Category Archives: Amino Acid Analysis

Amino Acid Analysis of Cell Culture Media

We have a new Method Abstract! MA371, Amino Acid Analysis of Cell Culture Media

Cell cultures are widely used to produce biopharmaceuticals and other biologically active compounds. The composition of the cell culture media affects the yield and structure of the desired products and must be carefully optimized. Cell culture media is typically composed of mixtures of amino acids, vitamins, carbohydrates, inorganic salts as well as different peptides, proteins and other compounds. As the cells grow, they consume nutrients and release target biopharmaceuticals as well as waste products.

Amino Acids serve as the building blocks of proteins, as well as intermediates in many metabolic pathways. Amino Acids are typically added to cell culture media to provide nutritional requirements for the cells. Monitoring and adjusting Amino Acid composition is an essential part of optimizing the manufacturing process to ensure high quality and optimum yield of the final product.

Amino Acid Analysis using cation-exchange chromatography with post-column Ninhydrin derivatization allows for easy determination of Amino Acid concentrations in many complex matrices, including cell culture media. The post-column method is very sensitive, reproducible and rugged. It has been and continues to be a method of choice for laboratories running biological samples, protein, peptides and foods analysis. Most chemical compounds present in the media do not interfere with analysis, so the majority of samples only need diluting with citric buffer and filtering before analysis. If serum is added to the media, then the proteins need to be precipitated using either Seraprep™ solution or ultrafiltration.

Pickering Laboratories, Inc. offers the complete solution for Amino Acid Analysis, including post-column derivatization instruments, columns, eluants, reagents and standards. The Pinnacle PCX derivatization system has a programmable column oven to allow for shorter run times and easy method optimization.

YChromatograms of Cell Culture Mediaou can download this application note, and many others from our website: www.pickeringlabs.com

METHOD
Analytical conditions
Column: High-efficiency Lithium cation-exchange column, 4.6 x 75 mm, Catalog Number 0354675T
Flow Rate: 0.55 mL/min
Mobile Phase: See method in Table 1

Post-Column Conditions
Post-column System: Pinnacle PCX
Reactor Volume: 0.5 mL
Reactor Temperature: 130 °C
Flow Rate: 0.3 mL/min
Detection: UV/VIS 570 nm for primary amino acids, 440 nm for secondary amino acids
Injection Volume: 10-50 uL

gradient table for AAA

2013 Pickering Laboratories North American Catalog

Our latest Catalog for North America is now available!

Click on the link to download the latest product Catalog from Pickering Laboratories, Inc.

Pickering Catalog: http://pickeringlabs-retentiontimes.com/wp-content/uploads/2013/08/2013_PickeringLabs_Catalog_NorthAmerica_LowRes.pdf

Inside our catalog you will find a complete product listing for all instruments and consumables we currently have available. From Sample Cleanup to Post-Column Derivatization, to artificial Perspiration and Saliva, we hope you find something you will find useful!

Pickering Laboratories in 2013

By Wendy Rasmussen

In recent years, we have released several new products and applications, and with still more on the horizon, it occurred to me that now would be a great time to summarize the Pickering of today – our mindset and our wide variety of products & applications.

Acai Berries
Acai Berries

No longer are we simply the “Post-Column Company”. We are the “Automated-Sample Antioxidants” company.  Think of us as the new “super fruit.” The Acai berry, or perhaps the new Chia Seeds (incidentally, we do have a post-column application for the identification & quantitation of  Antioxidants in a variety of matrices).

We are still very active, and we as a company plan to be here for many years to come. We are still the company founded on chemistry and a desire to to teach, to spread our technical expertise, to support our customers.

It’s been a few years now since Pickering began distributing and supporting our LCTech Product line.  The products have shown an ever increasing interest here in the US and in Canada (our official Sales Territory for this product line). We are very proud to offer these products and we hope we can develop it further in the future.

Historically, we have provided the back-end of an analysis (post-column derivatization). Nowadays, we can provide the front-end of analysis as well (the sample-cleanup).

In thinking about our product offerings, I realized that a simple list does not effectively show the scope of the products we have to offer in 2013 – primarily because we have a lot of overlap between products and product lines.  We’re not a vertical company in that regard.  I suppose one could say our product offerings are more circular in that many do not fall into a single distinct category.  I am a very visual person, and for me, a Venn diagram and our overall “product scale” really helped to understand and clarify our products:

Venn Diagram of Pickering Laboratories
Venn Diagram of Pickering Laboratories
General "Product Scale" for Pickering Offerings
General “Product Scale” for Pickering Offerings
For those of who like lists, you can find one Here, on Pickering’s website, and on LCTech’s Website

For any Questions, please feel free to contact us:

Pickering Laboratories, Inc.
Mountain View, California
Email: sales@pickeringlabs.com
Phone: (direct) 650-694-6700 or (toll-free) 800-654-3330

 

Image of Acai Berries:  http://acaiberryeducation.com/

Pickering Exhibits at Pittcon 2013

This year’s Pittcon was held in Philadelphia, Pennsylvania from March 17-21, 2013.

view of philly from the museum On display were Pinnacle PCX, EluVAC, and a video of the FREESTYLE Automated Sample Preparation Instrument.  The video was a new technique for us, and it was well received.

The video in our booth was a combination of the separate videos for each module of the FREESTYLE Automated Sample Preparation Instrument. You can view the originals here (they are very informative, and we highly recommend them): FREESTYLE VIDEOS

Wendy, Mike and David enjoyed visiting with existing and future customers, as well as our distributors from around the world, including our Canadian distributor, Chromatographic Specialties.

Before the show opened, we had the opportunity to explore the city.  Philadelphia is one of our oldest cities and is full of history and landmarks. Some of this editor’s favorites include Independence Hall and the surrounding neighborhoods, and the Art Museum (see more below).  On this trip, we explored Reading Terminal Market, and sampled that famous cuisine, the Philly Cheese steak Sandwich. I’m told it is not truly a Philly Steak sandwich unless you have it with Cheez Whiz. Any type of real cheese doesn’t count. I made that mistake on my first trip to Philadelphia, and the man behind the counter let me know it!  I wasn’t about to make that mistake again:

philly cheesesteak

The Philadelphia Museum of Art is known for its amazing collection of artwork (viewed on a previous visit), but is also made famous by the scene of Sylvester Stallone running up the stairs in the movie Rocky.  Since the author of this entry happened to be training for a race, she decided that a little cold wasn’t going to stop her. So she put on her iPod (Rocky Theme playing, of course), and ran from the hotel to the top of the steps and back (total distance: about 2.8miles).

view from the bottom

wendy philly top of steps

wendy rockyOn the return to the hotel, I came across a version of the LOVE sculpture, by American Artist Robert Indiana. I always thought it was was much bigger…

love sculpture

 

For those of you interested in learning more about the sculpture, click HERE

If you’re ever in Philadelphia, the Philadelphia Museum of Art is definitely worth a visit.

But if you can’t make the trip to Philadelphia, you could also stay home and watch Rocky…

Chromatograpy Quiz No. 12

Chromatography Quiz #11 Results

We would like to congratulate all six grand prize winners of our last newsletter’s Glyphosate Analysis Chromatography Quiz: Thomas Schneider from Suffolk County Water Authority, Hélène Lachance from Shur-Gain Nutreco in Canada, Jim Balk from DHHS Public Health Environmental Laboratory, Tom Phillips from Maryland Department of Agriculture, Matthew Hartz from Underwriters Laboratories, and Narjes Ghafoori from the LA County Environmental Toxicology Laboratory!!!

tea They have each won and will be receiving: a wooden chest of premium and specialty teas by Silkenty Tea (purchased from GiftTree.com)!  We would like to thank all of you for your submissions.

 

 

The correct answer for the modified Glyphosate chromatogram: Iron contamination has been introduced into the system and has fouled the column, resulting in poor peak shape and a retention time shift in AMPA.  If the problem worsened, the peak shapes would continue to deteriorate and eventually the Glyphosate peak could vanish completely.

The recommended action would be to reverse-flush the column with RESTORE™. The presence of polyvalent metal ions (especially iron) can rapidly lead to serious degradation of column performance.

Thank you!

Pickering Labs

Chromatography Quiz #12:

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 April 30, 2013 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 remain 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 Labs.  The chromatogram is a close-up of a customer’s reoccurring baseline noise, and the quiz question is: what is causing the noise?

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

Please use our website to view any reference Amino Acid chromatogram – this quiz is not application-specific per say, and as such any of our AAA methods could be applicable.  If you have any questions, please don’t hesitate to email Rebecca at rlsmith@pickeringlabs.com.

bad baseline chromatogram quiz 12

 

 

Midwest AOAC Meeting

This year’s Midwest AOAC meeting was held in St. Louis, Missouri from June 4-6. 
It was well-attended with a good technical program.

Gateway Arch in St. Louis, MO*

At this year’s meeting we hosted an Amino Acid workshop, in which attendees learned tips and troubleshooting techniques for amino acid analysis. All analytical methods were covered, including some discussions on sample preparation for amino acids.

The post-column analysis of amino acids is a tried and true technique. By derivatizing the amino acids post-column, any matrix components which might otherwise interfere with the derivatization steps are eliminated, leaving only the amino acids to react with the reagents. This leads to exceptional reproducibility, sensitivity and ruggedness.

Our Pinnacle PCX instrument is especially suited to amino acid analysis with its heated column oven (to perform temperature gradients), and pulse-free syringe pumps. More information can be found on our website: www.pickeringlabs.com, or by downloading one of the following:
MA380:  30-Minute Amino Acid Analysis of Hydrolyzed Samples
or our Amino Acid Brochure
View of St. Louis from the top of the Arch

* Interesting Fact: Gateway Arch is part of the Jefferson National Expansion Memorial. It was designed in 1947, but construction did not begin until 1965.  It opened to the public in 1967. http://en.wikipedia.org/wiki/Gateway_Arch

Chromatography Quiz #10

Chromatography Quiz #9 Results

We would like to congratulate the grand prize winner of our last newsletter’s Amino Acids Analysis Chromatography Quiz: Jim Balk from the State of Nebraska DHHS Public Health Environmental Laboratory!!!

He has won, and will shortly be receiving: an Amazon.com gift card!  Additionally, all participants will be receiving a gift card from Amazon.com!  Again, we would like to thank all of you for your submissions.  

The correct answer for the modified Amino Acids chromatogram: the sample has been prepared at the wrong pH.  The Aspartic Acid peak is misshapen and splitting, a clear indication that the sample is not sufficiently acidic. After checking the sample pH to determine the required correction, Li220 can be used to adjust to the appropriate pH. 

Thank you! 

Pickering Labs

Chromatography Quiz #10:

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 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). 

Hint: Quiz #8 was so popular that we’ve decided to run another hardware problem!

Amino Acid Analysis of Protein Hydrolysate

Pickering Standard: 012506H Protein Hydrolysate Standard, in 0.2 N Sodium citrate buffer pH 2.20, 0.25 µmole/mL, 10 µL injection

Pickering Column: 1193250 Standard Sodium Cation-exchange Column, 3.0 x 250 mm

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

Column Temperature: 48 °C

Flow rate: 0.3 mL/min

Eluant Gradient:

TIME
Na328 %
Na740 %
RG0011 %
0
100
0
0
10
100
0
0
32
0
100
0
56
0
100
0
56.1
0
0
100
58
0
0
100
58.1
100
0
0
70
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

Chromatogram to Troubleshoot:
Full Chromatogram:

Enlarged View:
Further Enlarged View:

Reference Chromatogram: Click HERE and scroll down to PN 1193250