Category Archives: Chromatography Quiz

Chromatography Quiz #37 — Get to know the Onyx PCX

Chromatography Quiz #36—Amino Acids bent outa shape—Winners

Pickering Labs would like to congratulate all of our winners for our previous newsletter’s Chromatography Quiz:

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Narjes Ghafoori from Los Angeles County Public Health Laboratory and Tom Schneider from Suffolk County Water Authority.

Winners will soon receive an Amazon eGift Card! ($100 value)

Congratulations to our quiz winners!

Thank you all for your submissions!

The Answer to Chromatography Quiz #36

The answer is column temperature was set too low. Column temperature has a big affect on retention time stability. That it is why it is important to use a column heater for your analytical methods. We have used this principle to our advantage with our column temperature gradient methods. Our fast amino acid methods use a column temperature gradient where the temperature is changed throughout the run. This has cut some of our older run times in half. Please note that these methods can only be run on our Pinnace or Onyx PCX. No other post-column systems have our repeatable temperature gradient capabilities. See our amino acid method abstracts for more details: https://www.pickeringlabs.com/library/method-abstracts-2/

Chromatography Quiz #37 – Get to know the Onyx PCX

Correctly answer the questions below and win a prize!  Simply email your answer and your full contact information to Rebecca at rsmith@pickeringlabs.com by March 15, 2021 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). Hint: Download the Onyx PCX Brochure here: https://www.pickeringlabs.com/wp-content/uploads/2020/01/Onyx-PCX-Brochure.pdf

 
Questions Select Answer
1. The Onyx PCX has a larger color display.  True or False
2. The Onyx PCX can be calibrated in the field. True or False
3. The Pinnacle PCX has a Journal for audit tracking.    True or False
4. The Pinnacle PCX is larger than the Onyx PCX.  True or False
5. The Onyx PCX heated reactor is backwards compatible. True or False
6. The Onyx PCX reagent tray can be used as secondary containment. True or False
7. The Onyx PCX only uses USB connection to the PC.   True or False
8. The Onyx PCX pump and valve motors are backwards compatible. True or False
9. The Onyx PCX syringe pump offers pulse free reagent flow. True or False
10. Older Pinnacle methods can be easily transferred to the Onyx PCX.  True or False
11. Both the Pinnacle and Onyx PCX have automated piston wash. True or False
12. Maintenance is easier with the Onyx PCX because of the larger size. True or False
13. The Pinnacle and Onyx PCX have the same relay connector. True or False
14. The Pinnacle and Onyx PCX feature quick change reactors.   True or False
15. The Pinnacle and Onyx PCX feature a column heater with temperature gradient capabilities. True or False

 

 

 

 

 

 

 

Chromatography Quiz #36 — Amino Acids Bent Outa Shape

Chromatography Quiz #35 – Bad Carbamate Chromatography

Pickering Labs would like to congratulate all of our winners for our previous newsletter’s Chromatography Quiz:

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Mark Ritari from Anatek Labs Inc., Jim Balk from Nebraska Public Health Environmental Lab, Josiah Hakala from Minnesota Department of Health, and Narjes Ghafoori from Los Angeles County Public Health Laboratory.

Winners will soon receive: Amazon eGift Card! ($100 value)

Congratulations to our quiz winners!

  

Thank you all for your submissions!

In this case, the cause for the bad chromatography was a damaged or old heated reactor coil. However, poor peak shape can be caused by many possible issues. Here is a beautifully summarized answer to the quiz from one of our winners:

Observation:  Peak Tailing observed for all target compounds- more extensive for earlier eluting chromatography peaks, carbaryl and 1-naphthol not as affected.  Extensive tailing will cause integration problems for choosing area or peak height as a response variable. Peak retention and selectivity not affected, compromise in resolution but peak separation is still present.  There are multiple causes of peak tailing.  Unwanted secondary interaction between the analyte and stationary phase can lead to peak shape problems leading to peak tailing and loss of efficiency (sensitivity).  Loss of resolution and difficulty with integration. One must have the proper instrument setup (detector time constant) and proper connections to eliminate extra column dead volume (connecting tubing, inline frits, guard column) to maintain column efficiency.  The presence of trace metals can cause increases stationary phase silanol activity, leading to peak tailing from secondary interaction – use type B silica columns.

Possible problems could be coating or obstruction on column frit of particles coating the column from sample matrix and is very common. This will affect all peaks and is causes a increase in backpressure. This can be prevented by filtering sample prior to HPLC.  Flushing with various solvents can help. If reverse flushing the column, replacing the guard column, and/or pre-column inline frit does not help, the column has been probably chemically damaged and should be replaced. Physical damage accompanied by pressure change, pressure shock, void present (bed deformation) in column bed will affect all peaks – the column will have to be replaced. I have injected 3-5 100uL 3% ChlorAC buffer in 30% methanol and running a gradient to 30% methanol (holding at 30% methanol for 30 minutes) and then 100% methanol for 1 hr at 1cc /min.  It is important to that a proper guard column and sample solvent be used for the best peak shape. The sample must be properly buffered for carbamate analysis and the guard column be the sample packing material as the analytical column.

Diagnosis /Prognosis

  1. Analytical column problems (peak shape/loss of efficiency) has multiple causes; problems can be related to all components in the HPLC system (pump, injector, column, detector, data system/integrator).
  2. Use column protection (In-line filters /Guard columns); filter samples, mobile phase compatible sample solvent with properly sample-clean-up.
  3. Peak tailing, broadening, and loss of efficiency may be cause by column secondary interaction-metal cation activated or free silanol residues, column contamination, column aging, column sample mass/ volume overload, and/or extra-column effects (extra-column dispersion)
  4. In this case peak retention/selectivity has been affected.  Some compromise in resolution has occurred because of peak tailing.
  5. Method development is important. Is the analytical column really the problem?  Troubleshooting HPLC system components is necessary. Replacing the column with a new column (high purity Type B silica) may eliminate HPLC system problem as the cause of bad chromatography.
  6. Peak tailing for all peaks may be a physical rather than a chemical problem. The initial problem occurred before analytes began migrating through the column. This type of peak shape problem occurs with a partially blocked or void at the head of the column. Replacing or installing in-line frit (0.5 um porosity) downstream from the autosampler is an easy and less expensive to solve this type of peak distortion problem.
  7. For the diagnosis of the “bad chromatogram”, the abnormal peak shape (peak broadening/tailing) is particularly evident for all peaks with less noticeable distortion for carbaryl /1-naphthol. If all peaks in the chromatogram are affected, the problem may be related to either the column or the system.  If only early eluting peaks are affected, then the problem lies within the fluid path (perhaps incorrect internal diameter of tubing, fittings, etc.).  Problems with single peaks may suggest a specific chemistry problem in my experience.  One also consider post-column broadening effects where extra-column band broadening or changes in the system are the most likely causes  

Chromatography Quiz #36 – Amino Acids Bent Outa Shape

Correctly answer the question below and win a prize! Simply email your answer and your full contact information to Rebecca at rlsmith@pickeringlabs.com by November 15, 2020 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).

Below you will find the method details for the Amino Acids method. What could be causing the poor peak shape?

Analytical column: 0354100T

Conditions

Post-column Conditions
Post-column System: Onyx PCX, Pinnacle PCX, or Vector PCX
Reactor 1: 1030 ºC, 0.5 mL
Reagent 1: Trione
Detection: 570nm
Injection Volume: 10ul

good and bad chromatogram

Black is a good chromatogram. Pink is the bad chromatogram.

Reference Chromatrogram:

Reference Chromatrogram

Bad Chromatogram:

Bad Chromatogram

 

 

 

 

 

Chromatography Quiz #35

Chromatography Quiz #34: “What the Brij?”- RESULTS

Pickering Labs would like to congratulate all of our winners for our previous newsletter’s Chromatography Word Puzzle:

Ben Covert from New Mexico State Laboratory Division, Jim Balk from Nebraska Public Health Environmental Lab, Naomi Herrera from New Mexico Department of Health, Joy Gottlieb from New Mexico Department of Health, Josiah Hakala from Minnesota Department of Health, Jiufeng Fan from GSK, Tom Schneider from Suffolk County Water Authority, Narjes Ghafoori from Los Angeles County Public Health Laboratory.

Winners will soon receive: Amazon eGift Card! ($40.00 Value) Happy online shopping!

Congratulations to our quiz winners!

Thank you all for your submissions!

Chromatography Quiz #35 – Bad Carbamate Chromatography

Correctly answer the question below and win a prize! Simply email your answer and your full contact information to Rebecca at rlsmith@pickeringlabs.com by June 15, 2020 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).

Below you will find the method details for the Carbamate Method. What could be causing the poor peak shape and low response?

Analytical Conditions
Column:

          Carbamate Column P/N 0846250
          (250 x 4.6 mm), C8, 5 um
Flow Rate: 
          1 mL/min
Column Temperature: 
          42 ºC
Mobile Phase: 
          see Table 1

Post-column Conditions
Post-column System:
 
          Pinnacle PCX or Vector PCX
Reactor 1: 
          100 ºC, 0.5 mL
Reactor 2: 
          Ambient, 0.1 mL
Reagent 1: 
          Hydrolysis Reagent CB130 or CB130.2
Reagent 2: 
          100 mg of OPA, 2 g of Thiofluor in
          950 mL of CB910
Detection: 
          FLD, Excitation 330 nm, Emission 465 nm
Injection Volume: 
          10-20 uL

Full method details from our website here.

 

 

 

Chromatography Quiz #34

Chromatography Quiz #33: “What the Brij?”- RESULTS

Pickering Labs would like to congratulate all of our winners for our previous newsletter’s I’m not seeing any peaks! Carbamates edition Quiz: Tom Schneider from Suffolk County Water Authority, Narjes Ghafoori from LA County Environmental Toxicology Lab, and Jiufeng Fan from Glaxo Smith Kline, and Dr. David Green from Pepperdine University.

Winners will soon receive: An HP Sprocket Portable Photo Printer! A fun-size, Bluetooth enabled, cherry-tomato, portable photo printer! Just in time for those holiday selfies with family and friends! Or even better, those festive ugly sweater parties with your colleagues! We’re looking forward to sharing plenty of selfies with Onyx, our 2020 influencer!

Congratulations to our quiz winners!

Thank you all for your submissions!

 

The correct answer for the “What the Brij” Quiz:

The Brij-35 prevents quenching of large molecules. When large molecules, like the Fumonisins derivatives, quench they fold in on themselves and can lead to a reduction in fluorescent signal.

Here is a lovely answer we received from one of our quiz entries, if you want to get more in depth:
The Brij-35 is a non-ionic surfactant that, as far as I’m concerned, is a nearly miracle additive to HPLC eluent buffers to improving retention characteristics of strongly adsorbed proteins from the column surface. In my experience, it has the added […] benefit of enhancing the fluorescence intensity of the fluorescent derivative produced in the post-column reactor. The fluorescence enhancement is due to the derivative being trapped in the non-polar core of the Brij micelle. This enhancement can be extremely significant: 100s x improvement in sensitivity. It is well-established that a non-polar chemical environment improves fluorescent quantum efficiency by stabilizing the HOMO-LUMO molecular orbitals.

Chromatography Word Puzzle #34

Correctly complete the word puzzle below and win a prize! Simply email your answer and your full contact information to Rebecca at rlsmith@pickeringlabs.com by January 31st, 2020 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).

Onyx
PCX
Pinnacle
New
Sign
Paint
Derivatization
Retirement
Pickering
Laboratories
Pittcon
Cheers
Product
Testing
Solutions
Reagents
Trione
Thiofluor
OPA
Press
Release

 

 

 

 

Chromatography Quiz #33

Chromatography Quiz #32: I’m not seeing any peaks! Carbamates edition – RESULTS

Pickering Labs would like to congratulate all of our winners for our previous newsletter’s I’m not seeing any peaks! Carbamates edition Quiz: Jim Balk from Nebraska Public Health Environmental Lab, Josiah Hakala from Minnesota Department of Health, Narjes Ghafoori from LA County Environmental Toxicology Lab, Tom Schneider from Suffolk County Water Authority, and Jiufeng Fan from Glaxo Smith Kline.

Winners will soon receive: A Tile Pro Combo from Amazon.com! Tile is a tiny Bluetooth tracker and easy-to-use app that helps you find everyday items in seconds. Sleek, durable and water-resistance, the Tile Pro seamlessly pairs with your smartphone. The easiest way to find your things!

Congratulations to our quiz winners!

Thank you all for your submissions! 

 

The correct answers for the “I’m not seeing any peaks!” Carbamates edition quiz:

The reactor temperature was set too low. In normal operation, the reactor temperature should be set to 100 °C for proper post-column reaction completion. The incomplete reaction will give you low response for all analytes except Carbaryl and 1-Naphthol. Carbaryl becomes 1-Naphthol after the first step in the post-column reaction and 1-Naphthol is naturally fluorescent and does not require the OPA reaction.

Chromatography Quiz 33 – What the Brij?!?

Correctly answer the question below and win a prize!  Simply email your answer and your full contact information to Rebecca at rlsmith@pickeringlabs.com by November 1, 2019 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). 

Below you will find the method details for the Fumonisins method. What is the purpose of adding 30% Brij to the post-column derivatizing reagent?

(Full method abstract(s) available on our website.)

 

 

 

Chromatography Quiz #32

Chromatography Quiz #31: Glyphosate Doublet Peak – RESULTS

Pickering Labs would like to congratulate all of our winners for our previous newsletter’s Glyphosate Doublet Quiz: Jim Balk from Nebraska Public Health Environmental Laboratory, Tom Schneider from Suffolk County Water Authority, Narjes Ghafoori from LA County Public Health Laboratories, and Jiufeng Fan from Glaxo Smith Kline.

Winners will soon receive: An All-New Amazon Kindle Whitepaper!!!  The thinnest, lightest Kindle Whitepaper yet, this waterproof e-reader is sure to help our winners unwind with some beachside or poolside relaxation this summer!  Happy reading!

Congratulations to our quiz winners!

Thank you all for your submissions! 

 

 

    .

The correct answers for the Glyphosate Doublet Peak quiz:

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 RestoreTM (pH 1.3) to the sample before injection will eliminate the ‘doublet’ and return proper peak shape. 

Chromatography Quiz #32 – I’m not seeing any peaks, Carbamates edition!

Identify the error made when running the Carbamates chromatogram below and win a prize! Simply email your answer and your full contact information to Rebecca at rlsmith@pickeringlabs.com by August 30, 2019 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). 

Good Chromatogram
HPLC Flow Rate: 1.0ml/min
Reagent Flow Rate: 0.3ml/min
Column Temp: 42C
Reactor Temp: 100C

Elution order:

Bad Chromatogram
HPLC Flow Rate: 1.0ml/min
Reagent Flow Rate: 0.3ml/min
Column Temp: 42C
Reactor Temp: 36C

 

 

 

 

Chromatography Quiz #30

Chromatography Quiz #29: Amino Acids Elevated Baseline – RESULTS

Pickering Labs would like to congratulate all of our winners for our previous newsletter’s Elevated Baseline Amino Acids Chromatogram: Tom Schneider from Suffolk County Water Authority, Narjes Ghafoori from LA County Environmental Toxicology Lab, and Dr. David Green from Pepperdine University!

Winners will soon receive: A Harvest Bundle of Gifts from www.HarryandDavid.com!

This bountiful harvest bundle includes: creamy Pumpkin Cheesecake, a beautiful Autumn Garden Party plant gift, and a Pumpkin-Shaped Gift Basket which features juicy pears, pumpkin bars, cranberry relish, and much more. It’s the perfect way to celebrate the changing of the seasons. Best of all, bundle gift items are sent individually to make the celebration last a little longer!

Congratulations to our quiz winners and we hope they look forward to receiving their gifts next week!

Thank you all for your submissions!
  
  
    
   

The correct answers for the Elevated Baseline Amino Acids Chromatogram are as follows:

The shift on the baseline is called an ammonia plateau and it is due to the presence of low-level amines and ammonia in the buffers. These compounds accumulate on the column during equilibration time and come out during the gradient in a form of a plateau. Since the buffers have low pH, these compounds are unavoidable but care should be taken to avoid excessive contamination that can cause the plateau to be too high. Amines are present even in the air and get dissolved in buffers as time goes by.

The issue usually comes from buffer A. Try and replace with a new lot if possible.

Below are some tips on how to minimize any potential problems:

  • Remove all filters from the ends of HPLC lines that go into the buffer bottles. All our products are filtered before bottling and these in-line filters only drag contamination from one bottle to another.
  • Replace open buffers on the instrument at least every two weeks. If you don’t use the full bottle in 2 weeks, pour half of the bottle into a clean glass bottle to put on the instrument and tightly cap the remaining portion to keep until future use.
  • Don’t flush column with water, use only Column Regenerant for cleaning the column.
  • Don’t use the first injection of the sequence for calculations since it usually has a different profile due to differences in equilibration time.
  • Program the needle wash between the runs to avoid carry over.
  • If you see unexpected peaks on your blank or other chromatograms make a fresh vial of the solution and run again to confirm the problem. Also run “No Injection” to see if the peaks are coming from the injected sample of from the baseline.
  • Flush HPLC periodically with 100% water, then 100 % methanol, then 100 % water with no column attached (!!!) to keep the lines clean.

Chromatography Quiz #30 – Aflatoxins Analysis, Decreased Signal:

Simply email your answer as well as your full contact information to Rebecca at rlsmith@pickeringlabs.com by December 21, 2018 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).

Aflatoxin analysis by photochemical derivatization is achieved with the parameters listed below:

Analytical Column: Mycotox Column, C18 4.6x250mm
HPLC Eluent: Sodium Phosphate buffer PN 1700-1108/Methanol/Acetonitrile (57/28/15)
Flow Rate: 1 ml/min
FLD: Excitation 365nm, Emission 430nm
UVE Photochemical reactor with 254nm UV light: 1.0ml knitted reaction coil.

 

What could contribute to a decrease in signal?