Category Archives: Chromatography Quiz

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?

 

 

 

 

 

 

Chromatography Quiz #29

Chromatography Quiz #28: 35th Anniversary Word Puzzle – Results

Pickering Labs would like to congratulate all of our winners for our previous newsletter’s 35th Anniversary Word Puzzle: Jim Balk from Nebraska DHHS Public Health Environmental Laboratory, Tom Schneider from Suffolk County Water Authority, Karissa Scroggins from North Coast Laboratories, Narjes Ghafoori from LA County Environmental Toxicology Lab, Joy Gottlieb from New Mexico Department of Health Scientific Lab Division, Hossein Hajipour from Texas Dept. of State Health Services Laboratories, and Widchuda Meeim from Thailand Bureau of Quality Control of Livestock Products.

Winners will soon receive a Packing Organizer Set from the Container Store! This colorful set of 6 packing cubes are durable and clearly labeled to help you organize your suitcase! Just in time as you plan for those summer getaways!

Congratulations to our quiz winners and happy packing!

Thank you all for your submissions! 


             

The correct answers for the Anniversary Word Puzzle are as follows:

Chromatography Quiz #29 – Amino Acids Elevated Baseline:

Simply email your answer as well as your full contact information to Rebecca at rlsmith@pickeringlabs.com by July 15, 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). 

Maroon: Good chromatogram

Blue: Elevated baseline

What could be contributing to the elevated baseline?

   

   

   

Chromatography Quiz #27: Only AMPA

Chromatography Quiz #26: Shifting Retention Times – Carbamates — Results

Pickering Labs would like to congratulate the winners of our last newsletter’s Shifting Retention Times–Carbamates Quiz: David Green from Pepperdine University, Jeff Fan from Cumberland Valley Analytical Services, Karissa Scroggins from North Coast Laboratories, Jim Balk from Nebraska DHHS Public Health Environmental Laboratory, Narjes Ghafoori from LA County Agricultural Commissioner Weights & Measure Environmental Toxicology Lab, Tom Schneider from Suffolk County Water Authority, and Ms. Widchuda Meeim from Thailand Bureau of Quality Control of Livestock Products.

They have each won and will shortly receive a Williams Sonoma BBQ Tools Set! Included in a stainless-steel case for easy storage, these sleek grilling tools are perfect for those upcoming summer cookouts!

Congrats to our quiz winners and happy grilling!

Thank you all for your submissions! 

The correct answer to the Sifting Retention Times – Carbamate Quiz:

Leaking proportioning valve. The leaking proportioning valve improperly mixed the method gradient and didn’t have enough methanol which caused the analytes to elute late.

Chromatography Quiz #27: Only AMPA

What is causing the bad chromatography in the example below?  Simply email your answer as well as your full contact information to Rebecca at rlsmith@pickeringlabs.com by September 1, 2017 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). 

Glyphosate Analysis – Only AMPA

Pinnacle or Vector PCX post-column instrument is being used, in a traditional HPLC setup as recommended by Pickering Laboratories. The quiz question: what is causing Glyphosate to disappear?  

Post-column conditions for carbamates analysis:

Reagent 1: GA116
Reagent 2: o-Phthalaldehyde and Thiofluor in GA104
Reactor: 36 °C, 0.5 mL
Reagent flow rate: 0.3 mL/min
Injection volume: 10uL

FLD Settings:

λex 330 nm
λem 465 nm

HPLC Flowrate: 0.4 mL/min
Column Temperature: 55°C


 

Good Chromatogram

Glyphosate Test Mix, 2.5ppm, 10µl Injection

Bad Chromatogram only shows AMPA peak. No Glyphosate peak can be detected.

Glyphosate Test Mix, 2.5ppm, 10µl Injection

 

Chromatography Quiz #26

Chromatography Quiz #25 – B@$eL!nE Noise! — Results

Pickering Labs would like to congratulate the winners of our last newsletter’s Baseline Noise Quiz: Joel Fray from Colorado Analytical Laboratories, Jim Balk from Nebraska DHHS Public Health Environmental Laboratory, Narjes Ghafoori from LA County Environmental Toxicology Lab, Hossein Hajipour from Texas Dept. of Health Services Laboratories, and Tom Schneider from Suffolk County Water Authority.

They have each won and will shortly receive a decadent Chocolate Dream Gift Basket! Created by Cherry Moon Farms, this lovely basket is the ultimate assortment of chocolate sweets that will surely delight! Congrats to our quiz winners and enjoy the goodies!

Thank you all for your submissions! 

The correct answer to the Amino Acids Baseline Noise Quiz:

The zoomed baseline exhibited sine wave with regular oscillations at frequency of about 6 sec. This type of pattern is characteristic to HPLC pump noise as reciprocating HPLC pump moves to deliver the flow of eluants through the column.  If pump is operating normally, the oscillations are very small and will not show as baseline noise. But if the pump has dirty/bad check valves or old/poorly installed seals, the noise increases. In our case, the HPLC pump check valves were bad and needed replacement. 

Chromatography Quiz #26: Shifting Retention Times – Carbamates

What is causing the retention time shift in the chromatogram below?  Simply email your answer as well as your full contact information to Rebecca at rlsmith@pickeringlabs.com by May 1st, 2017 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). 

Carbamates Analysis – Shifting Retention Times

Pinnacle PCX post-column instrument is being used, in a traditional HPLC setup as recommended by Pickering Laboratories. The quiz question: what is causing the shifting retention times? 

Post-column conditions for carbamates analysis:

Reagent 1: CB130
Reagent 2: CB910/Thiofluor/OPA
Reactor: 100 °C, 0.5 mL
Reagent flow rate: 0.3 mL/min
Injection volume: 10uL

FLD Settings:

λex 330 nm
λem 465 nm

HPLC Flowrate: 1.0 mL/min
Column Temperature: 42 °C

Good Chromatogram

Bad Chromatogram

 

Chromatography Quiz #25

Chromatography Quiz #24 Results

1-fit-bitPickering Labs would like to congratulate the winners of our last newsletter’s Glyphosate Quiz: David Green from Pepperdine University Natural Science Division, Jim Balk from the DHHS Public Health Environmental Laboratory, Narjes Ghafoori from LA County Environmental Toxicology Lab, Karissa Scroggins from North Coast Laboratories, Helene Lachance from Shur-Gain Nutreco, Tom Schneider from Suffolk County Water Authority, and Irene Taylor from Orange County Utilities Water Division Laboratory! 

They have each won and will shortly be receiving a: Fitbit Flex 2 wristband tracker! An ultra-slim, light-weight wristband for daily activity and sleep tracking! This wearable tech goody is also water and swim-proof! Happy tracking to our Quiz Winners!

Thank you all for your submissions!    

 

  

The correct answer to the Polyether Antibiotics Analysis quiz:

The correct answer to the Glyphosate Analysis Quiz was: we injected a sample at the wrong pH. The samples should be at pH 2 for Glyphosate analysis, especially at high injection volumes. A neutral pH sample can cause peak broadening and sometimes the Glyphosate peak elutes as a doublet. The later eluting AMPA peak is less affected by sample pH problems.

We received really great submissions on this quiz from our contestants that included other reasons for misshapen Glyphosate peaks, so we are included for a couple here for additional troubleshooting background/advice!  Nice work everyone!

From one submission, we learn that metal contamination can also affect peak shape: Glyphosate is a pretty good chelating agent through the phosphate end so contamination with transition metals, especially iron, will affect the peak shape of Glyphosate and AMPA with Glyphosate being the first peak impacted.  In the case of metal contamination Pickering recommends flushing the column with Restore, the solution formulated to remove transition metals and to bring the column back to “peak” performance.

Another scientist has pointed out a different common reason for Glyphosate peak shape problems and retention time shift: Extra-column band broadening. Extra-column band broadening will affect more dramatically the early eluting glyphosate peak. If all the peaks are affected in the chromatogram, it would suggest that a problem is related to either the system or the column. As this is not the case here and only the early eluting peaks are affected, it suggests that the problem could lay with the fluid path – fittings, void in guards or sample/mobile phase solvent, etc.  It is always a good idea to make sure all the tubing connections are made correctly; there are no bubbles in the lines or guard column and the injector stator/rotor seal are not worn or defective.

Chromatography Quiz #25: B@$eL!nE Noise!

What is causing the baseline noise in the chromatogram below?  Simply email your answer as well as your full contact information to Rebecca at rlsmith@pickeringlabs.com by January 1st, 2016 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 Laboratories. The quiz question: what is causing the baseline noise? 

Post-column conditions for Lithium Amino Acid analysis:
Reagent 1: Trione
Reactor: 130 °C, 0.5 mL
Reagent flow rate: 0.3 mL/min
Injection volume: 10uL

DAD Settings:
Signal = 570nm
Reference = Off

HPLC Flowrate: 0.35mL/min
Column Temperature: 37 °C

   

1-quiz-25

 

 

 

 

 

Chromatography Quiz #23

Chromatography Quiz #22 Results

Pickering Labs would like to congratulate our winners of our last newsletter’s AAA quiz: Narjes Ghafoori from LA County Environmental Toxicology Lab, Joy Gottlieb from New Mexico Department of Health Scientific Lab Division, Tom Schneider from Suffolk County Water Authority and Helene Lachance from Shur-Gain Nutreco!    

contest-prizeThey have each won and will shortly be receiving: a Picnic Basket Gift Tin from Harry & David!  From their webpage: Celebrate the season with this gourmet gift basket, featuring a wonderful assortment of snacks, including fruit, meat, cheese, and crackers perfect for picnics and lunches in the park. In addition to our remarkably juicy Royal Verano Pears, we're offering a range of picnic delights, like white cheddar cheese, hickory smoked summer sausage, peanut butter pretzels, sweet raspberry galettes, and more. Packaged in an exclusively designed picnic-ready tin, this gift is ready to help you make any occasion a special one

We hope our quiz winners enjoy their prizes and the springtime weather!

 

Thank you all for your submissions!  

 


The correct answer to the Carbamates Analysis quiz: 

The answer for last quarter’s quiz was: incorrectly prepared reagent.  We prepared our “hydrolysis reagent” with CB910 instead of CB130.  The hydrolysis reagent CB130 is at a pH of 12.5, which is much more basic than our OPA diluent CB910 at a pH of 9.1.  From our Carbamates Manual: The separated carbamates are first saponified by NaOH at 100°C to release an alcohol, carbonate, and methylamine.  In the second post-column reaction, methylamine reacts with OPA and Thiofluor to form the highly fluorescent derivative.  So, if there is insufficient NaOH present for the first reaction, some of the carbamates do not fully hydrolyze.  

Chromatography Quiz #23: Polyether Antibiotics Analysis

What caused the noise for the blue signal in the troubleshooting chromatogram below?  Simply email your multiple choice answer as well as your full contact information to Rebecca at rlsmith@pickeringlabs.com by July 1st, 2016 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). 

Polyether Antibiotics Analysis

Pinnacle PCX post-column instrument (two-pump) is being used in a traditional HPLC setup as recommended by Pickering Laboratories.  The reference chromatogram and troubleshooting chromatogram are both shown.

Narasin Standard: 2.5 µg/mL, 100uL injection

Pickering Column: 2381750, Polyether Column, C18, 4.6x250mm

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

Column Temperature: 40 °C
Flow rate: 0.7 mL/min
Isocratic: 90% Methanol, 10% of 5% Acetic Acid solution in water

Post-column conditions:

Reagent 1: Concentrated Sulfuric Acid / Methanol (4:96 v/v)
Reagent 2: 60g of Vanillin in 950mL of Methanol
Reactor 1: Ambient, 0.1mL
Reactor 2: 90 °C, 1.4mL
Reagent flow rates: 0.3 mL/min

Black (Reference) Signal:

DAD detector 520nm with bandwidth of 4nm
No reference wavelength
Sampling rate >0.10min (2.0 S response time) (2.5Hz)

Blue (Troubleshooting) Signal:

DAD detector 520nm with bandwidth of 4nm
Reference wavelength of 360nm
Sampling rate >0.05 min (1.0 S response time) (5HZ)
Can you identify the error made when running the chromatogram?

Multiple Choices:

A) Bad lamp
B) Reference Wavelength
C) Sampling rate
D) All of the above

Troubleshooting:

quiz-chart

 

 

 

 

Chromatography Quiz #22

Chromatography Quiz #21 Results

Pickering Labs would like to congratulate our winners of our last newsletter’s AAA quiz: Helene Lachance from Shur-Gain Nutreco, Joy Gottlieb from New Mexico Department of Health Scientific Lab Division, Tom Schneider from Suffolk County Water Authority, and Narjes Ghafoori from LA County Environmental Toxicology Lab!   

cq_21They have each won and will shortly be receiving: A fabulous Godiva gift basket from www.winecountrygiftbaskets.com!   “It’s easy to celebrate with this chocolate collection…!

We hope our quiz winners enjoy this infusion of February cheer.

Thank you all for your submissions!  

 
  

The correct answer to the Amino Acid Analysis quiz: 

Although we gave credit for answers indicating Trione oxidation, the correct answer was actually a reduced reactor temperature!  When the reaction was performed inside the heated reactor at a temperature of 120°C rather than 130°C, the peak areas are approximately 2/3 of their original size.  Similar results could also be expected with reduced dwell times or reactor volumes.

Chromatography Quiz #22: Carbamates Analysis

Identify the error made when running the Carbamates chromatogram below and win a prize!  Simply email your answer as well as your full contact information to Rebecca at rlsmith@pickeringlabs.com by March 15th, 2016 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).   

Carbamate Analysis for US EPA Method 531.1
Pinnacle PCX post-column instrument (two-pump) is being used in a traditional HPLC setup as recommended by Pickering Laboratories.  The reference chromatogram and troubleshooting chromatogram are both shown.  Can you identify the error made when running the chromatogram?

Pickering Standard: 1700-0063 Carbamate Test Mixture, 2.5 µg/mL, 10 µL injection

Pickering Column: 0846250 Carbamate Column, C8, 4.6 x 250 mm

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

Column Temperature: 42 °C

Flow rate: 1 mL/min

Eluent Gradient:

TIME (MIN)

WATER

MEOH, %

0

85

15

1

85 15

44

25 75

44.1

0 100

49

0 100
49.1 85 15
57 85 15

 

Post-column conditions:

Reagent 1: Hydrolysis reagent CB130

Reagent 2: 100 mg of OPA, 2 g Thiofluor in 950 mL of CB910

Reactor 1: 100 °C, 0.5 mL

Reactor 2: ambient. 0.1 mL

Reagent flow rates: 0.3 mL/min

Detection: Fluorometer λex 330 nm, λem 465 nm

Troubleshooting Chromatogram:

cq-22-1

Reference Chromatogram:

cq-22-2