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HPLC

Liquid Chromatography

ÖZET

The original development of HPLC used higher pressuresthan previously used ----High Pressure Liquid Chromatography

However, over the years the preferred term has become:

High Performance Liquid Chromatography

Advantages of HPLC

High resolution Speed Re-usable columns Great reproducibility Control of physical parameters

flow rate, polarity, packing efficiency, and particle size. Easy automation of instrument and data analysis.

HPLC Chromatograph of Muscadine Grape Juice

SOLVENTS

Includes both liquid phaseand solid materials (Buffers) dissolved in the liquid.

•Solvent properties affecting detection•Solvent properties affecting separation•Solvent properties affecting flow

•Viscosity•Miscibility

UV Cutoff -Solvent may interfere with detection

For peptide analysis UV = 215 nm. Solvents thatabsorb UV at this wavelength would not be goodcandidates for the mobile phase.

Refractive Index of Solvent vs Sample forRefractive Index detection (Carbohydrates)

Volatility needed for HPLC Mass Spectrometry(trifluoroacetic acid is a typical volatile buffer)

Solvent Properties Affecting Detection

BUFFERS

1)Buffers are needed to control the pH differences caused bythe sample matrix.

2)Buffers are used to control the ionization of compoundsand therefore their retention by the column.

Retention Time and pH in Reversed Phase

3 4 5 6 7 8 9pH

Rel

ativ

e R

eten

tio

n T

ime

pKa

partially charged

fully charged

not charged

When an acid or a base is ionized it becomes much less hydrophobic and will elute much earlier. Acids lose a proton and become ionized (negative charge) as pH increases. Bases on the other hand, gain a proton and acquire a positive charge as pH decreases.

BasicCompound

SOLVENT SELECTIVITY

The less time a compound spends in the stationary phase, the faster it will move through the column (less retention time).

If two compounds are added to the column, the ratio of theirretention times is called the selectivity.

The higher the selectivity, the better the separation.

Selectivity can be increased by adjustment of the mobile and stationary phases.

Solvent Selectivity TriangleRepresenting 3 “Polarity” factors 1) Each dot in the triangle

represent a different solvent2) Solvents can be groupedbased on their type of polarity3) Solvents and solvent mixturesare available for just about anyseparation you may desire.

Viscosity - resistance to flow

Difficult to force high viscosity solvents throughthe column.

Mixing solvents can drastically change viscosity

Viscosity of Water-Organic Solvent Mixtures

Viscosity vs. PressureThe higher the solvent viscosity, the harder it is for the

solvent to move through a column, and the more pressure inrequired to move the solvent at a specific velocity. The pressurerequired to move a solvent through a column can be estimated bythe following formula:

P = 250 L F / Dp2 Dc

2

Where P = pressure drop in psi. F = flow rate (mL/min)L = column length (cm) Dp = particle diameter (m)= solvent viscosity (cP) Dc = column diameter (cm)

P = 250 L F / Dp2 Dc

2

EXAMPLE

column length = 15 cm, column diameter =.5 cm, particle diameter = 5 m, flowrate = 2.0 mL/min

For water n = 1.0 250 x 15 x 1.0 x 2 / 52 x .52 = 7125/6.25 = 1200 psiFor methanol n = 0.54 250 x 15 x .54 x 2 / 52 x .52 = 2025/6.25 = 648 psiFor 60% water n = 1.9 250 x 15 x 1.9 x 2 / 52 x .52 = 7125/6.25 = 2280 psi 40% methanol

SOLVENTS

Water 190 1.00 10.2 ---Methanol 205 0.55 5.1 YTetrahydrofuran 212 0.55 4.0 YPropanol 210 2.3 4.0 YAcetonitrile 190 0.38 5.8 YHexane 195 0.31 0.1 NEthyl Acetate 256 0.45 4.4 NChloroform 245 0.57 4.1 N

UVCutoff Viscosity Polarity

Misciblewith

Water?

– ToxicityToxicity

– FlammabilityFlammability– Reactivity Reactivity solvent should not react with samplesolvent should not react with sample

– Cost Cost – Disposal Disposal can be more than purchase costcan be more than purchase cost

Peripheral Properties

Geometry of HPLC Columns

DiameterLengthParticle Size

What is the effect on pressure?

P = 250 L F / Dp2 Dc

2

Where P = pressure drop in psi. F = flow rate (mL/min)L = column length (cm) Dp = particle diameter (m)= solvent viscosity (cP) Dc = column diameter (cm)

Geometry of HPLC Columns

DiameterLengthParticle Size

What is the effect on Theoretical Plates?

What is the effect of column geometryon Theoretical Plates?

N=L/H where N is number of plates, H is plate height

and L is Column Length

Remember that separation is best on columns with highnumber of theoretical plates.

Therefore, doubling the column length will double Nbut this will double analysis time and pressure!

What is the effect of column geometryon Theoretical Plates?

Decreasing column diameter by half

halving the column diameter can also increase N slightly

For comparison purposes, let’s keep the mobile phasevelocity constant. Therefore, flow would be reduced 4Xand analysis won’t take any longer!

This reduces the amount of solvent used by 4X but alsoreduces the amount of sample that can be injected by 4X.

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What is the effect of column geometryon Theoretical Plates?

Decreasing particle size by half

However, halving the particle size can double N

Will increase pressure by 4X

Decreasing particle size and making the column half as long will keep N the same but cut sample time in half and solventuse in half.

In general small diameter columns withsmall particles are best for rapid separation,

….but require higher pressures, smaller samples, and can plug easier.

The problem with plugging should not be underestimatedand care should be exercised in keeping the sample, mobilephases, and columns CLEAN!