Electrophoresis for teaching

2.  Principle
 Factors affecting the distance of movement
 Application
 Polyacrylamide Gel Electrophoresis (PAGE)
 Hemoglobin Electrophoresis

3. Electrophoresis is a process
distinguishing and isolating different
compounds from each other.
It relies on the fact that charged
particles (molecules) can migrate in a
medium if the medium is subjected
to an electrical current.

5. 1- Net charge on the molecule:
Particles with negative net charges move toward the anode (positive
pole) where as particles with positive charges migrate toward the
cathode (negative pole).
2- Size and shape of the molecule:
Particles of identical net charge will be distinguished from each other
by their size. Heavier molecules will move slower than lighter ones.
3- Strength of the electrical field:
The higher the electrical current voltage the further distance travelled
and the faster the speed of the movement.
4- Supporting medium physical and chemical nature:
Some compounds need special medium, e.g., large polypeptides or
proteins are done in polyacrylamide gel where as nucleotide
oligomers are done in agarose and polyacrylamide gel.
5- Electrophoretic temperature:
Optimal temperature for migration must be used.

6.  Electrophoresis could be implemented on many
charged molecules:
Amino Acids, Polypeptide Chains, Proteins,
nucleotide oligomers, RNA, DNA, Phosphorus
sugars and any other ampholytes (molecules
whose net charge depends on the pH of the
surrounding medium).
 The medium and voltage power might change
from a compound to another depending on the
compound chemical nature and size.

7.  1- The identification of certain
molecules.
 2- The isolation of a certain
molecule.
 3- The molecular weight of
certain molecules.

8.  Used routinely in the analysis of single stranded
and double stranded DNA.
 Polyacrylamide is cross linked with TEMED to
form a porous gel, thus allowing movement of
DNA molecules.
 Separation of DNA is based on size.
For example DNA bands made of 1000-2000
base pairs (bp) can be resolved in 3.5% acrylamide
(W/V) where as bands of 6-100 bp are resolved
using a 20% acrylamide (W/V).
 Visualization of the bands could be done by
adding a dye such as Ethidium Bromide before
or after electrophoresis. Alternatively,
radioactively labeled DNA can be visualized by
autoradiography (X-ray film).

9.  The gel could be of a denaturing or
non-denaturing property.
 Denaturing polyacrylamide gels are
used mainly for sequencing of DNA
where as non-denaturing ones are
used to detect mutations.
 An even better and more sensitive
technique is "Denaturing-Gradient Gel
Electrophoresis". This technique is
sensitive enough to detect a single
base mutation out of a several
hundred long base pairs of DNA.

10. Principle
 Hemoglobin (Hgb) migrates according to net
charges of its constituent proteins and
polypeptide chains.
 Various types of hemoglobin can be
distinguished from one another according to
their movements. Some types of hemoglobin
might migrate identically therefore
manipulating pH can result in different
movements of such hemoglobins (Hgb's).

11. 1- Cellulose Acetate:
Performed under alkaline pH = 8.4 – 8.6.
2- Citrate Agar:
Performed under acidic pH = 6.0 – 6.2.
3- Globin Chain Electrophoresis:
Globin chains are separated from Hgb allowing individual
electrophoresis of the alpha and non-alpha chains.
4- Isoelecteric Focusing (IEF):
The pH of this technique varies according to the
constituents of the molecule. It could be between 3 and 10.
 The first two are used routinely especially in the diagnosis
of the common hemoglobin variants (hemoglobinopathies)
and Thalassemia. The latter two are used in special cases
when the first two techniques could not distinguish the
abnormal hemoglobin.

13. CAPILLARY ELECTROPHORESIS:
TECHNIQUE AND APPLICATION

14. System Schematic
Anode
+
Cathode
-
Applied Electric Field.
1000 Volts / Centimeter
Velocity = (Field Strength) (Electrophoretic Mobility)
Detector (UV, PDA or LIF)

15. Capillary Electrophoresis

16. Development of Capillary
Electrophoresis
• 1803 F.F. Reuss Clay Slab
• 1886 O. Lodge Zone Electrophoresis
• 1937 A. Tiselius Electrophoretic Cell
• 1967 S. Hjerten Rotating tubes (300
um)
• 1970 V. Neuhoff PAG filled
tubes
• 1979 Mikkers, Everaerts, Verheggen FZE
• 1981 Jorgenson and Lukags 75 um

17. Electroosmotic Flow
http://www.electrokinetic.co.uk/images/tech1.gif

18. Movement of Analyte
Analyte
 ν = µ E
 ν = velocity µ = electrophoretic mobility E = Electric field
 Electrophoretic mobility
 µ = q/[6πηr]
 q = charge η = solution viscosity r = radius
Electroosmotic flow
 νEOF = [ε/4πη]ζE
 ε = dielectric constant ζ = Zeta potential
Flow of migration
ν = [(μEO + μe)V]/L
V = potential L = length of capillary
Forensic Science International
77 (1996) 211 - 229

19. Injection of Sample
Current Analytical Chemistry. 2005, 1
http://www.calstatela.edu/dept/chem/gomez/pubs-pdf/flow-injection.pdf

20. Injection of Sample
• Injection is difficult due to sample size
• Electrokinetic Injection
– Differs by analyte
• Hydrodynamic
– Many parameters
Anal. Chem., 1997, 69 (15), pp 2952–2954

21. Injection of Samples
Anal. Chem.2001, 73,1974-1978

22. Injection of Sample
Current Analytical Chemistry. 2005, 1
http://www.calstatela.edu/dept/chem/gomez/pubs-pdf/flow-injection.pdf

23. Capillary Zone Electrophoresis
Separated by mass to charge ratio
Based on Electroosmotic Flow
Detectors:
 UV Detector – Beer’s Law
 Laser Fluorescence – Deriv.
 MS - electrospray
 Chemiluminescence
 Diode Array Detector
 Indirect
 Refractive Index
Compare with HPLC and GC
Neutral Compounds
Chiral Compounds

24. Increasing Path Length
http://www.chem.agilent.com/Library/technicaloverviews/Public/5989-9808EN.pdf

25. Beckman P/ACE System

26. Electrophoretic Mobility
Z
Uep = ---------------
6 n r
Where:
Z = Net charge on the analyte
n = Viscosity of the medium
r = Stoke’s Radius

27. Variables Governing Electrophoretic
Mobility
• Charge
• Radius*
• Mobility
*The Stoke’s radius is
related to molecular
mass. Heavier
molecules will have a
greater radius.
0
1
2
3
4
5
6
1 2 3 4 5
Charge
Radius
Viscosity

28. Electroosmotic Flow
(EOF)
This flow is a phenomena
resulting when a solution is
contained in a capillary with
fixed charges along its wall. This
is also known as the
Electroendosmotic Flow.

29. Where does the Electroosmotic flow come
from?
Anode
+
Cathode
-
Detector

30. Where does the Electroosmotic flow come
from?
Anode
+
Cathode
-
Applied Electric Field.
1000 Volts / Centimeter
Detector

31. Where does the Electroosmotic flow come
from?
Anode
+
Cathode
-
Detector
The interior wall of the capillary contains
charged sites that are created by the
ionization of Silanol groups on the fused
silica.

32. Where does the Electroosmotic flow come
from?

33. This is where the Electroosmotic flow
comes from.
What happens to the + cations when we turn on the power?

34. pH, Silanol Popaulation, and the rate of
EOF flow.
0
2
4
6
8
10
12
14
16
18
20
2
5
8
1
1
EOF
• At very low pH, not many
silanols are ionized and
the EOF is slow.
• As pH increases the
number of ionized sites
also increases. The EOF
speed rises steadily.
• At very high pH values, a
maximum number of
ionized sites is reached.
The EOF speed also
reaches a maximum.

35. The apparent velocity of any analyte
(u) will be a combination of its
electrophoretic velocity and its
movement in response to the EOF.
u = (Uep + Ueo) E

36. How does apparent velocity help us?
Analytes with a net positive charge will move faster than EOF
EOF
Analytes with no net charge will move at the same speed as the EOF.
(This is a useful tool that helps us to measure the EOF.)
EOF
Analytes with a net negative charge will move slower than EOF
EOF

37. Separation Efficiency
(apparent mobility) (Voltage)
N = ----------------------------------------
2 (diffusion coefficient)

38. Separation Efficiency (Y) and Diffusion
Coefficient (X)
• Note the very dramatic
drop in separation
efficiency with
increasing diffusion
coefficient.
• This means that in
some cases, there is no
real advantage over
conventional HPLC
for smaller molecules.

39. Injections
There are two principle methods:
• Pressure differential works by applying a pressure across the
capillary while it it is dipping into the sample solution.
• Electrokinetic injection works by applying a voltage and
allowing ions to migrate into the capillary because of their
charge.
Injection volumes are typically very small:
• Typically if injection volumes exceed 1% of the column
volume, separation efficiency severely suffers.
• Sample volume can be increased by focusing the ions inside
the capillary. This technique uses a combination of
additives to the medium and selectively applied charges.

40. Preconcentration to Increase Sensitivity
• Attached to front of
column
• Contains a selective
binding agent
• Allows several
capillary volumes to
pass
• Analytes of interest
are then eluted

41. Pressure and Electrokinetic Injections
• One additional advantage of electrokinetic injections is that
if appropriate conditions are set, extended injection times
allow analytes to be concentrated without overloading the
column.
+ + +
+
+ +
+
-
-
+

42. Setting up the Capillary Column
• Cut the ends cleanly.
• Load capillary into
the cartridge
• Place the clear
portion in the
detector window.

43. The Capillary Column’s Cartridge
• Allows the column to be
moved from vial to vial.
• Contains a cooling
medium.
• Contains gas and
vacuum connections.
• Holds electrodes that
place a charge on the
sample vials.

44. The Advantages of CE are:
• The number of theoretical plates is typically
in the hundreds of thousands.
• There is no mass transfer between mobile
and stationary phases as with HPLC and
GC, therefore the analytes remain in a
“plug” instead of spreading as a result of
laminar flow. (Peaks can still broaden
however.)
• Altering column conditions allows focusing
or concentration of samples.

45. Program
• General principles
• Safety
• Obtaining good results
• Example application, CE in drug discovery
• Running your samples on the Beckman
Coulter model P/ACE

46. SAFETY
• Chemical and Biological
• Remember that solvents will be flowing under high
pressure inside an electrically powered device.
• Aerosols may be generated, work in appropriate enclosure.
• Take all normal safety precautions when working with
toxic, pathogenic, or radioactive materials.
• Electrical
• Never remove covers and expose the electronics.
• Under certain conditions the chemist may have to be
grounded for protection against static electricity.
• Mechanical
• The CE unit features a robotic autosampler with many
moving parts and a sharp needle. Keep hands out of the
sample compartment while the unit is running.

47. Obtaining Reproducible (Good) Results
• Column condition.
• Composition and pH of the medium.
• Viscosity of the medium.
• Operating temperature.
• Adequate sample volume.
• Use of internal standards.

48. The pH must be tightly controlled to
obtain reproducible EOF flow.
0
2
4
6
8
10
12
14
16
18
20
2
5
8
1
1
EOF
• Remember that the
percentage of silanols that
are ionized is dependent
on the pH.

49. Column Condition
• As time goes on, certain
molecules will block or
otherwise neutralize the
ionized silanol sites.
This will change the
EOF and alter retention
times.
• It is also very important
to condition the column
properly before use.
Follow the directions in
the published method.

50. Internal Standards
• The main advantage of an internal standard is
that it is subject to the same conditions as the
analyte.

51. Program
• General principles
• Safety
• Obtaining good results
• Example application, CE in drug
discovery
• Running your samples on the Beckman
Coulter model P/ACE

52. Courtesy of Cetek Corporation

53. Courtesy of Cetek Corporation
20,000 Compounds Tested per Day
6,000,000 Tested since 1998

54. Other Applications
• Analysis of molecules that are not suited to
HPLC.
• Chiral separations of enantiomers.
• Determination of drug molecules in biological
fluids.
• Separating bacteria.
• Expect the unexpected.

55. Program
• General principles
• Safety
• Obtaining good results
• Example application, CE in drug discovery
• Running your samples on the Beckman
Coulter model P/ACE

57. MICELLAR ELECTROKINETIC
CHROMATOGRAPHY
UV
Neutral compounds
Comprable to HPLC

58. Capillary Electrochromatography
 Packed column with no pressure applied, only
electroosmotic pressure.

59. CAPILLARY GEL ELECTROPHORESIS
 Crosslinked vs. non crosslinked
 DNA sequencing
 Protein analysis
 Chirality possible
 EOF less desirable

60. CAPILLARY GEL ELECTROPHORESIS
http://www1.qiagen.com/Images/Catalog/2134.jpg

61. CAPILLARY ISOELECTRIC FOCUSING
http://www.targetdiscovery.com/~tdidocs/App_Note_5_200405.pdf

62. CAPILLARY ISOELECTRIC FOCUSING
 pH gradient
 Sample focusing and detection
 Movement of gradient towards the detector
 Zone broadening
 Not useful for chiral compounds

63. CAPILLARY ISOTACHOPHORESIS
 Two buffers form ionic zones
 Anions and Cations seperately
 Neutral compounds
 Used for concentration
 EOF less desirable

64. APPLICATIONS
CE and Analysis of Illicit Drugs

65. HPLC Heroin Analysis

66. HPLC Analysis of Heroin (SPE)
Fig. 2. (a) Representative total ion
chromatograms of all quantifiable
analytes spiked at LLQ level in
human plasma (5 ng/mL). The
intensity of the deuterated analytes
was above 2500 [cps]. (b)
Representative total ion
chromatograms of random chosen
patient’ plasma sample. (c) Total ion
chromatogram of a plasma sample
of a non-drug using volunteer. (A)
M3G and M3G-d3; (B) morphine
and morphine-d3; (C) M6G; (D) 6-
MAM; (E) heroin and heroin-d6;
(F) = methadone and methadone-
d9; (G) EMDP; (H) cocaine; (I)
benzoylecgonine.
DIODE ARRAY AND TRIPLE MS
5 ng/ml

67. FIRST PUBLISHED ANALYSIS OF ILLICIT DRUGS

68. FIRST PUBLISHED ANALYSIS OF ILLICIT DRUGS
Relative Standard Deviation
Migration 0.5%
Peak Area 4 – 8%
Twice as many peaks observed in Heroin analysis with MEKC
HPLC more sensitive
Smaller capillary did not help analysis with MEKC

69. LSD Analysis with Laser Fluorescence

70. LSD Analysis with Laser Fluorescence (0.2
ng/ml)

71. METHAMPHETAMINE ANALYSIS
50 μm Capillary with length of 40 cm
UV Detector
Electrophoresis 2006, 27, 4711–4716

72. METHAMPHETAMINE ANALYSIS
50 mL of 0.1 mol/L NaOH was added to 100 mL of urine
mixing by a vortex mixer for about 1 min.
1000 mL of ethyl acetate was pipetted in
continued mixing for 30 min.
centrifuged for 5 min at 5000 rpm.
the upper organic layer was carefully transferred to another polyethylene tube,
20 mL of 1.0 mol/L HCl was added
evaporated to dryness at 60oC
residues were then dissolved in 100 mL of doubly distilled water
Electrophoresis 2008, 29, 3999–4007

73. METHAMPHETAMINE ANALYSIS
50 mL of 0.1 mol/L NaOH was added to 100 mL of urine
mixing by a vortex mixer for about 1 min.
1000 mL of ethyl acetate was pipetted in
continued mixing for 30 min.
centrifuged for 5 min at 5000 rpm.
the upper organic layer was carefully transferred to another polyethylene tube,
20 mL of 1.0 mol/L HCl was added
evaporated to dryness at 60oC
residues were then dissolved in 100 mL of doubly distilled water
Electrophoresis 2008, 29, 3999–4007

74. ANALYSIS (LIQUID LIQUID EXTRACTION)
Electrophoresis 2008, 29, 4078–4087

75. ANALYSIS OF HAIR
Electrophoresis 1998, 19, 42-50

76. ANALYSIS OF BLOOD
Biomed. Chromatogr. 19: 737–742 (2005)

77. REFERENCES
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 Van Oss, C. Interfacial Forces in Aqueous Media. 1994: pg 145.
 Hjerten, S. Free Zone Electrophoresis. Chromatogr. Rev. 1937, 9: 122 – 219.
 Neuhoff, V., Wolf-Bernhard, S., and Sternbach, H. Micro-analysis of Pure Deoxyribonucleic-dependant Ribonucleic
Polymerase from E. Coli. Biochem. J. 1970, 117: 623 – 631.
 Camilleri, P. Capillary Electrophoresis: Theory and Practice. 2nd Edition. CRC Press. 1997: pgs 5-6.
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 Otto, M., Valcarcel, M. and Widmer, H. M. Analytical Chemistry. 2nd edition. Wiley. 2004: pgs 616-618.
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 Hashimoto, M. et. al. Microchip Capillary Electrophoresis using on-line chemiluminesce. J. Chrom. A. 2000, 867:271 –
279.
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 Paez, X. and Hernandez, L. Biomedical Applications of Capillary Electrophoresis with Laser Induced Fluorescence
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 Terabe, S. et. al. Electrokinetic seperations with Micellar Solutions and Open Tubular Capillaries. Anal. Chem. 1984,
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 http://www.chemistry.or.jp/gakujutu/bcsj/bc-cont/b98nov_gif/kea1009con.gif
 Altria, K. Capillary Electrophoresis Handbook: Principles, Operations, and Applications. Version 52. 1996: pgs 158 – 158.
 Chankvetadze, B. Capillary Electrophoresis in Chiral Analysis. 1997: pgs 43- 46.