USER'S GUIDE. Nanoflow Probe. Red Stripe. 3-4mm. ptfe Sleeve. To Injector. ZU1XC Fused Silica TTF115. Fused Silica. ptfe Sleeve

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1 USER'S GUIDE Nanoflow Probe 3-4mm Red Stripe ptfe Sleeve To Injector ZU1XC Fused Silica 20µm 90µm TTF115 ptfe Sleeve Fused Silica 25µm 375µm

2 Micromass UK Limited Floats Road Wythenshawe M23 9LZ Tel: Fax: Tudor Road Altrincham WA14 5RZ Tel: Fax: Warning There are high voltages and hot surfaces present throughout the mass spectrometer. Samples analysed may be toxic. Many of the instrument's components are susceptible to contamination by toxic deposits. Operational and maintenance procedures may require the use of corrosive and flammable agents. All procedures must be performed only by suitably trained personnel, aware of the inherent hazards and observing every precaution. This manual is a companion to the instrument's and Parts List, and to the MassLynx User s Guide. All information contained in these manuals is believed to be correct at the time of publication. The publishers and their agents shall not be liable for errors contained herein nor for incidental or consequential damages in connection with the furnishing, performance or use of this material. All product specifications, as well as the information contained in this manual, are subject to change without notice. Issue 2 Micromass Ltd Page 1

3 Contents Introduction 3 Preparation 4 Loading the Sample 6 Fused Silica Syringe Needle 6 Gel Loader Tip 6 Operation 7 Calculation of Flow Rate 9 Fused Silica Capillary Preparation 10 Operation 13 Consumable Spares 14 Fused Silica Capillary 14 Contents Page 2

4 Introduction The Micromass nanoflow probe allows electrospray ionisation to be performed in the flow rate range 5 to 1000 nanolitres per minute. There are two options for the spraying capillary, which can be alternately fitted to the probe: Borosilicate metal coated glass capillaries. Metal coated glass capillaries allow the lowest flow rates to be obtained although they are used for one sample only and then must be discarded. Fused silica capillaries. This option is suitable for flow injection analyses or for coupling to nano-hplc, and uses a pump to regulate the flow rate down to 100 nl/min. If a syringe pump is to be used, a gas -tight syringe is necessary to obtain correct flow rates without leakage. For a given sample concentration, the ion currents observed in nanoflow are comparable to those seen in normal flow rate electrospray. Great sensitivity gains are therefore observed when similar scan parameters are used, due to the great reductions in sample consumption. A list of consumable spare parts is included at the end of this document. Introduction Page 3

5 Preparation Referring to the instrument's, remove the counter electrode from the API source and clean the sample cone. Attach the alignment insert to the inner source. Reduce the source temperature to 30 C. Connect the source's drying gas inlet tube to the instrument's nebuliser gas supply. On Quattro 1, use the 1 /8" - 1 /16" adaptor to connect the bath gas line to the nebuliser flow regulator. Ensure that the exhaust tube from the inner source is not connected. Carefully remove the capillary from its case by lifting vertically while pressing down on the foam with two fingers. Caution: It is important to take great care when handling the tips as they are extremely fragile. Always handle using the square end of the capillary. Foam Capillary Page 4

6 Locate the Swagelok union in which the capillary is to be held and remove the knurled nut. Cut a 5mm length of conductive elastomer, if not already available. Pass, in turn, the knurled nut, the elastomer and the union, over the square end of the capillary. Secure the nut, finger tight, such that approximately 15mm of capillary protrudes from the nut as shown. Glass Capillary 15mm Blue Conductive Elastomer Blanking Nut 6mm ptfe 6-4mm Reducer Page 5

7 Loading the Sample There are five types of capillary available, each with a slightly different tip profile: Types A, C and E have been found to work optimally when using, for example, peptide samples. Types B and D work optimally for larger molecules or different solvent systems, such as 100% water + 2mM ammonium acetate. The capillary may be reused if the same sample is to be analysed, but to avoid cross contamination it is recommended that a new capillary be used for each sample. If possible, filter the sample using a 0.2µm Anopore filter. It has been found that beam stability improves when 50% methanol is used instead of 50% acetonitrile. The sample can now be loaded into the capillary using either a fused silica syringe needle or gel loader tips, as follows. Fused Silica Syringe Needle Gel Loader Tip Push the needle to the base of the tip before depressing the syringe plunger. A fused silica needle syringe is commercially available (Hamilton 75RNFS) which allows accurate volumes to be injected. Inject the sample to about half way. Hold the capillary vertically until the sample runs to the pointed end of the capillary. Gel loader tips allow accurate volumes to be injected without the possibility of cross contamination. Page 6

8 Operation Once the sample has been loaded into the capillary: Secure the Swagelok union to the probe. For Quattro 1, check that the distance from the probe stop to the probe tip is less than 257mm. For other instruments, check that the distance from the front of the probe body to the tip is less than 171mm. Referring to the diagram: On Quattro 1, attach the back pressure gas to the 1 /16" tubing using the ¼" to 1 /16" union provided. On other instruments, attach the nitrogen gas line to the red coupling at the back of the probe (in normal electrospray this would be the nebulising gas supply coupling). 0 to 2 bar (30 psi) Regulator 6mm ptfe Valve Tee 6mm ptfe 6mm - 4mm Reducer To Probe Nitrogen Quattro 1 6mm - 1/16" Reducer To Probe 1/16" ptfe Page 7

9 Insert the probe into the source. Set the back pressure to 70 to 140 mbar (1 to 2 psi). On the tune page, set Capillary and HV Lens to zero. Select. Set Capillary on the tune page to 1.5kV. An ion beam should now be present. Use the source parameters to tune as in normal flow rate electrospray. The normal operating flow rate for the bath gas is approximately 40 litres/hour. To improve beam stability and sensitivity: Adjust Capillary and/or the nitrogen back pressure. The capillary voltage should not need to be raised above 2.5kV and may even require reducing to achieve a stable beam. The nitrogen back pressure should not be raised above 1.4 bar (20 psi). If no ion beam is obtained, it may be possible to initiate the spray as follows: Reduce any back pressure to zero using the valve on the gas line and reset the regulator pressure to 70 to 140 mbar (1 to 2 psi). Remove the probe from the source. Using a tissue, touch very lightly on the tip. A drop of liquid may appear. Reinsert the probe and repeat the above procedure. Page 8

10 Calculation of Flow Rate If an unknown amount of sample is placed in the capillary, the amount of sample consumed and the average flow rate may be calculated as follows: Measure the internal diameter of the capillary (approximately 0.58mm). Measure the length (in mm) of liquid used during the run. By holding the capillary up to light it is possible to see the meniscus level of the liquid. Calculate the amount of sample consumed. mm³ is equivalent to µl. Knowing the duration of the run, calculate the average flow rate. Page 9

11 Fused Silica Capillary 3-4mm Red Stripe ptfe Sleeve To Injector Fused Silica 20µm 90µm ZU1XC TTF115 ptfe Sleeve Fused Silica 25µm 375µm Preparation Warning: Always wear eye protection when handling fused silica. Referring to the instrument's, remove the counter electrode from the API source and clean the sample cone. Attach the alignment insert to the inner source. Reduce the source temperature to 30 C. Ensure that the exhaust tube from the inner source is not connected. Cut approximately 600mm of the 25µm i.d. fused silica. The 25µm i.d. silica links the Valco injector on the top of the probe to the low dead volume union. Take care not to confuse the 25µm i.d. fused silica with the 75µm i.d. fused silica which is used to link the pump to the injector. Cut approximately 40mm of the 20µm i.d. fused silica. The 20µm i.d. silica is used as the probe tip. Fused Silica Capillary Page 10

12 It is recommended a white background is used when handling the 20µm i.d. fused silica. Cut approximately 30mm of the ptfe tubing. Cut approximately 30mm of the red stripe peek tubing. Locate the ultra low dead volume Valco union and the Valco plug cap. Remove the probe cover. Loosen the fingertight nuts in the union at the back of the probe. If fitted, remove the blanking nut from the Swagelok tee in the probe and the Swagelok adapter used in the borosilicate capillary option. Thread one end of the 25µm i.d. fused silica through the ptfe sleeve (TTF115). Set the correct pilot depth for the ptfe sleeve and a Valco ferrule using the plug cap provided. There is no need to overtighten when setting the pilot depth. Remove the ptfe sleeve from the plug cap and the nut from the ptfe sleeve. With a small amount of fused silica protruding, cut a small piece (approximately 2mm) off the end and then ensure that the fused silica is flush with the sleeve, using a scalpel blade to push it back. This ensures that the fused silica has a clean end. Using the plug cap again, set the correct pilot depth for the red stripe peek sleeving and a Valco ferrule. Remove the peek from the plug cap and the nut from the peek. Cut the peek so that approximately 5mm is protruding from the back end of the ferrule, and hence none can be seen when the nut is replaced. Thread the 20µm i.d. fused silica through the peek and ensure that the fused silica is flush with the peek sleeve using a scalpel blade. Attach the peek sleeved fused silica to one end of the low dead volume union using a normal Valco nut and attach the ptfe sleeved fused silica to the other end using the Valco adapter. Ensure both pieces of fused silica are tightly held. Thread the 25µm fused silica through the probe shaft and through the 0.023" i.d. ptfe sleeves (in 2 places) in the fingertight union at the probe end plate. Fused Silica Capillary Page 11

13 Screw the Valco adapter into the probe shaft (fingertight is adequate) and tighten the fingertight nuts to secure the fused silica. Cut down the 20µm i.d. fused silica such that 3 to 4mm protrudes from the nut. Attach the injector valve to the bracket and then the bracket to the probe. Replace the probe cover. The ports on the injector valve are as follows: C P W S column pump waste sample (attach the VISF port). Connect the pump to the injector using 75µm i.d. fused silica. For Quattro 1, adjust the probe stop such that approximately 10mm of the stop protrudes from the bracket. For other instruments, set the distance between the probe stop and the fused silica tip to 167mm. Ensure that any air bubbles are purged from the plumbing. This is effectively done by first purging up to and including the valve at a high flow rate (approximately 5 µl/min) with the 25µm i.d. fused silica disconnected. Reduce the flow to the required rate and attach the 25µm fused silica to the valve output. Insert the probe into the source. Fused Silica Capillary Page 12

14 Operation It has been found that beam stability improves when 50% methanol is used instead of 50% acetonitrile. The normal operating flow rate for the bath gas is approximately 40 litres/hour. On the tune page, set Capillary and HV Lens to zero. Select. A stable beam should be obtained with a Capillary voltage of ~2.5kV. It may be necessary to inject a known sample, for example gramicidin, to see a stable beam. However, there will be an upper and lower limit where the beam starts to go unstable with a difference between the two of approximately 800V. It has been found that the system operates efficiently when the capillary voltage is set to the mid point of these values. With time, the Capillary voltage needed to obtain a stable beam will start to rise. This usually means that the sample cone requires cleaning again with methanol. Fused Silica Capillary Page 13

15 Description Consumable Spares Ref. No. Borosilicate glass capillary, type A Borosilicate glass capillary, type B Borosilicate glass capillary, type C Borosilicate glass capillary, type D Borosilicate glass capillary, type E Blue conductive elastomer Fused Silica Capillary Description Ref. No. 25µm i.d. fused silica capillary µm i.d. fused silica capillary ptfe tubing TTF Red stripe peek tubing Ultra low dead volume Valco union Valco plug cap ptfe sleeve 0.023" i.d VISF port µm i.d. fused silica Consumable Spares Page 14