Agilent_MMILarge_Injection_Tutorial

Agilent_MMILarge_Injection_Tutorial
Agilent
Multimode Inlet
Large Volume
Injection Tutorial
Agilent Technologies
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© Agilent Technologies, Inc. 2009
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Manual Part Number
G3510-90020
Edition
First edition, May 2009
Printed in USA and China
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Agilent Multimode Inlet
Large Volume Injection Tutorial
Multimode Inlet Tutorial
Overview 4
Hot Splitless 5
Cold Splitless 6
Solvent Vent 7
Tutorial 8
Agilent Technologies
3
Overview
A growing number of researchers are exploring large
volume injection (LVI) techniques to improve existing
analyses. With traditional injection approaches in
capillary gas chromatography, most inlets and columns
can only handle 1-2 µL at a time. Attempts to increase
the injection volume lead to broadened and distorted
analyte peaks, large and long solvent peak tails, and
saturated or damaged detectors.
The desire to increase the injection volume is normally
to improve trace analysis. By introducing more of the
sample to the system, the mass of analyte reaching the
detector will be proportionately increased, resulting in
larger peak areas and peak heights. If the baseline noise
is kept constant, larger peak heights mean greater
signal to noise ratios and lower system detection limits.
An additional benefit of LVI is the ability to reduce the
amount of sample originally processed. For example,
suppose a water sample contains 1000 ng/L of
pollutant. If the current method extracts the pollutant
and reconstitutes it in 1 mL of solvent, the
concentration of analyte in the extract is 1000 ng/mL. A
1 µL injection of this extract puts 1 ng onto the column.
Now assume that LVI allows a 10 µL injection volume.
The researcher could start with 100 mL of sample,
extract it with less solvent, and reconstitute it in 1 mL.
A 10 µL injection puts 1 ng onto the column as before,
but starts with an order of magnitude less sample (and
likely, an order of magnitude less extraction solvent).
Another advantage of using LVI is the decrease in
solvent that actually reaches the detector. Usually, only
10-30% of the injection solvent actually enters the
column and makes it to the detector.
LVI can be applied to injection volumes ranging from a
few microliters up to 1 mL or more. In most LVI
approaches, the sample solvent is selectively
evaporated and removed from the inlet system before
the analytes are transferred to the separation column.
In this way, LVI is similar to nitrogen evaporation or
rotary evaporation of the solvent, with the added benefit
4
Large Volume Injection Tutorial
of being performed in the GC inlet rather than in a fume
hood. Analytes that would be lost during nitrogen
evaporation may be retained in the inlet and
successfully analyzed via LVI. Furthermore, the LVI
process can be automated and is reproducible. As in the
other evaporation techniques, the LVI approach is a
function of the solvent type, the inlet temperature, the
vent flow of evaporation gas, and the analyte boiling
point. In addition, the inlet pressure during evaporation
and the inlet liner have an impact on the rate of solvent
removal and analyte recovery. These parameters will be
described in more detail in the tutorial.
Hot Splitless
For most researchers considering LVI, their current
methods are using hot splitless injection. This proven
and reliable sample introduction method has worked
well for almost 40 years; however, it does present some
challenges to the sample integrity and to the method
developer. First, the inlet must be hot enough to flash
vaporize the solvent and analytes so that the resulting
vapor cloud can be transferred to the column. The inlet
liner volume must be sufficiently large to contain this
vapor cloud. If the liner volume is too small, the
vaporized sample can exit the liner and reach reactive
surfaces, leading to analyte loss. In addition, the
pressure wave generated by the vaporized sample can
push back against the incoming carrier gas and enter
sensitive pressure and flow control systems. Using the
Agilent pressure/flow calculator, a 1 µL injection of
acetone into an inlet at 240 °C and 14.5 psig expands to
288 µL of gas. Most inlet liners for standard
split/splitless inlets have a nominal volume of 1 mL. An
increase of injection volume to only 3.5 µL under these
conditions creates a vapor cloud of 1 mL which could
easily overflow the inlet liner.
Hot splitless injection also creates a challenging
environment for thermally unstable analytes.
Compounds such as the organochlorine pesticides DDT
and endrin can rearrange to form breakdown
compounds. This process is accelerated with the inlet
Large Volume Injection Tutorial
5
temperatures normally used to analyze them. Effective
chemical deactivation of the liner can minimize analyte
breakdown. However, high inlet temperatures can
decrease the lifetime of deactivated liners.
Another challenge created by hot splitless injection is
the opportunity for needle fractionation or analyte
discrimination. The needle temperature increases as
the sample is being transferred from the syringe to the
inlet because the needle is in contact with the septum.
The rise in needle temperature can cause the solvent to
“boil” away and deposit high boiling analytes inside the
needle. To avoid this fractionation problem, some
researchers load a solvent plug into the syringe first and
then draw up the desired sample volume. The thought is
that the solvent plug will wash any deposits into the
inlet. An effective way to address this problem is to
make a high speed injection. This minimizes the time
the needle is in contact with the septum and the time
the sample touches the needle. Even with these issues,
hot splitless injection can be made to work well. An
alternative approach, such as cold splitless can address
these concerns and improve the analysis results.
Cold Splitless
The Agilent Multimode Inlet (MMI) uses the same
liners and consumables as a standard split/splitless
inlet, making it compatible with existing hot split and
splitless methods. However, its temperature
programmability allows it to perform cold split and
splitless analyses as well. In cold splitless mode, the
MMI is cooled to a temperature below the normal
boiling point of the sample solvent so that when the
sample is injected, no vaporization takes place. The
injection is simply a liquid transfer from the syringe to
the inlet. Once the syringe is removed from the inlet, the
inlet is heated to vaporize the sample and transfer it to
the column. The solvent vaporizes first and moves to
column, allowing analyte focusing to take place as in
normal hot splitless injections. The analytes
subsequently vaporize and move to the column. The
main advantage is that the analytes vaporize at the
6
Large Volume Injection Tutorial
lowest possible inlet temperature, rather than at a
constant high temperature, minimizing thermal
degradation while still allowing a wide range of
analytes to vaporize. Cold splitless operations also do
not thermally stress the liner as harshly as hot splitless
does, prolonging its usable life. Cold splitless can also
extend the amount of sample that can be injected in
some cases. If a slow inlet temperature program is
used, the solvent can be vaporized slowly and not
overflow the liner volume. As long as the analytes can
be refocused on the column, slow inlet temperature
programs cause no detrimental effects to the
chromatography.
Solvent Vent
The solvent vent mode is how the MMI is able to do LVI.
In solvent vent mode, the inlet is kept at a low initial
temperature during sample application. Pneumatically,
the inlet is in split mode with a low inlet pressure. The
flow of gas through the inlet liner and out to vent
removes the evaporating solvent. The sample is
injected so that the incoming liquid is deposited on the
liner wall and the solvent evaporates at a similar rate.
Once the entire sample has been injected, the inlet
switches to a splitless mode for analyte transfer. The
inlet is then heated to vaporize the concentrated
sample and any remaining solvent and they are
transferred to the column. After a sufficient period to
ensure the sample transfer, the inlet is then switched to
a purge mode to allow any remaining material in the
inlet liner to be removed to waste. During the sample
injection and solvent venting period, the GC oven has
been held at an appropriate temperature to allow the
solvent to refocus the analytes on the column. When
this refocusing is complete, the oven is then
programmed to perform the separation.
Large Volume Injection Tutorial
7
Tutorial
You can choose to use a current hot splitless method to
follow this tutorial or the checkout sample that came
with your instrument. The tutorial will use the Flame
Ionization Detector (FID) MDL checkout sample (p/n
5188-5372) to demonstrate the method development
process. This sample contains four hydrocarbons (C13,
C14, C15, and C16) in isooctane. Flame ionization
detection is used as this will show you more of the LVI
behavior for analytes that elute closely to the solvent
and the solvent itself.
Step 1 - Hot Splitless
In order to calibrate your system for recovery
calculations, you will need to run your current method.
For your first step, simply run your sample by your
existing hot splitless method or use the conditions
below for the FID MDL alkane mix.
Column and sample
Type
HP-5, 30 m x 0.32 mm x 0.25 µm
(19091J-413)
Sample
FID MDL Checkout (5188-5372)
Column flow
4 mL/min
Column mode
Constant flow
MMI
8
Mode
Splitless
Inlet temperature
250 °C
Initial time
5 min
Rate 1
0 °C/min
Purge time
2 min
Purge flow
60 mL/min
Septum purge
3 mL/min
Large Volume Injection Tutorial
FID
Temperature
300 °C
H2 flow
30 mL/min
Air flow
400 mL/min
Makeup flow (N2)
25 mL/min
Lit offset
Typically 2 pA
Oven
Initial temperature
50 °C
Initial time
2 min
Rate 1
20 °C/min
Final temperature
200 °C
Final time
0 min
ALS
Sample washes
2
Sample pumps
6
Injection volume
1 µL
Syringe size
10 µL
PreInj Solvent A washes
3
PreInj Solvent B washes
3
PostInj Solvent A washes
3
PostInj Solvent B washes
3
Viscosity delay
0
Plunger speed
Fast
PreInjection dwell
0
PostInjection dwell
0
*Wash solvent — Isooctane
Data system
Data rate
Large Volume Injection Tutorial
20 Hz
9
You may want to run the sample 2-3 times to get an
average for the peak areas. Figure 1 shows the typical
results for the FID MDL sample under these conditions.
pA
C15 C16
500
400
300
200
C13 C14
100
0
0
2
Figure 1
4
6
8
min
Typical hot splitless FID MDL sample results
Step 2 - Cold Splitless
To make a cold splitless analysis, you will need to
change the inlet temperature. Set the inlet initial
temperature to 5-10 °C below the normal boiling point
of your sample solvent. Hold this temperature for 0.1
minutes, then program the inlet at 720 °C/min up to the
inlet temperature for the hot splitless method. See the
conditions below for the FID MDL method (only the
MMI conditions are given, the rest are all the same as
for hot splitless).
10
Large Volume Injection Tutorial
MMI
Mode
Splitless
Inlet temperature
90 °C
Initial time
0.1 min
Rate 1
720 °C
Final temperature
250 °C
Final time
5 min
Purge time
2 min
Purge flow
60 mL/min
Septum purge
3 mL/min
Compare the peak areas, peak widths, and peak shapes
for the hot and cold splitless modes. Figure 2 shows the
typical cold splitless results for the FID MDL sample. For
this sample, the results are almost identical between
hot and cold splitless.
pA
C15 C16
500
400
300
200
C13 C14
100
0
0
Figure 2
2
4
6
8
min
Typical cold splitless FID MDL sample results
Large Volume Injection Tutorial
11
Step 3 - Solvent Vent
Now change the MMI mode to PTV Solvent Vent.
Notice that the Solvent Elimination Calculator button
appears (Figure 3).
Figure 3
Accessing the Solvent Elimination Calculator
This calculator was designed to help you determine
reasonable starting conditions for your LVI method.
Click the Solvent Elimination Calculator button to start
the calculator. In the first screen (Figure 4 on page 13),
you are asked for several pieces of information. You
should be able to provide the sample solvent and your
desired injection volume. The third piece of information
is the boiling point of the earliest eluting analyte. If you
know this, select the temperature closest to the value;
otherwise, you can leave it at 150 °C as this will help
retain a wide range of analytes. For the FID MDL
12
Large Volume Injection Tutorial
sample, set the solvent to isooctane, the injection
volume to 5 µL, and the boiling point to 200 °C. Click
Next to go to the calculation screen.
Figure 4
Solvent Elimination Calculator
Figure 5 on page 14 shows the calculation screen.
Taking the information that you provided, the calculator
has used an initial set of instrument conditions to
determine the solvent elimination rate according to
fundamental theory. This “Elimination Rate” does not
account for other factors specific to LVI and is normally
too fast as determined from practical experience. The
“Suggested Injection Rate” does consider these factors
and is designed to leave a small amount of solvent in
the liner at the end of the venting period. This solvent
serves as a liquid “trap” for the more volatile analytes
and promotes their recovery. The “Suggested Vent
Time” is determined by dividing the injection volume by
the “Suggested Injection Rate”.
Large Volume Injection Tutorial
13
NO TE
Figure 5
14
For more information, see the LVI Method Help in your
instrument control software.
Solvent Elimination Calculator variables
Large Volume Injection Tutorial
The variables for determining elimination rate are
user-settable in the lower portion of the window. To
illustrate how these parameters interact, try the
following changes (marked in black) and record the
Elimination Rate value in Table 1.
Table 1
Elimination Rate worksheet
Inlet
temp
(°C)
Vent Flow
(mL/min)
Inject
Vol (µL)
Vent
Press
(psig)
Outlet
Press
(psig)
Solvent
Elimination
Rate
(µL/min)
60
100
5
5
0
Isooctane
137.64
40
100
5
5
0
Isooctane
60
50
5
5
0
Isooctane
60
100
5
2
0
Isooctane
60
100
5
5
2
Isooctane
60
100
5
5
0
Hexane
Note that a small change in inlet temperature has a
significant impact on elimination rate. Vent flow has a
linear effect such that a decrease by a factor of two in
vent flow gives an equal decrease in elimination rate.
As the vent pressure decreases, the elimination rate
increases. Bear in mind that the vent pressure also
impacts how much solvent reaches the column during
venting. As the vent pressure is increased, more solvent
is loaded onto the column before the analytes are
transferred. Finally, the solvent type, specifically its
normal boiling point, has a substantial impact on the
elimination rate.
To continue with the tutorial, change the calculator
values back to those shown in Figure 5 on page 14 and
listed below. Click Next to move to the method changes
screen (Figure 6 on page 16). The calculator “knows”
the syringe that is currently installed and will only allow
50% of that volume to be injected. If you ask for more,
the calculator will warn you that the system cannot
make the injection and give you a choice as to how to
proceed.
Large Volume Injection Tutorial
15
MMI
Figure 6
Mode
Solvent Vent
Inlet temperature
60 °C
Initial time
0.07 min
Rate 1
720 °C
Final temperature
250 °C
Final time
5 min
Vent flow
100 mL/min
Vent pressure
5 psig
Vent time
0.07 min
Method changes to download to the Method Editor
(Solvent Elimination Calculator)
This screen shows you all of the method changes that
will be downloaded to the Edit Parameters screen. You
can choose to accept or reject any of these parameters.
16
Large Volume Injection Tutorial
The Oven initial temperature and hold times are not
automatically checked in case your method requires
these values to be unchanged (e.g. you have a
Retention Time Locked method). For the FID MDL
sample, click Confirm and Copy and then Ok in the Edit
Parameters screen.
Run the analysis and compare the peak areas between
this run and your original hot splitless analysis. Figure 7
shows an overlay of these two runs. The dotted trace is
the original hot splitless injection result and the solid
trace is the solvent vent result.
C15
pA
C16
2000
1500
1000
500
C13
C14
0
6.5
Figure 7
7
7.5
8
8.5
9
min
Overlay of the original hot splitless injection result (dotted
line) and the solvent vent result (solid line)
Table 2 on page 18 compares the resulting peak areas
for the two runs. The peak widths for the analytes are
essentially the same for both runs. The result is that the
peaks are five times taller and show an increase of five
fold in signal to noise ratio.
Large Volume Injection Tutorial
17
Table 2
Resulting peak areas for hot splitless and solvent vent
runs
Inlet mode
Solvent
area
C13 area
C14 area
C15 area
C16 area
1 µL Hot splitless
17113114
56
56
555
554
5 µL Solvent vent
36859256
261
268
2622
2596
Solvent vent
recovery
44%
93%
96%
94%
94%
In Table 2, solvent vent recovery was calculated by
dividing the solvent vent run areas by five times the hot
splitless areas. For the analytes, the recoveries are
almost 100% and are almost identical, indicating that
the solvent vent conditions gave essentially a five-fold
improvement in instrument detection limits. Notice that
the solvent recovery is only 44%. This means that from
the 5 µL sample injection, only 2.2 µL entered the
column.
Let's extend this to larger injection volumes. Install a
larger syringe into the autosampler such as a 25 or
50 µL. Increase the injection volume to 10 µL and use
the Solvent Elimination Calculator to determine initial
conditions. For the FID MDL sample, the MMI
conditions are now:
MMI
18
Mode
Solvent Vent
Inlet temperature
60 °C
Initial time
0.15 min
Rate 1
720 °C
Final temperature
250 °C
Final time
5 min
Vent flow
100 mL/min
Vent pressure
5 psig
Vent time
0.15 min
Large Volume Injection Tutorial
Figure 8 shows a close-up of the analyte peaks with a
10 µL injection. The first two peaks are still reasonably
symmetrical but the last two peaks clearly show
fronting. This is due partially to column overload and to
the amount of solvent transferred to the column.
pA
C16
C15
3000
2000
1000
C14
C13
0
6.5
7
Figure 8
7.5
8
8.5
min
9
Analyte peaks using a 10 µL injection
Table 3 shows the recovery from the initial hot splitless
run. Notice that the 10 µL Solvent Vent recoveries are
slightly lower than the 5 µL Solvent Vent run shown in
Table 3. This is confirmed by the lower solvent recovery.
To improve this, the inlet temperature could be lowered
while keeping all other parameters the same or by
shortening the vent time slightly. In both cases, more
solvent would be left behind to help trap the C13. Of
these two approaches, the inlet temperature has a
larger effect on trapping the early eluting analytes.
Table 3
Recovery from the initial hot splitless run
Inlet mode
Solvent
area
C13 area
C14 area
C15 area
C16 area
1 µL Hot splitless
17113114
56
56
555
554
10 µL Solvent vent
59579040
493
510
5106
5208
Solvent vent
recovery
35%
88%
91%
92%
94%
Large Volume Injection Tutorial
19
To scale up to larger injection volumes, the easiest way
is to increase the vent time proportionally. You can use
the Solvent Elimination Calculator to explore this
relationship. For a 50 µL injection, a vent time of 0.75
minutes is needed. The injection parameters for the FID
MDL sample are given below. In order to avoid
overloading the column, the FID MDL sample was
diluted 1:10 in isooctane.
MMI
Mode
Solvent Vent
Inlet temperature
60 °C
Initial time
0.75 min
Rate 1
720 °C
Final temperature
250 °C
Final time
5 min
Vent flow
100 mL/min
Vent pressure
5 psig
Vent time
0.75 min
The resulting chromatogram is shown in Figure 9 on
page 21. The peak shapes are obviously distorted, a
result of too much solvent being transferred to the
column. You can fix such a problem in several ways.
Simply increasing the vent time even more will reduce
the amount of solvent in the column. Figure 10 on
page 22 shows the resulting chromatogram with a vent
time of 0.90 minutes instead of 0.75 minutes. The peak
shapes are greatly improved and are very similar to the
5 µL chromatogram shown in Figure 7 on page 17. Other
approaches to reduce the solvent being transferred to
the column are to increase the vent flow, decrease the
vent pressure, or increase the inlet temperature during
the vent period. The Solvent Elimination Calculator can
show you how much a change in the parameters will
affect the elimination rate. Two other approaches can
also help improve analyte recovery and peak shape.
Using a retention gap will aid in refocusing the analyte
20
Large Volume Injection Tutorial
peaks and improve peak shape. A second method is to
include some retaining material in the liner such as
glass wool or packing. Material in the liner aids in
holding the analytes during solvent venting and allows
more solvent to be vented. When you use retaining
material in the liner, you need to be aware of losing
analytes due to irreversible adsorption.
For more information and sample applications, please
refer to the Agilent Web site
(http://www.chem.agilent.com/en-US/Products/Instr
uments/gc/multimodeinlet/pages/default.aspx).
pA
2500
2000
1500
C16
C15
1000
500
C13
0
6.5
Figure 9
7
C14
7.5
8
8.5
9
min
50 µL injection of FID MDL sample diluted 1:10 in
isooctane
Large Volume Injection Tutorial
21
pA
2500
C16
C15
2000
1500
1000
500
C13
C14
0
6.5
Figure 10
22
7
7.5
8
8.5
9
min
50 µL injection resulting chromatogram with a vent time
of 0.90 minutes instead of 0.75 minutes
Large Volume Injection Tutorial
Large Volume Injection Tutorial
23
Large Volume Injection Tutorial
© Agilent Technologies, Inc. 2009
G3510-90020
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