Adjustment of Chromatographic Conditions as per BP / USP (for HPLC)

Allowable adjustments of Chromatographic Conditions as per BP / USP :

1.0 Adjustments / variations of operating conditions to meet system suitability:

• The extent to which the various parameters of a chromatographic test may be adjusted to satisfy the system suitability criteria without fundamentally modifying the method.
• Adjustment to chromatographic systems performed in order to comply with system suitability requirements are not to be made in order to compensate for column failure or system malfunction.
• If adjustments of operating conditions are necessary in order to meet system suitability requirements, each of the items in the following list is the maximum variation that can be considered, unless otherwise directed in the monograph.
• Adjustments are permitted 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’s specified in the official procedure.

2.0 Adjustments/ variations of operating conditions as per BP

• 2.1 Adjustments / variations of operating conditions for Gradient elution as per BP
  • Adjustment of chromatographic conditions for gradient systems requires greater caution than for isocratic systems.
  • Composition of the mobile phase/ gradient elution:
  • Minor adjustments of the composition of the mobile phase and the gradient are acceptable provided that;
    • System suitability requirement are fulfilled
    • The principle peak(s) elute within ±15% of the indicated retention time(s)
    • Final composition of the mobile phase is not weaker in elution power than the prescribed composition.
  • Where compliance with the system suitability requirements cannot be achieved, it is often preferable to consider to change the column.
  • pH of aqueous component of mobile phase: No adjustment permitted.
  • Concentration of salt in the buffer component of mobile phase: No adjustment permitted.
  • Flow rate: Adjustment is acceptable when changing the column dimension.
  • Column parameters
    • Stationary Phase: No change of the identity of the substitute of the stationary phase is permitted. (No replacement of C18 by C8).
    • Particle size: No adjustment permitted
    • Column dimension:
      • Length: ± 70%
      • Internal diameter: ±25%
    • When column dimensions are changed, the flow rate may be adjusted as necessary using the following equation.
    • F₂= F₁ x [l₂ x d² ₂/  l₁ x d² ₁]
    • Where,
      • F₁ = Flow rate indicated in monograph in milliliters/minutes
      • F₂ =  adjusted flow rate in milliliters/minutes.
      • L₁  = Length of the column indicated in the monograph in millimeter.
      • L₂  = Length of the column used in millimeter.
      • d1 = internal diameter indicated in the monograph in millimeter.
      • d2 = internal diameter used in millimeter.
  • Temperature: ± 5°C, where the operating temperature is specified, unless otherwise prescribed.
  • Detector wavelength: No adjustment permitted
  • Injection volume: May be decreased, provided detection and repeatability of the peaks to be determined are satisfactory, no increase permitted.

• 2.2 Adjustments / variations of operating conditions for Isocratic elution as per BP
  • Composition of the Mobile Phase:
    • The amount of minor solvent component may be adjusted ± 30% relative or ±2% absolute, whichever is larger.
    • (For minor component at 10% of the mobile phase of the mobile phase, a 30% relative adjustment allows a range of 7-13% whereas a 2% absolute adjustment allows a range of 8-12%, the relative value therefore being the larger.
    • For a minor component at b5% of the mobile phase, a 30% relative adjustment allows a range of 3.5-6.5% whereas a 2% absolute adjustment allows a range of 3-7 %, the absolute value being the larger in this case.)
    • No other component is altered by more than 10% absolute.
  • pH of aqueous component of mobile phase: pH ± 0.2, unless otherwise prescribed, or ± 1.0 pH when non-ionisable substances are to be examined.
  • Concentration of salt in the buffer component of mobile phase: ± 10%.
  • Flow rate: ± 50%: a larger adjustment is acceptable when changing the column dimensions.
  • Column parameters:
    • Stationary phase: No change of the identity of the substitute of the stationary phase is permitted. (No replacement of C18 by C8).
    • Particle size: Maximum reduction of 50%, no increase permitted.
    • Column dimension:
      • Length ± 70 %, Internal diameter ±25%
      • When column dimensions are changed the flow rate may be adjusted as necessary using the following equation.
      • F₂= F₁ x [l₂ x d² ₂/  l₁ x d² ₁]
      • Where
        • F₁ = Flow rate indicated in monograph in milliliters/minutes
        • F₂ = adjusted flow rate in milliliters/minutes
        • l₁ = Length of the column indicated in the monograph, in millimeter
        • l₂ = Length of the column used in millimeter
        • d₁ = internal diameter of the column indicated in the monograph in millimeter
        • d₂ = internal diameter of the column used in millimeter
    • Temperature: ±10°C where the operating temperature is specified, unless otherwise prescribed.
    • Detector wavelength : No adjustment permitted
    • Injection volume: May be decreased, provided detection and repeatability of the peaks to be determined and satisfactory, no increase permitted.

3.0 Adjustments/ variations of operating conditions as per USP

• pH of Mobile Phase: The pH of the aqueous buffer used in the preparation of the mobile phase can be adjusted to within ± 0.2 units of the value or range specified, applies to both gradient and isocratic separations.
• Concentration of Salts in Buffer: The concentration of the salts used in the preparation of the aqueous buffer used in the mobile phase can be adjusted to within ± 10 %, if the permitted pH variation is met, applies to both gradient and isocratic separations.
• Ratio of components in Mobile Phase: The following adjustment limits apply to minor components of the mobile phase (specified at 50 % or less). The amounts of these components can be adjusted by ± 30 % relative. However the change any component cannot exceed ± 10% absolute (i.e. in relation to the total mobile phase), whichever is larger. However the change in any component cannot exceed 10 % absolute, (i.e., in retention to the mobile phase). Adjustments can be made to one minor component in a tertiary mixture. Examples of adjustments for binary and tertiary mixtures are given below.
  • Binary mixture:
    • Specified ratio of 50:50 – 30 % of 50 is 15 % absolute, but this exceeds the maximum permitted change of ± 10 % absolute in either component. Therefore, the mobile phase ratio may be adjusted only within the range of 40:60 to  60:40.
    • Specified ratio of 2:98 – 30% of 2 is 0.6 % absolute. Therefore the maximum allowed adjustment is within the range of 1.4:98.6  or 26:97.4.
    • Tertiary mixture:
      • Specified ratio of 60:35:5 – For second component, 30 % of 35 is 10.5 % absolute, which exceeds the maximum permitted change of 10% absolute in any component. Therefore the second component may be adjusted only within the range of 25% – 45% absolute.
      • For third component, 30% of 5 is 1.5 absolute.
      • In all cases, a sufficient quantity of the first component is used to give a total of 100%. Therefore, mixture ranges of 50:45:5 – 70:25:5 or 58.5:35:6.5 – 61.5:35:3.5 would meet the requirement.
• Wavelength of UV-Visible detector: Deviations from the wavelengths specified in the procedure are not permitted. The procedure specified by the manufacturer, or another validated procedure, is to be used to verify that error in the detector wavelength is, at most ± 3 nm.
• Column Length: Refer below particle size point.
• Column inner diameter: Can be adjusted if the linear velocity is kept constant. Refer below flow rate point.
• Particle size:
  • For isocratic separations, the particle size and/or the length of the column may be modified provided that the ration of the column length (L) to the particle size (dp) remains constant or into the range between – 25% to + 50% of the prescribed L/dp ratio.
  • For gradient separations, changes in length, column inner diameter and particle size are not allowed.
• Flow rate:
  • When the particle size is changed, the flow rate may require adjustment, because smaller particle columns will require higher linear velocities for the same performance (as measured by reduced plate height). Flow rate changes for a both change in column diameter and particle size can be made by:
  • F₂= F₁ x [dc² ₂ x dp₁ /  dc² ₁ x dp₂  l₁ x d² ₂]
    • Where F₁ and F₂ are the flow rates for the original and modified conditions, respectively; dc₁ and dc₂ are the respective column diameters; and dp₁ and dp₂ are the particle sizes.
  • Additionally, the flow rate can be adjusted by ± 50% for isocratic only.
  • Change in flow rate, column diameter and particle size are not allowed for gradient separations.
• Injection volume: The injection volume can be adjusted as far 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 theoretical plates and resolution, applies to both gradient and isocratic separations.
• Column Temperature: The column temperature can be adjusted by as much as ± 10 %. Column thermostating is recommended to improve control and reproducibility of retention time, applies to both gradient and isocratic separations.

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