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HPLC. The best application fields of various chromatographic modes. GC Volatile, thermostable compounds LC Polar, non volatile. thermolabile EKC Ionic compounds. The role of interaction types in various chromatographic modes. Advantages of various chromatographic modes.

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Hplc

HPLC


The best application fields of various chromatographic modes

The best application fields of various chromatographic modes

GC

Volatile, thermostable compounds

LC

Polar, non volatile. thermolabile

EKC

Ionic compounds


The role of interaction types in various chromatographic modes

The role of interaction types in various chromatographic modes


Advantages of various chromatographic modes

Advantages of various chromatographic modes


Resolution as function of other chromatographic parameters

Resolution as function of other chromatographic parameters


Resolution efficiency selectivity

Resolution-efficiency- selectivity

HPLC can produce high selectivity,

but moderate efficieny (< 100 000 tp).

At least, α = 1.3 is required for baseline separation.


Band broadening in hplc

Band broadening in HPLC

The HPLC uses packed columns.

The diffusion processes are much slower in HPLC than GC.


Van deemter curve in hplc

Van Deemter curve in HPLC

The slow diffusion causes increasing HETP values

as function of linear flow of mobile phase.


Schematic view of high performance liquid chromatography hplc instrument

Schematic view of high performance liquid chromatography (HPLC) instrument

Degassing is important to gain smooth baseline.


An pp to date hplc instrument

An pp to date HPLC instrument

  • Pumps upto 300 bar

  • The degassing is important


Hplc

Pump

Pump head

Motor & Cam

Check valves

Plunger

Plunger seal


Pump of hplc instrument

Pump of HPLC instrument

Pulsation of system is decreased with two pumps,

working in opposite periods.


Gradient system

Gradient system

  • Isocratic system

    • Fixed (un-changeable) mixing ratio during analysis

  • Gradient system

    • Changeable mixing ratio during analysis

      • HPGE (High Pressure Gradient, mixing after pumps)

      • LPGE (Low Pressure Gradient, mixing before pumps)


Mobile phase pump with 4 eluents

Mobile phase pump with 4 eluents

Low Pressure Gradient


Aim of gradient problems in isocratic mode

Aim of gradient - problems in isocratic mode -

  • in isocratic mode

Methanol / water = 6 / 4

Long analysis time, low signal to noise ratio

Methanol / water = 8 / 2

Poor separations

(Column : ODS type)


Aim of gradient solution

95%

30%

Aim of gradient - solution -

  • Gradual change of the mixing ratio during analysis

Methanol concentration

in mobile phase

Short analysis time

&

Excellent separation, good signal to noise ratio


Polarity of eluents

Polarity of eluents


Rotary valve injection in hplcben

Rotary valve injection in HPLCben

The loop injector introduces exact volume of sample.


On line spe hplc arrangement

On-line SPE-HPLC arrangement

Precolumn is in the loop. Precolumn is good for sample concentration.


Hplc analyses of polar pesticides with precolumn concentration

HPLC analyses of polar pesticides with precolumn concentration


Integrated precoumn hplc

Integrated precoumn HPLC

The precolumn protect the main column, against thedepositionof matrixcomponents, and dissolution of stationary phase.

Main columns have 15-25 cm length and 2- 4,6mm I.D.


Dead volume

Dead volume

  • Dead volume may cause problems such as poor peak separations and poor reproducibility.

Male nut

Dead volume

Tube

Poor connection

Excellent connection


Sample vs hplc mode

Sample vs. HPLC mode


The diameters and porosity of sample influence of efficieny

The diameters and porosity of sample influence of efficieny

The efficiency increase with the decrease of packing diameter. However the mobile phase pressure has limits (~ 250 att), wichallows3-5 µmsize of packing material.

The increased porosity increased the loadability. However the deep holes are badly washed.

Spherical particles are the best.


Various hplc packings

Various HPLC packings

Goodnes: monolith > spherical > irregular


New type of packings

New type of packings

The limited depths of holes improves the efficiency.


New trend the use of 1 8 m diameter packings

New trend the use of 1.8 µm diameter packings

Very high pressure, short columns and fast analyses


Different molecular weight molecules requires different poremsizes

Different molecular weight molecules requires different poremsizes

Bigger molecules need bigger pore size..


Most frequently used hplc

Most frequently used HPLC


Normal phase reversed phase

Normal phase / Reversed phase


Retention order on reverse vs normal phase packings

Retention order on reverse vs. normal phase packings


Polarity of solvent

Polarity of solvent

The strongest mobile phase is hexane in reversed phase mode.

The strongest mobile phase is acetic acid in normal phase mode.


Bonded silica reversed phase hplc packing

Bonded silica(Reversed phase HPLC packing)

Revers phase s are used in 80 % of HPLC analyses.


Stationary phase

Stationary phase

Reversed phase packings:

  • C18

  • C8

  • C4

  • Cinao

  • Diol

    Normal

    Specials: chiral, ion exchange, gel

Increasing polarity→


Most frequently used hplc stationary phase c 18

Most frequently used HPLC stationary phase C18

Apolar compounds have big retention

Mobile phases are mixture of water, methanol acetonitrile.


Condition process of c 18 stationary phase

Condition process of C18 stationary phase

A methanol wash reqires for the activation of

C18 stationary phase.


Column polarity retention time

Column polarity - Retention time

OH

C18 (ODS)

weak

strong

CH3


Mobile phase polarity retention time

Mobile phase polarity - Retention time

Mobile phase: Methanol /Water

Methanol / Water

60 / 40

Methanol / Water

70 / 30

Methanol / Water

80 / 20


Influence of strength of mobile phes on c 18 stationary phase

Influence of strength of mobile phes on C18 stationary phase

A decrease of mobile phase strength results in

increases of resolution values and retention times.


Hplc analysis of basic herbicides

HPLC analysis of basic herbicides

Amines need specially deactivated packings


Ionic compounds analysed as ion pairs on c 18

Ionic compounds analysed as ion pairs on C18.


Cianopropyl stationary phases

Cianopropyl Stationary phases


Stationary phase vs sample

Stationary phase vs. sample


Normal phase adsorption chromatography

Normal phase,Adsorption chromatography

The molecules of sample is solved in mobile phase, but they touch only in the surface of stationary phase.


Ion excange chromatography

Ion excange chromatography

The ions of stationary phase interact with the oppositely

charged molecules of sample.


Ion chromatogram of anaions

Ion chromatogram of anaions

The stationary phase is anionic ionexchange resin.


Analysis of anions in ppb level using supressor

Analysis of anions in ppb level using supressor


Size excusion gel chromatography

Size excusion (gel) chromatography

The voluminous molecules elute fast because they are

excluded from the small diameter pores, therefore they

interact in less extent.


Size excusion gel chromatography1

Size excusion (gel) chromatography


Specially designes stationary phase for carbamate pesticides

Specially designes stationary phase for carbamate pesticides

Carbamate can not be analysed with GC,

because they are thermolabiles.


Molecular imprintesd mip stationary phases

Molecular imprintesd (MIP) stationary phases

They are very selective,

but low efficiency

packings


Various hplc detectors

Various HPLC detectors

Electrochemical S

Mass spectrometric U

Fluorescent S

Ultraviolett S

Refractive U

Light scaterringU

S, selective; U, univeral


Uv uv vis detector

Ein

A

C

UV/UV-VIS detector

C : Concentration

Cell

Eout

D2 / W Lamps

l

A= e·C·l= –log (Eout / Ein)

(A : Absorbance)


External standard

External standard

Area

Concentration

A1

Calibration curve

C1

A4

A2

A3

C2

Peak area

A2

A3

C3

A1

A4

C1

C2

C3

C4

C4

Concentration


Internal standard

Internal standard

Concentration

Area

Internal

Target

standard

A1

AIS

Calibration curve

C1

CIS

A4 /AIS

A2

AIS

A3 /AIS

C2

CIS

Area: Target / Internal standard

A2 /AIS

A3

AIS

C3

CIS

A1/AIS

A4

AIS

C1/CIS

C2 /CIS

C3 /CIS

C4 /CIS

C4

CIS

Concentration: Target / Internal standard


Diodarray dad uv vis detector

Diodarray (DAD) UV-VIS detector


Hplc uv detection of pesticides

HPLC-UV detection of pesticides


Recommended detection wave length for various functional groups

Recommended detection wave length for various functional groups


Light scattering hplc detector

Light scattering HPLC detector

Universal, sensitive


Refractive index detector rid 10a

Refractive index detector(RID-10A)

Photodiode

Reference

W Lamp

Sample


Ionization in hplc ms

Ionization in HPLC/MS


Lc ms ms is appropriate for compound identification

LC/MS-MS is appropriate for compound identification

First MS→Ionic adduct with soft ionization

Second MS→fragmentation with EI ionization


On line hplc ms coupling

On line HPLC/MS coupling


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