New Technologies to improve HPLC Compendial Pharmaceutical Methods

USP37-NF32_book imageAllowable Adjustments to Pharmacopoeia Methods for Isocratic and Gradient Separations

Effective August 2014, the United States Pharmacopeia and the National Formulary (USP-NF) published the latest revision to General Chapter <621> on Chromatography that further clarifies what “allowable adjustments” can be made to USP methods without having to revalidate these methods. In other words, any of these changes made to a particular method would still meet the original system suitability requirements. Understanding this latest revision is critical to improving productivity and cutting costs in any lab environment, yet it can be overwhelming to many less experienced chromatographers.

Generally these USP monographs were created for older generic drugs that are still manufactured and sold worldwide, and many of these methods were developed on older materials and are time-consuming to run. To increase productivity in the lab, improvements must be made to these methods in order to reduce these lengthy run times while still maintaining the system suitability requirements defined by the monograph.

Previous versions of USP <621> simply said that adjustments could be made to help meet system suitability; however, the extent of an “adjustment” was not defined. The 1999 revision then put restrictions on the amount of adjustment that could be made before revalidation was necessary. Nearly a decade later, the USP35-NF30 S2 (2007 revision) incorporated additional clarification to allowable adjustments, harmonized efforts with European Pharmacopoeia (Eur. Ph.), and also included a new tool to help determine column “equivalency”. Today, the latest USP37-NF32 S1 (2014 revision) provides a much clearer definition of what is allowable.

Before we dive into the redefined allowable adjustments, let’s review the definition of system suitability which is an integral part of HPLC methods and can be determined from a variety of parameters. Suitability verifies the system is adequate for the intended analysis. It is generally performed by replicate injections of suitable standard or other solution, as specified in method. Keep in mind, each HPLC method in a USP monograph may have its own specific system suitability requirements. And for these compendial methods, results are not valid unless system suitability requirements can be successfully met. Here is a quick review of some suitability parameters you will need to understand:

Resolution (Rs) ensures closely eluting compounds are resolved. It establishes resolving power of the system, where column efficiency contributes to resolution and column efficiency can affect sensitivity. Efficiency can be specified for system suitability, but it’s not a true measure of the resolving power of a system.

Diagram 1





Signal-to-Noise (S/N) is critical for accurate identification & quantitation. It is best if the noise measurement is taken before and after the peak of interest.

Diagram 2




H = height of the peak of interest from peak apex to a baseline extrapolated over a distance ≥5X times the peak width at half height (W1/2); h = difference between largest and smallest noise values observed over a distance ≥5X peak W1/2 and, if possible, situated equally around the peak of interest

Diagram 3







Symmetry Factor (As), also referred to as Tailing Factor (T), is a measure of peak symmetry and important for defining peak shape. Poor peak shape can affect quantitation and method precision. There are many different ways to measure this value (e.g. peak symmetry, peak asymmetry) yet most chromatography software will allow you to choose USP tailing.
Diagram 4



W0.05 = width of peak at 5% height f = distance from the peak maximum to the leading edge of the peak, the distance being measured at a point 5% of the peak height from the baseline
diagram 5






Allowable Adjustments and System Suitability

So now that we understand the critical method values, what are the “allowable adjustments” presented by the USP and how will they affect system suitability? First off, let’s break down the key points of USP <621> into the following:

  • Defines chromatographic terms and procedures
  • Allows for adjustments to methods
  • “in order to comply with system suitability…”
    • • when “it may be desirable to use a column with different dimensions…”
    • “only when suitable standards (including Reference Standards) are available for all compounds used in the suitability test and the adjustments or column change yields a chromatogram that meets all the system suitability requirements specified in the official procedure.”
    • “are not to be made in order to compensate for column failure or system malfunction”
  • Defines the maximum allowable adjustments
  • Adjustments may require additional validation
  • Multiple adjustments should be considered carefully
  • Addresses the continued trend toward <3 μm particles, superficially porous particles (i.e. core-shell particles), and fast LC / UHPLC

The maximum allowable adjustment is outlined in Figure 1, for isocratic methods, and Figure 2, for gradient methods. Adjustments may require verification, and consider multiple adjustments carefully as they can have a cumulative effect on system performance. Also, changes in chemical characteristics of stationary phase, such as a change from L1 (C18) to L7 (C8), is a modification and will require revalidation.


Mobile phase composition can be altered for isocratic methods following the allowable adjustments provided in Figure 1. Adjusting the minor component in the mobile phase can increase or decrease retention of the target analytes. However, for gradient elution (Figure 2) adjustments to the composition of the mobile phase may cause changes in selectivity and are not recommended.


Mobile phase pH of the aqueous buffer can be changed ± 0.2 units, which is applicable to both gradient and isocratic methods. Remember that the pH scale is logarithmic (1 unit = 10X increase), so a small change in pH can have a big impact on chromatographic performance. The main strategy is to ensure the mobile phase pH keeps the drug in one state; low pH can protonate acidic compounds (silanols), while high pH can neutralize basic compounds; avoid pH = pKa.

For buffer concentration, the minor ± 10% adjustment allowed by USP will most likely not make much difference in method performance. However, if buffer concentration is insufficient, pH can change within the mobile phase especially with large volume injections. Keep in mind, concentration is most critical at pH extremes of that buffer.

Column temperature is often a neglected method parameter and should always be controlled, even if the method calls for ambient temperature. Ambient temperature is relative and different throughout the year, in different labs, in different countries, and in different locations within a lab. Variations in temperature can cause shifts in retention time. Higher temperature can decrease mobile phase viscosity, improve column efficiency, lower column back pressure, and decrease analysis time. However, use caution as temperature changes may also affect selectivity.

Injection volume can be adjusted as much as desired as long as it is consistent with accepted precision, linearity, and detection limits. Note that excessive injection volume can lead to unacceptable band broadening, causing a reduction in column efficiency and resolution. These adjustments are applicable to both gradient and isocratic methods.

For isocratic separations only (not allowed for gradient separations), the ratio of column length (L) to the particle size (dp) must remain constant or within range between -25% to +50% of the prescribed L/dp ratio. Column ID can also be adjusted as long as the linear velocity is kept constant by adjusting flow rate (F), accordingly. A smaller column ID will help with solvent savings, reducing band broadening (sharper peaks), and increasing resolution (sharper peaks).

Flow rate can be adjusted ± 50% at any time for isocratic methods. Increasing flow is the easiest way to reduce run time. For example, 2X flow rate = ½ run time but with a slight decrease in resolution, so you need to make sure that the Rs still meets suitability requirements.

diagram 6




USP <621> and Guard Columns

Yes, in the latest revision guard columns are explicitly allowed. In HPLC procedures, a guard column may be used with the following requirements, unless otherwise is indicated in the individual monograph:

a. The length of the guard column must be ≤15% of the length of the analytical column,
b. The inner diameter must be the same or smaller than that of the analytical column, and
c. The packing material should be the same as the analytical column (e.g. silica) and contain the same bonded phase (e.g. C18).

In any case, all system suitability requirements specific in the official procedure must be met with the guard column installed.

But why would one want to use guard columns? Guards protect valuable analytical columns by removing particulates and strongly retained sample components that may accumulate on column, increase lifetime of analytical column, and maintain high column efficiencies, resolution, and peak shape. Figure 3 details the general criteria for when to replace guard columns or guard cartridge.

Upgrading from Fully Porous to Core-Shell 

Now let’s take a look at the allowable adjustments and how they can be incorporated with newer technologies on the market, such as moving to higher efficiency core-shell columns. What is it that sets core-shell columns apart from conventional, fully porous packed columns? The answer is simple: columns packed with coreshell particles will deliver significantly higher efficiency (N) than columns packed with fully-porous particles of the same diameter.1 To the end-user, this means that you are going to see narrower peaks, improved resolution, and shorter run times. Better chromatography results in higher productivity, which in turn results in greater cost savings.

Figure 4 shows the relationship between column efficiency, in plates per meter, as a function of particle size for fully porous and core-shell packed columns. As you can clearly see from this figure, columns packed with core-shell particles provide higher efficiency values than columns packed with fully porous particles of the same diameter. This means that, all other things being equal, using columns packed with core-shell particles will result in narrower analyte peaks than columns packed with fully porous particle of the same diameter. This, in turn, means that our ability to resolve closely-eluting compounds is greatly increased when using coreshell particles. Please also note that the pressure will simply be proportional to particle diameter, independent of whether it is fully porous or core-shell in morphology. Thus, the core-shell columns will generate the same pressure as fully porous particle of equivalent diameter.


Also, the efficiency value for the 1.3 μm and 1.7 μm particles taken were generated using 2.1 mm ID columns, while the efficiency values for larger particle columns were derived from 4.6 mm ID columns. This was done to reflect what the typical enduser will actually see in their methods, with most UHPLC methods being performed on narrow-ID columns while HPLC methods tend to be performed using 4.6 mm ID columns.

Caution should be taken when the adjustment results in a higher number of theoretical plates which generates smaller peak volumes, which may require adjustments to minimize extra-column band broadening by factors as instrument plumbing, detector cell volume and sampling rate, and injection volume. For gradient separations, changes in length, column inner diameter, and particle size are not allowed.

In conclusion, USP General Chapter <621> defines the “allowable adjustments” constrained within certain allowable values. These adjustments permit flexibility for users of compendial methods to greatly increase productivity in the lab by ultimately reducing run times while also minimizing solvent usage and cutting costs. System suitability must be met or revalidation will be required. The adjustments to particle size, column length, and column inner diameter are allowed for isocratic methods only. Upgrading to newer core-shell particle technology is a simple solution that can dramatically improve both isocratic and gradient separations. USP <621> also allows for guard columns as long as stated requirements for their use are met.

1. Gritti et al., Journal of Chromatography A, 1217 (2010) 1589

Michael Klein is a Brand Manager for HPLC & UHPLC Products at Phenomenex, Inc.

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