Summary of HPLC
HPLC Method Development
1.2.2 Separation Goals
The goals of HPLC separation need to be specified clearly. Some related questions that should be asked at the beginning of method development include:
Is the primary goal quantitative analysis, the detection of an (undesired) substance, the characterization of unknown sample components, or the isolation of purified material? The use of HPLC to isolate purified sample components for spectral identification or other purposes is discussed in Chapter 13.
Is it necessary to resolve all sample components? For example, it may be necessary to separate all degradants or impurities from a product for reliable content assay, but it may not be necessary to separate these degradants or impurities from each other. When the complete separation of a sample by means of a single HPLC run proves difficult, the separation of a smaller subset of sample components is usually much easier.
If quantitative analysis is requested, what levels of accuracy and precision are required? A precision of ±1 to 2% for major components of a sample is usually achievable, especially if sample pretreatment is not required. Means for improving assay precision are discussed in Chapter 14.
For how many different sample matrices should the method be designed? A particular compound may be present in different sample types (e.g., a raw material, one or more formulations, an environmental sample, etc.). Will more than one HPLC procedure be necessary? Is a single (or similar) procedure for all samples desirable?
How many samples will be analyzed at one time? When a large number of samples must be processed at the same time, run time becomes more important. Sometimes it is desirable to trade a decrease in sample resolution for a shorter run time [e.g., by shortening the column or increasing flow rate (Section 2.3.3.1)j. When the number of samples for analysis at one time is greater than 10, a run time of less than 20 mm often will be important.
What HPLC equipment and operator skills are present in the laboratory that will use the final method? Can the column be thermostated, and is an HPLC system for gradient elution available? Will the method be run on equipment of different design and manufacture [especially older models with increased extracolumn band broadening (Section 2.3.3.3)1? What HPLC experience and academic training do the operators have?
SAMPLE PRETREATMENT AND DETECTION Samples come in various forms?
Solutions ready for injection
Solutions that require dilution, buffering, addition of an internal standard, or other volumetric manipulation
Solids that must first be dissolved or extracted
Samples that require sample pretreatment to remove interferences and/or protect the column or equipment from damage
Selecting an HPLC Method and Initial Conditions
If HPLC is chosen for the separation, the next step (Fig. 1.3) is to classify the sample as regular or special.
We define regular samples as typical mixtures of small molecules (<2000 Da) that can be separated using more-or-less standardized starting conditions.
Improving the Separation
Separation or resolution (Section 2.2) is a primary requirement in quantitative HPLC analysis.
Usually, for samples containing five or fewer components, baseline resolution (Ri> 1.5) can be obtained easily for the bands of interest. This level of resolution favors maximum precision in reported results.
COMPLETING THE HPLC METHOD
The final procedure should meet all the goals that were defined at the beginning of method development. The method should also be robust in routine operation and usable by all laboratories and personnel for which it is intended.
Checking for Problems
CHAPTER II: BASIC OF SEPERATION
RESOLUTION: GENERAL CONSIDERATIONS
Chromatographers measure the quality of separations as in Fig. 2.la by the resolution R of adjacent bands. Two bands that overlap badly have a small value of R:
……….. Eq 2.1
Here t1 and t2 are the retention times of the first and second adjacent bands and W1 and W2 are their baseline bandwidths.
This measurement is somewhat awkward at first, which may make the corresponding determination of R imprecise. An alternative approach gives more reliable values of R: bandwidths at halfheight (W112 see Fig. 1.1., Appendix I) are measured for bands 1 and 2, W051 and W052.
Resolution can be estimated or measured in three different ways:
1. Calculations based on Eq. 2.1
2. Comparison with standard resolution curves
3. Calculations based on the valley between the two bands
RESOLUTION AS A FUNCTION OF CONDITIONS
The separation of any two bands in the chromatogram can be varied systematically by changing experimental conditions. Resolution R can be expressed in terms of three parameters (k, a, and N) which are directly related to experimental conditions:
Here k is the average retention factor for the two bands (formerly referred to as the capacity factor, k’), N is the column plate number, and a is the separation factor; a = k21k1, where k1 and k2 are values of k for adjacent bands 1 and 2.
Equation 2.3 is useful in method development because it classifies the dozen or so experimental variables into three categories: retention (k), column efficiency (N), and selectivity (a).
Changes in the mobile or stationary phases will generally affect both k and a but will have less effect on N. The column plate number N is primarily. dependent on column quality and can be varied by changing column conditions :
1. Flow rate 2. Column length 3. Particle size
A change in these conditions will not affect k or a as long as the mobile phase and stationary phase type are not changed.
In method development it is advisable first to change conditions that will optimize values of k and a, then (optionally) vary column conditions. In this way initial experiments can be used to obtain good values of k and a that will not change if only column conditions are varied further.
Effect of Solvent Strength
The effect of solvent strength on a reversed-phase separation is illustrated in Fig. 2.6 for the repetitive injection of a five-component sample with a change in mobile phase (varying percent methanol) between each injection.
In most cases, an intermediate solvent strength will be preferred so that 0.5 < k < 20 for all bands
This optimum value of % B (A is the weak and B the strong solvent component; see Table 2.1) can be determined by systematic trial-and-error experiments as in Fig. 2.6.
In evaluating successive method development experiments as in Fig. 2.6, it is important to know an approximate value of t0 for the HPLC system. A value of t0 can be estimated in various ways:
1. First significant baseline disturbance
2. Use of a very strong solvent as the mobile phase 3. Calculation from column dimensions
4. Injection of an unretained sample
Values of t0 can also be determined from Eq. 2.5 using an estimate of Vm (mL) from the length L (cm) and internal diameter d (cm) of the column:
Thus, for a 25 X 0.46-cm column, V,1 = 0.1 X 25 = 2.5 mL. If the flow rate is 1.5 mL/min, t0 2.5/1.5 = 1.67 mm (Eq. 2.5). Finally, an unretained compound can be injected, in which case its retention time equals t0.
When adjusting solvent strength, it is important to make rough estimates of k for the first and last bands in the chromatogram. The goal of solvent strength adjustment is to position all the bands within a k range of roughly 0.5 to 20 (0.5 < k < 20). This range in k will generally (not always!) avoid problems from the initial baseline disturbance overlapping the first band; when k > 0.5, early- eluting impurity bands are also less likely to overlap an analyte band. When k < 20, excessive broadening of the last band and run times that are too long will be avoided.
Optimizing Solvent- Type Selectivity.
This is illustrated in the hypothetical separations of Fig. 2.8. The first two experiments are designed to adjust solvent strength and the range of k values. It is advisable to start with a relatively strong mobile phase, 80% acetonitrile—water in this case. The sample is weakly retained (as expected) and leaves
the column quickly with poor resolution of the sample. The second experiment (40% ACN) provides adequate retention and resolution is improved. However, some band overlap occurs (bands 2/3 and 6/7) because of poor peak spacing. The organic solvent is then changed from acetonitrile (ACN) to methanol (50% MeOH) and a third run is carried out. Band spacing changes, but new band pairs are overlapped (3/4 and 5/6). By mixing these two mobile phases (equal volumes of 40% ACN and 50% MeOH), a final separation intermediate between the second and third runs (20% ACN + 25% MeOH) is obtained with acceptable resolution of all bands. The procedure of Fig. 2.8 can also be used when varying other conditions (e.g., pH, temperature, concentration of an ion-pair reagent, buffer, or other mobile-phase additive). In Chapters 6 to 9 we describe the general procedure of Fig. 2.8 in more detail and provide several (real) examples.
Effect of Column Plate Number
In other cases, further improvement in the separation is required. Equation 2.3 states that resolution increases for all bands when N is increased as long as values of k and a do not change.
So, if resolution needs to be improved after adjusting k and a values, an increase in N is one option. Conversely, if the separation has more resolution than required (R >> 2), this excess resolution can be traded for a shorter run time (by reducing column length and/or increasing flow
rate). An increase in N can always be achieved by increasing column length and/or reducing flow rate (but with an increase in run time).
The column plate number (N) increases with several factors:
1. Well-packed columns (column “quality”) 2. Longer columns
3. Lower flow rates (but not too low) 4. Smaller column-packing particles
5. Lower mobile-phase viscosity and higher temperature 6. Smaller sample molecules
7. Minimum extracolumn effects
DETECTION SENSITIVITY AND SELECTIVITY
UV DETECTION
3.2.1 General Considerations
Light from the lamp passes through a UV-transmitting flow cell connected to the column and impinges on a diode (or a phototube in older systems) that measures the light intensity I. Usually, light from the lamp is also directed to a reference diode for measurement of the original light intensity I. The detector electronics then convert the signal from the two diodes into absorbance A, which is transmitted to the data system:
Detection Selectivity between UV detection an fluorescence detection.
Choice of Wavelength
Sample Absorbance as a Function of Molecular Structure
wavelength chosen for UV detection must provide acceptable absorbance by the various analytes in the sample, combined with acceptable light transmittance by the mobile phase. For some samples it may also be important to elect a wavelength at which sample interferences have minimal absorption. igure 3.5, which shows the UV absorption spectra of dilute solutions of two compounds [amitryptiline (AMI) and imiprimine (IMI)], can be used to illustrate some of the considerations involved in the selection of a detection wavelength. If both compounds are of interest and maximum detection sensitivity is needed, detection at 210 nm might be a good choice.
The detector signal A is proportional to the molar absorptivity e of the compound of interest (Eq.
3.2). For UV detection to provide adequate sensitivity for the analysis of major sample components, E must be greater than 10 at some wavelength above 185 nm.
Aromatic compounds usually have e values above 1000 at wavelengths above 210 nm.
Mobile-Phase Absorbance as a Function of Composition.
One study indicates that baseline noise will increase significantly when A > 0.7 for the mobile phase.
UV Detection
Tables 3.2 to 3.4 may contain UV-absorbing impurities or develop absorption as a result of degradation when exposed to light and air. Therefore, the absorbance values in Tables 3.2 to 3.4 may represent maximum absorbances for HPLC-grade solvents. If the solvent absorbance is significantly greater than in Tables 3.2 to 3.4, the material is probably contaminated.
Signal, Noise, and Assay Precision
Signal (S) refers to the baseline-corrected absorbance of the analyte peak, and noise (N’) refers to the width of the baseline as illustrated in Fig. 3.3
Maximizing Signal/Noise Ratio for Better Assay Precision
When assay precision varies with analyte concentration, better precision can be obtained by increasing the S/N’ ratio. This can be achieved by either an increase in signal S or a decrease in noise N’. A maximum signal can be achieved by selecting the wavelength that gives maximum absorbance (e.g., = 210 nm for AMI in Fig. 3.5). Since noise does not vary much with wavelengths above 200 nm, the wavelength maximum also corresponds to maximum S/N’ ratio.
For wavelengths below 200 nm, noise increases rapidly (especially for detector lamps that have aged), and then the wavelength maximum may not be the same as the wavelength for maximum S/N’ value.
High-frequency noise can be removed by increasing the detector time constant or rise time (rise time 2T).
Diode-Array UV Detectors
diode-array detectors (DADs) allow simultaneous collection of chromatograms at different wavelengths during a single run. Following the run, a chromatogram at any desired wavelength (usually between 190 and 400 nm) can be displayed.
If the DAD is to confirm the presence of an overlapping peak successfully, the UV spectra of the two peaks must differ significantly, the relative concentration of one of the two peaks must fall within about 5 to 95% of the other, and the resolution of the two peaks must be greater than 0.3.
It must be emphasized that use of a DAD alone is by no means conclusive in establishing peak purity. Peak collection, followed by other qualitative analysis techniques, such as infrared (IR), nuclear magnetic resonance (NMR), mass spectroscopy, and so on, are often used to increase assurance of peak purity.
The use of a DAD is also important for peak tracking or the matching of peaks that contain the same compound between different experimental runs during method development.
SAMPLE PREPARATION
The aim of sample preparation is a sample aliquot that (1) is relatively free of interferences, (2) will not damage the column, and (3) is compatible with the intended HPLC method; that is, the sample solvent will dissolve in the mobile phase without affecting sample retention or resolution.
TYPES OF SAMPLES
PRELIMINARY PROCESSING OF SOLID AND SEMI-SOLID SAMPLES Reducing Sample Particle Size
It is desirable that solid samples be reduced in particle size since finely divided samples (1) are more homogeneous, allowing more representative sampling with greater precision and accuracy, and (2) dissolve faster and are easier to extract because of their greater surface area. Grinding with a mortar and pestle is recommended for many solid samples and most will withstand the thermal rigor of grinding. If the sample contains thermally labile or volatile compounds, it is important to minimize heating during the grinding process.
Drying the Sample
Inorganic samples such as soil should be heated at a temperature from 100°-110°C to ensure the removal of moisture. Sensitive biological compounds (e.g., enzymes) often are prepared in a coLd room at less than 4°C to minimize decomposition. Samples of such materials should be maintained at these low temperatures until the HPLC analysis step. Freeze-drying (lyophilization) often is used to preserve the ntegrity of heat-sensitive samples (especially biologicals). This is carried out by quick-freezing the sample, followed by removal of frozen water using sublimation under vacuum.
Filtration
Particulates should be removed prior to injection because of their adverse effect on column life.
There are several ways to remove the particulates, they are; fitration, centrifugation, and sedimentation. However, many HPLC users prefer disposable filters equipped with Luer fittings.
Here, the sample is placed in a syringe and filtered through the membrane using gentle pressure.
For most samples encountered in HPLC, filters in the range 0.25- to 2-µm nominal porosity are recommended.
4.4 SAMPLE PRETREATMENT FOR LIQUID SAMPLES Liquid—Liquid Extraction
Liquid—liquid extraction (LLE) is useful for separating analytes from interferences by partitioning the sample between two immiscible liquids or phases. One phase in LLE often is aqueous and the second phase an organic solvent. More-hydrophilic compounds prefer the polar aqueous phase, whereas morehydrophobic compounds will be found mainly in the organic solvent. Analytes extracted into the organic phase are easily recovered by evaporation of the solvent, while analytes extracted into the aqueous phase can often be injected directly onto a reversed-phase HPLC column. The following discussion assumes that an analyte-is extracted into the organic phase from an aqueous sample but similar approaches are used when the analyte is extracted into an aqueous phase. The The LLE organic solvent is chosen for the following characteristics:
Solid Phase Extraction