Introducing the PL-ELS 2100 & PL-ELS 2100 Ice Evaporative Light Scattering Detector Presentation to describe the state-of-the art developments in ELS detection, designed to meet the demands of small molecule analysis by sub-ambient ELSD.

First Impressions Compact, modern design Flat top Small (200x450x415mm) Operation via control panel and simple keypad Column connection at front of detector One exhaust tube at rear, one liquid waste port at front PL-ELS 2100 PL-ELS 2100 Ice

Gas at minimum flow rate (1.2 SLM) Simple Operation Two modes of operation STANDBY Power on LED off Heaters off Gas at minimum flow rate (1.2 SLM) Gas shuts off after 15 minutes RUN LED on Temperatures and gas flow controlled to set values

Principles of Operation The ELSD principle of operation employs three distinct stages : Nebulisation Evaporation Detection Gas ELSD employs three stages, of which the nebulisation is the most important. Get this wrong, it doesn’t matter how good the evaporator or optics are. Animation should start immediatley Eluent inlet Light Source Liquid waste

Nebuliser Range: ambient to 90°C Nebulisation Inside the nebuliser chamber, the liquid flow from the column is mixed with an inert gas (normally nitrogen) to form a fine plume of solvent droplets. The temperature controlled nebuliser is designed to give a uniform droplet size. Nebuliser Range: ambient to 90°C Droplet size determines the sensitivity of an ELSD. The larger the droplet, the larger the final particle will be at the optical stage and hence the greater the scattering. To achieve the ideal droplet size, the user can control the nebulisation gas and temperature (on most units). Higher neb gas flows- the smaller the droplets produced for a given solvent and flow rate.

Evaporation The eluent droplets pass through a narrow heated chamber where the eluent is removed by evaporation leaving a fine mist of analyte particles. The evaporator tube has an additional gas input to aid removal of the mobile phase PL-ELS 2100 Ice: 10-80°C PL-ELS 2100: 30-120°C The evaporation step must be effective enough to evaporate the large droplets formed during nebulisation. The evaporation is heated and adjustable by the user.

PL-ELS 2100 Design Feature for Ambient Temperature Operation Current ELS detectors have design features that allow low temperature operation with water. An ELSD designed to operate at low temperature in water has to overcome two major problems The length of drying time experienced at low temperatures for water, compared to higher temperatures Saturation of the evaporation tube (i.e 100% relative humidity), which prevents further evaporation Modern ELSDs can operate in water at ambient temperature and how they do this depends on the manufacturer. The primary problem ELSD manufacturers have of operating an ELSD at 30°C in water is the increase in drying time that is needed. Furthermore, at 30°C the surrounding gas within the evaporation tube will quickly become saturated (i.e. 100% Relative Humidity), preventing the evaporation of water. To overcome this, current ELSDs are designed to split the liquid flow between the nebulisation and evaporation stage, to prevent the evaporator from being saturated with solvent. Often this is still insufficient to effectively evaporate the water at ambient temperature, so droplet size has to be reduced by increasing gas flow to the nebuliser.

Evaporating Water at Ambient Temperature Consider, that a 20µm droplet of water takes ca. 2.5 times longer to dry at 30°C than at 50°C. So reducing droplet size will reduce the drying time: A Water droplet of 10µm dries 4x faster at 30°C than a 20µm droplet. However, decreasing droplet size reduces the sensitivity of an ELSD Consider, that a 20µm droplet of water takes ca. 2.5 times longer to dry at 30°C compared to 50°C. But if you reduce the droplet size: A water droplet of 10µm dries 4x faster than a 20µm droplet at 30°C. However, reducing droplet size reduces the sensitivity of an ELSD.

Drying time of water droplets as a function of evaporator temperature 10µm droplet dries 4x faster than 20µm droplet @ 30°C

Evaporating Water at Ambient Temperature By lowering the relative humidity (RH) within the evaporation tube, water can be evaporated at ambient temperature, without changing droplet size. The PL-ELS 2100 uses a stream of nitrogen gas during the evaporation step to lower the relative humidity Using this “evaporation gas” the PL-ELS 2100 can evaporate water at ambient temperature. A more effective approach to evaporate water at ambient temperature, without adjusting the nebulisation gas, is to reduce the relative humidity (RH), or vapour loading within the evaporation tube. So, a 20µm droplet of water can be dried ca. 2.9 times faster at 30% RH compared to 70% RH at a temperature of 30°C So by keeping the relative humidity low, aqueous eluents can be effectively dried without compromising sensitivity.

Drying Time of Water Droplets as a function of Relative Humidity (RH) 20µm droplet dries 3x faster at 30% RH compared to 70% RH @ 30°C A 20µm droplet of water can be dried ca. 2.9 times faster at 30% RH compared to 70% RH at a temperature of 30°C Changing the Vapour loading of the surrounding gas (I.e. relative humidity), drying time can be reduced in an ELSD without the need to raise temperature.

PL-ELS 2100 Ice Design Feature for Sub-Ambient Operation The PL-ELS 2100 Ice comprises an Integrated Cooled Evaporator in combination with the ‘evaporation gas’ technology to provide sub-ambient operation The ‘evaporation gas’ is increased to compensate for the increase in relative humidity at sub-ambient temperatures This approaches enables the PL-ELS 2100 Ice to operate as low as 10°C. The addition of gas to the evaporation step not only allows low-temperature operation with aqueous mobile phases, but also facilitates removal of water at sub-ambient temperatures. As the evaporator temperature is lowered, the evaporation gas can be increased to compensate for the decrease in vapour loading (i.e. increase in relative humidity) of the surrounding gas. This allows water to be evaporated at temperatures as low as 15°C

PL-ELS 2100 Ice Design Feature for Sub-Ambient Operation Aqueous eluents can be removed at 15°C Organic solvents (e.g. THF) can be evaporated as low as 10°C The benefit of operating below room temperature provides maximum sensitivity for low-molecular weight semi-volatile compounds. For organic solvents, such as THF, the ELSD can be operated as low as 10°C. Ideal for normal-phase HPLC of small molecules. The benefit of operating at sub-ambient temperature to provide maximum sensitivity for low-molecular weight semi-volatile compounds, is highlighted in the next slides.

PL-ELS 2100 Ice Design Feature for Sub-Ambient Operation Integrated cooled evaporator provides rapid heating/cooling 35°C to 10°C in 10 minutes 15°C to 80°C in 11 minutes Accurate temperature control of evaporator tube eliminates variations in semi-volatile response, due to ambient temperature variations For organic solvents, such as THF, the ELSD can be operated as low as 10°C. Ideal for normal-phase HPLC of small molecules. The benefit of operating at sub-ambient temperature to provide maximum sensitivity for low-molecular weight semi-volatile compounds, is highlighted in the next slides.

Detection The analyte particles enter an optical chamber where they are irradiated by a light source. The incident beam is scattered by the particles and the intensity of scattered light is proportional to the concentration of the analyte Having dried the droplet, the resulting particle is irradiated by a light source which then scatters the light. The amount of light scattered by a particle is related to the it’s size. For a fixed droplet size, the concentration determines the final particle size, hence the ELSD is responsive to concentration

Digital Signal Processing PL-ELS 2100/2100 Ice are equipped with digital signal processing for greater flexibility: PMT output or gain (1-10) Amplifies signal output by set value Smoothing (1-50) Adjustable according to peak width LED intensity (0-100) Light source can be adjusted according to column loading (e.g prep LC)

Advantages of Evaporative Light Scattering Detection Universal: responds to all compounds in the mobile phase Detects compounds that do not possess a UV chromophore (eg polymers, sugars)

Detects all components in a single run Advantages of Evaporative Light Scattering Detection: Pharmaceutical Mixture Detects all components in a single run Samples : 1. α-cyclodextrin 2. ß-cyclodextrin 3. Ibuprofen Column: Aquasil C18 5µm, 150x4.6mm Eluent A: Water Eluent B: Acetonitrile Gradient: 50-95% B in 5 mins Flow Rate: 1.0 ml/min Inj Vol: 20µl Detector: PL-ELS 2100 (neb=30°C , evap=50°C, gas=1.0 SLM) ELSD UV @ 220nm

Advantages of Evaporative Light Scattering Detection Universal: responds to all compounds in the mobile phase Detects compounds that do not possess a UV chromophore (eg polymers, sugars) Not dependent on spectroscopic properties of compound Produces more uniform detection sensitivity for analytes

Advantages of Evaporative Light Scattering Detection: Uniformity of Response Comparison of UV and ELS traces show that ELSD allows for a more uniform response than UV detection Column: PLRP-S 100Å 5µm, 150 x4.6mm Eluent A: 50% 0.1% TFA in Water : 50% 0.1% TFA in ACN Flow Rate: 1.0ml/min Inj Vol: 10µl Detector: PL-ELS 2100 (neb=30°C, evap=30°C, gas=1.4 SLM) UV-VIS @ 280nm

Advantages of Evaporative Light Scattering Detection Universal: responds to all compounds in the mobile phase Detects compounds that do not possess a UV chromophore (eg polymers, sugars) Not dependent on spectroscopic properties of analyte: Produces more uniform detection sensitivity for analytes Not susceptible to baseline drift during gradient elution, temperature or solvent pump fluctuations ELSD compatible with a much wider range of solvents compared to RI and UV

Fast Gradient, Fast Flow Rate Capability Sample: Indapamide (IND), Dibutyl phthalate (DBP) Column: PLRP-S 100Å 5µm, 50x4.6mm Eluent A: 0.05% TFA in Water Eluent B: 0.05% TFA in ACN Gradient: 5-95% B in 1 min Flow Rate: Increased from 2ml/min up to 5ml/min Detector: PL-ELS 2100 (neb=30°C, evap=30°C, gas=1.6 SLM) Note: IND is non-volatile, DBP is relatively volatile

Fast Gradient, Fast Flow Rate Capability 2ml/min 3ml/min 4ml/min 5ml/min IND DBP min

Fast Gradient, Fast Flow Rate Capability Stable baseline throughout the gradient PL-ELS 2100/2100 Ice can operate in 100% up to 5ml/min 2ml/min 3ml/min 4ml/min 5ml/min min

Similar operating principles to LC-MS Advantages of Evaporative Light Scattering Detection: Ideal Complement to LC-MS Similar operating principles to LC-MS Volatile buffers Favors lower flow rates (ie 0.2-0.5 ml/min) Can develop LC methods on PL-ELS 2100/2100 Ice then transfer to LC-MS ELSD can provide supporting information when used in tandem with LC-MS

Ideal Complement to LC-MS Sample Mixture of known 1:1 ratio LC-MS results show ratio to be 3:1 UV-Vis result show ratio to be 10:1 PL-ELS 2100 results show ratio to be 1:1 (Response independent of optical properties)

PL-ELS 2100/2100 Ice Sensitivity Limit of Detection LOD improved with lower eluent flow rates and smaller ID column Caffeine Loading 100ng 4.6 ID S/N = 4.4 Caffeine Loading 30ng 2.1 ID S/N = 11.5

Routine operation of PL-ELS 2100 & PL-ELS 2100 Ice FOR NON-VOLATILE SOLUTES Evaporator temperature can be set to 80-120°C PL-ELS 2100 Gas flow kept to a minimum to maximise S/N Compounds such as sugars, fats/oils, & surfactants. FOR SEMI-VOLATILE SOLUTES Evaporator temperature often set between 30-50°C PL-ELS 2100 or PL-ELS 2100 Ice Gas flow optimised according to mobile phase Water @ 30°C requires 1.6SLM of gas Pharmaceuticals (e.g. MW >300), antibiotics & nutraceuticals.

Routine operation of PL-ELS 2100 Ice FOR VOLATILE SOLUTES Sub-ambient Evaporator temperatures required - 10-30°C PL-ELS 2100 Ice Gas flow optimised for sub-ambient temperatures and mobile phase 100% Water @15°C requires 2.2SLM of gas Compounds with high vapour pressures, low MW pharmaceuticals, pesticides, hydrocarbons

Sub-Ambient Applications The S/N ratio for thermally sensitive compounds such as Acetanilide is improved at sub-ambient temperatures In addition, the response of compound’s with high boiling points and high volatility, such as Alkanes, is dramatically improved. If we focus on peak 1-acetanilide and look at the S/N improvement at sub-ambient temperature compared to 30°C. We see that the response of this compound improves 4-fold, as the temperature is halved. Which highlights the advantage of sub-ambient ELSD

Sub-Ambient Applications: Acetanilide (MW 135) 4x improvement in Signal to Noise ratio for acetanlide at 15°C compared to 30°C

Sub-Ambient Applications: Alkanes All three compounds have high boiling points that suggest they are easily detectable by ELS detection but as the figure highlights, even at 30°C, there is a distinct disparity in response between the three hydrocarbons. Operating the ELSD at 18°C dramatically improves the response of hexadecane and heptadecane, such that they saturate the detector signal. By contrast Eicosane is unaffected by the change in temperature. HPLC conditions: Conc:50µg/ml each –Flow injection analysis. Each alkane injected every 30 secs Column: PL-Gel Guard column- no retention occurred. 100% THF –1ml/min Inj vol: 10µL Neb 80°C Gas 1.1SLM

Improved accuracy of quantification at sub-ambient temperatures Quantity and purity analyses for pharmaceutical drugs typically use HPLC coupled with UV/MS/ELSD Only ELSD allows compound-independent calibration, provided gradient effects and physicochemical properties of analytes are accounted for. Volatility is the most important physicochemical property that limits the accuracy/sensitivity of ELSD. Hence, ELSD operating temperatures need to be as low as possible, to minimise volatility effects. The majority of compounds generated in the pharmaceutical industry via high throughput methods require purity measurements. However, there is no absolute fast and accurate method to do this due to the lack of reliable, well-characterised standards. The standard method of determining quantity and purity for pharmaceutical drugs involves HPLC coupled with UV detection and other detectors, such as mass spectrometry, evaporative light-scattering or chemi-luminescent nitrogen. Of these detectors, only the ELSD, with it’s universal response, allows compound-independent calibration, provided the variations in response due to gradient effects and physicochemical properties of the analytes are accounted for. The response change across a solvent gradient can be eliminated using 3-dimensional calibration plots2, however the difference in physicochemical properties between compounds can limit the accuracy of quantification by ELSD. For low molecular weight pharmaceuticals, the volatility difference between compounds is one of the overriding factors that determine the accuracy of measurement with an ELSD. To minimise these volatility differences between compounds, the operating temperature of the ELSD should be as low as possible.

Improved accuracy of quantification at sub-ambient temperatures Average Recovery 84% Average Recovery 28% A mass-balance experiment for four low-molecular weight semi-volatile compounds was carried out at 9 ELSD evaporator temperatures ranging from 16°C to 40°C. The amount of compound detected, at each evaporator temperature interval, was compared to their nominal value. The data is presented in figure 6. At a temperature of 30°C, which is typically the lowest operating temperature of modern ELSDs, the recovery of the four compounds ranged from 20% for acetanilide to 39% for ibuprofen. Whereas at 16°C, the recovery of all four compounds more than doubled, with a range of 62% for dipropyl phthalate to 113% for acetanilide. The data also shows how small changes in evaporator temperature (2-4°C) can affect the response of semi-volatile compounds. Consider methyl paraben: at 30°C only 28% of the nominal amount is detected, compared to 81% at 18°C. Therefore by running sub-ambient ELSD better recovery of semi-volatile For laboratories without a stable thermal environment, this will affect the ELSD performance for detecting these types of compounds Therefore, it is vital that the evaporator temperature on an ELSD is accurately controlled, especially at ambient temperatures and below. Thank Mark Taylor’s group at Pfizer UK for the data.

Improved accuracy of quantification for semi-volatile compounds At 30°C, the average recovery across the four compounds was 28% At 16°C, the average recovery across the four compounds was 84% By operating at sub-ambient temperatures the loss of semi-volatile analytes is minimised, hence the accuracy of quantification is improved. The data also shows how small changes (2-4°C) in evaporator temperature can have dramatic effects on the response of semi-volatile compounds. For laboratories without a stable thermal environment, it is vital that the evaporator temperature is accurately controlled

Ambient Applications: Pharmaceutical mixture Samples: 1. Acetanilide, 2. Indapamide, 3. Ibuprofen, 4. Dibutylphthalate Column: C18 5µm, 150x4.6mm Eluent A: 0.1% TFA in Water Eluent B: 0.1% TFA in ACN Gradient: 60-90%B in 5 mins Flow Rate: 1.0mL/min Detector: PL-ELS 2100 (neb and evap set at the same temperature, gas 1.8 SLM) Note: peak 2 is non-volatile, peaks 1, 3, 4 are relatively volatile A semi-volatile mixture of 4 compounds of equal concentration, should give equal responses on an ELSD. However, differences arise due to volatility differences between the four compounds. Peak 1 is the most volatile- acetanilide MW 135, BP 304°C Peak 2 is least volatile-indapamide MW 365, BP Not stated Peak 3 is Ibuprfoen MW 206 BP-not stated Peak 4 is dibutylphthalate MW 278, BP 340°C

Ambient Applications: Pharmaceutical mixture At high temperature, Peak 2 has better S/N but Peaks 1,3 & 4 are not detected At 30°C all four peaks are detected

Ambient Application: Herbicides Sample: Mixture of 10 Phenylurea herbicides Column: C18 5µm, 250x4.6mm Eluent A: Water Eluent B: Acetonitrile Gradient: 10-80% B in 40 mins Flow Rate: 0.7ml/min Inj Vol: 20µl Detector: PL-ELS 2100 (neb=25°C, evap & gas flow varied)

Environmental Application: Herbicides Sample integrity is preserved at lower operating temperatures 25°C

Non-Volatile Application: Triglycerides in Natural Oils Sample: 2mg/ml Starflower Oil Column: C18 5µm, 250x4.6mm Eluent A: ACN Eluent B: DCM Gradient: 30-50% B in 40 mins; 50-90% in 2 mins, hold for 3mins Flow Rate: 1.0ml/min Inj Vol: 20µl Detector: PL-ELS 2100 (neb=40°C, evap=70°C, gas=1.4 SLM)

PL-ELS 2100/2100 Ice: DMSO Transparency Sample: Pharmaceutical Mixture in DMSO Column: Thermo-Hypersil C8 5µm, 50x4.6mm Eluent A: Water Eluent B: Acetonitrile Gradient: 5-100% B in 5 mins Flow Rate: 1.0ml/min Inj Vol: 20µl Detector: PL-ELS 2100 Ice (neb=25°C, evap & gas flow varied)

DMSO Transparency: Ideal for High Throughput Screening Loss of volatile species occurs as temperature is increased

DMSO Transparency: Ideal for High Throughput Screening The design of the PL-ELS 2100 Ice makes it transparent to DMSO at 25-30°C Fast eluting compounds are not masked by DMSO peak. PL-ELS 2100: neb=25°C, evap=30°C

PL-ELS 2100/2100 Ice Control Software Control & Method Manager software supplied as standard Software control via a serial connection (RS232) on selectable COM port Method Manager utility allows the user to set up, edit and log defined methods which can then be downloaded to the PL-ELS 2100ICE The PL-ELS 2100/2100 Ice have 24bit digital outputs via the RS232 port to provide data acquisition via Galaxie™ chromatography data system

Benefits of Evaporative Light Scattering Detection Universal detector: independent of the optical properties of the solute Compatible with gradient elution Ideal for fast gradient elution - no solvent front removes the need for derivatization Compatible with chromatographic techniques such as MS and SFC Wide application area

Benefits of the PL-ELS 2100 The PL-ELS 2100/2100 Ice provides all the typical advantages of ELSD, plus: Compatible with eluent flow rates, up to 5ml/min Detection limit in the low nanogram range High sensitivity to semi-volatile compounds DMSO transparency at ambient temperature Extremely low dispersion for high resolution separations Software control Digital signal processing

Additional Benefits of the PL-ELS 2100 Ice Sub-ambient operation down to 10°C for maximum sensitivity to volatile compounds Active heating/cooling provides rapid equilibration between analyses. Improved accuracy of quantification.

PL-ELS 2100 Ice ‘Lock out’ Specs Sub-ambient operation down to 10°C High sensitivity to semi-volatile compounds DMSO transparency at ambient temperature Extremely low dispersion for high resolution separations Compatible with eluent flow rates, up to 5.0ml/min Rapid thermal equilibration Easy to use Extremely small footprint, stackable

PL-ELS 2100 Specifications Light source: LED 480nm Detector: Photomultiplier tube Nebuliser temperature: ambient to 90°C in 1°C increments Evaporator temperature: ambient to 120°C in 1°C increments Gas flow rate: up to 3.25 SLM Gas pressure: typical 60-100psi, maximum 100psi Eluent flow rate: up to 5 ml/min Output: Analogue: 0-1V FSD Digital: 24bit, 10Hz using serial connection Communications: serial I/O (RS232), contact closure, TTL Power requirements: 90/120V AC or 220/250V AC 50/60 Hz Dimensions: 200x450x415mm (wxdxh) Weight: 11kg Part No. PL0860-0110 (110v), PL0860-0240 (240v) 200mm 200mm 415mm 450mm 450mm

PL-ELS 2100 Ice Specifications Light source: LED 480nm Detector: Photomultiplier tube Nebuliser temperature: ambient to 90°C in 1°C increments Evaporator temperature: 10 to 80°C in 1°C increments Gas flow rate: up to 3.25 SLM Gas pressure: typical 60-100psi, maximum 100psi Eluent flow rate: up to 5 ml/min Output: Analogue: 0-1V FSD Digital: 24bit, 10Hz using serial connection Communications: serial I/O (RS232), contact closure, TTL Power requirements: 90/120V AC or 220/250V AC 50/60 Hz Dimensions: 200x450x415mm (wxdxh) Weight: 13kg Part No. PL0860-1110 (110v), PL0860-1240 (240v) 200mm 200mm 415mm 450mm 450mm