ch23. introduction to analytical separationschem.yonsei.ac.kr/~mhmoon/pdf/analchem/ch23.pdf · 23.1...

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


Ch23. Introduction to Analytical Separations

23.3


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



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



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


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



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



• 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



• Affinity chromatography: antibody immobilized on St. phase.only specific proteins trapped to antibody

23.10


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



• 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



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



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


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



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



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



23.18



• 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



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



4. Factors Affecting Resolution

)t

tfactor( separation )1(

4

NR

A

Bs ==γ−γ=

Doubling N

times increase

in Rs

2

23.21


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



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



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



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



• 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



5. Multiple flow path : Eddy diffusion, A term

tight packingregular shape, spherical betteruniform size: smaller the better

23.27



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



23.29



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



When silica surfaces (silanol) binds H-bond with polar soluteserious tailing

Silanization: blocking unreacted hydroxy group with TMS (trimethylsilyl group)

ch23. introduction to analytical separationschem.yonsei.ac.kr/~mhmoon/pdf/analchem/ch23.pdf · 23.1...

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