1. Instrumentation and Applications
HPLC
Instrumentation and Applications

2. Other names for HPLC
1- High Speed Liquid Chromatography
- As the separation is completed within few minutes.
2- High Performance Liquid Chromatography (HPLC)
3- High Resolution Liquid Chromatography (HRLC)
High performance is the result of many factors:
- Smaller particles of the stationary phase, uniform pore size, high pressure column slurry packing technique, accurate low volume of the sample injected, sensitive detector, and good pump system

3. Introduction
High pressure liquid chromatography (HPLC) is an advanced form of liquid chromatography used to separate the components of a mixture (Analytes).
In HPLC chromatography: the mixture is dissolved in a solvent (mobile phase) and then forced to flow through a chromatographic column under a high pressure. In the column, the mixture is resolved into its components.

4. The separation occurs because each component in the mixture interacts differently with the stationary phase. Molecules that interact strongly with the stationary phase (yellow component) will move slowly through the column, while the molecules that interact less strongly (blue component) will move rapidly through the column. The start to the detector

5. Why high pressure? In HPLC the stationary phase has two characters :
- Has small particles size (5- 10 µm).
- And packed under high pressure.
Reduction of the particle size of the stationary phase leads to:
- Leaving less space for the mobile phase to pass through.
- Decrease the flow rate of the liquid mobile phase.
- Thus pressure from 1000 to 5000 psi, pound per square inch (68 to 340 atm.) is applied to overcome the obstructive effect of the fine particles.

6. Classification of HPLC I- Types of HPLC according to the mechanism of separation
1- Adsorption chromatography
The stationary phase is an adsorbent and the separation is based on adsorption-desorption steps.
A- Normal phase chromatography
The stationary phase is strongly polar (e.g. silica gel) and the mobile phase is non polar such as (hexane or tetrahydrofuran).
Polar sample retained longer on the column.

7. B- Reversed Phase chromatography
The stationary phase is strongly non polar ( e.g. C-18 silica , hydrpophobic) while the mobile phase is polar (as a mixture of water and methanol or acetonitrile).

8. Large molecules Small molecules 2- Size exclusion chromatography.
The column is packed with material having controlled pore sizes and the sample is screened or filtered according to its molecular size, there is no interaction between solute and stationary phase. The large molecules rapidly washed through the column, the smaller molecules penetrate inside the pores and elute later.
Large molecules
Small molecules

9. 3- Ion exchange chromatography
The stationary phase has an ionically charged surface of opposite charge to the sample ions
- This technique is used only for ionic or ionisable samples.
Types of St. Ph
1- Anion exchange
resin
2- Cation exchange
Matrix: is polymer of styrene with divinyl benzene

10. Anaion exchange as:
strong anion as quaternary ammonium form Matrix- (NR3)+ -Cl-
- weak anion as Matrix-NH2(CH3)-Cl-
Cation exchange as:
sulfonic acid Matrix-(SO3)– H+ (strong).
And Matrix-COO- H+ (weak)
The stronger the charge on the sample, the stronger it will be attached to the ionic surface and thus the longer it will take to elute.
The mobile phase is an aqueous buffer, where the PH is adjusted to control elution time.

11. Choice of separation technique

12. II- HPLC can be divided into two main types according to the uses:
1- Analytical type: which is used
a- In identification and assay of the components in a mixture .
b- To know the number of components in a mixture (screening).
2- Preparative or semipreparative type: used in isolation and purification.

13. The difference between analytical and preparative HPLC
1- Dimensions of the column.
- Analytical, 1-6 mm i. d.
- Preparative up to 3 cm i. d.
2- Flow rate of mobile phase (pump).
For analytical HPLC pumps should has flow rates that range from 1 to 10 ml/min.
but for preparative HPLC, flow rates in excess of 100 ml/min.

14. 3- Injected volume of the sample
in analytical HPLC range from 20 uL to 1 mL,
but in preparative or semi preparative from 1 ml to 5 ml or more.
4- Size of the loop of injection port.

15. Chromatographic process
The process begins by:
Injecting the solute onto the column (zero time).
The separation occurs as the analyte and mobile phase are pumped through the column
Detection of components by detector is displayed on a chart or computer screen (chromatogram).

16. The advantages of HPLC 1- High speed 2- High resolution
3- High sensitivity
4- Re-usable column
5- No destruction of the components
6- The instrumentation are automatic, computerized
7- Sample is recovered completely
8- Quantitative work is more easily and most sensitive

17. Instrumentation of HPLC
HPLC instrument includes:
A- Reservoir for solvents (mobile phase)
B- High pressure pump
C- Sample inlet device
D- Column
E- Detector
F- Recorder

19. A- Reservoir for solvents (mobile phase)
-Mobile phase is usually organic or aqueous or mixture of both.
- Mobile phase is placed in bottles of glass.

20. Mobile phase Characters of mobile phase: 1- Pure 2 - Low viscosity
3-Chemically inert Low price
5- Compatible with detector
6- Solubility of the sample
Miscible with water, such as acetonitrile, methanol, or isopropanol.

21. Elution Techniques (Programinig)
1- Isocratic elution:
The mobile phase composition remains constant throughout the separation procedure.
2- Gradient elution:
The mobile phase composition is changed during the separation process.
Gradient elution is divided into two types:
A- Continuous (linear)
B- Discontinuous (stepwise)

22. Isocratic and gradient elution curves

23. Advantages of gradient elution technique
1- Shortening the time of analysis.
2- Reduces tailing, gives sharp peak.
3- Increases the sensitivity of analysis.
4- Decreases the retention of the later-eluting components so that they elute faster.

24. The selectivity of HPLC is affected by :
PH of mobile phase
The pH of the solvent (water) may be adjusted using phosphate or perchlorate or trifloroacetate acid or sulphate buffer.
The selectivity of HPLC is affected by :
1- Type of mobile phase, organic or aqueous.
2- The composition of the mobile phase, whether one solvent or more.
3- The pH of the mobile phase.

25. Effect of buffer used in separation of xanthene alkaloids
TFA
Incomplete separation
HClO4
No separation for 2 and 3
H2SO4
Best separation

26. Mobile Phase Composition Effect on Selectivity
30% MeCN
70% Water
45% MeOH
55% Water
Slow and better separation
Fast
Methanol and water give slow and better separation while use of actonitrile and water give fast and bad separation

27. % of Mobile phase B (MeOH) and separation selectivity
Low % of B gives
slow and slightly better separation
High % of B gives
fast and bad separation

28. Some solvents used in HPLC and their polarity
10.2
7.2
6.9
5.8
5.1
4.8
4.3
4.0
3.9
Water
Dimethyl sulfoxide
Ethylene glycol
Acetonitrile
Methanol
Acetone
Dioxane
Ethanol
Tetrahydrofuran
I-propanol
N.B. Chlorinated solvents do not used in HPLC to prevent rusting of stainless parts of the instruments

29. Treatment of mobile phase
A- Filtration before entering the column.
B- Degassing using degasser.
1- Heating with stirring
2- Applying vacuum,
3- Passing nitrogen or helium
4- Ultrasound
C- Pre-saturation with the stationary phase in case of liquid liquid chromatography.

30. B- Pump Function of the pump: Pump is used for forcing the
mobile phase through the
column
There are two types of pump:
1- Constant pressure pump
It is free from pulsation
resulting in smooth baseline
2- Constant flow pump
It is able to give constant flow rate of mobile phase
To column
Mobile phase

31. The main criteria for the pump
1- The pump should be capable of delivering accurate and pulse free flow rate (e.g. 5 ml/min). 2- The pump should be capable of delivering high volume of solvent. 3- The pump should be capable of delivering high pressure up to 5000 psi.

32. C- Sample inlet device (Injection port)
1- Manual injection
2-Automated injection

33. The injection port consists of
A- The injection valve.
B- The sample loop.
Manual injection
1- The sample is typically dissolved in the mobile phase.
2- It is drawn into a syringe and injected into the loop via injection valve.

34. Column in HPLC is either Different shapes for columns used in HPLC
D- Column
Column in HPLC is either
1- Analytical, 1-6 mm i. d.
2- Preparative up to 3 cm i. d.
Made from: Stainless
Shape: Straight
Length: Variable
Different shapes for columns used in HPLC

35. Other types of columns used in HPLC
Guard column:
1- Protect the analytical column
2- Organization of separation in HPLC

36. E- Detectors (Brain of HPLC)
Characters of detector
1- High sensitivity
2- Low noise (straight base line)
3-Wide range of response to different compounds
4- Unaffected by temperature or mobile phase
5- Non destructive to the compounds
6- Provides qualitative and quantitative information about the detected sample

37. Types of Detectors 1 -UV absorption detector
- It is the most sensitive, sensitive to ng of compound.
- The most widely used, it measure the UV absorption of the solute.

38. 2 - Refractive index detector
Not used in case of gradient elution
- Less sensitive
It measures the difference in RI between pure mobile phase and the column eluate (mobile phase + solute).

39. 3- Mass spectrometer detector
It is used with capillary column in analytical HPLC to give information about nature of the material by giving the mass spectrum of the material.
4- Fluorescence detector
- More sensitive than UV detector (1000 fold as UV)
It is used with compounds which are naturally fluorescent. Or compound which
can be converted to fluorescent
derivative.

40. 5- Photodiode array detector
- It is series of detectors each is responsible for receiving a different wavelength.
6-Flame ionization detector
Used for substances whose boiling point is higher than that of the mobile phase
- It is more sensitive than refractive index detector

41. Applications of HPLC
1- Isolation and purification of biologically active natural products
2- Control of synthetic reactions
Identification of intermediates and target compound.
3- Biosynthesis study
Detection of biogenetic intermediates and enzymes involved.
4-Control the microbiological process
Used for separation of antibiotic from broth mixture

42. 5- Pharmacokinetics study
Pharmacokinetic study comprises the measurement of drug metabolites concentration in body fluids, absorption, bioavailability and elimination of drugs
HPLC determines the drug and its metabolites in one step.
6- Stability test
Rapid method of analysis in stability test.
7- Quality control
HPLC is used to know the identity, purity and content of the ingredients (drugs, raw and pharmaceutical products,
8- Drugs metabolisms

43. Applications of HPLC in isolation and purification of natural products
I. Purification
refers to the process of separation or extraction the target compound from other compounds or contaminants.

44. 1- Separation of quinine and quinidine2
1- Quinidine
2- Qinine

45. 2- Separation of Xanthines alkaloids

46. 3- Separation of vitamin B-1, 2, 6
Column: Primesep
150x4.6 mm
Flow rate: 1ml/min
Detection: UV 280 nm
Mobile phase: MeCN/H2O (10/90)
With H3BO4 buffer
PH 3.0

47. 4- Separation of ascorbic acid and dehydro-ascorbic acid
Column: Primesep
50x4.6 mm
Flow rate: 1ml/min
Detection: UV
Mobile phase: MeCN/H2O (10/90)
With HCOOH buffer
0.1%.

48. 5- Separation of chloramphenicol from mixture of antibiotics

49. 6- Separation of mixture of alkaloids
1- Codeine
2- Strychnine
3- Papaverine
4- Quinine
5- Quinidine

50. II- Quantitative (assay) and qualitative determination of natural products
Quantification of compounds by HPLC
Is the process of determination of the unknown concentration of a compound in a known solution.
Identification of compound by HPLC through :
- Comparison of retention time with authentic
- Comparison of UV spectrum of the compound with that of the authentic.
- Comparison of the Mass spectrum with that of the authentic. -

51. Quantification of oroidin in Axinella damicornis sponge (assay)
Oroidin is known alkaloid isolated from sponge Axinella damicornis and it was identified by HPLC through the comparison of retention time and UV absorption with data base on HPLC.

52. Steps of assay
1- Quantification was done by injection of different known conc. of oroidin (authentic).
2- determination of the peak area for each concentration
3- Followed by drawing the standard curve (area under the peak against conc.).
4- Injection of known weight of the sponge extract and find the area under the peak.
5- From the standard curve find the corresponding concentration of oroidin in the injected weight.
6- Calculate the weight of oroidin in the sponge.

53. Standard curve of oroidin
160 mg was found to be the conc. of oroidin in one gram of the sponge