Академический Документы
Профессиональный Документы
Культура Документы
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
1. 2. 3.
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=1
n=2 n=3 n=4
B A
B A B A B
0/q Pq/q2
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
The expansion of the function (p+q)n is laborious for 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! r!(n-r)! prq(n-r)
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 = 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
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 = np
Standard deviation
= np
ELUTION CHROMATOGRAM
Concentration
time
Tr |
|
width
Random walk Reflects loss of efficiency Rate process controls zone width From plate theory : 2 = HL H is a measure of zone spreading and column efficiency ( height equivalent to theoretical plates) HETP = Length of the column no.of theoretical plates
1.
2.
3.
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
Hs-d = Cv
3.EDDY DIFFUSION
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
H=
1 + B + Cv 1/A + 1/Ev v
Fronting
tailing
detector signal
time
RETENTION TIME ( Rt ) RETENTION VOLUMN (Rv) ADJUSTED RETENTION VOLUMN (VR1) SELECTIVITY ( ) RESOLUTION ( Rs) EFFICIENCY(NUMBER OF PLATES)n HETP (H)
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 volume is the volume of carrier gas required to elute 50% of the component from the column
Retention volume = Retention time flow rate
Where VM is DEAD VOLUME of mobile phase Applying pressure drop correction to VR! Gives net retention volume
VN = j VR
SELECTIVITY (
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
(or)
RESOLUTION(Rs)
The degree of disengagement of two bands is resolution. In terms of width and diameter RS = dA dB
W
RS = 2 Rt 1 -Rt2 wA + wB RS = L . 16H R R
RS =
N 4
k k+1
-1
EFFICIANCY;NO.OF PLATES(n)
efficiency
If the no.of theoretical plates is high, the column is said to be highly efficient If the no.of theoretical plates is low , the column is said to be less efficient For GC columns, a value of 600/meter is sufficient But for HPLC , high values like 40,000 to 70,000/meter are recommended
HETP(H)
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 equation HETP = A + B + Cv v
INSTRUMENTATION
Carrier Gas Flow regulators and meters Sample injection system Columns & ovens Detectors
29
30
Column Oven
front view
Carrier gas
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 and hydrogen 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 why H, He are prepared for a carrier gas .
32
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 used
33
H2 inlet (detector)
Air inlet (detector))
N2 inlet(make-up gas)
34
Air Hydrogen
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.
36
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 ( 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 decrease as column T increases
37
FLOW REGULATORS
39
inlet tube
40
INJECTO R
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: 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
42
43
Micro syringe
44
1. Wash a syringe with acetone by filling the syringe completely and ejecting the waste acetone onto a paper towel. Wash 2-3 times. 2. Remove air bubbles in the syringe by rapidly moving the plunger up and down while the needle is in the sample. 3.Usually 1-2 mL of sample is injected into the GC.
45
COLUMN OVENS
Column ovens
Column temperature is very important in GC The column is ordinarily housed in a thermostatic 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 the degree of separation required. Roughly, a temperature equal to or slightly above the average boiling point of a sample results in a reasonable elution time (2 to 30 min).
47
COLUMNS
Columns
Two types of columns are used in gas chromatography, 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, glass, fused silica, or Teflon.
49
Column
Packed column-3m
Packed columns
Packed columns are fabricated from glass, metal (stainless steel, copper, aluminum), or teflon 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, or solid 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 of roughly 15 cm.
51
film (~30m) of a support materials, like diatomaceous earth Lower efficiency than WCOT, higher than packed column
0.25 mm
Packed
liquid coated silica particles (<100-300 mm
diameter) in glass tube best for large scale but slow and inefficient
Capillary/Open Tubular
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
53
54
56
DETECTOR S
Ideal
characters of detector
High sensitivity to even
small concentrtion linerity, ie, less response to low concentration &proportional response to high concentration Large linear dynamic range Useful at a range of temperatures
reproducibility Rapid response time Easy to use Stable, Predictable response Inexpensive operation from RT to 400 57 oC
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. Absolute Mass Detector(AMD) 10. Thermionic Detector(TD)
1. 2. 3. 4. 5. 6. 7. 8.
58
Most widely used, 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. Negative ions and electrons will be attracted to the jet anode. Organic compounds Produces ions and electrons pyrolyzed(temp of flame) burner tip and electrode.(fhv power) Ions &electrons move toward the collector less sensitive for non-hydrocarbon groups Insensitive to noncombustible gases(CO2, SO2, NO2 and H2O) Insensitive to functional group (carbonyl, alcohol, halogen and amine)
59
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 .
63
Six elements detect simultaneously . Determine the hetero atoms(H,P,S,O),silicon , heavy metals(Pb , Hg),tin, arsenic ,copper ,iron.
64
UV light (10.2 eV H2 or 11.7 eV Ar lamp) photo ionization of molecular current to flow between based electrodes Most sensitive for Aromatic and S, P easily photoionized molecules Linear range is high
65
filteres
photomultiplier
H2 + air
Column effluent
66
APPLICATIONS
67
Qualitative analysis:
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
68
Quantitative 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 . 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.unknown concentration is easily determined .
69
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)
70
Instrumental Analysis by Douglas A.Skoog , F.James Holler & Stanley R.Crouch. Text book of Pharmaceutical Analysis by Kenneth A.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