Gas Chromatograp

8/3/2019 Gas Chromatograp

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By

K Rakesh gupta


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INTRODUCTION

PRINCIPLE

THEORIES

PARAMETERS

INSTRUMENTATION

APPLICATIONS

REFERENCE


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CHROMATOGRAPHY: SEPARATION TECHNIQUE

: TWO PHASES are used

: MIKHAIL TSWETT invented

GAS CHROMATOGRAPHY: MARTIN AND JAMES:GAS as M.phase always

: solid or liquid S.Phase

Choice for THERMALLY STABLEand VOLATILEcompounds

TWO TYPES BASED ON STATIONARY PHASE

GSC:Stationary phase isSOLID

GLC:Stationary phase isLIQUID


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GSC:ADSORPTION

:RELATIVE AFFINITY

:More affinity towards S.P travels slowly-slow elution


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GLC

:PARTITION

: SOLUBILITY

:more partition coefficient travels slowly slow elution


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Mainly three theories are involved in GC

1. PLATE THEORY2. BAND BROADENING THEORY3. RATE THEORY


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Concept compared counter-current distribution

Plates are hypothetical lines where equilibration occur

Plates analogous to tubes in CCD (Catalytic Combustion

Detector)system

In plate theory two main terms are used as quantitativemeasures of chromaographic column efficiency

PLATE HEIGHT(HETP)PLATE COUNT or no. of PLATES(N)


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CALCULATION OF THE DISTRIBUTION

THROUGH 4 TRANSFERS:

Transfer non

Tube no.r = 0

Tube nor=1

Tube no:r=2

Tube no:r=4

Tube no:r=3

n=0 B

A

0/1

p/qn=1 B

A0/qPq/q2

p/0p2 /pq

n=2 BA

0/q2Pq2 /q3

pq/pq2p2 q/2pq2

p2/0p3 /p2 q

n=3 BA

0/q3Pq3 /q4

pq2 /2pq23p2q/3p2 q3

2p2 q/p2 q3p3q/3p2 q2

p3 /0p4 /p3 q

n=4 B 0/q4 Pq3 /3pq3 3p2q2/3p2q2 3p3q/p3 q p/0

Total after

4 transfers

q4 4pq3 6p2q2 4p3q p4


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The expansion of the function (p+q)n is laboriousfor large n, and an easier calculation is available.

The binomial expansion may be written

(p+q)n = qn + nqn-1p + n(n-1) qn-2 q2++ pn

2

which can be expressed

(p+q)n = nr=0 n! prq(n-r)

r!(n-r)!


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Binominal distribution expression for the fraction of totalsolute in rth plate after n mobile phase volumes havepassed into the column

pr q(n-r)r! (n-r)!

n = no.of mobile phase volumes passed into the column

r = plate number( 0,1,2,3,..r)

p = 1/(KU+1) = fraction of solute per plate in M.P at equilibrium

q = KU/(KU+1)= fraction of solute per plate in S.P at equilibria

Tnr = n! pr q(n-r)r! (n-r)!


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When n is large and p is not small (as in CCDS), binomial

distribution approaches normal (Gaussian) distribution

According to statistics MEAN is given as

Standard deviation

= np

= np


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ELUTION CHROMATOGRAM

Concentration

| Tr |time | width |


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CALCULATION FOR NO.OF PLATES(efficiency)

Length= velocity. time

= Rvtr Standard deviation = Rv

Where is zone-standard deviation Combining above equations

= L

tr

tr2 = L 2/ H since: 2 = HL

tr = r since:

=L/H

r = 16(tr/w)2


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Random walk

Reflects loss of efficiency

Rate process controls zone width

From plate theory : 2

= HLH is a measure of zone spreading and columnefficiency

( height equivalent to theoretical plates)

HETP = Length of the columnno.of theoretical plates


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1. LONGITUDINAL MOLECULARDIFFUSION

2. MASS TRANSFER(SORPTION-DESORPTION KINETICS)

3. EDDY DIFFUSION


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1.LONGITUDINAL MOLECULARDIFFUSION

L= vt

=2Dm t

=2Dm L/v

Hdiff =2Dm /v is empirical factor of value 0.6

Hdiff = 2Dm + 2s DS (1-R)/R

v

This is in the form of Hdiff = B/v

Where B is the function of molecular and chromatographic

properties


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2.MASS TRANSFER(SORPTION-DESORPTION KINETICS)

No.of random steps n = 2L/vta

True step length L =vta - Rvta or (1-R)vta According to random walk theory =Ln

=2(1-R)vta mm

Hs-d = Cv


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3.EDDY DIFFUSION

Flow and diffusion mechanism are coupled

Plate height contribution through flow and diffusion is notadditive ,but found to be

Heddy = 11/HF + 1/HD

H is independent of velocity and H is dependent onaverage velocity

Heddy = 11/A + 1/Ev

Where A and E include the partical diameter


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CALCULATION FOR HETP

Total plate height contribution from 3 process

H = Heddy + Hdiff + H(s-d)

Van Deemeter equation

H = A + B/v + Cv

H = 1 + B + Cv1/A + 1/Ev v


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Random walk Fronting tailing Quantitative measure

detector

signal

time

Fronting- saturation of S.P

Tailing-more active adsorption


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RETENTION TIME(Rt)

Retention time is the difference in the time between thepoint of injection and appearance of peak maxima

Rt is the time required for 50% of a component to beeluted from the column

Unites : min or sec

Retention time is also proportional to the distance movedon a chart paper, which can be measured in cm or mm


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RETENTION VOLUMN(VR)

Retention volume is the volume of carrier gasrequired to elute 50% of the component from thecolumn

Corrected retention volume

Where j is pressure drop correction factor

Pi and Po are inlet and outlet pressures

Retention volume = Retention time flow rate

VR0 = j VR

j = 3 . ( Pi / Po )2 - 1

2 . (Pi / Po )3 - 1


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ADJUSTED RETENTIONVOLUMN(VR!)

Adjusted retention volume is calculated as

Where VM is DEAD VOLUME of mobile phase Applying pressure drop correction to VR

! Gives

net retention volume

VR = VR - VM

VN = j VR


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SELECTIVITY( )

A way of improving resolution is to change the selectivityof the column by changing stationary and mobilephases

Selectivity is the ratio of partition coefficients

Selectivity term can be evaluated from the chromatogram

= VR,2 VM (or) = tr2 - tmV

R,1 V

Mtr1

- tm


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RESOLUTION(Rs)

The degree of disengagement of two bands is resolution.

In terms of width and diameter

In terms of time and width

In terms of zone of migration

In terms of , k ,Nwhere k is capacity factor

k=nS/nM

RS = dAdBW

RS = 2 Rt 1 -Rt2wA + wB

RS = L . R16H R

RS = N k -14 k+1


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EFFICIANCY;NO.OF PLATES(n)

Efficiency of column is expressed by the no. of theoreticalplates

If the no.of theoretical plates is high, the column is said tobe highly efficient

If the no.of theoretical plates is low , the column is said tobe less efficient

For GC columns, a value of 600/meter is sufficient

But for HPLC , high values like 40,000 to 70,000/meterare recommended

No.of theoretical plates efficiency


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HETP(H)

Decides the efficiency of separation

If HETP is less, the column is more efficient

If HETP is more, the column is less efficient

HETP = Length of column

no.of theoretical plates

HETP calculated by using Van Deemeter equationHETP = A + B + Cv

v

HETP 1EFFICIENCY


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INSTRUMENTATION

Carrier Gas

Flow regulators and meters

Sample injection system Columns & ovens

Detectors

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SCHEMATICDIAGRAMOF GAS CHROMATOGRAPH

G C


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Gas ChromatographComponents

FlameIonization

Detector

Column

Oven

Injection Port

top view

front view


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Carrier gas

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The mobile phase gas is called the carrier gas and

must be chemically inert.Sample component column detector

mobile phase gas

Helium ,argon ,nitrogen , carbon dioxide andhydrogen also used.

Selection of the best carrier gas very important ,because it effects both the column separation and

detector performance . The ratio of viscosity of diffusion coefficient

should be minimum for rapid analysis thats whyH, He are prepared for a carrier gas .


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Impurities in the carrier gas such as air water vapour

and trace gaseous hydrocarbons can cause sample

reaction, column character and affect the detector

performance.

The carrier gas system should contains a molecular

sieve to remove water and other impurities.

These gases are available in pressurized tanks.pressure regulators and flow meters are required to

control the flow rate of the gas.

The gases are supplied from the high pressure gas

cylinder , being stored at pressure up to 300psi

carrier gas should be better then 99.99%and 99.999%

is often used33


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Air inlet (detector))

H2 inlet (detector)N2 inlet(make-up gas)

He inlet

(carrier gas)


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Process Flow Schematic

Carrier gas(nitrogen orhelium)

Sample injection

Long Column (30 m)

Detector (flameionizationdetector or FID)

HydrogenAir


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Carrier Gas(mobile phase)

Requirements:

It should be inert and available at low cost High purity Easily available Less risk of explosion or fire hazards Pressure:

-Inlet 10 to 50 psi-packed column 25 to 150 mL/min.- capillary column 1 to 25 mL/min.

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Flow regulators & meters

Flow regulators are used to deliver the gaswith uniform pressure or flow rate

Flow rates of carrier gas:

Linear flow rate (cm/s): u = L/tr Volumetric flow rate (mL/min): u ( r2)L is length of column, it is retention time, r is the internal radius of column

Flow rate depends on type of column Packed column: 25-100 mL/min

Capillary column: 1 to 25 mL/min Flow rate will decreaseas column T increases

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FLOW REGULATORS


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Soap bubble flow meter

Aqueoussolution ofsoap ordetergent

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soap bubbles formedindicates the flow rate.

Glass tube with a inlet tubeat the bottom.

Rubber bulb-----store soapsolution

When the bulb is gently

pressed of soap solution isconverted into a bubble bythe pressure of a carrier gas&travel up.

Soap bubble meter


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inlet tube


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INJECTOR

Sample injection port


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Sample injection port Calibrated Micro syringes are used to

inject liquid sample

Purge :volatile components are

removed from sample by gentleheating

Rubber or silicone diaphragm(septum)

Sample port Temp: 50C

Packed Column: sample sizes-1 to 20

L

Capillary Column : 10 to 30 mL

splitter is used to deliver a fraction

of injection(1:50 to 1:500)

Avoid over loading

Slow injection & oversized samples

cause band spreading & poor

resolution

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Micro syringe


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1. Wash a syringe with acetone by filling

the syringe completely and ejecting thewaste acetone onto a papertowel. Wash 2-3 times.

2. Remove air bubbles in the syringe byrapidly moving the plunger up and downwhile the needle is in the sample.

3.Usually 1-2 mL of sample is injectedinto the GC.

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COLUMN OVENS


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Column temperature is very important in GC

The column is ordinarily housed in athermostatic oven.

they are usually formed as coils having

diameters of 10 to 30 cm. The optimum column temperature depends

upon the boiling point of the sample and thedegree of separation required.

Roughly, a temperature equal to or slightlyabove the average boiling point of a sampleresults in a reasonable elution time (2 to 30min).

Column ovens


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COLUMNS


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Columns

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Two types of columns are used in gaschromatography, packed and open

tubular or capillary.

Packed column length from less than 2m to 5 m

Capillary columns from few m to 100 m

They are constructed of stainless steel,glass, fused silica, or Teflon.


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Column

Types of columns

1.packed columns

2. Open tubular or capillary.

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Capillary column- 30mPacked column-3m


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Packed columns

Packed columns are fabricated from glass,metal (stainless steel, copper, aluminum), orteflon tubes that typically have

Lengths------ 2m to 3 m

Internal diameters ------- 2 to 4 mm. These tubes are densely packed with a

uniform, finely divided packing material, orsolid support, that is coated with a thin layer

(0.05 m) of the stationary liquid phase. In order to fit in a thermostatic oven, the tubes

are formed as coils having diameters ofroughly 15 cm. 51

Capillary (or)Open tubular


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Capillary (or)Open tubularColumns

1.Wall-coated open tubular (WCOT)

Capillary tubes coated with a thin layer ofstationary phase

Old: stainless steel, Al, Cu, plastic, glass.

2.Support-coated open tubular (SCOT)

Inner surface of the capillary is lined with a thinfilm (~30m) of a support materials, likediatomaceous earth

Lower efficiency than WCOT, higher than packed

column3.Fused-silica open tubular column (FSOT):Physical strength, low reactivity, flexibility. 0.32 to

0.25 mm


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Column Stationary Phases:

Packed liquid coated silica particles (


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The Stationary Phase

requirements for stationary phase

Low vapor pressure

Thermal stability

Low viscosity (for fast mass transfer)

High selectivity for compounds of interest

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DETECTOR


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DETECTORS Ideal characters of

detectorHigh sensitivity to even

small concentrtion linerity, ie, less response

to low concentration&proportional responseto high concentration

Large linear dynamicrange

Useful at a range oftemperatures

Good stability andreproducibility

Rapid response time

Easy to use

Stable, Predictableresponse

Inexpensive

operation from RT to 400

oC

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Types of detectors1. Thermal Conductivity Detector(TCD)2. Flame Ionization Detector(FID)3. Atomic Emission Detector(AED)4. Electron Capture Detector(ECD)

5. Nitrogen Phosphoroes Detector(NPD)6. Photo Ionization Detector(PID)7. Flame Photometric detector(FPD)8. Electrolytic conductivity detector (Hall

detector)9. Absolute Mass Detector(AMD)10. Thermionic Detector(TD)

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ame on za onDetector(FID)


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Detector(FID)Most widely used, Air-H2 flame

Number of ions depends onnumber of reduced (methylene)

carbons in a moleculeThe positive ions will be attracted tothe cylindrical cathode.

Negative ions and electrons will beattracted to the jet anode.

Organic compounds Producesions and electrons pyrolyzed(tempof flame) burner tip andelectrode.(fhv power)Ions &electrons move toward thecollector

less sensitive for non-hydrocarbongroups

Insensitive to noncombustiblegases(CO2, SO2, NO2 and H2O)Insensitive to functional group

(carbonyl, alcohol, halogen and amine)

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Thermal Conductivity Detector(TCD)


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Thermal Conductivity Detector(TCD)

Thermal conductivity detector cell

Arrangement of the twin detectors

Element(platinum, gold or tungsten wire)is electrically heated at constant power

Temperature depends on thermalconductivity (He & H)of surrounding gas.

Hydrogen and helium have higherthermal conductivity and carrier gas

provide best sensitivity

Six times greater than the Organiccompounds

Poorer sensitivity than FID, but more

universal

Advantages: simplicity, large range,inexpensive, linearity is excellent.organic & inorganic species

DA: low sensitivity ng/mL 60

Electron Capture Detectors (ECD)


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Electron Capture Detectors (ECD)

The sample elute from a column ispassed over a radioactive -

emitter(nickel-63) Selectively to halogen-containingorganic sample ,like pesticides and,polychlorinated biphenyls

Ni-63: radioactive -emitter-- electron --

ionization of carrier gas (N2)High electronegative group (halogen,peroxide, quinones and nitro group) in thesample capture the electronHighly selective and sensitive,

nondestructiveInsensitive to amines, alcohols andhydrocarbons

AD: High sensitivity, analyse thepolyhalogenated organic compounds

Small linear range61

Th i i d t t ( it h h d t t )


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Thermionic detector(nitrogen phosphorus detector)

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N or P containing organic compounds

phosphorus atom is approximately tentimes greater than to a nitrogen atom and104 to 106 larger than a carbon atom.

Compared with the FID , the thermionicdetector is approximately 500 times more

sensitive to phosphorus-containingcompounds and 50 times more sensitive tonitrogen bearing species.

Column effluant + H2 +air(hotgas)electrically hearted Rb2SiO4

(rubidium silicate)bead at 180 V plasma(600 800C ) ions to determinecompounds

useful for detecting and determining themany phosphorus-containing pesticides.

ATOMIC EMISSION DETECTOR


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Eluent(column) helium(carrier) water cooled microwave cavity helium plasma(high temp) characteristic atomic emissionspectra grating diode array optical emission spectrometer detect the element .

ATOMICEMISSIONDETECTOR


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Six elements detect simultaneously .Determine the hetero atoms(H,P,S,O),silicon , heavymetals(Pb , Hg),tin, arsenic ,copper ,iron.

PHOTO IONIZATION


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UV light (10.2 eV H2or 11.7 eV Ar lamp)photo ionization ofmolecular currentto flow betweenbased electrodes

Most sensitive forAromatic and S, Peasily

photoionizedmolecules

Linear range is high

PHOTO IONIZATIONDETECTOR(PID)

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Flame photometric detector


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p(FPD):

S and P compounds photomultiplier to view light of 394

nm for sulphur (H2 + air S2)measurement or 526 nm for

phosphorus (H2 + air HPOspecies) Filteres are used to isolate the

appropriate bands

Intensity is recordedphotometrically X-, N-, Sn , Cr, Se and Ge

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filteres

photomultiplier

H2 + air

Column effluent


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APPLICATIONS

Q lit ti l i


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Qualitative analysis:

Retention time data should be useful foridentification of mixtures

Comparing the retention time of the

sample as well as the standard Checking the purity of a compound:

compar the standard and sample

Additional peaks areobtained..impurities are

present.compound is not pure68

Quantitative


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analysis:

Direct comparison method:-comparing the area of the peak, peak height,

width of peak.

Calibration curves:-standards of varying concentration are used

determine peak areas .o Internal standard method:

-A known concentration of the internal standard isadded separately to the standard solution

-The peak area ratio of sample and internal

standard.unknown concentration is easilydetermined .

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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 ofcomponents(amino acids ,plant extracts,volatile oils)

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Instrumental Analysis by Douglas A.Skoog , F.JamesHoller & Stanley R.Crouch.

Text book of Pharmaceutical Analysis by KennethA.Connor

www.google .com

Text book of Pharmaceutical analysis by Dr.S.Ravi Sankar

Introduction to instrumental analysis by Robert D.Braun

Chromatograhic methods by Smith
http://www.google/http://www.google/


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