8-9. Basics of LC

T/F? Particles packed into an HPLC column are typically 2-5 mm in diameter. (1.3i)

False; 2-5 um, not mm

T/F? Particles packed into an HPLC column are typically 2-5 um in diameter. (3.3i)

True

List 3 differences that affect the fundamental nature of chromatographic separations in LC vs GC (beyond the phases). (1.5)

(All listed here)

1: Diffusion of molecules in liquid is 10^5x lower than in gas thus slower partitioning

2: Viscosity of m.p. Is higher in HPLC and thus higher flow resistance and backpressure issues

3: Compressibility of liquids is negligible (this is more of an issue for GC) bc no P drop across column in HPLC (it is constant)

4: HPLC uses primarily packed columns, whereas GC primarily uses open-tubular capillary columns

5: In HPLC, the m.p. has a strong effect on analyte retention; composition of m.p. Is changed over course of run.

Create a block diagram of HPLC instrumental components and provide examples for m.p. composition, s.p. type, and detector to perform a reverse (2.7)

Diagram: 

Solvent reservoirs -> Pumps -> Injector -> Column -> Detector -> Data Station

Mobile Phase: Water (weak) + Methanol (strong)

Column: C18 bonded

Detector: UV-Vis or MS

Why is it desirable to develop a chromatographic separation method where the separated analytes are characterized by 2 < k’ < 10? (3.4d)

k’ is a capacity/retention factor. It characterizes the relative amount of time an analyte spends in the s.p. vs m.p.

Formula:

k’ = (tR-t0)/t0 or t’R/t0

k’ > 2 is desirable so that analytes are well retained past t0, where many unretained interferences can elute. k’ = 2 means analyte spent 2x as much time in s.p. as it did in m.p.

k’ < 10 is desirable to avoid subjecting analytes to excessive band broadening. Analytes retained longer in the column will experience a loss of efficiency. Also, separations will take too long if k’ > 10.

Why is it not practical to use a standard wall-coated open tubular capillary GC column to perform HPLC separations? (4.5)

The mass transfer in the m.p. would be too high. The relatively small film of s.p. on the wall of the capillary would not provide for significant interaction with molecules present in the bulk m.p. In order to perform open-tubular HPLC, you must use capillaries having much smaller internal diameter (e.g. 10 um).

How can the control of column temperature affect HPLC separations? (5.4)

A column oven is commonly used to hold the column at a constant temperature (e.g. 30C) in order to improve precision. Temperature of the column can be increased (e.g. 50-90C), and this increases the kinetics of the separation process. It also reduces the viscosity of m.p. solvents. Thus, separations can be made faster without major increases in back pressure. Overall, efficiency can be increased by increasing column T.

An “HP-5” or “Rtx-5” GC column contains a stationary phase that is 5% propyl-functionalized polydimethylsiloxane. (6.2d)

False; “phenyl”-functionalized polydimethylsiloxane