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GAS CHROMATOGRAPHY

REAL PHARMACIST GROUP (RPG)

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Table Of Contents

• History & Introduction

• Advantages Of Gas Chromatography

• Components Of Gas Chromatography

• Detectors

• Gas Chromatography-Mass Spectrometry

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What Is Gas Chromatography?

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History

The father of modern gas chromatography is Nobel Prize winner John Porter Martin, who also developed the first liquid-gas chromatograph. (1950)

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Introduction

Gas chromatography – “It is a process of separating component(s) from the given crude drug by using a gaseous mobile phase”’

It involves a sample being vaporized and injected onto the head of the chromatographic column. The sample is transported through the column by the flow of inert, gaseous mobile phase. The column itself contains a liquid stationary phase which is adsorbed onto the surface of an inert solid.

Two major types• Gas-solid chromatography

(stationary phase: solid)• Gas-liquid chromatography

(stationary phase: immobilized liquid)

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Gas - Solid Chromatography (GSC)

The stationary phase, in this case, is a solid like silica or alumina.

It is the affinity of solutes towards adsorption onto the stationary phase which determines, in part, the retention time.

The mobile phase is, of course, a suitable carrier gas.

Most useful for the separation and analysis of gases like CH4, CO2, CO, ... etc.

The use of GSC in practice is considered marginal when compared to gas liquid chromatography.

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Gas - Liquid Chromatography (GLC)

The stationary phase is a liquid with very low volatility while the mobile phase is a suitable carrier gas.

GLC is the most widely used technique for separation of volatile species.

The presence of a wide variety of stationary phases with contrasting selectivities and easy column preparation add to the assets of GLC or simply GC.

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Advantages of Gas Chromatography

The technique has strong separation power and even complex mixture can be resolved into constituents

The sensitivity of the method is quite high

It gives good precision and accuracy

The analysis is completed in a short time

The cost of instrument is relatively low and its life is generally long

The technique is relatively suitable for routine analysis

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Types of Column

a) Open tubular (capillary) column

When the stationary phase (liquid or solid) is uniformly distributed on the interior surface of column it is called an open tubular column.

When the stationary phase is uniform film of few μm thick liquid

that is uniformly coat the inner capillary tubing. The type of column is capillary column.

They are further classified into three types i. Wall-coated open tubular column (WCOT)

ii. Support-coated open tubular column (SCOT) iii. Porous-layer open tubular column (PLOT)

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b).Packed Columns

These columns are fabricated from glass, stainless steel, copper, or other suitable tubes.

Stainless steel is the most common tubing used with internal diameters from 1-4 mm.

The column is packed with finely divided particles (<100-300 mm diameter), which is coated with stationary phase. However, glass tubes are also used for large-scale separations.

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Components Of Gas Chromatography

Carrier gas

Flow regulators & Flow meters

Injection devices

Columns

Temperature control devices

Detectors

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schematic diagram of a gas chromatograph

Syringe

Injector

Detector

Carrier Gas Cylinder Column

To Waste or Flow Meter

Flow Controller

Two-Stage Regulator

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Carrier Gas

» Hydrogen ( H2 )

o better thermal conductivity Disadvantage: it reacts with unsaturated compounds & inflammable

» Helium ( He)

o excellent thermal conductivityo it is expensive

» Nitrogen ( N2)

o reduced sensitivityo it is inexpensive

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Requirements Of A Carrier Gas

Inertness

Suitable for the detector

High purity

Easily available Cheap

Should not cause the risk of fire

Should give best column performance

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o Impurities in the carrier gas degrade the stationary phase. High-quality gases should be used, and even they should be passed through purifiers to remove O2, H2O, and traces of organic compounds prior to entering the column.

o Steel or copper tubing, rather than plastic or rubber tubing, should be used for gas lines because metals are less permeable to air and do not release volatile contaminants into the gas stream.

Impurities in the carrier gas

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Flow regulators & Flow meters

deliver the gas with uniform pressure/flow rate

flow meters:- Rota meter & Soap bubble flow meter Rota meter

placed before column inlet it has a glass tube with a float held on to a spring. the level of the float is determined by the flow rate of carrier gas

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Soap Bubble Meter Similar to Rota meter &

instead of a float, soap bubble formed indicates the flow rate

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Sample injection- Direct Injection

1)Direct injection : into heated port (>T oven) using micro syringe

(i) 1-20 uL packed column

(ii) 10-3 uL capillary column

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Sample Injection- Rotary Sample Valve With Sample Loop

Split injection:

o routine methodo 0.1-1 % sample to columno remainder to waste o The injector temperatureis kept high (for example,350 ) to promote fast evaporation.

o Quantitative analysis with split injection can be inaccurate because the split ratio is not reproducible from run to run.

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Split Ratio:-

o The proportion of sample that does not reach the column is called the split ratio and typically ranges from 50:1 to 600:1.

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Split less injection:

o all sample to columno best for quantitative analysiso only for trace analysis, low

[sample] o Injector temperature for

splitless injection is lower (220 ) than that for split injection, because the sample spends more time in the port and we do not want it to decompose.

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On-Column Injection

o On-column injection is used for samples that decompose above their boiling points and is preferred for quantitative analysis.

o Solution is injected directly into the column, without

going through a hot injector

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Gas Chromatography - Columns

o Important part of GCo Made up of glass or stainless steelo Glass column- inert , highly fragile

COLUMNS can be classified

Depending on its use

1. Analytical column 1-1.5 meters length & 3-6 mm d.m

2. Preparative column 3-6 meters length, 6-9mm d.m

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Depending on its naturea)-Packed columns o contain a finely divided, inert,

solid support material ( diatomaceous earth) coated with liquid stationary phase.

o Most packed columns are 1.5 - 10m in length and have an internal diameter of 2 - 4mm.

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b).Open tubular or Capillary column or Golay column

Long capillary tubing 30-90 M in lengthUniform & narrow d.m of 0.025 - 0.075 cmMade up of stainless steel & form of a coil

Disadvantage: more sample cannot loaded

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They are further classified into three types

i. Wall-coated Open Tubular Column

(WCOT)

ii. Support-coated Open Tubular

Column (SCOT)

iii. Porous-layer Open Tubular Column

(PLOT)

b).Open tubular or Capillary column or Golay column

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A:- Wall-coated Open Tubular Column (WCOT)

o Inner coated with the liquid stationary phase

o The most common type of wall coated open tubular column used is fused-silica, because it is stronger, inert, reliable, easy to use, and flexible.

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b:- Support-coated Open Tubular Column (SCOT)

o Inner layer is of Solid support coated with liquid stationary phase

o Open tubular column has a greater amount of stationary phase than the wall coated column, so it can handle a larger quantity of sample.

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C);- Porous-layer OpenTubular Column (PLOT)

o Inner layer is coated with stationary solid phase particles.

o The only difference b/w SCOT & PLOT is that a PLOT does not have a liquid stationary phase.

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Capillary columns advantages compared to packed columns1. higher resolution2. shorter analysis times3. greater sensitivity

Capillary columns disadvantage compared to packed columns

1. smaller sample capacity2. Need better experience

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Temperature Control Devices

Preheaters: convert sample into its vapour form, present along with injecting devices

Thermostatically controlled oven: temperature maintenance in a column is highly essential for efficient separation.

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Detectors

Definition:- “Detector, sensor, sensing element any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it in a distinctive manner”

Detectors can be grouped into:-

a)-concentration dependant detectors  & 

b)-mass flow dependant detectors

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o The signal from a concentration dependant detector is related to the concentration of solute in the detector, and does not usually destroy the sample Dilution of with make-up gas will lower the detectors response.

o Mass flow dependant detectors usually destroy the sample, and the signal is related to the rate at which solute molecules enter the detector. The response of a mass flow dependant detector is unaffected by make-up gas

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G C – IDEAL DETECTORS

Sensitive (10-8-10-15 g solute/s)

Operate at high Tempreture (0-400 °C)

Stable and reproducible

Linear response

Wide dynamic range

Fast response

Simple (reliable)

Nondestructive

Uniform response to all analytes

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Types Of Detectors

1. Thermal conductivity (TCD)

2. Flame Ionization Detector (FID)

3. Electron capture Detector (ECD)

4. Nitrogen-phosphorus Detector (NPD)

5. Flame photometric Detector (FPD)

6. Photo-ionization Detector (PID)

7. sulfur chemiluminescence detector

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1-Thermal conductivity (TCD)(Katharometer, Hot Wire Detector)

In the past, thermal conductivity detectors were most common in gas chromatography because they are simple and universal.

Principle:- “TCD is based upon changes in the thermal conductivity of the gas stream brought about by the presence of analyte molecules”

The sensing element of TCD is an electrically heated element whose temperature at constant electrical power depends upon the thermal conductivity of the surrounding gas.

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Helium is the carrier gas commonly used with a thermal conductivity detector.

Helium has the second highest thermal conductivity (after H2), so any analyte mixed with helium lowers the conductivity of the gas stream.

The heated element may be a fine platinum, gold, or tungsten wire or a semiconducting thermistor.

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Thermal Conductivity Basics

The TCD is a nondestructive, concentration sensing detector. A heated filament is cooled by the flow of carrier gas.

Flo

w

When the carrier gas is contaminated by sample , the cooling effect of the gas changes. The difference in cooling is used to generate the detector signal

Flo

w

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Advantages of Katharometer Linearity is good Applicable to most compounds Non destructive Simple & inexpensive

Disadvantages

Low sensitivity Affected by fluctuations in temperature and flow rate Biological samples cannot be analyzed

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2-Flame Ionization Detector

o It operates by the principle that by change in conductivity of the flame as the compound is burnt.

o The change in conductivity of the flame does not arise by simple ionization of the compound , it is partial or complete stripping of the compound to give charged hydrogen-deficient polymers or aggregates of carbon of low ionization potential

Carbon atoms (except carbonyl and carboxyl carbons) produce CH radicals, which are thought to produce CHO ions and electrons in the flame.

CH + O CHO + e

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Nitrogen gives best detection limit• signal proportional to number of susceptible carbon atoms• 100-fold better detection than thermal conductivity• 107 linear response range

Flame Ionization Detector

The flame ionization detector is sensitive enough for narrow-bore columns. It responds to most hydrocarbons and is insensitive to nonhydrocarbons such as H2, He, N2, O2,

CO, CO2, H2O, NH3, NO, H2S, and SiF4.

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FIDs are mass sensitive rather than conc. Sensitive

ADVANTAGES:

µg quantities of the solute can be detected Stable Responds to most of the organic compounds Linearity is excellent

DISADVANTAGE:

destroy the sample

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FID

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Remember that FID characteristics include:• Rugged

• Sensitive (10-13 g/s)

• Wide dynamic range (107)

• Signal depends on number of carbon atoms in organic analytes which is referred to as mass sensitive rather than concentration sensitive

• Weakly sensitive to carbonyl, amine, alcohol, amine groups

• Not sensitive to non-combustibles – H2O, CO2, SO2, NOx• Destructive

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3-Electron-Capture Detectors(ECD)

Principle:- “The electron-capture detector is selective in its response being highly sensitive to molecules containing electronegative functional groups such as halogens, peroxides, quinones, and nitro groups’’ Mechanism:- The detector consists of a cavity that contains two

electrodes and a radiation source that emits - radiation (e.g.63Ni, 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions.

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If a compound is present that contains electronegative atoms, those electrons are captured and negative ions are formed, and rate of electron collection decreases

The detector selective for compounds with atoms of high electron affinity.

APPLICATION:- This detector is frequently used in the analysis of chlorinated compounds e.g. – pesticides, polychlorinated biphenyls

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ECD

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Remember the following facts about ECD:

1. Electrons from a b-source ionize the carrier gas (nitrogen)

2. Organic molecules containing electronegative atoms capture electrons and decrease current3. Simple and reliable

4. Sensitive (10-15 g/s) to electronegative groups (halogens)

5. Largely non-destructive

6. Insensitive to amines, alcohols and hydrocarbons

7. Limited dynamic range (102)

8. Mass sensitive detector

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4-Nitrogen-phosphorus Detector(alkali flame detector)

The nitrogen-phosphorus detector, also called an alkali flame detector, is a modified flame ionization detector that is especially sensitive to compounds containing N and P. Its response to N and P is times greater than the response to carbon.

“It is particularly important for drug, pesticide, and herbicide analyses”.

Ions such as NO2, CN and PO2 , produced by these elements

when they contact a Rb2SO4 containing glass bead at the burner tip, create the current that is measured. N2 from air is inert to this detector and does not interfere.

The bead must be replaced periodically because RbSO4 is consumed shows a chromatogram from a nitrogen-phosphorus detector.

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Nitrogen-phosphorus Detector

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5-flame photometricdetector(FPD)

o A flame photometric detector measures optical emission from phosphorus, sulfur, lead, tin, or other selected elements.

o When eluate passes through a H2-air flame, as in the flame

ionization detector, excited atoms emit characteristic light.

Phosphorus emission at 536 nm or sulfur emission at 394 nm

can be isolated by a narrow-band interference filter and detected with a photomultiplier tube.

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FPD

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6-Photo-ionization Detector (PID)

o A photoionization detector uses a vacuum ultraviolet source to ionize aromatic and unsaturated compounds, with little response to saturated hydrocarbons or halocarbons.

o Electrons produced by the ionization are collected and measured.

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7-sulfur chemiluminescence detector

o A sulfur chemiluminescence detector takes exhaust from a flame ionization detector, in which sulfur has been oxidized to SO, and mixes it with ozone to form an excited state of that emits blue light and ultraviolet radiation.

o A nitrogen chemiluminescence detector is analogous. Combustion of eluate at 1800°C converts nitrogen into NO, which reacts with Ozone to create chemiluminescent product. The response to N is 107 times greater than the response of Cabon (C)

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Gas Chromatography-Mass Spectrometry (GC-MS)

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GC-MS Contents

A. GC-Mass Spectrometry Introduction

B. Mass Spectrometer & Their Components

C. Application Of GC-MS

I. Sample Preparation

II. Method Development in Gas Chromatography

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A:-Mass Spectrometry (MS)

Mass spectrometry (MS) is an analytical chemistry technique that helps identify the amount and type of chemicals present in a sample by measuring the mass-to-charge ratio and abundance of gas-phaseions

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Principle of GC-MS

The sample solution is injected into the GC inlet where it is vaporized and swept onto a chromatographic column by the carrier gas (usually helium).

The sample flows through the column and the compounds comprising the mixture of interest are separated by virtue of their relative interaction with the coating of the column (stationary phase) and the carrier gas (mobile phase).

The latter part of the column passes through a heated transfer line and ends at the entrance to ion source where compounds eluting from the column are converted to ions and detected according to their mass to charge m/z ratio

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B:-Mass Spectrometer & Their Components

“Mass spectrometry is a technique used for measuring the molecular weight and determining the molecular formula of an organic compound”

In general a mass spectrometer consists of

an ion source, High-vacuum system a mass-selective analyzer, and an ion collector

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C:-Application Of GC-MS

Petrochemical and hydrocarbons analysis

Geochemical research

Forensic (arson, explosives, drugs, unknowns) Environmental analysis

Pesticide analysis, food safety and quality

Pharmaceutical and drug analysis

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GC-MS can analyze the particles from a human body in order to help link a criminal to a crime. accelerant is significant evidence in a fire investigation because it suggests that the fire was set intentionally.

Criminal forensics

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Law enforcement

GC-MS is increasingly used for detection of illegal narcotics marijuana, cocaine, opioids Clinicians oxycodone and oxymorphone

Piperazines are not detectable by typical immunoassay testing, but they may be detectable via GC-MS

Sports anti-doping analysis

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Astrochemistry

Several GC-MS have left earth. Two were brought to Mars by the Viking program. Venera 11 and 12 and Pioneer Venus analysed the atmosphere of Venus with GC-MS.

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I. Sample Preparation

Sample preparation is the process of transforming a sample into a form that is suitable for analysis. This process might entail extracting analyte from a complex matrix, preconcentrating very dilute analytes to get a concentration high enough to measure, removing or masking interfering species, or chemically transforming (derivatizing) analyteinto a more convenient or more easily detected form.

Solid-phase microextraction Purge and trap Thermal desorption

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Solid-phase microextraction

Solid-phase microextraction is a method to extract compounds from liquids, air, or even sludge without using any solvent.16 The key component is a fused-silica fiber coated with a film of stationary phase similar to those used in gas chromatography.

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Purge and trap

Purge and trap is a method for removing volatile analytes from liquids or solids (such as groundwater or soil), concentrating the analytes, and introducing them into a gas chromatograph.

In contrast with solid-phase microextraction, which removes only a portion of analyte from the sample, the goal in purge and trap is to remove 100% of analyte from the sample. Quantitative removal of polar analytes from polar matrices can be difficult.

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Purge and trap

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Thermal desorption

Thermal desorption is a method to release volatile compounds from solid samples. Aweighed sample is placed in a steel or glass tube and held in place with glass wool. The sample is purged with carrier gas to

remove O2, which is vented to the air, not into the chromatography column.

The desorption tube is then connected to the chromatography column and heated to release volatile substances that are collected by cold trapping at the beginning of the column. The column is then heated rapidly to initiate chromatography.

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Thermal desorption

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II. Method Development in Gas Chromatography

Order Of Decision:-

1. Goal Of Analysis

2. Sample Preparation

3. Choosing the Detector

4. Selecting the Column

5. Choosing the Injection Method

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1. Goal Of Analysis

o What is required from the analysis? Is it qualitative identification of components in a mixture?

o Will you require high-resolution separation of everything or do you just need good resolution in a portion of the chromatogram?

o Can you sacrifice resolution to shorten the analysis time?

o Do you need quantitative analysis of one or many components?

o Do you need high precision?

o Will analytes be present in adequate concentration or do you needpreconcentration or a very sensitive detector for ultratrace analysis? How much can the Analysis cost?

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2. Sample Preparation

The key to successful chromatography of a complex sample is to clean it up before it ever sees the column.

solid-phase microextraction, purge and trap & thermal desorption to isolate volatile components from complex matrices.

Other methods include:- liquid extraction, supercritical fluid extraction, and solid-phase extraction,

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3. Choosing the Detector

The next step is to choose a detector. Do you need information about everything in the sample or do you want a detector that is specific for a particular element or a particular class of compounds?

The most general purpose detector for open tubular chromatography is a mass spectrometer.

1. Thermal conductivity (TCD)2. Flame Ionization Detector (FID)3. Electron capture Detector (ECD)4. Nitrogen-phosphorus Detector (NPD)5. Flame photometric Detector (FPD)6. Photo-ionization Detector (PID)

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4. Selecting the Column

The basic choices are the stationary phase, column diameter and length, and the thickness of stationary phase. A nonpolar stationary phase is most useful.

An intermediate polarity stationary phase will handle most separations that the nonpolar column cannot.

For highly polar compounds, a strongly polar column might be necessary.

Optical isomers and closely related geometric isomers require special stationary phases for separation.

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5. Choosing the Injection Method

The last major decision is how to inject the sample. Split injection:• concentrated sample• high resolution• dirty samples (use packed liner)• could cause thermal decomposition Splitless injection:• dilute sample• high resolution• requires solvent trapping or cold trapping On-column injection:• best for quantitative analysis• thermally sensitive compounds• low resolution

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Application Of Gas Chromatography

1)- Qualitative Analysis – by comparing the retention time or volume of the sample to the standard / by collecting the individual components as they emerge from the chromatograph and subsequently identifying these compounds by other method

2)- Quantitative Analysis- area under a single component elution peak is proportional to the quantity of the detected component/response factor of the detectors.

3)- Volatile Oils, official monograph gives chromatography profile for some drugs. E.g. to aid distinction between anise oil from star anise and that from Pimpinelle anisum

4)- Separation of fatty acids derived from fixed oils

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5)- Miscellaneous-analysis of foods like carbohydrates, proteins, lipids, vitamins, steroids, drug and pesticides residues, trace elements

6)- Pollutants like formaldehyde, carbon monoxide, benzene, DDT etc

7)- Dairy product analysis- rancidity

8)- Separation and identification of volatile materials, plastics, natural and synthetic polymers, paints, and microbiological samples

9)- Inorganic compound analysis

Application Of Gas Chromatography

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Forensic Pathology

gas chromatography is useful in identifying the individual elements and molecules present in a compound, it has been applied in forensic pathology to determine which fluids and compounds are present inside a human body after death

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Crime Scene Testing

Gas chromatography can also be used to test samples found at a crime scene, whether these be blood samples or fibre samples from clothing or other materials. This allows scientists to pinpoint exactly what (and perhaps who) was present at the scene of the crime

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Barbiturates & Benzodiazepines

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blood and serum

It can also be used for the volatile samples such as human breathe, blood, saliva and other secretions which contains organic volatiles and can be easily analyzed with Gas Chromatography.

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Cosmetics

Manufacturers of Cosmetics also use gas chromatography to effectively measure how much of each chemical is used for their products.

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Radioactive Isotopes

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pesticides and fungicides

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vitamins

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Volatile Oils

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References

1)-Quantitative Chemical Analysis 7th Edition Daniel C. HarrisPage # 528 ( Accessed at December 22,2014)

2)- Chromatographic methods 5th Edition By A. Braithwaite & F.J.Smith Page # 166 (Accessed at December 23,2014)

3)- Chemical Analysis Modern Instrumentation Methods and

Techniques 2nd Edition Francis Rouessac and Annick Rouessac Page # 31 (Accessed at December 24,2014)

4)- UNDERGRADUATE INSTRUMENTAL ANALYSIS 6th Edition By

James W. Robinson Page # 750 (December 26,2014)

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References

5)- VOGES Textbook Of Quantitaive Chemical Analysis By

MENDHAM. Page # 317,745 (December 27,2014)6)- Organic Spectroscopy & Chromatography By M.YOUNAS

7)- Mass Spectrometry Principles & Applications By ED Hoffmann & Stroobant Page # 217 (December 27,2014)8)- Mass Spectrometry By H-GROSS Page # 482

9)- Principles Of Instrumental Analysis By Holler & SkoogPage # 788 (Accessed at December 28,2014)

10)- Instrumental Techniques For Analytical Chemistry By FRAUT SELLER Page # 125 (Accessed at December 29,2014)

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References

11)- Dr.S. Ravi Sankar.Text book of pharmaceutical analysis:Rx Publication, Tiruneveli: Edition 1997,1999,2001.

12)- A Textbook Of Analytical Chemistry Mahinder Singh Page # 110 (Accessed at December 29,2014)

13)- Analytical Chemistry- A Modern Approach To Analytical Science

2nd Edition By M. Mermet, M.Otto Page # 536,553

14)-Analytical Chemistry By Gary D. Christian 6th EditionPage # 584,591,593 (December 30,2014)

15)-Chromatography CONCEPTS & CONTRASTS By James M. Miller Page # 141,165,278,285 (December 30,2014)

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References

16)-Forensic Applications Of Gas Chromatography By Michelle Groves Carlin.

17)- Amirav, A.Gordin, A. Poliak, M. Alon, T. and Fialkov, A. B. (2008),

"Gas Chromatography Mass Spectrometry with Supersonic Molecular Beams". Journal of Mass Spectrometry 43: 141–163.

18)- A. Hites and K. Biemann, Analytical Chemistry, 855

19)- H.F.Waltor,J.Reyes:Modern Chemical analysis and instrumentation.

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References

20)-Structural Identification Of Organic Compounds With Spectroscopic Techniques By Richard R.Ernst Page# 25221)- Introduction To Pharmaceutical Chemical Analysis By Steen Hansen Page # 191-20022)- G Chatwal,S anand.Ihstrumental Methods of Chemical analysis: Himalaya publishing house,Delhi:Edition June 1995.23)- www.GasChromatography/Slideshare.com

24)- www.Gas Chromatography/Authorstream.com

25)- www.GasChromatography/Orgchem.com

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Presented to:

Respected Mam Saharish & 3rd Prof (Eve)

Presentation Given by Members of:

‘‘Real Pharmacist Group (RPG)’’

Made By: Faizan Akram (Faizy)

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