LC•GC Europe - March 2001
GC connections
Strategies for GC
Optimization, Part I —
Setting Realistic
Expectations
John V. Hinshaw, ChromSource, Franklin, Tennessee, USA.
This month, John Hinshaw begins a multipart series about gas chromatograph performance optimization. In this
instalment he addresses questions of instrument capabilities and chromatographers’ expectations. In subsequent issues,
he will discuss adjustment of hardware settings, optimization of column parameters and data-handling issues.
Chromatographers have high expectations
of their instrumentation. They want to see
an instrument system deliver high
performance levels for their individual
samples, and if it does not, they want to
know why. Manufacturers write instrument
specifications for specific test and evaluation
samples under specific analytical conditions.
Real-world samples and instrument
conditions seldom closely resemble the test
set-up, so the manufacturers’ examples
often set analysts’ expectation levels too
high. Instrument specifications are better
thought of as a basis for comparing two or
more instrument systems before purchase,
much as car buyers might compare the
mileage rating or horsepower of several
competitive models.
Even if chromatographers were to verify
overall instrument system performance by
duplicating the manufacturer’s test protocols,
the instrument might not deliver that level
of performance for their specific samples.
They should consider any chromatographic
sample in terms of its relationship to the
instrument subcomponents — inlet,
column, detector and data handling —
when setting performance expectations. A
careful analysis of a sample as it passes
through each instrument subcomponent
before committing to a particular set of
instrument options and accessories will
help tremendously to reduce or eliminate
surprises and disappointments later.
Even when a system is already in use,
and its performance is less than desired,
this type of analysis will point out problem
areas that chromatographers can adjust
and optimize for the specific analytical
problem at hand.
Optimization Goals
Successful optimization requires realistic
expectations and a clear understanding of the
goals to be met. Optimization, whether of the
hardware or the gas chromatography (GC)
method, takes two forms. The first and
most common form involves adjustment of
operating parameters to achieve improved
performance. The second form buys better
performance through equipment or
column upgrades. Chromatographers
should expect their instrumentation to
deliver the performance of which it is
capable, but they should also realize that
going beyond that level might require
investment in upgraded or new
equipment. Strong interdependence of the
various instrument components on each
other often makes major upgrades
expensive or impractical, especially in GC.
For example, converting a packed-column
instrument to capillary-column capabilities
or going from conventional to high-speed
capillary column performance may require
the replacement of most of the instrument
components, from the pneumatics through
to the detector. In such situations, a new
instrument might be the best solution.
Alternatively, adding a selective detector or
an on-column inlet to an existing system
capable of accepting such new options is a
reasonable upgrade that adds capabilities
without requiring extensive modifications.
The best opportunities for hardware
optimization exist when instrumentation
fails to deliver desired performance goals
that lie within its capabilities. For example,
an improperly adjusted or configured
split–splitless inlet may compromise results’
accuracy and repeatability. Adjusting an
inlet’s operating conditions and
configuration might establish acceptable
performance but only if the inlet can
deliver that performance in the context of
the specific sample, its components and
their concentrations. In addition, the
column must accept the sample without
compromising resolution or quantification
as it comes from the inlet. Analytes must
be eluted from the column with mass
levels and peak profiles that do not exceed
the detector’s capabilities, and the datahandling system must process the raw
chromatographic data accurately and
consistently from run to run. Unrealistic
expectations cause a perceived problem
that requires either an adjustment of the
expectations or the installation of
hardware upgrades. The majority of
operational problems arise when one or
more of the analytical components fail to
deliver the performance of which they are
capable; improper set-up or operation
causes most of these difficulties.
Set Realistic Expectations
Often problems arise because analysts fail
to set performance expectations within the
capabilities of the instrument. Ideally,
analytical requirements will establish the
necessary level of instrument performance,
which will determine the feasibility for a
specific instrument package to meet the
analytical challenge. With an unlimited
budget, an analytical laboratory could
obtain various instruments, each suited to
a particular analysis or set of analyses. In
reality, however, instruments are often
tasked with samples that demand too
much. For instance, an instrument system
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might not have the necessary sensitivity, or chemically active
components might cause side effects in the column. Peaks may be
eluted too close together, or the pneumatic requirements for a
column may exceed an instrument’s capabilities. Concentrations of
all components of interest and the injected volume should fall
within the operating range of the instrument system. Inlet systems
and columns tolerate only a limited injection volume without
invoking specialized large-volume injection techniques. Polar and
chemically active components may encounter a restricted
operating range because of secondary effects from adsorption or
breakdown in the inlet or column. Column overloading affects
some components at lower amounts and temperatures than it
does at others.
Analysts sometimes try to force an instrument system to deliver a
separation that is outside its normal operating range, which usually
results in trouble. Knowing and understanding instrumentation
design and performance limits — and staying within those limits —
helps analysts avoid problems and understand difficulties when
they do occur. Setting realistic expectations requires a good
working knowledge of an instrument’s capabilities in terms of the
sample requirements as well as an understanding of the effects of
real-world samples and their matrices on long-term instrument
performance. Often chromatographers fail to examine the entire
sample path through the instrument from inlet to detector.
Evaluate the Operating Range
GC users should critically examine the relationship of their samples
to the operating range of their instrument system. If the sample
lies outside this range, a different injection technique, column or
detector may be appropriate. To determine how well matched the
sample and instrument are, first ascertain the highest and lowest
analyte concentrations in the sample, as well as the nature of any
chemically or thermally active components. Next, assess the upper
and lower limits within which the column will pass a sample
facilely without loss of trace-level components or loss of peak
resolution caused by overloading. Then obtain the manufacturer’s
detector specifications, which should list detector sensitivity or
minimum detectable amount, detector dynamic range and some
information about compatibility with packed or capillary columns.
With this information on hand, evaluate whether the sample
component amounts will exceed or undershoot the normal
operating ranges of the inlet, column and detector.
Inlets: Figure 1 illustrates the interrelationship between solute
amounts and inlet, column and detector considerations (1–3). The
dashed lines illustrate its use for 1 µL split injections at minimum
and maximum sample concentrations of 1 ng/µL (1 ppm) and
100 µg/µL (100 parts per thousand or 10%), respectively. This
range is a very wide sample dynamic range that analysts would
rarely encounter, but it will serve the purposes of this example.
Here, a split ratio of 100:1 reduces the minimum and maximum
amounts entering the column by a factor of 100 to 10 pg and
1 µg, respectively. A different split ratio will change the slope of
the slanted lines. To construct similar lines for other split-injection
situations, choose the minimum and maximum analyte amounts or
concentrations per microlitre of sample at the top of the figure on
the split scale. Draw two slanted lines that connect the minimum
and maximum injected analyte amounts on the split scale with the
corresponding amounts divided by the split ratio on the lower
splitless scale. Next, draw vertical lines connecting the lower ends of
the slanted lines on the lower splitless scale with the same amounts
on the bottom grams scale. In the figure, these lines are drawn at
1011 and 106 g, which correspond to the minimum and
maximum solute levels in the example sample after splitting 100:1.
LC•GC Europe - March 2001
For splitless injection, Figure 1 shows corresponding minimum
and maximum solute amounts of 100 fg/µL (100 parts per trillion)
and 10 ng/µL (10 ppm). To construct similar lines for other
samples, draw vertical lines connecting the minimum and
maximum solute amounts on the splitless scale on top with the
same amounts on the bottom grams scale and ignore the split
scale. In the figure, these lines are drawn at 1013 and 108 g,
which correspond to the minimum and maximum solute levels in
the example sample without splitting. For either split or splitless
injection, the left- and right-hand lines delineate the minimum and
maximum solute amounts entering the column from the inlet
system. If these limits do not lie within acceptable limits for the
column and detector, problems may arise with column
overloading, solute adsorption or decomposition, detector
sensitivity or detector overloading.
Analysts can adjust inlet parameters or the injection technique
itself to increase or decrease solute amounts entering the column.
If analyte levels entering the column are too high, increasing the
split ratio will adjust the amounts downward until they fall within
the range of operation of the rest of the instrument system. For
too-low levels, increasing the sample volume in splitless injection
will boost the amounts entering the column. Chromatographers
must observe certain operating limits to the split ratio and injected
volume, or they risk losing accuracy, precision and peak resolution.
Table 1 lists some of these parameters with suggested upper and
lower practical limits. Some specialized injection techniques such as
large-volume injection enable injection sizes of 100 µL or greater; a
previous “GC Connections” addressed this technique (4).
Columns: Now, move down to the Column diameter and film
thickness portion of the figure and find the column configuration
that is closest to the column in use. The grey areas on the lefthand side of each bar represent injected amounts less than 10 pg.
At these levels and less, solute adsorption and breakdown can
become a significant problem, not only in the column but also in
the inlet. Certain solutes — free acids and bases, alcohols and
other polar substances — will exhibit some degree of adsorption at
much higher levels. The extent of these effects depends strongly
on the chemical nature of the solute in question, column quality,
stationary-phase film thickness (df) and chemistry, injection
technique and inlet cleanliness and deactivation. It is sometimes
difficult to distinguish inlet and column effects without performing
an additional experiment on a separate inlet or column to
determine the problem source.
In the other direction, the right-hand sides of the column bars
represent typical solute amounts — the sample capacity — above
which significant peak distortion may detract from available peak
resolution. The lower limit for the stationary-phase film thickness
of 0.1 µm and the upper limits of 0.5, 1.0 or 5.0 µm represent a
sampling of commercially available columns. Speciality columns
may have other configurations. Column length also plays a role in
the sample capacity: longer columns tend to accept higher solute
amounts than do shorter ones. However, this effect is not large.
Column temperature is also important; columns will tolerate
greater solute amounts at higher temperatures.
Thicker stationary-phase films and larger column inner diameters
support higher solute amounts without distorting peak shapes. At
the same time, however, those columns deliver fewer theoretical
plates because of increased diffusion and resistance to mass
transfer in the larger mobile- and stationary-phase volumes. Gas
chromatographers usually choose first to adjust sample amounts
by changing the injection conditions. If that approach fails to
eliminate column overloading, they might have to use a thicker
film column.
LC•GC Europe - March 2001
Femtograms
Parts per trillion
1
10
100
require a working range of at least 20
binary bits to reproduce six orders of
magnitude: 220 1.05 106. A 24-bit
range is preferable: 224 1.68 107.
Even though they can output a very wide
signal range, digital systems rely on
analogue components close to the
detector itself, and so they are limited by
Picograms
Parts per billion
1
10
100
Nanograms
Parts per million
1
10
100
Micrograms
Parts per thousand
1
Percentage: 0.1%
10
100
1000
1%
10% 100%
Solute mass (g)
Splitless, direct,
on-column Split
Mass and
concentration
evidence of this situation, which must be
corrected to achieve good quantification.
A flame ionization detector, for example,
responds linearly over at least six orders of
magnitude. Purely analogue detector
electronics, however, may not cover such a
wide range without a means to change
range during an analysis. Digital electronics
1015 1014 1013 1012 1011 1010 109
108
107 106 105 104
103
Split 100:1
1015 1014 1013 1012 1011 1010 109
Splitless
Detector minimum detection Column diameter
limit and dynamic range
and film thickness
A larger inner diameter might solve the
problem, but this measure should be used
only as a last resort.
Detectors: To evaluate detector suitability,
follow the minimum and maximum solute
amount lines down to the detector section
of Figure 1. The minimum solute amount
should be significantly higher than the
minimum detection limit indicated in the
figure for the detector in use. The minimum
detection limits in the figure reflect solute
amounts that should produce at least a
10:1 signal-to-noise ratio (S/N) for the most
sensitive detector models, as listed in recent
manufacturers’ specifications. Not all
detectors will deliver this sensitivity, so be
sure to check the specifications for the
exact detector in use. Preferably, analysts
should measure individual detector
sensitivity and S/N with a known amount
of test substance so that they can
accurately gauge this lower limit. The
detector must be in good working order
as well. (See the June 2000 instalment
of “GC Connections” for a discussion of
S/N (1, 5).)
On the high end, most detectors — with
the exception of the electron-capture
detector and the mass spectrometry (MS)
detector in the single-ion monitoring mode
— produce linear responses at levels well
above the column overloading point for
capillary columns. Thus, the column will
usually impose an upper limit that lies within
the detector’s normal operating range. When
operating electron-capture detectors and MS
detectors in single-ion monitoring mode,
however, chromatographers need to be
aware that even though peak shapes can be
normal at higher solute levels, the detector
may not respond linearly to increasing
amounts. Here, as well, a careful evaluation
of a detector’s response across its intended
dynamic range for the specific sample in
question will reveal potential problems.
If the minimum and maximum solute
amounts lie outside a detector’s normal
operating range, consider adjusting the
amount of sample that enters the column by
modifying injection parameters such as split
ratio or injected amount. Sometimes it
makes more sense to change the injection
technique or even to consider a different
sample preparation procedure to bring the
minimum solute amounts up to useful levels.
Finally, not all detector electronics can
respond to the entire detector dynamic
output range at one setting. Usually this
response is not an issue unless the sample
itself spans a wide dynamic range of solute
concentration or the detector range and
attenuation settings are configured
incorrectly. Flat-topped peaks present strong
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108
107 106 105 104
dc
530 µ m
df
0.1 µ m
320 µ m
250 µ m
180 µ m
100 µ m
0.1 µ m
5.0 µ m
0.1 µ m 1.0 µ m
0.1 µ m 0.5 µ m
0.1 µ m 0.5 µ m
103
5.0 µ m
Thermal conductivity
Flame ionization
Nitrogen–phosphorus
Electron-capture
MS (scan)
MS (single-ion monitoring)
1015 1014 1013 1012 1011 1010 109 108
Solute mass (g)
107 106 105 104
103
Figure 1: GC dynamic range nomogram. Concentrations are expressed in grams per microlitre.
Column data are from references 2 and 3 and manufacturers’ information. Detector data are
from reference 4 and manufacturers’ aggregated 1999 specifications. Individual detector
models will vary — always obtain exact specifications from the manufacturer.
Table 1: Suggested Inlet Parameter Operating Limits.
Inlet Parameter
Range
Comments
Split vent flow
20–500 mL/min
High flows can cause undesirable
pressure drops in the inlet.
Injection size
Split
Splitless, on-column,
direct
0.5–1 µL
0.5–2 µL
Splitless vent time
30–90 s
Inlet pressure
Split–splitless
On-column, direct
35–700 kPa (5–100 psig)
7–700 kPa (1–100 psig)
Injections less than 0.5 µL can
compromise repeatability.
Injections greater than 1 or 2 µL can
compromise resolution and
quantification, depending on the
exact inlet hardware configuration.
Shorter times can affect
quantification. Longer times
are generally unnecessary.
Lower pressure can cause reduced
split vent flow. Higher pressures are
available with some pneumatic
systems.
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LC•GC Europe - March 2001
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the analogue electronics’ capabilities.
Schemes such as autoranging electronics
or logarithmic amplifiers help to increase
the analogue section’s performance.
Chromatographers need to be aware of
these issues to the extent that they can
identify a possible problem situation and
take corrective action as required.
Look at the Whole Picture
Figure 1 presents a way for chromatographers
to look at the whole instrument system in
the context of their samples. By following
the minimum and maximum solute
amounts through the inlet system, column
and detector, GC users can quickly
evaluate the suitability of their instrument
system for a particular analysis.
The high end: The example solute amount
lines in the figure delineate the upper and
lower limits of GC analysis on capillary
columns with standard split–splitless,
on-column or direct inlets. At the high end, a
10% sample will require split injection at a
100:1 ratio or more to bring solute amounts
to less than 1 µg for wide-bore column use.
A wide-bore, 530 µm i.d. column consumes
approximately 3–5 mL/min of carrier gas at
normal linear gas velocities; a 100:1 split
ratio on this type of column will need a
300–500 mL/min split vent flow. This
operation is possible if the column is long
enough to create a high enough pressure
drop so that the inlet system can deliver
the vent flow. The required pressure drops
of at least 20–35 kPa (3–5 psig) require
column lengths of 30 m or more for a
530 µm i.d. column. Shorter wide-bore
columns can cause problems if the split inlet
pressure is less than 20 kPa, because the
natural pressure drops through the inlet
system itself under high split flow-rates may
force the pressure at the column to be
greater. Depending on the inlet pneumatic
design, the column will operate at flow-rates
higher than apparent from the set point
pressure, or the inlet system will fail to reach
the pressure set point and will hold the
instrument system in an unready state.
If the analysis requires more resolution than
a wide-bore column can deliver, narrower
320 or 250 µm i.d. columns will require even
higher split ratios to bring solute amounts
down to their level. A 320 µm i.d. column
remains feasible, but the capability of an
inlet splitter to accommodate highly
concentrated samples for injection on
narrower bore columns is limited. Typical
flow-rates for a 250 µm i.d. column are in
the 1–2 mL/min range. At the maximum split
vent flow of 300–500 mL/min, this column
translates to split ratios of 150:1 to 500:1.
These split ratios are barely sufficient to
bring the solute amount entering the
column to the 500 ng level that a 250 µm
i.d. column with a relatively thick 1.0 µm
stationary-phase film will tolerate. The
more commonly used thin-film 250 µm i.d.
columns with 0.25–0.50 µm films require
another approach to bring solute amounts
to less than 100 ng. Very small liquid
sample injection volumes suffice, but
relative standard deviations for injected
amounts less than 0.5 µL tend to be worse
than for higher injected amounts. Most
chromatographers choose to dilute the
sample with a non-interfering solvent as a
way to bring solute concentrations to levels
that require less extreme split inlet vent
flow-rates.
Splitless, on-column or direct injection of
samples that contain 0.1–1 µg solute
amounts or less is practical on thick-film
320 µm or 530 µm i.d. columns, as shown
in Figure 1. Higher solute concentrations
will lead to column overloading. Narrower
bore columns, however, will not tolerate
those high solute amounts, so that sample
dilution again may be appropriate in this
situation. Ideally, split injection is the right
choice at such high concentrations, but
sometimes the sample requirements
dictate otherwise.
On the high end, only the flame
ionization and thermal-conductivity
detectors possess sufficient linearity at the
top of their ranges to make them useful
for these concentrated samples. The
selective and MS detectors will be severely
overloaded by solute amounts greater than
100 ng or greater than 10 ng in the
instance of the electron-capture detector.
Thus, even if the column tolerates high
solute levels, the detector requirements
might determine the best injection
technique, or they might dictate adding a
sample dilution step before injection to
bring injected quantities within the optimal
operating range.
The middle range: The overlap between
split and non-split injection techniques
covers approximately five orders of
magnitude. Chromatographers have a lot
more latitude for matching injection
techniques, column configurations and
detector ranges in this middle range —
from 10 pg to 100 ng. The injection
technique of choice will be split injection
for solute concentrations greater than
10 ng/µL and splitless, direct or on-column
injection for concentrations less than that
level. A direct or on-column inlet will work
at the upper end of this range, as well as
for situations in which a split inlet is not
available. Nearly all columns should
accommodate these solute levels without
much trouble. At the 10–100 pg level,
caution is merited when using split
injection for polar solutes or for those that
tend to decompose under the influence of
exposed active surfaces. Most selective
detectors, such as the nitrogen–
phosphorus, photoionization and flame
photometric detectors as well as the flame
ionization and MS detectors, function well
at these levels. Electron-capture detectors
and MS detectors in the single-ion
monitoring mode may experience detector
overload at the upper end of this range.
The low end: At the bottom end of the
mass range — less than 10 pg/µL — split
injection can reduce solute amounts to levels
that will experience problems with passage
through some columns or with detection
on many detectors. Splitless, direct or
on-column injection ensures that as much
solute as possible gets onto the column to
minimize problems arising from adsorption
and decomposition in the inlet or the
column. Remember, however, that too-large
injection volumes can cause solvent flooding
or other problems in the inlet or column that
lead to peak distortion and resolution loss.
Solutes in this low concentration range
benefit the most from additional sample
preparation steps that increase their
concentrations or from specialized largevolume injection techniques.
Column bleed can become a problem
with low-concentration solutes. Column
bleed appears as an increasing baseline
during temperature-programmed elution;
higher noise levels also result from column
bleed. The baseline offset and noise from
column bleed will reduce the signal-tonoise ratio, which will detract from a
detector’s available minimum detection
limit. Column bleed can also cause the
data-handling system to have trouble with
correctly assigning peak start, apex and end
points, which can cause reduced precision
and accuracy. Most column manufacturers
offer a line of high-performance, low-bleed
columns that chromatographers should
choose in this situation.
Detectors with high sensitivity, such as
electron-capture detectors or MS detectors
in the single-ion monitoring mode, should
have no difficulty operating in the low
range. Others, such as flame ionization and
nitrogen–phosphorus detectors, will be
hard pressed to perform well at levels less
than 1 pg. Analysts must take special care
to ensure system cleanliness and freedom
from gas contamination at these low
solute levels, no matter which detector
they use. Ferrules, O-rings and other seals
can contribute significantly to detector
background at these levels. Systems
LC•GC Europe - March 2001
previously used at high solute levels may
require a thorough cleaning before an
instrument will deliver its specified
performance on the low end. Carrier gas
and detector make-up or flame gases must
be ultra high-purity grade. Appropriate
pressure regulators, clean tubing and gas
filtration as close as possible to the
instrument are essential, even though the
gas supply is of high quality, because
minute leaks at gas line connections or
traces of organic material in the tubing or
regulators will destroy the best quality gas.
Conclusion
GC instruments accommodate an
extremely wide range of solute
concentrations. Although any one analysis
is unlikely to demand that the instrument
delivers high performance across its entire
operating range at once, the minimum and
maximum solute amounts in a sample
must lie within the available dynamic
ranges for the inlet, column and detector.
Straying outside the range of any one
subcomponent invites trouble with peak
resolution or analytical accuracy and
precision. The correct choice of sample
preparation and injection techniques,
matched with an appropriate column and
detector, helps ensure the reliability and
longevity of the instrument components
and the quality of the analytical results.
References
(1) J.V. Hinshaw, LC•GC Europe, 13(6), 410–417
(2000).
(2) K.J. Hyver, Ed., in High-Resolution Gas
Chromatography (Hewlett-Packard Co.,
Avondale, Pennsylvania, USA, 3rd ed., 1989),
1–21.
(3) W. Seferovic, J.V. Hinshaw Jr and L.S. Ettre,
J. Chromatogr. Sci., 24, 374–382 (1986).
(4) J.V. Hinshaw, LC•GC Int., 7(10), 560–563
(1994).
(5) H.M. McNair and James M. Miller, Basic Gas
Chromatography (John Wiley & Sons, Inc., New
York, USA, 1998),123.
“GC Connections” editor John V. Hinshaw is
president and principal scientist of
ChromSource, Franklin, Tennessee, USA, and
a member of the Editorial Advisory Board of
LC•GC Europe.
Direct correspondence about this column
to “GC Connections,” LC•GC Europe,
Advanstar House, Park West, Sealand Road,
Chester CH1 4RN, UK,
e-mail: [email protected]
For an ongoing discussion of GC issues with
John Hinshaw and other chromatographers,
visit the Chromatography Forum discussion
group at www.chromforum.com
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