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UNIVERSITY OF OSLO
© DEPARTMENT OF CHEMISTRY V11
KJM3420/4420-1
1
Analytisk kjemi – Separasjonsmetoder I
Chromatographic methods Electrophoretic methods Sample preparation Quantitative analysis
Textbook: KROMATOGRAFI (Greibrokk, Lundanes, Rasmussen) (in English: Quantitative Chemical Analysis (Harris)
UNIVERSITY OF OSLO
© DEPARTMENT OF CHEMISTRY V11
KJM3420/4420-1
2
teaching
Lectures: Tyge Greibrokk Lab.course: Elsa Lundanes Lab.course supervision: Hanne Hustoft Colloquies: Tyge Greibrokk
Wednesdays 09.15-11.00 in room Ø108 (or Ø 154) Fridays 09.15-11.00 in auditorium 3
UNIVERSITY OF OSLO
© DEPARTMENT OF CHEMISTRY V11
KJM3420/4420-1
3
CHROMATOGRAPHY
Separation methods chroma - color grafi - writing
PRINCIPLE: the compounds (analytes) to be separated are
distributed differently between two phases where one phase is mobile (MP) and the other is stationary (SP) (the compounds are transported by the mobile phase)
The “father” of chromatography:Tsvet (Tswett); 1903
UNIVERSITY OF OSLO
© DEPARTMENT OF CHEMISTRY V11
KJM3420/4420-1
4
When is chromatography used? separation of compounds
qualitative identification by UV, Fl, MS identification comparing tR with standards
quantitative determinations Using calibration curves (the injection technique must be under
control or internal standard used.) peak area peak height
UNIVERSITY OF OSLO
© DEPARTMENT OF CHEMISTRY V11
KJM3420/4420-1
5
Packed capillary LC-ELSD Comparison of three similar HALS trade products
Lowilite 62
Tinuvin 622
Uvisol 226
UNIVERSITY OF OSLO
© DEPARTMENT OF CHEMISTRY V11
KJM3420/4420-1
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PRINCIPLE ≠ TECHNIQUE (METHOD) Principles: (type of stationary phase)
adsorption partition chemical bonded SP ion exchange exclusion (gel filtration/GPC)
Techniques/methods: gas chromatography (GC)
the mobile phase is a gas supercritical fluid chromatography (SFC)
the mobile phase is a supercritical fluid liquid chromatography
the mobile phase is a liquid column:
• part. diam ≤ 10 µm (HPLC) • part. diam > 50 µm
thin layer chromatography (TLC)
UNIVERSITY OF OSLO
© DEPARTMENT OF CHEMISTRY V11
KJM3420/4420-1
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Technique/method
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© DEPARTMENT OF CHEMISTRY V11
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UNIVERSITY OF OSLO
© DEPARTMENT OF CHEMISTRY V11
KJM3420/4420-1
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UNIVERSITY OF OSLO
© DEPARTMENT OF CHEMISTRY V11
KJM3420/4420-1
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CHROMATOGRAPHY -separation methods
separation of a mixture of compounds is obtained if the compounds have different interaction with the stationary phase (and possibly with the mobile phase).
sample (A,B,C)
stationary phase (SP)
mobile phase (MP)
UNIVERSITY OF OSLO
© DEPARTMENT OF CHEMISTRY V11
KJM3420/4420-1
11
We want to separate the compounds A, B and C;
1. The compounds have different velocity if they have different interaction 2. The band width (b) of each compound increases with time (the migration length) 1 and 2 are characteristic properties of a chromatographic separation
BCAACB CBBAAC
b
t0 t1
C C C C C C C B B B B B B A A A A A A b
C C C C C C C B B
B B B B
A A A A A
A
b
t2
What happens when the compounds are applied on a column, and transported by the mobile phase through the column?
UNIVERSITY OF OSLO
© DEPARTMENT OF CHEMISTRY V11
KJM3420/4420-1
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1. Difference in migration velocity is caused by: the compounds A, B and C have different distribution between the
stationary phase (SP) and mobile phase (MP) (due to difference in interaction) The velocity of each compound (A, B or C) is determined by the fraction of
A molecules (or fraction of B or fraction of C) being in the mobile phase. (The A molecules are not moving when they are in the stationary phase)
A B C A A C A BB A B C B C A C CB
mobile phase molecule
Time t0
stationary phase
Time t1
C B BA A C B B A A C CC CB B A stationary phase
UNIVERSITY OF OSLO
© DEPARTMENT OF CHEMISTRY V11
KJM3420/4420-1
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The distribution between the stationary phase and the mobile phase (and thus the migration velocity) depends on:
1. composition of stationary phase 2. composition of mobile phase (LC and SFC) 3. temperature (mainly GC and SFC, but also LC)
Ideally, the distribution is not dependent on the (total) concentration of the analyte(s).
UNIVERSITY OF OSLO
© DEPARTMENT OF CHEMISTRY V11
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2. Band broadening Chromatogram:
always band broadening: molecules of the same kind have different (average) velocity and this
is caused by physical processes
without band broadening : not possible
UNIVERSITY OF OSLO
© DEPARTMENT OF CHEMISTRY V11
KJM3420/4420-1
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Physical processes giving band broadening:
Eddy ”diffusion” (dispersion) (resistance to) mass transport in MP (resistance to) mass transport in non-moving MP (resistance to) mass transport i SP
longitudinal diffusion (from high to low conc.)
UNIVERSITY OF OSLO
© DEPARTMENT OF CHEMISTRY V11
KJM3420/4420-1
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1. Eddy diffusion (dispersion)
different flow paths of MP
the MP moves at a higher speed in wide channels as compared to narrow channels
the band width increases when the compound moves downwards through the column
Start: xxxxxxxxxx xxxxxxxxxx b (band width)
XXX XXX
X X XX
XXX X X
X X
b
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© DEPARTMENT OF CHEMISTRY V11
KJM3420/4420-1
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X
X X
X X X
X
X
2. Mass transport in MP
the mobile phase moves slower close to a particle as compared to in the midstream (in the same flow path)
UNIVERSITY OF OSLO
© DEPARTMENT OF CHEMISTRY V11
KJM3420/4420-1
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3. Mass transport in stagnant MP
stagnant mobile phase the MP flow is low in the pores of the porous packing material; the flow rate
(velocity) depends on how far the MP penetrate the pore
start
X X
diffusion
Porous particle
Non-moving MP
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© DEPARTMENT OF CHEMISTRY V11
KJM3420/4420-1
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diffusion into the SP (or strongly adsorbed)
5. Longitudinal diffusion (conc. diffusion)
4. Mass transport in SP
X X
stationary phase
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© DEPARTMENT OF CHEMISTRY V11
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Retention parameters
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Relationship between tR and k
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© DEPARTMENT OF CHEMISTRY V11
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tR vs. VR
VR = tR · F (where the volumetric flow F is vol/time) Vm = tm · F (total amount of MP in the column) ⇒ VR = tR · Vm/tm = Vm(1+k)
VR is used occasionally in LC
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© DEPARTMENT OF CHEMISTRY V11
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Thin layer (and paper)
LX = distance moved by the compound from the start line L = distance moved by the mobile phase front from the start line
Rf = Lx/L = (ux·t)/(u·t) = ux/u = R = 1/(1+k)
Rf is called the retardation factor 0 ≤ Rf ≤ 1
Lx L
UNIVERSITY OF OSLO
© DEPARTMENT OF CHEMISTRY V11
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Band broadening
The standard deviation (σt) is a measure of the band broadening, but σt varies with time; we want a time independent parameter that describes the efficiency of the system:
DEF: N = (tR/ σt)2 - plate number (platetall)
For a given chromatographic system, N is approx. constant for the different analytes. We want little band broadening = high efficiency i.e. small σt or large N
Gaussian curves
UNIVERSITY OF OSLO
© DEPARTMENT OF CHEMISTRY V11
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25
W=4σ
0.607h N = 16 (tR/w)2
w0.5h
h/2
N = 5.54 (tR/w0.5h)2
w4.4%h
N = 25 (tR/w4.4%h)2
tR
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© DEPARTMENT OF CHEMISTRY V11
KJM3420/4420-1
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N is proportional with the column length; we often want to have a measure of the efficiency independent of column
length.
We define the plate height (platehøyden) H = σL2/L
σL2 is called the variance
H can be expressed by L and N as L/N ⇓ H = σL
2/L = L/N (column length pr. plate)
we want H to be as small as possible
UNIVERSITY OF OSLO
© DEPARTMENT OF CHEMISTRY V11
KJM3420/4420-1
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We also want to have a measure of the efficiency which is independent of
column dimensions and particle sizes:
Reduced plate height:
h=H/dp (for packed columns, mainly in HPLC)
h=H/dc (for open tubular columns, mainly in GC)
UNIVERSITY OF OSLO
© DEPARTMENT OF CHEMISTRY V11
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we want to express the ability to separate two or more components
requirements for separation: 1. t1 ≠ t2 2. sufficiently narrow bands
we introduce the concept resolution RS (oppløsningsevne) to get a quantitative measure
Resolution
UNIVERSITY OF OSLO
© DEPARTMENT OF CHEMISTRY V11
KJM3420/4420-1
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Resolution
Overlap of equally sized peaks
R= 1 2.33%
R= 1.5 0.14%
UNIVERSITY OF OSLO
© DEPARTMENT OF CHEMISTRY V11
KJM3420/4420-1
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for closely eluted compounds (k1≈ k2 = k)
where α = k2/k1 and is called separation factor
RS can be controlled by: α - the selectivity; by changing the SP and/or MP N – the efficiency; column length, part. size, MP flow, etc. k - LC: MP-strength GC: temperature SFC: density, temp., modifier
UNIVERSITY OF OSLO
© DEPARTMENT OF CHEMISTRY V11
KJM3420/4420-1
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Separation number (trennzahl, separasjonstall)
TZ = 1 + (tn+1 - tn)/(wn+1 - wn)
number of components which will fit between two consecutive homologues compounds with a resolution of RS = 1.2 (between the components)
mostly used in capillary GC
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© DEPARTMENT OF CHEMISTRY V11
KJM3420/4420-1
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Asymmetry
a and b are measured at 10% of h
As = b/a
LC: OK if AS < 1.5
a b
h