HPLC

1. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
DR. MANOJ ACHARYA
JUNIOR RESIDENT (1st YEAR)
DEPARTMENT OF BIOCHEMISTRY
BPKIHS

2. OUTLINE
◉ Introduction to HPLC
◉ Components of HPLC
◉ Modes of HPLC
◉ Different Detectors
◉ Parameters in HPLC
2

3. INTRODUCTION TO HPLC
 HPLC is an abbreviation for
High Performance Liquid Chromatography
(It has also been referred to as High Pressure LC)
 The history of HPLC:
 Beginning of the 60’s: Start of HPLC as High Pressure Liquid
Chromatography
 In 1969 A.D , there has been a very marked change in technique
of liquid column chromatography because of development of
HPLC by J. Calvin Giddings and István Halász.
 End of the 70’s improvements of column material and
instrumentation.
 Since 2006 new terms popped up like UPLC, RRLC etc.
3

4. HPLC is a separation technique that involves:
• The injection of a small volume of liquid sample
• into a tube (column) packed with tiny particles 3 to 5
micron (µm) in diameter called the stationary phase
• where individual components of the sample are moved
inside column by pressurizing the mobile phase (liquid),
through the column by high pressure delivered by a pump.
4

5. • Components are separated from one another by the column
packing that involves various chemical and/or physical
interactions between their molecules and the packing particles.
• These separated components are detected at the exit of the
column by a detector that measures their amount.
• An output from this detector is called a “liquid
chromatogram”.
• In principle, LC and HPLC work the same way except the speed,
efficiency, sensitivity and ease of operation of HPLC is vastly
superior.
5

6. Diagramatic representation of HPLC
6

7. Components of HPLC
Solvent reservoir
• A glass bottle having a lid and a tube which convey Mobile
Phase to degasser and then to pump.
• Mobile Phase: Organic solvents or an aqueous-organic
mixture or a buffer solution. (i.e 90% water and 10% organic
solvent)
• Mobile Phase should be free from dust and particulate
matter, otherwise they can lead to irregular pumping action,
damage seals and valves and ultimately blocks the column .
7

8. Degasser (To Degas)
• Problems caused by dissolved air (usually O2 and N2) in
mobile phase
• Unstable delivery by pump and disrupt flow and rate,
• More noise and large baseline drift in detector cell
• In order to avoid these problems the mobile phase must
be degassed either by subjecting mobile phase under
vacuum, distillation or ultrasonic stirring.
8

9. Pump:
• Pump forces a liquid (called the mobile phase) through
the liquid chromatograph at a specific flow rate,
expressed (cm3/min).
• Change in the pump settings will be able to change the
retention time of the compounds.
• Good pump should be capable of output at least 50 Mpa
and ideally their must be no pulses.
• Must have flow capability of 10cm3/min to 100cm3/min.
9

10. Pump Module – types:
• Isocratic pump - delivers constant mobile phase
composition
• solvent must be pre-mixed
• lowest cost pump
• Gradient pump - delivers variable mobile phase
composition.
10

11. Gradient vs. Isocratic Conditions
Isocratic mobile phase
 1 solvent or 1 mixture of solvent of constant
composition during elution of compounds.
• solvent composition remains constant with
time
• Best for simple separations
Gradient mobile phase
• 2 or more solvents programmed to change
composition of polarity during elution of
compounds.
• solvent composition increases with time.
• Best for the analysis of complex samples.
11

12. Injector:
• The injector serves to introduce the sample into the flow
stream of the mobile phase.
• Typical sample volumes are 5- to 20-microliters (µL).
• The injector must also be able to withstand the high
pressures of the liquid system.
12

13. Manual Injector:
1. User manually loads sample into the injector using a syringe .
And then turns the handle to inject sample into the flowing
mobile phase.
Auto sampler:
1. User loads vials with sample solution into the auto sampler
tray (100 samples)
2. And the auto sampler automatically
• measures sample volume,
• injects the sample,
• then flushes the injector to be ready for the next sample.
13

14. Column (Stationary phase)
• Proper choice of column is important for success in HPLC
• Considered the “heart of the chromatograph” the column’s
stationary phase separates the sample components of interest
using various physical and chemical parameters.
• The small particles inside the column are what cause the high
backpressure at normal flow rates.
• The pump must push hard to move the mobile phase through
the column and this resistance causes a high pressure within the
chromatograph.
14

15. 1. Conventional column:
• Made up of stainless steel
• Filled with stationary phase
• length: 3-25 cm long
• Diameter: 4.6mm
• Flow rate : 1-3 cm3/min
2. Microbore or open tubular column:
• Length: 25-50cm
• Internal diameter: 1-2mm
• Flow rate : 5-20mm3/min
• Stationary phase is coated inside wall of column.
15
Types of Column Tubes
The advantages of microbore columns are having capability of providing high
efficiency, high speed, and high mass sensitivity separations

16. Stationary Phase
◉ Phase in chromatography where the analytes are seperated
◉ Analytes moves through the column in stationary phase.
◉ This column is filled with solid adsorbent material called as a packing
material, that has diameter of approx. 3micro meter to 4 micro meter
◉ So after pressurizing the mobile phase the mobile phase can flow
through this column.
◉ The main principle is, Hydrophobic molecules has a strong affinity for a
hydrophobic stationary phase whereas, hydrophilic molecules will be
eluted first in hydrophobic stationary phase.
16

17. Silica Particle size
◉ Silica particle can be either spherical or irregular in shape.
◉ Silica particle with more spherical structure is expensive to
perform with but has better stacking property than irregular in
shape
◉ The particle size ranges from (3-10) microns in size
- Too small can lead to pressure build up
- Too large can lead to poor resolution.
17

18. Separation modes of HPLC
◉ The correct selection of the column packing and the mobile
phase are the most important factors in successful HPLC.
◉ four major separation modes:
1. Reversed-phase chromatography
2. Normal-phase or adsorption chromatography
3. Ion exchange chromatography
4. Size exclusion chromatography
18

19. Reverse phase chromatography(RPC):
• Retention by interaction of the stationary phase’s non-polar hydrocarbon chain
with non-polar sample molecules.
• Stationary phase:
The column packing is non-polar (e.g. C18, C8, C4, phenyl etc).
eg. octadecylsilane (C18) or octasilane bonded to silica(bonded phase).
• Mobile phase:
mobile phase polar consist of water and water-miscible organic solvent(methanol,
acetonitrile)
eg. Water/acetonitrile or water/methanol.
• Most frequently used mode in HPLC over 90% chromatographer.
19

20. ◉ Inside column there is a silica backbone with bunch of
alcohol group is bound.
◉ In reverse phase the silica base is modified by creating a
silica bond with long chain carbon, ( which is going to have
large alcohol chain coming off (C18) ), which will be non
polar.
◉ By modifying it into a reverse phase there may be a gap so
that is due to the steric hinderance, so at that condition
there is “ End Capping”.
20

21. End Capping
◉ Goal of Reverse column phase is to make stationary
phase more hydrophobic.
◉ So, in many reverse phase column have gaps in the alkyl
chain i.e leaving open silonol group.
◉ So it is solved with smaller steric molecule that cover up
the silanol group.
◉ This end capping helps to fix the tailing in the curve which is
obtained in the chromatogram.
21

22. Ion pair Chromatograpy
◉ What is ion-pair chromatography (IPC)?
 The addition of an ionic surfactant in order to affect retention and
selectivity of ionic compounds.
◉ Why do we need Ion-Pair HPLC?
 When a sample contains ionic components that tend to be very
hydrophilic, and so reversed-phase retention can be problematic or
vice versa in normal phase.
 PH is important factor in silica column as well.
 Many analytes can contain charges at various pH level depending on
Pka, which determines whether the analytes its in its ionic state or
neutral state.
 It is used in ion exchange HPLC (like buffers)
.
22

23.  Cation exchange resins:
Also called acidic ion exchangers.
Cation exchangers possess negatively charged groups and these
will attract positively charged cations.
 Anion exchange resins:
Also called basic ion exchangers
positively charged groups that will attract negatively charged
anions.
23

24. ◉ In reversed phase chromatography, ionic compounds are usually not
retained by hydrophobic stationary phase.
◉ By adding an ion-pair reagent with a ionic end and a hydrophobic tail,
the hydrophobic tail of the reagent gets retained by the stationary phase.
Thus an ion exchange group forms on the surface of the stationary phase.
◉ The samples ion exchanges with the counter ion of the ion-pair reagent
retained by the stationary phase, thus resulting in greater retention of the
sample.
24
(a)Bonded phase
(b)Stationary Phase
(c)Ion-pair reagent in mobile phase
(d)ion-pair reagent adsorbed to
Stationary phase
(e)Sample ion free in mobile Phase
(f)Sample retained on column by ion-
pair mechanism.

25. ◉ An ion pair reagent is added to enhance peak shape and
retention time.
◉ The ion pairing agent must be oppositely charged than the
analyte and must have good hydrophobicity.
◉ Ion-pairing chromatography (IPC) can be used for both
positively and negatively charged analytes.
 Negatively charged reagent can be used to retain positively
charged ionic bases.
 Positively charged reagent can be used to retain negatively
charged ionic acids.
Hydrophilic solute Hydrophobic ion-pair
(less retained in RPC) (more retained in RPC)
25

26. ◉ Typical ion pair matrices include:
26

27. 27

28. Retention Mechanism:
Two possible retention process
1. Partition model
2. Adsorption model
 Partition Model:. In this model, the ion-pairing agent is present in the
mobile phase. The analyte interacts with the ion-pairing agent in the
mobile phase first. It forms the ion-pair which is relatively non-polar
and partition into the stationary phase and get retained.
28

29.  Adsorption Model: The ion-
pairing agent present in the
mobile phase gets adsorbed
into the non-polar stationary
phase .
 As a result, the ion-pairing
reagent forms a ion-exchange
layer on the surface of the
stationary phase. The analyte
interacts with the ion-pairing
agent presented on the surface
to form ion-pair and gets
retained.
29

30. 30

31. Adsorption chromatography
◉ Based upon the principle that certain solid materials, collectively known as
adsorbents, have the ability to hold molecules at their surface.
• In this mode, the stationary phase is polar (e.g. silica gel, cyanopropyl-
bonded, amino-bonded, etc.)
mobile phase is non-polar (e.g. hexane, methylene chloride, ethyl
acetate)
• A solid such as silica gel is used as the stationary phase, and separation is
mainly in the degree of adsorption to its surface, are used to separate the
solutes.
• As eluent is constantly passed down the column, differences in these
binding strengths eventually lead to the separation of the analytes.
• Liquid-solid chromatography
31

32. Detector:
◉ The detector can detect the individual molecules that
comes out (elute) from the column.
◉ A detector serves to measure the amount of molecules so
that the biochemist can quantitatively analyze the sample
components.
◉ Detector provides an output to a recorder or computer that
results in the liquid chromatogram (i.e., the graph ).
◉ Detector is based on analyte or the sample under detection.
32

33. 33

34. Major Types of HPLC detectors
◉ Divided into two main Detectors
1) Based on Solute property
- UV Visible
- Electrochemical
- Fluorescence
2) Bulk Property
- Refractive index
- Conductivity
34

35. ULTRAVIOLET (UV) = Most popular and widely used.
- The solutes that contain a chromophore at the monitoring
wavelength, absorb the incident light as they pass through the
flow cell
- Amount of light absorbed produces a signal proportional to
the concentration of solute.
- Resulting change in this electrical signal is amplified and
directed to a recorder.
35

36. 36

37. 37
A typical PDA has a 512 diode to cover a wavelength of 190 to 800nm, with
each bandwidth of 2nm

38. Mass Spectroscopy
• A MS detector senses a compound eluting from the HPLC
column first by ionizing it then by measuring it’s mass or
fragmenting the molecule into smaller pieces
• An advantage of mass spectrometry detection is that it gives
idea for the identification of overlapping peaks.
• If there is a suspicion that a large peak is masking a smaller
peak then presence of a minor analyte can be confirmed by
selected ion monitoring provided that minor and major
analytes have a unique molecular ion or fragment ion.
38

39. 39

40. ◉ The ability of a compound or solvent to deflect light provides a way to detect
it.
◉ The RI is a measure of molecule’s ability to deflect light in a flowing mobile
phase in a flow cell relative to a static mobile phase contained in a reference
flow cell.
◉ The amount of deflection is proportional to concentration.
◉ The RI detector is considered to be a universal detector but it is not very
sensitive.
40
Refractive index (RI)detection

41. Fluorescence detectors
◉ Fluorescence detectors sense only those substances that fluoresce.
◉ Compared to UV-Vis detectors fluorescence detectors are highly sensitive and
selective .
◉ It is possible to detect even a presence of a single analyte molecule in flow
cell.
◉ Compounds having specific functional groups are excited by shorter
wavelength energy and emit higher wavelength radiation(energy emission)
which called fluorescence.
◉ Roughly about 15% of all compounds have a natural fluorescence. The
presence of conjugated pi-electrons especially in the aromatic components
gives the most intense fluorescent activity.
41

42. 42

43. Parameters in HPLC
1.Retention time
2.Retention volume
3.Seperation factor
4. Resolution
5.Theoretical number plate.
45

44. Retention time:
The time at which a specific analyte elutes (emerges from the
column) is called its retention time.
◉ The retention time tR for each analyte has two components,
◉ The first is the time it takes the analyte molecules to pass through the
free spaces between the particles of the matrix coated with the
stationary phase. This time is referred to as the dead time, tM
◉ The volume of the free space is referred to as the column void volume ,
V 0 .
◉ The second component is the time the stationary phase retains the
analyte, referred to as the adjusted retention time , t´R .
46

45. 47

46. Retention Factor
◉ One of most important parameters in column chromatography is
retention factor, k
◉ It is simply the additional time that analyte takes to elute from column
relative to an unretained or excluded analyte that does not interact with
stationary phase and which, by definition, has a k value of 0. Thus:
◉ Note that k has no units. It is apparent from this equation that if the
analyte spends an equal time in stationary and mobile phases, its tR
would equal 2 × t M and its k would thus be 1.
48

47. ◉ Theoretical Plate Number, N
column consist of number of adjacent zones in which there is
sufficient space for analyte to equilibrate between 2 phase.
Each zone is called theoretical plate.
49
W
W1/2
H1/2
H
R
W
2
16
= t
N

48. Efficiency:
Efficiency of a column is expressed by the theoretical
plates.
n = 16 tR
2/ w2
Where n = no of theoretical plates
tR= retention time
w = peak width at base
• tR and w are measured in common units (min or sec ,
cm or mm ).
• If no of theoretical plates is high, the column is said to be
highly efficient.
50

49. Column Efficiency Based on Theoretical Plate
Number
51
• If the retention times are
the same, the peak
width is smaller for the
one with the larger
theoretical plate number.
• If the peak width is the
same, the retention time is
longer for the one with the
larger theoretical plate
number.

50. RESOLUTION
◉ The success of a chromatographic separation is judged by the ability of
the system to resolve one analyte peak from another.
◉ Resolution ( R S) is defined as the ratio of the difference in retention
time (∆ tR) between the two peaks to the mean of their base widths ( w 1
and w 2 )
52

51. 53

52. Qualitative analysis
Identification of individual compounds in the sample:
 Most common parameter for compound Identification is its
retention time (time taken by specific compound to elute from the
column after injection).
 Depending on the detector used, compound Identification is also
based on the chemical structure, molecular weight or some other
molecular parameter.
54

53. Quantitative analysis
◉ Measurement of the amount of a compound in a
sample.
Two main ways to interpret a chromatogram (i.e. perform
quantification):
1. determination of the peak height.
2. determination of the peak area.
◉ In order to make a quantitative assessment of the compound, a
sample with a known amount of the compound of interest is
injected and its peak height or peak area is measured. In many
cases, there is a linear relationship between the height or area
and the amount of sample.
55

54. Preparative chromatograpy
◉ By collecting the chromatographic peaks at the exit of
the detector, and concentrating the compound (analyte)
by removing/evaporating the solvent, a pure substance
can be prepared for later use (e.g. organic synthesis,
clinical studies, toxicology studies, etc.).
◉ This methodology is called preparative chromatography
56

55. Trace Compound Analysis
◉ A trace compound is difficult to analyse due to its very low
concentration, usually less than 1% by weight, often parts
per million (ppm)..
◉ In a chromatogram trace substances can be difficult to
separate or detect, high resolution ,separations and very
sensitive detectors are required.
57

56. 58

57. 59

58. HPLC has been used for:
◉ HPLC provides a convenient and fast analytical approach in detection of
catecholamines in samples of urine or plasma using electrochemical or
fluorescence detection.
◉ HPLC has proved to be a valuable monitoring technique for glycaemic
control through accurate quantitative estimation of glycated
haemoglobins.
◉ HPLC is also used in routine estimation of vitamins, hormones and other
biomarkers
◉ also used to purify many proteins and peptides during investigative studies
and is used for large scale purification of protein.
◉ And manufacturing (e.g. during the production process of pharmaceutical
and biological products) purposes.
60

59. REFERENCES
◉ Wilson & Walker Principles and Techniques of Biochemistry and Molecular
Biology.7th .Edition.
◉ Wiley.Physical.Biochemistry.Principles.And.Applications.4th.Edition
◉ Ion Exchange Chromatography principles and methods – Pharmacia Fine
Chemicals.
◉ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1837629/
◉ Instrumental Liquid Chromatography - N A Parris
◉ Internet Sources
◉ HPLC - How to read Chromatogram Easy Explained - Simple Animation HD
61

60. 62