Category Archives: Chromatography Quiz

amino acid, amino acids, Chromatography Quiz

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
carbamate, Chromatography Quiz, post-column derivatization

Chromatography Quiz

Chromatography Quiz #7 Results
 

We would like to congratulate the grand prize winner of our last newsletter’s Amino Acid Analysis Chromatography Quiz: Jaime Lee Palmer from Underwriters Laboratories!!!

She has won, and will shortly be receiving: a gift card for Barnes and Noble!  Additionally, for this quiz all our participants will each be receiving a Smiley Face Sampler Box courtesy of The Popcorn Factory!  Again, we would like to thank all of you for your submissions. 
 
The correct answer for the modified Amino Acids chromatogram: the Trione reagent is oxidized.  Either the reagent has been improperly stored (not under Nitrogen), or has been in extended contact with air. 

Thank you! 
Pickering Labs

Chromatography Quiz #8:

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 January 31st 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). 

Carbamate Analysis for US EPA Method 531.1

Pickering Column: 1846250 Carbamate Column, C18, 4.6 x 250 mm

Sample: Actual customer’s 531.1 standard at 10ppb

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

Column Temperature: 42 °C

Flow rate: 1 mL/min

Eluant Gradient:
     

TIME
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  ex 330 nm,  em 465 nm

Full Chromatogram:

 





 

 Enlarged View:

 




 
Further Enlarged View:
  

For an example of a Good Carbamate Chromatogram, click here

Amino Acid Analysis, Chromatography Quiz

Chromatography Quiz No. 7

Chromatography Quiz #6 Results
We would like to congratulate the grand prize winners of our last newsletter’s Aflatoxins Analysis Chromatography Quiz: Matthew Hartz and Keena Njoroge from Underwriters Laboratories, Steven Moser from the Oklahoma Dept of Agriculture, Food & Forestry, and Holger Franz from Dionex Germany! 
They have won, and will shortly be receiving: a gift basket stuffed with Starbucks coffee, tea, and treats!  Additionally, for this quiz all of our participants will each be receiving a $20 gift card from Starbucks! Again, we would like to thank you all for your submissions.  
The correct answer for the modified Aflatoxins chromatogram: The Aflatoxins G1 and B1 must be derivatized in order to achieve maximum sensitivity.  The derivatization is achieved either by reaction with Iodine, or with a photochemical reactor.  In this case, we achieved the modified chromatogram by turning off the UVE prior to injection.  The same chromatogram would be noticed if the Iodine reagent was not pumping.  
Thank you! 
Pickering Labs
Chromatography Quiz #7:
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 August 31st 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 Protein Hydrolysate  
Pickering Standard: 012506H, 10 μL injected of 0.25 μmole/mL standard in both chromatograms
Pickering Analytical Column: 1154150 High-Efficiency Sodium Cation-Exchange Column (4.0 x 150 mm)
Pickering GARDTM Column Protection System: 1700-3102
Normal Operating Conditions: (for reference only, condition changes may be reflected in chromatogram)
Column Temperature: 48 °C
Flow rate: 0.40 mL/min
Eluent Gradient:
TIME
1700-0112 %
Na740 %
RG011 %
0
100
0
0
12
100
0
0
34
0
100
0
53
0
100
0
53.1
0
0
100
55
0
0
100
55.1
100
0
0
67
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
Amino Acids Chromatogram to Troubleshoot:
Amino Acids Reference Chromatogram:


aflatoxin, Chromatography Quiz

Chromatography Quiz No. 6

Chromatography Quiz #5 Results
We would like to congratulate the grand prize winner of our last newsletter’s Carbamate Chromatography Quiz: June Black from the Pennsylvania Department of Environmental Protection! 
She has won, and will shortly be receiving, an extensive California Wines Gift Basket!  Additionally, for this quiz all of our participants will each be receiving a $25 gift card from Bevmo! Again, we would like to thank you all for your submissions.  
The correct answer for the modified Carbamate chromatogram: The system was improperly equilibrated at a higher percentage of methanol (50:50 Water:Methanol) prior to injection.  After injection, the gradient program began with normal starting conditions (85:15 Water:Methanol). For the less retained compounds, the improper conditions on the column at the time of injection will push them through faster, causing coelutions.  Longer retained compounds are less affected by starting conditions of the column, and impacted more by the gradient (which was correct) so you see less of an effect on the later part of the chromatogram. 
Thank you! 
Pickering Labs
Chromatography Quiz #6:
Identify the error made when running the Aflatoxins 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 31st 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). 

Aflatoxins Analysis by AOAC Official Method 2005.08
LCTech Standard: 0.25 ng B2, G2 and 1.0 ng B1, G1 injected on-column in both chromatograms
LCTech Analytical Column: 150 x 4.6 mm; RP C18 (P/N 10522)
LCTech Guard Column: 8 x 4 mm (P/N 10523; holder P/N 10750)
Normal Operating Conditions: (for reference only, condition changes may be reflected in chromatogram)
Column Temperature: 36 °C
Mobile Phase: Water/methanol/acetonitrile 55/30/15
Isocratic Flow rate: 1.3 mL/min
Post-column conditions:
Photochemical Derivatization: 254nm UV low pressure lamp (LCTech UVE derivatizer, Pickering P/N 1100-3347)
Detection: Fluorometer:  ex 365 nm, em 460 nm

carbamate, Chromatography Quiz

Chromatography Quiz No. 5 and Results from Quiz 4

Chromatography Quiz #4 Results
Congratulations to the grand prize winner of our last newsletter’s Amino Acid Chromatography Quiz: Matthew Hartz from Underwriters Laboratories!

He has won, and will shortly be receiving, a $150 gift card to Dick’s Sporting Goods! Additionally, for this quiz our other participants will each be receiving a $20 gift card from Amazon.com. Thank you all for your submissions!

The correct answer for the modified Amino Acid chromatogram: the guard column has been contaminated with proteins. The dirty guard causes poor peak shape and loss of resolution on key pairs of amino acids. Replacing the guard will restore the chromatography.

Thank you!
Pickering Labs

Chromatography Quiz #5:

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 December 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).

Carbamate Analysis for US EPA Method 531.1

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

Pickering Column: 1846250 Carbamate Column, C18, 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
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 ex 330 nm, em 465 nm

For an example of a Good Carbamate Chromatogram, Click Here

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)