HPLC Injection Volume: What Should I Dilute It In and How Much Sample Can I Inject?

HPLC Injection Volume and Solution Tips: For best results, the choice of injection solution and amount must be carefully selected. Successful HPLC & LC-MS methods shall observe good chromatography fundamentals. 

• How much sample can I inject on my column? The HPLC injection volume must be carefully selected to avoid overloading the column and also maintain good quality peak shape (Good peak shapes, Gaussian are ideal, are preferred for accurate integration and quantitation). Too large an injection volume and the peak shape may be broad and result in co-elution, column fouling and/or poor reproducibility. Too low an injection volume may lead to no-detection, poor reproducibility and/or inaccurate integration. Choose an appropriate Injection Volume (and concentration) that is appropriate for the COLUMN and Method used (their is no universal answer as they depend on YOUR column and method). Start, by learning what your HPLC column's "dead" volume is (Determining the HPLC Column Volume Link here).  As a general guideline, keep the volume low and inject no more than ~ 1% of the column's dead volume (maximum for most columns is ~ 1 to 2 %, but if the peak shape is excellent, sometimes up to 3% is possible). The actual capacity will be different for different column support types, dimensions etc, so it is best not to guess. Use a volume that is within the injector's most accurate range (for most auto-injector's, the optimal range may be found away from the extreme limits, often between 20% and 80% of capacity, but please refer to the documentation for your injector for specifics). Once an acceptable volume has been identified, then you can vary the concentration to find the best sample load for your analysis conditions.
  
• NOTE: To find the true and correct answer to "How Much Can I Inject (Load) onto my column" requires that you conduct a 'Loading Study' [To run a loading study you will prepare a batch of samples of increasing concentrations levels which can be individually injected, then evaluated on YOUR column, using YOUR method. This is how we determine the MAXIMUM amount possible which can be loaded and still provide good quality results. All other methods are just estimates.

• What should I dilute my sample in? Dilute samples using the mobile phase solution (in the case of gradient compositions, use the "initial" mixture to avoid precipitation). Your sample(s) should be FULLY dissolved in the mobile phase and not in a solution that is chemically incompatible with the flow path or is "stronger" in elution strength than the initial mobile phase. The diluent should not interfere with the analysis or loading of the sample onto the column. Example: If your method is 100% aqueous, then do not inject the sample(s) in a solution that contains organic solvent (i.e. ACN). *Peak fronting, splitting, precipitation and/or distortion (broad shapes) may result from using a diluent that is stronger than the mobile phase.
  

• My sample solution is cloudy or has "stuff" floating in it. ONLY Inject sample solutions which are 100% fully dissolved, in-solution. Injecting samples which have precipitated out of the solution OR which are not fully dissolved in solution (100%) may result in line obstruction, clogging, column fouling and invalid data collection/results. Take the time to find a mobile phase that your sample fully dissolves in to avoid problems. Troubleshooting and repairing an HPLC system for clogs and/or column contamination is both time consuming and expensive.

• Filter sample solutions to prevent clogs and reduce column fouling. Make sure the sample is first fully dissolved in the solution and do not use a 'filtering' step as a cheat to remove undissolved sample. Filtering is used to protect the system from particles that we can not easily observe which may clog the system. Please refer to the article; "Syringe Filter Selection for HPLC or LC/MS samples"; for more information on filter selection.

• Improve Injector reproducibility: Leave the vial cap slightly loose so it does not make a full seal. *This prevents a vacuum forming inside the vial, resulting in injection volumes which may be lower than the selected volume. "Loose caps" can greatly improve accuracy and reproducibility when larger OR multiple volumes are injected from the same vial. Additionally, if the total sample vial volume is very small (i.e. ~ 200 ul), utilize a vial insert of the correct dimensions and type for improved accuracy. When using vial inserts, check that the needle height is correct for the vial insert used.  Do not use the entire sample volume! Never use more than 90% of the vial volume or air may be aspirated resulting in invalid data collection.
  

• Prevent sample carryover problems by regularly inspecting and servicing your HPLC injector (Manual valve and Autoinjector maintenance tips will be found at this LINK). Replace common wear parts such as rotary valve seals and needle seats on a regular basis (Do not "clean" and re-use seals). Carryover troubleshooting Tips will be found at this LINK.
  

•  Calibration Volumes for Quantitation: When creating a new calibration table for a group of standards, use the SAME VOLUME for each standard and vary the concentration ("calibration level") only with each vial. As we have seen, injection volume is a variable which may change peak shape and integration accuracy. If you inject the same volume of liquid for all standards (and samples too), then you remove this variable. Using the SAME injection volume for all standards and samples helps to reduce problems. *Note: Thought it may not be approved, if you thoroughly test varying the injection volume across the range used for the calibration to demonstrate no undesirable changes to peak shape, loss of resolution/separation, and it is reproducible and accurate for the analysis method, then you can vary injection volume. Link to: HPLC Calibration Article.
   

 

Please note that these are general guidelines only and the mode of chromatography (e.g. NP/RP/HILIC/SEC), scale (prep vs. analytical) and/or specific method used must be optimized for best results. Follow these basic guidelines to prevent analysis problems, prolong column and system lifetimes and increase reproducibility and accuracy.

External (ESTD) vs. Internal Standard (ISTD) Calibration in HPLC

Reliable quantitation of sample analytes using HPLC analysis requires accurate and reliable quantitation of a standard(s). For chromatography applications, we commonly use either an External Standard or an Internal Standard, as applicable, to insure reliable quantification of the sample.

• NOTE: A quick comment about calibration methods. Before you begin to create any calibration tables or analyze any standards/samples, please make sure that your current chromatography method follows good chromatography fundamentals. It must be selective for the sample type, retain the compound(s) with good K prime values, be reproducible and resolve apart all of the samples and possible impurities with near to perfectly symmetrical peak shapes. Your calibration results will only be as good as your original method. A poor quality method may not provide reliable results so be sure and spend as much time as possible developing the initial HPLC method to be as rugged and reliable as possible before starting any quantitation or calibration. *Poor quality method development is the number one reason for problems with quantitation.

Methods of Quantitation, Peak-height vs. Peak-area: Both types of response provide a measurement of the detector signal output. Proper and reproducible integration of the signal output is critical. Peak area is the most popular choice in chromatography, but peak height measurements can also be used if the peaks have near perfect symmetry (very rare, so peak area is far more reliable for integration). Whichever method you chose, you must use it consistently and document it well.

Definitions, External & Internal Standards: For most samples, there are two commonly used types of standards used. When known standards are run separately from the actual samples (in their own chromatogram) and their response is compared to that of the sample in another chromatogram, then we refer to this as an External Standard (ESTD). When the standard is added to the sample and analyzed at the same time we refer to this as an Internal Standard (ISTD). With an Internal Standard we are comparing the instrument's response to the sample to a reference standard with similar response characteristics, both run together.

External Standard (ESTD) Calibration Notes: The sample must fall within a range bracketed by the calibration solution. I suggest that you include a range which covers concentration values which are ~ 50% or more outside of the expected range. Dissolve the final calibration standards into the mobile phase (or a weaker solution) when preparing the injection vials from the stock solution. At least five (5) different concentration values should be used per order-of-magnitude (larger range = more stds). *Inject the same volume of solution (different concentration) for each calibration standard point ("level") onto the column. Do Not inject different volumes of solution from one std vial to create different concentrations. Plot peak response vs concentration. Ideally, you should have a linear response and the line will go through the origin (true zero intercept, ideally, though matrix effects/or the use on non std detectors such as the ELSD or CAD may require complex curve fits/formulas to describe the response). Once you have injected all of the standards, repeat the process again at least three more times (or use multiple injections) to determine overall reproducibility before constructing the final calibration table.

Internal Standard (ISTD) Calibration Notes: Internal standards are commonly used when many sample preparation steps are required before the sample can be injected onto the column. The internal standard may compensate for any losses during filtration or extraction. Selection of the Internal Standard is critical. Some of the characteristics of a good ISTD should include: It must be different than the sample, well resolved and must not elute where any sample peaks could be expected; It should not elute where any interfering matrix or other compounds could appear; It should have a similar linear response as the sample (Inject a fixed volume/concentration); Available in a high purity form from one or more commercial sources (certified method); Must be stable and not react with the sample or mobile phase solution. 

Add it to the samples before any extraction procedures. Base the amount of ISTD concentration such that it is between 1/3 and 1/2 of the expected concentration of the sample(s). The sample's target concentration range is a good value to use. *Because of these and other strict conditions, finding a suitable Internal Standard can take some time and testing.

HPLC to UHPLC Conversion Notes (Column Dimensions, Flow Rate, Injection Volume & System Dispersion)

The use of ultra-high performance liquid chromatography (UHPLC) columns to reduce analysis times and sometimes improve detection limits is a hot topic. UHPLC presents a number of new issues. The incorporation of smaller 1.9 to 3.0 micron particles and smaller frits will raise backpressures and increase system wear and tear. Smaller diameter lines are often used (I.D. of 0.12mm or less) which can increase blockages and clogs if you do not filter your mobile phase and samples through a 0.45 or 0.2 micron filters. Piston seals and valve rotors can wear out early due to the very high pressures, heating and stress imposed on them. You should monitor your HPLC system carefully over time and consider increasing the frequency of preventative maintenance and inspection services as well. However, the smaller particle sizes can provide better resolution in some applications so they are well worth evaluating.

I must answer twenty or so questions each week in the area of UHPLC. The most common questions deal with selection of an UHPLC column and making adjustments to a method for the changes which effect: (1) Column Dimensions; (2) Flow Rate; (3) Injection Volume and (4) System Dispersion. The good news is that some of these questions can be answered with some basic math while others just require a basic understanding of how the system works.

(1) COLUMN DIMENSIONS: Let's start by making things as simple and brief as possible (this is supposed to be a "hint & tip", not a thirty page article). When initially converting from a convention HPLC column (e.g. with 5 micron particles) to an UHPLC column (e.g. with 1.9 to 3 micron particles), initially select a column with the same I.D. and length for the calculation. This way only the particle size changes. *I like to change one variable at a time. If you would like to change the column length to take advantage of some of the increased efficiency (and decrease the pressure!) which results from smaller particles, then please refer to the following equation.

     EQUATION A:  'Lc2' = ('Lc1' * 'p2') / 'p1'

[ 'Lc1' = Length of Column #1 in mm; 'Lc2' = Length of Column #2 in mm; 'p1' = particle size of Column #1 in microns; 'p2' = particle size of Column #2 in microns].
                    
   Example: Column # 1 is a standard HPLC column;  4.6 mm x 250 mm (5u) Column. You want to find out the length of an equivalent column which uses 1.9 micron particles instead of the 5 micron particles.

   'Lc2' = (250 * 1.9) / 5 ; Answer is: 'Lc2' = 95 mm. *So a 10 cm long column would be a good choice here.


(2) FLOW RATE: Flow rate is directly proportional to column diameter and as we saw above in Equation A, the particle size can also affect it too. If you keep the column length and internal diameter the same, then the linear flow will be unchanged with the same particle size. A change to the particle size alone will change the flow rate as follows: 'Fc2' = 'Fc1' x ('p1'/'p2').

A change to a smaller diameter column to compensate for the improved efficiency will require a change to the original flow rate to preserve the linear velocity. Please refer to the following equation.

     EQUATION B:   'Fc2' = ('d2' / 'd1')^2 * 'Fc1'

['Fc1' = Flow Rate of Column #1 in ml/min; 'Fc2' = Flow Rate of Column #2 in ml/min; 'd1' = Column #1 Diameter in mm; 'd2' = Column #2 Diameter in mm].
                     
   Example: Column # 1 is a standard 4.6 mm ID Column. You want to find out what the linear flow rate should be if you use a smaller diameter column (2.1mm in this example).

   'Fc2' = (2.1/4.6)^2 * 1.000 ; Answer is: 'Fc2' = 0.208 ml/min. *A flow rate of 200 ul/min would be fine. 

However, one other factor should be considered. The optimum flow rate for sub 2.5u particles are often about double that of the "normal" linear flow rate used with conventional particles (>2.5u). Evidence for this has been shown through analysis of the van Deemter curve with the tiniest particles showing much flatter curves. Retention (K prime) can often be maintained by combining twice the normal flow rate and speeding up the gradient time by a factor of 2. So a method utilizing std sized particles with a linear flow rate of 0.200 ml/min might benefit from a faster flow rate of 0.400 ml/min and a twice as fast gradient composition change.


(3) INJECTION VOLUME: A change in the column dimension may require a change to the injection volume (note: "volume" and concentration are two different things. If the solution concentration remains the same and you inject less, the on-column sample concentration will also be less). The smaller the internal volume of the column, the smaller the injection volume. To calculate the linear change in volume, please refer to the following equation.

     EQUATION C:   'V2' = 'V1' * {('d2'^2 * 'L2') / ('d1'^2 * 'L1')}

['V1' = Injection Volume #1 in ul; 'V2' = Injection Volume #2 in ul; 'L1' = Column #1 Length in mm; 'L2' = Column #2 Length in mm; 'd1' = Column #1 Diameter in mm; 'd2' = Column #2 Diameter in mm].
                     
   Example: Current injection volume is 10 ul. Column # 1 is a standard 4.6 mm ID x 250 mm Column. You want to find out what the equivalent injection volume should be for a 2.1 mm ID x 150 mm column.

   'V2' = 10 * (2.1^2 * 150) / (4.6^2 * 250) ; Answer is: 'V2' = 1.25 ul.



(4) SYSTEM DISPERSION: When converting HPLC methods to "UHPLC" methods, few parameters effects the results obtained more than the HPLC system's System Dispersion. The volume of liquid that is contained between the injector needle and flow cell (with the column removed or by-passed) is know as the system dispersion volume. This volume is determined by how the specific HPLC is designed and plumbed. On most HPLC systems, it can be easily changed and optimized to fit the specific application desired and only requires that you have a solid understanding of how the HPLC system works. The choice of connection tubing ID and length, how the autoinjector is programmed, its loop size and the detector's flow cell volume all contribute to the system dispersion volume. In the same way that changes to the total column volume can effect the peak shape and resolution, the internal system dispersion volume also contributes to the results. 

With standard sized analytical columns (i.e. 4.6 x 250 mm), the typical HPLC's system volume is so small relative to the volume of the column (e.g. 100 ul system dispersion vs 2900 ul column volume, or 3.5%) that it does not negatively impact the chromatography. However, anytime we utilize a tiny HPLC column whose column volume is a fraction of that found in a standard column (i.e. 100 ul system dispersion vs 2.1 x 50 mm column with 120 ul volume), diffusion and band spreading can quickly become so significant that effective plate numbers are quickly reduced below values found on a standard sized column. As column volume decreases (and approaches the system volume) the total system dispersion volume must also decrease. In general, try and keep the system dispersion volume at or below 10% of the column dead volume. This is most easily accomplished by reducing the number of connections and fittings used, reducing the lengths of all tubing used, using much narrower ID tubing (e.g. 0.12 mm vs 0.17 mm ID), reducing flow cell volume and reducing the flow-through volume in the autoinjector (i.e. loop size, needle seat, etc). The injector is often the largest contributor to the system dispersion so concentrate efforts here (e.g. after injection, switch the loop out of the flow path).