Walk-Up LC-MS on the Agilent 1200/6130
Last updated Oct. 2014
Capabilities in Brief• Ionize molecules by electrospray ionization (ESI) or
atmospheric pressure chemical ionization (APCI)
• m/z 2-3000; 2500 or 5250 Da/sec scan speeds
• Analyze positive and negative ions in the same experiment
• Binary pump for LC gradient• 12 port inlet selection valve on A1 and B1 for method
development/non-standard HPLC solvents
• 6 column selector (loop injection or one of 5 columns)
• Diode array detector• Monitor up to 8 wavelengths simultaneously (190-950 Da)
• Record full UV-VIS spectra along with MS Data for LC runs
• See 1200/6130 Intro Document for more details
Sample Prep Notes
• Samples for ESI/APCI should be mg/L in concentration
• Samples for ESI need acidic/basic/chelating site to take a charge (acid, ester, amine, poly-ether, alcohol, amide)
• Samples for APCI need only be soluble in solvent
• NO PRECIPITATES or SOLIDS
• AVOID HIGH CONCENTRATION, EASILY CHARGED REAGENTS
• Tetraalkyl ammonium salts (TBAF), sulfonates (SDS)
• Remove/dilute these if at all possible if present in significant concetrations (e.g. liq.-liq. extraction)
ESI Solutions
• MSF provides 3 solutions for diluting ESI samples• 0.5% v/v HOAc, 10 µM NaOAc in MeOH
• 0.5% v/v HOAc, 10 µM NaOAc in 1:1 CHCl3:MeOH
• 10 µM NaOAc in 1:1 CHCl3:MeOH
• You may make your own solutions to fit your needs. Try to keep non-volatiles to a minimum (<50 µM) and use volatile, polar solvents for ESI
• These should make (M+H)+ or (M+Na)+ ions for the vast majority of polar organic compounds.
• They will also work for APCI samples• Users may make their own APCI solvents. Please use
volatile solvents and keep non-volatile components < 50 µM. One need NOT add salts/acids for APCI.
Basic Instructions to Run a Sample
• Make entry in online billing system
• Prepare sample(s) for analysis
• Enter sample(s) in Walkup LCMS software
• Place vial(s) in the indicated position(s) on the autosampler
• After run(s) is finished, process data and print report(s)
• Computer will email you when data are ready
Online Sample Submission
Click this
button to log
a sample
Use Your IU Login and Password
Submit a new job
Click this button
here to get to
the sample
entry screen
Fill out all the infoLoop injections, click
“self-run nominal
mass”
Scroll down and
indicate # of
samples for this
entry and click “I
Ran This Myself”
Record Sample Number for Later Use
Mass Spectrometer Sample Entry
Once Sample is logged into billing system, it may be put on the LCMS.
Go to the LCMS PC and look for either W icon in the task bar (top) or the “Agilent MassHunterWalkup Console” window (bottom) to proceed.
Clicking on the W icon brings up the Console window.
Click the “Submit Samples” button to put your samples in the queue.
Training Example: Chlorpramazine standard
• 5 µL of 1 g/L chlorpromazine in MeOH was added to 750 µL of the MeOH, 0.5% HOAc, 10 µM NaOAcsolution (final concentration ~7 mg/L)
• All solutions, tips, pipetters, and vials are found in A454
• Autosampler cap simply snaps on with a firm push
• A positive ion mode ESI/APCI-loop method was used to analyze this sample
• A chlorpromazine sample may be obtained from MSF staff if one needs to verify MS performance
Login Screen
No passwords required for
non-admins
Please limit usage to no more than 30 minutes of
continuous usage per submission during the day (8 am
until 6 pm). That is, 6 loop injections or 3 ten minute
LCMS runs.
Sample Description Screen
No “special” characters in “sample name”
column.
Special characters include: “ ! , @ $ # . ^ ? ;
These confuse the software (especially the . )
Choose
“Delayed”
priority to
queue
samples to
be analyzed
after 8:00
pm.
List of commonly used methods
We try to give method descriptive names.
Users may create their own methods (with MSF staff supervision)
Sample Placement Screen
An email will be sent when
the run is completed if this
is checked
Vial PlacementThe vials are arranged from 1-10 from front to back
Thus position 42 is the 2nd row in the 5th column (as shown here)
Note the software indicates the expected location with a green dot in the previous slide
Click finish when vial is placed and the instrument will perform the analysis.
Loop Injection Data Analysis
Data Analysis WindowWhen sample completes, pull up Data Analysis Window (Gray LC icon)
Data Analysis Screen
Make sure
Data
Analysis is
highlighted
Folder Navigation Window
Files are stored in subfolders based on Username and month recorded
The chlorpromazine data I just recorded are in jkarty\09-14
All data recorded in directory C:\Chem32\1\Walkup\user\Month-Year
All data are listed as “single runs” in the appropriate month
The files are accessible remotely and backed up every 6 months.
Files are in \\bl-chem-msf1200\Walkup for remote access (should work from any IUB or IUB VPN computer)
Older data get backed up into “year” directories (e.g. 2013\User\Month-Year)
Data File List
Lists all data files in a given directory. Simply double-click on a file to look at it.
Data files are named with “sample_counter” where sample is the sample info you put in the description screen earlier.
Chromatogram Window
The title section of each chromatogram indicates what data it contains.
DAD = Diode Array Detector, MSD = Mass Spectrometer Detector, TIC = total ion chromatogram, MM-ES = multimode Electrospray Ionization, MM-APCI = multimode atmospheric pressure chemical ionization, etc.
Toolbar Section
Mass Spectra are interacted with in the “Spectrum” toolbar. Make sure it is
highlighted
Used to average a set of
spectra together (use this one
most of the time)
Finds spectrum at apex of an
integrated peak (use this one
with LCMS data)
Any button’s function can be determined by hovering the pointer over it for a few
seconds.
Drop-down allows one to select
a single trace instead of seeing
them all (e.g. MSD1 TIC)
Magnify allows one to zoom in on a
particular region of a chromatogram
before extracting spectra
Left-click and drag across big peak @ 0.48 mins to average the spectra
Make sure “Average Spectra” tool
is selected
A new window containing the mass spectra opens
The header (in blue) indicates the retention
time window, TIC, and file responsible for
the spectrum
Left click and drag to draw a box
around a region to expand it.
The contents of the box will fill
the window when you let go of
the mouse button
Narrow region around m/z 319Use Exact Mass in Chemdraw to predict m/z values (don’t forget H+ or Na+)
m/z260 280 300 320 340 360 380 400
0
20
40
60
80
100
*MSD1 SPC, time=0.397:0.669 of C:\CHEM32\1\WALKUP\DATA\JKARTY\09-14\JK-2014-2-4_0215.D MM-ES, Pos, Scan, Frag: Var,
Max: 163871
335.1 257.0 279.1 302.1
322.1 377.1
320.2
321.1
301.1
319.1 S
NCl
HN
Chemical Formula: C17H20ClN2S+
Exact Mass: 319.10
Molecular Weight: 319.87
S
NCl
HN
Chemical Formula: C17H20ClN2OS+
Exact Mass: 335.10
Molecular Weight: 335.87
O
S
N
HN
Chemical Formula: C17H21N2OS+
Exact Mass: 301.14
Molecular Weight: 301.43
HO
Note no Cl in
isotope pattern!
Printing
To print a mass spectrum:
1. Make sure you have it
selected
2. Choose
File�Print�Selected
Window
3. PDF printer can be selected
from File�Printer Setup
1. Choose “DeskPDF”
LC-MS Data Analysis
LCMS Test Sample• Mixture of 4 compounds, propranolol, terfenadine,
verapamil, and niflumic acid• Available from MSF staff for LCMS performance testing
• Select 10_min_ POS_MIX method• A 10 minute experiment, both ESI and APCI data (m/z
150-1000) recorded in positive ion mode
• H2O-CH3OH gradient (10-100% MeOH in 7 min) for separation, 2.1x100 C18 column
• LCMS method can always be viewed by selecting “View Method…” from “Method” menu
Delayed LCMS Analyses
Use “delayed” priority if you need to run more than 2 LCMS
samples. They analyses will start AFTER 8:00 pm. This lets folks use
3 min loop injections throughout the day to monitor reactions.
LCMS Data Analysis Basics
• The controls (open file, zoom, extract spectrum, print, etc.) work the same
• Chromatograms that display a particular signal as a function of time are generated
• The TIC is not always the best way to find peaks in LCMS
• Use BPI or EIC to find particular ions in LCMS data
TIC, BPI, EIC, DAD Chromatograms• There are 4 basic chromatogram types for Agilent
LCMS data• TIC = total ion chromatogram
• Y-coordinate is the sum the intensities of ALL ions observed at any point in time
• BPC = base peak chromatogram• Y-coordinate is the intensity of the tallest peak at any point in
time
• EIC = extracted ion chromatogram• Y-coordinate is the intensity of a particular m/z at any point in
time
• DAD = diode array detector chromatogram
• Y-coordinate is the absorption at a particular UV-VIS wavelength as a function of time
Lilly Mix Data
UV aborbance, 254 nm
TIC, ESI
TIC, APCI
How to get EIC or BPCBPC is found at File�Overlay Base Peak
EIC is found in File�Extract ions
Don’t forget what MSD1 and MSD2 traces are (ESI and APCI in this case)
EIC notes
• Extract ion defaults to grabbing a range from 0.3 less than to 0.7 more than the mass entered. Thus, 260.2 will extract m/z 259.9 to m/z 260.9. The tenths place is important (260.2 instead of 260)
• Ion 1 and Ion 2 can be used to define a range of masses to extract (e.g. m/z 260-280 is extracted by setting Ion 1=260, Ion 2=280)
• If a specific set of ions is to be extracted repeatedly, one can add them to a custom method for future use (see Dr. Karty).
TIC, BPC, and EIC for Lilly mix
min1 2 3 4 5 6 7 8 9
100000
200000
300000
400000
500000
600000
700000
MSD1 TIC, MS File (C:\CHEM32\1\DATA\JAK\LILLYMIX 2014-08-28 15-54-50.D) MM-ES, Pos, Scan, Frag: Var, "ESI_POS"
0.1
12
1.1
74
1.8
85
2.3
25
2.8
44
3.1
05
3.4
51
3.7
05
3.9
34
4.5
24
4.9
29
5.5
01
5.7
18
7.0
23
7.3
01
8.2
70
9.1
03
min1 2 3 4 5 6 7 8 9
0
100000
200000
300000
400000
7.0
32
7.3
04
8.2
66
9.1
01
MSD1 BPC, MS File (C:\CHEM32\1\DATA\JAK\LILLYMIX 2014-08-28 15-54-50.D) MM-ES, Pos, Scan, Frag: Var, "ESI_POS"
min1 2 3 4 5 6 7 8 9
0250005000075000
100000125000150000175000
7.0
32
MSD1 260, EIC=259.9:260.9 (C:\CHEM32\1\DATA\JAK\LILLYMIX 2014-08-28 15-54-50.D) MM-ES, Pos, Scan, Frag: Var, "ESI_P
TIC � y = sum of all ions at any time point
BPC � y = intensity of most intense ion at any
time point
EIC260 � y = intensity @ m/z 260.2 (259.9-260.9)
at any time point
Note how rising baseline in TIC (likely multiple, low intensity contaminants coming off of the
column) is not present in the BPI or EIC. EIC only shows 1 peak.
ESI Mass Spectrum @ 7.04 min
m/z200 300 400 500 600 700 800 900
0
200
400
600
800
1000
*MSD1 SPC, time=7.044 of C:\CHEM32\1\DATA\JAK\LILLYMIX 2014-08-28 15-54-50.D MM-ES, Pos, Scan, Frag: Var, "ESI_POS"
Max: 193728
358.1 242.2 280.1 183.1
261.1
323.2
305.1
260.2
Base peak is 260.2, intensity = 193728
Total intensity is ~310,000
Likely propanalol (M+H)+