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2012-Fall version
Chapter 31
Characteristics of GLC 1. Sensitivity : mg ~ pg (103 ~ 109 g) 2. Versatility : from rare gases to liquids and solids in solution with 800~1000 MW 3. Speed of analysis : typically 5 ~ 30 min , complex 3 ~ 30 mixture separation 4. Reproducibility : qualitative Accuracy : quantitative
Fuel
Trap
Regulator
Split vent
Fuel
gas
Carrier
gas Basic components of Gas Chromatograph Carrier gas supply Sample introduction inlet Column and controlled-temperature oven Detector & oven Recorder
Ar
39.95
5.087
270.2 *
CO2
He H2 N2 O2 * at 99.6oC
44.01
4.00 2.016 28.01 32.00
5.06
39.85 49.94 7.18 7.427
197.2
234.1 104.6** 212.0 248.5***
** at 100.5oC
**** at 99.74oC
Using the correct carrier and detector gases are an important factor in installing a new GC. The five gases commonly used as carrier gas and detector fuels in capillary gas chromatography are helium, hydrogen, nitrogen, argon-methane, and air. The types of gases necessary are partly determined by the detection system used. Factors to consider for each individual gas are discussed below.
Carrier Gas Choice Carrier gases that exhibit a broad minimum on a van Deemter profile are essential in obtaining optimum performance. Volumetric flow through a capillary column is affected by temperature. When temperature programming from ambient to 300oC, the flow rate can decrease by 40 percent. A carrier gas that retains high efficiency over a wide range of flow rates and temperatures is essential in obtaining good resolution throughout a temperature programmed run. Figure 1 shows the van Deemter profile for hydrogen, helium, and nitrogen carrier gases.
Van Deemter curves for GC of n-C17H36 at 175oC, using N2, He, or H2 in a 0.25 mm diameter 25 m long wall coated column with OV-101 stationary phase
Hydrogen is the fastest carrier gas (uopt), with an optimum linear velocity of 40cm/sec, and exhibits the flattest van Deemter profile. Helium is the next best choice, with an optimum linear velocity of uopt = 20cm/sec. Nitrogen's performance is inferior with capillary columns because of its slow linear velocity, uopt = 12cm/sec. Argon-methane has a slower optimum linear velocity than nitrogen and is not recommended for use as a carrier gas with capillary columns. Air is not recommended as a carrier gas because it can cause stationary phase oxidation. With hydrogen and helium as carrier gases, the minimum H.E.T.P. values can be maintained over a broader range of linear velocities than with nitrogen, and high linear velocities can be used without sacrificing efficiency. Nitrogen is beneficial only when analyzing highly volatile gases under narrow temperature ranges where increasing stationary phase interaction is desirable. Otherwise, the use of N2 results in longer analysis times and a loss of resolution for compounds analyzed on a wide temperature range.
http://www.restekcorp.com/gcsetup/gcsetup3.htm
Carrier gases and detector fuel gases for use with various GC detectors
TCD Carrier Gases He H2 N2 Combustion/ Reaction Gases H2 O O O ECD O O FID O O O O NPD O O O FPD O O O O ELCD O O O PID O O O -
Air
Make-up Gases N2 He ArCH2
O O -
O O
O
O O -
O
O O -
O
O O -
O O -
http://www.restekcorp.com/gcsetup/gcsetup3.htm
Effect of impurities
- Impurities such as hydrocarbon, oxygen, water contribute to unwanted noise levels, excessive baseline drift. - Molecular sieve --- Moisture trap, Oxygen trap, Chemical filter
Effect of water on column efficiency - Carrier gas dryness is very important !! (use anhydrous sodium sulfate) - Water can and usually does react with some portion of the column. This results in loss of resolution and tends to produce asymmetric or tailing peaks. Unwanted components or ghost peaks may also appear. Another effect is a net loss of sensitivity.
Gas purifiers
The trap will remove any water vapor or oils that may have been introduced in the filling process since a number of gases are water pumped. The contaminants removed by the trap could otherwise interact with the column packing material to produce spurious peaks. In addition the contaminants can cause increased detector noise and drift.
The traps should be reconditioned (about twice a year ) by heating to 300 oC for 4~8 hr with a stream of gas passing through it or in a vacuum oven.
1
Gas cylinder
GC
ELCD reaction gas Hydrocarbon Packed column GC with FID or TCD Carrier Hydrocarbon, Moisture, Oxygen
Carrier
Flow requirements 1. Stable 2. Reproducible 3. Convenient The more constant the flow rates, the more precise and accurate the results. Flow controller ( Pressure controller ) --- to maintain precise and accurate flow rates
Gas generators
Pressure controllers 1. The second stage regulator on the cylinder 2. A pressure regulator mounted in the GC 3. A needle valve(variable restrictor) mounted in the GC 4. A fixed restrictor mounted in the GC
Flow measurement
1) Rotometer
The column flow rate is typically indicated by a rotometer . ( Calibrate equilibrium position indicating the flow )
Rotometer is operated by the volume of gas passing a ball in a tapered cell. 2) Bubble meter 3) Electronic flow sensor
Relationships between inside diameter, column length, mesh size, and carrier gas flow for packed column
Inside diameter
mm
Carrier flow
N2, ml/min He or H2, ml/min
2
3 4
100~120
100~120 80~100
80~100
80~100 60~80
8~15
15~30 30~60
15~30
30~60 60~100
1/8 in packed Inside diameter, mm Film thickness, m Phase volume ratio() Column length, m Flow rate, ml/min Effective plates(Neff) per meter Effective plate height (Heff),mm Typical sample size 2.2 5 15~30 1~2 20 2000 0.5
Air
40~80 ml/min
not necessary
Make-up(N2, He) 10~20 ml/min PID Make-up Sheath FPD Carrier Hydrogen Air 5~10 ml/min 30~40 ml/min 1~3 ml/min 85~100 ml/min 100~120 ml/min
Inlet requirements
1. Temperature controlled 2. Low volume (total swept by carrier ) 3. Inert construction
Column Overload
If too large an sample were allowed to enter small bore(capillary) columns column overload and a loss of resolving power would like occur.
Liner
All liners help protect the vaporized sample form contacting the metal wall of the inlet as sample flows onto the column. Deactivated glass wool may be used as an aid for sample vaporization, to minimize discrimination based on boiling point, and to provide a surface on which non0volatiles can be trapped. The simplest liner is a straight tube, which gives allaround good performance at low cost. Single-taper liners improve on a straight tube by minimizing sample vapor contact with metal at the bottom of the injection port, although they are somewhat more expensive. Liners are deactivated borosilicate glass, except quartz where noted. Liners are guaranteed inert for phenols, organic acids and bases.
Inlet configuration
1. Direct column inlet --- 1/8 " OD or larger column
sampling syringe is actually inserted into the end of the column needle guide / cap / spring / septum mounting holes / carrier gas in / inlet body column Swagelock ferrules / Swagelock nut 2. Splitter inlet --- open tubular column or less than 1/8" OD column
Because of the limited capacity for sample of these small bore columns and the difficulty of injecting extremely small volume samples, a large portion of the injected sample is vented to atmosphere by the inlet. septum / preheated carrier gas / mixing tube / restrictor buffer volume / tapered needle / gold gasket / column fitting
Injection port for split injection into an open tubular column. The glass liner is slowly contaminated by nonvolatile and decomposed samples and must be replaced periodically. For splitless injection, the glass liner is a atraight tube with no mixing chamber. For dirty samples, split injection is used and a packing material can be replaced inside the liner to adsorb undesirable components of the sample.
Split or on-column
Split 1) Simple 2) High column efficiency 3) Column may be protected On-column 1) Best accuracy 2) Thermolabile compounds 3) Trace analysis
Representative injection conditions for split, splitless, and on-column injection into an open tubular column.
Direct injector
1) Good sensitivity 2) Low column efficiency 3) Best for thick films, widebore column ( 0.53 mm )
Advantage of on-column
1) Best reproducibility : Quantitative results 2) No split, no loss of high boilers 3) "Cold" on-column injection available
Advantage of splitless
1) High sensitivity ( 95 % of sample on column ) 2) Solvent effect produces narrow sample bands 3) Same hardware as split injection
Disadvantage of splitless
1) Slow sample transfer to column 2) Must dilute sample with volatile solvent 3) Time consuming : must cool column 4) Poor for thermolabile compounds
Split and splitless injections of a solution containing 1 vol % methyl isobutyl ketone (bp 118 oC) and 1 vol % p-xylene (bp 138 oC) in dichloromethane (bp 40 oC) on a BP10 moderately polar cyanopropyl phenyl ,ethyl silicone open tubular column(0.22 mm I.d., 10 m long, 0.25 m, column temperature =75 oC).
Sampling Syringe
0 ~ 1 L --- the sample is totally confined to the needle 0 ~ 5 / 0 ~ 10 L needle / barrel / plunger
Solvent effect
t-1 t-2
time t-1 --- just after injection, solvent and sample are condensed in a long plug at the front of the column. The column temperature must be cold enough to condense the solvent. time t-2 --- after some time, the column temperature has been raised, most of the solvent has evaporated, and the solvent effect has left the sample molecules concentrated in a narrow band. As the column is further heated, the remaining solvent and sample molecules are rapidly vaporized --- resulting in high column efficiency and narrow peak.
Syringe for solid phase microextraction. Sampling by SPME and desorption of analyte from the coated fiber into a gas chromatograph.
Purge and trap apparatus for extracting volatile substances from a liquid or solid by flowing gas.
GC column
Parts of Column
1) Tubing material Stainless steel--- reactive ( steroids, amines, free acids ) Glass ------------ can be made inert, difficult handling Fused silica ---- flexible most inert most popular high resolution 2) Stationary phase Solid support --- carefully sized granular
Important column parameters 1) Inside diameter 2) Length 3) Film thickness 4) Stationary phase composition 5) Flow rate
Column diameter
i.d. Resolution Speed Capacity Ease
100 micrometer (narrow bore ) 250, 320 (mid bore) 530 (wide bore)
Column length N L
+++
++
+++
++
+
++
+
++
++
+++
+++
R L1/2
tR L
Column
24-foot 1/8" packed column wound on 6" coil 6-foot 1/4" packed column wound on 5" coil 60-meter 0.53mm metal wide bore capillary column wound on 3.5" coil 15-meter 0.53mm fused silica wide bore capillary column wound on 7" cage
Glass wool --- both ends of the column 1- (inlet side) 1/4 (detector side)
30-meter .25mm metal narrow bore capillary column wound on 3.5" coil
http://www.srigc.com/catalog/columns.htm
Fused silica
- High tensile strength - Flexible - Sheath of polyimide - Very inert
Tubing - Fused silica - Glass - Stainless steel Liquid phase coating WCOT - - - High resolution
Film thickness : 0.5 to 5.0 micrometer i.d. : 0.10, 0.25, 0.32, 0.53 mm Length : 10 to 60 m
Trennzahl(separation) number
per 25 m Optimum flow for N2, ml/min * Optimum flow for He, ml/min ** Optimum flow for H2, ml/min *** 40 0.5~1 1~2 2~4 35 0.8~1.5 1~2.5 3~7 25 2~4 5~10 8~15
* Optimum velocity is 10 to 15 cm/s for each column ** Optimum velocity is 25 cm/s for each column *** Optimum velocity is 35 cm/s for each column
Porous carbon stationary phase ( 2 m thick) on inside wall of fused silica open tubular column.
Theoretical plates(N/m)
Total plates length(N/m)
3000-5000
180000-300000
2000
4000
(Left) Gas chromatogram of alcohol mixture at 40 oC using packed column ( 2mm I.D., 76 cm long containing 20 % Carbowax 20 M on a Gas-Chrom R support and FID. (Right) Chromatogram of vapors from headspace of beer can, obtained with 0.25 mm diameter, 30 m long porous carbon column oerated at 30 oC for 2 min and then ramped up to 160 oC at 20 oC/min.
Component Separation with the Column < The process of separation >
A series of partitions : Dynamic In-and Out (or Stop-and-Go) All differential migration process. The most volatile components usually pass through the column first, the least volatile or highest boiling emerges last.
Analytes
Conditioning of the Column Once the column has been checked for proper installation and the absence of leaks, it is ready for conditioning. Heat the column to its isothermal, upper temperature limit (temperature limits listed below) or a temperature 10-20 oC above the highest operating temperature of your particular method. Do not exceed the upper limit or column damage will result. Heat the column rapidly - slow temperature programming is not necessary. After the column has reached the conditioning temperature, plot the baseline. Keep the baseline on scale so that it can be observed. The baseline should be elevated at first then start to drop after 510 minutes at the conditioning temperature. The baseline will continue to drop for 30-90 minutes then stabilize at a constant value. If the baseline does not stabilize after 2-3 hours or does not start to significantly decrease after 15-20 minutes, either a leak is present or a contamination problem exists. In either case, immediately cool the oven down below 40 oC and resolve the problem. Continued conditioning will result in column damage or the inability to obtain a stable baseline. Excessive conditioning of the column may result in a shortened lifetime. In general, polar stationary phases and thick film columns usually require longer times to stabilize than less polar and thinner film columns. GS PLOT columns require a different conditioning procedure than liquid stationary phase columns.
GS-Molesieve, GS-Alumina and GS-Q columns require special conditioning procedures. The following conditions are recommended. Column: Temperature: GS-Q: 250 oC for a minimum of 8 hours GS-Molesieve: 300 oC for 3 hours or 250 oC for 12 hours GS-Alumina: 200 oC for a minimum of 8 hours GS-Alumina and GS-Molesieve are susceptible to retention shifts from reversible absorption of water. If retention shifts are observed after analyzing high water content samples, recondition the column to remove any water trapped by the stationary phase. If the column is conditioned with the detector end disconnected, a small portion of the columns end may be damaged. Remove 10-20 cm of the exposed column end before installing the column into the detector.
Temperature Limits
The temperature limits define the range over which the column can be safely used. If the oven is operated below the lower temperature limit, poor separation and peak shape problems will be evident, but no column damage will occur. Upper temperature limits are usually given as two numbers. The first or lower temperature of the two is the isothermal limit. The column can be maintained at this temperature for indefinite periods of time. The second or higher temperature is the program limit. The column can be heated to this temperature for short periods of time (<10 minutes). Exceeding the upper temperature limits will significantly reduce column lifetime.
Column Dimethylpolysiloxane
-60 to 325/350 oC
-60 to 280/300 oC -60 to 360 oC -60 to 300/320 oC -60 to 260/280 oC -60 to 400 oC
DB-WAX DB-WAX; Megabore Base Deactivated Polyethylene Glycol CAM Carbowax 20M Carbowax
20 to 250/260 oC 20 to 230/240 oC
60 to 220/240 oC
60 to 220/240 oC
-60 to 325/350 oC -60 to 300/320 oC -60 to 260/280 oC -60 to 400 oC -60 to 325/350 oC -60 to 300/320 oC -60 to 325/350 oC
Temperature programming
With homologues, the retention time increases exponentially with the number of carbon. As retention time increases, width increase and the height decreases, making detection impossible after a few peaks have eluted. Since solubility of gas in a liquid decreases as temperatures goes up, we can reduce the retention of a compound by increasing column temperature.
General steps to create a program assuming that the separation is possible 1) Determine initial temperature and time based on best possible separation offirst few peaks 2) Report for the last few peaks to find the best final temperature and time 3) Experiment with various ramps to account for the rest of the components
Temperature programming
Factors to consider :
Must stay within Tmin/Tmax of column Other factors are found experimentally
A temperature program
Ex. 40 oC(5 min) 10oC/min 250oC(10 min) C B A
A: initial temperature and holding time B: ramp (oC/min) C: final temperature and holding time
Some GCs will allow for a more complex program.
3)
Sharpens peaks
Comparison of isothermal and programmed temperature chromatography. Each sample contains linear alkanes run on a 1.6 mm 6 m column containing 3% Apiezon L (liquid phase) on a 100/200 mesh VarAport 30 solid support with He flow rate of 10 ml/min.