Distorted Chromatographic Peaks Stephen J. Hawkes Oregon State University, Cowallis, OR 97331 Analytical texts usually give little attention to the subject of distorted peaks although their cause and cure are important to any student who will make use of chromatography. Completely symmetric peaks never occur even in theory although they are a close approximation for welldesigned chromatographs with well-chosen columns and uncom~licatedanalvtes. ~isckssionsin textbooks of chromatography tend to be either too analvtical to be com~rehensibleto service course students, e.g. ( I ) ,or too brief for them to conceptualize, e.g. (21, (31.
This review discusses the cause and cure of nonideal peaks in s i m ~ l terms e that can be conveved to students in analytical service courses who know little or no physical chemistw. More thoroueh and mathematical treatments have been published an2 are referenced below, but they tend to obscure what are basically simple phenomena. Large Sample If the sample injected is too large, a number of things may happen to cause skewing. First, it may fail to dissolve completely in the stationary phase or, in the case of a n adsorbent, will adsorb on every adsorption site and still have molecules that can find no place to adsorb. I n these cases some is swept forward that ideally should have dissolved causing a peak to "front" as in Figure 1.This and the less extreme variant of it that we describe next are the only causes of "fronting". Usually asymmetry occurs on the other side-the rear-of the peak. . . a situation called "tailing". A variant of this problem occurs when the sample dissolves or adsorbs completely but is so large that it changes the properties of the stationary phase while it is dissolved or adsorbed. This may cause either "fronting" or "tailing". The sample spreads out a s i t moves along the column so the c ~ n c ~ n t r a t i o gets n smaller. Thus, this problem is a t its worst a t the beginning - of the column near the injection port. Texts often deal with this by showing the nonideal isotherms that give nonideal peaks, e.g. (4,51. The bridge between these concepts is too difficult for most students, especially those who have no physical chemistry background. This problem can obviously be overcome by using a smaller sample. Alternatively, the conditions may be altered to keep more of the analyte in the mobile phase so the stationary phase is not overloaded. I n gas chromatography this means raising the temperature. A more powerful solvent is used a s mobile phase in liquid chromatography If it is convenient to change the column, then a column with more stationary liquid or a n adsorbent with greater surface area will resolve the problem. An example of column overload is given in (61, where nadolol in HPLC gives constant peak height above about 0.5 mg/mL while maintaining constant peak area. Heterogeneous Adsorption When a n adsorbent contains a n impurity t h a t is a stroneer adsorbent for the analvte. a tail results. This mav also cappen when the solid suppok for a liquid phase has adsorbent properties.
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Journal of Chemical Education
The cause may be understood by considering the extreme case of a n impurity that adsorbs the analyte permanently and never releases it. Most of the analyte passes through the column adsorbing and desorbing on the main adsorbent, producing a nearly symmetric peak but leaving behind a trail of molecules permanently adsorbed on the rare impurity sites. Now consider the less extreme case where the molecules are not permanently adsorbed but desorb much more slowly than from the main adsorbent sites. They are still left behind the main peak but do move along the column slowlv and will cause the main ~ e a to k trail asvm~toticallv . . into the hnsrlin6: ;IS in Figurc 2. This tall is sometimes so lonl: that n suhstmtial frxtion of rhc peak is hldden in a trailing portlon of the peak where the concentration is so from thl: bawlinr. Oh\+ weak that it 8s indistinm~iihable ously, this causes quantitative errors, but more insidiously it may cause ghost peaks if the flow is stopped. The adsorbed material slowly desorbs and when the flow is restarted it causes a new peak, which is smaller than the main peak and similarly tailed. Stopping the flow after the peak has eluted, restarting it and finding the tail a s a new peak is the only known method of detecting the problem. The effect can be reduced by including in the mobile phase a trace of some substance that adsorbs more strongly than the analyte. This covers the strong sites and prevents the analyte from absorbing on them. The mathematics of this phenomenon is given by Giddings ( I ) .
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Poor Desian of Flow Svstem If the inlet system has dead corners where there is no flow, sample may enter them by diffusion and remain there for whiie afteE most of the sample has been swept into the column. This causes a tail on the peak. This also happens when the diameter of the inlet or outlet system is too wide so that the regions of slower flow near the walls act in the same way a s the dead corners mentioned above.
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Figure 1. Fronted peak from column overload.
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Figure 2. Distorted peak from adsorption on two types of adsorption site on the same stationary phase.
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Figure 3. Double peak caused by chemical equilibrium between two forms of the same analyte. When using poorly designed chromatographs like this, the problem may be overcome by using a column with a very retentive stationary phase so that samples move slowly enough that the delay time from the dead pockets is too small to be important. The price is that analyses take longer to complete. This is mentioned by Giddings (2). Slow Electronics If the recorder or detector responds too slowly, the top of the peak will not be reached before the signal starts to go down and the tail of the oeak will be higher .. than it should he. This causes a tail on the peak and may causc poor resolution ol'the simals from two veaks. It also makes the retention time appear to be greater than it is because the peak maximum is later than the arrival of the zone center in the detector. Curiously, it has little effect on the area of the peak since the width goes up a s the height goes down and the effects nearly cancel. The effect is rare in modern chromatographs where the components have been designed to have a n adequate response time, but sometimes happens with the earliest peaks of a fast chromatogram. The effect is covered in basic texts on electronics, e.g. (7). The constant peak area is illustrated by the example in (6) cited earlier. Chemical Equilibrium When a substance changes its chemical form during an analysis the two forms have different retention times. If they are in equilibrium with each other the peaks are blurred together a s in Figure 3 or often simply blurred into one long trailing peak. Acids are the commonest cases of this. In gas chromatography they dimerize thus Z C H , C O ~ H(CH,CO,H), ~
eivine neaks for the monomeric CH.CO.H .. - and the dimer 1 i . In liquid chromatography they dissociate substantially if the pH is unfortuniitcly chosen thus C H , C O ~ HH+ ~ + CH,CO,-
and the CH3C0,H and the CH3C02 give separate peaks blurring into each other. A case where this is commonly seen is in the amino acids where the pH is changed during the analysis. The amino acids then adsorb differently on the stationary phase. I t is hard to make the pH changes a t the right time and to the right extent to avoid blurring peaks with the equilibrium phenomenon. This equilibration system is briefly discussed by Giddings ( I ) . Slow Injection If a sample evaporates too slowly i n a gas chromatograph the peak will form a tail. This is cured by a higher temperature in the inlet system, or a slower flow rate. Short or Inefficient Column The rear of a peak is in the column longer than the front and is exposed longer to the mechanisms that spread the
sample along the column. Inevitably this causes a tail on the oeak. The shorter the column the more aooarent this is. 1; many texts this is stated in the form t& the equak to Gauss' eauation for a normal distrition to a ~ e a tends bution a s retention time becomes large. E v e n cause of zone s ~ r e a d i n increases e this asvmmetrv becaus"e each one increases t c e extra broadening in thk rear of the oeak. Such imoerfections are listed bv Giddines e (2) and by ~ o o l eand ~ o o i (3). Nonlinear Detection System A peak that saturates the detection system or is high enough to reach the nonlinear portion of the detector's ranee will have a flattened ton. Examnles of nonlinear svsterns in HPLC are given in though with no i l l u s t r a t b of the resultine ~ e a skh a ~ ebecause . this was not the subject of the They sdow that the problem is cured by using a more linear system. Obviously, it also could be cured by a smaller sample.
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Exam Questions The following will test students' understanding rather than passive memorization. 1. What could you do to find out whether asymmetry were caused by (a) too large a peak, (b) heterogeneous equilibrium. i d slaw electronics? 2. ~hnr'wouldhe rhreFTrrt nfn slowrrflow rate on asymmetry of a peak caused hy ,a, slow rlcrtronirs, rb poor How
design, (c) heterogeneous adsorption, (dl slow injection, (4chemical equilibrium? 3. What alterations to the controls of a chromatograph could perhaps improve the symmetry of a peak? Under what circumstances would thev be successful? 4. \
