basic gas chromatography prepared by: mina s. buenafe

Basic Gas Basic Gas ChromatographyChromatography

Prepared by: Mina S. Buenafe

Gas Chromatography•Chromatography – A Very Brief History

•Definitions / Terminologies in GC

•Instrumentation Overview

•System Modules

•Mobile Phase (Carrier Gas)

•Inlets

•Stationary Phase(s)

•Columns (Packed and Capillary)

•Detector(s)

•Troubleshooting

Chromatography – A (Very) Brief History

W. Ramsey published his work on separation of mixture of gases and vapors on adsorbents like charcoal.

M. Tswett published his work on separation of plant pigments. He coined the term chromatography (literally translated as color writing) and scientifically described the process – earning him the title “Father of Chromatography”

IN THE EARLY 1900’S

IN THE EARLY 1940’s

A. Martin and R. Synge first suggested the possibilities of gas chromatography in a paper published in Biochem. J., v.35, 1358, (1941). Martin won a Nobel Prize for his work in Partition chromatography.

IN THE EARLY 1950’s

A. Martin and A. James published the epic paper describing the first gas chromatograph

Definition of Terms

Chromatography:

A physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary while the other moves in a definite direction

“Official” IUPAC definition

Definition of Terms

Chromatogram It is the output signal from

the detector of the instrument.

Distribution Constant (KC)

It is the tendency of a given component to be attracted to the stationary phase. This can be expressed in chemical terms as an equilibrium constant. Also called the partition coefficient (KP) or the distribution coefficient (KD)

Definition of Terms

KC = [A]S/[A]M

Mathematically, it is defined as the concentration of solute A in the stationary phase divided by its concentration in the mobile phase.

Definition of Terms

The attraction to the stationary phase can also be classified according to the type of sorption by the solute.

Adsorption: sorption on the surface of the stationary phaseAbsorption: sorption into the bulk of the stationary phase

(usually called ‘partition’ by chromatographers)

Definition of Terms

Retention Volume (VR)

It is usually defined as the distance between the point of injection to the peak maximum. It is the volume of the carrier gas necessary to elute the solute of interest.

Mathematically: VR = FC x tR

Where FC is the constant flow rate

tR is the retention time

Definition of Terms

Phase Ratio ()

For packed columns: Mobile Phase Volume Stationary Phase Volume

For capillary columns: rc/2df

Where rc is the radius of the column df is the thickness of the film

Definition of Terms

Retention Factor (k)It is the ratio of the amount of the solute

(NOT concentration) in the stationary phase to the amount in the mobile phase. It is also called capacity factor (k’), capacity ratio, or partition ratio

Mathematically: k = (WA)S/(WA)M = KC/

Also k = (tR - t0) = time in stationary phase

t0 time in carrier gas

k is temperature and flow dependent. Best separations occur when k is between 5 and 7

Definition of Terms

Theoretical Plates (N)

This is the most common measure of column efficiency in chromatography

N = 16(tR/Wb)2 = 5.54(tR/ Wh)2

Where Wb is the peak width at the base

Wh is the peak width at half-height

Definition of Terms

Height Equivalent to a Theoretical Plate (H)

This is a related parameter that also defines column efficiency. Also identified as HETP

Mathematically: H = L/N

Where L is the Column Length

(An efficient column will have a large N and a small H)

Separation Factor ()

It is a measure of relative distribution constants. Also known as selectivity and/or solvent efficiency.

Mathematically: = k2/k1 = (KC)2/(KC)1

It is dependent on: Chemical composition of the phase Partitioning between the two phases

Definition of Terms

Definition of Terms

Resolution (Rs)

It is the degree to which adjacent peaks are separated.

Mathematically: Rs = (tR)B – (tR)A

[(Wb)B + (Wb)A]/2

Also Rs =—L/H x k/(k+1) x -1/

Instrumentation Overview

Schematic of a Typical Gas Chromatograph

System ModulesCarrier Gas

Main purpose: carries the sample through the column

Secondary purpose: provides a suitable matrix for the detector to measure the sample component.

Detector Carrier Gas

Thermal Conductivity Helium

Flame Ionization Helium or Nitrogen

Electron Capture Very dry Nitrogen or Argon, 5% Methane

Carrier gases should be of high purity (minimum of 99.995%).

• Oxygen & water impurities can chemically attack the liquid phase of the column and destroy it.

• Trace water content can desorb other column contaminants and produce high detector background or ‘ghost peaks’.

• Trace hydrocarbon contents can cause high detector background with FID’s and limit detectability.


Flow Measurements and Control:

• Essential for column efficiency and qualitative analysis (e.g. reproducibility of retention times)

Average linear flow velocity (ū) in OT columns:

ū = L/tm

where L is column length in cm

tm is the retention time of an unretained peak (e.g. methane) in sec

To convert linear flow velocity to flow rate (Fc) in mL/min:

Fc = ū x r2 x 60sec/min


Effect of mobile phase (carrier gas) density on column efficiency. Van Deemter plots for the 3 common carrier gases for a column of capacity factor k’ = 7.90. The low density gases (H2 & He) have optimum efficiency at slightly higher flow rates than N2. The much lower slopes of H2 and He curves allow them to be used at higher flow rates (compared to N2) with very little loss of separation efficiency.

System Modules

Inlets

Inlets are the points of sample introduction

Ideal Sample Inlets for Column Type:

Packed Columns Capillary Columns

Flash Vaporizer Split

On-Column Splitless

On-Column

System ModulesInlets

Split Injector

Cross section of a typical split injector

The oldest, simplest, and easiest injection technique.

Advantages to Split Injection:

•High resolution separations

•Neat samples can be introduced.

•Dirty samples can be introduced by putting a deactivated glass wool plug in the liner to trap non-volatile components

Disadvantages:

•Trace analysis is limited

•Process sometimes discriminates between high molecular weight solutes so that the sample entering the column is not representative of the sample injected.

System Modules

Inlets

Splitless Injector

Cross section of a typical splitless injector

Samples have to be diluted in a volatile solvent and 1-5mL is injected in the heated injection port. Septum purge is essential in splitless injections.Advantages to Splitless Injection:

•Improved sensitivity over a split injector

Disadvantages:

•Time consuming

•Initial temperature and time of opening the split valve needs to be optimized.

•Not well suited for volatile compounds (boiling points of peaks of interest have to be about 30oC higher than solvent.

System Modules

Inlets

Other Types of Inlets:

• Direct Injection: involves injecting a small sample into a glass liner where vapors are carried directly into the column.

•On-Column Injection: inserting the precisely aligned needle into the capillary column and making injections inside the column.

•Flash Vaporization: involves heating the injection port to a temperature well above the boiling point to ensure rapid volatilization

•Static Headspace: concentrates the vapors over a solid or liquid sample (best for residual solvent analysis)

System Modules

Stationary Phase

Sub-classification of GC Techniques

• GSC: gas solid chromatography - stationary phase is solid

• GLC: gas liquid chromatography -stationary phase is liquid

System ModulesStationary Phase

Gas Solid Chromatography (GSC)

• Solids used are traditionally run in packed columns

• These solids should have small and uniform particle sizes (e.g. 80/100 mesh range)

Some Common GC Adsorbents

Commercial/Trade Names

Silica Gel Chromasil®, Porasil®

Activated Alumina Alumina F-1, Unibeads-A®

Zeolite Molecular Sieves MS 5A, MS 13X

Carbon Molecular Sieves Carbopack®, Carbotrap®, Carbograph®, Graphpac®

Porous Polymers Porapak®, HayeSep®, Chromosorb®

Tenax Polymers Texan TA®, Tenax GR®

•Some of these solids have been coated on the inside walls of capillary columns and are called “Support Coated Open Tubular” or SCOT columns.


One major application of Packed Column GSC is in Gas Analysis.

Reasons:

• Adsorbents provide high surface areas for maximum interaction with gases that may be difficult to retain on liquid stationary phase.

• Large samples can be accommodated, providing lower absolute detection limits.

• Some packed column GC’s can be configured to run below ambient temperature which will also increase the retention of the gaseous solutes.

• Unique combinations of multiple columns and/or valving make it possible to optimize analysis of a particular sample.

Packed Columns also provide the flexibility of allowing mixed packings for special applications (e.g. 5% Fluorcol on Carbopack B® for analysis of Freons)

System Modules

Gas Liquid Chromatography (GLC)

To use liquid as stationary phase,techniques were applied to hold the liquid in a column.

• For packed columns: liquid is coated onto a solid support, chosen for its high surface area and inertness. The coated support is then dry-packed into a column as tightly as possible.

• For capillary or open tubular (OT) columns: liquid is coated on the inside of the capillary. To make it adhere better, the liquid phase is often extensively cross-linked and sometimes chemically bonded to the fused silica surface.

Stationary Phase

Schematic representation of (a) packed column and (b) capillary column

System Modules

Stationary Phase


Requirements for the stationary liquid phase:

•Low vapor pressure

•Thermal stability

•(if possible) Low viscosity (for fast mass transfer)

•Should interact with the components of the sample to be analyzed (“Like dissolves like”)



Types of Capillary Columns (OT)

WCOT: Wall-coated open tubular column (provides the highest resolution of all OT’s – i.d.’s range from 0.1mm to 0.53mm and film thickness from 0.1 – 5.0)

PLOT: Porous layer open tubular column (less than 5% of all GC use these days)

SCOT: Surface-coated open tubular column (no longer available in fused silica)

Detectors

System Modules

The part of the system that ‘senses’ the effluents from the column and provides a record of the analysis in the form of a chromatogram. The signals are proportional to the quantity of each analyte.

System Modules

Detectors

FID: Flame Ionization Detector

The most common GC detector used.

The column effluent is burned in a small oxy-hydrogen flame producing some ions in the process. These ions are collected and form a small current that becomes the signals. When no sample is being burned, there should be little ionization, the small current is produced from impurities from the from the hydrogen and air supplies.

Hydrogen flow rate is commonly set to 40 – 45mL/min, Air, 350- 450mL/min, and for OT columns (with flows of about 1 mL/min), Make-Up gases is added to carrier gas (to make up the flow to 30mL/min)

System Modules

Detectors

TCD: Thermal Conductivity Detector

This is a differential detector that measures the thermal conductivity of the analyte in the carrier gas compared to the thermal conductivity of the pure gas. At least two cavities are required. These cavities are drilled into a metal block and each contain a hot wire or filament. The filaments are incorporated into a Wheatstone Bridge Circuit (for resistance measurements).

The choice of carrier gas will depend on the thermal conductivity of the analyte (H2 and He have highest TC’s, N2 gives rise to unusual peak shapes)

NPD: Nitrogen Phosphorus Detector

A bead of Rb or Cs is electrically heated when flame ionization occurs. The detector shows enhanced detectability for nitrogen-, phosphorus-, or halogen- containing samples.

System ModulesDetectors

System ModulesDetectors

MSD: Mass Spectrometric Detector

Analyte molecules are first ionized in order to be attracted or repelled by the proper magnetic or electrical fields.

Troubleshooting

• Retention Time Problems

• Resolution Problems

• Baseline Problems

Common GC Problems:

• Peak Problems

Retention Time Problems

Retention Time Shift

Possible Cause Solution Comments

Change in carrier velocity

Check the carrier gas velocity

All peaks will shift in the same direction by approximately the same amount

Change in column temperature

Check the column temperature

Not all peaks will shift by the same amount

Change in column dimensions

Verify the column identity

Large change in compound concentration

Try a different sample concentration

May also affect adjacent peaks. Sample overloading is corrected with an increased split ratio, sample dilution, or decreased injection volume.

Leak in the injector or column connection

Leak-check the injector and column installation

Usually accompanied by peak size change.

Retention Time Problems

Retention Time Shift


Blockage in a gas line

Clean or replace the plugged line

More common for the split line; also check flow-controllers and solenoids.

Septum leak Replace the septum Check for needle barb.

Sample solvent incompatibility

Change solvent.Use a retention gap.

For splitless injector.

Contamination Trim the column.

Solvent-rinse the column.

Remove ½ - 1 meter from the front of the column.Only for bonded and cross-linked phase.

Resolution ProblemsLoss of Resolution

Decrease in separation


Different column temperature

Check column temperature

Differences in other peaks will be visible

Different column dimensions or phase

Verify column identity Differences in other peaks will be visible

Co-elution with another peak

Change the column temperature

Decrease column temperature and check the appearance of a peak shoulder or tail.

Column contamination – resulting in a change in column selectivity

Trim the column

Solvent-rise the column


Resolution ProblemsLoss of Resolution

Increase in peak widthPossible Cause Solution Comments

Change in carrier gas velocity

Check carrier gas velocity

A change in retention time also occurs

Column contamination

Trim the column

Solvent-rise the column


Inlet liner contamination

Clean or replace liner

Change in the injector

Check the injector settings

Typical areas: split ratio, liner, temperature, injection volume

Change in the sample concentration

Try a different sample concentration

Peak width increases at higher concentration

Improper solvent effect lack of focusing

Lower oven temperature.Choose different solvent for better solvent/sample/phase polarity match.Use a retention gap

For splitless injection.

Baseline Problems• Excessive Column Bleed


Thermal damage to the column

Remove column from detector and bake-out overnight, reinstall and condition as usual

Use GC maximum temperature function

Oxygen damage to column

Columns damaged by oxygen will usually need to be replaced although an overnight bake-out may be attempted

Perform periodic leak checks. Change septa regularly. Use high quality carrier gases. Install and maintain oxygen traps

Chemical phase Chemical phase damage to columndamage to column

Remove ½ to 5 meters Remove ½ to 5 meters from the front of the from the front of the columncolumn

Perform sample prep to remove inorganic acids and bases from the sample. Install guard column and trim frequently. If acids or bases must be used, choose HCl or NH4OH, or an organic alternative.

Baseline Problems• Erratic Baseline (drift, wander)

Possible Cause

Solution Comments

Inlet contamination

Clean the injectorTry a condensation test; gas lines may also need cleaning. Take steps to prevent sample backflash (reduce injection volume, lower inlet temperature, use larger volume liner


Bake-out column.Solvent-rinse the column

Limit bake-out to 1 – 2 hoursOnly for bonded and cross-linked phases.Check for inlet contamination

Incompletely-conditioned column

Fully condition the column

More critical for trace analysis

Un-equilibrated detector

Allow the detector to stabilize

Some detectors may require up to 24 hours to fully stabilize

Change in carrier gas flow-rate during the temperature program

Normal in many cases

MS, TCD, and ECD respond to carrier gas flow rate changes


Contaminated gases Use appropriate purifier to remove contaminants

More of a problem for detector gases.

Column and inlet liner misaligned

Check installation of column end and inlet liner, adjust if necessary

Causes a baseline change after a large peak

Large leak at the septum during injection and for a short time thereafter

Replace septumUse smaller diameter needle

Causes a baseline change after a large peak.Common with large diameter needles.

Sample decomposing

Remove inlet liner and check cleanliness.Use new, deactivated liner or replace glass wool and packing.

Causes a baseline rise before and after a peak.

Baseline Problems• Erratic Baseline (drift, wander)

Baseline Problems·Noisy Baseline


Inlet contamination Clean the injector, replace liner, gold seal

Try a condensation test; gas lines may also need cleaning.

Column contamination Bake-out the column.

Solvent-rinse the column.

Limit bake-out to 1 – 2 hoursOnly for bonded and cross-linked phases.Check for inlet contamination

Detector contamination

Clean the detector. Usually the noise increases over time and not suddenly.

Contaminated or low quality gases

Replace spent gas purifier.Use purifiers to remove contaminants.Use better grade gases.

More of a problem for detector gases.

Column inserted too far into the detector

Reinstall the column. Consult the GC manual for the proper installation distance.

Baseline Problems·Noisy Baseline

Possible cause Solution Comments

Incorrect detector gas flow rates

Adjust the flow rates to the recommended values.

Consult the GC manual for appropriate flow rates

Leak when using an MS, ECD, or TCD

Find and eliminate the leak.

Usually at the column fittings or injector.

Old detector filament, lamp, or electron multiplier, NPD head

Replace appropriate part.

Septum degradation Replace septum. For high temperature applications, use appropriate septum

Baseline Problems·Ghost Peaks


Contaminants introduced with sample

Sample or solvent clean-up Contaminants in sample process or solvent.

Inlet contamination Clean the injector, replace liner, gold seal, and septum

Try a condensation test; gas lines may also need cleaning. Take steps to prevent sample backflash (reduce injection volume, lower inlet temperature, use larger volume liner)

Septum bleed Replace septum.Use a high quality septum appropriate for the inlet temperature.

Contamination of sample prior to introduction to the GC

Check sample handling steps for potential contamination sources: sample clean-up, handling, transfer, and storage

Semi-volatile contamination (peak widths will be broader than sample peaks with similar retention

Bake out column,Solvent-rinse the column.Check for contamination in the inlet, carrier gas, or carrier gas lines.

Limit bake-out to 1 – 2 hours.Only for bonded and cross-linked phases.

Peak Problems·Fronting Peaks


Column overloadReduce mass amount of the analyze to the column. Decrease injection volume, dilute sample, increase split ratio

Most common cause for fronting peaks.

Improper column installation.

Reinstall the column in the injector.

Consult the GC manual for the proper installation distance

Injection technique. Change technique.Usually related to erratic plunger depression or having sample in the syringe needle. Use an autosampler.

Compound very soluble in injection solvent

Change solvent. Using a retention gap may help.

Mixed sample solvent Change sample solvent.

Worse for solvents with large differences in polarity or boiling points.

Peak Problems·Tailing Peaks


Severe column contamination

Trim the column.Solvent rinse the column

Remove ½ - 1 meter from the front end of the column. Only for bonded or cross-linked phase.Check for inlet contamination. Tailing will sometimes increase with compound retention.

Active column Cut off 1-meter from the front end of the column.Replace column.

Only affects active compounds. Usually produces tailing that increases with retention.

Improper column installation, leak, or column end poorly cut

Re-cut and reinstall the column into the inlet.Replace ferrule.Confirm installation is leak-free

Make a clean square cut with a reliable cutting tool.Consult GC manual for the proper installation distance.More tailing for early eluting peaks.

Contaminated or active liner or gold seal

Use new, deactivated liner.Clean or replace gold seal.

Only affects active compounds.

Peak Problems


Solid particles in liner Clean or replace liner.

Needle hitting and breaking packing in inlet liner

Partially remove packing from liner or use without packing.

Solvent/column not compatible

Use a different solvent.

Use a retention gap.

More tailing for the early eluting peaks or those closest to the solvent front.3 – 5 meter retention gap is sufficient.

Split ratio too low Increase split ratio. Flow from split vent should be 20mL/min

Solvent effect violations for splitless or on-column injections

Decrease the initial column temperature to 10 - 25°C below solvent boiling point

Peak tailing decreases with retention.

Poor injection technique Change technique Usually related to erratic plunger depression or having sample in the syringe needle. Use an autosampler.

·Tailing Peaks

Peak Problems·Tailing Peaks


Inlet temperature too high Decrease inlet temperature by 50°C

Tailing generally worse for early eluters.

Inlet temperature too low Increase inlet temperature by 50°C

Tailing usually increases with retention

Dead volume in system Reduce dead volume. Transfer line connections, fused silica unions, etc.

Peak tailing decreases with retention.

Cold spots (condensation) Eliminate cold spots. Commonly at transfer lines.

Tailing usually increases with retention

Overloading of PLOT columns

Reduce the amount injected onto column.

Peak Problems·Split Peaks


Column installationReinstall the column in the injector

Consult the GC manual for the proper installation distance

Injection technique Change technique.Usually related to erratic plunger depression or having sample in the syringe needle. Use an autosampler.

Mixed sample solvent

Change sample solvent.

Worse for solvents with large differences in polarity or boiling points

Poor sample focusing

Use a retention gap For splitless, on-column, and PTV injectors

Solvent/column not compatible

Use a different solvent.Use a retention gap.

Peak Problems·Split Peaks


Sample degradation in injector (only some peaks show splitting)

Reduce inlet temperature.

Derivatize sample to make compounds thermally stable.Change to an on-column injector.

Peak broadening or tailing may occur if the temperature is too low.

Requires an on-column injector

Severe detector overload

Reduce the amount of sample on-column.

May only affect some peaks.

Peak Problems·Changes in Peak Size


Change in detector response

Check gas flow, temperature and settings.Check background level or noise

All peaks may not be equally affectedMay be caused by the system contamination, not the detector.

Change in the split ratio

Check the split ratio All peaks may not be equally affected.

Change in the purge activation time

Check the purge activation time

For splitless injectors

Change in injection volume

Check injection technique

Injection volumes are not linear.

Change in injector discrimination

Maintain the same injector parameters: flows, temperatures, liners, etc.

Most severe for spit injections. All peaks may not be equally affected

Peak Problems·Changes in Peak Size


Change in sample concentration

Check and verify sample concentration

May be caused by degradation, evaporation, or variances in sample temperature or pH

Leak in the syringe

Use a different syringe Sample leas past the plunger or around the needle; leaks are often not readily visible


Trim the column

Solvent-rinse the column

Remove ½ - 1 meter from the front of the column.Only for bonded and cross-linked phases

Column activity Trim or replace the column

Only affects active compounds


Co-elution Change column temperature or stationary phase

Decrease column temperature, and check for the appearance of peak shoulder or tail

Sample backflash

Inject less, use larger liner, or reduce the inlet temperature

Less solvent and higher flow rates are most helpful

Decomposition from inlet contamination

Clean the inlet, replace the liner, replace the gold seal

Only use deactivated liners and glass wool in the inlet.

Loss of sample prior to introduction into the GC

Check sample handling, sample preparation, transfer and storage

•Peak Problems·Changes in Peak Size

T H E E N D

basic gas chromatography prepared by: mina s. buenafe

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