HPLC Fundamentals


I. Short-Term Noise
Short-term noise, if detector related, is very small. Modern UV
detectors have noise specifications of about _+ 1 • 10 -s AU. More noisy
signals, like of Figure 7a, often originate with the low energy of an aging
UV lamp. UV lamps should be replaced regularly, after a specified number
of operating hours, or after a certain time period. Some short-term
noise is normal at high sensitivity.

Peak tailing & assymetry

  • Pharmaceutical industry uses the tailing factor (TF) which is determined by dividing the entire peak width ( at 5% of the height) by twice the front half-width.
  • Nonpharmaceutical laboratories often use the asymmetry factor( As )which is calculated by dividing the back half-width of the peak (at 10% of the peak height)by the front half-width.
  • If the peak is perfectly symmetric, the front and back half-widths will be the same, no matter where they are measured relative to the peak height, so for such peaks, TF ≡ As .
  • As tailing increases, however, the two numbers diverge, with A s growing faster than TF, but for peaks with a value less than 2 there is not a very noticeable difference.
  • There is no inherent value in using one technique versus the other for measuring peak shape; rather, it is important to choose one technique and use it to look for changes in peak shape over time.

(BEH)Ethylene Bridged Hybrid

(HSS) High Strength Silica

  • ACQUITY UPLC® HSS T3 Columns contain the first and only 100% silica particle intended for use in applications up to 15000 psi/1000 bar.
  • The ACQUITY UPLC® HSS particle is not an HPLC particle.
What is T3?
  • Many separation scientists prefer reversed-phased chromatographic approaches to retain polar compounds.
  • T3 particle technology is derived from C18 ligands and proprietary end-capping to achieve optimum characteristics suited for polar compound retention. 
  • Three stationary phase characteristics define the T3 columns for polar retention: 1. Optimized pore size. 2. Bonded alkyl functionality with superior low pH stability. 3. Optimized surface coverage and ligand density to  maximizes polar compound retention.
  • An aprotic solvent does not have an H atom bound to O or N, but it can participate in H-bonding with protic molecules. 
  • Acetone does not have an O-H group, but it has a C=O. group that can participate in H-bonding. So acetone is a polar aprotic solvent.
Retention Time((tR)
  • The time elapsed between the injection of sample component into the column (column inlet) and its detection (column end) is known as the retention time.
Capacity factor (k) (Retention factor)
  • The retention (or capacity) factor (k) is a means of measuring the retention of an analyte on the chromatographic column.
  • It measures the period of time that the sample component resides in stationary phase relative to time resides in the mobile phase.
  • It is calculated from Net Retention Time (tR‘) divided by the time for an unretained peak (t0).
  • A high k value indicates that the sample is highly retained and has spent a significant amount of time interacting with the stationary phase.
Selectivity (or Separation) Factor (α)
  • The selectivity factor (α) is the ability of the chromatographic system to ‘chemically’ distinguish between sample components.
  • It is ratio of the retention (capacity) factors (k) of the two peaks in question.
  • It can be visualized as the distance between the apicesof the two peaks. High α values indicate good separating power and a good separation between the apex of two peaks.
  • α is always more than 1. α ≥2  easy separation
  • α≤ 1.2  very difficult separation
Column Efficiency (Band broadening/ Peak Sharpness)
Peak Width/Sharpness
  • Peak width is an indication of peak sharpness and, an indication of the column efficiency .
  • After injection, a narrow chromatographic band is broaden during its movement through the column. The higher the band broadening, smaller the number of components can be separated in a given time and lower is the column efficiency.
  • Peak width is dependent on column length, flow rate, particle size.
  • Flow rate is the only parameter which can be changed from run to run on the same column.
  • Peak width increases with the square root of column length, hence we cannot just use a longer column to get better separation.
Theoretical plate number (N)
  • Theoretical plate number (N) is an index that indicates column efficiency.
  • In the absence of specific interactions or sample overloading, the chromatographic peak can be represented by a Gaussian curve with the standard deviation (s).
  • The ratio of standard deviation to the peak retention time (s/tR) is called the relative standard deviation, which is independent of the flow rate.
  • Square of the reciprocal value  N= (tR /σ)2 (for non-Guassian peak) has become the accepted expression of column efficiency. (Reason for using the second power is that it’s the variance, not the standard deviation that is basic measure of normal distribution).
  • In practice, it is more convenient to measure peak width either at the base line, or at the half height, and not at 0.609 of the peak height, which actually correspond to 2σ )
  • w is the peak width obtained by drawing tangents to the sides of the Gaussian curve at the inflection points and extrapolating the tangents to intercept the baseline.  w= 4σ, N= 16 (tR /W)2 or N= 5.54 (tR /W 0.5h)2

HETP (Height Equivalent to Theoretical Plate)or Plate Height ( h)
  • A more appropriate parameter for measuring efficiency is HETP (or h).
  • h=L/N and also variance per unit length (h= σ2/L)
  • Lower the HETP (and longer the column,) the higher the plate number, the more efficiently the chromatographic column is packed and more equilibrations between the stationary and mobile phases are possible and better the quality of the separation is.
  • Efficiency is theoretically related to the various kinetic processes that are involved in solute retention and transport in the column.
  • Van Deemter equation relates the effect of linear velocity on band broadening (HETP) and it comprises the three important factors that can result in band broadening:
  • HETP = A + B/u + Cu; Where: HETP = Plate Height, A = Eddy Diffusion, B/u = Longitudinal Diffusion, Cu = Mass Transfer.
  • Each column has an optimal flow rate to produce a minimum band broadening.
  • Smaller particles show
    • faster optimal flow rates.
    • minimal loss in efficiency even at faster flow rates as shown by less steep slope.



  • The resolution of a elution is a quantitative measure” of how well two elution peaks can be differentiated in a chromatographic separation.
  • Resolution is defined as the ratio of the center-to-center separation between two adjacent peaks to the average baseline width of those peaks. In other words, “how far apart, divided by how wide”. 
  • Resolution between two peaks depends upon three things:
    • 1. proper retention factor (k) – a function of the mobile phase strength),
    • 2. correct selectivity (α)– the chemical interaction provided by the stationary phase and mobile phase),
    • 3. enough efficiency (N)– a mechanical function of the column and packing dimensions).
  • The dead volume is the volume of an HPLC system between the point of injection to the point of detection, excluding the column.
  • The dwell volume (also called “gradient delay volume“) of a gradient or on-line mixing system is the volume of liquid contained in the system between the point where the gradient is formed and the point where the mobile phase enters the column.