Components of the HPLC System

An HPLC system (Figure 1.1) includes the following components. A description of the role and operating principle of each of these components is provided below; use the links to find the equivalent LabAlliance items in our catalog.

I) SOLVENT DELIVERY SYSTEM TO PROVIDE PULSE-FREE FLOWS

The requirements for an analytical HPLC pump include (i) pulse-free flows, (ii) flow rates ranging from 0.1-10 mL/min, (iii) accurate flow control with good reproducibility, (iv) generation of high pressure (up to 6000 psi), and (v) corrosion- and solvent- resistant components. Reciprocating pumps, which are currently used in about 90% of HPLC systems, consist of a small chamber into which the solvent is pumped by the back and forth motion of a motor-driven piston. Two check valves, which open and close alternately, control the direction and flow of solvent in and out of a cylinder. For single-piston pumps the use of specially designed cams permit very rapid refill times, producing a more continuous flow. The disadvantage of pulsed flows with reciprocating pumps is often overcome by using a pulse damper. The use of a dual-piston pump, which operates with the pistons moving out of phase with each other, offers a reasonable solution for pulse-free fluid delivery.

There are two options for gradient mobile phase operation - high pressure mixing which requires a pump for each solvent, and, low pressure mixing which requires only one pump (placed after a proportioning valve assembly that contains a multi-solvent inlet port). Programmable flow rate control is required for gradient operation in either mixing mode.

The incorporation of a mobile phase recycler, placed downstream of the detector, proves very useful for applications that involve the use of large amounts of solvents, as in semi-prep/prep applications. The recycling unit diverts effluent containing relatively pure mobile phase into a recycling container when the detector response is below the set threshold limit, and otherwise sends the detector effluent to waste.

LabAlliance offers a wide variety of options to meet solvent delivery requirements:

• Series I Plus metering pump with pulse-damper, 0.01 – 5.00 mL/min, 5000 psi
• Series II pump with pulse-damper, 0.01 – 9.99 mL/min, 6000 psi.
• Series III pump with pulse-damper, pressure limits, 0.01 – 9.99 mL/min, 6000 psi.
• Prep-100 pump for preparative applications, 0.1 – 100.0 mL/min, 4000 psi.
• Prep-24 dual-piston analytical/semi-prep pump, 0.01-24.00 mL/min, 10,000 psi.
• Flash-2000 pump for flash chromatography, 0.1-100.0 mL/min, 500 psi.
• Flash-24 dual-piston pump for low pressure LC, 0.01-24.00 mL/min, 2000 psi.
• Model-1500 pump, dual-piston analytical pump, 0.01-12.00 mL/min, 10,000 psi.
• Q-Grad high-pressure quaternary gradient pump, 0.01-5.00 mL/min, 5000 psi.
• Model CP, constant pressure pump, 10,000 psi.
• Prep System Keypad Controller, control of 2 Prep pumps, gradient, sample pump.

II) SAMPLE INJECTOR

A manual sample injector that is typically used comprises of a 6-port 2-position valve that includes a 20 or 100 µl fixed sample loop. In one configuration, the flow from the pump is sent directly into the column; when the position is switched the flow from the pump is diverted via the sample loop into the column, thereby performing a sample injection. Valves with electrically or pneumatically actuated position switches are also commercially available.

Automated sample injectors (autosamplers) that can store and sequentially inject multiple samples are popular in quality control applications and for high-throughput screening.

LabAlliance offers a low-cost Rheodyne-equivalent manual injector with position sensing switch for contact closure; also, variable volume and fixed loop autosamplers are available.

• EC1010 manual injector.
• LabAlliance Autosamplers.

III) HPLC COLUMN

In HPLC, the separation is effected by the difference in the relative affinity of the various compounds in the sample for the mobile phase and the stationary phase. A description of the various LC separation mechanisms has been presented in the HPLC theory section above.

LabAlliance offers a wide range of columns to cover most LC applications:

• For general reversed- and normal phase applications, use the ‘ST’ column series.
• For all ion-exchange applications, browse through the ‘SE’ column series.
• For size exclusion and gel columns, refer to the ‘JA’ column series.
• For economical strong ion-exchange columns, select the ‘OE’ brand C1 or A1 column.
• Bulk phases, semi-prep, and prep stationary phase materials are also available.

IV) DETECTOR FOR QUALITATIVE/QUANTITATIVE MEASUREMENT

The elution of a compound from the column is detected as a peak in the chromatogram. The retention time of the peak is used to identify the compound, and the peak height (or area) is proportional to the amount of the compound in the sample. The ideal detector has the following characteristics – good sensitivity, good stability, reproducibility, linear response over a few orders of magnitude, short response time, ease of operation, and is non-destructive in nature.

The HPLC detectors fall into a variety of categories based on the property of the sample that is detected.

UV/Vis Absorbance Detectors: Most modern UV/Vis detectors consist of a scanning spectrophotometer with grating optics. The independent or combined use of a Deuterium source (UV range, 190-360 nm) with a Tungsten source (visible range, 360-800 nm) provides a simple means of detecting absorbing species as they emerge from the column.

Several operational modes can be chosen –

1. the entire chromatogram may be obtained at a selected wavelength; or,
2. when the eluted peaks are suitably separated, different wavelengths may be selected for observing each peak (LabAlliance Model 201 detector); or,
3. simultaneous dual wavelength detection or the ratio of absorbances at two different wavelengths may be used for more accurate determinations, (LabAlliance Model 525 detector).

Examine the LabAlliance range of UV/Vis detectors to find one tailored for your application.

• Model 500 variable single-wavelength UV/Vis detector.
• Model 201 programmable UV/Vis detector.
• Model 525 dual-wavelength UV/Vis detector

PDA Detectors: The most powerful UV/Vis absorbance detectors in use today are photodiode-array (PDA) based instruments that permit very rapid collection of data over a selected spectral range. Thus, spectral data for each chromatographic peak can be collected and stored. This stored data may then be compared with the spectrum of a pure standard from a library - a spectral analysis study of peak purity. The PDA detector is very useful for the identification of components that are difficult to resolve (overlapping peaks) since the characteristic spectrum for each of the unresolved components is likely to be different.

• Model UV6000 PDA detector from LabAlliance, 190-800 nm.

Fluorescence Detectors: These instruments are most useful for the detection of components that exhibit a chemiluminescent property such as fluorescence or phosphorescence. They are more sensitive than UV absorbance detectors by at least one order of magnitude. Fluorescence is typically observed by detection of the grating-isolated emission radiation at a 90-degree angle to the excitation beam. Fluorescent compounds are typically encountered in the petroleum industry (PAH), pharmaceuticals and natural products (aflatoxins).

Often the number of fluorescing species can be enhanced by post-column derivatization (PCD) reaction of the eluted compounds (or pre-column derivatization reaction of the sample itself) with special reagents; e.g., analysis of amino acids, carbamates, glyphosates.

• Model 305 Ultrafluor scanning fluorescence detector; Xe excitation, 200-650 nm.
• Review the LabAlliance PCD systems with fluorescence detection – Amino Acids, Aflatoxins, Carbamates, Glyphosates, PAH.

Refractive Index (RI) Detector: RI detectors have the significant advantage of responding to nearly all solutes. The difference in the refractive index of the reference mobile phase versus the column effluent results in the detection of separated components as peaks on the chromatogram. Because of its extreme sensitivity to the mobile phase, this detector may not be used without adequate pulse-damping within the LC pump, nor is it suitable for gradient applications because of the changing mobile phase composition. The detection limits are usually lower than those observed with absorbance detectors.

• LabIndex-2000 RI detector; GPC software optional.
• Model 8110 RI detector, for analytical and semi-prep applications.

Electrochemical Detector: Detection based on amperometry is the most common electro-analytical method used in HPLC. Although these detectors have not yet been exploited to the same extent as optical detectors, they offer the advantage of wide applicability in addition to sensitivity. Potentially detectable organic functional groups by this method include hydrocarbons, olefins, amides, amines, diazo groups, nitro compounds, phenols, quinolines, halogens, ethers, esters, ketones, and aldehydes. The typical amperometric detector is made up of a simple thin-layer type of flow-through cell (1-5 microliter volume), and the working (indicator) electrode is usually one of glassy carbon, gold, or platinum.

Conductivity Detector: This instrument provides universal, reproducible, high-sensitivity detection of all charged species. This detector may be used with an HPLC system for the simple and reliable quantification of anions, cations, metals, organic acids, and surfactants down to the ppb level. The addition of a chemical suppressor between the column and conductivity detector serves to reduce the eluant conductivity, allowing the use of gradient elution and the determination of ppb levels with minimum baseline drift.

For a typical determination of low levels of anions, the eluant is converted to its weakly ionized low-conductivity acid (e.g., Na₂CO₃ to carbonic acid), reducing the background noise. At the same time, the analyte anions are converted to their corresponding high-conductivity acids (e.g., NaCl to HCl), increasing the relative analyte signal.

• LabAlliance Conductivity Detector.

V) DATA COLLECTION

A permanent record of the detector response may be obtained using a chart recorder or an integrator. Automated data and method storage, data processing, and reporting can be performed with a PC-based data collection package.

LabAlliance offers a variety of data collection options:

• EZChrom Software with selectable multi-channel, multi-instrument data collection package; binary gradient pump control option available.