GC By Faizy Final

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Anf-Pharma.blogspot.com Real Pharmacists Group (RPG)

Gas Chromatography

REAL PHARMACISTS

GROUP (RPG)

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Table Of Contents

History & Introduction

Advantages Of Gas Chromatography

Components Of Gas Chromatography

Detectors

Gas Chromatography-Mass

Spectrometry (GC-MS)

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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).

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)

Gas - Solid Chromatography (GSC)

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

alumina.

o It is the affinity of solutes towards adsorption onto the

stationary phase which determines, in part, the retention

time.

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

o Most useful for the separation and analysis of gases like

CH4, CO2 CO, ... etc.

o The use of GSC in practice is considered marginal when

compared to gas liquid chromatography.

o

o

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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 selectivity‟s and easy column preparation add

to the assets of GLC or simply GC.

o Advantages of Gas Chromatography

o The technique has strong separation power and even

complex mixture can be resolved into constituents.

o The sensitivity of the method is quite high.

o It gives good precision and accuracy.

o The analysis is completed in a short time.

o The cost of instrument is relatively low and its life is

generally long.

o The technique is relatively suitable for routine analysis.

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Types of Column

a):Open tubular (capillary) column:-

o When the stationary phase (liquid or solid) is uniformly

distributed on the interior surface of column it is called an

open tubular column.

o 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.

o 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).Packed Columns:-

o These columns are fabricated from glass, stainless steel,

copper, or other suitable tubes.

o Stainless steel is the most common tubing used with

internal diameters from 1-4 mm.

o 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

o Carrier gas

o Flow regulators & Flow meters

o Injection devices

o Columns

o Temperature control devices

o Detectors

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schematic diagram of a gas chromatograph:-

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Carrier Gas

Hydrogen ( H2

)

o better thermal conductivity

Disadvantage: it reacts with unsaturated compounds &

inflammable

Helium ( He)

o excellent thermal conductivity

o it is expensive

Nitrogen ( N2

)o reduced sensitivity o it is inexpensive

Requirements Of A Carrier Gas:- o Inertness

o Suitable for the detector

o High purity

o Easily available

o Cheap

o Should not cause the risk of fire

o Should give best column performance

o Impurities in the carrier gas:-

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.

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Flow regulators & Flow meters

deliver the gas with uniform pressure/flow rate

o 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

o Soap Bubble Meter:-

Similar to Rota meter & instead of

a float, soap bubble formed indicates the flow rate

Injection devices

1)-Split injection: o routine method o % sample to column

o remainder to waste

o The injector temperatureis kept high

(for example,350 ) to promote

fast evaporation

o Split Ratio:-

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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2)-Split less injection:

o all sample to column

o best for quantitative analysis

o only for trace analysis, low [sample]

o

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.

3)-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.

Gas Chromatography – Columns

o Important part of GC

o Made up of glass or stainless steel

o Glass column- inert , highly fragile

COLUMNS can be classified

o 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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o Depending on its nature:

a)-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

b).Open tubular or Capillary column

or Golay column

o Long capillary tubing 30-90 M in length

o Uniform & narrow d.m of 0.025 - 0.075 cm

o Made up of stainless steel & form of a coil

Disadvantage: more sample cannot loaded

o They are further classified into three types

a. Wall-coated Open Tubular Column (WCOT)

b. Support-coated Open Tubular Column (SCOT)

c. Porous-layer Open Tubular Column (PLOT)

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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

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.

c. Porous-layer Open Tubular 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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Gas Chromatography-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

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.

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.

G C – IDEAL DETECTORS

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

o Operate at high

o Tempreture (0-400 °C)

o Stable and reproducible

o Linear response

o Wide dynamic range

o Fast response

o Simple (reliable)

o Nondestructive

o 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

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.

o Helium is the carrier gas commonly used with a thermal

conductivity detector.

o Helium has the second highest thermal conductivity (after

H2), so any analyte mixed with helium lowers the

conductivity of the gas stream.

o The heated element may be a fine platinum, gold, or

tungsten wire or a semiconducting thermistor.

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Advantages of Katharometer:

o Linearity is good

o Applicable to most compounds

o Non destructive

o Simple & inexpensive

Disadvantages:

o Low sensitivity

o Affected by fluctuations in temperature and flow rate

o Biological samples cannot be analyzed

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.

o Carbon atoms (except carbonyl and carboxyl carbons)

produce CH radicals, which are thought to produce CHO

ions and electrons in the flame.

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CH + O CHO + e

o Nitrogen gives best detection limit

o signal proportional to number of susceptible

o carbon atoms

o 100-fold better detection than thermal conductivity

o 107 linear response range

o FIDs are mass sensitive rather than conc. Sensitive

ADVANTAGES:

o µg quantities of the solute can be detected

o Stable

o Responds to most of the organic compounds

o Linearity is excellent

DISADVANTAGE:

o destroy the sample

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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 emi - 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.

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 current

3. 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

APPLICATION:-

This detector is frequently used in the

analysis of chlorinated compound (Pesticides).

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4-Nitrogen-phosphorus

4-Nitrogen-phosphorus Detector

(alkali flame detector)

o 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.

o “It is particularly important for drug, pesticide, and

herbicide analyses”.

o 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.

o 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

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.

o 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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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

o are collected and measured.

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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GC-MS Contents

A. GC-Mass Spectrometry Introduction

B. Application Of GC-MS

C. Sample Preparation

D. Method Development in Gas Chromatography

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

Principle of GC-MS

o 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).

o 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).

o 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:-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

C. 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) analyte

into a more convenient or more easily detected form.

a)- Solid-phase micro extraction

b)- Purge and trap

c)- Thermal desorption

a)-Solid-phase micro extraction:

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.

b)- 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.

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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.

c)- 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.

D. 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

What is required from the analysis? Is it qualitative

identification of components in a mixture?

Will you require high-resolution separation of everything or

do you just need good resolution in a portion of the

chromatogram?

Can you sacrifice resolution to shorten the analysis time?

Do you need quantitative analysis of one or many

components?

Do you need high precision?

Will analytes be present in adequate concentration or do you

need

preconcentration or a very sensitive detector for ultratrace

analysis? How much can the Analysis cost?

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)

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.

5. Choosing the Injection Method

The last major decision is how to inject the sample.

Split injection:

o concentrated sample

o high resolution

o dirty samples (use packed liner)

o could cause thermal decomposition

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Splitless injection:

o dilute sample

o high resolution

o requires solvent trapping or cold trapping

On-column injection:

o best for quantitative analysis

o thermally sensitive compounds

o low resolution

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.

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

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8)- Separation and identification of volatile materials, plastics,

natural and synthetic polymers, paints, and microbiological

samples.

9)- Inorganic compound analysis.

10)- Manufacturers of Cosmetics also use gas chromatography

to effectively measure how much of each chemical is used for

their products.

11)- Barbiturates & Benzodiazepines

12)- 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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References

1)- Quantitative Chemical Analysis 7th Edition Daniel C. Harris

Page # 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)

5)- VOGES Textbook Of Quantitative 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 & Skoog

Page # 788 (Accessed at December 28,2014)

10)- Instrumental Techniques For Analytical Chemistry By

FRAUT SELLER Page # 125 (Accessed at December 29,2014)

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)

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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 Edition

Page # 584,591,593 (December 30, 2014)

15)- Chromatography CONCEPTS & CONTRASTS By James M.

Miller Page # 141,165,278,285 (December 30,2014)

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.

20)- Structural Identification Of Organic Compounds With

Spectroscopic Techniques By Richard R. Ernst Page# 252

21)- Introduction To Pharmaceutical Chemical Analysis By Steen

Hansen Page # 191-200

22)- G Chatwal,S anand. Instrumental 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

http://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)

Contributors:-

Ali Hammad Tariq

Zeeshan Saeed

For comments and mistakes correction please

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GC By Faizy Final