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

1. 2. 3.

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)

CALCULATION OF THE DISTRIBUTION THROUGH 4 TRANSFERS:

Transfer no Tube no. 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=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

Total after 4 transfers

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

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

CALCULATION FOR NO.OF PLATES (efficiency)

Length= velocity. time = Rvtr Standard deviation = Rv Where is zone-standard deviation

Combining above equations =L tr since: 2 = HL tr2 = L /2H since: =L/H tr = r r = 16(tr/w)2

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.

LONGITUDINAL MOLECULAR DIFFUSION MASS TRANSFER(SORPTIONDESORPTION KINETICS) EDDY DIFFUSION

2.

3.

1.LONGITUDINAL MOLECULAR DIFFUSION

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

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 =Ln =2(1-R)vta mm

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

CALCULATION FOR HETP

Total plate height contribution from 3 process H = Heddy + Hdiff + H(s-d)

H=

1 + B + Cv 1/A + 1/Ev v

Van Deemeter equation H = A + B/v + Cv

Random walk Quantitative measure

Fronting

tailing

detector signal

time

Fronting- saturation of S.P Tailing-more active adsorption

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

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

In terms of time and width In terms of zone of migration

RS = 2 Rt 1 -Rt2 wA + wB RS = L . 16H R R

In terms of , k ,N where k is capacity factor k=nS/nM

RS =

N 4

k k+1

-1

EFFICIANCY;NO.OF PLATES(n)

Efficiency of column is expressed by the no. of theoretical plates

No.of theoretical plates

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)

Decides the efficiency of separation

HETP 1 EFFICIENCY

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

SCHEMATIC DIAGRAM OF GAS CHROMATOGRAPH

30

Gas Chromatograph Components

top view Injection Port

Flame Ionization Detector

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)

He inlet (carrier gas)

34

Process Flow Schematic

Sample injection Carrier gas (nitrogen or helium) Detector (flame ionization detector or FID)

Air Hydrogen

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.
36

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 ( 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

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.

39

Soap bubble flow meter

inlet tube

40

INJECTO R

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: 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

Types of columns 1.packed columns 2. Open tubular or capillary.

Packed column-3m

Capillary column- 30m

50

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

Capillary (or)Open tubular Columns

1.Wall-coated open tubular (WCOT)

Capillary tubes coated with a thin layer of

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

2.Support-coated open tubular (SCOT)

Inner surface of the capillary is lined with a thin

film (~30m) of a support materials, like diatomaceous earth Lower efficiency than WCOT, higher than packed column

3.Fused-silica open tubular column (FSOT):

Physical strength, low reactivity, flexibility. 0.32 to

0.25 mm

Column Stationary Phases:

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

The Stationary Phase

requirements for stationary phase

Low vapor pressure Thermal stability Low viscosity (for fast mass transfer) High selectivity for compounds of interest

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

Good stability and

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

Flame Ionization Detector(FID)

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

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. Hydrogen and helium have higher thermal conductivity and carrier gas provide best sensitivity Six times greater than the Organic compounds Poorer sensitivity than FID, but more universal Advantages: simplicity, large range, inexpensive, linearity is excellent. organic & inorganic species

Thermal conductivity detector cell

Arrangement of the twin detectors

60

DA: low sensitivity ng/mL

Electron Capture Detectors (ECD)

The sample elute from a column is passed over a radioactive emitter(nickel-63) Selectively to halogen-containing organic 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 the sample capture the electron Highly selective and sensitive, nondestructive Insensitive to amines, alcohols and hydrocarbons AD: High sensitivity, analyse the polyhalogenated organic compounds Small linear range
61

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. Compared with the FID , the thermionic detector is approximately 500 times more sensitive to phosphorus-containing compounds and 50 times more sensitive to nitrogen bearing species. Column effluant + H2 +air(hot gas)electrically hearted Rb2SiO4 (rubidium silicate)bead at 180 V plasma (600 800C ) ions to determine compounds useful for detecting and determining the many phosphorus-containing pesticides.
62

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 .
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

PHOTO IONIZATION DETECTOR(PID)

65

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-, N-, Sn , Cr, Se and Ge

2

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