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)+