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Use of Small Particles in Ultra High Pressure Liquid Chromatography
L. Pereira1, C. Blythe1, R. Sherant2, H. Ritchie1
1Thermo Electron Corporation, Runcorn, UK,
2Thermo Electron Corporation, Bellefonte, PA
Abstract
Resolution and sensitivity
The work presented in this poster demonstrates how 1.9 µm particles facilitate higher resolution, A separation on a 200 x 2.1mm, 5 µm column was transferred to shorter columns packed with Figure 1 demonstrates the effect of particle size on the resolution of a mixture of 7 phenones, the higher sensitivity and faster analyses. The gains in resolution through manipulation of selectivity smaller particles to reduce analysis time (Figure 4). The 1.9 µm particles facilitate a decrease in run half height resolution of the two last eluting components is annotated. The Y-axes of the five (column chemistry) are also illustrated.
time from 6 to 0.5 minutes, whilst maintaining baseline resolution of the seven phenones.
chromatograms have been normalized to illustrate the gain in sensitivity when more efficient peaks are obtained. Introduction
FIGURE 2.
Effect uti
mnand sensi
geometr ti
, ity are i
particle n
ize an d as parti
d operati c
ndit s reduced.
ions on ru
and peak width (at baseline).
The aim of a chromatographic separation is to obtain good resolution in a short analysis time, i.e., FIGURE 2.
(R ) and sensitivity are increased as particle size is reduced.
s ) and sensitivity are increased as particle size is reduced.
resolution needs to be maximized while minimizing analysis time. Sub 2 µm particles have been developed as a chromatographic media to achieve high resolution of the sample components, fast Resolution (R ) is proportional to the square root of separation efficiency (N), as described by equation 1 which expresses resolution as a function of capacity factor (k), selectivity (α), and efficiency (N). Efficiency is, in turn, inversely proportional to the diameter of particle size (d ). Gradient: 65 to 95 %B in 1.5 min, hold for 1.5 min Unlike 5 and 3 µm particles, sub 2 µm particle packed columns can be operated over a wide flow rate range without loss of efficiency. The trade-off of using columns packed with such small particles and of operating at high flow rates is that the pressure drop (P) across the column increases. Equation 2 shows the dependency of P on particle size and flow rate. To operate under these conditions specialized equipment is required. FIGURE 1.
- selectivity, N – efficiency, k – retention factor L - column length (cm), η - viscosity (cP), F - flow rate (mL/min) d – particle diameter (µm), d – column internal diameter (cm) Selectivity
Column robustness and reproducibility
C18 like selectivity (Hypersil GOLD), polar endcapped C18 (Hypersil GOLD aQ) and TABLE 1. Operational parameters for N = 10000.
perfluorinated (Hypersil GOLD PFP) columns were used to separate a mixture of protease The stability of 1.9 µm Hypersil GOLD columns at high pressures is shown in Figure 5. After 100 inhibitors (anti-HIV drugs) and a mixture of aromatic amines (Figures 2 and 3 respectively). In the injections of a standard mixture at 11,500 psi the change in efficiency is 0.22%. Figure 6 shows the ∆P (bar)
batch-to-batch reproducibility of 5 batches (determined using the capacity factor of a standard first instance, several changes in elution order are observed with both the aQ and the PFP phases. For the aromatic amines, the elution order of the o-toluidine and 4,4-oxydianiline on the solute) and column-to-column reproducibility determined using the column efficiency (of 200 columns) and pressure drop across the column (8 columns).
FIGURE 5. Hypersil GOLD 1.9 µm, 50 x 2.1 mm column stability at 11,500 psi.
FIGURE 2. Effect of column chemistry (C18 selectivity, polar endcapped C18 and
pentafluorophenyl) on separation of protease inhibitors.
Column: Hypersil GOLD 1.9 µm, 50 x 2.1 mm Hypersil GOLD
Hypersil GOLD aQ
Hypersil GOLD PFP
165,183 N/m
Injection 1
165,563 N/m
A very effective way to increase the resolution of two chromatographic bands is to increase the selectivity factor (α) by manipulating column chemistry or mobile phase composition. 1.9 µm Injection 100
Hypersil GOLD™ columns are available in three chemistries, a C18 selectivity, a polar endcapped C18 and a perfluorinated phenyl that allow more flexibility when developing high resolution, fast methods. 1.9µm particle size Hypersil GOLD phases are based on very high purity silica using a new technique for processing particles with a very narrow size dispersion. This technology, FIGURE 6. Batch-to-batch and column-to-column reproducibility for Hypersil GOLD
combined with the latest in silica bonding and endcapping processes, enables the manufacturing of stationary phases which provide very symmetrical peak shapes, speed and resolution. Capacity factor: batch to batch
A process for packing 1.9 µm particles into robust and reproducible columns has also been Columns: Hypersil GOLD 1.9 µm, 50 x 2.1 mm Analytes: 1. Amprenavir
developed and optimized. Packing bed homogeneity at higher pressures has to be ensured to Mobile phase: H O + 0.1% Formic Acid / ACN + 0.1% Formic Acid 2. Nelfinavir
maintain efficiency, and column hardware and end-fittings had to be re-designed to withstand high Gradient: 0 – 2.2 mins @ 35% B, then to 100 % B @ 4 mins 3. Saquinavir
4. Ritonavir
5. Lopinavir
Detection: ESI +ve, scan 450 – 750amu (above mass chromatograms for [M+H]+) • Columns - Hypersil GOLD 12 µm, 200 x 2.1 mm; Hypersil GOLD 8 µm, 200 x 2.1 mm; Hypersil GOLD 5 µm, 200 x 2.1 mm; Hypersil GOLD 3 µm, 200 x 2.1 mm; Hypersil GOLD 1.9 µm, 200 x 2.1 Measure d press ure drop across column
Column reproducibility: measured plates/m
mm; Hypersil GOLD 1.9 µm, 100 x 2.1 mm; Hypersil GOLD 1.9 µm, 50 x 2.1 mm; Hypersil GOLD FIGURE 3. Effect of column chemistry (C18 selectivity, polar endcapped C18 and
aQ™ 1.9 µm, 50 x 2.1 mm; Hypersil GOLD PFP 1.9 µm, 50 x 2.1 mm (Thermo Electron si
p
10000
pentafluorophenyl) on separation of aromatic amines.
Hypersil GOLD
Hypersil GOLD aQ
Hypersil GOLD PFP
• Instrumentation - Finnigan™ Surveyor™; Finnigan LCQ™ Deca (Thermo Electron Corporation, Column numbe r
• U-HPLC system: Accela™ (Thermo Electron Corporation, San Jose, CA) Colum n num ber
• Mobile phase compositions, gradients, flow rates, solutes, temperatures and detection details are Conclusions
Method transfer to smaller column geometries packed with smaller particles (Figure 4A to C), was performed using the following equations for flow rate and gradient time adjustment: The work presented in this poster demonstrates that: 1.9 µm Hypersil GOLD columns provide very efficient peaks for high resolution, sensitive and a) Adjust flow rate (keep reduced linear velocity constant between original and new method) fast analysis; 12-fold reduction in analysis time can be achieved F - original flow rate; F - new flow rate (mL/min) 1.9 µm Hypersil GOLD column chemistries provide selectivity differences to facilitate method d - original column; d - new column ID (mm) development under generic mobile phase conditions The manufacturing process for 1.9 µm Hypersil GOLD columns is reproducible and the Columns: Hypersil GOLD 1.9 µm, 50 x 2.1 mm columns are stable when operated at high pressures b) Keep initial and final mobile phase composition, adjust gradient time Additional Information
t – gradient time in original method; t - gradient time in new method (min) V – original column volume; V – new column volume (mL) For additional information, please browse our website: www.thermo.com/columns.
F - original flow rate; F - new flow rate (mL/min) Hypersil GOLD, Hypersil GOLD aQ, Finnigan, Surveyor, LCQ and Accela are trademarks of Thermo Electron V – column void volume (mL); L – column length (cm); r – column radius (cm)

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