Category Archives: Post-column

Omelets and Pressure

By Saji George

There is a saying, “one cannot make an omelet without breaking a few eggs”. What do omelets and eggs have to do with post-column instrumentation? No direct correlation, but both have an underlying parameter that is important in achieving the final goal. Too much pressure on the egg and you will end up with egg shells in your omelet, too little pressure and there might not be an omelet.

In the case of the post-column derivatizer, there is a sweet spot (pressure range) that relates to a system that is working well. The system pressure is mostly comprised of the reagent pump(s) pressure(s) and gives an idea of how the overall system is performing. Too high and there is a constriction problem, too low and there is a leak in the flow path.

The pump pressure measures the pressure downstream from the pressure transducer. This includes the heated reactor, filters, unions, detector, and flow path tubing. All of these components have to be looked into when troubleshooting a high pressure problem. The best troubleshooting method is to remove one component at a time to look for the pressure drop; it is best to work backwards (start from the outlet of the detector). Components to pay attention to are: back pressure regulator, detector, ambient reactor, heated reactor, restrictors if there are any and last but not least the reagent filters. A significant drop (greater than 100 psi) in pressure when one of the components is removed would indicate that it is the source of the problem. The same thought process works for leaks also! Take a lint free towel and wipe all connections one by one, looking for areas of wetness that would indicate a leak.

Since summer is around the corner and graduations, admissions applications and final exams (to name a few) have been taken care of, please do take time to hit the sweet spot by enjoying the great outdoors!  

Replacing the Over-pressure Relief Valve Cartridge

Mixing-Manifold-1a 
Cleaning and Reassembly of the Over-pressure Relief Valve

  1. Remove the tubing connections to the Mixing Manifold. Use a 3/32” hex driver to remove the 2 screws holding the Mixing Manifold to the chassis. Use a 3/8” wrench to remove the end cap and discard the old Over-pressure Relief Valve Cartridge. Ultrasonicate the Mixing Manifold for at least 30 minutes. Rinse well with DI water.
      
      
     
  2. Connect the outlet of your HPLC pump to the Mixing Manifold inlet and pump 100% water at 0.5mL/min to verify the Mixing Manifold is not clogged. If the Mixing Manifold is still clogged after cleaning in an ultrasonicating bath, replace the Mixing Manifold Assembly (PN 1452-0040).
     
  3. Turn off the HPLC flow and make sure there is no pressure on the Mixing Manifold. Insert the new OPRV cartridge, green side down, and screw on the end cap to 20”lbs of torque. To approximate this level of torque, first finger tighten, then tighten an additional 1/8-1/4 turn with a 3/8” wrench.
     
  4. To verify the opening pressure of the Over-pressure Relief Valve, plug the two side inlets of the Mixing Manifold and turn on the HPLC pump to 0.5mL/min. Allow the pressure to slowly rise. The Over-pressure Relief Valve should open around 485psi. If the opening pressure is too low, tighten an additional 1/8 of a turn with a 3/8” wrench.

David Mazawa
david.mazawa@pickeringlabs.com
Technical Support Chemist
Pickering Laboratories, Inc.
1280 Space Park Way
Mountain View, CA 94043 USA
Phone: (650)694-6700 ext. 710
Fax: (650)968-0749

 

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

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/

Chromatography Quiz No. 2

Congratulations to the winners of our last newsletter’s Chromatography Quiz: Matthew Hartz, Jamie Palmer, and Keena Njoroge from Underwriters Laboratories, Sudheer Reddy from Chemtex, and Becky Canela from Environmental Laboratory Services!

They’ve each won, and will shortly be receiving from Gifttree.com, two dozen irresistible cookies in five flavors: White Chocolate Hazelnut, Snickerdoodle, Peanut Butter, Oatmeal Raisin, and Chocolate Chip.

The correct answer for the modified Carbamates chromatogram: we reversed the two reagents. The OPA reagent was pumped in the Reagent One position, and the Hydrolysis reagent was pumped in the Reagent Two position. Thus 1-Naphthol, which is naturally fluorescent, appears full-sized. The other Carbamate peaks have different sizes due to their varying rate of hydrolysis – the high pH of the OPA reagent will allow for some but not complete hydrolysis prior to detection.

Chromatography Quiz: Amino Acid Analysis

Identify the error made when running the Amino Acids chromatogram below and win a prize! Simply email your answer and your full contact information to Rebecca at rlsmith@pickeringlabs.com by March 1st in order to win. 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

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:

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, 570nm for primary amino acids, 440nm for secondary amino acids
Hint: Assume in this case that both Guard and Analytical column are good. To see a standard Amino Acid chromatogram, click here