Course goals: Course goals: Lecture 1 A brief introduction to chromatography. AM Quality parameters and optimization in Chromatography

Size: px

Start display at page:

Download "Course goals: Course goals: Lecture 1 A brief introduction to chromatography. AM Quality parameters and optimization in Chromatography"

Jared Welch
5 years ago
Views:

1 Emqal module: M Quality parameters and optimization in is a separation technique used for quantification of mixtures of analytes Svein.mjos@kj.uib.no Exercises and lectures can be found at Tswett's apparatus taken from his 1906 paper Michael Tswett Course goals: is today the most important analytical method in fields like: dicine There are many different forms of and chromatographic systems can be quite complex. The focus in this course will be basically on the chromatographic columns Injector Detector Pharmacy Food analysis Complex samples, many analytes in mixture Environmental studies Gas Liquid Course goals: There are large differences in the quality of the separations - Why? - How can we measure and optimize the separation? Lecture 1 brief introduction to 1

Course goals: t the end of the course you should be able to: Understand the basic concepts of separation (peak resolution, selectivity, efficiency) and which factors that will have influence on

Choose a suitable optimization strategy and apply the technique to optimize a system (e.g. experimental designs, simplex optimization).

2 Course goals: t the end of the course you should be able to: Understand the basic concepts of separation (peak resolution, selectivity, efficiency) and which factors that will have influence on separation in liquid and gas. Evaluate efficiency and selectivity of a chromatographic system using common parameters such as separation number, theoretical plates and peak capacity. Choose a suitable optimization strategy and apply the technique to optimize a system (e.g. experimental designs, simplex optimization). Use retention indices, relative retention, and other methods for standardization of chromatographic retention. In brief: Course goals: This course should help you to: Select the right columns in liquid and gas Select the right conditions for achieving optimal (or sufficient) chromatographic resolution Course contents: 1) Introduction & basic chromatographic principles 2) etention and phase distributions - Exercises 3) Efficiency, selectivity and resolution - Exercises 4) Band broadening - Exercises 5) Non-ideal conditions - Exercises 6) ptimization and monitoring Liquid/liquid Planar TLC (Normal phase) Paper Stationary phases Normal phase Densitometry Liquid efractive index Column Stationary phases UV/Vis ELSD Normal phase everse phase Ion exchange ther Fluorescense Mass spectrometry supercritical fluid Stationary phases Varying polarities Gas Column Flame ionization (FID) Mass spectrometry Electron capture (ECD) Thermal conductivity Liquid system Liquid system Flow mount Chromatogram 2

3 The principle Extraction is a separation technique that utilize different distribution between phases (like in extraction) Phase I Phase II Extraction Extraction Phase distribution of 20% 80% Dynamic equilibrium B Extraction Phase distribution of B Extraction Separation of a mixture B 50% 50% B (50% / 50%B) 3

4 Extraction Separation of a mixture B Extraction Separation of a mixture Start: 50% / 50%B Enrichment of B 29% 71% B Enrichment of 62% 38% B B Extraction leads to enrichment of and B relative to the other compound, but does not lead to complete separation Same analytes v = (20/100) v m 20% 80% Dynamic equilibrium 20% 80% Mobile phase Stationary phase The molecules moves with an average speed that is equal to the fraction of the molecules that is in the mobile phase multiplied with the speed of the mobile phase. Mobile phase (liquid) Mobile phase (liquid) B B Stationary phase (solid) The two phases and the two compounds have the same properties as in the extraction example. Stationary phase (solid) : B :

5 100% 100% B B B Complete separation of and B 80 Principle of separation 80 Principle of separation B B Same velocity Same velocity 80 Principle of separation 80 Principle of separation B B ll analytes travel with the same speed, and all analytes spend the same amount of time on the road. It It is the time spent in the stationary phase that determines how long time it it will take to get from to B. Spend more time in the stationary phase Spend less time in the stationary phase 5

80 Principle of separation The chromatogram has more affinity for the stationary phase than and will therefore spend more time in the stationary phase.

6 80 Principle of separation The chromatogram has more affinity for the stationary phase than and will therefore spend more time in the stationary phase. Spend more time in the stationary phase B Spend less time in the stationary phase Column mount etention time The chromatogram: etention time versus amount for each analyte The chromatogram The chromatogram The peaks are usually near gaussian (normal distribution) in shape. Chromatographic theory is based on gaussian peak shapes mount The amount of each compound is determined by the area of the peak mount etention time The chromatogram: etention time versus amount for each analyte The retention time gives information about the identity of the compound etention time The chromatogram: etention time versus amount for each analyte 21 (High Performance) Liquid (HPLC) Column with a liquid mobile phase and a solid stationary phase. The mobile phase is forced through the column by high pressure Liquid/liquid Planar TLC (Normal phase) Paper Stationary phases Normal phase Densitometry Liquid efractive index Column Stationary phases UV/Vis ELSD Normal phase everse phase Ion exchange ther Fluorescense Mass spectrometry supercritical fluid Stationary phases Varying polarities Gas Column Flame ionization (FID) Mass spectrometry Electron capture (ECD) Thermal conductivity 6

The equipment The column Typically 10-25 cm length and

6 mm The column Solvent flow Solvent flow Particles in

phase particle surrounded by mobile phase Solvent flow

(onepiece porous polymer) Usually silica ften with a

7 The equipment The column Typically cm length and internal diameter of mm The column Solvent flow Solvent flow Particles in column Solvent flow Solvent flow Solvent flow Stationary phase particle surrounded by mobile phase Solvent flow Solvent flow Usually particles, can also be monoliths (onepiece porous polymer) Usually silica ften with a modified surface Polar (normal phase) polar (reverse phase) Ion exchange ze exclusion Bio-affinity Traditional modes 7

Polar solid phases Polar solid phases Polar groups interact polar (organic) mobile phase lica, lumina Polar solid phases polar solid phases (eversed phase ) Normal phase Polar stationary phase Less

8 Polar solid phases Polar solid phases Polar groups interact polar (organic) mobile phase lica, lumina Polar solid phases polar solid phases (eversed phase ) Normal phase Polar stationary phase Less polar mobile phase Polar analytes are retained and have higher retention times than apolar analytes Polar groups interact Water-based mobile phase Polarity Particles are covered with apolar compounds (covalently bound to the particle) polar solid phases polar solid phases (eversed phase ) (eversed phase ) everse phase polar stationary phase Polar mobile phase (waterbased) polar analytes are retained and have higher retention times than polar analytes 8

polar solid phases polar solid phases (eversed phase ) (eversed phase ) Large number of

eversed phase separations are today the most common method.

of the phases can also be applied in the normal phase mode polar solid phases Ion exchange

ule of thumb: When separating compounds with different functional groups, use normal phase.

9 polar solid phases polar solid phases (eversed phase ) (eversed phase ) Large number of apolar phases The terms normal phase and reversed phase phase have historical origin. eversed phase separations are today the most common method. C18 (alkane, most common) Phenyl groups (pi-selectivity) Cyano (CN) groups mino groups Some of the phases can also be applied in the normal phase mode polar solid phases Ion exchange phases When to use normal phase and when to use reversed phase? ule of thumb: When separating compounds with different functional groups, use normal phase. When separating compounds with the same functional groups, use reversed phase. Ion exchange phases (eversed phase ) Ion exchange phases nion exchange phase Cations 9

10 Ion exchange phases Cation exchange phase nions Ion exchange phases Very useful for separation of ionized molecules, e.g. proteins and peptides The water-based mobile phase must be buffered ze exclusion phases ze exclusion phases Porous particle Small molecules can enter the pores ze exclusion phases Small molecules can enter the pores Large molecules are excluded Suitable for molecules above 2000 g/mol Small molecules have higher retention times than large molecules The solvents (eluents) 10

11 The solvents (eluents) Eluent strength (elution/solvent strength) For the same compound and the same stationary (solid) phase, a higher eluent strength will lead to higher portion of the analyte in the mobile phase. The solvents (eluents) Eluent strength (elution/solvent strength) Making the mobile phase more similar to the stationary phase will in general increase the eluent strength (Decreased polarity in reversed phase mode or increased polarity in normal phase mode increases eluent strength) Low eluent strength 20 % 80 % High eluent strength 50 % 50 % 20 % 80 % 50 % 50 % The solvents (eluents) Eluent strength (elution/solvent strength) Elution patterns typically follow an exponential function Too low eluent strength = too long time Too high eluent strength = too low resolution Solvent strength Detector response The solvents (eluents) Gradient elution: The first compounds eluted with low eluent strength, the last compounds eluted with high eluent strength Gradient elution is suitable for a large range of analyte properties Gas (GC) Liquid/liquid Liquid supercritical fluid Gas Column with a gas as mobile phase and a solid or liquid stationary phase. Planar TLC (Normal phase) Paper Stationary phases Normal phase Densitometry efractive index Column Stationary phases UV/Vis ELSD Normal phase everse phase Ion exchange ther Fluorescense Mass spectrometry Stationary phases Varying polarities Column Flame ionization (FID) Mass spectrometry Electron capture (ECD) Thermal conductivity 11

12 The principle of GC The columns are usually long narrow-bore open tubular columns where the stationary phase is coated on the walls The equipment Carrier gas (He / H 2 ) Injector Detector Mobile phase (Carrier gas) Capillary GC columns The mobile phase is a gas (Nitrogen, Helium, Hydrogen) is a viscous liquid, a polymer or solid particles Packed GC column The column in GC Length: typically m Cross-section Diameter: mm Packed columns in GC Used for gases and very volatile compounds. Because these have low volatility they are difficult to retain without a low phase ratio (V mobile phase / V stationary phase ) Low phase ratios are difficult to achieve with open tubular columns Phase thickness µm Capillary column Phase ratio, β = V m 0.25 diameter Typically: 250 V s film thickness (diameter 1000 times film thickness) Stationary phases in GC Stationary phases in GC is typically made of viscous polymers Stationary phases in GC Different -groups are used to vary the properties of the phases = thyl (CH 3 ) Ph = Phenyl (C 6 H 5 ) CN = Cyano (CN) Polysiloxane (silicone) Polyetylenglycol Polysiloxane (silicone) Ph CN Ph CN H CH 2 CH 2 CH 2 CH 2 H n n Ph CN 12

13 Stationary phases in GC Cross-linking is applied to increase thermal stability Stationary phases in GC The amounts of each analyte on a capillary GC column is typically in the ng to pg range Diameter: mm Phase thickness µm n The oven temperature The temperature in gas has the same rhole as eluent strength in liquid For the same compound and the same stationary (solid) phase, a higher eluent strength will lead to higher portion of the analyte in the mobile phase. The oven temperature Temperature effects Elution patterns follow an exponential function within a homologous series Too low temperature = too long time Mobile phase (Carrier gas) Low temperature Mobile phase (Carrier gas) High temperature Too high temperature = too low resolution The oven temperature The column in LC and GC Solvent strength Detector response Gradient elution: The first compounds eluted with low temperature, the last compounds eluted with high temperature Gradient elution is suitable for samples that have analytes with a large range of boiling points (e.g. crude oil) pen tubular Stationary phase coated on inner-wall of tube Basically GC (Particle) Packed Stationary phase coated on particles inside the column Basically LC Monolithic Semi-permeable one-piece packing Properties inbetween open tubular and packed Basically LC 13

14 Stationary phase interactions dsorption and absorption Partition (absorption) : nalytes are absorbed in the stationary phase Typical for GC, P-HPLC, HILIC ssumes unlimited availability of stationary phase dsorption : nalytes are adsorbed to the stationary phase Limited surface available. The analytes competes for the stationary phase Typical for NP LC on bare silica dsorption or absorption? eversed phase LC is usually regarded as absorption - The analyte is dissolved in the stationary phase - There is unlimited availability of stationary phase Normal phase LC is usually regarded as adsorption - The interaction with the stationary phase happens on the surface - There is limited surface available and competition for surface is a factor dsorption or absorption? With few exceptions GC is regarded as partition (absorption of analytes in the stationary phase) nalytes are dissolved in the stationary phase dsorption or absorption? Most methods are combinations of absorption and adsorption Much of the chromatographic theory is based on the assumption that absorption is the only significant effect (partition ) dsorption phenomena in absorption leads to non-ideal conditions (Lecture 5) 14

Chromatography. Gas Chromatography

Chromatography. Gas Chromatography Chromatography Chromatography is essentially the separation of a mixture into its component parts for qualitative and quantitative analysis. The basis of separation is the partitioning of the analyte mixture