Introduction Of UPLC

Ultra-performance liquid chromatography (UPLC), involves HPLC with very high pressures and columns having very small particle sizes.

Principle
The efficiency of HPLC increased as particle sizes of the column packing decreased from 10 [micro] m in the 1970s to 3.5 Nun in the 1990s. This is shown by lower values of HETP (height equivalent to a theoretical plate) for van Demeter plots of HETP (column efficiency) versus mobile phase flow rate in units of linear velocity ([mu], mm/s; Figure 1). hi this particle size range, and even down to 2.5 Nun particles used in shorter columns in the early 2000s, it was found that HETP decreases to a minimum value and then increases with increasing flow rate. However, with the 1.7 [micro] m particles used in UPLC, HETP is lowered compared to the larger particles and does not increase at higher flow rates. This allow faster separations to be carried out on shorter columns and/or with higher flow rates, leading to column increased resolution between specific peak pairs and increased peak capacity, defined as the number of peaks that can be separated with specified resolution in a given time interval.

Theory Of  UPLC
The chromatogram that depicts the elution of a solute is a graph relating the concentration of the solute in the mobile phase leaving the column to elapsed time. However, at a constant flow rate, the chromatogram will also relate the solute concentration to the volume of mobile phase passed through the column. In figure 1, is shown the elution of a single peak. The expression, f(v), is the elution curve equation and this will be derived using the plate theory.

Once the nature of f(v) identified, then by differentiating f(v) and equating to zero, the position of the peak maximum can be determined and an expression for the retention volume (Vr) obtained. The expression for (Vr) will disclose those factors that control solute retention.11
UPLC is a new separation technique with increased speed, sensitivity and resolution. The performance of a column can be measured in terms of the height equivalent to the theoretical plates (HETP or H), which is calculated from the column length (L) and the column efficiency, or number of theoretical plates (N). N is calculated from an analyst’s retention time (tR) and the standard deviation of the peak (σ).
                                     H = L/N    ------------------- (1)


                                    N=(tR/σ)2  -------------------- (2)

The van Deemter equation is the empirical formula that describes the relationship between linear flow velocity (μ) and column efficiency, where A, B, and C are constants related to the mechanistic components of dispersion.12

H = L/N = A + B/μ + Cμ---- (3)   (VAN DEEMTER EQUATION)

According to the van Deemter plot, column efficiency is inversely proportional to the particle size (dp) (Equation 4), so by decreasing the particle size there is an increase in efficiency. Since resolution is proportional to the square root of N (Equation 5), decreasing particle size increases resolution. Also, by using smaller particles, analysis time can be decreased without sacrificing resolution, because as particle size decreases, column length can also be reduced proportionally to keep column efficiency constant. By using the same HPLC mobile phase and flow rate, UPLC™ reduces peak width and produced taller peaks which increased the S/N 1.8 to 8 fold, improving both sensitivity and resolution.13-18

                               N α 1/dp --------------- (4)

                    R = √N/4(α-1/α)(k/k+1) --------------- (5)

 
Also according to the van Deemter plot, use of particles smaller than 2 μm produces no loss in column efficiency with increasing flow rates. However, by increasing flow rates to decrease analysis time, there is a corresponding increase in system pressure. As a result, a system capable of withstanding the proper pressures while still maintaining efficiency is required. As well, a mechanically stable column is needed.