CHROMATOGRAPHY - 2 Analytical - PowerPoint Presentation

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CHROMATOGRAPHY - 2

Analytical

Chemistry

II

Gas

Chromatography

Mobile phase: inert gases (helium, nitrogen, argon etc)Stationary phases: in capillary columnLiquid (siloxane polymers, polyethylene glycol)Solid (alumina, silicagel or carbon)Solid – liquid

Image: James W Zubrick, Gas Chromatography (Lab Manual)

Gas

Chromatography

In gas chromatography, the components of a vaporized sample are separated by being distributed between a mobile gaseous phase and a liquid or a solid stationary phase held in a column. In performing a gas chromatographic separation, the sample is vaporized and injected onto the head of a chromatographic column. Elution is brought about by the flow of an inert gaseous mobile phase.In contrast to most other types of chromatography, the mobile phase does not interact with molecules of the analyte. The only function of the mobile phase is to transport the analyte through the column.

Gas

Chromatography

In gas-liquid chromatography, while mobile phase (gas) wash away the analytes, they are separated owing to the different polarities that results in differences in interaction with

stationary phase.Gas moving with the pressure (unit mL/min)

Injection

unit

,

gasification at very high temperatureDerivatization for nonvolatile compounds

Görsel:https

://www.chromacademy.com/Headspace-GC.html Erişim tarihi: 28.02.2019

Derivatization

1-

trimethylsilylation: Alkoller, fenoller, steroidler, karbohidratlar ve amino asitlerin bu türevleri hazırlanır. Bu işlem için, trimetilklorsilan (TMCS) ve hekzametildisilazan (HMDS) karışımı kullanılır. RXH + (CH3)3SiCl → (CH3)

3SiXR + HCl 2 RXH + (CH3)3SiNHSi(CH3)3 → 2 (CH3)3SiXR + NH32-

Esterification

:

3-

Acylation

:

Stationary

phases

in gas chromatographyPacked columnsGlass or metal tubing.Typically 2 to 3 m long and have inside diameters of 2 to 4 mm.Packing material: siloxane polymers (methyl,phenyl and cyano

siloxane mixtures), polyethylene glycol

Capillary

columns

Fused

silica columnsoffer several important advantages over glass columns, such as physical strength, much lower reactivity toward sample componentsWall coated

open tubular columns (WCOT)Wall-coated columns are capillary tubes

coated with a thin layer of the liquid stationary phase.

Support

coated

open tubular columns (SCOT)the inner surface of the capillary is lined with a thin film (~

30 µm) of

a solid support material, such as diatomaceous earth, on which the liquid stationary phase is adsorbed. This type of column holds several times as much stationary phase as does a wall-coated column and thus has a greater sample capacity

Fused

silica

Liquid film

WCOT

Fused

silica

Liquid film

SCOT

Detectors

in

gas

chromatography

Column

Temperature

Choose considering the boiling point of the sampleReasonable elution times: 2 – 30 minTemperature of injection port must be higher than column temperature. GC-MS

Gas

Chromatography

Qualitative and quantitative analyses can be performed using retention times (tR) and peak area/height respectively.Comparison of GC and HPLC

GC

HPLC

Mobile

phase

is

gas. The number of gas

is limited and also it is impossible

to

change

the concentration of it.Mobile phase is liquid. There

are

lots of options available as a mobile phase and concentration

of it can be changed.

Samples can only be injected in gas phase. It is a disadvantage to

heat the samples

for gasification because some

substances can decompose at high temperatures.

Substances are dissolved in

liquid for analysis. There is no

need to derivatize

nonvolatile compounds. It is safe

to work with substances which are

decomposed at high temperatures. Preparative HPLC is also

available

for

obtaining

high

amounts

of

separated

substances

.

Columns

are

quite

long

(

upto

100 m !)

Columns

in HPLC

are

short

(10-30 cm).

This

shortens

the

separation

time.

Volatile

substances

can be

analyzed

easily

without

any

treatment

.

Pure

liquid

and

solutions

can

only

be

the

samples

in HPLC

S

ize

-exclusion chromatography

(SEC)Gel Permeation Chromatography (GPC)Gel Filtration Chromatography (GFC)

Mobile phases: Organic solventsMobile phases: Aqueous solventsSuitable

for

polymers

and substances dissolved in organic solventsSuitable for proteins and

substances dissolved in water.

Molecular

weight

determination

Column

packing

materials: polyacrylamide, de

xtran, agarose, silica

or crosslinked polystyrenesViscosity must be low

at working temperature. The

most used solvents are tetrahy

drofuran (THF) and toluen. Pumps: Piston

or peristaltic pumpsConcentration responsive

detectors: UV absoption (the most

sensitive one), refrac

tive index (RI) and differential

refractive index (DRI , the most

used), IR absorptMolecular weight responsive

detectors

:

light

scattering

detectors

(

Low-angle

laser

light

scattering

; LALLS

and

multiangle

laser

light

scattering

; MALLS)

S

ize

-exclusion chromatography

(SEC)Chromatograms show the decomposition of a mixture contains three compounds in time

Affinity

chromatography

Affinity chromatography is based on the biological interaction between antigen and anitbody, enzyme or substrate and ligand. A receptor is immobilized on a agarose gel.

A molecule from a sample can be purified or concetrated.Amount of some

componenets

can be

reduced

in a

mixture. Biological molecules that binds to specific compounds

can be determined (e.g. Pharmaceuticals).

Enzymes

can be

purified

or

concentrated in their solutions.

Image:

https://www.creative-biostructure.com/custom-affinity-chromatography-service-257.htm

Access date: 02.05.2019

Ion

-Exchange

Chromatography

Ion Exchange resinsCation exchange chromatography (separation of alkali compounds like amines)Anion exchange chromatography (compounds that contains negative ions like phosphate

, sulfonate and carboxylate)Used for substances with molecular

weight

lower

than 1500 g/mol, water soluble and ionizable.Parameters:

pH, temperature and ion

type

of mobile

phase

Suitable

for pharmaceuticals and their metabolites, serums

,

food

additives, vitamin mixtures, sugars.

Görsel:

https://www.lcgclabs.com/ion-exchange-chromatography/ Erişim tarihi: 28.02.2019

Ion

-

pair

chromatographyIonic compounds forms neutral ion-pair with ion-pairing reactive (counter ion) in the mobile phase. Hydrophobicity of formed ion-pair increases and results in increase in the

affinity toward stationary phase (non-polar). Separation occurs due to the different

affinities

of

different

ion-pairs. Acidic sample R–COO– + R’4N+ R–COO– + NR’

4 Basic sample

R–

+

NH

3

+ R’–SO

3– R–+NH3 –O3S –R’ https://www.youtube.com/watch?v=TSWP4igRwU0

Ion-pair

chromatography

Generally UV and fluorescence detectors are used. For high sensitivity mass spectrometry detectors are used.İon-pair reactives: Alkane sulfonates and quaternary ammonium

salts For separation of small inorganic and organic compoundsColumns can only be

used

for

once due to the contaminations Trifluoroacetic acid can be used for

biological molecules such as proteins

and

peptides

.

Monolithic

columns

in HPLCColumns with porous channelsThere is almost no dead volume in the column and the column is durable to 9 mL/min

flow rateBetter resolution and quick separationHigh back pressure– UHPLC -

1000  bar

High

speed

-

especially kinetic studies, analysis of highly decomposed substances

and stability studies Low

solvent

consumption

Column

length < 15 cmBroad pH rangeEffective in separation of

big

molecules due to the big channel

sizes

REFERENCES:

Skoog

DA, Holler FJ,

Nieman TA, Principles of Instrumental Analysis, Harcourt&Brace Company, USA, 1998.Khan JI, Kennedy TJ, Christian Jr DR, Basic Principles of Forensic Chemistry, Springer, New York, USA, 2012.Hage DS, Carr JD, Analytical Chemistry and quantitative analysis, Pearson Prentice

Hall, New Jersey, USA,2011.Onur F, Analitik Kimya II, Ankara Üniversitesi Eczacılık Fakültesi Yayın No:101