The best application fields of various chromatographic modes GC Volatile, thermostable compounds LC Polar, non volatile. thermolabile EKC Ionic compounds
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 • 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 Methanol / water = 6 / 4 Long analysis time, low signal to noise ratio Methanol / water = 8 / 2 Poor separations (Column : ODS type)
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
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 thedepositionof matrixcomponents, 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 Poor connection Excellent connection
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 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..
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 OH C18 (ODS) 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
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.
Size excusion (gel) chromatography The voluminous molecules elute fast because they are excluded from the small diameter pores, therefore they interact in less extent.