1. GC DERIVATIZATION :
GC DERIVATIZATION contents Derivatisation techniques: Applications of gas chromatography
What is derivatization? :
What is derivatization? What is GC Derivatization? Derivatization is theprocess of chemically modifying a compound to
produce a new compound which has properties that are suitable for analysis using a GC.
Slide 4:
Why Derivatize: To permit analysis of compounds not directly amenable to analysis due to, for example, inadequate volatility or
stability. Improvechromatographic behavior or detectability. Many compounds do not producea usable chromatograph
(i.e.multiple peaks, or one big blob), or the sample of interest goes undetected. As a result it may be necessary to derivatize the
compound before GC analysis is done. Derivatization is a useful tool allowing the use of GC and GC/MSto be done on samples
that would otherwise not be possible in various areas of chemistry such as medical, forensic, and environmental.
What Does DerivatizationAccomplish? :
What Does DerivatizationAccomplish? Increases volatility (i.e. sugars): Eliminates thepresence of polar OH, NH, & SH groups
Derivatization targets O,S, N and P functional groups (with hydrogens available. Increases detectability, I.e. steroids/ cholesterol
Increases stability. Enhances sensitivity for ECD (Electron CaptureDetection). The introduction of ECD detectable groups, such
as halogenated acyl groups, allows detection of previously undetectable compounds.
Types of Derivatization :
Types of Derivatization pre-column derivatization post-column derivatization Precolumn derivatisation: Components are
converted to volatile & thermo stable derivative Conditions - Pre column derivatisation Component ↓ volatile Compounds are
thermo labile ↓ tailing & improve separation
Post column derivatisation :
Post column derivatisation Improve responseshown by detector Components ionization / affinity towards electrons is increased
Pretreatment of solid support To overcometailing Generally doing separation of non polar components like esters, ethers…
TECHNIQUES OF DERIVATISATION :
TECHNIQUESOF DERIVATISATION SILYLATION ACYLATION PERFLOURO-ACYLATION ALKYLATION
ESTERIFICATION CONDENSATION CYCLISATION
Acylation :
Acylation Acylation reduces the polarity of amino, hydroxyl, and thiol groups and adds halogenated functionalities for ECD. In
comparison to silylating reagents, the acylating reagents target highly polar, multifunctional compounds, such as carbohydrates
and amino acids. Acylderivatives are formed with acyl anhydrides, acyl halides, and activated acyl amide reagents. The
anhydrides and acyl halides form acid by-products which must be removed before GC analysis.
CONT….. :
CONT….. Activated amide reagents, such as MBTFA, havetheadvantage of not yielding acid by-products. Fluorinated acyl
groups, going from trifluoracetyl to heptafluorobutyryl, can be used to increase retention times.
Acylating Reagents :
Acylating Reagents 1. Fluorinated Anhydrides:- TFAA- Trifluoroacetoic Anhydride PFPA- PentafluoropropionicAnhydride·
Most commonly used reagents, as derivatives are suitable for both FID and ECD. · Reacts with alcohols, amines, and phenols to
produce stable and highly volatile derivatives · The acid by-product should be removed, via a stream of nitrogen, before injection
onto column. Bases, such as triethylamine, can be added as an acid receptor and promotereactivity · Ability to adjust retention
times for ECD
Conti… :
Conti… 2. Fluoracylimidazoles TFAI- Trifluoroacetylimidazole PFPI- PentafluoropropanylimidazoleHFBI-
Heptafluorobutyrylimidazole· Usually a better choice for making ECD derivatives · React under mild conditions and their by -
products, theimidazole, is not acidic so it will not harm column. · Reagents are extremely sentive to water- will react violently to
it. · Works best with amines and hydroxy compounds
Cont.. :
Cont.. 3. MBTFA {N-methyl-bis(trifluoroacetamide)} · Reacts with primary and secondary amines, slowly with hydroxylgroups
and thiols. · Conditions are mild and theby-products arerelatively inert and are non acidic 4. PFBCI- Pentafluorobenzoyl
Chloride · Phenols most receptive site · Used for making derivatives of alcohols and secondary amines. Secondary amines will
react with this compound

2. Slide 14:
Ex: Esterification with n-propanol, acidic catalyst and benzene for remove water azeotropically, the ester were acylated with
acetic anhydride and finally derivatives extracted and diluted for GC.
Slide 15:
Esterification with n-propanol, acid catalyst and benzene removes water azeotropically. Later, Ester was acetylated with acetic
anhydride to yield the acetylated derivative.
Advantages and Disadvantages ofAcylation :
Advantages and Disadvantages ofAcylation Advantages: Addition of halogenated carbons increased detectability by ECD.
Derivatives are hydrolytically stable. Increased sensitivity by adding molecular weight Acylation can be used as a first step to
activate carboxylic acids prior to esterfication (alkylation).
Disadvantages :
Disadvantages Acylation derivatives can be difficult to prepare. Reaction products (acid by-products) often need to be removed
before analysis. Acylation reagents are moisture sensitive. Reagents are hazardous and odorous.
perflouro-Acylation :
perflouro-Acylation This group increases the mol.wt of thesample relative to theanalogous hydrocarbon. Best method to
increase the retention time. Eg. N-Triflouro acetic anhydride Direct acylation with Triflouro acetic anhydride in triflouro acetic
acid followed by methylation with diazomethane in methanol.
Alkylation :
Alkylation Alkylation reduces molecular polarity by replacing active hydrogens with an alkyl group. Thesereagents are used to
modify compounds with acidic hydrogens, such as carboxylic acids and phenols. These reagents make esters, ethers, alkyl amines
and alkyl amides. Reagents containing fluorinated benzoylgroups can be used for ECD. The principal reaction employed for
preparation of thesederivatives is nucleophilic displacement. Alkylation is used to modify compounds with acidic hydrogens,
such as carboxylic acids and phenols.
Slide 21:
Alkylation can be used alone to form esters, ethers and amides- or they can be used in conjunction with acylation or silylation. It
is generally used to convert organic acids into esters. As the acidity of the active hydrogen decreases, the strength of the
alkylating reagent must be increased. The harsher the reaction conditions or reagents, themore limited the selectivity and
applicability of this method.
Advantages Wide range of alkylation reagentsavailable Reaction conditions can vary from strongly acidic and strongly
basic. Some reactions can be done in aqueous solutions. Alkylation derivatives are generally stable.Disadvantages Limited
to amines andacidic hydroxyls. Reaction conditions are frequently severe. Reagents are often toxic. :
Advantages Wide range of alkylation reagents available Reaction conditions can vary from strongly acidic and strongly basic.
Some reactions can be done in aqueous solutions. Alkylation derivatives are generally stable.Disadvantages Limited to amines
and acidic hydroxyls. Reaction conditions are frequently severe. Reagents are often toxic.
esterification :
esterification Esterification: Esterification is used to preparederivatives of carboxyl group. The conversion of thecarboxyl group
to ester increases volatality by decreasing hydrogen bonding. Ex:- Analgesics, prostaglandins, aminoacids, & anti-inflammatory
agents. Derivatization by esterification can be carried out by using Fischer esterification procedure in which strongly acidic
conditions are present. R` - COOH + R -OH H+ R`- COOR + H2O BF3
Amino acids : E.x. Alanine, α-amino butyricacid, valine, leucine, isoleucine.1. α-chloromethyl esters: preparedby
treating the amino acid with a mixture of concentrated nitricacid and Hydrochloric acid.Aminoacid Chloro methyl
esterR– CH-NH2 – COOHHcl/ HNO3 R – CH – COOCH3 Cl 2. Methyl estersalts:Esterification of 1-leucine, 1-
methioninewith methanol & thionyl chloride. :
Amino acids : E.x. Alanine, α-amino butyricacid, valine, leucine, isoleucine.1. α-chloromethyl esters: prepared by treating the
amino acid with a mixture of concentrated nitric acid and Hydrochloric acid.Aminoacid Chloro methylesterR – CH-NH2 –
COOH Hcl/ HNO3 R – CH – COOCH3 Cl 2. Methylester salts:Esterification of 1-leucine, 1-methionine with methanol &
thionylchloride.
Silylation :
Silylation Silylation produces silylderivatives which are more volatile, less stable, and more thermally stable. Replaces active
hydrogens with a TMS(trimethylsilylgroup). Silylation occurs through nucleophilic attack (SN2). Thebetter theleaving group,
the better the siliylation. Silylation reagents will react with water and alcohols first. Care must be taken to ensure that both

3. sample and solvents are dry. Solvents should be as pureas possible. This will eliminate excessive peaks. Try using as little
solvent as possibleas this will prevent a large solvent peak.
Pyridine is the most commonly usedsolvent. Although pyridine may produce peak tailing, it is an acid scavanger and will
drive the reaction forward. In many cases, the needfor a solvent is eliminatedwith silylating reagents. (If a sample
readily dissolves in the reagent, it usually a sign that the derivatization is complete).. :
Pyridine is themost commonly used solvent. Although pyridinemay producepeak tailing, it is an acid scavanger and will drive
the reaction forward. In many cases, the need for a solvent is eliminated with silylating reagents. (If a sample readily dissolves in
the reagent, it usually a sign that thederivatization is complete)..
Ease of reactivity of functional groups towards silylation. Many reagents require heating (not in excess of 60 degrees C for
about 10-15 minutes, to prevent breakdown). Hindered products may require long term heating :
Ease of reactivity of functional groups towards silylation. Many reagents require heating (not in excess of 60 degrees C for about
10-15 minutes, to prevent breakdown). Hindered products may require long term heating
The ease of reactivity of the functional group toward silylationfollows the order:Alcohol > Phenol > Carboxyl > Amine >
AmideGeneralReaction MechanismR-OH+ (CH3)3 – Si - Cl R – O – Si - (CH3)3 + HCl :
The ease of reactivity of thefunctional group toward silylationfollows the order:Alcohol > Phenol > Carboxyl > Amine >
AmideGeneralReaction MechanismR-OH + (CH3)3 – Si - Cl R – O – Si - (CH3)3 + HCl Trimethylsilylether
Trimethylchlorosilane
Slide 33:
Silylating Reagents 1. HMDS(Hexamethyldisilane). · Weak donor, as it has symmetry · If used will attack only easily silylated
hydroxylgroups · Sometimes found in combination with TMCS2. TMCS(Trimethylchlorosilane). · Weak donor, again not
commonly used · Often found as a catalyst to increase TMSdonor potential· Bad by-product, HCL3. TMSI
(Trimethylsilylimidazole). · Not a weak donor, but it is selective (will not target N compounds) · Reacts readily with hydroxyls
but not with amines · Since it is selective, it will target the hydroxyls in wet sugars. It will derivatize the acid sites of amino acids,
and will leave the amino group free for fluorinated derivatization (ECD)
Slide 34:
4. BSA (Bistrimethylsilylacetamide). · First widely used silylating reagent · Strong silylating reagent- acetamide is a good
leaving group. Reacts under mild conditions and produces relatively stable by-products · Drawbacks:by-product, TMS-
acetamide, will sometimes producepeaks that overlap thoseof other volatile derivatives. BSA mixtures also oxidize to form
silicon dioxide, which can foul FID detectors TMS-DEA (Trimethylsilyldiethylamine). · Reagent is used for derivatizing amino
acids and carboxylic acids · Targets hindered compounds
Slide 35:
5. BSTFA (Bistrimethylsilyltrifluoroacetamide) · Developed by Gerhke in 1968 · Reacts similiarly to BSA but theleaving group
is trifluoroacetamide, so it acts faster and more completely than BSA · BSTFA is highly volatile, and produces by -products that
are more volatile than BSA by-products, thus thereis little interference with early eluting peaks · It can act as its own solvent ·
Combustion product silicon trifluoride, does not foul detectors
Slide 36:
Advantages and Disadvantages of Silylation Advantages Ability to silylatea wide variety of compounds Large number of
silylating reagents available. Easily prepared. Disadvantages Silylation Reagents are moisture sensitive Must useaprotic(no
protons available) organic solvents
Slide 37:
Condensation: If ketone or aldehyde is present in a sample, it is frequently derivatized to prevent hydrogen bonding due to
enolization & helps in resolution from an interfering substance. The most commonly used reagent is methoxylamine to protect
enolizable ketogroups in steroids by formation of methoximes. Cyclization: Cyclization is performed on compounds containing
two functional groups in close proximity so that 5 or 6 membered Heterocyclic rings can be formed.
Slide 38:
Heterocycles formed are ketals, boronates, triazines & phosphites. E.g: Cyclization of α – OH ketones (present in corticosteroids)
with formaldehyde forms bismethylene dioxy derivatives which are thermally stable & permit resolution of corticosterone from a
mixture of steroids.
applications :
applications Retention time data should be useful for identification of mixtures. Comparing theretention time of the sample as
well as thestandard. Checking the purity of a compound: compar thestandard and sample. Additional peaks are
obtained…..impurities are present….compound is not pure. Qualitative analysis:

4. Slide 40:
Quantitativeanalysis: Direct comparison method: -comparing the area of thepeak, peak height, width of peak. Calibration
curves: -standards of varying concentration are used determine peak areas . Internal standard method: -A known concentration of
the internal standard is added separately to thestandard solution -Thepeak area ratio of sample and internal standard….unknown
concentration is easily determined .
Slide 41:
Elemental analysis Determination of C,H ,O ,S and N . Determination of mixture of drugs Isolation and identification of drugs
Isolation and identification of mixture of components(amino acids ,plant extracts ,volatile oils) GS-MS is one of the most
powerfultool in siological and chemical studies. Other app… like Analysis of dairy prod.., aldehydes, ketones etc.. Which are
present in pharm..,Rancidity in fatty acids. Assay of drugs, purity of compounds, determination of foreign or related compounds.
PowerPoint Presentation:
GAS CHROMATOGRAPHY(GC) Gas chromatographyis the mostutilized of all the chromatographic techniques.GC
is mostwidelyused analytical technique in the world – Over 50 years in development – 2,000 instruments /yr –
25,000 in use – Worldwide market> $1billion GC is premier technique for separation and analysis ofvolatile
compounds – Gases,liquids,dissolved solids – Organic and inorganic materials – MW from 2 to > 1,000 Daltons.GC
has been applied for a wide variety of theoretical and practical problems in the separation and identification ofthe
components ofthe atmosphere ,gases liquids,drugs and commercial products.
TYPES OF GC GC is of vtwo types: Gas-liquid chromatography.(GLC) In GLC partition process occurs,thatis the
mobile phase is a gas and the stationaryphase is a thin layer of a non-volatile liquid bound to a solid support.It
involves the partition between the gas and an immobile liquid phase.2.Gas - Solid chromatography(GSC) GSC
utilizes the Solid adsorbentas the stationaryphase and an adsorption process takes place.It is based upon selective
adsorption ofsample on a solid surface.
HPLC GC:
HPLC GC Mobile phase changes Constanttemperature Compounds partition from the mobile phase based on
solubility.Elution is generallytime or volume dependentMobile phase is constantIncreasing temperature
Compounds partition from the mobile phase based on volatility. Elution is generallytemperature dependent
Differences between GC and HPLC
INSTRUMENTATION Many commercial variations are available,basicallyall gas chromatographs,whether GLC or
GSC, consists ofsix basic components.A carrier gas which is maintained ata high pressure and is delivered to the
instrumentata rapid and reproducible rate.A sample injection system.The separation column.One or more
detectors.Thermostatchambers for the temperature regulation ofcolumn and detectors.An amplification and
recorder system.
CARRIER GAS The mostwidelyused carrier gas are hydrogen, helium,nitrogen and air.Hydrogen- More advantage
as compared to other gases butis dangerous to use.It has better thermal conductivity , lower densityand greater
flow rates.Helium - It is used because ofits excellent thermal conductivity, inertness,low densityand it allows greater
flow rates.Nitrogen - It is inexpensive but gives reduced sensitivity. Air - It is used only when the oxygen in the air is
useful to the detector or separation.Oxygen is usuallyavoided since it oxidizes the stationary phase.
IDEAL conditions for carrier gas is as follows:It should be inertand not react with the sample,stationaryphase or
hardware.It should be suitable for the detector employed and type of sample to be analyzed. It should be readily
available in high purity. It should give bestcolumn performance consistence with required speed ofthe analysis.It
should be cheap and not cause the risk of fire or explosion hazards.
SAMPLE INTRODUCTION SYSTEM The sample introduction system is very important.very small amountofsample
is used.The sample – reproducible and mustvaporize it instantaneouslyso that the sample will enter the column as
a single slug.SOLIDS - samples mustbe dissolved in volatile liquids for introduction or may be introduced directly if
they can be liquefied.LIQUIDS-By using hypodermic syringe through a selfsealing rubber septum into a small inlet
chamber,which maybe heated to cause flash evaporation.GASES – Gas samples require a special gas sampling
valves for introduction into the carrier gas stream.
DETECTORS All the detectors effluently measure the changes in the composition arising from the variations in the
eluted components.When the carrier gas passes alone itgives zero signals.Butwhen the componentis detected
then a signal proportional to the concentration ofthat componentis produced.

5. Types of detectors are Thermal conductivity detector (TCD) Flame ionization detector (FID) Electron capture detector
(ECD) Up to 60 other types of detectors used… – IR (FTIR) – Atomic Emission Detector
:
IDEAL CHARACTERICTICS OF DETECTORS The sensitivityshould be high and withoutinstabilityat high
sensitivities.The volume should be low so that the compound eluted from the column in a small plug ofcarrier gas is
not diluted further within the detector itself.The response should be rapid and linear with concentration of compound.
The response should be unaffected by flow rate of carrier gas and temperature.The response should be fast.
THERMAL CONDUCTIVITY DETECTOR(KATHERTOMETER):
THERMAL CONDUCTIVITY DETECTOR(KATHERTOMETER) Principle:The thermal conductivity of the gas due to
resistance developed bytemperature.Electrical power is converted to heatin a resistantfilamentbythe increasing
temperature which causes heating.The filamentmayloose heatby radiation to a cooler surface and by conduction to
the molecules coming into contactwith it.
THERMAL CONDUCTIVITY DETECTOR
WORKING OF DETECTOR Conductivity of the carrier gas in the presence ofan organic compound.The tungsten
wires are heated electricallyand assume equilibrium conditions oftemperature and resistance.Wheatstone bridge
arrangement - signal,which is amplified and recorded.The sensitivityis low and affected by fluctuations of
temperature and flow rate.
Responds to all compounds Adequate sensitivity for many compounds Good linear range ofsignal Simple
construction Signal quite stable provided carrier gas flow rate, block temperature,and filamentpower are controlled
Nondestructive detection Thermal Conductivity Detector
FLAME IONISATION DETECTOR PRINCIPLE Based on the electrical conductivity of gases.At normal temperature
and pressure gases acts as insulators butwill become conductive ofions if electrons are present. Very small number
of ions can be detected on the basis ofconductivity.
WORKING Hydrogen is added with a capillaryjet if it was not used as carrier gas . The mixture is burnt in air (or
oxygen) in the detector. The platinum wire serves as one electrode ofthe cell and the collector is the other. A
sufficientpotential to collectall ions is used.This detector is remarkablyinsensitive to the presence ofwater vapour
or air in the carrier gas,and the background currentis low so that small quantities can be measured with proper
amplification.
Electron Capture Detector:
Electron Capture Detector The electron affinity of differentsubstances can be used as the basis for ionization
detection known as the electron capture detector. It depends to only those compounds whose molecules have an
affinity for electrons.E.g., Chlorinated compounds,alkyl lead,etc. It can also be employed for pesticide analysis
(subpicogram) and those which Acceptelectrons of carrier gas . On the contrary it responds very little to compounds
such as hydrocarbons.
Electron Capture Detector:
X - Ion recombination :X - + N 2 + = X + N 2 The “base line” will decrease and this decrease constitutes the signal.
Insecticides ,pesticides,vinyl chloride,and fluorocarbons  (e)  (e) = N 2 + + 2e , These N 2 + establish a “base line”
X (F, Cl and Br) containing sample +  particles).Ionization : N 2 (Nitrogen carrier gas) + Electron Capture Detector
ECD detects ions atthe exit of the gas from chromatographic column bythe anode electrode.( 3 H or 63 Ni which
emits
Other GC detectors � Nitrogen-Phosphorous Detector (NPD) Also know as the thermionic detector (TID) or alkali
flame detector. It is an FID tweaked for N-P cpds,and organics. � Flame Photometric Detector (FPD) FID tweaked
for S containing cpds. � Photoionization Detector (PID) UV ionization of organic analyte, coupled with high voltage
cathode and analode results in currentproportional to ionized organics.
Advantages of GC The technique has strong separation power and even complexmixture can be resolved into
constituents.The sensitivityof the method is quite high.It is micromethod and onlya few mg of the sample is

6. sufficientfor analysis.It gives good precision and accuracy.The analysis is completed in a shorttime. The costof
instrumentis relativelylow and its life is generallylong.The technique is suitable for routine analysis because the
operation of a gas chromatograph and related calculations do notrequire highlyskilled persons.GAS
CHROMATOGRAPH IS AT PRESENT ONE OF THE MOST AND WIDELY USED AND POWERFUL TOOL FOR
SEPERATION BECAUSE OF ITS SPEED, RESOLVING POWER AND EXTREME SENSITIVITY OF THE
TECHNIQUE.
DISADVANTAGES OF GC Limited to volatile samples Notsuitable for thermallylabile samples Some samples may
require intensive preparation Samples mustbe soluble and notreactwith the column.4. Requires spectroscopyto
confirm the peak identity
:
Instrumentation ofHPLC
Instrumentation:
Instrumentation Modern HPLC essentiallyconsistoffollowing main components:Solventdelivery systems Pumping
systems Sample Injector systems HPLC Column(s ) Detector Data System 9/22/2013 2
HPLC Instrumentation Overview:
HPLC Instrumentation Overview Principle Pattern An Example Solvent Reservoirs Controller SolventCabinet
Vacuum Degasser BinaryPump Auto sampler Column CompartmentDetector 9/22/2013 3
Solvent delivery systems:
Solvent delivery systems Continuouslyprovide eluent(solvent). Provide accurate mobile phas e compositions .
Includes solventreservoirs ,inletfilter , and degassing facilities which works in conjugation.9/22/2013 4
Solvent Reservoirs:
Solvent Reservoirs A good HPLC unit should have 3-4 solvent reservoirs to release eluentinto a mixing cham ber at
varying rate. Inert container for holding the solvent(mobile phase).9/22/2013 5
Inlet Filters:
Inlet Filters Type of filter. Stainless Steel or glass with 10 micron porosity. Removes particulates from solvent.
9/22/2013 6
Degassing System:
Degassing System Removed dissolved gases (such as oxygen and nitrogen).May consistofvacuum pump system,
a distillation system,a heating and stirring device, or a system for spearing.9/22/2013 7
Pumping System:
Pumping System C onstant,reproducible, and pulse free supplyof eluentto the HPLC column.Flow rate in between
0.1-10 cm 3 min -1 . Operating pressures from 3000 psi to 6000 psi.9/22/2013 8
Types of Pumping System:
Types of Pumping System Mainly three types Constantflow reciprocating pump Syringe (or displacement) type
pump.Pneumatic (or constantpressure) pump.9/22/2013 9
Constant flow reciprocating pump:
Constantflow reciprocating pump The term "reciprocating"describes anycontinuouslyrepeated backwards and
forwards motion . Widely used (~90% in HPLC system) type of pump.It gives a pulsating deliveryof the eluent.Pulse
damper is used to make the flow pulse free. Deliver solvent(s ) through reciprocating motion ofa piston in a hydraulic
chamber.Solvent is sucked during back stroke and gets deliver to the column in forward stroke. Flow rates of eluent
can be set by adjusting piston displacementin each stroke.9/22/2013 10
Reciprocating Pump Continuous… :

7. Reciprocating Pump Continuous… Advantages:Small internal volume (35-400 µL) High outputpressures up to
10,000 psi.Smartadaptability to gradientelution.Constantflow rates independentof column back pressure ,solvent
viscosity and temperature.9/22/2013 12
Syringe (ordisplacement) type pump :
Syringe (or displacement) type pump Consistoflarge syringe like chamber (capacityup to 500 Cm 2 ). Plunger
activated by screw-driven and hydraulic amplifier machine.Suitable for small bore column.9/22/2013 13
Displacement PumpContinuous… :
DisplacementPump Continuous… Advantages:Flow is independentofviscosity, back pressure.Deliver pulseless
flow. Provide pressure up to 78,000 psi.Disadvantages:CostlyLow flow rate (1 to 100 mL/min).Limited solvent
capacity. Inconvenience in frequent refilling i.e.in changing solvent9/22/2013 15
PneumaticPump:
Pneumatic Pump Gas is used to pressurize the mobile phase presentin a collapsible solventcontainer.9/22/2013 16
PneumaticPump Continuous…:
Pneumatic Pump Continuous… Advantages:Not very costly. Provide pulse free flow. Disadvantages:Produce
pressure onlyup to 2000 psi. Not suitable for gradientelution.Flow rate depends upon column back pressure,and
viscosity. Small capacity for filing of solvent. 9/22/2013 18
Sample Injection system:
Sample Injection system Introduce required sample volume accuratelyinto the HPLC system . Introduction of sample
withoutdepressurizing the system.Volume of sample mustbe very small (2 µL to 500 µL). Types of injection system:
Manual injection( Rheodyne / Valco injectors ) Automatic injection 9/22/2013 19
Manual Injector:
Manual Injector Also know as Rheodyne / Valco injectors .User manuallyloads sample into the injector using a
syringe.Overloading of column causes band broadening hence volume used mustbe very small (2 µL to 500 µL).
Sample should be introduce withoutdepressing the system.9/22/2013 20
Manual Injector :
Manual Injector Working: 9/22/2013 21
Automatic Injector System :
Automatic Injector System Also know as A utosampler . Programmed based sample deliverysystem.User loads vials
filled with sample solution into the autosampler tray (100 samples ).Autosampler automatically1.Measures the
appropriate sample volume,2.Injects the sample,3.F lushes the injector to be ready for the next sample ,etc ., until
all sample vials are processed.Also controls the sequence ofsamples for injection from vials.9/22/2013 22
HPLC Columns:
HPLC Columns Material:Stainless steel (highlypolished surface).External diameter:6.35 mm Internal diameter:4-5
mm ( usual 4.6 mm ) Length: 10-30 cm ( usual 25 cm ) Packing particles size ( 3 µm, 5µm,10 µm ) Stainless steel
frits or mess discs ( porosity< 2 µm ) retain packing material.9/22/2013 23
HPLC Columns Continuous…:
HPLC Columns Continuous… On the basis ofchromatographic objective HPLC column can be categorized as
follows:9/22/2013 28 Scale Chromatographic Objectives Analytical Information ( compound identification and
concentration) Semi-preparative Data and small amountofpurified compound[<0.5 g]Preparative Large amountof
purified compound [>0.5 g] Process (industrial) Manufacturing quantities ( g to kg)
Stationary Phase (column packing):
Stationary Phase (column packing) The stationaryphase is the substance fixed in place for the chromatography
procedure.The stationaryphase can be a solid,a liquid,or a bonded phase.B onded phase is a stationaryphase
that is covalently bonded to the supportparticles or to the inside wall ofthe column tubing . C hemically-modified
silicas ,unmodified silica or cross-linked co-polymers ofstyrene and divinyl benzene, commonlyused as stationary
phase.9/22/2013 29

8. Stationary Phase Continuous…:
Stationary Phase Continuous… S ilica particles as the basis ofthe support.Sizes 3 µm, 5 µm, and 10 µm (spherical
and regular in shape).Pore size normallyare in the 60 – 100 Å range.Pore size of 300 Å or larger being used for
larger biomolecules.Columns are packed using high-pressure to ensure thatthey are stable during use.9/22/2013
30
Mobile Phase:
Mobile Phase Also know as eluent. Solvent used in HPLC mustbe of HPLC grade i.e. Filtered using 0.2 μ m filter.
Eluting power of the mobile phase is determined byits overall polarity, stationaryphase polarity and the nature of the
sample components .For 'normal-phase‘ separations eluting power increases with increasing polarityof the solvent,
while for 'reverse-phase 'separations eluting power decreases with increasing solventpolarity. 9/22/2013 36
Detectors:
Detectors The detector refers to the instrumentused for qualitative and quantitative detection of analytes after
separation .Monitors the eluent as it emerges from column.E stablishing both the identity and concentration of
eluting components in the mobile phase stream.Characteristics ofdetectors:Adequate sensitivity ( 10 -8 to 10 -15 g
solute sec -1 ). Desired stabilityand reproducibility.Sort response time Minimal internal volume (minimize zone
broadening).9/22/2013 38
Detectors continuous…:
Detectors continuous… Solute propertydetectors:R espond to a particular physical or chemical characteristic ofthe
solute which should be ideallyand absolutelyindependentofthe mobile-phase being used.(a) UV - detectors ( b )
Fluorescence Detectors (c) Electrochemical detectors 9/22/2013 41
Refractive - index detector:
Refractive - index detector Also know as ‘RI-Detector’ and ‘Refract meter’.Based on refractive index measurement.
Determine change ofrefractive index of the eluantfrom the column with respectto pure mobile phase .Types: (a)
Deflection refractometer (b) Fresnel refractometer Referactive index (n) = c = speed oflightin vacuum v = speed of
lightin medium 9/22/2013 43
Refractive - index detector continuous…:
Refractive - index detector continuous… Advantages:Universal response Independentof flow rate. Disadvantages:
Less sensitivityTemperature dependent,stricttemperature control (±0.001 °C ). Not suitable for gradientelution.
9/22/2013 46
Conductivity detectors:
Conductivity detectors Conductivity measurementofeffluent. M ainly measure inorganic ions and small organic
substances,including organic acids and amines .Conductivity detector measures electronic resistance and
measured value is directlyproportional to the concentration of ions presentin the solution . Employed as a detector in
an ion chromatography.
Ultraviolet Detector:
Ultraviolet Detector B ased on the principle ofabsorption ofUV visible lightas the effluent from the column is passed
through a small flow cell placed in the radiation beam .H igh sensitivity(detection limitof about 1x10 -9 g mL -1 for
highly absorbing compounds ).Detector cells are generally1 mm diameter tubes with a 10 mm optical path length.
9/22/2013 49
Ultraviolet DetectorContinuous…:
Ultraviolet Detector Continuous… Ultravioletdetector are of fallowing types:F ixed-wavelength detector V ariable-
wavelength detector Photodiode-arraydetectors Fixed-wavelength detector:SimplestUV absorption detector.
Mercury lamp source, optical filters to selecta limited number ofwavelengths 220,250,254,280, 313, 334, 365,436,
and 546 nm.9/22/2013 50
Ultraviolet DetectorContinuous…:

9. Ultraviolet Detector Continuous… Variable-wavelength detector:Deuterium lamp (for UV region) or Tungsten filament
lightsource (for visible region) a diffraction grating monochromator for wavelength selection and a photomultiplier
detector. A llow monitoring atany wavelength within the working range of the detector. 9/22/2013 51
Fluorescence Detectors:
Fluorescence Detectors B ased on filter- fluorimeters or spectrofluorimeters .Flow cell has a capacity 10-25µL with a
narrow depth (1.07 mm) and large surface area for excitation-emission collection .The fluorescentradiation emitted
by the sample is usuallymeasured at90° to the incidentbeam .Simplestdetector:mercury excitation source,and
filters (one/more).Advanced detector: xenon source a nd a grating monochromator to isolate emitted fluorescent
radiation.9/22/2013 54
Electrochemical Detector:
Electrochemical Detector The term 'electrochemical detector'in HPLC normallyrefers to amperometric or coulometric
detectors.Measure the current associated with the oxidation or reduction of solutes.C omplete removal ofoxygen is
almostdifficult,therefore , electrochemical detection is normallybased upon the oxidation of the solute.A
mperometric detector is presentlyconsidered to be the bestelectrochemical detector.9/22/2013 57
Applications of HPLC:
Applications ofHPLC Used for both qualitative and quantitative analyses ofenvironmental,pharmaceutical ,
industrial,forensic,clinical,and consumer productsamples .A few typical examples:I solation ofnatural
pharmaceuticallyactive compounds C ontrol ofmicrobiological processes Assayof cephalosporins Assayof
frusemide Assayof theophylline A ssayof corticosteroids Assayof dichlorphenamide A ssayof human insulin
9/22/2013 61
Applications ofHPLC Continuous… Isolation ofnatural pharmaceuticallyactive compounds Chromatographic
Conditions :Column :Size-25 cm × 4.6 mm ID Adsorbent: Lichrosorb RP-8 Mobile-phase :Water/Acetonitrile-
GradientElution Detector : UV 254 nm Category of Natural Products Constituents Used as Alkaloids Morphine;
Codeine Analgesic,Antitussive Glycoside Digitalis glycosides Sennosides Cardiovascular diseases,Laxatives
9/22/2013 62
Applications ofHPLC Continuous… Control ofmicrobiological processes:Determine kinetics ofthe microbiological
process M onitoring ofthe on-going process Isolation and purification ofactive ingredients P urity control of active
constituents M onitoring derivatization reactions 9/22/2013 63
LAYER CHROMATOGRAPHY :
Rahul Patil HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY
Contents:
Contents Introduction.Principle of HPTLC. Difference between TLC & HPTLC. Steps involved in HPTLC. Material
used for plates.Mobile phase.Sample application.HPTLC Plate development.Applications of HPTLC.
Introduction:
Introduction Sophisticated form ofTLC. In 1973,Halpaap introduced first“Nano TLC plates’’ In 1977,the first major
HPTLC publication is “HPTLC-high”
Principle:
Principle Separation mayresultdue to adsorption or partition or by both phenomenon depending upon the nature of
adsorbents used on plates and solvents system used for development.
Fig. HPTLC Instrumentation:
Fig. HPTLC Instrumentation
Difference between HPTLC & TLC:
Difference between HPTLC & TLC HPTLC TLC Particle size 4-8 µm 5-20 µm Sorbent layer thickness 100 µm 250 µm
Efficiency High Low Separations 3-5cm 10-15cm Analysis time Faster Slower
Slide 7:
HPTLC TLC Developmentchamber Less amountofmobile phase More amountofmobile phase Sample spotting
Auto sample Manual spotting Scanning Visible/Fluoros-scanner scan the entire chromatogram Notpossible

10. Steps in HPTLC :
Steps in HPTLC Selection of chromatographic layer.Layer pre-washing.Preparation ofsample.Application of
sample.Chromatographic development.Detection of spot.Scanning and documentation ofchromatoplate .
Sample Preparation :
Sample Preparation For normal chromatography, solventshould be non-polar and volatile.For reversed
chromatography, polar solventis used for dissolving the sample.Sample and reference substances should be
dissolved in the same solventto ensure comparable distribution atstarting zones.
Application of sample :
Application of sample The selection ofsample application technique and device to be used depends primarilyon,
Sample volume No.of samples to be applied Required precision
Slide 11:
Micro syringes are preferred if automatic application devices are notavailable.Volume recommended for HPTLC -0.5-
5 μ l. Sample spotting should notbe excess or not low. Problem from overloading can be overcome by applying the
sample as band.
Some applicators used for application of sample :
Some applicators used for application ofsample Bycapillary tube,0.1-0.2μl volume sample spotis applied.By micro
syringes,1μl sample can applyeither as spotor band. By automatic sample applicator.By micro bulb pipette.
Sorbents usedin HPTLC :
Sorbents used in HPTLC Silica gel 60F, it analyses 80% of drugs.Aluminium oxide,itanalyses the basic substances
and steroids.Cellulose.Silica gel chemicallymodified in amino group and CN.
Plates usedin HPTLC:
Plates used in HPTLC Glass plates.Polyester/polyethylene plates.Aluminium plates.
Solvents usedforpre-washing :
Solvents used for pre-washing Methanol.Chloroform:Methanol (1:1) Chloroform:Methanol:Ammonia (90:10:1 )
Methylene chloride:Methanol ( 1:1 ) Ammonia solution (1%)
Pre-conditioning (ChamberSaturation) :
Pre-conditioning (Chamber Saturation) Time required for the saturation depends on the mobile phase.If unsaturated
chamber used for development,the solvent evaporates from the plate mainlyat the solvent front and it results in
increased R f values.
Mobile phase:
Mobile phase Solventcomposition expressed in v/v. Mobile phase should be ofhigh graded. Chemical properties ,
analyses and sorbentlayer factors should be considered while selection ofmobile phase.If possible mobile phase
containing more than 3 or 4 components should be avoided.
Slide 18:
Prevents contamination ofsolvents.Multi-componentofmobile phase once used is notrecommended for re-use.
Chemical reaction avoided between SP & MP. e.g. Acetic acid, Ammonia.
Methods of HPTLC development:
Methods of HPTLC developmentVertical development.Vario method development.Horizontal development.
Automatic multiple development.
Detection andvisualization:
Detection and visualization First spots detects under UV lightbecause itis non destructive.Fluorescentcompound
spots can be seen at 254nm or 366nm.For non fluorescentcompound spots,fluorescentstationaryphase (silica gel
GF) is used.Non UV absorbing compounds detects bydipping the plates in 0.1% iodine solution.

11. Factors influencing separation andresolution of spots :
Factors influencing separation and resolution ofspots Layer thickness.Mobile phase.Solventpurity Size of
developing chamber Sample volume to be spotted Size of initial spotSolvent level in chamber
Applications of HPTLC :
Applications ofHPTLC Pharmaceutical industry: quality control, purity check etc. Food analysis :quality control,
stabilitytesting etc. Clinical applications :metabolism studies,drug screening etc.Forensic : poisoning investigations.
1. The Mobile Phase
o The mobile phase of chromatography equipment is the substance that moves the sample
through the machine. In HPLC the mobile phase is a liquid made up of an organic solvent,
ultrapure water and other ingredients to ensure its compatibility with the sample. GC uses
gas for its mobile phase. Gases used include helium, nitrogen, argon or hydrogen,
depending on what is being analyzed.
The Columns
As samples travel over chromatography columns, the sample and mobile phase interact with
the column contents causing the components of the sample to elute at different time. HPLC
columns are typically four-to-six inch-long metal or glass tubes tightly packed with silica or
differing carbon chain lengths. GC systems have coiled capillary columns with interior walls
coated with various materials depending on the lab's needs. Stretched out, GC columns can
reach lengths of 100 feet.
The Samples
GC is used for volatile compounds (those that break down rapidly) while HPLC is better for
less volatile samples. If a sample contains salts or carries a charge, it must be analyzed using
HPLC, not GC.
Temperature Control
o GC columns are housed in an oven within the machine. A computer changes the
temperature while samples are analyzed. The higher the temperature, the faster the sample
elutes, but temperatures that are too high produce poor results. HPLC columns are kept at a
stable temperature (most often room temperature) at all times.
McLafferty Rearrangement
The McLafferty rearrangement is a characteristic fragmentation of the molecular ion of
a carbonyl compound containing at least one gamma hydrogen (see alpha hydrogen).
eg:

12. r Overhauser effect
From Wikipedia,the free encyclopedia
The Nuclear Overhauser Effect (NOE) is the transfer of nuclear spin polarization from one nuclear spin
population to another via cross-relaxation. It is a common phenomenon observed by nuclear magnetic
resonance (NMR) spectroscopy. The theoretical basis for the NOE was described and experimentally verified
by Anderson and Freeman in 1962.[1]
The NOE is an extension of the seminal work of American physicist Albert
Overhauser who in 1953 proposed that nuclear spin polarization could be enhanced by the microwave
irradiation of the conduction electrons in certain metals.[2]
The general Overhauser effect was first
demonstrated experimentally by T. R. Carver and C. P. Slichter, also in 1953.[3]
Another early explanation and
experimental observation of the NOE was by Kaiser in 1963 [4]
in an NMR experiment where the spin
polarization was transferred from one population of nuclear spins to another, rather than from electron spins to
nuclear spins. However, the theoretical basis and the applicable Solomon equations[5]
had already been
published by Ionel Solomon in 1955.[6]
Subsequent to its discovery, the NOE was shown to be highly useful in NMR spectroscopy for characterizing
and refining organic chemical structures.[7]
In this application, the NOE differs from the application of spin-spin
coupling in that the NOE occurs through space, not through chemical bonds. Thus, atoms that are in close
proximity to each other can give a NOE, whereas spin coupling is observed only when the atoms are
connected by 2–3 chemical bonds. The inter-atomic distances derived from the observed NOE can often help
to confirm a precise molecular conformation, i.e. the three-dimensional structure of a molecule. In 2002, Kurt
Wüthrich was awarded the Nobel Prize in Chemistry for demonstrating that the NOE could be exploited
using two-dimensional NMR spectroscopy to determine the three-dimensional structures of biological
macromolecules in solution.[8]
Some examples of two-dimensional NMR experimental techniques exploiting the NOE include:
 NOESY, Nuclear Overhauser Effect Spectroscopy, determination of the relative orientations of atoms in a
molecule, producing a three-dimensional structure
 HOESY, Heteronuclear Overhauser Effect Spectroscopy, or NOESY cross-correlation between atoms of
different elements
 ROESY, Rotational Frame Nuclear Overhauser Effect Spectroscopy, spin-locking the magnetization to
prevent it from going to zero, applied for molecules for whcich regular NOESY is not applicable
 TRNOE, Transferred Nuclear Overhauser Effect, measuring the NOE between two different molecules
interacting in the same solution, as in a ligand binding to a protein[9]

13.  DPFGSE-NOE, Double Pulsed Field Gradient Spin Echo NOE experiment, a transient experiment that
allows for suppression of strong signals and thus detection of very small NOEs
CHIRAL CHROMATOGRAPHY:
CHIRAL CHROMATOGRAPHY KARAN TRIVEDI 42
Introduction:- Chiral Chromatographyis a branch of chromatographythat is oriented towards the exclusive separation
of chiral substances.Certain stereoisomers thatdiffer only in the spatial arrangementoftheir atoms and in their
capacity for rotating the plane of polarized light are termed optically active or chiral and the individual isomers are
called enantiomers.Enantiomeric separations are achieved in chiral chromatographyby the judicious use ofchiral
phases.The mobile phase can be a gas or liquid giving rise to chiral gas chromatographyand chiral liquid
chromatography.43 KARAN TRIVEDI
Principal of Enantiomer separation:- On transfer of a pair of enantiomers into asymmetric environment. To
diastereomeric species are formed with distinctphysicochemical propertyprofile On the basis ofwhich physical
separation into individual enantiomers maybe achieve. 44 KARAN TRIVEDI
Chiral selector (SO):- Capable ofundergoing covalentor non-covalentinteraction with the individual enantiomer (
selectands,SAs ). Depending on the nature of the interaction stabilizing the respective diastereomer SO-SA species
45 KARAN TRIVEDI
Transformation ofthe SAs of interestinto covalent diastereomer byconversion with suitablyreactive SOs. Followed
by separation ofdiastereomeric product with achiral chromatographic techniques 46 KARAN TRIVEDI Direct
enantiomer separation Indirectenantiomer separation:- Chiral Derivatization agent(CDAs):-  Indirect enantiomer
separation Enantiomer separation strategies:-
Applicable only to enantiomer presenting a single or more butselectivelyaddressable functional group suitable for
derivatization. Direct enantiomer separation:- 1) Chiral mobile phase additive 2) chiral stationary phase mode 1)
Chiral mobile phase additive (CMPA): A combination of an chiral stationaryand a chiral mobile phase is employed.
On introduction of a mixture of enantiomers into this system.47 KARAN TRIVEDI
The individual of enantiomers form diastereomeric complexwith the chiral mobile phase additive.This diastereomeric
complexmay exhibit distinctassociation /dissociation rate,thermodynamics stability,and physiochemical property
therefore may be separated on an appropriate a chiral stationary phase.2) chiral stationary phase mode:Consists of
an inertchromatographic supportmatrixincorporating chemicallyor physicallyimmobilized SOspecies.48 KARAN
TRIVEDI
Incorporation into polymeric network by copolymerization or combination ofthis procedures.49 KARAN TRIVEDI
Physical fixation employing coating technique.  Covalentattachmenton to the surface of suitablyprefunctionalized
carrier materials. CSPs maybe created by a variety of SO a immobilization technique.
also used for gradientelution and variable temp. protocol.50 KARAN TRIVEDI used wide range of mobile phase
solventand modifiers.  stability of CSPs more and flexibility with respectto method optimization parameter. CSPs
provide several operational advantages over CMAs based enantiomers separation.
Carrier material modified with Chiral moieties -inorganic material mainlysilica gel modified on the surface. -organic
polymer network grafted with chiral molecules.51 KARAN TRIVEDIChiral Organic Polymer -Pure -Polymer coating
on inorganic support -Grafted polymer. Type-2 Classification ofChiral StationaryPhase:Type-1
imprinted material -imprinted polymer -inorganic material imprinted on the 52 KARAN TRIVEDIType-3
Polysaccharide based CSPs : -Applied as pure polymers in a form adequate for chromatographic purposes or as
coating on an inert achiral supportto confer mechanical stability.Polyacrylamide CSPs: -cross linked opticallyactive
polyacrylamides and polymethacrylamides constitute. -Improvementin the mechanical performance ofthese CSPs
was achived by polymerization of the acrylic monomer on the surface of silica gel,yielding a grafted polymer 53
KARAN TRIVEDIPolymeric phase
Polymeric chiral phase derived from Tartaric acid : -Prepared by polymerization of N,N’-diallyl derivatives of
tartardiamide and grafted onto silica gel. -As this phase are cross linked and bonded to silica gel they are insoluble in
organic solventand there is no limitation regarding the choice ofmobile phase. -Normal phase,reversed phase and
supercritical fluid condition have been applied. -They show good mechanical stabilitybut relatively high contentof
achiral silica gel in these CSPs reduces their loading capacity.54 KARAN TRIVEDI
Brush type CSPs Π -acidic and Π -basic phases : - Π -acceptor phases are derived from the amino acids
phenylglycine or leucine covalently or ironicallybonded to 3-aminopropyl silica gel. -These chiral phase are
commerciallyavailable for analytical or preparative separation ofenatiomers. -Amine chiral selector such as valine,

14. phenylalanine,tyrosine and 1,2-trans –diaminocyclohexane and 1,2-trans-diphenylethylene diamine.55 KARAN
TRIVEDI
Cyclodextrin based CSPs -It is cyclic olygosaccharides thatcan form inclusion complexin their highly hydrophobic
chiral cavity with a large variety of molecules. -The size of cavity differs for α -, β -, γ - cyclodextrins and the substitute
on cyclodextrin play a determining role in the ability to complexa defined molecule. -Cyclodextrin CSPs were
prepared by immobilizing CD in polymeric structures or on silica gel which give good performance on an analytical
scale.56 KARAN TRIVEDI
Chirobiotic CSPs -These phase have been prepared by covalent immobilization ofthe glycopeptides (vancomycin,
teicoplanin,and restocetin) to silica gel according to standard procedures. -The chiral selectors are characterized by
the presence ofseveral chiral cavities providing differentenvironments for enantioselective interaction.57 KARAN
TRIVEDI
Chiral ion-exchange stationaryphases -Developed a series ofanion- exchange CSPs based on quinine and quinidine
as chiral selectors. -This phase are perticularlyappropriate for the separation ofthe enantiomers ofchiral acidic
compounds.Aptamer type CSPs -DNA and RNA aptamer use as powerful targetspecific affinity probe for many
analytical application.58 KARAN TRIVEDI
Antibody type CSPs -Generation of sterioselective recombinantantibodyfragmentand which is immobilization to
polystyrene-based macro porous perfusion resin. -immobilized monoclonal anti-D and anti-L-phenylalanine antibodies
as target-specific CSPs -To avoide antibody denaturation enantiomer separation were carried outwith phosphate
buffer saline atpH 7.4 59 KARAN TRIVEDI
Application:- -Quinine and Quinidine -Atropine and Hyoscyamine -Cetrizine and Levo-cetrizine -Omeprazole and
Esomeprazole(more effective in GERD) -Dopamine and levodopamine -D-Amphetamine and Amphetamine -
Dextromethorphan and Levo-methorphan 60 KARAN TRIVEDI