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Mastering the Liquid Chromatography Autosampler: This Guide is All You Need

Mastering the Liquid Chromatography Autosampler: This Guide is All You Need

Liquid chromatography is one of the most commonly used large-scale analytical instruments in our laboratory, and the autosampler is one of the core modules of the liquid chromatograph.

1) Advantages

There are numerous advantages to using an autosampler, and here are some of the key ones:

  1. Increased Efficiency and Reduced Analysis Time: The autosampler significantly improves workflow efficiency and shortens the overall analysis time.

  2. Enhanced Precision and Throughput: It greatly enhances the precision and throughput of HPLC analysis.

  3. Labor Saving: With an autosampler, during batch processing, we can focus on other tasks that require manual attention.

  4. Advanced Features for Problem Solving: The autosampler comes with advanced features such as temperature control, sample dilution, overlapping injections, and online derivatization, which can solve many complex issues.

2) Common Autosampler Configurations

The three main autosampler designs are Pushed-loop, Pulled-loop, and Split-loop, each with its own advantages and disadvantages.

Load State Diagram for the Three Injection Methods

1.Pushed-loop Autosampler

  • Advantages: This method typically wastes less sample compared to the pulled-loop method, and it allows for easier system flushing, reducing carryover contamination.

  • Disadvantages: This method requires additional components such as an injection capillary and needle, which increases the likelihood of leaks and the need for replacements. Moreover, some sample loss can still occur when the sample remains in the injection capillary.

2. Pulled-loop Autosampler

  • Advantages: It has a simple structure, is easy to maintain and use, and has low operating costs.

  • Disadvantages: It tends to waste more sample, is challenging to clean thoroughly, and often leaves significant residuals.

3. Split-loop Autosampler

Also known as a needle-in-loop, flow-through needle, or integrated loop design, in this configuration, the injection needle is directly connected to the sample loop, and the sample flows through this loop into the chromatography column.

  • Advantages: This type of autosampler minimizes carryover contamination and prevents sample waste, as all the sample in the loop is carried by the mobile phase into the column. This makes it particularly suitable for applications involving small sample volumes or limited sample availability. Additionally, the split-loop autosampler has the shortest injection cycle among the three designs.

  • Disadvantages: The system requires a specialized integrated sample loop, which can be costly, and it has a limited capacity range. The split-loop injection system may also face issues with needle wear and leaks, as the needle is often subjected to high system pressures.

3) Common Injection Methods

  1. Full-Loop Injection

Full-Loop Injection refers to the method where the sample loop on the injector is completely filled with the sample. In this method, the injection volume is equal to the volume of the sample loop. Typically, the volume pushed (or pulled) into the sample loop is more than three times the loop volume to ensure that the loop is fully filled. This complete filling is necessary to entirely displace the mobile phase within the loop, eliminating wall effects and ensuring accuracy. As a result, full-loop injection offers high injection volume accuracy and repeatability.

  1. Partial-Loop Filling

Partial-Loop Filling, also known as micro-volume injection or partial filling, involves partially filling the sample loop with the sample. The remaining volume of the loop is filled with a wash solution. The partial injection volume is precisely controlled by a metering pump or syringe. Therefore, the repeatability of partial-loop injections depends on the precision of the metering pump. The injection volume should not exceed 50% of the loop's volume; exceeding this limit can result in inaccurate quantification.


Partial Injection & Full Injection Diagram

  •  Non-Loss Injection

Non-loss injection refers to a method where a syringe extracts a certain amount of sample into a buffer loop, and then this portion of the sample is pushed into the middle of the sample loop using a wash solution. The sample completely enters the chromatographic system for detection, ensuring no sample loss. This method is typically used when the sample amount is very small or precious, as it avoids any waste of the sample. The injection reproducibility is determined by the accuracy of the metering pump's infusion, usually less than 0.5%.

Non-Loss Injection:

  • 20 μL sample loop: 1~8 μL;
  • 50 μL sample loop: 1~20 μL.

Non-loss injection can achieve zero sample loss, but it is recommended that the wash solution matches the mobile phase as closely as possible. The reproducibility (RSD) is <0.5%.

  • Air-Gap Injection

In gas chromatography, there is a technique known as air-gap injection, also called sandwich injection or air-segmented injection. For micro-syringes with a volume greater than 10 μL and dead volume, directly injecting the sample into the gas chromatograph often leaves a small amount of sample (a few microliters) in the syringe needle. When the needle is inserted into the high-temperature vaporization chamber, the sample in the needle tip vaporizes first and enters the chromatographic column. Then, the rest of the sample is injected by pushing the plunger, resulting in what is effectively a two-step injection.

To overcome this issue, air-gap injection is used. Before sampling, a certain amount of air is first drawn into the syringe, followed by the sample, and then another segment of air is drawn. In the syringe, the sample is visibly sandwiched between two air segments.

This injection method is also suitable for liquid chromatography. An air segment of a certain volume can be used to reduce solvent consumption. The air segment is positioned in front of the wash solvent and will not be injected. If the sample is highly viscous, programming a larger wash volume and reducing the syringe speed may be necessary for better performance. Using the air-gap injection method can mitigate the effects of solvent interaction.


Air-Gap Injection Diagram

4) Auto Sampler Malfunctions and Maintenance Methods

(1) Needle Malfunction

Common issues with the injection needle include clogging caused by samples, buffer salts, or septum debris. This typically results in small peak areas, high peak area RSD, or no peak signal.

Solutions:

  • If the injection needle is clogged, it can be cleared with a metal needle tool or replaced with a new needle.
  • Select an appropriate filter membrane material when filtering samples.
  • Needle bending usually occurs when the needle hits the bottle cap or bottom. Adjust the injection needle height. If the needle is misaligned, calibrate the needle position. If the needle material is soft, consider using pre-slit septa.

(2) Tubing Malfunction

Tubing blockages are mainly caused by samples, buffer salts, or septum debris. Blockages often occur on the low-pressure side of the injection valve (the needle connection tubing), likely due to particulates, and this can result in reduced or missing peaks. The issue can be resolved by flushing or replacing the tubing.

(3) Syringe Malfunction

If the syringe leaks air or is cracked, it may result in insufficient sample being drawn or no sample being drawn at all. Tighten the screws on the sealing needle core or replace the syringe.