HPLC

The best application fields of various chromatographic modes

GCVolatile, thermostable compounds

LCPolar, non volatile. thermolabile

EKCIonic compounds

The role of interaction types in various chromatographic modes

Types GC SFC HPLC EKC

Dispersion ++++ +++ ++ +

- ++ ++ ++++ +

Dipole-dipole ++ ++ ++++ ++

Hydrogen bridge + ++ +++ +++

Ionic / / ++ ++++

Repulsion ++ ++ +++ ++

Advantages of various chromatographic modes

Tulajdonság GC SFC HPLC EKC

Efficiency ++++ +++ ++ ++++

Analyses temperature

+ +++ ++++ ++++

Variability of mobile phase

/ + ++++ +++

Speed of analyses ++++ ++ + +++

Sensitivity ++++ ++ +++ +

Established instrumentation

+++ + ++++ ++

Resolution as function of other chromatographic parameters

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

The HPLC uses packed columns.The diffusion processes are much slower in HPLC than GC.

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

Degassing is important to gain smooth baseline.

An pp to date HPLC instrument

• Pumps upto 300 bar

• The degassing is important

Pump

Motor & Cam

Plunger

Plunger seal

Check valves

Pump head

Pump of HPLC instrument

Pulsation of system is decreased with two pumps, working in opposite periods.

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

Low Pressure Gradient

Aim of gradient - problems in isocratic mode -

• in isocratic mode

Long analysis timeLong analysis time, low signal to noise ratio, low signal to noise ratio

Poor separationsPoor separations

Methanol / water = 6 / 4

Methanol / water = 8 / 2

(Column : ODS type)

Aim of gradient - solution -

• Gradual change of the mixing ratio during analysis

95%

30%

Methanol concentrationin mobile phase

Short analysis timeShort analysis time &&Excellent separationExcellent separation, good signal to noise ratio, good signal to noise ratio

Polarity of eluents

Rotary valve injection in HPLCben

The loop injector introduces exact volume of sample.

On-line SPE-HPLC arrangement

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

HPLC analyses of polar pesticides with precolumn concentration

Integrated precoumn HPLC

The precolumn protect the main column, against the deposition of matrix components, and dissolution of stationary phase.Main columns have 15-25 cm length and 2- 4,6mm I.D.

Dead volume

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

Tube

Male nut Dead volumeDead volume

Excellent connection Poor connection

Sample vs. HPLC mode

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), wich allows 3-5 µm size of packing material. The increased porosity increased the loadability. However the deep holes are badly washed. Spherical particles are the best.

Various HPLC packings

Goodnes: monolith > spherical > irregular

New type of packings

The limited depths of holes improves the efficiency.

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

Bigger molecules need bigger pore size..

Most frequently used HPLC

Normal phase / Reversed phase

Stationary phase Mobile phase

Normal phase

High polarity(hydrophilic)

Low polarity(hydrophobic)

Reversed phase

Low polarity(hydrophobic)

High polarity(hydrophilic)

Retention order on reverse vs. normal phase packings

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)

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

Stationary phase

Reversed phase packings: • C18

• C8

• C4

• Cinao• DiolNormal

Specials: chiral, ion exchange, gel

Increasing polarity→

Most frequently used HPLC stationary phase C18

Apolar compounds have big retention Mobile phases are mixture of water, methanol acetonitrile.

Condition process of C18 stationary phase

A methanol wash reqires for the activation of C18 stationary phase.

Column polarity - Retention time

C18 (ODS)

CH3

strongstrongweakweak

OH

Mobile phase polarity - Retention time

60 / 40

Mobile phase: Methanol /Water

80 / 20

70 / 30

Methanol / Water

Methanol / Water

Methanol / Water

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

Amines need specially deactivated packings

Ionic compounds analysed as ion pairs on C18.

Cianopropyl Stationary phases

Stationary phase vs. sample

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

The ions of stationary phase interact with the oppositely charged molecules of sample.

Ion chromatogram of anaions

The stationary phase is anionic ionexchange resin.

Analysis of anions in ppb level using supressor

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) chromatography

Specially designes stationary phase for carbamate pesticides

Carbamate can not be analysed with GC, because they are thermolabiles.

Molecular imprintesd (MIP) stationary phases

They are very selective, but low efficiency packings

Various HPLC detectors

Electrochemical S

Mass spectrometric U

Fluorescent S

Ultraviolett S

Refractive U

Light scaterring U

S, selective; U, univeral

UV/UV-VIS detector

A ·C·l= –log (Eout / Ein)

(A : Absorbance)

l

C : ConcentrationCell

Ein Eout

A

C

D2 / W Lamps

External standard

C1

C4

C3

C2

Concentration Area

A1

A2

A3

A4C1 C2 C3 C4

A1

A2

A3

A4

Concentration

Pea

k ar

ea

Calibration curveCalibration curve

Internal standard

C1

C4

C3

C2

ConcentrationArea

A1

A2

A3

A4C1/CIS C2 /CIS C3 /CIS C4 /CIS

A1/AIS

A2 /AIS

A3 /AIS

A4 /AIS

Concentration: Target / Internal standard

Are

a: T

arge

t /

Inte

rnal

sta

ndar

d Calibration curveCalibration curveTarget

Internal

CIS

CIS

CIS

CIS

AIS

AIS

AIS

AIS

standard

Diodarray (DAD) UV-VIS detector

HPLC-UV detection of pesticides

Recommended detection wave length for various functional groups

Light scattering HPLC detector

Universal, sensitive

Refractive index detector(RID-10A)

Sample

Reference

Photodiode

W Lamp

Ionization in HPLC/MS

LC/MS-MS is appropriate for compound identification

First MS→Ionic adduct with soft ionizationSecond MS→fragmentation with EI ionization

On line HPLC/MS coupling