INTRODUCTION:

Extensive research in biochemistry, drug discovery, environmental testing and even space research has increased the speed for high performance analytical equipment. Each new improvement offers much more than its predecessors. Greater emphasis on quality control in manufacturing processes and prospects of a vibrant recovering economy are also creating a positive impact.

Traditional analytical approaches including HPLC, GC, UV etc have become insufficient to complete the challenges in specific and sensitive analysis.

Advantages of advanced analytical instrumentation:

· Shorter analysis time

· Higher automation

· High-throughput

· Better reproducibility

· Reduction in contamination

SAMPLE PREPARATION:

Despite the development of highly efficient analytical instrumentation for the end point determination of analytes in pharmaceutical products and biological samples, sample pretreatment is usually necessary to extract, isolate and concentrate the analytes of interest from complex matrices.

In general, sampling and sample preparation steps constitute over 80 % of the total analysis time, and these steps are important in determining the success of analyzing compounds of interest in complex matrices such as biological samples. Therefore, it is not an exaggeration to say that the choice of an appropriate sample preparation method greatly influences the reliability and accuracy of the analysis.¹

Solid-Phase Extraction (SPE):

Solid-phase extraction (SPE) is a widely used sample preparation technique that is fast, safe, easy to perform, providing excellent recovery and reproducibility, automatable, and can be used with small amounts of sample and solvent.

Micro-extraction Techniques:

§ Solid-phase micro extraction (SPME)

§ On-line trace enrichment

§ Matrix solid-phase dispersion (MSPD)

§ Column Switching

§ Automated on-line SPE

SPE is an extraction technique based on the selective partitioning of one or more components between two phases. SPE is based on the partitioning of compounds between a liquid (sample) phase and solid (extraction) phase whereby the intermolecular forces between the phases influence retention and elution. Various SPE products are now available, such as column cartridges, disks, well plates and microfibres.

The new sample-preparation technique of in-tube SPME uses an open tubular capillary as an SPME device. It can be coupled on-line with HPLC or LC/MS. Although the technique using a GC capillary tube is also known as open-tubular trapping; it is coupled on-line with GC. In-tube SPME is suitable for automation and extraction. Desorption and injection can also be done continuously using a standard auto sampler.²

Liquid - Liquid Extraction (LLE):

LLE for drug analysis has recently become semi automated and multi well plates can now be used. A fully automated, high throughput LLE method for preparing biological samples using a 96-well LLE plate and a 96-channel robotic liquid handling workstation is also used now days. The extraction time is also relatively short. Liquid-phase micro extraction (LPME) is a new solvent minimized sample preparation technique that is quick, inexpensive and minimizes exposure to toxic organic solvents. It is compatible with capillary GC, CE and HPLC.

ANALYTICAL TECHNIQUES

High Performance Liquid Chromatography - (HPLC)

High-Performance Liquid Chromatography (HPLC) is one of the most powerful analysis tools widely used. Automation, integrating HPLC with powerful data acquisition and reduction system and laboratory robotics has further improved precision and accuracy of drug analysis by HPLC.

New Advances in HPLC:

· Monolithic reversed-phase (RP) silica columns have received significant attention for achieving faster analysis times using higher flow rates than are accessible with typical packed columns.

· High temperature liquid chromatography (HTLC) is performed at temperatures exceeding 100˚C. The main advantage of HTLC is that chromatographic run times can be greatly reduced.³

· Microchip HPLC System: It is conceivable that the need for HPLC miniaturization will lead to realization of constructing of a complete HPLC system on a single chip, in which unnecessary system fluidic swept/dead volume can be greatly reduced.

· Micro bore Liquid Chromatography and Nano Liquid Chromatography is latest technology in chromatographic field. These are useful in determination of impurity peaks, which are not detectable by conventional liquid chromatography. These techniques have important contribution in new drug discovery, bioavailability and bioequivalence studies, and toxicity studies.

Miceller Electro Kinetic Chromatography (MEKC)-It is mainly useful for separation of neutral molecule by miceller interaction with help of surfactant.

Over pressured Layer chromatography (OPLC):

Of the three forced flow planar chromatography techniques, the novel one is OPLC (Overpressured Layer chromatography). For this technique a specially prepared TLC plate is covered by a flexible inert sheet, which is subjected to over pressure and the mobile phase, is then pumped through the sorbent layer.

OPLC serves as a bridge between planar and liquid column chromatography, since it can be used either as a fully offline or fully online technique.⁴

Turbulent flow chromatography (TFC):

Turbulent flow chromatography (TFC) is an online application that enables quantitative determination without prior sample clean up. A tandem-mass spectrometric end point (TFC-MS/MS) is pre dominantly used for analysis of various biofluids. The ability to dilute and inject samples online with minimum intervention reduces the preparation time necessary and consequently offers a time saving advantage over more labor-intensive liquid-liquid extraction or solid phase extraction techniques. Recently, TFC operating in a dual column (sample extraction and isocratic focusing) mode has utilized a monolithic column compatible with high flow rate and low back pressures, to reduce cycle times to shorter than 1.5 min.⁵

Ultra Performance Liquid Chromatography (UPLC)

The recent introduction of ultra performance Liquid Chromatography (UPLC) instrumentation capable of providing liquid flow at pressures up to 1034 bar and the availability of columns packed with 1.7 mm particles able to withstand these pressures, extends the limits of what has been thus far achievable on commercial HPLC instrumentation.⁶

UPLC have made available reverse phase chromatography media with sub-2 mm particle size along with liquid handling systems that can operate such columns at much higher pressures. This technology offers significant theoretical advantages in resolution, speed, and sensitivity for analytical applications, particularly when coupled with mass spectrometric capable of high-speed acquisitions. Some experiments using UPLC-MS/MS and HPLC-MS/MS have shown that the UPLC-MS/MS improved cycle time by 50 – 100 % with increased sensitivity.⁷

Capillary Electrophoresis (CE):

Capillary electrophoresis has the advantage of decreased separation time, decreased sample consumption and increased resolution. On-chip capillary electrophoresis devices fabricated with semiconductor micro fabrication technology offer further advantages. Among these are miniaturizing devices, further reduction of analysis time, and the possibility of on-chip manipulation such as multi-channel capacity.⁸ Of particular note, is the development, which has occurred in combining the more advanced capillary-based electromigration separation techniques, such as capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF), capillary isotachophoresis (CIT), micellar electro kinetic chromatography (MEKC) and capillary electrochromatography (CEC), with ES/MS. The union of these electromigration schemes with MS detection provides a useful and sensitive analytical tool for the separation, quantitation and identification of biological, therapeutic, environmental and other important classes of chemical analytes.⁹

HYPHENATED TECHNIQUES:

A hyphenated technique in analytical chemistry is the marriage of two separate analytical techniques via appropriate interfaces.

In general, the term hyphenated technique ranges from the combination of separation- separation, separation-identification and identification-identification techniques.¹⁰

Gas chromatography-Mass spectrometry (GC-MS):

Advances in GC-MS technology have improved detection limit in complex samples. The ability to adjust the sensitivity requirement to match the analysis requirement makes the system useful for a wide range of application.

Vehicle –portable and man-portable GC-MS techniques are undergoing rapid development. Vehicle-portable GC-MS is expanding into area of temporally and spatially resolved mapping. ¹¹

Gas chromatography-Infra red spectroscopy (GC-IR):

GC-IR is a coupled technique in which the GC does the separating and FTIR does the identifying. It is used for separating and identifying the components of difficult mixtures.

GC-FTIR is used for samples that are very small physically. Detection limit of GC-FTIR systems are usually between 10 and 100 ng. ¹⁰

Due to complementary nature of IR and MS data, more GC peaks can be identified when GC-IR and GC-MS are used together in combination as GC-IR-MS.

Liquid chromatography-Mass spectrometry (LC – MS) and LC-MS/MS:

In pharmaceutical industry LC-MS has become method of choice in many stages of drug development. Recent advances in electro spray, thermo spray, and ion spray ionization techniques offer unique advantages of high detection sensitivity and specificity. Liquid secondary ion mass spectroscopy, laser mass spectroscopy with 600 MHz offer accurate determination of high mol.wt. (>10000 Da) proteins and peptides. Isotope pattern can be detected by this technique.

Atmospheric Pressure Laser Ionization:

Atmospheric Pressure Laser Ionization (APLI) is introduced as a powerful addition to existing atmospheric pressure (AP) ionization techniques, in particular APCI, ESI and APPI replacing the one step approach in APPI, with stepwise two photon ionization strongly enhancing the selectivity of the ionization process.

Furthermore, the photon flux during an ionization event is drastically increased over that of APPI, leading to very low detection limit. In addition, the APLI mechanism generally operates primarily directly on the analyte. This allows for very efficient ionization even of non-polar compounds.¹²

Capillary LC-MS/MS:

A novel capillary LC-MS/MS system combines advances in gradient delivery, flow control, sample injection, ion source, interface design and mass spectrometric detection. Sensitivity can be increased as much as 6 times compared to conventional LC-MS/MS system with 3 time’s faster throughput and dramatic reduction of solvent consumption.

Nanospray LC-MS:

Nanospray is the derivative of the electro spray LC-MS technique. Nanospray is more sensitive than ESI. Nanospray sprays on nL/min scale. It forms ions more readily. It also enhances sensitivity and increased ion signal. No heat is required for ionization. Wide range of liquid mixtures can be used from molecules to macromolecules. Nano LC-MS technique is commonly used for separating protein and peptide mixture. It has greater signal to noise ratio.

2-D online or offline liquid chromatography/ mass spectrometry:

It is state of the art for identification of proteins from complex proteome samples in many laboratories. Both 2-D liquid chromatography methods use two orthogonal liquid chromatography separation techniques. The most commonly used techniques are strong cation exchange chromatography for the first dimension and reversed phase separation for the second dimension.¹³

High Performance Liquid Chromatography - Nuclear Magnetic Resonance Spectroscopy (HPLC – NMR):

The combination of chromatographic separation techniques with NMR spectroscopy is one of the most powerful and timesaving methods for the separation and structural elucidation of unknown compounds and mixtures, especially for the structure elucidation of light and oxygen sensitive substances. The on-line LC-NMR technique also allows the continuous registration of time changes as they appear in the chromatographic run. Automated data acquisition and processing in HPLC-NMR improves speed and sensitivity of detection.¹⁴

FUTURE OF ANALYTICAL WORLD:

Currently the most techniques for trace element analysis include a combination of separation technique coupled with a detection technique that is more sensitive. Online coupling of two techniques have become increasingly common.

Environmental and biological research users are already using these types of instruments in varied applications. Commercially these equipments are expected to find demand as well. Research and manufacturing in areas like Chemical, petrochemical, food and beverages, plant and animal biochemistry, clinical chemistry etc will be more benefited by advanced analytical techniques.

CONCLUSION:

With the advancements in technological compatibility and integration, the need for hyphenated techniques has increased widely. Environmental and biological research users are already using these types of instruments in varied applications. Chemical and petrochemical, food and beverage, plant and animal biochemistry, clinical chemistry, etc., are all areas where research and manufacturing will be greatly benefited by these hyphenated techniques. Thus it can be concluded that “Advances in Analytical instrumentation” is bringing a new world to industry especially pharmaceutical with its varied applications.

REFERENCES:

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Received on 09.04.2009 Modified on 27.05.2009

Asian J. Research Chem. 2(3): July-Sept. 2009 page 235-238