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  • HPLC Troubleshooting Separations Retention Time, Efficiency and Peak Shape

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  • This essential guide examines the common causes of retention time drift, loss of chromatographic efficiency and peak shape issues – such as splitting, fronting, tailing and shouldering. Strategies for problem identification and calculation of critical chromatography performance indicators will be discussed.

    The degree to which these parameters are affected by mobile phase composition, temperature, sample solvent strength, and many other variables will be investigated alongside strategies for isolating the precise cause of the problem.

    Corrective and preventative actions will be described for the major causes of the symptoms observed. In Part II, July’s Essential Guide, we will focus on Selectivity, Resolution and Baseline issues

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  • Every separation in HPLC produces a chromatogram (we hope!). Broadly speaking, every analyte is represented by a single peak eluting at a particular time, according to its various physicochemical properties. Under ideal conditions, each peak should be narrow and symmetrical (Gaussian distribution).[1-8]

    A visual inspection of the chromatogram is often enough to highlight problems with a separation, however sometimes we need to be more quantitative to describe the extent of the issue. To this end, a set of parameters, the most popular being retention time, retention factor, selectivity, efficiency, asymmetry and tailing factor, have been developed and in this Essential Guide we shall be focusing on retention time, retention factor and efficiency

    Retention Time (tR)

    The analyte retention time can be defined as the elapsed time between sample injection and the time of elution of the peak maximum of that analyte. The non-retained mobile phase elutes at a time t0, which is known as the ‘hold up time’ or “dead time”.

    Chromatographic Parameters

    There are several ways to determine t0 including: 

    • The time at the baseline disturbance seen due to differences in  absorbance or refractive index as the injection solvent passes through  the detector • Retention time of Uracil (reverse phase) •Retention time of Hexane (normal phase) 

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  • Retention or Capacity Factor

    Isocratic Operation

    The retention factor (k’), also known as capacity factor, is a means of measuring the retention of an analyte on the chromatographic column. A high k’ value indicates that the sample is highly retained and has spent a significant amount of time interacting with the stationary phase

    The retention factor is equal to the ratio of retention time of the analyte on the column to the retention time of a non-retained compound. The non-retained compound has no affinity for the stationary phase and elutes with the solvent front at a time t0 .

    Retention factor is independent of some key variable factors including small flow rate variations and column dimensions. Because of this, it is a useful parameter when comparing retention of chromatographic peaks obtained using different HPLC systems and when converting conventional HPLC methods to UHPLC systems (and vice versa!). Chromatographers typically like to keep k’ values between 1 and 10 for good HPLC separations or 0.5 and 5 for methods developed on more modern UHPLC systems where highly efficient separations are the norm. Please do note however that there is the risk of peak overlap (poor resolution) when separating a moderate number of analytes at low k’ values.

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  • Gradient Operation

    One cannot assign a fixed k’ value to a compound when gradient elution is applied; k’ is the retention coefficient and  changes during gradient elution. 

    The equation for the gradient retention factor (k*) takes the form:

    Gradient retention factor (k*) is difficult to visualize as it differs from its isocratic counterpart (k’) and resembles more the profile of the gradient elution. It is effectively defined as the retention factor for an analyte that has migrated half  way down the HPLC column.

    The Retention Factor as a Means to Evaluate the State of an HPLC Separation

    The retention factor of peaks within a chromatogram can aid in the identification of many problems that are  associated with HPLC system components.  Variable retention factors may result from changes in mobile phase flow  rate, mobile phase composition, column stationary phase, and temperature.

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  • Efficiency

    The efficiency of a chromatographic peak is a measure of the dispersion of the analyte band as it travelled through the HPLC system and column. In an ideal world, chromatographic peaks would be pencil thin lines – however, due to dispersion effects the peaks take on their familiar “Gaussian” shape.

    The plate number (N) is a measure of the peak dispersion on the HPLC column, reflecting the column performance. N is derived from an analogy of Martin and Synge who likened column efficiency to fractional distillation, where the column is divided into Theoretical Plates.

    Each plate is the distance over which the sample components achieve one equilibration between the stationary and mobile phase in the column. Therefore, the more “theoretical” plates available within a column, the more equilibrations possible and the better quality the separation.

    Higher values for the Plate Number (N) are expected for subsequent peaks within a chromatogram. Later eluting peaks that look broad in comparison to early eluters may have a higher plate count. If this is not the case then your system contains a large extra-column dead volume which is dominating the diffusion process!

    For a fractionating tower of a given length (L), the higher the number of plates, the lower will be the distance between each plate, shown as plate height in the diagram. Therefore, for high efficiency separations, the plate number (N) will be high and the plate height (H) low. Note that plate height is often called – “Height Equivalent to a Theoretical Plate (HETP)”. Modern columns which employ are reduced particle size, sub 2 µm, enjoy an increased plate number (N) without increasing the length of the column (L) due to the reduction in distance between active sites, plate height (H)

    These two terms are related through the expression: H = L / N

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  • Figure 3. Factional distillation model of efficiency theoryca rlo

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  • The number of theoretical plates is often used to establish the efficacy of a column for a given method. The method developer may decide that a given method is no longer valid when the plate number falls below a predetermined value. At that time, the column would be replaced with a new one.

    Figure 4. Comparison of two chromatograms with the same selectivity and different efficiency (and resolution).ca rlo

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  • Efficiency as a Means to Evaluate the State of an HPLC Separation

    Peak efficiency within a chromatogram can aid in the identification of many problems that are associated with HPLC system components. Reduced efficiency may result from incorrect or mobile phase deterioration, incorrect or deteriorated column, increased dead volume, addition of a disproportionate length of tubing or wider I.D. tubing, etc.

    Peak Asymmetry

    In the ideal world all chromatographic peaks would be symmetrical (or Gaussian).

    However due to the effects of instrument dead-volume, adsorptive effects of the stationary phase and the quality of the column packing, peaks may often show a tailing behavior. Tailing describes a peak whose tail portion (distance ‘B’ in the diagram) is wider than the front portion (distance ‘A’ in the diagram). Also, if the sample concentration is too high or if the column is damaged and contains ‘channels’ then a fronting peak shape may occur.

    There are different ways of calculating the amount of peak tailing (or fronting). However, one of them, peak asymmetry (As), is probably the most commonly used, and is defined as:

    Where A and B are measured at 10% of the peak height.ca rlo

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  • Figure 5. Determination of Peak Asymmetry (AS) and examples of good and poor peak shape.

    The US Pharmacopeia (USP) recommends the use of a different measurement, the tailing factor (Tf), which has been defined as:

    Where A and B are measured at 5% of the peak height.

    In general terms, it doesn’t matter which measurement parameter is used (either As or Tf), as long as we are consistent in our measurement and are aware of the “acceptable” values for each measure. The definition of ‘acceptable’ peak tailing or fronting for the two different measurements is roughly the same - see Table 1.ca rlo

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  • Peak Distortion as a Means to Evaluate the State of an

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