Post on 19-Oct-2015
description
CM2192 Analytical
Experiment 1:
Gas Chromatography (GC) for Qualitative and Quantitative Analysis
Prepared by Dr Emelyn Tan
Type Stationary Phase Mobile Phase Sample
GC Liquid on solid or solid beads in column
Inert gas e.g. H2 He or N2
Mixed in mobile phase (gas)
Compound starts in mobile phase
Column Chromatography: stationary phase is held in a narrow tube and the mobile phase is forced through the tube by gravity or under pressure. - Column, GC, HPLC and SCFC.
Separation based on rate of analytes movement through stationary phase.
2
Schematic Diagram of GC
Figure 31-1 Skoog
T is regulated
Requirements for the Analyte: - Volatile (low boiling point, high vapour pressure) - Thermal stability
Separation depends on: a) Volatility (MAIN FACTOR):
Higher volatility, shorter retention time (tR).
b) Differential interaction between analytes and stationary phase:
Weaker interaction (opposite polarity), shorter tR.
No interaction with the mobile phase i.e. inert carrier gas.
3
Instrument Parameters
1. Column type Packed with Chromosorb W
Chromosorb W is a white, polar, crumbled, naturally occurring, soft siliceous sedimentary rock and diatomaceous earth (contains a type of hard-shelled algae).
2. Coating 20% Free Fatty Acid Phase (FFAP): polyethylene glycol substituted with terephthalic acid
3. Diameter of packing particle (mm) 10 to 200 m
4. Inner diameter (mm) 2 or 3 mm
5. Length (cm) 100 or 200 cm
6. Temperature isothermal at 150 C
7. Carrier gas N2 8. Linear flow rate (cm/s) varied
4
Flame Ionisation Detector (FID)
Effluent from the column is directed to H2-air flame. Organic compounds form cations and electrons when pyrolyzed at the temperature of the flame. Pyrolysis is a decomposition of organic material at elevated temperatures without the participation of oxygen.
Collector electrode will capture the charge carriers (ions and electrons), the resulting current is measured by a picoammeter.
The response is proportional to the number of the carbon atoms in the sample. This is related to the effective carbon number (ECN).
Sample in
Figure 31-8 Skoog
5
Plate Theory
Plate theory supposes that the chromatographic column contains a large number of separate layers or plates, of a given plate height.
This theory, which is adopted from a distillation column, is an assumption as there is actually insufficient time for equilibration in an chromatographic column.
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Figure 30F-2 Skoog
Column efficiency and separation improves:
as the plate count N increases
as the plate height H decreases
H
LN
N = plate count or number of theoretical plates
L = length of the column packing (cm) fixed
H = plate height or Height Equivalent of Theoretical Plate (HETP) (cm)
Column Efficiency, H
7
Because the chromatographic peaks are usually Gaussian, the column efficiency is reflected by the breath of the peaks i.e. the variance s2, per unit length of the column. The plate height i.e. column efficiency H:
L
H
2
Figure 30-11 Skoog
Plot showing distribution of molecules along the length of the column at the moment that the analyte peak reaches the end of the column/detector i.e. at the retention time, tR.
s2 = variance of Gaussian peak (cm2)
Gaussian Distribution
1s = 34%
t x uo = L
8
2
2
2
2
2
2
W
L16
)4(W
L
LN
No of theoretical plates:
Number of Theoretical Plates, N
H
LN
L
H
2
4W base, at width Peak
2
2
R
W
t16N tR
and W units are in minutes.
2
1/2
2
R
W
t5.54N
Peak width at height (PWHH), W1/2, is used when W is difficult to be accurately determined.
A larger N value represents better separation. Hence, a good separation is when narrow peaks are obtained.
W68% = 2s
linear flow rate (cm / min)
Confidence Level = 95.4%
Variables that affect Column Efficiency
A decrease of H relates to an increase in column efficiency and separation, less peak/band broadening.
Many variables affect H and various equations of H have been proposed that incorporate these complex physical interactions and effects.
Skoog
phase Mobile
phase Stationary
t
t k
Rate theory: The van Deemter equation
The column efficiency can be approximated by:
A: coefficient that describes multiple path effects (eddy diffusion)
B: longitudinal diffusion coefficient
CS: mass transfer coefficient for stationary phase
CM: mass transfer coefficient for mobile phase. At high flow rate, CM 0.
u: linear flow velocity (cm/s)
u )C (Cu
BAH Ms
10
In Plate theory, the number of theoretical plates, N, and plate height, H, is related closely to the efficiency of the chromatography column. Plate theory, however, does not provide any information on the effect of flow rate on H, whereas Rate theory does.
The Multiple Path Effects: Eddy Diffusion
A molecule can travel through a packed column using different paths, thus the residence times of each molecule is variable and band broadening occurs. This effect is called eddy diffusion.
The heterogeneity in axial velocities is related to:
- particle size and uniformity
- geometry of packing
Using small and uniform particles which give a
tighter and more consistent packing
will minimize this effect.
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Figure 30-14 Skoog
Molecule 2 will arrive later at B.
uCu
BAH s
Eddy Diffusion:
pd 2A
l is packing factor, ranging from 0.8 1
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The Multiple Path Effects: Eddy Diffusion
l is a constant accounting for the consistency of the packing. A more consistent packing gives a smaller value for l which range from 0.8 to 1.
dp is the average diameter of the packing particles.
Longitudinal Diffusion
In chromatography, the diffusion results in movement of the solute from the concentrated center of a band to less concentrated regions on both sides.
This results in band broadening and is more evident as time increases.
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uCu
BAH s
MD 2B
g is a constant related to the column packing which range from 0.6 to 0.8. DM is the solutes diffusion coefficient in the mobile phase.
Mass Transfer
The Cu term comes from the finite time required for solute to equilibrate between the two phases.
Thicker film on particles, smaller diffusion coefficient i.e. solute travels slower, larger Cs.
uCu
BAH s
S
2f
2S D
d
1)3(k
2kC
k: retention factor, tS/tM df: thickness of film on stationary phase DS: diffusion coefficient of solute on stationary phase
pd 2A MD 2B S
2f
2S D
d
1)3(k
2kC
uCu
BAH s
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van Deemter equation
NLH
tR and W units are in seconds. 2
1/2
2
R
W
t5.54N
Run GC at different carrier gas flow rates cm3/min 20 cm3/min
tR = 157.02 s
W1/2 = 11.99 s
N = 950.13
H = 200 / 950.13 = 0.210 cm
ruAuF 2oo uo = F / r
2
= (20/60) / 0.152
= 4.72 cm/s
Plot HETP (or H) vs. Carrier gas linear flow rate (or uo)
uCu
BAH s
Take 3 points, (uo, H), and solve simultaneously for A, B and C.
http://math.cowpi.com/systemsolver/3x3.html
Part 1: Determination of HETP Analyte: ethylbenzene
/min)(cm F 3
(cm/s) uo
)(cm A 2
4.72C4.72
BA0.21 s(4.72, 0.21)
(2.83, 0.19) (1.18, 0.19)
2.83C2.83
BA0.19 s
1.18C1.18
BA0.19 s
1 2 3
1 2 1.89C)2.83
B
4.72
B(0.02 s
1 3 3.54C)1.18
B
4.72
B(0.02 s
4 5
4 x (3.54/1.89) 4 x 1.873
3.54C)1.51
B
2.52
B(0.0375 s 6
6 5 0.048 B )1.18
B
4.72
B()
1.51
B
2.52
B(0.0175
0.014 C Sub B into 4, 5 or 6
Sub B and C into 1, 2 or 3 0.13 A 0.014u
u
0.0480.13H
(uo, H)
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uCu
BAH s
H (
cm)
u (cm/s)
B/u
A
Csu Mass transfer
Eddy diffusion
Longitudinal diffusion
Optimum linear flow rate and maximum column efficiency, when H and band broadening is minimum.
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Part 2: Qualitative and Quantitative Analysis
Sample A: equal volumes of toluene and ethylbenzene
Sample B: 3 mL cyclohexane, 4 mL of n-propanol and 3 mL o-xylene
Sample C: equal volumes of ethanol, n-propanol, n-butanol and n-pentanol
Sample D: ???
Sample Compound Density / g mL-1
Mr BP / oC
Vol / mL
Weight / g
Weight %
n Mole
% Peak area
%
A
toluene 0.8669 92.15 110.6 5 x 10-4 4.3345 x 10-4
50.01 4.704 x 10-6 53.55 49.69
ethylbenzene 0.8665 106.17 136.2 5 x 10-4 4.3325 x 10-4
49.99 4.081 x 10-6 46.45 50.31
Inject 1 mL
Weight = Density x Vol
% 100 xWeight Total
Weight% Weight
Mr
mn
% 100 xn Total
n% Mole
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Sample Compound Density / g mL-1
Mr BP / oC
Vol / mL
Weight / g
Weight %
n Mole
% Peak area
%
A
toluene 0.8669 92.15 110.6 5 x 10-4 4.3345 x 10-4
50.01 4.704 x 10-6 53.55 49.69
ethylbenzene 0.8665 106.17 136.2 5 x 10-4 4.3325 x 10-4
49.99 4.081 x 10-6 46.45 50.31
Sample A: toluene and ethylbenzene have similar polarity. Hence elution order is dependent on boiling point.
% 100 xarea peak Total
area Peak% area Peak
toluene ethylbenzene
Retention time, tR
20
Sample C: equal volumes of ethanol, n-propanol, n-butanol and n-pentanol
Retention Volume (mL) = volumetric flow rate (mL/min) x retention time (min)
Sample Compound No. of C tR Retention volume/ mL Logarithm of retention volume
C
ethanol 2 1.4
n-propanol 3 1.6
n-butanol 4 1.7
n-pentanol 5 1.9
y = 0.16x + 1.09 R = 0.9846
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2
0 1 2 3 4 5 6
Log
ret
enti
on
vo
lum
e (m
L)
Number of carbon atoms in the molecule
For n-hexanol : y = 0.16(6) + 1.09 = 2.05 Retention volume: 112 mL
Qualitative Analysis: identity of the analytes in Sample D can be determined by comparing the retention times, tR of the three unknown analytes in the Sample Ds chromatogram with the tR of nine known analytes in Sample A, B and C.
Sample A: equal volumes of toluene and ethylbenzene
Sample B: 3 mL cyclohexane, 4 mL of n-propanol and 3 mL o-xylene
Sample C: equal volumes of ethanol, n-propanol, n-butanol and n-pentanol
Sample D: ???
Quantitative Analysis:
C Sample in analyteof Volume xC Sample in analyteof area Peak
D Sample in analyteof area PeakD Sample in analyteof Volume
sample
standard standard sample
e.g. Analyte: n-propanol Volume of n-propanol in Sample D = L 0.5 L 0.25 x
10 x 2
10 x 45
5
% 100 xVolume Total
Volume% Vol
Volume area Peak
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Part 3: Effective Carbon Number
Flame Ionisation Detector (FID)
Current signal Number of ions produced
for organic compounds
Number of reduced carbon atoms in the
flame
Effective carbon number (ECN) = individual carbon atoms contributions + functional group contributions (Table 1-1 in lab manual).
a a
Atom Type ECN
C Aliphatic 1.0
C Aromatic 1.0
O primary alcohol 0.6
ECN: 6(1.0) + 1(1.0) = 7.0 e.g. toluene
ECN: 2(1.0) + 1(-0.6) = 1.4 e.g. ethanol
smallest ECN
Relative ECN ratio (expected): 7.0 / 1.4 = 5.00
Relative ECN ratio (expected): 1.4 / 1.4 = 1.00
Effective carbon number (ECN) (experimental) = n
area Peak
e.g. toluene
15
6-
10 x 1.56
)calculated y(previousl10 x 4.704
am)chromatogr (from .87317915310
tal)(experimen ECN
e.g. ethanol
14
6-
10 x 3.10
)calculated y(previousl10 x 4.274
am)chromatogr (from .41324364458
tal)(experimen ECN
smallest ECN (experimental)
Relative ECN ratio (experimental): 1.56 x 1015 / 3.10 x 1014 = 5.03
Relative ECN ratio (experimental): 3.10 x 1014 / 3.10 x 1014 = 1.00
Relative ECN ratio (expected): 7.0 / 1.4 = 5.00
Relative ECN ratio (expected): 1.4 / 1.4 = 1.00
% difference: 0.60 % % difference: 0.00 % 23