Phytochemistry 1

 When Mobile phase (carrier gas) pressed through column, the mixture
GC: Chromatographic technique used to separate volatile compounds.
GC Instrument:
components distribute themselves acc. to their partition coefficient between
mobile phase & stationary phase
 Compounds eluted acc. to their chemical composition, molecular weight &
velocity of compound.
 Detection carried out by very sensitive detector & then transferred to recorder
to draw the separated peaks.

Advantages of GC:
1) Resolving more than 150 mixed compounds in one experiment
2) Short time of analysis of sample
3) Give Qualitative & quantitative analysis
4) Make analysis of most of natural products in volatile form
5) Very small sample size required less than 0.1 mg

Definitions:
Retention Time: Time required for maximum solute peak to reach the detector
Retention Volume:
Volume of mobile phase required for maximum solute peak to reach the detector
Resolution Power of Column:
Relates the width of eluted peaks to the distance between maximum of peaks
Resolution Factor:
 Measure the degree of separation of adjacent peaks
 Determined by this equation:
𝒕𝑹𝟐−𝒕𝑹𝟏
Resolution Factor Rs = 𝟏
(𝑾𝟏−𝑾𝟐)
𝟐

GC divided into two types:

1) Gas - solid chromatography (GSQ when St. phase → Solid)
2) Gas - liquid chromatography (GLQ when the St. phase → Liquid)
Instrumentation:
1) Sample inlet system
Principle of GC: 2) Carrier gas source
 Sample mixture introduced as volatile liquid into head of column filled e` 3) Column & oven
stationary phase.
4) Detector
5) Recorder & amplifier

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Phytochemistry 2
7) High purity of gas: from:
 Sample (liquid or solid) dissolved in ether or chloroform, then introduced by  Water → so dried by molecular sieve tube in front of gas & just before
micro-syringe (l0μl -100μl) the column
N.B: Too much sample → causes overload of column → lower resolution  Dust & other impurities → using filter before the column
 For thermal conductivity detector → use 5-10μl of sample  Oxygen → using copper oxide tube before the column
 For high sensitivity detector → use 1μl of sample 8) Chemical inertness.
 Sample injected within 0.1 second.
N.B: Longer time e.g: one second causes several feeding (multiple feeding) to
the column e` the same sample w` will give multiple appearance of the same Types of Columns used in GC:
peak. A- Packed Column
 Stainless or Glass
Inlet Temperature:  Length: 1.5-10 m
 Temperature of injection port → 50 ᵒC higher than the boiling point of the  Diameter: 2-6 mm
least volatile component of sample.  St. Phase: solid
 Injection port has heater → gives 5-10 ᵒC over the temperature of column → GLC: finely divided inert solid support material e.g: diatomaceous earth
 Flow rate of gas: 10-100 ml gas/min → measured by flow meter while coated e` liquid st. phase.
pressure measured by mercury manometer → GSC: active adsorbent material e.g: Alumina
 Mechanism: Adsorption or may be coated e` liquid phase (Partition)
 Efficiency: 30.000 theoretical plates
 E.g: nitrogen, helium, argon, hydrogen & carbon dioxide  Sample size: from 10 microliter up to 20 microliters.
 Choice of carrier gas depend on types of detector Advantages: used for large size of sample without overloading

Characters of Carrier Gas:

1) Very low viscosity: give high flow rate e.g: H2
2) High thermal conductivity:
For thermal conductivity detector e. g: H2, helium
3) Diffusibility:
Gases e` high diffusion in stationary phase
e. g: H2, helium → cause lower column efficiency
Nitrogen & other heavy gases → give high efficiency
4) Ionization:
Gas as H2 has very high ionization (require least energy to ionization)
→ Useful for ionization type detectors
5) Compressibility: B- Capillary Column
When temp. increases → viscosity of gas increases → causes decrease in (Golay column) or (Open Tubular column)
velocity (decrease flow rate)  Glass.
So gas must be compressible to overcome this problem  Length: 30 m
6) Safety requirement:  Diameter: 0.2-1 mm
H2 → flammable & He → non-flammable  St. Phase: liquid → applied to the inner wall of capillary instead of being
adsorbed on support.
 Efficiency: 200.000 theoretical plates

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Phytochemistry 3
 Sample size: from 1 microliter Fused Silica Open Tubular: (FSOT)
 Need much less sample 10-3 ml (use split/split-less injection)  Much thinner walls than glass capillary columns
 Consists of outer flexible Polyimide coating → w` mechanically strong &
can be coiled
 e` Fused silica layer → less chemically reactive & narrow diameter (more
efficient)

Types of Capillary Column:

Support-Coated Open Tubular
(SCOT)
- Inner wall of capillary lined e` thin layer - Inner wall of capillary coated e`
of support material e.g: diatomaceous
earth coated e` liquid st. phase → GLC
Wall-Coated Open Tubular Column Temperature:
(WCOT)  Column temp must be controlled to within tenths of degree.
 Optimum column temperature → depend on the boiling point of sample.
liquid stationary phase → N.B:
Partition  When temp slightly above the average boiling point of sample → results in
elution time (2-30 min)
 Minimal temp give → good resolution, but ↑↑ elution times.
 If sample has wide boiling range, temperature programming can be useful.
 Column temp increased either:
a) Linear (Continuously):
Used when components of mix. → Few, & the B.P range → close
b) or Stepwise.
Used when components of mix. → Many, & the B.P range → wide

N.B: - SCOT columns less efficient than WCOT columns.

- Both types of capillary column are more efficient than packed columns.

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Phytochemistry 4

Factors affecting Column Performance:

1) Type of stationary phase.
2) Particle size.
Characters:
3) Reduction in sample size.
1) Chemically inert
4) High affinity of solute in carrier gas.
2) Porous
5) Velocity of carrier gas.
3) High thermal conductivity
6) Column diameter.
4) Good mechanical resistance
7) Column temperature.
5) No tendency for solute
8) Column length.
Types of Stationary Phase:
Measurement of Column Performance:  Alumina, Charcoal, Molecular sieves, Silica gel, Porous polymer,
1) Column efficiency:
Chromosorb-w & Chromosorb-p.
It is the number of theoretical plates
1) Alumina: Al2O3
 Powerful adsorbent
 Form hydrogen bond through hydroxyl groups formed on its surface by
2) Measurement of Resolution factor for two closely eluting peaks:
Measurement of degree of separation of two adjacent peaks hydration
𝒕𝑹𝟐−𝒕𝑹𝟏 2) Carbon Black: (Charcoal)
Resolution factor Rs = 𝟏
𝟐
(𝑾𝟏−𝑾𝟐)  Used for gas solid chromatography
3) Zeolites: ‫زي البالستيك‬
 The original Alumino-silicate molecular sieve
 Powerful adsorbent
 Adsorb water & CO2 so it must protected from atmosphere
4) Silica Gel:
 OH group is the main site of adsorption
5) Porous Polymer:
3) Measurement of Peak Asymmetry:  Styrene can polymerize to give porous beads
Peak Asymmetry factor (As) = BC/AC 6) Chromosorb-W (white):
 If value > 1 → Asymmetry Peak (Tailing)  Mixture of Sod. carbonate & Diatomite → ignited at 900 ᵒC to produce
 If value < 1 → Asymmetry Peak (Leading)
iron-sodium-silicate.
 If value = 1 → Symmetry Peak (Ideal value)
7) Chromosorb-P (pink):
 Mixture of Diatomites & Clay ‫ → طين‬heated at 900 ᵒC to produce
silicates e` excess of iron oxide.

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Phytochemistry 5

Characters:
- Liquid is directly packed in capillary column 1) Highly sensitive (10-7 μg)
- or Adsorbed on inert solid support 2) Fast responsibility to any change in eluted components.
Characters: 3) Linearity to concentration
1) Non-volatile: i.e: the intensity of response or the reading should have linear qualitative
i.e: liquid B.P must be higher, to prevent Bleeding (volatilization of liquid relationship to the concentration of component of effluent.
Stationary phase) & drifting of Baseline. 4) Simplicity.
5) Stability: i.e: withstand the large temp range during operation
6) Inert response.
7) Complete release of gas.

2) High thermal stability
3) Chemically inert
4) low viscosity
Non-specific & Non-destructive
Temperature Choice & Control:
 Control of temperature is very important:
 At high temp. → cause bleeding & affects column separation.
i.e: volatilization of liquid stationary phase w` destroy the column
 At low temp. → cause tailing & poorly peaks
 Different detectors will give different types of selectivity.
 Non-selective detector responds to all compounds except the carrier gas
 Selective detector responds to range of compounds e` common physical or
chemical property
 Specific detector respond to a single chemical compound
 Detectors grouped into: concentration dependent detectors & mass flow
dependent detectors.
Types of Detectors:
1) Thermal Conductivity Type (TCD)
2) Flame Ionization Detector (FID)
3) Electron Capture Detector (ECD)
4) Alkali Flame-Ionization Detector (AFID)
5) Flame Photometric Detector (FPD)
1) Thermal Conductivity Type: (TCD Katharometer)
Principle:
- This detector senses changes in the thermal conductivity of the column
effluent & compares it to a reference flow of carrier gas.
- Carrier Gas: Hydrogen or Helium.
- Metal wire (platinum or tungsten) having high temperature resistance, it is
put in metal glass or metal tube, wire is heated by constant electric current
- Temperature difference is established between the other wall of glass tube &
the hot wire, the difference in temp depends on the thermal conductivity of
the gas.
- Temperature difference is translated by the change in the resistance of wire &
measured by means of Wheatstone Bridge.
- To reduce the effects of gas rate to minimum, another wire is used as
reference & exposed only to pure carrier gas.
- Signal given by detector is then magnified by amplifier

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Phytochemistry 6
2) Flame Ionization Detector: (FID) 4) Electron Capture Detector: (ECD)

Specific
Destructive & Non-destructive
For organic compounds

- Particular selective detector that utilizes the ability of electrophilic

- Carrier gas: Nitrogen mixed e` hydrogen & air then burned in the detector
at a jet above which a collector electrode is placed
- When hydrogen alone is burned water & small electric current are produced
- When organic compounds are burned in the hydrogen flame → ions & CO2
are formed & the electric current increases acc. to the sum of the resulting
electrons & negative ions
H2 + O2 + RHCO2 → H2O + (ions) - current flow
N.B: The lack of response to water & air gases is useful in:
 There will be no need to remove them from the gas
- Detector sensitive to organic compounds
- While insensitive to water, CSs insecticide & inorganic compounds & gases
as: He, N2, CO2, NH2 (alkaloids peptides & amino acids)
- Disadvantage of FID: destroys the sample
compounds to absorb free electrons
- Ionisable carrier gas (Hydrogen) passes into ionization chamber w`
contains collector electrodes to collect free electrons that produced as a result
of ionization of carrier gas molecules by the β-particles
- The migration of electrons to the anode produces electric current (about 10-8
to 10-9 A)
- When electrophilic component (contain electronegative functional groups )
enters the chamber it captures the electrons to form stable negative
molecular ion or charged particles
AB + e- → (AB)- + or - energy
AB + e- → A + B- + or - energy
- Net result is decrease in electric current measured by detector w` displayed
as peak in chromatogram
- Uses:
 For halogenated compounds, phosphorous compounds
 For pesticides of chlorinated compounds
 N.B: Not used for Hydrocarbons (Carbohydrates) or Fatty acids
(No Electrophilicity)

Specific & Destructive

3) Alkali Flame-Ionization Detector: (AFID)
- Same as Flame-Ionization Detector, but used for compounds w` give poor Specific & Destructive
response e` normal Flame-Ionization Detector 5) Flame Photometric Detector: (FPD)
How to solve this problem?? - Used for compounds w` contain phosphorous or sulphur
- Electric current increases when certain hetero-compounds such as organic- - When burned in hydrogen flame give Chemiluminescence ‫ وميض‬as:
phosphorous, Halogen or Nitrogen compounds, seeded e` alkali metal salt HPO → 526 nm wave length
as Rubidium, Cesium or Potassium then burned together, so ↑↑ Response S2 → 394 nm wave length
- Uses: for alkaloids & pesticides - Uses: for pesticide & herbicides

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Phytochemistry 7

Programming of GLC: a) Sample dissolved in pyridine then add Trimethylchlorosilane

1) Isothermal Techniques: b) Mixture is stirred at room temperature for few minutes
- Temperature is fixed c) Mixture is then filtered off, dissolved in chloroform & injected into GC.
- Resolution is modified by several trials
2) Programmed Temperature Techniques: Factors affecting separation on GC:
Temperature is changed in two ways: 1) Sample (solvent used, concentration & weight)
a) Non-linear: temperature is changed by time, every 5, 10, 20 min. 2) Column (length, inner diameter, type of tubing & shape)
b) Linear: temperature is changed at uniform rate after the injection time. 3) Packing (nature & particle size)
3) Programmed Pressure: 4) Detector (nature & characters)
- Temperature is fixed & the change in flow rate only (i.e: ↑↑ pressure) 5) Injection chamber (ratio of splitting in relation to gas flow rate)
- Uses: for very sensitive compounds 6) Recorder (response time, chart speed)
- Advantages: 7) Temperature (of the injector, column & detector)
 Low temp. can be used → suitable for unstable compounds 8) Carrier gas (nature, inlet & outlet pressure & flow rate)
 Pressure is more controlled than temp. 9) Programming temperature
10) Flow rate
Sample Preparation for GC:
 Sample must be volatile (Volatile oils) Applications of GC:
 If Non-volatile compounds → converted into volatile by: Derivatization 1) Volatile oil analysis
a) Methylation 2) Free fatty acid (Fixed oil analysis)
b) Acetylation 3) Fatty acids methyl ester
c) Silylation 4) Carbohydrates
5) Hydrocarbons & Sterols
1) Methylation: 6) Pesticides determination
- Used for Acids or Alcohols, by addition of Methanol/ Sulfuric a` mixture to 7) Flavours & fragrances
compounds e` R-COOH or R-OH → ↓↓ Boling point 8) Food analysis
- Then refluxed for 2-4 hours. 9) Drug analysis
- The resulting methyl derivative is then distilled off & analyzed by GC. 10) Environmental analysis
R-COOH + MeOH / H → R-COOCH3 11) In forensic analysis of blood & urine alcohol (ethanol) levels
12) Fatty alcohols, Ethers & Ketones
2) Acetylation: 13) Amines & Amino acids
- Used for amines, alcohols & phenols to convert to volatile acetate d.v.s
- By addition of Pyridine & Acetic anhydride mixture to compounds &
stirring for few hours.
- The resulting acetate derivative is extracted e` chloroform, then evaporated
& dissolved in ether & injected to GC

3) Silylation:
- Used for acid, alcohol, amines, all types of carbohydrates, glycosides,
alkaloid, phenol, amino acids
- Reagents used: Trimethylchlorosilane Join our Facebook group:
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