Introduction to Small Molecule Analysis with the Agilent 1200/6130 LC-DAD-MSD system using Easy-Access and a basic guide to MSD ChemStation.

Transcription

1 Basic Info... 2 Quick Start Guide... 4 Sample Preparation... 5 Sample Guidelines... 5 Logging Sample into Online Billing System... 6 Multiple Samples on the Same Online Number Warning... 9 Performing an Analysis Using Easy-Access... 9 Entering Sample Data into Easy-Access... 9 Important sample entry notes: Loading Vials into the Autosampler Delaying Long Analyses Sample Disposal Warning: Basic Data Analysis in LC/MSD ChemStation Remote Access to LC-MS Data: Mapping a Network Drive Opening a Data File in ChemStation Basic ChemStation Navigation Printing Data Views in ChemStation The Integration Task The Calibration Task The Signal Task Spectrum Task Method and Run Control Window Troubleshooting and Obtaining Help Introduction to Small Molecule Analysis with the Agilent 1200/6130 LC-DAD-MSD system using Easy-Access and a basic guide to MSD ChemStation. Last updated August

2 Basic Info The Agilent 1200/6130 is a high performance liquid chromatograph with four different solvents (two can be used during any single analysis), a six port column selection valve, and with both a diode array detector (DAD, to record UV-VIS absorbance data) and a multi-mode electrospray ionization/atmospheric pressure chemical ionzation, quadrupole mass spectrometer (MSD). It can record UV-VIS absorbance spectra from nm and both positive and negative ions from m/z MSD stands for Mass Selective Detector (the ESI/APCI-quadrupole MS); DAD stands for Diode Array Detector (the UV-VIS detector). The MSD can be directed to selectively monitor up to 50 different discrete m/z ratios or of scan a wide range of masses repeatedly. The MSD has unit resolution (all features are ~0.5 m/z units wide) and has an accuracy of ±0.15 m/z. Under ideal conditions 50 pg of 0.4 ml/min in full scan mode and 1 pg of reserpine in select ion monitoring mode (SIM). Complete specifications can be found on Agilent s website: US/Products/Instruments/ms/6100SeriesQuadrupoleLCMSSystems/Pages/gp41725.as px. The standard 1200 series DAD can simultaneously monitor up to 8 separate UV-VIS wavelengths during the course of an analysis in addition to recording complete UV-VIS spectra at speeds of up to 20 Hz. The UV-VIS has an ultimate resolution of 1 nm and a wavelength accuracy of ±1 nm. Its absorbance drift is less than 1 mau/hr and it has a linear response to 2 AU. Complete specs can be found on Agilent s website: The instrument is located in A454 (door is by staircase to the LEFT of the elevator near A400). The same key is used to unlock A411 or A454. It is intended for walk-up loop injection and LC-MS analyses of small organic and inorganic compounds (typically under 2,000 Da). The current installed columns and solvents are found on the whiteboard in front of the instrument. Please hold LC-MS analyses longer than 20 minutes until AFTER 8:00 pm (see Delaying Long Analyses section below). If you have more than 8 quick samples (i.e. under 10 minutes each) to analyze at any given time, it is requested that you only submit 8 during regular business hours (9:00 am 8:00 pm). The additional samples should be delayed until AFTER 8:00 pm (see Delaying Long Analyses section below). This guarantees quick access to those needing to monitor reactions in progress by mass spectrometry. Prices for self-serve use (as of August 2009) are $7 for loop injection (under 3 minutes) and $10 for LC-MS (longer than 3 minutes, requiring an HPLC separation). Users will be allowed to create their own methods after they have demonstrated a sufficient level of proficiency to MSF staff. Talk to Dr. Karty or Angie about increasing permission levels. 2

3 LC/MSD ChemStation has EXCELLENT help information with step-by-step instructions, if necessary. Simply click the Help menu and select either Help Topics or Commands. Easy-Access has a rather incomplete help file, but this is not a problem for the casual user. It is normal for an MSD peak to appear about 0.1 minute AFTER its corresponding DAD peak. This is the time it takes for a compound to pass through the tubing between the two detectors, get ionized, mass selected, and then be detected by the MSD. 3

4 Quick Start Guide To run a sample, the following steps must be completed: Sample mg/ml in appropriate solvent (see Sample Preparation section) Sample logged into online billing system (see Online Database section) Click Submit New Sample button in Easy Access (see Performing an Analysis section) Enter number of samples (up to 8) and click OK Fill out all information in the table and click Load Samples Put the samples in the indicated position in the autosampler Click OK then wait for indicating samples have been analyzed Data are located in the single runs section of the following directory: D:\EZXData\username\month-year (see Opening a File section) Use Sigma button on Spectrum window in Data Analysis to extract mass spectra (See Data Analysis section) Click the mass spectrum window so it is blue, then select Print Selected Window from the File menu to print your data. (See Printing Data Views in ChemStation section) 4

5 Sample Preparation Sample Guidelines Samples should have an amine, alcohol, carboxylic acid, or ester group to support positive ionization (by protonation or sodium addition) by ESI. Sulfonic, phosphoric, and boronic acid groups can be deprotonated to make negative ions by ESI. APCI is a little harsher and can ionize many protected heteroatoms. Samples must have no particulate matter in them. Suspensions should be centrifuged or passed through a filter with a 1 µm or smaller pore size prior to ESI/APCI or LC-MS analysis. Prior to dilution, samples should be ~1 mg/ml in methanol, chloroform, acetonitrile, water, acetone, or other polar, volatile solvent. Alternatively, the sample could be made to the concentration specified in bold below using the solvents specified in the next step. Dilute the sample by adding 3 µl of sample to 750 µl of ESI solution. This can be done directly into an Agilent 12x45 mm autosampler vial. These vials are available in A454 or A411. Pre-made mass spectrometry solutions are methanol containing 0.5% v/v acetic acid and 5 µm sodium acetate or 1:1 chloroform:methanol, 0.5% v/v acetic acid, and 5 µm sodium acetate, and 1:1 chloroform:methanol with 5 µm sodium acetate (no acid) The solution actually injected into the LC-MS should be on the order of mg/ml analyte in ESI solution. Other valid solvent choices include water, acetone, acetonitrile, ethanol, isopropanol, chloroform and tetrahydrofuran (talk to Dr. Karty first about other solvents). Ideally, the solvent should be polar, volatile, and non-viscous. If the molecule under study has a permanent charge (organometallic complex, quaternary amine, etc.), non-viscous, volatile, relatively non-polar solvents (e.g. dichloromethane, fluorobenzene) can be used. Again, check with Dr. Karty prior to using non-polar solvents. Any solvent additives (e.g. buffers) should also be volatile. These should include a source or sink of protons and/or a source of an alkali cation. Suggested modifiers are weak, volatile acids and bases or salts of volatile, weak acids and bases. Valid additives include acetic acid, ammonium acetate, formic acid, ammonium formate, ammonia, triethylamine, methylamine, and ammonium bicarbonate. Ionic strength preferably should be less than 25 mm and should NEVER exceed 75 mm. Involatile alkali salts (e.g. sodium acetate) may be added at concentrations UNDER 5 µm to make M+Na cations. Be advised the trifluoroacetic acid (TFA) suppresses ionization because it is too strong of an acid (it tightly pairs with protonated basic sites in a molecule making a neutral complex). C18 reversed-phase HPLC is available for separating complex mixtures. It can also be used to clean up samples with significant contamination from buffers, supporting 5

6 electrolytes, or starting materials. Again, try to have the analyte be on the order or 0.05 mg/ml in the autosampler vial. If the supporting electrolytes or buffers contain quaternary amines, please try an extraction to remove the amines. Quaternary amines are detectable by mass spectrometry at extremely low concentrations (they are permanently charged) and can be difficult to remove from the source. The sample MUST be in a capped Agilent-brand 12x45 mm vial in order for the instrument to work properly. The solution level must be ABOVE the 0.5 ml mark in these vials so the needle will sample the liquid. The MSF has low-volume inserts that allow as little as 30 ul of liquid to be at a sufficient depth for analysis. Logging Sample into Online Billing System This part of the procedure can be completed from any computer with WWW access. First, browse to the MSF website, Then, click the blue icon in the center of the page labeled Online Submission Enter the sample submission system with your IU username and passphrase. Submit a new job by clicking the pink Submit a New Job icon. 6

7 Fill out the information on the top of the page. Enter in a sample name (should be what is on the vial), Faculty research group (from drop-down menu), and account number. Select the appropriate experiment type. For the Agilent 1200/6130, it is one of the two boxes farthest to the right. Select Self-run ESI for loop injections (less than 3 minutes, no HPLC column) analyses Select Self-run LC-MS for HPLC-MS analyses (longer than 3 minutes, requiring use of an LC column) 7

8 Select an ion type (Positive is the default, reserve Negative ionization analyses for extremely acidic molecules) Select the number of samples you intend to analyze in this batch (up to 39) and click the I Ran This Myself button. If all the steps above were followed correctly, you should end up at a screen that looks similar to this one: 8

9 The Sample Number (16828 in this case) is needed for the next step, be sure to record it somewhere. Multiple Samples on the Same Online Number Warning The online billing system treats all samples with the same Sample Number identically. Thus, if one had three LC-MS and three loop injections to perform, it is suggested that two separate online entries be made, one for the three LC-MS samples and the other for the three ESI loop samples. Performing an Analysis Using Easy-Access New users must have an Easy-Access login created by MSF staff prior to submitting a sample. Contact Dr. Karty (jkarty@indiana.edu) or Angie (asorg@indiana.edu) to set up an account. Entering Sample Data into Easy-Access Move the mouse to deactivate the screen-saver. Look for the Agilent Easy-Access window (screen-shot below). 9

10 If the Easy-Access window is hidden, try clicking on its tab in the taskbar along the bottom of the screen. If Easy-Access is not running, contact an MSF staff member Click the Submit New Sample button and the following window will appear. Enter your User Name and number of samples you intend to analyze into the appropriate fields then click OK. 10

11 Important sample entry notes: Fill out the table that comes up with all the appropriate information. Notice that the same Database # can be used for multiple samples (assuming you specified multiple samples in the online database in the previous section). Methods are selected using the drop-down menu inside the table. The # inj field indicates the number of replicates to be run from a vial (e.g. 3 for a triplicate quantitation experiment). The value is almost always set to 1. The sample name is used to generate the data file name. Make sure it is unique for each sample. There are several predefined methods that will satisfy the needs of most users. One can always view the experimental conditions for a given method in the Method and Run Control window. A more detailed discussion may be found in the Method and Run Control Window section of this document. Additional methods may be created at the request of users. The default injection volume should be used unless you feel there is a specific need to change it. Injection volumes can range from 1 µl to 100 µl. For example, a dilute sample may require a 50 µl injection as opposed to the 3 µl or 10 µl default volumes. 11

12 The UV(nm) field allows the user to specify a wavelength besides 254 nm to be monitored throughout the run. The absorbance trace from this wavelength will appear as Channel A when the data are analyzed (the 254 nm is channel B). Advanced users writing their own methods can specify other wavelengths to be monitored as chromatograms. The Description field MUST be filled in for a sample to be accepted. Click the Load Samples button when finished entering data. All data must be entered in within 8 minutes or Easy-Access will time-out and reset to its login screen. Loading Vials into the Autosampler Easy-Access will indicate the locations you are to put your samples. The positions in the autosampler tray will appear on the left-most column of the table and will be indicated by green dots in the picture on the left side of the image. The autosampler tray can be removed by lifting up and towards you. Be sure to put the vials into the positions indicated on the table. The tray should be put into the autosampler by snapping it into place. 12

13 Delaying Long Analyses If the method specified takes longer than 10 minutes, please check the Delay sample runs until after 8:00 pm box. The instrument will start these long data runs after 8:00 pm. Click the OK box when the autosampler tray is back in place with your samples in the correct positions. The yellow dots in the picture indicate samples that have already been completed; the blue dot represents the sample currently being analyzed, while the green dots correspond to samples still in the queue. Pink dots represent samples whose analyses failed for one reason or another (e.g. missing vial, power failure, etc.). Sample Disposal Warning: Unless MSF staff is asked to save them, all samples will be thrown away at our convenience. Let us know if your sample needs to be saved!! Easy-Access will send you an when it completes a batch, thus saving the user multiple trips back to A454 to find out the status of their sample. The instrument will put itself into standby mode six minutes after the last analysis is completed. Basic Data Analysis in LC/MSD ChemStation The MSF has 10 licenses of LC/MSD ChemStation for data review available for various research groups to install on their own computers. See Dr. Karty for the CD. 13

14 Data can be reviewed in A454 on the LC-MS computer (always let someone log a sample into Easy Access, however) or on a separate data review computer found to the left of the LC-MS computer, or remotely. A copy of ChemStation is installed on the computer to the left of the LC-MS computer in A454 and it always has the LC-MS data drive mounted as drive Q:\. Remote Access to LC-MS Data: Mapping a Network Drive The data directory on the 1200/6130 s computer is shared on the IUB network. The MSF suggests mapping the data drive over the network as follows: Make sure you are on a PC attached to IU s network either directly (if plugged into a wall jack on campus) or via IU s VPN. Pull up the Run option from the start bar. Type \\bl-chem-msf6130 in the dialog box that pops up. 14

15 You will then see the available resources on the LC-MS computer. Right click on the 6130_Data icon and select Map Network Drive. Select a drive letter (e.g. Q:\ for quadrupole or L:\ for LC-MS) and leave the Reconnect at logon box unchecked (unless you really need to save the 20 seconds it takes to redo this step each time your computer reboots). 15

16 The LC-MS computer s data hard drive will now have the letter you assigned it. The data are found in the Q:\EZXDATA\username directory (assuming Q was the assigned drive letter). Data for user mousemm is found in Q:\EZXDATA\mousemm. In the Q:\EZXDATA\username directory, the data are further sorted into directories based on the month and year the data were recorded (e.g for data recorded in October 2008). These folders are automatically created by Easy-Access. The data can either be copied to your local machine or you can let ChemStation access the files over the network (the latter option can be SLOW at times). The examples that follow assume the all data were copied into the C:\Chem32\1\EZXDATA\jkarty directory. Opening a Data File in ChemStation Make sure the offline copy (data review only) of ChemStation is open. If not, open if from the Start Bar. (Note: on the data review computer in A454, it is called Instrument 2 offline). 16

17 The ChemStation window will then appear in <30 seconds. 17

18 Make sure the Data Analysis tab is selected. If not, just click it so your screen is similar to the one shown above. Open your folder by double clicking on the appropriate icons in the data analysis tab on the left (jkarty/10-08 in the example below). The data are found in the Single Runs section under the appropriate month s folder. Alternatively, one can open a file by selecting Load Signal from the File menu. If the directory containing your data is not shown on the left, it can be added by selecting Preferences from the View menu. The directory is then added in the Paths tab. 18

19 Basic ChemStation Navigation The explanations that follow are all based the picture found below. Along the top, center of the frame is the navigation tool bar. It contains a list of available data files. The table can be sorted by any column heading. Click on the heading you wish to sort by (smart tip: sort by Date Time to find the newest files). Immediately below the data file descriptions is the Workspace Task Bar. This bar has four main choices which will be discussed a little further down. It has a drop-down menu that allows one to select which chromatogram(s) are shown in the window below. One can either select a single chromatogram or view all the chromatograms in the file. Below the Workspace Task Bar is a view of the individual chromatograms. A description of the data displayed in each window shown along the top (e.g. the middle trace is from the DAD detector and shows the 254 nm with a bandwidth of 4 nm). Additional chromatograms can be seen by using the slider bar to the right of the chromatogram windows. It should be noted that a brief description of almost any icon or toolbar can be obtained by moving the cursor over it and waiting a couple of seconds. The description will then appear in yellow next to the cursor. 19

20 Printing Data Views in ChemStation Printing works the same way no matter what task view is currently visible. The printing options are accessible from the File menu (see image below). Printer Setup allows one to select a printer and determine whether the document is printed in landscape or portrait orientation. Print Preview only works with reports, so it will be ignored in this manual. Most users will be able to ignore the Report printouts (the first several selections). These are often used with quantitative data using automatic integration and calibration routines that can be discussed with Dr. Karty. The two most-often used choices are Selected Window and All Windows The Print Selected Window option will print the highlighted view in a single window, and it will fill the entire page. It is suggested that you always click somewhere in the window you wish to print immediately prior to printing to ensure you get the printout desired. If no window was previously selected, a dialog box will appear inviting the user to select which particular open window will be printed. In the example below, there are three different windows open, a windows with two chromatograms, a window with a UV-VIS spectrum, and a window with a mass spectrum. If a printout of the mass spectrum was 20

21 the only thing wanted, you should select the third option, 79, Apex Mass Spectrum and this will be printed. The Integration Task This task bar selection allows one to control the parameters used in the integration of a chromatogram (i.e. the detection of chromatographic peaks and the calculations of their areas). This will be covered in a separate document on Advanced ChemStation Techniques. The Calibration Task This task bar selection works in tandem with the integration task to convert peak areas into concentrations and or amounts of specific analyte compounds. Again, this will be covered in the forthcoming Advanced ChemStation Techniques document. The Signal Task This selection is a powerful tool for creating views suitable for inclusion in a notebook or publication. The next few slides start with the following dataset. 21

22 One can see the display is a little cluttered with 4 different chromatogram traces. We can use the Delete Object from Chromatogram Display tool to remove some of them. This tool is 5 th from the right and looks like a chromatogram with a large X through it. The Delete Object from Chromatogram Display tool changes the pointer and now when the left mouse button is clicked, the chromatogram under the pointer will be deleted. Click anywhere in the chromatogram you wish to remove from view. The Delete Object from Chromatogram Display tool was used to remove the middle two traces from the display to yield the screen below. 22

23 Notice how the numbers are now much easier to read. If you need to reload a deleted signal, you will have to reload the entire data file as described above. The traces removed with this tool are still stored with the original data file. Simply reload the file to recover the hidden traces. An alternative method for selecting a single trace involves using the drop-down box in the center of the Signal Task Icon bar. Click on the drop-down box and you will see a list of available chromatograms. All Loaded Signals will display all chromatograms that are currently loaded (excluding one deleted using Delete Objects icon described above. Selecting any single trace will fill the window with ONLY that trace. The screen-shot below shows what happens when the MSD2 trace is selected from this drop-down menu. Notice how only one trace is visible. This tool works the same way in all Task views (Integration, Calibration, Signal, and Spectrum). 23

24 The two magnifying glass icons are relatively self-explanatory. They allow you to narrow or expand the X and Y axes displayed in a given window. Note, the Y-axes of the separate traces are somewhat linked, that is, if you double the vertical scale on one trace, the same doubling of the vertical scale will be seen in all other traces on the screen. A similar effect is seen with the X-axes. This feature can be deactivated if the Zoom Separate box is checked in the Graphics Signal Options dialog box. Similarly, the Signal Options dialog can be used to directly specify X and Y axis dimensions visible on the screen. The Graphics Signal Options dialog also allows one to specify the font, size, and rotation of the labels on the screen. The default is 8 pt, Arial, and

25 The image below shows the same data with the font set to 9 pt Arial with 0 rotation. The last two icons in the Signal task described in this document concern whether chromatograms are overlaid or displayed in separate windows. These two icons look like 3 colored balls either all in one box or in three separate boxes. 25

26 If you look at all the previous screenshots, you should notice that that the icon with the three balls in separate boxes has been depressed. This is the Display Separate Signals icon. Each data trace appears in its own window when this icon is selected. The icon with the three balls in the same windows is labeled Display Overlay of Signals. When this icon is selected, all loaded chromatograms are displayed in a single window with a common X-axis. The vertical scales can be specified using the Signal Options dialog box described earlier. The image below is overlays the DAD and TIC traces shown in the previous picture. The UV-VIS trace is in blue, the MS trace is in red. Notice the ~0.1 minute shift in the retention times due to the tubing between the DAD and the MSD. The overlay icons are ONLY available from the Signal Task window. Spectrum Task The spectrum task allows one to extract either mass spectrum or UV-VIS spectrum data from the chromatographic display. These spectra then appear in their own windows below the chromatograms. A view of the icons available in the Spectrum Task is below. 26

27 When working with UV-VIS and mass spectra, always select the mass spectrum FIRST. For some reason, ChemStation will delete the selected UV-VIS spectrum when a new mass spectrum is selected. A note about reference spectra in this view: MSF management may cover this feature in the ChemStation for Advanced Users document. It is suggested that novice users set these options to No Reference. The magnifying glass icons work the same as in the Signal Task. The most important icons in this view are three in the middle (the peak with a single arrow at a 45 degree angle, the peak with an arrow pointed downward, and the peak with a double-ended horizontal arrow). The icon with the arrow at a 45 degree angle is the Select Spectrum At Any Time Position tool. It allows the user to select a single mass spectrum anywhere in the chromatogram, regardless of whether or not the ChemStation Integrator indicates a peak. Do not click and drag across a region, or an overlay showing each spectrum in the region selected will appear. 27

28 This is the initial output from clicking the cursor at a point somewhere in the minute peak in the lower (mass spec TIC) trace. It shows a dominant (likely M+H), with a likely 13 C 557.3, and a probably M+Na (mass shifts of 22 often indicate sodium adducts). 28

29 This is the view generated after selecting a single point somewhere in the min peak in the upper (DAD) trace. Note how two new windows; the one labeled Original Spectrum(a) can be deleted as it relates to the referencing features that will NOT be covered in this document. Also, notice the title bars in each of the windows. These tell you where the data in a particular trace came from. For example, the DAD1 window title indicates that it is DAD data from retention time minutes and the maximum absorbance is 264 mau (mau = milliabsorance units or AU); the file from whence the data were taken is also indicated. The new windows can be resized (as these have been) for your convenience. Simply move the cursor to the edge of the window in question, then click and drag it to resize. The cursor will change to what looks like a double-headed arrow indicating which directions you can resize the window. Note, that when working with a file over a network (e.g. working from your office on a file still on the MSD s computer), that there can be a noticeable and frustrating delay when resizing windows. If your network connection is slow, copy the files to your local computer prior to analyzing the data. 29

30 The second spectrum selection icon (the one with the vertical arrow over a pair of peaks) is the Select Spectrum at Apex Position tool. It will show the data at the top (apex) of an integrated peak. This tool only works with data that have been processed with the integration tab. The most often used tool is the icon in the middle of the Spectrum task tray and it has a peak with a Greek letter Σ on it. This is the Average a Selected Set of Spectra tool. It allows a user to average a series of spectra together. Averaging spectra often improves the signal to noise ratio. The resulting spectrum is the average of all the scans selected. It works in either the DAD or MSD traces. The view above shows the average spectra taken by selecting the Average a Selected Set of Spectra tool, clicking and dragging across the minute peak in the TIC trace and the minute peak in the DAD1 trace. Note how the title bars of the two spectra indicate the range of retention times averaged ( min for the DAD trace) to generate the image. 30

31 Quantitation in ChemStation Quantitation in ChemStation can be performed using either DAD or MSD data. Again it is imperative that only a single wavelength or m/z ratio is used for quanititation to avoid errors caused by background noise or co-eluting species. Quantitation can be difficult to set up at first, but easy once a method has been optimized. Be sure to discuss any quantitation experiments with Dr. Karty if you are having difficulties. Extracting a Single m/z Chromatogram Above is a typical TIC and the extracted mass spectrum from the minute peak. Often, one wants to see when a particular ion was eluting from the column (not just when any ion came out), thus one can make what is called an extracted ion chromatogram (EIC). To do this, pick the desired mass (remember to include the ionizing H+ or Na+), then select Extract Ions from the File menu. 31

32 The following dialog box will appear You can then enter in masses where it says Ion 1. The drop box lets you select which TIC is used for the extraction. You can extract multiple ions by appending lines to the table. A new trace for each line is created. If an ions are put into both Ion 1 and Ion 2 in a row, the resulting chromatogram is the SUM of those two ions intensities as a function of time. The figure below is the EIC for from the MSD1 TIC. 32

33 The ion in question only came out in the 5.09 minute peak and nowhere else. Method and Run Control Window Coming soon Troubleshooting and Obtaining Help As stated before, MSD ChemStation has a rather complete Help file. Just select Help Topics from the Help menu along the top of the screen. Most answers can be found there. If the software does something strange or appears to be locked up, you can always Dr. Karty (jkarty@indiana.edu) or call him using one of the numbers on the chalkboard in A454. You can also a screen shot so Dr. Karty can see the error. Push the Print Screen key on the keyboard. This puts of a picture of the current screen into the Windows Clipboard. You can then paste the image into Word, PowerPoint, or directly into an . The MSD computer is on the web and you should be able to access IU WebMail to send Dr. Karty a screenshot. Please inform Dr. Karty or Angela if there is significant background contamination signal in your runs. This often arises from portions of previous samples coating the inside of the source (e.g. an intense ion at m/z 242 is most likely tetrabutylammonim ion from TBAF cleavage reactions). MSF staff will clean the source to remove the contamination as soon as possible. 33