Detail on Resolution (RS ), Selectivity, Column efficiency, Column Symmetry, Asymmetry Factor, or Tailing Factor
HPLC Resolution (RS )
- Resolution is a significant HPLC performance indicator usually assessed by how rapidly and how fully target components in a sample separate they move through a column.
- The difference in peak retention times is divided by the average peak width to determine resolution.
- The Resolution Equation can also be used to express resolution as a mixture of the components that influence it (separation, efficiency, and retention).
Void Volume (Vo)
- For column resolution, practically all HPLC modes require some type of selective retention by the stationary phase.
- To determine if a compound is retained, measure the retention time for an unretained solute at a particular flow rate and calculate the column void volume, Vo.
- The column void volume or void time for a particular flow rate will not change once it is understood. Resolution due to stationary phase interaction is impossible if a solute elutes near or at the vacuum.
Selectivity or Separation factor α (alpha)
- The ratio of k values for adjacent peaks (Two Peaks) is used to calculate selectivity or separation factor (alpha). So that k values are always equal to or larger than 1, the k for the latter peak is always placed in the numerator.
- HPLC has a selectivity of 1.1, which allows for a resolution of 1.5 with roughly 10,000 theoretical places. In a separation, the critical pair is defined as the adjacent solutes with the least value.
- The critical pair specifies the minimal number of plates required to obtain a given resolution, as well as the particle size and column length.
Column efficiency or Number of Theoretical Plates (N)
- Column efficiency, the rate of zone spreading measured by taking a ratio of retention time to peak width and squaring it, is usually expressed as N.
- When peak width is measured at half-height, the constant is 5.54.
- If the peak width is measured at base, a constant of 16 must be used to arrive at about the same N value.
- Retention time and peak width must be measured in the same units for a valid column efficiency determination.
What are the causes of reduced column efficiency?
- Band broadening outside of the column bed might be induced by laminar flow or drag caused by the tubing wall.
- Too much connection tubing or tubing with a big diameter can reduce perceived resolution greatly.
- Effects of chamber mixing
- If the sample spends too much time in this environment, an open tube becomes an outstanding mixer. This has the unfavourable impact of diminishing resolution and decreasing N. Making connections with very short pieces of small inner diameter (I.D.) tubing can reduce the effect.
- With small I.D. columns, extra caution is required. Extra-column dispersion is a common point of difference between HPLC systems.
- Because it has a more efficient(shorter/narrower) flow channel, one system will usually demonstrate higher efficiency with the same column.
- Eddy currents that can reason for sharp bends, variations in internal diameter (I.D), and irregularities or burrs in the flow pathway all can contribute to decrease column efficiency.
Peak Symmetry
- Column efficiency and, as a result, resolution are similarly affected by peak symmetry. Tailing peaks are frequently caused by highly absorptive or active locations.
- If such active sites are existing, columns may show good efficiency and resolution for neutral solutes but extremely poor efficiency and resolution for bases or acids.
- Peak broadening with an enlarged right half of the peak is a sign of solute tailing. Excessive column activity for a given solute isn’t always indicative of a column that has lost its stationary phase.
- It’s especially noticeable in new columns when the phase chemistry hasn’t been adjusted for the sample type or operating conditions.
Column Symmetry, Asymmetry Factor, or Tailing Factor
Asymmetry Factor (As)
- The ASTM (American Society for Testing and Materials) International standards organization indorses calculating column symmetry or asymmetry (As) as the back-to-front ratio of a bisected peak calculated at 10% of the height.
USP Tailing Factor (T) or Symmetry factor
- The front of a tailing peak is greater than 1.0, while the front of a fronting peak is less than 1.0.
- The United States Pharmacopeia (USP) recommends calculating tailing factor (T) as the back-to-front ratio of a bisected peak measured at 5% of height.
- The ratio is made by dividing the total width (w) by twice the front width .
where
W0.05= The width of the peak at 5% height
f= The distance from the peak maxi.to the leading edge of the peak
The distance is measured at a point 5% of the peak height from the baseline.
Asymmetry vs. USP Tailing Factor (T)
- For a symmetrical peak, both calculation methods yield a value of 1, however, the USP method yields smaller values for tailing and fronting than the ASTM method.
- Check the data system to identify which approach is being utilised for symmetry calculation if utilizing an automated method. Both calculating approaches are available in many data systems.
Retardation factor (RF)
- The ratio of the distance traveled by the center of the spot to the distance simultaneously traveled by the mobile phase and is used in planar chromatography.
RF = b/a
Retention factor (k):
- Also known as the “capacity factor (k′)”.
- K = (Amount of substance in Stationary phase) / ( Amount of substance in Mobile phase) Or
- K = (Time spend by a substance in Stationary phase) / ( Time spend by a substance in Mobile phase)
This all about :Column efficiency Column Symmetry Asymmetry Factor or Tailing Factor
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