Apat 2013 gc workshop 2

http://analysciences.com

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AnalySys Sciences

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Instrumental in your success



An Analytical chemist …

… tries to answer only two questions.

Given a sample …

What is it? Qualitative analysis

How much is it? Quantitative analysis

The evolution of analysis

1900‟s Manual titration 1 mg 10-3 0.001 gm

1920‟s TLC 1 µg 10-6 0.000001

1960‟s GC 1 ng 10-9 0.000000001

1980‟s HPLC 1 pg 10-12 0.0000000001

1990‟s GC-MS 1 fg 10-15 0.000000000000001

2008 LCMS 1 ag 10-18 0.000000000000000001

2013 FTMS 1 zg 10-21 0.000000000000000000001

Analytical Chemistry – The road ahead

Increased use of hyphenated techniques, like LC-MS, GC-FTIR

& LC-NMR.

Lower limits of detection.

“Walk-away” automation.

Intuitive software and data handling.

Increasing use single-point control systems via the Internet.

The Analytical Pharmacist in the 21st century

Full-time analytical chemist.

Part-time software engineer and EDP specialist.

AND…a knowledge of software platforms, data handling techniques and preferably, basic electronics.

Chromatography … An introduction

7http://analysciences.com

From: The Universal Etymological Dictionary, 1731

Chromatography … since Biblical times.So Moses brought Israel from the Red Sea, and they went

out in the wilderness of Shur …and found no water.

And when they came to Marah, they could not drink of the

waters of Marah, for they were bitter;

And the people murmured against Moses, saying, What

shall we drink?

And he cried unto the Lord and the Lord shewed him

a tree, which when he had cast into the waters, the

waters were made sweet.

Exodus,Chapter 15 §22–25 (King James Version).

Source: Article by Leslie Ettre.Ion exchange

chromatography?

http://www.chromatographyonline.com/lcgc/Column:+Milestones+in+Chromatography/Was-Moses-the-First-Chromatographer-Chromatography/ArticleStandard/Article/detail/393892

http://www.chromatographyonline.com/lcgc/Column:+Milestones+in+Chromatography/Was-Moses-the-First-Chromatographer-Chromatography/ArticleStandard/Article/detail/393892

110 years of modern chromatography

March 21, 1903

At the Warsaw Society of Natural Scientists,

Russian botanist, Mikhail Semenovich Tswett

presented the first official lecture on

chromatographic separation.

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Tswett, MS (1906) Physico-chemical studies on chlorophyll adsorptions. Berichte der Deutschen botanischen Gesellschaft, 24, 316-23

Tswett, MS (1906) Adsorption analysis and chromatographic method. Application to the chemistry of chlorophyll. Berichte der Deutschen

botanischen Gesellschaft, 24, 385 http://www.life.uiuc.edu/govindjee/Part2/34_Krasnovsky.pdf

http://web.lemoyne.edu/~giunta/tswett.html


http://www.life.uiuc.edu/govindjee/Part2/34_Krasnovsky.pdf

http://www.life.uiuc.edu/govindjee/Part2/34_Krasnovsky.pdf



When a chlorophyll solution in petrol ether is filtered through the column of an adsorbent …then the pigments will be separated from the top down in individual colored zones…the pigments which are adsorbed stronger will displace those which are retained more weakly.

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"Like light rays in the spectrum, the different components of a pigment mixture, obeying a law, are separated on the calcium carbonate column and can thus be qualitatively and quantitatively determined.

I call such a preparation a chromatogram and the corresponding method the chromatographic method."


Gas chromatography – the pioneers.

Erika Cremer, Univ of

Innsbruck, Austria,

1944, developed the

theory and use of gas

chromatography.

She was assisted by

her PhD student, Fritz

Prior.


Chromatography is …

“…a method in which the components of a

mixture are separated on an adsorbent

column in a flowing system". M.Tswett


A separation involving a mobile

phase, a stationary phase, and the

sample. The sample undergoes a

series of interactions between these

two phases, resulting in separation of

its components. Sample components

elute in increasing order of

interaction.

What interaction?

Adsorption

…analyte in mobile phase

adsorbed onto stationary phase.

Equilibration between the mobile

and stationary phase results in

separation.


Partition

…thin film of a liquid

stationary phase formed on a

solid support.

Solute molecules partition

between the mobile phase and

stationary phase.


Ion-exchange

Ion-ex resin is used to

covalently attach anions or

cations onto it. Solute ions of

the opposite charge are

attracted to the resin.

Example: Purification of hard

water.


Affinity

specific interaction between a

solute molecule and a

molecule that is immobilized

on a stationary phase. eg.

purification of

immunoglobulins.


Size Exclusiona porous gel separates

molecules by size.

Example: Purification of

enzymes or proteins.


Mobile phase

Gas

Gas-solid (Adsorption)

Gas-Liquid (Partition)

Liquid

TLC /Planar chromatography

Column chrom

HPLC

Supercritical fluid

SFC


Chromatography –

Modes

Mobile phase

Stationary phase

Eluate collection

Sample introduction

Detection

Chromatography

– the system

20

Stationary phase is

packed into a column,

or …

In the form of a thin

layer coated on a glass

or aluminium plate or

… In the form of a

thick sheet of paper.

A typical chromatogram


Y axis = Detector

response (usually in

millivolts)

X axis = retention time

(or volume)

A symmetrical peak is

known as a Gaussian

peak.

Some boring equations


Retention Volume / Time

Volume of mobile phase required to elute a particular

analyte from the stationary phase.

Time taken by an analyte to elute from the stationary

phase.

VR = tR x Fc

tR = Retention time

Fc = Flow rate


Retention Time

Dead Time/volumeRetention time / retention volume taken by an

unretained solute to elute from the system.

Represents the combined volume of tubings,

detector flow cell, injector loop, column volume.

Relative (corrected) retention time

0R Rt t t


Partition Co-efficient(Distribution / Adsorption co-efficient)

M

sCK

C


CS = concentration of the analyte in the

stationary phase.

CM = concentration in the mobile phase

Analytes in a sample mixture will separate

in a chromatographic system only if their K

values are significantly different.

Partition Ratio (Capacity Factor)

Measure of the time spent by a solute in the mobile phase, with respect to the stationary phase.

For baseline separation, K’ > 2


Relative retention (Selectivity / separation factor)

For baseline separation, a > 1.5

2

1

k

ka


Selectivity

Depends on

•Nature of the two phases

•Column temperature


Resolution

For baseline separation, Rs >2

2 1

1 2

2

R Rs

t tR

w w


Peak Width (4s)


Tailing factor (Asymmetry/ Skew factor)

BCAs

CA


Tailing factor - 2


System Suitability Parameters USP

Plate count > 2000 plates/meter

Tailing factor < 2

Resolution > 2

Partition ratio > 2

Relative retention > 1.5

Precision / repeatability RSD </= 1% for n >/= 5


Chromatography Theories

or… why a chromatography column will not do

what it’s told..


Plate theory Martin and Synge (1941)

Nobel in Chemistry, 1952 for “their

invention of partition chromatography”.

Chromatography column assumed to be

similar to a distillation column.

Separation occurs across a series of

theoretical plates.

Higher number of theoretical plates

improves column performance.


Plate theory explainedA distillation column is used for fractional distillation of liquid

mixtures. Higher surface area inside the column improves

distillation efficiency. This is done by putting in a series of

glass plates, with each plate containing glass beads or similar

packing material.

A chromatographic column is similar to a distillation column.

The packing inside the column is considered similar to the

packing inside a distillation column. There are no real plates

inside, hence „theoretical plates‟.

Hence, height equivalent to a theoretical plate (HETP). Higher

number of plates, higher separation efficiency.


Rate theory Dr JJ van Deemter (1956)

Plate theory does not explain band spreading and peak

broadening. Does not take into account packing material

properties, mobile phase flow rate and column geometry.

Rate theory takes into account various factors that cause

chromatographic peak broadening and reduction of

separation efficiency.

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van Deemter Equation

linear velocity ( flow rate)

CH A B

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van Deemter took into account several

factors that can affect HETP and column

performance. He formulated a mathematical

equation that defined the relationship

between various chromatographic factors and

HETP.

This equation made it possible to numerically

calculate column performance, design better

chromatography stationary phases and

improve separation efficiency.

A term – Multipath effect or Eddy diffusion

Analyte molecules take different paths

through the packing, leading to band

broadening

To reduce eddy diffusion, reduce

stationary phase particle size.

However, backpressure will increase.

In GC, backpressure is not a major

issue.


B term

Longitudinal diffusion / wall effect

Distortion of the mobile phase front, due to varying velocity across the column, especially at the column wallTo reduce wall effect, increase flow rate


C term – mass transfer resistance

Analytes remain trapped in stagnant pockets

in the packing. To improve mass transfer,

decrease mobile phase flow rate.


Van Deemter plot


What does it mean?

In practical terms, it means that for a given

stationary phase and for a given

chromatography column or plate, there is

one optimal mobile phase flow rate.

Increasing or decreasing flow rate might

have an adverse effect on performance.

For example: For an HPLC column with

4.6mm internal diameter and 150mm length,

packed with 5u, spherical particles, the

optimal flow rate is 1ml/min.

HETP Height Equivalent to a theoretical plate

2

2

4

16

2

5.54

R

R

LH

t

LH

t

s

s


Plate Count2

2

164

255

R

R

t

t

s

s

2

5.542

R

LN

H

t

s


Plate count – what it means.The plate count gives you an idea of the efficiency and separating power of a column.

Higher plate count for a given column implies better performance

(but does not guarantee it !)

Plate count is affected by:Nature of sample

Flow rate

Detector flow cell volume

Dead volume

Temperature

Detector settings / Data system settings.

Injector reproducibility, etc…

Be wary when comparing plate counts!!


A typical chromatogram


Y axis = Detector response (usually in millivolts)

X axis = retention time (or volume)

Quantitation in Chromatography

Area (height) under the peak is proportional to the injected amount.Proportionality constant is the response factor.


How is peak area determined?

Integration

Data system sub-divides

peak into small rectangles,

calculates area of each,

and adds them up.


Quantitation – External

standards

Inject known concentrations of the analyte using reference standards.

Analyse the test sample under the same conditions.

Plot a calibration curve of analyteconcentration v/s peak area (or height).


Internal Standards

Chemically similar to the analyte.

Added to the sample and external standards.

Same amount added to both.

Accounts for variations in injection volume

and other system variables.

Provides better precision.


Gas Chromatography


Gas Chromatography

Mobile phase is a gas

Used for volatile, heat stable samples only. eg.

Petroleum products, volatile oils, perfumeries.

… Or analytes that can be converted to

volatile derivatives, eg. amino acid silyl

derivatives, fatty acid methyl esters.


Why GC?

Minimal sample prep.

Fast analysis time. High separation

efficiency.

Easier to automate. Easier to upgrade

to hyphenated methods like GC-MS.

Lower capital costs and running costs.

Given a choice between HPLC and GC,

choose GC!


Restricted to analytes that are volatile

and thermo-stable … or to analytes that

can be derivatised.

Carrier gas

Filters/traps

Injector

Detector

Column oven

Column

Data system

GC Schematics


GC – Mobile phases / Carrier gases.

GC – Mobile phases

Helium is commonly used as a carrier gas. Nitrogen is also used.

Hydrogen is becoming a popular alternative to helium.

Gases are stored in high-pressure cylinders.

Gas flow is controlled by regulators.

Sometimes nitrogen and helium generators are used instead of cylinders.


Hydrogen as carrier gas.

H2 has low viscosity and high diffusivity.

Hence, faster analysis times.

Much cheaper than helium. Lower cost-

per-analysis.

Helium is extracted from natural gas.

Process is very expensive. Not eco-friendly.

Acute shortage of Helium.

H2 can be cheaply produced using H2

generators.


Gas manifolds

Gas manifolds are used to purify and

dehumidify the gases before they enter

the GC.

Dust filters, moisture traps, silica gel

pellets and molecular sieves are used.

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Sample

Introduction


Injector ports

Samples are injected through sealed, heated injection ports.

Injection volumes are very small, usually less than 5 μl.

Injectors should accurately deliver the vaporised sample on to the head of the GC column.


Packed column injector


Injector septum provides a leak-tight

seal.

Injector liner protects the inlet seal

from dirt and contaminants.

Inlet seal protects the GC column.

Injector body is heated by a

programmable heater system.

Used with capillary columns.

Injects small sample volumes.

(<1μl)

Splits the injection volume into

smaller volumes, by adjusting the

split ratio.


PTV injectorProgrammable temperature vaporising

injector.

Used for large sample volumes and

thermo-labile compounds

Instantly vaporises sample, upto 3000C

Highly reproducible and accurate.


Injector septa

Septa ensure a leak-tight seal

at the injection port.

Available in various materials

– teflon, rubber and silicone.


Sampling Valves

Used for continuous, reproducible injection

of gaseous samples.

Can be configured in several ways:

•Multiple column switching

•Detector switching

•Automated air sampling


Injector linersGlass liners are used inside the injector body.Protect the injector from sample debris.


GC – sample injection syringes

Septum piercing needle.

Available in various volumes,

from 1ul to 100ul.

Can be automated.


Autosamplers

Two types:Carousel

XYZ samplers

Can automate many tasks:Simple injection

Sample prep/derivatisation/filtration/

dilution/heating/cooling /weighing.


Autosamplers – pros & cons

Low cost-per-analysis.Reagent & solvent consumption is

reduced.

High reproducibility.Reliable results.

24/7/365 operation.Chemist is free of repetitive

manual tasks.

High capital costs.


GC – Stationary phases & columns


Packed columns


Made of SS, glass or copper tubing, filled with porous packing material, which may be coated with a viscous liquid phase.

Packed columns contain a finely divided, inert, solid support material (usually based on diatomaceous earth ) coated with liquid stationary phase. Most packed columns are 1.5 - 10m in length and have an internal diameter of 2 - 4mm.

Packed columns – phases.

The packing usually consists of an inert

porous material such as Celite (a

diatomaceous earth), or calcined Celite (in

the form of powdered fire brick) or a

synthetically polymeric resin.

Glass beads and molecular sieves are also

used.


Packed columns - KieselguhrPackings are treated with

dimethylchlorosilane to

remove active silanols.

Washed with HCl to

remove trace metals.


Diatomaceous earth or kieselguhr is soft, sedimentary rock that contains fossilised remains of diatoms (hard-shelled algae). It consists of 80-90% silica, and small amounts of alumina and iron oxide. It crumbles easily into a fine, white powder.

Celite is a brand name, owned by World Minerals Inc, a division of Imerys Filtration.

Chromosorb W = Untreated celite

Chromosorb P = Calcined celite

Chromosorb S = Celite calcined with sodium

carbonate.

Packed columns – Molecular sieves

Molecular sieves are synthetic

zeolites (complex alumino-silicates

of sodium, potassium or calcium)

of various pore sizes, usually 4 Å

or so.

Used for separation of fixed gases

like CO, CO2, CH4, Ar, H2, O2.


Packed columns – Polymeric packings

Macroporous, spherical,

ultrapure resins.

Used for difficult separations in

gas chromatography. Eg. Separation of H2S and H2O.

Separation of gas mixtures.

HayeSep is a popular brand.


Capillary columns Made from fused silica.

Have an internal diameter of a few

tenths of a millimeter, usually

0.32mm and 0.53 mm.

Length between 3m to 30m.

Capillary columns are more efficient

than packed columns.

Much higher plate counts >30,000

plates per meter.


Capillary columns - 2

Liquid stationary phase is

coated or chemically bonded to

the inner wall of the capillary.

Most common phases:

Polysiloxanes

Polyethylene glycols.


Separation mechanisms in GC

Partition: Analyte partitions between the carrier gas and a viscous stationary phase.

Adsorption: Analyteadsorbs/desorbs between the carrier gas and a solid stationary phase.


GC - Detection systems


Thermal Conductivity DetectorDetector cell contains a heated filament with an

applied current. As carrier gas containing solutes

passes through the cell, a change in the filament

current occurs. The current change is compared

against the current in a reference cell. The difference

is measured and a signal is generated. (Wheatstone

bridge principle).

Selectivity: All compounds except for the carrier gas

Sensitivity: 5-20 ng Linear range: 105-106

Temperature: 150-250°C


Flame Ionisation Detector

Analytes are burned in a hydrogen-air flame. Carbon

containing compounds produce ions that are attracted

to the collector. The number of ions hitting the

collector is measured and a signal is generated.

Selectivity: Compounds with C-H bonds.

Sensitivity: 0.1-10 ng. Linear range: 105-107

Gases: Combustion - hydrogen and air; Makeup -

helium or nitrogen. Temperature: 250-450°C.


Electron Capture

DetectorElectrons are supplied from a 63Ni foil

lining the detector cell. A current is

generated in the cell. Electronegative

compounds capture electrons, causing a

reduction in current. The amount of

current loss is indirectly measured and a

signal is generated.

Selectivity: Halogens, nitrates and

conjugated carbonyls.

Sensitivity: 0.1-10 pg

Temperature: 300-400°C


Pulsed discharge ionisation detector (PDID)

Pulsed DC discharge creates a plasma by ionising helium gas inside the detector body.

Charged helium plasma in turn ionises analytes eluting from the GC column.

This results in a current that is proportional to the amount of the analyte.


PDID - Advantages

Universal, non-destructive detector. Very sensitive, can detect analytes in the femtogram level (10-15).

Good alternative to electron-capture detector for pesticides and halogenated compounds, since it is non-radioactive.More sensitive than FID, and can be used in settings where a flame is not safe (like petroleum and gas analyses.)


Flame Photometric Detector.

Uses a photomultiplier tube to detect spectral lines of analytes, as they are burned in a flame. (like in a flame photometer).

Especially useful for sulfur and phosphorus compounds.


Photoionisation detector

UV lamp ionises analytes from the GC column eluent.

Useful for volatile organic compounds like polyaromatic hydrocarbons and inorganic species that are ionised in UV light.

Used for environmental pollutants.


Inside the GC


GC columns are mounted in

an oven.

Oven temperature can be

programmed.

Better separations are

achieved with temperature

programming.

Temperature programming – why.

In GC, analytes are separated according to boiling point and polarity.

Molecules with low boiling point will elute early from the GC column. Compounds

with high boiling point will elute later.

Analytes interact with the GC column. If the column is non-polar, analytes with

high polarity will travel faster through the column while more non-polar

compounds will be retained.

Isothermal GCIsothermal GC is not a good choice for samples containing analytes with varying boiling points. For example, petroleum products, silylated amino acids, methylated fatty acids.

In an isothermal GC analysis, the column temperature is constant. Fast eluting compounds may then appear as overlapping peaks and late eluting compounds will have long retention time and broad peak shape.

http://ull.chemistry.uakron.edu/chemsep/slide.php?Chapter=/chemsep/GC/&Last=100&Slide=56

Temperature programming

By varying column temperature over time, analytes with different boiling points can be separated.

Analysis time can be optimised.

http://ull.chemistry.uakron.edu/chemsep/slide.php?Chapter=/chemsep/GC/&Last=100&Slide=56

www.chem.agilent.com

Temperature ramping


Apat 2013 gc workshop 2

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