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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.

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slide2

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.
slide3

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

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

slide5

TLC vs High Performance Liquid Chromatography (HPLC)

HPLC Optimization

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

slide6

HPLC – Optimizing Separation

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

slide8

HPLC - Resolution

  • Resolution (RS) of a column provides a quantitative measure of its ability to separate two analytes

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

Rs =

slide9

HPLC - Resolution

Rs

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

slide10

HPLC - 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 (a): Also called separation or selectivity coefficient is defined as
  • a = 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, 4th ed. Suanders, 1992

slide13

HPLC - 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 (tR/W)2)

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

slide15

HPLC - 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 (tR/W)2)

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

slide17

HPLC - General Elution Problem

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

slide18

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

(TLC vs Normal Phase and Reverse Phase HPLC)

slide19

Reverse Phase HPLC

Skoog and Leary: Principals of Instrumental Analysis,

5th ed. Suanders, 1998

slide20

Normal Phase vs. Reverse Phase HPLC

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

slide21

RP-HPLC – Stationary Phase

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

slide22

RP-HPLC – Mobile Phase vs k’

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

slide23

RP-HPLC – Mobile Phase (k’, a)

Skoog and Leary: Principals of Instrumental Analysis,

5th ed. Suanders, 1998

slide24

RP-HPLC – Mobile Phase (a)

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

slide25

RP-HPLC - Example

Alltech Chromatography Sourcebook, 2004-04 catalog

slide26

RP-HPLC - Optimization

Alltech Chromatography Sourcebook, 2004-04 catalog

slide27

RP-HPLC – Gradient Elution

Alltech Chromatography Sourcebook, 2004-04 catalog

slide30

HPLC – Resolution vs Column Efficiency (N, H)

H = L / N

van Deemter Equation

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

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

slide31

HPLC - Column Efficiency

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

slide32

HPLC - Column Efficiency

van Deemter Equation

H = A + B/u +Cu

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

slide33

HPLC - Column Efficiency

H = A + B/u + Cu

A = 2l dp

  • l depends on particle size distribution, the narrower the distribution the smaller the l
  • dp = particle size
  • Independent of mobile phase flow rate
  • Also known as eddy diffusion

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

slide34

HPLC - Column Efficiencyparticle size

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

slide35

HPLC Column EfficiencyLongitudinal Diffusion (B)

H = A + B/u + Cu

B/u = 2gDM/u

  • g = constant depending on quality of packing
  • DM is the mobile phase diffusion coefficient
  • Inversely related to mobile phase flow rate
slide36

HPLC Column EfficiencyMass Transfer (Cs + Cm)

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

CS = fS(k’)df2/ DS

CM = fM(k’)dp2/ DM

  • DM is the mobile phase diffusion coefficient
  • DS is the stationary phase diffusion coefficient
  • df is film thickness
  • dp is particle size
  • Directly related to mobile phase flow rate

Skoog and Leary: Principals of Instrumental Analysis,

5th ed. Suanders, 1998

slide37

RP-HPLC – Variables

Alltech Chromatography Sourcebook, 2004-04 catalog

slide38

HPLC OF ANALGESICS - UV Detection

Standard Analgesics

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/min

UV detector set at 240 nm

2.82 min

Acetaminophen

1.48 min.

Aspirin

7.11 min.

Caffeine

1.35 min.

Ibuprofen

slide39

HPLC OF ANALGESICS - UV Detection

Area %

Aspirin 19.5%

Acetaminophen 50.0%

Caffeine 20.5%

Excedrin ES

250 mg aspirin

250 mg acetaminophen

65 mg caffeine

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

slide40

HPLC: Peak Area vs

Detector setting

UV Max

Aspirin 225, 296 nm

Acetaminophen 248 nm

Caffeine 272 nm

Area %

Aspirin 19.5%

Acetaminophen 50.0%

Caffeine 20.5%

Detector set at 240 nm

Detector set at 254 nm

Area %

Aspirin 7.3%

Acetaminophen 81.9%

Caffeine 10.8%

Detector set

at 280 nm

Area %

Aspirin 24.8%

Acetaminophen 39.3%

Caffeine 35.9%

slide41

HPLC – UV Detection

  • 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.
  • Detector set at 420 nm.
  • Detector set at 254 nm.
  • Detector set at 254 nm (0-3.5 min), 420 nm 3.5-15 min.

(A)

(B)

(C)