ikey Separation Device CONTENTS I. INTRODUCTION II. III. IV. ikey SEPARATION DEVICE FEATURES a. Analytical ikey and ikey HT b. PCA ikey c. Accessory ikeys d. ikey Component Description CONNECTING AND REMOVING THE ikey SEPARATION DEVICE a. Connecting the ikey to the ionkey Source b. Removing the ikey from the ionkey Source ikey SEPARATION DEVICE USAGE a. ph and Temperature Range b. Mobile Phases and Buffers c. PCA Channel Usage d. Sample Preparation V. CARING FOR THE ikey SEPARATION DEVICE a. Cleaning and Regeneration b. Storage VI. TROUBLESHOOTING ikey SEPARATION DEVICE PERFORMANCE a. Tips for Maximizing ikey Lifetimes b. Troubleshooting Questions c. Good Practices I. INTRODUCTION The Waters ikey Separation Device is a high-pressure, microfluidic device that provides UPLC separations with the ionkey/ms System. The ikey Separation Device eliminates manual fitting connections and minimizes extra-column dispersion and the potential for manual variability. The ikey Separation Device contains a ceramic substrate produced using a modified ceramic manufacturing process which contains a single analytical separation channel packed with stationary phase, an electrospray emitter, a column heater, and an integrated memory device. The PCA ikey is a separation device with an additional, unpacked fluid channel (the post-column addition channel). This unpacked channel allows for the addition of a modifier solvent post chromatographic separation. The effluents of the stationary phase channel and PCA channel are merged and collected at the inlet of the emitter. The integrated memory on the ikey Separation Device maintains the number of injections, maximum backpressure, and number of sample sets run. An integrated column heater allows the temperature of an ikey Separation Device to be controlled. The ikey Separation Device is manufactured to exacting specifications, providing outstanding peak symmetry for maximum sensitivity and accurate quantitation. Each ikey Separation Device is individually tested to ensure that it passes stringent quality control requirements. Compared to conventional chromatographic systems, successful day-to-day performance of the ionkey/ms System requires certain considerations. This document provides several essential recommendations for the successful use of the ikey Separation Device. The ikey Separation Device is the only analytical consumable device compatible with ionkey/ms System.
II. ikey SEPARATION DEVICE FEATURES a. Analytical ikey and ikey HT The analytical ikey Separation Device is available with a 150 µm I.D. (ikey) or 300 µm I.D. (ikey HT) separation channel, packed with sub-2- µm UPLC particles. An electrospray emitter is located at the end of the channel. A sheath is provided to protect the ikey Separation Device and its emitter from damage during handling. Left side of the ikey Separation Device Electrospray connection contact Electronic connections Fluidic ports (4) Electrospray emitter tip NanoFlow gas port Identification label Right side of the ikey Separation Device Notes label Orientation rib Fluidic path diagram Flow channel to emitter (50 mm shown) Injection inlet port Emitter connection port PCA fluidic port Outlet port (inactive) Infusion port (inactive) 2
b. PCA ikey Separation Device The PCA ikey Separation Device has a 150 µm separation channel as well as the post column addition (PCA) channel. This allows for mixing the mobile phase with a desired solvent post chromatographic separation. Both effluents are merged and collected at the inlet of the emitter. Separation channel (50 mm shown) Injection inlet port PCA fluidic port Outlet port (inactive) Post-column addition (PCA) channel Infusion port (inactive) Post-column addition of solvents can be used to enhance the electrospray process and increase sensitivity without affecting the chromatographic separation Applications of the PCA ikey Separation Device may include the post-column addition of DMSO (for peptides), organic and inorganic modifiers, isopropanol (for negative ionization), and matrix (for examination of ion suppression). c. Accessory ikeys Flow injection ikey Separation Device: This ikey Separation Device has no packing material. It enables one to inject a sample from the sample manager directly to the emitter on the ikey Separation Device without any chromatographic separation. The flow path is identical to the 50 mm analytical ikey Separation Device. Infusion ikey Separation Device: This ikey Separation Device enables calibrant infusion from the ionkey/ms fluidics. Additionally, it can also be used to divert flow to waste for solvent changeovers and idle flow conditions, when liquid flow into the source is not desired. Flow channel to emitter Flow channel to waste Injection inlet port PCA fluidic port Outlet port to waste Infusion port Diagnostic ikey Separation Device: This ikey Separation Device has no flow channels. It provides a seal at the inlet fitting of the ionkey Source for testing the fluidic integrity of the system. 3
d. ikey Separation Device Component Description Component Injection inlet port PCA fluidic port Infusion port Separation channel PCA channel NanoFlow gas inlet Electronic connections Electrospray connection contact Electrospray emitter tip Identification label Description Receives the injections and mobile-phase flow from the system. Fluid connection for the PCA channel. Connects to the MS fluidics. The separation channel is packed with sub-2-µm particles. The PCA (post-column addition) channel runs parallel to the separation channel. It is an open channel used to pass a modifier solvent through. The mobile phase and the modifier solvent are joined prior to the emitter tip. Connects to a supply of nitrogen from the mass spectrometer. The gas pressure is set in the mass spectrometer s tune window. Electronic connections for the heater and EEPROM device. Electrospray potential is applied to the ikey Separation Device emitter through a conductive pad. The emitter is housed within the ikey Separation Device. Electrospray occurs when liquid within the emitter is charged by a high-voltage connection. Identifies the specifications for the ikey Separation Device. 4
III. CONNECTING AND REMOVING THE ikey SEPARATION DEVICE a. Connecting the ikey Separation Device to the ionkey Source These procedures outline the steps necessary to connect your ikey Separation Device into the ionkey source. Connecting your ikey Separation Device properly is essential for operation. To insert the ikey Separation Device in the ionkey Source: The procedure described below applies to both, the standard single channel as well as the PCA ikey Separation Device. 1. Ensure the lever is vertical, and the unlock indicator is in the 12 o clock position. Unlock indicator in 12 o clock position Lever in vertical position 2. Remove the ikey Separation Device from the protective sheath. 3. Insert the ikey Separation Device fully in the ionkey Source s docking port with sufficient force until it reaches a hard stop and the base of the ikey Separation Device handle is nearly flush with the face plate on the source. The green STATUS LED on the source will flash indicating that the ikey Separation Device is inserted but not clamped. STOP FLOW STATUS PRESSURE ikey Separation Device 4. Rotate the lever counterclockwise 180 degrees until the lock indicator is in the 12 o clock position. Doing so locks the ikey Separation Device in place and engages the fluid, gas, and electronic connections. Once clamped, the high-voltage electrospray ionization (ESI) is connected for electrospray, and pressurization and temperature control are active. Lock indicator in 12 o clock position Lever in vertical position 5
b. Removing the ikey Separation Device from the ionkey Source CAUTION: Opening the locking lever on the ionkey System without first stopping flow and decompressing the fluidic pressure can damage the ikey Separation Device. This procedure applies to the standard as well as to the PCA ikey Separation Devices: 1. Stop the solvent flow and decompress the fluidic pressure on the ikey Separation Device using one of these methods. a. Press the STOP FLOW button on the front of the source. b. In the Console, select Sample Manager from the system tree, and then click > Maintain > Remove ikey. 2. Make sure the mobile phase flow is completely stopped and that the system pressure has decompressed below 100 psi. If the solvent is flowing, the PRESSURE LED is steady green. The PRESSURE LED blinks green as the system is decompressing. Wait until the light is turned off completely. If an ikey Separation Device is removed without first decompressing the system, a Removed under pressure error message will appear, indicating potential damage to the ikey Separation Device. 3. Rotate the lever clockwise 180 degrees to the vertical position with the unlock indicator in the 12 o clock position. Unlock indicator in 12 o clock position Lever in vertical position 4. Pull the ikey Separation Device from the docking port. 5. Place the ikey Separation Device in the protective sheath. WARNING: The electrospray emitter at the front end of the ikey Separation Device contains a very small metal tip. Like a needle, it can pierce skin. WARNING: If the ikey Separation Device had been run at high temperature, the metal rings near the fluidic ports on the side may be hot. Avoid touching them. 6
IV. ikey SEPARATION DEVICE USAGE To ensure the continued high performance of your ikey Separation Device, please follow these guidelines: a. ph and Temperature Range Please refer to the table below for the recommended ph range for mobile phases and buffers used in the ikey Separation Channel. When running UPLC separations at > ph7, the LC system needs to be equipped and prepared for high ph operation as described in literature number 715005272en. Solid phase ph Range and max. temperature BEH C 18, 130Å ph 1 10 BEH C 18, 300Å ph 1 10 BEH C 4, 300Å ph 1 10 CSH C 18, 130Å ph 1 10 HSS T3, 100Å ph 2 8 80 C (low ph) 60 C (high ph) 80 C (low ph) 60 C (high ph) 80 C (low ph) 50 C (high ph) 80 C (low ph) 45 C (high ph) 45 C (low ph) 45 C (high ph) Note: Working in combinations of extreme ph, temperature, and pressure may result in reduced ikey Separation Device lifetime. The PCA channel is compatible with post column addition solvents in a ph range from ph 1 10. 7
b. Mobile Phases and Buffers To maintain and ensure maximum ikey Separation Device performance and lifetime, only use MS grade solvents. Filter all aqueous buffers prior to use through a 0.2-μm filter. Solvents containing suspended particulate materials may clog the inlet of the ikey Separation Device. This may result in higher backpressure or distorted peak shape. A table of recommended buffers is listed below. Only prepare small quantities of mobile phase into fresh, clean bottles. Do not top off mobile phases. Exchange bottles and mobile phases after an extended period of time. See below for a list of recommended buffers: Additive/buffer pka Buffer range Volatility (±1 ph unit) Comments Acetic acid 4.76 Volatile Formic acid 3.75 Volatile Acetate (NH4CH2COOH) 4.76 3.76 5.76 Volatile Formate (NH4COOH) 3.75 2.75 4.75 Volatile Maximum buffering obtained when used with ammonium acetate salt. Used in 0.1 1.0% range. Maximum buffering obtained when used with ammonium formate salt. Used in 0.1 1.0% range. Used in the 1 10 mm range. Note that sodium or potassium salts are not volatile. Used in the 1 10 mm range. Note that sodium or potassium salts are not volatile. Additionally, the ikey Separation Device lifetime will vary depending upon the operative temperature and pressure as well the type and concentration of additive/buffer used. Caution: The use of ammonium fluoride additive to the PCA solvents may reduce the lifetime of the ikey Separation Device metal emitter tip. 8
c. PCA Channel Usage It is necessary that during usage of the PCA ikey Separation Device the post-column addition channel is always under positive pressure. The recommended flow rate of the PCA channel is between 0.1 and 2.0 µl/min. Without flow or with variable flow through the PCA channel, one may observe variability in signal response. Caution: To avoid potential ikey Separation Device damage, do not flow liquid through the PCA channel without flow on the separation channel. Caution: To avoid tube damage, the maximum pressure limit for PCA ikey Separation Device flow delivery is 5,000 psi (345 bar). Caution: To avoid potential ikey Separation Device damage, the maximum flow rate for the PCA ikey should be limited to a maximum of 5 µl/min. d. Sample Preparation Samples may contain buffers and residual reagents from approved digestion procedure. The sample must not contain other reagents, denaturants, detergents, lipids, and must be free of particulates. Samples containing hundreds of nano-gram or more of undigested protein per injection have been shown to limit or shorten column lifetimes, depending on the injected quantity and the column diameter. Therefore, it is essential that proper sample preparation protocols are followed. Below are examples of recommended protocols for preparing plasma. Protein precipitation >2:1 ACN:H 2 O Liquid/liquid extraction Digestion Affinity Solid-phase extraction Filter Centrifugation Dilution Sample Preparation This method effectively removes the majority of protein from solution, pay attention to final organic content. This method effectively extracts the analyte leaving the protein behind, pay attention to final organic content. Proteins are broken into smaller pieces that no longer present an issue. Small amounts of specific proteins are extracted from the sample for analysis This can be an effective way of preparing samples by removing residual protein. This is an issue when the analyte has similar properties to proteins (i.e., the methods used to isolate larger peptides often leave significant protein content in the final extract). If solid-phase extraction (SPE) is the sole sample preparation technique, multidimensional chromatography (i.e., trap and back-flush elution) is required. Filters will not remove protein that is in solution. Centrifugation will not remove protein that s in solution, unless it s been crashed with protein precipitation (PPT). It depends on the dilution ratio, and the diluent. In general small aqueous dilutions will not remove protein. Urine Bile Microsomes Plasma Organic extracts Sample Type Typically low protein content and does not require organic additive to crash proteins. Dilution and centrifugation is recommended. Typically low protein content and does not require organic additive to crash proteins. Dilution and centrifugation is recommended. Microsomal incubations contain proteins. Pay close attention to sample preparation to ensure that minimal protein remains in the final sample extract. Plasma typically contains high concentrations of protein. Pay close attention to sample preparation to ensure that minimal protein remains in the final sample extract. Injecting a large amount of undigested proteins onto the ikey Separation Device will result in compromised performance and shorter lifetimes. High amounts of organic affect the performance of any column, this effect is greater with small dimensions. 9
The use of multidimensional chromatography, specifically a trap and elution strategy, can provide further sample cleanup and facilitate injections of higher organic strength injection solvents without experiencing analyte breakthrough. This multidimensional chromatographic strategy is highly recommended when SPE is the sole sample preparation technique (i.e., no additional sample preparation technique is used to remove large amounts of undigested protein). It is preferable to prepare the sample in the initial mobile-phase conditions or a weaker solvent for the best peak shape and sensitivity. If the sample is not prepared in the mobile phase, ensure that the sample, solvent, and mobile phases are miscible in order to avoid sample and/or buffer precipitation. Consider filtering samples with a 0.2-μm membrane to remove particulates. If the sample is dissolved in a solvent that contains an organic modifier (e.g., acetonitrile, methanol, etc.) ensure that the membrane/filter material is compatible with the solvents in use. Alternatively, centrifuge the sample for 20 minutes at 8000 RPM, followed by the transfer of the supernatant to an appropriate Waters TruView LCMS Certified Vial could be considered. Below are two examples of commonly used sample preparation protocols: Protein precipitation (PPT)* a. Add acetonitrile to plasma at a ratio of 2:1 (acetonitrile: plasma). b. Vortex (mix) for 1 minute. c. Centrifuge at 5,000 relative centrifugal force (RCF) for 5 minutes. d. Remove supernatant. e. Perform SPE cleanup (optional).* f. Pipet into Waters TruView LCMS Certified Vial (e.g., part number 186005663CV). Liquid/Liquid Extraction (LLE)* 1. Add hexane to plasma at a ratio of 10:1 (hexane:plasma). 2. Vortex (mix) for 1 minute. 3. Centrifuge at 5,000 relative centrifugal force (RCF) for 5 minutes. 4. Remove supernatant and evaporate to dryness. 5. Reconstitute (i.e. initial gradient conditions solvents). 6. Centrifuge at 5,000 relative centrifugal force (RCF) for five minutes (optional). 7. Perform SPE cleanup (optional).* 8. Remove supernatant and pipet into Waters TruView LCMS Certified Vial (e.g., part number 186005663CV). *Additional sample cleanup steps using Oasis or Sep-Pak SPE Devices can provide cleaner samples, improved selectivity, higher sensitivity, and reduced matrix effects. 10
V. CARING FOR THE ikey SEPARATION DEVICE a. Cleaning and Regeneration Changes in peak shape, peak splitting, shouldering peaks, shifts in retention, change in resolution or increasing backpressure may indicate ikey Separation Device contamination. Flush with a neat organic solvent to remove the non-polar contaminant(s), taking care not to precipitate any buffered mobile-phase components. If this flushing procedure does not solve the problem, purge the ikey Separation Device with the following cleaning and regeneration procedures. Separation channel: Use a cleaning routine that matches the properties of the samples and stationary phase type and will solubilize the suspected contaminate. Flush with 20-column volumes of solvent at an intermediate temperature of 40 C. Return to the initial mobile-phase conditions by reversing the sequence. Purge the ikey Separation Device with a sequence of progressively more non-polar solvents (e.g., water, methanol, acetonitrile, isopropanol, etc.) and reverse the purging order to end the procedure at initial mobile-phase conditions. 25% water, 25% acetonitrile, 25% methanol, 25% isopropanol and 0.1% formic acid is a good all-round cleaning solvent. If ikey Separation Device performance has not improved after regeneration/cleaning procedures, contact your local Waters representative for additional support. PCA channel: Use a cleaning routine that matches the properties of the PCA solvent type used previously in the PCA channel to solubilize any b. Storage suspected contaminate (i.e. salts from buffers). Flush with 20 ikey Separation Device volumes of solvent at an intermediate temperature of 40 C. Return to the initial solvent conditions by reversing the sequence. ikey Separation Device: For periods longer than four days, store the reversed-phase ikey Separation Device in 100% acetonitrile. Do not store ikey Separation Devices in buffered eluents. If the mobile phase contained a buffer salt, flush the reversed-phase ikey Separation Device with 10 ikey Separation Device volumes of HPLC-grade water followed by 10 ikey Separation Device volumes of acetonitrile. Failure to perform this intermediate step could result in precipitation of the buffer salt in the column when 100% acetonitrile is introduced. Completely seal the ikey Separation Device to avoid solvent evaporation and drying out of the chromatographic bed. Note: If an ikey Separation Device has been run with a formate-containing mobile phase (e.g., ammonium formate, formic acid, etc.) and is purged with 100% acetonitrile, slightly longer equilibration times may be necessary when the ikey Separation Device is re-installed and re-wetted with that same formate-containing mobile phase. PCA ikey Separation Device: When stored, the PCA channel should always be purged of any buffered eluents and stored filled with an appropriate organic solvent. 11
VI. TROUBLESHOOTING ikey SEPARATION DEVICE PERFORMANCE The table below lists symptoms, causes, and solutions for troubleshooting the ikey Separation Device. Symptom Cause Solution Excessive baseline noise The emitter tip has residue on it Rinse the emitter tip in isopropanol. Poor LC quality such as retention time shift or peak width variance The ikey Separation Device does not fit into the ionkey Source s docking port Fluid leak The ikey Separation Device is upside down a. Tips for Maximizing ikey Separation Device Lifetimes To maximize ikey Separation Device lifetime, pay close attention to: Water quality (including water purification systems); Solvent quality; Mobile-phase preparation, storage, and age; Sample, buffer and mobile-phase solubles; Sample quality and preparation. Use the Fluidic Integrity Test within the console software in combination with the diagnostic ikey Separation Device to find and identify leaking components. Remove and orient the ikey Separation Device so that the four ports are on the left side. When problems arise, systematically troubleshoot potential causes one variable at a time in a systematic fashion. Always remember to: Discourage bacterial growth by minimizing the use of 100% aqueous mobile phases where possible; Discard and re-prepare aqueous mobile phase every 24 to 48 hours (if 100% aqueous mobile phase is required); Routinely maintain your water purification system to ensure it is functioning properly; Only use ultra-pure water (18 MΩ) and highest quality solvent possible; Consider improving sample preparation (e.g., SPE, filtration, centrifugation, etc.) when possible. Avoid when possible: 100% aqueous mobile phases; Do not use HPLC-grade bottled water use LC-MS-grade water and solvents only ; Do not top off mobile phases prepare small quantities of fresh mobile phase in appropriately sized bottles; Replace bottles when mobile phase is being replaced. Don t assume the ikey Separation Device is to blame: Investigate cause of ikey Separation Device failure; Monitor backpressure; Examine mobile-phase age, bacterial contamination, mobile phase precipitation, etc.; Examine sample quality; Examine injection solvent strength. Do not prepare excessive amounts of mobile phase: To reduce the chances of mobile-phase contamination or degradation, prepare enough mobile phase to last for 3 4 days. Alternatively, store excess bulk quantities in a refrigerated environment. 12
b. Troubleshooting Questions What is the age of your 100% aqueous mobile phase? Is the mobile phase filtered through a 0.2 μm membrane? Was the mobile phase prepared fresh or topped off? Is the water source of adequate quality? When was the last time the water system was serviced or was the bottle of water unopened? Is bacterial growth a possibility? If a neat standard is prepared in the initial mobile-phase conditions and injected, are the problems still observed? If the sample is additionally filtered/purified (i.e., SPE, filtration, etc.) is the problem still observed? Has the quality of the samples changed over time? c. Good Practices Do not operate at pressures that exceed 10,000 psi (690 bar). Do not apply electrospray potential to the emitter for an extended period of time without mobile-phase flow. Do not immerse in liquid. Do not touch any part of the ikey Separation Device other than the handle. Do not freeze. Flush with acetonitrile and store at room temperature when not in use. Use the ikey Separation Device sheath to protect device when not in use. Do not bend or pull the capillary connection tubing at the ionkey Source coupling. Avoid excess voltage (>4.0 kv) which can erode emitter over time. For a complete list of ikey Separation Devices, visit: www.waters.com/ ionkey. For the list of patents, visit www.waters.com/patents. Waters, The Science of What s Possible, UPLC, Oasis, and Sep-Pak are registered trademarks of Waters Corporation. TruView, ikey, NanoFlow, ionkey/ms, and ionkey are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. 2016 Waters Corporation. Produced in the U.S.A. October 2016 720004897EN IH-PDF Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com