Varian ELSD Technical Document -...

For Internal Use Only

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The Varian 380-LC & 385-LC Evaporative Light Scattering Detector

Technical Document

Polymer Laboratories, now a part of Varian Inc.Essex Road, Church Stretton, Shropshire SY6 6AX, UK


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Table of Contents

TABLE OF CONTENTS ...........................................................................................................................2 INTRODUCTION ......................................................................................................................................4

Principles of Operation ......................................................................................................................... 4 ELSD Theory ........................................................................................................................................ 5 Instrument Design ................................................................................................................................ 7

Nebuliser ..........................................................................................................................................7 Evaporator Design ...........................................................................................................................7 Optical Design..................................................................................................................................9 Hardware..........................................................................................................................................9 Firmware ........................................................................................................................................ 10

Instrument Controls ............................................................................................................................ 10 Display Screen ............................................................................................................................... 10 Keypad ........................................................................................................................................... 10 Sub- Menu Screen ......................................................................................................................... 11 Status Mode ................................................................................................................................... 11

STANDBY.................................................................................................................................. 11 RUN ........................................................................................................................................... 11

Error conditions .............................................................................................................................. 11 Operational Parameters ..................................................................................................................... 12

Method ........................................................................................................................................... 12 Loading a Method .......................................................................................................................... 12 Evaporator Temperature ................................................................................................................ 12 Nebuliser Temperature................................................................................................................... 13 Evaporator Gas Flow ..................................................................................................................... 13 Detector Gain (PMT) ...................................................................................................................... 13 Response Time (Smoothing).......................................................................................................... 13 Light Source Intensity (LED) .......................................................................................................... 13 Power Mode ................................................................................................................................... 13 Data Output Rate (Hz) ................................................................................................................... 14

Detector Control during an Injection ................................................................................................... 14 Timetable Operation....................................................................................................................... 14 Creating the Timetable................................................................................................................... 14 Starting & Stopping the Timetable.................................................................................................. 14

Connections........................................................................................................................................ 15 Power Connections ........................................................................................................................ 15 Extraction ....................................................................................................................................... 15 Gas Connection ............................................................................................................................. 15 Other Connections ......................................................................................................................... 15 Digital output .................................................................................................................................. 16 Analogue output ............................................................................................................................. 16 Connection to a Varian Star 800 Module Interface Box ................................................................. 16 Control I/O connector ..................................................................................................................... 17 Fluid Connection ............................................................................................................................ 17

Inlet ............................................................................................................................................ 17 Outlet ......................................................................................................................................... 17

Computer Control ............................................................................................................................... 17 Operating the Varian ELSD Software Utilities ................................................................................ 17

Control Software-Overview ........................................................................................................ 18 Control Software-System Test ................................................................................................... 18


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Method Editor Software-Overview ............................................................................................. 18 Controlling the Varian ELSD Using Galaxie ................................................................................... 18

Galaxie Status window............................................................................................................... 18 Status Overview......................................................................................................................... 20 Automating the Varian ELS detector.......................................................................................... 20

Varian 380-LC &385-LC Performance Characteristics ....................................................................... 21 Reproducibility................................................................................................................................ 21 Detector Noise ............................................................................................................................... 22 Signal Output ................................................................................................................................. 22 Sensitivity ....................................................................................................................................... 23 Linearity.......................................................................................................................................... 24 Uniformity of response ................................................................................................................... 25

Varian ELSD vs PL-ELS 2100 Performance ...................................................................................... 27 Best Practice for the Varian ELSD...................................................................................................... 28

Solvent recommendations.............................................................................................................. 28 Sample preparation........................................................................................................................ 29 Column Considerations .................................................................................................................. 29 Transferring ELSD Methods........................................................................................................... 29 Do’s and Don’ts of ELS Detection .................................................................................................. 29 Pumping systems........................................................................................................................... 29 Mobile phase priming ..................................................................................................................... 30

Maintenance ....................................................................................................................................... 30


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Introduction

The Varian Evaporative Light Scattering Detector is a universal detection technique for liquid chromatography. It is designed and built at the Polymer Laboratories Varian facility in Shropshire, UK. The unique design combines high sensitivity with low temperature operation.

Figure 1: Varian Evaporative Light-Scattering Detector The Varian ELSD has two models based on their operating temperature range:

Varian 380-LC --------------------- 25-120 °C

Varian 385-LC ---------------------- 10 –80 °C The technology of both ELSD models is near-identical and therefore all of the technical data referred to in this document apply to both models unless otherwise stated. The Varian ELSD is designed for analytical applications where compounds lack a UV chromophore or fluorophore, particularly in the pharmaceutical sector. The ELS detector is complementary to LC-MS as both techniques share similar chromatographic requirements.

Principles of Operation The operation of the ELSD consists of three consecutive steps, namely Nebulisation, Evaporation and Optical detection (see Figure 2). During the nebulisation stage, the mobile phase is passed through a fine needle and combined with a gas, such as Nitrogen or air, to form a plume of uniform aerosol droplets. The size and shape of the droplets* determine the sensitivity and repeatability of the detector. The droplet size distribution is influenced by changes in the nebulisation gas flow as well as other factors such as mobile phase composition. Large droplets are removed by condensation or collision with the chamber walls, and subsequently directed to a waste outlet. The nebulisation process can be heated independently of the evaporation tube, which is useful in minimising changes in mobile phase viscosity during gradient elution. The aerosol formed during nebulisation then passes through a heated coil or tube where the solvent is evaporated, to leave residual particles of the analyte. The temperature of this heated tube is controlled by the user and is commonly set according to the mobile phase composition or compound volatility. It is important to use

* Please Note, for purposes of clarity, droplet refers to a droplet of mobile phase liquid formed during nebulisation. Particle refers to the solid form of the analyte at the detection process.


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as low a temperature as possible for this step, to minimise sample volatilisation. Low temperature evaporation is required for low molecular weight pharmaceuticals. However, this type of compound is analysed in highly aqueous eluents, making their detection at ambient temperature difficult. To overcome this problem, the Varian ELSD uses additional dry gas during the evaporation step, to facilitate desolvation without increasing temperature. This 'evaporation gas' is controllable by the user according to the type of mobile phase. For example, high boiling point eluents, such as water, require higher evaporator gas flows than low boiling point solvents, such as dichloromethane. This technology enables the Varian 380-LC to evaporate water at ambient temperature (ca. 25 °C) at a flow rate of 5ml/min and the 385-LC to remove water at 10 °C.

Figure 2: General Schematic of an ELSD The analyte particle exits the drift tube and enters an optical chamber where the solute particles pass through a collimated light beam. The light source is typically a halogen lamp or a monochromatic laser-emitting diode. The light scattered by the particle is detected using a photomultiplier or photodiode, and the magnitude of the scattered light is dependent on the size of the particle formed. The size of the analyte particle is dependent on several factors, including sample concentration, physical properties of the mobile phase, gas flow and sample volatility. Typical detection limits lie in the range 1-50ng on-column, which can sometimes be 2-3 orders of magnitude lower than compared to UV. ELSD response is non-linear because its signal is related to the absolute quantity of the compound and independent of the analyte’s optical properties. Consequently, it does not obey Beer’s Law. A simple logarithmic manipulation of the data will produce a linear relationship. Therefore, some caution must be exercised for applications that demand a very low limit of detection or very high accuracy.

ELSD Theory There are four main processes by which the path of electromagnetic radiation or light can change direction, when passing through a medium containing a suspended particulate phase. These are shown in Figure 3.


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Figure 3:Scattering mechanisms in an ELSD The importance of each of these processes depends on the radius of the particle (r) compared to the wavelength

(λ) of the incident light. Rayleigh scattering is predominant when r/λ is < 5x10-2. When particle dimensions are greater than λ/20 they no longer behave as point sources, and Mie scattering becomes predominant. Once particle size approaches the wavelength of incident light then reflection and refraction begin to prevail. In order to decide which mechanism is predominantly responsible for the “scattering” observed in the Varian ELSD, an estimate of the size of the particles involved compared to the wavelength of the incident light can be made:

Du

QQa

0

0 45 1 5585

597 1000= +

⎛⎝⎜

⎞⎠⎟

⎛⎝⎜

⎞⎠⎟ =

σρ

μσ

. .

n D / n D a 3

a 2

where D0 = mean drop diameter

na = number of drops in the size range, with diameter D

σ = liquid surface tension ρ = liquid density μ = liquid viscosity u = relative velocity between the gas stream and the liquid stream Q = volumetric flow rate of liquid Qa = volumetric flow rate of gas The particulate size may be varied by altering the gas velocity, the eluent flow rate, the temperature of the nebuliser and the initial solute concentration Changes in the solute concentration and variations in the atomiser gas pressure influence the solute particle size. This relationship gives the instrument a maximum sensitivity around r/λ = 4. Detection declines rapidly when values for r/λ are above 5 or below 2.5. When r/λ < 2.5 the interference effects typical of Mie scattering cause the deflected light to be low in intensity at the measuring angles. As the particles increase in size, reflection and refraction become dominant and sensitivity increases. A further increase in the particle size causes the ratio of surface area to volume to decrease thus the sensitivity decreases. The distribution tails as diameter increases, the largest particle in a distribution generally reaching twice that of the mean. Consequently, although there is undoubtedly some Mie and Rayleigh scattering, the observed phenomena are predominantly due to reflection and refraction since the majority of the particles are larger than the incident wavelength.


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The relative importance of refraction and reflection can be understood by examining the effects of the incident light on a single spherical particle whose equilateral axis lies in the same plane as the photodetector and light source. With this configuration, refraction is of greater significance than reflection. The majority of organic compounds have refractive indices between 1.3 and 1.5. Changes in the refractive index within this range will not greatly affect the quantity of light reaching the detector. This accounts for similarities in the sensitivity of the instrument to various compounds. Therefore, the Varian ELS detector is useful as a universal detector, providing that the material under investigation is non-volatile under the operating conditions of the instrument.

Instrument Design Nebuliser The design and function of the nebuliser in the Varian ELS detector directly influences all subsequent stages of operation, consequently the nebuliser design is key to the performance of the Varian ELSD.

Figure 4: Varian ELSD Nebuliser cross-section The nebulisation stream can be categorised into three distinct aerosols; namely, primary, secondary and tertiary. The primary aerosol describes the plume formed straight after the nebuliser tip, whereas the tertiary aerosol is the portion of droplets/particles that reaches the detection cell (see Figure 4). Consequently, it is the properties of the tertiary aerosol that are important for ELSD performance, however the properties of the tertiary aerosol are dependent on the characteristics of the primary aerosol. The nebuliser in the Varian ELS detector is made of glass, as opposed to the stainless design in the PL-ELS 2100. The glass nebuliser is supplied by Glass Expansion®, and operates at a fixed gas flow of 0.3SLMs, as this is the optimum gas flow for the operation of the nebuliser. Changing nebuliser gas to reflect the mobile phase characteristics are not needed with the Varian 38-LC series as the nebuliser is highly efficient. Low viscosity solvents are nebulised as effectively as high viscosity solvents. The nebuliser can be heated to 90 °C, which can help in reducing viscosity changes across a solvent gradient. Typically the nebuliser temperature is set to the same value as the evaporator temperature, as this can improve signal-to-noise. It was found that higher temperatures on the glass nebuliser improved response. Even though the main design features of the PL-ELS 2100 are being retained in Varian ELSD, the glass nebuliser produces a different performance characteristic to the PL-ELS 2100. An overview of the performance characteristics is given in the “Detector Performance” section. Evaporator Design


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The technique of ELSD relies on removing solvent by evaporation prior to detection. For non-volatile compounds this approach is straightforward as the evaporator temperature can be set at, or close to, the boiling point of the mobile phase. In order to analyse semi-volatile species, the ELSD needs to operate at 30 °C, which makes the evaporation of solvents, such as water, very difficult. As Figure 5 shows, to dry a 10 µm water droplet, at 30 °C, takes 2.4 times longer than at 50 °C. So as you reduce the evaporation temperature, the evaporation tube needs to be longer, in order to remove the solvent. If the droplet size is doubled from 10 µm to 20 µm, the problem is even worse, because a 20 µm droplet takes 4 times longer to evaporate than a 10 µm droplet at 30 °C. The Varian ELSD has the shortest evaporation tube of other ELSDs on the market, so to remove solvents such as water, at ambient temperature, dry nitrogen gas is added during the evaporation step; this is referred to as “evaporation gas”. This evaporation gas reduces the relative humidity (vapour loading) of the surrounding solvent vapour in the evaporation tube; this in turn reduces the drying time. Figure 6 shows the affect that evaporation gas has on the drying time of water droplets at 30 °C. By reducing the relative humidity from 70% to 50% for a 20 µm droplet, the drying time is halved. So the addition of gas during the evaporation step allows the evaporation tube to be shorter, and facilitates low temperature operation at ambient temperature.

Figure 5: Drying time for water droplets at different evaporator temperatures

Figure 6: Drying time for water droplets at 30 °C under different relative humidities


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The evaporation gas is a user defined parameter and adjusted according to the mobile phase conditions. Optical Design The Varian ELS detector’s optical bench has been specifically designed to maximise sensitivity, whilst keeping the background noise to a minimum. The light source is a short-wavelength (480nm) Light-Emitting Diode (LED), which provides maximum sensitivity for small particles. The shorter the wavelength of light, the scattering process becomes more efficient because the smaller particles appear larger to the incident radiation; hence refraction and reflection predominates. The LED source is also a cool light source (unlike a halogen lamp), so internal heating of the instrument does not occur, which would otherwise prevent low temperature operation of the ELSD. It has a feedback monitor to maintain a constant output over the lifetime of ELSD. The focal path length of the LED is such that the light beam covers the largest area of the particle stream, consequently, the optics are ‘bent’ to accommodate this (see Figure 7). To maximise detection of the scattered light the Photo-multiplier (PMT) detector module is mounted as close as possible to the scattering region. The PMT is also arranged at the optimised angle of 120°C to the incoming radiation to ensure maximum sensitivity. The PMT and LED on the Varian ELSD have been “matched” to the same wavelength (480m), because the PMT is at its optimum sensitivity at this wavelength.

Figure 7: Cross-section of optical bench in the Varian ELSD To prevent condensation forming on the optical surfaces, the optical bench on the Varian ELS detecor is kept at a constant temperature of 50 °C and is “swept” by nitrogen gas at 0.4SLM. Hardware The ELSD is controlled using a single PCB, with surface mounted components. The main board controls all functions of the detector, with the exception of 385-LC, which has additional safety control on the Peltier module. The main board includes safety devices such as an internal vapour sensor as well as the RS232 communication ports. A DAC is mounted on the main PCB, plus additional electronics to provide digital signal processing.


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Firmware The Varian ELSD main PCB contains a main control PIC (microchip) and a second safety PIC. In total there are 4 pieces of firmware required to control the ELSD. The 385-LC has additional safety firmware on the Peltier module; hence the safety firmware on the 380-LC is different to the safety firmware on the 385-LC The firmware on the main control PIC is flash upgradeable via a bootloader, using the RS232 service port on the rear of the instrument. The version of firmware installed on the 380-LC & 385-LC at the time of release were:

Varian 380-LC v 22.02

Varian 385-LC v 22.04

Instrument Controls The Varian ELS detector can be used as a standalone detector via the front keypad and screen, as shown in Figure 8. Alternatively it can be controlled using a PC, running the following chromatography data systems (CDS):

Galaxie Agilent Chemstation Rev B EasyChrom Dionex Chromeleon

Display Screen The graphical interface on the front of the instrument displays the current method, status, evaporator temperature, nebuliser temperature, gas flow and output of the instrument. Operating parameters can be altered via the interactive menu bar at the bottom of the display.

Figure 8: Varian ELSD Display Screen Keypad The four arrows on the front of the instrument are used to navigate within the interactive menu bar. The AZ/Stop key has a dual function; it can be used to auto zero the ELSD at any time, unless a timetable is running. If the AZ/Stop key is pressed during an active timetable, the timetable will stop running and the ELSD will revert to STANDBY mode.

Main menu bar

To change the current settings, use the arrow keys to navigate across the interactive menu bar until the desired option is flashing. Using the up/down arrow keys alter the parameter to the desired setting. In order to action any changes, the cursor must be returned to the “Home” position ( or ).


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When the cursor is located in the “Home” position, the actual detector values are displayed in the main screen. If the instrument is controlled via PC software, then the home key will display a locked icon and the keypad will be disabled. To unlock the keypad, software control must be terminated. Sub- Menu Screen The sub-menu screen, which is accessed from the front screen by selecting the symbol and then pressing the

arrow key, allows changes to the following electronic parameters:- PMT Signal Gain SMTH Time Constant LED Light Source Intensity PWR MODE Status at Power up HZ Data Output Rate

Status Mode The Varian ELS detector can be operated in two modes; STANDBY and RUN, both of which are described below: To display the current mode and/or select a new mode, highlight the MODE function on the instrument display. The current mode will now be displayed on the screen. Using the keys, scroll up or down until the desired option is displayed. The instrument acknowledges the command by displaying the mode of operation in the top right hand corner of the screen. STANDBY The STANDBY mode is the “ground state” of the ELS detector, which is by default initiated automatically after power on (default can be changed using Power up option). In STANDBY mode the heaters are switched off, and the gas manifold valve is closed at power on. The STANDBY mode gives the user a control platform in which to set-up the operational parameters (gas flow, nebuliser and evaporator temperatures) before switching the unit into RUN mode. The instrument will default to STANDBY mode should an error occur on the instrument When the instrument is switched from RUN mode to STANDBY mode, following a command or error, then the gas management system is invoked and the gas flow set to a minimum flow of 1.2SLM for 15minutes before the gas manifold valve is closed. This minimum “blanket” gas is enough to nebuliser and evacuate solvent should the instrument default to STANDBY mode with solvent still flowing.

RUN The RUN mode is the detector’s operational mode. In this mode the instrument is controlled at the set temperatures and gas flow, and the system is fully operational. During heating or cooling the instrument will display NOT READY to show the system has not reached the set conditions. When the instrument has equilibrated READY will be displayed and the instrument is ready for use. Error conditions The Varian ELS detector is equipped with a number of sensors and error checking facilities to ensure safe operation. If an error is detected the instrument gives an audible warning and a visible description of the error


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condition. In event of any error condition, the unit defaults into the STANDBY mode in which the heaters, LED & gas are turned off. A complete list of instrument errors and remedial actions are given in the troubleshooting section of this manual. Clearing an Error Once the source of the problem has been corrected, the error message will be cleared automatically on the instrument display and remain in STANDBY mode. Select RUN mode to put the ELSD back into its operational state. If the problem has not been rectified the ELSD will repeatedly error when RUN mode is selected.

Operational Parameters Method The Varian ELS detector has the capacity to store 10 custom definable methods in memory. These methods comprise, evaporator & nebuliser temperature and gas flow, which can be optimized for specific applications. These 10 on-board methods are selected using the front keypad and screen, via the METHOD option. The Method Editor Software package is required to edit and download the custom methods. In addition to the 10 on-board methods, the Varian ELS detector has a method XXX that allows modification of the ELSD parameters to be made without the need for software control. Method XXX allows the detector to be used in standalone mode via the front screen and keypad. Loading a Method To load one of the 10 on-board methods, highlight METHOD. Using the keys scroll up or down to the required method number. The instrument will acknowledge the change by displaying the method number in the top left hand corner. Editing of this method is prohibited via the front keypad; this can only be carried out using the Method Editor Software (see section 2.4). The instrument is pre-loaded with 10 methods at the factory, as shown below:

Evaporator Temp (°C) Nebuliser Temp (°C) Gas Flow (SLM) Method Number Varian 380-LC Varian 385-LC Varian 380-

LC Varian 385-LC Varian 380-LC

Varian 385-LC 1 30 15 30 30 1.6 2.5 2 40 20 40 30 1.3 1.6 3 50 30 30 30 1.2 1.6 4 50 50 50 50 1.2 1.2 5 70 50 70 30 1.2 1.2 6 90 70 50 50 1.6 1.2 7 90 80 50 50 1.0 1.6 8 120 80 50 50 1.0 1.2 9 120 20 90 90 0.9 2.5 10 120 80 90 90 2.8 2.8

Table 1. Ten Preloaded Methods on Varian ELSD Evaporator Temperature The evaporator temperature is the most important setting on the ELS detector. This should be set according to the volatility of the compound(s) being analyzed. If the compound is non-volatile, e.g. sugars, then the evaporator temperature should be set to 80-90 °C If the compound is semi-volatile, or has a low molecular weight, e.g. pharmaceutical drug, then the evaporator temperature should be set between 20-30 °C. The evaporator temperature ranges for the Varian ELS models are:

Varian 380-LC……………………….OFF, 25-90 °C (1 °C increments) Varian 385-LC…………………...OFF, 10-80 °C (1 °C increments)

The default value is set to 30 °C


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Nebuliser Temperature The nebuliser temperature can be used to optimize signal response in addition to evaporator temperature. Higher nebuliser temperatures increase peak response, but the nebuliser temperature must not exceed the boiling point of the mobile phase. The nebuliser temperature range for both models is: Varian 380-LC & 385-LC……………OFF, 25-90 °C (1 °C increments) The default value is 30 °C Evaporator Gas Flow The evaporator gas flow is used to control the ELS detector’s evaporation process. The evaporator gas value is set according to the mobile phase composition, with higher gas flows (e.g. 1.6SLM) being used for aqueous eluent compared to those containing organic solvents. The higher the evaporator temperature the lower the evaporation gas setting required (e.g. 1.0-0.9SLM), regardless of mobile phase composition. Likewise, as the evaporator temperature is reduced to ambient and sub-ambient temperatures, the gas flow needs to be increased to compensate (e.g. 1.6-1.8SLM). The evaporation gas range for both models is: Varian 380-LC & 385-LC …………0.9-3.25SLM (0.05SLM increments) The default value is 1.6SLM Detector Gain (PMT) This parameter sets the factor by which the detector output signal is amplified. The gain setting does not change the sensitivity of the detector, but merely amplifies or divides the captured signal by the inputted factor. The gain can be adjusted from 1 to 10 in increments of 0.1. When setting the PMT (or Gain), both the signal and noise are simply amplified by the value set, so S/N values are unaffected. The raw signal output displayed on the parameter screen will reflect this increase or decrease in signal amplification. Please note that the instrument output displayed on the main operating screen is automatically zeroed to 10mV, following a PMT change, thus the recorded baseline position will remain unchanged. Confirmation of a PMT change will be the obvious by the change in baseline noise. Response Time (Smoothing) The data is continuously collected at a rate of 10 or 40 points per sec (10 & 40Hz), which can be averaged to produce a smoother output. The smoothing width is set to the number of data points over which the data is averaged and can be regarded as a digital time constant. The smoothing range is settable from 1-50, (in increments of 1) which translates to 0.1-5.0secs. For most HPLC applications the default value of 30 (3sec) is satisfactory. However for faster separations where peak widths <3sec, a setting of 1(0.1sec) is recommended. For GPC applications where peak widths can be >30sec, a value of 50 (5sec) is recommended. Light Source Intensity (LED) The Varian ELS detector’s LED intensity can be adjusted in order to bring the peak response back on-scale. The intensity range can be set between 0-100%, with the default factory setting being 100%, for maximum sensitivity. The LED setting is stored in memory and is retained even after a power on/off cycle. This feature is extremely useful for preparative chromatography where samples of high concentration can be analysed which would otherwise exceed the dynamic range of the detector. Power Mode The instrument can be configured from the front panel sub-menu, to start in either RUN or STANDBY mode when the unit is switched on via the rear power button. To configure the Power Mode, select the required Status Mode (i.e. STANDBY or RUN) you wish the unit to start-up in from the sub menu screen. Switch off the instrument and wait for at least 10 seconds before switching the unit back on, in order for the change to take affect. If RUN mode is selected as the desired Power mode, then the instrument will use the operating parameters stored in memory. In the unlikely event that the instrument encounters a fault during power-up the unit will automatically switch to STANDBY mode.


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Data Output Rate (Hz) The rate at which the Varian ELS detector outputs data can be selected from the sub-menu screen. A 10Hz output rate is selectable for standard LC applications, whilst a 40Hz output rate can be chosen for faster LC separations. The data rate is stored in memory and is retained even after a power on/off cycle. The default value is 10Hz.

Detector Control during an Injection The Varian ELS detector has the capability to change operational parameters in real-time, during a sample injection, using an on-board timetable. The on-board timetable is programmed using Dimension software Timetable Operation The Varian ELS detector can store in memory a series of time-based events, within a single timetable. This timetable allows the operational settings of the ELSD to be changed with respect to time. The temperatures, gas flow, LED, gain and smoothing parameters can all be configured, within this timetable, to change during a sample injection. The timetable can be used to program the gas flow, in order to compensate for the change in ELSD response across a solvent gradient. Alternatively, the timetable can be customized to create evaporator temperature gradients across a sample injection. The single timetable, stored on-board the ELSD, is only customizable using the DIMENSION software. Creating the Timetable In order to create or modify the on-board timetable, the DIMENSION software must be installed on your PC. The timetable software package allows you to create a timetable on a PC, which can subsequently be downloaded to the ELSD for later use. The ELSD can only store a single timetable in memory, so the timetable software can be used to create & save multiple timetables that can be downloaded individually at a later date. Starting & Stopping the Timetable The ELSD contains an internal timer that is used to trigger the time-based events stored within the timetable. The on-board timetable is triggered using a contact closure input via the remote start cable at the rear of the instrument When the timetable is initiated, the front panel of the ELSD will display “TTRUN” above the output, as shown in Figure 9.

1. Current runtime 2. Total runtime 3. On-board Timetable is active

Figure 9: ELSD Display during Timetable operation When the timetable is running, the current and the total run time are displayed, in minutes, at the top-centre of the ELSD display. When the timetable reaches the end of its run time, the ELSD will revert to RUN mode and be primed ready to start the timetable again. During an active timetable, where the evaporator temperature is being controlled, the status of the instrument will change from READY to NOT READY. This is normal behaviour and will not affect the running of the timetable.

2

1 3


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The STOP button on the instrument’s front keypad can be used to interrupt the active timetable whilst it is running. When the STOP button is pressed the instrument will revert to STANDBY mode.

Connections

Power Connections Before connecting the power cable, ensure the instrument voltage rating matches your local power supply.

Use only a supply with protective grounding.

The correct fuses should be installed.

For 115V (AC) or 230V (AC) use two 250V H 2A T 20 x 5 mm fuses

Note The unit is double-fused.

Extraction The PL-ELS Detector is provided with tubing for venting the exhaust gases and vapours, and so does not need to be placed in a fume cupboard. Instead, the exhaust hose provided must be attached to the rear of the unit and vented to a fume hood or other disposal unit. Ensure the exhaust hose has an upward slope from the detector so that any condensed solvent is collected in the waste bottle at the front of the unit and to prevent it accumulating in the tubing

Gas Connection The instrument should be supplied with clean, dry nitrogen gas between 60-100psi in pressure. For optimum performance a gas pressure of 60PSI is recommended. A convenient 4mm push-in connection for the gas source is provided at the rear of the instrument.

To prevent against unnecessary gas usage, a controlled gas shut off valve is integrated into the gas inlet manifold. This will only allow gas to pass into the instrument when the instrument is operating. Should the instrument default to a STANDBY mode the gas will reduce to a default value of 1.2SLM for 15mins before closing

Other Connections All power, signal and communication connections are made on the rear panel of the Varian ELS detector, see Figure 10. The connectors on the rear of the panel support communication configurations to a range of devices, such as autosamplers, pumps, valves and injectors.


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Figure 10: Front & Rear View of the Varian ELS Detector

Digital output The Varian ELS detector is fitted with a standard RS232 (DTE-DCE) 3-wire serial interface.

The serial RS232 connector provides a 24bit (10 or 40Hz) digital output for connection to a PC running a data acquisition package (e.g. Galaxie Chromatography software) or standalone control software. Further information on how to configure the PC for with Standalone or Galaxie software is given in sections 2.4 & 2.5, respectively.

If controlling the instrument from a PC, a free serial port is required. A USB port on the PC can be used but a USB-to-Serial adapter is required (PL0860-0620).

The Varian ELS detector is also fitted with a Service connector, located below the RS232 port, for flash upgrading firmware.

Analogue output The Varian ELS detector is supplied with an 1V analogue output cable (PL0890-0300) that allows data collection via an A/D interface, such as a Star MIB 800 module. The gold end fitting of the analogue cable plugs into the gold pin on the rear of the detector and the opposite end connects to the positive and negative inputs of the A/D interface.

Connection to a Varian Star 800 Module Interface Box The Star 800 Module Interface Box (MIB) provides analogue-to-digital signal conversion (ADC). Connections are made by connecting the analogue output cable to one of the analogue signal input ports on the middle right side of the Star 800 MIB. For more information about connecting your Varian PL-ELS Detector to a Star 800 MIB, refer to your Star 800 MIB documentation.

Liquid waste Outlet

Solvent Inlet

Gas Inlet

Detector Output (1V)

Serial RS232 connector

Firmware flash upgrade connector

I/O Connector


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Control I/O connector The Varian ELS detector can be connected to auxiliary equipment, such as an autosample, or pump via the 15pin I/O connector. The I/O connector can be configured in several ways to allow on-board timetable events to be triggered or to remotely auto-zero and shutdown the detector.

The ELS detector is equipped with 2 contact closures (normally-open) for stopping the operation of a pump if the unit reports an error condition.

The Varian ELS detector is equipped with one contact closure, which is normally open, and two TTL logic inputs, both active-low (with internal pull-up resistors to 5 V).

I/O description Pin number

Inputs Remote Start 14 & ground

Remote Standby 13 & ground

Remote A/Z 7 & ground

Output Pump stop contact closure – normally open 3 & 10

Ground (to case) 1, 5, 6, 11

Table 2: Control I/O connections

Fluid Connection

Inlet The eluent from the chromatography system is connected to the central front port of the Varian ELS detector via a low dead volume valco bulkhead connector.

The liquid inlet port is connected directly to the nebuliser by a short length (130 mm) of capillary tube giving a delay volume from port to nebuliser tip of ~5 µl. Outlet The waste eluent is expelled from the detector via the front outlet tube. The outlet tube should be connected to the 500 mL waste bottle using the supplied tygon tubing of 40 mm ID.

For long periods of use, it is recommended that a larger solvent container is used, to prevent the solvent overflowing.

Computer Control Operating the Varian ELSD Software Utilities These Windows™-based graphical interfaces offer a second and complementary level to total detector control. An intuitive single control panel provides simplistic control as well as a comprehensive monitoring system. Operational parameters can be easily manipulated, saved or loaded by using the flexible Methods Editor enabling rapid set-up and custom method archiving. A general description of the features available through the software utilities is given below, but for a more comprehensive guide on how to use the control software, please refer to the On-line help supplied with the software.


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Control Software-Overview Control of the Varian ELSD is achieved through the simple control window. Within the control window, the status of the detector is displayed, together with options to control the various instrument parameters. The control window is effectively divided into a Control, Status and Output section. For more information on operating the control software, please refer to the operation manual. Control Software-System Test The Control software has an option for testing the basic operation parameters of the ELSD to ensure the heaters and other hardware are operating within specification. When you select this feature (TOOLS / Run system test menu option), the software will run the ELSD through a number of internal diagnostic tests, and then compare the results with the expected values set by Varian, Polymer Laboratories. The test information can be subsequently printed to be kept as a record. The test takes approximately 2hours to complete and should only be used occasionally to check the instrument’s performance, or when a problem is encountered.

Method Editor Software-Overview The Varian ELSD can store 10 custom methods, which can be selected via the front of the instrument or via the control software. The method editor program allows the user to create, edit and download these custom methods for method development purposes or specific applications. The method editor program can be launched from the control software via this icon To change a custom method on the Varian ELSD, you must first create a custom "Method Set", (FILE/New), which can be subsequently downloaded to the detector. Each Method Set contains 10 custom methods, with each method containing three customisable parameters,

Evaporator Temperature Nebuliser Temperature Gas Flow

Single methods within a method set cannot be individually downloaded to the detector. All 10 methods of the Method Set must be downloaded simultaneously to the Detector. Each "Method Set" can be saved as an individual binary format file with a File Extension (.ELS). Controlling the Varian ELSD Using Galaxie In order to control the Varian ELS detector using Galaxie Chromatography software, the appropriate ELSD driver must be installed. Please refer to Galaxie User manual for instructions on how to install the Galaxie ELSD driver software. Galaxie Status window

Within Galaxie’s main menu screen, direct control of the instrument can be accessed via the button.


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In this status window, the detector’s output and control parameters (Nebuliser, evaporator, Gas flow, etc.) are shown with their current values, depending on the control method program. When the button is selected, the detector will begin heating to the set temperatures and gas flow. When the detector is heating the light will turn orange. When the detector has reached the set parameters, the light will turn green. When button is selected, the detector’s LED will turn off, along with the heaters. The gas flow will revert to a minimum value of 1.2SLM (section 2.3). When this function is active, the indicator light turns solid red. The detector can be auto zeroed from this screen at any time, by pressing the button. To set the detector parameters, click on the button to access the parameter configuration screen, as shown:

In the configuration men, the following detector parameters can be set and downloaded to the detector.

Parameter Operational Range Evaporator Temperature

Varian 380-LC Varian 385-LC

Ambient –120 °C

10-80 °C Nebuliser Temperature Ambient – 90 °C

Gas Flow (SLM) 0.9-3.25 Gain 1-10

Smoothing 1-50 (i.e. 0.1 to 5 secs) LED 0-100%

Note: Galaxie will automatically adjust the evaporator range according to the ELSD connected Once the detector parameters are set, press the button to download the values to the detector. The detector parameters cannot be adjusted during data acquisition. Connection status When the Varian ELS detector is connected to Galaxie™ Chromatography Software, the detector’s firmware version (e.g., 2.0.4) is shown at the top left corner of the configuration screen. If communication is lost, or no ELS detector is connected, “offline” is shown.


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Status Overview A general overview of the HPLC system and current status of the Varian 380-LC/385-LC detector can be

accessed via the button, within Galaxie’s main menu screen, The overview screen displays a graphical representation of the ELSD, along with the detector’s key parameters.

The status of the detector is indicated in the top left hand corner, with the green RUN light. When the Varian ELSD encounters an error it will switch to STANDBY mode, the status light will turn red and an error code message will be displayed on-screen. The error is also recorded in the Galaxie Events menu. A complete list of instrument errors and remedial actions are given in the troubleshooting section of this manual. Select RUN mode to clear the error and continue operating the detector. If the detector error is still present, the ELSD will show an error message as soon as the unit is put back into RUN mode. The current detector temperatures and output are also displayed in the screen, for easy reference and the detector can also be Auto zeroed from this overview screen.

Automating the Varian ELS detector Galaxie Software can be used to automate the operation of the Varian ELS detector, using methods and sequences. To create a Galaxie method or sequence, please refer to the Galaxie User’s Guide. In the Control section of the system method press the button to display the Varian ELSD control parameters. .

The method parameter window allows the ELS detector conditions to be changed according to a time-based program. For example, the gas flow parameter can be altered across a sample injection in order to control detector response. To create a time-based program, enter the time, when you want the change to take place, and the desired detector conditions across each line. Additional lines can be added, deleted or the whole table erased by using the

following buttons , respectively. The detector can also be autozeroed automatically between instrument methods if the Autozero on Method Change is selected. Alternatively, the instrument can be shutdown after an injection, or sequence if the Go to Standby at End of Run option is selected


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Varian 380-LC &385-LC Performance Characteristics The Varian ELSD like any other LC detector must meet certain performance standards in order to provide accurate and reliable results. The following section outlines the main performance characteristics of the Varian ELSD Reproducibility The use of a glass nebuliser within the instrument provides a very low variability between injections and between nebulisers.

Nebuliser S/N 81489 120-130 120-131 120-132 120-135 120-142 120-145 120-152 120-153 120-156 Average Peak response 1579.5 1436.3 1476.0 1448.4 1337.9 1453.0 1416.1 1429.7 1588.9 1241.3 Std Dev 67.46 11.10 26.63 24.67 16.91 26.54 12.96 16.47 32.47 10.20 %RSD 4.27 0.77 1.80 1.70 1.26 1.83 0.92 1.15 2.04 0.82 The average % variance across 10 nebulisers, for 10 injections of Glucose was 1.66%. Likewise the % variance BETWEEN nebulisers was only 5.38%, allowing nebulisers to be swapped between units without the need for re-optimisation or significant loss in performance.

Figure 11: %RSD of 10 Glass Nebulisers

The low RSD of the glass nebuliser also transfers to a low reproducibility for a complete LC stystem. 75 injections of Caffeine were made on a Varian LC system comprising 210 pump, 410 autosampler, Pursuit C18 column and 385-LC ELSD. This configuration gave a total system variance of 2.2%, see Figure 12.


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Figure 12: 75 injections of Caffeine using the Varian ELSD

Detector Noise The noise level of the Varian ELSD is measured under two conditions,

1) Gas flowing and no solvent flow 2) Gas Flowing with solvent flow

Under condition 1, the expected noise level is <0.1mV, whereas with solvent flow, the baseline noise will be <0.25mV. The noise level will increase above these levels if a mobile phase additive is present in the eluent, or if the temperatures are set too low. The noise level will be reduced if the peak smoothing setting is increased, or if the mobile phase is composed entirely of a highly volatile solvent, such as hexane. Signal Output The signal output rate on the ELSD is switchable between 10Hz (standard) and 40Hz. At 10Hz, the output rate is sufficient for most LC applications where peak widths >1sec. For LC separations where peak widths of <1sec are observed, such as fast LC methods, a data rate of 40Hz is required. Using 40Hz data rate the number of points across a 1sec peak are sufficient to properly characterise the shape, leading to higher resolution (see Figure 13)


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10

15

20

25

30

35

40

65 65.5 66 66.5 67 67.5 68 68.5 69

Time (sec)

10H

z E

LSD

dat

a (m

V)

11

16

21

26

31

36

41

40H

z E

LSD

dat

a (m

V)

10Hz Digital40Hz Digital

Figure 13: Comparison between 10Hz and 40Hz Output on Varian ELSD Sensitivity Five caffeine standards were obtained from Sigma-Aldrich (NIST accredited):

1. 500 µg/mL 2. 250 µg/mL 3. 125 µg/mL 4. 25 µg/mL 5. 5 µg/mL

These standards were injected at a solvent flow rate of 0.233 mL/min, to maximise the detection limit. The standards were also injected under different nebuliser configurations;

Glass neb 0.3SLM Glass neb 0.49SLM PL-ELS 2100 Nebuliser (0.5SLM)

The calibration curves for the three nebulisers configurations are shown in Figure 14, over the page. What the figure shows is that all three configurations give very similar results.


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Figure 14: Caffeine calibration for Glass and SS nebulisers under different gas flows.

Signal-to- Noise Caffeine Concentration (µg/ml) Glass Nebuliser Stainless Steel Neb

500 5515.0 5825.3 25 4170.6 3408.2 125 1782.2 1281.2 25 128.3 128.4 5 6.3 7.6

The S/N for the glass and SS nebulisers, for the lowest caffeine standard, are very close, with the Glass nebuliser being slightly worse than the SS neb. This higher LOD (or lower response) for the 5 µg/mL standard was because the peak width of the caffeine using the Glass nebuliser was broader than the SS neb. The dispersion characteristics of the glass neb have since been improved, so the Glass nebuliser should producer better S/N data. When higher gas flows are used with the Glass Nebuliser (i.e. 0.49SLM), the slope of the calibration increases and thus reduces the detection limit. Linearity

The Varian ELSD has a different linearity response than the PL-ELS 2100, as shown in Figure 14.The Varian ELSD has a linear range of 2-(3) orders of magnitude as shown in Figure 15, which is dependent on the analyte. Compounds of similar MW give similar calibration responses.


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Figure 15: Varian ELSD linearity for three pharmaceuticals, expressed with log axes. Uniformity of response The majority of pharmaceutical compounds possess a UV chromophore, hence UV detection is the first choice detection method. However, the use of a UV detector can limit the sensitivity and efficiency of a separation, because the optimum detection wavelength may not be compatible with the mobile phase eluent. In addition, different responses are obtained for different species at the same concentration due to differences in their extinction co-efficient. By contrast, since all analyte particles scatter light, the ELSD can detect all samples with high sensitivity and accuracy, irrespective of their chemistry or optical properties. Consequently, when developing chromatographic methods for active ingredients using ELSD, the analyst can select the appropriate solvent system to give the optimum separation without compromising the sensitivity or resolution. ELSD also provides a uniform response to all components, as highlighted in for Aspirin and Phenacetin. A solution containing equal quantities of Aspirin and Phenacetin produces an equal response for both compounds using the Varian ELS detector, whereas UV detection exhibits a lower response for Aspirin. This is due to a difference in extinction co-efficient between the two compounds. This example highlights the advantage of using ELSD when analysing mixtures of pharmaceutical compounds.


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Figure 16: Comparison of the uniformity of response between ELSD and UV/Vis for analysis of Aspirin and Phenacetin at equal concentrations.


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Varian ELSD vs PL-ELS 2100 Performance Nebuliser Characteristic PL-ELS 2100 Varian ELSD (380 & 385-LC) Optimum Nebuliser Gas flow

0.4-0.5SLM This is dependent on nebuliser. 0.5SLM is the fixed flow, but some nebulisers can take hours to configure.

0.3SLM Higher throughput of particles observed with higher gas flows >0.4SLM, but baseline noise also increases and hence S/N is poor

Nebuliser Temperature The 2100 shows little difference in performance as temperature is changed.

The Varian ELSD response is more dependent on temperature than the 2100. An increase in temperature significantly improves the response of the 220-this is useful for highly aqueous solvents.

Peak Shape (dispersion) The 2100 gives very sharp peak due to its low dispersion characteristics

Varian ELSD gives similar peak Internal volume is only 5.5 µL with Glass Expansion’s “LC” fittings kit

Sensitivity (Limit of Detection)

2100 seemed better for Glucose but worse for Caffeine. Depends on mobile phase composition

Generally similar to 2100, but depends on the compound. LOD was better for some analytes and worse for others when compared to 2100. Sensitivity can be improved with PMT gain voltage adjustments

Concentration response (Calibration)

2100 has a steeper calibration slope that results in a good response at high concentrations, but falls away quickly at lower concentrations.

Shallower calibration slope compared to 2100, which gives equal or better performance at the lower concentrations, but lower response at top end.

Uniformity of response

2100 response only changes 1.5-3.0x when going from aqueous to organic solvent; so has a shallow response across the gradient. However, combined with its calibration response, gives less uniform quantification than 380-LC.

Less uniform than 2100 across a solvent gradient. Higher response in organic compared to aqueous. However, combined with its shallow concentration response, appears to give a more uniform compound response.

Reproducibility (between injection)

3-6% 1.66%

Reproducibility (between Nebuliser)

Not measured; But known to be very high and a problem in production

5.4%. This provides a reliable interchange between Varian ELSD units.

Liquid flow rates <1ml/min Works well down to 200 µL/min but RSD worsens due to instability of plume. Imperfections on the needle tip are more pronounced at these flow rates.

Shows equal or better performance at flow rates around 200 µL. Caffeine response was equivalent or better than 2100. Other low flow rate versions available: 50µL/min-200µl/min The 50 µL/min nebuliser is to be tested. These are easily interchangeable

Liquid flow rates >1ml/min Compatible with flow rate up to 5 mL/min

Compatible with flow rate up to 5 mL/min

Operation below 25°C The 2100 gives droplets that can be evaporated water down at 18 °C.

The Varian ELSD allows operation down at 20 °C in Water, without problems.


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Different solvents Water: THF: ACN: MeOH: DCM: IPA: Additives (e.g TFA, TEA.)

No problems Problems over short-term No problems No problems No problems No problems No problems

Equivalent to 2100 No problems, probably even for long-term use. Equivalent to 2100 Equivalent to 2100 Equivalent to 2100 Equivalent to 2100 Equivalent/slightly better than 2100

QC procedure 2100 should produces a response of 300mV for Glucose solution

The Varian ELSD gives a 35% lower response, for the glucose solution compared to 2100, under same conditions. However, the QC procedure will need to be modified to increase the PMT gain voltage, to ensure equivalent/better sensitivity.

Table 3: Performance comparison between the PL-ELS 2100 and Varian ELSD

Best Practice for the Varian ELSD The Varian ELSD instrument should be thought of as a detector like any other designed for liquid chromatography. The main distinguishing feature is the ability to evaporate the solvent from the column eluent. Therefore, normal system set-up precautions should be remembered when starting to use the instrument. Any solvent intended for use with the Varian ELS detector should be fully miscible with any previously used in the liquid chromatograph; if there is any uncertainty, then a mutually miscible solvent should be run through the system as an intermediate liquid. The sample loop should also be flushed with miscible solvent where necessary. The intended eluent should be thoroughly degassed, contain no non-volatile salts or material and should be fully compatible with the column(s). All connections should be made with zero dead volume fittings and tubing with an I.D. ≤ 0.010”. The Varian ELSD requires nitrogen (purity >98%), capable of generating 60-100psi inlet pressure. If in-house nitrogen is not available then we recommend the use of a nitrogen generator to give a constant uninterrupted supply of high purity gas. Air can be used with non-flammable solvent systems. The eluent of choice should be fully volatile under the chosen detector parameters – any non-volatilised eluent will increase baseline noise and reduce sensitivity. The ELS detector is a destructive technique and must be placed last when used in series with other detectors. Solvent recommendations Any solvent intended for use with the Varian ELS detector should be thoroughly degassed, filtered (0.45µM) and fully compatible with the column(s). Solvents that are not properly degassed may cause problems at nebulisation leading to a poor reproducibility. Non-Volatile buffers are not compatible with the Varian ELS detector and should not be used. Only volatile mobile phase additives, such as those listed in Table 4 should be used with the ELS detector. Tetrahydrofuran (THF) stabilized with BHT, may increase the baseline noise level. Where possible unstabilised THF should be used with the ELS detector. If a non-volatile additive has been used with the Varian ELS detector, flush the instrument with an appropriate solvent overnight (e.g. Water) in order to remove the contaminant.

Mobile Phase Additive pKa pKb pH Range Bp (°C) Mp (°c) Acids Trifluoroacetic Acid (TFA) 0.3 13.70 72.4 -15.4 Formic Acid 3.75 10.25 100.7 8.3 Acetic Acid 4.75 9.25 116.0 16.6 Carbonic Acid 6.37 7.63 - - Bases Ammonia 9.25 4.75 -33.4 -77.7 Methylamine 10.66 3.34 -6.6 -94.0


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Ethylamine 10.81 3.19 16.6 -81.0 Triethylamine 11.01 2.99 89.3 -114.7 Buffers Ammonium Formate 3.8, 9.2 3.0-5.0 8.2-

10.2 120

Ammonium Acetate 4.8, 9.2 3.8-5.8 8.2-10.2

111

Ammonium Bicarbonate 6.3, 9.2, 10.3

6.8-11.3 106

Ion-Pair Reagent Pentafluoropropionic acid (PFPA) ~0.6 97 Heptafluorobutyric acid (HFBA) ~0.6 120 Nonafluoropropionic acid (NFPA) ~0.6 140 Tridecafluoroheptanoic acid ~0.6 175 Pentadecafluorooctanoic acid ~0.6 189

Table 4. Volatile mobile additives compatible with ELS detection Sample preparation Samples containing particulate matter should be filtered through a 0.45 µm filter prior to injection Column Considerations The ELS detector will detect all non-volatile components in the mobile phase, which includes column-packing material. Column packing material will become chemically and mechanically broken down over the lifetime of the column, causing particles to enter the ELSD. This column “shedding” will lead to extremely high baseline noise levels. Amino columns used with aqueous mobile phase are particularly prone to this type of shedding and should be checked regularly. To minimize column breakdown always follow the manufacturers instruction supplied with the column. Transferring ELSD Methods The direct transfer of ELSD operating conditions from other manufacturers’ ELS detectors, or other designs of ELSD (e.g. PL-ELS 1000) to the Varian ELSD will not provide equivalent performance. The operating temperatures of the PL-ELS detector are set according to the type of analyte and not the mobile phase composition as with other ELS detectors. For example, the PL-ELS 1000 has to operate at temperatures above 90 °C for a mobile phase of water; whereas the Varian ELSD can operate at 30 °C for the same mobile phase. Therefore the transfer of operating conditions from other models of ELSD to the Varian ELSD is not valid and the only way to ensure that the detector will provide the optimum analyte signal-to-noise is to follow the guidelines outlined in section 3.3. Do’s and Don’ts of ELS Detection

NEVER block the exhaust outlet as this causes increased back-pressure on the nebulisation chamber. High pressures on the internal chamber will lead to increased baseline noise and low sensitivity. NEVER allow the solvent waste outlet tube to become immersed in the waste solvent as this will create

back-pressure on the nebulisation chamber; leading to increased noise. When placing more than one HPLC detector in series, always place the ELS detector last. Only volatile mobile phase additives can be used the ELS. See Table 4. Volatile mobile additives

compatible with ELS detection(pg 29). The use of non-volatile buffers will lead to increased baseline noise levels.

Pumping systems

It is recommended to use a high-performance pumping system with no flow pulses to minimize nebulisation problems. Inconsistent solvent flow will result in poor reproducibility. For Varian Star 9000 series pumps and Varian 5000/5500 pumps or ProStar 210/215/218 pumps, make

sure the pump is operating at 10 MPa (1500 psi) of backpressure and that the pulse damper is functioning properly.


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The backpressure can be achieved either by the column itself or a coil of 0.005” ID tubing placed between the pump and the column. If pulse damping is needed, use the Varian Pulse Reduction Accessory (Part No. 0391970101) or the

Ultra High Pulse Reduction Accessory (Part No. 0391970102). For the Varian 212 & PrepStar SD1 pumps, no special operating pressure is required.

Mobile phase priming

The Varian ELS detector does not require any mobile phase priming, other than that required to prime the solvent through the pump, damper, injector, column, etc. It is recommended that priming of he LC system be performed without the ELS detector attached, to prevent non-volatile impurities contaminating the ELS detector. The mobile phase should be degassed and filtered, either by sparging with Helium or using an on-line

degasser (Part No. A6313).

Maintenance There is a potential for impurities to accumulate in the evaporation tube and nebuliser due to the nature of the instrument. Consequently, it is highly recommended that the detector is cleaned on a regular basis to prevent this build-up of contamination. The simplest was to prevent fouling of the instrument is to include a wash cycle in a sequence of injections, or to ensure clean mobile phase is flushed through the system prior to switching the unit off. If the instrument does become contaminated, a “steam clean” can be performed, whereby the instrument is heated to maximum temperatures and the gas flow is set to 3.0 SLMs. Water is then passed through the instrument for 16 hours (or overnight) to remove the contamination. Alternatively, the instrument can be flushed with a suitable solvent, if water isn’t applicable, for the same period of time.

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