introduction and principles of gas chromatography - …117).pdf · introduction and principles of...

Copyrights: Restek Corporation

Introduction and Principles of Gas Chromatography

Introduction and Principles of Gas Chromatography

Jaap de Zeeuw

Restek, Middelburg, The Netherlands

[email protected]


• Definition and Uses of Gas Chromatography

• GC Components and Types of Columns

• Factors Affecting Chromatographic Separation

• Basic Terminology and Theory


Which Industries Use Chromatography?Which Industries Use Chromatography?

• Chemical/Petrochemical

• Clinical/Forensic

• Consumer Products

• Environmental

• Food

• Pharmaceutical


Why Gas Chromatography?Why Gas Chromatography?

• Simple

• Cheap (can be automated)

• Short analysis times

• High Accuracy

• Qualitative and Quantitative analysis

• Applicable in % to ppb level


General• Nitrogen

• Hydrocarbons

• PCBs*

• Tars, Oils, waxes

• Radon

• Olefins

• Sulfur components

• Ethylene Glycol

• Aldehydes

• Phenols & cresols

• Amines

• Organic sulfur

• Organo metallic compounds

• Oxygenates

Which components do we see in Natural Gas?Which components do we see in Natural Gas?

* Ref: California proposed Public Gas Warning List


Important Separations in Natural Gas analysisImportant Separations in Natural Gas analysis

50 m x 0.32 mm CP-Sil 5 CB, 1.2 um

Helium, 160 kPa

40 °C, --> 200 °C, 5 °C/min

C8C9

C10C11

40 min

C6

C5

50 m x 0.32 mm CP-Sil 5 CB, 5 µm

40,2 °C

H2, 65 kPa; TCD

C4i-C4

C3

H2S

C2

CO2

CH4 + N2

10 min

•Methanol well separatedfrom matrix

•Symmetrical peak formethanol

•Methanol well separatedfrom matrix•Symmetrical peak for

methanol

Methanol 20 ppm

Natural gas peak

10 m x 0.53 mm CP-Lowox

150°C(2 min) -->200°C, 10 °C/min

Direct Injection, 50 µl

9 min

Hydrocarbons Nitrogen - methane

OxygenatesSulfur

RtxRtxRtxRtx----1 Thin1 Thin1 Thin1 Thin----film film film film Rt QRt QRt QRt Q----BONDBONDBONDBOND

RtxRtxRtxRtx----1 thick1 thick1 thick1 thick----filmfilmfilmfilm Polar phase (wax)Polar phase (wax)Polar phase (wax)Polar phase (wax)


IUPAC Definition of ChromatographyIUPAC Definition of Chromatography

“A physical method of separating sample components from a mixture by selective adsorption or partitioning of the analyte between two phases:

a “mobile phase and a stationary phase”


Chromatography PhasesChromatography Phases

Mobile Phases

• Liquids (methanol, water…)

• Changing dielectric strength, temperature, pH

• Gases (nitrogen, helium, hydrogen, argon)

Stationary Phases

• Solids (alumina, silica, polymers, carbon…)

• Adsorption chromatography

• Liquids (siloxanes, polyethylene glycols…)

• Partition (distribution) chromatography


stationary phasesstationary phases• Specialized polymer chemists

• Lowest specifications possible

• Innovation, unique phase technologies

High purity /viscosity polymers for low-bleed and stable stationary phases

Commercial phases are very dirty..


GC Components and Types of ColumnsGC Components and Types of Columns

• Components of a Gas Chromatograph

• Types of GC Columns

• Types of GC Capillary Columns


Components of a Gas ChromatographComponents of a Gas Chromatograph


12

Gas Purification EquipmentGas Purification Equipment

Triple Filter


Saturated O2 Indicator

New O2Indicator

Triple filter

O2 indicator

Triple filter

O2 indicator

Color change

New Filter Used Filter


Triple filter

H2O indicator

Triple filter

H2O indicator

Used FilterNew Filter

New H2O Indicator

Saturated H2O Indicator

Color change


Components of a Gas ChromatographComponents of a Gas Chromatograph


Types of GC columnsTypes of GC columns


Types of GC ColumnsTypes of GC Columns

Packed Capillary

Length, [meters] 1-6 5 - 150

ID, [millimeters] 0.53-4 0.1 - 0.53

Theoretical plates 5,000 (2m) 120,000 (30m)

Capacity [ng] 10,000 50 (0.25mm ID)

Amount of Liquid phase 1-30 % 0.1-7.0 μm

Price €100 €400 (30m, 0.25 mm ID)


Capillary Column MaterialsCapillary Column Materials

Fused Silica

• Synthetic, amorphous glass with very low (<1ppm) metallic oxide impurities

• Protective outer coating of polyimide resin imparts flexibility but coil diameter is limited

• Excellent inertness and useable up to approximately 380oC(400oC)

Metal Tubing-MXT

• MXT, stainless steel, surface coated, Siltek deactivated• Can be coiled to small diameter• Almost as inert as fused silica but useable up to approx. 450ºC


Types of GC Capillary Columns Types of GC Capillary Columns

WCOT (Wall Coated Open Tubular)• Partition chromatography

• Typical phases: Siloxanes and Polyethylene glycols

• 0.10 to 0.53mm internal diameters

PLOT (Porous Layer Open Tubular)• Adsorption chromatography

• Gases, light hydrocarbons/solvents analysis

• Adsorbents: molecular sieve, porous polymers, alumina (<1 um particle diameter)

• 0.25 to 0.53mm internal diameter


Compounds Amenable to Gas ChromatographyCompounds Amenable to Gas Chromatography

• Should be thermally stable

• Should be un-reactive and non-absorptive to chromatographic system

• Should be volatile at temperatures below 350-400°C

• Presence of polar groups reduces volatility


Sample TransferSample Transfer

Injection: how the sample is transferred to the column

• As a liquid via syringe

• Non-liquid techniques

• Purge & trap

• Headspace

• Gas sample loop

•NOTE: It is critical to get the sample into the column in a focused band…•NOTE: It is critical to get the sample into the column in a focused band…


Peak width of eluting componentPeak width of eluting component

σ injection + σ column + σ detection = Σ peak

+

++

+ =

=


Sample introductionSample introduction

Need to realize smallest possible sample band on the capillary column..

Injection Techniques commonly used:

• Split

• Splitless

• Thermal desorption (PTV)

• Headspace

• On-Column

% - 5 ppm levels

Small amount injected

Narrow injection band

ppm-ppb levels

Large amount injected

Must use a focusing mechanism..


Sample TransferHow to Get a Focused Initial Band

Sample TransferHow to Get a Focused Initial Band

• Analyte Focusing

• Higher boiling components

• Increase retention (stationary phases type, film and temperature)

• Solvent Focusing

• All components, but must elute later then solvent itself


Analyte focusingAnalyte focusing

• If analytes are high boiling, they will be focused by the retention of the stationary phase

• If analytes have lower boiling points this will show itself as “smeared” peaks


Analyte focusing: exampleAnalyte focusing: example

“Smeared” peaksNot enough retention for focusing

“focused” peaks


• In splitless injection some solvent condensation is required to create the ”solvent” effect.

• This solvent will trap (= focus) compounds and makes sure that a narrow band is formed.

• Realization: Oven temperature during injection must be 20°C below the Bp of the solvent.

Solvent-Focusing in splitless injection

Copyrights: Restek Corporation2 4 6 8 10

Splitless Injection at oven temperature 20°C BELOW BP of solventSplitless Injection at oven temperature 20°C BELOW BP of solvent

The “solvent effect” makes sure all peaks are focused


No Focusing:

Long Initial Sample Band, broad peaks

Focusing:

Correct solvent peak and narrow peaks

Focusing in Splitless injectionFocusing in Splitless injection

2 4 6 8 10


Split/Splitless injection systemSplit/Splitless injection system

Silicone septum


Split injection ConsiderationsSplit injection Considerations

• 1-3 mL/min into the column

• 10-200 mL/min in the inlet (mostly split vent flow)

Split Ratio:Column flow rate : 1 mL/min

Split vent flow rate : 100 mL/min

Split ratio = Column Flow rate = 1 Split-vent + Column flow rate 100+1

Split ratio ~ 1:100


0 10

25:1 Split

0 10

50:1 Split

Impact of Split Ratio Impact of Split Ratio

Increasing the split ratio decreases the peak area, if all other variables are equal.

Column Head Pressure

Total Flow Septum Purge

Split Vent Purge Vent

SPLIT


Factors impacting the separationFactors impacting the separation


Non-Column Factors Affecting SeparationNon-Column Factors Affecting Separation

• Carrier gas: type & linear velocity

• Temperature

• Injection bandwidth


Gas Carrier and Linear VelocityGas Carrier and Linear Velocity

Hydrogen : 40-50 cm/sec

Helium : 25-35 cm/sec

Nitrogen : 10-15 cm/sec


Carrier Gas and Linear VelocityCarrier Gas and Linear Velocity

• Isothermal Analysis

• Hydrogen is 2x faster than helium and 4x faster than nitrogen

• Temperature Programmed Analysis

• Need to optimize temperature program to get SAME elution temperatures;

• Also here Hydrogen is 2x faster then helium


Hydrogen is of interestHydrogen is of interest

• Fastest analysis

• Availability ( can be generated)

• Need less sample for same signal (maintenance)

Deal with safety issues

• Setting of constant flow (impossible to built up high H2 levels

• Use H2-detection (will cost $, but makes safety officer happy)

• Use metal (MXT) columns (also VERY inert)


Column lengthColumn length

10/15 m < 10 components and Fast analysis

25/30 m 10 - 20 components

50/60 m complex mixtures: > 20 components

Most widely used is 30m

Do we need extreme LONG columns?


Summary:Effect of Length

Summary:Effect of Length

• Retention time proportional to length in isothermal analysis but not proportional in temperature program analysis

• Gain in resolution is not double, but € are

The BETTER the

Chromatographer….. ... the SHORTERthe column..


Examples where we need LONG columnsExamples where we need LONG columns

• Detailed Hydrocarbon Analysis (>300 components)

• Bio-ethanol (to get isobutane-methanol separation)

• Separation of PCB isomers (209 congeners)

• Separation of Cis and Trans FAME isomers


Column Internal DiameterColumn Internal Diameter

0.53 mm

• High flow and loadability

• Direct injection via insert or valve (analyzer)

• TCD detection

• Retention gap for On-column

0.32 mm

• On-column injection;

• Thick films are possible

• Electronic Gas / Pressure Control

0.25 mm

• Ideal for split and splitless injection

• Relatively high plate number

0.18/0.15/0.10 mm

• Short analysis times

• Low bleed / GC x GC


Internal diameterInternal diameter

0.10mm 0.15/0.18mm 0.25mm 0.32mm 0.53 mm

Practically the following dimensions are used :

0.10 <1 %

0.15/0.18 mm 3 %

0.25 mm 45 %

0.32 mm 35 %

0.53 mm 10 %


Basic Terminology and TheoryBasic Terminology and Theory

• Resolution (R)

• Theoretical Plates (Neff)

• Height Equivalent to a Theoretical Plate (HETP)

• Phase Ratio (ß)

• Retention (Capacity) Factor (k)

• Retention Time (t)

• Column Selectivity Factor (α)


The Resolution RsThe Resolution Rs

� Quality of Separation between 2 Peaks

� Dimensionless

� Parameters:

- Selectivity

- Retention

- Efficiency


ResolutionResolution

Resolution depends on:

α : Selectivity

k’ : Retention Factor

Nth : Plate Number


Impact of Nth on resolutionImpact of Nth on resolution

Increase N:

Longer column

Smaller Internal diameter

Impact of Higher N using 2x

smaller diameter, same length


Impact of k on resolutionImpact of k on resolution

Increase k:

use thicker film (same column dimensions)

decrease oven temperature (ever 15C, k changes a factor 2)

CH4Impact of using 2x thicker film,

Same column dimensions and

temp.


Impact of α on resolutionImpact of α on resolution

Increase alpha:

use different stationary phase (same column dimensions)

CH4Impact of using different phase

with higher selectivity


Effective Theoretical Plates (Neff)Effective Theoretical Plates (Neff)

Dead

Time Wb

Neff = 16 ( )t'W

R2

b

t'R

•Adjusted Retention Time = Retention Time – Dead Time•Wb = Width between tangents of a peak at baseline intercept


Theoretical platesTheoretical plates

• This nr is the “number” of “touches” of a component with the stationary phase, while it moves through the column.

1 touch = 1 plate


One theoretical plate

Two Theoretical plates

Three Theoretical plates

Carrier gas


Theoretical platesTheoretical plates

• This nr is the “number” of “touches” of a component with the stationary phase, while it moves through the column.

• The more “touches”, the more plates, the better the separation

• Impacted by:

• Column diameter

• Column length

1 touch = 1 plate


Height Equivalent to a Theoretical Plate (h)Height Equivalent to a Theoretical Plate (h)

H depends on Flow and Column ID

Lower h = Improved Separation

=hL

N

Basic equation used in GC: Van Deemter equation


Van Deemter equationVan Deemter equation

H = A + B/u + Cu

H = height of a theoretical plate

U = average linear gas velocity

A, B, C = different contribution factors to peak broadening


The A - termThe A - term

Contribution to peak broadening due to different path length (eddy diffusion)

For capillary columns A = 0


The B - termThe B - term

Contribution to peak broadening due to multidirectional diffusion in the Gas phase

Indirect proportional to flow rate


The C - termThe C - term

Resistance to mass transfer in the liquid phase

Direct proportional to flow rate


Van Deemter: there is an optimal flowVan Deemter: there is an optimal flow


Van deemter: Gas Carrier and Linear VelocitiesVan deemter: Gas Carrier and Linear Velocities

1.0

0.6

0.2

HETP (

mm

)

Van Deemter Plot

Average Linear Velocity (cm/sec)

N2

He

H2

10 20 30 40 50 60 70


Stationary phase Film ThicknessKapacity factor “K” and RetentionStationary phase Film ThicknessKapacity factor “K” and Retention


Retention (Capacity) Factor : kRetention (Capacity) Factor : k

Practical the most effective separation occurs when the k value for an analyte is minimal 5.


Retention (k) can be influencedRetention (k) can be influenced

• By film thickness

Retention is LINEAR with film thickness

• By temperature

Every 15°C change in oven temp. the k will change about a factor 2


Film Thickness and Beta (phase ratio)Film Thickness and Beta (phase ratio)

0.25 μm 1.0 μm 3.0 μm

k is linear with Film thickness

6 min0.5 min 2 min


Phase Ratio (ß)Phase Ratio (ß)

β =column radius

2x film thickness

mobile phase volume

stationary phase volume =

Phase ratio is important if you want to change column internal diameter;For the most easy method conversions, one should try to keep the phase ratio the same


Film Thickness Effects : 0.25µm Rtx-1Film Thickness Effects : 0.25µm Rtx-1

2 4 6 [min]

1-6

78

9

30m, 0.32mm ID, 0.25µm Rtx-1

70ºC isothermal 1. 1-butanol 2. benzene 3. 2-pentanone 4. C7

5. 1-nitropropane 6. pyridine7. C8

8. C9

9. C10 5.5 min

K C10 = 4.5


1. 1-butanol 2. benzene 3. 2-pentanone 4. C7


8. C9

9. C10 19 min.

1-6

7

89

Film Thickness Effects: 1.0µm Rtx-1Film Thickness Effects: 1.0µm Rtx-1

K C10 = 18

30m, 0.32mm ID, 1.00µm Rtx-1

70ºC isothermal

[min]0 10 20


4 8 12 [min]

7

8

16 20 24

6

5

4

1,2,3

Film Thickness Effects : 3.0µm Rtx-1Film Thickness Effects : 3.0µm Rtx-1

Peak 9 elutes at 55 min..

K C10 = 54

1. 1-butanol 2. benzene 3. 2-pentanone 4. C7


8. C9

9. C10 55 min.

30m, 0.32mm ID, 3.0µm Rtx-1

70ºC isothermal

0


Stationary Phase: Column Selectivity (α)Stationary Phase: Column Selectivity (α)

• Interactions with the stationary phase

• pi – pi

• Van der Waals (london)

• Hydrogen bonding

• Depends upon the Chemical composition of the phase


Column SelectivityChemical Composition of PhasesColumn SelectivityChemical Composition of Phases

Rtx®-1 Stationary Phase100% dimethylpolysiloxane


Column SelectivityChemical Composition of PhasesColumn SelectivityChemical Composition of Phases

Rtx®-5 Stationary Phase5% diphenyl

95% dimethylpolysiloxane


Column Efficiency, Selectivity, and Peak SymmetryColumn Efficiency, Selectivity, and Peak Symmetry

Not Efficient, but Selective

Efficient and SelectiveEfficient, but not Selective

Not Efficient, not Selective


Examples of selectivity..


Selectivity via geometrySeparation of Para - en Meta XylenesSelectivity via geometrySeparation of Para - en Meta Xylenes

100 % methyl

Rtx-1

50 % phenyl

50 % methyl

Rtx-17

PEG

Rtx-WaxRt-TCEPSqualane

P

P

P + M P + MPMM

M

OO

O

O

O

Non-Polar Polar


Shape selectivityShape selectivity

Using selected cyclodextrins as modifiers

p-xylene

m-xylene

o-xylene


Basic rule in GC stationary phase..Basic rule in GC stationary phase..

Solubility of sample component in the stationary phase based upon “likes dissolve likes.”

“ choose a stationary phase that “looks like” the components you want to separate..”

Hydrocarbons 100% PDMS Rtx-1

Aromatic subst. Phenyl subst. PDMS Rtx-5, 17, 35 RT-Dioxins

Halogenates Arom. Fluorimated-phenyl Rtx-440, Cl-Pesticides, Rtx-200

Solvents Cyano /phenyl Rtx-1301/624

Alcohols PEG Stablewax

Double bonds Cyano propyl Rt 2330, 2460


(GC-MS) Presence of Diethylene Glycol and Ethylene Glycol in Toothpaste (GC-MS) Presence of Diethylene Glycol and Ethylene Glycol in Toothpaste

Column: Stablewax

Dissolves : ‘likes like”


Hydrocarbons on 100% PDMSHydrocarbons on 100% PDMSC

3n-C

4

n-C

5

n-C

6

n-C

7

n-C

8

n-C

9

n-C

10

0 50 100 150

Column : 100 x 0.25 mm Rtx-1 PONA CB, tuned 5%phenyl PDMS

Oven : 5°C, 10 min -> 50°C, 5°C/min, 54 min, --> 200°C, 1.3 °C/min

Carrier gas : He, 24 cm/s, 39.3 Psi; Injection Split, 1 : 150; Detection : FID;


PAHs using an Rxi-17Sil MS (30m x 0.25mm x 0.25μm)PAHs using an Rxi-17Sil MS (30m x 0.25mm x 0.25μm)

benzo(b

)flu

ora

nth

ene

benzo(k

)flu

ora

nth

ene benzo(j

)flu

ora

nth

ene

12

3

45

6

78

910

11

12/13

14,15,16

17

18

19

20

21/22

2324

25

26

27


Detection in GCDetection in GC

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