LECTURE 2

Advanced Separation Science Techniques Present and Future Separation Tools

Jack Henion, Ph.D. Emeritus Professor, Analytical Toxicology

Cornell University Ithaca, NY 14850

Lecture 2, Page 1

Contents

• HPLC

• HILIC

• Porous Graphitic Carbon

• UPLC

• Nano HPLC

• Capillary Electrophoresis (CE)

• Differential Mobility Spectrometry (DMS)

Lecture 2, Page 2

Schematic of HPLC System

Lecture 2, Page 3

The Resulting Chromatogram

tR

t0

minutes

wb

C

B A

Question: Is this an isocratic or gradient separation? What is the clue?

Lecture 2, Page 4

The Chromatographic Process

Least Retained Moderately Retained

Most Retained

Injected Sample Mixture

Lecture 2, Page 5

Retention and Differential Migration in LC

Mobile Phase

Equilibrium distribution of

compounds C and A

between stationary and

mobile phases

Cm Am

As Cs

S

Lecture 2, Page 6

Compound Polarity Differences Require Different Stationary Phases

‘Like Dissolves Like’ Choose a stationary phase that best

suits your sample mixture composition Lecture 2, Page 7

Comparison of Normal Phase vs.

Reversed-Phase Normal Phase

Reversed-Phase

Lecture 2, Page 8

The Five HPLC Modes Reversed-phase

ion exchange

ion pair

normal phase

size exclusion

Lecture 2, Page 9

HPLC

Pumping Systems

Injector

(Autosampler) Gradient Former

HPLC Pump(s)

HPLC Pump Characteristics: * Constructed of chemically inert materials. * Pulse-free and reproducible flow rate. * Flow rates from 0.05 mL/min to 2 mL/min. * Compatible with gradient elution. * Pressures up to 6000 psi. (400 bar).

Most common types of pumps: * Reciprocating pistons.

Key Issues: * Low pressure vs. high pressure mixing (helium sparging or no degassing). * Pulse dampner required which contributes delay to volume to gradient mixing. * Limitations regarding micro HPLC with gradients.

Lecture 2, Page 10

HPLC Column Dimensions

Lecture 2, Page 11

HPLC Column

HPLC Pump(s)

Injector

Detector

Column sizes: 4.6 mm i.d.: Flow = 1.0 mL/min 2.1 mm i.d.: Flow = 0.2 mL/min 1.0 mm i.d.: Flow = 0.050 mL/min 0.3 mm i.d.: Flow = 0.005 mL/min

0.075 mm i.d.: Flow = 0.0002 mL/min (200 nL/min)

Is the ‘condition’ of the bed packing important? Why Lecture 2, Page 12

Relationship Between Column Diameter and Particle Size

Lecture 2, Page 13

HPLC Resolution

Resolution is a function of three different factors:

1. Capacity factor, k’ 2. Column plate number, N. 3. Separation factor, alpha, or band spacing.

Changes in these three parameters are shown in figure.

Effect of different separation conditions on LC resolution (from J. W. Dolan and L.R. Snyder, “Troubleshooting LC/Systems”, Human Press, Clifton, NH 1989, p.97

Lecture 2, Page 14

Gradient HPLC System Two pumps and shorter run times

Lecture 2, Page 15

Key Parameters in HPLC Columns

• Column length – Long: (10-25 cm) gives more plates, more backpressure,

longer runs

– Short: (1-3 cm) gives fewer plates, lower backpressure, and short run times

• Column inside diameter (i.d.)

– Large: (4.6 mm and higher) employs higher mobile phase flow, reduced LC/MS sensitivity

– Small: (75 microns to 2.1 mm) employs lower mobile phase flow, higher sensitivity

Lecture 2, Page 16

Chromatographic Peak

Shape In Gradient Mode

HPLC

Contributed by: Tzipi Ben-Tzvi and Prof. Eli Grushka Institute of Chemistry The Hebrew University Jerusalem, Israel

Lecture 2, Page 17

Gradient

UA-velocity of mobile phase

U1=Ufront

U2=Urear

Lecture 2, Page 18

How Do We Characterize Peak Shape?

1.Tailing Factor = B/A

Lecture 2, Page 19

How Do We Characterize Peak Shape?

3

3/2

2( )

M

M2. Skew

Lecture 2, Page 20

Objectives

1. Comparing Peak shape in gradient and

isocratic separations

2. The dependence of peak shape on the time of gradient starts

3. The dependence of peak shape on the steepness of the gradient

Lecture 2, Page 21

Examples of peaks

-0.01

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

1900 1920 1940 1960 1980 2000 2020 2040 2060 2080 2100

Am

ou

nt

Transfer

Gradient starts after 570 transfers Skew=-0.34

-0.02

0

0.02

0.04

0.06

0.08

0.1

0.12

1000 1020 1040 1060 1080 1100 1120 1140 1160 1180 1200

Am

ou

nt

Transfer

Gradient starts after 30 transfers Skew=0.76

Lecture 2, Page 22

-0.01

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

1200 1700 2200

Am

ou

nt

Transfer

Grad start at 900

K=3 Skew2=0.259

K=1 Skew2=-1.16

K=0.5 Skew2=0.062

-0.01

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

1000 1500 2000

Am

ou

nt

Transfer

Grad start at 570

K=3 Skew2=-0.344

K=1 Skew2=-0.0262

K=0.5 Skew2=0.0432

-0.02

0

0.02

0.04

0.06

0.08

0.1

0.12

1050 1100 1150 1200

Am

ou

nt

Transfer

Grad start at 30

K=3 Skew2=0.706

K=1 Skew2=-0.134

K=0.5 Skew2=-0.032

Lecture 2, Page 23

Results – step gradient

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1842 1852 1862 1872 1882 1892 1902 1912

Am

ou

nt

Transfer

Gradient start after 900 transfers, Skew2=0.59

The chromatogram is in the limited range Lecture 2, Page 24

Conclusions 1. The peak shape gradient elution can be different than

in isocratic separations.

2. The peak shape is a function of the gradient starting point.

3. Step gradient influences the peak shape more than continuous gradient.

4. When the slope of the gradient is higher the absolute values of the skew are greater.

5. A more accurate picture about the peak shape is obtained with skew measured in the limited range.

Lecture 2, Page 25