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ORDERING INFORMATION

Good laboratory practices the include proper use of any HPLC column and using the Cogent Phenyl Hydride™ columns are no different. On this page you will learn how to get the best results from these Columns. Click HERE for General Instructions on using all Cogent TYPE-C™ Columns.


Start-Up Instructions:


SELECT THE MODE OF HPLC to use with Different Types of Compounds: 

RP - Reversed-Phase: 
Aromatic compounds containing one or more phenyl rings, phenols and conjugated double bonds like beta-carotene. Examples are: Ketorolac, Acetaminophen & beta-Carotene.
             Ketorolac Chemical Structure Graphic  Acetaminophen Chemical Structure Graphic  beta-Carotene Chemical Structure Graphic
                          Ketorolac                                Acetaminophen                                        beta-Carotene
      
                      
ANP - Aqueous Normal-Phase:  Compounds with two or more primary or secondary amines and two or more hydroxyl groups. Examples are: Luteolin 7-O-Glucoside & Ethambutol.
               
Luteolin 7-O-Glucoside Chemical Structure Graphic    Ethambutol Chemical Structure Graphic

                   Luteolin 7-O-Glucoside                 Ethambutol

BEFORE YOU USE THE COLUMN:
  • Completely purge the solvent lines of the previous mobile phase unless it is freshly prepared and identical to the one you will be using for the Phenyl Hydride™.
  • Purge the injection port to remove any residual compounds from previous analyses.
  • Equilibrate the column for 15 minutes with the starting mobile phase conditions for your method.
START-UP INSTRUCTIONS: 
  • Review Specifications (tolerance for pH, mobile phase and pressure) for these columns; Click HERE
  • When using "dirty" matrices or aggressive mobile phases, inline filters or guard columns should be used.
  • All solvents used must be a minimum HPLC grade and should be degassed prior to use as well as degassed inline by your instrument.
  • Buffers should be prepared fresh and removed from the system and column daily.
  • Before attaching the column to the instrument, purge the solvent lines of previous mobile phases unless it is identical to the one you will be using for this column and freshly prepared.
  • Purge the injection port to remove any residual compounds from previous analyses.
  • Install the new column according to the instrument instructions following "Good Laboratory Practices". Ensure the fittings and tubing are all properly connected.
  • Equilibrate the column for 30 minutes with a 50:50 mixture of organic solvent and water including any additives that will be used in your method.
  • For RP: start with your mobile phase at 50:50 organic/water. To increase the retention, increase the water content.
  • For ANP: start with your mobile phase at 50:50 organic/water. To increase the retention, increase the organic content.
  • Before removing the column from the instrument and preparing it for storage: fill it with a mobile phase of 90:10 organic/water.
  • To prevent "pressure shock" and damage to the column; be certain all "pressure" in the system is zero before disconnecting it from the instrument.


Method Development Tips for Phenyl Hydride™:

Before starting any method development it’s important to  follow the START-UP INSTRUCTIONS above and to have read:  How to Use TYPE-C™ Columns.


RP - REVERSED PHASE METHODS:
  • Neutral Compounds:
  • Use a mobile phase consisting of either acetonitrile or methanol for the organic component and DI water.  For most applications, the addition of 0.1% formic acid to both the DI water and the organic solvent is recommended. This addition of 0.1% formic acid, is essential when using mass spectrometry detection. If there are only a few components in the sample with a small range of hydrophobicity, start with a high amount of organic (70-90%) in the mobile phase and increase the amount of water in 10% increments until the desired separation is achieved. For more complex samples (numerous constituents or large ranges of hydrophobicity) set up a linear gradient from 90% to 10% water over 10 minutes.  Adjust the range of the mobile phase composition and steepness of the gradient (time) until the desired separation is achieved.
  • Acids: When analyzing acids in reversed-phase it is essential to acidify the mobile phase using 0.1% formic acid to make the target analyte neutral.  Follow the same protocol above for neutrals when developing either an isocratic or gradient method. 
  • Bases:   Many bases will have a substantial hydrophobic component and can be retained by the protocols described above. Some small bases are too polar to be retained in reversed-phase due to the positive charge on the amine group at low pH and are better analyzed in Aqueous Normal Phase (ANP).  Working in high pH is not recommended as it can damage the HPLC instrument and/or the stationary phase.

ANP - AQUEOUS NORMAL PHASE METHODS: 
  • AcidsTo take advantage of the polar properties of acids, they must be ionized. Start with a buffer of 10 mM ammonium formate or ammonium acetate at pH 6.5.  (Lower or higher molarity buffers can be used as well.) For samples with few components, start at 50% water/10mM buffer and increase the acetonitrile/10 mM buffer content in the mobile phase as needed to get the desired separation and retention. For more complex samples use a test gradient starting at 90% acetonitrile/10 mM buffer decreasing to 20% over 10 minutes.  Adjust as needed to get the desired resolution and retention.
  • Neutral Compounds:  Only polar neutrals are likely to be retained in ANP.  The same protocols for acids can be used for polar neutrals in ANP. 
  • Bases:   To take advantage of the polar properties of bases, they must be ionized. Start with a buffer of 0.1% formic acid or 0.2% acetic acid. Both isocratic and gradient protocols described above can be used for bases depending on whether the sample has a few components or is complex. 
Click HERE for Troubleshooting Tips.

Following Column Use:

  • Before removing the column, fill it with 90:10 organic/water mobile phase preparing it for storage.
  • Click HERE for complete instructions on How to Store Cogent TYPE-C™ Columns
  • To prevent "pressure shock" and damage to the column; be certain all "pressure" in the system is zero before disconnecting it from the instrument.
 

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