HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC). HIGH PERFORMANCE LIQUID CHROMATOGRAPHY High...

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY(HPLC)

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

• High Performance Liquid Chromatography (HPLC) is one of the most widely used techniques for identification, quantification and purification of mixtures of organic compounds.

• In HPLC, as in all chromatographic methods, components of a mixture are partitioned between an adsorbent (the stationary phase) and a solvent (the mobile phase).

• The stationary phase is made up of very small particles contained in a steel column. Due to the small particle size (3-5 um), pressure is required to force the mobile phase through the stationary phase.

• There are a wide variety of stationary phases available for HPLC. In this lab we will use a normal phase (Silica gel), although reverse phase (silica gel in which a 18 carbon hydrocarbon is covalently bound to the surface of the silica) columns are currently one of the most commonly used HPLC stationary phases.

http://www.chemistry.nmsu.edu/Instrumentation/Waters_HPLC_MS_TitlePg.html

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

http://www.labhut.com/education/flash/introduction07.php

TLC vs High Performance Liquid Chromatography (HPLC)HPLC Optimization

HPLC – Optimizing Separation

Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998

Schematic Presentation of a Chromatogram

HPLC - ResolutionHPLC - Resolution

• Resolution Resolution (R(RSS)) of a column provides a quantitative measure of its of a column provides a quantitative measure of its

ability to separate two analytesability to separate two analytes

Rs = Z /1/2(WA+WB)

Rs =

HPLC - ResolutionHPLC - Resolution

Rs

Skoog and Leary: Principals of Instrumental Analysis, 4 th ed. Suanders, 1992

HPLC - ResolutionHPLC - Resolution

Capacity Factor (k’): Also called retention factor. Is a measure for the position of a sample peak in the chromatogram.

k’ = (tR1-to)/to

• specific for a given compound and constant under constant conditions• A function of column and mobile phase chemistry• Primarily applicable under isocratic conditions• In general, a change in the k’ of one peak will move all peaks in the same direction.

Selectivity Factor (): Also called separation or selectivity coefficient is defined as

= k2’/k1’ = (tR2-to) / (tR1-to)

• A function of column and mobile phase chemistry• Primarily applicable under isocratic conditions• Changes in selectivity will affect different compounds in different ways.

Skoog and Leary: Principals of Instrumental Analysis, 4 th ed. Suanders, 1992

HPLC – Capacity Factor

HPLC – Selectivity Factor

HPLC - ResolutionHPLC - Resolution

Theoretical Plates (N): The number of theoretical plates characterizes the quality or efficiency of a column.

N = 5.54 [(tR) / w1/2]2 (N = 16 (t(N = 16 (tRR/W)/W)22))

Skoog and Leary: Principals of Instrumental Analysis, 4 th ed. Suanders, 1992

Phenomenex catalog, 1999

HPLC - ResolutionHPLC - Resolution

Theoretical Plates (N): The number of theoretical plates characterizes the quality or efficiency of a column.

N = 5.54 [(tR) / w1/2]2

(N = 16 (t(N = 16 (tRR/W)/W)22))

Plate Height (H): The height equivalent to a theoretical plate (HEPT = H)

H = L / N

Resolution (Rs) depends on the number of theoretical plates:

Rs =

Skoog and Leary: Principals of Instrumental Analysis, 4th ed. Suanders, 1992

Skoog and Leary: Principals of Instrumental Analysis, 4 th ed. Suanders, 1992

HPLC - General Elution ProblemHPLC - General Elution Problem

Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998

x

a

b

c

a b c

c b a

0

0

Time

Time

Normal Phase (SiO2)

Reverse Phase (C18)

Normal Phase (SiO2) TLC

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY(TLC vs Normal Phase and Reverse Phase HPLC)

Skoog and Leary: Principals of Instrumental Analysis, 5th ed. Suanders, 1998

Reverse Phase HPLC

Normal Phase vs. Reverse Phase HPLC

Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998

RP-HPLC – Stationary Phase

Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998

RP-HPLC – Mobile Phase vs k’

Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998

Skoog and Leary: Principals of Instrumental Analysis, 5th ed. Suanders, 1998

RP-HPLC – Mobile Phase (k’, )

RP-HPLC – Mobile Phase ()

Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998

RP-HPLC - Example

Alltech Chromatography Sourcebook, 2004-04 catalog

RP-HPLC - Optimization

Alltech Chromatography Sourcebook, 2004-04 catalog

RP-HPLC – Gradient Elution

Alltech Chromatography Sourcebook, 2004-04 catalog

Allt

ech

Ch

rom

ato

grap

hy S

ourc

eboo

k, 2

004-

04 c

ata

log

Alltech Chromatography Sourcebook, 2004-04 catalog

HPLC – Resolution vs Column Efficiency (N, H)HPLC – Resolution vs Column Efficiency (N, H)

van Deemter Equation H = A + B/u +(Cs + Cm)u

H = L / N

Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998

HPLC - Column EfficiencyHPLC - Column Efficiency

Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998

van Deemter Equation H = A + B/u +Cu

HPLC - Column EfficiencyHPLC - Column Efficiency

Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998

HPLC - Column EfficiencyHPLC - Column Efficiency

H = H = AA + B/ + B/uu + C + Cuu

A = 2A = 2 d dpp

1.1. depends on particle size distribution, the depends on particle size distribution, the narrower the distribution the smaller the narrower the distribution the smaller the

2.2. ddpp = particle size = particle size

3.3. Independent of mobile phase flow rateIndependent of mobile phase flow rate

4.4. Also known as eddy diffusionAlso known as eddy diffusion

Skoog and Leary: Principals of Instrumental Analysis, 5th ed. Suanders, 1998

HPLC - Column EfficiencyHPLC - Column Efficiencyparticle sizeparticle size

Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998

HPLC Column EfficiencyHPLC Column Efficiency

Longitudinal Diffusion (B)Longitudinal Diffusion (B)

H = A + H = A + B/B/uu + C + Cuu

B/u = 2B/u = 2DDMM/u/u

1. = constant depending on

quality of packing

2. DM is the mobile phase diffusion coefficient

3. Inversely related to mobile phase flow rate

HPLC Column EfficiencyHPLC Column Efficiency

Mass Transfer Mass Transfer (Cs + Cm)

H = A + B/u + (Cs + Cm)u

CS = fS(k’)df2 / DS

CM = fM(k’)dp2 / DM

• DDMM is the mobile phase is the mobile phase

diffusion coefficientdiffusion coefficient

• DDSS is the stationary phase is the stationary phase

diffusion coefficientdiffusion coefficient

• ddff is film thickness is film thickness

• ddpp is particle size is particle size

• Directly related to mobile Directly related to mobile phase flow ratephase flow rate

Skoog and Leary: Principals of Instrumental Analysis, 5th ed. Suanders, 1998

RP-HPLC – Variables

Alltech Chromatography Sourcebook, 2004-04 catalog

1.35 min.Ibuprofen

7.11 min.Caffeine

1.48 min.Aspirin

2.82 minAcetaminophen

Analgesic Retention

Time

Acetaminophen 2.82

Aspirin 1.48

Caffeine 7.11

Ibuprofen 1.35

Gradient = 0 min: 100% EtOAC (+ 0.2% HOAc) 3 min: 100% EtOAC (+ 0.2% HOAc) 5 min: 15% MeOH, 85% % EtOAc (+ 0.2% HOAc) 8 min: 15% MeOH, 85% % EtOAc (+ 0.2% HOAc) 10 min: 100% EtOAC (+ 0.2% HOAc)SiO2

Flow Rate = 1 mL/minUV detector set at 240 nm

HPLC OF ANALGESICS - UV Detection

Standard Analgesics

Question

The peak areas of aspirin and acetaminophen are very different, even though they are present in equal amounts (250mg/tablet) in Excedrin ES.

Caffeine is present at ~ ¼ the concentration of aspirin (65 mg/tablet vs. 250 mg/tablet), but it’s peak area is greater than the peak area of aspirin.

WHY? UV Absorbance of analgesics vs UV setting of detector

Area %Aspirin 19.5%Acetaminophen 50.0%Caffeine 20.5%

Excedrin ES250 mg aspirin250 mg acetaminophen65 mg caffeine

HPLC OF ANALGESICS - UV Detection

Detector set at 240 nm

Detector set at 254 nm

Detector set at 280 nm

UV MaxAspirin 225, 296 nmAcetaminophen 248 nmCaffeine 272 nm

Area %Aspirin 19.5%Acetaminophen 50.0%Caffeine 20.5%

Area %Aspirin 7.3%Acetaminophen 81.9%Caffeine 10.8%

Area %Aspirin 24.8%Acetaminophen 39.3%Caffeine 35.9%

HPLC: Peak Area vs Detector setting

Figure 2. HPLC (SiO2) of crude tumeric extract.

Gradient 0-2 min, 4% EtOAc/Hexane; 2-9 min 4 to 80% EtOAc;

9-11 min , 80% EtOAc/hexane; 11-13 min, 80 to 4% EtOAc/hex, 13-15 min, 4% EtOAc /hexane.

(A) Detector set at 420 nm. (B) Detector set at 254 nm. (C) Detector set at 254 nm (0-3.5 min), 420

nm 3.5-15 min.

(A)

(B)

(C)

HPLC – UV Detection