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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.
Type GC
Schematic Diagram of GC
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.
T is regulated
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. 4. 5. 6. 7. 8.
Diameter of packing particle (mm) 10 to 200 m Inner diameter (mm) 2 or 3 mm Length (cm) 100 or 200 cm Temperature isothermal at 150 C Carrier gas N2 Linear flow rate (cm/s) varied
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The response is proportional to the number of the carbon atoms in the sample. This is related to the effective carbon number (ECN).
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Sample in
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. 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 and separation improves: as the plate count N increases as the plate height H decreases
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L N H
Column Efficiency, H
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:
2 H L
s2 = variance of Gaussian peak (cm2) 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.
t x uo = L
Figure 30-11 Skoog
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L H
L2 L2 L2 N 2 16 2 2 (W 4) W
Peak width at height (PWHH), W1/2, is used when W is difficult to be accurately determined.
t N 5.54 R 2 W1/2
W68% = 2s
A larger N value represents better separation. Hence, a good separation is when narrow peaks are obtained.
Skoog
H A
B (Cs CM ) u u
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)
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B H A C su u
A 2 dp
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.
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Longitudinal Diffusion
B H A C su u
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.
B 2 DM
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.
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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.
B H A C su u
2k d2 f CS 3(k 1)2 DS
k: retention factor, tS/tM df: thickness of film on stationary phase DS: diffusion coefficient of solute on stationary phase
B H A C su u
B 2 DM
2k d2 f CS 3(k 1)2 DS
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A 2 dp
tR N 5.54 2 W1/2
L H N
F uo A uo r
2
uo (cm/s)
A (cm )
2
Plot HETP (or H) vs. Carrier gas linear flow rate (or uo)
B H A C su u
http://math.cowpi.com/systemsolver/3x3.html
(2.83, 0.19)
(1.18, 0.19) 12 13
3 4
B B ) C s 1.89 4.72 2.83 B B ) C s 3.54 4.72 1.18 B B ) C s 3.54 2.52 1.51 B 0.048
4 x (3.54/1.89) 4 x 1.873
0.0375 ( 0.0175 (
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Sub B into 4, 5 or 6
A 0.13
B H A C su u
H (cm)
Optimum linear flow rate and maximum column efficiency, when H and band broadening is minimum.
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Sample D: ???
Sample Compound toluene Density / g mL-1 0.8669 0.8665 Mr 92.15 106.17 BP / oC 110.6 136.2 Vol / mL 5 x 10-4 5 x 10-4 Weight /g 4.3345 x 10-4 4.3325 x 10-4 Weight % 50.01 49.99 n 4.704 x 10-6 4.081 x 10-6 Mole % 53.55 46.45 Peak area % 49.69 50.31
A
ethylbenzene
m Mr Mole % n
n x 100 % Total n
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Weight %
Sample
Compound toluene
Mr 92.15 106.17
BP / oC 110.6 136.2
A ethylbenzene
Sample A: toluene and ethylbenzene have similar polarity. Hence elution order is dependent on boiling point.
Peak area %
toluene
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 ethanol
n-propanol C n-butanol n-pentanol
2 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1 0
No. of C 2
3 4 5
tR
Retention volume/ mL
For n-hexanol :
y = 0.16x + 1.09 R = 0.9846
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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: ??? 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. Quantitative Analysis:
sample Peak area of analyte in Sample D Volume of analyte in Sample D x Volume of analyte in Sample C Peak area of analyte in Sample C sample standard standard e.g. Analyte: n-propanol
Vol %
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Effective carbon number (ECN) = individual carbon atoms contributions + functional group contributions (Table 1-1 in lab manual).
Atom Type ECN
C
C O
Aliphatic
Aromatic
1.0
1.0
e.g. toluene
e.g. ethanol
Effective carbon number (ECN) (experimental) = Peak area n e.g. toluene e.g. ethanol
ECN(experimen tal) 7317915310.8 (from chromatogr am) 4.704 x 10-6 (previousl y calculated) 1.56 x 1015
ECN(experimen tal) 1324364458.4 (from chromatogr am) 4.274 x 10-6 (previousl y calculated) 3.10 x 1014
smallest ECN (experimental) Relative ECN ratio (experimental): 3.10 x 1014 / 3.10 x 1014 = 1.00 Relative ECN ratio (expected): 1.4 / 1.4 = 1.00 % difference: 0.00 %
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Relative ECN ratio (experimental): 1.56 x 1015 / 3.10 x 1014 = 5.03 Relative ECN ratio (expected): 7.0 / 1.4 = 5.00 % difference: 0.60 %