1. No category

‫)‪(H-Class_setup_1-11-2012_Empower‬‬
‫‪Operating Waters’ H-Class UPLC in Empower‬‬
‫הכנת הממסים‬
‫החלקיקים בתוך קולונת ‪ UPLC‬הם קטנים בהרבה מאשר בקולונות ‪ ,HPLC‬וכן גם הפילטרים שלהן‪ ,‬לכן נא‬
‫לשים לב לטבלה הבאה‪:‬‬
‫‪Typical UPLC conditions:‬‬
‫‪• Flow rate: ~ 0.6 ml/mn‬‬
‫‪• Column I.D.: ~ 2 mm‬‬
‫‪• Inlet frit pore size: ~ 0.2 μ m‬‬
‫‪Typical HPLC conditions:‬‬
‫‪• Flow rate: ~ 1 ml/mn‬‬
‫‪• Column I.D.: ~ 4 mm‬‬
‫‪• Inlet frit pore size: ~ 2 μ m‬‬
‫לכן חלקיקים קטנים שלא מפריעים מיידית לעבודה ב‪ HPLC -‬עלולים להפריע לעבודה השוטפת ב ‪.UPLC‬‬
‫על כן‪:‬‬
‫יש לשים לב לדברים הבאים‪:‬‬
‫•‬
‫ממסים אולטרא‪-‬נקיים מתאימים ל ‪ UPLC‬והבופרים מסוננים בפילטר של ‪0.22μm‬‬
‫•‬
‫הבקבוקים חייבים להיות באיכות הגבוהה ביותר‪ :‬זכוכית ‪ TYPE 1‬או פיירקס‪ ,‬סגורים תמיד‪.‬‬
‫•‬
‫אין להשתמש בפאראפילם בכלל! )ראה תמונה( גם לא ב !!!‪HPLC‬‬
‫•‬
‫יש למנוע כניסת מזהמים ואבק מהאוויר על ידי שימוש בפילטרים על הפקקים )ראה בתמונות(‪.‬‬
‫בקבוקים סגורים עם פקקים‬
‫מומלץ להתקין על שולחן נמוך במיוחד כדי להגיע אל הממסים; ניתן להתקין מדף מיוחד עבורם כמו בתמונה‪.‬‬
‫‪Teflon corks with filters‬‬
‫‪High Quality Bottles‬‬
‫‪Sealing from dust‬‬
‫‪Low level shelf‬‬
‫‪UPLC grade solvents‬‬
‫‪Created by Dr. Shula Levin‬‬
‫‪12/5/2012‬‬
‫‪Page 1 of 32‬‬
‫‪DISCLAIMER:‬‬
‫!‪THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC‬‬
‫!‪IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING‬‬
‫‪http://www.forumsci.co.il/hplc/‬‬
Operating Waters’ H-Class UPLC in Empower
(H-Class_setup_1-11-2012_Empower)
Connecting the column
:‫הערה חשובה‬
!‫חשוב מאוד שהחיבורים יהיו מדויקים מאוד! כל חלל מיקרוסקופי בחיבורים גורם לעיוותים בצורת הפיקים‬
The column Thermal stabilizer and in-line filter are mandatory when Acquity columns are
used!
To connect the column in-line filter unit and the column inlet:
1. Remove the O-ring from the outlet end of the column in-line filter unit.
2. Remove the plug from the column inlet.
3. Push the outlet end of the column in-line filter unit into the column inlet until it stops.
4. Tighten the fitting manually until it is snug, and then add a quarter-turn.
5. Connect the eCord.
Page 2 of 32
12/5/2012
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
Operating Waters’ H-Class UPLC in Empower
(H-Class_setup_1-11-2012_Empower)
Remember: The Reusable Gold Compression Screw is adjustable. When an HPLC column
is used: Remove the in-line filter and connect the HPLC column directly to it.
All Connections of Nuts, guard columns or in-line filters should be perfect:
No voids! Bad connection will cause a bad peak shape:
:‫ וגם היא חייבת להיות מותקנת במדוייק‬In-line filter ‫ באה במקום ה‬GUARD ‫קולונת ה‬
0.040
0.030
AU
0.020
0.010
0.000
-0.010
5.605.705.805.906.006.106.206.306.40
Minutes
Caution: To avoid damaging the tubing, use only your fingers to tighten the adjustable fittings.
Various Connections
Page 3 of 32
12/5/2012
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
Operating Waters’ H-Class UPLC in Empower
Page 4 of 32
12/5/2012
(H-Class_setup_1-11-2012_Empower)
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
Operating Waters’ H-Class UPLC in Empower
(H-Class_setup_1-11-2012_Empower)
Connecting the eCord to the magnet will provide the following information: through the console->
Column:
The information is updated up to 50 lines only, so it should be printed periodically.
Page 5 of 32
12/5/2012
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
Operating Waters’ H-Class UPLC in Empower
(H-Class_setup_1-11-2012_Empower)
To connect the PEEK tubing from the column to the detector:
Flow out the solvent from the column before connecting it to the detector! Then attach the specific
tubing to the column outlet. Check the label on the flow cell inlet tubing to ensure it matches the
type of detector and flow-cell in your system.
Operating a light-guiding flow cell of the UV/PDA Detector
Light path in a light-guiding flow cell:
Caution: The operation and storage of a Waters® light-guiding flow cell is fundamentally
different than that of a conventional flow cell. A light-guiding flow cell requires careful
maintenance to ensure optimal performance.
General guidelines
… For extended storage, flush the flow cell with pure organic solvent and cap the inlet and
outlet ports.
Page 6 of 32
12/5/2012
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
Operating Waters’ H-Class UPLC in Empower
(H-Class_setup_1-11-2012_Empower)
… Do not power-on, operate, or ignite the lamp of the detector when there is no flow through
the cell, or when the flow cell is dry.
… Flush new columns for 15 minutes before connecting the flow cell, to minimize
contaminants that can leave deposits on the flow cell’s walls.
Shutdown considerations for light-guided flow cells
Less than 24 hours (more than 4
hours)
1. Continue to pump mobile phase through the
flow cell. You can reduce the flow rate to
conserve solvent.
2. Extinguish the detector lamp.
More than 24 hours
1. Extinguish the detector lamp.
2. Flush the flow cell with water.
3. Flush the flow cell with pure organic solvent
(such as methanol or acetonitrile).
4. Power off the system.
5. Cap the inlet and outlet ports of the flow cell to
preserve the cleanliness of the Teflon AF.
Page 7 of 32
12/5/2012
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
Operating Waters’ H-Class UPLC in Empower
(H-Class_setup_1-11-2012_Empower)
Starting up the system: Login-> Run Samples-> Select the project and the system and enter the
Run Samples screen:
Click on Console icon and the following window is opened: UPLC Console of the specific
system.
Each system component is controlled by the “Control” menu: Entire system’s control:
Pump (Solvent Manager):
Page 8 of 32
12/5/2012
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
Operating Waters’ H-Class UPLC in Empower
(H-Class_setup_1-11-2012_Empower)
Injector (Sample manager):
Detector:
:‫ לעבור דרך מים זמניים‬,HPLC ‫ כמו ב‬,‫המלצה לצעד ראשון כאשר משתמשים בבופרים בפאזה הנעה‬
‫כדאי לשטוף את הצינור של הבקבוק בו היה הבופר במים "זמניים" לפני שמעבירים אותו לבקבוק של הפאזה הנעה‬
.‫החדשה‬
Go to the Quaternary Solvent Manager and select: Control->Prime Solvents:
Page 9 of 32
12/5/2012
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
Operating Waters’ H-Class UPLC in Empower
(H-Class_setup_1-11-2012_Empower)
Example: the buffer was in both A and B: Transfer A and B to pure Water and start priming
them for 1 min with water, then transfer to the new buffers. Discard this water and use fresh
water for the washing reservoir.
Uncheck C and D if needed
Observe the flow coming out of the pump's waste.
USING THE SYSTEM STARTUP Tool
:‫ לבקבוק של הפאזה הנעה הטריה‬A, B ‫לאחר שטיפה במים אפשר להעביר את צינורות‬
Transfer the A and/or B inlets into the Mobile phase Bottle.
•
Go to the Console->Select to the ACQUITY UPLC SYSTEM->Control->Startup system.
•
Fill the values in the screens of the SYSTEM STARTUP.
Page 10 of 32
12/5/2012
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
Operating Waters’ H-Class UPLC in Empower
(H-Class_setup_1-11-2012_Empower)
The System Startup primes in parallel the following:
•
Purges marked solvents in the wizard: A; B; C; D;
•
Purges Wash solvents;
•
Primes Seal Wash solution.
•
Prepares the system to the next HPLC method (Equilibrate)
SYSTEM STARTUP !‫ למסך זה יש לשוניות‬:‫נא לשים לב‬
.‫כשהכתוב מודגש הלשונית פעילה‬
.‫הערכים כאן הם בגדר מינימום; ניתן להחליט להשתמש בערכים אחרים לפי מצב המערכת‬
The system will prime A, B, C, D for 2 min EACH at 4 ml/min. At the same time it will wash
the sample manager inside and outside.
Page 11 of 32
12/5/2012
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
‫)‪(H-Class_setup_1-11-2012_Empower‬‬
‫‪Operating Waters’ H-Class UPLC in Empower‬‬
‫כאשר משתמשים בלולאה של ‪ 100‬מיקרוליטר ומזרק של ‪ 250‬מיקרוליטר יש להכפיל את המספרים‪sec 30 :‬‬
‫עבור ה ‪ WASH‬ו – ‪ cycles 10‬עבור ה ‪.PURGE‬‬
‫כאשר מבצעים את השלב הזה של ‪ SM‬מומלץ מאוד לעקוב אחרי הציור שמתאר את התנהגות הלחץ של ה ‪Sample‬‬
‫‪ Manager‬בקונסול ולוודא שהיא אחידה בריבועים יפים‪ .‬בדוגמא שלמטה ההתנהגות לא כל כך מושלמת בהתחלה‪,‬‬
‫משתפרת לאחר מכן‪ ,‬אך לא לגמרי מושלמת‪.‬‬
‫כדי לתקן את זה מומלץ להעביר את נוזלי ה ‪ PURGE‬וה ‪ WASH‬אל מתנול נקי‪ ,‬לבצע שוב את ה ‪ PRIME‬ואז‬
‫להעבירם חזרה לנוזלי השיטה ולחזור על תהליך ה ‪ PRIME‬ולוודא שהציור מראה התנהגות אחידה של לחץ ה‬
‫‪.SAMPLE MANAGER‬‬
‫‪Created by Dr. Shula Levin‬‬
‫‪12/5/2012‬‬
‫‪Page 12 of 32‬‬
‫‪DISCLAIMER:‬‬
‫!‪THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC‬‬
‫!‪IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING‬‬
‫‪http://www.forumsci.co.il/hplc/‬‬
Operating Waters’ H-Class UPLC in Empower
(H-Class_setup_1-11-2012_Empower)
Optional Characterize:
Only if needle is replaced. NOT USED IN ROUTINE WORK:
The next screens prepare the system to work AFTER ALL STARTUP ENDS:
:‫ ולכן יש להכניס פרמטרים לפי השיטה שהולכים לעבוד בה‬:‫המסכים הבאים מכינים את המערכת לשיטה‬
:‫המשאבה )מומלץ להשתמש בקצב זרימה מינימלי של הנוזל הרצוי כדי לא לגרום לקפיצת לחץ‬
Page 13 of 32
12/5/2012
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
Operating Waters’ H-Class UPLC in Empower
(H-Class_setup_1-11-2012_Empower)
:‫האוטוסמפלר‬
:‫מנורת הגלאי‬
Click on START to enable all these processes simultaneously.
Page 14 of 32
12/5/2012
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
Operating Waters’ H-Class UPLC in Empower
(H-Class_setup_1-11-2012_Empower)
You can follow the pressure fluctuations in the pump:
:‫הערה‬
‫ על ידי בחירה של היחידות שבמערכת אחת אחת ואז שימוש‬,‫ בצורה ידנית‬:‫יש עוד דרכים להתחיל את המערכת‬
Run-Sample ‫ או להקליק בעכבר הימני על מסכי המערכת בחלון ה‬,‫ במסך הקונסול‬CONTROL ‫בתפריט של‬
.‫ולבחור פונקציות איתחול מכשיר‬
Check the following:
1. Tubing does not contain air
3. Flow in pumps' waste is stable
Page 15 of 32
12/5/2012
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
Operating Waters’ H-Class UPLC in Empower
(H-Class_setup_1-11-2012_Empower)
PREPARE TO RUN A SAMPLE: GO TO THE RUN SAMPLES WINDOW:
Click on the Flow link in the “Quaternary Solvent Manager” icon to set the flow rate:
‫ יש לפעול לפי הוראות מדריך‬,‫ אם היא חדשה לגמרי‬.‫יש הבדל בתחילת העבודה אם הקולונה חדשה לגמרי או לא‬
‫ ואז להניח לאצטוניטריל לזרום דרכה למשך כמה דקות לפחות בקצב הזרימה הגבוה ביותר‬.Care and Use guide
‫ בכל מקרה תמיד מתחילים בקצב זרימה נמוך‬.‫שאפשר כדי לשטוף שאריות ממנה לפני שמחברים אותה לגלאי כלשהו‬
.‫ ואז מעלים אותו עד למספר הנדרש‬0.1 mL/min -‫של לא יותר מ‬
When the column is Reversed Phase and NOT NEW, start with low flow rate through the column
with water or 10:90 Acetonitril:Water to remove remains of buffer if exist. Observe the
pressure closely. The pressure is an indicator for the status of the column and the solvents’
quality.
Page 16 of 32
12/5/2012
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
Operating Waters’ H-Class UPLC in Empower
(H-Class_setup_1-11-2012_Empower)
The pressure should not rise up and should be stabilized in a few minutes when the solvents are
UPLC grade.
Stop the water’s flow and select your mobile phase composition at 0.1 ml/min. Wait for the
pressure to stabilize
When stabilized, raise the pressure in steps of 0.2 ml/min. It should not take more than 1 min to
stabilize! If it takes longer, there might be a leak or some other irregularity in the system.
When the pressure is finally stabilized on the method’s flow rate, check the Instrument Method
before you set it up.
Page 17 of 32
12/5/2012
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
Operating Waters’ H-Class UPLC in Empower
(H-Class_setup_1-11-2012_Empower)
The Instrument Method Screens
Acquity Quaternary Solvent Manager
The quaternary solvent manager is capable of binary-ternary and quaternary gradients in a very
accurate manner. You can edit the solvents’ names
Page 18 of 32
12/5/2012
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
Operating Waters’ H-Class UPLC in Empower
(H-Class_setup_1-11-2012_Empower)
In the MISC tab you can set up acceleration of flow upon change in flow so that the pressure will
be built – up gradually.
The Pre-injection Volume is the parameter that enables the injection AFTER the start of the
gradient to compensate for the excessive delay volume relative to the Binary Acquity instrument,
so that methods that are transferred from the binary system can be transferred easily.
Page 19 of 32
12/5/2012
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
Operating Waters’ H-Class UPLC in Empower
(H-Class_setup_1-11-2012_Empower)
In advanced drivers there is a choice for adaptation to various delay volumes:
Various experimental channels can be collected for diagnostic purposes
Page 20 of 32
12/5/2012
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
Operating Waters’ H-Class UPLC in Empower
(H-Class_setup_1-11-2012_Empower)
Acquity Flow-Trough Needle (FTN) Sample Manager
The only parameters that you need to set are the pre/Post inject wash time, the column’s and
sample’s temperatures,
In the advanced button one can set the following:
Page 21 of 32
12/5/2012
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
Operating Waters’ H-Class UPLC in Empower
(H-Class_setup_1-11-2012_Empower)
Various experimental channels can be collected for diagnostic purposes
Page 22 of 32
12/5/2012
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
Operating Waters’ H-Class UPLC in Empower
(H-Class_setup_1-11-2012_Empower)
Automatic dilution can be done into the existing vials.
Page 23 of 32
12/5/2012
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
Operating Waters’ H-Class UPLC in Empower
(H-Class_setup_1-11-2012_Empower)
ACQ-TUV or PDA Detector:
Make sure that the right sampling rate was used in the detector and use the appropriate
combination between the sampling rates and filter times. A simple guideline is: make sure to have
about 40-50 points across peak (see Empower) for appropriate quantitation. It is possible to
collect both 3D data for spectral analysis and 2D data for quantitative analysis.
Page 24 of 32
12/5/2012
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
Operating Waters’ H-Class UPLC in Empower
(H-Class_setup_1-11-2012_Empower)
When using PDA in the 2D mode, make sure to use the 4.8 nm resolution to reduce noise. If you
need more information on the specific screen’s parameters just click on the help ? :
Page 25 of 32
12/5/2012
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
Operating Waters’ H-Class UPLC in Empower
(H-Class_setup_1-11-2012_Empower)
Only after you are sure that you can raise the flow without creating OVER-PRESSURE:
Click “Setup”, and then “Monitor” to observe the baseline go back to the console to observe
the pressure channel and pressure changes. Observe temperatures channels
Column of 2.1x100 mm should be stabilized in less than 10 min at flow rate of 0.4 ml/min if
column’s chemistry is simple and OK.
If not, check for leaks or bad column chemistry. Consider revising the protocol for washing
of columns (For example: make sure to use enough water wash between solvents and
buffers).
‫ מומלץ לשטוף את המערכת במים ולחזור לפאזה‬,‫ דקות יש בעיה ואין טעם לחכות‬10-‫אם זמן שיווי משקל ארוך מ‬
!‫הנעה‬
The system is ready when you see a full scale of x-10e-5 AU:
Make sure that baseline is stable, scale on ~10-5 AU in the TUV detector; pressures and
temperatures are stable
Running samples: Defining the Sample Plates
Page 26 of 32
12/5/2012
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
Operating Waters’ H-Class UPLC in Empower
(H-Class_setup_1-11-2012_Empower)
Go to the Run Samples and click on the Plates icon:
Left-click on the first line of the “Define Plate” screen. For the 2 mL vials use the following
ANSI-48Vial2mLHolder
You can use the Customize->Defaults to set this plate as the default plate.
Page 27 of 32
12/5/2012
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
Operating Waters’ H-Class UPLC in Empower
(H-Class_setup_1-11-2012_Empower)
Click on the plate number, select the plate of the first line in the sample set, and then select
“Append” or “Insert” so that the vial position is automatically entered in the right format: 1:A,1
and 2:A,1 etc.
Page 28 of 32
12/5/2012
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
Operating Waters’ H-Class UPLC in Empower
(H-Class_setup_1-11-2012_Empower)
TRANFER OF HPLC METHOD TO UPLC: Using the UPLC Calculator
The calculator is inside the INSTRUMENT METHOD.
Click on the calculator icon and the following screen appears where you fill in the details of
your HPLC or UPLC method. You can DRAG the gradient from the Instrument Method’s
Gradient Table into the calculator using the special icon at the top left of the table
Page 29 of 32
12/5/2012
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
Operating Waters’ H-Class UPLC in Empower
Page 30 of 32
12/5/2012
(H-Class_setup_1-11-2012_Empower)
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
Operating Waters’ H-Class UPLC in Empower
(H-Class_setup_1-11-2012_Empower)
Click on Calculate to get the new conditions
You can change the flow rate by clicking on the FLOW RATE icon, and the software will recalculate with the new flow-rate.
Page 31 of 32
12/5/2012
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/
Operating Waters’ H-Class UPLC in Empower
(H-Class_setup_1-11-2012_Empower)
You can drag back the new gradient into the gradient table in the instrument method by
dragging the icon at the top left of the table.
Page 32 of 32
12/5/2012
Created by Dr. Shula Levin
DISCLAIMER:
THIS GUIDE IS USED ONLY AS A HANDOUT DURING OFFICIAL TRAINING NEXT TO THE UPLC!
IT SHOULD NOT BE USED OR CONSIDERED AS A PROPER TRAINING!
http://www.forumsci.co.il/hplc/