSR), did the rap get hung on MSG? The answer is that tough, leathery cuts of meat are often tenderized with blends containing the digestive enzyme papain. These blends do usually include MSG as a flavor enhancer. Since the tenderizing effect is shallow, it is usually only used with thinly sliced meat which is typical in a Chinese kitchen. Catabolic enzymes like papain facilitate hydrolysis of peptide bonds. The reactive stew also brews side reactions, among which is the possible conversion of the amino acid L-Histidine into histamine, which is well-known to allergy sufferers.

Can you eat parmesan cheese, peas, corn, tomatoes or spinach? MSG is everywhere, even if you don’t eat. Do you think you’re allergic to MSG? Check again, perhaps there is something else…

amino acids, baseline noise, detector, glyphosate, Ninhydrin, outgassing, post-column derivatization, sine wave

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.

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