By K Rakesh gupta
INTRODUCTION PRINCIPLE THEORIES PARAMETERS INSTRUMENTATION APPLICATIONS REFERENCE
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 STABLE and VOLATILE compounds TWO TYPES BASED ON STATIONARY PHASE GSC: Stationary phase is SOLID GLC: Stationary phase is LIQUID
GSC :ADSORPTION :RELATIVE AFFINITY :More affinity towards S.P travels slowly-slow elution
GLC :PARTITION : SOLUBILITY : more partition coefficient travels slowly – slow elution
Mainly three theories are involved in GC
PLATE THEORY BAND BROADENING THEORY RATE THEORY
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 quantitative measures of chromaographic column efficiency PLATE HEIGHT(HETP) PLATE COUNT or no. of PLATES(N)
’n’ r=0 n=0 B A 0/1 p/q Tube no r=1 Tube no: r=2 Tube no: r=4 Tube no: r=3
n=2 n=3 n=4
B A B A B
0/q2 Pq2 /q3 0/q3 Pq3 /q4 0/q4 q4
p/0 p2 /pq
pq/pq 2p2 q/2pq2 pq2 /2pq2 3p2q/3p2 q3 Pq3 /3pq3 4pq3 p2/0 p3 /p2 q 2p2 q/p2 q 3p3q/3p2 q2 3p2q2/3p2q2 6p2q2 p3 /0 p4 /p3 q 3p3q/p3 q 4p3q p/0 p4
Total after 4 transfers
.CALCULATION OF THE DISTRIBUTION THROUGH 4 TRANSFERS:
Transfer no Tube no.
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 = n∑r=0 n! r!(n-r)! prq(n-r)
The expansion of the function (p+q)n is laborious for large „n‟. and an easier calculation is available.
Binominal distribution expression for the fraction of total solute in „rth‟ plate after „n‟ mobile phase volumes have passed into the column
pr q(n-r) Tnr =
n! pr q(n-r) r! (n-r)! (n-r)! r!
n = no.r) p = 1/(KU+1) = fraction of solute per plate in M.of mobile phase volumes passed into the column r = plate number( 0..2.P at equilibria
.….P at equilibrium q = KU/(KU+1)= fraction of solute per plate in S.3.1.
binomial distribution approaches normal (Gaussian) distribution According to statistic‟s MEAN is given as “ų” ų = np
Standard deviation “σ”
σ = np
When „n‟ is large and „p‟ is not small (as in CCDS).
CALCULATION FOR NO.OF PLATES (efficiency)
Length= velocity. time = Rvtr Standard deviation „σ‟ = Rvז Where „ „זis zone-standard deviation
Combining above equations σ =Lז tr since: σ2 = HL tr2 = L /2זH since: =זL/H tr = זr ½ r = 16(tr/w)2
of theoretical plates
Random walk Reflects loss of efficiency Rate process controls zone width From plate theory : σ2 = HL H is a measure of zone spreading and column efficiency ( height equivalent to theoretical plates) HETP = Length of the column no.
LONGITUDINAL MOLECULAR DIFFUSION MASS TRANSFER(SORPTIONDESORPTION KINETICS) EDDY DIFFUSION
1.LONGITUDINAL MOLECULAR DIFFUSION
σ²=2Dm t σ²=2Dm L/v Hdiff =2γDm /v
γ is empirical factor of value 0.6
Hdiff = 2γDm + 2γs DS (1-R)/R v This is in the form of Hdiff = B/v Where „B‟ is the function of molecular and chromatographic properties
2.MASS TRANSFER(SORPTIONDESORPTION KINETICS)
No.of random steps „n‟ = 2L/vta True step length „L‟ =vta .Rvta or (1-R)vta According to random walk theory σ²=L²n σ²=2(1-R)²vta mm
Hs-d = Cv
Flow and diffusion mechanism are coupled Plate height contribution through flow and diffusion is not additive .but found to be Heddy = 1 1/HF + 1/HD H is independent of velocity and H is dependent on average velocity Heddy = 1 1/A + 1/Ev Where A and E include the partical diameter
CALCULATION FOR HETP
Total plate height contribution from 3 process H = Heddy + Hdiff + H(s-d)
1 + B + Cv 1/A + 1/Ev v
Van Deemeter equation H = A + B/v + Cv
P Tailing-more active adsorption
.saturation of S.
Random walk Quantitative measure
RETENTION TIME ( Rt ) RETENTION VOLUMN (Rv) ADJUSTED RETENTION VOLUMN (VR1) SELECTIVITY ( ) RESOLUTION ( Rs) EFFICIENCY(NUMBER OF PLATES)’n’ HETP (H)
RETENTION TIME (Rt)
Retention time is the difference in the time between the point of injection and appearance of peak maxima Rt is the time required for 50% of a component to be eluted from the column Unites : min or sec Retention time is also proportional to the distance moved on a chart paper. which can be measured in cm or mm
RETENTION VOLUMN (VR)
Retention volume is the volume of carrier gas required to elute 50% of the component from the column
Retention volume = Retention time flow rate
Corrected retention volume
VR0 = j VR
Where „j‟ is pressure drop correction factor
j = 3 . ( Pi / Po )2 - 1 2 . (Pi / Po )3 - 1
Pi and Po are inlet and outlet pressures
ADJUSTED RETENTION VOLUMN (VR!)
Adjusted retention volume is calculated as
VR‟ = VR - VM
Where VM is DEAD VOLUME of mobile phase Applying pressure drop correction to VR! Gives “ net retention volume ”
VN = j VR‟
A way of improving resolution is to change the selectivity of the column – by changing stationary and mobile phases Selectivity is the ratio of partition coefficients Selectivity term can be evaluated from the chromatogram
= VR,2 – VM VR,1 – VM = tr2 - tm tr1 - tm
The degree of disengagement of two bands is resolution. 16H R R
In terms of .N where k is capacity factor k=nS/nM
. In terms of width and diameter RS = dA –dB
In terms of time and width In terms of zone of migration
RS = 2 Rt 1 -Rt2 wA + wB RS = L .
high values like 40.OF PLATES(n)
Efficiency of column is expressed by the no.000 to 70.of theoretical plates is low . the column is said to be highly efficient If the no.NO. of theoretical plates
No.EFFICIANCY.of theoretical plates
If the no. a value of 600/meter is sufficient But for HPLC .of theoretical plates is high.000/meter are recommended
. the column is said to be less efficient For GC columns.
Decides the efficiency of separation
HETP 1 EFFICIENCY
If HETP is less. the column is less efficient HETP = Length of column no. the column is more efficient If HETP is more.of theoretical plates HETP calculated by using Van Deemeter equation HETP = A + B + Cv v
Carrier Gas Flow regulators and meters Sample injection system Columns & ovens Detectors
SCHEMATIC DIAGRAM OF GAS CHROMATOGRAPH
Gas Chromatograph Components
top view Injection Port
Flame Ionization Detector
. He are prepared for a carrier gas .nitrogen . Selection of the best carrier gas very important . because it effects both the column separation and detector performance . Sample component column detector mobile phase gas Helium . carbon dioxide and hydrogen also used. The ratio of viscosity of diffusion coefficient should be minimum for rapid analysis that’s why H.Carrier gas
The mobile phase gas is called the carrier gas and must be chemically inert.argon .
The gases are supplied from the high pressure gas cylinder . being stored at pressure up to 300psi carrier gas should be better then 99. pressure regulators and flow meters are required to control the flow rate of the gas. These gases are available in pressurized tanks.999% is often used
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.99%and 99. The carrier gas system should contains a molecular sieve to remove water and other impurities.
H2 inlet (detector)
Air inlet (detector))
N2 inlet(make-up gas)
He inlet (carrier gas)
Process Flow Schematic
Sample injection Carrier gas (nitrogen or helium) Detector (flame ionization detector or FID)
Long Column (30 m)
.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.
Flow regulators & meters
Flow regulators are used to deliver the gas with uniform pressure or flow rate Flow rates of carrier gas: – Linear flow rate (cm/s): u = L/tr – Volumetric flow rate (mL/min): u (π r2)
L is length of column. 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 decrease as column T increases
. it is retention time.
Soap bubble flow meter
.Soap bubble meter
soap bubbles formed indicates the flow rate. Glass tube with a inlet tube at the bottom. Rubber bulb-----store soap solution When the bulb is gently pressed of soap solution is converted into a bubble by Aqueousof solution the pressure of a carrier gas soap or detergent &travel up.
Sample injection port
Calibrated Micro syringes are used to inject liquid sample Purge :volatile components are removed from sample by gentle heating Rubber or silicone diaphragm(septum) Sample port Temp: 50°C Packed Column: sample sizes-1 to 20 μL 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
Usually 1-2 mL of sample is injected into the GC. Wash a syringe with acetone by filling the syringe completely and ejecting the waste acetone onto a paper towel. Remove air bubbles in the syringe by rapidly moving the plunger up and down while the needle is in the sample.
. 3. 2. Wash 2-3 times.1.
. a temperature equal to or slightly above the average boiling point of a sample results in a reasonable elution time (2 to 30 min). they are usually formed as coils having diameters of 10 to 30 cm.Column ovens
Column temperature is very important in GC The column is ordinarily housed in a thermostatic oven. The optimum column temperature depends upon the boiling point of the sample and the degree of separation required.
or Teflon. glass. packed and open tubular or capillary.
. Packed column length from less than 2 m to 5 m Capillary columns from few m to 100 m They are constructed of stainless steel. fused silica.Columns
Two types of columns are used in gas chromatography.
Types of columns 1.packed columns 2.30m
. Open tubular or capillary.
the tubes are formed as coils having diameters of roughly 15 cm. copper.2m to 3 m Internal diameters ------. aluminum). or teflon tubes that typically have Lengths-----.Packed columns
Packed columns are fabricated from glass. that is coated with a thin layer (0. or solid support. These tubes are densely packed with a uniform.2 to 4 mm.
.05 m) of the stationary liquid phase. metal (stainless steel. finely divided packing material. In order to fit in a thermostatic oven.
2.Support-coated open tubular (SCOT)
Inner surface of the capillary is lined with a thin
film (~30μm) of a support materials. flexibility.Wall-coated open tubular (WCOT)
Capillary tubes coated with a thin layer of
stationary phase Old: stainless steel.25 mm
. higher than packed column
3. Al.Capillary (or)Open tubular Columns
1.Fused-silica open tubular column (FSOT):
Physical strength. plastic.32 to
0. Cu. low reactivity. like diatomaceous earth Lower efficiency than WCOT. glass. 0.
Column Stationary Phases:
liquid coated silica particles (<100-300 mm
diameter) in glass tube best for large scale but slow and inefficient
wall-coated (WCOT) <1 mm thick liquid coating
on inside of silica tube support-coated (SCOT) 30 mm thick coating of liquid coated support on inside of silica tube best for speed and efficiency but only small samples
The Stationary Phase
requirements for stationary phase
Low vapor pressure Thermal stability Low viscosity (for fast mass transfer) High selectivity for compounds of interest
characters of detector
High sensitivity to even
small concentrtion linerity. ie. Predictable response Inexpensive operation from RT to 400 57 oC
. less response to low concentration &proportional response to high concentration Large linear dynamic range Useful at a range of temperatures
Good stability and
reproducibility Rapid response time Easy to use Stable.
3. 2. 4. Absolute Mass Detector(AMD) 10. Thermionic Detector(TD)
1. 6. 8. 7.
.Types of detectors
Thermal Conductivity Detector(TCD) Flame Ionization Detector(FID) Atomic Emission Detector(AED) Electron Capture Detector(ECD) Nitrogen Phosphoroes Detector(NPD) Photo Ionization Detector(PID) Flame Photometric detector(FPD) Electrolytic conductivity detector (Hall detector) 9. 5.
(fhv power) Ions &electrons move toward the collector less sensitive for non-hydrocarbon groups Insensitive to noncombustible gases(CO2. halogen and amine)
. Air-H2 flame Number of ions depends on number of reduced (methylene) carbons in a molecule The positive ions will be attracted to the cylindrical cathode. alcohol. Negative ions and electrons will be attracted to the jet anode. NO2 and H2O) Insensitive to functional group (carbonyl. SO2. Organic compounds Produces ions and electrons pyrolyzed(temp of flame) burner tip and electrode.Flame Ionization Detector(FID)
Most widely used.
but more universal Advantages: simplicity. inexpensive. large range.Thermal Conductivity Detector(TCD)
Element(platinum. gold or tungsten wire) is electrically heated at constant power – Temperature depends on thermal conductivity (He & H)of surrounding gas. organic & inorganic species
Thermal conductivity detector cell
Arrangement of the twin detectors
DA: low sensitivity ng/mL
. linearity is excellent. Hydrogen and helium have higher thermal conductivity and carrier gas provide best sensitivity Six times greater than the Organic compounds Poorer sensitivity than FID.
peroxide. nondestructive Insensitive to amines.like pesticides and.Electron Capture Detectors (ECD)
The sample elute from a column is passed over a radioactive βemitter(nickel-63) Selectively to halogen-containing organic sample . quinones and nitro group) in the sample capture the electron Highly selective and sensitive. alcohols and hydrocarbons AD: High sensitivity. polychlorinated biphenyls Ni-63: radioactive β-emitter-.electron -ionization of carrier gas (N2) High electronegative group (halogen. analyse the polyhalogenated organic compounds Small linear range
Thermionic detector(nitrogen phosphorus detector)
N or P containing organic compounds phosphorus atom is approximately ten times greater than to a nitrogen atom and 104 to 106 larger than a carbon atom. Column effluant + H2 +air(hot gas)electrically hearted Rb2SiO4 (rubidium silicate)bead at 180 V plasma (600 – 800°C ) ions to determine compounds useful for detecting and determining the many phosphorus-containing pesticides. the thermionic detector is approximately 500 times more sensitive to phosphorus-containing compounds and 50 times more sensitive to nitrogen bearing species.
. Compared with the FID .
ATOMIC EMISSION DETECTOR
Eluent(column) helium(carrier) water cooled microwave cavity helium plasma(high temp) characteristic atomic emission spectra grating diode array optical emission spectrometer detect the element .
copper .S. arsenic .iron.Six elements detect simultaneously .P. heavy metals(Pb .silicon . Determine the hetero atoms(H. Hg).tin.O).
2 eV H2 or 11. P easily photoionized molecules Linear range is high
PHOTO IONIZATION DETECTOR(PID)
UV light (10.7 eV Ar lamp) photo ionization of molecular current to flow between based electrodes Most sensitive for Aromatic and S.
Flame photometric detector (FPD):
S and P – compounds photomultiplier to view light of 394 nm for sulphur (H2 + air S2) measurement or 526 nm for phosphorus (H + air HPO species) Filteres are used to isolate the appropriate bands Intensity is recorded photometrically X-. Se and Ge
H2 + air
. N-. Sn . Cr.
Retention time data should be useful for identification 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 are obtained…..impurities are present….compound is not pure
peak height. 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 the standard solution -The peak area ratio of sample and internal standard….Quantitative analysis:
Direct comparison method: -comparing the area of the peak.unknown concentration is easily determined . width of peak.
S and N .plant extracts .Elemental analysis
Determination of C.H .O . Determination of mixture of drugs Isolation and identification of drugs Isolation and identification of mixture of components(amino acids .volatile oils)
Skoog .google .
Instrumental Analysis by Douglas A. Text book of Pharmaceutical Analysis by Kenneth A.S.Ravi Sankar Introduction to instrumental analysis by Robert D.Connor www.com Text book of Pharmaceutical analysis by Dr. F.Braun Chromatograhic methods by Smith
.James Holler & Stanley R.Crouch.