Wednesday, December 29, 2010
Screening and Recruitment of Study Subjects in Clinical Research
Screening and Recruitment of Study Subjects
It is important that the Investigator resolves all questions from his/her staff concerning the interpretation of inclusion/exclusion criteria.
The Investigator should be able to dedicate time to the recruitment of suitable trial subjects – the consultation time for recruitment of each subject is likely to be longer than the time required for normal consultation.
The Investigator must ensure the unbiased selection of an adequate number of suitable study subjects as defined by the Protocol.
The Investigator must allow study subjects who meet the inclusion criteria the opportunity to decide for themselves whether or not to be entered into the study.
The Investigator must document the identification of subjects who entered trial screening by completing a subject screening/enrolment log.
Obtain Informed Consent from All Trial Subjects
The concept of obtaining informed consent is considered to be the heart of GCP. Informed consent is the process by which a study subject voluntarily confirms his/her willingness to participate in the trial. Only study subjects who have fully understood all aspects of their participation in the trial can make proper judgements and give their consent to participate in the trial.
Information on disease prevention and transmission must be provided to the study subjects for the whole of the trial period. Before any subject enters a trial, and before any study-related procedures begin, written informed consent must be obtained from the subject and/or his/her legally acceptable representative. In the case of a screening test which requires biological specimens to be collected prior to entering a trial, two types of consent form must be obtained, one for biological specimen collection and analysis, and the other for participation in the study after satisfactory laboratory results respecting the inclusion criteria have been obtained. Study subjects found ineligible at screening (for medical reasons) should receive supportive counselling, any necessary and available treatment and referral for continued counselling.
The Investigator can delegate the consent process to an appropriately qualified person; however, the Investigator should see the subject afterwards to ensure that the consent has been properly
obtained. Verbal and written information given to the trial subject should be in simple terms and in his/her first language. Medical terms should be avoided.
The Investigator/designated person should perform informed consent procedures fully with each subject during recruitment:
Ø The informed consent form should be personally dated and signed by the trial subject and/or his/her legally acceptable representative as well as the Investigator/designated person responsible for the informed consent procedures.
Ø If the study subject and/or legally acceptable representative is (are) unable to read, an impartial witness for the Investigator should be present during the entire informed consent discussion. After oral approval by the study subject and/or legally acceptable representative, the witness must sign and personally date the informed consent form and attest that the information was accurately explained and apparently understood, and that informed consent was given freely by the subject and/or legally acceptable representative. The subject and/or legally acceptable representative should personally sign and date the form if capable of doing so.
Ø The study subject and/or legally acceptable representative should be given a copy of the signed and dated informed consent form and any other written information.
Ø The original signed and dated informed consent form should be kept in the Investigator's File with the study subject’s data.
Trial subjects and/or their legally acceptable representatives should be kept informed throughout the trial of any new findings or information about the tested product which might be of consequence to their participation in the trial. They should receive updates of the signed and dated consent form as well as copies of any amendments to the written information. Updates of the original signed and dated consent form should be kept in the Investigator's File.
Study Subjects Data and Documents For Clinical Research
Study Subjects Data and Documents For Clinical Research
Ø All signed and dated informed consent forms (for enrolled and screened subjects).
Ø Study subject screening and/or enrolment log.
Ø Study subject identification list.
Ø Copy of all Case Report Forms (CRFs).
Ø Copy of the Serious Adverse Event form.
Ø Copy of documentation of CRF corrections.
Ø All study subjects source documents including laboratory results.
Ø Copy of all subjects CRFs retrieval certificate.
During the clinical research studies
The trial can be initiated (begin screening and/or enrolment of trial subjects) only after the Clinical Monitor has satisfactorily conducted a Trial Initiation Monitoring Visit and the TDR Clinical Coordinator has given written authorization.
Investigator’s File, Including Storage and Retention
On initiation of the study, the Investigator must prepare a file containing documents related to the trial. During the study, the Investigator is responsible for updating the File and regularly adding trial-related documents.
The Investigator should keep the File in a locked cabinet, in a secure area accessible only to the Investigator and authorized study staff. The Investigator File and associated source documents should be retained for the time agreed with TDR/sponsors. Patient identification codes should be kept for at least 15 years after completion of the trial. Written approval from sponsors must be obtained prior to destroying records.
The Investigator's File contains:
Ø Administrative and Regulatory Documents
Ø Composition of IEC/IRB.
Ø Local regulatory requirements.
Ø IEC/IRB and other authorities’ written approval for all documents (protocol, informed consent(s) and any written information including advertisements for recruitment of study subjects).
Ø IEC/IRB and other authorities’ written approval for protocol amendments.
Ø Correspondence with the Ethics Committee and the Authorities, including:
· Protocol submission.
· Amendment submission, if any.
· Protocol modification notification, if any.
· Interim report/written summaries of the trial, if applicable.
· Final Report/written summaries of the trial, if applicable.
· Product importation authorization.
Ø Correspondence about product importation.
Ø For studies under IND, a copy of the completed and signed Form FDA 1572.
Ø Investigator’ s and Co/Sub-investigators’ C.V.s
Ø New Investigator and Sub-investigators’ C.V.s, if appropriate.
Ø Authorized Staff Form (ASF).
Ø Technical Services Agreement (TSA) signed/dated by both parties.
Ø Signed confidentiality agreement.
Ø Signed agreement stating that products will not be used before the Trial Initiation
Ø Monitoring Visit has been made and approval from the TDR Clinical Coordinator obtained.
Ø Copy of the insurance certificate/other insurance.
Ø ICH GCP guideline.
Ø TDR/TDP investigator’ s SOPs.
Ø Study Archiving Form (copy).
Ø Copy of the Investigator's interim report/written summaries of the trial to the IEC/IRB and authorities, if applicable.
Ø Copy of the Investigator's final report/written summaries of the trial to the IEC/IRB and authorities, if applicable.
Correspondence and Monitoring
Ø Correspondence with TDR/sponsoring agencies (including the telephone call, E-mail etc).
Ø Notes of meetings with TDR/sponsoring agencies.
Ø Summary list of site visits (copy).
Ø Trial Initiation Monitoring Report (copy).
Ø Notification by Investigator to TDR/Sponsor of serious adverse event and related reports.
Ø Documentation of serious adverse event reporting by TDR/Sponsor to other investigators.
Ø Correspondence about important requests.
Ø Investigator interim report/summaries of the trial for TDR/sponsoring agencies, if applicable.
Ø Investigator final report/summary of the trial for TDR/sponsoring agencies, if applicable.
Trial Documents
Ø General documents
Ø Investigator’ s brochure, with updates, if any
Ø Approved protocol and amendments, signed and dated by the Investigator(s) and sponsoring agencies, and new protocol amendments, if any.
Ø Approved informed consent and any other written information including all translations, and advertisements for recruitment of study subjects.
Ø Informed consent procedure.
Ø Clinical Trial Final Report (if available during the Study Closeout Visit).
Data reporting
Ø Blank CRF.
Ø Blank SAE/UAE forms.
Ø Blank source document if not existing on site.
Ø Case Report Form completion procedure.
Ø Adverse event reporting procedure.
Ø Blank screening and enrolment log.
Product
Ø Product certificate/batch release.
Ø Certificate of extension of the batch expiry date, if applicable.
Ø Dispatch notes (original) and acknowledgement of receipt (copy) (in case of new delivery).
Ø Randomization list/envelope or acknowledgement of receipt.
Ø Randomization list/envelope or randomization list/envelope retrieval certificate.
Ø Code Breaking list/randomization envelope retrieval certificate.
Ø Subject assignment list.
Ø Product management procedure.
Ø Product exportation/importation authorization
Ø Product accountability log.
Ø Product management form.
Ø Return of unused products form, or product destruction certificate if destroyed on site.
Ø Temperature recording log, if appropriate (especially for vaccines/biologicals).
Ø Other products-related trial documents.
Laboratory specimens.
Ø Laboratory certification/normal ranges/update of normal values.
Ø Reactive dispatch note and acknowledgement of receipt.
Ø Specimen management procedures (collection, performing assay, storage, results).
Ø Subject specimen collection log.
Ø Shipment note, if appropriate.
Ø Temperature recording log, if appropriate (deep frozen samples).
Ø Record of retained laboratory specimens, if any, to document the location and identification of retained specimens if assays need to be repeated.
Ø Other laboratory specimen related trial documents.
Ø Specimen management procedures (collection, performing assay, storage, results).
Ø Subject specimen collection log.
Ø Shipment note, if appropriate.
Ø Temperature recording log, if appropriate (deep frozen samples).
Ø Record of retained laboratory specimens, if any, to document the location and identification of retained specimens if assays need to be repeated.
Ø Other laboratory specimen related trial documents.
Trial supplies/equipment
Ø Material/equipment dispatch note (original) and acknowledgement of receipt (copy).
Ø Return of trial material/equipment certificate (copy).
Ø Trial document dispatch note (original) and acknowledgement of receipt (copy) (CRFs, all trial logs).
Ø Return of trial document certificate (copy).
Probe Tuning :NMR
Probe Tuning :NMR
Probe tuning must be matched to the sample in order to minimize pulse widths and maximize sensitivity. Although the effects are small for routine proton spectra, they can be dramatic for more complex pulse sequences. Although the tuning changes between different organic solvents usually do not justify the effort to tune, if you are changing from an organic solvent to water or D2O, retuning the probe is usually worthwhile. If you are starting a long experiment, you will want to check the probe tuning.
The wobb command allows you to tune and match a probe in an easy way even when the coil is heavily mistuned and mismatched. First copy the parameters for the nucleus you wish to observe.
Now enter the acquisition window by clicking Acquire/Observe fid window and start the wobble routine by clicking on Acquire/Acquisition parameter setup/Tune probehead. Alternatively enter acqu and then wobb via the keyboard. The acquisition is started and after a few seconds the wobble curve is displayed and refreshed continuously. A vertical line is drawn at the center frequency
(SFOx) to provide optical information on the frequency which is to be tuned. The horizontal axis of the coordinate system is scaled in MHz and labeled accordingly. Useful information like nucleus, tuning frequency, frequency of the minimum of the wobble curve and wobble width, is displayed in the information window. Simultaneously the LED display on the preamplifier is set and refreshed accordingly. The wobble curve shows a dip downwards which changes while you turn the tune or match knobs of the probe. Example displays in Figure.
1. Turn the tune knob (tune sliders on the indirect probe) so that the dip moves towards the center of the screen. Keep on turning until the dip is exactly in the center across the vertical line.
2. Turn the match knob (match sliders on the indirect probe) so that the dip becomes deeper. Keep on turning until the base of the dip is at a minimum. This occurs at the zero level line for most probes.
3. On most probes the matching influences the tuning and vice versa, so repeat steps 1 and 2 until the dip is exactly in the center of the screen and its base at minimum level. Then the probe is tuned and matched.
When you are finished with tuning and matching stop wobb by activating the stop button or by entering stop via the keyboard.
Saturday, December 25, 2010
Prepare the required documents to be submitted to the IEC/IRB: according to ICH
Prepare the required documents to be submitted to the IEC/IRB:
Documents usually required by Ethics Committees
· Investigator Brochure and up-to-date safety information.
· Trial protocol (final version and amendments).
· Consent form(s) and subject information sheets.
· Subject recruitment procedures (e.g. advertisement
· Information on payment and compensation available to subjects.
· Current curriculum vitae for each investigator.
· Any other document requested by the IEC/IRB.
See ICH Guidelines 3.1.2
Obtain approval document from the Ethics Committee, which must identify the documents reviewed and state that the study is acceptable and can be initiated.
Send the approval document of the Ethics Committee, with a list of Committee members, to TDP/TDR/WHO as a supporting document for approval of the WHO Secretariat
Committee on Research Involving Human Subjects (SCRIHS).Prepare the application for Health Authority clearance in collaboration with TDR and other sponsoring agencies.
Prepare the application for product exportation/importation in collaboration with TDR and other sponsoring agencies.
If the IEC/IRB and others approve the trial, sign the final copy of the protocol and confirm in writing that he/she has read and understood, and will adhere to, the protocol, study procedures and ICH Good Clinical Practice, will collaborate with the monitor, and accords with TDR/sponsoring agencies on publications policy.
Submit requested documents to the Clinical Monitors, including:
1. Signed agreement to comply with this SOP (page 1).
2. Approved protocol, signed and dated.
3. Approved informed consent form and other subject information, advertisement (local language and English translation).
4. Investigator’ s and co-investigator’ s curriculum vitae (C.V.s).
5. Authorized Staff Form (ASF).
6. Product exportation/importation authorization.
7. Laboratory certification/list of normal laboratory ranges, dated and signed by Investigator.
8. Technical services agreement (TSA), signed and dated.
9. Signed agreement that the product will not be used before the Trial Initiation Monitoring Visit has been made and authorization obtained from the TDR Clinical Coordinator (if applicable).
10. Signed FDA 1572 form (if applicable, e.g. study under Investigational New Drug - IND).
Make sure that the local ethics committee fulfils the ICH GCP requirements. ICH GCP Composition and Operations of IEC/IRB
Make sure that the local ethics committee fulfils the ICH GCP requirements.
ICH GCP Composition and Operations of IEC/IRB
The IRB/IEC should determine the authority under which it is established and the composition (names and qualifications)of its members, which should consist of:
Ø A reasonable number of members who collectively have the qualifications and experience to review and evaluate the science, medical aspects and ethics of the proposed trial.
Ø At least five members.
Ø At least one member whose primary interest is in a non-scientific area.
Ø At least one member who is independent of the trial site.
An IEC/IRB may invite nonmembers with expertise in special areas to give assistance.
The Investigator may provide information on any aspect of the trial, but may not participate in the IEC/IRB deliberations, vote, or provide opinion.
Only members who participate in review and discussion of the protocol, and who are independent of the investigator and the sponsor, can vote or provide opinion.
The IEC/IRB should perform initial and continual reviews of the trials according to the written operating procedures, and maintain records of activities and minutes of meetings.
The IEC/IRB should notify promptly, and in writing, all trial-related decisions and opinions, specifying the reasons for each.
See ICH Guidelines 3.2
PRIOR TO INITIATION OF THE CLINICAL RESEARCH STUDY
PRIOR TO INITIATION OF THE CLINICAL RESEARCH STUDY
The Investigator should:
Be interested in the scientific aspects of the study and ensure that the study is responsive to the needs of public health within the country of the population in which it will be conducted.
Ensure the confidentiality of the product, the protocol and trial procedures by giving a confidentiality agreement in writing to TDP/TDR and/or sponsoring agencies.
Have sufficient time free from other obligations to prepare and conduct the trial. Clinical trials are time consuming and the Investigator should ensure that sufficient time can be dedicated to the study, including for informing and supervising study staff.
Review Investigator’s Brochure and any up-to-date information on the investigational product. The Investigator must be familiar with the product, including pre-clinical toxicology, pharmacology, pharmacokinetics and up-to-date clinical data.
Review and discuss in detail, the ICH GCP guidelines, investigators' SOPs and protocol with the Clinical Monitor. The Investigator should clearly define:
1. Factors that may alter the feasibility and acceptability of the trial.
2. An adequate recruitment rate for the trial by providing retrospective data on numbers of patients who would have satisfied the proposed entrance criteria during preceding time periods.
Make sure that the procedures stated in the study protocol are applicable in his/her centre and fully understood. The Investigator should ask the Clinical Monitor to clarify any points of possible misunderstanding.
Make sure that there are sufficient medical, paramedical and clerical staff to support the study and deal with foreseeable emergencies.
1. Provide a list of study personnel and functions in the study to the Clinical Monitor/Product Manager (Authorized Staff Form - ASF).
2. Provide curriculum vitae of the Sub-co Investigators in the responsible laboratory.
Make sure that the physical location and facilities are sufficient to allow the study to be undertaken efficiently. Ensure:
1. Confidentiality and safety conditions for trial subjects.
2. Adequate equipment/facilities for subject follow-up, examination and care.
3. Adequate facilities for product storage.
4. Adequate facilities for laboratory assay. The laboratory assay should be validated under good laboratory practice (GLP) principles.
5. Adequate facilities for retention of trial documents, ensuring confidentiality of all information about trial subjects and information supplied by TDR/sponsoring agencies.
Discuss the Case Report Form/SAE reporting forms and source documents in detail with the Clinical Monitor. Clearly define :
1. Who will be responsible for CRF completion.
2. Source documents/source data and access to source data.
Arrange archiving of trial documents according to GCP and regulatory requirements. It is important to check the duration of retention of patient records with the Institution's archive. In case the Institution's archive does not ensure retention of documents for the period of time requested by TDR/sponsor, the Investigator must arrange for the retention of the subjects’ source documents/records for the period requested by TDR/sponsor and regulatory requirements.
Finalize informed consent forms and associated trial subject information materials (advertisements); establish procedures for application for local clearance (e.g. dean of the institution) and Independent Ethical Committee (IEC)/Institutional Review Board (IRB) approval.
Clearly define how subjects will be approached and informed, who will inform them, and what material will be used. The informed consent form and all information (leaflet written in simple language, video) should be developed collaboratively with head members of the study population/ community to ensure methods are appropriate.
In case of the need for screening tests, including biological specimen collection, before entering a trial, two types of consent form should be developed: one for biological specimen collection and analysis, and one for participation in the study after obtaining satisfactory laboratory results and respecting inclusion criteria.
As a rule, the advertisement must not make reference to TDR or the compound, nor make any claims. Informed consent and advertisements must be submitted to TDR for review and must be included in documentation submitted to the Independent Ethical Committee and/or Institutional Review Board (IEC/IRB).
Operating procedure for NMR spectrometer : Bruker Avance DPX-300 NMR spectrometer
Note:
Unless otherwise specified, statements such as “click on calib” mean to move the cursor on top of the calib button on the screen, and click with the left-hand mouse button. Consider the left-hand mouse button the default; if another button is required, it will be explicitly stated.
Commands such as “Enter zg” mean to type zg followed by a return on the
keyboard. Commands must be followed by <return> before they are accepted.
Words written in bold Arial font, e.g., acqu, refer to buttons or pull down menus on the screen (items that can be selected with the mouse). Multilevel menu choices written like this, Windows/lock, mean choose the first menu choice by left clicking with the mouse. Choose the second menu choice from the menu that appears when the first choice is made.
Words written in bold Courier font, e.g., edsp, refer to commands that can be typed at the keyboard. Commands must be followed by <return> before they are accepted.
General Procedures For All Spectra
A. This manual is a condensed and incomplete account of instrument usage. It is in no way a substitute for reading the detailed instruction manuals available in the NMR room (M-02). Please get to know them. This manual is written in a similar format to the Tufts AM-300 manual, to facilitate the transition from the AM to the DPX.
B. All users should sign the logbook before they do anything else.
C. Log into the computer. The screen may be illuminated by hitting any key on the keyboard. You should see a Welcome to IRIS window. Enter your user name and password. When the UNIX shell window appears, type xwinnmr. The XWIN-NMR window will appear, as shown below.
D. Only the active window will accept typing. To make window active, position the mouse cursor inside it. To bring a window to the front, left click in its title bar.
E. Most commands are accessible from the menu bar. Alternatively, any command may be typed into the command line below the spectrum window. Most commands are the same as the AM-300.
F. Parameters may be changed by typing the abbreviation for that parameter followed by the (return) key (hereafter <rtn>. The computer will respond by popping a window with the current value of that parameter. Typing the new value of the parameter followed <rtn>by will replace the old value with the new value. Alternatively, groups of parameters can be edited with various ed commands. For example, eda presents a window of all acquisition parameters, edp presents a window of all processing parameters.
G. Sample control is provided through the BSMS (Bruker Smart Magnet System) control panel. To access the BSMS Panel choose Windows/BSMS Panel from the menu. Lock, Shim, and Sample panels are similar in appearance to the hardware buttons on the AM-300, except control is through a slider bar and +/- button, rather than through the knob. For all buttons the left mouse button decreases the value, the middle button increases. Step size is changed by left (decrease) or right (increase)clicking on the :2/*2 button
2. Loading the Sample
From the XWIN-NMR menu choose Windows/BSMS panel. Then click on the Shim button
-Click on the LOCK button to unlock the instrument. The color of the button will change from yellow to gray
- Click on the SPIN button to stop the spinning of the sample The color of the button will change from yellow to gray
- Click on the LIFT button. The standard sample should rise out of the probe on a stream of air. Remove the standard sample from the top of the probe. Remove the standard from the spinner.
-Place your sample tube in the appropriately sized spinner and adjust the precise depth of the tube within the spinner by using the depth gauge. Note that the depth gauge should be set at 10mm regardless of the tube size for a 10 mm probe. (and at 5 mm gauge for a 5 mm probe). Note: 10 mm tubes cannot be run in a 5 mm probe. For best shimming set tube 1mm below the appropriate line to assure a uniform sample in the active region of the probe.
- place your sample in the top of the probe. Do not release the sample unless you are sure it is supported on the air current.
- Click on the LIFT button again. Your sample should slowly lower into the tube as the air stream decreases. Click on the SPIN button.
B. The rate of the sample spinning should be 20-25 rps. To check this:
-Press the Sample button on the BSMS panel. Then click on the SPIN MEAS button on the Sample panel that pops up. To change the spin rate, click on the SPIN RATE button, and usethe slider bar to change the value.
3. Locking the Instrument
B. To lock the sample, type lock, then click on the solvent from the list that pops up. Lock power, phase, and gain will be adjusted automatically.
C. The lock can also be adjusted manually, using the buttons on the Lock panel from the BSMS panel. Note: Appropriate power and gain levels are different from the AM-300.
- Click on the LOCK GAIN button. Use the +/- button to adjust the lock gain until the trace is in the top two squares of the grid. Press the Z button. Use the +/- button to maximize the lock signal. Change the step size if needed. Repeat for the Z2 button. Continue to alternate between the these buttons maximizing the lock signal each time until no further improvement can be seen. You may have to adjust the Lock Gain if the trace goes off the screen.
-If the sample must be shimmed to highest standards, adjust the non-spinning shims by pressing the SPIN button and waiting until the sample stops spinning. Spin rate can be used to monitor this. Raise the lock level by adjusting the lock gain until the level is in the upper half of the screen. Now press the X button and use the +/- button to adjust the lock level to maximum. Then press the Y button and repeat this procedure until no further improvement is possible. Start the sample spinning again by pressing the SPIN button and repeat the Z and Z2 shimming as above.
Running a 1H Spectrum
B. Data files containing typical parameters for a proton spectrum are stored in the hard disk for easy access. To retrieve these:
Choose File/Copy/parameters from file from the menu, or type rpar. A list of standard parameter files will appear. Use the scroll bar to access the entire list. Click on proton. In the next window that pops up, click on Copy All.
Type eda to edit acquisition parameters. Always change Prosol to True by clicking on its value, which loads all default pulse lengths and also check that the solvent is correctly identified, otherwise calibration will be off. IMPORTANT: Failure to set Prosol to True will result in all pulse lengths not set by you will be of zero length and rga will go off scale trying to find a signal.
Change any other acquisition parameters desired, then click on SAVE. Acquisition, Ones you might want to change are
TD (time domain size)-Defines the number of data points of the fid.
NS (number of scans)
P1 (pulse length in microseconds)
SW, SWH (sweep width in ppm, Hz)
AQ (acquisition time in seconds)
RG (receiver gain)
B. You may wish to tune the probe before beginning acquisition. You can see this on another post Probe tuning.
C. The last parameter to be set is the receiver gain (rg) which adjusts the gain of the signal coming out of the probe. This will vary from sample to sample. To set this gain type: rga (receiver gain adjust). The computer will automatically take a few sample pulses and alter the RG between each. When the best value is found, the computer will type rga: finished. You may see this value which the computer has selected by typing: rg. If you want this value then type: <rtn>. If you wish to select another number then type in the new number and then type <rtn>. Do not increase the value, but you may decrease it.
6. Acquiring the Spectrum
B. The acquisition will automatically end when the number of scans taken equals ns. However if you wish to stop it prematurely or abort the run then you will have to click on the STOP button to the left of the FID in the acquisition window.
7. Transforming the Data
B. The time domain data can be processed by typing bc, then em, then ft (or type ef to do all three at once).
C. It is not necessary to wait for the acquisition to end before transforming the data. Type tr to transfer the data to disk at any time during the acquisition. Once written to disk, the data can be transformed. Time domain (fid) and frequency domain (spectrum) data are always stored in separate files. All data acquired so for can be processed while the acquisition continues in the background.
8. Manipulating the spectrum
B. If the automatic phase correction does not work well, click on the phase button to the left of the spectrum, then click on biggest. A marker will appear under the biggest peak in the spectrum. Left click and hold the PH0 button. Move the mouse vertically until the largest peak is phased correctly. Release the mouse button and click and hold the PH1 button. . Move the mouse vertically until the other peaks are phased correctly. Release the mouse button.
10. Setting the Reference
11. Changing SW and O1 for Increased Resolution
The default parameters display the spectral range 16 to –4 ppm. This range can be changed for greater resolution. Click the left mouse button somewhere in the spectral window to tie the cursor to the spectrum. Position the cursor at the left limit of the desired spectral width. Click the middle mouse button to set a marker at this frequency. Move the cursor to the desired right hand limit and click the middle mouse button to expand the spectrum. The expanded region now appears in the window. Click the left mouse button to release the cursor from the spectrum. Click on sw-sfo1 while the expanded region is displayed. This adjusts sw so that it has the same value as the expanded region and also adjusts o1 (and thus sfo1) so that the carrier frequency lies in the center of the expanded region. (You can verify these changes by checking the eda table.) Notice that by reducing the spectral width, the acquisition time aq is increased while the parameter fidres is reduced. Finally, now that the acquisition parameters are optimized, it is a good idea to repeat the automatic receiver gain adjustment (rga). Notice that since the spectral width has been changed, it may be necessary to readjust the phase correction.
B. To display the integral on the screen:
Choose Analysis/Manual integration from the menu, or simply click on the integrate button of the button panel at the left side of the XWIN-NMR window. Additional buttons, like those that control vertical and horizontal scale for the spectrum, will appear in the lower left. The upper part of the button panel is identical to the standard layout, and allows you to shift and scale the integral data on screen. In addition, there are three special sections headed by current:, all:, and mouse:. The command buttons in these sections work on the current integral marked by the user, on all integrals on the screen, and on the mouse sensitivity, respectively.
Defining integration regions
Move the cursor into the data area of the XWIN-NMR window. Click the left mouse button. The cursor is now bound to the spectrum, and moves along the spectrum trace when you move the mouse. It can be released from there by clicking the left button again. Clicking the middle button will mark the current position (the mark can be removed using the right button). Clicking the middle button a second time at a different cursor position will define the area between the mark and the current cursor position as the integration region, and the corresponding integral trace is displayed along with the value of the area under the integral. By default, the first integral region defined will be assigned the value 1.0. This procedure can be continued for all desired regions, and need not proceed left to right. Click the left button to release the cursor from the spectrum when you are finished.
In order to mark one of the defined integrals as current integral, move the cursor into the data area of the XWIN-NMR window. Click the left mouse button. The cursor is now bound to the spectrum, and moves along the spectrum trace when you move the mouse. Select the integral you want to make the current integral by moving the cursor under it, and release the cursor by clicking the left button again. The integral will be marked with an asterisk. All button panel commands in the section current: can now be applied to this (and only this) integral.
To calibrate the integrals, mark an integral as the current one, then click on calibrate. Enter the integral value in the dialog box.
When all integral regions have been defined, click on return. Click on save as ‘intrng’ and return to save the regions to a disk file.
13. Peak Picking
The most straightforward way to produce a peak list is through the command line. First display the Y axis in cm, if it is not already displayed. The Y button to the left of the spectrum window will toggle the y-axis, the YU button will change the units from cm to absolute. Choose the region for peak picking by setting the parameters f1p and f2p to the ppm values for the left and right limit, respectively. (If the x-axis is in Hz, use f1, f2 instead of f1p, f2p.) Set the parameter mi to the desired minimum intensity in cm. Type edo and check that CURPRIN is set to hplj5l. The command pp will produce a peak listing on the printer. Use pps to print to the screen.
14. Plotting the Spectrum
A straightforward way to plot 1D spectra is by using most of the plotting parameters found in the plot parameter file standard1D. Read in the file by choosing File/Copy/parameters from file from the menu, selecting standard1D from the menu of parameter file names, and then selecting plot from the menu of parameter file types that appears. Then click on Copy. This sets the plotting parameters to values appropriate for most 1D spectra. 1D and 2D Plotting Parameters’. For basic 1D spectra no changes need to be made within the parameter menu edg itself; however, the spectral region and the integral range must be defined, and the spectrum title must be written. To select the spectral region (full or expanded) to be plotted, first make sure the spectrum appears as desired on the screen, and then click DP1and simply hit return in response to the following three (3) questions:
F1 = <return>
F2 = <return>
Change y-scaling on display according to PSCAL?<return>
You may change the values of F1 and F2 if you wish.
Other plot parameters can be changed by typing edg (edit graphics) and changing the values, mostly yes/no, which determine whether integrals, parameters, etc appear on the plot. The edg window is analogous to the DPO command on the AM-300. Some plot features have a button labeled ed. Clicking on this button will open another window, with parameters that control the position and style of these features. If simply editing the yes/no parameters in the edg dialog box will not give the plot you want, it is usually easier to use XWIN-PLOT, rather than to change other plot parameters. XWIN-PLOT is a graphical plot layout program.
Next create a title for the spectrum. Enter setti to use the editor to open the title file. Write a title and save the file. The title must end with a <return>.
To preview the plot, as it will appear on paper, type view.
To plot the spectrum, type plot (provided the correct plotter is selected in
edo).
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