ch23. introduction to analytical separationschem.yonsei.ac.kr/~mhmoon/pdf/analchem/ch23.pdf · 23.1...
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23.1
Anal. Chem. by Prof. Myeong Hee Moon
Ch23. Introduction to Analytical Separations
Medical Issue : Measuring Silicones Leaking from Breast Implants
High molecular mass poly(dimethylsiloxane), PDMS, [(CH3)2SiO]n
: Used as GC stationary phase, gels in breast implants: 1~2% of silicones in breast implants are low-molecular-mass
when leaks, it travel through circulatory and lymph system todeposit in lipid rich tissues
23.2
Anal. Chem. by Prof. Myeong Hee Moon
Ch23. Introduction to Analytical Separations
23.3
Anal. Chem. by Prof. Myeong Hee Moon
23-1. Solvent Extraction
• Extraction : transfer of solute fromone phase to the other phase
Why to use extraction ?: to isolate or concentrate desired analyte.
- Organic solvent denser than water: CCl4, CH2Cl2, CHCl3
- Organic solvent lighter than water: diethylether, toluene
• Equilibrium in SoluteSolute: S in phase 1 2
Partition coefficient: acitivityA]S[
]S[
A
AK
i
1
2
S1
2
S
S =≅=
Phase 1: volume V1 ( water)Phase 2: V2 ( solvent, toluene)
23.4
Anal. Chem. by Prof. Myeong Hee Moon
23-1. Solvent Extraction
If, m: total # of molesq : fraction of S remaining in phase 1 molarity in phase 1
1V
mq ⋅=
1
2
1
2
V/qm
V/m)q1(
]S[
]S[K
−==
• Fraction remaining in phase 1after 1 extraction
∝+
= 21
1
KVV
Vq Partition coeff.
• If toluene removed, and add fresh toluene for complete extraction,
Fraction remaining in phase 12
⎟⎟⎠
⎞⎜⎜⎝
⎛+
=⋅=21
1
KVV
Vqq
Repeating results in the decrease of fraction remaining in water.
23.5
Anal. Chem. by Prof. Myeong Hee Moon
23-1. Solvent Extraction
1. pH effect
: if a solute is acidic or basic, solubility depends on pH(neutral mol dissolves in organic, charged species in aqueous)
pH is important in extraction
i.e.) Suppose conjugate acid: BH+ is soluble only in aqueous phase.
Distribution coefficient :1 phasein conc. total
2 phasein conc. total=D
11
2
]BH[]B[
]B[D ++=
1
2
]B[
]B[K =
]BH[
]H][B[Ka +
+
=
Ba
a K]H[K
KKD α⋅=
+⋅
= +αB: fraction of the weak base
in neutral form in aq.
To extract base in water, use HIGH pH to convert BH+ Bacid LOW pH
23.6
Anal. Chem. by Prof. Myeong Hee Moon
23-2 What is Chromatography ?
• Chromatography : similar to extraction
One phase : fixed – stationary phase : solid (liquid coated) particles packedThe other phase : moving – mobile phase : solvent filled, liq or gas
eluant
eluate
23.7
Anal. Chem. by Prof. Myeong Hee Moon
23-2 What is Chromatography ?
1. Types of Chromatography
• Adsorption : adsorption on solid surface
S.P.: solid (silica, alumina,etc)M.P.: Liq or Gas
stronger solute adsorption slow travel
• partition : similar to Liq-Liq extractionS.P.:GC- Liq st.ph. bonded to SiO2
of inner wall of capillaryLC- Liq st.ph. bonded to solid surface M.P.: Liq or Gas
23.8
Anal. Chem. by Prof. Myeong Hee Moon
23-2 What is Chromatography ?
• ion-exchange: similar to Liq-Liq extractionS.P.: ionic group attached on solid surface
-SO3-: attract cation
-NH4+ or –N(CH3)3
+ attract anionM.P.: Liq
• Molecular exclusion:gel filtration or gel permeation- separation by size. - Larger passes faster
23.9
Anal. Chem. by Prof. Myeong Hee Moon
23-2 What is Chromatography ?
• Affinity chromatography: antibody immobilized on St. phase.only specific proteins trapped to antibody
23.10
Anal. Chem. by Prof. Myeong Hee Moon
23-3. A plumber’s Point of View
for ex) column: id=0.60cm (r=0.30cm)M.P. occupies 20% in volumecolumn vol of each cm π(0.30)2 1cm = 0.283 mL- Volumetric flow rate = 0.30 mL/min.
0.30 mL / 0.0565 mL/cm = 5.3 cm5.3cm/min : linear flow rate
1. The chromatogram: graph showing the detector response as time
23.11
Anal. Chem. by Prof. Myeong Hee Moon
23-3. A plumber’s Point of View
• retention volume (tr) : time to reach detector from injector
• retention volume (Vr) : volume of M.P. to elute solute from the column
• dead time (tm) : passage time for mobiled phase through column
• adjusted retention time (tr’): tr’ = tr – tm
• relative retention (α)
for teo components 1, 2, tr2>tr1, α>1'r
'r
1
2
t
t=α
For each peak,
• capacity factor (k’) m
mr
t
tt'k
−= (or retention factor, cap. factor)
or partition ratio
M.P. in solute of time
S.P. in solute of time'k =
23.12
Anal. Chem. by Prof. Myeong Hee Moon
23-3. A plumber’s Point of View
mm
sS
VC
VC
M.P. in solute of moles
S.P. in solute of moles
M.P. in solute of time
S.P. in solute of time'k ===
tcoefficien partitionK]sample[
]sample[
C
C
m
S
m
S ⇒=⇒
m
'r
m
mr
m
S
t
t
t
tt
V
VK'k =
−==
'k
'k
t
t
1
2'r
'r
1
2 ==α
See example in P510
• retention volume (Vr)
rateflow volumetric :U UtV vvrr ×=
23.13
Anal. Chem. by Prof. Myeong Hee Moon
23-3. A plumber’s Point of View
2. Scaling Up
• chromatography for analytical purpose : get information• preparative use : collect fractions
Needs scale up
- Important in pharmaceutical industry - drug purification
i.e.) 2 mg out of 1.0-cm diameter columnHow large i.d. column needed
for separating 20 mg ?2
cm50.0
radius
mg2
mg20⎟⎠⎞
⎜⎝⎛= 1.58cm -- ~3cm in diameter
For flow rate, must be the same linear flow rateabout 10 times area increase
10 times higher volumetric flow rate
23.14
Anal. Chem. by Prof. Myeong Hee Moon
23-4. Efficiency of Separation
• How well separated ? by what differences to say it better or poor ?- time difference (far apart)- broadness : wider peak – poorer separation, information loss
Solute peak -- broadened as gaussian (due to diffusion)The longer separation, the broader the peak is
23.15
Anal. Chem. by Prof. Myeong Hee Moon
23-4. Efficiency of Separation
1. Resolution : measure of separation between two peaks
av2/1
r
av
r
av
rs w
t589.0
w
V
w
tR
⋅
Δ=
Δ=
Δ=
For quantitativeAnalysis
Rs>1.5
23.16
Anal. Chem. by Prof. Myeong Hee Moon
23-4. Efficiency of Separation
2. Diffusion Why becomes gaussian ? Due to diffusion
In a column, # of moles crossing /unit area/unit time : flux : J
dx
dcDJ)
sm
mol(Flux
2−=≡
⋅
D: diffusion coeff. (Table 23-1)dc/dx: conc. Gradient across a plane- sign: net flux is from high conc. to low conc.
23.17
Anal. Chem. by Prof. Myeong Hee Moon
23-4. Efficiency of Separation
23.18
Anal. Chem. by Prof. Myeong Hee Moon
23-4. Efficiency of Separation
• If a solute travels in a column with an infinitely sharp delta function concentration profile: solve out from flux equation
2Dt :dev std. )Dt4
xexp(
Dt4
mC
2
=σ−π
=C: mol/m3, x: distance along column
23.19
Anal. Chem. by Prof. Myeong Hee Moon
23-4. Efficiency of Separation
3. Plate height : a measure of column efficiency
xHxu
D2
u
x2D Dt2
xx
2 ⋅=⋅⎟⎟⎠
⎞⎜⎜⎝
⎛===σ Plate height
Height equivalent to theoretical platex x/ut =x
H2σ
=
• another meaning: length of column required for one eq. of solute bet. SP & MP
• By decreasing H better separation GC:~ 0.1~1mm, HPLC: ~10 μmC.E.: <1 μm
• for a solute emerging from column L
2
2
2
2
2 w
L16LLx
H
LN =
σ=
σ==
• Number of plates2
2/1
2r
2
2r
w
t55.5tN =
σ=
23.20
Anal. Chem. by Prof. Myeong Hee Moon
23-4. Efficiency of Separation
4. Factors Affecting Resolution
)t
tfactor( separation )1(
4
NR
A
Bs ==γ−γ=
Doubling N
times increase
in Rs
2
23.21
Anal. Chem. by Prof. Myeong Hee Moon
23-5. Why Band Spreads ?
So many factors influence band broadening.They re additive
∑σ=⋅⋅+σ+σ+σ=σ 2i
23
22
21
2obs
1. Broadening outside the column
When sample enters column, they are already in finite width through tubing.
Δt (in units of time),
22det
2inj )
12
t(Δ
=σ=σ
To minimize, reduce the dead space, tubing length etc
23.22
Anal. Chem. by Prof. Myeong Hee Moon
23-5. Why Band Spreads ?
2. Plate Height equation
Van Deemter Eq.
xx
Cuu
BAH ++≅
• Changing column, sp varies
packed column: A, B, C≠0OTC: A≠0CE: A=C=0
A: multiple path term oreddy diffusion
B: longitudinal diffusionC: mass transfer
Flow rate is critical
23.23
Anal. Chem. by Prof. Myeong Hee Moon
23-5. Why Band Spreads ?
3. Longitudinal Diffusion
Along the axis of column
• Raise ux H increased
xmm
2
u
x2D tD2 ==σ
• plate height due to L.D.
xx
m2
D u
B
u
2D
LH ≡=
σ=
Dm : diff. coeff. of solute in MP
23.24
Anal. Chem. by Prof. Myeong Hee Moon
23-5. Why Band Spreads ?
4. Finite Equilibration time between Phases : MASS TRANSFER
Cux: finite time for solute to reach Eq.
• plate height due to finite time
xmsxMT u)CC(CuH +==
s
2
2s D
d
)1'k(3
'k2C
+=
m
2
2
2
m D
r
)1'k(24
'k11'k61C
+++
=
r: column radiusd: thickness of SP
23.25
Anal. Chem. by Prof. Myeong Hee Moon
23-5. Why Band Spreads ?
• mass transfer term can be decreased by Tincrease of T increase of Ds
To increase T, SP must be stable
silica bases ? UnstableZrO2
23.26
Anal. Chem. by Prof. Myeong Hee Moon
23-5. Why Band Spreads ?
5. Multiple flow path : Eddy diffusion, A term
tight packingregular shape, spherical betteruniform size: smaller the better
23.27
Anal. Chem. by Prof. Myeong Hee Moon
23-5. Why Band Spreads ?
6. Advantage of Open Tubular Column (OTC)
In GC, OTC is used preferentially- higher resolution- shorter analysis time- increased sensitivity- low sample capacity
• OTC can be longer 100 times than the packed at same pressure
- if H is the same, N 10 times Rs- no band broadening by multiple paths- A term occupies ~half of H, upto ~30Ml/MIN.
IF A is deleted, N is doubled- Needs to be small radius
23.28
Anal. Chem. by Prof. Myeong Hee Moon
23-5. Why Band Spreads ?
23.29
Anal. Chem. by Prof. Myeong Hee Moon
23-5. Why Band Spreads ?
7. A Touch of Reality: Asymmetric band shapes
• ideal gaussian comeswhen K(=Cs/Cm) is independent ofconcentration
• but in real column,K changes as Cs, Cm increase
band skewed
i) Too much solute loaded ?overloaded
ii) When small quant. of solute retained more strongly thanlarge quantity
tailing
23.30
Anal. Chem. by Prof. Myeong Hee Moon
23-5. Why Band Spreads ?
When silica surfaces (silanol) binds H-bond with polar soluteserious tailing
Silanization: blocking unreacted hydroxy group with TMS (trimethylsilyl group)
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