Chromatography

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Chromatogra

Chromatography

phy Module

Module

Error and Error and Chemistry Chemistry Review Review  Atomic  Atomic Spectroscopy Spectroscopy (Elements

(Elements)) ChromatographyChromatography

Spectroscopy & Spectroscopy & Sensors Sensors

WEEK 4!

WEEK 4!

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retention time of analyte retention time of analyte 4

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chromatography of peaks chromatography of peaks

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Gas and Liqs and Liquiduid

Chromatography

Chromatography

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Basic Theory

Basic Theory

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Chromatography:

Chromatography:

Its Importance

Its Importance

Retention and Retention and Resolution Resolution Gas Gas Chromatography Chromatography Liquid Liquid Chromatography Chromatography The

Theory ory ofof

Chromatography

Chromatography

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Selecting and optimizing a Selecting and optimizing a chromatographic method chromatographic method

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(3)

Chromatography:

Chromatography:

Its Importance

Its Importance

Retention and Retention and Resolution Resolution Gas Gas Chromatography Chromatography Liquid Liquid Chromatography Chromatography The

Theory ory ofof

Chromatography

Chromatography

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Selecting and optimizing a Selecting and optimizing a chromatographic method chromatographic method

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Chromatograp

Chromatography

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Widespread Applications

Widespread Applications

Drugs on Dollars Drugs on Dollars Chocolate Chemicals Chocolate Chemicals Pharmaceutical Purity Pharmaceutical Purity

Chromatography is used for

Chromatography is used for identifying molecules, typically organicidentifying molecules, typically organic 5% of

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Definition of

Definition of

Chromatography

Chromatography

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chromatography

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suspension or as a vapor (as in gas chromatography)

suspension or as a vapor (as in gas chromatography)

through a medium in which the components move at

through a medium in which the components move at

different rates.

different rates.

Oxford English Dictionary Oxford English Dictionary

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The First Chromatogram ~ 1900

(Paper Chromatography)

• Chromatography: ‘Color’ and ‘graph’

• Early 1900s botanist: Tswett – separating leaf pigments

• Packed a column with chalk particles, different colors separated

• Example of a normal phase separation: polar column, non-polar mobile phase

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 Actual Chromatography:

Column Chromatography

1.Column loaded with silica/column medium

2.Eluting solvent added to compact silica layer and to remove air bubbles 3.Purple mixture as a thin layer is added to top of silica layer 

4.Eluting solvent added and eluted (purple layer separates into a red and blue layer) 5.Eluting solvent added and eluted (red and blue layers separate further)

6.Red layer collected (the faster moving layer) 7.Blue layer collected (the slower moving layer) 8.No more compounds are eluted, process ended

From Wikipedia Commons

Good youtube video visualizing analytes moving through mobile phases http://www.youtube.com/watch?v=SdYb6GgBQ7s

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The

Magic 

Boxes:

For Chromatography

 Amnt. Hexane  Amnt Heptane Inject Sample Here 20.34 05.34

1) Pumps for gas/liquid sample handling

2) Chromatography column itself 

3) Detection system

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Gas Chromatography:

The Box

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Liquid Chromatography:

The Box

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time    S    i  g  n   a    l  a    t    D  e    t  e  c    t  o  r

 A Chromatogram: The

Basics

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How to use

chromatograms

time    S    i  g  n   a    l  a    t    D  e    t  e  c    t  o  r time    S    i  g  n   a    l  a    t    D  e    t  e  c    t  o  r Quantitative

 Analysis Qualitative Analysis

Useful for identification

• Method calibrated with known material • The specific retention time is measured • Unknown sample identified

Useful for quantification

• Method calibrated with known material • The area under the peak signifies in

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Different Types of

Chromatography

Chromatography Liquid Chromatography Liquid-Solid Liquid-Liquid Gas Chromatography Gas-solid Gas-Liquid Stationary Phase Purple is the mobile phase Red is analyte

The primary names of different types of chromatography can come also from the nature of the analyte-column interaction

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 A fun

chromatogram

http://www.chem.agilent.com/en-US/Search/Library http://www.waters.com/waters/libraryList.htm?cid=511436&q=beer&locale=en_US&qTemp=beer 

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Chromatography:

Its Importance

Retention and Resolution Gas Chromatography Liquid Chromatography Theory of Chromatography

• Peak positions: retention • Peak widths: resolution

Week 4

Selecting and optimizing a chromatographic method

• The basics of chromatography

• Understanding the chromatogram

• Types of chromatography

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Retention Time:

Qualitative

Retention and Resolution Gas Chromatography Liquid Chromatography Theory of Chromatography

• Peak positions: retention • Peak widths: resolution

Week 4

Selecting and optimizing a chromatographic method

Chemical origins of retention

time

 Analyte chemistry as it moves

through a column

Linking analyte and column

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What dictates

retention times?

time    S    i  g  n   a    l  a    t    D  e    t  e  c    t  o  r

t

m

is the time for the mobile

phase to exit (earliest peak)

t

is the retention time of

analytes – can have more

than one

Mobile phase: tm

 Analyte peaks: t

 Analyte Retention Time t

 A 2 min

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Thinking About

Chromatography

Column

The mobile phase

elutes through the

column

Note: “Column” can be vague. Sometimes it is the entire part of the instrument containing the stationary phase, sometimes it is the stationary phase alone

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Eluent

Stationary Phase

(20)

 Analytes Partition Between

Mobile and Stationary Phases

Column = Phase 1 Mobile Phase = Phase 2

Take a snapshot:

x% analyte in phase 2

y% analyte in phase 1

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Partition: A Look

Inside

T=0 T=0 Signal T=4 T=4 T=2 T=2  Analyte A  Analyte B T=6 Signal

 Analyte A spends less time in the

stationary phase than Analyte B

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Practice

Questions

time    S    i  g  n   a    l  a    t    D  e    t  e  c    t  o  r

1 min A: 4 min B: 8 min

#1) What is the peak at 1 minute called? #2) Which analyte, A or B, spent more time in the stationary phase?

#3) What fraction of time did (A) and (B) spend in the stationary phase?

t

R

= t

S

+ t

M

So A spent 3 minutes in stationary phase Or ¾ = 75%

So B spent 7 minutes in stationary phase Or 7/8 = 88%

Solvent peak or mobile phase peak

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What controls the

partition?

T=0 T=0  Analyte A: Not POLAR  Analyte B: POLAR

“Like dissolves like”

Polar Column

Polar Column

Little ‘partition’

into a polar column

Definite partition into a polar column T=0 T=0  Analyte A: Not POLAR  Analyte B: POLAR Non-polar Column Non-Polar Column

Waxy column will

partition non-polar

molecules

Non-polar

column will NOT

partition much

polar material

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Reminder: Polarity

Non-polar 

Hydrophobic

Repels water 

Greasy

Waxy

Oily

Polar 

Hydrophilic

 Attracts water 

Wet

For your homework: need to look up ALL the names :>

1) 2) 3) 4) 6) 5)

(25)

 An Analogy: What happens

with polar and non-polar 

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Practice Questions

time    S    i  g  n   a    l  a    t    D  e    t  e  c    t  o  r

1 min A: 4 min B: 8 min

2) Which molecule is the most polar? Which molecule is the least polar?

C: 10 min 1) What fraction of time did A, B and C spend in the stationary phase?

This chromatogram was taken with a very polar column

 A: ¾ or 75 % B: 7/8 or 88% C: 9/10 or 90%

 A: LEAST polar  C: MOST polar 

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What’s Coming:

Instrumentation

http://www.chem.agilent.com/en-US/Search/Library http://www.waters.com/waters/libraryList.htm?cid=511436&q=beer&locale=en_US&qTemp=beer 

• What is the mobile phase? Stationary phase?:__________________________ 

• What types of molecules elute early:  _________________________ 

• Is the column polar or non-polar?  _________________________ 

 Helium, Ultra 2 

Polar

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Retention Time:

Qualitative

Retention and Resolution Gas Chromatography Liquid Chromatography

Selecting and optimizing a chromatographic method

Chemical origins of retention time

 Analyte chemistry as it moves through a

column

Linking analyte and column polarity to

(29)

Retention Time:

Quantitative

Retention and Resolution Gas Chromatography Liquid Chromatography Theory of Chromatography

• Peak positions: retention • Peak widths: resolution

Week 4

Selecting and optimizing a chromatographic method

Definition of the Partition

Coefficient of BIG K

Use of partition coefficient to

calculate retention times

Effect of column geometry on

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Separations in Liquids:

Partition Coefficients

 b a

]

Solute

[

]

Solute

[

 Solute 1 in phase A Solute 1 in phase B

The extent of extraction will depend on the volumes of each phase

b a b a

volume

inB

moles

volume

inA

moles

Solute

Solute

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1

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1

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 

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Octanol-Water Partition

Coefficients: An Example

GOUDARZI, Nasser and GOODARZI, Mohammad. QSPR study of partition coefficient (Ko/w) of some organic compounds using radial basic function-partial least square (RBF-PLS). J. Braz. Chem. Soc. [online]. 2010, vol.21, n.9 [cited 2013-06-01], pp. 1776-1783 . Available from: <http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0103-50532010000900027&lng=en&nrm=iso>. ISSN 0103-5053.

http://dx.doi.org/10.1590/S0103-50532010000900027. Kow is the ratio of a compound’s concentration in known voumes of n-octanol to concentration in

known volume water  This is 50%

shared

If you had 1 mill ion molecules of DDT only 1 might go to

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You know that the partition coefficient for hexane from your mobile

phase into your reversed phase column is 3. What fraction of the total

moles of hexane is present in the stationary phase at any given

moment? Note the mobile phase volume is 100X that of the stationary

phase volume.

 

 

Fraction = 1/(1+100/3)=.0291 or 2.9 %













Using Partition

Coefficients

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What are the typical fractions in the stationary phases for partition

coefficients into the stationary phase of .01 to 10 and V

s

/V

m

ranging from

.01 to .001?







Typical Trends via

Excel

Important take home: Even with high partition into the mobile phase, the bigger volume of the

mobile phase results in little material ‘in’ the stationary phase. K 100 Vm/Vs 1000 Vm/Vs 0.01 9.999E ‐05 9.9999E‐06 0.05 0.0004998 4.9998E ‐05 0.1 0.000999 9.999E ‐05 0.5 0.0049751 0.00049975 1 0.009901 0.000999 5 0.047619 0.00497512 10 0.0909091 0.00990099

In the original lecture notes, the column with 1000 Vm/Vs was too small by a factor of 10. Now its correct

(34)

How the Partition Coefficient

Dictates Retention Time

(35)

From retention time to

capacity factor 

Retention time: ttime needed for x to elute  Adjusted retention time: t – tm

Capacity or retention factor of a solute (little k)

Species not retained is ‘m’

Time it takes to elute is dead time and is ‘tm

What’s the challenge for solutes with large retention factors, above 50? What’s the challenge when solutes have small retention factors, less than 1

t

m

t

 

 1  1   

 1

  

 

 

  

(36)

Retention Time:

Quantitative

Retention and Resolution Gas Chromatography Liquid Chromatography

Selecting and optimizing a chromatographic method

(37)

Getting to retention time

Partition Coefficient (big K) Volumes of the stationary phase and mobile phase Capacity Factor (little k) Prediction of retention time Fractional time in stationary phase

  

 

 

  

 

 

(38)

Exampl e: A mixture of benzene, toluene and methane are injected into a gas

chromatograph. Methane gave a sharp s pike at 42 seconds whi le benzene requir ed 251 seconds and tol uene eluted in 333 seconds. Find the adjusted retention time and the capacity factor fo r each solute.

Practice finding little-k

Benzene: adj ret. Time = 251 – 42 = 209 sec

Toluene: adj ret. Time = 333 – 42 = 291 sec Little k =209/42 = 4.98

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Example: Solvent passes through a column in 3.0 minutes but a solute takes 9.0

minutes. (A) Calculate the capacity factor, k (B) What fraction of the time is the solute in the mobile phase? ( C) The volume of the stationary phase is 1.10th the volume of the mobile phase. Find the partition coefficient for this system.

Little k = (9 – 3 min)/(3) = 2

The fraction of time solute is in mobile is ratio of tm/tr = .333 Little k and Big K are related by k = KVs/Vm so:

K = kVm/Vs = 2 * 10 = 20

If you increase the partition coefficient between the stationary and mobile phase, what happens to little k? To the retention time?

If you increase the volume of the stationary phase, what happens to little k? to the retention time?

Then the solute spends more time in the stationary phase, K increases and little k increases, as does t

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Example: The open tubular column used in the last example has an ID of 250 microns and is coated with a layer of stationary phase 1 micron thick. Estimate the partition coefficient for benzene between the stationary and mobile phases.

We should calculate the relative volumes of the mobile and stationary phases – these are proportional to relative cross-sectional areas.

V-mobile=L*(124um)2= L*48,300 micron2

V-stationary=L*(125um)2-L*(124um)2= L*782 micron2

We know from the last example that k=5 for benzene in this column.

Figure

Updating...

References