ACQUITY UPLC Sample Manager - Fixed Loop Operator s Overview and Maintenance Information

Transcription

1 ACQUITY UPLC Sample Manager - Fixed Loop Operator s Overview and Maintenance Information Revision A Copyright Waters Corporation All rights reserved

2 Copyright notice WATERS CORPORATION. PRINTED IN THE UNITED STATES OF AMERICA AND IN IRELAND. ALL RIGHTS RESERVED. THIS DOCUMENT OR PARTS THEREOF MAY NOT BE REPRODUCED IN ANY FORM WITHOUT THE WRITTEN PERMISSION OF THE PUBLISHER. The information in this document is subject to change without notice and should not be construed as a commitment by Waters Corporation. Waters Corporation assumes no responsibility for any errors that may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall Waters Corporation be liable for incidental or consequential damages in connection with, or arising from, its use. ii

3 Table of Contents Copyright notice... ii Overview... 1 Location of SM-FL in the ACQUITY UPLC system... 1 Flow path through the sample management system... 2 SM-FL major components... 3 Functional systems... 6 Preparing for operation... 9 Installing the optional leak sensor... 9 Installing the waste tubing Priming the SM-FL Using the SM-FL Interface requirements Installation recommendations for fittings Selecting weak wash and strong wash solvents Washing the SM-FL needle Loading sample plates Sample chamber considerations Choosing needles and sample loops Choosing the sample syringe Choosing the sample syringe draw rate Choosing the needle height setting Recovering maximum sample from vials Revising a plate type Air gaps Selecting the optimum sample injection mode Load ahead option Loop offline option Diagnostic tests Resolving leak sensor errors Table of Contents iii

4 Maintaining the SM-FL Contacting Waters technical service Maintenance schedule Maintenance considerations Configuring maintenance warnings Replacing the leak sensor Replacing the sample needle Calibrating the needle s z axis Replacing the sample loop Replacing the sample syringe Replacing the wash syringes Replacing the injection valve cartridge Cleaning the instrument s exterior iv Table of Contents

5 Overview You can submit samples for analysis on the ACQUITY UPLC system by loading microtiter plates or vials onto the rotary sample tray of the sample manager-fixed loop (SM-FL). Using a fixed loop, the sample manager injects the samples it draws from the plates and vials onto a chromatographic column. Optional sample loops make possible a range of injection volumes. Location of SM-FL in the ACQUITY UPLC system The following diagram shows the location of the SM-FL in the ACQUITY UPLC system. Bottle tray Detector Column heater Sample Manager - Fixed Loop Solvent manager Overview 1

6 Tips: Use care when stacking or moving the SM-FL. Ensure the drip tray does not collide with any surface. Use care when installing and removing reusable fittings. See also: Column Compartments Operator's Overview and Maintenance Information on the ACQUITY UPLC System Documentation CD to avoid potential leaks or carryover. Flow path through the sample management system The following diagram shows how the SM-FL functions as part of the ACQUITY UPLC system. Drainage from column heater Mobile phase from solvent manager Degassed solvent from solvent manager Sample Inject valve To column Secondary drainage to solvent manager Secondary drainage to solvent manager Primary drainage to solvent manager 2

7 SM-FL major components The following diagrams show the SM-FL s major components. Front view with doors closed: On/off switch Power LED Run LED Sample compartment door Fluidics compartment door Overview 3

8 Front view, with doors open: Chamber temperature sensor Plate selector switch Access panel Location of column heater leak sensor Location of volume detection device Injection valve Sample syringe valve Sample syringe Sample tray Location of sample manager leak sensor 4

9 Sample compartment components visible with access panel removed: Compartment lighting Location of sample needle Sample needle carriage Location of column heater leak sensor cable connector Location of sample manager leak sensor cable connector SM-FL components: Component Access panel Compartment lighting Injection valve Volume detection device Description Removable panel (requires a TORX driver) that allows access to sample compartment components such as the seal assembly and needle carriage. LED that illuminates the sample compartment. The compartment light automatically turns on when the SM-FL s sample compartment door is opened and turns off when the door is closed. The light can also be turned off via the console to accommodate light-sensitive samples. A two-position, six-port injection valve. A sensor that detects air, versus liquid, in a clear tube, determining the volume of the installed sample needle and sample loop. Overview 5

10 SM-FL components: (Continued) Component Leak sensor Plate selector switch Sample syringe Sample syringe valve Sample needle Sample needle carriage Sample tray Chamber temperature sensor Description Continuously monitors the SM-FL for leaks and stops the system flow when its optical sensor detects about 1.5 ml of accumulated, leaked liquid in its surrounding reservoir. Toggle switch used to select either plate position 1 or plate position 2. Draws sample into the sample needle. A three-position, rotary-shear valve. Extracts sample from vials. Positions the sample needle in the sample compartment. Secures the sample plates or vial holders in place. Located on the upper guide rail toward the back of the sample chamber, to monitor sample environment. Functional systems RΘ positioning mechanism The RΘ (R-theta) positioning mechanism s two axes control the orientation of the sample plates within the sample compartment and the relative position of the sample needle carriage. The theta-rotary axis is a belt-driven shaft that rotates a pair of sample plates 360 from a reference point. The R-linear axis is the axis along which the sample needle carriage is oriented. The carriage runs from the rear-left corner to the front-right corner of the sample compartment. Injection system The injection flow path includes the assemblies required to aspirate a sample and deliver it to the column. The process involves the needle, sample loop, sample syringe and syringe valve, and injection valve. 6

11 Wash system The wash system uses a wash pump, wash solenoid, and weak-wash and strong-wash syringes to rinse areas that are contacted by the sample. The system uses two wash solvents: Strong needle-wash solvent, for flushing injected components from areas that are contacted by the sample. Strong needle-wash solvent is never injected along with the sample. Weak needle-wash solvent, for transporting sample to the sample needle and eliminating strong needle-wash solvent. Weak needle-wash solvent is injected with the sample in partial-loop, pressure-assist mode. The wash sequence consists of five primary operations: 1. The needle moves to the drain position and empties any excess sample into the drain port. The volume of wash solvent consumed during this flush depends on the size of the sample, air gap, and loop. 2. The needle remains in the drain position for rinsing with strong needle-wash solvent: If the specified volume of strong needle-wash solvent is fewer than three times the needle volume (about 150 µl), the entire wash volume is dispensed at the drain position. If the specified volume of strong wash exceeds three times the needle volume, the remainder (wash volume minus three times the needle volume) is dispensed at the combined injection and wash port. As the needle dispenses strong wash solvent, an equal amount of weak needle-wash solvent forces upward through the bottom of the combined injection and wash port. 3. The weak wash flushes the needle, removing from it any remaining traces of strong wash solvent. 4. The wash syringes are replenished with degassed wash solvent from the solvent reservoirs. 5. The needle is briefly rinsed at the drain when the sample loop returns to the load position, which happens at the start of a partial loop injection with pressure assist or a full loop mode injection. The brief rinsing ensures the loop, which is at system pressure rather than atmospheric Overview 7

12 pressure, is clear of any residual sample that could be expelled when the pressure is released. Flow path diagram: 8

13 Thermal system The thermal system maintains the set temperature in the sample compartment. Tips: You do not need to defrost the sample compartment. The SM-FL s fans stop circulating air whenever the sample compartment door is open. The sample tray rotates slowly when the system is idle, to help maintain a uniform temperature across the plates. Preparing for operation Note: The system is shipped with a 10-μL PEEK needle. If you are not using the default ACQUITY UPLC system configuration, which uses this needle, see page 49. Before you prepare the SM-FL for operation, prepare the solvent manager. Tip: For instructions on preparing the solvent manager, see ACQUITY UPLC Binary Solvent Manager Operator s Overview and Maintenance Information. Preparation of the SM-FL involves these steps: Installing the leak sensor Installing the waste tubing Priming the SM-FL Installing the optional leak sensor Warning: To avoid the harmful effects of personal contact with solvents, including inhalation, observe Good Laboratory Practice when you handle them. See the Material Safety Data Sheets for the solvents you use. Warning: To avoid personal contamination with biologically hazardous or toxic materials, wear clean, chemical-resistant, powder-free gloves when performing this procedure. Preparing for operation 9

14 Required materials Gloves: clean, powder-free, chemical-resistant Leak sensor To install the leak sensor: Caution: To avoid damaging electrical parts, never disconnect an electrical assembly while power is applied to an instrument. To completely interrupt power, set the power switch to Off, and then unplug the power cord from the AC source. Wait 10 seconds thereafter before you disconnect an assembly. 1. Power-off the SM-FL. 2. Open the fluidics compartment door. 3. Carefully unpack the new leak sensor. 4. Align the leak sensor s T-bar with the slot in the side of the leak sensor reservoir, and slide the leak sensor into place. TP02892 T-bar Slot in leak sensor reservoir Leak sensor installed in reservoir 5. Plug the leak sensor connector into the front of the instrument. 10

15 6. Power-on the SM-FL. 7. In the ACQUITY UPLC Console, select Sample Manager FL from the system tree. 8. In the SM-FL information window, click Control > Reset SM, to reset the SM-FL. Installing the waste tubing Caution: To prevent contamination to system components, wear clean, chemical-resistant, powder-free gloves when installing or removing the waste tubing. Required material Gloves: clean, powder-free, chemical-resistant ACQUITY UPLC I-Class System Accessories kit To install the waste tubing: 1. Attach the corrugated tubing, provided in the system accessories kit, to the process waste port (found on the lower drip tray of the sample manager). Preparing for operation 11

16 2. Route the tubing through the pass-through on the upper drip tray of the solvent manager. Process waste port Corrugated Teflon tubing Pass-through on upper drip tray of the solvent manager Front boss fitting on lower drip tray of the solvent manager 3. Slide the adapter onto the end of the corrugated Teflon tubing. 4. Connect the adapter to the front boss fitting on the lower drip tray of the solvent manager. Tip: For instructions on routing the solvent manager waste and vent lines, see the Binary Solvent Manager Operator s Overview and Maintenance Information. 12

17 Priming the SM-FL The priming process fills the sample needle with solvent, flushes new solvent through the injector lines, and/or purges air from the lines. You prime the system to accomplish these tasks: Prepare a new SM-FL for operation Prepare a SM-FL for operation after it has been idle for an extended period Change the solvent in the syringes Remove bubbles from the lines Ensure that the priming solvent is correctly composed and that it is high in quality and miscible with any other solvents used in your system. Use filters in all solvent reservoirs, and ensure the volumes of solvents are sufficient for priming. To prime the sample syringe: 1. In the ACQUITY UPLC Console, select Sample Manager from the system tree. Preparing for operation 13

18 Sample Manager information window: 2. Click Control > Prime Syringes. Alternative: Right-click in the SM-FL control panel, in the data application, and then click Prime. 3. In the Prime Syringes dialog box, select Sample syringe and both wash syringes. Tip: If you want only to remove air bubbles from the sample syringe, but do not want to prime the wash syringes, select Sample syringe only. However, do not select this option routinely. Priming all components at the same time is good practice. 4. Type the number of primes in the Number of cycles text box. Default: 1 Recommendation: Specify 5 to 7 primes when you are changing solvents. 14

19 5. Click OK to start priming. When the system status is Idle, priming is finished. Tip: Each prime takes approximately 2 to 4 minutes. Using the SM-FL Before running samples, Examine the injection valve, sample syringe, and all fittings for leaks. Tighten fittings as needed. Ensure both the sample compartment door and the fluidics compartment door are closed. Interface requirements Ventilation Allow at least 15.2 cm clearance at the rear and at least 1.3 cm clearance on the right-hand side of the SM-FL for ventilation. Drainage system Caution: Do not abort the sample manager priming sequence. Doing so can leave strong solvent in the sample needle, which can adversely affect chromatography. Allow clearance for fluid lines to pass along the right-hand side of the instrument (twelve inch OD tubes or four 1/6-inch OD tubes, for example). Ensure that the SM-FL can accept drainage from the column heater and provide a path to waste. Using the SM-FL 15

20 Installation recommendations for fittings Warning: To avoid personal contamination with biologically hazardous or toxic materials, wear clean, chemical-resistant, powder-free gloves when reinstalling fittings. Caution: When installing or removing a column, be sure that you turn the column itself and not the fitting (compression screw and ferrule). Otherwise, the active pre-heater tubing can turn with the fitting, potentially damaging the active pre-heater assembly. Most tubing connections use gold-plated compression screws and two-piece ferrules. See the diagram below for assembly orientation. Gold-plated compression fitting Ferrule with locking ring Compression screw Tubing The sample needle uses a PEEK compression screw and two-piece ferrules. The compression screw differs slightly, depending on the sample needle s outside diameter. See the diagram below for assembly orientation. PEEK sample needle fitting Locking ring (thin edge toward ferrule) Compression screw Tubing Ferrule The sample needle fitting is sometimes preset on the needle. Where it is not preset, assemble the fitting as shown above, ensuring that the thin wall of the 16

21 locking ring faces the flat-bottom ferrule. Doing so ensures the ferrule is correctly compressed onto the needle tubing. Recommendations: To prevent band spreading, ensure the tubing bottoms in its fitting hole before you tighten the compression screw. For easier accessibility, use long compression screws to attach tubes to the injector and vent valve. Perform the sample syringe leak test whenever you replace or loosen fittings during maintenance (see the ACQUITY UPLC online Help). Whenever you loosen fittings during maintenance, examine them for cracks, stripped threads, and deformations. Do not reuse stainless steel fittings more than six times. Required material Gloves: clean, powder-free, chemical-resistant When tightening system fittings, consult the following table. Installation recommendations for ACQUITY UPLC fittings: First use or re-installed First use Fitting Recommended tightening 1/4-28 flangeless with ferrule Finger-tight 1/4-28 flangeless with 2-piece ferrule Finger-tight Re-installed 1/4-28 flangeless with 2-piece ferrule Finger-tight Using the SM-FL 17

22 Installation recommendations for ACQUITY UPLC fittings: (Continued) First use or re-installed First use Fitting one-piece PEEK Finger-tight Stainless steel (gold-plated) with 2-piece stainless steel ferrule Recommended tightening Finger-tight, plus 3/4-turn using wrench 3/4-turn Re-installed Stainless steel (gold-plated) with 2-piece stainless steel ferrule (re-installed) Finger-tight, plus up to 1/6-turn using wrench 1/6-turn First use with unassembled ferrule PEEK fitting with flat-bottom ferrule Finger-tighten until first resistance is felt, then tighten an additional 1/4-turn using fingers or wrench. 1/4-turn 18

23 Installation recommendations for ACQUITY UPLC fittings: (Continued) First use with pre-set ferrule Fitting PEEK fitting with flat-bottom ferrule Recommended tightening Finger-tighten until first resistance is felt, then tighten an additional 1/4-turn using fingers or wrench. 1/4-turn Re-installed PEEK fitting with flat-bottom ferrule Finger-tighten until first resistance is felt, then tighten an additional 1/4-turn using fingers or wrench. 1/4-turn Using the SM-FL 19

24 Selecting weak wash and strong wash solvents For best performance, follow these guidelines when selecting wash solvents. Otherwise performance can be reduced, specifically Area/Height RSD and Linearity. Careful selection of wash solvents can also reduce carryover. The guidelines do not prohibit all other solvent combinations. Other combinations can be run with lower performance expectations or by manipulating default injection parameters. Use a weak wash solvent based on the sample and mobile phase chemistries of your application, making sure all solutions/buffers are miscible and soluble. Recommendation: For buffered aqueous, reversed-phase chromatographic conditions and MS applications, use a weak wash solvent of 100% water or 0% to 25% methanol or acetonitrile and a strong wash solvent of 50% to 100% methanol or acetonitrile. High sample concentrations can require other weak wash solvents. If your separation permits, Waters recommends adding a small amount of an organic solvent (~10%) to prevent microbial growth. See the Solvent Considerations appendix in the ACQUITY UPLC System Guide for further information about solvents. Caution: To avoid damage to the solenoid valve seats and seals in the solvent path, do not use a nonvolatile buffer as the weak wash or strong wash solvent. Tips: For best performance, the weak wash solvent must be similar or identical to your isocratic or initial gradient solvent conditions, excluding buffers. Do not use salt buffers in wash solvents. Choose a strong solvent at least equivalent to your final gradient conditions. A good starting point is a mixture of 25% water/25% acetonitrile/25% methanol/25% isopropanol. 20

25 Wash solvent effects: Property Organic species ph Concentration of strong solvent Sample diluent Wash volume ratio (weak to strong) Cycle times Effect As a general principle, strong and weak solvents must include the same organic species. Note that this is not always practicable. You can, however, use a 100% organic strong wash solvent. Adjust the ph of the strong and weak solvents for best peak shape and carryover performance. The strong solvent offers different selectivity than the weak wash solvent. The strong solvent must be no stronger than the concentration needed to reduce carryover to an acceptable level. The weak wash solvent will contact the sample, so match these as closely as possible. To offset adverse effects on peak shape caused by the matrix s composition, adjust the weak wash composition, especially when using the instrument in partial loop mode. Within a method, use a 3:1 ratio, weak wash to strong sufficient to ensure the weak wash flushes the strong from the needle and sample loop before sampling. When using a viscous strong solvent, use a 6:1 to 10:1 ratio, weak wash to strong. Higher viscosity wash solvents lengthen wash cycles. Washing the SM-FL needle Washing the needle is an optional procedure that flushes strong and/or weak wash solvent through the needle and injection port. Washing the needle removes contaminants from the inside and outside of the needle, the external piercing needle, and the injection port. You can also perform a needle wash to ascertain proper flow through the waste tubing and to confirm that the needle wash system is primed and properly operating. Using the SM-FL 21

26 Tip: Priming the system washes the sample needle, so whenever you prime the system, you can omit this procedure. To wash the SM-FL needle: 1. In the ACQUITY UPLC Console, select Sample Manager FL from the system tree. 2. In the SM-FL information window, click Control > Wash Needle. Alternative: Right-click the SM-FL control panel in the data application, and then click Wash Needle. 3. In the Strong Wash box, specify the volume for the strong wash solvent. Or, to omit strong wash solvent, enter 0 in the Strong Wash box, or leave it blank. Range: 0.0 through 99,999 μl Default: 0.0 μl Recommendation: 100 through 500 μl Tip: Using both a weak and strong wash solvent increases the wash time and solvent consumption because the system must be fully cleansed of the strong solvent before starting the next injection. Caution: To avoid strong wash solvent contacting the sample and contaminating it, use a sufficient quantity of weak wash solvent. 4. In the Weak Wash box, specify the volume for the weak wash solvent. Range: 1.0 through 99,999 μl Default: μl without strong wash or 500 μl with strong wash Recommendation: 200 through 500 μl or three times the strong wash volume. 5. Click OK. Result: The needle wash begins. When it ends, the status returns to idle. 22

27 To stop a needle wash routine before it finishes: In the SM-FL information window, click Control > Reset SM. Alternative: Right-click the SM-FL control panel in the data application, and then click Reset SM. Caution: Do not abort the sample needle wash sequence. Doing so can leave strong solvent in the sample needle, which can adversely affect chromatography. Loading sample plates The SM-FL is compatible with the ANSI standard well-plates, vial-trays, vials, and cap-mats/seals that are approved for use with the ACQUITY UPLC system. The SM-FL holds two ANSI/SBS plates that you load through the sample compartment door. Requirement: The plates you use must meet ANSI/SBS standards. Tip: Vial positions V1 through V4, located on the right-hand and left-hand sides of the sample tray, accommodate 4-mL vials. Contact Waters for inserts that allow you to use 2-mL vials in these positions. Observing vial and plate recommendations Waters recommends that you observe these usage guidelines for sample vials and plates in the SM-FL: Vials Use only Waters-certified vials. Ensure that vial holders conform to ANSI/SBS standards. Plates Use only XX-series plates and cap mats in the SM-FL. When selecting a new plate supplier, especially for 384-well plates, measure the plate size to ensure compatibility with Waters specifications for the SM-FL. To avoid warping plates, do not centrifuge them. Be aware that plates containing samples with high concentrations of organic solvent can give inconsistent results at room temperature. Using the SM-FL 23

28 Covers Use foil covers on vial plates whenever possible. Use pre-slit cap-mats/seals and vial caps. Use of non-pre-slit cap mats and vial caps can cause clogging in the wash lines. To prevent sample spillage or needle damage, use only Waters-approved covers on the sample vials. Warning: To avoid eye injury, wear safety glasses when loading sample plates. For more information about plates and vials, see Using Plates and Vials with ACQUITY UPLC Systems, part number Required material Safety glasses To load a sample plate: 1. Open the sample compartment door. 2. Press the plate selector switch on the top, center of the door frame to select plate position 1 or 2. Exception: If the SM-FL is accessing the sample tray when you select a new plate position, the selector switch does not operate and the instrument beeps once. The selector switch operates again after the SM-FL no longer accesses the sample tray. Tip: Press the plate selector switch twice to toggle between loading a sample plate and loading positions that accept 4-mL vials. 24

29 3. Pull the sample tray out. Position for a 4-mL vial (V1 through V4) Sample tray pulled out 4. Load the plate onto the tray so that well position A,1 is at the rear, left-hand corner, and the forward edge of the plate is behind the spring inside the front of the carrier. Tip: A represents the row number, 1 represents the vial position. A,1 well position Sample plate TP03232 Using the SM-FL 25

30 Sample plate vial positions: A B C D E F 5. Slide the tray in until it clicks into place. Sample tray handle Caution: To avoid damaging the sample needle, the sample plates must be positioned correctly and the sample tray must be fully engaged. 6. Close the sample compartment door. 26

31 Sample chamber considerations Warning: To avoid puncture wounds, keep hands or loose clothing clear of the needle assembly mechanism while it is moving. Note that the SM-FL beeps three times whenever the sample compartment door is open, and the needle assembly mechanism is about to move. Choosing needles and sample loops Sample needles Waters offers multiple needle sizes so you can choose the best option for your injection volume and sample viscosity. Waters recommends metal needles for samples known to be attracted to hydrophobic polymers and also when you use hexane and tetrahydrofuran. The following sample needles are available for the SM-FL: Needle size (μl) Material 10 PEEK (17-inch L inch ID) Default 10 PEEKsil 10 FEP with stainless steel tip 10 stainless steel 20 PEEK (17-inch L inch ID) 20 PEEKsil 20 FEP with stainless steel tip 20 stainless steel Tip: Using a smaller needle increases cycle times. Using the SM-FL 27

32 Follow these guidelines to select the most appropriate needle material for your application: Needle material PEEK PEEKsil Stainless steel FEP with stainless steel tip Recommended for General use Use with hydrophobic compounds Use with plates and capmats Use with plates and capmats Sample loops The sample loop holds the sample for injection. You install the sample loop between port 1 and port 4 on the injection valve. The following sample loops are available for the SM-FL: Loop size (μl) Minimum volume (μl) Maximum volume (μl) Sample loop volume is calculated based on loop length and an assumed internal diameter (ID), which can vary by as much as inch in stainless steel tubing. This variation in internal diameter across the length of the loop tubing can result in significant variation in the actual loop volume. 28

33 The table below illustrates the impact of variation in internal diameter for a 5-µL sample loop constructed of inch (nominal) ID tubing. Internal diameter (inch) Volume per inch length (μl) To observe the volume variation in the installed sample loop: 1. In the ACQUITY UPLC Console, click the Sample Manager view. 2. Select Maintain > Characterize > Needle and loop volumes. 3. Compare the measured and the nominal loop volumes. Modifying needle and sample loop configuration parameters To configure the system for a needle or sample loop size that differs from the one currently fitted: 1. In the ACQUITY UPLC Console, select Sample Manager FL from the system tree. 2. Select Configure > Volumes. Nominal to actual volume ratio 3. In the Volume Configuration dialog box, select the appropriate needle or sample loop size from the list, and then click OK. Needle and sample loop characterization Volume (μl) To ensure correct positioning of the sample in the sample loop, the sample manager characterizes the installed loop and needle. In doing so, it determines their actual volume, minimizing error associated with intrinsic variations in the volume of those components. The characterization time varies depending on the size of the installed loop and needle. To ensure accurate injection volumes, the sample manager slows the aspiration rate to 10 µl/min during characterization. It then determines the volume of the loop by locating the position of the aspirated air bubble twice, once with the loop offline and once with the loop online. For a 30-µL needle, Using the SM-FL 29

34 the air gap must displace about 40 µl of fluid to reach the volume detection device. The displacement process requires approximately four minutes for each mode, eight minutes in total. Because variations in the tolerance of the sample loop's internal diameter become more pronounced as the internal diameter of the loop tubing decreases, smaller loops can require more characterization time than larger loops. Solvent travels more slowly through the tubing of smaller loops, slowing the characterization process. UV-absorbing mobile phases, such as those containing TFA, can impede detection of the aspirated air gap, producing unsatisfactory characterization results. If the result of the characterization process is unsatisfactory, the sample manager repeats the process. Choosing the sample syringe The following sample syringe sizes are available to use in the SM-FL: 50 μl 100 μl Default 250 μl 500 μl Choose a syringe size that allows you to draw your desired total sample volume into the sample needle. Waters recommends using a sample syringe volume that is at least two times your sample volume. Modifying sample syringe configuration parameters To configure the system for a syringe size that differs from the one currently fitted: 1. In the ACQUITY UPLC Console, select Sample Manager FL from the system tree. 2. Select Configure > Volumes. 3. In the Volume Configuration dialog box, select the appropriate sample syringe size from the list, and then click OK. 30

35 Choosing the sample syringe draw rate The ideal syringe draw rate depends on sample volume, sample viscosity, and desired cycle time. The default draw rate varies depending on the volume of the sample loop and the needle installed in the device. Default draw rates: Draw rate (µl/minute) Loop volume (µl) 10-µL needle 20-µL needle (default) You can also specify the draw rate, if desired. The following tables list the maximum draw rates for common mobile phases. Maximum draw rates for 10-µL needle: Solvent 50% MeOH/ 50% H 2 O Draw rate (µl/minute) 1 µl loop 2 µl loop 5 µl loop 10 µlor larger loop % H 2 O % ACN % DMSO Using the SM-FL 31

36 Maximum draw rates for 20-µL needle: Solvent 50% MeOH/ 50% H 2 O Draw rate (µl/minute) 1 µl loop 2 µl loop 5 µl loop 10 µlor larger loop % H 2 O % ACN % DMSO Choosing the needle height setting The default needle penetration depth setting of 2.0 mm prevents you from reaching the bottom of the vial. Caution: To avoid damaging the needle, follow the guidelines in this section, and use the appropriate needle-height setting for your sample plates or vials. You can change the default needle placement (needle height) setting in the software in two places: the Dilution tab of the Sample Manager instrument method editor and the Advanced Settings dialog box. Set consistent values in both places. To aspirate more sample from the vial, decrease the value so that the needle tip is closer to the bottom of the vial. 32

37 Sample needle Vial depth 2 mm offset Recovering maximum sample from vials The current ANSI plate (48 vials) definition for the 2-mL Maximum Recovery Vials can leave some sample in the vial. If you want to recover the maximum amount of sample, change the needle placement setting. Tip: To modify needle placement for vials, click Instrument Method Editor > ACQ-FL > General tab > Advanced, and change the Needle Placement (from bottom) value. Vial type Waters Supplied Total Recovery Vial Waters Supplied Max. Recovery Vial Minimum needle placement (mm) Description 0.7 Screw Cap mm Clear Total Rec pre-slit PTFE/Silicone Septa 2.1 Screw Cap mm Clear Max Rec pre-slit PTFE/Silicone Septa Screw Cap mm Amber Max Rec pre-slit PTFE/Silicone Septa Part number C C C Using the SM-FL 33

38 Vial type Waters Supplied Flat Bottom Vial Minimum needle placement (mm) Description 0.1 Screw Cap mm Clear with pre-slit PTFE/Silicone Septa Screw Cap mm Amber pre-slit PTFE/Silicone Septa Screw Cap mm 750 µl PP pre-slit PTFE/Silicone Septa Screw Cap mm 300 µl PP pre-slit PTFE/Silicone Septa Part number C C See also: Waters Sample Vials and Accessories brochure on Revising a plate type The ANSI-48Tube0.65mLHolder plate type is defined in Empower software so that the needle does not use sample near the bottom of the vial, greatly increasing the residual volume. The plate type contains 0.65 ml tubes. To create a new plate type and correct the depth value: 1. In Empower software, open the Configure System window. 2. From the Empower Configuration tree, select Plate Types. 3. Select the plate type ANSI-48Tube0.65mLHolder. 4. From the menu, select File > Properties. 5. Under Plate Type, type a suitable name for the plate. 6. Change the Depth parameter to Click OK. Result: A copy of the plate definition is created with a new name and revised needle depth. 34

39 Air gaps If air gaps are required for your chromatography, you can specify an air-gap volume in the instrument method editor. The default air gap volume is dependent on the volume of the installed needle and the selected injection mode. Default air gap volume: Default air gap volume (µl) Needle volume Partial loop needle Partial loop mode Full loop mode overfill mode 10 µl µl To specify an air-gap volume: 1. In the instrument method editor, click the ACQ-FL tab and then the General tab. 2. Click Advanced. 3. In the Advanced Settings dialog box, mark the box for the air-gap option. 4. Specify volumes for your pre-aspirate and post-aspirate air gaps, and then click OK. Using the SM-FL 35

40 Selecting the optimum sample injection mode The sample manager supports three injection modes: Full loop Partial loop Partial loop needle overfill (PLNO) The injection process differs for each mode and thus, each mode has a different accuracy, visualized as a difference in peak area. Because the injection process differs, each mode delivers different amounts, albeit reproducibly and with linearity. The responses cannot be compared or plotted on the same calibration curve. Each injection mode has different advantages and can be used to improve performance, depending upon the individual goal of the analysis. Injection mode comparison: Injection mode Advantages Disadvantages Full loop Partial loop Best precision. Lowest dispersion. Lowest sample consumption. Good linearity and precision. Reduces cycle time slightly, compared to partial loop needle overfill mode. Increased sample consumption. Multiple sample loops are required for multiple injection volumes. Overfilling the loop lengthens the cycle time. Weak wash solvent, mobile phase, and sample are co-injected, and thus sample peak recoveries are method dependant. 36

41 Injection mode comparison: (Continued) Injection mode Advantages Disadvantages Partial loop needle overfill Full loop mode Good linearity, precision and accuracy. Yields consistent sample accuracy and peak areas from system to system and between sample loops. Offers lower dispersion than partial loop pressure assist mode injections. Sample and its diluent are exposed only to the weak wash and initial mobile phase. Only mobile phase and sample are injected. The sample is positioned at end of sample loop closest to column inlet. Increased sample consumption. Note: Under default conditions, uses 7 µl additional sample per injection. The full loop injection mode (recommended when using 1.0-mm I.D. columns) permits the least peak dispersion and delivers the highest precision of all the modes, particularly when you use it with smaller loops. When used with a 1-µL sample loop or a 2-µL sample loop, it is the best mode for plate-count studies. Because loop volume typically varies slightly, even for loops of the same nominal loop volume, area values can vary depending on which system an analysis is run. However, accuracy is determined by the tubing's internal diameter tolerance, not the injector performance. See page 28 for more information about loop volume variation. Using the SM-FL 37

42 Partial loop mode In partial loop mode, sample is drawn, placed between pressurized air gaps and then transferred to the center of the sample loop. Bracketing the sample volume with air gaps (bubbles) reduces peak dispersion resulting from laminar flow. To further reduce dispersion, and to retain the maximum amount of sample in loop, the sample is positioned with its leading edge at the end of the sample loop closest to the column. Use partial loop mode when analysis time takes precedence over any other concern, when the sample volume is very limited, or when the injection volume is large. In partial loop mode, weak wash solvent and sample are co-injected, so sample peak recoveries depend on the method used and on these conditions: Sample viscosity and the syringe draw rate The ratio of the sample volume to the loop volume. For best recovery, keep the ratio between 10% and 50% of the nominal loop volume. The ability of the sample manager to accurately characterize the volume of the sample needle, sample loop, and connecting tubing. To improve recovery, characterize the system using the solvents in the current method. Because the sample is co-injected with the weak wash solvent, ensure that the weak wash solvent does not interfere with the chromatography. Partial loop needle overfill mode In partial loop needle overfill mode, the sample needle draws extra sample to rinse the needle, ensuring that the volume placed into the sample loop is homogeneous. Partial loop needle overfill mode is the best general-purpose injection mode. It confers excellent linearity performance, and provides superior partial loop accuracy, precision, and linearity for a wide range of samples, including strong and weak acids and bases, hydrophilic, and hydrophobic compounds. The portion of the sample injected is the least diluted part of the sample volume. Only the initial mobile phase and sample are placed in the sample loop. Therefore, both the strong and weak wash can be used to ensure minimum carryover. This mode provides better recovery than partial loop pressure assist mode does. The loop is pre-rinsed, replacing the weak wash with extra sample. The 38

43 result is a less diluted sample than in partial loop pressure assist mode, where the sample slug is introduced by displacing the weak wash, diluting the sample. Partial loop needle overfill mode provides optimum performance when injection volumes are maintained within a range of 10% to 75% of the actual loop volume. The generally accepted practice for partial loop injection linearity restricts the injection volume selected to 50% nominal loop volume. Nevertheless, you can increase the usable loop volume to 75% of the actual loop volume by selecting the needle overfill technique. Doing so is based on achieving an injection-to-injection variability of 1% across the specified volume range. In addition, the correlation between specified injection volume and peak area is R2 > The following table provides the minimum and maximum injection volumes for each of the commonly used loops. Selecting the injection mode and loop volume: Sample injection volume range (µl) Loop volume Partial loop needle Partial loop mode Full loop mode overfill mode 1 µl 0.1 to.8 Not recommended 1 2 µl 0.2 to 1.5 Not recommended 2 5 µl 0.5 to 3.8 Not recommended 5 10 µl (default) 1.0 to to µl 2.0 to to µl a 5.0 to to µl a 10 to to µl a 25 to to a. You must install a 250- or 500-μL sample syringe in the sample manager when using this size sample loop. Using the SM-FL 39

44 Load ahead option The load-ahead option instructs the sample manager to aspirate the next sample in a sample list while a current sample is running, thus eliminating the majority of the overhead time (time in addition to the chromatographic analysis time) of an injection cycle. The Load Ahead option reduces cycle time without degrading chromatographic performance. Restrictions: Load Ahead Mode is designed for use with the Partial Loop with Needle Overfill injection mode. The first injection of a sample set and injection sets with different methods do not utilize load ahead mode. The following figures show a standard ACQUITY UPLC system cycle time and load-ahead cycle time. Standard ACQUITY UPLC system cycle time definition Standard injection mode and first injection in load ahead mode Total ACQUITY UPLC system cycle time System setup SM sample preparation and positioning Chromatographic run time SM wash time Start Sample injection End 40

45 ACQUITY UPLC system load-ahead cycle time definition Load ahead mode after the first injection Total ACQUITY UPLC system cycle time System setup and sample positioning into loop Chromatographic run time Buffer delay SM wash time SM sample preparation Start Sample injection End Tip: The buffer delay is a wait time that compensates for variations in the time it takes to load a sample. Because the first injection of each injection series does not use the load ahead injection option, the time savings depends on the number of consecutive injections made. If the wash time and the sample prep time are equal to or greater than the chromatographic run time then load ahead does not improve the cycle time. Loop offline option When using the full loop injection mode or the partial loop pressure assist injection mode, choose the Loop Offline option to specify an interval, in minutes, following an injection, where the needle and sample loop return to the load position. The Loop Offline option can also reduce the contribution of the sample loop to the dwell volume, if the loop is taken offline before the first change in the gradient. The dwell volume, however, is significant only in systems that use sample loops of volumes greater than 20 µl. The Loop Offline option is not available for injections using the partial loop with needle overfill mode. Using the SM-FL 41

46 Determining the correct time to take the loop offline Solutes that deposit in the sample loop because of poor solubility are not transferred to the column until the gradient composition dissolves the sample and flushes it onto the column. At that point, the solute's high retention factor (k') causes it to elute from the column in a single column volume. Choosing the correct time to take the sample loop offline ensures that all of the peaks are flushed from the loop. If you are taking the loop offline before the first gradient change, flush it with at least five loop volumes of solvent at the initial gradient composition to completely flush the sample onto the column. In this case, use the following formula to calculate the loop offline time: Loop offline time (min) = Nominal loop volume (μl) Flow rate (μl/min) 5 Example: For a system fitted with a 20-µL loop, in which the flow rate is 100 µl/min, the loop-offline time must be at least 1 minute. Loop offline time (min) = 20 (μl) 100 (μl/min) 5 Loop offline time (min) =.2 min 5 Loop offline time (min) = 1 min If you are taking the sample loop offline after the end of the gradient, ensure it is completely filled with solvent of the initial gradient composition before taking it offline. In addition, observe these considerations when determining the correct loop-offline time: If the strong wash volume has been significantly increased, the washing process is sometimes unfinished when the loop switches back. If such is the case, extend the loop-offline time. The loop offline time must not occur if the contents of the sample loop are of a higher concentration than the initial gradient conditions. 42

47 To choose the load-ahead and loop-offline options: 1. In the instrument method editor, click the ACQ-FL tab and then the General tab. 2. Select the boxes for the load-ahead and loop-offline options. 3. Specify a time interval for the loop-offline option, if necessary. Tip: In the SM-FL, a programmed gradient typically flows through all parts of the instrument that contact the sample. If you initiate the loop-offline option before the gradient reaches its final conditions, the highly organic portion of your gradient does not pass through the sample loop. As a result, the gradient can fail to remove all sample from the sample loop resulting in low sample recovery and an increased risk of carryover. Reducing carryover In a chromatographic system, any substance that creates unwanted peaks or excessive background noise is contamination. Carryover, a specific type of contamination, occurs when sample material remaining in the system after an injection appears as peaks in subsequent injections, compromising quantification. To optimize system performance, carryover must be minimized and held to an acceptable level (often, below the limits of detection). Carryover can result from incorrectly installed tubing, fittings, or other hardware or by selecting ineffective wash solvents. Take these actions to reduce carryover: Restrict sample loop usage to one system. When reinstalling the loop, adopt the same orientation as the original installation. A reversed sample loop can cause carryover when reinstalled. Ensure all high and low pressure tubing connections are properly set. Tubing must seat properly (that is, without gaps) in its fittings before you tighten them. Poorly seated connections create unnecessary space-reservoirs that retain sample, increasing carryover. When possible, partially fill sample wells or vials to minimize sample contact with the piercing needle. Ensure the needle is free of debris or residue. Inspect and clean the tubing that connects the piercing needle to the wash pump. During wash procedures, the tubing carries drawn wash solvent through the piercing needle. If it becomes clogged, the piercing needle goes unwashed. Using the SM-FL 43

48 Ensure the sample needle and air sensor tube are clean. Over time, they can become coated with sample to the extent that wash solvents fail to clean them adequately. Inspect the injection port and its O-ring for sample residue, which can cause carryover. When necessary, replace those components. Replace the injection valve at regular, preventive-maintenance intervals. Over time, the fittings and seals can become worn, causing carryover. Characterize the loop and needle volume after you change the method or solvents. A useful rule of thumb is to characterize the loop and needle volume at the start of each day. Avoid plate or vial sealing systems that use sticky substances, which can cause carryover. Follow the guidelines on page 20 when selecting wash solvents. Add the Cycle Inject Valve event to the method. The event actuates the injection valve during a run, pushing any residual material trapped within it into the sample loop and, at the end of the gradient, injecting that material. Diagnostic tests You can select the sample syringe leak test, which verifies the sample path is free of leaks, from the SM-FL s Maintain menu. The Maintain menu also lists these functions: Calibrating the needle s Z-axis, which calibrates the vertical position of the needle. Disabling motors, which you do before manually moving the sample tray and R-carriage. Parking the sample needle and injection valve, which you do before storing the system, or replacing a needle or valve. Replacing the needle, sample syringe, and wash syringes. See also: Binary Solvent Manager Operator s Overview and Maintenance Information document for information on the solvent manager s leak test. ACQUITY Console online Help for additional information about running diagnostic tests. 44

49 Resolving leak sensor errors The SM-FL can be fitted with two leak sensors, bottom and top: called the SM-FL leak sensor and the column heater leak sensor, respectively. After approximately 1.5 ml of liquid accumulates in the leak sensor reservoir, an alarm sounds indicating that the leak sensor detected a leak. Warning: To avoid personal contamination with biologically hazardous or toxic materials, wear clean, chemical-resistant, powder-free gloves when performing this procedure. Caution: To avoid scratching or damaging the leak sensor do not allow buffered solvents to accumulate and dry on it. do not submerge it in a cleaning bath. Required materials Cotton swabs Gloves: clean, powder-free, chemical-resistant Nonabrasive, lint-free wipes To resolve a leak sensor error: 1. In the ACQUITY UPLC Console s Leak Sensors dialog box, determine which of the SM-FL s 2 leak sensors detected a leak. 2. If the message reads Leak Detected, locate the source of the leak, and make the repairs necessary to stop it. If you need additional information, see the Column Compartments Operator s Overview and Maintenance Information document. Using the SM-FL 45