Chemistry Instrumental Analysis Lecture 25. Chem 4631

Transcription

1 Cheistry 4631 Instruental Analysis Lecture 25

2 History - Chroatography Originally the separation of color (in plant pigents) First deonstrated in 1906 by Michael Tswett (Russian botanist) used a colun of crushed chalk and poured leave extracts into it and separated color fractions (chlorophyll and carotene). Taylor and Urey 1938 developed ion-exchange chroatography to separate lithiu and potassiu isotopes on zeolites. Martin and Synge 1941 developed partition chroatography. They developed paper chroatography to separate aino acids using ninhydrin spray for detection.

3 History Chroatography, con t Jaes and Martin 1952 developed gas-liquid chroatography, separated aines and carboxylic acids using a gaseous obile phase. McWillia, Dewar and Harley Gas chroatography (GC) becae universally accepted with the invention of flae ionization detector. High perforance liquid chroatography was developed in sall stages by several researchers in the late 1960 s.

4 General Principles: Definition IUPAC Definition of Chroatography A ethod, used priarily for separation of the coponents of a saple in which the coponents are distributed between two phases, one of which is stationary while the other oves. The stationary phase ay be a solid, liquid supported on a solid, or a gel. The stationary phase ay be packed in a colun, spread as a layer, or distributed as a fil. The obile phase ay be gaseous or liquid. [Notice that the definition neglects supercritical fluid chroatography (SFC)].

5 General Principles:Classification Methods Classify by Mobile Phase Three areas: Liquid Chroatography (LC) Gas Chroatography (GC) Supercritical Fluid Chroatography (SFC) Further classify by specifying stationary phase, i.e. gas-solid, gas-liquid, etc. For LC: A nonpolar obile phase with polar stationary phase is called noral-phase chroatography. A polar obile phase with nonpolar stationary phase is called reversed-phase chroatography

6 General Principles:Classification Methods Classify by Technique Colun or Planar (TLC)

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8 General Principles:Classification Methods Classify by Separation Mechanis Adsorption solute and obile phase olecules copete for active sites on the surface of the solid stationary phase (the adsorbent). Partition partitioning of a solute between two iiscible liquids but one liquid is held stationary on a solid support. A solute in contact with two iiscible liquids will distribute itself between the according to its distribution coefficient, K. Ion-exchange Size exclusion physical sieving process (gel pereation is a size exclusion process). Affinity stationary phase is a bioactive liquid bonded to solid support. Antibody-antigen interactions, cheical, chiral, etc. Micellar or pseudophase odifier added to obile phase surfactants.

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10 Behavior of Solutes in Coluns: Paraeters Defined Reflects the distribution of a solute between the obile and stationary phases.

11 0 sec-injection Point M - Unretained Solute Peak A,B - Solute Peaks (Analyte) V r - Retention Volue-the volue of obile phase necessary to convey a solute band fro the point of injection, through the colun, to the detector. t r - retention tie of analyte to ove fro injection to detector V retention volue of the unretained solute t retention tie of unretained solute V r - adjusted retention volue t r - adjusted retention tie

12 Notice the retention tie an analyte spends in the syste is coposed of two coponents: t r and t t - is the tie it takes a solute to pass through the space occupied by the obile phase (dead space, colun void volue). t is the sae for all analytes in a given syste the solutes igrate with the sae velocity of the obile phase. t represents no separation process. The adjusted retention tie, t r - represents the tie the analyte spends retained by the stationary phase. t r represents the separation process or interaction of analyte with stationary phase.

13 Partition Coefficient, Distribution Coefficient K concentration of the analyte in the stationary and obile phase K C C C s concentration of the analyte in stationary phase C concentration of the analyte in obile phase K = 1 when the analyte is distributed equally s

14 Capacity Factor, Partition Ratio, k k solute partition ratio is an iportant paraeter routinely used in HPLC but not as uch in GC. k the ratio of the total aount of a solute in the stationary phase to the aount in the obile phase at equilibriu. k ' s

15 Relationship between k and K V s volue of the stationary phase s s s s s V V V V C C K / / s V V k K '

16 K k ' V V s The ratio V / V s is the phase ratio called β and is used to characterize coluns, especially in GC. β is a easure of the openness of the colun. K = k β relates the equilibriu distribution of the analyte within the colun to the therodynaic coefficient, K.

17 This probability can be related to tie, which is easier to easure. k ' t r t t t t ' r In practice, accurate easure of colun void volue is difficult in HPLC (difficult to find arker copounds), but for GC the practice is siple and accurate.

18 Retention Tie k' t r t t rearrange t r = t (1 + k ) and substitute for k K t r = t (1 + K V s / V ) V V k ' k ' K s V V s

19 Average linear obile phase velocity is u u = L / t L length of the colun t r L u ( 1 KV / V s ) equation used to predict the effect of change in colun length, linear velocity of obile phase, phase ratio and distribution coefficient on retention tie of an analyte. equation is only valid when the obile phase velocity is constant throughout the colun.

20 Colun Efficiency One goal of chroatography is to achieve sharp syetrical peaks, thus optiizing analyte separation and iproving detection. The sharpness of the peak represents the efficiency of the chroatographic colun (actually the entire syste). Two general approaches have been developed to easure colun efficiency plate theory and rate theory.

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22 Plate Theory Models a chroatographic colun as a series of narrow, discrete sections called theoretical plates. Assue that at each plate equilibriu of the analyte is established between obile and stationary phase. Moveent of analyte and obile phase is viewed as a series of transfers fro one plate to the next. Efficiency of a colun increases as the nuber of equilibrations (i.e. theoretical plates) increases.

23 Plate Theory Efficiency of a colun increases as the nuber of equilibrations (i.e. theoretical plates) increases. n - # of theoretical plates or plate nuber n theor easures the colun efficiency n t 5.54( w r 1/ 2 ) 2 (assues a Gaussian peak) tr n 16( w b 2 ) n is a diensionless quantity

24 Plate Theory N or N eff or n eff Effective plate nuber or nuber of effective theoretical plates. Used especially if void volue is large or for early eluting peaks. t t w ' r N 5. 54( 2 ) 5. 54( r 2 ) 1/ 2 t w 1/ 2 2 ' r r ( t t ) t N 16( 16( ) w w b b 2

25 Plate Theory If the peak is asyetrical then calculation of n is ore coplex. N A ( tr / w0. 1 s ) 2 (for asyetrical peak) A s asyetry factor w 0.1 peak width at 10% of peak height

26 Plate Theory Plate nuber and effective plate nuber depend on the length of the colun, so other paraeters have been developed. h or hetp or h etp the height equivalent to a theoretical plate or plate height h L n L length of the colun

27 Plate Theory H or HETP the effective plate height or height equivalent to an effective theoretical plate. H L N

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29 Assignent Hoework Chapter 23 Due Today Hoework Chapter 25: 1-8 and 10 Hoework Chapter 25 due 04/04/18 Read Chapter 26 Hoework Chapter 26: 1-17 HW Chapter 26 Due 4/06/18 Test 4 4/11/18

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