Clinical Chemistry (CHE221) Professor Hicks Lecture 12 Chromatography chromatography technique to separate mixtures each peak a different substance stationery phase = material mixtures comes in contact with that does not move mobile phase liquid or gas that washes mixture out of stationery phase Relative Response a chromatogram Retention Time (min) normal phase chromatography like a tug of war components of mixture stronger forces slower weaker forces faster mobile phase stationery phase components of mixture with stronger intermolecular forces stick better move slower raising temp (gas chromatography) moves all components faster 1
paper chromatography 1) mixture added to a solid material = stationary phase 2) washed out or eluted with a liquid solvent = mobile phase components elute (move up paper) at different rates paper chromatography movie keep a lid on it with no lid separation is poor with no lid the solvent evaporates fastest moving spots slow down towards top slower moving spots catch up elution takes much longer more left-right spreading out occurs eluted 90 minutes eluted 180 minutes 2
column chromatography larger amounts than paper chromatography mobile phase or eluting solvent mixture column of stationary phase separated components in eluting solvent width at half height describes how wide a peak is similar to standard deviation easy to measure graphically does not assume bell shaped curve ½ peak s height Resolution in order to identify peaks they must be resolved cannot overlap or almost overlap resolution = how close two peaks can be and still be distinguished 2 factors affect resolution 1) width at half height for the peaks involved 2) difference in retention time for the peaks involved both factors depend on experimental conditions column materials, temperature program, gas flow rates 3
Gas Chromatogram Relative Response Time (min) Gas Chromatogram Relative Response Time (min) Gas Chromatography numerous commercially available stationery phases (GC columns) many specialized for separating classes of compounds mobile phase inert gas often He stationery phase is liquid layer on surface of solid column material requires tiny amounts sample 4
separation in GC rate of movement depends on samples volatility (boiling point) typical starting temperature + 30 C of lowest bp components most volatile (lowest boiling compounds move fastest sometimes temperature is ramped to get lower components to come out in sequence Gas Chromatography area under a peak is proportional to the amount of compound present if a standard is run areas can be compared to calculate amount in unknown sample areas of different peaks are roughly quantitative % mass - depends on similarity of compounds and detectors type s Flame Ionization (FID) based on increase in conductivity from ions formed in flame -10 6 more sensitive than TCD but destroys sample Thermal Conductivity (TCD) based on changes in thermal conductivity as the sample occupies space instead of reference gas - does not destroy samples so mass spec can be run in tandem with GC 5
Flame Ionization (FID) conduction of electricity requires charged particles charged particles formed when compound burned in a flame amount of compound proportional to conductivity of flame as sample is burned Best method for C,H compounds poor detection if halogens or O in compound H 2 and Air mixed with sample at detector Flame Ionization data output (chart recorder or digital data file) C urrent ( m illia m p s ) Current versus Time 10 8 6 4 2 0 0 20 Time (s) GC column Flame Ionization Thermal Conductivity universal detector - it works on widest range of compounds every compound has a thermal conductivity different from He detector compares thermal conductivity of gas stream with sample to gas stream without sample changes in thermal conductivity changes in conductivity changes in current that flows while sample is in detector 6
Thermal Conductivity GC column Thermal Conductivity gas flow filament heat flows out sample conducts heat better than He cooling filament increase in conductivity increase in current flow more current flows through Current ( m illia m p s ) Current versus Time 10 8 6 4 2 0 0 20 Time (s) Types of Columns Packed Columns coiled tube filled with solid wetted with a high boiling liquid - diatomaceous earth coated with phenyl methyl silicone polymer is common Capillary Columns narrow tubes ( 1 mm OD 0.25 mm ID) with walls coated with stationery phase 0.1-1 m thick - higher resolution than packed columns Temperature Programming best separation often achieved by raising temperature after injection higher temperature gets the higher boiling components moving faster programs can be very detailed to squeeze out all the components with different retention times 7