Developments in Evaporative Light Scattering Detection for High Throughput HPLC Sam Azlein and Inga Henderson SofTA Corporation, 555 Burbank Street, Broomfield, CO 80020 (800)465-1106 www.softacorporation.com [email protected]

ABSTRACT The modern drug discovery techniques of the pharmaceutical industry have produced an enormous number of samples to be analyzed. This ever increasing sample volume has lead to the development of high throughput HPLC analysis. These fast HPLC methods employ short column lengths with small particle size to maintain analyte resolution. HPLC-MS has already proven to be an effective tool for characterizing pharmaceutical compounds, and currently is being optimized for speed. Evaporative light scattering detectors (ELSD) are used in parallel with many LC-MS systems to obtain concentration information for all compounds, irrespective of whether or not they are UV active. It is very important that the resulting chromatogram obtained from the ELSD keep up with the speed and resolution of the separation. The narrowest peak width possible is desired to maintain the sample throughput required by the pharmaceutical companies. ELSDs nebulize the column effluent, transforming it into an aerosol cloud. As this cloud travels through a heated zone within the instrument, the more volatile mobile phase evaporates, 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. Many commercially available ELSDs disperse the peak during the vaporization process, resulting in band broadening and reduced resolution. This presentation will compare the ability of these ELSDs to maintain the peak width eluting from the analytical column.

ELSD PRINCIPLES Commercial ELSDs all have substantial similarities with regard to how they operate, and also some notable differences. A thorough understanding of similarities and differences is helpful in understanding why different ELSDs have different response times. One of the purposes of this paper is to identify which these differences yield advantages related to high speed operation. All commercially available ELSDs use a pneumatic nebulizer to change the column effluent into an aerosol. However, the nebulizers are of different construction and materials. While cross flow nebulizers have been successfully used most, if not all, are now concentric flow. Stainless steel is the most popular material, though both glass and Teflon are commercially available. With a concentric flow nebulizer, the liquid path is centered in an annulus of high velocity gas, with Nitrogen and purified air being the most common. It is worth noting that the nebulizers vary widely with regard to how much gas they consume, the range being from approximately one to four SLPM. All commercially available ELSDs have a mechanism for sending part of the aerosol to waste, though the methods vary widely. One instrument compared in this paper uses a constricted aerosol flow path to separate larger droplets out. Another uses a plate impactor to accomplish the same goal. The SofTA 300 uses a thermal technique to divert part of the aerosol to waste. These three techniques for aerosol handling are shown in Figures 1,3, and 5. After dividing (if necessary) the aerosol cloud, the next stage is desolvation. Again, commercial instruments have significant differences here, but all possess a heated zone (drift tube) where the more volatile mobile phase is given an opportunity to become a gas through evaporation. The goal is to evaporate the mobile phase, while leaving the less volatile analyte in solid or liquid form.

The three examined instruments have quite different designs for the drift tubes. One has a relatively small ID coil of tubing, while the other two have larger I.D. and substantially shorter straight tubes. Drift tube design is of potential interest since mixing and dispersion of the aerosol cloud during evaporation could be a source of peak broadening. Finally, all ELSDs illuminate the remaining analyte droplets (or solid particles) and quantify the scattered light. Again, there are significant differences in how this illumination and detection are done. Two of the examined instruments use a laser, while one uses a halogen lamp. Two use photodiodes to measure the scattered light, while one uses a photomultiplier tube. Detection angles also vary, though this is thought to be unimportant to response time. (It may be a factor in sensitivity, or some other performance parameter.) While different sensors have different bandwidths (a measure of response speed), it is unlikely that these differences have anything to do with instrument response time. Both types of sensors are much faster than any chromatographic peak. For example, a photodiode can respond to a MHz signal, and peaks are at least several seconds. Therefore, sensor type is thought to be outside the subject of this paper.

EXPERIMENTAL CONDITIONS Because column performance changes over time and would be an additional variable, this experimental work was done with direct injection. We used a small (2 micro liter) injection valve to inject a relatively high concentration (1000 ng) of analyte (Sodium Benzoate). All work was done using 50/50 Methanol/Water. The peak width of three different ELSDs from three different manufacturers was then compared. SEE FIG. 2,4 &6. After obtaining “ out-of-the-box” results, one ELSD, the SofTA 300 was subjected to further experimentation to asses the possibility of improving detector speed. In particular, nebulizer design was modified, filter settings varied, and filter algorithms modified. SEE FIG. 7.

ELSD Under Test HPLC Pump@1ml/min.

50/50 MeOH/H20

2 ul Injection Valve

1.0 ml/min 1000 P.S.I.

Inlet

1000 P.S.I. Back Pressure Regulator

Drain

ELSD SETTINGS When possible, the recommended settings from the supplied manuals were used. In the case that the manual did not cover 50/50 Methanol Water, an experimental effort was made to determine best settings for each instrument. Our findings were that while the various settings for each ELSD made considerable difference in signal strength, they made almost none for peak width. The settings are summarized below in a table, and represent our conclusion for where each instrument performed the best in this test.

ELSD

Gas Setting

S.C. Temp.

D.T. Temp.

Filter

Other Gain 6

A

3.5 Bar

N/A

40° C

1

B

1.2 SLPM

N/A

45 °C

N/A

Gain 1 Impactor On

50 P.S.I.

10° C

45° C

2

Gain Normal

SofTA 300

ELSD A Construction and Performance ELSD A utilizes a constriction in the aerosol flow path to separate the large droplets out of the cloud generated by the nebulizer. This method of splitting has some natural variation with the mobile phase being used. It is claimed to split less when the mobile phase is organic, and more when the mobile phase is aqueous. In operation, the larger droplets impact the wall of the spray chamber and exit via a drain. Smaller droplets are able to stay in the gas flow path The instrument user has no control over the splitter, which precludes optimizing. However, it does result in simpler operation. SEE FIGURES 1 & 2

FIGURE 1 ELSD A CONSTRUCTION

ELSD B Construction and Performance ELSD B uses a plate impactor to separate large droplets. The manufacturer suggests the plate be activated for high flow rates, or for highly aqueous mobile phases. We found the instrument performed best with the plate active for our experimental flow of 1 ml/min of 50/50 MeOH/ Water. The user does have some control over this splitter action, but must make the choice between “ impactor on” and “ impactor off” operating modes. SEE FIGURES 3 & 4

FIGURE 3 ELSD B CONSTRUCTION

SofTA Corporation Model 300 ELSD Construction and Performance SofTA ELSDs utilize a thermal technique (Thermo-Split®) to split the aerosol cloud. The walls of the spray chamber can be heated or cooled: 1. When heated, the droplets on average shrink and more easily pass around the gentle bend at the end of the spray chamber. 2. When cooled, the droplets on average increase (by condensation) and become large enough for their mass to carry them into the wall of the spray chamber, and then exit via the drain. The SofTA splitter requires the most optimizing on the part of the user, but offers the chance to tune the instrument more precisely to a given mobile phase and flow rate. SEE FIGURES 5 & 6

Spray Chamber walls are heated.

FIGURE 5 SofTA ELSD 300 CONSTRUCTION

Spray Chamber walls are cooled.

SofTA Corporation Model 300 ELSD Performance after Modification In an effort to improve peak width performance, the nebulizer was modified with smaller internal passages, and all electronic filtering removed. In the case of this particular instrument, removing the filtering consisted of: 1. Setting the filter function to zero 2. Changing the operating software to remove the subtle filtering effects of averaging readings, etc. SEE FIGURE 7

DATA SUMMARY ELSD

A

B

SofTA (stock)

SofTA (modified)

Average Peak Width

14 seconds

9.4 seconds

6.1 seconds

5.1 seconds

CONCLUSIONS From the data, it is apparent that different ELSD designs give considerably different (3:1) peak widths. While all commercial ELSDs utilize similar principles of construction and operation, differences in spray chamber, nebulizer, filtering, and drift tube design all contribute to different instrument response times. It is generally becoming accepted that “ normal” HPLC is greater than 5 minute run time, “ fast” HPLC is between 1 and 5 minutes, and “ ultra-fast” is anything under one minute. If we assume that there could be 10 peaks of interest in a chromatogram, and that the peaks are evenly spaced, each peak would have about 6 seconds to present, and return to baseline for fast or ultra-fast chromatography. If an ELSD cannot respond this quickly, it is unsuitable for high speed work.