Webinar: How to be More Successful with your ACQUITY QDa Detector Eric van Beelen

Webinar: How to be More Successful with your ACQUITY QDa Detector Eric van Beelen 2015 Waters Corporation 1

We are here to address any Questions you may have That s the benefit of participating to the Live Session! 1. Post your Questions into the Q&A facility on the left 2. Refresh the Q&A window from time to time to view what it may contain Jonathan Scott Product Management ACQUITY QDa Tom Wheat Separations 2015 Waters Corporation 2

Webinar: How to be More Successful with your ACQUITY QDa Detector Eric van Beelen 2015 Waters Corporation 3

Outline ACQUITY QDa Detector an overview Mass Data Terminology Mobile Phase considerations Instrument setup Mode of operation Spectra interpretation Trouble shooting 2015 Waters Corporation 4

Outline ACQUITY QDa Detector an overview Mass Data Terminology Mobile Phase considerations Instrument setup Mode of operation Spectra interpretation Trouble shooting 2015 Waters Corporation 5

ACQUITY QDa Detector - Facts Hugely successful detector providing mass data to separation scientists where this was not before possible! Compact, intuitive and simple to use the most accessible mass detector available! Improving workflows and efficiency in every laboratory giving a guaranteed return on investment! 2015 Waters Corporation 6

ACQUITY QDa Detector - Facts 2 versions: Standard and Performance Mass range: 30-1250 m/z, 10,000 Da/sec scan speed Pre-Optimized Electrospray Ionisation ± 4 orders of dynamic range Compatible with all Waters HPLC/UPLC/UHPLC/UPC2/Purification systems Works with EMPOWER and MassLynx 2015 Waters Corporation 7

Outline ACQUITY QDa Detector an overview Mass Data Terminology Mobile Phase considerations Instrument optimization Mode of operation Spectra interpretation Trouble shooting 2015 Waters Corporation 8

Mass Measurement Dalton: The unit of atomic mass [M+H] + ion: The positively charged form of a molecule that is created when one proton is added during the ionisation process. The ion detected is one unit higher than the monoisotopic mass of the uncharged molecule. [M-H] _ ion: The negatively charged form of a molecule that is created when one proton is removed during the ionisation process. The ion detected is one unit lower then the monoisotopoc mass of the uncharged molecule 2015 Waters Corporation 9

Multiple Charging in ESI Mass spectrometers operate on the basis of mass-to-charge ratio (m/z). Single charge m/z = (M + H + )/1z Double charge m/z = (M + 2H + )/2z n charge m/z = (M + nh + )/nz Multiple charging extends the mass range of the mass spectrometer. 2015 Waters Corporation 10

Mass Measurement AVERAGE MASS: Based on average isotopic mass MONOISOPTIC MASS: The mass of an ion as the sum of the masses of the atoms in a molecule using the principle isotope for each element instead of the isotopic average mass. Formula Weight Considerations: Formula weights can include contributions of salts and/or hydrates. An ion associated with this molecular weight does not appear in the mass spectrum. 2015 Waters Corporation 11

Average vs Monoisotopic Mass monolinuron 2015 Waters Corporation 12

Average vs Monoisotopic Mass monolinuron 2015 Waters Corporation 13

Average vs Monoisotopic Mass monolinuron 2015 Waters Corporation 14

Formula Weight Considerations - Salts Formula weights can include contributions of salts and/or hydrates. An ion associated with this molecular weight does not appear in the mass spectrum. Clopidogrel Bisulfate C 16 H 16 Cl 2 NO 2 S H 2 SO 4 2015 Waters Corporation 15

Mass Spectrum Glossary Generic common names Electrospray ionization Cone voltage Probe temperature Capillary Voltage Single Ion Recording (SIR) Full Scan 2015 Waters Corporation 17

Electrospray Ionization (ESI) Ionization takes place at atmospheric pressure and has three stages: Formation of charged droplets Solvent evaporation and droplet fission Formation of gas-phase ions Generally produce (M+H) + or (M-H) - ions Produces little fragmentation Figure adapted from Andreas Dahlin showing positive ion mode 2015 Waters Corporation 18

Effect of Matrix on Analyte Response in ESI 1. Surface competition NH 4 + A NH 4 + NH 4 + AH + NH + 4 NH + 4 A AH + AH+OAc- NH 4 +OAc- A AH + NH 4 +OAc- AH + NH 4 + NH 4 + NH 4 + 2. Charge competition OAc - + AH + HOAc + A Competition between analyte and electrolyte ions for conversion to gas-phase ions decreases analyte response. Concentration Analyte response 2015 Waters Corporation 19

Effect of Flow Rate on Analyte Response in ESI ESI response may decrease with increasing flow rate. Droplet size increases with flow rate Increased gas flows and heater temperatures may be required to maintain sensitivity The ACQUITY QDa operates at flow rates up to 2mL/min Optimal flow rates are between 0.3 and 0.8 ml/min. 2015 Waters Corporation 20

Outline ACQUITY QDa Detector an overview Mass Data Terminology Mobile Phase considerations Instrument setup Mode of operation Spectra interpretation Trouble shooting 2015 Waters Corporation 21

Solvents & Buffers High purity solvents a recommended for best performance. Examples of recommended solvents/additives/buffers to be used with the ACQUITY QDa are the following: Solvents Water 100% Methanol 100% Acetonitrile 100% 2-Propanol (IPA) 100% Additives Formic acid <0.1% Acetic acid <0.1% Trifluoroacetic acid (TFA) <0.1% Ammonium hydroxide <0.1% Buffers Ammonium acetate <10mM Ammonium formate <10mM Ammonium bicarbonate <10 mm See the ACQUITY QDa Detector Overview and Maintenance Guide, reference USRM134766919 2015 Waters Corporation 22

Solvents & Buffers The choice of the UPLC/HPLC eluent is important when using a mass detector Many existing HPLC methods without MS detection use phosphate buffer in the mobile phase. This is not compatible with Electrospray Ionisation Non volatile buffers are not recommended to be used with the ACQUITY QDa detector even at levels lower than 1 mm This is NOT a limitation of the ACQUITY QDa or any Waters product. Any instrument will exhibit reduction in sensitivity and robustness if a non-volatile buffer is used with an electrospray source. 2015 Waters Corporation 23

Unsuitable Additives Non-volatile salts (like phosphate, borate and similar) Can deposit in source and plug capillaries (more cleaning/maintenance) Orthogonal spray interfaces can handle non-volatile additives without plugging, but frequent maintenance is still required. Surface-active agents (surfactants/detergents) Will suppress electrospray ionization Example SDS, nonylsulfate Inorganic acids (HNO3, HCl, H3PO4, H2SO4) Corrosive 2015 Waters Corporation 24 24

ph Considerations Analysis of basic compounds use positive ion mode Lower ph with an acid o Formic or acetic acid Analysis of acidic compounds use negative ion mode Raise ph with a base o Ammonium hydroxide Analysis of mixtures or unknowns - use positive/negative ion switching if available ph should be > 4 for the approach to be effective. 2015 Waters Corporation 25 25

Outline ACQUITY QDa Detector an overview Mass Data Terminology Mobile Phase considerations Instrument setup Mode of operation Spectra interpretation Trouble shooting 2015 Waters Corporation 26

Simply Turn on and... Use! Power LED (left) Green o Module on RAPID START-UP TO READY TIME Condition LED (right) Green o In Operate Flashing Green o Running Self Checks Flashing red o In Standby 2015 Waters Corporation 27

Simply Turn on and... Use! RAPID START-UP TO READY TIME Automatic calibration check following power ON provides data certainty with minimal user intervention 2015 Waters Corporation 28

Instrument Setup For routine analysis your instrument is ready to go. In rare occasions you might want to tweak some parameters Cone voltage Capillary Voltage Probe temperature 2015 Waters Corporation 29

Instrument Setup Cone Voltage Consider the following: Do not transfer the cone voltage value from any other instrument. Start at 15V in positive mode and 30V in negative mode. Higher Cone Voltages can lead to in source fragmentation Increase in 3V steps. Decrease in 2V steps. 2015 Waters Corporation 30

Instrument Setup Capillary Voltage Consider the following: Standard capillary voltage is 0.8 kv. Increase the voltage for flow rates lower than 0.3 ml/min or for high (>80%) organic methods. (Typically 1,5 kv) For negative ionisation method the cone voltage is normally at optimum value. 2015 Waters Corporation 31

Instrument Setup Probe Temperature Consider the following: Set the probe temperature to 600 ºC for most LC flow rates (QDa method default). Set the probe temperature to 300 ºC when the flow rate is less than 0.3 ml/min. This is an advanced mode option 2015 Waters Corporation 32

Outline ACQUITY QDa Detector an overview Mass Data Terminology Mobile Phase considerations Instrument setup Mode of operation Spectra interpretation Trouble shooting 2015 Waters Corporation 33

Modes of Operation You can acquire data using any of these operating modes: Scanning (MS Scan): Records all of the masses within a user-defined range at each time point in the chromatogram to produce qualitative information. An MS scan can be performed in positive (where only positively charged ions are transmitted and detected) or negative ion mode. This is similar to 3D PDA data. Selected ion recording (SIR): Records a single user-defined m/z value (positive or negative ion mode) at each time point in the chromatogram, with all other ions being rejected. This technique can introduce specificity and higher sensitivity into an analysis, and is most commonly used for quantitative analysis where lower limits of detection are required. This is similar to 2D PDA data. The ACQUITY QDa can performs Scanning and SIR acquisitions simultaneously in one experiment 2015 Waters Corporation 34

ACQUITY QDa Detector Scanning Enter the range of masses you wish to acquire. Maximum QDa mass range is 50-1250 Da Click to enable positive or negative ionization. Choose the sampling rate to ensure that you have enough points across the peak for the chromatography. Note that you can select a sampling rate faster than the scanning rate of the detector, but the actual sampling rate will not change. 2015 Waters Corporation 35

ACQUITY QDa Detector SIR SIR tab is used to specify specific masses to be acquired during the analysis. Specify a cone voltage for each compound entered. Enter the mass to be acquired. Remember to use [M+H]+ or [M-H]- for the mass to be monitored. 2015 Waters Corporation 36

ACQUITY QDa Detector 2015 Waters Corporation 37

In Source Fragmentation Peaks observed at lower m/z values to the analyte can correspond to fragments of the analyte, generated in the source. These fragments are characteristic of the analyte. In standard operation mode these are mostly absent. However at higher cone voltages you can force in source fragmentation and get additional information Monolinuron fragment ion -89 Monolinuron [M+H] + 2015 Waters Corporation 38

Sampling Rate - SIR SIR mode is often used for quantitative applications. Typically 12-15 points across the peak is sufficient, so for a 3 second peak select 5 Hz to achieve 15 points. There is no advantage to selecting sampling rates giving more than 15 points across a peak as the dwell time (time spent detecting the SIR channel) will decrease eventually reducing the signal to noise and data quality. For multiple (simultaneous or staggered function) experiments, the sampling rate is automatically reduced in the overlapping time regions so that the selected frequency is maintained for any given function. 2015 Waters Corporation 39

Sampling Rate - Scanning In MS Scanning mode, the slower the scan (lower frequency), the better quality the spectral data in terms of sensitivity and dynamic range. Typically 5-7 points a peak for qualitative experiments is sufficient. Select the smallest mass range which covers the compounds of interest to lower the scan speed. The achievable frequency in a MS Scan experiment is determined by the maximum scan speed (10,000 Da/s), the minimum scan time and the mass range as defined by the user. 2015 Waters Corporation 40

Sampling Rate Pos/Neg Switching The functions are automatically reordered to minimize the number of positive/negative switches per cycle, enabling the method to run all the positive and all the negative functions in 1 experiment. A warning will be displayed if a positive/negative switching experiment is set up with a frequency greater than 8 Hz (25 ms). In this example, functions 1, 3 and 5 would be run and then functions 2 and 4. This ensures there are only 2 switches per cycle which improves overall duty cycle reducing switching delays 2015 Waters Corporation 41

Outline ACQUITY QDa Detector an overview Mass Data Terminology Mobile Phase considerations Instrument setup Mode of operation Spectra interpretation Trouble shooting 2015 Waters Corporation 42

Spectral Interpretation - Dimers Spectral peaks can be observed at almost the double of the expected monoisotopic ion m/z value with its neutral counter part, for example M+[M+H] +. Monolinuron [M+H] + Monolinuron dimer M+[M+H] + Dimers are often formed at high concentrations, consider injecting less or diluting the sample 2015 Waters Corporation 43

Spectral Interpretation - Adducts These ions are formed by the association of metal ions, or other species contained in the eluent, with analytes in the ESI source. Adduct formation often occurs with lower quality solvents Sodium adduct of monolinuron +22 2015 Waters Corporation 44

Spectral Interpretation - Adducts Common adducts are listed here 2015 Waters Corporation 45

Spectral Interpretation Multiple Charge species Multiple charge ions normally are formed with larger molecules such as peptides. [M+2H] 2+ Doubly charged ion [M+H] + Singly charged ion. Angiotensin II peptide 1045 Da N charges: m/z =[M+nH]/n Peptides up to 7000 Da can be measured with multiple charge states visible on the ACQUITY QDa 2015 Waters Corporation 46

Digging into the data - EMPOWER If you have used PDA with Empower, you will already be familiar with navigating the 3D Review window. ACQUITY QDa full scan data uses many of the same tools. Data acquired using SIR mode is 2D data and is processed in the same manner as other 2D detectors (for example, UV, RI, ELSD) 2015 Waters Corporation 47

Mass Analysis Window Spectrum View Options Click the icon to open the data in the mass analysis window PDA and MS Spectra of each PDA integrated peak PDA Chromatogram - Integrated MS TIC not integrated MS XIC for all integrated peaks Mass Analysis Extraction Toolbar 2015 Waters Corporation 48

Outline ACQUITY QDa Detector an overview Mass Data Terminology Mobile Phase considerations Instrument setup Mode of operation Spectra interpretation Trouble shooting 2015 Waters Corporation 49

Trouble shooting Source Saturation Source saturation can occur when the ionization efficiency changes at higher concentrations, which results in non linearity Circled calibration points showing source saturation 2015 Waters Corporation 50

Trouble shooting Detector Saturation Detector saturation can occur when the signal arriving at the detector exceeds an upper finite level In centroid mode, you can indentify detector saturation when the intensity of an isotopic peak such as 13C, relative to the monoisotopic peak, is higher than theoretically predicted Saturated peak Unsaturated peak Apex of peak missing therefore annotated m/z value may not be accurate Observed relative intensity of 13 C peak higher than theoretical percentage Saturated peak in continuum mode showing flat top profile 2015 Waters Corporation 51

Trouble shooting The QDa software provide information if your sample aperture is blocked. This is measured with the change in turboflow power or your turbo pumps and gives you the following info. 2015 Waters Corporation 52

Trouble shooting Sample Aperture: As simple as replacing a detector lamp 2015 Waters Corporation 53

Trouble shooting Replacing the ESI sprayer No cutting or assembly required Finger tides connections 2015 Waters Corporation 54

1-Day Workshop Waters MS Headquarters (UK) Mass Detection in the Chromatography Laboratory Why & How? Wilmslow (Waters MS Headquarters near Manchester) November 19 th,2015 Involving Industry and Waters Experts Need more details? Select this option in our Follow-up Questionaire 2015 Waters Corporation 55

More information Reference Cards Starting point for using the ACQUITY QDa shipped with new QDa instruments and soon available on website 715004945 715004968 Class Room Courses: 750000833 ACQUITY QDa with EMPOWER 750000901 Operation of the ACQUITY QDa E-Learning Courses 750000862 Acquity QDa General Theory and Basic Use 2015 Waters Corporation 56

Thank you for your attention Jonathan Scott Tom Wheat eric_van_beelen@waters.com 2015 Waters Corporation 57