Analytisk kjemi – Separasjonsmetoder I · Textbook: KROMATOGRAFI (Greibrokk, Lundanes, Rasmussen)...

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 Analytisk kjemi – Separasjonsmetoder I

  Chromatographic methods   Electrophoretic methods   Sample preparation   Quantitative analysis

  Textbook: KROMATOGRAFI (Greibrokk, Lundanes, Rasmussen)   (in English: Quantitative Chemical Analysis (Harris)

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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

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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

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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

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Packed capillary LC-ELSD Comparison of three similar HALS trade products

Lowilite 62

Tinuvin 622

Uvisol 226

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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)

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Technique/method

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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)

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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?

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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

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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).

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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

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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.)

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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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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)

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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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  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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Retention parameters

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Relationship between tR and k

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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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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

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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

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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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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

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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)

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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

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Resolution

  Overlap of equally sized peaks

  R= 1 2.33%

  R= 1.5 0.14%

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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

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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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Asymmetry

  a and b are measured at 10% of h

As = b/a

LC: OK if AS < 1.5

a b

h