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you’re in > Crawford Scientific > Agilent Technologies > HPLC Columns > Agilent ZORBAX Method Development Strategy
 

Agilent ZORBAX HPLC Method Development Strategy

Chromatographic resolution between two or more peaks depends upon three factors; column efficiency, selectivity and retention. ZORBAX columns have a wide variety of bonded phases for optimizing selectivity and retention - the key to the best resolution.
These columns are available in a variety of particle sizes - from 1.8-7µm to provide the efficiency needed for any separation.

Start method development at low pH (pH 2-4)
With so many choices available, how do you select a column for method development? The recommended starting point for method development is using a buffered low pH mobile phase - around pH 2-3. Using a low pH mobile phase results in the best peak shap for basic compounds on silica-based columns. At low pH, the silanols on the silica are fully protonated so positively charged basic compounds do not interact strongly. The result is good peak shape. Many acidic compounds are non-charged, maximizing their retention at low pH. These observations are key advantages to method development at low pH. For standard analytical work start method development with acetonitrile as the mobile phase organic modifier and 25 - 50 mM phosphate buffer (pH 2-3) as the aqueous component. This provides good pH control, necessary for the most reproducible analyses of ionizable compounds.

Choose ZORBAX Eclipse Plus first for best peak shape
Select ZORBAX Eclipse Plus C18 or C8 columns first for method development at low pH. Eclipse Plus columns are the newest addition to the Eclipse family and use improved silica and bonding technologies to provide good peak shape for basic compounds. Eclipse Plus columns can be used from pH 2-9 providing method development flexibility. They are stable down to pH 2 making them an ideal choice for initial method development.

Optimize solvents and bonded phases at low pH
The initial method development steps may lead very quickly to a satisfactory separation. But if more optimization is needed, the acetonitrile can be replaced by methanol or tetrahydrofuran and the separation re-optimized. This step may lead to a satisfactory solution, but if still more optimization is needed, the column bonded phase can be changed. At low pH there are many bonded phase choices available for optimization. These include the Eclipse Plus phases as well as the Eclipse XDB family with C18, C8, Phenyl and CN. Alternate choices include six different StableBond bonded phases: SB-C18, SB-C8, SB-Phenyl, SB-CN, SB-C3, and SB-Aq. It may be necessary at low pH to improve the retention of acidic compounds. For these situations, lower the pH even further, down to pH 1-2, and use StableBond columns. These columns provide the greatest stability at very low pH and provide many selectivity options for achieving the highest resolution separations.

Method Development at Mid pH (4-9) ZORBAX Eclipse Plus
There are some samples that may not be resolved at low pH or may have better solubility and stability at mid pH. While still using the Eclipse Plus C18 column, the mid pH range can be used for method development. The Eclipse Plus column is stable up to pH 9 so it is equally reliable at mid pH. These double endcapped columns have two key advantages – good peak shape at low and mid pH, as well as sufficient bonded phase density to protect the column from silica degradation from pH 6-9. At mid pH, basic compounds (e.g., amines) may still have a positive charge and the silanols on the silica surface may have a negative charge. Therefore covering as many silanols as possible leads to the best peak shape at mid pH. This makes the Eclipse Plus C18 the best starting choice for a column at mid pH. Phosphate buffer is usually the first choice for mobile phase modifier at pH 7 because its buffer range is pH 6.1-8.1. A second choice for mid pH is acetate buffer since it buffers from pH 3.8-5.8 and its volatility makes it a good choice for LC/MS compatibility.

Alternate selectivities – ZORBAX Eclipse XDB-Phenyl, CN and Bonus-RP
The method development process at mid pH mimics the process at low pH with optimization of the organic modifier and selecting an alternate bonded phase if resolution is not achieved after that step. The alternate bonded phases at mid pH are the Eclipse XDB-Phenyl, Eclipse XDB-CN and Bonus-RP. They provide very different selectivities for many samples and the method development process is followed again. The Bonus-RP column has a polar embedded amide group that provides different selectivity for many samples, provides good peak shape for basic compounds and allows the column to be used with up to 100% aqueous mobile phases.

Method development at high pH (9-12) ZORBAX Extend-C18 columns
At low or mid pH, some separations of basic compounds may still not have enough retention or the desired selectivity. For these samples, high pH separations may be appropriate. Until recently, high pH separations on silica were avoided because of short column lifetimes, due to dissolution of the underlying silica gel. Newer column technologies, i.e. the ZORBAX Extend-C18, can protect the silica from dissolution, so a reasonable column lifetime can be achieved and the selectivity advantages of high pH can be explored. The mobile phase buffer choices at high pH with the Extend-C18 column are organic buffers like triethylamine and ammonium hydroxide. These buffers are best used with methanol as the organic modifier to extend the column lifetime at high pH.

 
ZORBAX columns have been carefully designed for optimum sample resolution and longest column lifetimes for the low, mid, and high pH ranges.
 
Agilent ZORBAX HPLC pH Method Development Strategy
 

The figure below defines these pH regions and highlights the benefits of working in the different pH regions.

Low pH 1-3 - Region A

  • Start method development at low pH, where silanols on a RP-HPLC column are protonated. This minimizes peak tailing by eliminating silanol/base interactions.
  • At low pH basic compounds are protonated, and their retention may be reduced.
  • Acidic compounds may be protonated and have increased retention.
  • Retention times are usually stable with small changes in pH, producing a robust method.
  • Volatile mobile phase additives, such as formic acid or trifluoroacetic acid (TFA), are often used at low pH with LC/MS.

Mid pH 3 - 8 - Region B

  • Develop methods at pH’s at least 1 pH unit different from pKa to minimize changes in retention with pH .
  • Some silica surface SiOH becomes SiO- at pH 4 - 6, tailing interactions may be possible.
  • Minimize interactions by selecting an endcapped column, using additives such as TEA (less desirable) or using "polar" bonded phases.
  • As pH increases, silica dissolution is decreased by endcapping .
  • Silica breakdown prevented by innovative bonding chemistry and use of Rx-SIL.

High pH - Region C

  • In this region, basic compounds may be in their free base form.
  • Increased retention; and resolution of basic compounds are likely.
  • Retention changes little in this region - thus robust methods can be developed.
  • Silica breakdown prevented by innovative column chemistry, use of RX-SIL and optimum mobile phase.
  • Ammonium hydroxide is a volatile mobile phase buffer for this region.
  • Changing to Common Column Diameters to Maintain Equivalent Linear Velocity for Different Column IDs
  • The equation beneath the chart helps determine the flow rate for the smaller column ID if the column you are using differs from those listed here. The goal is to keep the same linear velocity when you scale down to keep peaks eluting in the same order. Or if you are just starting with one of the smaller columns, then you know what corresponds to a typical flow rate. For a capillary column, we are using flow rates in the low μL/min range and for nano columns the flow rates are down to nL/min – very low flow rates.
 

For additional method development guidelines, review the section on method development from low to high pH.

 
For additional method development guidelines, review the section on method development looking at the molecular weight aspect



 
 
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