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  • Dr. Babasaheb Ambedkar Marathwada University.sir Sayyed college Aurangabad Maharashtra India .


UPLC is an advance technique of liquid chromatography where it takes advantage of innovation in various technologies such as instrumentation and particle size to achieve dramatic increases in resolution, speed and sensitivity of the liquid chromatography. It operates at higher pressure than that used in HPLC and uses fine particles (less than 2.5μm) & mobile phases at high linear velocities. UPLC Technology is now applied throughout the world produce quality data with reproducible and robust methods as compared to the conventional HPLC. UPLC can be hyphenated with other techniques such as Mass spectrometer (MS), Ion chromatograph (IC), Nuclear magnetic resonance spectrometer (NMR) and Infrared spectrometer (IR) etc. This technique provides unique end-to-end solutions for all industries and has found application in various fields such as pharmaceutical, food, environmental, forensic, toxicology and pesticide. Vol 5, Issue 3, 2016.
Shaikh et al. World Journal of Pharmaceutical Research
Dr. Sayyed Hussain and Tabrez Shaikh*
Sir Sayyed College, P.G. Department of Chemistry, Roshangate, P.B.No.89, Aurangabad,
UPLC is an advance technique of liquid chromatography where it takes
advantage of innovation in various technologies such as
instrumentation and particle size to achieve dramatic increases in
resolution, speed and sensitivity of the liquid chromatography. It
operates at higher pressure than that used in HPLC and uses fine
particles (less than 2.5µm) & mobile phases at high linear velocities.
UPLC Technology is now applied throughout the world produce
quality data with reproducible and robust methods as compared to the
conventional HPLC. UPLC can be hyphenated with other techniques
such as Mass spectrometer (MS), Ion chromatograph (IC), Nuclear
magnetic resonance spectrometer (NMR) and Infrared spectrometer
(IR) etc. This technique provides unique end-to-end solutions for all industries and has found
application in various fields such as pharmaceutical, food, environmental, forensic,
toxicology and pesticide.
KEYWORD: UPLC, Theory, Detectors, Advantage, Applications.
From past 30 year HPLC is the predominant technique to use for analysis in Laboratory but
due to significant advances and innovation in instrumentation, detector design, data
processing and particle size technology, leads to the development of Ultra Performance
Liquid Chromatography (UPLC). Principle of UPLC basically remains same only with the
help of technology; it achieves dramatic increases in resolution, speed and sensitivity of the
liquid chromatography. UPLC Technology is applied throughout the world for providing
World Journal of Pharmaceutical Research
SJIF Impact Factor 6.805
Volume 5, Issue 3, 387-394. Review Article ISSN 2277 7105
Article Received on
22 Dec 2015,
Revised on 13 Jan 2016,
Accepted on 03 Feb 2016
*Correspondence for
Tabrez Shaikh
Sir Sayyed College, P.G.
Department of Chemistry,
Roshangate, P.B.No.89,
Aurangabad, India. Vol 5, Issue 3, 2016.
Shaikh et al. World Journal of Pharmaceutical Research
advantage of time, cost and quality. Beside this it is also reproducible result and robust
method as compared to conventional HPLC.
Separation in chromatography can be explained by Van Deemter equation which gives
relation of resolving power of chromatographic column with various flows and Kinetic mass
HETP = A + B/u + Cu,
HETP is height equivalent to theoretical plate
A is eddy diffusion
B is longitudinal diffusion
C is mass transfer and
u is linear velocity (flow rate)
Eddy diffusion (A) is caused by a turbulence in the solute flow path and is mainly unaffected
by flow rate, but changes with particle size. Smaller the particle size less is the eddy
diffusion. Longitudinal diffusion (B) is related the movement of an analyte molecule outward
from the center to the edges of its band. Thus higher the lineary velocities will limit the
outward distribution, keeping the band tighter. Mass transfer (C) is the movement of analyte,
or transfer of its mass, between the mobile and stationary phases. Through this type of
diffusion, increased flows have been observed to widen analyte bands or lower peak
efficiencies. [2]
Fig. 1 - Van Deemter plots for various particle sizes Vol 5, Issue 3, 2016.
Shaikh et al. World Journal of Pharmaceutical Research
Fig-1 indicates that the decrease in particle size results in an increase in efficiency of column
and on the other hand increase in linear velocity (flow rate) increase the efficiency for the
column for particle size less than 1.9 µm and after the optimized flow it remains same, while
for column with particle size greater than 1.9µm, efficiency again decrease after certain
optimized flow. [3]
Therefore, using smaller particles, speed and peak capacity (number of peaks resolved per
unit time) can be extended to new limits also decrease in the internal diameter of the column
will require a less flow rate which is known as Ultra Performance. Larger diameter columns
require higher flow rates, and thus larger volumes of mobile phase, to reach the desired linear
velocity therefore UPLC have column with less diameter.
The Basic principle and instrumentation of UPLC system remains same as that of HPLC but
only differs in upgrading instrumentation and hardware. The UPLC System consists of a
binary solvent system, sample manager, column manager and detector. The solvent manager
uses two flow pumps to deliver a parallel binary gradient which is mixed under high pressure.
[4] Degassing system degasses the mobile phase, which is selected by valve up to four solvent.
UPLC system can withstand pressure of about 15,000 psi (about 1000 bar) to take full
advantage of the sub-2-mm particles.[5] The sample manager is also having advance
technology where sample temperature can be taken to as low as 0°C [6] whereas column
manger can manage the column temperature up to 90°C using high temperature ultra
performance liquid chromatography (UPLC), it is possible to drastically decrease the analysis
time without loss in efficiency.[7]
Fig. 2 Schematic diagram of UPLC Vol 5, Issue 3, 2016.
Shaikh et al. World Journal of Pharmaceutical Research
Detectors such as UV/Visible, Photodiode array (PDA), Evaporative light scattering (ELS),
Refractive index (RI) and Fluorescence (FLR) are commonly used with UPLC. Beside these
detectors, capability of UPLC can be greatly increased by hyphenating instrument with other
technique such as mass spectrometer, ion chromatograph, nuclear magnetic resonance
spectrometer, inductive coupled plasma-mass spectrometer and Infrared spectrometer.
Ultra-violet/visible (UV)
This detector are used for organic compound which absorb the light in the range of 190 to
800 nm This detector can be tuned to specific wavelengths in UV or Visible range for
detection. It provides performance benefits for both routine and complex analyses in
pharmaceutical, life science, environmental, agricultural and petrochemical applications.
Photodiode Array (PDA) detector
This detector offers simultaneous advanced optical detection in the range of 190 to 800 nm. It
provides unprecedented trace impurity detection and quantitation with spectral analysis
capabilities. Definitive compound identification and co-elution detection with simultaneous
2D and 3D operation. This detector finds major application in drug discovery and
pharmaceutical development.
Fluorescence (FLR) Detector
For sensitivity and selectivity to fluorescence-based applications, this detector is used. It
extends the benefits of UPLC technology for the analysis of polynuclear aromatic
hydrocarbons (PAHs), drugs of abuse, and vitamins any component with chemiluminescent
properties, such as fluorescence or phosphorescence.
Refractive index (RI) detector
RI is a universal detector which is used where chemical is having no or limited UV
absorbance. These include alcohols, sugars, fatty acids, excipents, raw material and
pharmaceutical drug products. Beside this characterization of low molecular weight polymers
also finds application on UPLC. Main disadvantage of this detector is that it lacks sensitivity.
Mass detector (MS)
UPLC can be coupled with mass spectrometrer (MS) and tandem mass spectrometer (MS-
MS) detector which find application in various fields and is used for identification, Vol 5, Issue 3, 2016.
Shaikh et al. World Journal of Pharmaceutical Research
quantitation and mass analysis of materials. Also, structural elucidation of unknown molecule
can be found out by fragmentation. This detector have various mass analyzers depending
upon their application some of analyzer are Single quadrupole, Triple quadrupoles (Tandem),
Ion trap and Time of flight (TOF). These detectors provide very high sensitivity, selectivity
and time resolution.
Beside these detector many other detectors can be hyphenated to UPLC such as Infrared (IR),
Inductive Coupled plasma mass spectrometry (ICP-MS), nuclear magnetic resonance (NMR)
and Evaporative light scattering detector (ELSD), Electrochemical detector (EC)
UPLC has many advantages over conventional HPLC of which major advantages are speed,
quality and cost of analysis. Due to development of particle size technology and the use of
sub micron particle size in column packing, significantly reduced in the analysis run time
which result in the development of faster methods and faster analysis of samples. Compound
separated on UPLC are more resolved as compared to conventional HPLC also peak capacity
is also increased (number of peaks resolved per unit time). Sensitivity of the method is
increased up to 3- 5 fold. Band spreading is reduced due to the population of analyte
molecule is more concentrated in UPLC resulting in a higher plate count. Following figure-3
show the comparison of UPLC and HPLC chromatogram.
Fig. 3 Chromatographic separation comparison on UPLC and HPLC Vol 5, Issue 3, 2016.
Shaikh et al. World Journal of Pharmaceutical Research
The time spent in optimizing and validating new methods is greatly reduced with the use of
UPLC. Mobile phase solvent consumption for the analysis is greatly reduced due to low flow
rate and short run time. Reduces process cycle times, so that more product can be produced
with existing resources. Real time analysis by UPLC reduced the cost of failure of product
and process control. UPLC system can be hyphenated with various techniques for which it
finds application in vast areas. Conventional HPLC are nowadays are replaced with UPLC
considering greater commercial benefit, superior sensitivity, resolution, speed and sample
Pharmaceutical analysis
UPLC finds major application in pharmaceutical analysis. Methods use in drug substances
and drug product analysis should be well developed and validated and these processes are
much time consuming on conventional HPLC. UPLC gives scope for development and
validation of analytical methods in less time. In the drug development impurity profiling is
major activity where detecting and quantifying impurities in drug substance and drug product
can be done by UPLC as it has good resolving power, reproducibility, efficiency and short
time. As UPLC provides faster analysis of samples, this advantage can be use to monitor the
inprocess and real time samples of reaction monitoring where very short time is required to
control the processes thus saving the cost of failure. UPLC also finds application in
dissolution testing, which is one of very important test in the formulation of drug product.
This test requires uniformity reliability and batch to batch reproducibility. Metabolites studies
are required in the new drug development process where main metabolite is determined and
identified as rapidly as possible in the drug discovery phase. UPLC is capable of determining
and identifying metabolite and biomarker structure elucidation.
Environmental analysis
Environmental sample requires innovative techniques to detect and identify the chemical
contamination. UPLC provides the analysis of these samples with less time, cost and more
information about sample content. Some applications of UPLC are analysis of organic
component in soil, air, hazardous wastes, drinking water, wastewater, pesticide residue and
perfluorinated compounds (PFCs) analysis. Vol 5, Issue 3, 2016.
Shaikh et al. World Journal of Pharmaceutical Research
Food analysis
Food manufacturer are always in search of a compressive solution for food testing thus UPLC
decrease operation cost, increase productivity and provide identification of diverse chemicals
in a food sample, thus providing public safety. Beside this it also offers quality and
consistency of the product. UPLC is also applied for food profiling, identification of natural
toxins, vitamins and pesticide residue in food product.
Forensic and Toxicological
UPLC finds good application in the identification of drug of abuse from blood, urine and oral
samples. Several cannabinoids, opioids, barbiturates can be identified and analysed by
UPLC. The combination of UPLC with various instruments gives the unique benefit of drug
screening with excellent sensitivity and accuracy at trace level.
UPLC is a powerful emerging technique which by use of advances in instrumentation and
particle technology increases the productivity for pharmaceutical and other industries. This
technique provides the better resolution for separating component, high sensitivity for the
analysis of low concentration component and reduces time of analysis. As solvent
consumption for this technique is less it also reduces the cost of analysis. The method can be
well developed and validated in less time on this system. This technique can be easily
hyphenated with various other techniques such as mass spectrometry, UPLC finds versatile
application for impurity profiling, metabolite identification, dissolution testing and process
control analysis in pharmaceutical, food, environment, forensic and Toxicological areas.
1. Swartz, Michael E. "UPLC™: an introduction and review." Journal of Liquid
Chromatography & Related Technologies 2005; 28(7-8): 1253-1263
2. Zhang, Bin, Xiaofeng Li, and Bing Yan. "Advances in HPLC detection - towards
universal detection." Analytical and bioanalytical chemistry 2008; 390(1): 299-301.
3. Wren, Stephen AC, and Pierre Tchelitcheff. "Use of ultra-performance liquid
chromatography in pharmaceutical development." Journal of Chromatography A 2006;
1119(1): 140-146.
4. Swartz, Michael E., and Brian Murphy. "New frontiers in chromatography." Am. Lab
2005; 37: 22-27. Vol 5, Issue 3, 2016.
Shaikh et al. World Journal of Pharmaceutical Research
5. Wu, Naijun, and Richard Thompson. "Fast and efficient separations using reversed phase
liquid chromatography." Journal of liquid chromatography & related technologies 2006;
29(7-8): 949-988.
6. Wales, Thomas E., et al. "High-speed and high-resolution UPLC separation at zero
degrees Celsius." Analytical chemistry 2008; 80.17: 6815-6820.
7. Nguyen, Dao T-T., et al. "High throughput liquid chromatography with sub-2μm particles
at high pressure and high temperature." Journal of Chromatography A 2007; 1167.1:
... Kromatografi Cair Kinerja Ultra-Massa Tandem (UPLC) adalah teknik lanjutan kromatografi cair yang memanfaatkan inovasi dalam berbagai teknologi seperti instrumentasi dan ukuran partikel untuk mencapai peningkatan dramatis dalam resolusi, kecepatan, dan sensitivitas kromatografi cair. Ini beroperasi pada tekanan yang lebih tinggi daripada yang digunakan dalam HPLC dan menggunakan partikel halus (kurang dari 2,5μm) & fase gerak pada kecepatan linier tinggi (Hussain et al., 2014). ...
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Paracetamol merupakan obat pereda nyeri dan analgesik yang paling sering digunakan oleh masyarakat. Pemeriksaan konsentrasi obat dalam sampel biologis dengan menggunakan metode yang tepat diperlukan untuk memastikan kualitas obat dan mengoptimalkan terapi obat. Oleh karena itu, studi literatur ini dilakukan dengan tujuan untuk menentukan kadar paracetamol dalam sampel biologis dan menguji validitasnya dengan berbagai metode. Penelitian literatur dilakukan dengan mencari beberapa artikel jurnal penelitian yang sudah diterbitkan pada tahun 2013-2023, yang dipilih sesuai dengan kriteria inklusi dan eksklusi. Diperoleh 6 jurnal mengenai validasi metode analisis penetapan kadar paracetamol dalam sampel biologis dengan berbagai metode seperti Kromatografi Gas Spektrometri Massa (GC-MS), Kromatografi Cair Spektrometri Massa Tandem (LC-MS/MS), Spektrometri massa-kromatografi cair kinerja tinggi (HPLC-MS), dan Kromatografi Cair Kinerja Sangat Tinggi Spektrometri Massa (UHPLC-MS). Dari tinjauan diatas dapat disimpulkan bahwa metode analisis senyawa parasetamol dalam sampel biologis dapat dilakukan dengan keempat metode tersebut dimana hasil dari metode analisis yaitu akurasi, selektivitas, linieritas, presisi, LOD, dan LOQ semuanya memenuhi persyaratan atau kriteria yang ditetapkan.
... Higher sample throughput with more information per sample may decrease the time to market, an important driving force in today's pharmaceutical industry 7,8 . UPLC is equipped with detectors such as Tunable Ultraviolet detector, Photodiode array detector, Fluorescence detector, Refractive index detector and mass detector 9,10 . ...
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Objectives: Various analytical techniques are applied in pharmaceutical field to estimate the quality of active pharmaceutical ingredients, amount of drug in biological fluids and in formulations. The aim of this review article is to provide utmost existing analytical methods for analysis of dihydropyridines based calcium channel blockers for estimation of Amlodipine, Lacidipine, Isradipine, Nifedipine, Felodipine, and Cilnidipine in pure form, biological fluids (like Human Plasma, Human Serum, Human Urine etc.,) and its related formulations including novel formulations. Dihydropyridines based Calcium Channel blockers is a major chemical class of drugs used in the treatment of hypertension and various coronary artery diseases. Evidence acquisition: Current analytical techniques available for active pharmaceutical ingredients and its related formulations are tabulated with extensive method conditions which can be used in analysis of dihydropyridines based calcium channel blockers drugs outlined from official pharmacopoeias and other relevant research articles. Conclusion: Various analytical techniques such as HPLC, HPTLC, UPLC, GC, LC-MS, and GC-MS are involved. This review assists in appropriate selection of analytical technique, solvent, mobile phase, column, detector based on available analytical instruments and chemicals, by referring tabulated extensive method conditions. It can be implemented in quality control and quality assurance department for quality assessment of diverse pharmaceutical formulations.
... However, quantitative analytical methods of cyclophosphamide and 4-OHCP in DBS simultaneously using UPLC-MS/MS have never been done before. UPLC is a technical advancement of liquid chromatography where there are innovations in instrumentation and particle size to improve the resolution, speed, and sensitivity of the method [8]. 4-OHCP metabolites are unstable in biological fluids such as in plasma, which has a half-life of 6 min [7]; this can be overcome by performing derivatization procedures prior to analysis [7,9,10]. ...
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Objective: To develop the method for the simultaneous analysis of cyclophosphamide and 4-hydroxycyclophosphamide (4-OHCP) in Dried Blood Spot (DBS) using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) and its application in breast cancer patients for therapeutic drug monitoring. Methods: Sample preparation used protein precipitation with methanol and acetonitrile (2:1 v/v). The separation was conducted using 1.7μm (2.1 x 100 mm) Waters AcquityTM UPLC C18 column; mobile phase consists of 0.01% formic acid and methanol (50:50 v/v) with isocratic elution, column temperature 30 °C, flow rate 0.3 ml/min and hexamethylphosphoramide (HMP) used as an internal standard. Analysis was performed by a triple quadrupole mass spectrometry with a positive ion mode of Electrospray Ionization. Cyclophosphamide was detected at m/z 260.968>139.978, 4-OHCP at m/z 338.011>224.979, and HMP at m/z 180.17>92.08. The method was applied to quantify cyclophosphamide and 4-OHCP in DBS of breast cancer patients. Blood samples were collected at 2 and 4 h after cyclophosphamide administration for therapeutic drug monitoring. Results: The method was linear in the range of 50–30.000 ng/ml for cyclophosphamide and 10–1000 ng/ml for 4-OHCP. Lower Limit of Quantification (LLOQ) concentration of cyclophosphamide was 50 ng/ml and 4-OHCP was 10 ng/ml. Accuracy and precision within-run and between-run met the requirements with % diff and CV, not exceeding ±15% and not more than ±20% for LLOQ concentration. The results from DBS samples of cancer patients showed that the level of cyclophosphamide was in the range of 6045.980 ng/ml to 37024.403 ng/ml and 4-OHCP was in the range 33.155 ng/ml to 246.362 ng/ml. Conclusion: The developed method met the requirements of all validation parameters under the Guideline on Bioanalytical Method Validation by the European Medicines Agency in 2011. Method can be applied on DBS of cancer patients and the results showed that cyclophosphamide and 4-OHCP was detected on 17 samples of breast cancer patients. This can be one of the parameters for therapeutic drug monitoring.
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Background Cyclophosphamide (CPA) is a cytotoxic prodrug that needs to be metabolized by cytochromes P450 enzymes, like CYP2B6. Unfortunately, CYP2B6 is a very polymorphic enzyme and can cause a change in 3-hydroxypropyl mercapturic acid (3-HPMA), the most found CYP metabolite in urine levels. Change in 3-HPMA levels can also indicate the level change in its precursor, acrolein, which is responsible for the hematuria incidence after CPA administration. This review’s purpose is to obtain a conclusion about the optimal 3-HPMA analysis method in urine after the administration of cyclophosphamide using liquid chromatography-tandem mass spectrometry (LC-MS/MS) through literature review from previous studies. Also, this review was written to examine the relationship between levels of 3-HPMA in urine, polymorphisms of CYP2B6 enzymes, and the incidence of hematuria after cyclophosphamide administration in cancer patients. Methods Major databases, such as Universitas Indonesia’s library database ScienceDirect, PubMed/Medline, Frontiers Media, and Google Scholar database, were used to find both published and unpublished studies without a time limit until 2020. Studies on pharmacokinetics, pharmacodynamics, drug therapy monitoring of cyclophosphamide, bioanalysis, and polymerase chain reaction (PCR) published in Indonesian and English were included. Meanwhile, non-related studies or studies written in other languages besides Indonesian and English were excluded. Two independent reviewers screened the titles, abstracts, and full-text manuscripts. Data obtained from eligible sources were used to answer the purpose of this review in a narrative form. Results The authors found 436 related studies from various databases and websites. Then, the authors narrowed it down into 62 pieces of literature by removing the duplicates and reviewing the abstracts and full-text manuscripts. Out of 62 sources, the authors found 30 studies that explained 3-HPMA analysis using LC/MS-MS, CYP2B6 polymorphisms, and hematuria occurrences. The authors used those 30 studies to build a conclusion regarding the purpose of this study. We strengthened the results with some additional information from the other 32 eligible sources. Conclusions The authors conclude that according to literature searches from previous studies, the optimal 3-HPMA analysis method in urine after cyclophosphamide administration using LC-MS/MS is using triple quadrupole LC-MS/MS; source of positive ion electrospray ionization (ESI); mobile phase combination of 0.1% formic acid in water (A) - 0.1% formic acid in acetonitrile (90:10 v/v) (B); the Acquity® BEH C18 column (2.1 x 100 mm; 1.7 μm); injection volume of 10 μl; flow rate of 0.2 ml/minute; gradient elution method. Detection was carried out using mass spectrometry with m/z ratio of 222.10 > 90 for 3-HPMA and m/z 164.10 > 122 for n-acetylcysteine (NAC). The optimal sample preparation method is acidification and dilution ratio of 1:5 v/v. Also, there is a relationship between 3-HPMA levels, CYP2B6 polymorphisms, and the occurrences of hematuria after the administration of cyclophosphamide, which is a type of CYP2B6 polymorph, namely CYP2B6*6, can increase cyclophosphamide hydroxylation so that it can increase the levels of acrolein and 3-HPMA, as its metabolites, and risk of hematuria. Ethics and dissemination This research does not use human participants, human data, or human tissue for being directly studied for the review. Therefore, ethics approval and consent to participate are not applicable. Registration This research has not been registered yet.
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The advances made in the field of high performance liquid chromatography (HPLC) detection has influenced the general progress and the extent of application of HPLC technology. HPLC detection has become the most widely applied analytical separation techniques because of its superior performance and reliability, especially in the pharmaceutical, environmental, forensic, clinical, food and flavor sciences. Extensive investigations into the use of refractive index (RI) detectors, evaporative light scattering detectors (ELSD) and chemiluminescence nitrogen detectors (CLND) as universal detectors for HPLC have been performed. ELSD detectors detect all the compounds that are less volatile than the mobile phase. ELSD can therefore offer distinct advantages over conventional UV or RI detection, particularly for gradient separations. The response of the ELSD is related to the absolute quantity of the compound and is independent of the analytes' optical properties.
The Ultra Performance Liquid Chromatography (UPLC™), capable to run separations using shorter columns and higher flow rates for increased speed with superior resolution and sensitivity, is presented. In order to provide enhanced mechanical stability to UPLC, a second generation bridged ethane hybrid (BEH) technology, ACQUITY BEH, was developed. All ACQUITY BEH columns are equipped with eCord™ microchip technology and facilitates the manufacturing informations, quality control tests, and certificate of analysis for each columns. The ACQUITY UPLC™ system helps in e-CORD databases updating and in order to maintain a complete column history, provide real-time informations about number of injections and pressure.
Liquid chromatography has seen a dramatic increase in speed and efficiency over the past decade. The advances in separation speed have been mostly related to the development of column technology and instrumentation. The column technology includes small uniform particles, monolithic columns, and thermally stable phases with various bonding chemistries. Relatively short columns packed with sub‐2 µm particles provide high speed and efficient separations. The high porosity and small skeleton size of monolithic columns permit operation at high flow rates on relatively long columns, using a conventional LC system for high speed and high efficiency separation. The new instrumentation is associated with ultra‐high pressure pump systems and high temperature systems. Ultra‐high or very high pressure pump systems have been used to overcome the high pressure drop generated by small particles. High temperature liquid chromatography allows fast separation using high linear velocities and stable stationary phases. In this review, ultra‐high pressure liquid chromatography, ultra‐performance liquid chromatography, monolithic columns, and high temperature liquid chromatography are discussed as means for attaining fast and efficient separations. Minutes to sub‐minute separations for various samples are demonstrated using these technologies.
Ultra performance liquid chromatography™ (UPLC) takes advantage of technological strides made in particle chemistry performance, system optimization, detector design, and data processing and control. Using sub‐2 µm particles and mobile phases at high linear velocities, and instrumentation that operates at higher pressures than those used in HPLC, dramatic increases in resolution, sensitivity, and speed of analysis can be obtained. This new category of analytical separation science retains the practicality and principles of HPLC while creating a step‐function improvement in chromatographic performance.This review introduces the theory of UPLC, and summarizes some of the most recent work in the field.
The conformational properties of proteins can be probed with hydrogen/deuterium exchange mass spectrometry (HXMS). In order to maintain the deuterium label during LC/MS analyses, chromatographic separation must be done rapidly (usually in under 8-10 min) and at 0 degrees C. Traditional RP-HPLC with approximately 3-mum particles has shown generally poor chromatographic performance under these conditions and thereby has been prohibitive for HXMS analyses of larger proteins and many protein complexes. Ultraperformance liquid chromatography (UPLC) employs particles smaller than 2 mum in diameter to achieve superior resolution, speed, and sensitivity as compared to HPLC. UPLC has previously been shown to be compatible with the fast separation and low temperature requirements of HXMS. Here we present construction and validation of a custom UPLC system for HXMS. The system is based on the Waters nanoACQUITY platform and contains a Peltier-cooled module that houses the injection and switching valves, online pepsin digestion column, and C-18 analytical separation column. Single proteins in excess of 95 kDa and a four-protein mixture in excess of 250 kDa have been used to validate the performance of this new system. Near-baseline resolution was achieved in 6-min separations at 0 degrees C and displayed a median chromatographic peak width of approximately 2.7 s at half-height. Deuterium recovery was similar to that obtained using a conventional HPLC and ice bath. This new system represents a significant advancement in HXMS technology that is expected to make the technique more accessible and mainstream in the near future.
Ultra-performance liquid chromatography (UPLC) has been investigated as an alternative to HPLC for the analysis of pharmaceutical development compounds. We present data on three compounds showing that significant reductions in separation time can be achieved without compromising the separation quality. Results from precision and comparative studies indicate that UPLC is a suitable technique for routine pharmaceutical analysis.
In this study, ultra performance liquid chromatography (UPLC) using pressures up to 1,000 bar and columns packed with sub-2 microm particles has been combined with high temperature mobile phase conditions (up to 90 degrees C). By using high temperature ultra performance liquid chromatography (HT-UPLC), it is possible to drastically decrease the analysis time without loss in efficiency. The stability and chromatographic behavior of sub-2 microm particles were evaluated at high temperature and high pressure. The chromatographic support remained stable after 500 injections (equivalent to 7,500 column volumes) and plate height curves demonstrated the capability of HT-UPLC to obtain fast separations. For example, a separation of nine doping agents was performed in less than 1 min with sub-2 microm particles at 90 degrees C. Furthermore, a shorter column (30 mm length) was used and allowed a separation of eight pharmaceutical compounds in only 40s.