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Disturbances (Continued)
Wander:
1. Contaminated carrier gas if using
isothermal conditions.
Solution: Change the carrier gas or 63,.,1*
SPIKING 12,6(
NOISE :$1'(5
2))
WANDER
use (change) carrier gas impu- rity
traps (pg 25).
2. Contaminated gas chromatograph.
Solution: Clean the injector
and / or gas lines. Solvent rinse the
column (pg 26).
3. Poor control of the carrier gas or
'5,)7 2)) 6(7
OFFSET
detector gas flows. DRIFT:$1'(5 '5,)7 SLIPPERY
Solution: Clean, repair or WHEN WET
change the flow controller. '
or Sizes (Continued)
7. Very low or no carrier gas flow.
Solution: Immediately lower
the column temperature to
35-40C. Measure and verify the
carrier gas flow rate (pg 17). TAILING
7$,/,1* FRONTING
)5217,1* SPLIT
63/,7
Check for leaks.
8. Poor injection technique (usu - 5. Mixed sample solvent for splitless 4. Large changes in the sample
ally too slow of an injection). or on-column injections. concentration.
Solution: Change injection Solution: Use a single solvent for Solution: Adjust or compensate for
technique. sample injections (pg 20). the concentration change.
Ferrules
4 mm Splitless Liner
Pyrolyzed compounds can build
up on liner walls. This buildup
causes clogging and sample ad-
ADM 1000 Model Flow meter sorption, which can result in a
nonrepresentative chromatogram.
Hamilton Cemented
Needle
Be sure to have a clean, working
syringe. Problems can sometimes
be traced to the autosamplers.
J&W offers a complete line of
4 mm Splitless Liner
Hamilton Syringes.
Stationary Phase
The stationary phase is a polymer Another widely used stationary
that is coated onto the inner wall of phase is polyethylene glycol
the fused silica tubing. The Æ
(Figure 3). Carbowax 20M is one of
thickness, uniformity and chemi- cal the most widely used polyethylene
nature of the stationary phase are glycols to be used as a gas chro-
(Not to Scale)
extremely important. It is the matographic phase. The major dis-
stationary phase that has the great est advantage to polyethylene glycol
Figure 1 influence on the separations phases is their high susceptibility to
Fused Silica Tubing obtained. structural damage by oxygen at
elevated temperatures. Damage
The fused silica used to manufac- occurs at lower temperatures and
ture capillary columns is synthetic lower oxygen levels than most
quartz typically containing less polysiloxane stationary phases. The
than 1 ppm metallic impurities. R high polarity and unique separa-
Blanks (preforms) of fused silica tion characteristics of polyethylene
are drawn through a furnace at a
carefully metered rate. Laser mi-
crometers are used to ensure a
[ O Si ] n glycol stationary phases are useful;
thus, the liabilities are tolerated.
constant tube diameter. As part of R A newer class of capillary column
the column manufacturing pro- contains a gas-solid adsorption
cess, the inner surface of the tub-
R = CH3 methyl type of stationary phase. These
ing is purified and deactivated.
CH2CH2CH2CN cyanopropyl columns are often called porous
This process is used to minimize
CH2CH2CF3 trifluoropropyl layer open tubular or PLOT col-
chemical activity (unwanted umns. PLOT columns contain a
inter- actions between the tubing layer of solid particles coated onto the
and inner walls of the fused silica
the injected sample) and to create a tubing. Instead of a gas-liquid par-
phenyl
chemically uniform surface for titioning process between the in- jected
the stationary phase. sample and stationary phase, a gas-
solid adsorption process
Figure 2 occurs. Examples of PLOT
stationary phases include polysty-
rene, aluminium oxide and
molecu- lar sieve.
Phone 031 972 3152 © 2005 MSP 7
GC Re f e rence
1
Tridecane k = 6.3 Tridecane k = 5.7
a. b.
Figure 4
Phone 031 972 3152 © 2005 MSP 9
GC Re f e rence
a. Tridecane k = 5.7
b.
Tridecane k = 20.5
Figure 5
* Capacity for solutes that are well matched with the stationary phase. Amounts are for each component (single
peak) in the sample and not the amount of the entire sample. Capacity is defined as the amount where peak
broadening by 10% at half-height occurs. If the inner diameter you are using is not listed, please call MSP
for additional information.
** Standard.
Temperature Limits time, use any column at the lowest The bleed trace is a record of the
reasonable temperature. There is a baseline rise during a temperature
All stationary phases have upper direct relationship between in- program. The rise is caused by the
and lower temperature limits creased column lifetime and increased normal degradation of
which define the temperature lower operating temperatures. the stationary phase at higher col-
range over which the column can Leaving the column oven at lower umn temperatures. For a blank run
be safely used. The lower tem- temperatures when the column is (no injection), there are several
perature limit is the point where not in use will extend its lifetime. important characteristics for the
the properties of the stationary bleed trace:
phase are not conducive to chro- 1. The baseline rise starts about
matography. At or below the lower Column Bleed 30-40°C below the isothermal
temperature limit, the column will Column bleed is defined as the upper temperature limit of the
exhibit poor separation and peak normal background signal caused by column.
shape problems. Permanent dam- the elution of stationary phase 2. The baseline remains relatively
age will not result if the column is (polymer) degradation products. level before the rise begins and
exposed to temperatures below These degradation products are after the maximum temperatur e
the lower limit. Heating the col- always present and are not neces- is reached and held.
umn above the lower temperature sarily a sign of a damaged column. 3. There are no discrete peak s
limit will restore its performance. present.
Every column, regardless of the
source or quality, will exhibit som e
Exceeding the upper temperature column bleed. The amount of nor- The presence of discrete peaks in a
limit will result in accelerated mal column bleed will increase blank run indicates that the inlet o r
degradation of the stationary front portion of the column is con -
with increasing film thickness,
phase. Upper temperature limits taminated. Stationary phase degra -
column diameter and length. Polar
are usually given as two numbers. dation is a continuous process an d
phases will usually exhibit slightly
The first or lower temperature is not an isolated, one-time event .
higher bleed levels than nonpolar A single point introduction o f
called the isothermal limit. This is phases. A bleed trace (normal back-
the temperatu re at w hich the col- ground) is shown in Figure 6. Ap- compound(s) into the column i s
umn can be maintained for indefi- proximately 10 ng of tetradecane required to obtain peaks. Sinc e
nite periods of time. The second or was injected into the column to use stationary phase degradation is a
higher number is called the as a scale. continuous process and peak gener -
temperature program limit. The
column should not be used at this Normal Bleed Profile
temperature for prolonged periods
(>10 min). The upper temperature
limits are not precise thresholds, Column: DB-5
but temperatures where column 30 m x 0.25 mm I.D., 0.25 µm
J&W P/N: 122-5032
lifetime is substantially decreased. Carrier: Helium at 40 cm/sec
Exceeding the upper temperature for Oven: 100-320C at 20/min
320C for 10 min
short periods of time will not result in Injector: Split 1:100, 250C
instant phase damage; Detector: FID, 300C
Nitrogen makeup gas at
however, the rate of column deg- 30mL/min
radation increases with increasing
(Peak represents approximately 10 ng of tetradecane)
temperatures.
be cut. Attempting to cut through the possible contact of the tubing with small amounts of the Snoop may
fused silica will result in an sharp edges such as column iden- aspirate into the leak and contami- nate
uneven and unsatisfactory cut. After tification tags, oven walls, column the injector, detector or col-
scribing the polyimide, grasp the hangers, etc. are eliminated. The umn. Using a nonretained
column on each side of the scribe. forced air currents inside the GC compound to verify the injector
By pulling along the tubing length and oven will cause movement of the
bending away from the scribe point, a column, and these contact points
clean cut will be obtained. This may will abrade the column. This will
seem awkward at first, eventually lead to column break-
but after several attempts, the pro- cess
will become quite routine. The end of
the column should be
examined with a 10-20X magnifier to
verify that a clean and straight cut has
been obtained.
H (mm)
0.8
generates the highest efficiency He
Use the highest purity carrier gas
for a column. The curves also 0.6
for maximum column life. The
show that using a linear velocity 0.4
H2
use of impurity traps (water and
that is too low or high will result 0.2 oxygen) on the gas lines is
in a rapid loss of column highly recommended to extend
efficiency. 10 20 30 40 50 60 70 80 90
column lifetime and to improve
Average Linear Velocity (cm/sec)
D517
detector sensitivities. The
Usually a linear velocity that is Figure 7 slightly higher cost of high
greater than the value purity gases will be offset by
correspond- ing to the minimum greatest column efficiency, but the
longer column and trap life.
minimum in the van Deemter
in the van Table 4 lists recommended
curves occurs over a very narrow
Deemter curve is used. A value minimum purities for carrier and
range and at a low linear velocity.
1.5-2 times the µopt (called the detector gases.
OPGV or optimal practical gas Substantial analysis speed must be
sacrificed for optimal resolution
velocity) is the point where maxi-
mum column efficiency per unit when using nitrogen. For helium
and hydrogen, high linear veloci-
time is obtained. Setting the linear
ties can be used to reduce analysis
velocity at a higher value also
compensates for the decrease in
times without sacrificing a large Makeup Gas
linear velocity with increasing amount of efficiency. The faster Most commercially available GC
flow rates also sweep the injector detectors require 30-40 mL / min
column temperature as encoun-
tered when using a temperature faster, which improves the sample total gas flows for best sensitivity
program. introduction process. Helium, and and peak shape. Carrier flows for
especially hydrogen, provide the capillary columns range from less
best resolution when the analytes than 1 mL / min to over 10 mL / min.
With helium and hydrogen as
elute over a wide temperature These flow rates are well below the
carrier gases, the minimum in the van
Deemter curves occurs over a much range. Nitrogen is not recom- range where most detectors will
broader range and at higher linear mended as a carrier gas for capil- exhibit optimal performance. To
velocities than with nitro- gen. Using lary columns even though it supplement the carrier gas flows,
nitrogen provides the makeup gas is added at the column exit
to obtain a total gas flow of 30-
Recommended Linear Velocities for 30 Meter Columns
40 mL / min into the detector. The
makeup gas can be the same as the
Column Linear Velocity Flow Rate carrier gas or a different gas de-
Diameter (cm/sec) (mL/min) pending on the type of detector
(mm)
being used. The makeup gas is
independent of the flame gases in
He H2 He H2
combustion type detectors (e.g., FID,
0.18 30-45 45-60 0.5-0.7 0.7-0.9
NPD and FPD). For some de-
0.25 30-45 45-60 0.9-1.3 1.3-1.8
tectors, such as the ECD, the
0.32 30-45 45-60 1.4-2.2 2.2-2.9
moder- ating or auxiliary gas may
0.45 30-45 45-60 2.9-4.3 4.3-5.7
act as the makeup gas. Exact
0.53 30-45 45-60 4.0-6.0 6.0-7.9
recommenda-
tions for makeup gas flows and
Table 3 type can be found in the instruction
Recommended Minimum Gas Purities manual for the GC being used.
hydrogen 99.99% carrier
99.95% FID
helium 99.995% carrier
nitrogen 99.999% carrier and ECD moderating gas
99.95% makeup
Table 4
active compounds may occur will be smaller than the more vola-
Capillary GC Injectors when they come in contact with tile compounds. The longer the
the metal surfaces. sample spends in the heated injec-
There are two goals of sample in- tor, the less severe the discrimina-
troduction. One is to introduce the Backflash problems can be tion.
sample into the column such that it minimized by:
1. Using a septum purge with Septum Purge
will occupy the shortest length of split / splitless injectors.
column. The shorter the sample Most split / splitless capillary injec-
band is at the beginning of the 2. Using small injection volumes. tors have a septum purge function.
process, the sharper and more The septum purge minimizes the
narrow the peaks will be on the 3. Using large volume injector amount of septum bleed materials that
chromatogram. The end result is liners. may contaminate the GC sys-
more sensitivity and better 4. Using the optimal injector tem. The septum purge gas sweeps the
resolu- tion. The second goal is to temperature. bottom face of the septum and carries
have the contaminants out
the composition of the sample Injector Temperatures through the septum purge vent. The
introduced into the column be as septum purge flow is usually be-
The injector temperature should be
close to the composition of the tween 0.5 and 5 mL / min. High or
just hot enough to insure "in- stant"
sample before the injection. There higher than optimum septum purge
vaporization of the entire
should be no sample degradation or flows may result in the loss of some
sample without degrading any of the
adsorptive losses occuring or caused of the more volatile sample compo-
sample components. If the
by the injector. nents. The septum purge function is
injector temperature is too low,
not essential for good chromato-
carry over problems, incomplete
Backflash graphic results; however, any
sample vaporization or broad
septum bleed problems may be
With the exception of on-column peaks (especially the solvent front)
minimized.
injection, all injectors utilize va- will be obtained. If the injector
porization to introduce the sample temperature is too high, excessive
into the capillary column. The backflash or sample degradation may
injected sample is rapidly occur. Using an injector tem- perature
vapor- ized in the heated above the upper tempera- ture limit of
injector, and a gas (the carrier the column stationary phase will not
gas) flowing damage the col-
through the injector carries the umn. For most samples, 250 °C is a
sample into the column. One good injector temperature. Some
prob- lem that vaporization experimentation may be
injection warranted to obtain the smallest
techniques have is backflash. amount of backflash and the maxi-
Upon sample vaporization, the mum amount of sample
gaseous sample will expand to fill the vaporization.
injector liner volume.
Backflash is when the vaporized Inlet Discrimination
sample expands beyond the capac- Upon injection, the less volatile
ity of the liner volume and into the sample components will not va-
injector body. Since the sample porize as rapidly as the more
now occupies a larger volume, it volatile sample components. Im-
takes longer for the sample to be mediately following injection, the
carried into the column. A large vaporized sample has a greater
and tailing solvent front is ob- proportion of the more volatile
tained. If the vaporized sample compounds than the less volatile
comes in contact with cold spots like compounds. Thus, more of the
the septum and gas inlets of the volatile compounds are intro-
injector, small amounts of the
duced into the column. This effect is
sample may condense. This con- called discrimination. The peaks for
densation can result in carry over the less volatile compounds
problems (ghost peaks) on subse-
quent injections. The metal
injector is not inert and loss of
18 © 2005 MSP FAX 031 971 4643
Re f e rence GC
Injection Techniques Split Ratio: The amount of sample Applications involving highly
entering the column will be depen- concentrated samples or very
There are four major capillary dent on the carrier gas flows into small diameter columns may re-
injection techniques - split, splitless, on- the column and out of the split vent. quire the use of higher split ratios.
column and Megabore direct. By measuring the column flow and the
Note: Split ratio can be directly measured
Nearly every standard capillary split vent flow, the amount of using ADM flowmeters.
injector is capable of split and sample "splitting" that occurs can be
splitless injections. On-column calculated. This value is called the Equation 4: Split ratio
injections require a dedicated split ratio. The split ratio is nor-
capillary on-column injector. mally reported with the column
split vent flow
Megabore injections utilize flow rate normalized to 1. The split column flow
= split ratio
packed column injectors that have ratio is determined using Equation
been converted to a Megabore 4. The split ratio can be used to Example:
injector. estimate the amount of sample Column flow = 2 mL/mi n
entering the column. A split ratio of Split vent flow = 100 mL/mi n
Split Injection 1:50 would indicate that one part of the
Split injection is very simple and sample enters into the column
and 50 parts are discarded out of 100
the most common of the capillary Split ratio =
2
= 50
injection techniques. The highest the split vent. Therefore, 1 / 51 of the
resolution and system efficiencies total sample theoretically makes it into Therefore, the split ratio is 1:50.
are obtained with split injections. the column. Typical split ratios range
Split injections are used for highly from 1:10 to 1:100.
concentrated samples with typical
per component concentrations of 0.1-
10 µg /µ L. Injection volumes of 1-2
µL are normally used, but vol- umes
up to 5 µL can be used with- out
significant problems.
Injection Techniques
Splitless Injection
Splitless injections are used for
trace level analyses or when the per
component amounts are no more
than approximately 200 ng. Splitless
injections are slightly more
complex than split injections and are
subject to several restrictions and
conditions.
Figure 12 Figure 11
'
Column Performance
Columns can be easily rinsed Chromatogram
Contamination using a rinse kit (see Figure 13).
One of the most common causes of Before rinsing the column, break Definitions
off one-half meter from the injec- Partition Ratio (k)
column performance degradation
is contamination. Usually these tor end. Place the detector end of The partition ratio (k) is a measure of
contaminants originate from the the column into the rinse kit. Fill how much time a solute spends in the
injected samples. Semivolatile or the vial with the appropriate sol- stationary phase relative to
nonvolatile sample components vent. Apply 10-15 psi pressure to the time it spends in the mobile
accumulate in the injector and force the solvent through the col- phase (carrier gas). All solutes
column after repeated injections of the umn. For columns with 0.18-0.32 spend the same amount of time in
sample. Dirty samples will mm I.D., use 4-5 mL of each sol- the mobile phase, thus the partition
contaminate the GC system at a vent. For larger I.D. columns, use ratio is directly related to retention
faster rate than clean samples. Loss 8-10 mL of each solvent. Each caused by the stationary phase. It is a
of resolution or separation, solvent should stay in the column unitless number and can be calcu-
peak shape problems and / or base- for at least 10 min utes. The p revi- lated using Equation 5. The parti- tion
line disturbances (artificial bleed) are ous solvent does not have to ratio is a better measure of retention
common symptoms of column com- pletely vacate the column than the actual solute retention time.
contamination. Active compounds before rinsing with the next The partition ratiois inversely
such as carboxylic acids, amines, solvent. proportional to column
phenols and diols are particularly temperatures. This means that re-
affected by contamination. After the last solvent has left the tention increases as the column
column, allow the pressurizing gas temperature is decreased and reten-
Solvent rinsing a capillary column to flow through the column for 5-10 tion decreases as the column tem-
will remove most contaminants minutes. Install the col- umn into the perature is increased.
and restore column performance. injector of the GC
A column must have a bonded and and let carrier gas flow through the Equation 5: Partition Ratio (k)
cross-linked stationary phase to column for 5-10 minutes. In- stall
be solvent rinsed. the column in the detector and
check for leaks. Heat the column tR - tM t ’R
A good general column rinsing at k= =
tM tM
can be accomplished by using 2-3°C / min u ntil the normal con di-
methanol, methylene chloride, and tioning temperature is reached.
where
hexane in series. Other Condition the column as usual. t R = solute retention time
solvent choices will work, but t M = retention time of a non
they should meet the following retained compound
criteria: tíR = adjusted retention time
Figure 13
Column Test Standards high molecular weight materials), or column and cleaning the injector.
an extremely damaged station- ary
One of the best means to evaluate the phase. Gas flow problems Acids and Bases
performance of a capillary include dead volume, leaks in the
column is by analyzing a Usually substituted phenols (acid) and
injector, improper installation of
proper- ly designed test anilines (base) are used to
the column, broken or improperly
standard. Test measure a columnís behavior
installed injector liner, backflash and
standards contain compounds that toward acidic or basic com-
poorly cut column ends.
have a range of functional groups. By pounds, respectively. The presence of
Straight chain hydrocarbons are
evaluating the shape, size and peak tailing for either peak
used to calculate retention indices.
retention of the various test stan- indicates that there is reversible
They may also be used to deter-
dard components, a substantial adsorption and that the column (or
mine the number of theoretical
amount of information is obtained perhaps the injector liner) is
plates or coating efficiency.
about the column and possibly the exhibiting acidic or basic charac-
chromatographic system. Every J&W teristics. The acid peak will tail if the
Alcohols
column is tested using a spe- cially column is too basic, and the
designed test standard. The actual Hydroxyl groups easily interact base peak will tail if the column is
chromatogram is included with every with any material or species in the too acidic. Columns should exhib-
column (see Figure 14 on page 275). carrier gas flow path that can hy- it a ìneutralî character if they are
It is important to save this test drogen bond. Ideally the only to be applicable for a wide range
chromatogram for future reference. interactions occurring is with the of analyses. The height of the acid
The column performance stationary phase. A tailing alcohol and base peaks are also compared
chromatogram provides peak often indicates column to the height of a hydrocarbon
additional information about the activ- ity. Silanol groups present peak. Hydrocarbons are not sus-
column such as temperature limits, in the column or injector liners ceptible to adsorption, thus they
actual dimensions and serial are a often serve as reference peaks. The
numbers. source of this activity. An oxidized ratio of the peak height of the acid
stationary phase will also cause an d base to a hy d rocarbon is cal-
Hydrocarbons tailing alcohol peaks. In most cases, culated. A reduction in the height
stationary phase damage and the ratio indicates that the column is
Hydrocarbon peaks are the stan- resulting active sites are irreversibly adsorbing the corre-
dard to which all other peaks are permanent and cannot be reversed sponding acid or base.
compared. Due to the lack of func- without substantial effort. Alcohol
tionality, hydrocarbons can only peak tailing can also be caused by Others
interact with the stationary phase. contamination. Sample residues
Hydrocarbon peaks should be Polynuclear aromatic hydrocar-
from previous injections or other
sharp and symmetrical. Mal- bons (PAHs) or fatty acid methyl
contaminants will interact with the
formed hydrocarbon peaks are esters (FAMEs) are often included in
alcohols resulting in tailing peaks.
due to gas flow problems, poor test mixtures. They may be
These contaminants can be re-
injection technique, column used to calculate the number of
moved by solvent rinsing the
con- tamination (especially theoretical plates, coating
solid or efficiencies, retention ( k) or
retention indices.