HPLC Theory Flashcards by Clodagh cultra

What is the mobile phase?

-Liquid solvent that flows through the column carrying the analytes
-controls retention time
-separates compounds based on interactions with stationary phase

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What is the role of the degasser?

Filtration of mobile phase to remove particulates and dust

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What is the injection?

-Manual injection
-Hand-operated valve
-Sample loaded into fixed-volume loop
-Manually switched

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What is the autosampler?

-samples placed in vials
-Software controls injection volume and timing
-injects automatically

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What are the different detectors?

-Mass spec
-Diode array detector

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What can cause a fluctuating baseline?

Lamp needs replacing
-Mobile phase absorbing UV light

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What can cause low pressure?

-Air in HPLC column
-Column is leaking

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What can cause bad fluctuation greater than 1psi?

-Air in injector, pump and lines

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What are the stationary phase interactions?

-Polar
Dipole–dipole, hydrogen bonding
Important in normal phase

-Nonpolar
van der Waals
Important in reverse phase

-Ionic interactions
Attraction between charged species

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What is reverse-phase chromatography?

-stationary phase = non-polar
-C18 column

-polar compounds low retention time
-non-polar compounds long retention time

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What is normal phase chromatography?

-stationary phase made of silica with polar functional groups
-polar compounds long retention time
-non-polar compounds low retention time

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What are the 2 parts of the mobile phase?

-A = Aqueous
-B = Organic

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What is the aqueous phase of the mobile phase?

-Water
-Additives -control peak shape, pH, separation and MS

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What is the organic phase of the mobile phase?

-Organic solvents
-Less polar and help compounds move

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What occurs when changing the ratio of A and B in the mobile phase?

-Gradient run
-Starts as mainly water and increases organic solvent
-helps separate complex mixtures

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What happens when the ratio of A and B are fixed in the mobile phase?

-used for simple mixtures
-always 70%water 30%organic

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What do Gaussian peaks indicate?

-Efficient mass transfer
-Minimal tailing or fronting
-Easier intergration and better quantitation

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How do you calculate the Partition Co-efficient (K)?

k = Cs/Cm

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What are the values for the Partition Co-efficient (K)?

Cs = concentration of solute in SP
Cm = concentration of solute in MP
K = how much an analyte prefers the SP

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What does a high/low Partition Co-efficient (K) indicate?

Low K = quicker elution, prefers MP
High K = slower elution, prefers SP
Differences in K = separation

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What is the ideal isotherm?

-Linear relationship between analyte conc in the MP and SP
-Constant K
-No peak distortion

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What results from a constant K?

-solute partitions evenly
-minimised band broadening
-symmetric peak

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What results from a non-constant K?

-Column overload
-Peaks tail or front
-Resolution and quantitation suffer

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What is the retention factor (K’)?

Study These Flashcards

How long a compound is retained on the column

How do you calculate the retention factor (K')?

K' = tR - t0/t0

What are the values of the retention factor (K')?

tR = Retention time of analyte t0 = Dead time, time for unretained compounds to elute K' = how long an analyte is retained relative to an unretained compound

What is the other calculation for retention time (K')?

tR = t0 (1+K')

What does a small K' indicate?

-1 or less -weak interaction -elutes fast

What is the ideal K' time?

-1-5 -good separation

What does a high K' value indicate?

-strong interaction -takes too long -peaks broaden

What is the selectivity factor (a)?

how differently 2 compounds interact with the stationary phase

What is the formula for the selectivity factor (a)?

a = tR2/tR1 = K'2/K'1

What are the values for the selectivity factor (a)?

tR1 = retention time of early eluting peak tR2 = Retention time of later eluting peak

What does a larger selectivity factor (a) indicate?

-easier to separate the peaks

What is column efficiency (N)?

N = How efficiently a column separates compounds More N = better separation

What is the calculation for column efficiency (N)?

N = 16 (tR/Wb)2 or N=5.54(tR/Wh/2)2

What are the values for column efficiency (N)?

tR = retention time of peak Wb = peak width at baseline W1/2 = Peak width at half height

What is column efficiency (HETP)?

Height Equivalent of a Theoretical Plate -Lower HETP = narrow peak = better separation

What is the calculation for column efficiency (HETP)?

HETP = L/N

What are the values for column efficiency (HETP)?

L = length of column N = number of plates

What does the Resolution (Rs) of peaks depend on?

-Efficiency (N) -Selectivity (a) -Retention (k)

What is the calculation for Resolution (Rs)?

Rs = |N a-1 K' / 4a 1+K'

What is the other calculation for Resolution (Rs)?

Rs = 2(tR2-tR1)/ Wb1 + Wb2

What is the Resolution (Rs) value when 2 peaks are said to be resolved?

>1.5

What is Eddie diffusion?

-Each particle takes a diff path which varies in time -Leads to peak broadening

What is Longtitudinal diffusion?

Low mobile phase flow rate leads to more diffusion and more broadening

What is resistance to mass transfer?

-Particle interacts with SP, no longer moves in MP and interacts with SP at the same time -Causes peak broadening

What is the Van Deemter equation?

Gives optimum flow rate to get the highest efficiency of separation

What is the calculation for the Van Deemter equation?

HETP = A + B/ u + Cu

What are the values for the Van Deemter equation?

-A = Eddy diffusion -B = Longtitudinal diffusion -C = Resistance to mass transfer -U = Mobile phase velocity

How to optimise Resolution (Rs)?

-Change column chemistry -Change composition of MP -Change the flow rate -Change dimensions of column

What causes peak tailing?

-Too much sample saturates SP -Sample solvent polarity matches SP -Mobile phase pH too high

How do you calculate the tailing factor?

Tf = W0.05 / 2f W0.05 = width of peak 5% it's height f = distance between peak max and edge at W0.05

How do you correct peak tailing?

-Reduce concentration or injection volume -Lower organic content of sample solvent -Replace column -Lower pH of MP

What causes peak fronting?

-Too much sample -Sample solvent polarity matches SP -Low column temperature -Column deterioration -MP pH too high

How do you correct peak fronting?

-Reduce conc or injection volume -Increase organic content of sample solvent -Increase column temp -Lower pH of MP

What causes peak splitting?

-MP pH too high -Sample solvent incompatible with MP -Dirty guard column -Column inlet contamination

How to correct peak splitting?

-Lower pH of MP -Use MP as sample solvent -Wash column with solvent or replace

HPLC Theory Flashcards

(58 cards)