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 GC SFC HPLC EKC Dispersion ++++ +++ ++ +  -  Dipole-dipole Hydrogen bridge Ionic / Repulsion A királis felismerő kölcsönhatásoknak legjobban megfelelő módot érdemes kiválasztani. GC a funkciós csoport nélküli szénhidrogén enatiomerek elválasztására is alkalmas.

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 Pump head Motor & Cam Check valves Plunger Plunger seal

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

Gradient system Isocratic system Gradient 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 - 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 - Gradual change of the mixing ratio during analysis 95% 30% Methanol concentration in mobile phase Short analysis time & Excellent separation, 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. Male nut Dead volume Tube 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

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 Diol Normal 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) OH weak strong CH3

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 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 Ein Eout l A A = e·C·l = –log (Eout / Ein) C C : Concentration Cell Ein Eout A C D2 / W Lamps l A = e·C·l = –log (Eout / Ein) (A : Absorbance)

External standard Calibration curve Area Concentration A1 C1 A4 A2 A3 Peak area A2 A3 C3 A1 A4 C4 C1 C2 C3 C4 Concentration

Internal standard Calibration curve Concentration Internal Area Target 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

HPLC-UV detection of pesticides

Recommended detection wave length for various functional groups

Light scattering HPLC detector Universal, sensitive

Refractive index detector (RID-10A) Photodiode Reference W Lamp Sample

Ionization in HPLC/MS

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