SofTA Corporation Pittcon 2005 Technical Presentation

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Developments in Evaporative Light Scattering Detection for Gradient Elutions of Small Molecules Inga Henderson, SofTA Corporation, Miyako Kawakatsu, M&S Instruments Trading Inc., Hiroyuki Kaiho, M&S Instruments Trading Inc., Sam Azlein, SofTA Corporation SofTA Corporation, 555 Burbank Street, Broomfield, CO 80020 (800)465-1106 www.softacorporation.com [email protected] Thermo-split is a trademark of SofTA Corporation.

Abstract Many HPLC separations depend on gradient elution to create greater resolution between early eluting peaks and reduce the retention of later eluting peaks thus improving productivity by shortening run times and improving resolution. Evaporative Light Scattering Detection (ELSD) responds to all compounds that are less volatile than the HPLC mobile phase and are often described as a “ universal” detector. However components eluting under gradient conditions often do not respond with equivalent mass. Unlike some HPLC detectors, Evaporative Light Scattering Detectors (ELSD) do not respond to changes in mobile phase composition with baseline drift and noise but the resulting peak area can differ dramatically with changes in mobile phase composition. During gradient elutions the composition of the mobile phase can affect the size and uniformity of the droplets. Lower viscosity mobile phases result in smaller droplet sizes. Conversely higher viscosities produce larger droplets. Changes in the droplets can significantly impact sensitivity and reproducibility. This presentation will compare three different methods of vapor phase control; flow restriction, plate barrier, and Thermo-Split™ to illustrate the correlation between droplet size control and mass response during gradient elution.

ELSD Method of Operation ELSDs employs a unique method of detection. The process involves three steps. First, the column eluent is transformed into an aerosol cloud with pneumatic nebulization. The mobile phase in the vapor is then evaporated leaving a smaller cloud of analyte particles. These particles pass through a beam of light, scattering some of the light, which is converted into an electronic signal.

Nebulization Nebulization transforms the liquid phase leaving the column into an aerosol cloud of fine droplets. The size and uniformity of the droplets are extremely important in achieving sensitivity and reproducibility. All the ELSDs tested use a concentric gas type nebulizer and a constant flow of an inert gas.

Evaporation The aerosol cloud is propelled through the heated evaporation tube assisted by the carrier gas. In the evaporation tube the solvent is volatilized to produce particles or droplets of pure analyte. The temperature of the drift tube is set at the temperature required to evaporate the solvent. The temperature is kept as low as possible to avoid evaporation of the analyte.

Detection The particles emerging from the evaporation tube enter the optical cell, where the sample particles pass through a beam of light. The particles scatter the light. A light trap is located opposite the laser to collect the light not scattered by particles. The quantity of light detected is proportional to the solute concentration and solute particle size distribution.

Experimental Conditions Table 1 lists the details used for comparing the effects of vapor phase control on baseline and peak shape during gradient elution. Manual injections were made approximately every minute to evaluate the ELSD response as the mobile phase composition changed. The results obtained from the three detectors are shown in Figures 1, 2, and 3. Mobile Phase Gradient Flowrate Analyte Injection Volume Backpressure ELSD Conditions SofTA ELSD Model 300 ELSD “ A” ELSD “ B”

A: Water B: Methanol 10% B to 90% B in 10 minutes 1mL/min Sodium Benzoate in water (5ng/µL) 20µL Maintained at approx. 1200psi with a pressure regulator between pump and injection valve. Spray Chamber 25°C Drift Tube 65°C, 50psi N2 40°C, 3.5 Bar N2, Gain 12, Filter 1 Impactor ON, 40°C, 1.4 SLPM N2, Gain 16

Table 1: HPLC Conditions

Discussion Nebulization and Droplet Size The ELSDs in this study use different styles of concentric pneumatic nebulizers to create the vapor. This study did not address the differences in nebulization, but it can be assumed that all nebulizers are affected by changes in mobile phase composition. The widely used equation to measure droplet size, the Sauter Mean Diameter, predicts that droplets size will be significantly affected by the viscosity and surface tension of the liquid. Lower viscosity liquids produce smaller droplets. Conversely higher viscosities produce larger droplets. The viscosity of the mobile phase at Time 0 (90% water/10% methanol) in the experiment would be higher in viscosity then the mixture at 10 minutes (10% water/90% water). However, water mixed with methanol has significantly higher viscosities then either of the pure solvent. The viscosity of the water methanol mixture is greatest at 40% methanol or at 6.25 minutes in this experiment. At this time the viscosity is three times that of methanol alone and 2x that of water. See Table 2. With this background information it can be assumed that the droplets size leaving the nebulizer increases until 6.25 minutes and then decreases during this experiment. At the end of the gradient the droplet size is the smallest.

Viscosity of Water Methanol Mixtures

Viscocity [mPa s]

2

1.5

1

0.5 10

30

50

70

90

% Methanol in Water

Table 2: Numeric values after J. W. Dolan and L.R. Snyder, Troubleshooting LC Systems, Humana Press, Clifton, 1989, p.85.

Vapor Phase Control The evaporative light scattering detectors in this study use one of three different types of vapor phase control. Each produced significantly different results under the gradient condition tested. To handle flow rates and mobile phases common in HPLC, all ELSDs need a way to divert part of the aerosol cloud to waste. One of the earliest successful approaches to aerosol splitting involved a nebulization chamber with a flow restriction and sharp turn. The larger particles in the cloud would fail to make the turn, hit the wall, and ultimately run out a drain. This is the type of vapor phase control used by ELSD “ A” in this study. ELSD “ B” uses a plate barrier to control the amount of vapor entering the drift tube. For high flow rates, or aqueous mobile phases, the plate is placed perpendicular to the flow. Larger droplets hit the plate, condense, and go out a drain. Smaller droplets make it through the annular space and continue on to the evaporative zone. When less severe conditions are encountered, the plate is turned parallel to the flow, allowing all droplets to pass. The SofTA ELSD uses the patent pending Thermo-Split technology for vapor phase control. ThermoSplit technology provides the ability to vary the split ratio smoothly over a wide range. The Thermo-Split chamber combines a gentle bend with temperature controlled (heated or cooled) walls. For easily evaporated mobile phases, the walls are heated. As the droplets traverse the chamber, they partially evaporate, shifting the particle size distribution low enough for essentially all the droplets to negotiate the bend. When highly organic mobile phases are used, the Thermo-Split chamber is used at ambient or elevated temperatures. Under these conditions a majority of the droplets pass through the chamber and are carried into the evaporative zone. For difficult to evaporate mobile phases, or high flow rates, the walls of the Thermo-Split chamber are cooled. When the droplets exiting the nebulizer encounter a cooled environment, they partially condenses into larger droplets whose momentum carries them into the wall and down the drain. By making the walls suitably cold, 99+% of an aqueous stream can be diverted away from the evaporative zone.

Results The gradient chosen for this experiment begins with 90% water, a more difficult mobile phase to evaporate. Each of the ELSDs was operated at the conditions required to accommodate this mobile phase.

Vapor Phase Control by Flow Restriction The ELSD “ A” , Figure 1, was also affected by a change in mobile phase composition. Because the sharp turn is always present to control the vapor, ELSD “ B” was operated under temperature and gas flow conditions that produced the least noise with the beginning mobile phase. In this case as the mobile phase becomes more viscous and the droplets size increases the baseline begins to climb. The signal may be increasing because the larger droplets are not being completely evaporated in the drift tube. As the droplet size decreases due to the reduction in mobile phase viscosity, the signal begins to decrease. The peak shape of the analyte remained fairly consistent over the entire gradient.

Plate Barrier Vapor Phase Control ELSD “ B” can operated with the plate barrier in either the parallel or perpendicular position. To accommodate the water in the starting mobile phase, the plate had to be in the perpendicular positioned. If the plate was in the parallel position the vapor did not evaporate and saturated the output. During this experiment, ELSD “ B” , showed significant baseline drift during the gradient. See Figure 2. The baseline signal decreased as the water decreased in the mobile phase mixture. At this point the droplets size becomes larger, impacted the plate, and was diverted to waste, decreasing the amount of vapor entering the drift tube. After 6 minutes, as the droplets began to get smaller and navigate around the plate, the baseline began to climb again. The peak shape deteriorated when the methanol concentration exceeded 50%. The peaks begin to broaden because the analyte is distributed among a larger number of smaller droplets.

SofTA Thermo-Split Technology If the method specified an isocratic elution, the SofTA ELSD Thermo-Split temperature would be set to a sub ambient temperature for 90% water or at an elevated temperature if 90% methanol were used. Since the elution was performed as a gradient, a moderate Thermo-split temperature of 25°C was selected. The SofTA ELSD, Figure 3, exhibited a smooth baseline signal over the entire gradient and demonstrated superior signal to noise results. It can be seen that the peak height decreased and the peak width increased with an increase in methanol in the mobile phase. The peaks begin to broaden because the analyte is distributed among a larger number of smaller droplets. However, the resulting peak area deviation was with in 10% of the mean until the last peak at 82% methanol. Peak 1 2 3 4 5 6 7 8 9 10 11

Time 0.18 0.93 1.93 2.93 3.96 5.02 5.98 6.96 7.99 9.02 9.78

% Water Height Area 89 11468 25247 83 13238 28564 75 14823 28017 67 11744 28111 58 13391 35169 50 11479 30274 42 11234 31316 34 8805 26673 26 8080 30857 18 6132 30317 12 4353 22227 Table 3: SofTA ELSD Gradient Analysis

PW 0.11 0.15 0.16 0.15 0.20 0.22 0.22 0.17 0.20 0.24 0.22

Conclusions The SofTA ELSD with Thermo-split vapor phase control has been shown to produce a more stable baseline and superior signal to noise under that gradient conditions tested. Controlling the vapor phase with a flow restriction or a plate barrier did not produce a stable baseline during the gradient elution.

Figure 1: ELSD “A” Gradient Profile

Figure 2: ELSD “B” Gradient Profile

Figure 3: SofTA ELSD Gradient Profile