1. business and industrial
2. energy
3. oil
4. diesel fuel

How to get the most
out of your GCMS
analysis
Marcus Kim, Ph.D.
GCMS Product Specialist/
p
Applications Engineer
g , ON
Mississauga,
marcus.kim@agilent.com
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Gas Chromatography- some considerations
High Purity He essential!
5.0 = 99.999% purity
5.5 = 99.9995% purity
BUT compressed gas cylinders are usually
only certified to 10% of their original pressure
(i.e. 200 psi for a 2000 psi tank). At
press res belo
pressures
below 10% the g
guarantees
arantees on gas
purities are not applicable.
Major contaminants ~ O2, H2O,
O hydrocarbons
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Sample Preparation
Garbage in- Garbage out ….into your mass spectrometer
Capacity of capillary columns
30m, 0.25mm column has analyte capacity of
25-300 ng (for most common columns)
Remove matrix
Eliminate active sites and contamination
Active sites in GC can cause poor peak shapes
and contamination can result in ghost peaks and
reduced sensitivity
Upper concentration for MS is ~100ppm
Less is more with mass spectrometry.
Contamination can occur with highly concentrated
samples and injection of too much sample can
result in clipped peaks or nonlinear performance
due to saturation.
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Concentrate analytes
Remove water
Volatilize
analytes
and salts
marcus.kim@agilent.com
Syringe
needle
The problem with water
Liner
Solvent
Approximate
vapour volume ((μ
μL)
Water
Isooctane
n-Hexane
Toluene
Methylene Chloride
Methanol
Carrier
1010
110
140
170
285
450
Vent
Sample
Analyte
Solvent
1μLof liquid vapourized at 250 ˚C and 20psi
Download Agilent GC pressure/flow calculator app
Liner diameter
2mm liner
4mm liner
Theoretical
volume
236 μL
942 μL
Column
Actual
volume
118 μL
471 μL
From Grob, Split and Splitless Injection, 3rd ed.
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The problem with water
Heated zone
Transfer line
to mass spec
p
Injector
Cold,
C
ld att room
temperature
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Column
marcus.kim@agilent.com
The problem with water
• Water has very high surface tension, nonpolar stationary phase has very
low surface tension. Water also has high boiling point so some water will
pass through the column as a liquid. Resulting in band broadening and split
peaks
• Salts can travel far into the column and be active sites for future injections
• Polar stationary phases (wax) can swell with water and could be damaged
and
d ttakes
k llong titimes to
t equilibrate
ilib t
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The solution with water
Headspace
Liquid/Liquid Extraction
Solid Phase Extraction
Purge and Trap
SPME (Solid Phase MicroExtraction)
Fused silica fibre
Stationary phase
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SPME can directly sample the liquid
water or can sample headspace
Organics will preferentially partition
into the SPME coating and
concentrate. No water is carried
into the injection port
Addition of salt and pH adjustments
can greatly enhance SPME
recoveries
Chromatography is very clean with
almost no noise
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SPME headspace of crushed raspberries
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Agilent 7697A Headspace Autosampler
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Agilent 7697A Headspace Autosampler
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Agilent
g
5990-7907EN brochure
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Liquid/Liquid Extraction
Transfer analytes from a GC incompatible solvent (aqueous) to a GC compatible
solvent (organic) …Luke Method
Organic
g
layer
y
Analytes partition
Water- add salts
and adjust pH
Advantages
• quick
• easy
• efficient
Disadvantages
• Forms emulsions
• Disposal of large quantities of organic solvents
• less than quantitative recoveries
• Does
D
nott remove allll matrix
t i components
t and
d
interferences
• Labour intensive
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Solid Phase Extraction
• SPE is a mini-column for extracting
g and
concentrating analytes of interest
• Superior to LLE because over 60 different
chemistries and tunable selectivity of the organic
solvent mixtures of the eluents gives SPE higher
selectivity and versatility for higher yield
recoveries
• Much cleaner extracts- better removal of
interferences and matrix components
• Much lower quantities of organic solvents used
and disposed
• Capable of being used in automated processes
Agilent brochure: 5989-9334EN
5989 9334EN
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Multimodal SPE
p
for veryy complex
samples
Advantages
Disadvantages
Very selective
Effective with variety of matrix
High recoveries
Highly reproducible
Complex/difficult to master
Lengthy method development
Costly
So many choices
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Solid Phase Extraction Discs
• Fits on the end of a plastic syringe
• Faster flow rates than cartridges due to larger cross-sectional area
• Used for trace organics in drinking water, waste water etc… (EPA 1664)
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QUECHERS QUick Easy CHeap Effective Rugged Safe
QuEChERS
• Based on partitioning/extraction
• SPE material in a free flowing form absorbs interferents: dispersive
• Rapidly being adopted worldwide for GCMS and LCMS sample preparation
• Substantially decreases cost per sample and increases throughput
Luke method, traditional SPE,
or GPC
QuEChERS
QuEChERS Benefits!
Estimated Time to process
6 samples
l ((min)
i )
120
30
4x faster
Solvent Used (mL)
90 mL
10mL
9 x less solvent
Chlorinated Waste (mL)
30 mL
none
safer, greener, less
costly
Glassware/ specialized
equipment
Funnel, water bath, 200mL
containers, evaporator, etc.
None
No additional
supplies needed
M. Anastassiades et al., 2003, J. AOAC Int, 86:412–431
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QUECHERS
(www.quechers.com)
• Unbuffered method first published in 2003
• 2 validated versions
• AOAC official method 2007.01
• EN official method 15662
• All 3 methods give excellent results: average 98% recoveries with 10% RSDs
• Unbuffered method has a negative effect on few pH-dependent pesticides
• Original QuEChERS method (unbuffered)
• 4 or 6 g MgSO4, 1 or 1.5 g NaCl
• AOAC method 2007.01 (AOAC)
(
)
• 6 g MgSO4, 1.5 g Na Acetate
• EN method 15662 (CEN)
g
1 g NaCl, 1 g NaCitrate, 0.5 g disodium citrate sesquihydrate
q y
• 4 g MgSO4,
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GCMS
or
LCMS
Agilent QUECHERS Brochure: 5990-3562EN
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QUECHERS
• Quechers is a very popular technique because it is fast, easy and cheap;
however, it does not clean the samples as well as SPE. Quechers is just
good enough for most applications
Agilent sells complete quechers kits for different matrices
General fruits & vegetables
Fruits & vegetables with
f t and
fats
d waxes
Pigmented fruits &
vegetables
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Highly pigmented fruits &
vegetables
Fruits & vegetables with
pigments
i
t and
d ffatt
Other food methods
marcus.kim@agilent.com
QUECHERS Beyond Fruits and Veggies
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Agilent Ultra Inert Technology
All that time spent optimizing the sample preparation, inlet parameters to
get sensitivity
g
y is worth nothing
g if the p
peaks start tailing
g or disappearing!
pp
g
Active analytes adsorbing on the column stops productivity in its tracks.
Start with Ultra Inert liners and column for guaranteed performance upon
i t ll ti
installation
Ultra-Inert Brochure 5990-8532EN
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Challenges and Needs of Today’s Laboratories
• Challenges
• Needs
– Qualification/quantification of trace samples
– Lower detection limits
– Keep instrument up and running
– Improved stability in GC or GC/MS system
Lower Detection Limit
Reduce noise
Injection system (septa
(septa, liners
liners, connections)
Carrier gas and detector gases
Leaks
Temperature setting
Stationary phase and column bleed
Increase signal
Sample concentration
Sample size
Inert injection and detection port sleeves/liner
Gas velocity or temp program rate
Column inertness
• Only when a column exhibits both low bleed and low activity are results reliable.
– Low bleed increases the signal
signal-to-noise
to noise ratio
ratio, but if any of the analyte is adsorbed by
active sites in the column, the results are flawed.
– If the column is well deactivated but the bleed is high, some of the signal generated by the
analytes
y
is smothered byy the bleed signal.
g
Again,
g
the results are flawed.
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How important is Column inertness to overall Flowpath
Inertness?
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Ultra-inert Liners- Endrin, DDT breakdown test
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EPA 8270- 2,4,-Dinitrophenol
• Complex mixture of 92 acid/base/neutrals semivolatiles
• Nitrophenols show lower response factors at low concentrations with poor
linearity and will fail the run if the flow path is active
2,4-dinitrophenol
p
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Column Activity
• Traditional deactivation technology only caps ~40-65% of silanols
• Gaps in surface coverage due to bulky TMS moieties and tight fused
silica lattice
Interaction causes
tailing
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DB-5ms and HP-5ms Ultra-Inert Columns
• Polymeric deactivation technology
• “Binds at multiple points with many available silanols”
• The deactivation blanket sterically hinders active silanols making them
less available
• Optimized column chemistry and manufacturing for exceptionally low bleed
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Ultra Inert Test Mix - DB-5ms Ultra Inert vs. competitors
pA
2
18
5
3
16
1.
1-Propionic acid
2.
1-Octene
3.
n-Octane
8
14
12
4 Pi li
4-Picoline
5.
n-Nonane
6.
Trimethyl phosphate
7.
1,2-Pentanediol
18
8.
n-Propylbenzene
n
Propylbenzene
16
9.
1-Heptanol
14
10
9
10
4
11
4
1
7
8
6
pA 0
20
10. 3-Octanone
12
11. n-Decane
10
2
5
4
2
3
6
6
4
8
10
1
9
10
11
Competitor
Column
7
6
0
2
4
pA
27.5
4
2
25
6
8
10
min
5
3
22.5
Agilent J&W DBDB
5ms Ultra Inert
8
20
1
17.5
9
10
15
12.5
6
10
0
of 64
min
8
8
Page 31
Competitor
Column
2
4
11 30m x 0.25mm x
0.25um
(P/N 122-5532UI)
7
6
8
10
min
marcus.kim@agilent.com
Analyte protectants
-Si-OH
-Si-OH
-Si-O-Si-O-Si-O-Si-O-
Liner or
column
Free
silanol
• Free silanol groups are potential active sites that will
hold on to analytes or matrix components
• Calibration standards made up in pure solvent vs.
matrix matched calibrations can have differing
responses
• Free silanol sites can be blocked by the matrix
resulting in higher response of analytes in matrix
• Use of cal stds in solvent can result in
overestimation of analytes in matrix
TMS
• Gradual accumulation of matrix non-volatiles
non volatiles will
deactivated result in matrix induced diminishment and will
negatively affect ruggedness
M. Anastassiades et al., 2005, Anal. Chem, 77:8129-8137
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Analyte protectants
• Matrix matching difficult when analyzing
multiple food types
• Extent of effect is still governed by
analyte concentration and matrix
composition
• EU requires matrix matched standards
for multiresidue pesticide analysis in foods
• FDA and EPA do not permit matrix
matched standardization for enforcement
Reproduced with permission from ACS
Hydrogen bonding and volatility
(retention time coverage) most
important when choosing an AP
• Analyte protectants protect coinjected
analytes from degradation and/or
absorption in the GC
• Analyte protectants are added to both
th calibration
the
lib ti and
d extracts
t t ffor even
response
M. Anastassiades et al., 2005, Anal. Chem, 77:8129-8137
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Analyte protectants
Reproduced with permission
from ACS
M. Anastassiades et al., 2005, Anal. Chem, 77:8129-8137
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Reproduced with
permission from ACS
M. Anastassiades et al., 2005, Anal. Chem, 77:8129-8137
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Ok what other factors affect my GCMS method?
Ok…what
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Influence of Injection Efficiency
Short
Concentrated
Solute Bands
Long
Diffuse
Same column,, same amount injected,
j
, same chromatographic
g p
conditions
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Split Injector
Major Variables
Split ratio -
determines amount of sample onto column and
y of injection
j
((sensitivity
y vs p
peak shape)
p )
efficiency
Liner - influences efficiency of vaporization/discrimination
Temperature - hot enough to vaporize sample without
d
degradation
d ti or causing
i backflash
b kfl h
Injection volume - typically 1
1-3uL,
3uL increasing it does not have
as much of an effect as one might think
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Split Liner
Packed with Glass Wool
C10
Peak Area Ratio
n-C40/n-C10 = 0.64
C40
C10
Without Glass Wool Packing
Peak Area Ratio
n-C
n
C40/n
/n-C
C10 = 0.37
0 37
C40
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Split Liners
Pro
Cheap
Pro
Less
Pro
Very little discrimination
discrimination
Con
MW
discrimination
Exposure to
metal surface
at bottom
Straight
tube
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Prevents non-volatiles
from reaching column
No exposure
to hot metal
Con
Con
Expensive!
Potential
active sites
Not for very dirty samples
Single taper
with
glass wool
Jennings
cup
marcus.kim@agilent.com
GLASS WOOL
Placement in Liner
Near centre of liner:
• Provide thermal bulk and complete volatilization of sample (minimize sample
discrimination)
• Trap non-volatile and septum particles before they reach the column
• Wipe any residual sample from the needle and preventing residue buildup at the
septum
Near bottom of liner:
• Helps in volatilization of high MW components
• Trap non-volatile components
Glass wool is generally not
recommended for the
following compounds:
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phenols
organic acids
pesticides
g y acidic
slightly
drugs of abuse
reactive polar compounds
thermally labile
marcus.kim@agilent.com
Split Injector - 200:1 vs 5:1
Compaction
p
and focusing
g of analytes
y
tighter
g
in 200:1 split
p situation
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Split Injector
Injection Volume
2
1 µL
2
3 µL
4
4
5
5
1
1
1
3
2
3
Time (min.)
4
5
6
1
3
2
3
Time (min.)
4
5
6
DB-1, 15 m x 0.25 mm I.D., 0.25 µm (part # 122-1012)
60°C ffor 1 min,
i 60
60-180°C
180°C att 20°/
20°/min;
i Helium
H li
att 30 cm/sec
/
1. n-heptane 2. toluene 3. n-decane 4. n-butylbenzene 5. n-tridecane
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Splitless Injector
Major Variables
Purge activation time - determines amount of sample onto column and
efficiencyy of injection
j
Liner - preventing backflash more critical than vaporization (double tapered type
recommended))
Injection volume - typically 1uL or less (backflash)
Injection speed- the slower, the better!
Temperature – long residence times allow for lower temps
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Splitless Injector
Sample Re-focusing
Sample re-focusing improves efficiency
X
Use low column temperature to refocus solvent called the solvent effect
Use cold trapping
Retention gap greatly improves sample focusing
Use Pressure pulse when possible
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Splitless Liners
Pro
Cheap
Con
MW
discrimination
Exposure to
metal surface
at bottom
Straight
tube
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Pro
Pro
Improves
sample
transfer
Decrease sample
backflash
No exposure
to hot metal
Less
discrimination
Con
Con
Potential
sample
backflash
Single taper
Cannot be
packed with wool
Double
taper
Pro
Ultimate
sensitivity
Con
Especially
sensitive to
injection
parameters
Direct
connect liner
marcus.kim@agilent.com
Splitless Injector
Injection Volume
1 µL
3
2
4
1
3 µL
3
2
4
1
2
4
Time (min.)
6
8
2
4
Time (min.)
6
8
DB-1, 15 m x 0.25 mm I.D., 0.25 µm (part # 122-1012)
60°C ffor 1 min,
i 60
60-180°C
180°C att 20°/
20°/min;
i Helium
H li
att 30 cm/sec
/
1. n-decane 2. n-dodecane 3. n-tetradecane 4. n-hexadecane
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Splitless Injector
Solvent Effect
1.
Solvent and
solutes
Initial column temperature at
least 10°C below sample
solvent boiling point
Required to obtain good peak
shapes unless cold trapping
occurs
Rule of thumb, if solute BP
>150°C above initial column
temperature, the solute will
cold trap
Cold trapping has greater
efficiency than solvent effect
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2.
Solvent film
3.
4.
marcus.kim@agilent.com
Splitless Injector
Initial Column Temperature
Hexane Solvent (BP = 68-69°C)
Solvent Effect
Cold Trapping
50°C
70°C
3
3
2
4
4
1
2
1
2
4
Time (min.)
6
8
2
4
Time (min.)
6
8
DB-1, 15 m x 0.25 mm I.D., 0.25 µm (part # 122-1012)
50°C
50
C or 70°C
70 C for 0
0.5
5 min
min, to 210°C
210 C at 20°/min;
20 /min; Helium at 30 cm/sec
1. n-decane 2. n-dodecane 3. n-tetradecane 4. n-hexadecane
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Splitless Injector
Reverse Solvent Effect
Hexane
5
4
Methanol
6
4
1
5 6
2
2
3
3
1
0
1
2
3
Time (min.)
4
5
6
1
2
3
Time (min.)
4
5
6
DB-1, 15 m x 0.25 mm I.D., 0.25 µm (part # 122-1012)
50°C
50
C for 1 min
min, 50
50-210
210°C
C at 20°/min;
20 /min; Helium at 30 cm/sec
1. 1,3-DCP 2. 3-hexanol 3. butyl acetate 4. 1-heptanol 5. 3-octanone 6. 1,2-dichlorobenzene
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Retention Gap
Also Called A Guard Column
Injector
Detector
Deactivated
Fused Silica
Tubing
Union
Column
Usuallyy 2-10 meters long
g and same diameter as the column
(or larger if needed)
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Splitless Injector
3mx0
0.25
25 mm II.D.
D Retention Gap
Hexane
4
Methanol
1
5 6
4
5 6
2
23
1
0
1
2
3
Time (min.)
4
5
6
1
2
3
Time (min.)
3
4
5
6
DB-1, 15 m x 0.25 mm I.D., 0.25 µm (part # 122-1012)
50°C for 1 min
min, 50
50-210°C
210°C at 20°/min; Helium at 30 cm/sec
1. 1,3-DCP 2. 3-hexanol 3. butyl acetate 4. 1-heptanol 5. 3-octanone 6. 1,2-dichlorobenzene
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Pressure Pulse Splitless Injection
Pressure Pulse contains sample expansion and transfers
analytes to the column faster.
Pulsed Splitless
- sample containment more critical than in split injection
- sharper peaks than in traditional splitless injection
- two new parameters to set:
- pulse pressure and pulse time
Typical starting point
- Pulse pressure = double to triple resting pressure
- Tie pulse time to purge time
- Fast injection rate
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Split vs. Splitless Injection Technique - Summary
SPLIT:
SPLITLESS:
-Best Injection Efficiency
-Poor Injection efficiency
-Less sensitive
-solvent effect
Prone to discrimination
-Prone
retention gap
-retention
-Proper liner choice more important
-pressure pulse
-Good for Trace level detection
-Solvent/column polarity match more
critical
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What about the Mass Spec?
• The sample prep has been optimized, ultra inert liners and columns are
used, analyte protectants have been added, the injection parameters have
been optimized…is there anything else?
Fenitrothion
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Source contamination
voltage
+
+
+
+
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Organic layer
voltage
+
+
+
+
+
+
EI - a dirty source is
indicated by increased
EM voltage after tune
(~+400 V)
Also look for 502
abundance
CI – a dirty source will
typically not tune
marcus.kim@agilent.com
Large filament and drawout plate
hole to maximize flux of electrons
into and ions out of the source
Filament hole
Tiny
y filament and drawout p
plate
hole to maximize pressure of CI
gases inside source
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EI filament
Loss in sensitivity in CI not always due to
contaminated source. Filament sag is common
and can result in signal loss.
Use EI filament for CI analysis
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CI filament
marcus.kim@agilent.com
When to do source maintenance
Maintain a control chart (Levey-Jennings graph) of a known standard to monitor
instrument performance.
Do an injection everyday (weekly) and look for trends or outliers. Change inlet
g source maintenance.
liner first before doing
CI is more prone to source contamination than EI
Source/inlet maintenance ultimately up to cleanliness of samples!
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New 7693A Automatic Liquid Sampling System
New Injector with 16
Vi l Positions
Vial
P iti
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New Tray with 150
Vi l Positions
Vial
P iti
marcus.kim@agilent.com
Sample Prep Programming Flexibility
Sandwich injections ( up to 3 layers with air gap)
• Examples of Simple liquid manipulation
• ISTD addition
• Reconstitution
• Mixing (Requires Bar Code Reader / Heater/ Mixer option)
• Dilution
• Derivatization
• In Vial Extraction
Ambient
H d
Headspace
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In-vial
E t
Extraction
ti
Small-Volume
Sampling
S
li
Derivitization
Dilution
Internal Standard
Additi
Addition
Heating/Mixing
Bar
B Code
C d
marcus.kim@agilent.com
Sample workbench extraction and purification of PCBs from
waste oil
Waste oil added to acid
silica and SAX
Add more hexane and
internal standard
Mix at 4000
RPM for 5 min
Transfer to vial
containing Silica
Mix at 4000
RPM for 5 min
Transfer to vial and
inject
Agilent publication 5990-9164EN
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GC-MS/MS MRM TICs of 5 aliquots prepared by the
workbench
Workup and extraction is
automated and FAST!
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Summary/Conclusions
• Garbage in/Garbage out
– Sample Preparation extremely important
– SPE gives cleanest extracts
– Q
QuEChERS
EChERS gives
i
extracts
t t that
th t are just
j t clean
l
enough
h but
b t is
i ffast,
t robust
b t and
d
CHEAP!
– Do NOT inject water into the GC
– Backflush!
B kfl h!
• Use ultra-inert liners and columns whenever possible for best results
– Matrix matching can resolve matrix induced enhancement problems
– Use Analyte Protectants when possible
• Injector Dynamics key to Good chromatography
– Focus! Focus! Focus!
– Solvent effect, cold trap, retention gap or pressure pulse for splitless focusing
• Automation of common sample prep steps with the 7693A and workbench
– increases productivity and decreases operator variability
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marcus.kim@agilent.com