Ultra Performance Liquid Chromatograph

By: Pharma Tips | Views: 6960 | Date: 17-Jun-2010

Chromatography is the science, which includes the studies of separation of molecules based on differences in their structure and/or composition. Chromatographic separations can be carried out using a ` of supports, including

Chromatography is the science, which includes the studies of separation of molecules based on differences in their structure and/or composition. Chromatographic separations can be carried out using a ` of supports, including
Immobilized silica on glass plates (thin layer chromatography)
Volatile gases (gas chromatography), paper (paper chromatography)
Liquids, which may incorporate hydrophilic, insoluble molecules (liquid chromatography).

Ultra Performance Liquid Chromatograph
1.2.1 Ion-Exchange Chromatography 
Proteins are made up of twenty common amino acids. Some of these amino acids possess side groups ("R" groups), which are either positively or negatively charged. A comparison of the overall number of positive and negative charges will give a clue as to the nature of the protein. If the protein has more positive charges than negative charges, it is said to be a basic protein. If the negative charges are greater than the positive charges, the protein is acidic. When the protein contains a predominance of ionic charges, it can be bound to a support that carries the opposite charge. A basic protein, which is positively charged, will bind to a support, which is negatively charged. An acidic protein, which is negatively charged, will bind to a positive support. The use of ion-exchange chromatography, then, allows molecules to be separated based upon their charge. Families of molecules (acidic, basics and neutrals) can be easily separated by this technique. This is perhaps the most frequently used chromatographic technique used for protein purification.5 
1.2.2 Hydrophobic Interaction Chromatography ("HIC") 
Not all of the common amino acids found in proteins are charged molecules. There are some amino acids that contain hydrocarbon side-chains, which are not charged and therefore cannot be purified by the same principles involved in ion-exchange chromatography. These hydrophobic ("water-hating") amino acids are usually buried away in the inside of the protein as it folds into its biologically active conformation. However, there is usually some distribution of these hydrophobic residues on the surface of the molecule. Since most of the hydrophobic groups are not on the surface, the use of HIC allows a much greater selectivity than is observed for ion-exchange chromatography. These hydrophobic amino acids can bind on a support, which contains immobilized hydrophobic groups. It should be noted that these HIC supports work by a "clustering" effect; no covalent or ionic bonds are formed or shared when these molecules associate. 
1.2.3 Gel-Filtration Chromatography 6
This technique separates proteins based on size and shape. The support for gel-filtration chromatography is beads, which contain holes, called "pores," of given sizes. Larger molecules, which can't penetrate the pores, move around the beads and migrate through the spaces, which separate the beads faster than the smaller molecules, which may penetrate the pores. This is the only chromatographic technique, which does not involve binding of the protein to a support. 
1.2.4 Affinity Chromatography7 
This is the most powerful technique available to the chromatographer. It is the only technique, which can potentially allow a one-step purification of the target molecule. In order to work, a specific ligand (a molecule which recognizes the target protein) must be immobilized on a support in such a way that allows it to bind to the target molecule. A classic example of this would be the use of an 
immobilized protein to capture it's receptor (the reverse would also work). This technique has the potential to be used for the purification of any protein, provided that a specific ligand is available. Ligand availability and the cost of the specialized media are usually prohibitive at large-scale. 

1.3 References
1)  Zuiderwerg,F.J. and Klinkenberg,A., Chem.Eng.Sci. vol-5, 1956,271.
2) www.google.com.
3) www.pubmed.com
4) www.accessexcellence.org
5) chromatographyonline.findpharma.com
6) www.environmental-expert.com
7)  Jerkovitch,A.D., Mellors,J.S.  and  Jorgenson, J.W.,LCGC , Vol-21,2003,7.

Ultra-performance liquid chromatography (UPLC), involves HPLC with very high pressures and columns having very small particle sizes.

2.1 Principle1-5 
The efficiency of HPLC increased as particle sizes of the column packing decreased from 10 [micro] m in the 1970s to 3.5 Nun in the 1990s. This is shown by lower values of HETP (height equivalent to a theoretical plate) for van Demeter plots of HETP (column efficiency) versus mobile phase flow rate in units of linear velocity ([mu], mm/s; Figure 1). hi this particle size range, and even down to 2.5 Nun particles used in shorter columns in the early 2000s, it was found that HETP decreases to a minimum value and then increases with increasing flow rate. However, with the 1.7 [micro] m particles used in UPLC, HETP is lowered compared to the larger particles and does not increase at higher flow rates. This allow faster separations to be carried out on shorter columns and/or with higher flow rates, leading to column increased resolution between specific peak pairs and increased peak capacity, defined as the number of peaks that can be separated with specified resolution in a given time interval. 

2.2 Theory Of  UPLC6-10
The chromatogram that depicts the elution of a solute is a graph relating the concentration of the solute in the mobile phase leaving the column to elapsed time. However, at a constant flow rate, the chromatogram will also relate the solute concentration to the volume of mobile phase passed through the column. In figure 1, is shown the elution of a single peak. The expression, f(v), is the elution curve equation and this will be derived using the plate theory.

Figure 1. The Elution Curve of a Single Peak
Once the nature of f(v) identified, then by differentiating f(v) and equating to zero, the position of the peak maximum can be determined and an expression for the retention volume (Vr) obtained. The expression for (Vr) will disclose those factors that control solute retention.11
UPLC is a new separation technique with increased speed, sensitivity and resolution. The performance of a column can be measured in terms of the height equivalent to the theoretical plates (HETP or H), which is calculated from the column length (L) and the column efficiency, or number of theoretical plates (N). N is calculated from an analyst’s retention time (tR) and the standard deviation of the peak (σ).
H = L/N    ------------------- (1)

N=(tR/σ)2  -------------------- (2)

The van Deemter equation is the empirical formula that describes the relationship between linear flow velocity (μ) and column efficiency, where A, B, and C are constants related to the mechanistic components of dispersion.12

H = L/N = A + B/μ + Cμ---- (3)   (VAN DEEMTER EQUATION)

According to the van Deemter plot, column efficiency is inversely proportional to the particle size (dp) (Equation 4), so by decreasing the particle size there is an increase in efficiency. Since resolution is proportional to the square root of N (Equation 5), decreasing particle size increases resolution. Also, by using smaller particles, analysis time can be decreased without sacrificing resolution, because as particle size decreases, column length can also be reduced proportionally to keep column efficiency constant. By using the same HPLC mobile phase and flow rate, UPLC™ reduces peak width and produced taller peaks which increased the S/N 1.8 to 8 fold, improving both sensitivity and resolution.13-18 

N α 1/dp --------------- (4)

R = √N/4(α-1/α)(k/k+1) --------------- (5)

Also according to the van Deemter plot, use of particles smaller than 2 μm produces no loss in column efficiency with increasing flow rates. However, by increasing flow rates to decrease analysis time, there is a corresponding increase in system pressure. As a result, a system capable of withstanding the proper pressures while still maintaining efficiency is required. As well, a mechanically stable column is needed. 
2.3 References
1) Lippert,A.D. and  Lee,M.L., J. Chromotogr.,Vol-911,2001,1.
2)  Swartz,M.E., J. Liq. Chromatogr., Vol-91,2001,1.
3)Allanson,J.P.,Biddlecombe,R.A.,Jones,A.E,Pleasance,S.,Rapid Communication,Mass Spectrom., Vol-10,1998,811.
4) Ayrton,J., Dear,G.J.,  Leavens,W.J.,  MallettD.N., Plumb,R.S. and  Dickins,M.,Rapid Communication Mass Spectrom., Vol-12, 1998,217–224.
5) Ayrton,J., Dear,G.J., Leavens,W.J. and D.N,Plumb,R.S., J. Chromatogr.,Vol-10, 1998,243–254.
6)  Jermal,M. and  Xia,Y., Rapid Communication Mass Spectrom.
Vol-13, 1999, 97
7)Plumb,R.S., Dear,G.J., Mallett D.N. and Ayrton,J., Rapid Communication Mass Spectrom.  Vol-15,2001,986-993.
8) Bungay. and  Henry R., BASIC Biochemical Engineering, 1993. 
9) Harris M. and Daniel C., Quantitative Chemical Analysis, 1995. 
10)Brown,D. and Superti-Furga, J.Drug Discovery Tech. Vol-8,2003,1067-1077.
11) Smith,G.A., Rawls, C.A. and  Kunka, R.L.,J.Pharmaceutical analysis,
Vol- 21,2002,154-158.
12)  Needham S.R. and  Wehr,T.R., LCGC,Vol-14,2001,244-249.
13)  Lars, Y. and  Honore,H.S., J. Chromatogr., Vol-102,2003,59-67
14) D.A. McLoughlin, T.V. Olah.  and J.D. Gilbert, J. Pharm. Biomed. Anal. Vol-102, 2003, 59-67.
15)  Hoke, S.H., Tomlinson, J.A., Bolden R.D., Morand, K.l., Pinkston,J.D. and Wehmeyer,K.R., Anaytical. Chemistry, Vol-73, 2001, 3083-3088.
16) Zhang,Y.H.,  Gong,X.Y.,  Zhang,H.M.,  Larock, R.C.and  Yeung,E.S.,
J. Comb. Chemistery, Vol-2,2000,450-452.
17) Zhou,C., Jin,Y.,  Kenseth,J.R.,  Stella,M, Wehmeyer,K.R. and  Heineman,W.R., J. Pharmaceutical science,Vol-94,2005,576-589.  
18)  Rajan,A.,  Mullen,J.,  Bhatnagar,N.,  Dubey,A., Niemz,A.,  ChakravartiB., and  Chakravarti,D.N., J.Pharmaceutical science, ,Vol-9,2004,312-317

no. Reported method Method Description
Development and validation of UPLC method for determination of primaquine phosphate and its impurities. UPLC For high speed separation
Column: BEH C18, 50*2.1mm, 1.7um 
Runtime: 5min

2 The ultra-performance liquid chromatography (UPLC) analysis of phenolics in four plant species. UPLC Detector: PDA detector 
Software: Empower Pro 2.0 
Column:  UPLC BEH C18 column 
(1.7mm, 50mm ´ 2.1mm)  
Mobile phase: Acetic acid: Water: ACN.         
(0.1: 0.1: 40%)
Flow rate: 0. 35 mL/min.
3 A rapid method for simultaneous determination of 15 flavonoids in Epimedium using pressurized liquid extraction and ultra-performance liquid chromatography

UPLC Column: UPLC BEH C18 column 
(50mm × 2.1 mm I.D., 1.7 μm) Gradient elution:Acetic acid aqueous 
Solution & ACN (50 Mm)
Runtime: 12 min
LOD: lower than 0.13ng on column
LOQ:  lower than 0.52 ng on column.

4 An Improved UPLC Method for Rapid Analysis of Levofloxacin in Human Plasma UPLC Column: C18 column 100 × 2.1 mm i.d., 
1.7 µm 
Mobile Phase: Triethylamine:Buffer:ACN 
(88:12 (v/v): pH 3:0. 4%)
Pressure:  11,000 psi (758 bar) 
Flow-rate:  0.3 mL min−1. 
UV detection: 300 nm.
Retention time: Levofloxacin-3.4 min 
Enoxacin -2.8 min.       
Run-time: 5 min. 

3.1 References
1)Larkin,F., Jouranal of pharmaceutical and  Biomedical Analysis,
2)Stochmal, A. and Janada, B., Institute of Soil Science and Plant Cultivation, Department of Biochemistry (IUNG), Czartoryskich 8, Vol- 46,Issue2,2008,
3)Szajwaj,B., Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University, Beijing 100083, Chine.
4)Janada,B., Department of Pharmacology, China Pharmaceutical University, Nanjing 210009, Chine.

4.1 Instrumentation Parts   
FIGURE 2: The schematic diagram of UPLC is:
4.1.1 Pump1
(1) Reciprocating pump   (2) Pneumatic pump
4.1.2 Gradient mixers6
4.1.3 Columns11
4.1.4 Sample injector14
4.1.5 Optical detectors15
4.1.6 Combination of detectors16-19

4.1.1. Pump1,2
There are two types of pumps:
1. Reciprocating pump   
2. Pneumatic pump
1. Reciprocating pump: There are two general types of reciprocating pumps.  The piston pump and the diaphragm pump. These types of pump operate by using a reciprocating piston or diaphragm.   The liquid enters a pumping chamber via an inlet valve and is pushed out via a outlet valve by piston Reciprocating pumps are generally very efficient and are suitable for very high   flows. This type of pump is self-priming as it can draw liquid from a level below the suction flange even if the suction pipe is not evacuated. The pump delivers reliable discharge flows and accurate quantities of fluid. The reciprocating pump is not tolerant to solid particles and delivers a highly pulsed flow.   If a smooth flow is required then the system has to include additional features such as accumulators to provide even flows. Reciprocating pumps designed for delivering high pressures must include methods for releasing excessive fluid pressures.   The pumps should include for built in relief valves or relief valves should be included in the fluid circuit, which cannot be isolated from the pump. 

(i) Piston Pumps /Plunger pumps3 
A piston pump can be based on a single piston or, more likely, multiple parallel pistons.   The pistons are reciprocated using cams or crankshafts.   The stroke is generally adjustable.  This type of pump can deliver heads of up to 1000 bar.   The largest sizes of piston pumps can deliver flows of 40m3 /hr. In practice these pumps are more likely to be used for metering low flow rate fluids at more modest pressures in laboratories and chemical process plants.   Piston pumps are not generally suitable for transferring toxic or explosive media.

(ii) Diaphragm Pumps
There are two types of diaphragm pumps. The hydraulically operated diaphragm metering pumps and the air actuated type.
(iii) Hydraulically operated diaphragm pump 4
The hydraulically operated diaphragm-metering pump is used for similar duties as the piston pump.   It has some significant advantages compared to the piston pump in that the design does not require glands or piston seals. The diaphragm in the hydraulically operated diaphragm pump shown below is actuated using a plunger pump arrangement. This provides full support of the diaphragm allowing high-pressure operation.    The pump can include for duplex diaphragms with the interface being monitored for failure of the diaphragm in contact with the fluid.   This type of pump can be used for pumping toxic and explosive fluids.   The pump can deliver heads of up to 700 bar and transfer flows of up 20 m 3 /hr.  These pumps require continuous monitoring as the diaphragm is under high fatigue loading and the inlet and outlet valves are subject to erosion and blocking.   Under a high quality maintenance regime these pumps are very reliable.

(iv) Air Operated Pump 
The air operated pump is generally a low cost workhorse pump used for transferring any type of liquid including sludge.   The inlet and outlet valves are often low cost easily replaced flap or ball valves.   The pump is comprises two circular chambers each split by a large electrometric diaphragm.   The two diaphragm centers are mechanically coupled together with a shaft.   An interlocked valve admits air pressure to one side of one of the chambers and exhausts the air from the opposite side of the other chamber.   This causes both diaphragms to move.One diaphragm pushing fluid out through a non-return valve. The other diaphragm drawing fluid in through a non-return valve. On completion of a full stroke the valve reverses the air supply and exhaust directions causing the diaphragms to move back.   The diaphragm, which was pushing fluid out of the pump, now sucks fluid and the diaphragm admitting fluid now pushes fluid out.   The system is double acting.
The pump capacity is limited by the air pressure available (generally 7 bar) and the design of the diaphragm.  An electrometric diaphragm has a limited life and will only operate for a few million cycles.    A flow rate of about 40 m3 /hr is a reasonable maximum achievable flow with a larger pump. For any air operated diaphragm pump the higher the flow the lower the discharge head possible.
2. Pneumatic pump5
The pneumatic pump is double piston pump, one piston having a relatively large diameter and the other a relatively small diameter. The two pistons are connected together and fit inside two connected cylinders. The smaller cylinder is fitted with inlet and outlet non-return valves. The larger piston is driven by compressed air(the gas alternately driving the piston in one direction and then the other) and actuates the smaller piston which pumps the liquid. The system acts as a pressure amplifier, as the output pressure from the pump with the smaller piston will be equal to the pressure applied to the larger piston times the ratio of the cross-sectional area of the larger piston to that of the smaller piston. This type of piston was originally used for normal liquid chromatography separations but was found to be noisy and produced strong flow pulses that destabilized the detector. It is now used almost exclusively for slurry packing liquid chromatography columns. It is the simplest type of pump that can be designed to provide exceedingly high pressures. 

4.1.2 Gradient mixers6
Gradient mixers must provide a very precise control of solvent composition to maintain a reproducible gradient profile. This can be complicated in HPLC by the small elution volumes required by many systems. It is much more difficult to produce a constant gradient when mixing small volumes then when mixing large volumes. For low pressure systems this requires great precision in the operation of the miniature mixing valves used and low dispersion flows throughout the mixer.For multi-pump high pressure systems it requires a very precise control of the flow rate while making very small changes of the flow rate. 

4.1.3 Columns7
There are two types of columns :
1.Analytical column
2.Guard column
1. Analytical column8
Different columns used in UPLC are:
Pro C18, Pro C8, Hydrosphere C18,YMC30 , YMC basic ,
2. Guard column9
A guard column and retention gap is the same thing, but they serve different purposes. Both are 1-10 meters of deactivated fused silica tubing attached to the front of the column. Deactivated fused silica tubing does not contain any stationary phase; however, the surface is deactivated to minimize solute interactions. A suitable union is used to attach the tubing to the column. In most cases, the diameter of the retention gap or guard column should be the same as the column. If the tubing sizes are different, it is better to have a larger diameter guard column or retention gap than a smaller one. Guard columns are used when samples contain non-volatile residues that may contaminate a column. The non-volatile residues deposit in the guard column and not in the column. This greatly reduces the interaction between the residues and the sample since the guard column does not retain the solutes (because it contains no stationary phase). Also, the residues do not coat the stationary phase which often results in poor peak shapes. Periodic cutting or trimming of the guard column is usually required upon a build-up of residues. Guard columns are often 5-10 meters in length to allow substantial trimming before the entire guard column has to replaced. The onset of peak shape problems is the usual indicator that the guard column needs trimming or changing.
Guards for 4 mm ID analytical columns:
Art. No. Description

CHIRAL-AGP guard cartridge
10x4 mm, 2-pack


CHIRAL-CBH guard cartridge
10x4 mm, 2-pack


CHIRAL-HSA guard catridge
10x4 mm, 2-pack

4.1.4 Sample injector
The function of the injector is to place the sample into the high-pressure flow in as narrow volume as possible so that the sample enters the column as a homogeneous, low-volume plug. To minimize spreading of the injected volume during transport to the column, the shortest possible length of tubing should be used from the injector to the column. This HP 1090 Chromatograph has both a manual injector and an auto injector. The first one allows you to do only a single injection and the second can be programmed to do up 99 injections in a sequence. The sample injection valve is a six-port rotary valve designed for high performance liquid chromatography. When an injection is started, an air actuator rotates the valve: solvent goes directly to the column; and the injector needle is connected to the syringe. The air pressure lifts the needle and the vial is moved into position beneath the needle. Then, the needle is lowered to the vial. The sample is drawn up into a sample loop by the syringe, metered by a stepper motor. The needle is raised a second time to allow the vial to move away. Then, the needle is lowered a second time and the air actuator reverses the valve, reconnecting the sample loop to the solvent flow. The entire sample is flushed out of the injector, reaching the column as an undiluted plug. Finally, the syringe stepper-motor moves the syringe plunger to the end of the syringe sending the remaining solvent to the waste

4.1.5 Optical detectors11
Detectors mostly used in this system are TUV, PDA, or ELS optical detector or combination of three.
(1) TUV detector12,13
The TUV (tunable ultraviolet) optical detector is a two-channel, ultraviolet/visible (UV/Vis) absorbance detector designed for use in the UPLC system. The detector, controlled by Empower software for LC applications or Mass Lynx software for LC/MS applications, operates as an integral part of the system.The detector offers two flow cell options. The analytical cell, with a volume of 500 neon liters and a path length of 10 mm, and high sensitivity flow cell, with a volume of 2.4 micro liters and a 25 mm path length, both utilize the flow cell technology. The TUV detector operates at wavelengths ranging from 190 to 700 nm.
(2) PDA detector14
The PDA (photodiode array) optical detector is an ultraviolet/visible light (UV/Vis) spectrophotometer that operates between 190 and 500 nm. The detector offers two flow cell options. The analytical cell, with a volume of 500 nanoliters and a path length of 10 mm, and high sensitivity flow cell, with a volume of 2.4 micro liters and a 25 mm path length, both utilize the flow cell technology.
(3) ELS detector15
ELS detector is an evaporative light scattering detector designed for use in the UPLC system. This can be controlled by Empower or MassLynx software.

4.1.6 Combination of detectors16

(i) UPLC / Evaporative Light Scattering (ELS) detector 

Designed for optimal UPLC/ELS performance in a small footprint. 
(ii) UPLC / Fluorescence (FLR) detector

Provides sensitive, selective detection for UPLC-based fluorescence applications. 

(iii) UPLC/ Photo Diode Array (PDA) detector17

Allows us to detect and quantify lower concentrations of sample analysts and compare spectra across wavelengths and broad concentration ranges. 

(iv) UPLC PDA eλ Detector  
Quantitative compounds with visible wavelength maxima up to 800 nm and achieve higher sensitivity levels with high resolution spectra for peak identification and purity analysis. 

(v) UPLC/ Tunable Ultraviolet (TUV) Detector18          
A dual-wavelength ultraviolet/visible detector optimized for Ultra Performance LC (UPLC). It offers optimal linearity, resolution and sensitivity for UPLC/UV separations.

(vi) SQD19 
Combines the resolution, sensitivity, and speed of Ultra Performance technology with single quadrupled MS detection. 

(vii) Tunable Quadrapole Detector (TQD) 
A bench top, tandem quadrupole MS detector for UPLC/MS/MS and HPLC/MS/MS applications. 

4.2 Refrences
3) Said,A.S., J. Chemical  Engineering,Vol-2,1956,477.
4) Martin,A.J. and  Synge,R.L.M., J. Biochem,Vol-35,1941,1358.
5) James A.T. and Martin,A.J., J.Biochem,Vol-50,1952,579.
6) Engelhardt,H.,  Muller,H. and  Dreyer,B., J Chromatographia,
7) Knox J.H. and  Kaliszan,R.J.,  J.  Chromatogr.,Vol-349,1985,211.
8)  Smith R.J. and C. S. Nieass,  J. Liq. Chromatogr.,Vol-3, 1986,1387.
9)  Scott R.P.W. and  Kucera, P., J.  Chromatogr.,Vol-15,1987,69.
10) McCormick,R.M. and Karger,B.R., J. Analytical chemistry,
11) Scott R.P. and Simpson,C.F., J. Chem. Soc.,Vol-15,1980,69.
12)Alhedai,A.,  Martire D.E. and  Scott R.P., J.Analyst.,Vol-43, 1989,898.
13) Scott., R. P. W.  and C. E. Reese, J. Chromatogr.,Vol-114,1989,869.
14) Purnell, J.H. and Bohemen,J., J. Chem. Soc. Vol-2,1961,2030.
15)  Desty D.H.and  Goldup A., J.Gas Chromatography, V0l-3,1960,162.
16)  Giddings,J.C., J.The Dynamics of Chromatography, Vol-22,1965,265.
17)  Giddings,J.C.,  J. Chromatogr. Science,Vol-1,1960,19
18)  Scott,R.P., J. Anaytical Chemistry., Vol-44,1962,1198.
19)  Davis, J.M. and Giddings,J.c., J.  Analytical  Chemistry,V0l-8,1983,203.

5.1 Advantages of UPLC

 High resolution
 High speed
 It is up to 9 times faster, has up to twice the resolution and three times the sensitivity than that of HPLC
 Quick and ease of instrument handling.
 High sensitivity.
 Even neon particles can be separated easily.

5.2 Application of UPLC

 From injection of a poly-drug reference standard and whole blood extract, separation and identification of amphetamine, methamphetamine, ephedrine, pseudoephedrine, phentermine, MDA, MDMA, MDEA and ketamine in less than 3 min using the UPLC method

 Uplc is used for studying hydrolysis kinetics of CL-20 and related energetic compounds.

 Simultaneous Determination of Tetracycline’s and Quinolones Antibiotics in Egg can be done by Ultra-Performance Liquid Chromatography–Electro spray Tandem Mass Spectrometry

 Determination of Coumarone in Food can also be done using Ultra-Performance Liquid Chromatography-Electro spray-Tandem Mass Spectrometry .

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