Sample injection methods in high-performance liquid chromatography (HPLC) can have an impact on reproducibility, throughput, sample and solvent use, carryover and more. Due to the importance of this step in the HPLC process, more laboratories are turning to automated sample injection methods to overcome the limitations of manual injection.
HPLC autosamplers greatly improve the precision and throughput of HPLC processes, and these modules have grown more advanced over the years, with capabilities for temperature control, sample dilution, overlapping injections for faster cycle times, and compatibility with ultrahigh-performance liquid chromatography (UHPLC).
There are a range of autosamplers available for HPLC with differing designs, specifications and features, and selecting an HPLC autosampler should involve assessing these factors in relation to the requirements of your application. This HPLC autosamplers buyer’s guide breaks down key things to consider when shopping for an autosampler.
Pulled Loop, Pushed Loop and Split Loop Designs
Three main autosampler designs, with regard to the method of introducing the sample to the column, are the pulled loop, pushed loop and split loop designs, each with their own advantages and disadvantages.
The pulled loop design, also known as pull-to-fill, is the simplest but least popular method in modern instruments. In this type of autosampler, the sample is pulled directly into the sample loop and introduced to the column from the other end when the injection valve is switched to the inject position. Due to its simplicity, as it does not require any special additional tubing or a separate needle port, the pull-to-fill autosampler is typically less costly and requires less part replacements over time. However, this method results in more sample waste, as not all of the sample pulled into the sample loops enters the column, and carryover is of greater concern as washing the pulled-loop system is more difficult than with other designs. Utilizing wash vials between samples reduces carryover but decreases the throughput of the system.1
The pushed loop design, also called push-to-fill, uses a similar mechanism to manual injection. In this type of autosampler, the sample is first pulled into tubing that is separate from the sample loop, referred to as the aspiration capillary. Then, the needle is inserted into a low-pressure needle port and the sample is pushed into the sample loop, and subsequently introduced into the column when the injection valve moves to the inject position. This method tends to waste less sample than pull-to-fill and allows for easier flushing of the system to reduce carryover. However, it requires additional parts – the aspiration capillary and needle port – which increases the possibility of leaks and replacements, and some sample can still be lost when retained in the aspiration capillary.
The split loop design, also referred to as the needle-in-loop, flow-through needle or integrated-loop design, involves the use of a needle that is directly connected to the portion of the sample loop through which the sample flows into the column. The integrated needle first pulls the sample into the loop and then is firmly placed inside a high-pressure needle port. When the injection valve is switched to the inject position, the sample is dispensed at system pressure through the loop and needle into the column with the mobile phase. This type of autosampler experiences the least carryover and eliminates sample waste as all of the sample pulled into the loop is swept up with the mobile phase. This makes it especially useful for applications where small samples sizes are used, or limited sample is available. Needle-in-loop autosamplers also tend to have the fastest injection cycles of the three designs.2
However, these systems require the use of special integrated sample loops, which can be more expensive and have a limited volume range, with a typical maximum volume of 100 µL. This allows for less flexibility than pulled/pushed loop methods, with which sample volume capacity can be adjusted by simply interchanging any loop size.1 Split loop systems can also run into problems with needle port wear and leakage, as the port is regularly subjected to high system pressure. Purchasers of flow-through needle autosamplers will want to ensure the system is designed to withstand the pressures used in their application, and will need to keep in mind that the needle port should be regularly checked for damage.
Vial-to-Needle, Needle-to-Vial and Carousel Designs
Another design difference between autosamplers is the robotic movement used to bring together the needle and sample. Three main variations are vial-to-needle configurations, needle-to-vial configurations and carousel configurations.2
In a vial-to-needle design, a robotic arm retrieves each sample vial from the tray and positions it below the needle (and over any needle port), requiring the needle to simply move up and down to draw in the sample or perform an injection. One of the benefits of this design is that the needle requires minimal displacement, reducing strain on components such as the sample loop and needle port, which makes this format especially useful in split loop autosamplers. Additionally, the robotic arm can be used to shake and homogenize the sample. However, this design can only be used with vials and not microplates, and the vial-to-needle technique may be slower than other methods, as each vial must be moved and replaced in the tray.
A needle-to-vial format using a static sample tray requires the needle to move in x, y and z directions to access each sample. This configuration is typically faster than designs that use a separate robotic arm to move the sample, takes up less space within the sample compartment and is compatible with microplates. However, there is more risk of wear on the tubes and needle port due to increased movement of the sampling needle, as well as greater demands on the drive mechanisms. Additionally, the static tray does not provide a way to homogenize the samples before aspiration, which can only be done by programming the needle to mix the sample with withdrawal and release steps.2
A third design is the sample carousel, which incorporates a rotating sample tray that positions the vials or microplate wells under the sampling needle. Due to the movement of the carousel placing the samples in the correct y position, the needle only needs to move in the x and z positions to access each sample. This results in less mechanical strain than designs using static trays. Carousels also provide some of the fastest injection cycle times, are compatible with microplates and allow a degree of sample homogenization due to the rotating movement. However, carousels take up significant space, with unused space in the middle of the carousel, which means reduced sample capacity and less efficient temperature control inside the sample compartment.2
Sample Capacity and Compatibility
The sample capacity of the autosampler – i.e. how many samples it can hold at once – is a major factor in throughput, and labs should select autosamplers with capacities corresponding to their needs. Capacities can range from a couple dozen samples to thousands of samples for systems that use microtiter plates and tray changers. Buyers should ensure the autosampler selected fits the vial and microplate sizes suitable for their application. Carousel-type autosamplers typically have lower sample capacity while needle-to-vial configurations support the highest capacity; keep in mind that vial-to-needle autosamplers do not support microplates.
Tray changers, or called plate changers or rack changers, are typically offered as an optional add-on. These modules can multiply the capacity of an autosampler by automatically changing out vial trays or microplates, expanding the number of samples that can be continuously analyzed. This accessory is a great option for labs that want to maximize throughput, or start out with a smaller capacity and eventually expand their capacity as sample volume grows. However, keep in mind that many tray changers are only compatible with specific autosampler models, so consider the add-on options before purchasing the autosampler, even if you don’t plan to purchase these add-ons right away.
Compatibility with biological samples is also a concern when purchasing an HPLC autosampler, as not all autosampler components are made with bio-inert surfaces. If you are using HPLC to analyze biological samples, look for autosamplers that use biocompatible materials in the sample flow path such as polyether ether ketone (PEEK), ceramics or titanium, and avoid those that use metals such as stainless steel, which is less bio-inert than titanium. Some autosamplers are specifically designed for use with biological samples.
Injection Volume and Precision
One of the great benefits of autosamplers is their superior precision over manual injection. Precision is related to the variation in injection volume between numerous samples, whereas accuracy describes the relationship between the intended injection volume and actual injection volume. When a system is calibrated correctly, precision is more important than accuracy, as most LC methods prioritize consistent injection volumes rather than specific volumes of sample.3 Therefore, look for data demonstrating the autosampler’s precision and reproducibility rather than looking solely at accuracy. Flow-through needle autosamplers tend to demonstrate the highest precision, as 100% of the sample is carried out of the split loop by the mobile phase.2
The range of injection volumes offered by the autosampler should fall in line with the intended application. On the lower end, injection volumes under 1 µL enable advanced micro, nano and UHPLC applications, as well as analysis of limited-volume samples and samples in highly miniaturized microplates. On the high end, injection volumes up to several milliliters allow autosampling for preparative applications. Pulled and pushed loop systems often support a wide range of injection volumes using exchangeable samples loops and partial loops modes, whereas split loops systems are more limited based on the availability of integrated loop sizes, but provide greater precision for smaller-volume injections. For especially high injection volume needs, autosamplers specifically designed for preparative systems may be desired.
Other Factors to Consider
Temperature Control
Autosamplers may include a thermostat to support temperature control within the sample compartment; consider whether the temperature range of the thermostat fits the needs of your experiments, and whether the thermostat is integrated or is available as an optional add-on. Carousel-type autosamplers may be more challenging to regulate, with the risk of a temperature gradient from the center to the outer ring of the carousel.
Footprint
Consider the amount of bench space you’re able to dedicate to the autosampler and look for stackable models for greater space savings.
Overlapped Injection and Dual Injection
Overlapped injection increases throughput by injecting the next sample while the previous sample is being analyzed, rather than waiting for the full analysis to be complete before the next injection is initiated. While autosamplers with overlapped injection capabilities can save significant time, it is important to program the instrument to ensure no overlap between target peaks.
Dual injection involves the use of one autosampler to simultaneously inject samples into two separate flow paths, allowing complementary chromatography methods (using separate pumps, columns and detectors) to be run in parallel. This capability removes the need to run complementary methods on the same sample separately at separate times, or to split the sample into separate vials to be run on separate autosamplers.
Automated Sample Preparation
Some advanced autosamplers also have the capability to perform automated sample preparation tasks, such as dilutions, mixing and derivatization. While these features may add on to the cost of the instrument, consider whether this additional automation would be more cost-effective than manual preparation or purchasing a separate sample preparation device.
HPLC Autosampler Manufacturers to Consider:
- Agilent Technologies
- HTA srl.
- JASCO
- KNAUER
- Shimadzu Scientific Instruments
- Thermo Fisher Scientific
- Waters Corporation
References
- Dolan, J.W. Autosamplers, Part I — Design Features. LCGC North America. 2001, 19 (4), 386-391. https://www.chromatographyonline.com/view/autosamplers-part-i-design-features
- Steiner, F.; Paul, C.; Dong, M.W. HPLC Autosamplers: Perspectives, Principles, and Practices. LCGC North America. 2019, 37 (8), 514-529. https://www.chromatographyonline.com/view/hplc-autosamplers-perspectives-principles-and-practices
- Dolan, J.W. How Does It Work? Part III: Autosamplers. LCGC North America. 2016, 34 (7), 472-478. https://www.chromatographyonline.com/view/how-does-it-work-part-iii-autosamplers